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 9

Expression of ER chaperone calreticulin is increased in Myh9&10-cKO mouse kidneys and partially colocalizes with UMOD-positive vesicles.

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Expression of ER chaperone calreticulin is increased in Myh9&10-cKO ...
(A–D) Images represent PFA-fixed, 9-week-old control and cKO kidney sections stained to visualize ER chaperone protein calreticulin (CALR) along with UMOD and DAPI in the TAL tubules. (A) Cross section of tubules from control kidney shows expression and localization pattern of CALR (red) along with UMOD (green). (B) Enlarged region from A (white box) reveals that in control kidneys, UMOD localizes to the apical membrane of the TAL tubule, and CALR localizes to the nuclear membrane as well as intracellular punctate structures. (C) Dilated tubules from the cKO kidney sections show excessive UMOD accumulated within the cells; colocalization of vesicular structures positive for CALR along with UMOD is apparent in some areas inside the cell. (D) Enlarged region from C (white box) depicts the partial colocalization between CALR and UMOD-positive vesicles around the nucleus in the TAL cells. Asterisks (*) in all images denote the lumen, which is dilated in the cKO kidneys. Scale bar: 10 μm. Images are representative of n ≥ 3 kidneys for control and cKO samples. (E) Whole-kidney lysates from cKO and control mice from 9-week and 12-week cohorts were subjected to immunoblot analysis to detect protein levels of CALR and TFRC (loading control). Immunoblot analysis detected a ~50 kDa band for CALR in control and cKO kidneys. Intensity of the CALR bands was increased in the 12-week-old cKO samples, but not in 9-week samples. “L” marks the ladder lanes, molecular weight labels indicate the corresponding bands. (F) The bar graph shows that calreticulin expression is increased in cKO kidneys and is statistically significant at the 12-week time point (P value = 0.002 by multiple t test, 2-tailed; n = 3 samples per group at each time point). Error bars represent standard deviation.