Rhbg and Rhcg, the Putative Ammonia Transporters, Are Expressed in the Same Cells in the Distal Nephron

ARTICLES J Am Soc Nephrol 14: 545–554, 2003 RhBG and RhCG, the Putative Ammonia Transporters, Are Expressed in the Same Cells in the Distal Nephron FABIENNE QUENTIN,* DOMINIQUE ELADARI,*† LYDIE CHEVAL,‡ CLAUDE LOPEZ,§ DOMINIQUE GOOSSENS,§ YVES COLIN,§ JEAN-PIERRE CARTRON,§ MICHEL PAILLARD,*† and RE´ GINE CHAMBREY* *Institut National de la Santeét de la Recherche Me´dicale Unite´356, Institut Fe´de´ratif de Recherche 58, Universite´Pierre et Marie Curie, Paris, France; †Hoˆpital Europeén Georges Pompidou, Assistance Publique- Hoˆpitaux de Paris, Paris, France; ‡Centre National de la Recherche Scientifique FRE 2468, Paris, France; and §Institut National de la Santeét de la Recherche Me´dicale Unite´76, Institut National de la Transfusion Sanguine, Paris, France. Abstract. Two nonerythroid homologs of the blood group Rh RhBG expression in distal nephron segments within the cortical proteins, RhCG and RhBG, which share homologies with specific labyrinth, medullary rays, and outer and inner medulla. RhBG ammonia transporters in primitive organisms and plants, could expression was restricted to the basolateral membrane of epithelial represent members of a new family of proteins involved in am- cells. The same localization was observed in rat and mouse monia transport in the mammalian kidney. Consistent with this kidney. RT-PCR analysis on microdissected rat nephron segments hypothesis, the expression of RhCG was recently reported at the confirmed that RhBG mRNAs were chiefly expressed in CNT apical pole of all connecting tubule (CNT) cells as well as in and cortical and outer medullary CD. Double immunostaining intercalated cells of collecting duct (CD). To assess the localiza- with RhCG demonstrated that RhBG and RhCG were coex- tion along the nephron of RhBG, polyclonal antibodies against the pressed in the same cells, but with a basolateral and apical local- Rh type B glycoprotein were generated. In immunoblot experi- ization, respectively. In conclusion, RhBG and RhCG are present ments, a specific polypeptide of Mr approximately 50 kD was in a major site of ammonia secretion in the kidney, i.e., the CNT detected in rat kidney cortex and in outer and inner medulla and CD, in agreement with their putative role in ammonium membrane fractions. Immunocytochemical studies revealed transport. Ammonium is the preferred nitrogen source of bacteria, fungi, the result of NH3 diffusion, indirectly driven by (coupled to) ϩ ϩ and plants; however, in mammals, NH4 is synthesized as an active H secretion (for review, see reference 7). NH3 is end product of nitrogen and has to be eliminated. Renal excre- believed to pass freely through lipid bilayers down its concen- ϩ ϩ tion of NH4 also allows the elimination of the daily acid load, tration gradient generated by the pH gradient, whereas NH4 is provided merely by amino acid metabolism (1). Many studies orders of magnitude less permeant. However, NH may not be ϩ 3 have focused on NH4 transport mechanisms in the mamma- so lipid soluble, as its partition coefficient between chloroform lian kidney but at variance with primitive organisms, in which and water is only 0.04 (8); accordingly, some particular cell ammonium transport is mediated by the Mep/Amt (for Methyl- membranes appear to be virtually impermeable to NH3 (9,10). ammonium Permease/Ammonium Transporters) superfamily In addition, recent pieces of evidence have been provided that members (2–4), no specific ammonium transport protein has ϩ transport across cell membranes of other “freely permeant” been described (1,5). NH4 transport through mammalian cell ϩ volatile solutes such as CO2 could be accelerated by specific membranes is rather believed to occur by substitution of NH4 channel proteins (for review, see reference 11). for Hϩ or Kϩ on diverse cation transporters such as the New insight in ammonium transport mechanisms has been Naϩ/Hϩ exchangers, the Naϩ/Kϩ/2ClϪ cotransporters, and the ϩ ϩ recently provided by studies focusing on the red blood cell Na /K -ATPase (6). It has also been suggested that it could be Rhesus (Rh) proteins, the major targets of hemolytic disease of the newborn. The Rh complex is defined as the association of Received October 1, 2002. Accepted November 8, 2002. membrane polypeptides that includes the non-glycosylated Correspondence to Dr. Re´gine Chambrey, Unite´ INSERM 356, Institut de RhD and RhCE proteins, which carry the Rh antigens, and the Recherche des Cordeliers, 15 rue de l’Ecole de Me´decine, 75270 Paris Cedex RhAG glycoprotein (Rhesus associated glycoprotein), which is 06, France. Phone: 33-1-44-41-37-10; Fax: 33-1-44-41-37-17 E-mail: [email protected] required for cell surface expression of the whole complex 1046-6673/1403-0545 (12,13). Their membrane topology and the sequence homology Journal of the American Society of Nephrology between RhAG and the Mep/Amt protein family, followed by Copyright © 2003 by the American Society of Nephrology the consecutive cloning of two nonerythroid homologs, RhBG DOI: 10.1097/01.ASN.0000050413.43662.55 and RhCG, found to be expressed highly, but not exclusively, 546 Journal of the American Society of Nephrology J Am Soc Nephrol 14: 545–554, 2003 in the kidney, have raised the possibility that RhAG and its measurement. RNAs were extracted according to the technique pre- nonerythroid homologs may represent mammalian equivalents viously described (22) and briefly summarized below. Pools of tu- of Mep/Amt proteins (14–16), an hypothesis strengthened by bules were transferred with 10 ␮l of microdissection solution in 400 ␮ two functional studies strongly suggesting that RhAG and l of denaturing solution (4 M guanidium thiocyanate, 25 mM sodium ␤ RhCG could be involved in ammonium transport (17,18). citrate [pH 7.0], 0.1 M -mercaptoethanol, and 0.5% sarcosyl) containing 20 ␮g of yeast RNA used as carrier. After phenol-chloroform Although the recent study by Westhoff et al. (18) using Xe- extraction and isopropyl alcohol precipitation, the RNA pellets were nopus oocyte expression system concludes that RhAG protein ϩ ϩ ϩ dried under vacuum and dissolved in RNA dilution buffer (10 mM could mediate NH4 /H exchange, whether NH4 is preferen- Tris [pH 7.6], 1 mM ethylenediamine tetra-acetic acid, 2 mM dithio- tially transported by the renal Rh proteins over NH3 remains threitol, and 40 U/ml Rnasin; Promega, Madison, WI) at a dilution undetermined. We recently reported the expression of RhCG in corresponding to 1-mm tubular length or 1 glomerulus/3 ␮l of dilution the apical membrane of connecting tubule and collecting duct buffer. cells in the rat and demonstrated that RhCG was coexpressed with the Hϩ-ATPase in intercalated cells of the collecting duct (19). This localization favors the hypothesis that Rh proteins RT-PCR Analyses of mRNAs Encoding RhBG ϩ The presence of mRNAs encoding RhBG along the nephron was may mediate net NH3 diffusion through membranes, as NH4 secretion by the collecting ducts is believed to result from determined by RT-PCR. Primers were designed from the rat EST coupled Hϩ active secretion by the Hϩ-ATPase, along with (AW920339) showing homologies with the 5' coding region of human RhBG mRNA (NM_020407). NH “passive diffusion” (20). However, definitive demonstra- 3 The sense (5'-TTCACACATTTGGGGCCTACTT-3') and anti- tion of NH3 transport by the Rh proteins remains difficult sense (5'-CTGTCGGAGCGGAGTTGAAA-3') primers correspond to because of the absence of models of Rh protein inactivation bases 205 to 226 and 352 to 375 of the rat EST, respectively. and the difficulties of expressing these proteins at sufficient RT was carried out for 45 min at 45°C in a final volume of 50 ␮l level in heterologous systems of expression. in the presence of mRNA corresponding to 0.5 mm of nephron No study has rendered a complete description of the distri- segment or to 0.5 glomerulus, antisense primers (6.25 pmol) and bution along the nephron of the second renal Rh homolog, Moloney Murine Leukemia virus reverse transcriptase (200 U). RT RhBG. We therefore investigated RhBG expression from glo- was also carried out in the absence of reverse transcriptase as negative meruli down to inner medullary collecting duct cells by spe- control. ␮ cific nephron segments RT-PCR and immunohistochemically PCR was subsequently carried out in a final volume of 100 l after ␣32 ␮ by using polyclonal antibodies raised against peptides of the addition of sense primer (6.25 pmol), ( P)dCTP (5 Ci/sample; 6000 Ci/mmol), and Taq Polymerase (1.25 U). Samples were submit- Rh type B glycoprotein. ted to 31 cycles of three temperature steps: 95°C for 30 s, 56°C for 30 s, and 72°C for 1 min. During the final cycle, the elongation step Materials and Methods lasted 5 min. Animals Aliquots of 16 ␮l of each RT-PCR sample were electrophoresed Experiments were performed on male Sprague-Dawley rats weigh- through 2% agarose slab gels. The gels were fixed in 10% acetic acid, ing 200 to 300 g or on male mice (Iffa-Credo, L’Arbresle, France). All dried under vacuum at 70°C, and submitted to PhosphorImager (Mo- animals had access to a standard laboratory diet and water ad libitum. lecular Dynamics) to determine the intensity of the signal. For each Animals were anesthetized with intraperitoneal injection of sodium animal and each experiment, the signal intensity (arbitrary units) of pentobarbital, 5 mg/100 g body wt. the different samples was expressed as percent of the signal detected in the CCD. Results are presented as mean Ϯ SEM from different animals. Isolation of Nephron Segments and RNA Extraction The left kidney was perfused with microdissection solution (see composition below) containing 0.16% (wt/vol) collagenase (Serva, Antibodies Heidelberg, Germany). The microdissected solution was prepared Two rabbit polyclonal antibodies were raised against the Rh type B from sterile Hank’s solution (Eurobio, France) supplemented with 1 glycoprotein.

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