Abstract: TH-PO1020

The A-Splice Variant of NBCe1 Is Necessary for Basal and Acidosis-Stimulated Renal Ammonia Metabolism

Session Information

  • Acid Base: Basic
    November 02, 2017 | Location: Hall H, Morial Convention Center
    Abstract Time: 10:00 AM - 10:00 AM

Category: Fluid, Electrolytes, and Acid-Base

  • 701 Acid-Base: Basic

Authors

  • Lee, Hyun-Wook, University of Florida , Gainesville, Florida, United States
  • Osis, Gunars, University of Florida , Gainesville, Florida, United States
  • Harris, Autumn N, University of Florida , Gainesville, Florida, United States
  • Fang, Lijuan, University of Florida , Gainesville, Florida, United States
  • Holmes, Heather L., Mayo Clinic, Rochester, Minnesota, United States
  • Rossano, Adam Joseph, Mayo Clinic, Rochester, Minnesota, United States
  • Romero, Michael F., Mayo Clinic, Rochester, Minnesota, United States
  • Verlander, Jill W., University of Florida , Gainesville, Florida, United States
  • Weiner, I. David, University of Florida , Gainesville, Florida, United States
Background

Renal ammonia excretion is the largest component of net acid excretion during both basal conditions and metabolic acidosis. Proximal tubule (PT) ammonia generation is critical for normal renal ammonia excretion, but the mechanisms through which external stimuli alter PT ammonia metabolism are incompletely understood. This study’s purpose was to determine the role of the predominant proximal tubule NBCe1 (SLC4A4) splice variant, NBCe1-A, in ammonia metabolism under basal conditions and in response to metabolic acidosis.

Methods

We used mice with specific deletion of the NBCe1-A splice variant generated using TALEN gene editing. Mice were fed normal diet or were acid-loaded by adding HCl to chow. All studies compared homozygous deletion (KO) mice with wild-type (WT) littermates.

Results

Under basal conditions, KO mice had spontaneous metabolic acidosis, consistent with proximal RTA from impaired proximal tubule bicarbonate reabsorption. Despite this acidosis, urinary ammonia excretion was not elevated in KO mice. Urine pH was lower in KO than WT mice, indicating that the failure of basal acidosis-stimulated ammonia excretion was not due to impaired urine acidification. Immunoblots and immunohistochemistry (IHC) showed KO mice, despite their acidosis, expressed less phosphate-dependent glutaminase (PDG) and phosphoenolpyruvate carboxykinase (PEPCK), key ammonia generating enzymes, throughout the entire PT, and expressed more glutamine synthetase (GS) in cortical PT segments than did WT. After acid-loading, the ability to increase ammonia excretion was impaired significantly, by ~70%, in KO mice. Immunoblots and IHC showed less change in PEPCK, PDG and GS in the proximal convoluted tubule (PCT) of KO than WT mice. However, in the proximal straight tubule (PST) in the outer medulla, where in the normal mouse NBCe1-A expression is less than in the PCT, PDG and PEPCK upregulation and GS downregulation were greater in KO than in WT mice.

Conclusion

(1) NBCe1-A is a key protein in the signaling pathway through which PCT ammonia metabolism is regulated during basal conditions and metabolic acidosis; and, (2) In the PST, one or more additional mechanisms enable responsiveness to exogenous acid-loading, but not to the spontaneous metabolic acidosis, in KO mice.

Funding

  • NIDDK Support