Identification of RhBG Mutations that Impair NH3/NH4+ Transport in Patients with Chronic Kidney Disease

Background: Acid-base disturbances are common in many illnesses, and metabolic acidosis is a major complication in patients with chronic kidney disease (CKD). The kidneys respond to acidosis by increasing urinary excretion of total ammonia (NH 3 /NH 4 + ) and to a lesser extent titratable acids. In the collecting duct, total ammonia is secreted by the NH 3 /NH 4 + transporters, RhBG and RhCG. RhBG transports NH 3 and NH 4 + at the basolateral membrane of intercalated cells. In CKD patients, the increase in ammonium excretion in response to acidosis is markedly impaired. Recent studies suggest that the reduced ability of the kidney to excrete ammonium is likely a cause of detrimental renal outcome in CKD and faster progression towards end-stage renal disease. Hypothesis: This study aims to identify loss of function mutations of RhBG (nsSNPs) that impair NH 3 /NH 4 + transport which may contribute to the deleterious effect of acidosis in CKD. Methods: We used Chronic Renal Insufficiency Cohort (CRIC) study to select SNPs of RhBG that are present in CKD patients but not in controls, or have a significant odds ratio (OR). Transport studies were conducted on Xenopus oocytes expressing Rh proteins or mutants. Wild-type RhBG (RhBG-WT) or RhBG-mutants were expressed by microinjecting oocytes with respective mRNA. H 2 O-injected oocytes served as negative controls. Each oocyte was perfused with solutions containing 5 mM NH 3 /NH 4 + or 5 mM MA/MA + (methylamine/methylammonium, a surrogate of NH 3 /NH 4 + ) to test RhBG function. Using ion-selective microelectrodes, we measured changes in intracellular pH, whole cell currents or surface pH to assess transport of NH 3 or NH 4 + . Results: Two RhBG mutations, G148R and G148W, which are only present in the patient group from CRIC cohort, significantly inhibited transport of NH 3 and NH 4 + (and MA/MA + ) compared with RhBG-WT (p<0.05) in Xenopus oocytes. Two other mutations, G86S and G86C, positively associated with CKD patients (OR=4.65, p<0.05), showed different inhibition patterns: G86S maintained the ability to transport NH 4 + , NH 3 and MA + while G86C inhibited NH 3 and NH 4 + transport but MA + transport was less affected compared with RhBG-WT. Both G86S and G86C showed a 50% decrease in MA transport (p<0.05) that was still significantly higher than negative control. Conclusions: We discovered two RhBG mutations, G148R and G148W, in CKD patients that completely inhibited the transport of NH 3 /NH 4 + . The other two mutations, G86S and G86C, partially inhibited transport, and G86C was more deleterious than G86S in its inhibitory effect on NH 4 + and NH 3 transport. Significance: This study is the first to identify, in a cohort of CKD patients, 4 mutations with functional effects on NH 3 /NH 4 + transport. This work provides the foundation to further investigate whether naturally occurring genetic variations that impair ammonia secretion are associated with fast CKD progression under acidosis conditions. Funding grants: NIH-U54 GM104940, Paul Teschan Research, NIH-R01, Carol Lavin Bernick, Tulane institutional This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.