The experiments described in this proposal are a continuation of studies to determine the mechanism of cyclosporine A (CsA) nephrotoxicity. The in vivo administration of CsA to the Sprague-Dawley rat results in a tissue specific, dose-dependent inhibition of renal microsomal protein synthesis at the level of translation elongation. Renal cytoplasm from rats treated in vivo with CsA contains translation inhibitor, which inhibits translation when added to microsomes from tissues of untreated animals. Renal cytoplasm containing translation inhibitor was found to contain CsA and CsA metabolites M1 and M17. The addition of CsA metabolites M1 or M17, but not CsA itself, to renal microsomes in vitro produced translation inhibition. The translation inhibitor was shown to be >5kD by gel filtration chromatography. It is proposed that renal peptidil prolyl cis-trans isomerase (PPlase) plays a role in polypeptide folding during translation, and that CsA nephrotoxicity arises from an inhibition of PPlase activity. It is further proposed that translation inhibition is due to the renal formation or uptake of CsA metabolites M1 or M17, which inhibit translation after binding to a renal isoform of PPlase (cyclophilin), or to renal eukaryotic elongation factor eEF-2. The experiments outlined in this proposal seek to identify the >5kD inhibitor of renal translation and to determine the mechanism of its action.
The specific aims of the proposal are: (1) to isolate and identify the >5kD inhibitor of renal translation produced after in vivo CsA, (2) to examine renal proteins synthesized during various stages of CsA induced renal translation inhibition to determine if translation is inhibited at the stage of elongation, (3) to determine if formation of the >5kD renal translation inhibitor is correlated with renal formation or uptake of CsA metabolites M1 or M17, and (4) to determine if the >5kD renal translation inhibitor is CsA A metabolite M1 or M17 bound to renal PPlase or eEF-2. Purified renal PPlase will be examined for folding activity during protein synthesis, and PPlase will also be examined for ATPase or GTPase activity. The ability of CsA and CsA metabolites M1 and M17 to alter the phosphorylation state and activity of eEF-2 will be determined. These experiments will provide insights into the tissue-specific actions of PPlase and the role of PPlase in translation. The experiments will examine the interaction of CsA metabolites with PPlase and eEF-2, and determine their effects on ribosomal translation. If individual CsA metabolites are found to inhibit renal protein synthesis, CsA analogues can be synthesized from which these metabolites cannot be formed. These studies will provide new ways to reduce or circumvent CsA toxicity, and thereby increase the effectiveness of CsA in immunomodulation.
