A K Salahudeen1, H Huang, P Patel, J K Jenkins. 1. Department of Medicine, University of Mississippi Medical Center, Jackson 39216-4505, USA. asalahudeen@medicine.umsmed.edu
Abstract
BACKGROUND: The recent observation that cold storage of kidneys and tubular cells causes marked increase in free radical-catalyzed F2-isoprostanes suggests that radicals might be formed during cold storage. As cold temperature is associated with reduced metabolic and enzymic activity, the notion that cold temperature causes free radical production appeared less tenable. The objective was, therefore, to seek direct evidence for the free radical production during the cold storage of human renal tubular cells, and to define the roles of extrinsic and intrinsic antioxidants in cold-induced cell injury. METHODS: Human renal tubular cells were cold-stored at 4 degrees C for varying duration in University of Wisconsin solution and subjected to mRNA analysis, biochemical measurements, and cytoprotective studies. RESULTS: Cold storage caused a time-dependent reduction in glutathione levels, and an increase in the formation superoxide, hydrogen peroxide, and hydroxyl radicals. Cold-induced lactate dehydrogenase (LDH) release, ATP depletion, DNA damage, and membrane degradation were suppressed with the inclusion of antioxidant 2-methyl aminochroman or deferroxamine. The cells that were structurally protected with antioxidants were also intact functionally, as they had significantly improved cell proliferation. To examine the effect of cold on intrinsic antioxidant gene expression, antioxidant mRNA levels were analyzed using reverse transcription-polymerase chain reaction. The gene expression of mitochondrial Mn-superoxide dismutase (SOD), but not of cytosolic Cu,Zn-SOD or of glutathione peroxidase expression increased with cold exposure. The oxidant-sensitive gene heme oxygenase I increased slightly with 48-hr cold storage. CONCLUSIONS: Cold storage of human tubular cells causes marked increase in free radicals. These are likely of mitochondrial origin as there is a differential inducement of Mn-SOD gene, and are causal to cold-induced cell injury as extrinsic antioxidants abrogated the injury. Our findings support the strategy of adding antioxidants to preservation solutions or the strategy of pre-conditioning the organs to oxidative stress to minimize cold storage-induced organ damage.
BACKGROUND: The recent observation that cold storage of kidneys and tubular cells causes marked increase in free radical-catalyzed F2-isoprostanes suggests that radicals might be formed during cold storage. As cold temperature is associated with reduced metabolic and enzymic activity, the notion that cold temperature causes free radical production appeared less tenable. The objective was, therefore, to seek direct evidence for the free radical production during the cold storage of human renal tubular cells, and to define the roles of extrinsic and intrinsic antioxidants in cold-induced cell injury. METHODS:Human renal tubular cells were cold-stored at 4 degrees C for varying duration in University of Wisconsin solution and subjected to mRNA analysis, biochemical measurements, and cytoprotective studies. RESULTS: Cold storage caused a time-dependent reduction in glutathione levels, and an increase in the formation superoxide, hydrogen peroxide, and hydroxyl radicals. Cold-induced lactate dehydrogenase (LDH) release, ATP depletion, DNA damage, and membrane degradation were suppressed with the inclusion of antioxidant 2-methyl aminochroman or deferroxamine. The cells that were structurally protected with antioxidants were also intact functionally, as they had significantly improved cell proliferation. To examine the effect of cold on intrinsic antioxidant gene expression, antioxidant mRNA levels were analyzed using reverse transcription-polymerase chain reaction. The gene expression of mitochondrial Mn-superoxide dismutase (SOD), but not of cytosolic Cu,Zn-SOD or of glutathione peroxidase expression increased with cold exposure. The oxidant-sensitive gene heme oxygenase I increased slightly with 48-hr cold storage. CONCLUSIONS: Cold storage of human tubular cells causes marked increase in free radicals. These are likely of mitochondrial origin as there is a differential inducement of Mn-SOD gene, and are causal to cold-induced cell injury as extrinsic antioxidants abrogated the injury. Our findings support the strategy of adding antioxidants to preservation solutions or the strategy of pre-conditioning the organs to oxidative stress to minimize cold storage-induced organ damage.
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