Functional Characterization of an Ammonia Transporter RhBG Mutation Identified in Diabetic Kidney Disease Patients

Metabolic acidosis is a major complication of CKD (chronic kidney disease) and DKD (diabetic kidney disease). The kidney responds to metabolic acidosis by eliminating acid, primarily by producing and excreting NH4+ in the urine. Two Rh glycoproteins, RhBG and RhCG, expressed in the collecting duct are directly involved in NH3/NH4+transport. We previously determined that RhBG transports both NH4+ and NH3 across the basolateral membrane of intercalated cells. Impaired NH3/NH4+ production and/or transport may be a factor in increasing acid load and worsening kidney disease. In type 2 diabetes, net endogenous acid production is significantly elevated, and the proportion of acid excreted as NH4+ is decreased even after accounting for diet and renal function. This is significant given that the association between acidosis and faster decline in GFR is now well established. In DKD, impaired NH4+ excretion as a potential risk factor for progression of kidney disease remains poorly understood. Specifically, the genetic risk factors contributing to DKD are unclear. Hypothesis: Defined RhBG variant(s) linked to DKD cause impaired NH3/NH4+ transport that may contribute to DKD development and progression. Methods: We conducted whole-exome sequencing (WES) analysis using data from A therosclerosis R isk i n C ommunities (ARIC) study and the C hronic R enal I nsuffciency C ohort (CRIC) study. We selected DKD patients with the most rapid GFR decline and compared them with: 1) subjects without kidney disease (healthy control); 2) diabetic patients without CKD (DM control); 3) CKD patients without diabetes (CKD control). This approach will identify single nucleotide variants (SNVs) of RhBG associated with DKD. This will also determine if the detected variants contribute to the development of kidney disease per se and whether they contribute to the development of diabetes and/or fast GFR decline. To study function, we expressed RhBG variants (and wild-type RhBG) in Xenopus oocytes by microinjecting oocytes with the respective mRNA. The effect of defined SNV of RhBG on NH3/NH4+ transport was assessed from measurements of changes in intracellular pH (pHi), membrane potential (Vm), whole cell currents (I) or surface pH (pHs) induced by exposing the oocytes to bath solutions containing NH4Cl or methyl ammonium chloride (MA/MA+). Results: We identified an SNV, rs3748569 (minor allele frequency MAF > 0.05), that was significantly associated with DKD. Significance was determined compared to DM control (OR=1.38, p=0.0026) and healthy control (OR=1.26, p=0.01). This variant has also been predicted to be deleterious by 4 bioinformatic tools. Our electrophysiological measurements showed that the resultant amino acid switch, G315R, completely inhibited transport of NH3 and NH4+ (p<0.001) and the transport of MA and MA+ (p<0.0001), compared with RhBG-WT. Conclusions: WES results suggest that this naturally occurring RhBG mutation, G315R, contributes to DKD occurrence. This effect is through the development of kidney disease and less likely due to development of diabetes. It is therefore likely a general CKD risk factor. Inhibition of NH3/NH4+ transport by this mutation is a contributing factor to its deleterious effect. This work provides experimental validation of computational predictions concerning CKD and diabetes. NIH-U54 GM104940, Paul Teschan Research, NIH-R01, Carol Lavin Bernick, Tulane institutional. This is the full abstract presented at the American Physiology Summit 2024 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.