Search

Cullin-RING-ligases (CRLs) are a group of E3 ubiquitin ligases that mediate the regulated degradation of proteins. All CRLs are regulated by the COP9 signalosome (CSN), which interacts with the CRL and turns off ubiquitin ligase activity. To investigate the role of impaired CSN dysfunction in the kidney, we inactivated the CSN by deleting Jab1 (the key CSN catalytic subunit) along the entire nephron. Even though the CSN is expressed all along the nephron, these mice showed remodeling only along the distal nephron, with shortening of the distal convoluted tubule (DCT) and a large reduction in DCT-specific proteins. The DCT is known to remodel due to changes in dietary salt intaked, drug administration, and human disease. Yet, the cellular and molecular basis for this plasticity is unclear. We hypothesized that CSN dysfunction causes de-differentiation of DCT cells leading to DCT remodeling. To test our hypothesis, inducible DCT-specific NCC-Cre-ERT2 mice were bred to INTACT (Isolation of Nuclei TAgged in specific Cell Types, which fluorescently labels nuclei) and Jab1 floxed mice to generate DCT-INTACT- Jab1 -/- mice. One adult male DCT-INTACT- Jab1 -/- mouse and one DCT-INTACT control mouse were treated with tamoxifen to induce nuclear GFP expression and to induce deletion of Jab1 only in DCT cells. Six-weeks post treatment, nuclei from one kidney cortex was isolated and the DCT was enriched using Fluorescence-Activated Nuclei Sorting (FANS). GFP-positive nuclei were used for single-nucleus RNA-sequencing (NovaSeq). Initial results showed that unbiased clustering and UMAP visualization revealed 7 clusters with most cells from the DCT (99.0% for control and 97.6% for Jab1 -/- mice), indicating that the enrichment process was highly successful. Both the control and Jab1 -/- mouse had similar proportions of DCT1 and DCT2 cells; however, we identified two specialized cell types (dDCT1 and dDCT2) which comprised a much larger proportion of the cells in the Jab1 -/- mouse (dDCT1: 2.9% in control vs 41.1% in Jab1 -/-; dDCT2: 2.0% in control vs 13.9% in Jab1 -/-). These cells were highly de-differentiated with lower expression of the canonical DCT identifier genes such as Egf and Umod for DCT1, Calb1 and S100g for DCT2, as well as Slc12a3. Both dDCT1 and dDCT2 cell populations had induced expression of the disease- or carcinoma-related genes Ano3, Nrg1, and Pvt1. In conclusion, Jab1 deletion causes de-differentiation of a population of DCT cells. Further examination of CSN dysfunction via DCT-enriched single-nucleus RNA-sequencing could lead to a better understanding of the molecular mechanisms involved in DCT remodeling. NIH K01DK120790, NIH R01DK51496, Foundation LeDucq: Transatlantic Network of Excellence 17CVD05 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.

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.

Cullin-RING ligases are a family of E3 ubiquitin ligases that control cellular processes through regulated degradation. Cullin 3 targets with-no-lysine kinase 4 (WNK4), a kinase that activates the Na(+)-Cl(−) cotransporter (NCC), the main pathway for Na(+) reabsorption in the distal convoluted tubule (DCT). Mutations in the cullin 3 gene lead to familial hyperkalemic hypertension by increasing WNK4 abundance. The constitutive photomorphogenesis 9 (COP9) signalosome (CSN) regulates the activity of cullin-RING ligases by removing the ubiquitin-like protein neural precursor cell expressed developmentally downregulated protein 8. Genetic deletion of the catalytically active CSN subunit, Jab1, along the nephron in mice (KS-Jab1(−/−)) led to increased WNK4 abundance; however, NCC abundance was substantially reduced. We hypothesized that the reduction in NCC resulted from a cortical injury that led to hypoplasia of the segment, which counteracted WNK4 activation of NCC. To test this, we studied KS-Jab1(−/−) mice at weekly intervals over a period of 3 wk. The results showed that NCC abundance was unchanged until 3 wk after Jab1 deletion, at which time other DCT-specific proteins were also reduced. The kidney injury markers kidney injury molecule-1 and neutrophil gelatinase-associated lipocalin demonstrated kidney injury immediately after Jab1 deletion; however, the damage was initially limited to the medulla. The injury progressed and expanded into the cortex 3 wk after Jab1 deletion coinciding with loss of the DCT. The data indicate that nephron-specific disruption of the cullin-RING ligase system results in a complex progression of tubule injury that leads to hypoplasia of the DCT. NEW & NOTEWORTHY Cullin 3 (CUL3) targets with-no-lysine-kinase 4 (WNK4), which activates Na(+)-Cl(−) cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Renal-specific genetic deletion of the constitutive photomorphogenesis 9 signalosome, an upstream regulator of CUL3, resulted in a reduction of NCC due to DCT hypoplasia, which coincided with cortical kidney injury. The data indicate that nephron-specific disruption of the cullin-RING ligase system results in a complex progression of tubule injury leading to hypoplasia of the DCT.

Cl−-sensitive with-no-lysine kinase (WNK) plays a key role in regulating the thiazide-sensitive Na+-Cl−cotransporter (NCC) in the distal convoluted tubule (DCT). Cl−enters DCT cells through NCC and leaves the cell across the basolateral membrane via the Cl−channel ClC-K2 or K+-Cl−cotransporter (KCC). While KCC is electroneutral, Cl−exit via ClC-K2 is electrogenic. Therefore, an alteration in DCT basolateral K+channel activity is expected to influence Cl−movement across the basolateral membrane. Although a role for intracellular Cl−in the regulation of WNK and NCC has been established, intracellular Cl−concentrations ([Cl−]i) have not been directly measured in the mammalian DCT. Therefore, to measure [Cl−]iin DCT cells, we generated a transgenic mouse model expressing an optogenetic kidney-specific Cl-Sensor and measured Cl−fluorescent imaging in the isolated DCT. Basal measurements indicated that the mean [Cl−]iwas ~7 mM. Stimulation of Cl−exit with low-Cl−hypotonic solutions decreased [Cl−]i, whereas inhibition of KCC by DIOA or inhibition of ClC-K2 by NPPB increased [Cl−]i, suggesting roles for both KCC and ClC-K2 in the modulation of [Cl−]i. Blockade of basolateral K+channels (Kir4.1/5.1) with barium significantly increased [Cl−]i. Finally, a decrease in extracellular K+concentration transiently decreased [Cl−]i, whereas raising extracellular K+transiently increased [Cl−]i, further suggesting a role for Kir4.1/5.1 in the regulation of [Cl−]i. We conclude that the alteration in ClC-K2, KCC, and Kir4.1/5.1 activity influences [Cl−]iin the DCT.

BACKGROUND: Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) gene cause the disease familial hyperkalemic hypertension (FHHt). In the kidney, mutant CUL3 (CUL3-Δ9) increases abundance of With-No-Lysine (K) Kinase 4 (WNK4), inappropriately activating sterile 20/SPS-1–related proline/alanine-rich kinase (SPAK), which then phosphorylates and hyperactivates the Na(+)Cl(–) cotransporter (NCC). The precise mechanism by which CUL3-Δ9 causes FHHt is unclear. We tested the hypothesis that reduced abundance of CUL3 and of Kelch-like 3 (KLHL3), the CUL3 substrate adaptor for WNK4, is mechanistically important. Because JAB1, an enzyme that inhibits CUL3 activity by removing the ubiquitin-like protein NEDD8, cannot interact with CUL3-Δ9, we also determined whether Jab1 disruption mimicked the effects of CUL3-Δ9 expression. METHODS: We used an inducible renal tubule-specific system to generate several mouse models expressing CUL3-Δ9, mice heterozygous for both CUL3 and KLHL3 (Cul3(+/−)/Klhl3(+/−)), and mice with short-term Jab1 disruption (to avoid renal injury associated with long-term disruption). RESULTS: Renal KLHL3 was higher in Cul3(−/−) mice, but lower in Cul3(−/−/Δ9) mice and in the Cul3(+/−/Δ9) FHHt model, suggesting KLHL3 is a target for both WT and mutant CUL3. Cul3(+/−)/Klhl3(+/−) mice displayed increased WNK4-SPAK activation and phospho-NCC abundance and an FHHt-like phenotype with increased plasma [K(+)] and salt-sensitive blood pressure. Short-term Jab1 disruption in mice lowered the abundance of CUL3 and KLHL3 and increased the abundance of WNK4 and phospho-NCC. CONCLUSIONS: Jab1(−/−) mice and Cul3(+/−)/Klhl3(+/−) mice recapitulated the effects of CUL3-Δ9 expression on WNK4-SPAK-NCC. Our data suggest degradation of both KLHL3 and CUL3 plays a central mechanistic role in CUL3-Δ9–mediated FHHt.

In the distal kidney tubule, the steroid hormone aldosterone regulates sodium reabsorption via the epithelial sodium channel (ENaC). Most studies seeking to identify ENaC-regulating aldosterone-induced proteins have used transcriptional profiling of cultured cells. To identify salt-sensitive transcripts in an in vivo model, we used low-NaCl or high-NaCl diet to stimulate or suppress endogenous aldosterone, in combination with magnetic- and fluorescence-activated cell sorting to isolate distal tubule cells from mouse kidney for transcriptional profiling. Of the differentially expressed transcripts, 162 were more abundant in distal tubule cells isolated from mice fed low-NaCl diet, and 161 were more abundant in distal tubule cells isolated from mice fed high-NaCl diet. Enrichment analysis of Gene Ontology biological process terms identified multiple statistically overrepresented pathways among the differentially expressed transcripts that were more abundant in distal tubule cells isolated from mice fed low-NaCl diet, including ion transmembrane transport, regulation of growth, and negative regulation of apoptosis. Analysis of Gene Ontology molecular function terms identified differentially expressed transcription factors, transmembrane transporters, kinases, and G protein-coupled receptors. Finally, comparison with a recently published study of gene expression changes in distal tubule cells in response to administration of aldosterone identified 18 differentially expressed genes in common between the two experiments. When expression of these genes was measured in cortical collecting ducts microdissected from mice fed low-NaCl or high-NaCl diet, eight were differentially expressed. These genes are likely to be regulated directly by aldosterone and may provide insight into aldosterone signaling to ENaC in the distal tubule.

Phosphorylation of the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) is altered rapidly in response to changes in extracellular K(+) concentration ([K(+)]). High extracellular [K(+)] is believed to activate specific phosphatases to dephosphorylate NCC, thereby reducing its activity. This process is defective in the human disease familial hyperkalemic hypertension, in which extracellular [K(+)] fails to dephosphorylate NCC, suggesting an interplay between NCC-activating and NCC-inactivating switches. Here, we explored the role of STE20/SPS1-related proline-alanine-rich protein kinase (SPAK) and intracellular Cl(−) concentration in the rapid effects of extracellular K(+) on NCC phosphorylation. SPAK was found to be rapidly dephosphorylated in vitro in human embryonic kidney cells and ex vivo in kidney slices by high [K(+)]. Acute high-K(+) challenge resulted in DCT1-specific SPAK dephosphorylation in vivo and dissolution of SPAK puncta. In line with the postulate of interplay between activating and inactivating switches, we found that the “on” switch, represented by with no lysine kinase 4 (WNK4)-SPAK, must be turned off for rapid NCC dephosphorylation by high [K(+)]. Longer-term WNK-SPAK-mediated stimulation, however, altered the sensitivity of the system, as it attenuated rapid NCC dephosphorylation due to acute K(+) loading. Although blockade of protein phosphatase (PP)1 increased NCC phosphorylation at baseline, neither PP1 nor PP3, singly or in combination, was essential for NCC dephosphorylation. Overall, our data suggest that NCC phosphorylation is regulated by a dynamic equilibrium between activating kinases and inactivating phosphatases, with kinase inactivation playing a key role in the rapid NCC dephosphorylation by high extracellular K(+). NEW & NOTEWORTHY Although a great deal is known about mechanisms by which thiazide-sensitive NaCl cotransporter is phosphorylated and activated, much less is known about dephosphorylation. Here, we show that rapid dephosphorylation by high K(+) depends on the Cl(−) sensitivity of with no lysine kinase 4 and the rapid dephosphorylation of STE20/SPS1-related proline-alanine-rich protein kinase, primarily along the early distal convoluted tubule.

Background Previous studies have described a ‘renal potassium switch’ within the distal nephron that turns on the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT) in response to low potassium intake and off in response to high potassium intake. Studies using genetically modified mice indicate that decreased NCC activity is associated with decreased DCT length and mass; increased NCC activity is associated with increased, DCT length and mass. The aim of our study was to test whether dietary potassium intake causes the DCT remodeling physiologically. Methods Male C57Bl/6 mice were provided either control potassium diet or low potassium diet for four weeks and blood and kidneys were harvested. To determine the length of the DCT in three dimensions, we used Ethyl-cinnamate-based optical clearing, combined with whole-mount immunolabeling, confocal microscopy and three-dimensional morphometric analysis. Results Mice on low potassium diet for four weeks were severely hypokalemic (plasma potassium Conclusion Our results indicate that the DCT remodels physiologically to maintain potassium homeostasis. Additional animals are currently being studied.

Abstract Eating more potassium may reduce blood pressure and the occurrence of other cardiovascular diseases by actions on various systems, including the vasculature, the sympathetic nervous system, systemic metabolism, and body fluid volume. Among these, the kidney plays a major role in the potassium-rich diet–mediated blood pressure reduction. Purpose of Review To provide an overview of recent discoveries about the mechanisms by which a potassium-rich diet leads to natriuresis. Recent Findings Although the distal convoluted tubule (DCT) is a short part of the nephron that reabsorbs salt, via the sodium-chloride cotransporter (NCC), it is highly sensitive to changes in plasma potassium concentration. Activation or inhibition of NCC raises or lowers blood pressure. Recent work suggests that extracellular potassium concentration is sensed by the DCT via intracellular chloride concentration which regulates WNK kinases in the DCT. Summary High-potassium diet targets NCC in the DCT, resulting in natriuresis and fluid volume reduction, which are protective from hypertension and other cardiovascular problems.

Calcineurin inhibitors (CNIs) are immunosuppressive drugs used to prevent graft rejection after organ transplant. Common side effects include renal magnesium wasting and hypomagnesemia, which may contribute to new‐onset diabetes mellitus, and hypercalciuria, which may contribute to post‐transplant osteoporosis. Previous work suggested that CNIs reduce the abundance of key divalent cation transport proteins, expressed along the distal convoluted tubule, causing renal magnesium and calcium wasting. It has not been clear, however, whether these effects are specific for the distal convoluted tubule, and whether these represent off‐target toxic drug effects, or result from inhibition of calcineurin. The CNI tacrolimus can inhibit calcineurin only when it binds with the immunophilin, FKBP12; we previously generated mice in which FKBP12 could be deleted along the nephron, to test whether calcineurin inhibition is involved, these mice are normal at baseline. Here, we confirmed that tacrolimus‐treated control mice developed hypomagnesemia and urinary calcium wasting, with decreased protein and mRNA abundance of key magnesium and calcium transport proteins (NCX‐1 and Calbindin‐D28k). However, qPCR also showed decreased mRNA expression of NCX‐1 and Calbindin‐D28k, and TRPM6. In contrast, KS‐FKBP12−/− mice treated with tacrolimus were completely protected from these effects. These results indicate that tacrolimus affects calcium and magnesium transport along the distal convoluted tubule and strongly suggests that inhibition of the phosphatase, calcineurin, is directly involved., We showed that deletion of the tacrolimus‐binding protein, necessary for tacrolimus to inhibit calcineurin, completely prevented the effects on divalent ion metabolism. This was associated with protection of transport proteins in the distal convoluted tubule.

With no lysine kinase 4 (WNK4) is essential to activate the thiazide-sensitive NaCl cotransporter (NCC) along the distal convoluted tubule, an effect central to the phenotype of familial hyperkalemic hypertension. Although effects on potassium and sodium channels along the connecting and collecting tubules have also been documented, WNK4 is typically believed to have little role in modulating sodium chloride reabsorption along the thick ascending limb of the loop of Henle. Yet wnk4−/−mice (knockout mice lacking WNK4) do not demonstrate the hypocalciuria typical of pure distal convoluted tubule dysfunction. Here, we tested the hypothesis that WNK4 also modulates bumetanide-sensitive Na-K-2Cl cotransporter (NKCC2) function along the thick ascending limb. We confirmed that w nk4−/−mice are hypokalemic and waste sodium chloride, but are also normocalciuric. Results from Western blots suggested that the phosphorylated forms of both NCC and NKCC2 were in lower abundance in wnk4−/−mice than in controls. This finding was confirmed by immunofluorescence microscopy. Although the initial response to furosemide was similar in wnk4−/−mice and controls, the response was lower in the knockout mice when reabsorption along the distal convoluted tubule was inhibited. Using HEK293 cells, we showed that WNK4 increases the abundance of phosphorylated NKCC2. More supporting evidence that WNK4 may modulate NKCC2 emerges from a mouse model of WNK4-mediated familial hyperkalemic hypertension in which more phosphorylated NKCC2 is present than in controls. These data indicate that WNK4, in addition to modulating NCC, also modulates NKCC2, contributing to its physiological function in vivo.

The discovery of new genetic mutations that cause hypertension has illuminated previously unrecognized physiological pathways. One such regulatory pathway was identified when mutations in with no lysine kinase (WNK)4, Kelch-like 3 ( KLHL3), and cullin 3 ( CUL3) were shown to cause the disease familial hyperkalemic hypertension (FHHt). Mutations in all three genes upregulate the NaCl cotransporter (NCC) due to an impaired ability to degrade WNK protein through the cullin-RING-ligase (CRL) ubiquitin-proteasome system. The CUL3 FHHt mutations cause the most severe phenotype, yet the precise mechanism by which these mutations cause the disease has not been established and current proposed models are controversial. New data have identified a possible novel mechanism involving dysregulation of CUL3 activity by the COP9 signalosome (CSN). The CSN interaction with mutant CUL3 is diminished, causing hyperneddylation of the CRL. Recent work has shown that direct renal CSN impairment mimics some aspects of the CUL3 mutation, including lower KLHL3 abundance and activation of the WNK-NCC pathway. Furthermore, in vitro and in vivo studies of CSN inhibition have shown selective degradation of CRL substrate adaptors via auto-ubiquitination, allowing substrate accumulation. In this review, we will focus on recent research that highlights the role of the CSN role in CUL3 mutations that cause FHHt. We will also highlight how these results inform other recent studies of CSN dysfunction.

WNK lysine-deficient protein kinase 4 (WNK4) is an important regulator of renal salt handling. Mutations in its gene cause pseudohypoaldosteronism type II, mainly arising from overactivation of the renal Na(+)/Cl(−) cotransporter (NCC). In addition to full-length WNK4, we have observed faster migrating bands (between 95 and 130 kDa) in Western blots of kidney lysates. Therefore, we hypothesized that these could correspond to uncharacterized WNK4 variants. Here, using several WNK4 antibodies and WNK4(−/−) mice as controls, we showed that these bands indeed correspond to short WNK4 variants that are not observed in other tissue lysates. LC-MS/MS confirmed these bands as WNK4 variants that lack C-terminal segments. In HEK293 cells, truncation of WNK4's C terminus at several positions increased its kinase activity toward Ste20-related proline/alanine-rich kinase (SPAK), unless the truncated segment included the SPAK-binding site. Of note, this gain-of-function effect was due to the loss of a protein phosphatase 1 (PP1)-binding site in WNK4. Cotransfection with PP1 resulted in WNK4 dephosphorylation, an activity that was abrogated in the PP1-binding site WNK4 mutant. The electrophoretic mobility of the in vivo short variants of renal WNK4 suggested that they lack the SPAK-binding site and thus may not behave as constitutively active kinases toward SPAK. Finally, we show that at least one of the WNK4 short variants may be produced by proteolysis involving a Zn(2+)-dependent metalloprotease, as recombinant full-length WNK4 was cleaved when incubated with kidney lysate.

Kir4.1 in the distal convoluted tubule plays a key role in sensing plasma potassium and in modulating the thiazide-sensitive sodium-chloride cotransporter (NCC). Here we tested whether dietary potassium intake modulates Kir4.1 and whether this is essential for mediating the effect of potassium diet on NCC. High potassium intake inhibited the basolateral 40 pS potassium channel (a Kir4.1/5.1 heterotetramer) in the distal convoluted tubule, decreased basolateral potassium conductance, and depolarized the distal convoluted tubule membrane in Kcnj10flox/flox mice, herein referred to as control mice. In contrast, low potassium intake activated Kir4.1, increased potassium currents, and hyperpolarized the distal convoluted tubule membrane. These effects of dietary potassium intake on the basolateral potassium conductance and membrane potential in the distal convoluted tubule were completely absent in inducible kidney-specific Kir4.1 knockout mice. Furthermore, high potassium intake decreased, whereas low potassium intake increased the abundance of NCC expression only in the control but not in kidney-specific Kir4.1 knockout mice. Renal clearance studies demonstrated that low potassium augmented, while high potassium diminished, hydrochlorothiazide-induced natriuresis in control mice. Disruption of Kir4.1 significantly increased basal urinary sodium excretion but it abolished the natriuretic effect of hydrochlorothiazide. Finally, hypokalemia and metabolic alkalosis in kidney-specific Kir4.1 knockout mice were exacerbated by potassium restriction and only partially corrected by a high-potassium diet. Thus, Kir4.1 plays an essential role in mediating the effect of dietary potassium intake on NCC activity and potassium homeostasis.

The mammalian distal convoluted tubule (DCT) makes an important contribution to potassium homeostasis by modulating NaCl transport. The thiazide-sensitive Na+/Cl− cotransporter (NCC) is activated by low potassium intake and by hypokalemia. Coupled with suppression of aldosterone secretion, activation of NCC helps to retain potassium by increasing electroneutral NaCl reabsorption, therefore reducing Na+/K+ exchange. Yet the mechanisms by which DCT cells sense plasma potassium concentration and transmit the information to the apical membrane are not clear. Here, we tested the hypothesis that the potassium channel Kir4.1 is the potassium sensor of DCT cells. We generated mice in which Kir4.1 could be deleted in the kidney after the mice are fully developed. Deletion of Kir4.1 in these mice led to moderate salt wasting, low BP, and profound potassium wasting. Basolateral membranes of DCT cells were depolarized, nearly devoid of conductive potassium transport, and unresponsive to plasma potassium concentration. Although renal WNK4 abundance increased after Kir4.1 deletion, NCC abundance and function decreased, suggesting that membrane depolarization uncouples WNK kinases from NCC. Together, these results indicate that Kir4.1 mediates potassium sensing by DCT cells and couples this signal to apical transport processes.

Background: Nrf2 is postulated to play a protective role in oxidative stress-induced kidney injury; conversely, multiple studies have shown that aberrant Nrf2 activity can be damaging. Nrf2 abundance is determined by cullin-RING ligases (CRLs), which control the regulated degradation of proteins, through ubiquitin proteasome system. CRLs, in turn, are regulated by the COP9 Signalosome (CSN). We previously reported that nephron-specific CSN disruption (KS-Jab1-/- mice) causes progressive renal injury, with increased Nrf2. Here, we tested the hypothesis that direct Nrf2 accumulation exacerbates kidney injury in this model, using KS-Jab1 and Nrf2 double knock out mice (DKO). Methods: The Pax8-rtTA mouse system was used to generate inducible mice with genetic deletion of the catalytically active CSN subunit, Jab1, only along the nephron (KS-Jab1-/-). KS-Jab1-/- were bred to mice with global and constitutive deletion of Nrf2 (DKO). Kidney damage was evaluated at 1 week (early) and 8 weeks (late) after Jab1 deletion. Results: KS-Jab1-/- showed an increase in Nrf2 activity which was attenuated in DKO at both time points. Early KS-Jab1-/- demonstrated an increase in blood urea nitrogen (BUN; 34 ± 2 vs. 26 ± 1 mg/dl in controls), and kidney wgt (6.8 ± 0.3 vs. 5.3 ± 0.2 mg/g·BW in controls). Analysis of the proximal tubule injury marker KIM-1 (kidney injury molecule-1) via Western blot revealed an increase in KS-Jab1-/- compared to controls. Furthermore, immunofluorescent staining using antibodies against the proliferation marker Ki-67 demonstrated an increase in the number of proliferating cells located in kidney medulla (208 ± 27 vs. 42 ± 4 cells/field in controls). Nrf2 deletion in early DKO significantly attenuated the rise in BUN (29 ± 2 mg/dl), KIM-1 abundance, and medullary Ki-67 positive cells (96 ± 13 cells/field). Kidney wgt trended lower (6.0 ± 0.5 mg/g·BW), but was not significantly different. Late DKO showed no significant differences in BUN, kidney wgt, or KIM-1 abundance compared to time-matched KS-Jab1-/-. Conclusions: Disrupting the CRL pathway in mouse tubules, which leads to kidney damage, also increases Nrf2 activity. Nrf2 deficiency reduces kidney injury after Jab1 deletion early, but not at later time points. The results suggest that treatment via modulation of Nrf2 activity in progressive kidney disease may have unexpected effects.

Calcineurin inhibitors (CNIs) are immunosuppressive drugs used to prevent graft rejection after organ transplant. Adverse effects of CNIs include renal magnesium wasting and hypomagnesemia, which may contribute to the development of new-onset diabetes mellitus after kidney transplantation, and hypercalciuria, which may implicate CNIs in post-transplant osteoporosis. The distal convoluted tubule (DCT) is a key site of magnesium and calcium reabsorption by transcellular transport in the kidney. Tacrolimus is a CNI that must bind to its immunophilin, FKBP12, to inhibit the phosphatase calcineurin and impart immunosuppressive effects. Here, we determined the direct role of calcineurin in magnesium and calcium homeostasis and tested whether effects are specific to the DCT. Female and male inducible kidney-specific FKBP12 knockout (KS-FKBP12-/-) mice and control littermates received daily subcutaneous injections of tacrolimus (3 mg∙kg-1 body weight) or vehicle for 18 days. Tacrolimus-treated control mice developed low plasma magnesium concentration and urinary calcium wasting. Western blot analysis demonstrated decreased abundance of key magnesium and calcium transport proteins (NCX-1 and Calbindin-D28k) and qPCR showed decreased mRNA expression of NCX-1 and Calbindin-D28k in addition to TRPM6, an apical magnesium transport channel in the DCT, in tacrolimus-treated control mouse kidney. In contrast, KS-FKBP12-/- mice did not develop changes in these proteins. Gene transcripts associated with magnesium absorption in the thick ascending limb, Claudin 16 and Claudin 19, were not affected by tacrolimus treatment. These results suggest that calcineurin is a key regulator of magnesium and calcium transport proteins in DCT cells., This poster was presented at Oregon Health and Science University at Research Week (RW) on May 13, 2019 by Brittany D.K. Gratreak.

The cullin 3 mutant that causes human familial hyperkalemic hypertension increases WNK abundance, activating the thiazide-sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Previous work determined that the mutation impairs binding to the deneddylase COP9 signalosome (CSN), which might contribute to the disease pathogenesis. In vivo disruption of the CSN using a nephron-specific, inducible mouse model in which the catalytic subunit, JAB1, is deleted (KS-Jab1-/- mice), resulted, however, in a mixed phenotype with WNK activation, but decreased NCC abundance. To test the hypothesis that the effects on NCC are secondary to tubule damage or remodeling, mice were given doxycycline in their drinking water for 3 weeks (w), to induce genetic deletion of Jab1, and were analyzed at 0 w, 1 w, 2 w and 3 w post treatment (PT). Western blotting for WNK4 showed an increase in protein abundance at 0 w PT compared to control, and a significantly higher abundance at each subsequent time point. Consistent with WNK4 activation, the abundance of phosphorylated NCC (pNCC) was greater at 0 w, 1 w, 2 w PT. Total NCC abundance was unchanged at 0 w, 1 w, and 2 w PT, but was significantly lower at 3 w PT. To examine if the late effect on NCC results from DCT remodeling, we used kidney clearing (ethyl cinnamate) with confocal imaging, using antibodies against pNCC to capture three dimensional images of the DCT and measure tubule length. At 0 w PT, DCT length was unchanged compared to control (423.3 ± 25.64 vs. 406.5 ± 21.2 μm), but was significantly shorter in mice at 3 w PT (276.1 ± 18.1 μm, P < 0.01). Along with a shorter DCT length, the number of pNCC-positive tubules was substantially lower at 3 w PT compared to control, suggesting tubule dropout. Immunofluorescent staining for kidney injury molecule-1 (KIM-1) showed similar intensity in the cortex at 0 w and 3 w PT compared to control, indicating that the long-term effects of CSN disruption on the DCT is not caused by tubule damage. However, KIM-1 staining intensity was high in the outer medulla at 0 w and remained so at 3 w PT, suggesting tubule damage at this site. This may have contributed to increased urine output of KS-Jab1-/- mice (control: 5.28 ± 0.29 vs. 0 w: 9.16 ± 1.83 ml/d, P < 0.01) and mitigated the FHHt phenotype. Quantitative real-time PCR measurements demonstrated that mRNA expression for the DCT markers NCC and parvalbumin showed a linear decrease over time (NCC 18% of control at 3 w PT; parvalbumin 8% of control). Interestingly, mRNA expression for the proximal tubule marker, NHE3, and thick ascending limb marker, NKCC2, was not significantly different at 0 w and 1 w PT, however, at 2 w and 3 w PT mRNA levels were approximately 50% of control, suggesting that nephron remodeling/tubule dropout is not limited to the DCT. The results show that in vivo disruption of the CSN mimics many aspects of familial hyperkalemic hypertension, but also leads to tubule damage and nephron remodeling; the latter is likely absent in human disease, owing to its autosomal dominant pathogenesis., This poster was presented at the Experimental Biology (EB) Conference on April 6-9, 2019 by Ryan J. Cornelius.

Background The kidney plays a key role in the regulation of K+ excretion and K+ homeostasis. Previous studies have demonstrated that the thiazide-sensitive Na-Cl cotransporter (NCC) in the distal convoluted tubule (DCT) plays an important role in the regulation of renal K+ excretion by controlling sodium and volume delivery to the distal nephron. It has been shown that NCC is inhibited when K+ intake is high and activated when dietary K+ intake is low. It is now generally accepted that WNKs (with-no-lysine kinases) plays a major role in NCC phosphorylation and activation. A large body of evidence has demonstrated that WNK1 and WNK4 are chloride-sensitive kinases, which are inhibited by high intracellular chloride concentration ([Cl-]i) and activated by low [Cl-]i. It has also been suggested that membrane voltage changes by extracellular K+ concentrations are responsible for altering the [Cl-]i thereby affecting WNK activity. The aim of our study is to test the hypothesis that changes in the basolateral cell voltage alter the [Cl-]i in the DCT. Methods To determine the [Cl-]i in the DCT, we have used isolated single DCT tubule of transgenic mice expressing Cl-sensor, a chloride-sensitive fluorescent protein modified from Chlomeleon. The Cl-sensor includes both yellow and cyan fluorescent moieties, allowing ratiometric estimation of [Cl-]i. Results We first measured the [Cl-]i in the isolated DCT by establishing the calibration curve. Using this calibration curve, we estimated that basal DCT [Cl-]i is 7± 2.3mM (n=18) . Changing the extracellular potassium concentration from 10 mM to 2 mM decreases the [Cl-]i. To test the role of the basolateral Kir4.1/Kir5.1 of the DCT in altering [Cl-]i, we examined whether inhibition of the basolateral K+ channels with Ba2+ would increase the [Cl-]i since Ba2+ has been shown to depolarize DCT membrane. Indeed, Inhibition of Kir4.1/Kir5.1 significantly increased the [Cl-]i in the DCT bathed in a solution containing 2 mM K+. Conclusion Our results indicate that the intracellular Cl- concentrations of DCT cells are low at baseline and that the depolarization increases whereas hyperpolarization decreases the intracellular Cl- concentrations. Thus, changes in DCT voltage are associated with alteration of intracellular Cl- concentrations. &nbsp

Excess aldosterone is an important contributor to hypertension and cardiovascular disease. Conversely, low circulating aldosterone causes salt wasting and hypotension. Aldosterone activates mineralocorticoid receptors (MRs) to increase epithelial sodium channel (ENaC) activity. However, aldosterone may also stimulate the thiazide-sensitive Na(+)-Cl(-) cotransporter (NCC). Here, we generated mice in which MRs could be deleted along the nephron to test this hypothesis. These kidney-specific MR-knockout mice exhibited salt wasting, low BP, and hyperkalemia. Notably, we found evidence of deficient apical orientation and cleavage of ENaC, despite the salt wasting. Although these mice also exhibited deficient NCC activity, NCC could be stimulated by restricting dietary potassium, which also returned BP to control levels. Together, these results indicate that MRs regulate ENaC directly, but modulation of NCC is mediated by secondary changes in plasma potassium concentration. Electrolyte balance and BP seem to be determined, therefore, by a delicate interplay between direct and indirect mineralocorticoid actions in the distal nephron.

Familial hyperkalemic hypertension is caused by mutations in with-no-lysine kinases (WNKs) or in proteins that mediate their degradation, kelch-like 3 (KLHL3) and cullin 3 (CUL3). Although the mechanisms by which WNK and KLHL3 mutations cause the disease are now clear, the effects of the disease-causing CUL3Δ403–459 mutation remain controversial. Possible mechanisms, including hyperneddylation, altered ubiquitin ligase activity, decreased association with the COP9 signalosome (CSN), and increased association with and degradation of KLHL3 have all been postulated. Here, we systematically evaluated the effects of Cul3Δ403–459 using cultured kidney cells. We first identified that the catalytically active CSN subunit jun activation domain-binding protein-1 (JAB1) does not associate with the deleted Cul3 4-helix bundle domain but instead with the adjacent α/β1 domain, suggesting that altered protein folding underlies the impaired binding. Inhibition of deneddylation with JAB1 siRNA increased Cul3 neddylation and decreased KLHL3 abundance, similar to the Cul3 mutant. We next determined that KLHL3 degradation has both ubiquitin ligase-dependent and -independent components. Proteasomal KLHL3 degradation was enhanced by Cul3Δ403–459; however, autophagic degradation was also upregulated by this Cul3 mutant. Finally, to evaluate whether deficient substrate adaptor was responsible for the disease, we restored KLHL3 to wild-type (WT) Cul3 levels. In the absence of WT Cul3, WNK4 was not degraded, demonstrating that Cul3Δ403–459 itself cannot degrade WNK4; conversely, when WT Cul3 was present, as in diseased humans, WNK4 degradation was restored. In conclusion, deletion of exon 9 from Cul3 generates a protein that is itself ubiquitin-ligase defective but also capable of enhanced autophagocytic KLHL3 degradation, thereby exerting dominant-negative effects on the WT allele.

Background The familial hyperkalemic hypertension (FHHt) cullin 3 (CUL3) mutant does not degrade WNK kinases normally, thereby leading to thiazide-sensitive Na-Cl cotransporter (NCC) activation. CUL3 mutant (CUL3 Δ 9) does not bind normally to the COP9 signalosome (CSN), a deneddylase involved in regulating cullin-RING ligases. CUL3 Δ 9 also caused increased degradation of the CUL3-WNK substrate adaptor kelch-like 3 (KLHL3). Here, we sought to determine how defective CSN action contributes to the CUL3 Δ 9 phenotype. Methods The Pax8/LC1 mouse system was used to generate mice in which the catalytically active CSN subunit, Jab1 , was deleted only along the nephron, after full development (KS- Jab1 −/− ). Results Western blot analysis demonstrated that Jab1 deletion increased the abundance of neddylated CUL3. Moreover, total CUL3 expression was reduced, suggesting decreased CUL3 stability. KLHL3 was almost completely absent in KS- Jab1 −/− mice. Conversely, the protein abundances of WNK1, WNK4, and SPAK kinases were substantially higher. Activation of WNK4, SPAK, and OSR1 was indicated by higher phosphorylated protein levels and translocation of the proteins into puncta, as observed by immunofluorescence. The ratio of phosphorylated NCC to total NCC was also higher. Surprisingly, NCC protein abundance was low, likely contributing to hypokalemia and Na + and K + wasting. Additionally, long-term Jab1 deletion resulted in kidney damage. Conclusions Together, the results indicate that deficient CSN binding contributes importantly to the FHHt phenotype. Although defective CUL3 Δ 9-faciliated WNK4 degradation likely contributes, dominant effects on KLHL3 may be a second factor that is necessary for the phenotype.

Tacrolimus is a widely used immunosuppressive drug that inhibits the phosphatase calcineurin when bound to the 12 kDa FK506-binding protein (FKBP12). When this binding occurs in T cells, it leads to immunosuppression. Tacrolimus also causes side effects, however, such as hypertension and hyperkalemia. Previously, we reported that tacrolimus stimulates the renal thiazide-sensitive sodium chloride cotransporter (NCC), which is necessary for the development of hypertension. However, it was unclear if tacrolimus-induced hypertension resulted from tacrolimus effects in renal epithelial cells directly or in extrarenal tissues, and whether inhibition of calcineurin was required. To address these questions, we developed a mouse model in which FKBP12 could be deleted along the nephron. FKBP12 disruption alone did not cause phenotypic effects. When treated with tacrolimus, however, BP and the renal abundance of phosphorylated NCC were lower in mice lacking FKBP12 along the nephron than in control mice. Mice lacking FKBP12 along the nephron also maintained a normal relationship between plasma potassium levels and the abundance of phosphorylated NCC with tacrolimus treatment. In cultured cells, tacrolimus inhibited dephosphorylation of NCC. Together, these results suggest that tacrolimus causes hypertension predominantly by inhibiting calcineurin directly in cells expressing NCC, indicating thiazide diuretics may be particularly effective for lowering BP in tacrolimus-treated patients with hypertension.

The with-no-lysine (K) kinases, WNK1 and WNK4, are key regulators of blood pressure. Their mutations lead to familial hyperkalemic hypertension (FHHt), associated with an activation of the Na-Cl cotransporter (NCC). Although it is clear that WNK4 mutants activate NCC via Ste20 proline-alanine–rich kinase, the mechanisms responsible for WNK1-related FHHt and alterations in NCC activity are not as clear. We tested whether WNK1 modulates NCC through WNK4, as predicted by some models, by crossing our recently developed WNK1-FHHt mice ( WNK1 +/FHHt ) with WNK4 −/− mice. Surprisingly, the activated NCC, hypertension, and hyperkalemia of WNK1 +/FHHt mice remain in the absence of WNK4. We demonstrate that WNK1 powerfully stimulates NCC in a WNK4-independent and Ste20 proline-alanine–rich kinase–dependent manner. Moreover, WNK4 decreases the WNK1 and WNK3-mediated activation of NCC. Finally, the formation of oligomers of WNK kinases through their C-terminal coiled-coil domain is essential for their activity toward NCC. In conclusion, WNK kinases form a network in which WNK4 associates with WNK1 and WNK3 to regulate NCC.

The sodium chloride cotransporter (NCC) is the primary target of thiazides diuretics, drugs used commonly for long-term hypertension therapy. Thiazides also completely reverse the signs of familial hyperkalemic hypertension (FHHt), suggesting that the primary defect in FHHt is increased NCC activity. To test whether increased NCC abundance alone is sufficient to generate the FHHt phenotype, we generated NCC transgenic mice; surprisingly, these mice did not display an FHHt-like phenotype. Systolic blood pressures of NCC transgenic mice did not differ from those of wild-type mice, even after dietary salt loading. NCC transgenic mice also did not display hyperkalemia or hypercalciuria, even when challenged with dietary electrolyte manipulation. Administration of fludrocortisone to NCC transgenic mice, to stimulate NCC, resulted in an increase in systolic blood pressure equivalent to that of wild-type mice (approximately 20 mm Hg). Although total NCC abundance was increased in the transgenic animals, phosphorylated (activated) NCC was not, suggesting that the defect in FHHt involves either activation of ion transport pathways other than NCC, or else direct activation of NCC, in addition to an increase in NCC abundance.

Calcineurin inhibitors (CNIs) are immunosuppressive drugs that are used widely to prevent rejection of transplanted organs and to treat autoimmune disease. Hypertension and renal tubule dysfunction, including hyperkalemia, hypercalciuria and acidosis, often complicate their use(1,2). These side effects resemble familial hyperkalemic hypertension, a genetic disease characterized by overactivity of the renal sodium chloride cotransporter (NCC) and caused by mutations in genes encoding WNK kinases. We hypothesized that CNIs induce hypertension by stimulating NCC. In wild-type mice, the CNI tacrolimus caused salt-sensitive hypertension and increased the abundance of phosphorylated NCC and the NCC-regulatory kinases WNK3, WNK4 and SPAK. We demonstrated the functional importance of NCC in this response by showing that tacrolimus did not affect blood pressure in NCC-knockout mice, whereas the hypertensive response to tacrolimus was exaggerated in mice overexpressing NCC. Moreover, hydrochlorothiazide, an NCC-blocking drug, reversed tacrolimus-induced hypertension. These observations were extended to humans by showing that kidney transplant recipients treated with tacrolimus had a greater fractional chloride excretion in response to bendroflumethiazide, another NCC-blocking drug, than individuals not treated with tacrolimus; renal NCC abundance was also greater. Together, these findings indicate that tacrolimus-induced chronic hypertension is mediated largely by NCC activation, and suggest that inexpensive and well-tolerated thiazide diuretics may be especially effective in preventing the complications of CNI treatment.

SummaryThe renal thick ascending limb (TAL) and distal convoluted tubule (DCT) play central roles in salt homeostasis and blood pressure regulation. An emerging model suggests that bumetanide- and thiazide-sensitive NaCl transporters (NKCC2 and NCC) along these segments are phosphorylated and activated by WNK kinases, via SPAK and OSR1. Here, we show that a kidney-specific SPAK isoform, which lacks the kinase domain, inhibits phosphorylation of NCC and NKCC2 by full-length SPAK in vitro. Kidney-specific SPAK is highly expressed along the TAL, whereas full-length SPAK is more highly expressed along the DCT. As predicted from the differential expression, SPAK knockout in animals has divergent effects along TAL and DCT, with increased phosphorylated NKCC2 along TAL and decreased phosphorylated NCC along DCT. In mice, extracellular fluid volume depletion shifts SPAK isoform abundance to favor NaCl retention along both segments, indicating that a SPAK isoform switch modulates sodium avidity along the distal nephron.

The with no lysine (WNK) kinases comprise a novel branch of the human kinome that plays a central role in regulating renal sodium, potassium, and chloride transport, and, therefore, blood pressure. Mutations of two WNK kinases, WNK1 and WNK4, cause familial hyperkalemic hypertension (Gordon’s syndrome or Type II pseudohypoaldosteronism), a rare monogenic disease. Many aspects of WNK action have been elucidated during the past seven years. WNKs are all expressed along a short segment of renal distal tubule, where they modulate the activity of a wide variety of transport proteins. These diverse effects, however, make it difficult to describe an integrated model of WNK function within the kidney. Recently, work in vivo and in vitro has begun to clarify this picture. The present review emphasizes recent insights into mechanism by which WNK kinases interact to modulate sodium and potassium transport along the aldosterone-sensitive distal nephron. We describe a potential mechanism by which WNK4 mutations convert the action of WNK4 from inhibiting renal sodium chloride retention to stimulating it, thereby affecting both blood pressure and potassium balance. An explanation for how WNK kinases can alter the effects of aldosterone from primarily kaliuretic to primarily sodium chloride retentive, according to physiological need, is also described.

The cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-gated chloride channel. WNK kinases are widely expressed modulators of ion transport. WNK1 and WNK4, two WNK kinases that are mutated in familial hyperkalemic hypertension (FHHt), are co-expressed with CFTR in several organs, raising the possibility that WNK kinases might alter CFTR activity in vivo or that CFTR could be involved in the pathogenesis of FHHt. Here, we report that WNK1 co-localizes with CFTR protein in pulmonary epithelial cells. Co-expression of WNK1 or WNK4 with CFTR in Xenopus laevis oocytes suppresses chloride channel activity. The effect of WNK4 is dose dependent and occurs, at least in part, by reducing CFTR protein abundance at the plasma membrane. This effect is independent of WNK4 kinase activity. In contrast, the effect of WNK1 on CFTR activity requires intact WNK1 kinase activity. Moreover WNK1 and WNK4 exhibit additive CFTR inhibition. Previous reports suggest that patients with FHHt exhibit mild changes in nasal potential difference that resemble the more severe changes that occur in cystic fibrosis. We report that the FHHt-causing mutant WNK4 Q562E is a more potent inhibitor of CFTR activity than is the wild-type WNK4. Taken together, these results suggest that WNK1 and WNK4 may modulate CFTR activity; they further suggest that WNK kinases may be potential therapeutic targets for cystic fibrosis.

To investigate the protective effect of Danxuetong injection (DXT, a combination of Danshen and Xueshuantong injections) against testicular ischemia-reperfusion injury following testis torsion/detorsion in rats.Thirty-two 4-week-old healthy male SD rats were randomly divided into four groups of equal number: sham operation, normal saline, single DXT injection, and successive DXT injection. The rat models of testicular ischemia-reperfusion injury were established by 2-hour 720-degree torsion/detorsion of the unilateral testis. At 6 weeks after modeling, the rats were killed and their testes were harvested for measure- ment of testicular coefficients, sperm counts, sperm motility, and the levels of total anti-oxidative capacity (T-AOC) , superoxide dismutase (SOD) , nitric oxide synthase (NOS) , and malondialdehyde ( MDA) in the testis tissue.Compared with the rats of the normal saline group, those of the single DXT injection and successive DXT injection groups showed significant increases in the testicular coefficient (0.11 ± 0.03 vs 0.35 ± 0.04 and 0.40 ± 0.06, P0.05), sperm count ([0.46 ± 0.10] vs [1.44 ± 0.50] and [3.00 ± 1.28] x10(9)/ml, P0.05), sperm motility ([13.63 ± 14.04] vs [39.63 ± 5.04] and [76.31 ± 3.67]%, P0.05), the activity of SOD (72.76 ± 5.58 vs 116.25 ± 8.83 and 133.20 ± 13.84, P0.05), and the level of T-AOC (5.58 ± 1.07 vs 13.34 ± 5.81 and 19.21 ± 5.69, P0.05), but a remarkable decrease in the content of MDA (42.38 ± 8.94 vs 20.94 ± 5.65 and 15.02 ± 1.03, P0. 05) in the injured testes.DXT can effectively rid the testis tissue of oxygen free radicals, improve sperm count and motility by antioxidation, and protect the testis tissue of prepubertal rats against testicular ischemia-reperfusion injury after testis torsion/detorsion. It also has a protective effect on the contralateral testis, and successive injection has a better effect than single injection of DXT.

Mutations in WNK1 and WNK4, and in components of the Cullin-Ring Ligase system, kelch-like 3 (KLHL3) and Cullin 3 (CUL3), can cause the rare hereditary disease, Familial Hyperkalemic Hypertension (FHHt). The disease is characterized by overactivity of the renal sodium chloride cotransporter (NCC), which is phosphorylated and activated by the WNK-stimulated Ste20-type kinases, SPAK and OSR1. WNK kinases themselves can be targeted for ubiquitination and degradataion by the CUL3-KLHL3 E3 ubiquitin ligase complex. It is unclear, however, why there are significant differences in phenotypic severity among FHHt patients with mutations in different genes. It was reported that kelch-like 2 (KLHL2), a homolog of KLHL3, can also target WNK kinases for ubiquitation and degradation, and may play a special role in the systemic vasculature. Our recent study revealed the disease mutant CUL3 exhibits enhanced degradation of its adaptor protein KLHL3, potentially resulting in accumulation of WNK kinases secondarily. To investigate if KLHL2 plays a role in FHHt, we studied the effect of wild type and FHHt mutant CUL3 on degradation of KLHL2 and WNK kinase proteins in HEK293 cells. Although CUL3 facilitates KLHL2 degradation, the disease mutant CUL3 is more active in this regard. KLHL2 facilitated the degradation of wild type but not disease mutant WNK4 protein. These results suggest that KLHL2 likely plays a role in the pathogenesis of FHHt, and aggravates the phenotype caused by mutations in CUL3 and WNK4.

Dietary potassium deficiency activates thiazide-sensitive sodium chloride cotransport along the distal nephron. This may explain, in part, the hypertension and cardiovascular mortality observed in individuals who consume a low potassium diet. Recent data suggest plasma potassium affects the distal nephron directly by influencing intracellular chloride, an inhibitor of the With no lysine kinase (WNK)-Ste20p-related proline-and alanine-rich kinase (SPAK) pathway. Since previous studies used extreme dietary manipulations, we sought to determine if the relationship between potassium and NCC is physiologically relevant and clarify the mechanisms involved. We report that modest changes in both dietary and plasma potassium affect the thiazide-sensitive sodium-chloride cotransporter, NCC, in vivo. Kinase assay studies showed that chloride inhibits WNK4 kinase activity at lower concentrations than it inhibits activity of WNK1 or WNK3. Also, chloride inhibited WNK4 within the range of distal cell chloride. Mutation of a previously identified WNK chloride-binding motif converted WNK4 effects on SPAK from inhibitory to stimulatory in mammalian cells. Disruption of this motif in WNKs 1, 3 and 4 had different effects on NCC, consistent with the three WNKs having different chloride sensitivities. Thus, potassium effects on NCC are graded within the physiological range, which explains how unique chloride-sensing properties of WNK4 enable kinase mediating effects of potassium on NCC in vivo.