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The disease familial hyperkalemic hypertension (FHHt; also known as Gordon syndrome) is caused by aberrant accumulation of with-no-lysine kinase (WNK4) activating the NaCl cotransporter (NCC) in the distal convoluted tubule (DCT) of the kidney. Mutations in cullin 3 (CUL3) cause FHHt by disrupting interaction with the deneddylase COP9 signalosome (CSN). Deletion of Cul3 or Jab1 (the catalytically active CSN subunit) along the entire nephron causes a partial FHHt phenotype with activation of the WNK4-STE20/SPS1-related proline/alanine-rich kinase (SPAK)-NCC pathway. However, progressive kidney injury likely prevents hypertension, hyperkalemia, and hyperchloremic metabolic acidosis associated with FHHt. We hypothesized that DCT-specific deletion would more closely model the disease. We used Slc12a3-Cre-ERT2 mice to delete Cul3 (DCT-Cul3−/−) or Jab1 (DCT-Jab1−/−) only in the DCT and examined the mice after short- and long-term deletion. Short-term DCT-specific knockout of both Cul3 and Jab1 mice caused elevated WNK4, pSPAKS373, and pNCCT53 abundance. However, neither model demonstrated changes in plasma K+, Cl−, or total CO2, even though no injury was present. Long-term DCT-Jab1−/− mice showed significantly lower NCC and parvalbumin abundance and a higher abundance of kidney injury molecule-1, a marker of proximal tubule injury. No injury or reduction in NCC or parvalbumin was observed in long-term DCT-Cul3−/− mice. In summary, the prevention of injury outside the DCT did not lead to a complete FHHt phenotype despite activation of the WNK4-SPAK-NCC pathway, possibly due to insufficient NCC activation. Chronically, only DCT-Jab1−/− mice developed tubule injury and atrophy of the DCT, suggesting a direct JAB1 effect or dysregulation of other cullins as mechanisms for injury. NEW & NOTEWORTHY: CUL3 degrades WNK4, which prevents activation of NCC in the DCT. CSN regulation of CUL3 is impaired in the disease FHHt, causing accumulation of WNK4. Short-term DCT-specific disruption of CUL3 or the CSN in mice resulted in activation of the WNK4-SPAK-NCC pathway but not hyperkalemic metabolic acidosis found in FHHt. Tubule injury was observed only after long-term CSN disruption. The data suggest that disruption of other cullins may be the cause for the injury. [ABSTRACT FROM AUTHOR]

The with-no-lysine kinase 4 (WNK4)-sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK)/oxidative stress-responsive kinase 1 (OSR1) pathway mediates activating phosphorylation of the furosemide-sensitive Na+-K+-2Cl- cotransporter (NKCC2) and the thiazide-sensitive NaCl cotransporter (NCC). The commonly used pT96/pT101-pNKCC2 antibody cross-reacts with pT53-NCC in mice on the C57BL/6 background due to a five amino acid deletion. We generated a new C57BL/6-specific pNKCC2 antibody (anti-pT96-NKCC2) and tested the hypothesis that the WNK4-SPAK/OSR1 pathway strongly regulates the phosphorylation of NCC but not NKCC2. In C57BL/6 mice, anti-pT96-NKCC2 detected pNKCC2 and did not cross-react with NCC. Abundances of pT96-NKCC2 and pT53-NCC were evaluated in Wnk4-/-, Osr1-/-, Spak-/-, and Osr1-/-/Spak-/- mice and in several models of the disease familial hyperkalemic hypertension (FHHt) in which the CUL3-KLHL3 ubiquitin ligase complex that promotes WNK4 degradation is dysregulated (Cul3+/-/Δ9-Klhl3-/-, and Klhl3R528H/R528H). All mice were on the C57BL/6 background. In Wnk4-/- mice, pT53-NCC was almost absent but pT96-NKCC2 was only slightly lower. pT53-NCC was almost absent in Spak-/- and Osr1-/-/Spak-/- mice, but pT96-NKCC2 abundance did not differ from controls. pT96-NKCC2/total NKCC2 was slightly lower in Osr1-/- and Osr1-/-/Spak-/- mice. WNK4 expression colocalized not only with NCC but also with NKCC2 in Klhl3-/- mice, but pT96-NKCC2 abundance was unchanged. Consistent with this, furosemide-induced urinary Na+ excretion following thiazide treatment was similar between Klhl3-/- and controls. pT96-NKCC2 abundance was also unchanged in the other FHHt mouse models. Our data show that disruption of the WNK4-SPAK/OSR1 pathway only mildly affects NKCC2 phosphorylation, suggesting a role for other kinases in NKCC2 activation. In FHHt models NKCC2 phosphorylation is unchanged despite higher WNK4 abundance, explaining the thiazide sensitivity of FHHt. [ABSTRACT FROM AUTHOR]

The study of rare monogenic forms of hypertension has led to the elucidation of important physiological pathways controlling blood pressure. Mutations in several genes cause familial hyperkalemic hypertension (also known as Gordon syndrome or pseudohypoaldosteronism type II). The most severe form of familial hyperkalemic hypertension is caused by mutations in CUL3 , encoding CUL3 (Cullin 3)—a scaffold protein in an E3 ubiquitin ligase complex that tags substrates for proteasomal degradation. In the kidney, CUL3 mutations cause accumulation of the substrate WNK (with-no-lysine [K]) kinase and ultimately hyperactivation of the renal NaCl cotransporter—the target of the first-line antihypertensive thiazide diuretics. The precise mechanisms by which mutant CUL3 causes WNK kinase accumulation have been unclear, but several functional defects are likely to contribute. The hypertension seen in familial hyperkalemic hypertension also results from effects exerted by mutant CUL3 on several pathways in vascular smooth muscle and endothelium that modulate vascular tone. This review summarizes the mechanisms by which wild type and mutant CUL3 modulate blood pressure through effects on the kidney and vasculature, potential effects in the central nervous system and heart, and future directions for investigation. [ABSTRACT FROM AUTHOR]

Mutations in the ubiquitin ligase scaffold protein cullin 3 (CUL3) cause the disease familial hyperkalemic hypertension (FHHt). We recently reported that in the kidney, aberrant mutant CUL3 (CUL3-D9) activity lowers the abundance of CUL3-D9 and Kelch-like 3, the CUL3 substrate adaptor for with-no-lysine kinase 4 (WNK4) and that this is mechanistically important. However, whether CUL3-D9 exerts additional effects on other targets that may alter renal function is unclear. Here, we sought to determine 1) whether CUL3-D9 expression can rescue the phenotype of renal tubule-specific Cul3 knockout mice, and 2) whether CUL3-D9 expression affects other CUL3 substrates. Using an inducible renal tubule-specific system, we studied two CUL3-D9-expressing mouse models: Cul3 knockout (Cul3-/-/D9) and Cul3 heterozygous background (Cul3-/-/D9, FHHt model). The effects of CUL3-D9 in these mice were compared with Cul3-/- and Cul3+/- mice. Similar to Cul3-/- mice, Cul3-/-/D9 mice displayed polyuria with loss of aquaporin 2 and collecting duct injury; proximal tubule injury also occurred. CUL3-D9 did not promote degradation of two CUL3 targets that accumulate in the Cul3-/- kidney: high-molecular-weight (HMW) cyclin E and NAD(P)H:quinone oxidoreductase 1 (NQO1) [a surrogate for the CUL3-Kelch-like ECH-associated protein 1 (KEAP1) substrate nuclear factor erythroid-2-related factor 2]. Since CUL3-D9 expression cannot rescue the Cul3-/- phenotype, our data suggest that CUL3-D9 cannot normally function in ubiquitin ligase complexes. In Cul3+/-/D9 mice, KEAP1 abundance did not differ but NQO1 abundance was higher, suggesting adaptor sequestration by CUL3-D9 in vivo. Together, our results provide evidence that in the kidney, CUL3-D9 completely lacks normal activity and can trap CUL3 substrate adaptors in inactive complexes. [ABSTRACT FROM AUTHOR]

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 Naþ-Cl-cotransporter (NCC), the main pathway for Naþ 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. [ABSTRACT FROM AUTHOR]

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 R26RTdTomato 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

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 wnk4-/- 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. [ABSTRACT FROM AUTHOR]

A low-Na+, high-K+ diet (LNaHK) is considered a healthier alternative to the "Western" high-Na+ diet. Because the mechanism for K+ secretion involves Na+ reabsorptive exchange for secreted K+ in the distal nephron, it is not understood how K+ is eliminated with such low Na+ intake. Animals on a LNaHK diet produce an alkaline load, high urinary flows, and markedly elevated plasma ANG II and aldosterone levels to maintain their K+ balance. Recent studies have revealed a potential mechanism involving the actions of alkalosis, urinary flow, elevated ANG II, and aldosterone on two types of K+ channels, renal outer medullary K+ and large-conductance K+ channels, located in principal and intercalated cells. Here, we review these recent advances. [ABSTRACT FROM AUTHOR]

In many circumstances, the pathogenesis of distal renal tubular acidosis (dRTA) is not understood. In the present study, we report that a mouse model lacking the electrogenic Na+ -HCO3- cotransporter [NBCe2/Slc4a5; NBCe2 knockout (KO) mice] developed dRTA after an oral acid challenge. NBCe2 expression was identified in the connecting tubule (CNT) of wild-type mice, and its expression was significantly increased after acid loading. NBCe2 KO mice did not have dRTA when on a standard mouse diet. However, after acid loading, NBCe2 KO mice exhibited complete features of dRTA, characterized by insufficient urinary acidification, hyperchloremic hypokalemic metabolic acidosis, and hypercalciuria. Additional experiments showed that NBCe2 KO mice had decreased luminal transepithelial potential in the CNT, as revealed by micropuncture. Further immunofluorescence and Western blot experiments found that NBCe2 KO mice had increased expression of H+ -ATPase B1 in the plasma membrane. These results showed that NBCe2 KO mice with acid loading developed increased urinary K+ and Ca2+ wasting due to decreased luminal transepithelial potential in the CNT. NBCe2 KO mice compensated to maintain systemic pH by increasing H+ -ATPase in the plasma membrane. Therefore, defects in NBCe2 can cause dRTA, and NBCe2 has an important role to regulate urinary acidification and the transport of K+ and Ca2+ in the distal nephron. [ABSTRACT FROM AUTHOR]

The gene SLC4A5 encodes the Na(+)-HCO3 (-) cotransporter electrogenic 2, which is located in the distal nephron. Genetically deleting Na(+)-HCO3 (-) cotransporter electrogenic 2 (knockout) causes Na(+)-retention and hypertension, a phenotype that is diminished with alkali loading. We performed experiments with acid-loaded mice and determined whether overactive epithelial Na(+) channels (ENaC) or the Na(+)-Cl(-) cotransporter causes the Na(+) retention and hypertension in knockout. In untreated mice, the mean arterial pressure was higher in knockout, compared with wild-type (WT); however, treatment with amiloride, a blocker of ENaC, abolished this difference. In contrast, hydrochlorothiazide, an inhibitor of Na(+)-Cl(-) cotransporter, decreased mean arterial pressure in WT, but not knockout. Western blots showed that quantity of plasmalemmal full-length ENaC-α was significantly higher in knockout than in WT. Amiloride treatment caused a 2-fold greater increase in Na(+) excretion in knockout, compared with WT. In knockout, but not WT, amiloride treatment decreased plasma [Na(+)] and urinary K(+) excretion, but increased hematocrit and plasma [K(+)] significantly. Micropuncture with microelectrodes showed that the [K(+)] was significantly higher and the transepithelial potential (Vte) was significantly lower in the late distal tubule of the knockout compared with WT. The reduced Vte in knockout was amiloride sensitive and therefore revealed an upregulation of electrogenic ENaC-mediated Na(+) reabsorption in this segment. These results show that, in the absence of Na(+)-HCO3 (-) cotransporter electrogenic 2 in the late distal tubule, acid-loaded mice exhibit disinhibition of ENaC-mediated Na(+) reabsorption, which results in Na(+) retention, K(+) wasting, and hypertension. [ABSTRACT FROM AUTHOR]

The gene SLC4A5 encodes the Na+-HCO3 cotransporter electrogenic 2, which is located in the distal nephron. Genetically deleting Na+-HCO3 cotransporter electrogenic 2 (knockout) causes Na+-retention and hypertension, a phenotype that is diminished with alkali loading. We performed experiments with acid-loaded mice and determined whether overactive epithelial Na+ channels (ENaC) or the Na+-Cl- cotransporter causes the Na+ retention and hypertension in knockout. In untreated mice, the mean arterial pressure was higher in knockout, compared with wild-type (WT); however, treatment with amiloride, a blocker of ENaC, abolished this difference. In contrast, hydrochlorothiazide, an inhibitor of Na+-Cl- cotransporter, decreased mean arterial pressure in WT, but not knockout. Western blots showed that quantity of plasmalemmal full-length ENaC-α was significantly higher in knockout than in WT. Amiloride treatment caused a 2-fold greater increase in Na+ excretion in knockout, compared with WT. In knockout, but not WT, amiloride treatment decreased plasma [Na+] and urinary K+ excretion, but increased hematocrit and plasma [K+] significantly. Micropuncture with microelectrodes showed that the [K+] was significantly higher and the transepithelial potential (Vte) was significantly lower in the late distal tubule of the knockout compared with WT. The reduced Vte in knockout was amiloride sensitive and therefore revealed an upregulation of electrogenic ENaC-mediated Na+ reabsorption in this segment. These results show that, in the absence of Na+-HCO3 cotransporter electrogenic 2 in the late distal tubule, acid-loaded mice exhibit disinhibition of ENaC-mediated Na+ reabsorption, which results in Na+ retention, K+ wasting, and hypertension. [ABSTRACT FROM AUTHOR]

A low Na, high K diet (LNaHK) is associated with a low rate of cardiovascular (CV) disease in many societies. Part of the benefit of LNaHK relies on its diuretic effects; however, the role of aldosterone (aldo) in the diuresis is not understood. LNaHK mice exhibit an increase in renal K secretion that is dependent on the large, Ca-activated K channel, (BK-α with accessory BK-β4; BK-α/β4). We hypothesized that aldo causes an osmotic diuresis by increasing BK-α/β4-mediated K secretion in LNaHK mice. We found that the plasma aldo concentration (P[aldo]) was elevated by 10-fold in LNaHK mice compared with control diet (Con) mice. We subjected LNaHK mice to either sham surgery (sham), adrenalectomy (ADX) with low aldo replacement (ADX-LA), or ADX with high aldo replacement (ADX-HA). Compared to sham, the urinary flow, K excretion rate, transtubular K gradient (TTKG), and BK-α and BK-β4 expressions, were decreased in ADX-LA, but not different in ADX-HA. BK-β4 knockout (β4KO) and WT mice exhibited similar K clearance and TTKG in the ADX-LA groups; however, in sham and ADX-HA, the K clearance and TTKG of β4KO were less than WT. In response to amiloride treatment, the osmolar clearance was increased in WT Con, decreased in WT LNaHK, and unchanged in β4KO LNaHK. These data show that the high P[aldo] of LNaHK mice is necessary to generate a high rate of BK-α/β4-mediated K secretion, which creates an osmotic diuresis that may contribute to a reduction in CV disease. [ABSTRACT FROM AUTHOR]

R2679 --> 614.3 --> With‐no‐lysine kinase 4 (WNK4) activates the NaCl cotransporter (NCC) which is the main pathway for sodium reabsorption in the distal convoluted tubule (DCT) of the kidney. The cullin‐RING ubiquitin ligase cullin 3 (CUL3) regulates WNK4 abundance via interaction through the substrate adaptor KLHL3. The monogenic disease familial hyperkalemic hypertension (FHHt) is caused by mutations in this pathway. Mutations in KLHL3 and WNK4 mainly disrupt formation of this complex, whereas, the CUL3 mutations impair binding to the COP9 signalosome (CSN), a deneddylase and upstream regulator of cullin‐RING ligases. Since FHHt is mostly a disease that affects the DCT, we proposed that DCT‐specific knockdown of the main CSN subunit, JAB1, would phenocopy FHHt. We deleted Jab1 in the DCT using the tamoxifen inducible NCC‐Cre‐ERT2 mouse model (DCT‐Jab1‐/‐). After five days of tamoxifen administration, proximal tubule, thick ascending limb, DCT, and connecting tubule/collecting duct segments were individually dissected and isolated to analyze protein abundance of each specific segment via Western blot. JAB1 and CUL3 protein abundance were substantially reduced in the DCT of DCT‐Jab1‐/‐ mice, whereas, WNK4 and phosphorylated NCC (pNCC) abundance were markedly elevated. Western blot analysis of cortical kidney tissue showed lower KLHL3 abundance and significantly higher abundance of phosphorylated SPAK (pSPAK), the intermediary kinase between WNK4 and NCC. Immunofluorescent staining of WNK4 and pSPAK showed increased expression and puncta formation in DCT1 and DCT2 tubules of DCT‐Jab1‐/‐ mice. This was not observed in other tubule segments that express WNK4 or pSPAK, such as the thick ascending limb and connecting tubule/collecting duct. Although there was obvious activation of the WNK4‐SPAK‐NCC pathway DCT‐Jab1‐/‐ mice, plasma analysis showed no significant differences in potassium, chloride or TCO2. However, challenging the DCT‐Jab1‐/‐ mice with a high potassium diet for seven days exaggerated the differences in pSPAK and pNCC protein abundance and led to a significant increase in plasma potassium. The results indicate that DCT‐specific disruption of JAB1 recapitulates many aspects of FHHt, including decreased KLHL3 and CUL3 abundance which leads to activation of the WNK4‐NCC pathway and a tendency toward hyperkalemia. These results implicate defective CSN binding as a key component of CUL3‐mediated familial hyperkalemic hypertension. [ABSTRACT FROM AUTHOR]

In the distal nephron, the large-conductance Ca-activated K (BK) channel, comprised of a pore-forming-α (BK-α) and the BK-β4 subunit, promotes K excretion when mice are maintained on a high-K alkaline diet (HK-alk). We examined whether BK-β4 and the acid-base status regulate apical membrane expression of BK-α in the cortical (CCD) and medullary collecting ducts (MCD) using immunohistochemical analysis (IHC) and Western blot. With the use of IHC, BK-α of mice on acontrol diet localized mostly cytoplasmically in intercalated cells (IC) of the CCD and in the perinuclear region of both principle cells (PC) and IC of the MCD. HK-alk wild-type mice (WT), but not BK-β4 knockout mice (β4KO), exhibited increased apical BK-α in both the CCD and MCD. When given a high-K acidic diet (HK-Cl), BK-α expression increased but remained cytoplasmic in the CCD and perinuclear in the MCD of both WT and β4KO. Western blot confirmed that total BK-α expression was enhanced by either HK-alk or HK-Cl but only increased in the plasma membrane with HK-alk. Compared with controls, mice drinking NaHCO3 water exhibited more apical BK-α and total cellular BK-β4. Spironolactone given to mice on HK-alk significantly reduced K secretion and decreased total cellular BK-α but did not affect cellular BK-β4 and apical BK-α. Experiments with MDCK-C11 cells indicated that BK-β4 stabilizes surface BK-α by inhibiting degradation through a lysosomal pathway. These data suggest that aldosterone mediates a high-K-induced increase in BK-α and urinary alkalinization increases BK-β4 expression, which promotes the apical localization of BK-α. [ABSTRACT FROM AUTHOR]

Ca-activated K channels (BK), which are stimulated by high distal nephron flow, are utilized during high-K conditions to remove excess K. Because BK predominantly reside with BK-β4 in acid/base-transporting intercalated cells (IC), we determined whether BK-β4 knockout mice (β4KO) exhibit deficient K excretion when consuming a high-K alkaline diet (HK-alk) vs. high-K chloride diet (HK-Cl). When wild type (WT) were placed on HK-alk, but not HK-Cl, renal BK-β4 expression increased (Western blot). When WT and β4KO were placed on HK-Cl, plasma K concentration ([K]) was elevated compared with control K diets; however, K excretion was not different between WT and β4KO. When HK-alk was consumed, the plasma [K] was lower and K clearance was greater in WT compared with β4KO. The urine was alkaline in mice on HK-alk; however, urinary pH was not different between WT and β4KO. Immunohistochemical analysis of pendrin and V-ATPase revealed the same increases in β-IC, comparing WT and β4KO on HK-alk. We found an amiloride-sensitive reduction in Na excretion in β4KO, compared with WT, on HK-alk, indicating enhanced Na reabsorption as a compensatory mechanism to secrete K. Treating mice with an alkaline, Na-deficient, high-K diet (LNaHK) to minimize Na reabsorption exaggerated the defective K handling of β4KO. When WT on LNaHK were given NH4Cl in the drinking water, K excretion was reduced to the magnitude of β4KO on LNaHK. These results show that WT, but not β4KO, efficiently excretes K on HK-alk but not on HK-Cl and suggest that BK-α/β4-mediated K secretion is promoted by bicarbonaturia. [ABSTRACT FROM AUTHOR]

Increased flow in the distal nephron induces K secretion through the large-conductance, calcium-activated K channel (BK), which is primarily expressed in intercalated cells (IC). Since flow also increases ATP release from IC, we hypothesized that purinergic signaling has a role in shear stress (π; 10 dynes/cm²) -induced, BK-dependent, K efflux. We found that 10 μM ATP led to increased IC Ca concentration, which was significantly reduced in the presence of the P² receptor blocker suramin or calcium-free buffer. ATP also produced BK-dependent K efflux, and IC volume decrease. Suramin inhibited T-induced K efflux, suggesting that K efflux is at least partially dependent on purinergic signaling. BK-β4 small interfering (si) RNA, but not nontarget siRNA, decreased ATP secretion and both ATP-dependent and π-induced K efflux. Similarly, carbenoxolone (25 μM), which blocks connexins, putative AlP pathways, blocked T-induced K efflux and ATP secretion. Compared with BK-β4-/- mice, wild-type mice with high distal flows exhibited significantly more urinary ATP excretion. These data demonstrate coupled electrochemical efflux between K and ATP as part of the mechanism for π-induced ATP release in IC. [ABSTRACT FROM AUTHOR]

The rare disease Familial Hyperkalemic Hypertension (FHHt) is caused by mutations in the genes encoding Cullin 3 (CUL3), Kelch-Like 3 (KLHL3), and two members of the With-No-Lysine [K] (WNK) kinase family, WNK1 and WNK4. In the kidney, these mutations ultimately cause hyperactivation of NCC along the renal distal convoluted tubule. Hypertension results from increased NaCl retention, and hyperkalemia by impaired K + secretion by downstream nephron segments. CUL3 and KLHL3 are now known to form a ubiquitin ligase complex that promotes proteasomal degradation of WNK kinases, which activate downstream kinases that phosphorylate and thus activate NCC. For CUL3, potent effects on the vasculature that contribute to the more severe hypertensive phenotype have also been identified. Here we outline the in vitro and in vivo studies that led to the discovery of the molecular pathways regulating NCC and vascular tone, and how FHHt-causing mutations disrupt these pathways. Potential mechanisms for variability in disease severity related to differential effects of each mutation on the kidney and vasculature are described, and other possible effects of the mutant proteins beyond the kidney and vasculature are explored. © 2024 American Physiological Society. Compr Physiol 14:5839-5874, 2024., (Copyright © 2024 American Physiological Society. All rights reserved.)

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., (Copyright © 2022 by the American Society of Nephrology.)

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., (Copyright © 2018 by the American Society of Nephrology.)