Abstract: TH-OR059

Interference with COP9 Signalosome Mimics FHHt Effects on WNK/NCC Signaling

Session Information

Category: Hypertension

  • 1102 Hypertension: Basic and Experimental - Renal Causes and Consequences

Authors

  • Cornelius, Ryan J., Oregon Health and Science University, Portland, Oregon, United States
  • Yang, Chao-Ling, Oregon Health and Science University, Portland, Oregon, United States
  • Ellison, David H., Oregon Health and Science University, Portland, Oregon, United States
Background

The Familial Hyperkalemic Hypertension (FHHt) cullin 3 (CUL3) mutant is unable to degrade WNK kinases normally, activating the thiazide-sensitive NaCl cotransporter (NCC). Previous work showed that the CUL3 mutant 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.

Methods

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-/-).

Results

JAB1 protein abundance was 62% lower in KS-JAB1-/- kidney vs WT. Western blot analysis demonstrated that loss of JAB1 caused enhanced neddylation of CUL3. Moreover, total CUL3 expression was reduced by 37%, indicating decreased CUL3 stability, as 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. KS-JAB1-/- mice were hypokalemic ([K+]: 3.20 ± 0.07 vs 3.55 ± 0.06 mM, P < 0.01), hyperchloremic ([Cl-]: 117.5 ± 1.7 vs 112.7 ± 1.3 mM; P < 0.05), and acidemic ([TCO]: 11.83 ± 0.87 vs 18.00 ± 0.57 mM, P < 0.001). Urine output was approximately twice as high in KS-JAB1-/- (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.

Conclusion

CUL3 in KS-JAB1-/- mice was highly neddylated, with increased WNK protein abundance, consistent with impaired degradation; Na+ and K+ wasting was observed, however, due to decreased levels of total NCC. In conclusion, KS-JAB1-/- mice mimic many, but not all, aspects of the FHHt phenotype.

Funding

  • NIDDK Support