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

Shroom3 在天然肾和同种异体移植肾的管状细胞和足细胞中的二分作用

• 批准号: 10015268
• 负责人: Madhav C Menon
• 金额: $ 38.14万
• 依托单位: ICAHN SCHOOL OF MEDICINE AT MOUNT SINAI
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-10 至 2020-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10015268
• 关键词: Actins; Adult; Albuminuria; Allografting; Amino Acid Motifs; Binding; Biopsy; Blood Vessels; Cell Size; Cells; Chronic Kidney Failure; Cytoskeletal Modeling; Cytoskeleton; Data; Development; Diffuse; Disease; Disease Progression; Drug Targeting; End stage renal failure; Enhancers; Etiology; F-actin-binding proteins; FYN gene; Fibrosis; Focal Segmental Glomerulosclerosis; Foot Process; GTP-Binding Protein alpha Subunits, Gs; Genetic Transcription; Genomics; Glomerular Filtration Rate; Histologic; Histology; Human; Human Genome; Impairment; In Vitro; Injury; Kidney; Kidney Diseases; Link; Mediating; Modeling; Morphology; Mus; Mutation; Nephrectomy; Nephrotic Syndrome; Outcome; Pathway interactions; Patients; Phenotype; Phosphorylation; Phosphotransferases; Production; Proteins; Proteinuria; Regulation; Renal Tissue; Renal function; Rho-associated kinase; Role; Signal Transduction; Single Nucleotide Polymorphism; Susceptibility Gene; TCF7L2 gene; Tertiary Protein Structure; Testing; Transforming Growth Factor beta; Transgenic Organisms; Translations; Tubular formation; Variant; Work; base; biophysical properties; cell type; cohort; comparative; disease phenotype; experimental study; genome wide association study; glomerulosclerosis; human data; in vivo; inhibitor/antagonist; interstitial; kidney allograft; kidney fibrosis; knock-down; morphometry; mutant; nephrin; novel; novel therapeutics; overexpression; podocyte; post-transplant; protective effect; src-Family Kinases; transcriptome

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.

项目摘要：慢性肾脏病(CKD)被确定为肾功能下降，由 肾小球滤过率(EGFR)和/或蛋白尿的存在。通过组织学在本地和 CKD以肾间质纤维化、血管内膜纤维化和肾小球纤维化为主要特征。 硬化症，反映出不同的肾间室受损。全基因组关联研究(GWAS)已经 确定了CKD的候选易感基因座。GWA型变异与慢性肾脏病关联的机制基础 在很大程度上没有描述，阻碍了GWAS信息的翻译，以开发新的治疗方法 CKD。我们先前在供体肾脏中发现了一个与CKD相关的SHROOM3基因座-Rs17319721。 SHROOM3表达增加(通过TCF7L2依赖的转录)并促进移植肾纤维化 (IF/TA)，通过转化生长因子-β-1信号转导。这些数据与Shroom3在 肾小球发育。一贯地，在肾小球和非肾小球之间可以看到相反的联系。 肾小球SHROOM3与慢性肾脏病肾功能的关系。在同种异体移植中，173198721卢比的纯合性 A/A与肾小球滤过率降低有关，与移植后2年尿蛋白减少有关。 Gwas数据还显示，Rs17319721与蛋白尿减少但EGFR减少有关。至 我们利用可诱导的Shroom3基因敲除小鼠观察了这些二分性效应的机制。 通过击倒小管Shroom3减少肾脏纤维化。在肾小管细胞中，Rho激酶(ROCK)抑制剂或 Shroom3中岩石结合(ASD2-)结构域的缺失减少了促纤维化信号。相反，肾小球-，但 非小管状Shroom3基因敲除，诱导蛋白尿伴弥漫性足细胞足突消失 足细胞丢失。这种表型类似于人类微小病变病(MCD)。在足细胞中，我们识别出 &证实了SHROOM3与FYN(一种Src激酶)通过关键的Src同源-3结合而发生的新的相互作用 结构域，与其ASD2-结构域不同。在体外和体内，需要Shroom3-Fyn相互作用才能激活 足细胞中的Fyn激酶及其下游的Nephin磷酸化和肌动蛋白细胞骨架。这部小说 机制解释了Shroom3(&Rs17319721)对成人蛋白尿的保护作用。我们 假设SHROOM3在肾小管细胞和足细胞中具有二分性作用，这是由 不同的蛋白质模体。我们将通过三个目标来检验我们的假设。在AIM-1中，我们将超越ASD2- 结构域缺陷的Shroom3在体外/体内通过Shroom3确认ASD2结构域依赖的促纤维化信号。 在AIM-2中，Fyn结合突变体Shroom3将在体内过表达以确认足细胞损伤和表型。 由于Fyn激活受损与MCD有关，而Shroom3基因敲除抑制了Fyn，在AIM-3中，我们 将通过比较肾小球研究Shroom3-Fyn相互作用与人类MCD的相关性 形态计量学和基因组学利用来自肾病综合征海王星联盟的人类数据。这 这项工作对于开发针对CKD和IF/TA纤维化的区域特异性Shroom3抑制剂至关重要。