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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

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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 4

Evidence for increased Na+-K+-2Cl– cotransporter (NKCC2) activation and reduced Na+ flux through the epithelial sodium channel (ENaC) in CUL3-Δ9–mediated FHHt.

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Evidence for increased Na+-K+-2Cl– cotransporter (NKCC2) activation and ...
(A) Western blotting of whole kidney lysate showed that control CUL3-Het mice had similar abundances of NKCC2 phosphorylated threonines 96 and 101 (pNKCC2) and total NKCC2 (tNKCC2), and a similar pNKCC2/tNKCC2 ratio. (B) CUL3-Het/Δ9 mice displayed significantly increased abundance of pNKCC2 than controls (*P = 0.0009, 2-tailed unpaired t test). tNKCC2 did not differ, but pNKCC2/tNKCC2 was significantly higher in CUL3-Het/Δ9 mice compared with controls (*P = 0.0005, 2-tailed unpaired t test). For quantification, densitometric values were normalized using Coomassie stained gels (see Supplemental Figure 1 for details). (C) To determine whether Na+ flux through ENaC was altered in CUL3-Het/Δ9 mice, an amiloride response test was performed. Vehicle (0.09% saline) was injected and urine collected for 6 hours; the next day, amiloride (40 μg 25 g−1 body weight) was injected followed by 6-hour urine collection. The difference in Na+ or K+ excretion between vehicle and amiloride injection was then calculated. Compared with controls and CUL3-Het mice, CUL3-Het/Δ9 mice displayed blunted natriuretic responses to amiloride. *P < 0.05 for control versus CUL3-Het/Δ9 and for CUL3-Het versus CUL3-Het/Δ9. One-way ANOVA, Tukey’s multiple comparison test; values in parentheses indicate n.

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