DCT‐specific COP9 Signalosome Deletion Activates the WNK4‐NCC Pathway and Mimics Familial Hyperkalemic Hypertension

Abstract

With‐no‐lysine kinase 4 (WNK4) activates the NaCl cotransporter (NCC) which is the main pathway for sodium reabsorption in the distal convoluted tubule (DCT) of the kidney. The cullin‐RING ubiquitin ligase cullin 3 (CUL3) regulates WNK4 abundance via interaction through the substrate adaptor KLHL3. The monogenic disease familial hyperkalemic hypertension (FHHt) is caused by mutations in this pathway. Mutations in KLHL3 and WNK4 mainly disrupt formation of this complex, whereas, the CUL3 mutations impair binding to the COP9 signalosome (CSN), a deneddylase and upstream regulator of cullin‐RING ligases. Since FHHt is mostly a disease that affects the DCT, we proposed that DCT‐specific knockdown of the main CSN subunit, JAB1, would phenocopy FHHt. We deleted Jab1 in the DCT using the tamoxifen inducible NCC‐Cre‐ERT2 mouse model (DCT‐Jab1‐/‐). After five days of tamoxifen administration, proximal tubule, thick ascending limb, DCT, and connecting tubule/collecting duct segments were individually dissected and isolated to analyze protein abundance of each specific segment via Western blot. JAB1 and CUL3 protein abundance were substantially reduced in the DCT of DCT‐Jab1‐/‐ mice, whereas, WNK4 and phosphorylated NCC (pNCC) abundance were markedly elevated. Western blot analysis of cortical kidney tissue showed lower KLHL3 abundance and significantly higher abundance of phosphorylated SPAK (pSPAK), the intermediary kinase between WNK4 and NCC. Immunofluorescent staining of WNK4 and pSPAK showed increased expression and puncta formation in DCT1 and DCT2 tubules of DCT‐Jab1‐/‐ mice. This was not observed in other tubule segments that express WNK4 or pSPAK, such as the thick ascending limb and connecting tubule/collecting duct. Although there was obvious activation of the WNK4‐SPAK‐NCC pathway DCT‐Jab1‐/‐ mice, plasma analysis showed no significant differences in potassium, chloride or TCO2. However, challenging the DCT‐Jab1‐/‐ mice with a high potassium diet for seven days exaggerated the differences in pSPAK and pNCC protein abundance and led to a significant increase in plasma potassium. The results indicate that DCT‐specific disruption of JAB1 recapitulates many aspects of FHHt, including decreased KLHL3 and CUL3 abundance which leads to activation of the WNK4‐NCC pathway and a tendency toward hyperkalemia. These results implicate defective CSN binding as a key component of CUL3‐mediated familial hyperkalemic hypertension.