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。     

不同降压药对醛固酮输注大鼠足细胞损伤的影响

目的 研究醛固酮（ALD）对肾小球足细胞的损伤作用并探讨醛固酮拮抗剂依普利酮（EPL）、氨氯地平（CCB）和替米沙坦（ARB）对ALD所致损伤的影响及机制。 方法 30只SD大鼠被随机分为对照组（CTL组）、ALD输注组以及ALD输注并用EPL、CCB或ARB治疗组。以1.5 μg/h输注ALD造模，用EPL（100 mg·kg-1·d-1）、CCB（10 mg·kg-1·d-1） 和ARB（3 mg·kg-1·d-1）分别灌胃治疗。检测28 d内血压及尿白蛋白排泄率（UAER）。于28 d处死动物，检测血浆ALD、AngⅡ、血钾、钠、Scr；观察肾脏光镜和电镜病理改变；TUNEL法检测细胞凋亡；免疫组化和RT-PCR法检测nephrin表达。 结果 （1）与CTL组相比，ALD组大鼠7 d时血压开始升高，28 d达高峰（P < 0.01）；EPL组血压和UAER均显著低于同时间点ALD组（P < 0.01）；CCB组UAER显著高于同时间点EPL组，但与ALD组差异无统计学意义。（2）ARB组血浆AngⅡ水平显著高于ALD组、CCB组和EPL组（P < 0.01）。（3）ALD组肾小球出现系膜细胞增殖、系膜外基质增多、足突融合和微绒毛化等病理改变。EPL组肾小球损伤评分和凋亡率显著低于ALD组、CCB组和ARB组（分别P < 0.05，P < 0.01）。（4）ALD组nephrin mRNA和蛋白表达均高于CTL组，EPL组nephrin蛋白和mRNA表达低于ALD组（P < 0.01）。 结论 ALD输注可诱导肾小球足细胞损伤。CCB和ARB能显著降低血压，但不能减轻ALD所致损伤。EPL可通过非血流动力学作用部分阻断ALD的损伤效应。     

敲低CD2相关蛋白的表达对足细胞黏附和胞质伸展功能的影响

目的 观察敲低足细胞CD2相关蛋白（CD2AP）表达对细胞黏附和胞质伸展功能的影响，并探讨其机制。 方法 用RPMI 1640培养基33℃下培养小鼠未分化足细胞系，转染针对CD2AP的小分子干扰RNA（siRNA），设无特异靶位点的scrambing 序列即control siRNA转染组作对照。48 h后将转染的足细胞制备成单细胞悬液，接种于预铺有Ⅳ型胶原蛋白的96孔板内，33℃下培养90 min后检测足细胞的贴壁率和细胞的伸展面积；流式细胞仪检测抑制CD2AP后足细胞的凋亡率以及在不同氨基核苷嘌呤霉素（PAN）刺激下足细胞的凋亡率；激光共聚焦显微镜下检测F肌动蛋白（F-actin）的分布变化；Western印迹和免疫共沉淀检测nephrin蛋白的表达及其磷酸化水平。 结果 转染CD2AP siRNA足细胞的黏附率为41.72%±6.07%，显著低于对照组64.46%±8.53%（P < 0.05）；细胞伸展的面积>200 μm2的比例（55.86％）亦显著低于对照组（73.61％）。转染CD2AP siRNA后48 h足细胞的凋亡率高于对照组[（5.73±0.61）％比（3.26±0.45）％，P < 0.05]。100 mg/L的PAN能明显诱导足细胞的凋亡，减少足细胞的黏附率（P < 0.05）。敲低CD2AP的表达后足细胞F-actin的分布发生明显的变化，nephrin蛋白表达和磷酸化水平下降（P < 0.05）。 结论 敲低CD2AP的表达使足细胞易于凋亡，影响细胞的黏附功能。足细胞骨架蛋白的紊乱和nephrin信号通路的抑制可能是足细胞黏附和伸展功能下降的机制。     

血管紧张素Ⅱ灌注诱导nephrin表达改变与足细胞凋亡

目的 研究血管紧张素Ⅱ（AngⅡ）灌注对大鼠足细胞裂隙膜分子nephrin表达及足细胞凋亡的影响，以及探讨AngⅡ引起蛋白尿及肾小球硬化的机制。方法 36只雄性Sprague Dawley大鼠分为AngⅡ灌注组(400 ng&#8226;kg-1&#8226;min-1)、生理盐水灌注组和正常对照组，测定28 d内大鼠血压及尿蛋白。分别于14、28 d处死动物取肾，观察组织学改变，并用免疫荧光、免疫电镜检测nephrin分布。RT-PCR及Western印迹法分别检测nephrin mRNA及蛋白表达。TUNEL法检测足细胞凋亡。结果 (1) AngⅡ灌注组大鼠血压升高，14 d达峰值并维持该水平至28 d；AngⅡ灌注7 d即出现蛋白尿，并持续增加。(2) AngⅡ灌注14 d时，足细胞裂隙膜变窄；灌注28 d时，足突增宽及节段性融合，部分足细胞有凋亡小体形成，少数肾小球出现节段性硬化。TUNEL法检测发现足细胞凋亡[（2.7±1.6）个/肾小球切面]，凋亡数与蛋白尿量呈正相关（r = 0.86，P < 0.01）。(3) AngⅡ灌注14 d时，肾皮质nephrin mRNA及蛋白表达上调（P < 0.05）。nephrin由正常的沿毛细血管袢线状分布向粗颗粒、团块状分布模式转变。AngⅡ灌注28 d时，肾皮质nephrin mRNA及蛋白表达下降（P < 0.05），且nephrin蛋白表达与足细胞凋亡数呈负相关（r = －0.63，P < 0.01）。 结论 AngⅡ灌注诱导的nephrin表达及分布改变可能导致了足细胞凋亡及肾小球硬化的发生与发展。     

活性维生素D改变FSGS大鼠肾小球整合素连接激酶的表达

目的观察整合素连接激酶（ILK）在局灶节段性肾小球硬化（FSGS）大鼠肾小球的表达以及活性维生素D[1，25-（OH2D3]对其表达的影响。方法SD大鼠随机分为对照组、模型组和治疗组，每组10只。采用左肾摘除、阿霉素重复注射诱导FSGS大鼠模型。治疗组皮下埋置渗透性微量泵，给予1，25-（OH）：D30．03ng·g^-1·d^-1，连用8周。检测3组大鼠尿蛋白、尿足细胞。血清白蛋白（SA）及半胱氨酸蛋白酶抑制剂（CysC），测定。肾小球硬化指数（GSI），电镜检测每视野足细胞数、足突平均宽度，间接免疫荧光检测肾小球ILK蛋白的表达，WT-1免疫组化染色观察每个肾小球足细胞数目。结果①与对照组相比较，模型组大鼠尿蛋白、尿足细胞、Cyst、GSI明显增加，SA、肾小球足细胞数目减少，足突宽度增加，肾小球ILK表达明显降低；②与模型组相比较，治疗组尿蛋白、尿足细胞排泄明显减少，GSI明显降低，肾小球足细胞数目增多，足突宽度减小，肾小球ILK表达明显增加。结论1，25-（OH2）D3可增加照；S大鼠肾小球ILK的表达，减少足细胞脱落，维持肾小球足细胞数量。     

大剂量螺内酯对自发性高血压大鼠肾脏纤维化的影响

目的 观察大剂量螺内酯对自发性高血压大鼠（SHR）肾脏纤维化的影响。 方法 8周龄的雄性SHR 24只随机分为低剂量和大剂量螺内酯干预组[分别为20和100 mg&#8226;kg-1&#8226;d-1螺内酯灌胃]和高血压对照组，同时设同源正常对照组京都大鼠（WKY）8只。干预8周，检测收缩压、尿蛋白、血白蛋白、钾、钠、Scr和肾组织及血浆醛固酮水平。肾组织切片分别行HE和Masson染色，以评价肾小球损伤及肾小球内胶原沉积情况。免疫组化SABC法检测肾组织TGF-β1和醛固酮受体蛋白表达。RT-PCR检测肾组织TGF-β1和醛固酮受体mRNA水平。 结果 与高血压组大鼠相比，低剂量螺内酯干预后，尿蛋白减少（P < 0.05），血白蛋白升高（P < 0.05），血浆和肾组织醛固酮水平降低，但差异无统计学意义；大剂量螺内酯干预后，血压没有显著改变，尿蛋白显著升高[（27.3±4.5）比（24.5±3.2） mg/d， P < 0.05]，血白蛋白显著减少[（20.2±4.2）比（22.7±3.5） g/L, P < 0.05]，血浆和肾组织醛固酮水平显著升高[肾组织（28.3±1.5）比（22.2±0.6） ng/g， P < 0.05]。与高血压组比较，低剂量螺内酯干预后，蛋白管型增多、管周炎性细胞浸润均减少（P < 0.05）；大剂量螺内酯干预后，蛋白管型、小管扩张加重，管周炎性细胞浸润明显增多（P < 0.05），肾小球内胶原形成亦明显增多（P < 0.05）。与高血压组大鼠比较，低剂量螺内酯干预后，肾组织醛固酮受体mRNA和蛋白表达均无显著改变，TGF-β1 mRNA和蛋白的表达显著减少（P < 0.05）；大剂量螺内酯干预后，肾组织醛固酮受体及TGF-β1 mRNA和蛋白的表达均显著升高(P < 0.05)。 结论 大剂量螺内酯可以加重高血压肾脏纤维化，可能是通过上调醛固酮及其受体表达实现的。     

巢蛋白在足突广泛融合的肾小球中表达下调及与蛋白尿程度的相关性

目的 观察中间丝蛋白类的巢蛋白（nestin）在足突广泛融合的肾小球中的表达及其与足突病变动态过程和蛋白尿产生的关系。 方法 免疫组化法检测nestin在人正常肾组织及微小病变肾组织中的表达。构建氨基核苷嘌呤霉素肾病大鼠模型，应用免疫组化、荧光实时定量PCR、Western印迹法检测注射嘌呤霉素后第1、4、10、20天大鼠肾小球中nestin的分布与表达；电镜观察肾脏足细胞改变，测定尿蛋白量（24 h）。分析nestin的变化与蛋白尿的相关性。 结果 免疫组化显示nestin在人类微小病变肾小球中的表达较正常组织显著下调（0.93±0.08 比 1.65±0.12，P < 0.05）。在嘌呤霉素损伤足细胞早期，肾小球nestin的表达曾有一过性增加（mRNA和蛋白水平分别为对照组的1.23倍和1.48倍，P < 0.05），随后持续下降。nestin的mRNA水平在嘌呤霉素注射后第4天时降至对照组的35.8%；第10天时为对照组的12.1%（均P < 0.01）；病变好转后开始上升，恢复为对照组的65.8%（P < 0.05）。Western印迹检测nestin蛋白改变也有类似的趋势，嘌呤霉素注射后第4 天，nestin蛋白水平有所下降，为对照组的77.0%（P < 0.05）；至大量蛋白尿的第10天，nestin蛋白水平仅为对照组的58.0%（P < 0.05）；而随着病变的恢复，嘌呤霉素注射后第20天时nestin蛋白量恢复为对照的83.4%。Pearson相关分析结果显示，注射嘌呤霉素后nestin mRNA（r = －0.667，P < 0.05）及蛋白（r = －0.621，P < 0.05）表达与尿蛋白量(24 h)均呈负相关。 结论 在以足突广泛融合为特征的肾脏病变中，肾小球中间丝蛋白nestin表达显著减少，并与蛋白尿程度呈负相关，提示nestin可能参与了足细胞形态和功能的维持。     

IgA肾病患者血清IgA1与系膜细胞共培养上清诱导足细胞凋亡

目的 探讨IgA肾病(IgAN)患者血清IgA1与系膜细胞共培养上清对足细胞凋亡的影响。 方法 用Jacalin 亲和层析柱和Sephacryl S-200 分子筛纯化蛋白。单体IgA1（mIgA1）热聚合为聚合体IgA1（aIgA1）。实验分为患者上清组、健康上清组和对照组，系膜细胞分别与IgAN患者的aIgA1、健康对照的aIgA1和5%胎牛血清共培养，收集上清，与同步化的足细胞作用。流式细胞仪检测细胞凋亡情况。实时定量PCR 检测凋亡相关基因Bcl-2、Bax、Fas和Fas-L表达情况。 结果 患者上清可诱导足细胞凋亡，其凋亡率显著高于健康上清组和对照组[（28.5±5.9）%比（22.5±5.8）%、（20.5±4.5）%， 均P < 0.05]。患者上清可诱导足细胞Fas mRNA 升高，为对照组的1.89倍（P < 0.05）， 而Bcl-2 mRNA下调为对照组的72%（P < 0.05）。患者上清组的AngⅡ和TGF-β1水平均高于健康上清组[（13.2±3.4） ng/L比（8.2±2.3） ng/L，P < 0.05；（15.4±3.4） ng/L比（10.8±3.2） ng/L，P < 0.05]。 结论 IgAN患者血清IgA1与系膜细胞共培养上清可诱导足细胞凋亡，可能参与IgAN的进展。     

JAK/STAT通路在金属蛋白酶1组织抑制剂抑制肾小球系膜细胞凋亡中的作用

目的 探讨金属蛋白酶1组织抑制剂（TIMP-1）抑制大鼠肾小球系膜细胞（RMC）凋亡与Janus激酶/信号转导和转录激活子（JAK/STAT）通路的关系。 方法 用无血清培养基体外培养pcDNA3空载体、人正义、反义TIMP-1基因重组真核表达载体转染RMC。根据是否加JAK2特异性抑制剂AG490刺激24 h,将细胞分为未转染组、未转染＋AG490组、空载体组、空载体＋AG490组、正义组、正义＋AG490组、反义组和反义＋AG490组。另外设正常培养条件下的RMC作为正常对照组。应用流式细胞技术检测各组RMC的凋亡率。RT-PCR检测TIMP-1、bcl-xl、cyclin D1、p27kip1和JAK2 mRNA的表达。Western印迹检测胞质中JAK2、STAT3、STAT5及其相应磷酸化蛋白(p-JAK2、p-STAT3、p-STAT5)的表达。 结果 未转染组、正义组及反义组RMC凋亡率分别为（10.59±0.96）％、（7.08±0.43）％和（21.91±0.25）％，各组间差异有统计学意义(P < 0.05或P < 0.01)。在未加AG490的各组RMC中,bcl-xl和cyclin D1 mRNA在正义组中表达最高，反义组中最低，p27kip1 mRNA在反义组中表达最高。加入AG490后，各组细胞的凋亡率均显著增加（P < 0.01）；TIMP-1、bcl-xl和cyclin D1 mRNA表达均减少；p27kip1 mRNA表达均增加。在未加AG490的各组细胞中，p-JAK2、p-STAT3和p-STAT5在正义组中表达最高，反义组中最低。加入AG490后上述蛋白表达均减少，且正义＋AG490组最高，反义＋AG490组最低。 结论 TIMP-1表达受JAK/STAT信号通路调控，后者可通过上调前者的表达抑制RMC凋亡；TIMP-1通过JAK/STAT信号通路抑制RMC凋亡。 bcl-xl、cyclin D1和p27kip1参与了上述过程。     

雷公藤内酯醇干预高糖影响小鼠肾脏足细胞Nephrin蛋白表达的研究

目的：通过观察雷公藤内酯醇（triptolide,TP）及对照药物缬沙坦（valsartan,Val）干预高糖环境培养的足细胞肾小球足细胞裂孔隔膜（GPSD）核心蛋白nephrin的表达,来深入探讨雷公藤治疗糖尿病肾病（DN）蛋白尿的分子生物学机制。方法：（1）将培养成熟的足细胞分为对照组1、对照组2、模型组、雷公藤内酯醇干预组及缬沙坦干预组（干预组均设低、中、高3个剂量）,培养24h后以RT-PCR法检测各组足细胞nephrin mRNA的表达。（2）将培养成熟的足细胞设为对照组、模型组、雷公藤内酯醇干预组及缬沙坦干预组,培养24h后行间接免疫荧光法检测nephrin蛋白的表达。结果：（1）与模型组相比,不同剂量TP和Val组均能提高nephrin mRNA表达（P〈0.05）,并且中剂量TP组和高剂量Val组效果最好（P〈0.05）。（2）与对照组相比,间接免疫荧光法可见模型组nephrin表达下降。经TP和Val干预24h后,TP和Val组足细胞nephrin蛋白的表达均有一定程度提高。结论：雷公藤内酯醇和缬沙坦均能上调高糖诱导的足细胞nephrin的表达下降,这可能是雷公藤及缬沙坦在临床上有效治疗DN蛋白尿的机制之一。     

1,25(OH)2D3 ameliorates high glucose-induced podocyte injury via PI3K/p-Akt signalling pathway

Objective To investigate the effect of 1,25(OH)2D3 on high glucose induced podocyte injury and its signal transduction mechanism. Methods Differentiated mouse podocytes were exposed to normal glucose, high glucose, and different concentrations of 1,25(OH)2D3 or LY294002 (a selective PI3K inhibitor) for 24 h. PCR and immunofluorescent staining were used to detect nephrin, podocin, and desmin. Western blotting was used to detect protein expression of nephrin, podocin, desmin, PI3K, Akt and p-Akt. Results Compared with high glucose group, 1,25(OH)2D3 (100 nmol/L and 1000 nmol/L) significantly up-regulated the expression of podocin and nephrin in podocytes induced by high glucose (P＜0.05). Meanwhile, 1,25(OH)2D3 (100 nmol/L) significantly reduced the expression of desmin (P＜0.05). PI3K and p-Akt were obviously reduced in high glucose group. In the presence of 1,25(OH)2D3, the trends were reversed. However the above effects of 1,25(OH)2D3 were abolished when p-Akt was blocked by the PI3K inhibitor LY294002. Conclusions 1,25 (OH)2D3 can inhibit high glucose-induced podocyte injury through PI3K/p-Akt signaling pathway.     

cAMP/PKA signal activation prevents chemical-induced podocyte injury

Objective To investigate the role of activated cylic AMP(cAMP) signaling in chemical-induced podocyte injury. Methods Eight-weeks-old male BalB/C mice were randomly divided into three groups: control group, Adriamycin (ADR) group and Forskolin+ADR group. ADR nephropathy models were established by tail intravenous injection,and part of them were injected Forskolin, an agonist of adenylate cyclase, intraperitoneally. Phosphorylation of cAMP response element binding protein (CREB) was detected by laser confocal microscopy,morphology of foot processes were determined with transmission electron microscope, and WT-1 expression in glomeruli were detected by immunohistochemistry. Conditionally immortalized podocytes were treated with puromycin aminonucleoside (PAN), Exchange protein directly activated by cAMP (Epac) agonist 8-pCPT-2-O-Me-cAMP (2Me),protein kinase A (PKA) antagonist H89 and its agonist pCPT-cAMP(pCPT). Western blot was used to detect the expression levels of Epac, caspase3 and cleaved caspase3. PKA activity was assayed using cAMP-dependent protein kinase detection system. Cell viability was determined by a cell count kit and podocyte apoptosis was estimated by TUNEL staining. Mitochondrial membrane potential was evaluated by JC-1 staining. Results （1）Compared with ADR group, the urine albumin decreased significantly (P＜0.05) among Forskolin+ADR group and the WT-1 positive cells per glomerulus increased obviously (P＜0.05). （2）PAN decreased podocyte number in a time-dependent manner (P＜0.05), pre-treatment with pCPT obviously inhibited PAN induced podocyte decrease (P＜0.05), but H89 prevented the effect of pCPT in a dose-dependent manner (P＜0.05). （3）JC-1 staining showed that the percentage of podocyte with green fluorescence for control, PAN and pCPT+PAN group were (12.67±2.15)%, (31.35±4.60)% and (16.96 ± 2.51)%respectively (P＜0.05), and pretreatment with H89 inhibited the effect of pCPT (P＜0.05). （4）PAN promoted podocyte apoptosis and cleaved caspase3 expression (P＜0.05), and pretreatment with pCPT significantly prevented PAN-induced podocyte apoptosis and cleaved caspase3 expression (P＜0.05). Conclusions cAMP signaling activation ameliorated podocyte injury in ADR mice and PAN-induced podocyte apoptosis, and cAMP/PKA pathway may mediate these processes.     

Tacrolimus protects podocytes by up-regulating autophagy in type 2 diabetic model rats

Objective To assess the effects of tacrolimus (FK506) on podocyte in type 2 diabetic model rats and to explore the potential mechanism. Methods The model rats were fed with high fat and high sugar food and combining with a low-dose of streptozotocin (STZ). They were then randomly divided into a diabetic mellitus group (DM group) and a FK506 group. A normal control group (NC group) was also set. The rats in FK506 group were given with 0.5 mg?kg-1?d-1 FK506 for 8 weeks. The biochemical parameters were measured. The changes of renal pathology and ultrastructure of podocyte were observed by the light and electron microscopy. The expression of nephrin and LC3-Ⅱ was determined by immunohistochemistry and Western blotting. Results (1) Compared with those in NC group, KW/BW, systolic blood pressure (SBP), fasting blood glucose (FBG), triglyceride (TG), total cholesterol (TC), urinary albumin excretion rate (UAE) and creatinine clearance rate (Ccr) in DM group were significantly increased (all P＜0.05). And the KW/BW, UAE and Ccr were decreased in FK506 group compared to those in DM group (all P＜0.05), while other parameters had no significant difference (all P＞0.05). (2) Compared with those in NC group, the glomerular volume, mesangial cell proliferation and accumulation of mesangial matrix were increased, and the foot process became disorder and fusion in DM group, while these changes were significantly reduced in FK506 group. (3) Compared with that in NC group, the expression of nephrin and LC3-Ⅱ was decreased in DM group (all P＜0.05), and both of parameters were higher in FK506 group than those in DM group (all P＜0.05). Conclusion FK506 may enhance podocyte autophagy in type 2 diabetic model rats and attenuate podocyte injury.     

Fluvastatin ameliorates podocyte injury in proteinuric rats via modulation of excessive Rho signaling

Statins have been reported to confer renoprotection in several experimental models of renal disease through pleiotropic actions. The roles of statins in glomerular podocytes have not been explored. The objective of this study was to evaluate the effects of fluvastatin on podocyte and tubulointerstitial injury in puromycin aminonucleoside (PAN)-induced nephrosis. PAN induced massive proteinuria and serum creatinine elevation on day 7, which were significantly suppressed by fluvastatin. Immunofluorescence studies of podocyte-associated proteins nephrin and podocin revealed diminished and discontinuous staining patterns in rats with PAN nephrosis, indicating severe podocyte injury. Fluvastatin treatment dramatically mitigated the abnormal staining profiles. Reduction of nephrin expression by PAN and its reversal by fluvastatin were confirmed by quantitative analyses. By electron microscopy, effacement of foot processes was ameliorated in fluvastatin-treated rats. Fluvastatin also mitigated tubulointerstitial damage in PAN nephrosis, with the repression of PAN-induced NF-kappaB and activator protein-1 activation in the kidneys. In addition, expression of activated membrane-bound small GTPase RhoA was markedly increased in the glomeruli of PAN nephrosis, which was inhibited by fluvastatin treatment. In cultured podocytes, fluvastatin suppressed PAN-evoked activation of RhoA and actin cytoskeletal reorganization. Furthermore, fasudil, a specific Rho-kinase inhibitor, successfully ameliorated PAN-induced podocyte damage and proteinuria. In summary, fluvastatin alleviated podocyte and tubulointerstitial injury in PAN nephrosis. The beneficial effects of fluvastatin on podocytes can be attributable to direct modulation of excessive RhoA activity. Our data suggest a therapeutic role for statins in clinical conditions that are relevant to podocyte injury.     

Effects of bone marrow-derived mesenchymal stem cells on glomerular podocyte injured by lipopolysaccharide

Objective To observe the effects of bone marrow-derived mesenchymal stem cells (BMSC) on glomerular podocyte injured by lipopolysaccharide (LPS) and the expression of related protein. Methods Podocytes are divided into control group, BMSC group, LPS group and LPS plus BMSC group. After 24 hours of intervention, observing each experimental group podocyte form under inverted phase contrast microscope;detecting the expressions of mRNA and protein of nephrin, CD2AP, synaptopodin, and TRPC6 by RT-PCR and Western-blot. Results Compared with control group, expressions of nephrin, CD2AP, and synaptopodin in LPS group decreased (P＜0.05) while that of TRPC6 increased (P＜0.05); compared with LPS group, expressions of nephrin, CD2AP, and synaptopodin in LPS+MSC group increased (P＜0.05) while that of TRPC6 decreased (P＜0.05). Conclusion BMSC may relieve LPS-induced podocyte injury.     

活性维生素D通过调节糖尿病肾病大鼠巨噬细胞M1及M2表型活化防治足细胞损伤

目的 探讨活性维生素D（VD）能否通过调节肾组织巨噬细胞M1 及M2 表型活化从而防治糖尿病肾病（DN）大鼠足细胞损伤。 方法 利用腹腔注射链脲菌素（STZ）建立糖尿病大鼠模型。将SD 雄性大鼠按随机数字表法分为4 组：对照组1（NC-1 组,n=8）、对照组2（NC-2 组,n=8,对照+骨化三醇0.1 μg·kg^-1·d^-1 灌胃）、DN 组（n=24）、DN+VD 干预组（VD 组,n=24,DN+骨化三醇0.1 μg·kg^-1·d^-1 灌胃）,定期检测血糖、体质量,收集尿标本,分别于干预后8周、14周、18周末处死动物,检测Scr、BUN和尿蛋白变化;PAS染色观察肾脏病理改变;免疫组化法检测肾组织CD68+巨噬细胞浸润数量;Western 印迹法检测nephrin、podocin、CD68 以及M1巨噬细胞特异性标志物诱导型氮氧化物合酶（iNOS）、肿瘤坏死因子α（TNF-α）和M2巨噬细胞特异性标志物CD163、精氨酸酶1（Arg-1）、甘露糖受体（MR）表达。 结果 （1）与两对照组相比,DN 组Scr、BUN、24 h 尿蛋白量及肾小球系膜基质增生程度显著增加（P＜0.05）,podocin、nephrin蛋白表达下降（P＜0.05）。VD干预后能明显改善上述病理现象（均P＜0.05）。（2）与两对照组相比,DN组肾组织CD68⁺巨噬细胞浸润数量明显增加,呈时间依赖性。VD干预后能显著减少CD68⁺巨噬细胞浸润数量（P＜0.05）。（3）进一步确定肾组织巨噬细胞M1、M2活化表型发现,8周、14周、18周末DN组iNOS、TNF-α蛋白表达较对照组显著升高（均P＜0.05）,VD干预后能明显抑制同期DN肾组织iNOS、TNF-α表达（均P＜0.05）;8周、14周末VD组CD163、Arg-1、MR蛋白表达与DN组相比差异无统计学意义（均P＞0.05）,而18周末VD组CD163、Arg-1、MR蛋白表达较DN 组显著升高（均P＜0.05）,CD163/CD68 蛋白表达比例亦显著增加（P＜0.05）。（4）相关分析显示,M1 标志物iNOS 与nephrin、podocin 蛋白表达均呈负相关（r＝-0.707,P＜0.01;r＝-0.712,P＜0.01）;M2标志物CD163与nephrin、podocin蛋白表达均呈正相关（r＝0.627,P＜0.01;r＝0.613,P＜0.01）。 结论 活性维生素D具有调节巨噬细胞表型活化的能力,通过抑制巨噬细胞M1型活化并增强M2型活化,进而发挥足细胞保护作用。     

Effects and the possible mechanism of histone deacetylase inhibitor SAHA on the interstitial fibrosis induced by diabetes

Objective To explore the effects and possible mechanism of histone deacetylase inhibitor SAHA on the interstitial fibrosis induced by diabetes. Methods The SD rats were divided into three groups: control group (Con, n=9), diabetes mellitus (DM) group (n=9) and SAHA treatment group (n=9). The diabetic rat model was established by injecting streptozotocin (STZ) through tail vein. After 8 weeks, the SAHA treatment group rats were fed with a SAHA solution (25 mg?kg-1?d-1) by gastric gavage. After 16 weeks, all rats were sacrificed to detect relevant biochemical parameters, and observe the changes of pathomorphology in kidney. In addition, immunohistochemistry staining and Western blotting were employed to detect the protein expressions of transforming growth factor-β1 (TGF-β1), Smad2, Smad3, p-Smad2, p-Smad3, Smad7, collagen-Ⅰ and collagen-Ⅲ, respectively. Results Compared with Con group, the levels of blood glucose (BG), urinary trace albumin/urinary creatinine (ACR), triglyceride (TG) and total cholesterol (TC) in the diabetic group were all increased significantly (all P＜0.05), the protein expressions of TGF-β1, p-Smad2, p-Smad3, collagen-Ⅰ and collagen-Ⅲ in kidney were all increased in diabetic group (all P＜0.05), and the expression of Smad7 was significantly reduced (P＜0.05). Compared with DM group, the levels of ACR was reduced, the renal fibrosis was alleviated, the protein expressions of TGF-β1, p-Smad2, p-Smad3, collagen-Ⅰ and collagen-Ⅲ in SAHA group were all decreased (all P＜0.05), and the expression of Smad7 was increased significantly (P＜0.05). Conclusion SAHA may restore the protein level of Smad7 by enhancing protein stability, then promote the moderate transduction of TGF-β1/Smads signaling pathway, which reduce the fibrosis of renal tubules in diabetic rats.     

Effect of long-term low-dose 1α, 25-dihydroxy vitamin D3 on the expression of aquaporin 2 in kidneys of uremic rats

Objective To investigate the effect of long-term low-dose 1α, 25-dihydroxy vitamin D3 [1,25(OH)2D3] on rat kidney aquaporin (AQP) 2 expression in 5/6 nephrectomized rats. Methods Twelve Sprague-Dawley rats underwent 5/6 nephrectomy surgery were divided into model group (n＝6) and 1,25(OH)2D3 group (n＝6) randomly; sham-operated rats only received the renal capsule stripping (control group, n＝6). Rats in 1,25(OH)2D3 group received 1,25(OH)2D3 (3 ng•100 g-1•d-1, ip) for 24 weeks. Serum and 24-hour urine specimens were collected for measurement of serum creatinine, arginine vasopressin (AVP) and urine protein. Animals were sacrificed at week 24 and kidneys were removed for routine pathological, immunohistochemistry and immunoblotting analysis. Results At week 24, plasma AVP level in 1,25(OH)2D3 and model group was much higher than that in control group (all P＜0.05), with no significant differences between the former two groups (P＞0.05). Lower serum creatinine and urinary protein were presented in 1,25(OH)2D3 group compared with the model group rats at week 24 (P＜0.05). Renal medullar fibrosis and inflammatory cell infiltration were improved significantly in 1,25(OH)2D3 group compared with model group (P＜0.01, P＜0.05). Immunohistochemistry analysis revealed abundant AQP2 and p-AQP2 expressed in the renal medulla of sham group, mainly in apical membrane of collecting duct cells. AQP2 expression in model group was down-regulated (P＜0.05) and p-AQP2 expression in apical membrane was reduced. AQP2 expression in 1,25(OH)2D3 group increased compared with model group, with increased p-AQP2 expression in apical membrane. Western blotting revealed same results of these expressions (all P＜0.05). Correlation analysis showed a negative correlation of AQP2 expression with urine volume, medullary fibrosis, and inflammatory cell infiltration (P＜0.05). Conclusion Long-term low-dose 1,25(OH)2D3 improves AQP2 expression and response to AVP in collecting duct, which may involve in the anti-polyuric effect of 1,25(OH)2D3 in uremic rat.     

Attenuation of high glucose induced podocyte injury by ursolic acid up-regulating autophagy level

Objective To investigate the effects of ursolic acid (UA) on autophagy and podocyte injury induced by high glucose. Methods Conditionally immortalized murine podocyte were cultured in high glucose, the effect of PI3K inhibitor LY294002 and ursolic acid treatment were observed. The miR-21 expression was detected using RT-qPCR. The activation of PTEN-PI3K/Akt/mTOR pathway, expression of autophagy-related protein and podocyte marker protein were determined by Western blot. Immunofluorescence staining showed the expression of podocyte marker protein and endogenous accumulation of LC3. Autophagosomes were observed using electron microscopy. Results Compared with normal control group,the cells exposed to high glucose condition showed down-regulated synaptopodin, podocin and nephrin expression (P＜0.01), up-regulated miR-21 expression (P＜0.01), down-regulated PTEN expression (P＜0.01), up-regulated p85-P13K, phospho(p)-Akt, p-mTOR,p62/SQSTMI, expression and down-regulated LC3II and Beclin1 expression (all P＜0.01). Ursolic acid and LY294002 promoted synaptopodin, podocin and nephrin expression (all P＜0.01), up-regulated LC3II, Beclin1 expression and down-regulated p62/SQSTM1 expression (all P＜0.01), down-regulated p85-PI3K, p-Akt, p-mTOR expression (all P＜0.01). However, LY294002 did not affect the expression of miR-21 and PTEN. Ursolic acid inhibited miR-21 expression and upregulated PTEN level. Conclusions The podocyte injury is associated with defective autophagy level under high glucose condition. Ursolic acid could reduce podocyte injury by increasing autophagy level via inhibition of miR-21 expression and PTEN/Akt/mTOR pathway.