Kidney Week

Abstract: FR-OR074

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

Session Information

• Genetics and Kidney Diseases: Beyond PKD
  October 26, 2018 | Location: 33C, San Diego Convention Center
  Abstract Time: 05:42 PM - 05:54 PM

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

Gentzon Hall, MD, PhD



• Kovalik, Maria Eugenia, Duke University, Durham, North Carolina, United States

Maria Eugenia Kovalik,

• Gregory, Olivia G., Duke University Medical Center, Durham, North Carolina, United States

Olivia G. Gregory,

• Lane, Brandon M., Duke University Medical Center, Durham, North Carolina, United States

Brandon M. Lane,

• Lagas, Maxwell S., University of Arizona, Phoenix, Arizona, United States

Maxwell S. Lagas,

• Wu, Guanghong, Duke University Medical Center, Durham, North Carolina, United States

Guanghong Wu,

• Chryst-Stangl, Megan, Duke Molecular Physiology Institute, Durham, North Carolina, United States

Megan Chryst-Stangl,

• Wang, Liming, Duke University Medical Center, Durham, North Carolina, United States

Liming Wang,

• Spurney, Robert F., Duke University Medical Center, Durham, North Carolina, United States

Robert F. Spurney,

• Gbadegesin, Rasheed A., Duke University Medical Center, Durham, North Carolina, United States

Rasheed A. Gbadegesin,

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 LMX1β_R246Q-induced glomerulopathy. We hypothesized that LMX1β_R246Q 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 LMX1β_WT and LMX1β_R246Q-overexpressing podocyte lines to uncover potential therapeutic targets.

Methods

We conducted RNA-seq, qPCR, immunoblot, immunofluorescence, and scratch wound healing assays in LMX1β_WT and LMX1β_R246Q-overexpressing podocytes to evaluate the effects of the LMX1β_R246Q mutation on podocyte dysmotility.

Results

We detected significantly reduced Chloride Intracellular Channel 5 (CLIC5) expression in LMX1β_R246Q podocytes relative to LMX1β_WT 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 LMX1β_R246Q podocytes. LMX1β_R246Q podocytes displayed decreased wound closure, β1-integrin expression, FAK phosphorylation and paxillin activation. Focal adhesion density was increased in LMX1β_R246Q 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 LMX1β_R246Q podocytes. To determine the effect of NO/sGC/PKG pathway agonists on podocyte motility, we treated LMX1β_R246Q-overexpressing podocytes with vardenafil (PDE5 inhibitor), cinaciguat (sGC activator) or riociguat (sGC stimulator). These NO/sGC/PKG agonists ameliorated podocyte dysmotility.

Conclusion

The LMX1β_R246Q 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 LMX1β_R246Q-induced podocyte dysmotility.

Funding

• NIDDK Support