Regulation of NKCC2 Ubiquitination by baSubstrate Recognition Adaptor FBXL13 in Thick Ascending Limbs
Authors: Ares Gustavo 
Published: May 2024

NaCl reabsorption by the thick ascending limb (TAL) is mediated by the Na/K/2Cl cotransporter (NKCC2). Nitric Oxide and atrial natriuretic peptide decrease NaCl absorption in the TAL by increasing the second messenger cyclic guanosine monophosphate (cGMP). We recently showed that NKCC2 is constitutively ubiquitinated, and cGMP enhances the rate of ubiquitination. The cGMP-dependent increase in NKCC2 ubiquitination is mediated by the CRL (Cullin-Ring-E3 ubiquitin Ligase) complex, which is the largest family of E3 ubiquitin ligase in mammals. By using a targeted proteomics approach, we identified a substrate recognition adaptor F-Box leucine-rich repeat 13 (FBXL13) that bind the NH2-terminus portion of NKCC2. FBXL13 is expressed in rats TALs and co-immunoprecipitates with NKCC2. Therefore, we hypothesized that the substrate recognition adaptor FBXL13 mediates NKCC2 ubiquitination. To test our hypothesis, we generated a global FBXL13 knockout mice by crossing FBXL13-Flox with a global Cre mice. We confirmed the deletion of FBXL13 in tail by PCR and in TALs by Western blot. First, we studied whether deletion of FBXL13 affects total NKCC2 expression. For this, TALs from FBXL13-Flox and FBXL13-KO mice were lysed, and equal amount of protein resolved by SDS-PAGE and detected by Western blot. We found that total NKCC2 expression in FBXL13-KO mice is increased by 73% (FBXL13-Flox: 100% vs. FBXL13-KO mice: 173 ± 37%, p<0.05). These data suggest that NKCC2 has been accumulated and not degraded. We tested whether FBXL13 mediates the cGMP-dependent increase on NKCC2 ubiquitination. TALs from FBXL13-Flox control mice and FBXL13-KO mice were incubated at 37°C during proteasome inhibition with MG132 and treated with vehicle or db-cGMP 500μM. Ubiquitinated proteins were pulled down and NKCC2 measured by Western blot. We found that cGMP increased NKCC2 ubiquitination by 24 ± 10% in FBXL13-Flox mice, whereas this effect was completely absent in FBXL13-KO mice (-14 ± 12%, p<0.05). These data suggest that FBXL13 mediates the cGMP-dependent increase in NKCC2 ubiquitination in native TALs. Finally, we study the role of FBXL13 on renal function by measuring bumetanide-induced natriuresis. Bumetanide is a loop diuretic that specifically inhibits NKCC2. We observed that the FBXL13-KO mice has higher bumetanide-induced Na+ excretion (Flox: 88.8 ± 6.6 μmol Na+ vs. FBXL13-KO: 124.0 ± 14.6 μmol Na+, p<0.05). These data are in alignment with the results previous since higher bumetanide-induced natriuresis response would result with higher NKCC2 expression. We conclude that the cGMP-dependent increase in NKCC2 ubiquitination is mediated by the novel substrate recognition adaptor FBXL13. To our knowledge, this is the first evidence showing a substrate recognition adaptor FBXL13 that interacts with NKCC2 in native TALs. NIH - 1K01DK123192. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.