Podocyte foot process broadening in experimental diabetic nephropathy: amelioration with renin-angiotensin blockade

Aims/hypothesis. Changes in podocyte number and morphology have been implicated in the pathogenesis of proteinuria and the progression of human and experimental kidney disease. This study sought to examine podocyte foot process and slit pore architecture in experimental diabetic nephropathy and to determine whether such changes were modified with renoprotective intervention by blockade of the renin-angiotensin system. Methods. The number of filtration slits per 100 μm of glomerular basement membrane was assessed by transmission electron microscopy and quantitated histomorphometrically in control animals and in rats with 24 weeks of streptozotocin-induced diabetes. Diabetic rats were either untreated or received the angiotensin converting enzyme inhibitor ramipril, or the angiotensin II type 1 receptor antagonist, valsartan. Results. When compared with control animals, diabetes was associated with a decrease in the number of slit pores per unit length of glomerular basement membrane, indicative of podocyte foot process broadening. Both ramipril and valsartan attenuated these ultrastructural changes to a similar degree. These differences remained after correcting for glomerular volume as a possible confounding variable. Conclusion/interpretation. Preservation of podocyte architecture could contribute to the renoprotective effects of renin-angiotensin system blockade in diabetic nephropathy. [Diabetologia (2001) 44: 878–882] Received: 19 January 2001 and in revised form: 28 March 2001     

通过基因敲低探讨多个足细胞分子作用及分子间反应

目的 研究足细胞裂孔隔膜(SD)复合体分子nephrin､podocin和CD2AP,以及足细胞骨架蛋白α-辅肌动蛋白(actinin)-4的作用及分子间反应｡方法 针对nephrin､ podocin､CD2AP和α-actinin-4 mRNA序列分别设计并构建2个特异RNA干扰质粒-psiRNA-hH1GFPzeo,分别导入小鼠足细胞系MPC5以 “敲低”其表达｡免疫荧光染色观察其分布方式｡半定量RT-PCR和免疫蛋白印迹检测其mRNA和蛋白表达｡ 结果 (1) podocin敲低组(siPod966和siPod54):未检测到podocin及nephrin mRNA,其蛋白分别下降了92%､79%及82%､67%｡而CD2AP mRNA和蛋白分别增加了62%､42%及71%､46%｡α-actinin-4无变化｡(2)nephrin敲低组(siNep492):未检测到nephrin mRNA和蛋白｡而CD2AP mRNA和蛋白分别增加了35%､48%｡Podocin和α-actinin-4无变化｡(3)CD2AP敲低组(siCda744和siCda21):未检测到CD2AP mRNA,其蛋白分别下降了92%和83%｡Nephrin mRNA和蛋白分别下降了60%､48%及76%､72%;而podocin mRNA和蛋白分别增加了38%､22%及56%､44%｡Α-actinin-4无变化｡(4)α-actinin-4敲低组(siAct1790和siAct319):α-actinin-4和nephrin 的mRNA分别下降了69%､58%及64%､49%;蛋白分别下降了81%和55%以及71%､64%｡而podocin以及CD2AP mRNA分别增加了50%､34%及45%､28%;蛋白分别增加了64%､46%及65%､42%｡(5)敲低nephrin､podocin和CD2AP后,这些表达量降低的分子的分布发生了明显改变,即以核周为主;而相应分子敲低后引起的podocin和CD2AP表达增加,其分布亦主要以核周染色增强为主｡α-actinin-4即使表达降低,分布亦无变化,仍呈细丝状分布于胞质及足细胞伸出的突起中｡结论 (1)在SD复合体分子中,nephrin可能具有相对独立的作用｡(2) α-actinin-4对nephrin､podocin和CD2AP有直接或间接的作用｡(3)足细胞分子间的作用和联系不总是“一致的”,可能是“单向的”､也可能是“双向的”｡(4)nephrin､podocin､CD2AP和α-actinin-4在足细胞的分布有赖于其表达量的正常及正常的分子间反应｡     

Role of autophagy in high glucose-induced podocyte lipid droplet accumulation

Objective To investigate the role of autophagy in high glucose-induced podocyte lipid droplet metabolism. Methods (1) Cultured, conditionally immortalized human podocytes (HPC) were divided into normal control group, high glucose group and mannitol group. Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation; Protein level of SREBP-1 was analyzed by Western blotting. (2) HPC were cultured and divided into normal control group, high glucose group, high glucose+3-methyladenine (3-MA) group, and mannitol group. Acridine orange staining was used to observe the formation of autophagosomes. Western blotting was used to detect the protein levels of beclin-1 and LC3-II/LC3-I. Oil red O staining and oil red O staining extraction assay was used to observe the degree of lipid accumulation; Western blotting was used to analyze the expression of SREBP-1. Results (1) Compared with the normal control group, the lipid accumulation in the high glucose group was increased and the lipid metabolism related molecule SREBP-1 was up-regulated (P＜0.05); There was no significant difference between the normal control group and the mannitol group in lipid accumulation (P＞0.05). (2) Compared with the normal group, the number of autophagosomes was increased and autophagy-related proteins beclin-1 and LC3-II/LC3-I were up-regulated in high glucose group (all P＜0.05). After intervened with 3-methyladenine, a significant decrease in autophagosomes was observed; Protein levels of autophagy-related proteins beclin-1 and LC3-II/LC3-I were decreased (all P＜0.05); The lipid droplets in the high glucose+3-MA group was decreased and lipid metabolism related molecule SREBP-1 was down-regulated (all P＜0.05). Conclusion Autophagy may be involved in the process of high-glucose-induced podocyte lipid accumulation by affecting SREBP-1 expression, and inhibition of autophagy can alleviate the high-glucose-induced podocyte lipid accumulation.     

Roles of AKAP1 in high-glucose induced mitochondrial fission in podocytes

Objective To investigate the roles of A kinase anchoring protein1(AKAP1)in high-glucose induced mitochondrial fission in podocytes. Methods Conditionally immortalized human podocytes were cultured in serum-free medium for 24 hours, and then exposed to different glucose concentration conditions in different time periods. The protein expressions of AKAP1 were observed by immunofluorescence, and AKAP1, dynamin related protein1 (Drp1) and phospho Ser 637-Drp1 (p-Drp1) were analyzed by Western blotting. AKAP1 siRNA was transfected to block AKAP1 expression.Podocytes were then divided into normal control group (5 mmol/L glucose), hypertonic group (30 mmol/L mannitol+5 mmol/L glucose), high glucose group (35 mmol/L glucose), and high glucose+AKAP1 siRNA group. Mitochondrial morphological changes were assessed by mitotracker red staining. Podocyte apoptosis was assessed by flow cytometry. Results Compared with normal group, high-glucose induced more podocytes apoptosis (P＜0.05), more mitochondrial fission with decreased aspect ratio and form factor (all P＜0.05). Upregulated AKAP1 protein level, and increased ratio of p-Drp1/Drp1 (all P＜0.05) in time and concentration dependent manners were also observed. Compared with high glucose group, transfection of AKAP1 siRNA showed less apoptosis (P＜0.05), less mitochondrial fission with increased aspect ratio and form factor (all P＜0.05), and down-regulated AKAP1 protein level as well as p-Drp1/Drp1 ratio (all P＜0.05). Conclusion High glucose induced mitochondrial fission might be induced through AKAP1-Drp1 pathway.     

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。     

全反式维甲酸对高糖培养的人肾小球足细胞synaptopodin蛋白表达的影响

目的 探讨高糖培养的人肾小球足细胞synaptopodin蛋白表达的变化以及全反式维甲酸对其表达的干预效应.方法 以体外培养的条件性永生人肾小球足细胞为研究对象,平均分为正常糖(NG)组、高糖(HG)组、HG+5tmol/L全反式维甲酸干预组、HG+ 15 μmol/L全反式维甲酸干预组.蛋白质印迹法检测HG组足细胞培养0、3、6、9d时和其他各组足细胞培养9 dsynaptopodin蛋白的表达.结果 HG组足细胞培养3、6、9d时synaptopodin蛋白表达灰度值与培养0d时比较明显降低[(1.15±0.04)％、(0.78±0.04)％、(0.41±0.05)％比(1.49±0.07)％,P＜0.05].培养9d后,与NG组比较,HG组、HG+5μmol/L维甲酸干预组、HG+ 15 μmol/L维甲酸干预组足细胞synaptopodin蛋白表达灰度值均明显下降[(0.41±0.05)％、(0.86±0.04)％、(0.90±0.07)％比(1.47 ±0.08)％,均P＜0.05].结论 高糖可以使体外培养的足细胞synaptopodin蛋白表达下降,全反式维甲酸具有潜在的保护足细胞和防治糖尿病肾病的作用.     

α-actinin-4在糖尿病肾病大鼠的表达及其与足细胞数目的相关性

目的：探讨糖尿病肾病发展过程中α-actinin-4的表达变化及其与足细胞数目的关系。方法：建立STZ诱导的糖尿病大鼠模型,应用免疫组化及RT-PCR方法观察在糖尿病肾病发展过程中α-actinin-4的蛋白及mRNA表达变化,以及应用WT-1来标记足细胞核,观察足细胞数目的变化。结果：8周时,糖尿病组染色强度较正常组明显增高（P〈0．01）,12周时变化更为明显（P〈0．01）。α-actinin-4的mRNA的表达水平在各时间点与组化结果相同。8周时,糖尿病组足细胞数目较正常组明显降低（P〈0．05）,12周时变化更为明显（P〈0．01）。α-actinin-4与足细胞数目呈负相关关系（r=-0．957,P〈0．01）,足细胞数目与尿蛋白呈负相关（r=-0．95,P〈0．01）。结论：α-actinin-4在糖尿病肾病发展过程中表达增高,并与足细胞数目呈负相关α-actinin-4的表达增高与尿蛋白的发生密切相关。     

A case of glomerulopathy showing podocytic infolding in association with Sjögren’s syndrome and primary biliary cirrhosis

A 49-year-old man was admitted to our hospital with mild proteinuria. Prior to admission, he had been diagnosed as having Sjögren’s syndrome in association with primary biliary cirrhosis. Examination of a renal biopsy under light microscopy revealed diffuse and global mesangial cell proliferation and a spike and/or bubbling formation of the glomerular basement membrane (GBM), resembling membranoproliferative glomerulonephritis. In contrast, immunofluorescent studies showed marked immunoglobulin and complement depositions in the mesangial areas; however, only faint granular IgG and IgA deposition was observed along the GBM. Interestingly, electron microscopy revealed that a microtubular structure, derived from podocytes, was present in the GBM. We present a case of glomerulopathy showing podocytic infolding in association with Sjögren’s syndrome and primary biliary cirrhosis.     

霉酚酸酯及缬沙坦对糖尿病大鼠足细胞的保护机制研究

目的探讨霉酚酸酯、缬沙坦及2者联合应用对糖尿病。肾病（DN）大鼠足细胞损伤的保护作用。方法雄性Wistar大鼠行右肾切除后，腹腔注射链脲佐菌素（STZ，65mg／kg）建立糖尿病模型。将实验动物随机分为右。肾切除对照组（NC）、糖尿病组（DM）、霉酚酸酯治疗组（M）、缬沙坦治疗组（V）、缬沙坦和霉酚酸酯联合治疗组（V＋M）。治疗组分别给予霉酚酸酯15mg·kg^-1·d^-1，缬沙坦40mg·kg^-1·d^-1；联合治疗组为上述两组之和。检测各组8周末的左肾质量／体质量比值、尿蛋白量（24h）、血糖（Glu）、Scr。光镜及电镜观察肾组织形态学变化。免疫组化检测肾组织中nephrin、结蛋白（desmin）及单核细胞趋化因子1（MCP-1）蛋白表达。实时PCR测定肾组织中nephrin及MCP-1mRNA表达。结果与NC组相比，DM组大鼠血糖、尿蛋白量及左肾质量／体质量比值均显著上升（P〈0．01）；肾小球硬化指数（GSI）及肾间质损害加重（P〈0．01）；肾组织内MCP-1、desmin蛋白表达均显著上调（P〈0．01）。与DM组比较，M组、V组及V＋M组上述指标除Glu、Scr外，均明显改善（P〈0．05或P〈0．01）。与NC组（100％）相比，DM组nephrinmRNA表达下调（78％，P〈0.05）；各治疗组nephrinmRNA表达增加，以M组增加最明显（134％，P〈0．01）。与NC组（100％）相比，DM组MCP-1mRNA表达明显上调（251％，P〈0.05）；各治疗组明显降低，以M组最显著（126％，P〈0．01）。nephrinmRNA与MCP-1mRNA表达呈负相关（r=-0，86。P〈0．01）。尿蛋白量（24h）与MCP-1mRNA呈正相关fr=0．82，P〈0．01）；与nephrinmRNA呈负相关（r=-0．78，P〈0．01）。结论霉酚酸酯及缬沙坦均能下调糖尿病大鼠肾组织中desmin及MCP-1基因及蛋白的表达，上调nephrin基因及蛋白表达，降低尿蛋白量，预防肾损伤。联合治疗不优于单一治疗。霉酚酸酯可能通过抗炎性反应减轻足细胞损伤，减少蛋白尿，对早期DN大鼠具有明显的肾保护作用。     

糖肾宁对糖尿病肾病大鼠肾组织nephrin、desmin表达的影响

目的探讨糖肾宁对链脲佐菌素诱导的糖尿病大鼠肾小球足细胞损伤的影响。方法8周龄SPF级雄性SD大鼠46只,随机选择10只为对照组,其余大鼠予大剂量STZ腹腔注射建立糖尿病模型。其中34只大鼠血糖≥16.7 mmol/L视为糖尿病模型建立成功,随机分为模型组、缬沙坦组、糖肾宁组,予糖肾宁及缬沙坦干预12周,测定空腹血糖、血清尿素氮、血清肌酐、24小时尿蛋白,足细胞裂孔隔膜蛋白nephrin、骨架蛋白desmin的蛋白或基因表达。结果与对照组比较,模型组大鼠空腹血糖、血清尿素氮、血清肌酐、24小时尿蛋白均明显升高(P0.05),desmin蛋白、基因表达上调(P0.05)、nephrin mRNA表达下调(P0.05);与模型组比较,糖肾宁、缬沙坦干预后,desmin蛋白、基因表达下调(P0.05)、nephrin mRNA表达上调(P0.05),血清尿素氮、血清肌酐、24小时尿蛋白显著降低(P0.05),但血糖无明显变化。结论糖肾宁干预链脲佐菌素诱导的糖尿病肾病大鼠可减少肾小球足细胞损伤,降低蛋白尿、保护肾功能。