Perilipin2 inhibits diabetic nephropathy-induced podocyte apoptosis by activating the PPARγ signaling pathway

Podocyte apoptosis plays a pivotal role in the pathogenesis of diabetic nephropathy (DN). The main purpose of this study was to investigate the effects of perilipin2 on high glucose (HG)-induced podocyte apoptosis and associated mechanisms. Differentially expressed genes (DEGs) in BTBR ob/ob mice vs. nondiabetic mice kidneys were obtained from GSE106841 dataset and picked out using the ‘limma’ package. The protein-protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes (STRING) and was visualized by Cytoscape. Perilipin2 was a hub gene using the cytoHubba plug-in from Cytoscape. Gene ontology (GO) analysis revealed that the 126 overlapping DEGs were mainly enriched in ‘oxidation reduction’ [biological process, (BP)], metal ion binding’ [molecular function, (MF)] and ‘extracellular region’ [cellular component, (CC)]. KEGG pathway analysis revealed that perilipin2 was mainly involved in ‘PPAR signaling pathway’. DN inhibited perilipin2 expression and PPARγ expression, as by both in vitro and in vivo studies. In vitro experiments demonstrated that perilipin2 inhibition could not only reduced PPARγ expression in podocytes, it could also promote the apoptosis, and inhibit the viability in HG treated podocytes using western blot, CCK8 and flow cytometry assays. Perilipin2 overexpression reversed the effects of HG on inhibiting podocalyxin, nephrin, precursor (pro)-caspase-3/-9 and PPARγ protein expression and increasing cleaved caspase-3/-9 protein expression. Furthermore, the functions of perilipin2 overexpression reversing HG-induced podocyte apoptosis were inhibited by PPARγ inhibitor. In conclusion, the functions of DN-induced podocyte apoptosis were inhibited by activation of the PPARγ signaling pathway caused by perilipin2 overexpression.     

缬沙坦联合辛伐他汀对早期糖尿病肾病大鼠肾脏的保护作用

目的 探讨降脂药辛伐他汀及血管紧张素Ⅱ受体拮抗剂类药物缬沙坦对早期糖尿病肾病大鼠肾脏的保护作用.方法 用链脲佐菌素(STZ)诱导糖尿病肾病大鼠模型,将SD大鼠随机分成5组,每组10只:正常对照组(C组)、糖尿病肾病组(D组)、缬沙坦组(X组)、辛伐他汀组(Z组)和缬沙坦及辛伐他汀联合组(L组).5周末检测5组大鼠血糖(BG)、糖化血红蛋白(HbA1c)、总胆固醇(TC)、甘油三酯(TG)、血尿素氮(BUN)、肌酐(SCr)、肌酐清除率(Ccr)、尿白蛋白排泄率(UAER)等指标的变化;利用透射电子显微镜观察肾脏足细胞超微病理结构.结果 D组与C组比较,BG[分别为(20.3±3.2)、(6.1±0.4)mmol/L]、HbA1c[分别为(7.18±0.47)%、(3.37±0.15)%]、TC[分别为(2.69±0.35)、(1.28±0.24)mmol/L]、TG[分别为(3.09±0.37)、(1.18±0.25)mmol/L]明显升高(P均＜0.05);D组Ccr[(0.89±0.19)ml/min]较C组[(1.27±0.33)ml/min]和X、Z、L组显著下降(P＜0.05),D组大鼠UAER[(19.87±3.85)μg/24 h]明显高于C组[(3.67±1.01)μg/24 h](P＜0.05),X、Z、L组大鼠UAER显著低于D组(P＜0.05),而L组改善尤其显著(P＜0.05);D组的足细胞足突严重融合,X、Z、L组仅少量足突融合较D组改善,而L组改善尤其显著.结论 缬沙坦及辛伐他汀单用及联合应用,尤其是联合用药对早期糖尿病大鼠肾脏有保护作用.

Abstract：

Objective To explore the protection of valsartan combined with simvastatin on kidney in early diabetic nephropathy rats. Methods Diabetic nephropathy rats model were induced by streptozocin (STZ) ,the experimental rats were randomly divided into 5 groups: control (group C), diabetic nephropathy (group D) ,diabetes treated with valsartan (group X) ,diabetes treated with simvastatin (group Z) ,and diabetes treated with combined valsartan and simvastatin ( group L). Blood glucose (BG), HbA1c, blood cholesterol ( TC), trigalloylglycerol ( TG ), blood ureanitrogen ( BUN ), serum creatinine (SCr) , urinary albumin excretion rate (UAER) were measured, and the podocyte ultrastructure was observed by transmission electronic microscopy. Results The levels of BG, HbA1c,TC,TG and UAER in group D increased significantly compared togroup C(BG:[20.3 ±3.2]mmol/L vs [6.1 -±0. 4]mmol/L;HbA1c:[7.18 ±0.47]% vs [3.37 ±0. 15]% ;TC: [2. 69 ±0. 35] mmol/L vs [1.28 ±0. 24] mmol/L;TG: [3.09 ±0. 37] mmol/L vs [1.18 ±0. 25]mmol/L) (P ＜ 0. 05 ). Creatinine clearance rates (Ccr) in group D ( [0. 89 ± 0. 19] ml/min ) decreased significantly compared to group C( [1.27 ±0. 33] ml/min) ,as well as group X,Z and L( Ps ＜ 0. 05 ). UAER in group D was significantly higher than that in group C ( [19. 87 ±3. 85] μg/24 h vs [3. 67 ± 1.01] μg/24 h) (P ＜ 0. 05 ), as well as group X, Z and L ( P ＜ 0. 05 ), and the improvement in group L was particularly significant ( P ＜ 0. 05 ). The projections of podocyte in group D severely syncretized, there were slightly improvement in group X, Z and L compared to group D, and the improvement in group L was remarkable. Conclusion The treatment with valsartan, simvastatin and their combination will effectively protect the kidney in early diabetic nephropathy rats,and the effect of using the combination therapy is much better.     

氟伐他汀对高糖诱导人足细胞血管内皮生长因子表达的影响及机制

目的探讨氟伐他汀对高糖诱导的人足细胞血管内皮生长因子(VEGF)表达的影响及机制。方法体外培养人足细胞,随机分为A组(正常糖组)、B组(高糖组)、C组(高糖+氟伐他汀10-7mol/L组)、D组(高糖+氟伐他汀10-6mol/L组)、E组(高糖+氟伐他汀10-5mol/L组)和F组[高糖+胞外信号调节激酶(ERK)抑制剂PD98059组]。用Western blot检测VEGF和磷酸化ERK1/2(pERK1/2)蛋白表达。结果 B组VEGF、pERK1/2表达较A组明显升高,并呈时间依赖性(P<0.05)。F组VEGF、pERK1/2表达较B组显著降低(P<0.01)。与B组比较,C、D、E组VEGF及pERK1/2表达亦明显减少,并呈浓度依赖性(P<0.05)。结论高糖刺激体外培养人足细胞VEGF表达增加,氟伐他汀可减少高糖诱导的VEGF表达,其机制可能与氟伐他汀抑制ERK信号通路有关。     

TRPC6与肾脏疾病关系的研究进展

瞬时受体电位阳离子通道蛋白6（TRPC6）是由TRPC6基因编码的瞬时受体超家族成员之一,在人体内各组织或器官广泛表达,在肾小球足细胞也有表达。TRPC6与podocin、nephrin、ACTN4及CD2AP等多种裂孔隔膜（SD）蛋白相互作用共同维系肾小球足细胞的结构及功能。多因素作用导致肾小球足细胞损伤引起SD的足突融合是肾脏疾病最主要的形态学改变。该文就TRPC6生物学功能及其对肾脏疾病的影响作一简述。     

FK506对早期糖尿病肾病大鼠足细胞损伤的保护作用

目的探讨FK506对早期糖尿病肾病大鼠肾小球足细胞损伤的保护作用。方法将38只正常雄性SD大鼠随机分为正常对照组(N组)8只、糖尿病肾病组(DN组)10只、FK506治疗组(F组)10只、洛汀新治疗组(L组)10只,其中DN组、F组、L组应用链脲佐菌素(STZ)60 mg/kg腹腔注射建立糖尿病大鼠模型。F组成模4周后给予FK506 1 mg/(kg.d)灌胃,L组成模4周后给予洛汀新10 mg/(kg.d)灌胃,DN组与N组4周后给予等量蒸馏水灌胃,每4周末监测各组大鼠血糖(BS)、体质量(BW)、24 h尿蛋白定量,药物干预8周后测定各组大鼠的血肌酐(SCr)、尿素氮(BUN)、肾质量/体质量(KW/BW),在光镜、电镜下观察肾脏病理组织学改变,应用West-ern blot印迹检测足细胞特异标记物Nephrin的蛋白表达。结果①与N组比较,DN组大鼠BS、SCr、BUN、KW/BW、24 h尿蛋白定量均明显上升(P<0.05);与DN组比较,F组和L组上述指标除BS外均明显降低(P<0.05),且F组和L组之间差异无统计学意义(P>0.05);②光镜下,肾组织病理学观察N组无明显异常,DN组可见明显的肾小球体积增大,肾小球系膜细胞增生,系膜区增宽,基底膜增厚;F组、L组较DN组病理改变明显减轻(P<0.05),且F组和L组之间病理改变无显著性差异(P>0.05);③电镜下,DN组肾小球基底膜显著增宽,足突排列紊乱,足突增宽、融合,F组、L组较DN组上述病变有所减轻(P<0.05);④Western blot结果显示,DN组肾组织Nephrin蛋白表达较N组下降60.1%(P<0.05),F组和L组可明显恢复肾组织Nephrin蛋白的表达(P<0.05)。结论 FK506可能部分通过恢复糖尿病大鼠肾组织Nephrin蛋白表达、维护足细胞结构和功能的完整而发挥减少尿蛋白、延缓DN进展的作用。     

环氧合酶-2基因敲低对足细胞相关蛋白表达的影响

目的:观察足细胞环氧合酶-2(COX-2)基因敲低对足细胞相关蛋白表达的影响。方法:以条件永生性小鼠足细胞株为研究对象,分为足细胞正常对照组、足细胞COX-2基因敲低组和足细胞阴性转染组,用流式细胞仪检测足细胞凋亡水平,用Western bolt检测COX-2、Nephrin、Podocin、CD2相关蛋白(CD2AP)的表达水平。结果:与足细胞正常对照组及阴性转染组相比,足细胞COX-2基因敲低组COX-2蛋白表达明显降低、细胞凋亡率明显增高,而Nephrin、Podocin、CD2AP蛋白表达则显著降低。结论:足细胞COX-2基因敲低可以导致足细胞凋亡,且足细胞Nephrin、Podocin、CD2AP蛋白的表达下调。     

脂质对小鼠肾足细胞增生影响

目的观察脂质对体外培养小鼠肾足细胞增生的影响。方法用不同浓度的低密度脂蛋白（LDL）和氧化低密度脂蛋白（OX—LDL）对体外培养的小鼠肾足细胞进行不同时间的刺激干预,然后用四唑盐（MTT）法检测足细胞增生。结果LDL刺激足细胞24、48h后,从最低剂量6．25μg／ml组到25μg／ml组呈现剂量依赖性OD值增高（与正常组比较,P〈0．01）;24h25μg／ml组达到刺激足细胞增生的峰值、48h12．5μg／ml组达到刺激足细胞增生的峰值。OX—LDL刺激24h后,从12．5μg／ml组到100p,g／ml组呈现剂量依赖性OD值增高（与正常组比较,P〈0．01）。结论脂质LDL和OX—LDL在一定的剂量和时间条件下可以刺激足细胞增生。     

霉酚酸酯对微小病变肾病治疗作用研究

目的以阿霉素肾病大鼠建立微小病变肾病模型,以霉酚酸酯(MMF)进行干预,检测nephrin的表达,研究霉酚酸酯对微小病变肾病的治疗作用及其机制。方法SD大鼠18只,分为肾病模型组、MMF治疗组、对照组。模型组、治疗组大鼠尾静脉一次性注入阿霉素7.5mg/kg,治疗组大鼠于注药次日开始使用MMF20mg/(kg·d)灌胃。对照组大鼠尾静脉一次性注入等量生理盐水。每组于第28天各取大鼠6只,检测尿蛋白、血生化指标;并用免疫组化、Westernblot方法,检测肾组织内nephrin蛋白水平。结果肾病组尿蛋白第28天达高峰;血清清蛋白明显降低(P<0.01);治疗组第28天尿蛋白、血清甘油三酯、胆固醇较肾病组减少(P<0.01),血清清蛋白较肾病组增加(P<0.01)。免疫组化、Westernblot结果提示肾病组较对照组第28天nephrin蛋白表达升高(P<0.01);治疗组第28天nephrin蛋白表达较肾病组下调(P<0.01)。结论阿霉素诱发的微小病变肾病发生与nephrin表达异常有关,霉酚酸酯可以减少蛋白尿,延缓微小病变型大鼠肾损伤,其作用与影响nephrin的表达有关。