Regulation of glomerular matrix proteins via podocytic and endothelial-cell derived microRNAs

蛋白尿是肾小球疾病的标志。我们确定了肾连蛋白（NPNT）作为细胞外基质蛋白的肾小球滤过屏障的重要。足细胞microRNA-378 a-3 p（miR-378 a-3 p）和内皮miR-192调节足细胞NPNT表达，并在膜性肾小球肾炎中上调。miR-378 a-3 p或miR-192的过表达以及npnt的敲低导致蛋白尿、水肿、足细胞消失和肾小球基底膜增宽的表型。此外，我们希望通过足细胞和内皮细胞衍生的miRs分析肾小球基质蛋白的调节。我们将研究npnt的肾小球功能，并产生足细胞特异性npnt敲除小鼠，将分析肾小球损伤的自发或应激相关发展。使用不同的Npnt构建体（有和没有特异性miR结合位点），我们将解决miR-378 a-3 p过表达后肾小球变化在多大程度上是由于npnt敲低或其他miR靶点的问题。为了鉴定由miR-378 a-3 p和miR-192转录后控制的其他基质相关靶标，我们将在miR转染人足细胞和肾小球内皮细胞后进行比较微阵列。最后，我们将研究miR-378 a-3 p和miR-192的抑制剂的治疗潜力，以分析这些miR的抑制是否具有保护作用。

Proteinuria is a hallmark of glomerular disease. We identified nephronectin (NPNT) as an extracellular matrix protein important for the glomerular filtration barrier. Podocyte microRNA-378a-3p (miR-378a-3p) and endothelial miR-192 regulate podocyte NPNT expression and are upregulated in membranous glomerulonephritis. Overexpression of miR-378a-3p or miR-192 as well as knockdown of npnt caused a phenotype with proteinuria, edema, podocyte effacement and widening of the glomerular basement membrane. Furthermore, we want to analyze the regulation of glomerular matrix proteins via podocyte and endothelial cell derived miRs.We will investigate the glomerular function of npnt and generate podocyte specific npnt knockout mice that will be analyzed regarding spontaneous or stress related development of glomerular damage. Using different Npnt constructs with and without the specific miR binding site we will solve the question to what degree the glomerular changes after miR-378a-3p overexpression are due to npnt knockdown or other miR targets.We will use fluorescent dextrans with different molecular weights to characterize the increase of glomerular permeability after npnt knockdown. To identify other matrix associated targets that are post-transcriptionally controlled by miR-378a-3p and miR-192, we will perform comparative microarrays after miR transfection of human podocytes and glomerular endothelial cells. Finally, we will investigate the therapeutic potential of antagomirs of miR-378a-3p and miR-192 to analyze if inhibition of these miRs has a protective effect.