Search

Renal fibrosis results in the progressive renal dysfunction and leads to chronic kidney disease (CKD) and ultimately end-stage renal disease. Asiaticoside was reported to regulate synaptopodin, desmin, nephrin and podocin levels in adriamycin-induced nephropathy of rats. In this study, we found out that asiaticoside inhibited renal fibrosis in vitro and in vivo. Additionally, miR-142-5p was upregulated in in vitro and in vivo models of CKD. MiR-142-5p promoted the levels of collagen I, collagen IV and fibronectin proteins. Additionally, miR-142-5p overexpression partly rescued the protective effect of asiaticoside on renal fibrosis. Mechanistically, miR-142-5p inhibited ACTN4 levels by binding with its 3´untranslated region (UTR), and further reduced its translation. Treatment of aisaticoside decreased miR-142-5p levels and increased ACTN4 levels. Rescue assays revealed that ACTN4 overexpression partially rescued the effect of miR-142-5p on renal fibrosis. Asiaticoside mitigated renal fibrosis by regulating the miR-142-5p/ACTN4 axis. In conclusion, asiaticoside inhibits renal fibrosis by regulating the miR-142-5p/ACTN4 axis. This novel discovery suggested that asiaticoside may serve as a potential medicine for renal fibrosis improvement. This article is protected by copyright. All rights reserved.

Metabolic syndrome (MetS) increases the risk of kidney disease. In SHRSP.Z-Leprfa /IzmDmcr (SHRSP.ZF) rats with MetS, protease-activated receptor 2 (PAR2)-mediated vasorelaxation is preserved in the aorta at 20 weeks of age (weeks) via enhancement of nitric oxide production but impaired at 30 weeks by oxidative stress. However, impairment of PAR2-mediated vasorelaxation of renal arteries and its possible implications for kidney disease are unclear. We used organ baths to assess PAR2-mediated vasorelaxation of isolated renal arteries, colorimetric methods to measure urinary protein levels as an index of renal function, and western blot to determine expression of PAR2 and nephrin proteins in the kidneys of SHRSP.ZF rats at 10, 20, and 30 weeks. We assessed renal arteries and kidney function for effects of orally administered GB88, a pathway-dependent PAR2 antagonist, from 10 to 18 weeks, and azilsartan, an angiotensin II type 1 receptor blocker, from 13 to 23 weeks. PAR2-mediated vasorelaxation was slightly lower at 20 weeks and attenuated significantly at 30 weeks compared with those at 10 weeks. Urinary protein levels were increased at 20 and 30 weeks. Decreased protein expression of PAR2 and nephrin in the kidney were observed at 30 weeks. Administration of GB88 increased blood pressure (BP) and proteinuria. Azilsartan reduced the high BP and the impaired PAR2-mediated vasorelaxation, but did not restore the increase in urinary protein levels and decreased PAR2 and nephrin protein expression in the kidney. PAR2 activation in the kidney may be associated with maintenance of BP and urinary protein excretion in MetS.

Mesangial lesions and podocyte injury are essential manifestations of the progression of diabetic kidney disease (DKD). Although cross-communication between mesangial cells (MCs) and podocytes has recently been suggested by the results of single-nucleus RNA sequencing analyses, the molecular mechanisms and role in disease progression remain elusive. Our cDNA microarray data of diabetic mouse glomeruli suggested the involvement of endoplasmic reticulum (ER) stress in DKD pathophysiology. In vitro experiments revealed the suppression of the ER-associated degradation (ERAD) pathway and induction of apoptosis in podocytes that were stimulated with the supernatant of MCs cultured in high glucose conditions. In diabetic mice, ERAD inhibition resulted in exacerbated albuminuria, increased apoptosis in podocytes, and reduced nephrin expression associated with the downregulation of ERAD-related biomolecules. Flow cytometry analysis of podocytes isolated from MafB (a transcription factor known to be expressed in macrophages and podocytes)-GFP knock-in mice revealed that ERAD inhibition resulted in decreased nephrin phosphorylation. These findings suggest that an intraglomerular cross talk between MCs and podocytes can inhibit physiological ERAD processes and suppress the phosphorylation of nephrin in podocytes, which thereby lead to podocyte injury under diabetic conditions. Therapeutic intervention of the ERAD pathway through the cross talk between these cells is potentially a novel strategy for DKD.

Podocyte injury is the primary cause of glomerular injury in diabetic nephropathy (DN). Advanced oxidation protein products (AOPPs), the triggers and markers of oxidative stress in DN, have been linked to podocyte damage. However, the underlying mechanism is not yet clear. Here, we investigated the potential role of FOXO3a, a key transcription factor in the response to stress, in mediating AOPPs-induced podocyte injury. We found that FOXO3a expression was increased in the glomeruli of kidney biopsies from patients with DN and it was positively correlated with proteinuria. The serum from patients with DN significantly increased FOXO3a and its downstream genes FasL and Bim, thereby inducing the high level of cleaved caspase3 and the loss of nephrin and podocin expressions in podocytes. Blockade of AOPPs signaling by a neutralizing antibody against the receptor of advanced glycation end products (αRAGE) abolished the effect of DN serum on podocytes, confirming the pathogenic role of AOPPs in DN serum. Downregulation of FOXO3a decreased AOPPs-induced podocyte apoptosis and restored the levels of podocyte markers nephrin and podocin, and upregulation of FOXO3a exacerbated these changes in podocytes after AOPPs treatment. Furthermore, FOXO3a specifically activated proapoptotic genes in podocytes only in the presence of AOPPs. Mechanistically, AOPPs increased the FOXO3a protein levels by inhibiting their autophagic degradation in a ROS/mTOR-dependent manner. Moreover AOPPs activated the accumulated FOXO3a by maintaining FOXO3a in the nucleus, and this process was dependent on ROS-mediated AKT signaling deactivation. These studies suggest that FOXO3a plays a critical role in mediating AOPPs-induced podocyte injury and reveal a new mechanistic linkage of oxidative stress, FOXO3a activation and podocyte injury in DN.

Cisplatin (CP), an anticancer drug, often causes kidney damage. However, the mechanism of CP‐induced acute kidney injury (AKI) is not completely understood. AKI was induced by intravenous injection (i.v.) of cisplatin at doses of 5, 8, and 10 mg/kg. Anemoside B4 (B4) (20 mg/kg, i.m.) and dexamethasone (DXM) (0.5 mg/kg, i.v.) were used for AKI treatment. Biochemical indicators were assessed using an automatic biochemical analyzer, protein expression was analyzed by western blotting, and morphological changes in the kidney were examined by PAS staining. The serum creatinine (Cre) and blood urea nitrogen (BUN) levels did not change significantly in the first 2 days but abruptly increased on the third day after CP injection. The serum albumin (ALB) and total protein (TP) levels decreased in both a time‐ and dose‐dependent manner. The urine protein level increased, the clearing rate of Cre decreased distinctly, and morphologic changes appeared in a dose‐dependent manner. The protein expression of p53/caspase‐3, NLRP3, IL‐6, and TNF‐α was obviously upregulated on day 3; concurrently, nephrin and podocin were downregulated. The expression of LC3II and p62 was upregulated significantly as the CP dose increased. B4 and DXM obviously decreased the BUN and Cre levels after 3 or 5 days of treatment. AKI appeared distinctly in a time‐dependent manner at 2 to 5 days after the administration of 5 mg/kg CP and in a dose‐dependent manner upon the administration of 5, 8, and 10 mg/kg CP. The third day was a significant time point for renal deterioration, and treatment with B4 and DXM within the first 3 days provided significant protection against AKI.

Long-term kidney transplant (KT) survival has remained relatively stagnant. Protocol biopsy studies suggest that glomerulosclerosis is a significant contributor to long-term graft failure. We previously demonstrated that podocyte loss in the 1st year post-transplantation predicted long-term allograft survival. However, whether increased podocyte loss continues over the lifespan of a KT remains unclear. We performed a cross-sectional analysis of 1,182 urine samples from 260 KT recipients up to 19-years after transplantation. Urine pellet mRNAs were assayed for podocyte (NPHS2/podocin and nephrin/NPHS1), distal tubule (aquaporin2), and profibrotic cytokine (TGFbeta1). Multivariable generalized estimating equations were used to obtain "population-averaged" effects for these markers over time post-KT. Consistent with early stresses both podocyte and tubular markers increased immediately post-KT. However, only podocyte markers continued to increase long-term. A role for hypertrophic stresses in driving podocyte loss over time is implied by their association with donor BMI, recipient BMI and donor-recipient BMI mismatch at transplantation. Furthermore, urine pellet podocin mRNA was associated with urine TGFbeta1, proteinuria and reduced eGFR, thereby linking podocyte injury to allograft fibrosis and survival. In conclusion we observed that podocyte loss continues long-term post-KT suggesting an important role in driving late graft loss. This article is protected by copyright. All rights reserved.

Aims/Introduction Amelioration of renal impairment is the key to diabetic nephropathy (DN) therapy. The progression of DN is closely related to podocyte dysfunction, but the detailed mechanism has not yet been clarified. The present study aimed to explore the renal impairment amelioration effect of berberine and related mechanisms targeting podocyte dysfunction under the diabetic state. Materials and Methods Streptozotocin (35 mg/kg) was used to develop a DN rat model together with a high‐glucose/high‐lipid diet. Renal functional parameters and glomerular ultrastructure changes were recorded. The alterations of phosphatidylinositol 3‐kinase (PI3K), protein kinase B (Akt) and phosphorylated Akt in the kidney cortex were determined by western blot. Meanwhile, podocyte dysfunction was induced and treated with berberine and LY294002. After that, podocyte adhesion functional parameters, protein biomarker and the alterations of the PI3K–Akt pathway were detected. Results Berberine reduces the increased levels of biochemical indicators, and significantly improves the abnormal expression of PI3K, Akt and phosphorylated Akt in a rat kidney model. In vitro, a costimulating factor could obviously reduce the podocyte adhesion activity, including decreased expression of nephrin, podocin and adhesion molecule α3β1 levels, to induce podocyte dysfunction, and the trends were markedly reversed by berberine and LY294002 therapy. Furthermore, reduction of PI3K and phosphorylated Akt levels were observed in the berberine (30 and 60 μmol/L) and LY294002 (40 μmol/L) treatment group, but the Akt protein expression showed little change. Conclusions Berberine could be a promising antidiabetic nephropathy drug through ameliorating renal impairment and inhibiting podocyte dysfunction in diabetic rats, and the underlying molecular mechanisms might be involved in the regulation of the PI3K–Akt signaling pathway., The phosphatidylinositol 3‐kinase–protein kinase B signaling pathway in podocytes might be an important therapeutic target in diabetic nephropathy treatment. Berberine will be a promising antidiabetic nephropathy drug.

New findings What is the central question of this study? Can a single bone marrow mononuclear cell (BMMC) transplant into the subcapsular region of kidney improve cellular communication and adhesion, while restoring renal tissue cytoarchitecture and function during renovascular hypertension? What is the main finding and its importance? The BMMC transplantation restored connexin 40 expression and led to recovery of N- and E-cadherin levels within 15 days. It was observed, for the first time, that BMMC transplantation restores expression of nephrin, a component of the glomerular filtration barrier related to podocytes and the glomerular basal membrane. Abstract Stem cell therapy has emerged as a potential treatment for renal diseases owing to the regenerative potential of stem cells. However, a better understanding of the morphological and functional changes of damaged renal cells in the presence of transplanted stem cells is needed. The aim of this study was to investigate cell-cell communication and adhesion in renal parenchyma, with analysis of fibrosis, to evaluate renal morphology and function after bone marrow mononuclear cell (BMMC) transplantation in two-kidney-one-clip rats. The BMMC therapy significantly decreased blood pressure and renin expression, improved renal morphology and restored the glomerular filtration barrier, with remodelling of podocytes. In addition, there was a reduction in fibrosis, and connexin 40 and nephrin expression were significantly increased after 7 and 15 days of transplantation. Plasma creatinine, urea and total protein levels were restored, and proteinuria was reduced. Furthermore, N- and E-cadherin expression was increased soon after BMMC therapy. Green fluorescent protein-positive BMMCs were found in the renal cortex 24 and 48 h after transplantation into the renal subcapsule, and at 7 and 15 days after transplantation, these cells were observed throughout the renal medulla, indicating cellular migration. Therefore, these data suggest that transplanted BMMCs improve cell-cell communication and adhesion between damaged cells, which is accompanied by a recovery of renal morphology and function.

Dynamin plays an essential role in maintaining the structure and function of the glomerular filtration barrier. Specifically, dynamin regulates the actin cytoskeleton and the turnover of nephrin in podocytes, and knocking down dynamin expression causes proteinuria. Moreover, promoting dynamin oligomerization with Bis-T-23 restores podocyte function and reduces proteinuria in several animal models of chronic kidney disease. Thus, dynamin is a promising therapeutic target for treating chronic kidney disease. Here, we investigated the pathophysiological role of dynamin under proteinuric circumstances in a rat model and in humans. We found that glomerular Dnm2 and Dnm1 mRNA levels are increased prior to the onset of proteinuria in a rat model of spontaneous proteinuria. Also, in zebrafish embryos, we confirm that knocking down dynamin translation results in proteinuria. Finally, we show that the glomerular expression of dynamin and cathepsin L protein is increased in several human proteinuric kidney diseases. We propose that the increased expression of glomerular dynamin reflects an exhausted attempt to maintain and/or restore integrity of the glomerular filtration barrier. These results confirm that dynamin plays an important role in maintaining the glomerular filtration barrier, and they support the notion that dynamin is a promising therapeutic target in proteinuric kidney disease. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Background and purpose The endocannabinoid (EC) system has been implicated in the pathogenesis of diabetic nephropathy (DN). We investigated the effects of peripheral blockade of the cannabinoid CB1 receptor as an add-on treatment to ACE-inhibition in type 1 diabetic mice (DM) with established albuminuria. Experimental approach Renal functional parameters (albumin excretion rate, creatinine clearance), tubular injury, renal structure, both EC and CB receptor levels and markers of podocyte dysfunction, fibrosis and inflammation were studied in streptozotocin-induced DM treated for 14 weeks with vehicle, the ACE-inhibitor perindopril (2 mg·kg-1 ·day-1 ), peripherally-restricted CB1 receptor antagonist AM6545 (10 mg·kg-1 ·day-1 ) or both. Treatments began at 8 weeks after diabetes onset, when early DN is established. Key results CB1 receptors were overexpressed in DM and neither perindopril nor AM6545 altered this effect, while both drugs abolished diabetes-induced overexpression of angiotensin AT1 receptors. Single treatment with either AM6545 or perindopril significantly reduced progression of albuminuria, down-regulation of nephrin and podocin, inflammation and expression of markers of fibrosis. However, reversal of albuminuria was only observed in mice administered both treatments. The ability of the combination therapy to completely abolish slit diaphragm protein loss, monocyte infiltration, overexpression of inflammatory markers and favour macrophage polarization towards an M2 phenotype may explain this greater efficacy. In vitro experiments confirmed that CB1 receptor activation directly inhibits retinoic acid-induced nephrin expression in podocytes and IL-4-induced M2 polarization in macrophages. Conclusion and implications Peripheral CB1 receptor blockade used as add-on treatment to ACE-inhibition reverses albuminuria, nephrin loss and inflammation in DM.

Diabetic nephropathy is one of the leading causes of end-stage renal disease and creates heavy healthcare burdens globally. Dysfunction of mesangial cells and podocytes contributes to diabetic nephropathy. Dysregulation of signaling involved in renal development and regeneration may cause diabetic kidney damages. Growing evidences suggest the importance of dysregulated dickkopf-1 (DKK1)/Wnt/ β-catenin signaling pathways in the pathogenesis of diabetic glomerular injuries. The inhibition of Wnt signaling in injured mesangial cells is likely attributed to the high glucose-induced Ras/Rac1 dependent superoxide formation. When DKK1, the cellular inhibitor of Wnt signaling, binds to the Kremen-2 receptor, depositions of extracellular matrix increase in the mesangium of diabetic kidneys. Additionally, reactivation of Notch-1 signaling has been implicated in podocytopathy during diabetic proteinuria development. Knocking down Notch-1 alleviates vascular endothelial growth factor (VEGF) expression, nephrin repression and proteinuria in diabetic kidneys. It is also found that epigenetic modulations by histone deacetylase 4 (HDAC4) and miR-29a could lead to diabetic nephropathy. High glucose increases the expression of HDAC4, which causes deacetylation with subsequent ubiquitination of nephrin. Overexpression of miR-29a in diabetic transgenic mice would decrease the expression of HDAC4 and stabilize nephrin. Surprisingly, reprogramming or reactivation of signaling involved in renal development or regeneration often brings about diabetic glomerular sclerosis in mesangial cells and podocytes. Better knowledge about modifications of embryonic stem cell signaling will have a chance to implement strategically focused pharmacological research programs aiming to the development of new drugs for diabetic kidney injuries.

Aim This study aimed to evaluate the molecular mechanism mitigating progress of chronic nephropathy by mesenchymal stem cells (MSCs). Methods Rats were divided into normal control (Normal), adriamycin (ADR)+vehicle (CON), and ADR+MSC (MSC) groups. Nephropathy was induced by ADR (4 mg/kg) and MSCs (2 × 106 ) were injected. Rats were euthanized 1 or 6 weeks after ADR injection. NF-kB, MAPKs, inflammation, oxidative stress, profibrotic molecules, and nephrin expression were evaluated. Electron and light microscopy were used for structural analysis. MSCs were co-cultured with renal tubular epithelial cells or splenocytes to evaluate relation with oxidative stress and inflammatory molecules RESULTS: Adriamycin treatment upregulated inflammation, oxidative stress, and profibrotic molecules; this was mitigated by MSCs. Glomerulosclerosis and interstitial fibrosis were observed in ADR-treated groups, and were more prominent in the CON group than in the MSC group. Fusion of foot processes and loss of slit diaphragms were also more prominent in the CON group than in the MSC group. In vitro, MSCs reduced oxidative stress related molecules, inflammatory cytokines, and NF-kB transcription. MSC- or ADR-induced regulation of NF-kB transcriptional activity was confirmed by a luciferase reporter assay. Conclusions Mesenchymal stem cells attenuate ADR-induced nephropathy by diminishing oxidative stress and inflammation via downregulation of NF-kB.

Aims Nephrotic syndrome (NS) is a major manifestation of lupus nephritis (LN). The dysregulation of podocytes, the glomerular basement membrane (GBM) and endothelial cells (ECs) results in proteinuria in glomerular diseases. The aim of our study was to clarify whether the dysregulation of these barriers is associated with NS in proliferative LN and membranous LN. Methods and results Fifty-six patients with NS, including minimal change NS in 15, primary membranous nephropathy (PMN) in 13, class III/IV LN in 15, and class V LN in 13, were enrolled in this study. Subjects with idiopathic haematuria were assigned as controls. Glomerular expression of Wilms tumour protein 1 (WT1), nephrin, synaptopodin and podocalyxin was evaluated by immunohistochemistry (IHC) and real-time quantitative reverse transcription polymerase chain reaction. EC injury was evaluated by CD31 immunostaining and electron microscopy (EM). Reduced expression of WT1, nephrin and synaptopodin was found in PMN, class III/IV LN and class V LN as compared with controls by IHC and mRNA analysis. Reduced expression of these molecules was not different between class III/IV LN and class V LN. Reduced numbers of CD31-positive ECs were found in class III/IV LN as compared with class V LN. EC injury showing subendothelial widening on EM was apparent in class III/IV LN as compared with class V LN. Foot process effacement was found only along the GBM showing EC injury in class III/IV LN. Conclusions Our study suggests that coexistence of podocyte and EC injury may lead to NS in proliferative LN. Podocyte damage alone leads to NS in membranous LN.

Liraglutide, a human long‐lasting GLP‐1 analogue, is currently regarded as a powerful treatment option for type 2 diabetes. Apart from glucoregulatory and insulinotropic actions, liraglutide increases β‐cell mass through stimulation of β‐cell proliferation and islet neogenesis, as well as inhibition of β‐cell apoptosis. However, the underline molecular mechanisms have not been fully characterized. In this study, we investigated the mechanism by which liraglutide preserves islet β‐cells in an animal model of overt diabetes, the obese db/db mice, and protects a mouse pancreatic β‐cell line (βTC‐6 cells) against apoptosis. Treatment of 12‐week‐old diabetic mice with liraglutide for 2 weeks had no appreciable effects on blood non‐fasting glucose concentration, islet insulin content and body weight. However, morphological and biochemical examination of diabetic mouse pancreatic islets demonstrated that liraglutide restores islet size, reduces islet β‐cell apoptosis and improves nephrin expression, a protein involved in β‐cell survival signalling. Our results indicated that liraglutide protects βTC‐6 cells from serum withdrawal‐induced apoptosis through inhibition of caspase‐3 activation. The molecular mechanism of the anti‐apoptotic action of liraglutide in βTC‐6‐cells comprises stimulation of PI3‐kinase‐dependent AKT phosphorylation leading to the phosphorylation, hence inactivation of the pro‐apoptotic protein BAD and inhibition of FoxO1 transcription factor. In conclusion, we provided evidence that the GLP‐1 analogue liraglutide exerts important beneficial effects on pancreatic islet architecture and β‐cell survival by protecting cells against apoptosis. These findings extend our understanding of the actions of liraglutide and further support the use of GLP‐1R agonists in the treatment of patients with type 2 diabetes.

Aims To determine the specific role of podocyte-expressed cannabinoid-1 receptor (CB1R) in the development of diabetic nephropathy (DN), relative to CB1R in other renal cell types. Material and methods We developed a mouse model with a podocyte-specific deletion of CB1R (pCB1Rko) and challenged this model with streptozotocin (STZ)-induced type-1 DN. We also assessed the podocyte response to high glucose in vitro and its effects on CB1R activation. Results High glucose exposure for 48h led to an increase in CB1R gene expression (CNR1) and endocannabinoid production in cultured human podocytes. This was associated with podocyte injury reflected by decreased podocin and nephrin expression. These changes could be prevented by Cnr1-silencing, thus identifying CB1R as a key player in podocyte injury. After 12 weeks of chronic hyperglycemia, STZ-treated pCB1Rko mice showed similar elevated blood glucose as their wild-type littermates. However, they displayed less albuminuria and less podocyte loss than STZ-treated wild-type mice. Unexpectedly, pCB1Rko mice also have milder tubular dysfunction, fibrosis and reduction of cortical microcirculation compared to wild-type controls, which is partly mediated by podocyte-derived endocannabinoids acting via CB1R on proximal tubular cells. Conclusions Activation of CB1R in podocytes contributes to both glomerular and tubular dysfunction in type-1 DN, which highlights the therapeutic potential of peripheral CB1R blockade.

Angiotensin II type 2 receptor (AT2 R) deficiency in AT2 R knockout (KO) mice has been linked to congenital abnormalities of the kidney and urinary tract; however, the mechanisms by which this occurs are poorly understood. In this study, we examined whether AT2 R deficiency impaired glomerulogenesis and mediated podocyte loss/dysfunction in vivo and in vitro. Nephrin-cyan fluorescent protein (CFP)-transgenic (Tg) and Nephrin/AT2 RKO mice were used to assess glomerulogenesis, while wild-type and AT2 RKO mice were used to evaluate maturation of podocyte morphology/function. Immortalized mouse podocytes (mPODs) were employed for in vitro studies. AT2 R deficiency resulted in diminished glomerulogenesis in E15 embryos, but had no impact on actual nephron number in neonates. Pups lacking AT2 R displayed features of renal dysplasia with lower glomerular tuft volume and podocyte numbers. In vivo and in vitro studies demonstrated that loss of AT2 R was associated with elevated NADPH oxidase 4 levels, which in turn stimulated ectopic hedgehog interacting protein (Hhip) gene expression in podocytes. Consequently, ectopic Hhip expression activation either triggers caspase-3 and p53-related apoptotic processes resulting in podocyte loss, or activates TGFβ1-Smad2/3 cascades and α-SMA expression to transform differentiated podocytes to undifferentiated podocyte-derived fibrotic cells. We analyzed HHIP expression in the kidney disease database (Nephroseq) and then validated this using HHIP immunohistochemistry staining of human kidney biopsies (controls versus focal segmental glomerulosclerosis). In conclusion, loss of AT2 R is associated with podocyte loss/dysfunction and is mediated, at least in part, via augmented ectopic Hhip expression in podocytes. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Aim: Hyperfiltration is a cause of podocyturia and occurs physiologically in the kidney of pregnant women. Podocyturia is increased in preeclamptic pregnancies, but it is unclear whether there is also any increase in uncomplicated pregnancies. This study was performed to examine whether podocyturia and urine aquaporin 2 mRNA expression are increased in healthy pregnant women (PW) compared to healthy non-pregnant women (NPW). Methods: Eleven urines obtained from 11 NPW and longitudinal 76 urines from 40 PW with uncomplicated pregnancies (median number [range] of urine samples/person, 2 [1 - 3]) were studied. Determination of protein and creatinine concentrations and number of cells in urine other than blood cells, and quantitative analyses of the mRNA expression of aquaporin 2 (AQP2-mRNA), podocin (Pod-mRNA), and nephrin (Nep-mRNA) were performed using RT-PCR in pelleted urine samples. Podocyturia was monitored with urine Pod- and Nep-mRNA expression levels normalized relative to creatinine. Results: Urine cell density and urine AQP2-, Pod-, and Nep-mRNA expression normalized relative to creatinine were significantly higher in PW than NPW. The number of cells per milligram of creatinine was significantly positively correlated with expression of all three mRNAs with correlation coefficients (R-value) of 0.442, 0.481, and 0.561 for Pod-, Nep-, and AQP2-mRNA, respectively. AQP2-mRNA expression was strongly (R > 0.8) positively correlated with both Pod- and Nep-mRNA expression. Conclusion: Podocyturia monitored by Pod- and Nep-mRNA expression and urine cells expressing AQP2-mRNA were increased in uncomplicated pregnancies compared to healthy non-pregnant women. Urine cells expressing AQP2-mRNA increased with increasing podocyturia in healthy women.

AIM Hyperfiltration is a cause of podocyturia and occurs physiologically in the kidney of pregnant women. Podocyturia is increased in preeclamptic pregnancies, but it is unclear whether there is also any increase in uncomplicated pregnancies. This study was performed to examine whether podocyturia and urine aquaporin 2 mRNA expression are increased in healthy pregnant women (PW) compared to healthy non-pregnant women (NPW). METHODS Eleven urines obtained from 11 NPW and longitudinal 76 urines from 40 PW with uncomplicated pregnancies (median number [range] of urine samples/person, 2 [1 - 3]) were studied. Determination of protein and creatinine concentrations and number of cells in urine other than blood cells, and quantitative analyses of the mRNA expression of aquaporin 2 (AQP2-mRNA), podocin (Pod-mRNA), and nephrin (Nep-mRNA) were performed using RT-PCR in pelleted urine samples. Podocyturia was monitored with urine Pod- and Nep-mRNA expression levels normalized relative to creatinine. RESULTS Urine cell density and urine AQP2-, Pod-, and Nep-mRNA expression normalized relative to creatinine were significantly higher in PW than NPW. The number of cells per milligram of creatinine was significantly positively correlated with expression of all three mRNAs with correlation coefficients (R-value) of 0.442, 0.481, and 0.561 for Pod-, Nep-, and AQP2-mRNA, respectively. AQP2-mRNA expression was strongly (R > 0.8) positively correlated with both Pod- and Nep-mRNA expression. CONCLUSION Podocyturia monitored by Pod- and Nep-mRNA expression and urine cells expressing AQP2-mRNA were increased in uncomplicated pregnancies compared to healthy non-pregnant women. Urine cells expressing AQP2-mRNA increased with increasing podocyturia in healthy women.

Podocyte dysfunction results in glomerular diseases accounted for 90% of end‐stage kidney disease. The evolutionarily conserved Notch signalling makes a crucial contribution in podocyte development and function. However, the underlying mechanism of Notch pathway modulating podocyte differentiation remains less obvious. Autophagy, reported to be related with Notch signalling pathways in different animal models, is regarded as a possible participant during podocyte differentiation. Here, we found the dynamic changes of Notch1 were coincided with autophagy: they both increased during kidney development and podocyte differentiation. Intriguingly, when Notch signalling was down‐regulated by DAPT, autophagy was greatly diminished, and differentiation was also impaired. Further, to better understand the relationship between Notch signalling and autophagy in podocyte differentiation, rapamycin was added to enhance autophagy levels in DAPT‐treated cells, and as a result, nephrin was recovered and DAPT‐induced injury was ameliorated. Therefore, we put forward that autophagy is involved in kidney development and podocyte differentiation regulated by Notch signalling.

Aim This study was aimed at examining the effects of treatment with rituximab, a chimeric monoclonal antibody against the protein CD20, in a B-lymphocyte independent adriamycin-induced rat model of nephrotic syndrome. Rituximab is an emerging rescue therapy used in patients with complicated nephrotic syndrome and, therefore, we sought to elucidate the apparent B-lymphocyte independent mechanism underlying its anti-proteinuric effect. Methods Adriamycin-induced nephropathy was established in Wistar rats by intravenously injecting 10 mg/kg of adriamycin, which were then treated with rituximab or purified human IgG weekly and euthanized on day 28. Proteinuria, glomerular expression of sphingomyelin phosphodiesterase acid-like 3b protein, and podocyte-related proteins were examined using immunofluorescence staining and a reverse transcription-polymerase chain reaction. Results Rituximab treatment of rats with adriamycin-induced nephropathy significantly reduced urinary protein excretion 14 to 28 days after induction of disease, compared with those treated with purified normal human IgG. Furthermore, rituximab treatment also prevented the reduction of glomerular nephrin and podocin expression on day 28. Double-immunofluorescence staining revealed that after in vivo administration, rituximab was bound to the glomeruli, which also expressed synaptopodin or sphingomyelin phosphodiesterase, acid-like 3b. Moreover, sphingomyelin phosphodiesterase, acid-like 3b expression was significantly decreased on day 28 of adriamycin-induced nephropathy, which was also prevented by rituximab. Conclusions This study demonstrated that rituximab directly affected glomerular podocytes and ameliorated proteinuria in adriamycin-induced nephropathy in rats. Furthermore, protection of podocyte function by rituximab may be mediated by direct modulation of a sphingomyelin phosphodiesterase, acid-like 3b-dependent mechanism.

Background and Purpose Therapeutic options to treat glomerulopathies, the main cause of chronic kidney disease, are limited. Podocyte dedifferentiation is a major event in the pathogenesis of glomerulopathies. The goal of the present study was therefore to develop an assay to monitor podocyte differentiation suited for compound screening. Experimental Approach We isolated and cultured glomeruli from transgenic mice, expressing cyan fluorescent protein (CFP) under control of the promoter of nephrin, a marker of podocyte differentiation. Mean CFP fluorescence intensity per glomerulus (MFG) was determined by summation of all glomerular voxels from confocal z-stacks in the absence and presence of pharmaceutical compounds. Key Results In untreated cultured glomeruli, MFG remained fairly stable during the first 5 days, when foot processes were already effaced, and the level of many podocyte-specific proteins was only mildly affected as revealed by proteomics. Between day 6 and 9, MFG decreased to almost zero. The decrease of MFG was paralleled by a decrease in CFP and nephrin expression as determined by RT-PCR, Western blot and proteomics. Puromycin aminonucleoside (PAN), which damages podocytes, concentration-dependently induced a complete loss of MFG (IC50 = 0.3 µg/ml). Dexamethasone (25 μM) and pioglitazone (10 μM) markedly attenuated the effect of 0.6 µg/ml PAN on MFG. Conclusion and Implications In summary, we established a novel assay to assess the effect of pharmaceutical compounds on the differentiation of podocytes in situ. Our assay is suited for compound screening to identify drugs for the treatment of glomerulopathies. This article is protected by copyright. All rights reserved.

Aim Podocyte depletion in the kidney is associated with end-stage kidney disease (ESKD). Pre-eclampsia (PE) increases the risk of ESKD in later life. This study was performed to determine whether nephrinuria (soluble nephrin in the urine) is correlated with proteinuria and/or podocyturia (podocytes in the urine) in PE women. Methods Eighty-three urine samples, consisting of 45 and 38 samples from 27 normotensive and nine PE women, respectively, underwent simultaneous determination of nephrin, protein, and creatinine concentrations in the urine supernatant and quantitative analysis of podocyte-specific protein mRNA expression. This included podocin (Pod-mRNA) and nephrin (Nep-mRNA), using real-time polymerase chain reaction in the pelleted urine. Nephrinuria and proteinuria were corrected by creatinine concentration. Pod- and Nep-mRNA expression levels were corrected by GAPDH. Results Nephrinuria, proteinuria, Pod-mRNA expression, and Nep-mRNA expression all increased with advancing gestation in PE women, while not in normotensive women. The nephrinuria was strongly correlated with proteinuria (R = 0.901, P < 0.001), Pod-mRNA expression level (R = 0.824, P

Background Previous studies have found that metformin provided some reno-protection for diabetic renal damage. In the present study ,we observed the effects of different doses of metformin on expression of renal tissue nephrin in type 2 diabetes model rats and mechanism of its possible protective effect in kidney podocytes was explored. Methods Type 2 diabetes (T2DM) model rats were induced by a high-fat diet combined with low dose of streptozotocin. Diabetic rats were treated with different doses of metformin for 8 weeks. At the end of the study, urinary and blood sample were collected for measuring corresponding indices. Light microscopy and transmission electron microscopy were used to observe morphologic changes. The protein expression of renal tissue nephrin was assayed by immunohistochemical staining. Real-time polymerase chain reaction was used to detect the mRNA expression of renal tissue nephrin. Results Compared with T2DM model rats, ratios of urinary albumin and nephrin, glomerular basement membrane thickness (GBMT) and foot process fusion rate (FRFT) ameliorated significantly, nephrin protein and mRNA expression of renal tissue increased markedly in rats, which were treated with different doses of metformin. Conclusion Metformin can protect the kidney podocytes by adjusting the renal tissue nephrin expression in type 2 diabetes mellitus model of rats in a dose dependent manner. The significant finding (s) of the study: different dose of metformin treatment reduces the excretion of urinary albumin and nephrin, ameliorates the level of GBMT and FRFP and up -regulates the expression of renal tissue nephrin protein and mRNA in a varying degree. This study adds: Metformin may play a role in protecting the kidney podocytes via up-regulating the renal tissue nephrin expression in type 2 diabetes mellitus model of rats.

Aim Autosomal recessive mutations in NPHS1 gene are a common cause of congenital nephrotic syndrome (CNS). The disorder is characterized by massive proteinuria that manifests in utero or in the neonatal period during the first 3 months of life. NPHS1 encodes nephrin, a member of the immunoglobulin family of cell adhesion molecules and the main protein expressed at the renal slit diaphragm. Currently, there are approximately 250 mutations described in the NPHS1 gene distributed among all nephrin domains. The main objective of this study was to perform the analysis of the NPHS1 gene in patients with congenital nephrotic syndrome in order to determine the molecular cause of the disease. Methods Direct sequencing of NPHS1 gene in four children was performed. Results Each patient was heterozygous for two pathogenic mutations disclosing the molecular cause of the disease in 100% of the cases. We identified six different mutations, consisting of one in-frame deletion, one frameshift, and four missense substitutions. The p.Val736Met mutation that is described here for the first time was considered pathogenic by different mutation predictive algorithms. Regardless of the type of mutation, three patients had a bad outcome and died Conclusions Despite the small size of the cohort, this study contributed to the increasing number of deleterious mutations in the NPHS1 gene by describing a new mutation. Also, since we identified NPHS1 pathogenic mutations as the cause of the disease in all cases analyzed, it might be a frequent cause of CNS in the South Eastern region of Brazil, although the analysis of a larger sample is required to obtain more indicative epidemiological data.

Preclinical Research Vanillic acid (VA) is a dihydroxybenzoic acid derivative widely used as a flavoring agent. It has chemopreventive effects on experimentally-induced carcinogenesis and in ulcerative colitis. The object of the present study was to investigate the effects of VA, alone and in combination with methylprednisolone (MP), on cationic bovine serum albumin (cBSA induced immune-complex glomerulonephritis in female BALB/c mice. Pre-immunization was carried out with cBSA in BALB/c mice and repeated (cBSA, 13 mg/kg, 3 times/week, i.v.) for 6 weeks to induce glomerulonephritis which was confirmed by the presence of severe proteinuria. The effect of VA (50, 100, and 200 mg/kg, p.o.) and its combination with MP (12.5 mg/kg, p.o.) was assessed in the nephrotic disease model. Treatment with VA decreased inflammatory nephrotic injury as evidenced by decreased proteinuria, serum creatinine, blood urea nitrogen, serum IgG1 and TNF-α levels. Co-administration of VA with MP showed an improvement in the immunohistochemistry of glomerular nephrin and podocin. The present results indicate that VA has a nephroprotective effect in the management of autoimmune nephritis. Drug Dev Res 77 : 171-179, 2016. © 2016 Wiley Periodicals, Inc.

Objective The aim of this study was to correlate different degrees of excess weight with the expression of podocyte-associated messenger RNAs (mRNAs) in urine. Methods The sample comprised 83 patients with overweight or obesity class I, II, or III and 18 healthy controls. The expression of nephrin, podocin, podocalyxin, α-actinin-4, α3β1integrin, vascular endothelial growth factor, and transforming growth factor-beta (TGF-β1) mRNA in urine was quantified with the real-time polymerase chain reaction. mRNA expression was correlated with body mass index, the metabolic syndrome, albuminuria, and inflammation. Results Adults with obesity class III had higher levels of serum lipids, glucose, HbA1C, insulin resistance, and C-reactive protein (P

Accumulating evidence has suggested that podocytes undergo epithelial-mesenchymal transition (EMT) in diabetic nephropathy (DN). However, the underlying mechanisms of EMT in podocyte are not well understood. PI3K/Akt pathway is involved in the progression of DN. In the present study, we demonstrated that PI3K/Akt pathway was activated in podocytes exposed to high glucose conditions, accompanied by down-regulation of the podocalyxin (PCX) and nephrin expression and up-regulation of the desmin and α-smooth muscle actin (α-SMA) expression. Inhibition of PI3K/Akt pathway by chemical LY294002 or Phosphase and tensin homology deleted on chromosome ten (PTEN) prevented the phenotypic transition. These findings indicate that PTEN/PI3K/Akt pathway mediates high glucose-induced phenotypic transition in podocytes.

Objective This study aimed to investigate the structural changes in the slit diaphragm, caused by early diabetes, and the nephroprotective effect of C-peptide. Methods Streptozotocin-induced type 1 diabetic Wistar rats were divided into control, control plus C-peptide, early diabetes, and early diabetes plus C-peptide groups. C-peptide was infused into rats for one day. The slit diaphragm component proteins podocin, CD2AP, and nephrin, were stained immunofluorescently and their distribution quantitatively analyzed by determining the overlapping area ratio. The interfoot process gap length was measured from electron microscopic images. Results Diabetic duration correlated best with the area ratio of podocin and CD2AP (r = 0.626), followed by other protein combinations, showing progressive change in the slit diaphragm structure. C-peptide-treatment did not alter area ratios. The interfoot process gap length was wider in diabetic rats (p

Background Proteinuria is typically accompanied by structural and compositional changes of the foot processes and of the slit diaphragms between podocytes. CD2-associated protein (CD2AP) in podocytes serves as an adaptor protein binding to nephrin and podocin, anchoring these slit diaphragm proteins to actin filaments of podocyte cytoskeleton and sending signals inward or outward. Methods In the present study, we prepared streptozotocin-induced diabetic renal tissues and cultured podocytes in diabetic conditions to investigate podocyte phenotypical changes, including quantitative and distributional changes of CD2AP protein and search for the signalling mechanisms in diabetic conditions. We prepared cultured rat glomerular epithelial cells and mouse podocytes to study how high glucose and advanced glycosylation end products (AGE) induce phenotypical changes of cultured podocyte, under (1) normal glucose (5 mM, = control), (2) high glucose (30 mM), (3) AGE-added or (4) high glucose plus AGE-added conditions. Results According to diabetic duration, density of CD2AP in renal tissue of experimental diabetic nephropathy became conglomerulated and diminished. In cultured podocytes, CD2AP co-localized with nephrin and zonula occludens-1 by confocal imaging. High glucose and high glucose plus AGE induced the relocalization and concentration of CD2AP at internal cytoplasmic and perinuclear areas of podocytes. High glucose plus AGE-added condition also decreased CD2AP protein amount and its mRNA expression compared with normal glucose or osmotic control conditions. In addition, LY294002, a phosphoinositide 3-kinase inhibitor, prevented the quantitative and distributional changes of CD2AP induced by high glucose and AGE. Conclusions These findings suggest that diabetic conditions induce the phenotypical changes of podocyte CD2AP possibly via phosphoinositide 3-kinase/Akt signalling. Copyright © 2014 John Wiley & Sons, Ltd.

Bisphenol-A, a chemical used in the production of the plastic lining of food and beverage containers, can be found in significant levels in human fluids. Recently, bisphenol-A has been associated with low-grade albuminuria in adults as well as in children. Since glomerular epithelial cells (podocytes) are commonly affected in proteinuric conditions, herein we explored the effects of bisphenol-A on podocytes in vitro and in vivo. On cultured podocytes we first observed that bisphenol-A?at low or high concentrations?(10?nM and 100?nM, respectively) was able to induce hypertrophy, diminish viability, and promote apoptosis. We also found an increase in the protein expression of TGF-?1 and its receptor, the cyclin-dependent kinase inhibitor p27Kip1, as well as collagen-IV, while observing a diminished expression of the slit diaphragm proteins nephrin and podocin. Furthermore, mice intraperitoneally injected with bisphenol-A (50?mg/Kg for 5 weeks) displayed an increase in urinary albumin excretion and endogenous creatinine clearance. Renal histology showed mesangial expansion. At ultrastructural level, podocytes displayed an enlargement of both cytoplasm and foot processes as well as the presence of condensed chromatin, suggesting apoptosis. Furthermore, immunohistochemistry for WT-1 (specific podocyte marker) and the TUNEL technique showed podocytopenia as well as the presence of apoptosis, respectively. In conclusion, our data demonstrate that Bisphenol-A exposure promotes a podocytopathy with proteinuria, glomerular hyperfiltration and podocytopenia. Further studies are needed to clarify the potential role of bisphenol-A in the pathogenesis as well as in the progression of renal diseases., Ministerio de Ciencia e Innovación, Instituto de Salud Carlos III, The Eugenio Rodríguez Pascual Foundation

Objective Obesity-related glomerulopathy is characterized initially by glomerular hyperfiltration with hypertrophy and then development of proteinuria. Putative mechanisms include endothelial dysfunction and filtration barrier injury due to oxidant stress and immune activation. There has been recent interest in targeting dipeptidyl peptidase 4 (DPP4) enzyme due to increasing role in non-enzymatic cellular processes. Methods The Zucker obese (ZO) rat (aged 8 weeks) fed a normal chow or diet containing the DPP4 inhibitor linagliptin for 8 weeks (83 mg/kg rat chow) was utilized. Results Compared to lean controls, there were increases in plasma DPP4 activity along with proteinuria in ZO rats. ZO rats further displayed increases in glomerular size and podocyte foot process effacement. These findings occurred in parallel with decreased endothelial stromal-derived factor-1α (SDF-1α), increased oxidant markers, and tyrosine phosphorylation of nephrin and serine phosphorylation of the mammalian target of rapamycin (mTOR). DPP4 inhibition improved proteinuria along with filtration barrier remodeling, circulating and kidney tissue DPP4 activity, increased active glucagon like peptide-1 (GLP-1) as well as SDF-1α, and improved oxidant markers and the podocyte-specific protein nephrin. Conclusions These data support a role for DPP4 in glomerular filtration function and targeting DPP4 with inhibition improves oxidant stress-related glomerulopathy and associated proteinuria.

Aim Heparin, a highly sulfated glycosaminoglycan, has been shown to have a renoprotective effect on renal diseases, but its mechanisms remain to be elucidated. In this study, we examined the effect of heparin on podocytes by using primary cultured podocytes positive for podocyte-specific markers including podocin and podocalyxin. Methods Podocytes were cultured from highly purified glomeruli isolated by the method with renal perfusion with magnetic beads and digestion of collagenase. Podocyte-specific gene expressions and proteins were examined by real-time polymerase chain reaction (PCR), Western blotting and immunofluorescence microscopy. Results Real-time PCR showed that addition of heparin to the culture media significantly upregulated most of the podocyte-specific genes in a dose-dependent and time-dependent manner. Western blotting showed a marked increase in protein levels of nephrin and podocin. Podocin localization at cell–cell contact sites became conspicuous in the presence of heparin. The effect of heparin was observed even in culture media deprived of bovine foetal serum. Heparan sulfate, less sulfated than heparin, and hyaluronan did not show such effects, but sulfated dextran did markedly. Conclusion Heparin acts on cultured podocytes to increase podocyte-specific gene expressions. A high degree of sulfation is crucial for the effect of heparin.

PAI-1R, a mutant plasminogen activator inhibitor (PAI-1) that reduces endogenous PAI-1 activity has been shown to inhibit albuminuria and reduce glomerulosclerosis in experimental diabetes. The mechanism of albuminuria reduction is unclear. This study sought to determine whether the administration of PAI-1R protected podocyte from injury directly, thereby reducing albuminuria in the db/db mouse, a model of type 2 diabetes. Untreated uninephrectomized db/db mice developed significant mesangial matrix expansion and albuminuria at week 22 of age, associated with segmental podocyte foot process effacement, reduction of renal nephrin, podocin and zonula occludin-1 (ZO-1) production and induction of renal desmin and B7-1 generation. In contrast, treatment with PAI-1R at 0.5 mg/kg BW through peritoneal injection, twice daily from weeks 20 to 22 reduced glomerular matrix accumulation, fibronectin and collagen production and albuminuria by 36%, 62%, 65% and 31% respectively (p

Congenital nephrotic syndrome (CNS) is a rare disease inherited as an autosomally recessive trait and defined as proteinuria manifesting at birth or in the first 3 months of life. The classical form is the Finnish type of CNS (CNF), which is caused by mutations in the nephrin gene (NPHS1). The classical findings include prematurity, large placenta and massive proteinuria. Minor cardiac findings have been reported as a minor functional disorder but CNS with major cardiac malformation is rare. Here we report the case of a Turkish child with CNS with small indel mutation (c.614_621delCACCCCGGinsTT) in exon 6 of NPHS1 and also major cardiac malformation who did not develop end-stage renal disease until the age of 5 years.

Gremlin is a bone morphogenic protein (BMP) antagonist and is elevated in diabetic kidney tissues. In the early course of diabetic nephropathy (DN), podocyte are injured. We studied the protein and gene expression of gremlin in mice podocytes cultured in hyperglycemia ambient. The role of gremlin on podocyte injury and the likely signaling pathways involved were determined. Expression of gremlin was visualized by confocal microscopy. Recombinant mouse gremlin and small interfering RNA (siRNA) targeting to gremlin1 identified the role played by gremlin on podocytes. Study of canonical (smad2/3) and non-canonical (p38MAPK and JNK1/2) transforming growth factor beta (TGFβ)/smad mediated signaling revealed the putative signaling mechanisms involved. Smad2/3 siRNA and TGFβ receptor inhibition (SB431542) were used to probe canonical TGFβ/smad signaling in gremlin-induced podocyte injury. Apoptosis of podocytes was measured by TUNEL assay. Gremlin expression was enhanced in high glucose cultured mouse podocytes, and was localized predominantly in the cytoplasm and negligibly on the cell membrane. Not only expression of nephrin and synaptopodin were decreased on treatment with gremlin, but also synaptopodin rearrangement and nephrin relocalization were evident. Knockdown gremlin1 or smad2/3 by siRNA, and inhibition of TGFβR (SB431542) attenuated podocyte injury. Inhibition of canonical TGF-β signal blocked the injury of gremlin on podocytes. In conclusion, gremlin was clearly elevated in high glucose cultured mouse podocytes, and likely employed endogenous canonical TGFβ1/Smad signaling to induce podocyte injury. Knockdown gremlin1 by siRNA may be clinically useful in the attenuation of podocyte injury.

Background: The DNA-binding transcription factor Wilms' Tumor Suppressor-1 (WT1) plays an essential role in nephron progenitor differentiation during renal development. We previously used Wt1 chromatin-immunoprecipitation coupled to microarray (ChIP-chip) to identify novel Wt1 target genes that may regulate nephrogenesis in vivo. We discovered that all three members of the SoxC subfamily, namely, Sox4, Sox11, and Sox12, are bound by Wt1 in mouse embryonic kidneys in vivo. SoxC genes play master roles in determining neuronal and mesenchymal progenitor cell fate in a multitude of developmental processes, but their function in the developing kidney is largely unknown. Results: Here we show that all three SoxC genes are expressed in the nephrogenic lineages during renal development. Conditional ablation of Sox4 in nephron progenitors and their cellular descendants (Sox4nephron- mice) results in a significant reduction in nephron endowment. By postnatal day (P)7, Sox4nephron- renal corpuscles exhibit reduced numbers of Wt1+ podocytes together with loss of expression of the slit diaphragm protein nephrin. Sox4nephron- mice develop early-onset proteinacious glomerular injury within 2 weeks of birth progressing to end-stage renal failure within 5–9 months. Conclusions: Collectively, our results demonstrate an essential requirement of Sox4 for normal renal development in vivo. Developmental Dynamics 242:790–799, 2013. © 2013 Wiley Periodicals, Inc.

Diabetic nephropathy (DN) is the major life-threatening complication of diabetes. Abnormal permeability of glomerular basement membrane plays an important role in DN pathogenesis. This study was performed to assess the effect of arctiin, the lignan constituent from Arctium lappa L., on metabolic profile and aggravation of renal lesions in a rat model of streptozotocin (STZ)-induced DN. STZ-induced diabetic rats were treated with arctiin at the dosage of 60 or 40 mg/kg/day via intraperitoneal injection for 8 weeks. Blood glucose and 24-h urinary albumin content were measured, and kidney histopathological changes were monitored. RT-PCR and immunohistochemistry were used to detect the mRNA and protein levels of nephrin, podocin and heparanase (HPSE) in the kidney cortex of rats, respectively. Treatment with arctiin significantly decreased the levels of 24-h urinary albumin, prevented the sclerosis of glomeruli and effectively restored the glomerular filtration barrier damage by up-regulating the expression of nephrin and podocin and down-regulating HPSE level. Our studies suggest that arctiin might be beneficial for DN. The effects of arctiin on attenuating albuminuria and glomerulosclerosis are possibly mediated by regulating the expression of nephrin and podocin and HPSE in STZ-induced diabetic rats.

The slit diaphragm (SD) is an intercellular junction between renal glomerular epithelial cells (podocytes) that is essential for permselectivity in glomerular ultrafiltration. The SD components, nephrin and Neph1, assemble a signaling complex in a tyrosine phosphorylation dependent manner, and regulate the unique actin cytoskeleton of podocytes. Mutations in the NPHS1 gene that encodes nephrin cause congenital nephrotic syndrome (CNS), which is characterized by the loss of the SD and massive proteinuria. Recently, we have identified the expression of the transmembrane glycoprotein signal regulatory protein α (SIRPα) at the SD. In the present study, we analyzed the expression of SIRPα in developing kidneys, in kidneys from CNS patients and in proteinuric rat models. The possibility that SIRPα interacts with known SD proteins was also investigated. SIRPα was concentrated at the SD junction during the maturation of intercellular junctions. In the glomeruli of CNS patients carrying mutations in NPHS1, where SD formation is disrupted, the expression of SIRPα as well as Neph1 and nephrin was significantly decreased, indicating that SIRPα is closely associated with the nephrin complex. Indeed, SIRPα formed hetero-oligomers with nephrin in cultured cells and in glomeruli. Furthermore, the cytoplasmic domain of SIRPα was highly phosphorylated in normal glomeruli, and its phosphorylation was dramatically decreased upon podocyte injury in vivo. Thus, SIRPα interacts with nephrin at the SD, and its phosphorylation is dynamically regulated in proteinuric states. Our data provide new molecular insights into the phosphorylation events triggered by podocyte injury.

Chronic renal failure involves the progressive loss of renal parenchymal cells. For example, Alport syndrome develops from mutated type IV collagen that fosters the digestion of glomerular basement membranes and podocyte loss, followed by progressive glomerulosclerosis, ie Alport nephropathy. Here we show that autosomal recessive Alport nephropathy in collagen 4a3-deficient mice is associated with increased intrarenal expression of the pro-apoptotic cytokine tumour necrosis factor-alpha (TNF-α) in glomerular cells including podocytes as well as in infiltrating leukocytes. We therefore hypothesized that TNF-α contributes to Alport glomerulosclerosis by inducing podocyte apoptosis. To address this issue, we treated 4-week-old collagen 4a3-deficient mice with either vehicle or the TNF-α antagonist etanercept for a period of 5 weeks. Etanercept treatment prolonged mean survival from 68 to 81 days as compared to vehicle-treated mice. The beneficial effect of etanercept on survival was associated with a significant improvement of the glomerulosclerosis score, proteinuria, and the glomerular filtration rate at 9 weeks of age. Etanercept treatment specifically reduced the numbers of apoptotic podocytes, increased total podocyte counts, and increased the renal mRNA expression of nephrin and podocin without affecting markers of renal inflammation. TNF-α-induced podocyte loss is a previously unrecognized pathological mechanism of Alport glomerulosclerosis, and TNF-α blockade might be a therapeutic option to delay the progression of Alport nephropathy and potentially of other forms of glomerulosclerosis.

In the gel model for the glomerular (and peritubular) capillary membrane, the integrity of the membrane is supposed to result from the fluid reabsorption induced by the osmotic action of the counter-ions attracted to negative fixed charges, increasing the gel pressure such that it becomes the same as in the capillaries. From this point on, the gel will be unaffected by the high capillary pressure. The same fluid reabsorption will also suspend the fibrils in the matrix such that they form a series of grids composed of, for example, horizontal fibrils spaced similarly from one another. The model thereby explains the well-known phenomenon of a uniform 'pore' size, although slits rather than pores constitute the transport routes. The model also explains the fact that the plasma proteins are free to move in the membrane matrix, which is the consequence of a recent finding that a major restriction to albumin is offered by a unique protein, nephrin, located between the podocytes in Bowman's space cells. A large molecule, which may become trapped in a slit between two fibrils, will thus push out the positive counter-ions whereby the charges become free and hence repel one another, widening the slit such that the molecule is free to move in any direction. It is furthermore concluded that the restriction to proteins is also dependent on the width of the slits closest to plasma.

Aim: Podocytes provide a slit diaphragm to inhibit proteinuria, and nephrin between podocytes functions as a barrier during glomerular filtration. Hepatocyte growth factor (HGF) can improve proteinuria in rodents with various renal injuries, but little is known about the role of HGF in podocyte-based events during glomerulonephritis. In the present study, we examined whether and how nephrin expression is sustained by podocytes during the HGF-mediated attenuation of albuminuria. Methods: Lipopolysaccharide (LPS)-treated mice were used as an animal model of albuminuria. We evaluated the effect of HGF on slit proteins using immunohistochemistry, western blotting and real-time polymerase chain reaction. Results: Albuminuria occurred 36 h after LPS treatment in mice. This albuminuria did not involve podocyte loss, but was associated with a decrease in nephrin and its key anchor, synaptopodin. In these processes, c-Met tyrosine phosphorylation, which represented HGF signal activation, occurred in glomerular cells including podocytes. When recombinant HGF was administrated to nephritic mice, c-Met tyrosine phosphorylation became evident in podocytes. The enhancement of the HGF-c-Met signal was associated with increases in nephrin and synaptopodin. An electron microscopic examination revealed that LPS induced the foot process effacement of podocytes, while HGF injections suppressed the foot process injury. Overall, albuminuria was attenuated in the LPS-treated mice after HGF administration. Conclusion: HGF protects podocytes from a loss of nephrin, at least in part, through maintaining synaptopodin. As a result, HGF was shown to sustain foot process structure, and albuminuria was attenuated under inflammation.

Recent findings of podocyte shedding/podocyturia highlight the central significance of podocyte injury in preeclampsia, a hypertensive disorder unique to human pregnancy. To test the hypothesis that oxidative stress contributes to kidney podocyte injury in preeclampsia, we specifically examined expression and distribution of antioxidant CuZn‐SOD with nephrin and podoplanin in shed podocytes from women with preeclampsia. Human podocyte AB 8/13 cells served as control. We found that CuZn‐SOD was localized at the front/outreach region of nephrin at the cell periphery (foot process areas) in control podocytes and expression of CuZn‐SOD, nephrin, and podoplanin were all dislocated or lost in shed podocytes from preeclamptic patients. We further tested oxidative stress‐induced nephrin shedding in podocytes, in which AB 8/13 podocytes were cultured under lowered oxygen condition (2%O2) or treated with hypoxic mimicking agent cobalt chloride. Our results showed that reduced nephrin and podoplanin expression were associated with downregulation of CuZn‐SOD expression in podocytes when cells were cultured under lowered oxygen or hypoxic conditions. Nephrin shed in urinary specimen from preeclamptic women was also determined by immunoprecipitation/immunoblotting. The molecular sizes of nephrin that corresponded to that were lost when cells were cultured under hypoxic conditions. We concluded that increased oxidative stress plays a significant role in inducing podocyte protein shedding in preeclampsia.

Summary 1. Nephropathy and elevated plasma aldosterone concentrations (PAC) have been observed in (pro)renin receptor transgenic (TG) rats. In the present study, we hypothesized that PAC and/or mineralocorticoid receptor contribute to the nephropathy of TG rats. To test this hypothesis, the effects of a high-sodium (8% NaCl) diet and heminephrectomy on PAC were examined. 2. Feeding of the high-sodium diet for 12 weeks similarly decreased PAC in TG and wild-type (WT) rats. Heminephrectomy further reduced PAC in TG rats fed a high-sodium diet, but had no effect on PAC in WT rats fed a high-sodium diet. 3. Next, the effects of eplerenone (125 mg/kg per day) and dietary salt restriction (0.36% NaCl diet) on proteinuria and renal morphology were examined in rats fed a high-sodium diet or subjected to heminephrectomy. Both eplerenone and dietary sodium restriction significantly reduced the arterial pressure of TG rats, but had no effect in WT rats. In TG rats, treatment with eplerenone significantly decreased urinary protein excretion, but dietary sodium restriction did not. 4. Nephrin and podocin mRNA levels, as determined by real-time quantitative reverse transcription–polymerase chain reaction, were significantly lower in TG rats than in WT rats. In TG rats, eplerenone treatment significantly increased nephrin mRNA levels, but not podocin mRNA levels. Dietary salt restriction significantly increased mRNA levels of both nephrin and podocin. Although zonula occludens (ZO)-1 mRNA levels were similar in both WT and TG rats, eplerenone treatment significantly decreased ZO-1 mRNA levels in TG rats. 5. The results of the present study suggest that the improvement in proteinuria following eplerenone treatment is independent of its effects on sodium balance and may be mediated by effects on the expression of slit diaphragm proteins.

Aim: Several proteins constituting the slit diaphragm are considered important for maintaining capillary wall permselectivity. Early intervention with blockers of angiotensin II receptors (AR) and mineralocorticoid receptors (MR) is effective against proteinuria in models of chronic hypertensive and protein-induced renal damage. However, the effects of AR and/or MR blockers in a model of acute nephrotic syndrome remain unknown. The effects of AR and MR blockers were examined in puromycin aminonucleoside (PAN)-treated rats. Methods: Six week old male Sprague–Dawley (SD) rats were injected with PAN or vehicle and assigned to groups as follows: vehicle (group C); PAN (group P); PAN followed 3 days later by administration of the MR blocker, eplerenone (group MR), and by the AR blocker, losartan (group AR). Blood pressure and urinary protein excretion were measured and all rats were killed for immunohistochemical investigation on day 14 after PAN administration. Results: Blood pressure did not change throughout the study period. Proteinuria was decreased in groups MR and AR compared with group P (on day 14 after PAN administration, respectively; group P vs AR, P

Background Glomerulopathy is an increasingly identified complication in young patients with sickle cell disease (SCD). Hyperfiltration and albuminuria followed by declining glomerular filtration rates and eventual end-stage renal disease (ESRD) is assumed to be the typical progression of glomerular disease. There are only a few reported biomarkers to identify early-stage renal disease in SCD. Procedures We detail the renal profile of 101 children with SCD in Malawi and propose a novel urinary biomarker for the identification of early renal disease. Results Among children with sickle cell anemia, 24.8% had a urine albumin-creatinine ratio of 30 mg/g or above. In univariate analysis, only patients with higher urinary nephrin, a urinary marker of glomerular injury, had significantly greater odds of having albuminuria. In multivariable analysis, nephrin remained significantly associated with albuminuria. A nephrin-creatinine ratio (NCR) cut-point of 622 ng/mg, the 50th percentile, was associated with a 45.8 times greater odds of having albuminuria in children with nephrinuria above this value. Further analysis revealed this urinary NCR cut-point to have 96% sensitivity, 64% specificity, 47% positive predictive value, and 98% negative predictive value for the presence of albuminuria. Conclusions These data suggest that a substantial number of children with SCD in Malawi have renal disease and could be at risk for worsening nephropathy and ESRD as they age. Our data suggest that urinary nephrin could be utilized as an early marker of glomerular disease in SCD.

A lot of mutations of podocin, a key protein of podocyte slit diaphragm (SD), have been found both in hereditary and sporadic focal segmental glomeruloscleorosis (FSGS). Nevertheless, the mechanisms of podocyte injury induced by mutant podocins are still unclear. A compound heterozygous podocin mutation was identified in our FSGS patient, leading to a truncated (podocin (V165X)) and a missense mutant protein (podocin (R168H)), respectively. Here, it was explored whether and how both mutant podocins induce podocyte injury in the in vitro cultured podocyte cell line. Our results showed that podocin (R168H) induced more significant podocyte apoptosis and expression changes in more podocyte molecules than podocin (V165X). Podocyte injury caused by the normal localized podocin(V165X) was effectively inhibited by TRPC6 knockdown. The abnormal retention of podocin(R168H) in endoplasmic reticulum (ER) resulted in the mis-localizations of other critical SD molecules nephrin, CD2AP and TRPC6, and significantly up-regulated ER stress markers Bip/grp78, p-PERK and caspase-12. These results implicated that podocin (R168H) and podocin (V165X) induced different degrees of podocyte injury, which might be resulted from different molecular mechanisms. Our findings provided some possible clues for further exploring the pharmacological targets to the proteinuria induced by different mutant podocins.

1. Accumulating evidence suggests that vitamin D and its analogues are renoprotective. However, the precise mechanisms and the molecular targets by which active vitamin D exerts its beneficial effects remain obscure. The objective of the present study was to evaluate the effect of active vitamin D on rats with puromycin aminonucleoside (PAN) nephropathy, a model that is characterized by predominant podocyte injury. 2. The PAN nephropathy rats were created by a single intravenous injection of 100 mg/kg PAN. Changes in renal pathology and podocyte numbers were observed. Real-time polymerase chain reaction (PCR) was performed to examine mRNA expression of nephrin, transforming growth factor (TGF)-beta1 and bone morphogenetic protein (BMP)-7. Protein expression of nephrin, TGF-beta1, BMP-7 and p-Smad2/3 and p-Smad1/5/8 was examined by immunofluorescence, immunohistochemistry and western blotting, respectively. Rats were treated with 1,25(OH)(2)D(3) by gastric gavage at a dose of 2.5 microg/kg per day, starting 2 days before PAN injection and continuing throughout the experiment. 3. A single injection of PAN induced massive proteinuria and elevated serum creatinine on Day 7, both of which were significantly suppressed by 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)). Immunofluorescence and real-time PCR of the podocyte-associated protein nephrin revealed reduced and discontinuous staining and this change was reversed by 1,25(OH)(2)D(3). In PAN nephropathy rats, TGF-beta1 and p-Smad2/3 expression was upregulated, whereas that of BMP-7 and p-Smad1/5/8 was downregulated. Treatment with 1,25(OH)(2)D(3) significantly restored BMP-7/Smad signalling while suppressing TGF-beta1/Smad signalling. 4. In conclusion, 1,25(OH)(2)D(3) can ameliorate podocyte damage and proteinuria induced by PAN. The beneficial effects of 1,25(OH)(2)D(3) on podocytes may be attributable, in part, to direct modulation of TGF-beta1/BMP-7 signalling.

Emerging evidences show that CD2-associated protein (CD2AP) is involved in podocyte injury and the pathogenesis of proteinuria. However, the exact molecular mechanism by which CD2AP exerts its biological function is elusive. We knocked down CD2AP gene by target siRNA in conditionally immortalized mouse podocytes, which showed lowered cell adhesion and spreading ability (P < 0.05). At the same time, cell cycle was arrested in G2/M phase (P < 0.05), and pathologic nuclear division could easily be seen in CD2AP siRNA-transfected podocytes. The proliferation of podocytes were also inhibited significantly by CD2AP siRNA transfection (P < 0.05). Further study revealed disordered distributions of F-actin, as well as lowered nephrin expression and phosphorylation in podocytes. These data suggest that CD2AP may play a crucial role in maintaining the normal function of podocytes and lowered CD2AP causes podocyte injury by disrupting the cytoskeleton and disturbing the nephrin-CD2AP signaling pathway.