Mutant Cullin 3 causes familial hyperkalemic hypertension via dominant effects
Mohammed Z. Ferdaus, … , Curt D. Sigmund, James A. McCormick
Mohammed Z. Ferdaus, … , Curt D. Sigmund, James A. McCormick
Published December 21, 2017
Citation Information: JCI Insight. 2017;2(24):e96700. https://doi.org/10.1172/jci.insight.96700.
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Research Article Nephrology

Mutant Cullin 3 causes familial hyperkalemic hypertension via dominant effects

Abstract

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 WNK4 and 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-positive segments 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.

Authors

Mohammed Z. Ferdaus, Lauren N. Miller, Larry N. Agbor, Turgay Saritas, Jeffrey D. Singer, Curt D. Sigmund, James A. McCormick

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Figure 5

CUL3-Het/Δ9 mice display increased plasma [K+] and blood pressure, and expression of CUL3-Δ9 on a WT background also activates Na+-Cl– cotransporter (NCC).

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CUL3-Het/Δ9 mice display increased plasma [K+] and blood pressure, and e...
(A) Plasma [K+] did not differ between control and CUL3-Het mice, but it was significantly higher in CUL3-Het/Δ9 mice (adjusted P values, *P < 0.0001 versus control, and #P = 0.004 versus CUL3-Het. One-way ANOVA, Tukey’s multiple comparison test). Panel to right shows normalization of plasma [K+] in CUL3-Het/Δ9 mice by administration of hydrochlorothiazide (HCTZ) for 3 days. (B) Radiotelemetric blood pressure measurement revealed higher systolic blood pressure (SBP) in CUL3-Het/Δ9 compared with CUL3-Het mice on a normal (0.49% NaCl) diet. Left, trace showing 1 hour average values; right, mean of the hourly averages over 4 dark periods, ± SEM (*P < 0.0001, 2-tailed unpaired t test). (C) Western blotting of whole kidney lysate showed that, compared with WT (CUL3-WT) mice, WT mice expressing CUL3-Δ9 (CUL3-WT/Δ9) displayed significantly higher abundances of With-No-Lysine [K] Kinase (WNK4) (*P = 0.01, lane marked # was not used in analysis), phosphorylated NCC (pNCC) (*P = 0.02), and phosphorylated Na+-K+-2Cl– cotransporter (pNKCC2) (*P = 0.03). Note correlation of tdTomato abundance, which may reflect CUL3-Δ9 expression, with abundances of WNK4, pNCC, and total NCC (tNCC). Densitometric values were normalized using Coomassie stained gels (see Supplemental Figure 1 for details). Values in parentheses indicate n; statistical tests in B and C are 2-tailed unpaired t tests.