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Abstract: FR-PO926

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

Session Information

Category: Glomerular Diseases

  • 1204 Podocyte Biology

Authors

  • Elimam, Hanan, McGill University, Montreal, Quebec, Canada
  • Guillemette, Julie, McGill University, Montreal, Quebec, Canada
  • Papillon, Joan, McGill University, Montreal, Quebec, Canada
  • Cybulsky, Andrey V., McGill University, Montreal, Quebec, Canada
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.