Date of Award

2012

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biomedical and Pharmaceutical Sciences

College

College of Graduate Studies

First Advisor

Rick G. Schnellmann

Second Advisor

Craig C. Beeson

Third Advisor

P. Darwin Bell

Fourth Advisor

John J. Lemasters

Abstract

Acute kidney injury and chronic kidney disease are becoming more prevalent and have high morbidity and mortality, and despite several therapies targeted towards treating or preventing kidney injury, outcomes have been unchanged for many years. Calcium­independent phospholipase A2y (iPLA2γ) is one of seven Ca2+-independent PLA2 enzymes and represents a potential therapeutic target in kidney disease because of its unique subcellular localization and its potential as the putative membrane remodeling/repair enzyme. Studies in our laboratory suggest iPLA2γ is cytoprotective during oxidant injury by preventing and/or repairing oxidant-induced lipid peroxidation and pointed to ER-iPLA2γ as the major component of membrane repair and remodeling. The goals of these studies are to understand the role of ER-iPLA2γ in oxidant-induced ER lipid peroxidation, Ca2+ release, loss of lipid homeostasis, and renal cell death. Our studies in isolated microsomes (ER) provide evidence that inhibition of ER­iPLA2γ results in the potentiation of oxidant-induced lipid peroxidation. Studies in renal proximal tubule cells confirmed that iPLA2γ is protective of oxidant-induced lipid peroxidation and genetic ablation of iPLA2γ in mice resulted in oxidant injury (lipid peroxidation and mitochondrial DNA damage). To determine the role of ER-iPLA2γ in oxidant-induced Ca2+ release we developed assays to measure ER Ca2+ release from isolated microsomes and in RPTC using Ca2+-sensitive fluorophores. These studies showed ER- iPLA2γ prevented oxidant-induced ER Ca2+ release. To determine the role of iPLA2γ in oxidant-induced loss of lipid homeostasis, we employed electron spray ionization-mass spectrometry (ESI-MS) to measure lipid changes (phospholipids and fatty acids) in isolated microsomes, RPTC, and iPLA2γKO mouse urine. These studies provide evidence that ER-iPLA2γ mediates oxidant-induced fatty acid release (oxidized and/or unsaturated) in isolated microsomes; iPLA2γ inhibition results in the build-up several phospholipids within renal cell membranes; and genetic ablation of iPLA2γ in mice results 1n decreased phosphatidylcholine containing phospholipids and increased fatty acids with methyl or keto additions (likely lipid hydroperoxides) in mouse urine. Our studies in RPTC demonstrated ER-iPLA2γ protects renal cell from oxidant-induced necrotic cell death by preventing ER membrane disruption and subsequent Ca2+ release. We report the first in vivo studies suggesting that genetic ablation of iPLA2γ results in kidney oxidant injury, induces compensatory mitochondrial changes and upregulation of antioxidant stress pathways, however does not result measurable loss of kidney function. Together these data support our previous findings that iPLA2γ is cytoprotective in renal cells and provide evidence that iPLA2γ is critical for the prevention and/or repair of lipid peroxidation in oxidative kidney injury.

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