Lack of Role of NHE4 in Renal Ammonia Metabolism

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Background: Renal ammonia absorption by the thick ascending limb of Henle’s loop (TAL) is a critical step in renal ammonia handling and excretion. The Na+/H+ exchanger, isoform 4 (NHE4) has been proposed to have a critical role in TAL ammonia reabsorption by acting as a basolateral Na+/NH4+ exchanger mediating basolateral NH4+ exit. This study’s purpose was to determine whether NHE4 gene deletion alters renal ammonia metabolism. Methods: We backcrossed homozygous NHE4 KO mice (Jax Labs, MMRRC stock #34266) with 129S6/SvEv mice (Taconic Biosciences, 129VE), which matches the ES cell used to generate NHE4 KO mice, to obtain heterozygous NHE4 KO mice. We then bred heterozygous dams and sires. All studies compared homozygous deletion ~4-month-old mice with wild-type (WT) littermates. Both male and female mice were studied. Results: Under basal conditions, serum Na+, K+,and HCO3- did not differ between WT and KO mice. KO mice had a urine pH that was significantly more acidic than WT mice and also exhibited significantly increased urinary ammonia, titratable acid, and net acid excretion. Immunoblot analysis showed that NHE4 KO increased the expression of phosphoenolpyruvate carboxykinase (PEPCK), a critical ammonia-generating enzyme. It decreased the expression of glutamine synthetase (GS), a critical ammonia-recycling enzyme in cortical proximal tubule segments. After acid-loading for seven days, neither urine pH and ammonia excretion changes nor serum Na+, K+,and HCO3- differed significantly between WT and NHE4 KO mice. Thus, NHE4 deletion-induced increases in ammonia excretion involve increased PEPCK expression and decreased GS expression, and its deletion does not alter the response to acid-loading. Because of this lack of effect of NHE4 KO to inhibit ammonia metabolism, we examined its expression using KO mice as a negative control. Both immunoblot analysis and immunohistochemistry showed no detectable NHE4 expression. snRNA-seq analysis of WT mouse kidney showed no significant NHE4 expression in TAL1 or TAL2 cells or cortical TAL. Conclusion: We conclude that: 1) NHE4 is neither expressed in the TAL nor does it contribute to renal ammonia metabolism; and (2) extrarenal roles of NHE4 may induce mild metabolic acidosis upon its deletion that leads to a compensatory stimulation of ammonia metabolism. Funding: NIDDK Support

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