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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 6

With-No-Lysine [K] Kinase (WNK4) and Sterile 20 (STE20)/SPS-1–related proline/alanine-rich kinase (SPAK) localize to puncta in CUL3-Het/Δ9 mice, but not in Na+-K+-2Cl– cotransporter–positive (NKCC2-positive) segments.

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With-No-Lysine [K] Kinase (WNK4) and Sterile 20 (STE20)/SPS-1–related pr...
(A) Immunoflourescence for WNK4 revealed weak signal in cortex in control and CUL3-Het mice. In CUL3-Het/Δ9 mice, a strong WNK4 signal (red) was observed in large cytoplasmic puncta. (B) Immunofluorescence for SPAK (green) showed its localization at the apical membrane and in small cytoplasmic puncta in cortical segments in control and CUL3-Het mice. In CUL3-Het/Δ9 mice, the majority was localized in large cytoplasmic puncta. (C) Colocalization studies in sections from CUL3-Het/Δ9 mice showed that WNK4 and SPAK puncta did not colocalize with NKCC2 along the cortical thick ascending limb (cTAL). Colocalization with parvalbumin (PV) showed WNK4 and SPAK puncta were present along early distal convoluted tubule (DCT), DCT1. Colocalization with phosphorylated Na+-Cl– cotransporter (pNCC) showed that WNK4 and SPAK puncta were present along both DCT1 and late DCT (DCT2), since no pNCC-positive tubules lacking puncta were observed. However, some NCC-negative segments contained puncta. While calbindin (CB) is also expressed along DCT2, colocalization of puncta with CB but not pNCC indicates expression along connecting tubule (CNT) and/or cortical collecting duct (CCD). Representative of 8 independent experiments. In A–C, scale bars: 50 μm.

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