Podocyte cell communication through microRNA-containing exosomes and autophagy in membranous glomerulonephritis

原发性膜性肾小球肾炎（MGN）是由自身抗体结合足细胞抗原引起的。MGN中最常见的可检测自身抗体是抗磷脂酶A2受体抗体（PLA 2 R-ab）。然而，它仍然是未知的自身抗体如何到达上皮下空间在MGN，因为肾小球滤过屏障通常是不可渗透的抗体。肾小球滤过屏障由特化的肾小球内皮细胞（GEC）、GBM和足细胞组成。肾粘连蛋白（NPNT）是一种由GEC来源的miR-192调控的足细胞细胞外基质蛋白。最近，我们可以表明，敲低Npnt在斑马鱼引起水肿，蛋白尿，足细胞消失和结构变化的GBM类似MGN。此外，NPNT在MGN患者中下调。在本项目中，我们希望研究通过含miR的exosomes和自噬在MGN中的细胞间通讯中的作用。我们推测，GEC衍生的miR-192被包装在外泌体中，并穿过GBM到达足细胞，在足细胞中被足细胞丝状伪足吸收并下调NPNT。我们希望研究在足细胞单一培养中，在我们的3D肾小球共培养模型中，外泌体中包装的miR的影响，并分析各种斑马鱼模型中基于miR的肾小球细胞-细胞通信的功能方面，包括具有荧光自噬体和体内吗啉代的转基因斑马鱼系，以敲除自噬所必需的基因。我们希望干扰自噬与外泌体释放之间的关系，并研究自噬，外泌体释放和外泌体摄取之间的关系，这些关系将通过创新方法进行研究，例如体内和体外pH依赖性荧光外泌体标记，自噬通量测量，以及扫描离子电导显微镜（SICM）和SEM。我们希望研究NPNT调节miR-192的作用，不仅在外泌体和miR-介导的细胞间通讯，而且在自噬和内溶酶体途径的足细胞在体内和体外的情况下。在此背景下，我们将评估尿miR-192作为MGN的非侵袭性生物标志物。细胞外蛋白NPNT和自噬之间的关系迄今尚未研究。这一方面将在细胞培养装置中进行研究，但也在我们的足细胞特异性Npnt敲除小鼠中进行研究。我们将研究足细胞特异性敲除Npnt如何促进MGN的THSD 7A小鼠模型中的自身抗体通过，此外，我们将使用转基因PLA 2 R小鼠来研究Npnt、miR和自噬在MGN中的作用。最后，我们将在足细胞特异性Npnt基因敲除小鼠中用链脲佐菌素治疗，以探讨miRs和NPNT在糖尿病肾病和MGN之间的潜在联系。我们希望这项研究将有助于我们更好地了解miRs和自噬在MGN中的病理生理作用，并为未来的临床应用提供潜在的非侵入性生物标志物和治疗靶点。

Primary membranous glomerulonephritis (MGN) is caused by autoantibodies binding to podocyte antigens. The most frequent autoantibody detectable in MGN is the anti-phospholipase A2 receptor antibody (PLA2R-ab). However, it remains unknown how autoantibodies reach the subepithelial space in MGN because the glomerular filtration barrier is usually impermeable for antibodies. The glomerular filtration barrier is composed of specialized glomerular endothelial cells (GEC), the GBM and podocytes. Nephronectin (NPNT) is a podocyte extracellular matrix protein regulated by GEC-derived miR-192. Recently, we could show that knockdown of Npnt in zebrafish induces edema, proteinuria, podocyte effacement and structural changes in the GBM resembling MGN. Furthermore, NPNT was downregulated in patients with MGN.In this project, we would like to investigate the role of cell-cell communication through miR containing exosomes and autophagy in MGN. We speculate that GEC-derived miR-192 is packed in exosomes and crosses the GBM to reach podocytes where it is taken up by podocyte filopodia and down regulates NPNT. We want to study effects of miRs packed in exosomes in podocyte monoculture, in our 3D glomerular co-culture model and analyze functional aspects of miR-based glomerular cell-cell communication in various zebrafish models, including a transgenic zebrafish line with fluorescent autophagosomes and in vivo morpholinos to knock down genes essential for autophagy. We would like to interfere pharmacologically with exosome release and study the relationship between autophagy, exosome release and exosome uptake that will be studied with innovative methods such as pH-dependent fluorescent exosome labelling in vivo and in vitro, autophagy flux measurements, as well as scanning ion conductance microscopy (SICM) and SEM. We would like to investigate the role of NPNT regulating miR-192 not only in exosome- and miR-mediated cell-cell communication, but also in the context of autophagy and endolysosomal pathways in podocytes in vivo and in vitro. In this context, we will evaluate urinary miR-192 as non-invasive biomarker for MGN.The relationship between the extracellular protein NPNT and autophagy has not been investigated so far. This aspect will be studied in a cell culture setup, but also in our podocyte-specific Npnt knockout mice. We will investigate how podocyte specific knockout of Npnt facilitates autoantibody passage in a THSD7A mouse model for MGN and in addition, we will us the transgenic PLA2R mouse to investigate the role of Npnt, miRs and autophagy in MGN. Finally, a potential link between diabetic nephropathy and MGN through miRs and NPNT will be addressed in podocyte-specific Npnt knock out mice treated with Streptozotocin.We hope that this research will help us to better understand the pathophysiological role of miRs and autophagy in MGN with potential relevance for non-invasive biomarker and therapeutic targets for future clinical use.