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

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.