K+ deficiency stimulates renal salt reuptake via the Na+-Cl− cotransporter (NCC) of the distal convoluted tubule (DCT), thereby reducing K+ losses in downstream nephron segments while increasing NaCl retention and blood pressure. NCC activation is mediated by a kinase cascade involving with no lysine (WNK) kinases upstream of Ste20-related proline-alanine-rich kinase (SPAK) and oxidative stress-responsive kinase-1 (OSR1). In K+ deficiency, WNKs and SPAK/OSR1 concentrate in spherical cytoplasmic domains in the DCT termed “WNK bodies,” the significance of which is undetermined. By feeding diets of varying salt and K+ content to mice and using genetically engineered mouse lines, we aimed to clarify whether WNK bodies contribute to WNK-SPAK/OSR1-NCC signaling. Phosphorylated SPAK/OSR1 was present both at the apical membrane and in WNK bodies within 12 h of dietary K+ deprivation, and it was promptly suppressed by K+ loading. In WNK4-deficient mice, however, larger WNK bodies formed, containing unphosphorylated WNK1, SPAK, and OSR1. This suggests that WNK4 is the primary active WNK isoform in WNK bodies and catalyzes SPAK/OSR1 phosphorylation therein. We further examined mice carrying a kidney-specific deletion of the basolateral K+ channel-forming protein Kir4.1, which is required for the DCT to sense plasma K+ concentration. These mice displayed remnant mosaic expression of Kir4.1 in the DCT, and upon K+ deprivation, WNK bodies developed only in Kir4.1-expressing cells. We postulate a model of DCT function in which NCC activity is modulated by plasma K+ concentration via WNK4-SPAK/OSR1 interactions within WNK bodies.

Hypokalemia contributes to the progression of chronic kidney disease, although a definitive pathophysiological theory to explain this remains to be established. K+ deficiency results in profound alterations in renal epithelial transport. These include an increase in salt reabsorption via the Na+-Cl− cotransporter (NCC) of the distal convoluted tubule (DCT), which minimizes electroneutral K+ loss in downstream nephron segments. In experimental conditions of dietary K+ depletion, punctate structures in the DCT containing crucial NCC-regulating kinases have been discovered in the murine DCT and termed “WNK bodies,” referring to their component, with no K (lysine) kinases (WNKs). We hypothesized that in humans, WNK bodies occur in hypokalemia as well. Renal needle biopsies of patients with chronic hypokalemic nephropathy and appropriate controls were examined by histological stains and immunofluorescence. Segment- and organelle-specific marker proteins were used to characterize the intrarenal and subcellular distribution of established WNK body constituents, namely, WNKs and Ste20-related proline-alanine-rich kinase (SPAK). In both patients with hypokalemia, WNKs and SPAK concentrated in non-membrane-bound cytoplasmic regions in the DCT, consistent with prior descriptions of WNK bodies. The putative WNK bodies were located in the perinuclear region close to, but not within, the endoplasmic reticulum. They were closely adjacent to microtubules but not clustered in aggresomes. Notably, we provide the first report of WNK bodies, which are functionally challenging structures associated with K+ deficiency, in human patients.

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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.

The extent of NaCl reabsorption in the early distal convoluted tubule (DCT1) via the apical Na+‐Cl− cotransporter (NCC) defines the sodium load of the ensuing late DCT (DCT2) and connecting tubule (CNT). DCT2 and CNT express the epithelial sodium channel (ENaC) and potassium Kir1.1 channel (ROMK) to perform electrogenic sodium‐dependent potassium secretion into the urine. Kidney‐specific deletion of Kir4.1 (Ks‐Kir4.1−/−) causes profound hypokalemia in mice, which may result from reduced DCT function and increased sodium load of DCT2/CNT, because potassium sensing via Kir4.1 is a critical step for NCC activation. Since Kir4.1 expression extends throughout DCT2 and CNT, we hypothesized that the deletion of Kir4.1 may also affect the expression and activity of ENaC and ROMK. To address this hypothesis we examined ENaC and ROMK functions by measuring the amiloride‐sensitive Na+ currents and TPNQ‐sensitive K+ currents in the DCT2/CNT of wild‐type (WT) and Ks‐Kir4.1−/− mice fed regular or K+‐deficient diets for three days. Cellular distribution of ENaC and ROMK was studied by immunofluorescence. K+‐deficient diet decreased amiloride‐sensitive Na currents from 205 pA to 120 pA at −60 mV and decreased TPNQ‐sensitive K currents from 1100 pA to 600 pA at −40 mV in WT animals. Ks‐Kir4.1−/− mice showed increased baseline Na+ (520 pA at −60 mV) and K+ currents (1600 pA at −40 mV). Feeding Ks‐Kir4.1−/− mice with K+‐deficient diet failed to inhibit ENaC and ROMK. Localization experiments revealed substantially increased ROMK signal intensities along DCT2 and CNT, which were identified by double immunostaining for parvalbumin, calbindin, or aquaporin 2. Moreover, DCT2 exhibited significant elongation in Ks‐Kir4.1−/− mice indicating nephron remodeling. We conclude that Kir4.1 regulates ENaC and ROMK functions in the DCT2 and CNT. Their upregulation in Ks‐Kir4.1−/− mice likely contributes to renal K wasting. Support or Funding Information Deutsche Forschungsgemeinschaft grants MU2924/2‐2, BA700/22‐2, SFB 1365; National Institute of Health grants DK 54983 and DK 115366; Fondation LeDucq and NIDDK R0151496; Russian Academic Excellence project “5–100”.

ObjectiveRenal salt retention due to excessive function of the renal NaCl cotransporter (NCC) along the distal convoluted tubule (DCT) plays a central role in the pathogenesis of hypertension. The Western type diet with high salt and low potassium content (HS/LK) has been shown to increase NCC activity by stimulating its phosphorylation via a kinase cascade comprising with‐no‐lysine kinases (WNKs) and a Ste20‐related kinase (SPAK). WNKs and SPAK have been shown to accumulate in perinuclear aggregates in response to potassium depletion, but the functional significance of these aggregates remained obscure. These structures may in part reflect a degradative pathway but may serve pooling functions as well.MethodsMice received HS/LK or control diets for 10 days. WNK1‐transfected DCT cells were exposed to vehicle, hyperosmotic stress, or potassium depletion. WNK1, WNK4, SPAK, phospho‐S383 SPAK, and various autophagy markers were studied by immunofluorescence and immunoblotting. Electron microscopy was applied for ultrastructural analysis.ResultsHS/LK diet induced accumulation of WNKs and SPAK in the form of perinuclear protein aggregates within DCT cells. These aggregates were rare in DCT of mice treated with control diet and virtually absent after administration of high potassium diet. The autophagy marker ATG5 was present in the aggregates and the 20S proteasome signal was intensified adjacently, whereas standard lysosomal markers showed no significant association with the punctate WNK/SPAK signal. Ultrastructural analysis of DCT in kidneys of mice treated with HS/LK revealed abundant aggresomes adjacent to rough endoplasmic reticulum and clathrin‐coated vesicles. Evaluation of cultured DCT cells transfected with WNK1 or KS‐WNK1 revealed formation of similar aggresomes in response to potassium depletion, whereas hyperosmotic stress induced formation of autophagosomes. Pretreatment of cells with proteasome inhibitor MG‐132 or autophagy/lysosome inhibitor bafilomycin A1 prior to potassium depletion substantially augmented the size of WNK1‐containing aggresomes.ConclusionChallenging of DCT epithelium induces the formation of perinuclear aggresomes which pool the components of the kinase cascade controlling NCC activity. From there, WNKs and SPAK may either be degraded via proteasomes or lysosomes, or reintroduced into the signal chain.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.