Kidney Week

Abstract: FR-PO739

Mechanisms and Implications of Podocyte Autophagy in Alport Syndrome

Session Information

• Glomerular Diseases: Mechanisms and Podocyte Biology
  October 25, 2024 | Location: Exhibit Hall, Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1401 Glomerular Diseases: Mechanisms, including Podocyte Biology

Authors

• Njeim, Rachel, University of Miami Miller School of Medicine, Miami, Florida, United States

Rachel Njeim, PharmD, PhD

• Ficarella, Maria, University of Miami Miller School of Medicine, Miami, Florida, United States

Maria Ficarella, MD

• Fontanella, Antonio Miguel, University of Miami Miller School of Medicine, Miami, Florida, United States

Antonio Miguel Fontanella

• Gye, Haley, University of Miami Miller School of Medicine, Miami, Florida, United States

Haley Gye

• Kim, Jin Ju, University of Miami Miller School of Medicine, Miami, Florida, United States

Jin Ju Kim, PhD

• Mitrofanova, Alla, University of Miami Miller School of Medicine, Miami, Florida, United States

Alla Mitrofanova, PhD

• Ali, Hassan, University of Miami Miller School of Medicine, Miami, Florida, United States

Hassan Ali, PhD

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

Sandra M. Merscher, PhD

• Fornoni, Alessia, University of Miami Miller School of Medicine, Miami, Florida, United States

Alessia Fornoni, MD, PhD, FASN

Background

Alport syndrome (AS) is a hereditary disease affecting the glomerular basement membrane, leading to renal fibrosis and end stage renal disease. We have previously shown that the accumulation of lipid droplets (LD) in podocytes contributes to the pathogenesis of proteinuric kidney diseases, including AS. Chaperone-mediated autophagy (CMA) is a selective lysosomal pathway for the degradation of cytosolic proteins. Recent studies suggest that CMA also plays a crucial role in LD breakdown, a process termed chaperone-mediated lipophagy (CML). We hypothesized that dysregulated autophagy in AS exacerbates LD accumulation in podocytes, thus driving disease progression.

Methods

Publicly available CKD and AS patient RNASeq datasets (NephroSeq, GSE134011) were used to analyze differential gene expression of various autophagy markers. Autophagy genes were determined by QRT-PCR and Western blot analysis of proteins isolated from kidney cortices and immortalized podocytes derived from wildtype (WT) and Col4a3 knockout (AS) mice. To investigate lipophagy, podocytes were treated with Lalistat, a lysosomal acid lipase inhibitor, and LD accumulation was assessed. Unbiased phenotypic profiling (Cell Painting) will be employed to further investigate the role of autophagy in WT and AS podocytes treated with autophagy inducers.

Results

Transcriptomic analysis showed altered expression of various autophagy genes. LD accumulation was significantly increased in AS compared to WT podocytes. PLIN2 protein expression was significantly increased in LD isolated from AS compared to WT podocytes. mRNA and protein expression of the rate-limiting enzyme of CMA, LAMP2A, was significantly increased in AS podocytes and in kidney cortices of AS mice, suggesting activation of CML. LC3II/I and GATE16 expression were significantly increased and p62 significantly decreased in AS, further supporting activation of autophagy in AS. Treatment of AS podocytes with Lalistat further increased LD accumulation in AS podocytes, indicating that lipophagic flux is efficient in AS podocytes and that the observed LD accumulation may be attributed to lysosomal LD accumulation.

Conclusion

Taken together, our findings suggest that autophagy and CML are functional in AS but are not sufficient to reduce LD accumulation and prevent podocyte lipotoxicity.

Funding

• NIDDK Support