Reduced podocin expression in minimal change disease and focal segmental glomerulosclerosis is related to the level of proteinuria

Background

Glomerular podocyte molecules are involved in the pathogenesis of congenital nephrotic syndrome. However, their role in primary nephrotic syndrome is not clear. This study investigated the expression of nephrin, podocin and synaptopodin in primary nephrotic syndrome.

Methods

Eighty-seven patients with primary nephrotic syndrome including minimal change disease (MCD), focal segmental glomerulosclerosis (FSGS), membranous nephropathy (MN) and membranoproliferative glomerulonephritis Type I (MPGN) were included in the study. Glomerular expression of nephrin, podocin and synaptopodin was studied in renal biopsies by immunofluorescence and immunohistochemistry. Correlation of expression with clinical and biochemical parameters was performed.

Results

The pattern of expression for all podocyte proteins in controls was uniform fine granular along the capillary walls towards the visceral epithelial cell aspect. Glomerular expression of nephrin was present in all renal biopsies and was similar to that in controls. Glomerular synaptopodin expression was seen in all MN and MPGN patients, while it was seen in 74 % (17/23) MCD and 93.5 % (29/31) FSGS. Reduced synaptopodin expression showed no correlation with clinical and biochemical factors. Podocin expression was present in 5/23 MCD (22 %), 3/31 FSGS (9.6 %), 13/17 MN (76.4 %) and 13/16 MPGN (81 %) patients. The reduced expression of podocin significantly correlated with the degree of proteinuria (p = 0.032). No correlation with age, gender and serum creatinine level was observed.

Conclusion

Reduction of glomerular podocin expression found in MCD and FSGS is related to the amount of proteinuria. Our findings suggest that alteration in podocyte phenotype may not be a primary event and may reflect the degree of podocyte injury in primary nephrotic syndrome.     

IgA肾病中足细胞损伤及其与蛋白尿的关系

目的 观察 IgA肾病（IgAN）患者足细胞损伤的各种表现，探讨其与蛋白尿的关系。 方法 收集35例伴有明显蛋白尿[尿蛋白量（24 h）>1.0 g]的IgAN患者肾活检组织作研究；以8例肾错构瘤患者术后切除肾和肾癌患者术后远离癌旁肾组织为正常对照。免疫组化方法观察肾组织细胞周期调节蛋白（p21、p27）、足细胞结构蛋白（nestin）、足细胞数目 （WT1）。用显微切割方法取出肾小球，通过实时定量PCR方法检测整合素（integrin）β1、nephrin和α辅肌动蛋白4（α-actinin 4）水平。电镜观察足细胞超微结构的改变。根据足细胞数目密度(Nv, n×106/μm3)将35例IgAN患者分为足细胞数目减少组（ Nv<52.49×106/μm3，n = 15）和足细胞数目正常组（Nv≥52.49×106/μm3，n = 20）。随访蛋白尿的转归情况，共18个月。 结果 （1）与正常对照组比较，IgAN患者肾小球内个别足细胞重新表达p21，而足细胞p27的表达明显降低（0.71±0.12比0.91±0.07，P < 0.05）。（2）IgAN患者足细胞nestin 蛋白表达比正常对照显著降低（13.40%±0.04%比 17.60%±0.04%，P < 0.05）；肾小球内integrin-β1 mRNA表达显著升高（12.54±5.20比1.02±0.30，P < 0.05），而nephrin及α-actinin4 mRNA无明显改变。（3）电镜下观察到明显的足突融合和足细胞从基底膜脱落。（4）IgAN患者足细胞数目密度比正常对照组显著减少(161.27±225.92比323.22±138.12，P < 0.05)，且与Lee氏分级相关。（5）足细胞数目密度、integrin-β1 mRNA与肾穿刺当时的尿蛋白量（24 h）呈负相关(r = -0.4483、-0.840, 均P < 0.05)。足细胞数目减少组较足细胞数目正常组的蛋白尿下降程度明显减少（P < 0.05）。 结论 伴蛋白尿的IgAN中存在足细胞的损伤，表现为足细胞周期调节蛋白、结构蛋白的改变，足突的融合及足细胞数目的减少，而足细胞损伤及足细胞数目减少会影响蛋白尿的发生和发展。     

Selective impairment of gene expression and assembly of nephrin in human diabetic nephropathy

BACKGROUND: Recent disclosure of podocyte proteins has unraveled previously rather mysterious mechanisms that govern glomerular perm-selectivity in health and disease. Here we addressed the role of nephrin, CD2-associated protein (CD2AP), and podocin together with the integrity of the slit diaphragm in the pathogenesis of proteinuria of patients with diabetes and nephropathy. METHODS: Nephrin mRNA and protein expression were evaluated in parallel in adult diabetic patients by in situ hybridization and immunohistochemistry. For comparison, nondiabetic patients with minimal change nephrosis and normal control patients were evaluated. CD2AP and podocin expression by immunohistochemistry was also assessed. The filtration slit was analyzed by morphometry and transmission electron microscopy. RESULTS: Extracellular nephrin mRNA and protein were markedly reduced in diabetic patients. No changes were found in patients with minimal change versus controls. CD2AP and podocin were comparable in all subjects. Ultrastructural analysis showed in diabetic patients a remarkable reduction in the percentage of electron dense slit diaphragms, despite a frequency of the filtration slits comparable to control patients. CONCLUSION: Down-regulation of nephrin and loss of the electron dense structure of slit diaphragm indicate a novel mechanism accounting for proteinuria in diabetic nephropathy. To the extent that glomerular protein trafficking contributes to renal disease progression, our findings may have clinical relevance. Reduction of nephrin in the context of normal expression of CD2AP and podocin can be taken reasonably as a specific marker of renal disease in diabetes. Therapies targeted at correcting podocyte nephrin might be of value for diabetic medicine.     

Evaluation of Renal Clinicopathological Changes in IgA Nephropathy by Urinary Podocytes Excretion and Podocalyxin Expression

Objective: To explore the association of urinary podocyte excretion and renal expression of podocyte-specific marker podocalyxin (PCX) with clinicopathological changes in immunoglobulin A nephropathy (IgAN). Methods: Morning urine samples from IgAN patients and healthy controls were collected. The expression of glomerular PCX was quantified in 50 IgAN patients diagnosed by renal biopsy. IgAN was classified based on the Lee’s Grading system and scored according to the Katafuchi semiquantitative criteria. Morphological evaluation of podocyte was determined by electron microscopy. Results: The amount of urinary podocytes in the IgAN patients was significantly higher than that in the healthy controls (p < 0.01). Pairwise comparison among Lee’s grades of IgAN showed that the median of urinary podocytes in Lee’s I–II group was lower than that in Lee’s III, IV, and V groups (p < 0.05); group III lower than group V (p < 0.05). The positive rate of urinary podocytes was the highest in Lee’s IV and V groups (100%), and lowest in Lee’s I–II group (55%). Multiple comparison among groups of Lee’s grades of IgAN showed that the glomerular PCX expression in Lee’s I–II group was higher than that in Lee’s III, IV, and V groups (p < 0.05); groups III and IV higher than group V (p < 0.05). The amount of urinary podocytes in IgAN patients was negatively correlated with PCX expression (r = ?0.702, p < 0.01), but positively correlated with 24-h urinary protein (r = 0.465, p < 0.01) and glomerular (r = 0.233, p < 0.01) and renal tubular pathological scores (r = 0.307, p < 0.05). The glomerular PCX expression was negatively correlated with 24-h urinary protein (r = ?0.367, p < 0.05) and glomerular (r = ?0.560, p < 0.05) and tubular pathological scores (r = ?0.377, p < 0.05). Electron microscopy showed significant changes in podocytes of IgAN, especially in the foot process. Conclusion: The amount of urinary podocyte can reflect the loss of podocytes in renal tissue, which may be a marker of IgAN progression.     

Podocyte injury induced by mesangial-derived cytokines in IgA nephropathy

Background. We have previously documented that human mesangialcell (HMC)-derived tumour necrosis factor- (TNF-) is an importantmediator involved in the glomerulo-tubular communication inthe development of interstitial damage in IgA nephropathy (IgAN).With the strategic position of podocytes, we further examinedthe function of podocytes in IgAN. Methods. Podocyte markers were examined in renal tissues byimmunofluorescence. In vitro experiments were conducted withpodocytes cultured with polymeric IgA (pIgA) or conditionedmedium prepared from HMC incubated with pIgA (IgA–HMCconditioned medium). Results. Glomerular immunostaining for nephrin or ezrin wassignificantly weaker in patients with IgAN. The immunostainingof IgA and nephrin was distinctly separate with no co-localization.In vitro experiments revealed no effect of pIgA on the expressionof these podocyte proteins as IgA from IgAN patients did notbind to podocytes. In contrast, IgA conditioned medium preparedfrom IgAN patients down-regulated the expression of these podocyteproteins as well as other podocyte markers (podocin and synaptopodin)in cultured podocytes. The mRNA expression of nephrin, erzin,podocin but not synaptopodin correlated with the degree of proteinuriaand creatinine clearance. The down-regulation was reproduciblein podocytes cultured with TNF- or transforming growth factor-β(TGF-β) at concentration comparable to that in the IgA–HMCconditioned medium. The expression of these podocyte proteinswas restored partially with a neutralizing antibody againstTNF- or TGF-β and fully with combination of both antibodies. Conclusion. Our finding suggests podocyte markers are reducedin IgAN. An in vitro study implicates that humoral factors (predominantlyTNF- and TGF-β) released from mesangial cells are likelyto alter the glomerular permeability in the event of proteinuriaand tubulointerstitial injury in IgAN.     

足细胞与Alport综合征蛋白尿的关系

目的 探讨足细胞与Alport综合征（AS）蛋白尿的关系。 方法 AS 患者21例,男13例,女8例,根据24 h尿蛋白量将患者分3组, 10例<30 mg/kg为轻度蛋白尿组, 4例30~50 mg/kg为中度蛋白尿组,7例>50 mg/kg为重度蛋白尿组。正常肾组织对照3例。电镜下根据平均足突宽度=л/4×（Σ基底膜长度/Σ足突个数）,计算每例患者足突宽度。分析足突宽度与蛋白尿关系。用免疫组化方法分析肾组织中裂孔隔膜分子nephrin、podocin和细胞骨架分子synaptopodin的表达。 结果 AS患者肾小球足细胞足突宽度（420~2270 nm）与24 h尿蛋白量呈正相关（r = 0.765,P < 0.01）。轻度蛋白尿组足突宽度[475（420~900 nm）]显著低于重度蛋白尿组[1520（480~2270） nm]（P < 0.05）。重度蛋白尿组患儿nephrin和podocin表达分布发生改变;表现为弥漫足突融合者synaptopodin表达分布发生改变。2例蛋白尿病程较短（1年）患儿无弥漫足突融合,synaptopodin分布正常,但nephrin和podocin分布异常。 结论 肾小球足细胞足突融合、裂孔隔膜及足细胞骨架分子参与AS患儿大量蛋白尿的发生,裂孔隔膜损伤似乎早于足细胞骨架改变。对蛋白尿早期干预可能有助于延缓疾病进展。     

Podocyte proteins in Galloway-Mowat syndrome

Galloway-Mowat syndrome is an autosomal recessive disorder characterized by early onset nephrotic syndrome and central nervous system anomalies. Mutations in podocyte proteins, such as nephrin, α-actinin 4, and podocin, are associated with proteinuria and nephrotic syndrome. The genetic defect in Galloway-Mowat syndrome is as yet unknown. We postulated that in Galloway-Mowat syndrome the mutation would be in a protein that is expressed both in podocytes and neurons, such as synaptopodin, GLEPP1, or nephrin. We therefore analyzed kidney tissue from normal children (n=3), children with congenital nephrotic syndrome of the Finnish type (CNF, n=3), minimal change disease (MCD, n=3), focal segmental glomerulosclerosis (FSGS, n=3), and Galloway-Mowat syndrome (n=4) by immunohistochemistry for expression of synaptopodin, GLEPP1, intracellular domain of nephrin (nephrin-I), and extracellular domain of nephrin (nephrin-E). Synaptopodin, GLEPP1, and nephrin were strongly expressed in normal kidney tissue. Nephrin was absent, and synaptopodin and GLEPP1 expression were decreased in CNF. The expression of all three proteins was reduced in MCD and FSGS; the decrease in expression being more marked in FSGS. Synaptopodin, GLEPP1, and nephrin expression was present, although reduced in Galloway-Mowat syndrome. We conclude that the reduced expression of synaptopodin, GLEPP1, and nephrin in Galloway- Mowat syndrome is a secondary phenomenon related to the proteinuria, and hence synaptopodin, GLEPP1, and nephrin are probably not the proteins mutated in Galloway-Mowat syndrome. Received: 27 April 2001 / Revised: 15 June 2001 / Accepted: 18 June 2001     

Therapy with atorvastatin versus rosuvastatin reduces urinary podocytes,podocyte-associated molecules,and proximal tubule dysfunction biomarkers in patients with type 2 diabetes mellitus: a pilot study

Background: Diabetic nephropathy is a severe complication of Type 2 diabetes. Tubular lesions may play an important role in its early stages. The aim of our study was to determine if atorvastatin protects the podocytes and the proximal tubule in patients with Type 2 diabetes.

Methods: A total of 63 patients with Type 2 diabetes completed this 6-months prospective pilot study. They were randomized to continue rosuvastatin therapy (control group) or to be administered an equipotent dose of atorvastatin (intervention group), and were assessed regarding urinary podocytes, podocyte-associated molecules, and biomarkers of proximal tubule dysfunction.

Results: The patients from the intervention group presented a significant reduction in podocyturia (from 7.0 to 4.0 cells/ml, p?p?p?1-microglobulin (from 10.0 to 8.3?mg/g, p?p?p?Conclusions: In patients with Type 2 diabetes, atorvastatin exerts favorable effects on the kidney. There is a correlation between the evolution of the podocytes and of the proximal tubule biomarkers, supporting the hypothesis that the glomerular changes parallel proximal tubule dysfunction in the early stages of diabetic nephropathy.     

Reduction of VEGF-A and CTGF expression in diabetic nephropathy is associated with podocyte loss

Micro-vascular and renal complications in diabetic patients are a considerable clinical challenge. In a previous study, we found a significant decrease in vascular endothelial growth factor A (VEGF-A) mRNA levels in glomeruli from patients with diabetic nephropathy (DN). We now set out to investigate the relationship between reduced VEGF-A and connective tissue growth factor (CTGF) expression levels, the number of podocytes, and the extent of interstitial fibrosis. Laser capture microdissection was applied to obtain glomerular RNA from 28 patients with DN and 22 controls. mRNA levels of VEGF-A, CTGF, nephrin, podocin, and Wilms tumor1 (WT1) were measured using real-time polymerase chain reaction. Protein expression was evaluated using immuno-stainings for VEGF-A and CTGF, as well as markers for podocytes (WT1) and endothelial cells (CD31). We found a significant decrease in glomerular mRNA levels for VEGF-A (2.5 times), CTGF (1.6), nephrin (2.8), podocin (3.3), and WT1 (1.7) in patients with DN. There was a significant correlation between expression of podocyte markers and VEGF-A mRNA levels, and an inverse correlation between podocin message and the extent of interstitial fibrosis. CD31-positive area was significantly decreased (3.2 times) in patients with DN. Reduction of angiogenic factors correlated with the extent of interstitial fibrosis. This downregulation was related to a reduction of podocytes in DN. The results may suggest that downregulation of VEGF-A and CTGF in DN is a result of podocyte loss.     

Effects of low protein diet supplemented with ketoacids on local renin - angiotensin system enzyme activities and podocytes loss in patients with diabetic nephropathy

Objective To explore the effects of low-protein diet supplemented with ketoacids on podocytes as well as local RAS in the kidney of patients with diabetic nephropathy. Methods A tal of 61 patients with T2DN and CKD stages 3-4 were included. All the patients were randomly divided into two groups: low protein group (0.6 g•kg BW-1•d-1 and 30 kcal•kg BW-1•d-1, LPD) and LPD +Ketoacids (KA) group (0.6 g•kg BW-1•d-1, 30 kcal kg BW-1•d-1, and Ketoacids 100 mg•kg BW-1• d-1). Blood and 24 h urine samples were collected at baseline and every 3 months for routine examination to evaluate the efficacy of LPD + KA diet. Podocytes loss was evaluated by mRNA expression of nephrin, podocin, and synaptopodin in urine at baseline and every 3 months. Urinary angiotensinogen was detected by ELISA at baseline and every 3 months. Results After 12 months of follow-up, there were no significant difference statistically in declines of GFR between LPD and LPD+ KA group (P＞0.05). Compared with LPD group, proteinuria in LPD+KA group was decreased [(0.43± 0.35) vs (0.15±0.36) g/24 h, P＜0.01]. Patients in two groups were both in good nutritional condition, and KA independently increased the levels of serum albumin and prealbumin (all P＜0.05). The urinary angiotensinogen/creatinine ratio was correlated with GFR (r＝-0.437, P＝0.001), serum creatinine (r＝-0.733, P＝0.000) and proteinuria (r＝-0.851, P＝0.000); while urinary mRNA expression of podocyte markers (podocin and synaptopodin were correlated with proteinuria (r＝0.340, P＝0.012; r＝0.333, P＝0.014; respectively), but had no correlation with GFR and serum creatinine. After 12 months of follow-up, the urinary angiotensinogen/creatinine ratio in LPD+KA group was lower than in LPD group (P＜0.05). The mRNA expression of podocin and synaptopodin were lower in LPD+ KA group compared with LPD group in urinary sediment (P＜0.05). Further correlation analysis suggested that the change of urinary expression of podocin and synaptopodin had a modest but significant correlation with the change urinary angiotensinogen creatinine ratio change (r＝0.305, P＝ 0.026; r＝0.281, P＝0.04, respectively) after treatment for one year. Conclusions Low protein diet supplemented with Ketoacids (LPD + KA) is associated with amelioration of proteinuria, meanwhile nutrition status remains well. The mechanism of these effects may be explained by the role of LPD+KA diet in reducing urine podocyte loss and lowering the angiotensinogen level in the urine.     

Cloning of rat homologue of podocin: expression in proteinuric states and in developing glomeruli

Podocin is identified as a product of the gene mutated in a patient with autosomal recessive steroid-resistant nephrotic syndrome. Although podocin is reported to be located at the slit diaphragm area, the precise role of podocin for maintaining the barrier function of the slit diaphragm has not been clearly elucidated. A rat homologue of podocin was cloned, and the expression of podocin was investigated and then compared with the nephrin and the ZO-1 expressions in rat experimental proteinuric models and in developing glomeruli. Amino acid sequences of rat and human podocin are highly homologous (84.3% identity). The domain structure of podocin is also highly conserved between rat and human. The mRNA expression for podocin was detected in glomeruli and the nerve tissues. The localization of podocin has close proximity to that of nephrin in normal adult rat glomeruli. Podocin staining was restricted to the basal side of the podocyte of the early developing stage, whereas nephrin staining was detected on the basolateral surface of podocyte. The redistribution of podocin was observed in the anti-nephrin antibody (ANA)-induced nephropathy and puromycin aminonucleoside (PAN) nephropathy. The redistribution of podocin paralleled with nephrin in ANA nephropathy but not in PAN nephropathy. Podocin is observed at the site of tight junction newly formed in proteinuric state in PAN nephropathy. It is postulated that podocin is one of the critical components of a slit diaphragm for maintaining the barrier function of the glomerular capillary wall.     

三种不同药物作用下足细胞分子的变化

目的 从足细胞分子的角度探讨抗蛋白尿药物作用的细胞分子机制。方法 建立阿霉素肾病大鼠模型。注射阿霉素后次日分别给予利生普利、泼尼松以及全反式维甲酸(ATRA)干预蛋白尿。注射阿霉素后第3、7、14、28 天每组处死6 只大鼠，留取肾脏标本。应用间接免疫荧光染色、实时PCR、Western印迹分别检测各个时间点nephrin、podocin、CD2相关蛋白(AP)、α辅肌动蛋白(actinin)-4 的分布、mRNA 和蛋白表达量的变化。应用免疫沉淀检测nephrin 与podocin、nephrin与CD2AP 分子间作用以及nephrin 磷酸化水平。结果 与对照组相比，第14天时肾病组尿蛋白显著增加(P < 0.01)。与肾病组相比，利生普利、泼尼松和ATRA干预后均显著降低了蛋白尿( P < 0.05)， 减轻了足突融合。通过分析不同时间点足细胞分子的表达，显示3种干预药物均引起了nephrin、podocin、CD2AP 表达的变化，维持了正常的nephrin 磷酸化水平，而且利生普利和泼尼松首先抑制了podocin 分子，而ATRA首先抑制了CD2AP 分子的异常变化。与此同时，nephrin、podocin、CD2AP 和α-actinin-4 分子的分布在干预后也趋于正常。此外，无论在肾病组还是干预组大鼠，nephrin 与podocin、nephrin 与CD2AP 分子间一直保持着共沉淀关系。 结论 利生普利、泼尼松和ATRA 都通过稳定重要的足细胞分子nephrin、podocin、CD2AP 来发挥它们的抗蛋白尿作用。     

Triptolide protects podocytes from puromycin aminonucleoside induced injury in vivo and in vitro

Extracts of Tripterygium wilfordii Hook F have been used to treat glomerulonephritis for more than 30 years in China with dramatic antiproteinuric effects. Triptolide, a diterpene triepoxide, is one of the major active components of these extracts. To clarify its antiproteinuric effects we induced podocyte injury by puromycin aminonucleoside. Triptolide effectively reduced the proteinuria induced by puromycin in nephrotic rats without reducing the glomerular filtration rate. The antiproteinuric effect was associated with improvement in the foot process effacement, a decrease in the podocyte injury marker desmin as well as the restoration of nephrin and podocin expression and distribution. In cultured mouse podocytes triptolide pretreatment prevented the puromycin-induced disruption of the actin cytoskeleton and microfilament-associated synaptopodin while protecting nephrin and podocin expression. Triptolide suppressed reactive oxygen species generation and p38 mitogen-activated protein kinase activation while restoring RhoA signaling activity. These results show that triptolide ameliorates puromycin aminonucleoside-mediated podocyte injury in vivo and in vitro.     

Essential role of integrin-linked kinase in podocyte biology: Bridging the integrin and slit diaphragm signaling

Integrin-linked kinase (ILK) has been implicated in the pathogenesis of proteinuria and congenital nephrotic syndrome. However, the function of ILK in glomerular podocyte in a physiologic setting remains unknown. In this study, a mouse model was generated in which ILK gene was selectively disrupted in podocytes by using the Cre-LoxP system. Podocyte-specific ablation of ILK resulted in heavy albuminuria, glomerulosclerosis, and kidney failure, which led to animal death beginning at 10 wk of age. Podocyte detachment and apoptosis were not observed at 4 wk of age, when albuminuria became prominent, indicating that they are not the initial cause of proteinuria. Electron microscopy revealed an early foot process effacement, as well as morphologic abnormality, in ILK-deficient podocytes. ILK deficiency caused an aberrant distribution of nephrin and alpha-actinin-4 in podocytes, whereas the localization of podocin and synaptopodin remained relatively intact. Co-immunoprecipitation demonstrated that ILK physically interacted with nephrin to form a ternary complex, and alpha-actinin-4 participated in ILK/nephrin complex formation. Therefore, ILK plays an essential role in specifying nephrin and alpha-actinin-4 distribution and in maintaining the slit diaphragm integrity and podocyte architecture. These results also illustrate that the integrin and slit diaphragm signals in podocytes are intrinsically coupled through an ILK-dependent mechanism.     

The effect of acteoside on puromycin nephropathy and podocyte injury model

Objective To explore the effect of acteoside (one of the ingredients of Total Glycosides Extracted from Rehmannia capsules) on treatment of proteinuria and protection of podocytes. Methods In this study, puromycin nephropathy rat model was successfully established. After detecting the degree of proteinuria, the expression of podocyte injury markers and the degree of podocyte foot process fusion were investigated by electron microscope. In addition, puromycin treated podocyte injury model was also successfully established in vitro. Podocyte viability, migration, cytoskeleton and injury marker were detected. Results In vivo study showed that acteoside could effectively reduce proteinuria (P＜0.05), restore the expression of podocyte injury markers such as nephrin and synaptopodin (all P＜0.05), and alleviate the degree of podocyte foot process fusion. In vitro study showed that acteoside could effectively restored podocyte viability (P＜0.05), reduce abnormal migration ability (P＜0.05), protecte cytoskeleton and restore the expression of podocyte injury marker nephrin (P＜0.05). Conclusions This study confirms that acteoside can reduce the degree of proteinuria in puromycin nephropathy rat model in vivo and alleviate the degree of podocyte injury in vitro as well as enrich the molecular mechanism of Total Glycosides Extracted from Rehmannia capsules in treatment of proteinuria.     

Vitamin D/vitamin D receptor/Atg16L1 axis maintains podocyte autophagy and survival in diabetic kidney disease

ObjectiveTo investigate the effect of vitamin D/vitamin D receptor (VDR)/Atg16L1 signaling on podocyte autophagy and survival in diabetic nephropathy.MethodsDiabetic rat models were induced by intraperitoneal injection of streptozotocin (STZ) (60 mg/kg) and treated with and without gavage of 0.1 μg/kg/d active vitamin D3 (aVitD3; 1,25- OH vitamin D3) and kidney tissues assessed by histopathology and immunohistochemistry. The murine podocyte cell line MPC-5 was cultured under hyperglycemic conditions in the absence or presence of 100 nmol/L calcitriol to investigate podocyte injury and autophagy. Cell survival rates were analyzed using Cell Counting Kit-8 (CCK-8) assays and the numbers of autophagosomes were determined after transduction with the mRFP-GFP-LC3 autophagy reporter construct. The expression of autophagy-related proteins (LC3-II, beclin-1, Atg16L1) and podocyte-related proteins (nephrin, podocin, synaptopodin, and desmin) was determined by Western blotting.ResultsVDR expression and autophagy were decreased in diabetic nephropathy. Calcitriol treatment repressed renal injury in rat diabetic kidneys and reduced high glucose-induced damage to cultured podocytes. Mechanistically, Atg16L1 was identified as a functional target of VDR, and siRNA-mediated knockdown of VDR and Atg16L1 blocked the protective effects of aVitD3 against podocyte damage.ConclusionAutophagy protects podocytes from damage in DN and is modulated by VitD3/VDR signaling and downstream regulation of Atg16L1 expression.