Abstract 046: High Fructose Intake Increases Phosphorylation and Trafficking of the Na/K/2Cl Cotransporter (NKCC22) In Rat Thick Ascending Limbs

Abstract

A third of the US population consumes 20-40% of their caloric intake from added sugars with half of those calories from fructose. Fructose consumption is linked to salt-sensitive hypertension in humans and rodents. We found that feeding Sprague-Dawley rats 20% fructose in their drinking water did not increase blood pressure unless a high salt diet was added. The thick ascending limb (TAL) reabsorbs 25% of filtered NaCl, primarily via NKCC2. NKCC2 activity is increased by enhanced expression at the apical membrane and phosphorylation at Thr 96/101 . We found enhanced NKCC2 activity and trafficking in genetic models of salt-sensitive hypertension. However, the effect of fructose on NKCC2 regulation is unknown. Thus, we hypothesized that a fructose enriched diet stimulates NKCC2 activity. Sprague-Dawley rats were fed control diet or 20% fructose in drinking water for 1 week. TALs were isolated and phosphorylated and total NKCC2 was measured by surface biotinylation followed by western blot. A fructose-enriched diet increased surface-to-intracellular NKCC2 ratio by 60 ± 23% ( p< 0.05) and increased NKCC2 phosphorylation at Thr 96/101 by 8.02 ± 2.67 fold ( p <0.05). NKCC2 phosphorylation at the plasma membrane was also increased by 4.5 fold ( p <0.05). Total NKCC2 expression was reduced by 40.1 ± 8.9% ( p< 0.05). Since phosphorylation of NKCC2 at Thr 96/101 is mediated by STE20- and SPS1-related proline and alanine-rich kinases (SPAK) and oxidative stress-responsive kinase 1 (OSR1), we studied whether fructose stimulates expression and/or activity of SPAK/OSR1. Total SPAK/OSR1 expression was not enhanced by one week of a fructose diet. However, phosphorylation at Ser373 was enhanced by 2.8 ± 0.3 fold ( p< 0.05). We concluded that a fructose enriched diet increases phosphorylation and trafficking of NKCC2, enhancing its accumulation at the apical membrane. Moreover, a fructose diet increased SPAK/OSR1 kinases phosphorylation, suggesting they may be responsible for the enhanced NKCC2 phosphorylation. Our data suggest that a fructose-enriched diet promotes salt-sensitive hypertension in part by stimulating NKCC2 and TAL-dependent NaCl reabsorption.