The effect of H2S on ischemia/reperfusion injury in the kidney. An in vitro and ex vivo approach

Background Kidney transplantation is a life saving therapy for patients suffering end-stage renal disease. Since there is a donor shortage, acceptance of suboptimal donors has taken place in order to bridge the gap. During the three phases of transplantation, organ retrieval, preservation and reperfusion, ischemia and reperfusion cause injury in the organ (ischemia/reperfusion injury (IRI)). In order to improve short-term outcome after transplantation, new approaches for organ procurement and preservation are necessary. Maintaining organ viability during preservation is a key element for optimal graft function and survival. Addition of hydrogen sulfide (H2S) to the preservation solution might be a way to achieve this since several studies have demonstrated that H2S protects from IRI in various tissues. It lowers metabolic demand by blockage of cytochrome c (complex IV) in the electron transport chain, and thereby reversibly induces a hypometabolic state. In this study we aimed to research the effect of H2S on IRI in the kidney in an ex vivo setting at ±15°C by usage of rat kidneys from NHB donor rats. Furthermore, we tried to develop an in vitro IRI model in a cold ischemia setting using proximal tubular kidney cells, Normal Rat Kidney (NRK-52E), and Human Kidney-2 (HK-2) cells. Materials and methods The cells were placed in an environment lacking nutrients (PBS). A layer of mineral oil was used to induce ischemia in the cells and NaHS and Na2S were added to serve as H2S donor. After this the cells were exposed to a warm (37°C) and cold (4°C) environment to induce respectively warm (organ retrieval) and cold ischemia (preservation). Reperfusion was simulated by removal of the oil and replacement of the PBS with fresh medium. Subsequently, the effect of H2S was determined by measuring cell viability. We used the ECOPS and donor rat kidneys for the ex vivo experiments to determine if H2S treatment after organ procurement could be a protective agent in NHB donation. Four different groups were studied. Results The in vitro experiments did not show a clear effect of H2S on cell viability. The ex vivo experiments demonstrated that the treatment of rat kidneys with H2S in an ECOPS-setup does not cause an extra protective effect. Conclusion The in vitro IRI model in its current design is not reproducible at this moment and therefore not applicable to determine the effect of H2S on cell viability in NRK-52E and HK-2 cells after IRI. To see if addition of H2S to preservation solution might be a way to enhance kidney preservation and thereby the outcome of transplantation, further research is necessary before a definite conclusion is drawn.