David E. Stec

Carbon monoxide (CO) protects renal tubular epithelial cells against cold-rewarm apoptosis

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, nu2009=u20095; 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 (nu2009=u20094). These results suggest that increased levels of CO alone can protect against cold-rewarm-induced apoptosis.

Fenoldopam preconditioning: Role of heme oxygenase-1 in protecting human tubular cells and rodent kidneys against cold-hypoxic injury

Background. Kidneys from brain-dead donors are cold preserved until transplanted. However, prolonged cold storage can contribute to allograft failure. Studies suggest that donor preconditioning with dopaminergics may reduce cold-ischemic transplant injury, but whether heme oxygenase (HO)-1 induction is an underlying mechanism is not known. Objective. To test whether preconditioning with fenoldopam (FD) induce HO-1 and protect kidneys against cold storage injury and whether HO-1 plays a role in protection. Method. We used human renal proximal tubular epithelial cells, rat kidney transplants, and HO-1(−/−) null mice kidneys. Results. FD preconditioning of cells for 4 hr significantly protected against cell death from 24-hr cold hypoxia and was associated with a dose-dependent increase in HO-1 expression. In a syngeneic rat kidney transplant model, FD preconditioning for 18 hr markedly increased kidney HO-1 expression and protected kidneys against 24-hr cold-ischemic transplant injury. To test the role of HO-1, renal proximal tubular epithelial cells were treated with HO-1 small interfering RNA, followed by FD-preconditioning. Small interfering RNA inhibited the HO-1 messenger RNA expression and reversed the FD protection. Suspension of kidneys of HO-1(−/−) null and wild-type mice preconditioned with FD or saline were subjected to 24- and 48-hr cold storage. N-acetyl glucosaminidase, a specific tubular injury marker, was significantly lower in FD-preconditioned wild-type kidneys, but not in HO-1 null kidneys, suggesting a role for HO-1 in FDs preconditioning. Conclusion. Our data suggest HO-1 induction as an underlying mechanism for FD preconditioning and support the idea of testing FD preconditioning in the clinical setting. Studies are required to determine the optimum FD-preconditioning protocol.

The Modulatory Role of Heme Oxygenase on Subpressor Angiotensin II-Induced Hypertension and Renal Injury

Angiotensin II (AngII) causes hypertension (HTN) and promotes renal injury while simultaneously inducing reno-protective enzymes like heme oxygenase-1 (HO-1). We examined the modulatory role of HO on sub-pressor angiotensin II (SP-AngII) induced renal inflammation and injury. We first tested whether the SP-AngII-induced renal dysfunction, inflammation and injury are exacerbated by either preventing (chronic HO-1 inhibition) or reversing (late HO-1 inhibition) SP-AngII-induced HO (using tin protoporphyrin; SnPP). We next examined whether additional chronic or late induction of SP-AngII-induced HO (using cobalt protoporphyrin; CoPP), prevents or ameliorates renal damage. We found that neither chronic nor late SnPP altered blood pressure. Chronic SnPP worsened SP-AngII-induced renal dysfunction, inflammation, injury and fibrosis, whereas late SnPP worsened renal dysfunction but not inflammation. Chronic CoPP prevented HTN, renal dysfunction, inflammation and fibrosis, but surprisingly, not the NGAL levels (renal injury marker). Late CoPP did not significantly alter SP-AngII-induced HTN, renal inflammation or injury, but improved renal function. Thus, we conclude (a) endogenous HO may be an essential determining factor in SP-AngII induced renal inflammation, injury and fibrosis, (b) part of HOs renoprotection may be independent of blood pressure changes; and (c) further induction of HO-1 protects against renal injury, suggesting a possible therapeutic target.