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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.

Hypomagnesemia is associated with reduced kidney function and life-threatening complications and sustains hypokalemia. The distal convoluted tubule (DCT) determines final urinary Mg2+excretion and, via activity of the Na+-Cl−cotransporter (NCC), also plays a key role in K+homeostasis by metering Na+delivery to distal segments. Little is known about the mechanisms by which plasma Mg2+concentration regulates NCC activity and how low-plasma Mg2+concentration and K+concentration interact to modulate NCC activity. To address this, we performed dietary manipulation studies in mice. Compared with normal diet, abundances of total NCC and phosphorylated NCC (pNCC) were lower after short-term (3 days) or long-term (14 days) dietary Mg2+restriction. Altered NCC activation is unlikely to play a role, since we also observed lower total NCC abundance in mice lacking the two NCC-activating kinases, STE20/SPS-1-related proline/alanine-rich kinase and oxidative stress response kinase-1, after Mg2+restriction. The E3 ubiquitin-protein ligase NEDD4-2 regulates NCC abundance during dietary NaCl loading or K+restriction. Mg2+restriction did not lower total NCC abundance in inducible nephron-specific neuronal precursor cell developmentally downregulated 4-2 (NEDD4-2) knockout mice. Total NCC and pNCC abundances were similar after short-term Mg2+or combined Mg2+-K+restriction but were dramatically lower compared with a low-K+diet. Therefore, sustained NCC downregulation may serve a mechanism that enhances distal Na+delivery during states of hypomagnesemia, maintaining hypokalemia. Similar results were obtained with long-term Mg2+-K+restriction, but, surprisingly, NCC was not activated after long-term K+restriction despite lower plasma K+concentration, suggesting significant differences in distal tubule adaptation to acute or chronic K+restriction.

Manipulation of host ubiquitin signaling is becoming an increasingly apparent evolutionary strategy among bacterial and viral pathogens. By removing host ubiquitin signals, for example, invading pathogens can inactivate immune response pathways and evade detection. The ovarian tumor (OTU) family of deubiquitinases regulates diverse ubiquitin signals in humans. Viral pathogens have also extensively co‐opted the OTU fold to subvert host signaling, but the extent to which bacteria utilize the OTU fold was unknown. We have predicted and validated a set of OTU deubiquitinases encoded by several classes of pathogenic bacteria. Biochemical assays highlight the ubiquitin and polyubiquitin linkage specificities of these bacterial deubiquitinases. By determining the ubiquitin‐bound structures of two examples, we demonstrate the novel strategies that have evolved to both thread an OTU fold and recognize a ubiquitin substrate. With these new examples, we perform the first cross‐kingdom structural analysis of the OTU fold that highlights commonalities among distantly related OTU deubiquitinases., Prediction and validation of pathogenic bacterial DUBs and insights into their structures and (poly)ubiquitin linkage specificities allows for first cross‐kingdom comparison of the OTU deubiquitination enzyme fold.

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.

Hypomagnesemia is associated with reduced kidney function and life-threatening complications and sustains hypokalemia. The distal convoluted tubule (DCT) determines final urinary Mg(2+) excretion and, via activity of the Na(+)-Cl(−) cotransporter (NCC), also plays a key role in K(+) homeostasis by metering Na(+) delivery to distal segments. Little is known about the mechanisms by which plasma Mg(2+) concentration regulates NCC activity and how low-plasma Mg(2+) concentration and K(+) concentration interact to modulate NCC activity. To address this, we performed dietary manipulation studies in mice. Compared with normal diet, abundances of total NCC and phosphorylated NCC (pNCC) were lower after short-term (3 days) or long-term (14 days) dietary Mg(2+) restriction. Altered NCC activation is unlikely to play a role, since we also observed lower total NCC abundance in mice lacking the two NCC-activating kinases, STE20/SPS-1-related proline/alanine-rich kinase and oxidative stress response kinase-1, after Mg(2+) restriction. The E3 ubiquitin-protein ligase NEDD4-2 regulates NCC abundance during dietary NaCl loading or K(+) restriction. Mg(2+) restriction did not lower total NCC abundance in inducible nephron-specific neuronal precursor cell developmentally downregulated 4-2 (NEDD4-2) knockout mice. Total NCC and pNCC abundances were similar after short-term Mg(2+) or combined Mg(2+)-K(+) restriction but were dramatically lower compared with a low-K(+) diet. Therefore, sustained NCC downregulation may serve a mechanism that enhances distal Na(+) delivery during states of hypomagnesemia, maintaining hypokalemia. Similar results were obtained with long-term Mg(2+)-K(+) restriction, but, surprisingly, NCC was not activated after long-term K(+) restriction despite lower plasma K(+) concentration, suggesting significant differences in distal tubule adaptation to acute or chronic K(+) restriction.

Mutations in the ubiquitin ligase scaffold protein Cullin 3 (CUL3) cause the disease familial hyperkalemic hypertension (FHHt). In the kidney, mutant CUL3 (CUL3-Δ9) increases abundance of With-No-Lysine [K] Kinase 4 (WNK4), with excessive activation of the downstream Sterile 20 (STE20)/SPS-1–related proline/alanine-rich kinase (SPAK) increasing phosphorylation of the Na+-Cl– cotransporter (NCC). CUL3-Δ9 promotes its own degradation via autoubiquitination, leading to the hypothesis that Cul3 haploinsufficiency causes FHHt. To directly test this, we generated Cul3 heterozygous mice (CUL3-Het), and Cul3 heterozygotes also expressing CUL3-Δ9 (CUL3-Het/Δ9), using an inducible renal epithelial–specific system. Endogenous CUL3 was reduced to 50% in both models, and consistent with autoubiquitination, CUL3-Δ9 protein was undetectable in CUL3-Het/Δ9 kidneys unless primary renal epithelia cells were cultured. Abundances of WNK4and phosphorylated NCC did not differ between control and CUL3-Het mice, but they were elevated in CUL3-Het/Δ9 mice, which also displayed higher plasma [K+] and blood pressure. Abundance of phosphorylated Na+-K+-2Cl– cotransporter (NKCC2) was also increased, which may contribute to the severity of CUL3-Δ9–mediated FHHt. WNK4 and SPAK localized to puncta in NCC-positivesegments but not in NKCC2-positive segments, suggesting differential effects of CUL3-Δ9. These results indicate that Cul3 haploinsufficiency does not cause FHHt, but dominant effects of CUL3-Δ9 are required.