TNF inhibits NKCC2 phosphorylation by a calcineurin-dependent pathway.

We previously demonstrated that TNF inhibits NKCC2 phosphorylation in the thick ascending limb (TAL); however, the underlying mechanism remains unclear. We tested the hypothesis that the induction of calcineurin (CN) activity and the expression of CN isoforms contribute to the mechanism by which TNF inhibits phospho-NKCC2 (pNKCC2) expression. CN activity increased by approximately 2-fold in primary cultures of medullary (m)TAL cells challenged with mouse recombinant TNF. In contrast, silencing TNF production in mTAL cells using lentivirus U6-TNF-ex4 reduced CN activity. pNKCC2 expression decreased in mTAL cells challenged with TNF whereas inhibition of CN activity with cyclosporine A (CsA) increased pNKCC2 expression. Although mTAL cells express both the calcineurin A subunit (CNA) a and b isoforms, only CNA b isoform mRNA increased after mTAL cells were challenged with TNF. In vivo, both TNF and CNA b expression increased in outer medulla (OM) from mice given 1% NaCl in the drinking water for 7 days and intrarenal lentivirus silencing of TNF selectively reduced expression of CNA b. Intrarenal injection of a lentivirus that specifically silenced CNA b (U6-CNAb-ex6) increased pNKCC2 expression and attenuated the inhibitory effects of TNF on pNKCC2 expression in freshly isolated TAL tubules. Collectively, the study is the first to demonstrate that TNF increases CN activity and specifically induces β-isoform expression in the kidney. Since NKCC2 is a known target of the CNA b isoform, these findings suggest that a CN-dependent signaling pathway involving this isoform contributes to the mechanism by which TNF inhibits pNKCC2 expression.