Mouse Models Expressing Mutant CUL3 Provide Insight into the Mechanism of Familial Hyperkalemic Hypertension (FHHT) and Reveal Additional Functional Defects

The pathogenesis of essential hypertension is complex and multifactorial. Studying Mendelian forms of hypertension like Familial Hyperkalemic Hypertension (FHHt) is a powerful approach to identify and understand pathways that may contribute. Cullin‐3 ( Cul3 ) mutations cause the most severe form of FHHt. Cullin 3 (CUL3) along with a substrate adaptor and RING ligase forms a complex that ubiquitinates proteins for proteasomal degradation. In FHHt, mutant CUL3 (CUL3‐D9) activity impairs With‐No‐lysine [K] kinase 4 (WNK4) degradation, leading to increased phosphorylation and thus activation of the Na + Cl − cotransporter (NCC) along the renal distal convoluted tubule (DCT). We previously reported that CUL3‐D9 degrades the WNK4 adaptor KLHL3 in vitro , and a recent study suggests this occurs in vivo . This depletion of KLHL3 could prevent WNK4 degradation, providing a mechanism by which CUL3‐D9 leads to FHHt. To confirm that CUL3‐D9 degrades KLHL3 in vivo and determine whether it has any other activities, we studied inducible renal epithelial‐specific Cul3 knock‐out (CUL3‐KO) mice and CUL3‐KO mice expressing CUL3‐D9 (CUL3‐KO/D9). Western blotting and immunofluorescence showed that CUL3‐KO mice had higher KLHL3 abundance compared with controls, while CUL3‐KO/D9 had similar KLHL3 abundance to controls. CUL3‐KO and CUL3‐KO/D9 mice also had higher WNK4 abundance compared with controls. These data suggest that KLHL3 is a substrate of wild‐type CUL3, and that CUL3‐D9 is an active form of CUL3 since it can prevent KLHL3 accumulation in CUL3‐KO mice. However, CUL3‐D9 cannot promote WNK4 degradation and did not rescue the chronic kidney disease CUL3‐KO phenotype, suggesting a generalized loss‐of‐function of CUL3‐D9‐containing ubiquitin ligase complexes. A model of FHHt, inducible renal epithelia‐specific heterozygous Cul3 mice with CUL3‐D9 expression (CUL3‐Het/D9), further confirmed this observation with significantly lower KLHL3 abundance compared with control and heterozygous Cul3 mice. To investigate effects of CUL3‐D9 on other CUL3 targets, we investigated the abundance of another CUL3 adaptor protein, Kelch‐like ECH Associated Protein 1 (Keap1), the adaptor for Nuclear factor erythroid 2‐related factor 2 (NRF2). Both CUL3‐KO/D9 and CUL3‐Het/D9 had similar Keap 1 abundance compared with controls, but NAD(P)H Quinone Dehydrogenase 1 (NQO1), a surrogate for NRF2 activity, was significantly higher. These data suggest that in addition to effects on KLHL3/WNK4, CUL3‐D9 forms a dysfunctional complex with Keap1, leading to increased NRF2 activity. In conclusion, we confirm that CUL3‐D9 degrades its adaptor KLHL3 in vivo resulting in WNK4 accumulation, and also show that CUL3‐D9 cannot form normally‐functioning complexes with other substrate adaptors. Support or Funding Information NIH grants DK098141 and DK117903 to JAM, and AHA 17POST33670206 to MZF