Dichotomous roles of Shroom3 in Tubular cells and Podocytes in native and allograft kidneys

项目总结：慢性肾脏病（CKD）被确定为肾功能下降， 肾小球滤过率（eGFR）和/或由蛋白尿的存在表示。按组织学， 在同种异体移植肾中，CKD的特征在于肾间质纤维化、血管内膜纤维化和肾小球- 硬化，反映了不同肾室的损伤。全基因组关联研究（GWAS） 确定了CKD的候选易感基因座。GWAS变异与CKD相关的机制基础 大部分未被描述，阻碍了GWAS信息的翻译，用于开发新的治疗方法， CKD。我们之前的研究表明，在供体肾中，CKD相关的SHROOM 3基因座-Rs 17319721， 增加SHROOM 3表达（通过TCF 7 L2依赖性转录）并促进肾移植物纤维化 (IF/TA），通过TGF-β1信号传导。这些数据与Shroom 3在细胞凋亡中的保护作用的证据形成对比。 肾小球发育一致的是，在肾小球和非肾小球之间观察到相反的联系。 肾小球SHROOM 3和CKD活检中的肾功能。在同种异体移植物中，Rs 173198721处的纯合性即 A/A与较低的GFR相关，与移植后2年的白蛋白尿减少相关。 GWAS数据还显示，Rs 17319721与白蛋白尿减少但eGFR降低相关。到 为了研究这些二分效应的机制，我们使用了诱导型Shroom 3基因敲除小鼠，并观察到 用管状Shroom 3敲低减少肾纤维化。在肾小管细胞中，Rho激酶（ROCK）抑制剂或 Shroom 3中的ROCK结合（ASD 2-）结构域缺失减少了促纤维化信号传导。相反，肾小球-，但 而不是肾小管-Shroom 3敲低，诱导蛋白尿伴弥漫性足细胞足突消失， 足细胞丢失这种表型与人类微小病变病（MCD）相似。在足细胞中，我们发现 并证实了SHROOM 3与FYN（一种Src激酶）通过关键的Src同源性-3结合的新型相互作用 域，不同于其ASD 2域。在体外和体内，Shroom 3-Fyn相互作用是激活所必需的 Fyn激酶和下游的Nephrin磷酸化和肌动蛋白细胞骨架在足细胞。这本小说 该机制解释了Shroom 3（& Rs 17319721）对成人蛋白尿的保护作用。我们 假设SHROOM 3在肾小管细胞和足细胞中具有二分作用，其由 不同的蛋白质基序。我们将通过三个目标来检验我们的假设。在aim-1中，我们将过度表达ASD 2- 结构域缺陷Shroom 3的体外/体内研究，以证实Shroom 3的ASD 2结构域依赖性促纤维化信号传导。 在aim-2中，Fyn结合突变体Shroom 3将过表达以确认体内足细胞损伤和表型。 由于受损的Fyn激活与MCD相关，Shroom 3敲低抑制Fyn，在aim-3中，我们 我们将通过比较肾小球的形态学来研究Shroom 3-Fyn相互作用与人MCD的相关性。 形态测量学和基因组学，利用来自肾病综合征NEPTUNE协会的人类数据。这 这项工作对于开发针对CKD和IF/TA中纤维化的结构域特异性Shroom 3抑制剂至关重要。

Project Summary: Chronic kidney disease (CKD) is identified as decline in renal function estimated by glomerular filtration rate (eGFR) and/or signified by the presence of proteinuria. By histology in native and allograft kidneys, CKD is characterized by renal interstitial fibrosis, vascular intimal fibrosis and glomerulo- sclerosis, reflecting damage to different renal compartments. Genome-wide association studies (GWAS) have identified candidate susceptibility loci for CKD. The mechanistic basis of GWAS-variant associations with CKD are largely undescribed, hindering translation of GWAS information for the development of novel therapies for CKD. We previously showed that a CKD-associated SHROOM3 locus - Rs17319721, in the donor kidney increased SHROOM3 expression (by TCF7L2-dependent transcription) and promoted renal allograft fibrosis (IF/TA), through TGF-β1 signaling. These data contrast with evidence of a protective role for Shroom3 in glomerular development. Consistently, opposing associations were seen between glomerular- and non- glomerular SHROOM3, and renal function in CKD biopsies. In allografts, homozygosity at Rs173198721 ie A/A, which associated with lower GFR, was associated with reduced albuminuria by 2-years post-transplant. GWAS data also showed that Rs17319721 was associated with reduced albuminuria but reduced eGFR. To study mechanism of these dichotomous effects, we used inducible Shroom3 knockdown mice and observed reduced renal fibrosis with tubular Shroom3 knockdown. In tubular cells, Rho kinase (ROCK) inhibitors or ROCK-binding (ASD2-) domain deletion in Shroom3 reduced profibrotic signaling. Conversely, glomerular-, but not tubular-Shroom3 knockdown, induced albuminuria with diffuse podocyte foot process effacement without podocyte loss. This phenotype is similar to human minimal change disease (MCD). In podocytes, we identified & confirmed the novel interaction of SHROOM3 with FYN (a Src kinase) via a critical Src homology-3 binding domain, distinct from its ASD2-domain. In vitro and in vivo, Shroom3-Fyn interaction was required for activation of Fyn kinase and downstream Nephrin phosphorylation and actin cytoskeleton in podocytes. This novel mechanism explains the protective effect of Shroom3 (& Rs17319721) on proteinuria in adults. We hypothesize that SHROOM3 has dichotomous roles in renal tubular cells and podocytes, that are mediated by distinct protein motifs. We will test our hypothesis by three aims. In aim-1, we will overeprexpress ASD2- domain deficient Shroom3 in vitro/in vivo to confirm ASD2-domain dependent profibrotic signaling by Shroom3. In aim-2, Fyn-binding mutant Shroom3 will be overexpressed to confirm podocyte injury and phenotype in vivo. Since impaired Fyn activation is associated with MCD, and Shroom3 knockdown inhibited Fyn, in aim-3, we will investigate the relevance of Shroom3-Fyn interaction to human MCD by comparative glomerular morphometry and genomics utilizing human data from the Nephrotic syndrome NEPTUNE consortium. This work is essential to developing domain-specific Shroom3 inhibitors for fibrosis in CKD and IF/TA.