Cells of the renal inner medulla are challenged with high sodium und urea concentrations. The cells have developed mechanisms to prevent tonicity induced damage. The activity of the transcription factor tonicity enhanced binding protein (TonEBP) also called NFAT5 is induced by increased tonicity. This leads to the expression of so called osmoprotective genes. Several studies suggest that cyclopsorin A (CsA) inhibits the activation of TonEBP thereby mediating a part of its nephrotoxic action. We used primary cultured inner medullary collecting duct (IMCD) cells to study the effect of CsA on changes in gene expression and cell viability during adaptation on changes in extracellular tonicity. In one experimental setup the IMCD-cells were cultivated either at 300, 600 or 900 mosmol/kg, left untreated or incubated for 24h with CsA, were used for analysis. To analyze if CsA influences the adaptation on changes in osmolarity in the second experimental setup IMCD-cells cultivated at 300 mosmol/kg were challenged for 24h with 600 mosmol/kg and cells cultivated at 600 mosmol/kg were challenged for 24h with 900 mosmol/kg, again with or without CsA incubation. Apoptosis was measured by flow cytometry using Anexin-5 as marker. Gene expression analysis was performed using microarray and real time PCR analysis. The first experimental setup showed that the fraction of apoptotic cells was not different when cells were cultivated at 300 or 600 mosmol/kg in the presence of CsA compared to the untreated group with the same osmolarity. But the number of Anexin-5 positive cells was 2 times higher when cells were cultivated at 900 mosmol/kg in the presence of CsA compared to untreated cells. This indicates that with an increase in osmotic stress CsA leads to an increase in apoptosis. The second experimental setup showed that the fraction of apoptotic cells was not different when cells were shifted from 300 to 600 mosmol/kg for 24h in the presence of CsA compared to cells shifted without CsA. But increased apoptosis was observed when the cells were shifted from 600 to 900 mosmol/kg in the presence of CsA compared to cells shifted without CsA. Immunoflourescence studies showed no changes in TonEBP localization by CsA. In cells shifted from 600 to 900 mosmol/kg in the presence of CsA more than 100 genes were differentially expressed compared to the group shifted without CsA. Pathway analysis showed that expression of TNFa and genes involved in the TNFa pathway were induced in the presence of CsA. Real time PCR analysis validated the increased expression of CsA induced TNFa. The study shows that CsA induced toxicity in the collecting duct might be induced via TNF pathway in this nephron segment.