The calcineurin inhibitors, (CIs) cyclosporinen, and tacrolimus are the basis of most immunosuppressive protocols after organ transplantation. The propensity of these agents to ultimately damage the very organs they were intended to protect, especially the kidney, was always recognized, but largely tolerated due to their impressive ability to improve short-term outcomes. With the target-of-rapamycin (TOR) inhibitor sirolimus, an equally potent immunosuppressant that itself is lacking the most important side effects of Cls, such as nephrotoxicity and neurotoxicity, has become available. The combination of sirolimus with CIs is attractive since it results in synergistic immunosuppressive activity. Although devoid of nephrotoxicity when administered alone, sirolimus unexpectedly enhanced cyclosporine nephrotoxicity in clinical studies. The exact biochemical mechanisms underlying immunosuppressant toxicity alone and in combination is still poorly understood. Based on our previous work, we hypothesize that both, CI-induced mitochondrial dysfunction in blood vessel endothelium leading to vasoconstriction and direct negative effects on mitochondrial kidney energy metabolism, cause CI nephrotoxicity and that TOR inhibitors enhance CI nephrotoxicity by enhancing the negative effects of CIs on mitochondrial energy metabolism. To test our hypothesis, we propose to systematically study the effect of calcineurin inhibitors and/or sirolimus on kidney and arterial endothelial cell metabolism in the rat in vivo using magnetic resonance spectroscopy (MRS) and to correlate those with histological, functional and molecular changes known to be typical for CI nephrotoxicity. We will identify the biochemical mechanisms using MRS, will evaluate the role of cyclophilin and calcineurin in the observed changes, will compare different in vivo kidney toxicity models, will evaluate the contribution of pharmacodynamic and pharmacokinetic drug interactions when calcineurin and TOR inhibitors are combined and will evaluate differences in the negative effects of the study drugs and their combinations on transplant kidneys (exposed to ischemia/reperfusion) versus non-transplant kidneys. The results of our studies (a) will give important new insights in the biochemical mechanisms underlying toxicity of immunosuppressants and their combinations, (b) will identify surrogate markers for immunosuppressant toxicity (e.g. the potential use of isoprostanes for clinical pharmacodynamic monitoring of CI toxicity) and (c) will propose assays to test interactions of immunosuppressants in terms of toxicity during pre-clinical development. ? ?
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