NHE3 in the thick ascending limb is required for sustained but not acute furosemide-induced urinary acidification

Abstract

The Na+/H+ exchanger isoform 3 (NHE3) facilitates Na+ reabsorption and H+ secretion by the kidneys. Despite stronger NHE3 abundance in the thick ascending limb (TAL) compared to the S1 and S2 segments of the proximal tubule, the role of NHE3 in the TAL is poorly understood. To investigate the role of NHE3 in the TAL, we generated and phenotyped TAL-specific NHE3 knockout mice (NHE3TAL-KO). Compared to control mice, NHE3TAL-KO mice did not show significant differences in body weight, blood pH or plasma Na+, K+ or Cl- levels. Fluid intake trended to be higher and urine osmolality was significantly lower in NHE3TAL-KO mice. Despite a similar GFR, NHE3TAL-KO mice had a greater urinary K+/creatinine ratio. One proposed role of NHE3 relates to furosemide-induced urinary acidification. Acute bolus treatment with furosemide under anesthesia did not result in differences in the dose dependence of urinary flow rate, Cl- excretion or maximal urinary acidification between genotypes; however, in contrast to control mice, urinary pH returned immediately towards baseline levels in NHE3TAL-KO mice. Chronic furosemide treatment reduced urine osmolality similarly in both genotypes but metabolic alkalosis, hypokalemia and calciuresis were absent in NHE3TAL-KO mice. Compared to vehicle, chronic furosemide treatment in control mice resulted in greater NKCC2 and lower Npt2a abundances, effects that were absent in NHE3TAL-KO mice. In summary, NHE3 in the TAL plays a role for the sustained acidification effect of furosemide. Consistent with this, long-term treatment with furosemide did not result in metabolic alkalosis in NHE3TAL-KO mice.