Calcineurin inhibitor (CI) therapy has been associated with chronic nephrotoxicity, which limits its long-term utility for suppression of allograft rejection. In order to understand the mechanisms of the toxicity, we analyzed gene expression changes that underlie the development of CI immunosuppressant-mediated nephrotoxicity in male Sprague-Dawley rats dosed daily with cyclosporine (CsA; 2.5 or 25 mg/kg/day), FK506 (0.6 or 6 mg/kg/day), or rapamycin (1 or 10 mg/kg/day) for 1, 7, 14, or 28 days. A significant increase in blood urea nitrogen was observed in animals treated with CsA (high) or FK506 (high) for 14 and 28 days. Histopathological examination revealed tubular basophilia and mineralization in animals given CsA (high) or FK506 (low and high). We identified a group of genes whose expression in rat kidney is correlated with CI-induced kidney injury. Among these genes are two genes, Slc12a3 and kidney-specific Wnk1 (KS-Wnk1), that are known to be involved in sodium transport in the distal nephrons and could potentially be involved in the mechanism of CI-induced nephrotoxicity. The downregulation of NCC (the Na-Cl cotransporter coded by Slc12a3) in rat kidney following CI treatment was confirmed by immunohistochemical staining, and the downregulation of KS-Wnk1 was confirmed by quantitative real-time-polymerase chain reaction (qRT-PCR). We hypothesize that decreased expression of Slc12a3 and KS-Wnk1 could alter the sodium chloride reabsorption in the distal tubules and contribute to the prolonged activation of the renin-angiotensin system, a demonstrated contributor to the development of CI-induced nephrotoxicity in both animal models and clinical settings. Therefore, if validated as biomarkers in humans, SLC12A3 and KS-WNK1 could potentially be useful in the early detection and reduction of CI-related nephrotoxicity in immunosuppressed transplant patients when monitoring the health of kidney xenographs in clinical practice.