RAPA is a new and potent macrolide immunosuppressant which is now being used in drug combination strategies with CsA. CsA has significantly improved allograft survival. However, it can be associated with nephrotoxicity, characterized by tubulointerstitial fibrosis and tubular atrophy, which may be due, in part, to the profibrogenic effect of TGF-β1. Induction of TGF-β1 expression by CsA has been shown in numerous cell lines, and in animal models. We have previously shown that CsA enhances TGF-β1 expression in IRPTC in a dose-dependent manner (100 to 5000 ng/ml). Recently, in a salt-depleted rat model, coadministration of RAPA and CsA appeared to increase CsA-induced nephrotoxicity. The present studies explored whether RAPA potentiates CsA-induced TGF-β1 expression. At confluence, IRPTC were placed in defined serum free media × 24hr and treated with RAPA(10;50;100; or 200 nM) or CsA (500ng/ml), RAPA (200nM) or CsA + RAPA (500ng/ml and 200nM) × 24 hr. Cells were harvested after 24hr for northern blotting (25μg mRNA) and hybridized with a TGF-β1 cDNA probe and then normalized by hyrdization to a cDNA for GAPDH. RAPA, even at a dose of 10 ng/ml increased the expression of TGF-β1 (1.3 fold). RAPA alone or in combination with CsA increased TGFβ1 mRNA expression without apparent synergy at the doses used. Table

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Since TGF-β1 may increase fibrogenesis, additive effects between CsA and RAPA on TGF-β1 expression may be associated with higher nephrotoxicity with implications for long-term allograft survival and function.