Regulation of NKCC2 ubiquitination by Neddylation of Cullin‐Ring Ligases in Thick Ascending Limb
Authors: Ares Gustavo 
Published: May 2022

Hypertension and Vascular Research Division, Department of Internal Medicine, Henry Ford Hospital, Detroit, Michigan. Wayne State University, Physiology department, Detroit, Michigan.

The Na/K/2Cl cotransporter (NKCC2) reabsorbs ~30% of the filtered NaCl by the glomeruli. Nitric Oxide and atrial natriuretic peptide decrease NaCl reabsorption in thick ascending limbs (TALs) by increasing the second messenger cyclic guanosine monophosphate (cGMP). cGMP decreases surface NKCC2 levels by increasing NKCC2 ubiquitination and proteasomal degradation. The Cullin‐RING Ligases (CRLs) are the largest family of E3 ubiquitin ligases, responsible for ~20% of protein ubiquitination in mammals. Since the CRLs requires neddylation for its activation, we hypothesized that neddylation of CRLs is required for the cGMP‐dependent increase on NKCC2 ubiquitination in TALs. To test our hypothesis, we pulled down ubiquitinated proteins and biotinylated surface proteins from Sprague‐Dawley (SD) rats and blotted for NKCC2. First, we look at the expression of members of the cullin family and found expression of cullin‐1, ‐2, ‐3, ‐4 and ‐5 in TALs from SD rats. Since activity of CRLs is dynamically regulated by neddylation, we blocked cullin neddylation using (MLN4924, 1 µM) and studied whether cGMP increases NKCC2 ubiquitination. To measure ubiquitination of NKCC2, TALs suspensions were treated with proteasomal inhibitor (MG132, 20 µM). TALs was divided in 4 aliquots and incubated at 37°C with vehicle or MLN4924 for 10 minutes and treated with vehicle or db‐cGMP 500 µM at 37°C for an extra 50 minutes. We found that blockade of cullin neddylation, inhibits the cGMP‐dependent increase in ubiquitinated NKCC2 (baseline: 100%; db‐cGMP 500 µM: 162.5 ± 10.3%; MLN4924: 75.1 ± 20.2%; MLN4924 + db‐cGMP 500 µM: 96.8 ± 19.6 %, p<0.05). As expected, neddylation of cullins was blunted in MLN4924 treated samples (baseline: 100%; db‐cGMP: 120.3 ± 24.4%; MLN4924: 11.0 ± 3.5%; MLN4924 + db‐cGMP: 11.4 ± 5.8 %, p<0.05). These data indicate that neddylation of the CRLs mediates the cGMP‐dependent increase in NKCC2 neddylation. NKCC2 activity is directly related to surface NKCC2 expression. Therefore, we study whether blockade of cullin neddylation prevents the cGMP‐dependent decrease in surface NKCC2 expression. TALs suspensions were divided in 4 aliquots. TALs were incubated at 37°C with vehicle or MLN4924 for 10 minutes and then treated with vehicle or db‐cGMP 500 µM at 37°C for an extra 20 minutes. We found that blockade of cullin neddylation, inhibits the cGMP‐dependent decrease in surface NKCC2 expression. (baseline: 100%; db‐cGMP: 81.0 ± 3.4%; MLN4924: 96.4 ± 2.2%; MLN4924 + db‐cGMP: 99.8 ± 9.3 %, p<0.04). These data indicate that cullin neddylation is required for the cGMP‐dependent decrease in surface NKCC2 expression. To our knowledge, this is the first evidence pointing to the E3 ubiquitin ligase complex involved in the ubiquitination of NKCC2 in native TALs. Identifying the E3 ubiquitin ligases that regulates NKCC2 activity may provide new targets for the development of specific loops diuretics.