ABSTRACT: The use of calcineurin inhibitor (CI) immunosuppressants has significantly improved the early allograft survival rate in organ transplantation. However, CI therapy has been associated with chronic nephrotoxicity, which limits their long-term utility. In order to understand the mechanisms of the toxicity, we analyzed the gene expression changes that underlie the development of CI immunosuppressant-mediated nephrotoxicity, in male Sprague-Dawley (SD) rats dosed daily with cyclosporine (CsA), FK506 or rapamycin (Rapa) for 1 to 28 days. We identified a group of genes, whose expression in rat kidney is quantitatively correlated with CI-induced kidney injury as observed in changes in blood urea nitrogen (BUN) levels and kidney histopathology. These genes include both up-regulated genes, such as Ren1 and Klks3, and down-regulated genes, such as Calb1, Egf, NCC, and kidney specific Wnk1 (KS-Wnk1). Using the down-regulated genes alone we successfully predicted CI immunosuppressant-mediated kidney injury in rats following 7 days of treatment. Among these genes are two mechanism-related genes, NCC and KS-Wnk1, both of which are involved in the sodium transport in the distal nephrons. The down-regulation of both genes at the mRNA and protein level in rat kidney following CI treatment was confirmed by quantitative RT-PCR and immunohistochemical staining, respectively. We hypothesize that decreased expression of NCC may cause reduced sodium chloride reabsorption in the distal tubules, and contribute to the prolonged activation of the Renin-Angiotensin-System (RAS), a demonstrated contributor to the development of CI-induced nephrotoxicity in both animal models and clinical settings. Therefore, NCC and KS-Wnk1 could potentially be used as biomarkers for early detection and prevention of CI-related nephrotoxicity in clinical practice. Male SD rats approximately 5-7 weeks old were maintained on certified rodent chow and dosed i.p. with olive oil, CsA (CAS # 59865-13-3, 2.5 or 25 mg/kg/day), FK506 (CAS # 104987-11-3, 0.6 or 6 mg/kg/day), or Rapa (CAS # 53123-88-9, 1 or 10 mg/kg/day) (LC Laboratories, Woburn, MA) for 1, 7, 14 or 28 consecutive days. Blood was collected at the terminal necropsy (24 hours after 1, 7, 14 or 28 days of treatment) for clinical chemistry analysis. Each kidney was cut in half (one cross section and the other longitudinal). One half of the left kidney and one half of the right kidney were used for histopathological evaluation and the remaining portions of the kidney were processed for gene expression analysis. Total RNA was isolated from rat kidney using QIAGEN RNeasy kits (QIAGEN Inc., Valencia, CA) following manufacture’s instruction. The rat genome array (RAT 230 2.0) GeneChip (Affymetrix, Santa Clara, CA) was used for gene expression profiling.
