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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 2

CUL3 distribution shifts from the nucleus to cytoplasm in CUL3-Het/Δ9 mice in the distal nephron.

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CUL3 distribution shifts from the nucleus to cytoplasm in CUL3-Het/Δ9 mi...
Immunofluorescence for CUL3 was performed using an antibody validated in kidney-specific CUL3-KO mice. (A) In CUL3-Het mice, CUL3 displayed strong nuclear localization in most tubules, including early distal convoluted tubule (DCT1), confirmed by colocalization with parvalbumin (PV). In CUL3-Het/Δ9 mice, CUL3 still localized to nuclei in most segments but appeared more diffuse in PV-positive segments, suggesting redistribution from the nucleus to the cytoplasm. (B) A similar redistribution was seen in tubules expressing calbindin (CB) which is also expressed along late distal convoluted tubule (DCT2), connecting tubule (CNT), and cortical collecting duct (CCD). In (A and B) PV- and CB-positive tubules are outlined on CUL3 images to assist with identification. Representatives of 2 independent experiments. Scale bar: 50 μm.

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