SPAK disruption increases the glomerular filtration rate

Abstract

The thick ascending limb (TAL) of the kidney reabsorbs approximately 25% of filtered NaCl via its luminal Na‐K‐Cl cotransporter (NKCC2). In specialized TAL cells constituting the macula densa (MD), NKCC2 serves to sense the luminal NaCl concentration for paracrine adjustments of the glomerular filtration rate (GFR) to the needs of the body. The catalytically active full‐length (FL) SPAK and OSR1 kinases activate NKCC2 by phosphorylation. In addition to the FL‐SPAK, TAL cells express truncated SPAK variants (SPAK2 and KS‐SPAK) arising from alternative splicing or proteolytic cleavage. SPAK2 and KS‐SPAK exert strong dominant‐negative effects on FL‐SPAK/OSR1‐dependent NKCC2 phosphorylation. This study addresses the role of SPAK variants in regulation of NKCC2 in MD cells with respect to the modulation of GFR.SPAK variants, OSR1, NKCC2 and phospho‐NKCC2 were analyzed in kidneys of wild type (WT) and SPAK‐deficient (SPAK−/−) mice and cultured TAL and MD cells using immunohistochemistry, immunoblotting and quantitative PCR. Key juxtaglomerular components involved in GFR regulation such as cyclooxygenase 2 (COX2), nitric oxide synthase 1 (NOS1) and renin were compared between the two mouse genotypes. Effects of SPAK deficiency on GFR were evaluated by transcutaneous assessment of FITC‐sinistrin clearance in freely moving mice.Immunohistochemical analysis revealed that NKCC2 phosphorylation levels were significantly higher in TAL cells of SPAK−/− kidneys compared to WT, supporting the presence of the dominant‐negative SPAK2 and KS‐SPAK isoforms in WT TAL cells. In contrast, NKCC2 phosphorylation was not significantly altered in SPAK‐deficient MD cells, suggesting minor effects of truncated SPAK variants on NKCC2 and a redundant role of FL‐SPAK in this cell type. In line with this, immunoblotting of lysates from cultured TAL and MD cells showed predominant abundance of SPAK2 and KS‐SPAK in TAL cells, whereas MD cells mainly expressed FL‐SPAK. Both TAL and MD cells showed substantial OSR1 levels, which may explain the FL‐SPAK redundancy. Juxtaglomerular COX2, NOS1 and renin levels as well as the FITC‐sinistrin clearance were substantially higher in SPAK−/− which might reflect increased NKCC2‐mediated salt reabsorption in preceding TAL and preserved NKCC2‐mediated sensing of luminal NaCl in MD cells.Our results describe differential expression of SPAK variants in TAL and MD cells and demonstrate that SPAK disruption stimulates GFR likely due increased NKCC2 activity in TAL prior to MD resulting in reduced NaCl concentration at the MD site.Support or Funding InformationFunding: DFG German Research Foundation Fellowship (332853055) and Else Kröner‐Fresenius Stiftung (2015_A197) to TS, R01DK098141 to JAM; DFG Grant MU2924/2‐2This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.