Long‐term Disruption of the COP9 Signalosome Decreases NCC Abundance Due to Remodeling of the DCT

The cullin 3 mutant that causes human familial hyperkalemic hypertension increases WNK abundance, activating the thiazide‐sensitive NaCl cotransporter (NCC) in the distal convoluted tubule (DCT). Previous work determined that the mutation impairs binding to the deneddylase COP9 signalosome (CSN), which might contribute to the disease pathogenesis. In vivo disruption of the CSN using a nephron‐specific, inducible mouse model in which the catalytic subunit, JAB1, is deleted (KS‐ Jab1 −/− mice), resulted, however, in a mixed phenotype with WNK activation, but decreased NCC abundance. To test the hypothesis that the effects on NCC are secondary to tubule damage or remodeling, mice were given doxycycline in their drinking water for 3 weeks (w), to induce genetic deletion of Jab1 , and were analyzed at 0 w, 1 w, 2 w and 3 w post treatment (PT). Western blotting for WNK4 showed an increase in protein abundance at 0 w PT compared to control, and a significantly higher abundance at each subsequent time point. Consistent with WNK4 activation, the abundance of phosphorylated NCC (pNCC) was greater at 0 w, 1 w, 2 w PT. Total NCC abundance was unchanged at 0 w, 1 w, and 2 w PT, but was significantly lower at 3 w PT. To examine if the late effect on NCC results from DCT remodeling, we used kidney clearing (ethyl cinnamate) with confocal imaging, using antibodies against pNCC to capture three dimensional images of the DCT and measure tubule length. At 0 w PT, DCT length was unchanged compared to control (423.3 ± 25.64 vs. 406.5 ± 21.2 μm), but was significantly shorter in mice at 3 w PT (276.1 ± 18.1 μm, P < 0.01). Along with a shorter DCT length, the number of pNCC‐positive tubules was substantially lower at 3 w PT compared to control, suggesting tubule dropout. Immunofluorescent staining for kidney injury molecule‐1 (KIM‐1) showed similar intensity in the cortex at 0 w and 3 w PT compared to control, indicating that the long‐term effects of CSN disruption on the DCT is not caused by tubule damage. However, KIM‐1 staining intensity was high in the outer medulla at 0 w and remained so at 3 w PT, suggesting tubule damage at this site. This may have contributed to increased urine output of KS‐ Jab1 −/− mice (control: 5.28 ± 0.29 vs. 0 w: 9.16 ± 1.83 ml/d, P < 0.01) and mitigated the FHHt phenotype. Quantitative real‐time PCR measurements demonstrated that mRNA expression for the DCT markers NCC and parvalbumin showed a linear decrease over time (NCC 18% of control at 3 w PT; parvalbumin 8% of control). Interestingly, mRNA expression for the proximal tubule marker, NHE3, and thick ascending limb marker, NKCC2, was not significantly different at 0 w and 1 w PT, however, at 2 w and 3 w PT mRNA levels were approximately 50% of control, suggesting that nephron remodeling/tubule dropout is not limited to the DCT. The results show that in vivo disruption of the CSN mimics many aspects of familial hyperkalemic hypertension, but also leads to tubule damage and nephron remodeling; the latter is likely absent in human disease, owing to its autosomal dominant pathogenesis. Support or Funding Information NIH R01 DK51496 (D.H. Ellison and C‐L. Yang), T32 DK067864 (D.H. Ellison), and NIH F32 DK112531 (R.J. Cornelius), Department of Veteran Affairs Merit Review grant 1I01BX002228‐01A1 (D.H. Ellison) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .