Generation and Characterization of Thick Ascending Limb‐Specific NHE3 Knockout Mice

Abstract

The Na+/H+ exchanger 3 (NHE3) mediates Na+ absorption in the intestine and reabsorption in the kidney, thereby playing a key role in fluid homeostasis and blood pressure regulation. NHE3 is expressed in the luminal membrane of the renal proximal tubule (PT), where the majority of renal Na+ reabsorption (~67%) occurs. In addition, NHE3 is expressed substantially in the thick ascending limb (TAL). Whole body NHE3 knockout mice exhibit hypovolemia, hypotension, diarrhea, metabolic acidosis and renal Na+ wasting. Studies in kidney‐specific (PT and TAL) NHE3 knockout mice showed a rather mild phenotype in terms of Na+ homeostasis and no acid‐base phenotype (Kidney International; 2017,92:397–414). To determine the physiological role of NHE3 in the TAL we generated a novel tamoxifen (Tam)‐inducible TAL‐specific NHE3 knockout (NHE3TAL‐KO) mouse model by crossing NHE3loxlox (Control, Con) with uromodulin Cre (UmodCreERT2) mice. After Tam administration (3 days, 67 mg/kg via oral gavage), TAL‐specific NHE3 knockout was confirmed by quantitative RT‐qPCR for NHE3 in microdissected inner medulla/inner stripe outer medulla (ISOM) and renal cortex and by immunofluorescent labeling with NHE3 (PT and TAL), NKCC2 (TAL marker) and lotus tetragonolobus lectin (LTL, PT marker). NHE3 mRNA levels in NHE3TAL‐KO mice (n=8) were reduced by 30% in the renal cortex and by 94% in the ISOM. Control mice showed colocalization of NHE3 with LTL and NKCC2; in contrast, no immunofluorescence was observed for NHE3 in the TAL of NHE3TAL‐KO mice. NHE3TAL‐KO mice were born at predicted Mendelian frequencies, appeared grossly indistinguishable from Con mice, and developed normally. Two weeks after Tam administration, NHE3TAL‐KO mice (n=11) showed a ~25% lower urine osmolality compared to Con mice (n=10) (2224±90 versus 1826±93 mOsm/kg, P<0.05) associated with ~20% higher water intake (5.3±0.2 versus 6.3±0.3 mL/day, P<0.05). Blood pH was slightly but significantly increased in NHE3TAL‐KO mice (n=20) versus Con mice (n=19) (7.39±0.01 versus 7.37±0.01, P<0.05), which was associated with a tendency of higher blood HCO3− in NHE3TAL‐KO mice (26.1±0.4 versus 25.5±0.3 mmol, P=0.07). However, no differences were observed for urinary pH (6.1±0.1 vs 6.2±0.1, NS), urinary Na+/creatinine (37±5 vs 41±5 mmol/mmol, NS), K+/creatinine (81±6 vs 94±7 mmol/mmol, NS), plasma Na+ (147±1 versus 147±1 mmol, NS) or plasma K+ (4.2±0.1 vs 4.4±0.1 mmol, NS). In conclusion, we successfully generated TAL‐specific NHE3 knockout mouse and found that NHE3 in the TAL contributes to urinary concentrating ability and acid‐base regulation. This mouse model will further be useful to delineate the TAL‐specific role of NHE3 for renal function.Support or Funding InformationTR is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (1R01DK110621) and American Heart Association Transformational Research Award (19TPA34850116), LT was supported by an American Heart Association Postdoctoral Fellowship (19POST34400026) and JX was supported by an American Heart Association Predoctoral Fellowship (18PRE33990236).