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

基本信息

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
项目类别：
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

项目摘要

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.

项目摘要：慢性肾脏病（CKD）被确定为肾功能下降。肾小球滤过率（EGFR）和/或通过蛋白尿的签名。通过组织学在本地和同种异体肾脏，CKD的特征是肾纤维化，血管内膜纤维化和肾小球 - 硬化，反映了对不同肾脏室的损害。全基因组关联研究（GWAS）具有确定CKD的候选易感性位置。 GWAS变量与CKD的机械基础在很大程度上没有描述，阻碍了GWAS信息的翻译，以开发新的疗法 CKD。我们以前证明了与CKD相关的Shroom3基因座-RS17319721，在供体肾脏中增加了Shroom3表达（通过TCF7L2依赖性转录）并促进肾脏同种异体纤维化（如果/ta），通过TGF-β1信号传导。这些数据与Shroom3在肾小球发育。始终如一地，在肾小球和非 - CKD活检中的肾小球舒适3和肾功能。在Alloycrafts中，纯合子在RS173198721 IE 与较低GFR相关的A/A与移植后2年减少了蛋白尿有关。 GWAS数据还显示，RS17319721与减少的蛋白尿相关，但减少了EGFR。到这些二分法效应的研究机制，我们使用了诱导的shroom3敲低小鼠并观察到肾小管3敲低减少肾纤维化。在管状细胞中，Rho激酶（岩石）抑制剂或 shroom3中的岩石结合（asd2-）域删除降低了纤维化信号传导。相反，肾小球 - 但是不是管状 - 棚架3敲击，诱导蛋白尿，没有弥漫性足细胞工艺能量足细胞损失。该表型类似于人类最小变化疾病（MCD）。在足细胞中，我们确定了并通过关键的SRC同源3结合确认了Shroom3与FYN（SRC激酶）的新型相互作用域，与其ASD2域不同。在体外和体内，激活需要Shroom3-Fyn相互作用 Fyn激酶和下游肾素磷酸化和肌动蛋白细胞骨架中的脑骨骼。这本小说机制解释了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作用，这些作用是由不同的蛋白质基序。我们将以三个目标检验我们的假设。在AIM-1中，我们将过度重视ASD2- 缺乏域的shroom3体外/体内，以证实shroom3的依赖性纤维化信号3。在AIM-2中，Fyn结合突变体Shroom3将过表达以确认体内的足细胞损伤和表型。由于Fyn激活受损与MCD有关，而Shroom3敲低抑制了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MCD的相关性使用肾病综合征神经财团的人类数据的形态计量学和基因组学。这工作对于开发针对CKD和IF/ta的纤维化的域特异性舒适抑制剂至关重要。