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/项目摘要肌动蛋白相关运动蛋白非肌肉肌球蛋白II同工型A（NM2A）的突变，由MyH9编码，具有至少有三分之一的患者与肾脏疾病有关。以前的工作着重于 MyH9在足细胞肌动蛋白细胞骨架中。我们最近的工作已经对MYH9和MYH10基因发挥了关键作用在成年小鼠肾上皮中。成年小鼠肾脏中MYH9和10的诱导性，有条件的淘汰（CKO）上皮细胞导致渐进的管状疾病，并在较厚的上升肢（TAL）中具有转运缺陷。 MYH9和10蛋白的损失导致沿着GPI锚定蛋白尿素蛋白（UMOD）的运输沿随着ER应力和展开的蛋白质反应途径的上调，促进了管状损伤和疾病 MYH9和10 CKO小鼠。此外，Na+ K+ 2Cl-共转运蛋白（NKCC2）不定位于顶膜我们观察到MYH9和10 CKO小鼠肾脏中NKCC2蛋白水平的逐渐下降。单一旁系同源肾小管特异性MYH9-PT CKO小鼠也会患上中等管状肾脏疾病小鼠中的MYH9-CKO不会导致肾脏疾病。在这里，我们建议确定特定细胞类型的角色对于TAL上皮和足细胞中的MYH9及其对肾脏疾病的贡献。我们创造了小说，永生的TAL细胞培养系统可以长期体外货物运输研究。我们将利用单个 Paralog MyH9和MyH10泛肾小管（PT）CKO，TAL特异性CKO和Podocyte特异性MYH9-CKO 小鼠模型可以在两个不同的肾脏中解剖NM2的机械和生理作用细胞类型。将测试高盐饮食和性依赖性变异对疾病病理学的影响。鼠标在足细胞和TAL上皮中都有MyH9-CKO的模型将被表征以确定协同的病理效应，可以更好地对MYH9-RD的严重形式进行建模，并完全失去功能。我们提出的工作将发现NM2电动机蛋白在专业货物运输中的关键作用，并将确定与MYH9突变有关的新型机制相关肾脏疾病和其他与TAL相关的机制肾脏疾病。