Vitamin D Ameliorates Podocyte Injury by Enhancing Autophagy Activity in Diabetic Kidney Disease.

INTRODUCTION: Restoration of podocyte autophagy is considered as a feasible strategy for the treatment of diabetic kidney disease (DKD). This study aimed at investigating the protective effect and potential mechanism of vitamin D on podocyte injury of DKD. METHODS: Type 2 diabetic db/db mice received intraperitoneal injections of vitamin D analog paricalcitol 400 ng/kg per day for 16 weeks. Immortalized mouse podocytes were cultured in high glucose (HG) medium with active vitamin D3 calcitriol or autophagy inhibitor 3-methyladenine. Renal function and urine albumin creatinine ratio were assessed at week 24. HE, PAS staining, and electron microscopy were used to evaluate renal histopathology and morphological changes. Immunohistochemistry, immunofluorescence, and Western blot were used to evaluate protein expression of nephrin and podocin in kidney tissue and podocytes. The expression of autophagy-related proteins (LC3, Beclin-1, Vps34) and apoptosis-related proteins (cleaved caspase-3, Bax) was determined by Western blotting. Podocyte apoptosis was further evaluated by using flow cytometer. RESULTS: Albuminuria in a db/db mouse model was markedly attenuated after treatment with paricalcitol. This was accompanied by alleviation of mesangial matrix expansion and podocyte injury. Besides, the impaired autophagy in podocytes under diabetic conditions was also markedly enhanced after paricalcitol or calcitriol treatment, accompanied by restored decreased podocyte slit diaphragm proteins podocin and nephrin. Furthermore, the protective effect of calcitriol against HG-induced podocyte apoptosis could be abated by autophagy inhibitor 3-methyladenine. CONCLUSION: Vitamin D ameliorates podocyte injury of DKD by enhancing podocyte autophagy activity, which may become a potential candidate autophagy activator for the therapeutic interventions for DKD.

Receptor activator of NF-κB mediates podocyte injury in diabetic nephropathy.

Receptor activator of NF-κB (RANK) expression is increased in podocytes of patients with diabetic nephropathy. However, the relevance of RANK to diabetic nephropathy pathobiology remains unclear. Here, to evaluate the role of podocyte RANK in the development of diabetic nephropathy, we generated a mouse model of podocyte-specific RANK depletion (RANK-/-Cre T), and a model of podocyte-specific RANK overexpression (RANK TG), and induced diabetes in these mice with streptozotocin. We found that podocyte RANK depletion alleviated albuminuria, mesangial matrix expansion, and basement membrane thickening, while RANK overexpression aggravated these indices in streptozotocin-treated mice. Moreover, streptozotocin-triggered oxidative stress was increased in RANK overexpression but decreased in the RANK depleted mice. Particularly, the expression of NADPH oxidase 4, and its obligate partner, P22phox, were enhanced in RANK overexpression, but reduced in RANK depleted mice. In parallel, the transcription factor p65 was increased in the podocyte nuclei of RANK overexpressing mice but decreased in the RANK depleted mice. The relevant findings were largely replicated with high glucose-treated podocytes in vitro. Mechanistically, p65 could bind to the promoter regions of NADPH oxidase 4 and P22phox, and increased their respective gene promoter activity in podocytes, dependent on the levels of RANK. Taken together, these findings suggested that high glucose induced RANK in podocytes and caused the increase of NADPH oxidase 4 and P22phox via p65, possibly together with the cytokines TNF- α, MAC-2 and IL-1 ß, resulting in podocyte injury. Thus, we found that podocyte RANK was induced in the diabetic milieu and RANK mediated the development of diabetic nephropathy, likely by promoting glomerular oxidative stress and proinflammatory cytokine production.

Associations between endothelial nitric oxide synthase polymorphisms and risk of diabetic nephropathy: an updated meta-analysis.

BACKGROUND: Genetic polymorphism of endothelial nitric oxide synthase (eNOS) has been implicated in the risk of diabetic nephropathy (DN), but the published findings were inconsistent. We performed a comprehensive meta-analysis to derive a more precise estimation of the association between genetic polymorphisms of eNOS and the risk of DN. METHODS: Six online database were researched on the associations between polymorphisms of eNOS (T786C, G894T, 4b/4a) and DN risk. PRISMA statement and Hardy-Weinberg equilibrium assessment were used in this report. Odds ratio and 95% confidence interval were estimated based on the following genetic contrast/models: allelic model, dominant model, recessive model and co-dominant model. The publication bias and sensitivity analysis were also performed to guarantee the statistical power. RESULTS: A total of 49 case-control studies with 11,990/9754/5131 participants for DN/DM/HC group were eligible for meta-analysis (7/25/31 studies for T786C/G984T/4b/a). For the eNOS-T786C, C allele showed a weak association between C allele and DN risk in DN/T2DM group. For eNOS-G894T, there was an association between T allele and DN risk in the global, Asian and African population in DN/T2DM group. For the eNOS-4b/4a, 4a allele was found contributing significantly to increased DN risk in the global population. CONCLUSION: Our comprehensive meta-analysis suggests that three polymorphisms of eNOS may be the increased risk factors of DN development, especially in Asian population and T2DM group.

1,25-Dihydroxyvitamin D(3) prevents puromycin aminonucleoside-induced apoptosis of glomerular podocytes by activating the phosphatidylinositol 3-kinase/Akt-signaling pathway.

BACKGROUND: Accumulating evidence suggests that vitamin D and its analogs reduce proteinuria and slow the decline in kidney function in chronic kidney disease. Given a rich literature identifying podocyte apoptosis as an early step in the pathophysiological progression to proteinuria and glomerulosclerosis, we hypothesized that vitamin D protects podocytes from undergoing apoptosis. METHODS: A rat model of podocyte apoptosis was created by a single intravenous injection of 100 mg x kg(-1) puromycin aminonucleoside (PAN) and received either solvent or 1,25(OH)(2)D(3) treatment. Proteinuria, podocyte apoptosis, the expression of nephrin protein and mRNA, TGF-beta/Smad and phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway were evaluated, respectively. RESULTS: PAN induced massive proteinuria, serum creatinine elevation and podocyte apoptosis in PAN nephropathy rats, which was associated with the loss of nephrin, an adhesion molecule specific for the glomerular slit and the reduced of p-Akt/Akt ratio. Moreover, PAN induced foot process retraction, redistribution of nephrin and the activation of TGF-beta/Smad-signaling pathway. Compared with PAN nephropathy rats, 1,25(OH)(2)D(3) significantly prevented loss of nephrin, foot process retraction and podocyte apoptosis by stimulating Akt phosphorylation and suppressing TGF-beta/Smad-signaling pathway. CONCLUSION: 1,25(OH)(2)D(3) reduced the PAN-induced podocyte apoptosis and loss of nephrin in PAN nephropathy rat. The anti-apoptotic effects of 1,25(OH)(2)D(3 )on podocytes may be partly attributable to activation of a PI3K/Akt survival pathway.

Podocyte injury is suppressed by 1,25-dihydroxyvitamin D via modulation of transforming growth factor-beta 1/bone morphogenetic protein-7 signalling in puromycin aminonucleoside nephropathy rats.

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.

Differential expression and therapeutic efficacy of microRNA-346 in diabetic nephropathy mice.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease, in which the SMAD signaling pathway plays an important role. The aim of the present study was to identify differentially expressed microRNAs (miRNAs) during the progression of DN and to investigate a selected miRNA in relation to SMAD3/4 and its therapeutic efficacy. The miRNA microarray was used to identify differentially expressed miRNAs in db/db DN mice. Reverse transcription-quantitative polymerase chain reaction and immunoblot analyses were used to detect SMAD3/4 expression. The development of DN in the db/db mice was demonstrated by glucose dysregulation and typical morphological changes in the kidney. miRNA-346 (miR-346) was identified as one of the differentially expressed miRNAs. The expression of SMAD3/4 was significantly attenuated by miR-346 administration and the therapeutic effects of miR-346 were observed in the DN mouse models. miR-346 was identified as a negative regulator of SMAD3/4. SMAD3/4 was upregulated in the renal tissue of db/db mice. The administration of miR-346 attenuated the SMAD3/4 expression in renal tissue and ameliorated the renal function and glomerular histology in DN mice. This study paves the way for clinical studies of miR-346 in DN.

[Renal expression of RANK-RANKL in a rat model of puromycin aminonucleoside nephropathy].

OBJECTIVE: To investigate RANK-RANKL expression in the kidneys of a rat model of puromycin aminonucleoside nephropathy (PAN). METHODS: Thirty-six SD rats were randomly divided into PAN model group and normal control group. PAN was induced by a single intravenous injection of 100 mg/kg puromycin aminonucleoside. Serum creatinine and 24-hour urinary protein were measured on days 3, 7, and 14 after the injection, and renal pathologies were assessed with optical and immune transmission electron microscopy. The expression of RANK and RANKL in the kidneys was examined using reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. RESULTS: The PAN model rats showed massive proteinuria and elevated serum creatinine on day 3, which peaked on day 7. RANK-RANKL protein and mRNA expressions in PAN model group was higher than those in the control group. In the PAN rats, RANK was expressed mainly on the top cell membrane and in the cytoplasm of renal podocytes with a significantly increased expression level compared with that in the control group. CONCLUSION: The PAN rat model shows aberrant RANK and RANKL expressions in the podocytes, indicating their contribution to podocyte injury in PAN.

Vascular endothelial growth factor gene polymorphisms and renal cell carcinoma: A systematic review and meta-analysis.

Renal cell carcinoma (RCC) accounts for 3% of all cancer-related mortalities in adults. The risk factors for the development of RCC remain under investigation. Vascular endothelial growth factor (VEGF) is a key mediator of angiogenesis and is crucial for the development and metastasis of tumors, including RCC. VEGF gene polymorphisms may alter VEGF protein concentrations, affect the process of angiogenesis and may be involved in inter-individual variation in carcinogenesis. In the present study, a systematic review and meta-analysis were performed based on published case-control studies in order to estimate the association between VEGF gene polymorphisms and the susceptibility to RCC. A total of five studies that involved eight polymorphisms and were published between January 2000 and December 2012 were identified from PubMed. The results of this systematic review and meta-analysis indicate that the VEGF 936C/T, 1612G/A, -1154G/A, -2549I/D, -460T/C and 405G/C gene polymorphisms are not associated with the risk of RCC. There was no polymorphism in 702C/T and RCC and the -2578C/A gene polymorphism may be associated with an increased risk of RCC. However, due to the limitations of the present study, further high quality case-control studies are warranted to confirm these findings.

Receptor activator of NF-kappaB and podocytes: towards a function of a novel receptor-ligand pair in the survival response of podocyte injury.

BACKGROUND: Glomerulosclerosis correlates with reduction in podocyte number that occurs through mechanisms which include apoptosis. Podocyte injury or podocyte loss in the renal glomerulus has been proposed as the crucial mechanism in the development of glomerulosclerosis. However, the mechanism by which podocytes respond to injury is poorly understood. TNF and TNF receptor superfamilies are important in the pathogenesis of podocyte injury and apoptosis. The ligand of receptor activator of NF-kappaB (RANKL) and receptor activator of NF-kappaB (RANK) are members of the TNF and receptor superfamilies. We investigated whether RANK-RANKL is a receptor-ligand complex for podocytes responding to injury. METHODOLOGY/PRINCIPAL FINDINGS: In this study, RANKL and RANK were examined in human podocyte diseases and a rat model of puromycin aminonucleoside nephrosis (PAN). Compared with controls, RANK and RANKL were increased in both human podocyte diseases and the rat PAN model; double immunofluorescence staining revealed that RANK protein expression was mainly attributed to podocytes. Immunoelectron microscopy showed that RANK was localized predominantly at the top of the foot process membrane and the cytoplasm of rat podocyte. In addition, RANK was upregulated in mouse podocytes in vitro after injury induced by puromycin aminonucleoside (PA). Knockdown of RANK expression by small interference RNA (siRNA) exacerbated podocyte apoptosis induced by PA. However, RANKL inhibited significantly the apoptosis of podocytes induced by PA. CONCLUSIONS/SIGNIFICANCE: These findings suggest the increase in RANK-RANKL expression is a response to podocyte injury, and RANK-RANKL may be a novel receptor-ligand complex for the survival response during podocyte injury.

Gypenosides inhibit renal fibrosis by regulating expression of related genes in rats with unilateral ureteral obstruction.

BACKGROUND: Transforming growth factor ß1 (TGF-ß1), connective tissue growth factor (CTGF) and Smad7 are potent components of fibrogenesis-related signal transduction pathways. Renal fibrosis is the major pathological change in the rat models with unilateral ureteral obstruction (UUO). Investigating the effects of gypenosides (GPs) on the expression of fibrogenesis-related genes in the UUO model may lead to the development of effective therapy for renal diseases. METHODS: Rats were randomly divided into 3 experimental groups: (i) sham operation rats treated with saline (sham group), (ii) UUO model rats treated with saline (control group) and (iii) UUO model rats treated with GPs (GPs group). Blood urea nitrogen and serum creatinine were detected as the measurement of renal function. UUO-treated kidney tissues were taken for assessment of renal damage index and determination of related gene expression through immunohistochemistry and RT-PCR. RESULTS: UUO-induced tubulointerstitial damage and fibrosis were attenuated by the application of GPs (day 3 and day 7, p<0.01; day 14, p<0.05). The expression of TGF-ß1 and CTGF was significantly reduced with GPs treatment (TGF-ß1, p<0.01; CTGF, p<0.05). Smad7 expression was elevated with GPs treatment at days 7 and 14 (p<0.01). GPs' protective effects on renal function were also demonstrated with this UUO model. CONCLUSIONS: These results suggest that UUO-induced tubulointerstitial fibrosis can be effectively attenuated by GPs application. GPs-mediated down-regulation of TGF-ß1 and CTGF and up-regulation of Smad7 are essential for their effects of antifibrogenesis.

[Effect of bone morphogenic protein 7 on nephrin expression and distribution in diabetic rat kidneys].

OBJECTIVE: To evaluate the effect of bone morphogenic protein 7 (BMP-7) on nephrin expression and distribution in diabetic rat kidneys. METHODS: Twenty rats with diabetes mellitus (DM) induced by streptozotocin (STZ) injection were randomly divided into DM model group and BMP-7 treatment group, with another 10 normal rats serving as the normal control group. The rats in BMP-7 group received intraperitoneal human recombinant BMP-7 injections at 30 microg/kg twice a week for 24 consecutive weeks, while normal saline was administered in rats of the other two groups. Blood glucose and 24 hour urinary protein and creatinine (Ccr) were measured at 8, 16 and 24 weeks, and the rats were sacrificed at 24 weeks to obtain the renal tissues for detecting the expression and distribution of nephrin using immunofluorescence assay and RT-PCR and for examining the expressions of transforming growth factor-beta1 (TGF-beta1) and WT1 using immunohistochemistry. RESULTS: Compared with the normal control group, the DM model group showed significantly increased 24 hour urinary protein, kidney to body weight ratio and TGF-beta1 expression, but had lowered Ccr, glomerular podocyte number and nephrin expression. The linear distribution of nephrin along the capillary loops as found in the normal control group became granular in the kidney of diabetic rats. The rats in BMP-7 group showed less urinary protein excretion, lower TGF-beta1 expression and greater glomerular podocyte number than those in the DM group, and the expression and distribution of nephrin remained normal in the kidney. CONCLUSION: Administration of BMP-7 can significantly suppress the down-regulation of nephrin expression and maintain its normal distribution in the podocytes in diabetic rats possibly in association with a direct suppression of TGF-betasignaling.