(727.17) Thick ascending limb specific inactivation of Myh9 and Myh10 results in progressive kidney disease with sexually dimorphic sodium transport regulation

Poster Board Number: E210

Karla Otterpohl (Sanford Research), Brook Busselman (Sanford Research), Jenna Zimmerman (Sanford Research), Kameswaran Surendran (Sanford Research), Indra Chandrasekar (Sanford Research)

Mutations in MYH9 gene that encodes for actin associated molecular motor nonmuscle myosin II (NM2) result in kidney disease in some patients. Based on our previous work that demonstrated a novel role for NM2 motors, we hypothesized that NM2 genes might play a critical role in renal epithelial transport. To test our hypothesis, we generated pan renal tubular conditional knockout (cKO) of Myh9amp;10 in adult mice that resulted in progressive kidney disease of TAL origin with aberrant transport of TAL cargoes UMOD and NKCC2 (Otterpohl et al; 2020). To confirm the cell-autonomous role for NM2 proteins in adult TAL epithelium, we generated a TAL-specific cKO mouse model of Myh9amp;10 using a TAL specific cre transgenic mice, UMOD-gt;CreERT2. Cre transgene was induced at 5 weeks of age with tamoxifen injections, age matched control and cKO littermates were part of every cohort. The mice underwent metabolic, histologic, immunohistochemical, and biochemical analysis at varying time points until moribund. The TAL-specific Myh9amp;10 cKO mice develop progressive kidney disease indicated by increase in BUN and serum creatinine as well as increasing levels of the tubular injury marker, NGAL. Our results show that Myh9amp;10 TAL- cKO females had significant loss of weight at 12 weeks, the female cKO animals became moribund between 13 – 14 weeks, while the male cohorts survive up to 25 weeks. Histology confirmed the tubulointerstitial disease in Myh9amp;10 TAL-cKO with dilated tubules and cellular infiltration. Urine pH declined starting at 9-weeks of age in females and 12-week in males. Interestingly, female cKO mice have hypernatremia at 12-weeks of age compared to male mice which maintain normal sodium levels. Since the pan renal tubular Myh9amp;10 cKO exhibited loss of NKCC2 leading to salt wasting, this result was unexpected. We analyzed NKCC2 protein levels in kidney lysates, loss of NKCC2 was evident in both female and male Myh9amp;10 TAL-cKO. We also observe significant decline in serum potassium levels in 7.5-week females. We speculated that the hypernatremia in TAL-Myh9amp;10 cKO females is due to tubular adaptation in the distal nephron and the collecting duct. Characterization of sodium cotransporters and channels in distal nephron and collecting duct segment revealed an increase in NCC/pNCC levels in males and a decrease in females. Immunostaining experiments showed increase in ENaC gamma expression in the cortical collecting ducts of the females. This could potentially explain the hypernatremia and early morbidity observed in Myh9amp;10 TAL-cKO females. We speculate that females upregulate ENaC gamma to compensate for loss of NKCC2, instead males upregulate NCC/pNCC that prevents their early lethality. These experiments confirm the critical role for Myh9amp;10 proteins in TAL tubule and identify a novel, sex specific tubular adaptation mechanisms. NM2 motor regulating the unique, TAL-specific sodium cotransporter is intriguing and warrants further analysis. Our model also provides a good resource for understanding the sex-specific differences of sodium handling and potentially clarify mechanisms underlying Type I Bartter Syndrome.