Role of the interaction of slit membrane, podocyte and glomerular basement membrane in pathogenesis of glomerular kidney diseases such as Alport's syndrome

肾小球滤过屏障的突变，如IV型胶原(Col4A3/4/5，肾小球基底膜)或podocin(Nphs2，裂隙横隔膜)导致蛋白尿、肾脏纤维化和终末期肾病的早期开始。杂合子COL4A3/4突变携带者发展为薄基底膜肾病，在以后的生活中只有血尿。最近，青少年被描述为早期蛋白尿和肾功能衰竭，只有杂合型IV型胶原突变。在这些患者中发现了额外的podocin基因多态。在这个项目中，计划利用双杂合Col4A3+/-//Nphs2+/R140Q-小鼠来研究可能的致病相关性。我们假设在肾小球基底膜(GBM)和裂隙隔膜(SD)之间存在通过足细胞受体的相互作用。这一环节对滤过屏障的动态平衡和稳定性至关重要。我们想把重点放在修饰基因，如podocin，对发展成TBMN的Col4A3小鼠的影响。我们的第一个结果显示，在这些小鼠中，早期蛋白尿以及病理性足细胞足突消失。具体地说，我们想要重点研究高滤过对COL4A3+/-/Nphs2+/R140Q-小鼠单肾切除后肾脏纤维化发展的影响。此外，还将研究单肾切除后蛋白尿前和蛋白尿期间应用血管紧张素转换酶抑制剂的效果。这些问题应在临床参数上进行研究，如以肾功能衰竭为主要终点的生存时间，蛋白尿/蛋白尿的发展，以及肾脏的组织学和超微结构分析。此外，还将通过免疫组织化学、实时荧光定量聚合酶链式反应和Western Blot研究体内和体外对GBM和SD的组成及其受体的描述。这也将用我们在不同疾病阶段建立的超微结构免疫组织化学进行研究。我们对GBM-和SD-发现的第一个结果表明，双杂合子小鼠在足细胞、其GBM和SD之间的联系的基本机制以及它们的动态平衡方面产生了重要的见解。我们的项目对阿尔波特综合征和局灶节段性肾小球硬化等人类疾病具有重要的临床参考价值。预期的结果也可能为糖尿病肾病或肾小球肾炎等更常见的肾脏疾病的治疗选择提供新的基本理解。

Mutations in the glomerular filtration barrier like collagen type IV (COL4A3/4/5, glomerular basement membrane) or podocin (Nphs2, slit diaphragm) lead to an early begin of proteinuria, kidney fibrosis and end-stage-renal disease. Heterozygous COL4A3/4-mutation-carriers develop a thin basement membrane nephropathy with only hematuria later in life. Recently, adolescents have been described developing early proteinuria and kidney failure with only heterozygous collagen type IV-mutations. In these patients additional podocin-polymorphism were found. In this project, a possible pathogenetic correlation is planned to be investigated with help of double-heterozygous COL4A3+/-//Nphs2+/R140Q-mice. We hypothesize that between the glomerular basement membrane (GBM) and slit diaphragm (SD) an interaction exists via the podocytes' receptors. This link is essential for homeostasis and stability of the filtration barrier. We want to focus on the influence of a modifier-gene, like podocin, on the COL4A3-mice who develop TBMN. Our first results show an early proteinuria as well as pathological podocyte foot process effacement in these mice. Specifically, we want to focus on the influence of hyperfiltration on kidney fibrosis development due to uninephrectomy in COL4A3+/-//Nphs2+/R140Q-mice. Furthermore, the effect of ACE-inhibitor therapy after uninephrectomy before and during proteinuria will be investigated. These questions shall be studied on clinical parameters like survival time till kidney failure as primary end point, development of proteinuria/albuminuria, as well as histological and ultrastructural analysis of the kidneys. Furthermore, description of the GBM-, SD-composition and their receptors will be studied, in vivo and in vitro by immunohistochemistry, real-time PCR and Western Blot. This will also be studied with our well-established ultrastructural immunogoldhistochemistry during different disease stages. Our first results of the GBM- and SD-findings demonstrate that the double-heterozygous mice give rise to important insights in basic mechanisms of the link between podocyte, its GBM and SD, as well as their homeostasis. Our project has a significant clinical reference for human diseases like Alport Syndrome and focal segmental glomerulosclerosis. The expected results could also offer a new basic understanding for treatment options of more frequent kidney diseases like diabetic nephropathy or glomerulonephritis.