The apical membrane localization and trafficking of uromodulin (UMOD) is critically dependent on nonmuscle myosin II isoforms Myh9 and Myh10 in mouse thick ascending limb epithelial cells

Abstract

Mutations that alter the trafficking and function of uromodulin, a membrane associated GPI anchored protein expressed in the TAL segment, result in autosomal dominant tubulointerstitial kidney disease (ADTKD)1. Proper localization and function of GPI‐anchored proteins requires intact ER to Golgi to apical membrane transport. Recently Nonmuscle myosin II (NM2) have been linked to vesicular transport2, and we have identified a novel function for NM2 isoforms Myh9 (NM2A) and Myh10 (NM2B) in regulating membrane remodeling in mammalian cells3. Due to the differential expression and unique localization of NM2 isoforms in the murine renal tubules4, we hypothesized that NM2 isoforms are involved in the specialized transport pathways of membrane associated proteins in the TAL epithelial cells. Conditional genetic knockout of Myh9 & Myh10 within the renal tubules of mice results in initial mis‐localization and gradual loss of the TAL‐specific protein: Uromodulin. These mice develop rapidly progressing tubulointerstitial kidney disease and are moribund by 5–6 months of age. In order to understand the mechanisms underlying the mis‐localization of the Uromodulin, we generated an immortalized TAL cell line from the adult kidneys of Pax8‐>rtTA; Tet‐o‐cre; Myh9F/F; Myh10F/F mice crossed to mice harboring Immorto +/Tg transgene. These cells can be maintained at 33C indefinitely due to the expression of the temperature sensitive SV40 T‐antigen transgene. To promote polarization and endogenous expression of uromodulin and other membrane‐associated proteins in these cells, we have modified a recently established culture protocol with cells grown on polyester membranes at 37C on an orbital shaker5. We observe robust expression of endogenous uromodulin detected by immunohistochemistry and western blot analysis. Furthermore, induction of Myh9 and Myh10 inactivation by doxycycline treatment of these cells results in altered levels of uromodulin in the cells possibly indicating changes in N‐glycosylation of uromodulin along the ER‐Golgi transport pathway. Loss of NM2 also decreases the intracellular sodium levels in the knockout cells, observed using sodium green™ indicator. Our studies indicate a novel relationship between NM2 isoforms and uromodulin transport along the TAL segment and are suggestive of a role for MYH9 and MYH10 in tubulointerstitial kidney disease associated with abnormal renal epithelial transport.Support or Funding InformationFunding support from NIH Grant P20GM103620 and Sanford Research Program Funds.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.