Abstract 025: RNAseq And Bioinformatics Identify New Pathways In The Thick Ascending Limb (TAL) Involved In Fructose Induced Salt Sensitive Hypertension

Abstract

Elevated fructose consumption is associated with hypertension in humans. In normal Sprague Dawley rats, a high fructose intake (20% fructose drinking water) induced salt sensitive hypertension. We found that fructose directly stimulates the apical Na/K/2Cl cotransporter NKCC2 in the thick ascending limb (TAL), and when consumed for more than 7 days, increases NKCC2 phosphorylation at Thre 96,101. SPAK and OSR1 kinases are known to phosphorylate NKCC2, but it is not known how fructose activates SPAK or OSR1. We hypothesized that fructose intake activates several signaling cascades in TALs that selectively stimulate SPAK. Using an antibody that recognizes activated SPAK and OSR1 (P-Ser 373 SPAK; Ser325 in OSR1), we found that 20% fructose (14 d) enhanced SPAK/OSR1 phosphorylation by 179±32% (p<0.05). This effect was specific for SPAK because phosphorylation of OSR1, immunoprecipitated from TALs, was not increased by fructose. To study the mechanism of SPAK activation, we isolated TALs from rats on control or 20% fructose diet for (14 d, n=6) and performed RNAseq. Over 12,000 genes were statistically compared and 500 were significantly different (p<0.05). Expression of some of the most highly upregulated genes (PRKRIP1 and SOCS3) was confirmed by Western blot in TALs. However, expression of upstream SPAK activators was unchanged by fructose (WNK1/3/4). We developed a targeted bioinformatics approach that included angiotensin signaling, nitric oxide and lipid metabolism, plus protein-protein interaction networks for SPAK. We identified PRKCQ (PKC Theta), a kinase upstream of SPAK, that was upregulated in TALs by fructose. In fructose fed rats, PRKCQ expression was 31±11% higher than control, as was Phospho-PRKCQ (223±45% increase, p<0.05). We then measured systolic BP (SBP) by telemetry in Dahl SS rats with genetic deletion of SPAK (SPAK KO). In WT Dahl SS rats, 4 weeks of fructose intake increased SBP by 25±4 mmHg (p<0.05), whereas in SPAK KO the increase in SBP was reduced (11±4 mmHg, p<0.05). We concluded that high fructose intake induces several prohypertensive signaling pathways in the TAL that lead to SPAK and NKCC2 activation. We identified PRKCQ as a potential activator of SPAK. Fructose induced hypertension in Dahl SS rats was in part mediated by SPAK.