The Role of Protein Degradation Systems for Glomerular Protein Homeostasis

三种类型的肾小球细胞形成功能性合胞体，实现肾过滤。其中，足细胞和内皮细胞被肾小球基底膜分开，并最终组成三层肾小球滤过屏障（GFB），这被认为是赋予大小选择性和电荷选择性的性质。肾小球系膜细胞占据GFB之间的空间，提供结构支持，并通过收缩减少肾小球表面积间接参与滤过。肾小球损伤的典型特征是来源于血浆的蛋白质以上皮下、内皮下和系膜沉积物的形式沉积。在生理条件下，肾小球蛋白质沉积是可以忽略的，即使GFB对血浆蛋白质的渗透性只是部分的，这表明预防机制必须存在。这些机制的损害可导致病理性肾小球蛋白沉积，这反过来又会影响肾小球功能。肾小球蛋白质稳态的基础是未知的，部分原因是蛋白质降解系统的复杂性和肾小球合胞体的复杂性。在这个提议中，我们假设，蛋白质的摄取和细胞内降解通过两个主要的降解系统，即泛素-蛋白酶体系统（UPS）和自噬体溶酶体系统（ALS）在肾小球细胞中相互作用，并有助于维持肾小球过滤器的完整性，在细胞特异性的方式。初步研究表明，内和细胞外的蛋白质稳态的系膜和内皮细胞主要依赖于一个完整的ALS，而在第一线的足细胞依赖于UPS。因此，UPS的抑制促进上皮下IgG沉积和蛋白尿，而ALS抑制导致系膜IgG沉积。为了剖析UPS和ALS对于肾小球细胞内和细胞外蛋白质稳态的未知生理和病理生理重要性，我们定义了三个目标，这些目标将通过肾小球细胞特异性分析的新技术来解决。(1)定义肾小球细胞中UPS和ALS的差异细胞特异性基础活性。(2)临床应用蛋白酶体抑制剂对肾小球细胞蛋白质稳态影响的比较分析。(3)未受攻击的肾小球合胞体和IgG暴露后遗传细胞特异性蛋白酶体和溶酶体损伤后果的比较分析。这些结果将为理解肾小球蛋白沉积的机制、理解关于蛋白酶体抑制剂的药物不良反应的相互矛盾的数据以及涉及UPS和ALS蛋白的突变的不同肾小球表型提供缺失的基础。

Three types of glomerular cells, which form a functional syncytium, achieve renal filtration. Of these, podocytes and endothelial cells are separated by the glomerular basement membrane and ultimately compose the three-layered glomerular filtration barrier (GFB), which is thought to impart both size-selective and charge-selective properties. Mesangial cells occupy the space inbetween the GFB to provide structural support and to indirectly participate in filtration by reducing the glomerular surface area by contraction. Typical for glomerular injury is the deposition of proteins originating from the plasma in form of subepithelial, subendothelial and mesangial deposits. Under physiologic conditions glomerular deposition of protein is neglectable even though permeability of the GFB to plasma protein is only partial, suggesting that preventive mechanisms must exist. Impairment of these mechanisms could result in pathologic glomerular protein deposition, which in turn would affect glomerular function. The basis of glomerular protein homeostasis is unknown partly due to the complexity of protein degradation systems and due to the complexity of the glomerular syncytium. In this proposal we hypothesize, that protein uptake and intracellular degradation through the two major degradative systems, namely the ubiquitin-proteasomal system (UPS) and the autophagosomal lysosomal system (ALS) interplay in glomerular cells and contribute to maintain the integrity of the glomerular filter in a cell-specific manner. Preliminary investigations demonstrate that intra- and extracellular protein homeostasis of mesangial and endothelial cells predominantly depends on an intact ALS, whereas podocytes in first line depend on the UPS. Thereby, inhibition of the UPS promotes subepithelial IgG deposition and proteinuria whereas ALS inhibition results in mesangial IgG deposition. To dissect the unknown physiologic and pathophysiologic importance of the UPS and ALS for the intra- and extracellular protein homeostasis of glomerular cells we defined three aims which will be addressed with novel techniques of glomerular cell-specific analyses. (1) Define the differential cell-specific basal activity of the UPS and the ALS in glomerular cells. (2) Comparative analyses of the consequence of clinically used proteasomal inhibitors on the protein homeostasis of glomerular cells. (3) Comparative analyses of the consequence of genetic cell-specific proteasomal and lysosomal impairment for the unchallenged glomerular syncytium and after IgG exposition. The results will provide the missing basis for i.e. understanding the mechanisms of glomerular protein deposition, for understanding conflicting data on adverse drug effects of proteasomal inhibitors, and of the different glomerular phenotypes of mutations involving UPS and ALS proteins.