Regulation and function of NFAT5 in medullary thick ascending limb (mTAL) cells

Abstract

The molecular mechanism and function of NFAT5 in mTAL cells has not been fully explored. In this study,RT‐PCR analysis was performed on primary cultures of mouse mTAL cells and freshly isolated mTAL tubules to determine which Na+K+2Cl‐ cotransporter type 2 (NKCC2) isoforms are present. The results show that primary cultures of mTAL cells and freshly isolated mTAL tubules express NKCC2 isoforms A and F, but not B, the expression of which is restricted to the macula densa. The data also indicate that the abundance of NKCC2A and NFAT5 mRNA significantly increased when mTAL cells were exposed to a change in osmolality from 300 to 500 mosmol/kg H2O, produced with NaCl for 2 or 4 hours, respectively. Moreover, transient transfection of cells with shRNA NKCC2A prevented the increase in NFAT5 in response to hypertonic stress, suggesting a role for NKCC2A in the regulation of NFAT5 in these cells. DNA degradation was attenuated and cell viability improved with an NFAT5 overexpression vector, but not empty vector, when osmolality was changed from 300 to 500 mosmol/kg H2O for 48 hr. Overexpression of DN‐NFAT5 attenuated calcium sensing receptor‐mediated Cl‐ influx by 42%, suggesting that a NFAT5‐dependent mechanism contributes to the regulation of Cl‐ transport in mTAL cells. Elucidating NFAT5‐dependent pathways in mTAL cells will be crucial to understanding mechanisms that regulate extracellular fluid volume and salt balance.