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Renal ischemia: implications in mitochondrial function and cardiolipin structure
Date Issued |
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2023-04-19 |
Clinical and preclinical research
Bibliogr.: p. 64
Introduction Cardiolipin is a unique phospholipid found in mitochondrial membranes, and is important in optimising the structure and function of OXPHOS complexes and participates in mitochondrial autophagy and cell apoptosis [1]. Four fatty acids in cardiolipin structure make up a variety of its forms, which are specific to different organs. The location of cardiolipin at major ROS-producing sites and its polyunsaturated fatty acids makes this phospholipid sensitive to oxidation [2]. Changes in cardiolipin structure followed by mitochondrial and cellular dysfunction is observed in many pathologies, especially during oxidative stress in ischemia/reperfusion injury [3]. Little is known about renal cardiolipin and its alterations during renal ischemia and reperfusion, which is not only a part of renal disease pathophysiology but is also induced during surgical procedures such as removal of cancer. Aim The aim of this study was to investigate the effect of ischemia in vitro and in vivo on mitochondrial function and cardiolipin alterations. Methods Adult Wistar male rats were used as in vivo ischemia model – under anesthesia the abdomen was opened and renal blood vessels were clamped for 30-60 minutes, kidneys were harvested for mitochondrial extraction using differential centrifugation. Human renal proximal tubule epithelial cells (RPTEC/TERT1) were used for in vitro hypoxia studies. Cells were placed in a hypoxic chamber (2 % oxygen), the growth medium was replaced with medium that was kept in the hypoxic chamber overnight. For reoxygenation the cells were replaced in standard incubating conditions with fresh medium change. Cell viability and proliferation were assessed by fluorescence microscopy using PI and Hoechst 33342 dyes. Mitochondrial respiration was assessed using Oroboros Oxygraph-2k. Cardiolipin analysis was performed using UPLCMS. Results are presented as means ± standard deviation of 3-5 experiments. Results It was determined that major renal cardiolipin species in rat kidneys were tetralinoleoyl (CL (C18:2)4) and trilinoleoyl-mono-oleoyl cardiolipins (CL(C18:2)3/C18:1). After 30, 40 and 60 min in vivo renal ischemia the amounts of these cardiolipins decreased on average by 46, 68 and 75 % respectively, this was led by a decrease in mitochondrial respiration rates. Mass spectrometry revealed that CL (C18:2)4 is oxidized by adding 1-8 extra oxygen atoms and the level of these oxidized forms significantly increased about 7-foldafter 40 minutes of in vivo ischemia. In another set of experiments, we determined that the major cardiolipin species in human renal RPTEC cells was CL (C18:1)2/C18:2/C16:1, although significant amounts of CL (C18:2)4 and CL(C18:2)3/C18:1 were detected. After 24-hour hypoxia the amounts of CL (C18:2)4 andCL (C18:2)3/C18:1 decreased 30 % and 78 % respectively, however the dominant cardiolipin amount increased 2,5 times. In addition, 24-hour hypoxia significantly reduced mitochondrial respiration rates in phosphorylation and uncoupled states on average by 29 %, the respiratory control index was reduced by52 %. No changes in cell viability were observed after 24-hour hypoxia, however cell proliferation was reduced by 28 %. Conclusions In this study it was shown that there are differences in cardiolipin composition between rat kidney mitochondria and human renal proximal tubule cells. It was also shown that ischemia/hypoxia induces mitochondrial dysfunction and cardiolipin alterations in rat kidney mitochondria as well as in human renal cells.