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

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 至少有三分之一的患者与肾脏疾病有关。以前的工作主要集中在 Myh9在足细胞肌动蛋白细胞骨架中。我们最近的工作确定了Myh9和Myh10基因的关键作用 在成年小鼠的肾上皮细胞中。成年小鼠肾脏中可诱导的Myh9&10条件性基因敲除(CKO) 上皮细胞导致进行性肾小管疾病，并伴有粗大升肢(TAL)的运输缺陷。 Myh9&10蛋白的丢失导致GPI锚定蛋白尿调蛋白(UMOD)的非调控转运 随着内质网应激和未折叠蛋白反应通路的上调，促进肾小管损伤和疾病 Myh9&10只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蛋白水平逐渐下降。单对角线 肾小管特异性Myh9-PT CKO小鼠也会发生中度肾小管疾病，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特定角色 MYH9在TAL上皮和足细胞中的表达及其在肾脏疾病中的作用。我们创造了小说， 永生化的TAL细胞培养系统，使长期的体外货物运输研究成为可能。我们将利用单曲 Paralog Myh9和Myh10全肾小管(PT)CKO、TAL特异性CKO和足细胞特异性Myh9-CKO 解剖NM2在两个不同肾脏中的机制和生理作用的小鼠模型 单元类型。将测试高盐饮食和性别差异对疾病病理的影响。一只老鼠 在足细胞和TAL上皮细胞中都含有Myh9-CKO的模型将被鉴定以确定 协同病理效应，这将更好地模拟完全丧失功能的严重形式的Myh9-RD。 我们拟议的工作将揭示NM2马达蛋白在专门的货物运输中的关键作用，并将 确定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。

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.