Loss of activity of the NaCl Cotransporter (NCC) in Gitelman syndrome and with chronic thiazide treatment results in hypomagnesemia but the underlying mechanism remains unresolved. The prevailing hypothesis is that it results indirectly from distal convoluted tubule (DCT) atrophy lowering magnesium (Mg)-reabsorbing capacity by this segment. We tested the hypothesis that NCC inhibition directly disrupts a cellular mechanism related to Mg handling independent of DCT atrophy. We used the NCC-Cre+−/−- INTACT+/- mouse line to allow enrichment of DCT nuclei via Cre-dependent GFP expression in DCT nuclei. Eight- to 10-week-old male mice were given 50 mg/kg/day metolazone (MTZ) orally in food for 4 days; littermate controls receiving only vehicle. After nuclei isolation, GFP+ events (DCT nuclei) were sorted by fluorescence assisted nuclei sorting. Purified DCT nuclei were sequenced using 10X Chromium controller. Reads were then analyzed using a standard transcriptomic bioinformatics pipeline with Seurat. Tubule morphometrics using optical tissue clearing was performed on kidneys from animals receiving the same treatment. MTZ-treated mice had lower plasma Mg compared to controls recapitulating thiazide-induced hypomagnesemia. Our snRNAseq dataset containing 45,953 cells from 3 MTZ-treated and 3 control mice showed clear separation into early DCT (DCT1) and late DCT (DCT2) based on canonical segment markers. Gene expression analysis showed the DCT1 contained a magnesium cassette consisting of known magnesiotropic genes Trpm6, Trpm7, Egf, Umod, Prox1, Fxyd2, Cnnm2, and Slc41a3 so we focused subsequent analyses on DCT1. NCC inhibition caused DCT1 degeneration as indicated by lower mean DCT1 score of DCT1 cells and a greater proportion of DCT1 cells with low differentiation state in the MTZ group. Similarly, MTZ-treated samples had lower DCT1/DCT2 cell proportion in our snRNAseq dataset and morphometrics revelead significantly shorter DCT1 tubules implying that the degeneration process eventually leads to DCT1 atrophy. Using the aggregate expression of the magnesium cassette genes, we computationally defined a Mg score as a surrogate marker for Mg reabsorption capacity for each individual cells. The average Mg score was lower in the MTZ-treated group compared to the controls. Our data supports the long-standing hypothesis that hypomagnesemia is a secondary consequence of the loss of effective surface area for Mg reabsorption. Additionally, we uncovered that even at the single cell level, the Mg handling capacity of the cell decreases as a result of NCC inhibition implying that a disruption in sodium reabsorption disrupts expression of the Mg reabsorption machinery. NIH DK51496, NIH DK054983, NIDDK DK132066, VA 1I01BX002228, LeDucq Foundation, DOST-PCHRD-ASTHRDP. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Low potassium (K) intake has adverse health effects, including hypertension, osteoporosis, and nephrolithiasis. Dietary K restriction rapidly activates a K switch in the distal nephron to maintain homeostasis by reducing K excretion and activating the sodium chloride cotransporter (NCC). The distal convoluted tubule (DCT) is morphologically and functionally divided into DCT1 and DCT2, with each segment specialized for a different function. Here, we define the differences between DCT1 and DCT2 transcriptionally, examine their unique responses to potassium restriction, and detrimental physiological responses in mice.The K restriction effects were examined at 3 time points, overnight (acute), 4 days (short-term), and 4 weeks (chronic). Electrophysiology was used to assess effects of acute K deprivation on basolateral K channel (Kir4.1/5.1) activity. To determine effects of K restriction for 4 days, the DCT-targeted single-nucleus RNA-sequencing (snRNAseq) approach was applied. Female NCC-Cre-INTACT (PMID: 26087164) mice were provided either normal (NK) or K deficient (KD) diet for 4 days (short-term) and kidneys were snap-frozen for targeted snRNAseq (10X Chromium, NovaSeq) (3 mice per diet, 10,000 nuclei per mouse). Effects of chronic K-restriction were assessed by serum electrolytes, tissue clearing, and western blot.Acute K restriction stimulated basolateral K channel activity in DCT1, but not in DCT2, indicating that DCT1 and DCT2 respond differently. Although both DCT1 and DCT2 express NCC, snRNAseq data showed that DCT1 and DCT2 have distinct transcriptional signatures, with DCT1 expressing genes involved in magnesium reabsorption, and DCT2 expressing genes involved in calcium reabsorption and electrogenic sodium channel (ENaC). Four days of K restriction switched the DCT from a K-wasting state to K-sparing state, shown by reduction of gENaC ( Scnn1g) and Kallikrein ( Klk1) expression. Furthermore, K restriction decreased the expression of the calcium-handling genes Slc8a1, Calb and Vdr. When prolonged, K restriction led to increases in NCC, reduction in ENaC cleavage, and elongation of the DCT. It also led to calcium wasting (plasma iCa in NK, 1.38 ± 0.03, n=7; KD, 1.24 ± 0.01 mM, n=6) and reduction in calbindin D-28k protein.Our results suggest that DCT1 and DCT2 both express NCC, but have distinct transcriptomic signatures with unique roles in magnesium and calcium transport, respectively. Dietary K restriction reprograms DCT2 cells to be more like DCT1 cells, thereby maintaining K balance but contributing to calcium wasting as a complication. NIH DK51496, NIH DK054983, VA 1I01BX002228, LeDucq Fondation This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Thiazide diuretics lower urinary calcium excretion and are used to prevent calcium stone recurrence. Thiazides inhibit the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Hypocalciuria also occurs in Gitelman syndrome, caused by mutations in SLC12A3, encoding NCC. The mechanism by which loss of NCC function affects calcium handling remains very controversial. The two prevailing hypotheses invoked are 1) hypocalciuria results from volume-dependent increases in proximal tubule reabsorption; 2) thiazides activate calcium reabsorption along the DCT. Here we combined single nucleus RNA sequencing with physiologic analysis to unravel the causes of hypocalciuria.Eight to 10-week old male mice were given 50 mg/kg/day metolazone (MTZ) orally in food for 4 days. Littermates receiving only vehicle were used as controls. As DCT cells comprise a small percentage of the kidney mass, we used the NCC-Cre +/- - INTACT +/- mouse line to enrich for DCT via Cre-dependent GFP expression in DCT nuclei. After nuclei isolation, GFP+ events (DCT nuclei) were enriched by fluorescence assisted nuclei sorting. The purified DCT nuclei were sequenced using 10X Chromium controller. Reads were then analyzed using a standard transcriptomic bioinformatics pipeline with Seurat.The MTZ-treated mice showed lower urinary calcium excretion compared to controls recapitulating thiazide-induced hypocalciuria. Our snRNAseq integrated dataset contained 51,876 mapped genes across 45,953 samples from 3 MTZ-treated and 3 control mice. Roughly 99% of the nuclei were singlets expressing Slc12a3 showing successful enrichment for the DCT. Dimensional reduction analysis and projection confirmed two clusters, DCT1 and DCT2, based on canonical segment markers consistent with previously published transcriptomic atlases. Gene expression analysis showed the DCT2 contained a calcium cassette consisting of the known calciotropic genes Trpv5, Calb1, S100g, Slc8a1, Ryr2, Vdr, Casr, and Atp2b1. These genes were also expressed in the connecting tubule (CNT), as documented using the Calb1-Cre-INTACT system. From this set of genes, we defined a calcium score computed from the pooled average expression of all these genes in a single cell. Although all calcium cassette genes, and the average calcium score, were lower in the MTZ-treated group, the proportion of DCT2/DCT1 cells increased (0.27 ± 0.03 vs 0.35 ± 0.03; p = 0.027). These data suggest that lower calcium delivery to the DCT reduces calcium cassette expression per cell, but that distal fractional reabsorption is nonetheless increased owing to hypertrophy and elongation of the calcium transporting segments, DCT2 and CNT.We propose a modified mechanism to account for thiazide -induced hypocalciuria: 1) volume-dependent increase in proximal tubule reabsorption; 2) increased fractional (not absolute) DCT2 reabsorption owing to structural remodeling of DCT2 and CNT providing more surface area for calcium reabsorption. Funding: NIH DK51496, NIH DK054983, VA 1I01BX002228, LeDucq Foundation, DOST-PCHRD-ASTHRDP This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background: The furosemide-sensitive Na+-K+-2Cl− cotransporter (NKCC2) reabsorbs 20% of filtered Na+ along the thick ascending limb (TAL), while and thiazide-sensitive NaCl cotransporter (NCC) reabsorbs 5-10% along the distal convoluted tubule (DCT). The WNK4-SPAK/OxSR1 pathway mediates NKCC2 phosphorylation at threonines 96 and 101 (pT96/pT101) and NCC phosphorylation at threonine 53 (pT53). This activates both cotransporters. A recent study reported that the commonly used pT96/pT101 pNKCC2 antibody cross-reacts with pT53 NCC in mice on the C57BL/6 background due to a 5 amino acid deletion, calling into question some previous findings regarding WNK4-SPAK/OxSR1 regulation of NKCC2. Although WNK4 is highly expressed along both TAL and DCT, dysregulation of WNK4-SPAK/OxSR1 pathway specifically causes Familial Hyperkalemic Hypertension (FHHt), which is exquisitely sensitive to thiazide diuretics, and thus a disease of NCC dysregulation. Using a new pNKCC2 antibody specific in C57BL/6 mice, we tested the hypothesis that the WNK4-SPAK/OxSR1 pathway more strongly activates NCC than it does NKCC2. Methods: A new pT96 NKCC2 antibody was validated in C57BL/6, 129Sv, and Slc12a3 −/− mice. The abundances of pT96 NKCC2 and pT53 NCC were evaluated in Wnk4 −/−, Oxsr1 −/−, Spak −/− , Oxsr1 −/−/ Spak −/− mice, and several FHHt model, Cul3 +/−/Δ9, Klhl3 −/−, and Klhl3 R528H/R528H mice. NKCC2 activity was confirmed by furosemide response test with thiazide pre-treatment in Klhl3 −/− mice. Both male and female were used and all genetically modified strains were on the C57BL/6 background. Results: The pT96 NKCC2 antibody detected pNKCC2 in C57BL/6 mice but not 129Sv mice, and did not cross-react with phosphorylated NCC. pT53 NCC was almost absent but pT96 NKCC2 was only slightly lower in Wnk4 −/− mice. pT53 NCC was almost absent with Spak deletion ( Spak −/− and Oxsr1 −/−/ Spak −/− mice) but pT96 NKCC2 abundance did not differ from controls. Oxsr1 deletion ( Oxsr1 −/− and Oxsr1 −/−/ Spak −/− mice) led to a modest lowering of pT96 NKCC2/total NKCC2 ratio but not pT96 NKCC2 abundance. Interestingly, immunofluorescence revealed that WNK4 expression was increased along not only DCT but also cortical TAL in Klhl3 −/− mice, but pT96 NKCC2 abundance was not changed. pT53 NCC abundance, but not pT96 NKCC2 abundance, was higher in two other FHHt mouse models with higher WNK4 ( Cul3 +/−/Δ9 and Klhl3 R528H/R528H mice). Consistent with no difference in pT96 NKCC2 abundance, urine Na+ excretion after furosemide injection following thiazide treatment was similar between Klhl3 −/− and control mice. Conclusions: NCC is strongly phosphorylated by the WNK4-SPAK pathway, whereas NKCC2 is only mildly phosphorylated by the WNK4-OxSR1 pathway, suggesting another kinase can phosphorylate NKCC2. In FHHt models with Cul3 or Klhl3 mutations, NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide-sensitivity of FHHt. Y.M. received a postdoctoral award from the Uehara Foundation; J.A.M. is funded by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK098141; M.C.B. is funded by CONACyT, Mexico, Grant 101720. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

BACKGROUND:Kir4.2 and Kir4.1 play a role in regulating membrane transport in the proximal tubule (PT) and in the distal-convoluted-tubule (DCT), respectively. METHODS:We generated kidney-tubule-specific-AT1aR-knockout (Ks-AT1aR-KO) mice to examine whether renal AT1aR regulates Kir4.2 and Kir4.1. RESULTS:Ks-AT1aR-KO mice had a lower systolic blood pressure than Agtr1aflox/flox (control) mice. Ks-AT1aR-KO mice had a lower expression of NHE3 (Na+/H+-exchanger 3) and Kir4.2, a major Kir-channel in PT, than Agtr1aflox/flox mice. Whole-cell recording also demonstrated that the membrane potential in PT of Ks-AT1aR-KO mice was lesser negative than Agtr1aflox/flox mice. The expression of Kir4.1 and Kir5.1, Kir4.1/Kir5.1-mediated K+ currents of DCT and DCT membrane potential in Ks-AT1aR-KO mice, were similar to Agtr1aflox/flox mice. However, angiotensin II perfusion for 7 days hyperpolarized the membrane potential in PT and DCT of the control mice but not in Ks-AT1aR-KO mice, while angiotensin II perfusion did not change the expression of Kir4.1, Kir4.2, and Kir5.1. Deletion of AT1aR did not significantly affect the expression of αENaC (epithelial Na+ channel) and βENaC but increased cleaved γENaC expression. Patch-clamp experiments demonstrated that deletion of AT1aR increased amiloride-sensitive Na+-currents in the cortical-collecting duct but not in late-DCT. However, tertiapin-Q sensitive renal outer medullary potassium channel currents were similar in both genotypes. CONCLUSIONS:AT1aR determines the baseline membrane potential of PT by controlling Kir4.2 expression/activity but AT1aR is not required for determining the baseline membrane potential of the DCT and Kir4.1/Kir5.1 activity/expression. However, AT1aR is required for angiotensin II-induced hyperpolarization of basolateral membrane of PT and DCT. Deletion of AT1aR had no effect on baseline renal outer medullary potassium channel activity but increased ENaC activity in the CCD.

Thiazide sensitive sodium‐chloride cotransporter (NCC) is a major salt transport pathway in the apical membrane of the nephron distal convoluted tubule. While we know the importance of its function in maintaining sodium‐potassium homeostasis, the regulation of NCC is complicated and the mechanisms are not well understood. The aim of this study is to evaluate the role of mineralocorticoid receptors (MRs) in the regulation of NCC. Here, we generated DCT‐specific MRs Knockout (KO) mice, in which MRs were deleted in cells expressing NCC, taking advantage of a cre recombinase under the control of the NCC gene. Under standard and Low‐Na+ diets, these DCT‐specific MRs‐KO mice display normal Na+/K+ balance but exhibit problems with Cl‐ regulation. These KO mice presented hypochloremia under normal diet but unexpectedly, hyperchloremia after 6d on low Na+ diet. The expression of NCC and phosphorylated NCC were both decreased in KO mice compared to that in control mice, under both standard and low‐Na+ diet. This decrease in NCC protein levels is related to a decrease in transcription levels. Under standard diet, decreased NCC expression is compensated by an increase in αENaC and pendrin expression, which is not the case under low‐salt diet. Under a low‐K+ diet, KO mice exhibit hypokalemia and volume depletion with increased blood CO2 levels and Renin mRNA levels. NCC expression and its phosphorylation are both increased in control and KO groups under low‐K+ diet compared to standard diet but, the levels of NCC protein and mRNA are still decreased in the KO group compared to the control group mice. Taken together, these results demonstrate that MRs is not necessary for the complete regulation of NCC under low‐Na+ and low‐K+ diet. On the other hand, MRs appears to be indispensable to assure proper NCC expression in adult DCT renal tubules.

Abstract Background and Aims Chronic hypokalemia causes kidney fibrosis with cystic lesions and arterial hypertension. In contrast, potassium-rich diet lowers blood pressure. The acute effects of hypo- and hyperkalemia on heart and kidney are not well understood. Method Wild-type mice were fed with low (LK), normal (NK) and high (HK) potassium diet for 4 and 20 days. Kidneys were examined for site of acute injury, inflammation and fibrosis. Blood analysis of electrolytes and kidney parameters were analyzed. Echocardiography and ECG were used to assess heart function. Further, KCNJ10 knockout mice were used to investigate kidney damage in a genetically induced hypokalemia model. Results Proximal tubule injury as detected by KIM-1+ staining and yH2AX+ DNA-damage was observed after 4 and 20 days of LK diet. Injury was associated with strong Ki-67+ proliferation of proximal tubule cells. No injury was detected in mice on NK and HK diet. After 20 days of LK diet, F4/80+ inflammation and aSMA+ extracellular matrix accumulation, typical for fibrosis, were observed. LK mice developed polyurie, volume depletion, loss of body weight and high BUN. Lower cardiac output and signs of myocardial stress was seen in echocardiography and ECG. Consistent with WT mice on LK diet, KCNJ10 knockout mice developed same pattern of kidney injury. Nine months after deletion of KCNJ10, cysts were observed in the proximal tubule in outer medzulla. Conclusion Acute hypokalemia causes kidney injury and myocardial stress. Cystic lesions originate from late proximal tubule. Hypokalemia should be corrected rapidly to stop progression into kidney fibrosis.

Cre-lox technology has revolutionized research in renal physiology by allowing site-specific genetic recombination in individual nephron segments. The distal convoluted tubule (DCT), consisting of distinct early (DCT1) and late (DCT2) segments, plays a central role in Na+ and K+ homeostasis. The only established Cre line targeting the DCT is Pvalb-Cre, which is limited by noninducibility, activity along DCT1 only, and activity in neurons. Here, we report the characterization of the first Cre line specific to the entire DCT. CRISPR/Cas9 targeting was used to introduce a tamoxifen-inducible IRES-Cre-ERT2 cassette downstream of the coding region of the Slc12a3 gene encoding the NaCl cotransporter (NCC). The resulting Slc12a3-Cre-ERT2 mice were crossed with R26R-YFP reporter mice, which revealed minimal leakiness with 6.3% of NCC-positive cells expressing yellow fluorescent protein (YFP) in the absence of tamoxifen. After tamoxifen injection, YFP expression was observed in 91.2% of NCC-positive cells and only in NCC-positive cells, revealing high recombination efficiency and DCT specificity. Crossing to R26R-TdTomato mice revealed higher leakiness (64.5%), suggesting differential sensitivity of the floxed site. Western blot analysis revealed no differences in abundances of total NCC or the active phosphorylated form of NCC in Slc12a3-Cre-ERT2 mice of either sex compared with controls. Plasma K+ and Mg2+ concentrations and thiazide-sensitive Na+ and K+ excretion did not differ in Slc12a3-Cre-ERT2 mice compared with controls when sex matched. These data suggest genetic modification had no obvious effect on NCC function. Slc12a3-Cre-ERT2 mice are the first line generated demonstrating inducible Cre recombinase activity along the entire DCT and will be a useful tool to study DCT function.

The pathogenesis of essential hypertension is complex and multifactorial. Studying Mendelian forms of hypertension like Familial Hyperkalemic Hypertension (FHHt) is a powerful approach to identify and understand pathways that may contribute. Cullin‐3 (Cul3) mutations cause the most severe form of FHHt. Cullin 3 (CUL3) along with a substrate adaptor and RING ligase forms a complex that ubiquitinates proteins for proteasomal degradation. In FHHt, mutant CUL3 (CUL3‐D9) activity impairs With‐No‐lysine [K] kinase 4 (WNK4) degradation, leading to increased phosphorylation and thus activation of the Na+ Cl− cotransporter (NCC) along the renal distal convoluted tubule (DCT). We previously reported that CUL3‐D9 degrades the WNK4 adaptor KLHL3 in vitro, and a recent study suggests this occurs in vivo. This depletion of KLHL3 could prevent WNK4 degradation, providing a mechanism by which CUL3‐D9 leads to FHHt. To confirm that CUL3‐D9 degrades KLHL3 in vivo and determine whether it has any other activities, we studied inducible renal epithelial‐specific Cul3 knock‐out (CUL3‐KO) mice and CUL3‐KO mice expressing CUL3‐D9 (CUL3‐KO/D9). Western blotting and immunofluorescence showed that CUL3‐KO mice had higher KLHL3 abundance compared with controls, while CUL3‐KO/D9 had similar KLHL3 abundance to controls. CUL3‐KO and CUL3‐KO/D9 mice also had higher WNK4 abundance compared with controls. These data suggest that KLHL3 is a substrate of wild‐type CUL3, and that CUL3‐D9 is an active form of CUL3 since it can prevent KLHL3 accumulation in CUL3‐KO mice. However, CUL3‐D9 cannot promote WNK4 degradation and did not rescue the chronic kidney disease CUL3‐KO phenotype, suggesting a generalized loss‐of‐function of CUL3‐D9‐containing ubiquitin ligase complexes. A model of FHHt, inducible renal epithelia‐specific heterozygous Cul3 mice with CUL3‐D9 expression (CUL3‐Het/D9), further confirmed this observation with significantly lower KLHL3 abundance compared with control and heterozygous Cul3 mice. To investigate effects of CUL3‐D9 on other CUL3 targets, we investigated the abundance of another CUL3 adaptor protein, Kelch‐like ECH Associated Protein 1 (Keap1), the adaptor for Nuclear factor erythroid 2‐related factor 2 (NRF2). Both CUL3‐KO/D9 and CUL3‐Het/D9 had similar Keap 1 abundance compared with controls, but NAD(P)H Quinone Dehydrogenase 1 (NQO1), a surrogate for NRF2 activity, was significantly higher. These data suggest that in addition to effects on KLHL3/WNK4, CUL3‐D9 forms a dysfunctional complex with Keap1, leading to increased NRF2 activity. In conclusion, we confirm that CUL3‐D9 degrades its adaptor KLHL3 in vivo resulting in WNK4 accumulation, and also show that CUL3‐D9 cannot form normally‐functioning complexes with other substrate adaptors.Support or Funding InformationNIH grants DK098141 and DK117903 to JAM, and AHA 17POST33670206 to MZF

Calcineurin inhibitors (CnI) such as Cyclosporin A (CsA) are instrumental for immunosuppression after organ transplantation but may cause serious neurologic side effects, including seizures.Neuronal excitability depends on intact Cl− homeostasis. Generation of hyperpolarizing synaptic responses to GABA and glycine requires a Cl− gradient across the cell membrane with a low [Cl−]i, mainly established by K+‐Cl− cotransporter 2 (KCC2) and to a lesser extent Na+‐K+‐Clcotransporter 1 (NKCC1); both are cation‐coupled chloride cotransporters (CCCs). Calcineurin has been implicated in the regulation of CCCs, whereas impaired CCC function is a well‐known condition in human pharmacoresistant epilepsy. Therefore, we hypothesized that CsA affects KCC2 or NKCC1 functions, thus causing neuronal hyperexcitability.In ex vivo intracellular recordings with sharp microelectrodes, Wistar rat pyramidal neurons in neocortical slices responded to CsA (5 μM for 1h) with a less negative GABAA reversal potential (+7.2 mV) and prolonged Cl− extrusion time after iontophoretic Cl− loading (+3.9 s). 2‐photon fluorescence lifetime imaging in presence of Cl− sensitive dye MQAE showed an increased [Cl−]i in layer V neurons (+6.9 mM), suggesting reduced KCC2 activity. Co‐immunoprecipitation studies in rodent brain tissue suggested interactions of calcineurin Aβ (CnAβ) with KCC2 and NKCC1.In vivo, CsA administration to rats (5–25 mg/kg i.p.) enhanced levels of inhibitory tyrosine KCC2 phosphorylation at short term (4h; +172%) and reduced levels of activating S940‐KCC2 phosphorylation in the long term (14d; −61%). Reduced phospho‐S940‐KCC2 levels were observed in further models of calcineurin inhibition such as genetic CnAβ‐deficiency in mice (−78%) or CsA treatment of zebrafish larvae (10 μM in water: −54% after 24h). In contrast, NKCC1 and its activating kinase SPAK were stimulated upon calcineurin inhibition in rodents. Similar data were obtained in Drosophila melanogaster.Our results provide evidence that CnI attenuate KCC2 function but may stimulate NKCC1 leading to elevated [Cl−]i. These effects may increase neuronal excitability and contribute to CnI neurological adverse effects. The benefits of KCC2 activators or SPAK inhibitors for enhancing chloride extrusion in patients experiencing CnI neurotoxicity deserve further characterization.Support or Funding InformationThis study was supported by Deutsche Forschergemeinschaft.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

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 Information Funding: DFG German Research Foundation Fellowship (332853055) and Else Kröner‐Fresenius Stiftung (2015_A197) to TS, R01DK098141 to JAM; DFG Grant MU2924/2‐2 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Tubular remodeling can be induced by various stimuli and plays a critical role in functional adaptation. For instance, hypokalemia is associated with increased kidney mass caused by cellular hypertrophy and hyperplasia. However, accurate quantitative analysis of tubular remodeling is still lacking since most kidney morphometry was performed with two‐dimensional techniques. Here, we combined optical clearing and advanced light microscopy as a new approach for three‐dimensional analysis of tubular remodeling in mouse kidneys, upon dietary potassium (K+) restriction.Adult C57BL/6J mice were fed for 3 days with K+‐deficient diet (approx. 0.002% K+) or normal K+ diet (1.2% K+) and were injected with Bromodeoxyuridine (BrdU) intraperitoneally before kidneys were perfusion‐fixed 4 h later. Kidneys were cut into 1.0–1.5 mm thick slices and stained for BrdU then optically cleared with Ethyl Cinnamate. Confocal microscopy was used to identify the total number of BrdU positive cells per unit volume. In addition, 4 μm sections were used for co‐staining of BrdU with segment‐specific antibodies to localize proliferating cells. Whole blood was collected via terminal cardiac puncture and analyzed using i‐STAT analyzer (Abbot Point of Care Inc.).As expected, serum K+ levels were significantly lower in mice on K+ ‐deficient diet than in mice on normal diet (3.7 ± 0.1 mmol/L vs. 4.2 ± 0.1 mmol/L, p<0.005, n=6–7). After optical clearing, confocal microscopy allowed high‐resolution imaging of BrdU positive cells within the whole thick kidney slice. Quantification of BrdU positive cells revealed that the cell proliferation rate was low in control mice, but that K+ restriction strongly induced cell proliferation (3748 ± 392 cells/mm3 vs. 1089 ± 341 cells/mm3 p<0.05, n=3). Co‐localization using thin sections and segment‐specific markers revealed that this increase in proliferation was restricted to the S3 segment of the proximal tubule and outer medullary collecting duct intercalated cells.In this study, we validated a combination of optical clearing and advanced light microscopy as a new toolbox for imaging and quantification of tubular remodeling, which will decrease our reliance on biased two‐dimensional morphometric techniques and time‐consuming stereological approaches. Our analysis revealed that dietary K+ restriction caused preferential hyperplasia in S3 segments and in intercalated cells. The former observation might reflect a functional adaptation after low‐K+ induced injury of proximal tubules, whereas the latter likely represent a compensatory response to increase renal potassium uptake.Support or Funding InformationDFG German Research Foundation Fellowship (332853055) and Else Kröner‐Fresenius Stiftung (2015_A197) to TS, Humboldt Foundation and National Health and Medical Research Council of Australia Research Fellowships to VGP, R01DK098141 to JAM, and R01DK054983 to DHE.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Familial Hyperkalemic Hypertension (FHHt) is caused by mutations in the With‐No‐Lysine [K] kinases WNK1 and WNK4, or in components of a ubiquitin ligase complex that regulates WNK abundance. In FHHt, increased WNK abundance ultimately leads to increased phosphorylation and thus activity of the Na+–Cl− cotransporter (NCC) along the distal convoluted tubule. The resulting excessive Na+ reabsorption raises extracellular fluid volume and thus blood pressure (BP), and may decrease K+ secretion along the distal nephron by reducing Na+ delivery to the epithelial sodium channel.The complex that tags WNK kinases for proteasomal degradation consists of the scaffold protein Cullin 3 (CUL3), an adaptor protein (KLHL3) and a RING ligase. Mutations in both Cul3 and KLHL3 also cause FHHt, with Cul3 mutations causing the most severe form. FHHt‐causing Cul3 mutations are autosomal dominant and occur at splice sites, leading to the generation of a CUL3 mRNA lacking exon 9, translated to a form of CUL3 with deletion of 57 internal amino acids (403–459). The role of this altered CUL3 (CUL3‐Δ9) in FHHt is still unclear. It was recently reported that CUL3‐Δ9 promotes its own degradation, but not that of wild type (WT) CUL3, with a net result that total CUL3 levels are reduced to 50% of normal. Thus, it was proposed that CUL3 haploinsufficiency is sufficient to cause FHHt.To test this hypothesis, we compared mice heterozygous for Cul3 (CUL3‐Het) with mice heterozygous for Cul3, but also expressing CUL3‐Δ9 (CUL3‐Het/Δ9). The Pax8‐LC1 system, which permits doxycycline‐inducible CRE‐mediated recombination in renal epithelia, was used. To generate CUL3‐Het or CUL3‐Het/Δ9, Pax8‐LC1/Cul3fl/fl mice were bred with WT mice, or with mice carrying a lox‐STOP‐lox/CUL3‐Δ9 transgene, respectively. Recombination was induced with 2 mg/ml doxycycline in 5% sucrose water (vehicle) for 2 weeks.In CUL3‐Het mice, CUL3 abundance was ~50% lower than in controls, but the abundances of WNK4, phosphorylated NCC (pNCC), total NCC (tNCC), phosphorylated Na+–K+–2Cl−cotransporter 2 (pNKCC2), and total NKCC2 (tNKCC2) did not differ. BP, determined by telemetry, and plasma [K+] did also not differ. In CUL3‐Het/Δ9 mice WT CUL3 abundance was also ~50% lower than in controls. Consistent with a previous report that CUL3‐Δ9 promotes its own degradation, CUL3‐Δ9 expression was not detected by Western blot. However, clear cortical and medullary expression of the fluorophore tdTomato, translated from the same transcript as CUL3‐Δ9 via an internal ribosome entry site, confirmed transgene expression in CUL3‐Het/Δ9 mice, but not in vehicle‐treated mice. Abundances of WNK4 (191%), pNCC (591%), tNCC (154%), and pNKCC2 (340%) were significantly higher in CUL3‐Het/Δ9 mice than in controls (100%). Consistent with an FHHt phenotype, plasma [K+] was higher in CUL3‐Het/Δ9 mice than in controls (4.73 mM vs 4.20 mM, p<0.05). Directly comparing CUL3‐Het and CUL3‐Het/Δ9 mice revealed significantly higher pNCC/tNCC in CUL3‐Het/Δ9 mice. These data suggest that a unique function of CUL3‐Δ9 itself, rather than haploinsufficiency due to autodegradation, plays a key role in the pathogenesis of FHHt.Support or Funding InformationNIH

Familial Hyperkalemic Hypertension (FHHt) is caused by mutations in genes that regulate activity of the renal sodium chloride cotransporter (NCC), including the kinases WNK1 and WNK4, and Cullin3 (Cul3) and KLHL3, members of a ring ubiquitin ligase complex. To examine the physiological roles of Cul3 and how mutations in it cause FHHt, we generated Cul3‐/‐ mice. An inducible system that disrupts Cul3 specifically along the entire nephron was used since complete Cul3 disruption is embryonic lethal. Western blots showed nearly complete absence of Cul3 in Cul3‐/‐ kidneys. Cul3‐/‐ mice displayed volume contraction, hypochloremia and alkalosis, and elevated plasma aldosterone. Blood pressure, measured by radiotelemetry, was similar in both groups on normal NaCl diet, but during NaCl restriction, Cul3‐/‐ mice displayed a progressive reduction in blood pressure. After 9 days, the 24h mean blood pressure pressure was 18 mmHg lower in Cul3‐/‐ mice. Expression of total and phospho‐NCC were slightly elevated in Cul3‐/‐ mice on normal diet, but dietary NaCl restriction did not cause their levels to increase to the same extent as it did in wild types. WNK4 expression was dramatically increased in Cul3‐/‐ mice on normal and low NaCl diets, while WNK1 and WNK3 were slightly elevated. In conclusion, these data suggest that loss of Cul3 activity along the nephron does not mimic FHHt, but leads to a mild salt‐wasting phenotype.Grant Funding Source: KO1 DK076617

The Na‐K‐2Cl cotransporter (NKCC2) of the thick ascending limb (TAL) and the Na‐Cl cotransporter (NCC) of the distal convoluted tubule (DCT) are critical for renal salt handling. Activation of these transporters by vasopressin (AVP) includes their N‐terminal phosphorylation. Little is currently known about the kinases that mediate this action of AVP. Two homologous Ste20‐like kinases, SPAK and OSR1, can phosphorylate the cotransporters directly. In this process, full‐length SPAK variant (FL‐SPAK) and OSR1 interact with a truncated isoform, KS‐SPAK, which has inhibitory effects. Our study tested the hypothesis that SPAK is an essential component of the AVP stimulatory pathway.Short‐ and long‐term effects of desmopressin (dDAVP), a V2 receptor‐specific agonist, on the kinases and transporters were evaluated in wild type and SPAK‐deficient mice and in AVP‐deficient rats.SPAK variants displayed prominent regulatory changes along TAL and DCT along with activation of the cotransporters, whereas OSR1 was less involved. The KS‐ and FL‐SPAK variants were modulated by AVP for their selective interaction with NKCC2 in control of its activation, whereas the phosphorylation of NCC was essentially governed by FL‐SPAK alone.In sum, our data specify how SPAK may serve as a hallmark kinase in modulating Na+ reabsorption along the distal nephron under the endocrine control of AVP.

In the present study, we investigated the activity of the thiazide-sensitive NCC (Na(+)-Cl(-) co-transporter) in experimental metabolic syndrome and the role of insulin in NCC activation. Renal responses to the NCC inhibitor HCTZ (hydrochlorothiazide), as a measure of NCC activity in vivo, were studied in 12-week-old ZO (Zucker obese) rats, a model of the metabolic syndrome, and in ZL (Zucker lean) control animals, together with renal NCC expression and molecular markers of NCC activity, such as localization and phosphorylation. Effects of insulin were studied further in mammalian cell lines with inducible and endogenous expression of this molecule. ZO rats displayed marked hyperinsulinaemia, but no differences in plasma aldosterone, compared with ZL rats. In ZO rats, natriuretic and diuretic responses to NCC inhibition with HCTZ were enhanced compared with ZL rats, and were associated with a decrease in BP (blood pressure). ZO rats displayed enhanced Thr(53) NCC phosphorylation and predominant membrane localization of both total and phosphorylated NCC, together with a different profile in expression of SPAK (Ste20-related proline/alanine-rich kinase) isoforms, and lower expression of WNK4. In vitro, insulin induced NCC phosphorylation, which was blocked by a PI3K (phosphoinositide 3-kinase) inhibitor. Insulin-induced reduction in WNK4 expression was also observed, but delayed compared with the time course of NCC phosphorylation. In summary, we report increased NCC activity in hyperinsulinaemic rodents in conjunction with the SPAK expression profile consistent with NCC activation and reduced WNK4, as well as an ability of insulin to induce NCC stimulatory phosphorylation in vitro. Together, these findings indicate that hyperinsulinaemia is an important driving force of NCC activity in the metabolic syndrome with possible consequences for BP regulation.

Aim of the present study was to elucidate the role of the sterile 20/SPS1-related proline/alanine-rich kinase (SPAK) during the vasopressin-induced phosphorylation of the renal Na+,K+,Cl−- (NKCC2) and Na+,Cl−- (NCC) cotransporters. To this end adult SPAK-knockout mice and wildtype mice (WT) were treated with the vasopressin V2 receptor agonist desmopressin (dDAVP, 1ng/kg bodyweight, 30 min) or vehicle. Abundance and localization of total and phosphorylated NKCC2 and NCC were determined by Western blot, immunofluorescence labelling and immunoelectron microscopy. During steady state SPAK −/− mice showed markedly increased levels of phosphorylated NKCC2 (+320±40% vs. controls, p < .05) whereas total NKCC2 levels were unchanged. Levels of total and of phosphorylated NCC were reduced (−72±18% for NCC, −58±13% for pS71-NCC vs. controls, p <.05). dDAVP treatment significantly increased SPAK phosphorylation at S383 in WT mice (+89±32% vs. controls, p < .05) and NKCC2 phosphorylation to a similar extent in WT (+55% ) and SPAK−/− mice (+55%). In contrast, dDAVP-induced increases of NCC phosphorylation were only detectable in WT (+86% for pS71-NCC and +163% for pT53-NCC) but not in SPAK−/− mice. In conclusion, this study provides in-vivo evidence for divergent roles of SPAK in TAL and DCT. Work was supported by the German Research Foundation (FOR667).