A 3D tri‐culture system reveals that activin receptor‐like kinase 5 and connective tissue growth factor drive human glomerulosclerosis

Glomerular scarring, known as glomerulosclerosis, occurs in many chronic kidney diseases and involves interaction between glomerular endothelial cells (GECs), podocytes, and mesangial cells (MCs), leading to signals that promote extracellular matrix deposition and endothelial cell dysfunction and loss. We describe a 3D tri‐culture system to model human glomerulosclerosis. In 3D monoculture, each cell type alters its phenotype in response to TGFβ, which has been implicated as an important mediator of glomerulosclerosis. GECs form a lumenized vascular network, which regresses in response to TGFβ. MCs respond to TGFβ by forming glomerulosclerotic‐like nodules with matrix deposition. TGFβ treatment of podocytes does not alter cell morphology but increases connective tissue growth factor (CTGF) expression. BMP7 prevents TGFβ‐induced GEC network regression, whereas TGFβ‐induced MC nodule formation is prevented by SMAD3 siRNA knockdown or ALK5 inhibitors but not BMP7, and increased phospho‐SMAD3 was observed in human glomerulosclerosis. In 3D tri‐culture, GECs, podocytes, and MCs form a vascular network in which GECs and podocytes interact intimately within a matrix containing MCs. TGFβ treatment induces formation of nodules, but combined inhibition of ALK5 and CTGF is required to prevent TGFβ‐induced nodule formation in tri‐cellular cultures. Identification of therapeutic targets for glomerulosclerosis depends on the 3D culture of all three glomerular cells. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Loss of PTEN promotes podocyte cytoskeletal rearrangement,aggravating diabetic nephropathy

In diabetic nephropathy (DN), podocyte cytoskeletal rearrangement occurs followed by podocyte effacement and the development of proteinuria. PTEN (phosphatase and tensin homologue) is a ubiquitously expressed phosphatase that plays a critical role in cell proliferation, cytoskeletal rearrangement, and motility. In mouse models of diabetes mellitus, PTEN expression is reportedly decreased in mesangial cells, contributing to expansion of the mesangial matrix, but how PTEN in the podocyte influences the development of DN is unknown. We observed that PTEN expression is down‐regulated in the podocytes of diabetic db/db mice and patients with DN. In cultured podocytes, PTEN inhibition caused actin cytoskeletal rearrangement and this response was associated with unbalanced activation of the small GTPases Rac1/Cdc42 and RhoA. In mice treated with PTEN inhibitor, actin cytoskeletal rearrangement occurred in podocytes and was accompanied by increased albumin excretion. We also created mice with an inducible deletion of PTEN selectively in podocytes. These mice exhibited increased albumin excretion and moderate foot process effacement. When the mice were challenged with a high fat diet, podocyte‐specific knockout of PTEN resulted in substantially increased proteinuria and glomeruloclerosis compared to control mice fed a high fat diet or mice with PTEN deletion fed a normal diet. These results indicate that PTEN is involved in the regulation of cytoskeletal rearrangement in podocytes and that loss of PTEN predisposes to the development of proteinuria and DN. Copyright © 2015 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

α‐Actinin‐4 is involved in the process by which dexamethasone protects actin cytoskeleton stabilization from adriamycin‐induced podocyte injury

Aim: Glucocorticoid therapy has been used in childhood nephrotic syndrome since the 1950s, where the characteristic change is effacement of the actin‐rich foot process of glomerular podocytes. Recent studies have shown that glucocorticoids, in addition to their general immunosuppressive and anti‐inflammatory effects, have a direct effect on podocytes, regulate some apoptotic factors, and increase the stability of actin filaments. However, the precise mechanism(s) underlying the protective effects of glucocorticoids on podocytes remain unclear. It is known that adriamycin (ADR) can induce podocyte foot process effacement and trigger massive proteinuria in rodent models. However, few reports have examined the direct role of ADR in podocyte actin rearrangement in vitro. In this study, we investigated how ADR directly induced podocyte actin cytoskeleton rearrangement and further analyzed how dexamethasone prevented such injury. Methods: We used confocal microscopy to assess podocyte actin rearrangement. Western blot analysis and real‐time polymerase chain reaction were performed to measure the protein and mRNA levels of α‐actinin‐4. Results: We demonstrated that there was a time‐dependent ADR‐induced podocyte actin rearrangement with less than 12 h of ADR treatment in cultured podocytes. Dexamethasone could protect podocytes from ADR‐induced injury and also stabilize the expression of α‐actinin‐4. Conclusion: This study showed that dexamethasone had direct effects on podocytes: α‐actinin‐4 may be one of the potential target molecules.     

Dipping your feet in the water: podocytes in urine

Podocyte injury and loss plays an important role in the pathogenesis and progression of many kidney diseases. Studies have shown that podocyte-related markers and products can be detected in the urine of patients with glomerular diseases such as focal segmental glomerulosclerosis, IgA nephropathy, lupus nephritis, diabetic nephropathy and pre-eclampsia. Therefore, detecting the loss of podocytes in the urine provides a useful noninvasive technique of gathering information about the disease type and/or activity of glomerular diseases. Currently, urine podocyte-related protein markers, mRNA, microRNA and exosomes have been used with varying degrees of success to study glomerular diseases. The determination of urinary podocyte loss may become an important noninvasive tool in the evaluation of glomerular diseases.     

糖尿病肾病肾小球滤过膜超微结构改变及其与蛋白尿的关系

目的:观察2型糖尿病肾病(2TDN)患者肾小球滤过膜超微结构变化特点及与蛋白尿的关系。方法:将明确诊断的2TDN患者分为微量白蛋白尿组(尿白蛋白/24h30～300mg);蛋白尿组(尿蛋白0.5～2.0g/24h)和大量蛋白尿组(尿蛋白>3.5g/24h)。收集三组患者临床指标,并分别观察各组患者肾活检组织学及肾小球滤过膜超微结构变化,采用形态学计量分析方法分别测算肾小球体积、肾小球足细胞和内皮细胞的相对密度、绝对数目和足细胞足突宽度及肾小球基膜(GBM)厚度。结果:三组患者的年龄、性别、BMI、血压和糖尿病及肾脏病病程均无统计学差异。2TDN患者肾组织超微结构定量分析结果证实:(1)2TDN患者在微量白蛋白尿期肾小球毛细血管袢内皮细胞数目就已增加(实验组vs对照组P<0.01),内皮细胞相对密度则维持在一定水平(各组间P>0.05);(2)GBM改变包括三层结构消失、GBM增厚等,这些改变在微量白蛋白尿期就已发生,且GBM与病程密切相关(蛋白尿组,r=0.538,P<0.05)。(3)微量白蛋白尿组患者肾小球足细胞相对密度下降(微量白蛋白尿组vs对照组P<0.01)、足细胞足突宽度增加(微量白蛋白尿组vs对照组P<0.05)。随病程进展,足细胞相对密度和绝对数目呈进行性减少(微量白蛋白尿组vs大量蛋白尿组P<0.05)。(4)2TDN患者蛋白尿水平与肾小球足细胞数目(大量蛋白尿组,r=0.648,P<0.01)及GBM厚度(蛋白尿组,r=0.538,P<0.05)相关。结论:DN患者肾小球滤过膜超微结构发生了系列变化,这些变化与蛋白尿的发生、发展有着内在联系。     

Nephrin,podocin及α-actinin在小鼠肾小球足细胞系的表达与分布

目的 :探讨nephrin ,podocin及α actinin在小鼠肾小球足细胞系 (MPC5)的表达与分布 ,为进一步研究上述分子间的相互作用建立稳定的实验平台。　 方法 :培养小鼠MPC5,以γ 干扰素诱导在 33℃传代增生 ,继而在37℃培养两周使细胞分化成熟以用于实验研究。相差显微镜观察足细胞形态 ;免疫荧光染色观察nephrin ,podocin ,α actinin及WT1在足细胞的分布 ;RT PCR检测足细胞nephrin ,podocin及α actinin 4的mRNA ;免疫蛋白印迹检测nephrin ,podocin ,α actinin及WT1蛋白。　 结果 :成熟足细胞呈星形且有突起形成。免疫荧光及免疫蛋白印迹显示足细胞特异性的表达WT1分子。免疫荧光染色可见nephrin和podocin在足细胞内均呈线状分布于胞膜表面 ,α actinin呈细丝状分布于胞质内及伸出的突起中。在mRNA水平及蛋白水平均检测到nephrin ,podocin和α actinin 4表达 ,其蛋白大小分别为 180KDa,4 5KDa和 10 0KDa。　 结论 :首次在mRNA水平及蛋白水平证实了小鼠MPC5能够表达nephrin ,podocin及α actinin ,为进一步研究这些分子间的相互作用奠定了基础。     

阿米洛利抑制尿激酶受体表达及减少蛋白尿的机制

目的：观察阿米洛利在经典的5/6肾切除大鼠模型中对蛋白尿的影响,探讨其影响蛋白尿的机制。方法构建经典的5/6肾切除大鼠模型,实验分为3组：假手术对照组（Sham）、5/6肾切除组（NTX）、5/6肾切除+阿米洛利干预组（NTX+阿米洛利）。采用考马斯亮蓝法检测各组24 h尿蛋白,利用免疫荧光共聚焦、实时荧光定量PCR等技术,检测足细胞尿激酶受体（uPAR）蛋白和uPAR mRNA的表达情况。结果第2周时,与假手术对照组（Sham）相比较,5/6肾切除组（NTX）24 h尿蛋白明显升高（47.50±28.05 mg vs 14.28±3.8 mg,P=0.023）,有统计学意义;与NTX组相比,NTX+阿米洛利24 h尿蛋白无统计学意义（51.56±21.03 mg vs 47.50±28.05 mg,P=0.748）。第12周时,与NTX组（188.31±29.82）mg相比,NTX+阿米洛利组（121.37±31.14）mg、Sham组（21.32±8.59）mg 24 h尿蛋白增加量均进一步减少,具有统计学意义[P值分别为0.001,0.000]。激光共聚焦显微镜观察,与Sham组相比较,NTX组PLAUR mRNA表达明显增高（9.74±1.44 vs 1.01±0.13,P<0.01）,有统计学意义;与NTX组相比较, NTX+阿米洛利mRNA表达明显降低（9.74±1.44 vs 5.01±1.36,P<0.01）,有统计学意义。结论阿米洛利可能通过抑制受损足细胞uPAR的表达,从而起到降蛋白尿、延缓肾小球硬化的作用。     

The Renin-Angiotensin-aldosterone system in podocytes

The renin-angiotensin-aldosterone system (RAAS) plays a critical role in kidney function and its inhibition reduces proteinuria and preserves kidney function in patients with chronic kidney disease. Recent studies have shown that podocytes generate many components of the RAAS and they express receptors of RAAS, including angiotensin II, mineralocorticoid, and prorenin receptors. Crucial functions of podocytes, such as contraction, apoptosis, autophagocytosis, and cytoskeletal organization, have been shown to be regulated by the angiotensin II type 1 receptors. An activation of the glomerular RAAS and protection from podocyte injury by RAAS inhibitors have been shown in many glomerular diseases. Exploring the interaction between the local RAAS and the signaling involved in RAAS activation in podocytes will lead to new therapeutic strategies of podocyte protection.     

Fabry病的临床表现及肾脏病理学特征

目的 :探讨Fabry病患者临床表现及肾组织形态学特点。　 方法 :总结 9例Fabry病患者临床表现和肾组织形态学特点。同时对肾小球足细胞数定量分析 ,并借助足细胞特殊标记物WT1对足细胞的密度及其缺失分布特点进行观察。　 结果 :①患者以年轻男性为主 ,多有肾脏病家族史。②多伴有Fabry病肾外表现 ,表现为皮肤血管角质瘤、少汗、低热、肢端感觉异常等症状。心脏受累发生率较高 ,多表现为房室传导阻滞、PR间期缩短、ST段和T波的异常、左心室高电压等。③均无明显角膜混浊、视网膜动脉曲张等Fabry病常见眼部病变。④肾脏病变表现为轻至中度蛋白尿。部分患者伴轻微镜下血尿。肾小管间质损伤较突出。⑤肾组织形态学改变表现为光镜下肾小球足细胞弥漫空泡变性 ;甲苯胺蓝染色显示足细胞浆内含大量嗜甲苯胺蓝的蓝色颗粒状物 ;电镜下嗜锇性、同心圆样髓样小体在胞浆中堆积。多数患者小管间质中重度病变。肾间质血管病变现象较为突出。⑥肾小球足细胞计数及足细胞密度均明显低于正常 (P <0 0 1) ,WT1表达缺失呈节段分布 ,缺失部位多位于周边袢和有节段硬化病变处。　 结论 :①Fabry病以男性青年多见 ,多有肾脏损害的家族史。②多数患者存在肾外表现 ,心脏病变发生率较高。③无明显Fabry病特征性眼部病变 ,无明显     

Adrenomedullin protects against oxidative stress-induced podocyte injury as an endogenous antioxidant.

BACKGROUND: We previously reported that puromycin aminonucleoside (PAN) increased adrenomedullin (AM) secretion and AM mRNA expression in podocytes, through overproduction of oxidative stress. To clarify the cytoprotective role of AM as antioxidative and antiapoptotic substance in podocytes, we investigated the effect of exogenous AM and AM antagonist on PAN-induced apoptosis in conditionally immortalized murine podocytes. METHODS: The expression of AM, RAMP 2 and RAMP 3 was investigated using real-time PCR, western blotting analysis and immunofluorescence microscopy. Reactive oxygen species (ROS) production was measured by CM-H(2)DCFDA fluorescence intensity method. The percentage of apoptotic cells was measured by Hoechst 33342 staining. RESULTS: PAN (100 microg/ml) significantly (P < 0.01) increased ROS production, associated with an increase in apoptosis; the percentage of apoptotic cells is 5.3% + 0.05% (P < 0.01) with 36 h treatment of PAN compared to 0.24 + 0.16% with no treatment. Several antioxidants could markedly reduce PAN-induced apoptosis in cultured podocytes, suggesting that PAN-induced apoptosis might be attributable to the overproduction of ROS. Accordingly, the administration of exogenous AM (10(-6) M) could significantly reduce not only ROS production via a PKA-dependent pathway, but also the resultant apoptosis induced by PAN. AM antagonists, CGRP8-37, augmented PAN-induced apoptosis, associated with increased ROS production, 2.2- and 2.3-Fold, respectively. RAMP 2 and RAMP 3 could be detected in podocytes and glomeruli. CONCLUSIONS: This suggests that ROS-induced up-regulation of AM with PAN could counteract ROS-induced apoptosis, by the suppression of ROS production. Therefore, AM might have the endogenous antioxidant potential to protect against ROS-induced podocyte injury.