Renal CUL3 Haploinsufficiency is Not Sufficient to Cause Familial Hyperkalemic Hypertension

Familial Hyperkalemic Hypertension (FHHt) is caused by mutations in the With‐No‐Lysine [K] kinases WNK1 and WNK4, or in components of a ubiquitin ligase complex that regulates WNK abundance. In FHHt, increased WNK abundance ultimately leads to increased phosphorylation and thus activity of the Na + –Cl − cotransporter (NCC) along the distal convoluted tubule. The resulting excessive Na + reabsorption raises extracellular fluid volume and thus blood pressure (BP), and may decrease K + secretion along the distal nephron by reducing Na + delivery to the epithelial sodium channel. The complex that tags WNK kinases for proteasomal degradation consists of the scaffold protein Cullin 3 (CUL3), an adaptor protein (KLHL3) and a RING ligase. Mutations in both Cul3 and KLHL3 also cause FHHt, with Cul3 mutations causing the most severe form. FHHt‐causing Cul3 mutations are autosomal dominant and occur at splice sites, leading to the generation of a CUL3 mRNA lacking exon 9, translated to a form of CUL3 with deletion of 57 internal amino acids (403–459). The role of this altered CUL3 (CUL3‐Δ9) in FHHt is still unclear. It was recently reported that CUL3‐Δ9 promotes its own degradation, but not that of wild type (WT) CUL3, with a net result that total CUL3 levels are reduced to 50% of normal. Thus, it was proposed that CUL3 haploinsufficiency is sufficient to cause FHHt. To test this hypothesis, we compared mice heterozygous for Cul3 (CUL3‐Het) with mice heterozygous for Cul3 , but also expressing CUL3‐Δ9 (CUL3‐Het/Δ9). The Pax8‐LC1 system, which permits doxycycline‐inducible CRE‐mediated recombination in renal epithelia, was used. To generate CUL3‐Het or CUL3‐Het/Δ9, Pax8‐LC1/ Cul3 fl/fl mice were bred with WT mice, or with mice carrying a lox‐STOP‐lox/CUL3‐Δ9 transgene, respectively. Recombination was induced with 2 mg/ml doxycycline in 5% sucrose water (vehicle) for 2 weeks. In CUL3‐Het mice, CUL3 abundance was ~50% lower than in controls, but the abundances of WNK4, phosphorylated NCC (pNCC), total NCC (tNCC), phosphorylated Na + –K + –2Cl − cotransporter 2 (pNKCC2), and total NKCC2 (tNKCC2) did not differ. BP, determined by telemetry, and plasma [K + ] did also not differ. In CUL3‐Het/Δ9 mice WT CUL3 abundance was also ~50% lower than in controls. Consistent with a previous report that CUL3‐Δ9 promotes its own degradation, CUL3‐Δ9 expression was not detected by Western blot. However, clear cortical and medullary expression of the fluorophore tdTomato, translated from the same transcript as CUL3‐Δ9 via an internal ribosome entry site, confirmed transgene expression in CUL3‐Het/Δ9 mice, but not in vehicle‐treated mice. Abundances of WNK4 (191%), pNCC (591%), tNCC (154%), and pNKCC2 (340%) were significantly higher in CUL3‐Het/Δ9 mice than in controls (100%). Consistent with an FHHt phenotype, plasma [K + ] was higher in CUL3‐Het/Δ9 mice than in controls (4.73 mM vs 4.20 mM, p<0.05). Directly comparing CUL3‐Het and CUL3‐Het/Δ9 mice revealed significantly higher pNCC/tNCC in CUL3‐Het/Δ9 mice. These data suggest that a unique function of CUL3‐Δ9 itself, rather than haploinsufficiency due to autodegradation, plays a key role in the pathogenesis of FHHt. Support or Funding Information NIH