NBCe1-A Regulates Proximal Tubule Ammonia Metabolism under Basal Conditions and in Response to Metabolic Acidosis

Abstract

Renal ammonia metabolism is the primary mechanism through which the kidneys maintain acid-base homeostasis, but the molecular mechanisms regulating renal ammonia generation are unclear. In these studies, we evaluated the role of the proximal tubule basolateral plasma membrane electrogenic sodium bicarbonate cotransporter 1 variant A (NBCe1-A) in this process. Deletion of the NBCe1-A gene caused severe spontaneous metabolic acidosis in mice. Despite this metabolic acidosis, which normally causes a dramatic increase in ammonia excretion, absolute urinary ammonia concentration was unaltered. Additionally, NBCe1-A deletion almost completely blocked the ability to increase ammonia excretion after exogenous acid loading. Under basal conditions and during acid loading, urine pH was more acidic in mice with NBCe1-A deletion than in wild-type controls, indicating that the abnormal ammonia excretion was not caused by a primary failure of urine acidification. Instead, NBCe1-A deletion altered the expression levels of multiple enzymes involved in proximal tubule ammonia generation, including phosphate-dependent glutaminase, phosphoenolpyruvate carboxykinase, and glutamine synthetase, under basal conditions and after exogenous acid loading. Deletion of NBCe1-A did not impair expression of key proteins involved in collecting duct ammonia secretion. These studies demonstrate that the integral membrane protein NBCe1-A has a critical role in basal and acidosis-stimulated ammonia metabolism through the regulation of proximal tubule ammonia-metabolizing enzymes.

Expression of the B splice variant of NBCe1 (SLC4A4) in the mouse kidney

“…Antibodies specific to NBCe1-A (␣-333) and antibodies directed against NBCe1-B (␣-332) were described previously (50). We demonstrated the specificity of the NBCe1-A antibody recently in studies using NBCe1-A KO mice (33).…”

“…We used mice with a NBCe1-A deletion that we reported recently (33). This mouse has an 11-bp deletion in the 5= portion of the coding sequence specific to NBCe1-A (and NBCe1-D) that is not present in the coding sequence of NBCe1-B, NBCe1-C, or NBCe1-E (33).…”

“…NBCe1-A knockout (KO) mice were genotyped using tail-clip samples as we described recently (33). Briefly, DNA was amplified using the primers: 5=-ACCTCAGCTCATCTGAGGG-3= and 5=-CACGGGTCAGCCACTTAA-3=.…”

“…We then used mice with NBCe1-A deletion (33) to facilitate identification of alternative NBCe1 splice variant protein expression in the mouse kidney. These mice do not have early mortality and grow to adulthood.…”

“…These mice do not have early mortality and grow to adulthood. They exhibit spontaneous metabolic acidosis, are normokalemic, and have an intact, even exaggerated, ability to acidify their urine (33). First, we performed immunohistochemistry studies using an antibody that recognizes all NBCe1 splice variants.…”