Combined Kelch-like 3 and Cullin 3 Degradation is a Central Mechanism in Familial Hyperkalemic Hypertension in Mice.

Abstract

Mutations in the ubiquitin ligase scaffold protein Cullin 3 (<i>CUL3</i>) gene cause the disease familial hyperkalemic hypertension (FHHt). In the kidney, mutant <i>CUL3</i> (<i>CUL3-Δ9</i>) increases abundance of With-No-Lysine (K) Kinase 4 (WNK4), inappropriately activating sterile 20/SPS-1-related proline/alanine-rich kinase (SPAK), which then phosphorylates and hyperactivates the Na⁺Cl^- cotransporter (NCC). The precise mechanism by which <i>CUL3-Δ9</i> causes FHHt is unclear. We tested the hypothesis that reduced abundance of CUL3 and of Kelch-like 3 (KLHL3), the CUL3 substrate adaptor for WNK4, is mechanistically important. Because JAB1, an enzyme that inhibits CUL3 activity by removing the ubiquitin-like protein NEDD8, cannot interact with CUL3-Δ9, we also determined whether <i>Jab1</i> disruption mimicked the effects of CUL3-Δ9 expression. We used an inducible renal tubule-specific system to generate several mouse models expressing <i>CUL3-Δ9</i>, mice heterozygous for both <i>CUL3</i> and <i>KLHL3</i> (<i>Cul3^+/-/Klhl3^+/-</i> ), and mice with short-term <i>Jab1</i> disruption (to avoid renal injury associated with long-term disruption). Renal KLHL3 was higher in <i>Cul3^-/-</i> mice, but lower in <i>Cul3^-/-/Δ9</i> mice and in the <i>Cul3^+/-/Δ9</i> FHHt model, suggesting KLHL3 is a target for both WT and mutant <i>CUL3</i>. <i>Cul3^+/-/Klhl3^+/-</i> mice displayed increased WNK4-SPAK activation and phospho-NCC abundance and an FHHt-like phenotype with increased plasma [K⁺] and salt-sensitive blood pressure. Short-term <i>Jab1</i> disruption in mice lowered the abundance of CUL3 and KLHL3 and increased the abundance of WNK4 and phospho-NCC. <i>Jab1^-/-</i> mice and <i>Cul3^+/-/Klhl3^+/-</i> mice recapitulated the effects of CUL3-Δ9 expression on WNK4-SPAK-NCC. Our data suggest degradation of both KLHL3 and CUL3 plays a central mechanistic role in CUL3-Δ9-mediated FHHt.