ALMS1 (Alstrom Syndrome 1), a new interacting protein of NKCC2, regulates apical NKCC2 trafficking, urinary concentration and blood pressure

Abstract

NaCl absorption by the Thick Ascending Limb (TAL) is mediated by the apical Na/K/2Cl cotransporter, NKCC2. Increased NKCC2 activity and apical trafficking are associated to hypertension. However, only few proteins are known to bind and regulate NKCC2 trafficking. A 150 amino acid region in the carboxyl terminus of NKCC2 (C‐NKCC2) was shown to be important for apical trafficking of NKCC2. We hypothesized that proteins which bind to this C‐terminus region in NKCC2 play a role in regulating NKCC2 trafficking in the TAL. To identify new TAL proteins that bind C‐NKCC2, we performed a proteomics‐based screening of TAL proteins which interacted with Glutathione‐S‐Transferase‐C‐NKCC2, and identified Alstrom syndrome 1 (ALMS1) as an interacting partner. ALMS1 has been linked to human hypertension and renal function in Genome Wide Association Studies. Thus, we hypothesized that ALMS1 is involved in NKCC2 trafficking in the TAL, sodium reabsorption and blood pressure regulation. First, we confirmed that ALMS1 is expressed in TALs by Western blot and immuno‐labeling of isolated perfused TALs. To study the role of ALMS1 we obtained ALMS1 KO rats in collaboration with the Genome Editing Rat Resource Consortium. To study the effect of ALMS1 deletion on NKCC2 trafficking we isolated outer medullary TALs and measured surface and total NKCC2 expression. In TALs from ALMS1 KO, the percentage of total NKCC2 at the surface was higher compared to WT (13.8 ± 1.2% vs 9.1 ± 1.0%, p<0.05, n=6). Total NKCC2 expression was not different between strains. Urine osmolality was 45% higher in ALMS1 KO rats (2800±37 vs. 1927±167 mOsm/kg H2O, p<0.001, n=5), whereas urinary volume was lower in ALMS1 KO (ALMS1: 10.1±0.5 vs. WT: 14.4± 1.7 ml/day, p< 0.05, n=5). Water intake was also lower in ALMS1 KO rats (18.4±1.1 vs. 28.2±1.5 ml/day, p<0.001, n=5). At three months of age, ALMS1 KO rats had higher mean arterial pressure (ALMS1: 141±5 vs WT: 99 ±6 mmHg, p< 0.001). Combined, these data indicate that ALMS1 binds and regulates NKCC2 trafficking and suggest that higher urine concentration is in part due to increased NKCC2 activity in the TAL of ALMS1 KO rats. We conclude that ALMS1 plays a role in NKCC2 trafficking and regulates blood pressure. The mechanism causing hypertension in ALMS1 KO rats may involve an increase in NKCC2 activity and higher sodium reabsorption by the TAL.