Abstract 064: Hypokalemia Induced By Loss Of Kir5.1 Initiates WNK-SPAK/OSR1 Signaling In Dahl Salt-sensitive Rats

Abstract

Background: Hypokalemia promotes Na + reabsorption via the Na + -Cl - (NCC) cotransporter of the distal convoluted tubule (DCT), which reduces the K + losses in downstream nephron segments and increases NaCl retention and blood pressure. In dietary K + depletion, activation of NCC is mediated by the with no lysine (K) kinase (WNK)-Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1) pathway. Our previous work suggested that deletion of Kcnj16 (the gene that encodes K ir 5.1) in Dahl salt-sensitive (SS) rats (SS Kcnj16-/- ) resulted in hypokalemia with a compensatory increase in expression and activity of NCC. Here, we hypothesized that hypokalemia induced by loss of the K ir 5.1 subunit initiates WNK-SPAK/OSR1 signaling. Methods: 8 to 12-week-old male SS WT and SS Kcnj16-/- rats were separated into 3 groups (N=5 per group) and fed different diets for 3 weeks: SS WT (0.4% NaCl); SS Kcnj16-/- (0.4% NaCl); SS Kcnj16-/- (4% NaCl+2% KCl). At the end of the experiment, kidneys were flushed and processed for Western blot, immunofluorescence, and electron microscopy analysis. Results: Electron micrographs exhibited WNK body clusters in SS Kcnj16-/- compared to SS WT rats. Immunofluorescence staining showed dramatically increased expression of both SPAK and phospho-SPAK (pSPAK) in DCTs of SS Kcnj16-/- rats, where pSPAK colocalized with NCC. Similarly, Western blot analysis revealed significantly elevated expression of total SPAK (3.32± 0.18 vs. 1.00 ± 0.19, P <0.0001) and pSPAK (2.09 ± 0.51 vs. 1.00 ± 0.19, P <0.001) in the kidneys of SS Kcnj16/- compared to SS WT rats. Moreover, supplementing the SS Kcnj16-/- rats with high Na + and high K + (4% NaCl+2% KCl) reverses the expression and activity of SPAK. Conclusion: Our results indicate that hypokalemia resulting from loss of K ir 5.1 subunit stimulates WNK-SPAK/OSR1 signaling. Furthermore, the fact that dietary K + supplementation overturned the altered expression of this pathway implies that plasma K + level is responsible for the activation of the WNK-SPAK/OSR1 pathway.