Conditional Myh9 and Myh10 inactivation in adult mouse renal epithelium results in progressive kidney disease
Karla L. Otterpohl, Brook W. Busselman, Ishara Ratnayake, Ryan G. Hart, Kimberly R. Hart, Claire M. Evans, Carrie L. Phillips, Jordan R. Beach, Phil Ahrenkiel, Bruce A. Molitoris, Kameswaran Surendran, Indra Chandrasekar
Karla L. Otterpohl, Brook W. Busselman, Ishara Ratnayake, Ryan G. Hart, Kimberly R. Hart, Claire M. Evans, Carrie L. Phillips, Jordan R. Beach, Phil Ahrenkiel, Bruce A. Molitoris, Kameswaran Surendran, Indra Chandrasekar
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Research Article Cell biology Nephrology

Conditional Myh9 and Myh10 inactivation in adult mouse renal epithelium results in progressive kidney disease

Abstract

Actin-associated nonmuscle myosin II (NM2) motor proteins play critical roles in a myriad of cellular functions, including endocytosis and organelle transport pathways. Cell type–specific expression and unique subcellular localization of the NM2 proteins, encoded by the Myh9 and Myh10 genes, in the mouse kidney tubules led us to hypothesize that these proteins have specialized functional roles within the renal epithelium. Inducible conditional knockout (cKO) of Myh9 and Myh10 in the renal tubules of adult mice resulted in progressive kidney disease. Prior to overt renal tubular injury, we observed intracellular accumulation of the glycosylphosphatidylinositol-anchored protein uromodulin (UMOD) and gradual loss of Na+ K+ 2Cl– cotransporter from the apical membrane of the thick ascending limb epithelia. The UMOD accumulation coincided with expansion of endoplasmic reticulum (ER) tubules and activation of ER stress and unfolded protein response pathways in Myh9&10-cKO kidneys. We conclude that NM2 proteins are required for localization and transport of UMOD and loss of function results in accumulation of UMOD and ER stress–mediated progressive renal tubulointerstitial disease. These observations establish cell type–specific role(s) for NM2 proteins in regulation of specialized renal epithelial transport pathways and reveal the possibility that human kidney disease associated with MYH9 mutations could be of renal epithelial origin.

Authors

Karla L. Otterpohl, Brook W. Busselman, Ishara Ratnayake, Ryan G. Hart, Kimberly R. Hart, Claire M. Evans, Carrie L. Phillips, Jordan R. Beach, Phil Ahrenkiel, Bruce A. Molitoris, Kameswaran Surendran, Indra Chandrasekar

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Figure 3

Loss of MYH9 and MYH10 does not affect the localization of proximal tubule–associated sodium cotransporters NHE3 and SGLT2.

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Loss of MYH9 and MYH10 does not affect the localization of proximal tubu...
(A–F) Bouin’s-fixed kidney sections from 9-week-old cKO mice and control littermates were stained using NHE3 antibody along with Oregon green–488–wheat germ agglutinin (WGA) and DAPI. (A–C) NHE3 (red) localizes to the PCT brush borders and does not colocalize with WGA (A and B); intracellular NHE3 staining was also observed. (D–F) NHE3 staining was reduced in cKO mouse kidney sections, and partial loss of expression along the apical membrane was observed in some tubules with loss of brush border (F, white arrowhead). Adjacent tubular segments that are severely dilated are denoted with asterisks (D–F). (G–L) Kidney sections from 9-week-old cKO mice and control littermates were stained using an SGLT2 antibody and WGA. (G–I) Control kidney sections show positive staining for SGLT2 (red) along the brush borders. (J–L) In the cKO kidneys, SGLT2 expression varied between tubules. Some tubules showed decreased expression (white arrowhead), while other tubules maintained SGLT2 expression (white arrows). Asterisks (*) mark adjacent tubular segments that are severely dilated (J and K). The white dotted squares (B, E, H, and K) denote regions enlarged in C, F, I, and L. Scale bar: 10 μm. Images are representative of n ≥ 3 kidneys for control and cKO samples. (M and N) Graphs represent MFI for NHE3 (M) and SGLT2 (N) that show statistically significant reduction in staining intensity in cKO mice. Control tubules (n = 96 for NHE3 and n = 69 for SGLT2), cKO tubules (n = 108 for NHE3 and n = 102 for SGLT2). Error bars show standard deviation of samples. *P < 0.0001, calculated by unpaired 2-tailed t test.