Carbon Monoxide (CO) Protects Renal Tubular Epithelial Cells against Cold-Rewarm Apoptosis

Abstract

Recent studies have suggested that carbon monoxide (CO) inhalation can reduce ischemia-reperfusion injury of kidneys. The purpose of the present study was to determine whether the direct application of CO using tricarbonylchloro (glycinato) ruthenium II (CORM3) would reduce cold-rewarm-associated apoptosis in renal tubular epithelial (RPTE) cells. RPTE cells were subjected to 48 hours of cold followed by 24 hours of rewarming with increasing concentrations (0–500 μM) of CORM3. CORM3 (100 μM) reduced apoptosis as determined by the TUNEL method from 21.6 ± 5.2 to 5.8 ± 1.1 % (untreated vs. treated, n = 5; p < 0.001). We subsequently observed that the incubation of RPTE cells with CORM3 induced heme oxygenase (HO)-1 gene expression. As HO-1 itself can confer protection against cold rewarm injury, we investigated the role of HO-1 in the protective actions of CORM3 using siRNA oligonucleotides directed against HO-1. CORM3 treatment of RPTE cells caused a 4.9- fold increase in HO-1 gene expression as determined by real time PCR. Prior treatment of RPTE cells with siRNAs against HO-1 was able to completely abolish the CORM3 mediated induction of HO-1 mRNA and protein. The abolition of HO-induction with siRNAs did reduce CORM3-mediated protection against cold rewarm-induced apoptosis; however, CORM3 was able to significantly protect RPTE cells against cold-rewarm injury: apoptosis was 33.7 ± 0.9% vs. 15.4 ± 0.5% vs. 62.8 ± 1.5% vs. 23.5 ± 3.4 in control cold-rewarm vs. cold-rewarm + CORM3 (100 μM) vs. cold-rewarm + HO-1 siRNA vs. cold-rewarm + CORM3 (100 μM) + HO-1 siRNA (n = 4). These results suggest that increased levels of CO alone can protect against cold-rewarm-induced apoptosis.