Regulation of NKCC2 isoforms and blood pressure by tumor necrosis factor-alpha

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We previously showed that tumor necrosis factor-alpha (TNF) produced within the kidney is part of a system that regulates renal function and the blood pressure (BP) response to increases in dietary salt intake via inhibition of the Na+-K+-2Cl- (NKCC2) cotransporter. Thus, we developed two mouse models in which TNF has been genetically deleted in the: 1) thick ascending limb of Henle’s loop (TAL), and 2) distal nephron downstream of the proximal tubule (PT), which will be applied to determine the role and mechanisms of TNF produced by renal epithelial cells as part of an emerging intratubular TNF system that attenuates increases in BP induced by high salt (HS) intake. A complementary approach, using PT- and TAL-specific TNF silencing lentivirus constructs, will be used to specifically inhibit TNF production by these nephron segments to define the contribution of TNF derived from renal epithelial cells to the regulatory effects of this cytokine in the kidney. The genetic and lentivirus approaches also will be used to determine the molecular mechanisms by which TNF regulates NKCC2 phosphorylation and isoform expression, renal function, and BP. Preliminary data indicate that TNF, via activation of TNF receptor 1 (TNFR1), inhibits phospho-NKCC2 (pNKCC2) expression by a mechanism involving activation of the serine/threonine phosphatase, calcineurin (CN). The effects of TNF on CN in the kidney have not been studied, thus experiments will address TNF-dependent increases in CN activity as well as expression of the catalytic subunit CNAb and regulatory subunit CNB. The genetic and lentivirus strategies will be adapted to determine the effects of salt intake on TNFR1-dependent CN-mediated inhibition of pNKCC2 expression, electrolyte excretion, and the BP response to HS intake. Fine-tuning of NKCC2 function is dependent upon the NKCC2A and NKCC2B isoforms, which are strategically localized along the mammalian TAL and contribute to regulatory functions in response to high and low salt conditions, respectively. TNF inhibits the expression of these isoforms suggesting a role for this cytokine in both the mTAL and cTAL/MD segments of the TAL. Moreover, TNF regulates renal function involving these isoforms in a manner that limits reabsorption of NaCl. However, the molecular mechanism by which TNF suppresses NKCC2A and NKCC2B mRNA accumulation in response to high and low salt intake, respectively, has not been determined. miRNA profiling of the TAL in combination with preliminary data have identified miRNAs that regulate NKCC2 isoform mRNA abundance, phosphorylation and BP, the first data to show NKCC2 function is regulated by a miRNA-dependent mechanism. TAL-specific lentivirus manipulations will link miRNA-195 expression induced by TNF derived from the TAL to a NKCC2A-dependent mechanism that attenuates BP in mice ingesting HS. Collectively, the studies will reveal mechanisms by which an intratubular regulatory system, in which TNF produced by renal tubular epithelial cells in response to increases in salt intake, regulates NKCC2 isoform expression, phosphorylation via CN, electrolyte excretion, and contributes to BP homeostasis.