The SGLT2 inhibitor dapagliflozin improves kidney function in glycogen storage disease XI.

Glycogen storage disease XI, also known as Fanconi-Bickel syndrome (FBS), is a rare autosomal recessive disorder caused by mutations in the SLC2A2 gene that encodes the glucose-facilitated transporter type 2 (GLUT2). Patients develop a life-threatening renal proximal tubule dysfunction for which no treatment is available apart from electrolyte replacement. To investigate the renal pathogenesis of FBS, SLC2A2 expression was ablated in mouse kidney and HK-2 proximal tubule cells. GLUT2Pax8Cre+ mice developed time-dependent glycogen accumulation in proximal tubule cells and recapitulated the renal Fanconi phenotype seen in patients. In vitro suppression of GLUT2 impaired lysosomal autophagy as shown by transcriptomic and biochemical analysis. However, this effect was reversed by exposure to a low glucose concentration, suggesting that GLUT2 facilitates the homeostasis of key cellular pathways in proximal tubule cells by preventing glucose toxicity. To investigate whether targeting proximal tubule glucose influx can limit glycogen accumulation and correct symptoms in vivo, we treated mice with the selective SGLT2 inhibitor dapagliflozin. Dapagliflozin reduced glycogen accumulation and improved metabolic acidosis and phosphaturia in the animals by normalizing the expression of Napi2a and NHE3 transporters. In addition, in a patient with FBS, dapagliflozin was safe, improved serum potassium and phosphate concentrations, and reduced glycogen content in urinary shed cells. Overall, this study provides proof of concept for dapagliflozin as a potentially suitable therapy for FBS.

What is the significance of end-stage renal disease risk estimation in living kidney donors?

Two end-stage renal disease (ESRD) risk calculators were recently developed by Grams et al., and Ibrahim et al. to calculate ESRD risk before donation among living kidney donors. However, those calculators have never been studied among potential donors for whom donation was refused due to medical contraindications and compared to a group of donors. We compared 15-year and lifetime ESRD risk of donors and nondonors due to medical cause as estimated by those two calculators. Nondonors due to medical cause (n = 27) had a significantly higher 15-year ESRD risk compared to donors (n = 288) with both calculators (0.25 vs. 0.14, P < 0.001 for that developed by Grams et al. and 2.21 vs. 1.43, P = 0.002 for that developed by Ibrahim et al.). On the contrary, lifetime ESRD risk was not significantly different between the two groups. At both times (15 years and lifetime), we observed a significant overlap of ESRD risk between the two groups. ESRD risk calculators could be complementary to standard screening strategy but cannot be used alone to accept or decline donation.

Use of computed tomography assessed kidney length to predict split renal GFR in living kidney donors.

OBJECTIVES: Screening of living kidney donors may require scintigraphy to split glomerular filtration rate (GFR). To determine the usefulness of computed tomography (CT) to split GFR, we compared scintigraphy-split GFR to CT-split GFR. We evaluated CT-split GFR as a screening test to detect scintigraphy-split GFR lower than 40 mL/min/1.73 m2/kidney. METHODS: This was a monocentric retrospective study on 346 potential living donors who had GFR measurement, renal scintigraphy, and CT. We predicted GFR for each kidney by splitting GFR using the following formula: Volume-split GFR for a given kidney = measured GFR*[volume of this kidney/(volume of this kidney + volume of the opposite kidney)]. The same formula was used for length-split GFR. We compared length- and volume-split GFR to scintigraphy-split GFR at donation and with a 4-year follow-up. RESULTS: A better correlation was observed between length-split GFR and scintigraphy-split GFR (r = 0.92) than between volume-split GFR and scintigraphy-split GFR (r = 0.89). A length-split GFR threshold of 45 mL/min/1.73 m2/kidney had a sensitivity of 100 % and a specificity of 75 % to detect scintigraphy-split GFR less than 40 mL/min/1.73 m2/kidney. Both techniques with their respective thresholds detected living donors with similar eGFR evolution during follow-up. CONCLUSION: Length-split GFR can be used to detect patients requiring scintigraphy. KEY POINTS: • Excellent correlation between kidney length and scintigraphy predicted GFR • Kidney length screening detects all donors with GFR lower than 40 mL/min/1.73 m 2 • Kidney length screening can replace scintigraphy screening.

Intercalated Cell Depletion and Vacuolar H+-ATPase Mistargeting in an Ae1 R607H Knockin Model.

Distal nephron acid secretion is mediated by highly specialized type A intercalated cells (A-ICs), which contain vacuolar H+-ATPase (V-type ATPase)-rich vesicles that fuse with the apical plasma membrane on demand. Intracellular bicarbonate generated by luminal H+ secretion is removed by the basolateral anion-exchanger AE1. Chronically reduced renal acid excretion in distal renal tubular acidosis (dRTA) may lead to nephrocalcinosis and renal failure. Studies in MDCK monolayers led to the proposal of a dominant-negative trafficking mechanism to explain AE1-associated dominant dRTA. To test this hypothesis in vivo, we generated an Ae1 R607H knockin mouse, which corresponds to the most common dominant dRTA mutation in human AE1, R589H. Compared with wild-type mice, heterozygous and homozygous R607H knockin mice displayed incomplete dRTA characterized by compensatory upregulation of the Na+/HCO3- cotransporter NBCn1. Red blood cell Ae1-mediated anion-exchange activity and surface polypeptide expression did not change. Mutant mice expressed far less Ae1 in A-ICs, but basolateral targeting of the mutant protein was preserved. Notably, mutant mice also exhibited reduced expression of V-type ATPase and compromised targeting of this proton pump to the plasma membrane upon acid challenge. Accumulation of p62- and ubiquitin-positive material in A-ICs of knockin mice suggested a defect in the degradative pathway, which may explain the observed loss of A-ICs. R607H knockin did not affect type B intercalated cells. We propose that reduced basolateral anion-exchange activity in A-ICs inhibits trafficking and regulation of V-type ATPase, compromising luminal H+ secretion and possibly lysosomal acidification.

Renal Atp6ap2/(Pro)renin Receptor Is Required for Normal Vacuolar H+-ATPase Function but Not for the Renin-Angiotensin System.

ATPase H+-transporting lysosomal accessory protein 2 (Atp6ap2), also known as the (pro)renin receptor, is a type 1 transmembrane protein and an accessory subunit of the vacuolar H+-ATPase (V-ATPase) that may also function within the renin-angiotensin system. However, the contribution of Atp6ap2 to renin-angiotensin-dependent functions remains unconfirmed. Using mice with an inducible conditional deletion of Atp6ap2 in mouse renal epithelial cells, we found that decreased V-ATPase expression and activity in the intercalated cells of the collecting duct impaired acid-base regulation by the kidney. In addition, these mice suffered from marked polyuria resistant to desmopressin administration. Immunoblotting revealed downregulation of the medullary Na+-K+-2Cl- cotransporter NKCC2 in these mice compared with wild-type mice, an effect accompanied by a hypotonic medullary interstitium and impaired countercurrent multiplication. This phenotype correlated with strong autophagic defects in epithelial cells of medullary tubules. Notably, cells with high accumulation of the autophagosomal substrate p62 displayed the strongest reduction of NKCC2 expression. Finally, nephron-specific Atp6ap2 depletion did not affect angiotensin II production, angiotensin II-dependent BP regulation, or sodium handling in the kidney. Taken together, our results show that nephron-specific deletion of Atp6ap2 does not affect the renin-angiotensin system but causes a combination of renal concentration defects and distal renal tubular acidosis as a result of impaired V-ATPase activity.

Long term outcomes of transplantation using kidneys from expanded criteria donors: prospective, population based cohort study.

OBJECTIVES: To assess the long term outcomes of transplantation using expanded criteria donors (ECD; donors aged ≥ 60 years or aged 50-59 years with vascular comorbidities) and assess the main determinants of its prognosis. DESIGN: Prospective, population based cohort study. SETTING: Four French referral centres. PARTICIPANTS: Consecutive patients who underwent kidney transplantation between January 2004 and January 2011, and were followed up to May 2014. A validation cohort included patients from another four referral centres in France who underwent kidney transplantation between January 2002 and December 2011. MAIN OUTCOME MEASURES: Long term kidney allograft survival, based on systematic assessment of donor, recipient, and transplant clinical characteristics; preimplantation biopsy; and circulating levels of donor specific anti-HLA (human leucocyte antigen) antibody (DSA) at baseline. RESULTS: The study included 6891 patients (2763 in the principal cohort, 4128 in the validation cohort). Of 2763 transplantations performed, 916 (33.2%) used ECD kidneys. Overall, patients receiving ECD transplants had lower allograft survival after seven years than patients receiving transplants from standard criteria donors (SCD; 80% v 88%, P<0.001). Patients receiving ECD transplants who presented with circulating DSA at the time of transplantation had worse allograft survival after seven years than patients receiving ECD kidneys without circulating DSA at transplantation (44% v 85%, P < 0.001). After adjusting for donor, recipient, and transplant characteristics, as well as preimplantation biopsy findings and baseline immunological parameters, the main independent determinants of long term allograft loss were identified as allocation of ECDs (hazard ratio 1.84 (95% confidence interval 1.5 to 2.3); P < 0.001), presence of circulating DSA on the day of transplantation (3.00 (2.3 to 3.9); P < 0.001), and longer cold ischaemia time (> 12 h; 1.53 (1.1 to 2.1); P = 0.011). Recipients of ECD kidneys with circulating DSA showed a 5.6-fold increased risk of graft loss compared with all other transplant therapies (P < 0.001). ECD allograft survival at seven years significantly improved with screening and transplantation in the absence of circulating DSA (P < 0.001) and with shorter (<12 h) cold ischaemia time (P=0.030), respectively. This strategy achieved ECD graft survival comparable to that of patients receiving an SCD transplant overall, translating to a 544.6 allograft life years saved during the nine years of study inclusion time. CONCLUSIONS: Circulating DSA and cold ischaemia time are the main independent determinants of outcome from ECD transplantation. Allocation policies to avoid DSA and reduction of cold ischaemia time to increase efficacy could promote wider implement of ECD transplantation in the context of organ shortage and improve its prognosis.

Renal ß-intercalated cells maintain body fluid and electrolyte balance.

Inactivation of the B1 proton pump subunit (ATP6V1B1) in intercalated cells (ICs) leads to type I distal renal tubular acidosis (dRTA), a disease associated with salt- and potassium-losing nephropathy. Here we show that mice deficient in ATP6V1B1 (Atp6v1b1-/- mice) displayed renal loss of NaCl, K+, and water, causing hypovolemia, hypokalemia, and polyuria. We demonstrated that NaCl loss originated from the cortical collecting duct, where activity of both the epithelial sodium channel (ENaC) and the pendrin/Na(+)-driven chloride/bicarbonate exchanger (pendrin/NDCBE) transport system was impaired. ENaC was appropriately increased in the medullary collecting duct, suggesting a localized inhibition in the cortex. We detected high urinary prostaglandin E2 (PGE2) and ATP levels in Atp6v1b1-/- mice. Inhibition of PGE2 synthesis in vivo restored ENaC protein levels specifically in the cortex. It also normalized protein levels of the large conductance calcium-activated potassium channel and the water channel aquaporin 2, and improved polyuria and hypokalemia in mutant mice. Furthermore, pharmacological inactivation of the proton pump in ß-ICs induced release of PGE2 through activation of calcium-coupled purinergic receptors. In the present study, we identified ATP-triggered PGE2 paracrine signaling originating from ß-ICs as a mechanism in the development of the hydroelectrolytic imbalance associated with dRTA. Our data indicate that in addition to principal cells, ICs are also critical in maintaining sodium balance and, hence, normal vascular volume and blood pressure.

Renal intercalated cells are rather energized by a proton than a sodium pump.

The Na(+) concentration of the intracellular milieu is very low compared with the extracellular medium. Transport of Na(+) along this gradient is used to fuel secondary transport of many solutes, and thus plays a major role for most cell functions including the control of cell volume and resting membrane potential. Because of a continuous leak, Na(+) has to be permanently removed from the intracellular milieu, a process that is thought to be exclusively mediated by the Na(+)/K(+)-ATPase in animal cells. Here, we show that intercalated cells of the mouse kidney are an exception to this general rule. By an approach combining two-photon imaging of isolated renal tubules, physiological studies, and genetically engineered animals, we demonstrate that inhibition of the H(+) vacuolar-type ATPase (V-ATPase) caused drastic cell swelling and depolarization, and also inhibited the NaCl absorption pathway that we recently discovered in intercalated cells. In contrast, pharmacological blockade of the Na(+)/K(+)-ATPase had no effects. Basolateral NaCl exit from ß-intercalated cells was independent of the Na(+)/K(+)-ATPase but critically relied on the presence of the basolateral ion transporter anion exchanger 4. We conclude that not all animal cells critically rely on the sodium pump as the unique bioenergizer, but can be replaced by the H(+) V-ATPase in renal intercalated cells. This concept is likely to apply to other animal cell types characterized by plasma membrane expression of the H(+) V-ATPase.

PTH-independent regulation of blood calcium concentration by the calcium-sensing receptor.

Tight regulation of calcium levels is required for many critical biological functions. The Ca2+-sensing receptor (CaSR) expressed by parathyroid cells controls blood calcium concentration by regulating parathyroid hormone (PTH) secretion. However, CaSR is also expressed in other organs, such as the kidney, but the importance of extraparathyroid CaSR in calcium metabolism remains unknown. Here, we investigated the role of extraparathyroid CaSR using thyroparathyroidectomized, PTH-supplemented rats. Chronic inhibition of CaSR selectively increased renal tubular calcium absorption and blood calcium concentration independent of PTH secretion change and without altering intestinal calcium absorption. CaSR inhibition increased blood calcium concentration in animals pretreated with a bisphosphonate, indicating that the increase did not result from release of bone calcium. Kidney CaSR was expressed primarily in the thick ascending limb of the loop of Henle (TAL). As measured by in vitro microperfusion of cortical TAL, CaSR inhibitors increased calcium reabsorption and paracellular pathway permeability but did not change NaCl reabsorption. We conclude that CaSR is a direct determinant of blood calcium concentration, independent of PTH, and modulates renal tubular calcium transport in the TAL via the permeability of the paracellular pathway. These findings suggest that CaSR inhibitors may provide a new specific treatment for disorders related to impaired PTH secretion, such as primary hypoparathyroidism.

A new look at electrolyte transport in the distal tubule.

The distal nephron plays a critical role in the renal control of homeostasis. Until very recently most studies focused on the control of Na(+), K(+), and water balance by principal cells of the collecting duct and the regulation of solute and water by hormones from the renin-angiotensin-aldosterone system and by antidiuretic hormone. However, recent studies have revealed the unexpected importance of renal intercalated cells, a subtype of cells present in the connecting tubule and collecting ducts. Such cells were thought initially to be involved exclusively in acid-base regulation. However, it is clear now that intercalated cells absorb NaCl and K(+) and hence may participate in the regulation of blood pressure and potassium balance. The second paradigm-challenging concept we highlight is the emerging importance of local paracrine factors that play a critical role in the renal control of water and electrolyte balance.

Deletion of hensin/DMBT1 blocks conversion of beta- to alpha-intercalated cells and induces distal renal tubular acidosis.

Acid-base transport in the renal collecting tubule is mediated by two canonical cell types: the ß-intercalated cell secretes HCO(3) by an apical Cl:HCO(3) named pendrin and a basolateral vacuolar (V)-ATPase. Acid secretion is mediated by the α-intercalated cell, which has an apical V-ATPase and a basolateral Cl:HCO(3) exchanger (kAE1). We previously suggested that the ß-cell converts to the α-cell in response to acid feeding, a process that depended on the secretion and deposition of an extracellular matrix protein termed hensin (DMBT1). Here, we show that deletion of hensin from intercalated cells results in the absence of typical α-intercalated cells and the consequent development of complete distal renal tubular acidosis (dRTA). Essentially all of the intercalated cells in the cortex of the mutant mice are canonical ß-type cells, with apical pendrin and basolateral or diffuse/bipolar V-ATPase. In the medulla, however, a previously undescribed cell type has been uncovered, which resembles the cortical ß-intercalated cell in ultrastructure, but does not express pendrin. Polymerization and deposition of hensin (in response to acidosis) requires the activation of ß1 integrin, and deletion of this gene from the intercalated cell caused a phenotype that was identical to the deletion of hensin itself, supporting its critical role in hensin function. Because previous studies suggested that the conversion of ß- to α-intercalated cells is a manifestation of terminal differentiation, the present results demonstrate that this differentiation proceeds from HCO(3) secreting to acid secreting phenotypes, a process that requires deposition of hensin in the ECM.

Tissue kallikrein permits early renal adaptation to potassium load.

Tissue kallikrein (TK) is a serine protease synthetized in renal tubular cells located upstream from the collecting duct where renal potassium balance is regulated. Because secretion of TK is promoted by K+ intake, we hypothesized that this enzyme might regulate plasma K+ concentration ([K+]). We showed in wild-type mice that renal K+ and TK excretion increase in parallel after a single meal, representing an acute K+ load, whereas aldosterone secretion is not modified. Using aldosterone synthase-deficient mice, we confirmed that the control of TK secretion is aldosterone-independent. Mice with TK gene disruption (TK-/-) were used to assess the impact of the enzyme on plasma [K+]. A single large feeding did not lead to any significant change in plasma [K+] in TK+/+, whereas TK-/- mice became hyperkalemic. We next examined the impact of TK disruption on K+ transport in isolated cortical collecting ducts (CCDs) microperfused in vitro. We found that CCDs isolated from TK-/- mice exhibit net transepithelial K+ absorption because of abnormal activation of the colonic H+,K+-ATPase in the intercalated cells. Finally, in CCDs isolated from TK-/- mice and microperfused in vitro, the addition of TK to the perfusate but not to the peritubular bath caused a 70% inhibition of H+,K+-ATPase activity. In conclusion, we have identified the serine protease TK as a unique kalliuretic factor that protects against hyperkalemia after a dietary K+ load.

Low bone mineral density and high incidences of fractures and vitamin D deficiency in 52 pediatric cancer survivors.

OBJECTIVE: To evaluate bone mineral density (BMD), fractures, and vitamin D deficiency in pediatric patients in complete remission of solid tumor; and to identify risk factors for these three abnormalities. STUDY DESIGN: Data were collected prospectively after completion of cancer treatment. Hormonal and vitamin D deficiencies were treated. The patients were evaluated again 1 year later. PATIENTS: 52 consecutive patients, 30 boys and 22 girls. Among them, 21 completed the second evaluation. MEASUREMENTS: A clinical examination, nutritional assessment, and laboratory workup were performed. BMD was measured by absorptiometry. RESULTS: Calcium intake was inadequate in 75% of patients and vitamin D reserves were low in 61.5%. BMD was low at the spine in 32.7%, and at the femur in 24% of patients. Spinal and femoral BMD Z-scores correlated significantly with each other. Femoral BMD Z-score showed significant positive correlations with changes in body mass index, urinary calcium/creatinine ratio, and time since treatment completion, and a significant negative correlation with treatment duration. Fractures were noted in 10 patients but were not correlated with BMD. In the 21 re-evaluated patients, no significant improvements were found in calcium intake, vitamin D status, or BMD Z-score. CONCLUSIONS: Survivors of childhood solid cancer have high rates of insufficient calcium intake, vitamin D deficiency, low bone mass and fractures.

Genetic ablation of Rhbg in the mouse does not impair renal ammonium excretion.

NH(4)(+) transport by the distal nephron and NH(4)(+) detoxification by the liver are critical for achieving regulation of acid-base balance and to avoid hyperammonemic hepatic encephalopathy, respectively. Therefore, it has been proposed that rhesus type B glycoprotein (Rhbg), a member of the Mep/Amt/Rh NH(3) channel superfamily, may be involved in some forms of distal tubular acidosis and congenital hyperammonemia. We have tested this hypothesis by inactivating the RHbg gene in the mouse by insertional mutagenesis. Histochemical studies analyses confirmed that RHbg knockout (KO) mice did not express Rhbg protein. Under basal conditions, the KO mice did not exhibit encephalopathy and survived well. They did not exhibit hallmarks of distal tubular acidosis because neither acid-base status, serum potassium concentration, nor bone mineral density was altered by RHbg disruption. They did not have hyperammonemia or disturbed hepatic NH(3) metabolism. Moreover, the KO mice adapted to a chronic acid-loading challenge by increasing urinary NH(4)(+) excretion as well as their wild-type controls. Finally, transepithelial NH(3) diffusive permeability, or NH(3) and NH(4)(+) entry across the basolateral membrane of cortical collecting duct cells, measured by in vitro microperfusion of collecting duct from KO and wild-type mice, was identical with no apparent effect of the absence of Rhbg protein. We conclude that Rhbg is not a critical determinant of NH(4)(+) excretion by the kidney and of NH(4)(+) detoxification by the liver in vivo.

The ammonium transporter RhBG: requirement of a tyrosine-based signal and ankyrin-G for basolateral targeting and membrane anchorage in polarized kidney epithelial cells.

RhBG is a nonerythroid member of the Rhesus (Rh) protein family, mainly expressed in the kidney and belonging to the Amt/Mep/Rh superfamily of ammonium transporters. The epithelial expression of renal RhBG is restricted to the basolateral membrane of the connecting tubule and collecting duct cells. We report here that sorting and anchoring of RhBG to the basolateral plasma membrane require a cis-tyrosine-based signal and an association with ankyrin-G, respectively. First, we show by using a model of polarized epithelial Madin-Darby canine kidney cells that the targeting of transfected RhBG depends on a YED motif localized in the cytoplasmic C terminus of the protein. Second, we reveal by yeast two-hybrid analysis a direct interaction between an FLD determinant in the cytoplasmic C-terminal tail of RhBG and the third and fourth repeat domains of ankyrin-G. The biological relevance of this interaction is supported by two observations. (i) RhBG and ankyrin-G were colocalized in vivo in the basolateral domain of epithelial cells from the distal nephron by immunohistochemistry on kidney sections. (ii) The disruption of the FLD-binding motif impaired the membrane expression of RhBG leading to retention on cytoplasmic structures in transfected Madin-Darby canine kidney cells. Mutation of both targeting signal and ankyrin-G-binding site resulted in the same cell surface but nonpolarized expression pattern as observed for the protein mutated on the targeting signal alone, suggesting the existence of a close relationship between sorting and anchoring of RhBG to the basolateral domain of epithelial cells.

Regulation of the Cl-/HCO3- exchanger AE2 in rat thick ascending limb of Henle's loop in response to changes in acid-base and sodium balance.

The Cl(-)/HCO(3)(-) exchanger AE2 is believed to be involved in transcellular bicarbonate reabsorption that occurs in the thick ascending limb of Henle's loop (TAL). The purpose of this study was to test whether chronic changes in acid-base status and sodium intake regulate AE2 polypeptide abundance in the TAL of the rat. Rats were subjected to 6 d of loading with NaCl, NH(4)Cl, NaHCO(3), KCl, or KHCO(3). AE2 protein abundance was estimated by semiquantitative immunoblotting in renal membrane fractions isolated from the cortex and the outer medulla of treated and control rats. In the renal cortex, AE2 abundance was markedly increased in response to oral loading with NH(4)Cl or with NaCl. In contrast, AE2 abundance was unchanged in response to loading with KCl or with NaHCO(3) and was decreased by loading with KHCO(3). The response of AE2 in the outer medulla differed from that in the cortex in that HCO(3)(-) loading increased AE2 abundance when administered with Na(+) but had no effect when administered with K(+). Immunohistochemistry revealed that NaCl loading increased AE2 abundance in the basolateral membrane of both the cortical and the medullary TAL. In contrast, NH(4)Cl loading increased AE2 abundance only in the cortical TAL but not in the medullary TAL. These results suggest that regulation of the basolateral Cl(-)/HCO(3)(-) exchanger AE2 plays an important role in the adaptation of bicarbonate absorption in the TAL during chronic acid-base disturbances and high sodium intake. The present study also emphasizes the contribution of cortical TAL adaptation in the renal regulation of acid-base status.

RhBG and RhCG, the putative ammonia transporters, are expressed in the same cells in the distal nephron.

Two nonerythroid homologs of the blood group Rh proteins, RhCG and RhBG, which share homologies with specific ammonia transporters in primitive organisms and plants, could represent members of a new family of proteins involved in ammonia transport in the mammalian kidney. Consistent with this hypothesis, the expression of RhCG was recently reported at the apical pole of all connecting tubule (CNT) cells as well as in intercalated cells of collecting duct (CD). To assess the localization along the nephron of RhBG, polyclonal antibodies against the Rh type B glycoprotein were generated. In immunoblot experiments, a specific polypeptide of Mr approximately 50 kD was detected in rat kidney cortex and in outer and inner medulla membrane fractions. Immunocytochemical studies revealed RhBG expression in distal nephron segments within the cortical labyrinth, medullary rays, and outer and inner medulla. RhBG expression was restricted to the basolateral membrane of epithelial cells. The same localization was observed in rat and mouse kidney. RT-PCR analysis on microdissected rat nephron segments confirmed that RhBG mRNAs were chiefly expressed in CNT and cortical and outer medullary CD. Double immunostaining with RhCG demonstrated that RhBG and RhCG were coexpressed in the same cells, but with a basolateral and apical localization, respectively. In conclusion, RhBG and RhCG are present in a major site of ammonia secretion in the kidney, i.e., the CNT and CD, in agreement with their putative role in ammonium transport.