Uncovering cargo and cell type specific molecular mechanisms of renal tubular epithelial transport

揭示肾小管上皮运输的货物和细胞类型特异性分子机制

基本信息

批准号：
10705236
负责人：
Indra Chandrasekar
金额：
$ 37.73万
依托单位：
SANFORD RESEARCH/USD
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-09-15 至 2027-04-30
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10705236
关键词：
Actins Address Adult Architecture Biochemical Biological Models Cell Culture System Cell model Cell physiology Cells Chronic Kidney Failure Code Complex Cytoskeleton Defect Diet Disease Dissection Endoplasmic Reticulum Epithelial Cells Epithelium Epstein&apos s syndrome Exhibits Functional disorder Genes Human In Vitro Individual Injury Kidney Kidney Diseases Knockout Mice Limb structure Mediating Membrane Membrane Proteins Modeling Molecular Motor Mus Mutation Myosin Type II Nonmuscle Myosin Type IIA Organelles Pathologic Pathology Pathway interactions Patients Physiological Play Point Mutation Protein Isoforms Proteins Proteinuria Renal tubule structure Research Role Severity of illness Sex Differences Site Structure System Testing Thick Tubular formation UMOD gene Up-Regulation Validation Variant Work apical membrane autosomal dominant mutation cell type comparative conditional knockout dietary salt disease phenotype endoplasmic reticulum stress experimental study functional loss glomerular function glomerulosclerosis high salt diet human disease in vivo in vivo Model insight kidney cell loss of function mouse model non-muscle myosin novel paralogous gene podocyte renal epithelium response sex superresolution imaging symporter trafficking

项目摘要

ABSTRACT/PROJECT SUMMARY Mutations in actin associated motor protein nonmuscle myosin II isoform A (NM2A), encoded by MYH9, have been associated with kidney disease in at least one-third of the patients. Previous work focused on the role of Myh9 in podocyte actin cytoskeleton. Our recent work has established a critical role for Myh9 and Myh10 genes in the adult mouse renal epithelium. Inducible, conditional knockout (cKO) of Myh9&10 in adult mouse renal epithelial cells resulted in progressive tubular disease with transport defects in the thick ascending limb (TAL). Loss of Myh9&10 proteins resulted in deregulated transport of GPI-anchored protein uromodulin (UMOD), along with upregulation of ER stress and unfolded protein response pathways, promoting tubular injury and disease in Myh9&10 cKO mice. In addition, Na+ K+ 2Cl- cotransporter (NKCC2) does not localize to the apical membrane and we observe a progressive decline in NKCC2 protein levels in Myh9&10 cKO mouse kidneys. Single paralog renal tubule specific Myh9-PT cKO mice also develop moderate tubular kidney disease, but podocyte-specific Myh9-cKO in mice does not lead to kidney disease. Here, we propose to determine the cell type specific roles for Myh9 in TAL epithelium and podocytes and their contribution to kidney disease. We have generated novel, immortalized TAL cell culture system to enable long-term in vitro cargo transport studies. We will utilize the single paralog Myh9 and Myh10 pan-renal tubular (PT) cKO, TAL-specific cKO and podocyte-specific Myh9-cKO mouse models to enable dissection of the mechanistic and physiological roles for NM2 in two different kidney cell types. Effects of high salt diet and sex-dependent variations on disease pathology will be tested. A mouse model harboring Myh9-cKO in both podocytes and TAL epithelium will be characterized to determine the synergistic pathological effect, that will better model a severe form of MYH9-RD with complete loss of function. Our proposed work will uncover the critical roles for NM2 motor proteins in specialized cargo transport and will identify novel mechanisms involved in MYH9 mutation associated kidney diseases and other TAL-associated kidney disorders.

摘要/项目摘要由 MYH9 编码的肌动蛋白相关运动蛋白非肌肉肌球蛋白 II 亚型 A (NM2A) 的突变至少三分之一的患者与肾脏疾病有关。以前的工作重点是角色足细胞肌动蛋白细胞骨架中的 Myh9。我们最近的工作确定了 Myh9 和 Myh10 基因的关键作用在成年小鼠肾上皮中。成年小鼠肾脏中 Myh9 和 10 的诱导条件敲除 (cKO) 上皮细胞导致进行性肾小管疾病，并伴有粗升肢（TAL）运输缺陷。 Myh9 和 10 蛋白的丢失导致 GPI 锚定蛋白尿调节素 (UMOD) 的运输失调，上调 ER 应激和未折叠蛋白反应途径，促进肾小管损伤和疾病 Myh9&10 cKO 小鼠。此外，Na+ K+ 2Cl- 协同转运蛋白 (NKCC2) 并不定位于顶膜我们观察到 Myh9&10 cKO 小鼠肾脏中 NKCC2 蛋白水平逐渐下降。单旁系同源物肾小管特异性 Myh9-PT cKO 小鼠也会出现中度肾小管疾病，但足细胞特异性小鼠体内的 Myh9-cKO 不会导致肾脏疾病。在这里，我们建议确定细胞类型的具体作用 TAL 上皮和足细胞中的 Myh9 及其对肾脏疾病的贡献。我们已经生成了小说，永生化 TAL 细胞培养系统可实现长期体外货物运输研究。我们将利用单一 paralog Myh9 和 Myh10 泛肾小管 (PT) cKO、TAL 特异性 cKO 和足细胞特异性 Myh9-cKO 小鼠模型能够剖析 NM2 在两个不同肾脏中的机制和生理作用细胞类型。将测试高盐饮食和性别依赖性变异对疾病病理学的影响。一只老鼠足细胞和 TAL 上皮细胞中均含有 Myh9-cKO 的模型将被表征以确定协同病理效应，将更好地模拟功能完全丧失的严重形式的 MYH9-RD。我们提出的工作将揭示 NM2 运动蛋白在专门货物运输中的关键作用，并将确定涉及 MYH9 突变相关肾脏疾病和其他 TAL 相关疾病的新机制肾脏疾病。