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

摘要/项目总结 由MYH 9编码的肌动蛋白相关运动蛋白非肌肉肌球蛋白II亚型A（NM 2A）的突变， 至少有三分之一的患者与肾脏疾病有关。以前的工作重点是 足细胞肌动蛋白细胞骨架中的myh 9。我们最近的工作已经确定了Myh 9和Myh 10基因的关键作用 在成年小鼠的肾上皮中。成年小鼠肾脏中Myh 9和10的诱导性条件性敲除（cKO） 上皮细胞导致进行性肾小管疾病伴粗升支转运缺陷（TAL）。 Myh 9和10蛋白的缺失导致GPI锚定蛋白尿调素（UMOD）的转运失调，沿着 与ER应激和未折叠蛋白反应途径的上调，促进肾小管损伤和疾病， Myh 9和10 cKO小鼠。此外，Na+ K+ 2Cl-协同转运蛋白（NKCC 2）不定位于顶膜 并且我们观察到Myh 9和10 cKO小鼠肾脏中NKCC 2蛋白水平的进行性下降。单身派对 肾小管特异性Myh 9-PT cKO小鼠也发生中度肾小管性肾病，但足细胞特异性Myh 9-PT cKO小鼠也发生中度肾小管性肾病。 小鼠中的Myh 9-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， 对于TAL上皮和足细胞中的Myh 9及其对肾脏疾病的贡献。我们创造了新的， 永生化TAL细胞培养系统，以实现长期的体外货物转运研究。我们将利用单一的 Parkinson Myh 9和Myh 10泛肾小管（PT）cKO、TAL特异性cKO和足细胞特异性Myh 9-cKO 小鼠模型，以便能够解剖两种不同肾脏中NM 2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。鼠标 将表征在足细胞和TAL上皮中均含有Myh 9-cKO的模型，以确定 协同病理学作用，这将更好地模拟具有完全功能丧失的MYH 9-RD的严重形式。 我们提出的工作将揭示NM 2马达蛋白在专门的货物运输中的关键作用， 鉴定MYH 9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.