Kidney Week

Abstract: FR-PO926

Genetic Ablation of Calcium-Independent Phospholipase A₂γ Exacerbates Glomerular Injury in Adriamycin Nephrosis in Mice

Session Information

• Glomerular Diseases: Podocyte Biology - II
  November 08, 2019 | Location: Exhibit Hall, Walter E. Washington Convention Center
  Abstract Time: 10:00 AM - 12:00 PM

Category: Glomerular Diseases

• 1204 Podocyte Biology

Authors

• Elimam, Hanan, McGill University, Montreal, Quebec, Canada

Hanan Elimam,

• Guillemette, Julie, McGill University, Montreal, Quebec, Canada

Julie Guillemette,

• Papillon, Joan, McGill University, Montreal, Quebec, Canada

Joan Papillon,

• Cybulsky, Andrey V., McGill University, Montreal, Quebec, Canada

Andrey V. Cybulsky,

Background

Genetic ablation of calcium-independent phospholipase A₂γ (iPLA₂γ) in mice resulted in marked damage of mitochondria and enhanced autophagy in glomerular visceral epithelial cells (GECs) or podocytes. iPLA₂γ knockout (KO) GECs in culture show mitochondrial dysfunction and enhanced autophagy. The present study addresses the role of iPLA₂γ in glomerular injury, focusing on mitochondrial function and autophagy.

Methods

Adriamycin nephrosis was induced in wild type (WT) or iPLA₂γ KO mice (age 3.5-4.5 months) by a single intravenous injection of adriamycin (12 mg/kg). Cultured WT or iPLA₂γ KO GECs were transfected with mito-YFP (to label mitochondria), RFP-LC3 (to label autophagosomes) and RFP-LAMP1 (to label lysosomes). Colocalization of fluorescent signals was measured by the Pearson correlation coefficient.

Results

In adriamycin nephrosis, deletion of iPLA₂γ exacerbated albuminuria and reduced podocyte number (WT1 counts). Glomerular LC3-II increased and p62 decreased in adriamycin-treated iPLA₂γ KO mice, compared with treated control. In cultured iPLA₂γ KO GECs, LC3-II was increased, compared with WT cells (by immunoblotting). After transfection of WT and KO GECs with RFP-LC3, RFP-LC3-II puncta and puncta area were enhanced in KO cells, consistent with greater autophagy. iPLA₂γ KO GECs showed increased phosphorylation of AMP kinase (pAMPK), in keeping with mitochondrial dysfunction and lower cell ATP levels. Adriamycin further stimulated pAMPK and LC3-II. For comparison, induction of mitochondrial dysfunction with carbonyl cyanide m-chlorophenylhydrazone (CCCP) also increased pAMPK and LC3-II. There was greater colocalization of mito-YFP with RFP-LC3 and with RFP-LAMP1 in iPLA₂γ KO GECs, compared with WT, indicating enhanced mitophagy in KO. Adriamycin and CCCP increased colocalization of mito-YFP with RFP-LC3 and with RFP-LAMP1.

Conclusion

iPLA₂γ has a cytoprotective function in the normal glomerulus and in glomerulopathy, as deletion of iPLA₂γ leads to mitochondrial damage and impaired ATP production. Deletion of iPLA₂γ induces autophagy and mitophagy. Understanding how iPLA₂γ maintains mitochondrial integrity and attenuates mitochondrial damage in glomerular diseases is essential for development of novel therapeutic approaches to glomerular injury and proteinuria.