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Kidney Week

Abstract: FR-OR074

The LMX1βR246Q Mutation Causes FSGS through Dysregulation of Chloride Intracellular Channel Expression and Focal Adhesion Dynamics

Session Information

Category: Genetic Diseases of the Kidney

  • 1002 Genetic Diseases of the Kidney: Non-Cystic

Authors

  • Hall, Gentzon, Duke University Medical Center, Durham, North Carolina, United States
  • Kovalik, Maria Eugenia, Duke University, Durham, North Carolina, United States
  • Gregory, Olivia G., Duke University Medical Center, Durham, North Carolina, United States
  • Lane, Brandon M., Duke University Medical Center, Durham, North Carolina, United States
  • Lagas, Maxwell S., University of Arizona, Phoenix, Arizona, United States
  • Wu, Guanghong, Duke University Medical Center, Durham, North Carolina, United States
  • Chryst-Stangl, Megan, Duke Molecular Physiology Institute, Durham, North Carolina, United States
  • Wang, Liming, Duke University Medical Center, Durham, North Carolina, United States
  • Spurney, Robert F., Duke University Medical Center, Durham, North Carolina, United States
  • Gbadegesin, Rasheed A., Duke University Medical Center, Durham, North Carolina, United States
Background

We previously reported a heterozygous missense mutation of LMX1β as a cause of nail-platella-like renal disease in two families with hereditary FSGS. Currently, there are no targeted therapies for LMX1βR246Q-induced glomerulopathy. We hypothesized that LMX1βR246Q disrupts the expression of disease-relevant molecular targets that may be amenable to pharmacotherapy. To test this hypothesis, we performed an unbiased, RNA-seq analysis in our established LMX1βWT and LMX1βR246Q-overexpressing podocyte lines to uncover potential therapeutic targets.

Methods

We conducted RNA-seq, qPCR, immunoblot, immunofluorescence, and scratch wound healing assays in LMX1βWT and LMX1βR246Q-overexpressing podocytes to evaluate the effects of the LMX1βR246Q mutation on podocyte dysmotility.

Results

We detected significantly reduced Chloride Intracellular Channel 5 (CLIC5) expression in LMX1βR246Q podocytes relative to LMX1βWT controls by RNA-seq and qPCR. CLIC5 protein expression was similarly reduced as was the expression of CLIC3 and CLIC4. Given the established roles of CLIC proteins in the regulation of foot process architecture (i.e. CLIC5) and β1-integrin recycling (i.e. CLIC3 and CLIC4), we evaluated motility in LMX1βR246Q podocytes. LMX1βR246Q podocytes displayed decreased wound closure, β1-integrin expression, FAK phosphorylation and paxillin activation. Focal adhesion density was increased in LMX1βR246Q podocytes relative to controls as evidenced by an increase in VASP staining by immunofluorescence. Gene expression of PKG II, an upstream activator of VASP, was also significantly downregulated by RNA-seq and qPCR in LMX1βR246Q podocytes. To determine the effect of NO/sGC/PKG pathway agonists on podocyte motility, we treated LMX1βR246Q-overexpressing podocytes with vardenafil (PDE5 inhibitor), cinaciguat (sGC activator) or riociguat (sGC stimulator). These NO/sGC/PKG agonists ameliorated podocyte dysmotility.

Conclusion

The LMX1βR246Q mutation may cause FSGS, in part, through the dysregulation of CLIC-mediated foot process architecture and β1- integrin recycling and through impaired of PKG-mediated VASP activation in focal adhesions. NO/sGC/PKG pathway agonists may be effective in ameliorating LMX1βR246Q-induced podocyte dysmotility.

Funding

  • NIDDK Support