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：慢性肾病（CKD）被确定为肾功能下降，估计为 肾小球滤过率（eGFR）和/或以蛋白尿的存在为标志。通过组织学在本地和 CKD的特点是肾间质纤维化、血管内膜纤维化和肾小球纤维化。 硬化，反映不同肾区的损伤。全基因组关联研究（GWAS） 确定了 CKD 的候选易感位点。 GWAS 变异与 CKD 关联的机制基础 很大程度上没有被描述，阻碍了 GWAS 信息的翻译，以开发新疗法 慢性肾病。我们之前表明，供体肾脏中存在 CKD 相关的 SHROOM3 位点 - Rs17319721 增加 SHROOM3 表达（通过 TCF7L2 依赖性转录）并促进肾同种异体移植纤维化 (IF/TA)，通过 TGF-β1 信号传导。这些数据与 Shroom3 的保护作用证据形成对比 肾小球发育。一致地，肾小球性和非肾小球性之间存在相反的关联。 肾小球 SHROOM3 和 CKD 活检中的肾功能。在同种异体移植物中，Rs173198721 处的纯合性即 A/A 与较低的 GFR 相关，与移植后 2 年白蛋白尿的减少相关。 GWAS 数据还显示，Rs17319721 与白蛋白尿减少有关，但与 eGFR 减少有关。到 为了研究这些二分效应的机制，我们使用诱导型 Shroom3 敲低小鼠并观察 通过敲除肾小管 Shroom3 减少肾纤维化。在肾小管细胞中，Rho 激酶 (ROCK) 抑制剂或 Shroom3 中 ROCK 结合 (ASD2-) 结构域的缺失减少了促纤维化信号传导。相反，肾小球-，但是 不是管状Shroom3敲低，诱导蛋白尿，伴有弥漫性足细胞足突消失，但没有 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 & 通过关键的 Src 同源性 3 结合证实了 SHROOM3 与 FYN（一种 Src 激酶）的新型相互作用 域，不同于其 ASD2 域。在体外和体内，Shroom3-Fyn 相互作用是激活所必需的 Fyn 激酶和下游 Nephrin 磷酸化和足细胞中肌动蛋白细胞骨架的影响。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作用，其介导 独特的蛋白质基序。我们将通过三个目标来检验我们的假设。在目标 1 中，我们将过度表达 ASD2- 体外/体内的 Shroom3 结构域缺陷，以确认 Shroom3 的 ASD2 结构域依赖性促纤维化信号传导。 在 goal-2 中，Fyn 结合突变体 Shroom3 将过表达，以确认足细胞损伤和体内表型。 由于 Fyn 激活受损与 MCD 相关，而 Shroom3 敲低会抑制 Fyn，因此在 goal-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 利用来自肾病综合征 NEPTUNE 联盟的人类数据进行形态测量和基因组学。这 这项工作对于开发针对 CKD 和 IF/TA 纤维化的特定领域 Shroom3 抑制剂至关重要。