Different effects of CsA and FK506 on aquaporin-2 abundance in rat primary cultured collecting duct cells

Abstract

Calcineurin (Cn) inhibitors (CnI) such as cyclosporine A (CsA) and FK506 are nephrotoxic immunosuppressant drugs, which decrease tubular function. Here, we examined the direct effect of CnI on aquaporin-2 (AQP2) expression in rat primary cultured inner medullary collecting duct cells. CsA (0.5-5 μM) but not FK 506 (0.01-1 μM) decreased expression of AQP2 protein and messenger RNA (mRNA) in a concentration and time dependent manner, without affecting mRNA stability. This effect was observed despite similar inhibition of Cn activity by both CnI, thereby suggesting that the CsA-dependent decrease in AQP2 expression was Cn independent. Another inhibitor of cyclophilin A, the primary intracellular target of CsA, had no effect on AQP2 expression. In order to investigate the mechanism of decreased AQP2 transcription, we studied activation status of two suggested transcriptional regulators of AQP2, cAMP-responsive element binding protein (CREB), and tonicity enhancer binding protein (TonEBP). Localization of TonEBP, as well as TonEBP-mediated gene transcription, was not affected by CsA. Phosphorylation of CREB at an activating phosphorylation site (S133) was decreased by CsA, but not by FK506. However, both CnI did not affect cellular cAMP levels. We show that CsA decreases transcription of AQP2, a process that is in part independent of Cn or cyclophilin A and suggests dependence on decreased activity of CREB.

The kidney‐specific expression of genes can be modulated by the extracellular osmolality

“…Our goal was to identify genes whose renal expression is affected by differences in the extracellular osmolality by using primary cultured IMCD cells (14, 16, 17). We used an experimental setup with 10 different cell culture conditions, with 3 biologic replicates for each condition, and performed microarray analysis (Supplemental Fig.…”

“…Functional analysis was performed with Ingenuity Pathway Analysis (Qiagen) software. Real‐time PCR was performed as described before (16, 17). The results and the primers are listed in Supplemental Table S1.…”

“…To verify this hypothesis, we performed global gene expression profiling to identify changes that are induced by hyperosmolality in primary cultured rat inner medullary collecting duct (IMCD) cells (14, 15). The advantage of this system is that the cells in culture still express collecting‐duct‐specific genes, such as Aqp2–4, TonEBP, epithelial sodium channel (ENaC), renal outer medullary K channel (Romk), and UTA2 (14, 16, 17). In this cell model, we observed massive changes in gene expression after changes in medium osmolality.…”

Different Effects of Tacrolimus on Innate and Adaptive Immune Cells in the Allograft Transplantation

“…Both CsA and FK506 inhibit phosphatase calcineurin action [47][48][49][50]. Calcineurin regulates the transport of nuclear factor of activated T cells, which is a transcription factor that regulates lymphokine gene transcription [51]. CsA and FK506 exert their cellular effects on the action of calcineurin through different cytoplasmic receptors, as CsA binds to cyclophilins and FK506 binds to FK-binding proteins [52].…”

Calcineurin-inhibition Results in Upregulation of Local Renin and Subsequent Vascular Endothelial Growth Factor Production in Renal Collecting Ducts

“…None of them addressed other potential anatomical sources of the elevated serum renin, like the CD, although, the effect of CNIs on CD segment is known. 30,31 …”