The lack of increase in titratable acid excretion with acid loading is similar to that we have observed previously (4,27) == Rhcg and Rhbg expression in response to metabolic acidosis. it was significantly less than in C mice ondays 15. Urine pH was significantly more acidic in CD-Rhbg/Rhcg-KO mice ondays 1,3, and5of acid loading. Metabolic acidosis increased phosphenolpyruvate carboxykinase (PEPCK) and Na+/H+exchanger NHE-3 and decreased glutamine synthetase (GS) expression in both genotypes, and these changes were significantly greater in CD-Rhbg/Rhcg-KO than in C mice. We conclude that1) Rhbg and Rhcg are critically important in the renal response to metabolic acidosis;2) the significantly greater changes in PEPCK, NHE-3, and GS expression in acid-loaded CD-Rhbg/Rhcg-KO compared with acid-loaded C LRP12 antibody mice cause the role of Rhbg and Rhcg to be underestimated quantitatively; and3) in mice with intact Rhbg and Rhcg expression, metabolic acidosis does not induce maximal changes in PEPCK, NHE-3, and GS expression despite the presence of persistent metabolic acidosis. Keywords:acid-base, ammonia, collecting duct renal ammonia1excretion isa major mechanism through which the kidneys contribute to acid-base homeostasis and involves integrated contributions of intrarenal ammoniagenesis and renal epithelial cell ammonia transport. Our understanding of the molecular mechanisms of ammonia transport has undergone fundamental changes in the past several years. In particular, the previous paradigm that NH3is in diffusion equilibrium in the kidney and that NH4+is not transported, i.e., that it is trapped, is being replaced by one in which both NH3and NH4+are transported across plasma membranes by specific proteins, and that regulated expression and function of these proteins is integral to acid-base homeostasis (47,49). The Rhesus (Rh) glycoproteins, Rh B glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), have an important role in renal epithelial cell ammonia transport. Rhbg and Rhcg are ammonia-specific transporters (2,31,53) and are expressed in the distal convoluted tubule (DCT), connecting segments (CNT), initial collecting tubule, cortical collecting duct (CCD), outer medullary collecting duct (OMCD), and inner medullary collecting duct (IMCD) (11,18,37,41,46). Notably, these sites are responsible for secreting the majority Avitinib (AC0010) of urinary ammonia (16,38). Recent studies showed that individual deletion from the kidney of either Rhbg (4) or Rhcg (5,27,28) inhibited both renal ammonia excretion and the maintenance of acid-base homeostasis in response to experimental metabolic acidosis. However, because both Rhbg and Rhcg are expressed in the basolateral plasma membrane of renal epithelial cells (7,18,24,40), adaptive changes in transport mediated by one of these ammonia transporter family members might compensate, at least partially, for the lack of transport by the other when only a single ammonia transporter family member is deleted. Thus the purpose of this study was to examine the effect of genetic deletion of both Rhbg and Rhcg from the collecting duct on basal ammonia and acid-base homeostasis and on the renal response to metabolic acidosis. We generated mice with combined collecting duct-specific deletion of both Rhbg and Rhcg using Cre-loxP techniques. We then examined basal acid-base homeostasis and ammonia excretion, the response to HCl-induced metabolic acidosis, and the adaptive changes in several other proteins involved in renal ammonia metabolism. Our results show several important findings. First, Rhbg and Rhcg are critically important in the renal response to metabolic acidosis. Second, there are significantly greater changes in phosphenolpyruvate carboxykinase (PEPCK), glutamine synthetase (GS), and NHE-3 expression in acid-loaded CD-Rhbg/Rhcg-knockout (KO) compared with acid-loaded mice with intact Rhbg and Rhcg expression. This indicates the role of Rhbg and Rhcg in ammonia excretion is underestimated quantitatively, due to exaggerated changes in other proteins involved in renal ammonia metabolism. Third, in mice with intact Rhbg and Rhcg expression, acidosis-induced changes in PEPCK, GS, and NHE-3 expression are submaximal, despite the presence of persistent metabolic acidosis. == METHODS == == == == Animals. == Avitinib (AC0010) We generated mice with combined collecting duct-specific Rhbg and Rhcg deletion using Cre-loxP techniques. Briefly, we bred mice with floxed Rhcg alleles and expressing Cre-recombinase under control of the Ksp-cadherin promoter (Ksp-Cre) (27,29) with mice with floxed Rhbg alleles (3,4), and by selective breeding of offspring generated mice with floxed Rhbg and Rhcg alleles that also expressed Ksp-Cre. We genotyped all mice used in these studies using tail-clip samples, as we have described previously (3,4,2729). Mice used in the current studies were offspring of mating mice with floxed Rhbg Avitinib (AC0010) and Rhcg alleles and expressing Ksp-Cre with mice with floxed Rhbg and Rhcg alleles that did not express Ksp-Cre. Animal breeding was performed in the University of Florida College of Medicine Cancer and Genetics Transgenic Animal Core Facility by trained personnel. All animal studies were approved by the University of Florida College of Medicine and the North Florida/South Georgia Veterans Health System Institutional Animal Care and Use Committee. ==.