Am J Physiol Renal Physiol 306: F721–F723, 2014;
doi:10.1152/ajprenal.00681.2013.
Editorial Focus
Two Rhesus protein ammonia transporters team up to eliminate ammonium
into urine
Carsten A. Wagner and Soline Bourgeois
Institute of Physiology, University of Zurich, Zurich, Switzerland
Submitted 19 December 2013; accepted in final form 9 January 2014
Address for reprint requests and other correspondence: C. A Wagner,
Institute of Physiology, Univ. of Zurich, Winterthurerstrasse 190, CH-8057
Zurich, Switzerland (e-mail: [email protected]).
http://www.ajprenal.org
However, mice lacking the NKCC1 cotransporter have no
problem excreting ammonium into urine, suggesting that either
NKCC1, the only Na⫹-K⫹-2Cl⫺ cotransporter expressed in the
collecting duct, is not involved in ammonium transport in vivo,
or that its absence can be fully compensated (12, 13).
The discovery that members of the Rhesus family of membrane proteins, RhAG, RhBG, and RhCG, can transport NH3/
NH⫹
4 suggested that these proteins may participate in renal and
extrarenal ammonium transport (8). RhAG is absent from the
kidney, whereas RhBG and RhCG are highly expressed in the
kidney collecting duct. RhCG is found in all type A intercalated cells (Fig. 1) and segment-specific cells of the initial
collecting duct system at both the luminal and basolateral
membranes. RhBG is found in the same cells as RhCG but
exclusively at the basolateral membrane (14). The functional
relevance of these two proteins was first revealed in a series of
mouse studies with complete or partial deletions of Rhbg or
Rhcg KO (2). These studies showed that complete deletion of
RhCG leads to a strong reduction in urinary ammonium excretion and severe metabolic acidosis. Mice with partial deletion suffer also from reduced ammonium excretion (14). Moreover, microperfusion experiments demonstrated that luminal
NH3 permeability was drastically reduced and that total transepithelial NH3 transport abolished (2, 3). Thus Rhcg is absolutely required for urinary ammonium excretion by the collecting duct and is likely the only NH3 permeation pathway in the
luminal membrane.
But what about the basolateral uptake pathway? In the case
of RhBG, two different mouse models have been generated, a
complete and a partial knockout (KO). The total Rhbg KO
exhibits no impairment of urinary ammonium excretion; normal ability to regulate systemic acid-base homeostasis and to
transport NH3 or NH⫹
4 across the basolateral membrane was
observed (4). In contrast, a mouse model with partial Rhbg
deletion had mildly reduced urinary ammonium excretion (1).
A major difference between both reports was the amount of
acid loaded, which was much higher in the partial Rhbg KO
mouse model, suggesting that Rhbg may become limiting only
under very extreme conditions.
Now, in a report published in a recent issue of the American
Journal of Physiology-Renal Physiology, a mouse model lacking both Rhcg and Rhbg is presented (7). Deletion was
achieved with Ksp-Cre mice driven by the cadherin promoter,
which has no or only little activity in the late distal convoluted
tubule and connecting tubule, leaving Rhbg and Rhcg expression intact in these early parts of the collecting duct system.
Nevertheless, these mice excrete ⬃30 –50% less ammonium
than their wild-type controls. This relatively mild phenotype
may be due to a massive compensatory increase in key molecules of proximal tubular ammoniagenesis such as PDG and
PEPCK as well as the NHE3 Na⫹/H⫹exchanger. Unfortu-
1931-857X/14 Copyright © 2014 the American Physiological Society
F721
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the de novo synthesis of bicarbonate consumed by metabolism and the excretion of acid.
Ammonium is eventually excreted into urine after accumulation in the medullary interstitium. The process of ammonium
excretion was long thought to be a mostly passive process
mediated by simple diffusion of NH3 and NH⫹
4 and driven by
urinary acidification and consequent trapping of ammonium in
urine. The discovery of Rhesus proteins capable of transporting
NH3 has revolutionized our view on renal ammonium excretion. The kidney expresses two members of this family, Rh B
Glycoprotein (RhBG) and Rh G Glycoprotein (RhCG), and
whereas RhCG is found both in apical and basolateral membranes of cells along the collecting duct, RhBG is restricted to
the basolateral membrane of the same cells. New work presented in a recent issue of the American Journal of PhysiologyRenal Physiology (7) demonstrates that combined deletion of
both transporters causes a more severe defect in urinary ammonium excretion than single deletion of each transporter,
suggesting that both transporters cooperate in final urinary
elimination of ammonium.
Renal ammoniagenesis is critical for the maintenance of
acid-base homeostasis and the renal defense against acidosis.
Proximal tubular glutamine metabolism can yield two molecules of NH3 and HCO⫺
3 allowing the buffering of acids
generated by metabolism (5). The synthesis of ammonia involves several steps catalyzed by mitochondrial phosphatedependent glutaminase and glutamate dehydrogenase and fuels
␣-ketoglutarate into gluconeogenesis where phospho-enol pyruvate carboxy kinase (PEPCK) acts as a key enzyme. At
physiological pH, most of the NH3 will bind a proton and
thereby contribute to the elimination of acid as well as to the
reabsorption of bicarbonate. A major fraction of NH⫹
4 excreted
into the proximal tubule lumen is reabsorbed by the Na-K-2Cl
cotransporter (NKCC2) in the thick ascending limb of the loop
of Henle and accumulated in the medullary interstitium, possibly by binding to sulfatides (9).
The final excretion of ammonium into urine occurs at the
level of the collecting duct system. NH3/NH⫹
4 is taken up from
the interstitium and secreted into urine where NH3 recombines
with protons secreted by H⫹-ATPases (and H⫹-K⫹-ATPases),
and is finally trapped as NH⫹
4 . Ammonium to be excreted from
the interstitium into urine must pass two plasma membranes,
the basolateral and luminal membranes of cells lining the
collecting duct (11). In vitro microperfusion experiments demonstrated that passage of ammonium across the basolateral
membrane can be mediated by Na⫹-K⫹-2Cl⫺-cotransporters
and Na⫹-K⫹-ATPases where NH⫹
4 substitutes for potassium.
RENAL AMMONIAGENESIS SERVES
Editorial Focus
F722
Interstitium
Urine
Na+/K+-ATPase
Na+
NH4+
K+(NH4+)
NKCC1
Na+
K+(NH4+)
2Cl-
NH4+
RhBG
NH3
NH3
RhCG
NH3
H+
NH3
HCO3Cl-
HCO3-
CO2
AE1
RhCG
NH3
H+
NH3
H+
H+
H+-ATPase
K+
H+
H+/K+-ATPase
CO2 + H2O
NH4+
nately, the effect on thick ascending limb NKCC2 activity and
expression and medullary ammonium accumulation was not
tested.
Do these experiments explain basolateral ammonium transport? Conclusions are difficult. Since complete deletion of
Rhcg decreased NH3 transport by the collecting duct from
acidotic animals by ⬎80%, an additional deletion of Rhbg
would likely not aggravate the phenotype. Thus the relative
contribution of each single transporter is difficult to infer from
the combined partial deletion of Rhbg and Rhcg. It is certainly
more powerful than only the partial deletion of Rhbg, but
whether it has a more severe impact than the partial deletion of
Rhcg is hard to tell and probably not to be expected. Direct
measurement of basolateral transport activities would be a way
of shedding some light on this question. In mice with complete
Rhcg KO, basolateral NH3 permeability is drastically reduced
in the absence of sodium, indicating that Rhcg is the main
mediator of basolateral NH3 uptake, leaving little space for
Rhbg (3). Direct measurements would also help to answer
another question: what is the substrate of Rhbg in vivo, NH3
and/or NH⫹
4 , CO2? For a long time, this has been an open
question for Rhcg, and three lines of evidence eventually
demonstrated that NH3 but not NH⫹
4 is the likely substrate: the
crystal structure of the related bacterial Amt protein and human
RhCG suggest a permeation pathway not permeable for
charged molecules; direct reconstitution of RhCG in liposomes; and microperfusion experiments with collecting ducts
from Rhcg KO mice demonstrated NH3 but not NH4⫹ transport
(10). Based on the high similarity of RhBG and RhCG transport of NH3 by RhBG is likely but not proven. Moreover, a
recent paper demonstrated the capability of Rhesus proteins
expressed in Xenopus oocytes to also mediate CO2 permeability similar to distant relatives of Rhesus proteins in algae and
that RhBG induced more CO2 than RhCG (6). Whether this is
relevant in native tissues and in vivo remains to be clarified.
Thus the double KO mouse model is a very valuable tool
that could provide some additional insights into the complex
handling of ammonium by the kidney. However, many experiments will depend on almost extinct and cumbersome tech-
niques such in vitro microperfusion that can still provide many
new insights. The fact that double KO mice are able to adapt
might also suggest that there is cross talk between the collecting duct and the upstream segments such as proximal tubule
and thick ascending limb in adapting renal ammoniagenesis
and ammonium excretion. If this depends on systemic acidbase homeostasis, e.g., the degree of acidosis, or is mediated by
some other signals remains to be further explored.
GRANTS
This work was supported by the Swiss National Science Foundation
(31003A-138143).
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the authors.
AUTHOR CONTRIBUTIONS
Author contributions: C.A.W. drafted manuscript; C.A.W. and S.B. edited
and revised manuscript; C.A.W. and S.B. approved final version of manuscript.
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Fig. 1. Schematic model of an acid-secretory type A
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Editorial Focus
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