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 mechanism that regulates renal function and the blood pressure (BP) response to increases in dietary salt intake. Our recent studies suggest that TNF effects in the kidney are evident in the medullary (m) thick ascending limb (TAL), proximal tubule (PT), and cortical (c) TAL/macula densa (MD) regions of the nephron. However, the cellular sources within the kidney that produce the TNF that accounts for these effects have not been determined, nor have the molecular mechanisms been identified. Thus, we developed two mouse models in which TNF has been genetically deleted in the: 1) TAL, and 2) distal nephron downstream of the PT, which will be used to understand the role 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. We also have tailored a complementary approach, using PT- and TAL-specific TNF silencing lentivirus constructs, to specifically inhibit TNF production by these cell types. The genetic and lentivirus approaches will be used in tandem to determine the mechanism by which TNF regulates Na+-K+-2Cl- (NKCC2) phosphorylation and isoform expression, renal function, and BP. Preliminary data suggest 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 have not been explored in the kidney, 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. The NKCC2A and NKCC2B isoforms 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 both isoforms suggesting a role for this cytokine in both the mTAL and cTAL/MD segments of the TAL. We previously showed that in each instance, TNF regulates renal function involving these isoforms in a manner that limits reabsorption of NaCl. However, the molecular mechanism by which TNF suppresses both NKCC2A and NKCC2B mRNA in response to high and low salt intake, respectively, has not been determined. Previous miRNA profiling of the TAL in combination with new preliminary data have identified 3 candidate miRNAs that regulate NKCC2 isoform mRNA abundance. For instance, miRNA-195 expression is induced by TNF derived from the TAL and inhibits NKCC2A mRNA accumulation and pNKCC2 expression in mice ingesting HS. Collectively, the studies will define a novel intratubular regulatory system in which TNF production by renal tubular epithelial cells, in response to increases in salt intake, regulates NKCC2 isoform expression and function and contributes to BP homeostasis.