Kidney Week

Abstract: TH-PO1095

Lanosterol Synthase Genetic Variant rs2254524 V642L and Its Role in the Pathogenesis of Kidney Disease

Session Information

• Glomerular Diseases: Podocyte Biology - I
  November 07, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1204 Podocyte Biology

Authors

• Macrina, Lorenza, University of Miami, Miami, Florida, United States

Lorenza Macrina,

• Manunta, Paolo, San Raffaele Scientific Institute, Milan, Milano, Italy

Paolo Manunta,

• Molina David, Judith T., University of Miami, Miami, Florida, United States

Judith T. Molina David,

• Citterio, Lorena, San Raffaele Scientific Institute, Milan, Milano, Italy

Lorena Citterio,

• Faienza, Sipontina, San Raffaele Scientific Institute, Milan, Milano, Italy

Sipontina Faienza,

• Simonini, Marco, San Raffaele Scientific Institute, Milan, Milano, Italy

Marco Simonini,

• Varona Santos, Javier T., University of Miami, Miami, Florida, United States

Javier T. Varona Santos,

• Merscher, Sandra M., University of Miami, Miami, Florida, United States

Sandra M. Merscher,

• Fornoni, Alessia, University of Miami, Miami, Florida, United States

Alessia Fornoni,

Background

A single nucleotide polymorphism in the Lanosterol Synthase (LSS) gene, rs2254524, has been recently associated with an increased susceptibility to renal damage and increased Endogenous Ouabain (EO) content in human kidney. Our hypothesis is that the presence of the genetic variant rs2254524 causes increased susceptibility to EO-mediated glomerular damage and impairs cholesterol metabolism leading to podocyte injury.

Methods

Two immortalized human podocyte cell lines, one homozygous (LSS AA) the other heterozygous (LSS AC) for the LSS mutant variant, were used in this study. LSS expression in podocytes was verified by RT-PCR, Western blot and immunofluorescence. Apoptosis and cytotoxicity were assessed 5 days post-treatment with EO using the ApoTox-Glo Triplex Assay. The number of lipid droplets (LDs) per cell was assessed by Nile Red staining after 24h of treatment with EO 10^-9 M, puromycin 100 μg/ml and TNFα 100 ng/ml, alone and in combination with CoCl₂ 100 μM.

Results

We demonstrate for the first time that LSS is expressed in human podocytes. Dose-dependent cytotoxicity of EO was observed only in LSS AA podocytes while dose-dependent apoptosis occurred in both, LSS AA and AC podocyte cell lines. Using in vitro models of podocyte injury (e.g. exposure to puromycin or inflammatory cytokine TNFα alone and in combination with CoCl₂), we demonstrated that LSS AA podocytes are more susceptible to LDs accumulation when compared to LSS AC podocytes. However, there was no increase in LDs when cells were exposed to EO.

Conclusion

Our data indicate that individuals that carry two LSS mutant variants (rs2254524) are more susceptible to EO-mediated podocyte cytotoxicity. Moreover, the mutation might render podocytes susceptibleto accumulate LDs under certain stress conditions which may contribute to podocyte injury.

Funding

• NIDDK Support