Protease-activated receptor 2 (PAR2) is a seven-transmembrane, G-protein-coupled receptor that is activated by coagulation proteases such as factor VIIa and factor Xa and other serine proteases. It is a potential therapeutic target for kidney injury, as it enhances inflammatory and fibrotic responses via the nuclear factor-kappa B and mitogen-activated protein kinase cascades. The body of knowledge regarding the role of PAR2 in kidney disease is currently growing, and its role in various kidney disease models, such as acute kidney injury, renal fibrosis, diabetic kidney disease, aging, and thrombotic microangiopathy, has been reported. Here, we review the literature to better understand the various aspects of PAR2 in kidney disease.

AA amyloidosis is a rare renal complication of Waldenström's macroglobulinemia/lymphoplasmacytic lymphoma (WM/LPL). A 66-year-old man with WM/LPL presented with nephrotic syndrome. A renal biopsy showed AA amyloidosis. Chemotherapy resulted in the remission of hematologic and nephrotic syndromes. Two years into follow-up, he became infected with COVID-19 and had massive proteinuria, despite no relapse of WM/LPL. A second renal biopsy confirmed a diagnosis of AA amyloidosis. However, increased prednisolone did not improve proteinuria. The patient ultimately died of cryptococcal meningitis. This case highlights the diverse spectrum of renal involvement in monoclonal IgM-secreting diseases and difficulty in managing fatal complications.

BACKGROUND: Chronic kidney disease (CKD) causes progressive and irreversible damage to the kidneys. Renal biopsies are essential for diagnosing the etiology and prognosis of CKD, while accurate quantification of tubulo-interstitial injuries from whole slide images (WSIs) of renal biopsy specimens is challenging with visual inspection alone. METHODS: We develop a deep learning-based method named DLRS to quantify interstitial fibrosis and inflammatory cell infiltration as tubulo-interstitial injury scores, from WSIs of renal biopsy specimens. DLRS segments WSIs into non-tissue areas, glomeruli, tubules, interstitium, and arteries, and detects interstitial nuclei. It then quantifies these tubulo-interstitial injury scores using the segmented tissues and detected nuclei. RESULTS: Applied to WSIs from 71 Japanese CKD patients with diabetic nephropathy or benign nephrosclerosis, DLRS-derived scores show concordance with nephrologists' evaluations. Notably, the DLRS-derived fibrosis score has a higher correlation with the estimated glomerular filtration rate (eGFR) at biopsy than scores from nephrologists' evaluations. Validated on WSIs from 28 Japanese tubulointerstitial nephritis patients and 49 European-ancestry patients with nephrosclerosis, DLRS-derived scores show a significant correlation with eGFR. In an expanded analysis of 238 Japanese CKD patients, including 167 from another hospital, deviations in eGFR from expected values based on DLRS-derived scores correlate with annual eGFR decline after biopsy. Inclusion of these deviations and DLRS-derived fibrosis scores improve predictions of the annual eGFR decline. CONCLUSIONS: DLRS-derived tubulo-interstitial injury scores are concordant with nephrologists' evaluations and correlated with eGFR across different populations and institutions. The effectiveness of DLRS-derived scores for predicting annual eGFR decline highlights the potential of DLRS as a predictor of renal prognosis.

Anemia is a hallmark of chronic kidney disease (CKD), worsens with disease progression, and profoundly affects a patient's well-being. Major pathogenic factors are inadequate kidney erythropoietin (EPO) production and absolute and functional iron deficiency. The 2 mainstays of current anemia treatment are a) replacement therapy with recombinant EPO or 1 of its glycosylated derivatives, administered subcutaneously or intravenously, and b) intravenous (IV) iron injections. Over the past 5 years, hypoxia-inducible factor (HIF)-prolyl hydroxylase inhibitors (HIF-PHIs) have been approved in many countries for the management of anemia in both nondialysis and dialysis-dependent patients with CKD. Due to cardiovascular safety concerns, only 2 HIF-PHIs, daprodustat and vadadustat, have been approved for marketing in the United States, and only for patients on maintenance dialysis. HIF-PHIs are oral agents that are effective at improving and maintaining hemoglobin levels by activating HIF signaling in anemic patients with CKD. They stimulate the production of endogenous EPO, increase total iron-binding capacity through their direct effects on transferrin gene transcription, lower plasma hepcidin indirectly, and have beneficial effects on red blood cell parameters. Here, we discuss the mechanisms of action and pharmacologic properties of different HIF-PHIs. We discuss unwanted on-target and off-target effects, review cardiovascular and other safety concerns, and provide a benefit/risk-based perspective on how this new class of oral drugs might impact current anemia management in CKD. A clinical case is presented that highlights the clinical complexities and therapeutic challenges in managing anemia in CKD.

Low birthweight (LBW) increases the risk of adult-onset diseases, including kidney diseases, with intergenerational consequences; however, the underlying mechanisms and effective interventions are unclear. To examine the cross-generational effects of LBW, we established an LBW mouse model through reduced uterine perfusion pressure (RUPP) and investigated the therapeutic potential of tadalafil, a phosphodiesterase 5 inhibitor, on LBW-associated consequences. RUPP-pups (R1) had lower fetal and birth weights, delayed renal development, and fewer glomeruli than Sham-pups. In adulthood, R1 mice exhibited persistently fewer glomeruli and elevated blood pressure, while Tadalafil-R1 mice showed reduced hypertension in both sexes and improved renal pathological changes in males. Additionally, pregnant R1 mice displayed inadequate gestational liver hypertrophy, impaired hepatic purine metabolism, and diminished placental angiogenesis, resulting in fetal growth restriction in the subsequent generation. These findings underscore the lasting impact of LBW on adult health and future generations and suggest tadalafil's potential to mitigate LBW-associated risks.

This study aimed to focus on the role of radiologists in the diagnosis and management of adrenal lesions, particularly primary aldosteronism (PA) and secondary hypertension. As hypertension affects more than one-third of the population in Japan, identifying secondary causes such as PA and adrenal lesions is crucial. Establishing a radiological differential diagnosis of adrenal lesions using advanced imaging techniques, such as computed tomography and magnetic resonance imaging, is crucial. Knowledge of the imaging findings of various benign and malignant adrenal lesions, such as adrenocortical adenomas, cortisol-producing lesions, pheochromocytomas, adrenocortical carcinoma, malignant lymphoma, and metastatic tumors, is necessary. Adrenal venous sampling (AVS) plays a crucial role in accurately localizing aldosterone hypersecretion in PA, especially when imaging fails to provide a clear diagnosis. This paper details the technical aspects of AVS, emphasizing catheterization techniques, anatomical considerations, and the importance of preprocedural imaging for successful sampling. Furthermore, we explore segmental adrenal venous sampling (SAVS), a more refined technique that samples specific adrenal tributary veins, offering enhanced diagnostic accuracy, particularly for microadenomas or challenging cases that may be missed with conventional AVS. The methodology for performing SAVS, along with the interpretation criteria for successful sampling and lateralization, is also outlined. Furthermore, radiologists have initiated treatments for unilateral PA, such as radiofrequency ablation, and play an integral role in the management of adrenal lesions. Collaborative approaches across clinical departments are required to enhance patient management in medical care involving the adrenal gland.

BACKGROUND: This study aimed to assess the prognosis of people with chronic kidney disease (CKD) in Japan using the Kidney Disease: Improving Global Outcomes (KDIGO) heatmap. METHODS: The prognoses of individuals with estimated glomerular filtration rates (eGFR) <90 mL/min/1.73 m2 were evaluated based on the KDIGO heatmap using an electronic medical record database in Japan. The primary outcome was major adverse cardiovascular events (MACE), a composite of myocardial infarction (MI), stroke, heart failure (HF) hospitalization and in-hospital death (referred to as MACE1). Additionally, ad hoc MACE2 (MI hospitalization, stroke hospitalization, HF hospitalization and in-hospital death) was examined. The secondary outcome was the renal outcome. RESULTS: Of the 543 606 individuals included, the mean age was 61.6 ± 15.3 years, 50.1% were male and 40.9% lacked urine protein results. The risk of MACEs increased independently with both eGFR decline and increasing proteinuria from the early KDIGO stages: hazard ratios (95% confidence interval) of MACE1 and MACE2, compared with G2A1 were 1.16 (1.12-1.20) and 1.17 (1.11-1.23), respectively, for G3aA1, and 1.17 (1.12-1.21) and 1.35 (1.28-1.43), respectively, for G2A2. This increased up to 2.83 (2.54-3.15) and 3.43 (3.00-3.93), respectively, for G5A3. Risks of renal outcomes also increased with CKD progression. CONCLUSIONS: This study is the first to demonstrate the applicability of the KDIGO heatmap in assessing cardiovascular and renal risk in Japan. The risk increased from the early stages of CKD, indicating the importance of early diagnosis and intervention through appropriate testing.

INTRODUCTION: The breakthrough in erythropoietin-stimulating agents in the 1990s improved the prognosis and treatment of complications in chronic kidney disease patients and renal anemia. Discovery of the novel molecular mechanisms for hypoxia-inducible factor (HIF) transcription factor under hypoxic conditions has led to the development of oral drugs, HIF-Prolyl Hydroxylase inhibitors (HIF-PHIs), that constantly activate erythropoietin by inhibiting prolyl hydroxylase. HIF-PHIs have gained rapid approval in Asian countries, including Japan, with six distinct types entering clinical application. AREAS COVERED: This article provides a comprehensive review of the latest literature, with a particular focus on the effectiveness and safety of vadadustat. EXPERT OPINION: A phase 3, randomized, open-label, clinical trial (PRO2TECT) demonstrated that vadadustat had the prespecified non-inferiority for hematologic efficacy as compared with darbepoetin alfa in non-dialysis-dependent patients not previously treated with ESA. However, vadadustat did not show non-inferiority in major adverse cardiovascular events in the non-US/non-Europe patients. It may partly because of imbalances of the baseline eGFR level in those countries. In dialysis-dependent patients, a phase 3 clinical trial (INNO2VATE) showed vadadustat was non-inferior to darbepoietin alfa in cardiovascular safety and maintenance of hemoglobin levels. Adverse events including cancer, retinopathy, thrombosis, and vascular calcification should be evaluated in future clinical studies.

BACKGROUND: The beneficial effects of oral supplements with alkalinizing agents in patients with chronic kidney disease (CKD) have been limited to the severe stages. We investigated whether two types of supplements, sodium bicarbonate (SB) and potassium citrate/sodium citrate (PCSC), could maintain renal function in patients with mild-stage CKD. METHODS: This was a single-center, open-labeled, randomized cohort trial. Study participants with CKD stages G2, G3a, and G3b were enrolled between March 2013 and January 2019 and randomly assigned by stratification according to age, sex, estimated glomerular filtration rate (eGFR), and diabetes. They were followed up for 6 months (short-term study) for the primary endpoints and extended to 2 years (long-term study) for the secondary endpoints. Supplementary doses were adjusted to achieve an early morning urinary pH of 6.8-7.2. We observed renal dysfunction or new-onset cerebrovascular disease and evaluated urinary surrogate markers for renal injury. RESULTS: Overall, 101 participants were registered and allocated to three groups: standard (n = 32), SB (n = 34), and PCSC (n = 35). Two patients in the standard group attained the primary endpoints (renal stones and overt proteinuria) but were not statistically significant. There was one patient in the standard reduced eGFR during the long-term study (p = 0.042 by ANOVA). SB increased proteinuria (p = 0.0139, baseline vs. 6 months), whereas PCSC significantly reduced proteinuria (p = 0.0061, baseline vs. 1 year, or p = 0.0186, vs. 2 years) and urinary excretion of 8-hydroxy-2'-deoxyguanosine (p = 0.0481, baseline vs. 6 months). CONCLUSION: This study is the first to report supplementation of PCSC reduced intrarenal oxidative stress in patients with mild-stage CKD.

To maintain the oxygen supply, the production of red blood cells (erythrocytes) is promoted under low-oxygen conditions (hypoxia). Oxygen is carried by hemoglobin in erythrocytes, in which the majority of the essential element iron in the body is contained. Because iron metabolism is strictly controlled in a semi-closed recycling system to protect cells from oxidative stress caused by iron, hypoxia-inducible erythropoiesis is closely coordinated by regulatory systems that mobilize stored iron for hemoglobin synthesis. The erythroid growth factor erythropoietin (EPO) is mainly secreted by interstitial fibroblasts in the renal cortex, which are known as renal EPO-producing (REP) cells, and promotes erythropoiesis and iron mobilization. Intriguingly, EPO production is strongly induced by hypoxia through iron-dependent pathways in REP cells. Here, we summarize recent studies on the network mechanisms linking hypoxia-inducible EPO production, erythropoiesis and iron metabolism. Additionally, we introduce disease mechanisms related to disorders in the network mediated by REP cell functions. Furthermore, we propose future studies regarding the application of renal cells derived from the urine of kidney disease patients to investigate the molecular pathology of chronic kidney disease and develop precise and personalized medicine for kidney disease.

CONTEXT: Detecting patients with surgically curable aldosterone-producing adenoma (APA) among hypertensive individuals is clinically pivotal. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) is the ideal method of measuring plasma aldosterone concentration (PAC) because of the inaccuracy of conventional chemiluminescent enzyme immunoassay (CLEIA). However, LC-MS/MS is expensive and requires expertise. We have developed a novel noncompetitive CLEIA (NC-CLEIA) for measuring PAC in 30 minutes. OBJECTIVE: This work aimed to validate NC-CLEIA PAC measurements by comparing them with LC-MS/MS measurements and determining screening cutoffs for both measurements detecting APA. METHODS: We retrospectively measured PAC using LC-MS/MS and NC-CLEIA in 133 patients with APA, 100 with bilateral hyperaldosteronism, and 111 with essential hypertension to explore the accuracy of NC-CLEIA PAC measurements by comparing with LC-MS/MS measurements and determined the cutoffs for detecting APA. RESULTS: Passing-Bablok analysis revealed that the values by NC-CLEIA (the regression slope, intercept, and correlation coefficient were 0.962, -0.043, and 0.994, respectively) were significantly correlated and equivalent to those by LC-MS/MS. Bland-Altman plot analysis of NC-CLEIA and LC-MS/MS also demonstrated smaller systemic errors (a bias of -0.348 ng/dL with limits of agreement of -4.390 and 3.694 within a 95% CI) in NC-CLEIA than LC-MS/MS. The receiver operating characteristic analysis demonstrated that cutoff values for aldosterone/renin activity ratio obtained by LC-MS/MS and NC-CLEIA were 31.2 and 31.5 (ng/dL per ng/mL/hour), with a sensitivity of 91.0% and 90.2% and specificity of 75.4% and 76.8%, respectively, to differentiate APA from non-APA. CONCLUSION: This newly developed NC-CLEIA for measuring PAC could serve as a clinically reliable alternative to LC-MS/MS.

The measurement evolution enabled more accurate evaluation of aldosterone production in hypertensive patients. However, the cut-off values for novel assays have been not sufficiently validated. The present study was undertaken to validate the novel chemiluminescent enzyme immunoassay for aldosterone in conjunction with other methods. Moreover, we also aimed to establish a new cut-off value for primary aldosteronism in the captopril challenge test using the novel assay. First, we collected 390 plasma samples, in which aldosterone levels measured using liquid chromatography-mass spectrometry ranged between 0.18 and 1346 ng/dL. The novel chemiluminescent enzyme immunoassay showed identical correlation of plasma aldosterone with liquid chromatography-mass spectrometry, in contrast to conventional radioimmunoassay. Further, we enrolled 299 and 39 patients with primary aldosteronism and essential hypertension, respectively. Plasma aldosterone concentrations measured using the novel assay were lower than those measured by radioimmunoassay, which resulted in decreased aldosterone-to-renin ratios. Subsequently, positive results of the captopril challenge test based on radioimmunoassay turned into "negative" based on the novel assay in 45% patients with primary aldosteronism, using the conventional cut-off value (aldosterone-to-renin activity ratio > 20 ng/dL per ng/mL/h). Receiver operating characteristic curve analysis demonstrated that aldosterone-to-renin activity ratios > 8.2 ng/dL per ng/mL/h in the novel assay was compatible with the conventional diagnosis (sensitivity, 0.874; specificity, 0.980). Our study indicates the great measurement accuracy of the novel chemiluminescent enzyme immunoassay for aldosterone, and the importance of measurement-adjusted cut-offs in the diagnosis of primary aldosteronism.

Chronic kidney disease (CKD) guidelines recommend early identification and intervention to delay the progression of CKD. The Kidney Disease: Improving Global Outcomes (KDIGO) heatmap is widely used for risk evaluation in CKD management; however, real-world evidence on clinical characteristics based on the KDIGO heatmap remains limited worldwide including Japan. In order to understand the management of CKD including its diagnostic rates in a Japanese clinical setting on the basis of KDIGO heatmap, we utilized a medical record database that contains estimated glomerular filtration rate (eGFR) and urine protein data. Adult individuals (≥ 18 years) with two eGFR results of < 90 mL/min/1.73 m2, 90-360 days apart, were included. Approximately half of patients (452,996/788,059) had proteinuria test results and 6.9% (54,073) had quantitative results. CKD diagnosis rate in patients without proteinuria data was 5.9%, with a lower rate (2.9%) in stage G2; the corresponding rates with quantitative test results were 43.5% and 31.3%, respectively. The most frequent comorbidities were hypertension, diabetes, and cardiovascular disease, and their prevalence increased as the eGFR and proteinuria stages progressed. This study revealed a low rate of proteinuria assessment, especially using quantitative methods, and diagnosis in individuals with suspected CKD. With emerging treatment options to prevent CKD progression and complication onset, there is a need for early evaluation and diagnosis of CKD.

BACKGROUND: In systemic lupus erythematosus (SLE), autoreactive B cells are thought to develop by-passing immune checkpoints and contribute to its pathogenesis. Toll-like receptor (TLR) 7 and 9 signaling have been implicated in their development and differentiation. Although some B cell subpopulations such as T-bet + double negative 2 (DN2) cells have been identified as autoreactive in the past few years, because the upregulated surface markers of those cells are not exclusive to them, it is still challenging to specifically target autoreactive B cells in SLE patients. METHODS: Our preliminary expression analysis revealed that phospholipase D4 (PLD4) is exclusively expressed in plasmacytoid dendritic cells (pDCs) and B cells in peripheral blood mononuclear cells (PBMCs) samples. Monoclonal antibodies against human PLD4 were generated, and flow cytometry analyses were conducted for PBMCs from 23 healthy donors (HDs) and 40 patients with SLE. In vitro cell culture was also performed to study the conditions that induce PLD4 in B cells from HDs. Finally, recombinant antibodies were synthesized from subpopulations of PLD4 + B cells from a patient with SLE, and their antinuclear activity was measured through enzyme-linked immunosorbent assay. RESULTS: pDCs from both groups showed comparable frequency of surface PLD4 expression. PLD4 + B cells accounted for only a few percent of HD B cells, whereas they were significantly expanded in patients with SLE (2.1% ± 0.4% vs. 10.8% ± 1.2%, P < 0.005). A subpopulation within PLD4 + B cells whose cell size was comparable to CD38 + CD43 + plasmablasts was defined as "PLD4 + blasts," and their frequencies were significantly correlated with those of plasmablasts (P < 0.005). PLD4 + blasts phenotypically overlapped with double negative 2 (DN2) cells, and, in line with this, their frequencies were significantly correlated with several clinical markers of SLE. In vitro assay using healthy PBMCs demonstrated that TLR7 or TLR9 stimulation was sufficient to induce PLD4 on the surface of the B cells. Finally, two out of three recombinant antibodies synthesized from PLD4 + blasts showed antinuclear activity. CONCLUSION: PLD4 + B cells, especially "blastic" ones, are likely autoreactive B cells undergoing TLR stimulation. Therefore, PLD4 is a promising target marker in SLE treatment.

Epidemiological studies have shown that patients who recovered from acute kidney injury (AKI) may subsequently develop chronic kidney disease (CKD). AKI is primarily caused by renal hypoxia, and it causes epigenetic alterations, known as hypoxic memory. 3-Deazaneplanocin A (Dznep), an inhibitor of histone modification, suppresses renal fibrosis and the expression of tissue inhibitor of metalloproteinases-2 (TIMP2), a profibrotic factor, in mouse ischemia-reperfusion models. The current study investigated the epigenetic regulation of TIMP2 in human kidney 2 (HK-2) cells. The expression of TIMP2 was upregulated in HK-2 cells under hypoxic conditions and was suppressed by Dznep. ChIP-qPCR showed that Dznep reduced the amount of H3K4me3 at the promoter region of the TIMP2 gene under hypoxic condition. Formaldehyde-assisted isolation of regulatory elements-qPCR of the TIMP2 gene showed that Dznep reduced open chromatin area. In addition, based on ChIP-qPCR of hypoxia-inducible factor 1 alpha (HIF1α), Dznep inhibited the binding of HIF1α to the TIMP2 gene under hypoxic conditions. The reporter assays for the binding region of HIF1α showed enhanced transcriptional activity by hypoxia. Dznep suppresses the expression of TIMP2 under hypoxic conditions by inhibiting the binding of HIF1α to the TIMP2 gene.

Epigenetic modifications such as DNA methylation, histone modifications, and chromatin structures in the kidney contribute towards the progression of chronic kidney disease (CKD). In this study, the role of chromatin remodeling factor inositol requiring 80 (INO80) was investigated. Although INO80 regulates transcription by altering the chromatin structure at the nucleosome level, its role in the kidney remains unknown. We demonstrated that the expression of INO80 in impaired kidneys decreased in rats with unilateral urethral obstruction. We investigated INO80 expression in a proximal tubular cell line and observed that its expression decreased under hypoxic condition. Additionally, INO80 knockdown promoted apoptosis, suggesting that INO80 plays a role in inhibiting tubular cell apoptosis. We identified downstream target genes of INO80 via genome-wide analysis using RNA-sequences and found that the expression of apoptosis-related genes, such as TP53 and E2F1, and pro-apoptotic genes, such as PMAIP1, increased upon INO80 knockdown. ChIP-qPCR of the loci of PMAIP1 showed that the amount of H2A.Z. increased instead of decreasing the amount of H2A when INO80 was knocked down. These results indicated that INO80 plays a role in the exchange of H2A.Z. for H2A in the promoter region of PMAIP1 in tubular cells to inhibit apoptosis during CKD progression.

Sensitive biomarkers can enhance the diagnosis, prognosis, and surveillance of chronic kidney disease (CKD), such as diabetic kidney disease (DKD). Plasma growth differentiation factor 15 (GDF15) levels are a novel biomarker for mitochondria-associated diseases; however, it may not be a useful indicator for CKD as its levels increase with declining renal function. This study explores urinary GDF15's potential as a marker for CKD. The plasma and urinary GDF15 as well as 15 uremic toxins were measured in 103 patients with CKD. The relationship between the urinary GDF15-creatinine ratio and the uremic toxins and other clinical characteristics was investigated. Urinary GDF15-creatinine ratios were less related to renal function and uremic toxin levels compared to plasma GDF15. Additionally, the ratios were significantly higher in patients with CKD patients with diabetes (p = 0.0012) and reduced with statin treatment. In a different retrospective DKD cohort study (U-CARE, n = 342), multiple and logistic regression analyses revealed that the baseline urinary GDF15-creatinine ratios predicted a decline in estimated glomerular filtration rate (eGFR) over 2 years. Compared to the plasma GDF15 level, the urinary GDF15-creatinine ratio is less dependent on renal function and sensitively fluctuates with diabetes and statin treatment. It may serve as a good prognostic marker for renal function decline in patients with DKD similar to the urine albumin-creatinine ratio.

Chronic hypoxia in the renal tubular interstitium has been reported to contribute to the progression of chronic kidney disease (CKD). Recently, long non-coding RNAs (lncRNAs) have been shown to be involved in various pathological conditions, including hypoxia, one of which is the MIR210 host gene (MIR210HG). To elucidate the function of MIR210HG in renal hypoxia, we exposed primary cultured renal proximal tubular epithelial cells to hypoxia and examined the temporal profile of MIR210HG expression and the role of MIR210HG interaction with hypoxia-inducible factor1α (HIF1α, encoded by HIF1A). MIR210HG expression was induced by hypoxia. HIF1A silencing and cobalt chloride exposure showed that MIR210HG expression in hypoxia is HIF1α dependent. MIR210HG silencing significantly reduced both the mRNA and protein levels of HIF1α, pointing to positive feedback regulation. To further investigate the details of this regulation, we turned to the in-silico miRNA targets of MIR210HG. We found that miR-93-5p levels increased when MIR210HG was knocked down. We then showed that miR-93-5p reduced the expression of HIF1A mRNA and MIR210HG. Furthermore, a dual luciferase assay confirmed that miR-93-5p binds to MIR210HG and HIF1A 3' UTR, inhibiting their expression. In conclusion, the lncRNA MIR210HG is induced shortly after hypoxia, and it promotes HIF1α expression by competing for miR-93-5p and inhibiting it. MIR210HG plays a crucial role in the biological response to hypoxia in renal tubular epithelial cells.

Diabetic kidney disease (DKD) is the leading cause of chronic kidney disease, including end-stage kidney disease, and increases the risk of cardiovascular mortality. Although the treatment options for DKD, including angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, sodium-glucose cotransporter 2 inhibitors, and mineralocorticoid receptor antagonists, have advanced, their efficacy is still limited. Thus, a deeper understanding of the molecular mechanisms of DKD onset and progression is necessary for the development of new and innovative treatments for DKD. The complex pathogenesis of DKD includes various different pathways, and the mechanisms of DKD can be broadly classified into inflammatory, fibrotic, metabolic, and hemodynamic factors. Here, we summarize the recent findings in basic research, focusing on each factor and recent advances in the treatment of DKD. Collective evidence from basic and clinical research studies is helpful for understanding the definitive mechanisms of DKD and their regulatory systems. Further comprehensive exploration is warranted to advance our knowledge of the pathogenesis of DKD and establish novel treatments and preventive strategies.

BACKGROUND: Interstitial nephritis is a common cause of renal failure. Gallium-67 scintigraphy is reportedly useful for diagnosing interstitial nephritis; however, its ability to assess disease activity remains unknown. We aimed to analyze the relationship between the renal uptake of gallium-67 and the disease activity in interstitial nephritis. METHODS: We retrospectively analyzed the data of patients who underwent gallium-67 scintigraphy at a hospital in Tokyo. The renal uptake adjusted for the soft tissues beneath the kidneys was semi-quantitatively evaluated. We compared the renal uptake levels between patients clinically diagnosed with and without interstitial nephritis. Among those undergoing renal biopsy, we evaluated the predictive ability of gallium-67 scintigraphy and analyzed the renal uptake levels regarding the disease activity through a histopathological analysis. RESULTS: We included 143 patients; among them, 30, 17, and 96 patients were clinically diagnosed with interstitial nephritis, other kidney diseases, and non-kidney diseases, respectively. The renal uptake of gallium-67 was the highest among patients with interstitial nephritis. Among the 25 patients who underwent renal biopsy, 15 were pathologically diagnosed with interstitial nephritis. The renal uptake levels showed a high discriminative ability (C-statistic: 0.83). Furthermore, net reclassification improvement with the addition of gallium-67 scintigraphy to N-acetyl-β-D-glucosaminidase for the prediction of interstitial nephritis was 1.14. Histopathological analysis revealed a positive correlation between renal uptake and inflammation in the cortex and peritubular capillaries. CONCLUSIONS: This study confirmed the diagnostic value and potential usefulness of gallium-67 scintigraphy for evaluating interstitial nephritis.

Podocytes, localized in the glomerulus, are a prognostic factor of proteinuria in kidney disease and are exposed to distinct physiological stimuli from basal to apical filtration flow. Research studies on drug discovery and disease modeling for glomerulopathy have developed a glomerulus-on-a-chip and studied podocyte mechanobiology to realize alternative methods to animal experiments. However, the effect of filtration stimulus on podocytes has remained unclear. Herein, we report the successful development of a user-friendly filtration culture device and system that can precisely control the filtration flow using air pressure control by incorporating a commercially available culture insert. It allows mouse podocytes to be cultured under filtration conditions for three days with a guarantee of maintaining the integrity of the podocyte layer. Using our system, this study demonstrated that podocyte damage caused by hyperfiltration resulting from glomerular hypertension, a common pathophysiology of many glomerulopathies, was successfully recapitulated and that filtration stimulus promotes the maturation of podocytes in terms of their morphology and gene expression. Furthermore, we demonstrated that filtration stimulus induced different drug responsiveness in podocytes than those seen under static conditions, and that the difference in drug responsiveness was dependent on the pharmacological mechanism. Overall, this study has revealed differentiating and pharmacodynamic properties of filtration stimulus and brings new insights into the research field of podocyte mechanobiology towards the realization of glomerulus-on-a-chip.

Chronic kidney disease (CKD) affects kidney cancer patients' mortality. However, the underlying mechanism remains unknown. M2-like macrophages have pro-tumor functions, also exist in injured kidney, and promote kidney fibrosis. Thus, it is suspected that M2-like macrophages in injured kidney induce the pro-tumor microenvironment leading to kidney cancer progression. We found that M2-like macrophages present in the injured kidney promoted kidney cancer progression and induced resistance to anti-PD1 antibody through its pro-tumor function and inhibition of CD8+ T cell infiltration. RNA-seq revealed Slc7a11 was upregulated in M2-like macrophages. Inhibition of Slc7a11 with sulfasalazine inhibited the pro-tumor function of M2-like macrophages and synergized with anti-PD1 antibody. Moreover, SLC7A11-positive macrophages were associated with poor prognosis among kidney cancer patients. Collectively, this study dissects the characteristic microenvironment in the injured kidney that contributed to kidney cancer progression and anti-PD1 antibody resistance. This insight offers promising combination therapy with anti-PD1 antibody and macrophage targeted therapy.

For the past 3 decades, erythropoiesis-stimulating agents (ESA) in conjunction with iron supplementation has been the mainstay of treatment for anemia in chronic kidney disease (CKD). Although ESAs are well-established and highly efficacious treatment, clinical trials demonstrated that the use of ESAs with a high hemoglobin (Hb) target was associated with increased risk of cardiovascular events. This safety concern raised considerable interest in developing an alternative therapeutic strategy. Hypoxia-inducible factor-prolyl hydroxylase inhibitors (HIF-PHIs) are such novel agents to treat anemia in CKD. They stimulate endogenous erythropoietin production via HIF activation and thereby induce erythropoiesis. At least 6 small-molecule HIF-PHIs have been developed to date. The phase 3 clinical trials demonstrated that their effects were noninferior to ESAs. HIF-PHIs may have several advantages over the conventional treatment, such as oral route of administration and their ability to raise Hb levels in patients with chronic inflammation. Although many of the phase 3 clinical trials demonstrated that HIF-PHIs were noninferior to placebo or ESAs with respect to cardiovascular safety, one of the compounds failed to meet the prespecified noninferiority criterion in non-dialysis-dependent CKD patients, and some studies of another HIF-PHI indicated potential risks for thromboembolic events. While the regulatory agencies of some countries including Japan and the European Union concluded that roxadustat, one of the HIF-PHIs, had a favorable benefit-risk profile, the U.S. Food and Drug Administration decided not to approve the drug because of safety reasons. In order to establish the optimal anemia management in CKD, further studies are needed to evaluate important aspects of HIF-PHIs, such as long-term safety, appropriate Hb target, and the types of patients who would gain benefits from these new drugs.

Diabetic kidney disease is the leading cause of end-stage kidney disease, and it remains a major challenge. Many factors, such as glomerular hyperfiltration, oxidative stress, inflammation, hypoxia, and epigenetics, are associated with the progression of diabetic kidney disease; however, the whole mechanism is not yet completely understood. No specific treatment for diabetic kidney disease has been established, so new approaches are being explored extensively. Sodium-glucose cotransporter 2 inhibitors have shown renoprotective effects in several human clinical trials. Glucagon-like peptide 1 receptor agonists and mineralocorticoid receptor antagonists have been reported to be effective in diabetic kidney disease, and novel therapeutic candidates are also being examined. In the TSUBAKI trial, a nuclear factor erythroid 2-related factor 2 activator, bardoxolone methyl, improved the glomerular filtration rate of diabetic kidney disease patients. Similarly, new agents that act in the oxidative stress and inflammation pathways are of major interest, such as pentoxifylline, apoptosis signal-regulating kinase-1 inhibitors, C-C chemokine receptor 2 inhibitors, and Janus kinase-1/2 inhibitors. Endothelin-1 receptor A antagonists and soluble guanylate cyclase stimulators are also expected to affect renal hemodynamics. Some preclinical studies suggest that hypoxia-inducible factor prolyl hydroxylase inhibitors, which influence multiple inflammations and oxidative stress pathways, reduce albuminuria in diabetic kidney disease. Advanced glycation end-product inhibitors and treatments related to epigenetics have also shown promise as potential diabetic kidney disease treatments in preclinical studies. The discovery of new targets could provide new therapeutic options for overcoming diabetic kidney disease.

Cholinergic anti-inflammatory pathway (CAP) describes a neuronal-inflammatory reflex centered on systemic cytokine regulation by α7 nicotinic acetylcholine receptor (α7nAChR) activation of spleen-residue macrophage. However, the CAP mechanism attenuating distal tissue inflammation, inducing a low level of systemic inflammation, is lesser known. In this study, we hypothesized that CAP regulates monocyte accessibility by influencing their adhesion to endothelial cells. Using RNA-seq analysis, we identified that α1,3-Fucosyltransferase 7 (FucT-VII), the enzyme required for processing selectin ligands, was significantly downregulated by α7nAChR agonist among other cell-cell adhesion genes. The α7nAChR agonist inhibited monocytic cell line U-937 binding to P-selectin and adhesion to endothelial cells. Furthermore, α7nAChR agonist selectivity was confirmed by α7nAChR knockdown assays, showing that FUT7 inhibition and adhesion attenuation by the agonist was abolished by siRNA targeting α7nAChR encoding gene. Consistently, FUT7 knockdown inhibited the adhesive properties of U-937 and prevented them to adhere to endothelial cells. Overexpression of FUT7 also abrogated the adhesion attenuation induced by GTS-21 indicating that FUT7 inhibition was sufficient for inhibiting adhesion by α7nAChR activation. Our work demonstrated that α7nAChR activation regulates monocyte adhesion to endothelial cells through FUT7 inhibition, providing a novel insight into the CAP mechanism.

TRPM2 is a Ca2+-permeable cationic channel and serves as an oxidative stress sensor.TRPM2 deletion was harmful in renal ischemia-reperfusion injury, whereas TRPM2 deletion mitigated kidney fibrosis.Our findings suggest the role of TRPM2 in kidney diseases is context dependent.

INTRODUCTION: Anemia is one of the major complications of chronic kidney disease (CKD). Erythropoiesis-stimulating agents (ESAs) have been the mainstay of renal anemia treatment. However, there are several safety drawbacks, and a safer and more effective alternative treatment has been sought. AREAS COVERED: Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) have been developed as a novel orally active therapeutic agent for renal anemia. HIF-PHIs stimulate endogenous EPO and optimize iron utilization. Roxadustat is a first-in-class HIF-PHI for the treatment of anemia in CKD patients approved in China, Japan, South Korea, and Chile. The authors herein evaluate the pharmacology of roxadustat and give their expert perspectives on its use. EXPERT OPINION: Phase 3 clinical trials have demonstrated that roxadustat effectively increases and maintains hemoglobin (Hb) levels in both nondialysis-dependent and dialysis-dependent CKD patients. Roxadustat also improved iron metabolism and reduced intravenous (IV) iron requirements. However, pooled analyses of phase 3 studies have revealed frequent thromboembolic events in the roxadustat group, which might be attributed to rapid changes in Hb and inadequate iron supplementation. Roxadustat is an attractive alternative treatment especially for patients with ESA hyporesponsive due to impaired iron utilization, and so appropriate selection of target patients and its proper use are crucially important.

Formation of processes in podocytes is regarded as the hallmark of maturity and normal physical condition for the cell. There are many accumulated findings about molecular mechanisms that cause retraction of podocyte processes; however, there is little knowledge of the positive mechanisms that promote process formation in vitro, and most previous reports about this topic have been limited to low-density cultures. Here, we found that process formation can be induced in 100% confluent cultures of conditionally immortalized podocytes in mouse, rat, and human species by combining serum depletion and Y-27632 ROCK inhibitor supplementation on the scaffold of laminin-521(L521). We noted the cytoskeletal reorganization of the radial extension pattern of vimentin filaments and downregulation of actin stress fiber formation under that condition. We also found that additional standard amount of serum, depletion of ROCK inhibitor, or slight mismatch of the scaffold as laminin-511(L511) hinder process formation. These findings suggest that the combination of reduced serum, podocyte-specific scaffold, and intracellular signaling to reduce the overexpression of ROCK are required factors for process formation.

Diabetic kidney disease (DKD) is a major cause of end-stage kidney disease, and it is crucial to understand the pathophysiology of DKD. The control of blood glucose levels by various glucose-lowering drugs, the common use of inhibitors of the renin-angiotensin system, and the aging of patients with diabetes can alter the disease course of DKD. Moreover, metabolic changes and associated atherosclerosis play a major role in the etiology of DKD. The pathophysiology of DKD is largely attributed to the disruption of various cellular stress responses due to metabolic changes, especially an increase in oxidative stress. Therefore, many antioxidants have been studied as therapeutic agents. Recently, it has been found that NRF2, a master regulator of oxidative stress, plays a major role in the pathogenesis of DKD and bardoxolone methyl, an activator of NRF2, has attracted attention as a drug that increases the estimated glomerular filtration rate in patients with DKD. This review outlines the altered stress responses of cellular organelles in DKD, their involvement in the pathogenesis of DKD, and discusses strategies for developing therapeutic agents, especially bardoxolone methyl.

Diabetic kidney disease (DKD) is a chronic complication of diabetes mellitus which may eventually lead to end-stage kidney disease (ESKD). Despite improvements in glycaemic control and blood pressure management with renin-angiotensin-aldosterone system (RAAS) blockade, the current therapy cannot completely halt DKD progression to ESKD in some patients. DKD is a heterogeneous disease entity in terms of its clinical manifestations, histopathology and the rate of progression, which makes it difficult to develop effective therapeutics. It was formerly considered that albuminuria preceded kidney function decline in DKD, but recent epidemiological studies revealed that a distinct group of patients presented kidney dysfunction without developing albuminuria. Other comorbidities, such as hypertension, obesity and gout, also affect the clinical course of DKD. The pathophysiology of DKD is complex and multifactorial, involving both metabolic and haemodynamic factors. These induce activation of intracellular signalling pathways, oxidative stress, hypoxia, dysregulated autophagy and epigenetic changes, which result in kidney inflammation and fibrosis. Recently, two groups of antidiabetic drugs, sodium-glucose cotransporter 2 (SGLT2) inhibitors and glucagon-like peptide-1 (GLP-1) receptor agonists, were demonstrated to provide renoprotection on top of their glucose-lowering effects. Several other therapeutic agents are also being developed and evaluated in clinical trials.

In patients with chronic kidney disease, skeletal muscle dysfunction is associated with mortality. Uremic sarcopenia is caused by ageing, malnutrition, and chronic inflammation, but the molecular mechanism and potential therapeutics have not been fully elucidated yet. We hypothesize that accumulated uremic toxins might exert a direct deteriorative effect on skeletal muscle and explore the pharmacological treatment in experimental animal and culture cell models. The mice intraperitoneally injected with indoxyl sulfate (IS) after unilateral nephrectomy displayed an elevation of IS concentration in skeletal muscle and a reduction of instantaneous muscle strength, along with the predominant loss of fast-twitch myofibers and intramuscular reactive oxygen species (ROS) generation. The addition of IS in the culture media decreased the size of fully differentiated mouse C2C12 myotubes as well. ROS accumulation and mitochondrial dysfunction were also noted. Next, the effect of the β2-adrenergic receptor (β2-AR) agonist, clenbuterol, was evaluated as a potential treatment for uremic sarcopenia. In mice injected with IS, clenbuterol treatment increased the muscle mass and restored the tissue ROS level but failed to improve muscle weakness. In C2C12 myotubes stimulated with IS, although β2-AR activation also attenuated myotube size reduction and ROS accumulation as did other anti-oxidant reagents, it failed to augment the mitochondrial membrane potential. In conclusion, IS provokes muscular strength loss (uremic dynapenia), ROS generation, and mitochondrial impairment. Although the β2-AR agonist can increase the muscular mass with ROS reduction, development of therapeutic interventions for restoring skeletal muscle function is still awaited.

Chronic hypoxia in the renal tubulointerstitium plays a key role in the progression of chronic kidney disease (CKD). It is therefore important to investigate tubular hypoxia and the activity of hypoxia-inducible factor (HIF)-1α in response to hypoxia. Rarefaction of the peritubular capillary causes hypoperfusion in CKD; however, the effect of hypoperfusion on HIFs has rarely been investigated. We induced hypoperfusion caused by coverslip placement in human kidney-2 cells, and observed an oxygen gradient under the coverslip. Immunocytochemistry of HIF-1α showed a doughnut-shaped formation on the edge of a pimonidazole-positive area, which we named the "HIF-ring". The oxygen tension of the HIF-ring was estimated to be between approximately 4 mmHg and 20 mmHg. This result was not compatible with those of past research showing HIF-1α accumulation in the anoxic range with homogeneous oxygen tension. We further observed the presence of a pH gradient under a coverslip, as well as a shift of the HIF ring due to changes in the pH of the culture medium, suggesting that the HIF ring was formed by suppression of HIF-1α related to low pH. This research demonstrated that HIF-1α activation mimics the physiological state in cultured cells with hypoperfusion.

Diabetic kidney disease (DKD) is the major cause of end-stage kidney disease. However, only renin-angiotensin system inhibitor with multidisciplinary treatments is effective for DKD. In 2019, sodium-glucose cotransporter 2 (SGLT2) inhibitor showed efficacy against DKD in Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) trial, adding a new treatment option. However, the progression of DKD has not been completely controlled. The patients with transient exposure to hyperglycemia develop diabetic complications, including DKD, even after normalization of their blood glucose. Temporary hyperglycemia causes advanced glycation end product (AGE) accumulations and epigenetic changes as metabolic memory. The drugs that improve metabolic memory are awaited, and AGE inhibitors and histone modification inhibitors are the focus of clinical and basic research. In addition, incretin-related drugs showed a renoprotective ability in many clinical trials, and these trials with renal outcome as their primary endpoint are currently ongoing. Hypoxia-inducible factor prolyl hydroxylase inhibitors recently approved for renal anemia may be renoprotective since they improve tubulointerstitial hypoxia. Furthermore, NF-E2-related factor 2 activators improved the glomerular filtration rate of DKD patients in Bardoxolone Methyl Treatment: Renal Function in chronic kidney disease/Type 2 Diabetes (BEAM) trial and Phase II Study of Bardoxolone Methyl in Patients with Chronic Kidney Disease and Type 2 Diabetes (TSUBAKI) trial. Thus, following SGLT2 inhibitor, numerous novel drugs could be utilized in treating DKD. Future studies are expected to provide new insights.

Anemia is a major complication of chronic kidney disease (CKD), which mainly results from appropriate erythropoietin production impairment. Prolyl hydroxylase domain (PHD) inhibitors are currently being developed and approved in some countries as a new treatment for CKD patients with anemia due to the stabilization of intracellular hypoxia-inducible factor (HIF) 1α and HIF2α by PHD inhibition. Daprodustat is one of the orally administrated small-molecule HIF-PH inhibitors, leading to an increase in erythropoietin production, which is regulated by HIF. Also, daprodustat is expected to improve iron metabolism. Recently, several clinical trials showed its efficacy and safety in both hemodialysis- and non-hemodialysis- dependent CKD patients. In addition, some international Phase 3 studies are underway to confirm these effects and reveal the safety profile. This article summarizes the development process and results of each clinical trial.

Anemia is a significant complication of chronic kidney disease (CKD), caused by erythropoietin deficiency and reduced iron availability. Erythropoiesis-stimulating agents have been used with iron supplementation to treat anemia; however, they are associated with some problems. Hypoxia-inducible factor prolyl hydroxylase inhibitor (HIF-PHI) is a promising new class of oral therapy for the treatment of anemia associated with CKD. HIF-PHI inhibits HIF-prolyl hydroxylase enzymes and results in the HIF-α accumulation, which leads to increased expression of HIF-responsive genes, including erythropoietin and vascular endothelial growth factor (VEGF). HIF stimulates endogenous erythropoietin production and also reduces circulating hepcidin concentrations, resulting in improved anemia. Many clinical trials demonstrate that HIF-PHI improves anemia in patients with CKD and on dialysis. In addition to treating anemia, HIF-PHI may have multiple potential effects. Several animal experiments show that HIF-PHI protects against ischemic kidney damage that progresses to CKD and also improves metabolic disorders and ameliorates cardiovascular complications. In contrast, malignant tumor and retinopathy should be carefully evaluated due to theoretical concerns that HIF stabilization may result in increased VEGF protein expression. Some adverse events such as shunt occlusion reported in large clinical trials also need attention and warrant further investigations.

PURPOSE OF REVIEW: Hypoxia-inducible factor prolyl hydroxylase inhibitors (HIF-PHIs) are orally active small molecules and are launched as novel therapeutic agents for anemia in chronic kidney disease (CKD). In contrast to conventional exogenous erythropoietin (EPO) administration, HIF-PHIs stimulate endogenous EPO production and improve iron metabolism via stabilization of hypoxia-inducible factor (HIF). This review summarizes the mechanism of action, the results of clinical trials, and future perspectives of HIF-PHIs. RECENT FINDINGS: Six HIF-PHIs are currently under phase III studies, some of which have been already completed. According to the results of clinical trials, HIF-PHIs increased and maintained hemoglobin levels in both nondialysis-dependent and dialysis-dependent CKD patients with physiological EPO concentrations. HIF-PHIs also improved iron utilization and were comparably effective regardless of underlying inflammation and iron status. SUMMARY: HIF-PHIs have several advantages including oral administration, physiological EPO secretion, and improved iron utilization. Undoubtedly, HIF-PHIs will pave the new way in the field of treatment of anemia in CKD, but it should be noted that HIFs have pleiotropic effects on a plethora of cellular functions, which might lead to either beneficial or undesirable off-target effects. Intensive postmarketing surveillance is crucially important to identify unexpected consequences.

Hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitors, also known as HIF stabilizers, increase endogenous erythropoietin production and serve as novel therapeutic agents against anemia in chronic kidney disease. HIF induces the expression of various genes related to energy metabolism as an adaptive response to hypoxia. However, it remains obscure how the metabolic reprogramming in renal tissue by HIF stabilization affects the pathophysiology of kidney diseases. Previous studies suggest that systemic metabolic disorders such as hyperglycemia and dyslipidemia cause alterations of renal metabolism, leading to renal dysfunction including diabetic kidney disease. Here, we analyze the effects of enarodustat (JTZ-951), an oral HIF stabilizer, on renal energy metabolism in the early stages of diabetic kidney disease, using streptozotocin-induced diabetic rats and alloxan-induced diabetic mice. Transcriptome analysis revealed that enarodustat counteracts the alterations in diabetic renal metabolism. Transcriptome analysis showed that fatty acid and amino acid metabolisms were upregulated in diabetic renal tissue and downregulated by enarodustat, whereas glucose metabolism was upregulated. These symmetric changes were confirmed by metabolome analysis. Whereas glycolysis and tricarboxylic acid cycle metabolites were accumulated and amino acids reduced in renal tissue of diabetic animals, these metabolic disturbances were mitigated by enarodustat. Furthermore, enarodustat increased the glutathione to glutathione disulfide ratio and relieved oxidative stress in renal tissue of diabetic animals. Thus, HIF stabilization counteracts alterations in renal energy metabolism occurring in incipient diabetic kidney disease.

Hypoxia-inducible factor (HIF) mediates protection via hypoxic preconditioning in both, in vitro and in vivo ischemia models. However, the underlying mechanism remains largely unknown. Prolyl hydroxylase domain proteins serve as the main HIF regulator via hydroxylation of HIFα leading to its degradation. At present, prolyl hydroxylase inhibitors including enarodustat are under clinical trials for the treatment of renal anemia. In an in vitro model of ischemia produced by oxygen-glucose deprivation of renal proximal tubule cells in culture, enarodustat treatment and siRNA knockdown of prolyl hydroxylase 2, but not of prolyl hydroxylase 1 or prolyl hydroxylase 3, significantly increased the cell viability and reduced the levels of reactive oxygen species. These effects were offset by the simultaneous knockdown of HIF1α. In another in vitro ischemia model induced by the blockade of oxidative phosphorylation with rotenone/antimycin A, enarodustat-enhanced glycogen storage prolonged glycolysis and delayed ATP depletion. Although autophagy is another possible mechanism of prolyl hydroxylase inhibition-induced cytoprotection, gene knockout of a key autophagy associated protein, Atg5, did not affect the protection. Enarodustat increased the expression of several enzymes involved in glycogen synthesis, including phosphoglucomutase 1, glycogen synthase 1, and 1,4-α glucan branching enzyme. Increased glycogen served as substrate for ATP and NADP production and augmented reduction of glutathione. Inhibition of glycogen synthase 1 and glutathione reductase nullified enarodustat's protective effect. Enarodustat also protected the kidneys in a rat ischemia reperfusion injury model and the protection was partially abrogated by inhibiting glycogenolysis. Thus, prolyl hydroxylase inhibition protects the kidney from ischemia via upregulation of glycogen synthesis.

BACKGROUND: Prolyl hydroxylase domain (PHD) inhibitors, which stimulate erythropoietin production through the activation of hypoxia-inducible factor (HIF), are novel therapeutic agents used for treating renal anemia. Several PHD inhibitors, including enarodustat, are currently undergoing phase 2 or phase 3 clinical trials. Because HIF regulates a broad spectrum of genes, PHD inhibitors are expected to have other effects in addition to erythropoiesis, such as protection against metabolic disorders. However, whether such beneficial effects would extend to metabolic disorder-related kidney disease is largely unknown. METHODS: We administered enarodustat or vehicle without enarodustat in feed to diabetic black and tan brachyury (BTBR) ob/ob mice from 4 to 22 weeks of age. To elucidate molecular changes induced by enarodustat, we performed transcriptome analysis of isolated glomeruli and in vitro experiments using murine mesangial cells. RESULTS: Compared with BTBR ob/ob mice that received only vehicle, BTBR ob/ob mice treated with enarodustat displayed lower body weight, reduced blood glucose levels with improved insulin sensitivity, lower total cholesterol levels, higher adiponectin levels, and less adipose tissue, as well as a tendency for lower macrophage infiltration. Enarodustat-treated mice also exhibited reduced albuminuria and amelioration of glomerular epithelial and endothelial damage. Transcriptome analysis of isolated glomeruli revealed reduced expression of C-C motif chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) in enarodustat-treated mice compared with the vehicle-only group, accompanied by reduced glomerular macrophage infiltration. In vitro experiments demonstrated that both local HIF-1 activation and restoration of adiponectin by enarodustat contributed to CCL2/MCP-1 reduction in mesangial cells. CONCLUSIONS: These results indicate that the PHD inhibitor enarodustat has potential renoprotective effects in addition to its potential to protect against metabolic disorders.

OBJECTIVE: Glomerular filtration rate (GFR) decreases without or prior to the development of albuminuria in many patients with diabetes. Therefore, albuminuria and/or a low GFR in patients with diabetes is referred to as diabetic kidney disease (DKD). A certain proportion of patients with diabetes show a rapid progressive decline in renal function in a unidirectional manner and are termed early decliners. This study aimed to elucidate the prevalence of DKD and early decliners and clarify their risk factors. RESEARCH DESIGN AND METHODS: This combination cross-sectional and cohort study included 2385 patients with diabetes from 15 hospitals. We defined DKD as a urinary albumin to creatinine ratio (ACR) ≥30 mg/gCr and/or estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m². We classified patients into four groups based on the presence or absence of albuminuria and a decrease in eGFR to reveal the risk factors for DKD. We also performed a trajectory analysis and specified the prevalence and risk factors of early decliners with sequential eGFR data of 1955 patients in five facilities. RESULTS: Of our cohort, 52% had DKD. Above all, 12% with a low eGFR but no albuminuria had no traditional risk factors, such as elevated glycated hemoglobin, elevated blood pressure, or diabetic retinopathy in contrast to patients with albuminuria but normal eGFR. Additionally, 14% of our patients were early decliners. Older age, higher basal eGFR, higher ACR, and higher systolic blood pressure were significantly associated with early decliners. CONCLUSIONS: The prevalence of DKD in this cohort was larger than ever reported. By testing eGFR yearly and identifying risk factors in the early phase of diabetes, we can identify patients at high risk of developing end-stage renal disease.

Cardiovascular disease (CVD) is the main cause of death in patients with kidney disease. Hypoxia plays a crucial role in the progression of chronic kidney disease (CKD) and cardiovascular disease, which is associated with fibrosis, inflammation, and oxidative injury. Previous studies have indicated that prolyl hydroxylase (PHD) inhibitors, stabilizers of hypoxia-inducible factors (HIFs), can be used to treat acute organ injuries such as renal ischemia-reperfusion, myocardial infarction, and, in some contexts, CKD. However, the effects of PHD inhibitors on cardiovascular complications in CKD remain unknown. In the present study, we investigated whether HIF activation has a beneficial effect on kidney and cardiovascular outcomes in the remnant kidney model. We used the 5/6 nephrectomy model with the nitric oxide synthase inhibitor Nω-nitro-l-arginine (20 mg/L in the drinking water). Rats received diet with 0.005% enarodustat (PHD inhibitor) or vehicle for 8 wk starting 2 wk before 5/6 nephrectomy. Activation of HIF by the PHD inhibitor reduced cardiac hypertrophy and ameliorated myocardial fibrosis in association with restored capillary density and improvement in mitochondrial morphology. With regard to kidneys, enarodustat ameliorated fibrosis in association with reduced proinflammatory cytokine expression, reduced apoptosis, and restored capillary density, even though renal endpoints such as proteinuria and serum creatinine levels were not significantly affected by enarodustat, except for blood urea nitrogen levels at 4 wk. In addition, cardiac hypertrophy marker genes, including atrial natriuretic peptide, were suppressed in P19CL6 cells treated with enarodustat. These findings suggest that PHD inhibitors might show beneficial effects in cardiovascular complications caused by CKD.

PURPOSE OF REVIEW: Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor which regulates a wider range of downstream pathways than previously thought. This review focuses on the novel findings about the internal regulatory mechanisms of Nrf2, the expanding understanding of its role in maintaining cellular homeostasis and the attempts to broaden the clinical application of its activators. RECENT FINDINGS: Nrf2 is in charge of the maintenance of cellular homeostasis under stress and there exist the internal regulatory mechanisms for Nrf2 which have recently been elucidated. New downstream pathways of Nrf2 have been discovered, including the defense against ferroptosis, the latest concept of cell death. Several Nrf2 activators are at various stages of clinical development and are being tested in clinical trials for chronic kidney disease (CKD) including diabetic kidney disease, Alport syndrome, autosomal dominant polycystic kidney disease and focal segmental glomerulosclerosis. SUMMARY: Nrf2 has been gathering attention as an emerging treatment target of chronic diseases which have oxidative stress and inflammation as their pathogenesis including CKD. Basic and clinical studies are under way to establish its role as a target for treatment of those diseases.

Some preceding studies have provided evidence that hypoxia-inducible factor (HIF)-prolyl hydroxylase (PH) inhibitors have therapeutic potential against tubular interstitial fibrosis (TIF). Recently, transformation of renal interstitial fibroblasts (RIFs) into α-smooth muscle actin-positive myofibroblasts with loss of their hypoxia-inducible erythropoietin (EPO) expression has been hypothesized as the central mechanism responsible for TIF with renal anemia (the RIF hypothesis). These reports have suggested that HIF-PH inhibitors may suppress TIF via suppressing transformation of RIFs. However, the direct effect of HIF-PH inhibitors on transformation of RIFs has not been demonstrated because there has been no appropriate assay system. Here, we established a novel in vitro model of the transformation of RIFs. This model expresses key phenotypic changes such as transformation of RIFs accompanied by loss of their hypoxia-inducible EPO expression, as proposed by the RIF hypothesis. Using this model, we demonstrated that JTZ-951, a newly developed HIF-PH inhibitor, stabilized HIF protein in RIFs, suppressed transformation of RIFs, and maintained their hypoxia-inducible EPO expression. JTZ-951 also suppressed the expression of FGF2, FGF7, and FGF18, which are upregulated during transformation of RIFs. Furthermore, expression of Fgf2, Fgf7, and Fgf18 was correlated with TIF in an animal model of TIF. We also demonstrated that not only FGF2, which is a well-known growth-promoting factor, but also FGF18 promoted proliferation of RIFs. These data suggest that JTZ-951 has therapeutic potential against TIF with renal anemia. Furthermore, FGF2, FGF7, and FGF18, which faithfully reflect the anti-TIF effects of JTZ-951, have potential as TIF biomarkers.

Tubulointerstitial hypoxia negatively influences the balance between injury and repair, and serves as a final common pathway in chronic kidney disease (CKD). Studies on erythropoietin (EPO) transcription led to the identification of hypoxia inducible factors (HIFs) and their key regulators, prolyl hydroxylases (PHDs). Based on these, several small molecule PHD inhibitors are developed for the treatment of anemia in CKD, which are currently in phase II/III clinical trials. In addition to treating anemia, application of PHD inhibitors may have several potential implications; there is a promising view that activation of the HIF signaling might protect the ischemic kidney from injury. This is extensively tested in multiple acute kidney injury models, whereas knowledge is limited in the context of CKD. Some studies demonstrate the protective effects of ameliorating inflammation and reducing oxidative stress, whereas negative consequences of sustained HIF activation, such as renal fibrosis and aggravation of polycystic kidney disease, are also reported. Recent human clinical studies reported amelioration in glucose and lipid metabolism, which may be beneficial for the treatment of metabolic kidney disorders. Renal consequences of PHD inhibitors are likely determined by multiple systemic effects of sustained HIF activation and may thus differ depending on the clinical context and the pathological stages.

Acute kidney injury (AKI) is characterized by mitochondrial dysfunction and activation of the innate immune system. The cyclic GMP-AMP synthase (cGAS) stimulator of interferon genes (STING) pathway detects cytosolic DNA and induces innate immunity. Here, we investigate the role of mitochondrial damage and subsequent activation of the cGAS-STING pathway using a genetically engineered animal model of cisplatin-induced AKI and cultured tubular cells. Cisplatin induced mtDNA leakage into the cytosol-probably through BCL-2-like protein 4 (BAX) pores in the mitochondrial outer membrane-in tubules, with subsequent activation of the cGAS-STING pathway, thereby triggering inflammation and AKI progression, which is improved in STING-deficient mice. STING knockdown in cultured tubular cells ameliorates inflammatory responses induced by cisplatin. mtDNA depletion and repletion studies support tubular inflammatory responses via the cGAS-STING signal activation by cytosolic mtDNA. Therefore, we conclude that mitochondrial dysfunction and subsequent activation of the mtDNA-cGAS-STING pathway is a critical regulator of kidney injury.

JTZ-951 (enarodustat) is an oral hypoxia-inducible factor (HIF) prolyl hydroxylase inhibitor. JTZ-951 has inhibitory activities on human HIF-prolyl hydroxylase 1-3, but not on various receptors or enzymes. In Hep3B cells, JTZ-951 increased HIF-1α and HIF-2α protein levels, erythropoietin (EPO) mRNA levels, and EPO production. In normal rats, after a single oral dose of JTZ-951, the hepatic and renal EPO mRNA levels and plasma EPO concentrations were also increased. In 5/6-nephrectomized rats, repeated oral doses of JTZ-951 once daily or intermittent dosing showed the erythropoiesis stimulating effect. The administration of JTZ-951 at a high dose increased plasma vascular endothelial growth factor (VEGF) levels; however, retinal VEGF mRNA levels and the retinal vascular permeability were not changed. Finally, we evaluated the effect of JTZ-951 in a colorectal cancer cell-inoculated mouse model. Although JTZ-951 at a high dose increased the plasma VEGF, it had no effect on tumor growth. In summary, JTZ-951 induces erythropoiesis without affecting VEGF function. Therefore, it is expected that JTZ-951 will be a new oral candidate that increases and maintains hemoglobin concentrations in renal anemia patients.

The epidemic of obesity and its complications is rapidly increasing worldwide. Recent drug discoveries established the utility of prolyl hydroxylase domain (PHD) inhibitors as stabilizers of hypoxia-inducible factors (HIFs) in vivo, which are currently in human clinical studies for the treatment of anemia in chronic kidney disease (CKD). These studies suggest a role for PHD inhibitors in ameliorating obesity and hyperlipidemia. We hypothesized that HIF activation using a PHD inhibitor, JTZ-951, protects from obesity-related diseases in the white adipose tissue (WAT), liver, and kidney in mice fed with high-fat diet (HFD). Eight-week-old, C57BL/6J mice were fed with HFD for 20 weeks with or without JTZ-951(0.005%; mixed in chow). Body weight and plasma non-high-density lipoprotein (HDL) cholesterol levels were significantly lower in the JTZ-951 group as compared with the vehicle group. PHD inhibition improved liver steatosis, macrophage infiltration into WAT and adipocyte fibrosis. In the kidney, PHD inhibition reduced albuminuria. Histologically, the number of F4/80- positive infiltrating macrophages and mesangial expansion were milder in the JTZ-951 group. Relative mRNA expression of adiponectin in WAT was higher in the JTZ-951-treated group and inversely correlated with hepatic steatosis score, adipocyte macrophage aggregation, and albuminuria. Activation of HIF ameliorates multiple obesity-related consequences in mice with HFD. The results of the present study offer the promising view that pharmacological PHD inhibition may be beneficial for the treatment of obesity-related diseases that can be ameliorated by weight loss.

The sympathetic nervous system is critical in maintaining the homeostasis of renal functions. However, its three-dimensional (3D) structures in the kidney have not been elucidated due to limitation of conventional imaging methods. CUBIC (Clear, Unobstructed Brain/Body Imaging Cocktails and Computational analysis) is a newly developed tissue-clearing technique, which enables whole-organ 3D imaging without thin-sectioning. Comprehensive 3D imaging by CUBIC found that sympathetic nerves are primarily distributed around arteries in the mouse kidney. Notably, the sympathetic innervation density was significantly decreased 10 days after ischemia-reperfusion injury (voluminal ratio of innervation area to kidney) by about 70%. Moreover, norepinephrine levels in kidney tissue (output of sympathetic nerves) were significantly reduced in injured kidneys by 77%, confirming sympathetic denervation after ischemia-reperfusion injury. Time-course imaging indicated that innervation partially recovered although overall denervation persisted 28 days after injury, indicating a continuous sympathetic nervous abnormality during the progression of chronic kidney disease. Thus, CUBIC-kidney, the 3D imaging analysis, can be a strong imaging tool, providing comprehensive, macroscopic perspectives for kidney research.

BACKGROUND: This study aims to describe the regional variance in prescriptions for chronic kidney disease (CKD) medications and to analyze regional characteristics to identify the sources of these differences by utilizing the National Database of Health Insurance Claims, which provides more than 95% of nationwide claim information for Japan. METHODS: Data regarding the total claimed amount for phosphate binders (PBs), erythropoiesis-stimulating agents (ESAs), carbonaceous adsorbents, and potassium-lowering agents in fiscal year 2015 were obtained. Correlation coefficients were calculated for the claimed amount of these drugs per CKD patient and social and medical variables, including the percentage of the population aged ≥ 65 years and the numbers of hospital beds and certified nephrologists. Subsequent multiple regression analysis was performed using explanatory factors affecting the amount of PB or ESA prescriptions per CKD patient. RESULTS: The total claimed amounts were 585,485,115 for PBs and 2,373,777 for ESAs. Variations in the claimed amount per CKD patient among 47 prefectures were 4.9-, 6.1-, 6.6-, and 6.0-fold for PBs, ESAs, carbonaceous adsorbents and potassium-lowering agents, respectively. The number of nephrologists per CKD patient was positively correlated with the prescribed amount for PBs and ESAs per CKD patient, and independently associated with the prescribed amount also in regression analysis. CONCLUSION: Substantial regional variation in CKD-related drug prescriptions exists even within a uniform health care system. The number of certified nephrologists was associated with the prescribed amount for PBs and ESAs. Further studies are needed to clarify whether geographic distribution of certified nephrologist may affect clinical practice pattern.

Tubulointerstitial fibrosis is a strong predictor of progression in patients with chronic kidney disease, and is often accompanied by lipid accumulation in renal tubules. However, the molecular mechanisms modulating the relationship between lipotoxicity and tubulointerstitial fibrosis remain obscure. ATF6α, a transcription factor of the unfolded protein response, is reported to be an upstream regulator of fatty acid metabolism. Owing to their high energy demand, proximal tubular cells (PTCs) use fatty acids as their main energy source. We therefore hypothesized that ATF6α regulates PTC fatty acid metabolism, contributing to lipotoxicity-induced tubulointerstitial fibrosis. Overexpression of activated ATF6α transcriptionally downregulated peroxisome proliferator-activated receptor-α (PPARα), the master regulator of lipid metabolism, leading to reduced activity of fatty acid β-oxidation and cytosolic accumulation of lipid droplets in a human PTC line (HK-2). ATF6α-induced lipid accumulation caused mitochondrial dysfunction, enhanced apoptosis, and increased expression of connective tissue growth factor (CTGF), as well as reduced cell viability. Atf6α-/- mice had sustained expression of PPARα and less tubular lipid accumulation following unilateral ischemia-reperfusion injury (uIRI), resulting in the amelioration of apoptosis; reduced expression of CTGF, α-smooth muscle actin, and collagen I; and less tubulointerstitial fibrosis. Administration of fenofibrate, a PPARα agonist, reduced lipid accumulation and tubulointerstitial fibrosis in the uIRI model. Taken together, these findings suggest that ATF6α deranges fatty acid metabolism in PTCs, which leads to lipotoxicity-mediated apoptosis and CTGF upregulation, both of which promote tubulointerstitial fibrosis.

In the original publication of the article, two sentences in the "Results" section were published incorrectly. The corrected texts are provided below.

BACKGROUND: This post-marketing surveillance (PMS) study evaluated the safety and effectiveness of long-term darbepoetin alfa (darbepoetin) for the treatment of renal anemia in Japanese non-dialysis chronic kidney disease patients. METHODS: Patients were treated with darbepoetin and followed up for 3 years. Adverse events (AEs), adverse drug reactions (ADRs), hemoglobin (Hb) levels, and renal function were assessed. Patients were stratified by Hb level at the time of occurrence of cardiovascular-related AEs. Statistical analyses were performed to explore factors affecting the occurrence of AEs, cardiovascular-related AEs, and composite renal endpoints. RESULTS: In the safety analysis set (5547 patients), AEs and ADRs occurred in 44.4 and 7.1% of patients, respectively. Cardiovascular-related AEs were observed in 12.6% of the overall population. The proportion of patients who presented cardiovascular-related AEs was lower among those with a higher Hb level at the time of occurrence. In the effectiveness analysis set (5024 patients), mean Hb levels remained between 10.0 and 10.6 g/dL (Weeks 4-156). Three months after darbepoetin administration, patients with Hb ≥ 11 g/dL presented fewer composite renal endpoints than those with Hb < 11 g/dL (p = 0.0013), and the cumulative proportion of renal survival was higher in those with Hb ≥ 11 g/dL vs. Hb < 11 g/dL (p < 0.0001). CONCLUSIONS: This PMS study showed the safety and effectiveness of long-term use of darbepoetin in a large number of patients. Patients with Hb ≥ 11 g/dL presented fewer composite renal endpoints than those with Hb < 11 g/dL, without an increase in the incidence of cardiovascular-related AEs.

Regeneration of a functional kidney from pluripotent stem cells (PSCs) is challenging because of its complex structure. Kidneys are derived from embryonic metanephros, which are composed of three progenitor cells: nephron progenitors, ureteric bud, and stromal progenitors. Nephron progenitors and ureteric bud have been induced successfully from PSCs as a result of the understanding of their detailed developmental process through cell-lineage tracing analysis. Moreover, these induced progenitors can be used to reconstruct the three-dimensional (3D) structure of kidneys in vitro, including glomeruli with podocytes, renal tubules, and the branching ureters. Induction of the remaining renal progenitors (that is, stromal progenitors from PSCs and the further maturation of reconstructed kidneys) needs to be studied extensively to regenerate functional and sophisticated kidneys from PSCs. In addition to the proper induction of renal progenitors, new bioengineering methods such as decellularization and 3D bioprinting and the recent advancements in the regeneration of kidneys in other species are promising leads for regenerating the complex spatial arrangement of kidneys, including the vascular network and urinary excretion pathway in humans.

BACKGROUND: Hypoxia-inducible factor (HIF) stabilizers, also known as inhibitors of HIF prolyl hydroxylase domain (PHD) inhibitors enzymes, are novel small-molecule agents to treat renal anemia. They increase endogenous erythropoietin (EPO) production by stabilizing HIF. This review focuses on the mechanisms by which PHD inhibitors ameliorate anemia in chronic kidney disease (CKD) and summarizes the current clinical experience with and prospects for these drugs. SUMMARY: Anemia is a serious complication of CKD and is an independent risk factor for congestive heart failure. Appropriate treatment of anemia is important in the management of advanced stage CKD, as it might help to extend life expectancy and improve the physical function of patients with CKD. However, at present, adverse effects of treatment, such as thromboembolic events, as well as high therapeutic cost have a negative impact on society. PHD inhibitors stabilize the transcription factor HIF, increasing the expression of downstream target genes, including EPO and enzymes involved in iron metabolism, resulting in increased EPO production and improved iron utilization. Key Messages: The potential advantages of PHD inhibitors over conventional EPO-based therapies include a more physiologic response to renal anemia, noninvasive oral administration, and lower cost. Phase III trials of more than 5 PHD inhibitors are ongoing, with overall demonstration of success in increasing hemoglobin levels. In this review, we focus on the mechanisms of PHD inhibitors in improving renal anemia in CKD and summarize the current clinical findings regarding these drugs.

It is unclear whether long-term sodium-glucose cotransporter 2 (SGLT2) inhibition such as that during the treatment of diabetes has deleterious effects on the kidney. Therefore, we first sought to determine whether abnormal glucose metabolism occurs in the kidneys of 22-week-old BTBR ob/ob diabetic mice. Second, the cumulative effect of chronic SGLT2 inhibition by ipragliflozin and 30% calorie restriction, either of which lowered blood glucose to a similar extent, on renal glucose metabolism was evaluated. Mass spectrometry-based metabolomics demonstrated that these diabetic mice exhibited an abnormal elevation in the renal pools of tricarboxylic acid cycle metabolites. This was almost completely nullified by SGLT2 inhibition and calorie restriction. Moreover, imaging mass spectrometry indicated an increased level of the tricarboxylic acid cycle intermediate, citrate, in the cortex of the diabetic mice. SGLT2 inhibition as well as calorie restriction almost completely eliminated citrate accumulation in the cortex. Furthermore, imaging mass spectrometry revealed that the accumulation of oxidized glutathione in the cortex of the kidneys, prominent in the glomeruli, was also canceled by SGLT2 inhibition and calorie restriction. Effects of these beneficial interventions were consistent with improvements in glomerular damage, such as albuminuria, glomerular hyperfiltration, and mesangial expansion. Tubulointerstitial macrophage infiltration and fibrosis were ameliorated only by calorie restriction, which may have been due to autophagy activation, which was observed only with calorie restriction. Thus, chronic SGLT2 inhibition is efficient in normalizing the levels of accumulated tricarboxylic acid cycle intermediates and increased oxidative stress in the kidneys of diabetic mice.

End-stage renal disease is a leading cause of morbidity and mortality worldwide. The prevalence of the disease and the number of patients who receive renal replacement therapy are expected to increase in the next decade. Accumulating evidence suggests that chronic hypoxia in the tubulointerstitium represents the final common pathway to end-stage renal failure, and that reactive oxygen species (ROS) and reactive nitrogen species (RNS) are the key players in kidney injury. However, ROS and RNS that exceed the physiological levels associated with the pathophysiology of most kidney diseases. The molecules that comprise ROS and RNS play an important role in regulating solute and water reabsorption in the kidney, which is vital for maintaining electrolyte homeostasis and the volume of extracellular fluid. This article reviews the physiological and pathophysiological role of ROS and RNS in normal kidney function and in various kidney diseases.

Lipotoxicity plays an important role in the progression of chronic kidney damage via various mechanisms, such as endoplasmic reticulum stress. Several studies proposed renal lipotoxicity in glomerular and tubular cells but the effect of lipid on renal erythropoietin (EPO)-producing (REP) cells in the interstitium has not been elucidated. Since renal anemia is caused by derangement of EPO production in REP cells, we evaluated the effect of palmitate, a representative long-chain saturated fatty acid, on EPO production and the endoplasmic reticulum stress pathway. EPO production was suppressed by palmitate (palmitate-conjugated bovine serum albumin [BSA]) or a high palmitate diet, but not oleic acid-conjugated BSA or a high oleic acid diet, especially under cobalt-induced pseudo-hypoxia both in vitro and in vivo. Importantly, suppression of EPO production was not induced by a decrease in transcription factor HIF activity, while it was significantly associated with endoplasmic reticulum stress, particularly transcription factor ATF4 activation, which suppresses 3'-enhancer activity of the EPO gene. ATF4 knockdown by siRNA significantly attenuated the suppressive effect of palmitate on EPO production. Studies utilizing inherited super-anemic mice (ISAM) mated with EPO-Cre mice (ISAM-REC mice) for lineage-labeling of REP cells showed that ATF4 activation by palmitate suppressed EPO production in REP cells. Laser capture microdissection confirmed ATF4 activation in the interstitial area of ISAM-REC mice treated with palmitate-conjugated BSA. Thus, endoplasmic reticulum stress induced by palmitate suppressed EPO expression by REP cells in a manner independent of HIF activation. The link between endoplasmic reticulum stress, dyslipidemia, and hypoxia may contribute to development and progression of anemia in CKD.

PURPOSE OF REVIEW: Small-molecule inhibitors of prolyl hydroxylase domain enzymes (PHD inhibitors) are novel renal anemia therapies that increase endogenous erythropoietin (EPO) production by stabilizing hypoxia-inducible factor (HIF). This review summarizes recent findings and future perspectives of PHD inhibitors (HIF stabilizers) in chronic kidney disease (CKD)-associated anemia. RECENT FINDINGS: Clinical trials have demonstrated that HIF stabilizers effectively increase hemoglobin levels of both nondialysis and dialysis CKD patients without causing serious adverse effects. HIF stabilizers not only restore EPO production but also optimize iron metabolism by reducing hepcidin levels. Considering the pleiotropic roles of the PHD-HIF pathway, HIF stabilizers might have both advantageous and disadvantageous effects in humans, in addition to erythropoiesis. Results of studies in animal models have suggested that HIF stabilizers alleviate ischemia-reperfusion injury and play protective roles against metabolic diseases. In contrast, a theoretical concern exists regarding the potential for tumorigenesis due to HIF stabilization. SUMMARY: At least five HIF stabilizers are now in phase III trials and may appear on the market in 1-2 years. The long-term effects and safety of HIF stabilization should be carefully examined in future basic and clinical studies.

Sodium-glucose cotransporter 2 (SGLT2), which is specifically expressed on the apical side of proximal tubular cells, is involved in the reabsorption of most of the glucose filtered by the glomeruli, and its inhibitors are gaining publicity as potent antihyperglycemic drugs. In some clinical trials, SGLT2 inhibitors exerted cardiovascular and kidney protective effects, which appeared to be partly independent of the original glucose-lowering effect. SGLT2 inhibitors have both direct and indirect renoprotective effects. The direct effects involve the suppression of hyperplasia/hypertrophy, inflammation, and fibrosis in the proximal tubular cells, utilization of ketone bodies, restored tubuloglomerular feedback, decreased oxygen consumption, improvement in anemia, and preconditioning against ischemia/reperfusion. The indirect effects involve a reduction in insulin levels and resistance, uric acid concentration, body weight, and blood pressure. However, safety concerns remain, including consequences of an enhanced glucose load in the lower nephron, leg amputation, bone fractures, and therapeutic efficacy in patients with advanced chronic kidney disease.

Renal tubulointerstitial hypoxia is recognized as a final common pathway of chronic kidney disease and is considered a promising drug target. However, hypoxia in the tubules is not well examined because of limited detection methods. Here, we devised a method to visualize renal tubular oxygen tension with spatial resolution at a cellular level using the cell-penetrating phosphorescent probe, BTPDM1 (an iridium-based cationic lipophilic dye), and confocal phosphorescence lifetime imaging microscopy to precisely assess renal hypoxia. Imaging with BTPDM1 revealed an oxygen gradient between S1 and S2 segments in mouse kidney. We also demonstrated that our microscopy system can detect subtle changes of hypoxemia and reoxygenation, and the acquired phosphorescence lifetime can be converted to partial pressure of oxygen. This new method allows, for the first time, visualization of intravital oxygen gradients at the renal surface with high spatial resolution. Thus, the confocal phosphorescence lifetime imaging microscopy platform, combined with BTPDM1, will promote an accurate understanding of tissue hypoxia, including renal hypoxia.

Cardiovascular disease is a common and serious complication in patients with chronic kidney disease (CKD). One of the fundamental functions of the cardiovascular system is oxygen delivery, therefore cardiovascular disease inherently is linked to insufficient tissue oxygenation. Advances in our knowledge of cellular oxygen sensing by a family of prolyl hydroxylases (PHDs) and their role in regulating hypoxia-inducible factors (HIFs) have led to the discovery of PHD inhibitors as HIF stabilizers. Several small-molecule PHD inhibitors are currently in clinical trials for the treatment of anemia in CKD. An additional advantage of PHD inhibition may be found in the potential impact on cardiovascular consequences associated with CKD. Several preclinical studies have suggested a potential benefit of HIF activation in myocardial infarction, cardiac remodeling, atherosclerosis, and peripheral artery disease. Ameliorating glucose and lipid metabolism and lowering blood pressure may also contribute to cardiovascular protection. On the other hand, the broad spectrum of HIF-dependent functions also may include unwanted side effects. Clinical application of PHD inhibitors therefore necessitates careful evaluation of the net systemic effect of HIF activation.

Recent epidemiologic studies revealed a correlation between acute kidney injury (AKI) episodes and the progression to chronic kidney disease (CKD). Although the severity and duration of the initial insult likely predict the development of CKD, information regarding tissue markers predictive of early development of renal fibrosis is limited. We investigated key markers in fibrotic kidney in rats and mice. Seven- to eight-week-old male Sprague-Dawley rats underwent bilateral ischemia-reperfusion injury (IRI). Kidney tissues were collected to determine the markers correlated with the severity of kidney fibrosis. In a separate set, a specific chemokine (C-C motif) receptor 2 (CCR2) inhibitor, RS-102895, was administered to 9-week-old male C57BL/6J mice that underwent unilateral IRI (9.2 mg/kg/day in drinking water for 17 days) to investigate whether blockade of the monocyte chemotactic protein-1 (MCP-1) signaling was sufficient to prevent fibrosis. Among candidate tissue markers, neutrophil gelatinase-associated lipocalin (NGAL) and MCP-1 mRNA expressions were correlated with kidney fibrosis. Studies on macrophage polarity showed that mRNA expression of M2, but not M1 macrophage markers, were correlated with acute-phase serum creatinine and fibrosis. Pharmacological blockade of the MCP-1-CCR2 signaling downregulated CCR2, which was insufficient to improve fibrosis in mouse unilateral IRI model, suggesting that additional, redundant pathways contribute to fibrosis. These findings suggested that tissue NGAL expression and M2 macrophage markers are promising markers that show severity of kidney fibrosis. Mechanistic involvement of these markers in CKD pathogenesis warrant additional investigation.

Tubulointerstitial fibrosis has been recently reported to be caused by the collapse of the epigenetic regulation of kidney diseases. We examined whether pharmacological inhibition of histone modification is effective against renal fibrosis. DZNep (3-deazaneplanocin A) was originally developed as an anti-cancer drug to inhibit the repressive histone mark, H3K27me3. We used a model of chronic tubulointerstitial fibrosis induced by unilateral ischaemia/reperfusion and administered DZNep intravenously to the mice for 8 weeks. We found DZNep contributes to the reduction of tubulointerstitial fibrosis. We selected only tubular cells from in vivo samples using laser-capture microdissection because epigenetic regulation is specific to the cell types, and we focused on the changes in the tubular cells. We performed a genome-wide analysis of tubular cells using high-throughput sequencing (RNA-seq) to identify novel epigenetic factors associated with renal fibrosis. We found that pro-fibrotic genes such as COL3A1 (collagen type 3a1) and TIMP2 (tissue inhibitor of metalloproteinase 2) were suppressed by DZNep in vivo. In addition, pro-fibrotic genes such as COL4A1 (collagen type 4a1), TIMP2 and MMP14 were down-regulated by DZNep in vitro. In conclusion, we found that pharmacological epigenetic modification by DZNep decreased the expression levels of fibrogenic genes in tubular cells and inhibited tubulointerstitial fibrosis.

Insufficient oxygenation during pregnancy negatively influences kidney development, which likely serves as a predisposing factor in chronic kidney disease at later stages in life. In this issue of Kidney International, Kobayashi et al. demonstrate that deletion of prolyl hydroxylase 2 and 3, 2 of the major oxygen sensors, in the FoxD1 lineage cells reduces kidney size and inhibits nephrogenesis in mice. Temporospatial expression pattern and studies on additional knockouts suggest the involvement of hypoxia-inducible factor 2.

There are major gaps between our growing knowledge of effective treatments for chronic kidney disease (CKD), and the delivery of evidence-based therapies to populations around the world. Although there remains a need for new, effective therapies, current evidence suggests that many patients with CKD are yet to fully realize the benefits of blood pressure-lowering drugs (with and without reducing proteinuria with renin-angiotensin system blockade), wider use of statins to reduce atherosclerotic cardiovascular disease events, and better glycemic control in both type 1 and type 2 diabetes. There are many barriers to optimizing evidence-based nephrology care around the world, including access to health care, affordability of treatments, consumer attitudes and circumstances, the dissemination of appropriate knowledge, the availability of expertise and structural impediments in the delivery of health care. Further investment in implementation science that addresses the major barriers to effective care in a cost-effective manner could yield both local and global benefits.

The focus of this article is to define goals and resulting action plans that can be collectively embraced by interested stakeholders to facilitate new therapeutic approaches to mitigate chronic kidney disease progression. The specific goals include identifying druggable targets, increasing the capacity for preclinical and early clinical development, broadening the availability of new therapeutic approaches, and increasing investment in the development of new therapies to limit chronic kidney disease. Key deliverables include the establishment of new regional, national, and global consortia; development of clinical trial networks; and creation of programs to support the temporary mutual movement of scientists between academia and the biotechnology and pharmaceutical sector. Other deliverables include cataloging and maintaining up-to-date records to collate progress in renal research and development, inventorying the capacity of research and clinical networks, and describing methods to ensure novel drug development.

Anemia is a common complication of chronic kidney disease and is mainly caused by the inability of injured kidneys to produce adequate amounts of erythropoietin. Studies elucidating the regulation of erythropoietin production led to the identification of hypoxia-inducible factor (HIF), which activates the transcription of genes that mediate adaptive responses to hypoxia. HIF is a heterodimer that consists of an α and β subunit. While HIF-β is constitutively expressed, HIF-α is subjected to ubiquitination and proteasomal degradation under normoxic conditions. This process is mediated by prolyl hydroxylase domain proteins, the inhibition of which results in an increased expression of hypoxia-induced genes, including erythropoietin. These findings led to the development of prolyl hydroxylase domain inhibitors as novel therapeutic agents against anemia in chronic kidney disease. Prolyl hydroxylase domain inhibition improves iron metabolism, which also contributes to erythropoiesis. To date, at least 6 small-molecule inhibitors of the prolyl hydroxylase domain have been tested in humans, and clinical trials have shown that they are effective without causing serious adverse events. However, there is a theoretical concern that the systemic activation of HIF could also induce deleterious effects such as tumorigenesis and severe pulmonary hypertension, which demands careful assessments in future clinical studies.

Obesity is a risk factor for many diseases including diabetes, cancer, cardiovascular disease, and chronic kidney disease. Obesity is characterized by the expansion of white adipose tissue (WAT). Hypertrophy and hyperplasia of adipocytes cause tissue hypoxia followed by inflammation and fibrosis. Its trigger, preadipocyte differentiation into mature adipocytes, is finely regulated by transcription factors, signal molecules, and cofactors. We found that echinomycin, a potent HIF-1 inhibitor, completely inhibited adipogenesis in 3T3-L1 WAT preadipocytes by affecting the early phase of mitotic clonal expansion. The dose required to exert the effect was surprisingly low and the time was short. Interestingly, its inhibitory effect was independent of HIF-1 pathways. Time-course DNA microarray analysis of drug-treated and untreated preadipocytes extracted a major transcription factor, CCAAT/enhancer-protein β, as a key target of echinomycin. Echinomycin also inhibited adipogenesis and body weight gain in high fat diet mice. These findings highlight a novel role of echinomycin in suppressing adipocyte differentiation and offer a new therapeutic strategy against obesity and diabetes.

Vascular adhesion protein-1 (VAP-1) is a unique molecule since it acts as an adhesion molecule as well as an ectoenzyme catalyzing oxidative deamination of primary amines and generates hydrogen peroxide in the extracellular space. While VAP-1 is implicated in various inflammatory diseases, its role in acute kidney injury is less characterized. Here we studied VAP-1 expression in the kidney and the effect of its inhibition in a rat model of renal ischemia/reperfusion injury. VAP-1 was predominantly expressed in pericytes, which released enzymatically active enzyme. In vivo, a specific VAP-1 inhibitor, RTU-1096, significantly ameliorated rat renal ischemia/reperfusion injury and decreased neutrophil infiltration measured 12 hours after injury without altering macrophage or T lymphocyte populations. The protective effect of VAP-1 inhibition was lost in neutrophil-depleted rats, suggesting its inhibition ameliorated renal ischemia/reperfusion injury by suppressing neutrophil infiltration. To investigate whether hydrogen peroxide generated by VAP-1 enzyme reaction enhances neutrophil infiltration, we conducted an under-agarose migration assay with purified human neutrophils. Recombinant human VAP-1 significantly induced neutrophil migration, which was almost completely inhibited by RTU-1096 or catalase. Thus, VAP-1 plays a critical role in the pathophysiology of renal ischemia/reperfusion injury by enhancement of neutrophil infiltration generating a local hydrogen peroxide gradient. Hence, VAP-1 inhibition may be a novel therapy in ischemic acute kidney injury.

Diabetic kidney disease (DKD) is a worldwide public health problem. The definition of DKD is under discussion. Although the term DKD was originally defined as 'kidney disease specific to diabetes,' DKD frequently means chronic kidney disease with diabetes mellitus and includes not only classical diabetic nephropathy, but also kidney dysfunction as a result of nephrosclerosis and other causes. Metabolic memory plays a crucial role in the progression of various complications of diabetes, including DKD. The mechanisms of metabolic memory in DKD are supposed to include advanced glycation end-products, deoxyribonucleic acid methylation, histone modifications and non-coding ribonucleic acid including micro ribonucleic acid. Regardless of the presence of diabetes mellitus, the final common pathway in chronic kidney disease is chronic kidney hypoxia, which influences epigenetic processes, including deoxyribonucleic acid methylation, histone modification, and conformational changes in micro ribonucleic acid and chromatin. Therefore, hypoxia and oxidative stress are appropriate targets of therapies against DKD. Prolyl hydroxylase domain inhibitor enhances the defensive mechanisms against hypoxia. Bardoxolone methyl protects against oxidative stress, and can even reverse impaired renal function; a phase 2 trial with considerable attention to heart complications is currently ongoing in Japan.

Chronic tubulointerstitial hypoxia plays an important role as the final common pathway to end-stage renal disease. HIF-1 (hypoxia-inducible factor-1) is a master transcriptional factor under hypoxia, regulating downstream target genes. Genome-wide analysis of HIF-1 binding sites using high-throughput sequencers has clarified various kinds of downstream targets and made it possible to demonstrate the novel roles of HIF-1. Our aim of this study is to identify novel HIF-1 downstream epigenetic targets which may play important roles in the kidney. Immortalized tubular cell lines (HK2; human kidney-2) and primary cultured cells (RPTEC; renal proximal tubular cell lines) were exposed to 1% hypoxia for 24-72 h. We performed RNA-seq to clarify the expression of mRNA and long non-coding RNA (lncRNA). We also examined ChIP-seq to identify HIF-1 binding sites under hypoxia. RNA-seq identified 44 lncRNAs which are up-regulated under hypoxic condition in both cells. ChIP-seq analysis demonstrated that HIF-1 also binds to the lncRNAs under hypoxia. The expression of novel lncRNA, DARS-AS1 (aspartyl-tRNA synthetase anti-sense 1), is up-regulated only under hypoxia and HIF-1 binds to its promoter region, which includes two hypoxia-responsive elements. Its expression is also up-regulated with cobalt chloride exposure, while it is not under hypoxia when HIF-1 is knocked down by siRNA To clarify the biological roles of DARS-AS1, we measured the activity of caspase 3/7 using anti-sense oligo of DARS-AS1. Knockdown of DARS-AS1 deteriorated apoptotic cell death. In conclusion, we identified the novel lncRNAs regulated by HIF-1 under hypoxia and clarified that DARS-AS1 plays an important role in inhibiting apoptotic cell death in renal tubular cells.

Renal fibrosis is characterized by tubular cell atrophy and accumulation of extracellular matrix. Fibroblast activation becomes evident in areas surrounding atrophic tubules, with rarefaction of peritubular capillaries. Tubulointerstitial hypoxia is the final common pathway in progressive kidney disease. Hypoxia suppresses tubular epithelial growth and leads to failure of remodeling by facilitating dedifferentiation and apoptosis. Profibrotic factors such as transforming growth factor-β (TGF-β) mediate fibroblast activation, and recruited leukocytes, which appear in hypoxic areas, contribute to fibrosis. While resident renal cells adapt to the hypoxic environment via upregulation of relevant genes by hypoxia-inducible factor (HIF) family members, hypoxic adaptation via HIF may not be sufficient in chronic kidney disease (CKD) due to multiple factors. Thus, restoration of HIF-mediated responses may contribute to amelioration of CKD pathology. Studies to date have reported that HIF activation reduces inflammation and oxidative stress and ameliorates injury by decreasing tubular cell apoptosis and restoring peritubular capillary network. Prolyl hydroxylase domain (PHD) inhibitors that specifically activate HIF are currently evaluated for the treatment of renal anemia and may be effective for the treatment of CKD.

AST-120 (kremezin; Kureha Chemical, Tokyo, Japan) is an oral spherical carbonaceous adsorbent, which was approved for clinical use in Japanese chronic kidney disease (CKD) patients in 1991. It adsorbs indole, the precursor of indoxyl sulfate, in the intestines and prevents indoxyl sulfate production. Indoxyl sulfate, initially identified as a major uremic toxin that causes uremic symptoms, contributes to CKD progression. Since AST-120 decreases serum indoxyl sulfate in a dose-dependent manner, multicenter prospective trials have been conducted in Japan in the 1980s; these trials were mostly in favor of the efficacy of AST-120 in delaying the initiation of dialysis in patients with advanced stage CKD. Many animal studies support the effects of AST-120 on renal outcomes as well as on cardiovascular complications. However, there are yet no reports that unequivocally demonstrate the improvement of hard renal endpoints and/or cardiovascular endpoints. This commentary briefly reviews the major outcomes of the recent clinical trials on AST-120.

Chronic kidney disease (CKD) is a major public health problem. Accumulating evidence suggests that CKD aggravates renal hypoxia, and in turn, renal hypoxia accelerates CKD progression. To eliminate this vicious cycle, hypoxia-related therapies, such as hypoxia-inducible factor (HIF) activation (prolyl hydroxylase domain inhibition) or NF-E2-related factor 2 activation, are currently under investigation. Clinical studies have revealed heterogeneity in renal oxygenation; therefore, the detection of patients with more hypoxic kidneys can be used to identify likely responders to hypoxia-oriented therapies. In this review, we provide a detailed description of current hypoxia detection methods. HIF degradation correlates with the intracellular oxygen concentration; thus, methods that can detect intracellular oxygen tension changes are desirable. The use of a microelectrode is a classical technique that is superior in quantitative performance; however, its high invasiveness and the fact that it reflects the extracellular oxygen tension are disadvantages. Pimonidazole protein adduct immunohistochemistry and HIF activation detection reflect intracellular oxygen tension, but these techniques yield qualitative data. Blood oxygen level-dependent magnetic resonance imaging has the advantage of low invasiveness, high quantitative performance, and application in clinical use, but its biggest disadvantage is that it measures only deoxyhemoglobin concentrations. Phosphorescence lifetime measurement is a relatively novel in vivo oxygen sensing technique that has the advantage of being quantitative; however, it has several disadvantages, such as toxicity of the phosphorescent dye and the inability to assess deeper tissues. Understanding the advantages and disadvantages of these hypoxia detection methods will help researchers precisely assess renal hypoxia and develop new therapeutics against renal hypoxia-associated CKD.

Previously acute kidney injury (AKI) had been believed to be a transient event, and recovery from AKI had been thought to lead to no consequences. However, recent epidemiological studies have shown that even if there is complete recovery of the kidney function, AKI can eventually result in chronic kidney disease (CKD) and eventually in end-stage kidney disease in the long term. Transition of AKI to CKD is mediated by multiple mechanisms, including aberrant cell cycle arrest and hypoxia. Hypoxia of the kidney is induced by rarefaction of the peritubular capillaries after AKI episodes, and induces inflammation and fibrosis. It should also be noted that epigenetic changes are closely related to hypoxia, and epigenetic changes induced by hypoxia, called "hypoxic memory" can explain the AKI-to-CKD transition in the long term after complete recovery from the initial AKI episode. Targeting hypoxia and subsequent epigenetic changes are promising strategies to block the transition from AKI to CKD.

Studies over the last two decades have established tubulointerstitial hypoxia as a final common pathway leading to end-stage kidney disease (ESKD). Chronic kidney disease (CKD) is frequently associated with various degrees of hypoxic injury in distinct tubular segments, depending on the etiology and pathological stages, which constitutes an intricate link among inflammation, oxidative stress and fibrosis. Resident cells in the kidney are equipped with mechanisms through which they cope with hypoxia. Here, transcription of genes by hypoxia-inducible factors (HIFs) plays a central role. In the ischemic kidney, HIF-1 is expressed in tubular and glomerular epithelial cells and in papillary interstitial cells, whereas HIF-2 is expressed in endothelial cells and interstitial fibroblasts. There is ample evidence that HIF protects the kidney from acute ischemic damage. In CKD, studies suggest that the function of HIF may be suppressed because of factors, such as oxidative stress and uremia, which may underlie the pathogenesis of both CKD and co-existing problems, such as renal anemia. Based on these observations, efforts are in progress to test whether restoration and activation of HIF might protect the kidney from CKD. Initial studies using non-specific or supraphysiological HIF activation suggested that the role of HIF may be multifactorial and depend on pathological context. On the other hand, specific HIF stabilizers, such as prolyl hydroxylase (PHD) inhibitors, are being developed for the treatment of renal anemia. Application of these compounds in experimental CKD may override those previous findings and provide deeper insight into the roles of hypoxia and oxygen-sensing pathways.

The number of patients with kidney failure has increased in recent years. Different factors contribute to the progression of chronic kidney disease, including glomerular sclerosis, atherosclerosis of the renal arteries and tubulointerstitial fibrosis. Tubulointerstitial injury is induced by hypoxia and other inflammatory signals, leading to fibroblast activation. Technological advances using high-throughput sequencing has enabled the determination of the expression profile of almost all genes, revealing that gene expression is intricately regulated by DNA methylation, histone modification, changes in chromosome conformation, long non-coding RNAs and microRNAs. These epigenetic modifications are stored as cellular epigenetic memory. Epigenetic memory leads to adult-onset disease or ageing in the long term and may possibly play an important role in the kidney disease process. Herein we emphasize the importance of clarifying the molecular mechanisms underlying epigenetic modifications because this may lead to the development of new therapeutic targets in kidney disease.

Tubulointerstitial hypoxia plays a critical role in the pathogenesis of kidney injury, and hypoxia-inducible factor (HIF)-1 is a master regulator of cellular adaptation to hypoxia. Aside from oxygen molecules, factors that modify HIF-1 expression and functional operation remain obscure. Therefore, we sought to identify novel HIF-1-regulating genes in kidney. A short-hairpin RNA library consisting of 150 hypoxia-inducible genes was derived from a microarray analysis of the rat renal artery stenosis model screened for the effect on HIF-1 response. We report that CCAAT/enhancer-binding protein δ (CEBPD), a transcription factor and inflammatory response gene, is a novel HIF-1 regulator in kidney. CEBPD was induced in the nuclei of tubular epithelial cells in both acute and chronic hypoxic kidneys. In turn, CEBPD induction augmented HIF-1α expression and its transcriptional activity. Mechanistically, CEBPD directly bound to the HIF-1α promoter and enhanced its transcription. Notably, CEBPD was rapidly induced by inflammatory cytokines, such as IL-1β in a nuclear factor-κB-dependent manner, which not only increased HIF-1α expression during hypoxia, but was also indispensable for the non-hypoxic induction of HIF-1α. Thus our study provides novel insight into HIF-1 regulation in tubular epithelial cells and offers a potential hypoxia and inflammation link relevant in both acute and chronic kidney diseases.

BACKGROUND: Accumulating evidence has demonstrated that renal hypoxia has a crucial role in the pathogenesis of acute kidney injury (AKI), chronic kidney disease (CKD), and AKI-to-CKD transition, ultimately culminating in end-stage kidney disease. Renal hypoxia in progressive CKD is intricately linked to persisting capillary loss, which is mainly due to dysregulated angiogenesis. SUMMARY: In CKD, hypoxia-inducible factor (HIF) accumulates in the ischemic tubulointerstitium but fails to sufficiently stimulate angiogenic responses, partly because of blunted activation of HIF, which is best exemplified in diabetic kidney disease. In addition, vascular endothelial growth factor (VEGF) expression is downregulated, possibly because injured tubules are not able to express sufficient VEGF and inflammatory circumstances inhibit VEGF expression. The upregulation of antiangiogenic factors and the incompetence of endothelial progenitor cells (EPCs) may also play some roles in the inadequacy of capillary restoration. Administration of VEGF or angiopoietin-1 maintains peritubular capillaries in several kidney diseases; however, administration of a single angiogenic factor may lead to the formation of abnormal vessels and induce inflammation, resulting in worsening of hypoxia and tubulointerstitial fibrosis. HIF stabilization, which aims to achieve the formation of mature and stable vessels by inducing coordinated angiogenesis, is a promising strategy. Given that the effect of systemic HIF activation is highly context-dependent, further studies are needed to elucidate the precise roles of HIF in various kidney diseases. The adoptive transfer of EPCs or mesenchymal stem cells (MSCs) is a fascinating alternative strategy to restore the peritubular capillaries. KEY MESSAGE: Suppressed HIF activation and VEGF expression may be responsible for the dysregulated angiogenesis in progressive CKD. Administration of a single angiogenic factor can cause abnormal vessel formation and inflammation, leading to a detrimental result. Although further studies are warranted, HIF stabilization and adoptive transfer of EPCs or MSCs appear to be promising strategies to restore normal capillaries.

Dipeptidyl peptidase (DPP)-4 is an enzyme that cleaves and inactivates incretin hormones capable of stimulating insulin secretion from pancreatic β-cells. DPP-4 inhibitors are now widely used for the treatment of type 2 diabetes. Experimental studies have suggested a renoprotective role of DPP-4 inhibitors in various models of diabetic kidney disease, which may be independent of lowering blood glucose levels. In the present study, we examined the effect of DPP-4 inhibitors in the rat Thy-1 glomerulonephritis model, a nondiabetic glomerular injury model. Rats were injected with OX-7 (1.2 mg/kg iv) and treated with the DPP-4 inhibitor alogliptin (20 mg·kg(-1)·day(-1)) or vehicle for 7 days orally by gavage. Alogliptin significantly reduced the number of CD68-positive inflammatory macrophages in the kidney, which was associated with a nonsignificant tendency to ameliorate glomerular injury and reduce proteinuria. Another DPP-4 inhibitor, anagliptin (300 mg·kg(-1)·day(-1) mixed with food) and a glucagon-like peptide-1 receptor agonist, exendin-4 (10 mg/kg sc), similarly reduced CD68-positive macrophage infiltration to the kidney. Furthermore, ex vivo transmigration assays using peritoneal macrophages revealed that exendin-4, but not alogliptin, dose dependently reduced monocyte chemotactic protein-1-stimulated macrophage infiltration. These data suggest that DPP-4 inhibitors reduced macrophage infiltration directly via glucagon-like peptide-1-dependent signaling in the rat Thy-1 nephritis model and indicate that the control of inflammation by DPP-4 inhibitors is useful for the treatment of nondiabetic kidney disease models.

Patients with advanced chronic kidney disease are exposed to uremic toxins. In addition to causing uremic symptoms, uremic toxins accelerate the progression of renal failure. Indoxyl sulfate (IS) increases oxygen consumption in tubules, aggravating hypoxia of the kidney, and progression of the kidney disease. IS also induces endoplasmic reticulum stress and thereby contributes the progression of cellular damages in tubular epithelial cells. Hypoxia-inducible factor (HIF) is a master transcriptional regulator of adaptive responses against hypoxia and regulates expression of erythropoietin (EPO). IS suppresses EPO expression via HIF-dependent and HIF-independent manner. IS impedes the recruitment of transcriptional coactivators to HIF via upregulation of Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 through a mechanism of posttranscriptional messenger RNA stabilization. Furthermore, IS induces activating transcription factor 4 via endoplasmic reticulum stress, decreasing EPO expression. Although erythropoiesis-stimulating agent (ESA) resistance is generally defined as lack of responses to exogenous ESA administration, suppression of endogenous production of EPO under uremic conditions may aggravate ESA resistance. Uremia is associated with increased formation of advanced glycation end products (AGE). Studies of transgenic rats overexpressing glyoxalse 1 (GLO1), which detoxifies precursors of advanced glycation end products, demonstrated that glycative stress causes renal senescence and vascular endothelial dysfunction. Glycative stress also suppresses HIF activation making the kidney susceptible to hypoxia as a final common pathway to end-stage kidney disease.

Chronic kidney disease (CKD) is defined as any condition that causes reduced kidney function over a period of time. Fibrosis, tubular atrophy and interstitial inflammation are the hallmark of pathological features in CKD. Regardless of initial insult, CKD has some common pathways leading CKD to end-stage kidney disease, including hypoxia in the tubulointerstitium and proteinuria. Recent advances in genome editing technologies and stem cell research give great insights to understand the pathogenesis of CKD, including identifications of the origins of renal myofibroblasts and tubular epithelial cells upon injury. Environmental factors such as hypoxia, oxidative stress, and epigenetic factors in relation to CKD are also discussed.

Iron deficiency is a major cause of hyporesponsiveness to erythropoiesis-stimulating agents (ESAs) and is often observed in chronic kidney disease (CKD) patients with anemia. With iron supplementation, ESA doses can be decreased, resulting in lower treatment costs and possibly lower cardiovascular risks that are associated with high-dose ESA therapy. The 2012 Kidney Disease: Improving Global Outcomes Guideline specified ferritin ≤ 500 ng/ml and transferrin saturation (TSAT) ≤ 30% as thresholds of iron parameters for CKD patients. However, long-term safety (in terms of mortality, cardiovascular/infection risk and tissue deposition) of high-dose intravenous iron supplementation with such high target levels of ferritin/TSAT has not been confirmed. Recently, there has been increase in the use of intravenous iron and average ferritin levels in dialysis patients in the United States. Clinical trials conducted so far have been underpowered to conclusively establish the long-term safety of intravenous iron supplementation. Results from observational studies are conflicting, and many experimental studies have even shown negative effects of intravenous iron. Clearly, randomized clinical trials are urgently needed, studying various doses of intravenous iron, with sufficient patient numbers and longer observation periods, to investigate mortality, cardiovascular effects and infection risks of this treatment. Until the long-term safety of iron supplementation at high doses is established, a more prudent decision on iron supplementation with lower target levels of ferritin/TSAT seems reasonable, in light of the decades of experience with ESA that has shown that definitive clinical outcomes have been dissociated from surrogate outcomes (especially hemoglobin concentration).

Renal anemia is caused by the deficiency of endogenous erythropoietin (Epo) due to renal dysfunction. We think that it is possible to slow the progression of chronic kidney disease (CKD) in case we initiate Epo early in pre-dialysis patients, especially in the non-diabetic population. Erythropoiesis stimulating agent (ESA) treatments targeting mild anemia (10-12 g/dl) can decrease the risk of occurrence of cardiovascular disease (CVD) in patients with hypertension, diabetes mellitus and congestive heart failure. As the large randomized controlled trials such as Cardiovascular Risk Reduction by Early Anemia Treatment with Epoetin Beta, Correction of Hemoglobin and Outcomes in Renal Insufficiency and Trial to Reduce Cardiovascular Events with Aranesp Thearpy in the Western countries suggested, we do not recommend high doses of ESA to achieve the target hemoglobin (Hb) level. The target Hb of >13 g/dl might lead to increase in the risk of CVD although maintaining a high Hb of >12 g/dl without ESA is not harmful for CKD patients. It is desirable to determine the target Hb in dialysis patients depending on their ages. Renal anemia should be monitored constantly to start ESA and iron replacement therapy at an appropriate time, while avoiding their excess in order to minimize the occurrence of CVD and other complications. Taken all the international guidelines and our clinical experiences together, we should consider administration of ESA when the Hb level becomes <11 g/dl in pre-dialysis patients and <10 g/dl in dialysis patients.

Recent clinical and animal studies have shown that acute kidney injury (AKI), even if followed by complete recovery of renal function, can eventually result in chronic kidney disease (CKD). Renal hypoxia is emerging as a key player in the pathophysiology of the AKI-to-CKD transition. Capillary rarefaction after AKI episodes induces renal hypoxia, which can in turn profoundly affect tubular epithelial cells, (myo)fibroblasts, and inflammatory cells, culminating in tubulointerstitial fibrosis, i.e., progression to CKD. Damaged tubular epithelial cells that fail to redifferentiate might supply a decreased amount of vascular endothelial growth factor and contribute to capillary rarefaction, thus aggravating hypoxia and forming a vicious cycle. Mounting evidence also shows that epigenetic changes are closely related to renal hypoxia in the pathophysiology of CKD progression. Animal experiments suggest that targeting hypoxia is a promising strategy to block the transition from AKI to CKD. However, the precise mechanisms by which hypoxia induces the AKI-to-CKD transition and by which hypoxia-inducible factor activation can exert a protective effect in this context should be clarified in further studies.

Chronic kidney disease (CKD) is placing an increasing burden on patients and societies because no decisive therapy has been established. Tubulointerstitial lesions accompanied by fibrosis, inflammatory cells, and capillary rarefaction not only characterize, but also aggravate renal dysfunction in CKD. In this setting, renal cells, particularly tubular cells, suffer from hypoxia caused by the imbalance of blood perfusion and oxygen demand despite their adaptive responses represented by upregulation of hypoxia-inducible factors (HIFs). Fibrosis is a pathological state characterized by excess extracellular matrix (ECM) deposition, which is also a hallmark and causative factor of many chronic diseases including CKD. Recent studies have suggested that the dominant origin of ECM-producing myofibroblasts (MFs) may be pericytes, which are indispensable cells for maintaining proper capillary functions, as they wrap capillaries and stabilize them through a fine-tuned interplay with endothelial cells. During fibrosis, pericytes are activated and detach from capillaries before conversion into MFs, which compromises capillaries and worsens hypoxia. We also discuss how hypoxia and HIFs affect fibrogenesis. Given that hypoxia is caused by insufficient angiogenesis and that fibrosis results from pericyte loss, restoration of pericytes should be an intriguing target for overcoming both hypoxia and fibrosis. We propose the deactivation of MFs to recover lost pericytes as a promising therapy for CKD.

Incretin-based drugs, i.e., glucagon-like peptide-1 (GLP-1) receptor agonists and dipeptidyl peptidase-4 (DPP-4) inhibitors, are widely used for the treatment of type 2 diabetes. In addition to the primary role of incretins in stimulating insulin secretion from pancreatic β-cells, they have extra pancreatic functions beyond glycemic control. Indeed, recent studies highlight the potential beneficial effects of incretin-based therapy in diabetic kidney disease (DKD). Experimental studies using various diabetic models suggest that incretins protect the vascular endothelium from injury by binding to GLP-1 receptors, thereby ameliorating oxidative stress and the local inflammatory response, which reduces albuminuria and inhibits glomerular sclerosis. In addition, there is some evidence that GLP-1 receptor agonists and DPP-4 inhibitors mediate sodium excretion and diuresis to lower blood pressure. The pleiotropic actions of DPP-4 inhibitors are ascribed primarily to their effects on GLP-1 signaling, but other substrates of DPP-4, such as brain natriuretic peptide and stromal-derived factor-1α, may have roles. In this review, we summarize recent studies of the roles of incretin-based therapy in ameliorating DKD and its complications.

Chloride secretion driven by calcium-dependent chloride channels has a major impact on cyst enlargement in polycystic kidney disease. Buchholz et al. studied the functional role of anoctamin 1 (ANO1), a recently identified calcium-activated chloride channel, in kidney cyst growth, using principal-like cells forming cysts within a collagen matrix and an embryonic kidney cyst model. Their results support a potential role for this chloride channel in cyst generation.

PURPOSE OF REVIEW: Chronic hypoxia in the tubulointerstitium has been recognized as a final common pathway that leads to the development of end-stage renal disease. Hypoxia-inducible factor (HIF), a master regulator of the adaptive response against hypoxia, is involved in the pathogenesis of chronic kidney disease (CKD). This review focuses on HIF and novel therapeutic strategies targeting HIF. RECENT FINDINGS: Although HIF upregulation is beneficial against hypoxic kidney injury, it may be harmful under certain pathological conditions. Recent advances in epigenetic changes provide an additional layer of complexity to our understanding of gene regulation in response to hypoxia, which is most likely involved in the progression of CKD. On the basis of this novel knowledge, the pharmacological activation and modulation of HIF is emerging as a novel therapeutic target. SUMMARY: HIF plays a crucial role in the pathophysiology of CKD. The underlying molecular mechanisms, including epigenetics, have been thoroughly investigated. On the basis of the experimental data available to date, the pharmacological activation of HIF is likely a novel promising therapy for CKD.

Chronic hypoxia in the tubulointerstitium serves as a final common pathway in progressive renal disease. Circumstantial evidence suggests that hypoxia-inducible factor (HIF)-1 in the ischemic tubules may be functionally inhibited in a chronic kidney disease (CKD) milieu. In this study, we hypothesized that indoxyl sulfate (IS), a uremic toxin, impairs the cellular hypoxic response. In human kidney (HK-2) proximal tubular cells, IS reduced the hypoxic induction of HIF-1 target genes. This effect was not associated with quantitative changes in the HIF-1α protein, but with functional impairment of the HIF-1α C-terminal transactivation domain (CTAD). Among factors that impeded the recruitment of transcriptional coactivators to the HIF-1αCTAD, IS markedly up-regulated Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2) through a mechanism of post-transcriptional mRNA stabilization involving the extracellular signal-regulated kinase (ERK) 1/2 pathway. In vivo, disproportionate expression of HIF target genes was demonstrated in several CKD models, which was offset by an oral adsorbent, AST-120. Furthermore, administration of indole reduced the induction of angiogenic, hypoxia-inducible genes in rats with experimental heart failure. Results of these studies reveal a novel role of IS in modulating the transcriptional response of HIF-1 and provide insight into molecular mechanisms underlying progressive nephropathies as well as cardiovascular complications.

INTRODUCTION: Anemia occurs in various chronic diseases and its treatment is dramatically improved after the appearance of erythropoiesis-stimulating agents (ESA). However, there are several problems regarding the use of ESA including: i) invasiveness, ii) high cost and iii) ESA resistance. Therefore, there is a need to develop small molecule drugs which can improve these problems. Hypoxia-inducible factor (HIF) plays a key role in regulating erythropoietin production. HIF stabilizers, particularly, prolyl hydroxylase domain-containing protein (PHD) inhibitors, have emerged as small molecule-based anti-anemia medicine. AREAS COVERED: This article discusses the current status of PHD inhibitors and the pros and cons of currently tested methods. Specifically, the article reviews the advantages of structure-based drug design in the development of PHD inhibitors and looks at future perspectives within the field. EXPERT OPINION: Despite the fact that structure-based drug design has dramatically improved drug discovery, testing on humans is still one of the most time-consuming parts of drug discovery and one that is not accelerated by structural approaches. Exploratory clinical trials, first-in-man studies have emerged as a new strategy for preclinical and clinical development of drugs. Exploratory clinical trials will not only reduce the time and cost in preclinical trials but also provide important information on candidate drug's pharmacological effects in humans. Exploratory clinical trials may be a potential alternative strategy for the drug discovery in the future.

Hypoxia-inducible factor (HIF) is a transcriptional master regulator that takes control of the gene expressions under hypoxia. Several lines of evidence have shown that chronic hypoxia in tubulointerstitium results in irreversible renal disease. Recently, HIF1 was reported to organize a cluster of histone-modifying enzymes by binding to their promoter regions in various kinds of cell lines. However, its function in renal disease remains largely unknown. We focused on the epigenetic regulation on the progression of chronic kidney disease and have reviewed the latest knowledge in this area with special emphasis on the involvement of HIF. For example, a set of HIF1 downstream target genes also were reported to be regulated by cooperative combination of HIF1 and histone demethylase. We suggest a novel epigenetic pathway that affects the final common pathway to end-stage renal disease in addition to the tubulointerstitial hypoxia. We emphasize the importance of figuring out the epigenetic mechanisms of renal failure to find the novel therapeutic approach of chronic kidney disease.

Hypoxia plays a crucial role in many pathophysiological conditions, including cancer biology, and hypoxia-inducible factor (HIF) regulates transcriptional responses under hypoxia. To elucidate the cellular responses to hypoxia, we performed chromatin immunoprecipitation with deep sequencing in combination with microarray analysis and identified HIF-1 targets. We focused on one of the novel targets, sperm-associated antigen 4 (SPAG4), whose function was unknown. SPAG4, an HIF-1-specific target, is up-regulated in various cultured cells under hypoxia. Examination of SPAG4 expression using a tissue microarray consisting of 190 human renal cell carcinoma (RCC) samples revealed that SPAG4 is an independent prognostic factor of cancer-specific mortality. Live-cell imaging revealed localization of SPAG4 at the intercellular bridge in telophase. We also studied cells in which SPAG4 was knocked down. Hypoxia enhances tetraploidy, which disturbs cell proliferation, and knockdown of SPAG4 increased tetraploid formation and decreased cell proliferation under both normoxic and hypoxic conditions. Studies using deletion mutants of SPAG4 also suggested the involvement of SPAG4 in cytokinesis. Microarray analysis confirmed dysregulation of cytokinesis-related genes by knockdown of SPAG4. In conclusion, SPAG4 is an independent prognostic factor in RCC and plays a crucial role in cytokinesis to defend against hypoxia-induced tetraploid formation. This defensive mechanism may promote survival of cancer cells under hypoxic conditions, thus leading to poor prognosis.

Loss of glomerular function associated with the presence of tubulointerstitial lesions, which are characterized by peritubular capillary loss, is a common finding in progressive renal disorders. Dysregulated expression of angiogenic factors (such as vascular endothelial growth factor [VEGF] and angiopoietins) and endogenous angiogenic inhibitors (such as thrombospondin-1, angiostatin and endostatin) underlie these conditions and negatively influence the balance between capillary formation and regression, resulting in capillary rarefaction. Recent studies have provided unequivocal evidence for a pathogenic role of tubulointerstitial hypoxia and the involvement of hypoxia-inducible transcription factors in the advanced stages of chronic kidney disease. The mainstay of potential angiogenic therapies is the application of angiogenic factors with the primary aim of ameliorating reduced oxygenation in the ischaemic tubulointerstitium. However, this strategy is strongly associated with inflammation and changes in vascular permeability. For example, supraphysiological expression of VEGF results in glomerular expansion and proteinuria, whereas VEGF blockade using neutralizing antibodies can cause hypertension and thrombotic microangiopathy. These effects highlight the importance of tight regulation of angiogenic factors and inhibitors. Novel therapeutic approaches that target vascular maturation and normalization are now being developed to protect kidneys from capillary rarefaction and hypoxic injury.

Hypoxia upregulates the hypoxia-inducible factor (HIF) pathway and the endoplasmic reticulum (ER) stress signal, unfolded protein response (UPR). The cross talk of both signals affects the pathogenic alteration by hypoxia. Here we showed that ER stress induced by tunicamycin or thapsigargin suppressed inducible (CoCl(2) or hypoxia) transcription of erythropoietin (EPO), a representative HIF target gene, in HepG2. This suppression was inversely correlated with UPR activation, as estimated by expression of the UPR regulator glucose-regulated protein 78, and restored by an ER stress inhibitor, salubrinal, in association with normalization of the UPR state. Importantly, the decreased EPO expression was also observed in HepG2 overexpressing UPR activating transcription factor (ATF)4. Overexpression of mutated ATF4 that lacks the transcriptional activity did not alter EPO transcriptional regulation. Transcriptional activity of the EPO 3'-enhancer, which is mainly regulated by HIF, was abolished by both ER stressors and ATF4 overexpression, while nuclear HIF accumulation or expression of other HIF target genes was not suppressed by ER stress. Chromatin immunoprecipitation analysis identified a novel ATF4 binding site (TGACCTCT) within the EPO 3'-enhancer region, suggesting a distinct role for ATF4 in UPR-dependent suppression of the enhancer. Induction of ER stress in rat liver and kidney by tunicamycin decreased the hepatic and renal mRNA and plasma level of EPO. Collectively, ER stress selectively impairs the transcriptional activity of EPO but not of other HIF target genes. This effect is mediated by suppression of EPO 3'-enhancer activity via ATF4 without any direct effect on HIF, indicating that UPR contributes to oxygen-sensing regulation of EPO.

Anthracycline chemotherapeutic agents of the topoisomerase inhibitor family are widely used for the treatment of various tumors. Although targeted tumor tissues are generally situated in a hypoxic environment, the connection between efficacy of anthracycline agents and cellular hypoxia response has not been investigated in depth. Here, we report that doxorubicin (DXR) impairs the transcriptional response of the hypoxia-inducible factor (HIF) by inhibiting the binding of the HIF heterodimer to the consensus -RCGTG- enhancer element. This pleiotropic effect retarded migration of von Hippel-Lindau (VHL)-defective renal cell carcinoma and that of VHL-competent renal cell carcinoma in hypoxia. This effect was accompanied by a coordinated down-regulation of HIF target lysyl oxidase (LOX) family members LOX, LOX-like2 (LOXL2), and LOXL4. Furthermore, DXR suppressed HIF target genes in tumor xenografts, inhibited cardiac induction of HIF targets in rats with acute anemia, and impaired the angiogenic response in the isoproterenol-induced heart failure model, which may account for the clinical fragility of doxorubicin cardiomyopathy. Collectively, these findings highlight the impaired hypoxia response by anthracycline agents affecting both tumors and organs of the cancer host and offer a promising opportunity to develop HIF inhibitors using DXR as a chemical template.

Erythropoietin (EPO) is an essential glycoprotein that facilitates red blood cell maturation from erythroid progenitors and mediates erythropoiesis. The use of recombinant human EPO (rhEPO) dramatically changed management of anemic patients with chronic kidney disease and improved their quality of life. EPO is mainly produced in the fetal liver and the postnatal kidney, although the molecular determinants for tissue-specific expression are elusive. Meanwhile, recent advances established a role of hypoxia-inducible factors (HIF) in transcriptionally upregulating EPO in hypoxia. Out of three HIF- isoforms, HIF-2 appears to play a central role. Prolyl hydroxylase domain-containing protein (PHD), a key regulator connecting oxygen availability and HIF-α expression, is also involved in hypoxic induction of EPO mRNA and the precise roles of PHD paralogs in erythropoiesis are now beginning to be uncovered. On the other hand, widespread expression of EPO receptors (EPOR) beyond erythroid progenitors led to the discovery of non-hematopoietic, pleiotropic roles of EPO in the brain, the heart and the kidney. The precise signal transduction pathways of pleiotropic EPO remain unclear, but carbamylated EPO, which fails to bind to the canonical EPOR homodimers or transduce the JAK2-STAT5 signaling, conferred organ protection through multimeric receptors composed of EPO-R and the common β subunit (βCR). The clinical benefit of normalization of anemia in pre-dialysis CKD by EPO therapy is controversial and recent large-scale, randomized-controlled trials do not favor normalization of anemia by EPO in improving cardiovascular as well as renal outcomes. The optimal EPO therapy should be determined based on the clinical context of individual patients.

Because kidneys consume a large amount of oxygen and are relatively inefficient in oxygen uptake, they are susceptible to hypoxia, especially in patients with advanced chronic kidney disease accompanied by loss of peritubular capillaries. Accumulating evidence suggests that chronic tubulointerstitial hypoxia acts as a final common pathway leading to end-stage renal disease. Some biologically active uremic retention molecules, considered as uremic toxins, accumulate as the renal function declines, and at this moment, more than 90 bioactive uremic toxins have been identified. Uremic toxins per se have been proven to accelerate the progression of renal failure. However, the causal relationship between uremic toxin and tubulointerstitial hypoxia remains unclear. Our studies provided direct evidence that uremic toxin dysregulates oxygen metabolism in the kidney. Indoxyl sulfate (IS), a representative protein-bound uremic toxin, increased oxygen consumption in proximal renal tubules, decreased renal oxygenation, and consequently aggravated hypoxia in the remnant rat kidneys. The increase in tubular oxygen consumption by IS was dependent on sodium-potassium adenosine triphosphatase and oxidative stress. Our work also indicated a possible connection between IS and the desensitization of the oxygen-sensing mechanism in erythropoietin-producing cells, which may partly explain inadequate erythropoietin production in hypoxic kidneys of end-stage renal disease patients. Studies of uremic toxins will open a new avenue in development of novel therapeutic approaches of kidney disease.

BACKGROUND: Oxidative stress and endoplasmic reticulum (ER) stress play a crucial role in tubular damage in both acute kidney injury (AKI) and chronic kidney disease (CKD). While the pathophysiological contribution of microRNAs (miRNA) to renal damage has also been highlighted, the effect of miRNA on renal damage under oxidative and ER stresses conditions remains elusive. METHODS: We assessed changes in miRNA expression in the cultured renal tubular cell line HK-2 under hypoxia-reoxygenation-induced oxidative stress or ER stress using miRNA microarray assay and real-time RT-PCR. The pathophysiological effect of miRNA was evaluated by cell survival rate, intracellular reactive oxygen species (ROS) level, and anti-oxidant enzyme expression in miRNA-inhibited HK-2 or miRNA-overexpressed HK-2 under these stress conditions. The target gene of miRNA was identified by 3'-UTR-luciferase assay. RESULTS: We identified 8 and 10 miRNAs whose expression was significantly altered by oxidative and ER stresses, respectively. Among these, expression of miR-205 was markedly decreased in both stress conditions. Functional analysis revealed that decreased miR-205 led to an increase in cell susceptibility to oxidative and ER stresses, and that this increase was associated with the induction of intracellular ROS and suppression of anti-oxidant enzymes. While increased miR-205 by itself made no change in cell growth or morphology, cell viability under oxidative or ER stress conditions was partially restored. Further, miR-205 bound to the 3'-UTR of the prolyl hydroxylase 1 (PHD1/EGLN2) gene and suppressed the transcription level of EGLN2, which modulates both intracellular ROS level and ER stress state. CONCLUSIONS: miR-205 serves a protective role against both oxidative and ER stresses via the suppression of EGLN2 and subsequent decrease in intracellular ROS. miR-205 may represent a novel therapeutic target in AKI and CKD associated with oxidative or ER stress in tubules.

Advanced chronic kidney disease (CKD) patients encounter anemia through insufficient erythropoietin (EPO) production by peritubular fibroblasts. Recent studies showed an increase in EPO production by pharmacological activation of hypoxia-inducible transcription factors (HIFs) in dialysis patients, suggesting that desensitization of the oxygen-sensing mechanism is responsible for the development of renal anemia. Our recent work demonstrated that indoxyl sulfate (IS), a uremic toxin, dysregulates oxygen metabolism in tubular cells. Here we provide evidence of an additional property that IS impairs oxygen sensing in EPO-producing cells. HepG2 cells were stimulated with cobalt chloride (CoCl(2)) or hypoxia under varying concentrations of IS. EPO mRNA was evaluated by quantitative PCR. Nuclear accumulation of HIF-α was evaluated by western blotting. Transcriptional activity of HIF was checked by hypoxia-responsive element (HRE)-luciferase reporter assay. The impact of IS was further evaluated in vivo by administering rats with indole, a metabolic precursor of IS, and subjecting them to CoCl(2) stimulation, in which renal EPO mRNA as well as plasma EPO levels were measured by quantitative PCR and enzyme-linked immunosorbent assay, respectively. Although IS induced cellular toxicity at relatively high concentrations (2.5 mM), EPO mRNA expression was significantly suppressed by IS at concentrations below cytotoxic ranges. In HepG2 cells, IS treatment decreased nuclear accumulation of HIF-α proteins and suppressed HRE-luciferase activity following hypoxia. Furthermore, administration of rats with indole suppressed renal EPO mRNA expression and plasma EPO levels, corroborating in vitro findings. Results of the present study provide a possible connection between a uremic toxin and the desensitization of the oxygen-sensing mechanism in EPO-producing cells, which may partly explain inadequate EPO production in hypoxic kidneys of CKD patients.

PURPOSE OF REVIEW: Incretin mimetics, such as glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), as well as dipeptidyl peptidase-IV (DPP-IV) inhibitors, are used in the treatment of type 2 diabetes mellitus (T2DM). In addition to stimulating insulin secretion from pancreatic β cells, incretins have apparent extrapancreatic functions beyond glycemic control. This review summarizes the recent findings regarding the blood-pressure-lowering effects of incretins and DPP-IV inhibitors in patients who are obese, diabetic, or have metabolic syndrome. RECENT FINDINGS: Clinical studies have indicated that GLP-1 and its analogues lower blood pressure in patients with T2DM, particularly in patients with moderate-to-severe hypertension. DPP-IV inhibitors also appear to elicit a similar blood-pressure-lowering effect. In animal models of salt-sensitive hypertension, incretins appear to induce their antihypertensive effects by inhibiting the proximal tubular sodium reabsorption, and thereby increasing urinary excretion of sodium. These data suggest that the local actions of incretins may be via their key role in regulating natriuresis and lowering blood pressure. SUMMARY: Incretin mimetics and DPP-IV inhibitors are a novel class of antihypertensive drugs with natriuretic properties. They can be used in the treatment of salt-sensitive hypertension, which is characterized by edema.

Injury to the interstitium of the kidney is regarded as a common pathway leading to end-stage renal insufficiency, regardless of etiology. Tubular interstitial nephritis is characterized histologically by inflammatory changes in the tubulointerstitial compartment, such as interstitial edema, leukocyte infiltration, accumulation of extracellular matrix proteins, tubular dilation and atrophy. Acute interstitial nephritis is often associated with use of drugs, such as β-lactam antibiotics and non-steroidal anti-inflammatory drugs, and is likely mediated through allergic mechanisms. On the other hand, chronic progressive tubular interstitial nephritis has a much more diverse etiology, ranging from infection and drugs to immune-mediated, hematologic, metabolic and hereditary disorders. Experimental studies in the past decade have focused mainly on common factors and mechanisms underlying chronic tubulointerstitial injury, such as activation of peritubular fibroblasts, leukocyte infiltration, release of inflammatory cytokines and growth factors at affected regions, epithelial-mesenchymal transition of tubular epithelium, and apoptosis. The execution of each is mediated by a number of local stimuli, such as filtered albumin, chronic hypoxia and oxidative stress, in addition to cytokines and growth factors. This chapter provides an overview of acute and chronic tubular interstitial nephritis, according to clinical manifestations of the disease. It also provides insight into common pathways underlying chronic tubular interstitial nephritis based on recent advances in translational and experimental research.

The hypoxia response regulated primarily by hypoxia-inducible factor (HIF) influences metabolism, cell survival, and angiogenesis to maintain biological homeostasis. In addition to the traditional transcriptional regulation by HIF, recent studies have shown that epigenetic modulation such as histone methylation, acetylation, and DNA methylation could change the regulation of the response to hypoxia. Eukaryotic chromatin is known to be modified by multiple post-translational histone methylation and demethylation, which result in the chromatin conformation change to adapt to hypoxic stimuli. Interestingly, some of the histone demethylase enzymes, which have the Jumonji domain-containing family, require oxygen to function and are induced by hypoxia in an HIF-1-dependent manner. Recent studies have demonstrated that histone modifiers play important roles in the hypoxic environment such as that in cancer cells and that they may become new therapeutic targets for cancer patients. It may lead to finding a new therapy for cancer to clarify a new epigenetic mechanism by HIF and histone demethylase such as JMJD1A (KDM3A) under hypoxia.

Cytoglobin (Cygb), a novel member of the globin superfamily, is expressed by fibroblasts in various organs. However, its function remains unknown. Because of its localization, we speculated that a biological role of Cygb may be related to fibrogenesis. To clarify the role of Cygb in kidney fibrosis, we employed the remnant kidney model in rats. Immunohistochemical analysis showed an increase in Cygb expression in parallel with disease progression. To investigate the functional consequence of Cygb upregulation, we established transgenic rats overexpressing rat Cygb. Overexpression of Cygb improved histological injury, preserved renal function, and ameliorated fibrosis, as estimated by the accumulation of collagen I and IV as well as Masson trichrome staining. These protective effects of Cygb were associated with a decrease in nitrotyrosine deposition in the kidney and urinary 8-hydroxy-2'-deoxyguanosine (8-OHdG) excretion as a marker of oxidative stress. We also performed in vitro studies utilizing a rat kidney fibroblast cell line transiently overexpressing Cygb, an inducible kidney cell transfected with Cygb, and primary cultured fibroblasts isolated from the kidneys of the transgenic rats. These different experimental systems consistently showed that Cygb inhibited collagen synthesis. Furthermore, mutant disruption of heme in Cygb that impaired its antioxidant properties led to the loss of antifibrotic effects, suggesting that Cygb reduces fibrosis via a radical scavenging function. In conclusion, we showed that Cygb plays an important role in protection of the kidney against fibrosis via the amelioration of oxidative stress both in vitro and in vivo. Cygb might represent a good therapeutic target in chronic kidney disease.

Uremic toxins can deteriorate renal function, but little is known about its mechanism. Because tubular injury is central to progression of chronic kidney disease (CKD), we investigated the effects of a representative uremic toxin indoxyl sulfate (IS) on tubular cells. IS induced endoplasmic reticulum (ER) stress in cultured human proximal tubular cells, demonstrated by the increase in C/EBP homologous protein (CHOP) in the immunoblots. Moreover, administration of an oral adsorbent AST-120 reduced serum IS concentration and decreased tubular expression of CHOP in immunohistochemistry in 5/6-nephretomized, CKD model, rats. Furthermore, we disclosed that IS inhibited proliferation of tubular cells in 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium and 5-bromo-2'-deoxyuridine assay, whereas the results of trypan blue exclusion and lactate dehydrogenase assay showed that IS did not promote cell death. This inhibition was mitigated by small interfering (si) RNA against CHOP. Furthermore, IS increased the cyclin-dependent kinase inhibitor p21(WAF1/CIP1) (p21). Surprisingly, this was mediated by the inflammatory cytokine interleukin (IL)-6, the expression of which was decreased by siRNA against activating transcription factor 4, another ER stress marker; however, the induction of IL-6 and p21 by IS was not suppressed by siRNA targeted to CHOP, suggesting that they were downstream of ER stress, but independent of CHOP. Moreover, we found that their upregulation was dependent on ERK, using the ERK pathway inhibitor U-0126. Collectively, we demonstrated that IS induced ER stress in tubular cells and inhibited cell proliferation via two pathways downstream of ER stress, namely CHOP and ERK-IL-6-p21. These are possible targets for suppressing progression of CKD.

In addition to causing uremic symptoms, uremic toxins accelerate the progression of renal failure. To elucidate the pathophysiology of uremic states, we investigated the effect of indoxyl sulfate (IS), a representative uremic toxin, on oxygen metabolism in tubular cells. We demonstrated an increase in oxygen consumption by IS in freshly isolated rat and human proximal tubules. Studies utilizing ouabain, the Na-K-ATPase inhibitor, and apocynin, the NADPH oxidase inhibitor, as well as the in vivo gene-silencing approach to knock down p22(phox) showed that the increase in tubular oxygen consumption by IS is dependent on Na-K-ATPase and oxidative stress. We investigated whether the enhanced oxygen consumption led to subsequent hypoxia of the kidney. An increase in serum IS concentrations in rats administered indole was associated with a decrease in renal oxygenation (8 h). The remnant kidney in rats developed hypoxia at 16 wk. Treatment of the rats with AST-120, an oral adsorbent that removes uremic toxins, reduced serum IS levels and improved oxygenation of the kidney. Amelioration of hypoxia in the remnant kidney was associated with better renal functions and less histological injury. Reduction of serum IS levels also led to a decrease in oxidative stress in the kidney. Our ex vivo and in vivo studies implicated that uremic states may deteriorate renal dysfunction via dysregulating oxygen metabolism in tubular cells. The abnormal oxygen metabolism in tubular cells by uremic toxins was, at least in part, mediated by oxidative stress.

PURPOSE OF REVIEW: Tubulointerstitial hypoxia in the kidney has been considered a hallmark of injury and mediator of disease progression. This review focuses on hypoxia-inducible factor (HIF)-1, a master transcription factor in cellular adaptation to hypoxia. RECENT FINDINGS: HIF-1alpha is expressed in the hypoxic tubular epithelium as well in as the papillary interstitium and glomerular epithelial cells. Although HIF-1 plays a protective role in a number of acute kidney injury models when overexpressed, its activation in chronic kidney disease results in multiple phenotypic changes, depending on the pathological context. SUMMARY: Hypoxia, especially HIF-1, is a critical mediator in the pathogenesis of chronic kidney disease. Underlying molecular mechanisms, together with responsible HIF target genes, are currently under investigation.

HIF (hypoxia-inducible factor)-3alpha is the third member of the HIF transcription factor family. Whereas HIF-1alpha and -2alpha play critical roles in the cellular and systemic adaptation to hypoxia, little is known about the regulation and function of HIF-3alpha. At least five different splice variants may be expressed from the human HIF-3alpha locus that are suggested to exert primarily negative regulatory effects on hypoxic gene induction. In the present paper, we report that hypoxia induces the human HIF-3alpha gene at the transcriptional level in a HIF-1-dependent manner. HIF-3alpha2 and HIF-3alpha4 transcripts, the HIF-3alpha splice variants expressed in Caki-1 renal carcinoma cells, rapidly increased after exposure to hypoxia or chemical hypoxia mimetics. siRNA (small interfering RNA)-mediated HIF-alpha knockdown demonstrated that HIF-3alpha is a specific target gene of HIF-1alpha, but is not affected by HIF-2alpha knockdown. In contrast with HIF-1alpha and HIF-2alpha, HIF-3alpha is not regulated at the level of protein stability. HIF-3alpha protein could be detected under normoxia in the cytoplasm and nuclei, but increased under hypoxic conditions. Promoter analyses and chromatin immunoprecipitation experiments localized a functional hypoxia-responsive element 5' to the transcriptional start of HIF-3alpha2. siRNA-mediated knockdown of HIF-3alpha increased transactivation of a HIF-driven reporter construct and mRNA expression of lysyl oxidase. Immunohistochemistry revealed an overlap of HIF-1alpha-positive and HIF-3alpha-positive areas in human renal cell carcinomas. These findings shed light on a novel aspect of HIF-3alpha as a HIF-1 target gene and point to a possible role as a modulator of hypoxic gene induction.

Recent studies underscore that chronic hypoxia in the tubulointerstitium is a final common pathway to progression to end-stage renal failure regardless of etiology. We used microarray analysis of rat kidneys made hypoxic by unilateral renal artery stenosis to measure transcriptomic events and clarify pathophysiological mechanisms of renal injury induced by chronic hypoxia. Many genes were upregulated in the kidney by chronic hypoxia, but we focused on metallothionein due to its antioxidative properties. Using tubular epithelial cells transfected with a reporter construct of luciferase, driven by the hypoxia-responsive elements (HRE), we found that addition of metallothionein to the culture media increased luciferase activity. This was associated with upregulation of the target genes of hypoxia-inducible factor (HIF), such as vascular endothelial growth factor and glucose transporter-1. Stimulation of the HIF-HRE pathway by metallothionein was confirmed by metallothionein overexpression. Hypoxia and exogenous metallothionein increased HIF-1alpha protein without changes in its mRNA levels, suggesting protein stabilization. Upregulation of the HIF-HRE system by metallothionein was associated with phosphorylation of ERK but not Akt. MEK inhibition and rapamycin decreased metallothionein-induced HIF activity. Our study shows that upregulation of metallothionein expression by hypoxia activates the HIF-HRE system through the ERK/mTOR pathway and may be a novel defense against hypoxia.

Hypoxia-inducible factor (HIF) is a heterodimeric transcription factor composed of an oxygen-dependent alpha-subunit and constitutively expressed beta subunit, which plays a central role in cellular adaptation to hypoxia by transcriptionally upregulating its target genes involved in angiogenesis, erythropoiesis, glycolysis, and so on. Recent studies demonstrated that hypoxia in the tubulointerstitium is involved in the pathology of progressive renal diseases and that HIF, which is activated in experimental kidney diseases, may serve to protect tubulointerstitium from the ischemic insult. The expression of HIF alpha-chains is post-translationally regulated and hydroxylation at one or two of the conserved proline residues by prolyl-hydroxylase domains (PHDs) is a critical step for the oxygen-dependent recruitment of the von Hippel-Lindau gene product (pVHL), a recognition component of the E3 ubiquitin ligase complex, and degradation of HIF-alpha. Conversely, modalities to inhibit the enzymatic activities of PHDs have been shown to activate HIF irrespective of oxygenation status and are regarded as candidate targets of pharmacological approaches against chronic kidney diseases characterized by hypoxia.

Reduction of vascular endothelial growth factor (VEGF) expression plays a crucial role in chronic kidney disease (CKD). In order to clarify a cause of VEGF suppression in CKD, we examined an interaction between proteinuria and VEGF. Rat proximal tubular cells were subjected to hypoxia with or without albumin to mimic proteinuric conditions, and VEGF expression was assessed by real-time quantitative PCR and enzyme-linked immunosorbent assays. Albumin significantly reduced VEGF expression under hypoxia. Luciferase activity controlled by hypoxia-responsive element (HRE) was suppressed by albumin, demonstrating suppression of the hypoxia-inducible factor (HIF)/HRE pathway. Studies utilizing a proteasome inhibitor and a prolyl hydroxylase inhibitor showed that mechanisms of HIF/HRE pathway suppression by albumin load did not involve degradation of HIF protein levels. Further, albumin did not change HIF mRNA levels. Our data, for the first time, suggest a clear 'link' between proteinuria and hypoxia, the two principal pathogenic factors for CKD progression.

Central to cellular responses to hypoxic environment is the hypoxia-inducible factor (HIF) transcriptional control system. A role for HIF-2alpha was investigated in a model of renal ischemia-reperfusion injury (IRI) associated with oxidative stress using HIF-2alpha knockdown mice. In these mice, HIF-2alpha expression was approximately one half that of wild-type mice, whereas HIF-1alpha expression was equivalent. HIF-2alpha knockdown mice were more susceptible to renal IRI, as indicated by elevated blood urea nitrogen levels and semiquantitative histologic analysis. Immunostaining with markers of oxidative stress showed enhanced oxidative stress in the kidney of HIF-2alpha knockdown mice, which was associated with peritubular capillary loss. Real-time quantitative PCR analysis showed decreased expression of antioxidative stress genes in the HIF-2alpha knockdown kidneys. Studies that used small interference RNA confirmed regulation of the antioxidative stress genes in cultured endothelial cells. Although HIF-2alpha knockdown mice were anemic, serum erythropoietin levels were not significantly increased, reflecting inappropriate response to anemia as a result of HIF-2alpha knockdown. Experiments that used hemodiluted mice with renal ischemia demonstrated that anemia of this degree did not affect susceptibility to ischemia. Knockdown of HIF-2alpha in inflammatory cells by bone marrow transplantation experiments demonstrated that HIF-2alpha in inflammatory cells did not contribute to susceptibility to renal IRI. Restoration of HIF-2alpha in endothelium by intercrossing with Tie1-Cre mice ameliorated renal injury by IRI, demonstrating a specific role of endothelial HIF-2alpha. These results suggest that HIF-2alpha in the endothelium has a protective role against ischemia of the kidney via amelioration of oxidative stress.

Since the first description of a protective effect of hypoxic preconditioning in the heart, the principle of reducing tissue injury in response to ischemia by prior exposure to hypoxia was confirmed in a number of cells and organs. However, despite impressive preclinical results, hypoxic preconditioning has so far failed to reach clinical application. Nevertheless, it remains of significant interest to induce genes that are normally activated during hypoxia and ischemia as part of an endogenous escape mechanism prior to or during the early phase of an ischemic insult. This approach has recently been greatly facilitated by the identification of hypoxia-inducible factors (HIFs), transcription factors that operate as a master switch in the cellular response to hypoxia. Far more than 100 target genes are regulated by HIF, including genes such as erythropoietin and hemoxygenase-1, which have been shown to be tissue-protective. The identification of small molecule inhibitors of the oxygen-sensing HIF-prolyl hydroxlases now offers the possibility to mimic the hypoxic response by pharmacological stabilization of HIF in order to achieve organ protection. Oxygen-independent activation of HIF is therefore a promising therapeutic strategy for the prevention of organ injury and failure.

Renal senescence is characterized by interstitial fibrosis and loss of peritubular capillaries. In this study, we provided evidence of tubulointerstitial hypoxia and the operation of hypoxia-inducible factor (HIF) in the aging kidney. Using two distinct methods, pimonidazole immunostaining and the expression of the "hypoxia-responsive" reporter of the transgenic rats, we identified the age-related expansion of hypoxia in all areas of the kidney. Expansion was most prominent in the cortex. Clusters of hypoxic tubules were observed in the superficial cortical zones, areas adjacent to the outer nephrons and expanded in the medullary rays. The degree of hypoxia was positively correlated with the age-related tubulointerstitial injury (R(2) = 0.88, p <.01), which was associated with the upregulation of HIF-regulated genes, such as vascular endothelial growth factor (VEGF) and glucose transporter-1 (GLUT1) (real-time polymerase chain reaction). These findings point to the involvement of hypoxia and highlight the pathological relevance of HIF and its target genes in the aging kidney.

Tissue hypoxia occurs when local metabolism is disturbed by an imbalance between oxygen supply and consumption. This condition can lead to a variety of serious ischemic disorders, including a number of important cardiovascular diseases. In the search for therapeutic approaches, focused modalities which specifically target hypoxia have been particularly sought. These efforts would profit from the ability to utilize the mechanisms by which cells adjust to hypoxic conditions. At the center of the cellular response to hypoxia is hypoxia-inducible factor, HIF. This factor is composed of two subunits, an oxygen-sensitive HIF-alpha subunit and a constitutively expressed HIF-beta subunit. Intracellular accumulation of HIF induces the coordinated expression of a number of adaptive genes against hypoxic insult. Because activation of HIF is a promising therapeutic modality for ischemic cardiovascular disease, recent studies have focused on the development of HIF stimulators. HIF levels are regulated by prolyl hydroxylation and asparaginyl hydroxylation of the HIF-alpha subunit. To date, a single HIF asparaginyl hydroxylase has been identified, factor inhibiting HIF (FIH), whereas the mammalian genome encodes three closely related proteins that have HIF prolyl hydroxylase activity, PHD1, PHD2 and PHD3. Recent patents have disclosed methods for identifying modulators of HIF or PHD as well as novel compounds with properties of HIF modulation or prolyl hydroxylase inhibition. This review highlights the identification of novel HIF stabilizers as specific molecularly targeted therapies against cardiovascular disease.

Vascular endothelial growth factor (VEGF) is an important survival factor for endothelial cells in hypoxic environments. High glucose regulates certain aspects of VEGF expression in various cell types, including proximal tubular cells. Thus, ambient glucose levels may modulate the progression of chronic kidney disease, especially diabetic nephropathy. Immortalized rat proximal tubular cells (IRPTC) were cultured for 24 h under hypoxic conditions (1% O(2)), with or without high d-glucose (25 mM), or with or without high l-glucose (25 mM). Controls included culture in normoxic conditions and normal d-glucose (5.5 mM). VEGF mRNA expression was assessed by real-time quantitative PCR, and VEGF protein in the supernatant was assessed by ELISA. Hypoxia increased VEGF expression. This response was significantly blunted by high d-glucose (1.98 +/- 0.11- versus 2.65 +/- 0.27-fold increase for VEGF mRNA expression, 252.8 +/- 14.7 versus 324.0 +/- 11.5 pg/10(5) cells for VEGF protein; P < 0.05 both) but not by high l-glucose. It is interesting that hydrogen peroxide also blunted this response, whereas alpha-tocopherol restored the VEGF response to hypoxia in the presence of high d-glucose. For determination of involvement of the hypoxia-inducible factor (HIF)/hypoxia-responsible element (HRE) pathway, IRPTC that were stably transfected with HRE-luciferase were cultured under the previous conditions. High d-glucose also reduced luciferase activity under hypoxia, whereas alpha-tocopherol restored activity. In vivo experiments using streptozotocin-induced diabetic rats confirmed that hyperglycemia blunted HIF-HRE pathway activation. Insulin treatment restored activation of the HIF-HRE pathway in streptozotocin-induced diabetic rats. In conclusion, high glucose blunts VEGF response to hypoxia in IRPTC. This effect is mediated by the oxidative stress-regulated HIF-HRE pathway.

BACKGROUND: We previously demonstrated that chronic hypoxia has pivotal roles in the progression of tubulointerstitial injury from the early stage of the uninephrectomized Thy1 nephritis model. We have also shown that pretreatment of cobalt confers renoprotection in the ischemia/reperfusion (I/R) injury, in association with the up-regulation of hypoxia-inducible factor (HIF)-regulated genes. Here, we tested the hypothesis that cobalt administration not only attenuates acute ischemic insult, but also ameliorates tubulointerstitial injury secondary to chronic hypoxia. METHODS: We applied sustained cobalt treatment to the uninephrectomized Thy1 nephritis model at 3 to 5 weeks, when tubular hypoxia appeared. Histologic evaluation, including glomerular and peritubular capillary networks, was made at 8 weeks. HIF activation was confirmed by real-time polymerase chain reaction (PCR) analyses for HIF-regulated genes, such as erythropoietin (EPO), vascular endothelial growth factor (VEGF), and heme oxygenase 1 (HO-1). Up-regulation of HIF-1alpha and HIF-regulated genes was also verified by Western blotting analysis. To elucidate responsible mechanisms of cobalt in the amelioration of tubuloniterstitial injury, terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick end labeling (TUNEL) staining was conducted at 5 weeks. A combination therapy with angiotensin receptor blocker (ARB), olmesartan, was also challenged. RESULTS: Although the intervention did not change glomerular structural damage or urinary protein excretion rate, tubulointerstitial injury was improved in cobalt-treated animals when compared with the vehicle-treated group. The amelioration was associated with the parallel up-regulation of renoprotective, HIF-regulated gene expression. TUNEL staining revealed that the number of apoptotic cells was reduced in the cortex by cobalt administration, suggesting that renoprotection was achieved partly through its antiapoptotic properties. Furthermore, it was demonstrated that cobalt treatment exerts additional renoprotective effects with the ARB treatment in this model. CONCLUSION: Maneuvers to activate HIF in the ischemic tubulointerstitium will be a new direction to future therapeutic strategies.

Hypoxia-inducible factor (HIF)-1 is a transcription factor mediating cellular response to hypoxia. Although it is expressed in tubular cells of the ischemic kidney, its functional role is not fully clarified in the pathological context. In this study, we investigated a role of HIF in tubular cell apoptosis induced by cisplatin. HIF-1alpha was expressed in tubular cells in the outer medulla 3 days after cisplatin (6 mg/kg) administration. With the in vivo administration of cobalt to activate HIF, the number of apoptotic renal tubular cells became much smaller in the outer medulla, compared with the vehicle group. We also examined the functional role of HIF-1 in vitro using immortalized rat proximal tubular cells (IRPTC). In hypoxia, IRPTC that express dominant-negative (dn) HIF-1alpha showed impaired survival in cisplatin injury at variable doses (25-100 microM, 24 h), which was not obvious in normoxia. The observed difference in cell viability in hypoxia was associated with the increased number of apoptotic cells in dnHIF-1alpha clones (Hoechst 33258 staining). Studies on intracellular signaling revealed that the degree of cytochrome c release, dissipation of mitochondrial membrane potentials, and caspase-9 activity were all more prominent in dnHIF-1alpha clones than in control IRPTC, pointing to the accelerated signaling of mitochondrial pathways. We propose that HIF-1 mediates cytoprotection against cisplatin injury in hypoxic renal tubular cells, by reducing the number of apoptotic cells through stabilization of mitochondrial membrane integrity and suppression of apoptosis signaling. A possibility was suggested that activation of HIF-1 could be a new promising therapeutic target for hypoxic renal diseases.

Tubulointerstitial hypoxia has been implicated in a number of progressive renal diseases, and several lines of evidence indicate that the administration of angiogenic growth factors ameliorates tubulointerstitial injury. We hypothesized that induction of hypoxia-inducible factors (HIF) mediates renoprotection by their angiogenic properties. At 5-9 weeks after subtotal nephrectomy, cobalt was administered to rats to activate HIF. Histological evaluation demonstrated that the tubulointerstitial injury was significantly ameliorated in animals that received cobalt (score: 2.51+/-0.12 (cobalt) vs 3.21+/-0.24 (vehicle), P<0.05). Furthermore, animals receiving cobalt had fewer vimentin- and TdT-mediated dUTP nick-end labeling (TUNEL)-positive tubular cells. The renoprotective effect of cobalt was associated with the preservation of peritubular capillary networks (rarefaction index: 13.7+/-0.4 (cobalt) vs 18.6+/-0.9 (vehicle), P<0.01). This improvement in capillary networks was accompanied by an increased number of proliferating (PCNA-positive) glomerular and peritubular endothelial cells. The angiogenesis produced by this method was not accompanied by an increase in vascular permeability. Furthermore, in vitro experiments clarified that HIF-1 in tubular epithelial cells promotes proliferation of endothelial cells and that HIF-2 overexpressed in renal endothelial cells mediates migration and network formation. Collectively, these findings demonstrate a renoprotective role of HIF through angiogenesis and provide a rationale for therapeutic approaches to target HIF for activation.

BACKGROUND: Activation of hypoxia-inducible factor-1 (HIF-1) is the primary defensive mechanism against hypoxia. HIF-1 activation generally occurs in pathologic disruption of tissue oxygenation. However, a biologic role of HIF-1 in the medulla of the kidney, which is considered perpetually hypoxic under physiologic conditions due to its unique circulation, remains to be elucidated. METHODS: The expression of HIF-1alpha was detected by immunohistochemical analysis. Functional studies of HIF in medulla were carried out by gene transfer of various plasmids by retrograde injection via ureter. RESULTS: Our immunohistochemical analysis detected HIF-1alpha in the inner stripe and the inner medulla of normal rats. Water deprivation increased the number of HIF-1alpha-positive cells, which may be mediated by an increase in medullar workload and a decrease in local blood flow. To perform functional studies, we performed gene transfer. Efficient expression of the transgene was confirmed using an enhanced green fluorescent protein (E-GFP) expressing vector. Our histologic and immunoblotting analysis detected the transgene product at the inner medulla and the inner stripe 48 hours after injection. Administration of negative-dominant HIF induced severe damage in the medulla of normal rats. In contrast, gene transfer of constitutively active HIF (HIF/VP16) induced expression of various HIF-regulated genes and protected the medulla against ischemic insults. CONCLUSION: Our studies demonstrated a crucial role of HIF in the renal medulla under normal and hypoxic circumstances.

Despite the increasing need to identify and quantify tissue oxygenation at the cellular level, relatively few methods have been available. In this study, we developed a new hypoxia-responsive reporter vector using a hypoxia-responsive element of the 5' vascular endothelial growth factor untranslated region and generated a novel hypoxia-sensing transgenic rat. We then applied this animal model to the detection of tubulointerstitial hypoxia in the diseased kidney. With this model, we were able to identify diffuse cortical hypoxia in the puromycin aminonucleoside-induced nephrotic syndrome and focal and segmental hypoxia in the remnant kidney model. Expression of the hypoxia-responsive transgene increased throughout the observation period, reaching 2.2-fold at 2 weeks in the puromycin aminonucleoside model and 2.6-fold at 4 weeks in the remnant kidney model, whereas that of vascular endothelial growth factor showed a mild decrease, reflecting distinct behaviors of the two genes. The degree of hypoxia showed a positive correlation with microscopic tubulointerstitial injury in both models. Finally, we identified the localization of proliferating cell nuclear antigen-positive, ED-1-positive, and terminal dUTP nick-end labeled-positive cells in the hypoxic cortical area in the remnant kidney model. We propose here a possible pathological tie between chronic tubulointerstitial hypoxia and progressive glomerular diseases.

In hypoxia, ATP depletion causes cellular Ca(2+) increase, mitochondrial injury, and apoptosis in renal tubular cells. However, the molecular basis of these observations is incompletely delineated. IRPTC, a rat renal proximal tubular cell line, was treated with antimycin A, and disturbances in cytoplasmic calcium ([Ca(2+)]c) and mitochondrial calcium ion concentration ([Ca(2+)]m), dissipation of mitochondrial membrane potential (DeltaPsi(m)), cytochrome c release, and resultant apoptosis were examined. Pharmacologic targeting of L-type Ca(2+) channels in vitro and in vivo was used to clarify the involvement of voltage-dependent Ca(2+) channels during this process. In vitro studies indicated that ATP depletion-induced apoptosis was preceded by increased [Ca(2+)]c and [Ca(2+)]m before activation of mitochondrial signaling. Antagonizing L-type Ca(2+) channels offset these findings, suggesting [Ca(2+)]c and [Ca(2+)]m involvement. Azelnidipine administration ameliorated cellular and mitochondrial Ca(2+) accumulation, mitochondrial permeability transition, cytochrome c release, caspase-9 activation, and resultant apoptosis (15.8 +/- 0.8% versus 8.9 +/- 0.7%; P < 0.01). Similar effects of azelnidipine were substantiated in an in vivo ischemia/reperfusion injury model. There were fewer terminal-deoxynucleotidyl transferase mediated dUTP nick-end labeling-positive cells in the azelnidipine-treated group (0.322 +/- 0.038/tubule) as compared with the vehicle-treated group (0.450 +/- 0.041; P < 0.05), although the antiapoptotic effect was smaller in vivo than in vitro, partly as a result of distinct levels of Bax expression. It is proposed that voltage-dependent Ca(2+) channels are involved in cellular and mitochondrial accumulation of Ca(2+) subsequent to ATP depletion and play an important role in regulating mitochondrial permeability transition, cytochrome c release, caspase activation, and apoptosis.

Chronic hypoxia likely plays a pivotal role in chronic renal disease, but the specifics of its involvement remain unclear. To elucidate how chronic hypoxia occurs and whether hypoxia participates in the progression of renal disease, the authors established an irreversible glomerulonephritis model induced by uninephrectomy and repeated anti-Thy-1 antibody injections. Glomerulosclerosis with microvascular obliteration was complete at 2 wk after antibody injection and was not restored until 11 wk. Tubulointerstitial injury was mild at 2 wk and was gradually exacerbated until 11 wk, a pattern that was in accordance with the loss of peritubular capillaries. Immunohistochemical analysis using pimonidazole revealed the augmentation of hypoxia in the cortex before the aggravation of tubulointerstitial injury and subsequent peritubular capillary loss. The preexistence of hypoxia implies that it had substantial participation in the progression of tubulointerstitial injury. To test whether blood flow was inhibited in diseased kidneys, capillaries with intact blood flow were identified by tail vein injection of biotinylated lectin specific to endothelial cells. The renal microvasculature was well recognized by lectin in the controls, whereas lectin binding to peritubular capillaries was strikingly decreased in diseased kidneys, suggesting a disturbance of blood flow. Intravital microscopy analysis confirmed that blood flow in peritubular capillaries was decreased by approximately 40% in the disease group compared with the controls. In conclusion, stagnation of blood flow in peritubular capillaries induced chronic hypoxia at an early stage in this model, which was followed by progressive tubulointerstitial injury and a loss of peritubular capillaries.

The remnant kidney model is a mainstay in the study of progressive renal disease. The earliest changes in this model result from glomerular hemodynamic alterations. Given that progressive renal disease is the result of subsequent interstitial damage initiated by undetermined pathogenic factors, the authors investigated the role of hypoxia as a pathogenic factor in tubulointerstitial damage after renal ablation in rats. Cortical tissue hypoxia in the early phase (4 and 7 d) in remnant kidney rats, sham-operated rats, and animals treated with the angiotensin II receptor blocker (ARB) olmesartan (10 mg/kg per d) was assessed by uptake of a hypoxic probe, pimonidazole, expression of HIF-1alpha, and by increased transcription of hypoxia-responsive genes. Physiologic perfusion status of the postglomerular peritubular capillary network was evaluated by lectin perfusion and Hoechst 33342 diffusion techniques. Results showed that the number of hypoxic tubules was markedly increased 4 and 7 d after nephron loss. These findings antedated any histologic evidence of tubulointerstitial damage. The hypoxic state persisted until interstitial damage developed. These results were confirmed using HIF-1alpha immunoprecipitation and increase of hypoxia-responsive genes. Pathologic studies of the vasculature demonstrated significant functional changes that generated a hypoxic milieu. ARB treatment prevented vascular changes and ameliorated tubular hypoxia. These results suggest that the initial tubulointerstitial hypoxia in remnant kidney model plays a pathogenic role in the subsequent development of tubulointerstitial injury. The initial hypoxia in this model was dependent on activation of the renin-angiotensin system and hemodynamic alterations after nephron loss.

Immune complex deposition is associated with the accumulation of neutrophils, which play an important role in the various immune-mediated diseases. A novel anti-inflammatory agent, the lipoxin A (LXA) analogue (15-epi-16-(FPhO)-LXA-Me)), a stable synthetic analogue of aspirin-triggered 15-epi-lipoxin A4 (ATLa), was used in experimental anti-glomerular basement membrane (GBM) antibody nephritis in mice. ATLa was administered before the induction of the disease, and 2 h later, the animals were killed. ATLa reduced the infiltrating neutrophils and nitrotyrosine staining in glomeruli. Subsequent changes of gene expression in the early phase were evaluated, and 5674 genes were present under the basal conditions in kidneys from normal mice; 54 upregulated genes and 25 downregulated genes were detected in anti-GBM nephritis. Eighteen of these upregulated genes were those induced by IFN-gamma. Real-time quantitative PCR analysis confirmed the results of the microarrays. To investigate a role of IFN-gamma in neutrophil infiltration, anti-GBM nephritis was induced in IFN-gamma knockout mice. The number of infiltrating neutrophils in these mice did not differ from those in wild-type mice. Also examined were CD11b expression on neutrophils from mice treated with ATLa by flow cytometry, but suppression of this adhesion molecule was not observed. Neutrophil infiltration was successfully inhibited by ATLa in the early phase of murine anti-GBM nephritis. Microarray analysis detected the change of mRNA expression in anti-GBM nephritis and demonstrated amelioration of various genes by ATLa, which may provide a clue to the development of novel therapeutic approaches in immune renal injury.

BACKGROUND: Tubulointerstitial fibrosis leads to progressive kidney disease and, ultimately, may result in end-stage renal disease (ESRD). Myofibroblasts, which express alpha-smooth muscle actin (alpha-SMA) in their cytoplasm, regulate renal fibrogenesis. Recent studies suggest that certain interstitial myofibroblasts derive from renal tubular cells that have undergone epithelial-mesenchymal transformation (EMT) (transdifferentiation). However, the role(s) of hypoxia, which is involved in progressive kidney disease, on tubular EMT remains unclear. METHODS: Immortalized rat proximal tubular cells (IRPTC) were cultured in normobaric hypoxia (1% O2) for 3, 6, or 15 days, with match control in normoxic conditions. alpha-SMA, vimentin, and desmin chosen as markers of EMT were measured by immunocytochemistry and immunoblots collagen I production and cell motility were chosen as functional assays. Various concentrations of cobaltous chloride (CoCl2) were used as hypoxic mimickers. In vivo studies were carried out in a chronic ischemic kidney model. RESULTS: Immunohistochemical studies revealed increased expression of alpha-SMA. Striking morphologic changes were detected after 6 days of hypoxia for alpha-SMA-positive fibroblast-like cells (SMA + fib) and after 15 days for alpha-SMA-positive myofibroblast-like cells (SMA + myo). Immunoblots confirmed these findings. Collagen I production increased in a time-dependent manner parallel to alpha-SMA expression. Cell motility assays demonstrated that transformed cells had higher migratory capacity than normal tubular cells. Cobaltous salt also induced alpha-SMA and collagen I synthesis. Chronic ischemic kidney revealed in vivo tubular EMT at day 7. CONCLUSION: Hypoxia can induce tubular EMT. This process may play an important role in progression of kidney disease.

BACKGROUND: Glomerular endothelial cells (GENs) play a key role in the preservation and reconstruction of the glomerular capillary network following injury, thus maintaining the tissue oxygenation. Accumulating evidence has shown that failure to maintain the microcirculation leads to irreversible glomerular injury and glomerular sclerosis. In this regard, the behavior of endothelial cells in a hypoxic milieu is of interest. METHODS: We exposed cultured GENs to hypoxia and observed apoptosis by annexin V assay. We examined mitochondrial signaling, focusing on Bcl2 and Bax by real-time polymerase chain reaction (PCR), immunocytochemistry, and immunoprecipitation. Furthermore, we examined the response to hypoxia in an overexpression model of Bcl2. RESULTS: Hypoxic treatment induced apoptosis in 12.8%+/- 1.1% of GENs at 24 hours, and in 19.8%+/- 0.9% at 24 hours followed by 8 hours of reoxygenation. The expression of Bcl2 mRNA decreased to 0.45- +/- 0.15-fold at 24 hours, whereas that of Bax increased to 7.3- +/- 1.2-fold 1 hour after reoxygenation, accompanied by translocation from the cytosol to mitochondria. These changes were associated with a decrease in mitochondrial membrane potentials and an increase in caspase-9 activity. Both overexpression of Bcl2 and inhibition of Bax protected GENs from hypoxic injury. CONCLUSION: We conclude that changes of quantity and localization of Bcl2 and Bax contribute to hypoxia-mediated apoptosis of GENs in vitro. Further investigation into glomerular endothelial cell injury and intracellular signaling in a hypoxic milieu is required to better understand and ultimately prevent progression of chronic kidney disease.

Chronic hypoxia is a major contributor to tubulointerstitial injury in various renal diseases and apoptosis is apparently involved. Although many studies report hypoxia-induced apoptosis in cultured tubular cells, information has been limited in proximal tubular cells, those from the most susceptible portion of renal tubules against hypoxia. This study was to confirm a role for apoptosis in hypoxic proximal tubular cells and to investigate its association with HIF-1. Temperature-sensitive SV40-immortalized rat proximal tubular cells (IRPTCs) showed apoptosis in 21.9+/-2.9% by hypoxia (0.2% O(2), 48h), with alterations in mitochondrial signaling such as Bcl2 and caspase-9. Bax mRNA was unaffected during the process. However, treating IRPTCs at the nonpermissive temperature showed an upregulation of Bax by hypoxia, which was abrogated by overexpressing dominant-negative HIF-1alpha. These findings extend previous reports on hypoxia-mediated tubular cell apoptosis and demonstrate the possible involvement of HIF-1 as an upstream molecule of Bax.

Hypoxia in the tubulointerstitium has been thought to play pivotal roles in the pathophysiology of acute renal failure and the progression of chronic kidney disease. Pre-induction of hypoxia-inducible and renoprotective gene expression may protect subsequent ischemic injury. This study evaluated the efficacy of cobalt, which inhibits HIF-1 degradation and increases the expression level of hypoxia-related genes, in an acute ischemic tubulointerstitial injury model of rats. Ischemic renal injury was induced by 45-min clamping of renal pedicles with contralateral nephrectomy. Elevation of serum creatinine and morphologic injury after the ischemic insult was observed. Administration of cobalt chloride afforded striking functional improvement (mean +/- SEM creatinine in mg/dl: Co treatment group, 2.14 +/- 1.21; control, 3.69 +/- 1.43; P < 0.05) associated with amelioration of tubulointerstitial damage. Cobalt treatment also reduced macrophage infiltration significantly. In the kidney of rats treated with cobalt, mRNA levels of several genes that serve for tissue protection, such as HO-1, EPO, Glut-1, and VEGF, were increased before ischemic injury. Upregulation of HO-1 by cobalt was confirmed at the protein level. Subcutaneous injection of cobalt also ameliorated ischemic injury, which was associated with upregulation of renal HIF-1alpha protein expression. These results suggest that protection against hypoxic tubulointerstitial injury by cobalt administration is mediated by induction of renoprotective gene expression. HIF induction is one possible and attractive explanation for the observed effects.