Kidney‐Specific COP9 Signalosome (CSN) Deletion Mimics FHHt Effects on WNK/NCC Signaling

Abstract

The Familial Hyperkalemic Hypertension (FHHt) cullin 3 (CUL3) mutant is unable to degrade WNK kinases normally, activating the thiazide‐sensitive NaCl cotransporter (NCC). Previous in vitro work showed that the CUL3 mutant has enhanced neddylation and does not bind to the COP9 signalosome (CSN), a deneddylase involved in regulating cullin‐RING ligases. We sought to determine whether this impaired binding was the cause of the increased WNK protein abundance in FHHt by inhibiting the CSN in vivo. The Pax8 mouse system was used to generate mice in which the catalytically active CSN subunit, JAB1, was deleted only along the nephron, after full development (KS‐Jab1−/−). JAB1 protein abundance was 62% lower in KS‐Jab1−/− kidney vs control. Western blot analysis demonstrated that loss of JAB1 caused enhanced neddylation of CUL3. Moreover, total CUL3 abundance was reduced by 37%, indicating decreased CUL3 stability, which is consistent with results reported by other groups for the FHHt CUL3 mutant. As expected, KLHL3 protein abundance was lower and total WNK1 and WNK4 protein abundance were higher. Phosphorylated WNK4 levels and the ratio of pNCC to total NCC were also higher, but, surprisingly, total NCC protein abundance was dramatically reduced. Consistent with the low total NCC, KS‐Jab1−/− mice did not develop an FHHt phenotype. Blood pressure was not significantly different between the two groups. KS‐Jab1−/− mice had lower plasma [K+] (3.20 ± 0.07 vs 3.55 ± 0.06 mM, P < 0.01), lower plasma bicarbonate ([TCO2]: 11.83 ± 0.87 vs 18.00 ± 0.57 mM, P < 0.001), and higher plasma [Cl−] (117.5 ± 1.7 vs 112.7 ± 1.3 mM; P < 0.05) compared to control mice. Urine output was approximately twice as high in KS‐Jab1−/− mice (9.4 ± 1.6 vs 4.7 ± 0.50 ml/day, P < 0.05), and there was Na+ and K+ wasting (Na+ clearance: 0.135 ± 0.013 vs 0.088 ± 0.006 ml/day/g, P < 0.01; K+ clearance: 4.90 ± 0.40 vs 3.68 ± 0.28 ml/day/g, P < 0.05). As we reported with CUL3 deletion, chronic JAB1 deletion also led to kidney tubule damage, as determined by H&E staining. In conclusion, KS‐Jab1−/− mice resemble many aspects of the FHHt CUL3 mutant, including changes in KLHL3 and WNK abundance, and increased phosphorylation of WNK and NCC; however, due to decreased levels of total NCC, the mice do not mimic all aspects of the FHHt phenotype.Support or Funding InformationNIH R01 DK51496, NIH T32 DK067864, VA 1I01BX002228‐01A1, AHA 16POST3064003, NIH F32 DK112531This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.