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

Abstract

Genetic ablation of calcium-independent phospholipase A2γ (iPLA2γ) in mice results in marked damage of mitochondria and enhanced autophagy in glomerular visceral epithelial cells (GECs) or podocytes. The present study addresses the role of iPLA2γ in glomerular injury. In adriamycin nephrosis, deletion of iPLA2γ exacerbated albuminuria and reduced podocyte number. Glomerular LC3-II increased and p62 decreased in adriamycin-treated iPLA2γ knockout (KO) mice, compared with treated control, in keeping with increased autophagy in KO. iPLA2γ KO GECs in culture also demonstrated increased autophagy, compared with control GECs. iPLA2γ KO GECs showed a reduced oxygen consumption rate and increased phosphorylation of AMP kinase (pAMPK), consistent with mitochondrial dysfunction. Adriamycin further stimulated pAMPK and autophagy. After co-transfection of GECs with mito-YFP (to label mitochondria) and RFP-LC3 (to label autophagosomes), or RFP-LAMP1 (to label lysosomes), there was greater colocalization of mito-YFP with RFP-LC3-II and with RFP-LAMP1 in iPLA2γ KO GECs, compared with WT, indicating enhanced mitophagy in KO. Adriamycin increased mitophagy in WT cells. Thus, iPLA2γ has a cytoprotective function in the normal glomerulus and in glomerulopathy, as deletion of iPLA2γ leads to mitochondrial damage and impaired energy homeostasis, as well as autophagy and mitophagy.

Role of IRE1α in podocyte proteostasis and mitochondrial health

“…In cultured GECs, maximal and ATP-coupled mitochondrial OCR were reduced by IRE1α deletion even under basal conditions, consistent with a report in another cell line 43 . The importance of resting mitochondrial metabolism for podocyte homeostasis remains to be determined 44 ; nonetheless, in the context of disease, mitochondrial function appears to be essential for podocyte health 45,46 . Of note, after incubation with adriamycin, ATP-linked respiration was further reduced in IRE1α KO GECs, consistent with the prominent mitochondrial injury in podocytes of adriamycin-treated IRE1α KO mice.…”

“…Adult male IRE1α KO mice and control littermates (11-13 weeks old) received adriamycin (doxorubicin; MilliporeSigma) in a single injection through the tail vein at a dose that induces only modest injury in control mice (18 mg/kg) 45,50 . Untreated mice were also littermates.…”

“…Untreated mice were also littermates. The number of mice required for this study was based on our previous experience with studies of adriamycin nephrosis 45,50 . Urine collections were performed weekly for 4 weeks until the mice were sacrificed.…”

“…Images were captured in a Zeiss Axio Observer fluorescence microscope with visual output connected to an AxioCam MRm monochrome camera (Carl Zeiss AG; Toronto, ON). Quantification of immunofluorescence intensity was described previously 45,50 .…”

Autophagy Function and Regulation in Kidney Disease

“…In a sepsis model of cecal ligation and puncture, nicotinamide adenine dinucleotide NAD-dependent protein deacetylase SIRT3, a member of the mammalian sirtuin family of proteins, provided protection from sepsis-induced AKI through AMPK/mTOR-regulated autophagy [100]. In adriamycin-treated calcium-independent phospholipase A2 (iPLA2)γ knockout (KO) mice, both AMPK activation and autophagy were increased compared with treated control mice [101]. Heme oxygenase-1 (HO-1)-mediated autophagy is AMPK dependent and has provided protection against high -glucose-induced podocyte apoptosis [102].…”