The Role of the Renal Ammonia Transporter Rhcg in Metabolic Responses to Dietary Protein

BASIC RESEARCH www.jasn.org The Role of the Renal Ammonia Transporter Rhcg in Metabolic Responses to Dietary Protein † † † Lisa Bounoure,* Davide Ruffoni, Ralph Müller, Gisela Anna Kuhn, Soline Bourgeois,* Olivier Devuyst,* and Carsten A. Wagner* *Institute of Physiology and Zurich Center for Integrative Human Physiology, University of Zurich, Zurich, Switzerland; and †Institute for Biomechanics, ETH Zurich, Zurich, Switzerland ABSTRACT High dietary protein imposes a metabolic acid load requiring excretion and buffering by the kidney. Impaired acid excretion in CKD, with potential metabolic acidosis, may contribute to the progression of CKD. Here, we investigated the renal adaptive response of acid excretory pathways in mice to high- protein diets containing normal or low amounts of acid-producing sulfur amino acids (SAA) and examined how this adaption requires the RhCG ammonia transporter. Diets rich in SAA stimulated expression of + enzymes and transporters involved in mediating NH4 reabsorption in the thick ascending limb of the loop of Henle. The SAA-rich diet increased diuresis paralleled by downregulation of aquaporin-2 (AQP2) water + channels. The absence of Rhcg transiently reduced NH4 excretion, stimulated the ammoniagenic path- 2 way more strongly, and further enhanced diuresis by exacerbating the downregulation of the Na+/K+/2Cl cotransporter (NKCC2) and AQP2, with less phosphorylation of AQP2 at serine 256. The high protein acid load affected bone turnover, as indicated by higher Ca2+ and deoxypyridinoline excretion, phenomena exaggerated in the absence of Rhcg. In animals receiving a high-protein diet with low SAA content, the + kidney excreted alkaline urine, with low levels of NH4 and no change in bone metabolism. Thus, the acid load associated with high-protein diets causes a concerted response of various nephron segments to + excrete acid, mostly in the form of NH4 , that requires Rhcg. Furthermore, bone metabolism is altered by a high-protein acidogenic diet, presumably to buffer the acid load. J Am Soc Nephrol 25: 2040–2052, 2014. doi: 10.1681/ASN.2013050466 The kidney is the central organ that excretes acid and remained an open question.15–23 In kidney disease, replenishesbicarbonatebufferusedby metabolism.1,2 high animal protein may accelerate decay of renal The importance of the kidney in acid-base balance is function; thus, current protocols strongly suggest demonstrated by inherited and acquired renal diseases, that patients with CKD reduce animal protein in- reducing its ability to excrete acid and reabsorb and take.24–26 2 3–5 synthesize bicarbonate (HCO3 ). Recent studies Dietary protein intake and its metabolism can suggest that the progression of CKD is delayed by provide a major acid load, but the metabolic acid alkalinizing therapies aiming to reduce the meta- load depends on the nature and composition of bolic acidosis occurring with the disease.6–10 proteins. Proteins rich in sulfur-amino acids (SAA; In a Western diet, protein intake exceeds by up to 50% the recommended average daily consumption of 0.8 g of protein per kg per day; most protein is Received May 7, 2013. Accepted January 20, 2014. from animal sources, which are rich in sulfur- Published online ahead of print. Publication date available at 11,12 containing acidogenic amino acids. Diets high www.jasn.org. in animal protein have gained additional popularity Correspondence: Dr. Carsten A. Wagner and Dr. Soline Bourgeois, 13,14 in the context of obesity and its treatment. Ad- Institute of Physiology, University of Zurich, Winterthurerstrasse 190, verse effects of high protein intake on many organs CH-8057 Zurich, Switzerland. Email: [email protected] or have been described. Whether high-protein diets [email protected] negatively affect bone or kidney function has Copyright © 2014 by the American Society of Nephrology 2040 ISSN : 1046-6673/2509-2040 JAmSocNephrol25: 2040–2052, 2014 www.jasn.org BASIC RESEARCH i.e., cysteine and methionine) release protons (H+) and sulfate urinary excretion of titratable acids (Supplemental Figure 1C, 22 + (SO4 ) during metabolism and cause increased renal acid Supplemental Table 1). Urinary NH4 excretion was markedly + 22 and NH4 excretion paralleled by high urinary SO4 and urea increased in the HC groups whereas it decreased in the HS removal.27,28 Plant proteins, such as soy protein, contain only groups (Table 1, Supplemental Table 1, Figure 1E, Supplemen- small amounts of SAA and consequently cause a milder acid or tal Figure 1D). Rhcg deletion delayed the increase in urinary 19,29,30 + even alkaline load. The acid content of a high-protein diet NH4 excretion in response to the HC acid challenge (Figure 2 2 has been linked to the development of tubular-interstitial injury 1E). At days 2 and 4 of the HC diet, respectively, Rhcg / secondary to augmented intrinsic acid production provoked by exhibited a 38.5%60.1% and 41.4%60.1% decrease in ex- 29,31,32 + +/+ endothelin-stimulated enhanced aldosterone activity. creted urinary NH4 compared with Rhcg mice (Table 1). 2/2 The renal ammonia (NH3) transporter RhCG is critical to At day 9, Rhcg adapted to the HC diet acid load and ex- + 33–36 + +/+ eliminate NH4 and maintain systemic acid-base balance. creted similar amounts of NH4 ,asdidRhcg mice. The HS + RhCG is localized in most cells along the collecting duct and diet did not induce any difference in urinary NH4 excretion mediates basolateral uptake of NH3 and final excretion into in all three genotypes (Supplemental Figure 1D). Taken to- urine. Its expression is stimulated by acidosis in mice, and gether, these data confirm that a diet containing high amounts + Rhcg becomes rate-limiting for urinary NH4 excretion dur- of SAA causes a metabolic acid load and demonstrate that its 33,36–38 ingstrongacidloads(NH4Cl or HCl loading). elimination depends partially on the presence of the ammonia Here we examined the effect of high-protein diets contain- transporter Rhcg. ing acidogenic SAA(casein diet) or those almost devoid of these amino acids (soy protein diet) on renal mechanisms mediating HC Diet Stimulates Ammoniagenesis in the Proximal ammoniagenesis and excretion of the acid load. Our data Tubule demonstrate a concerted response of various nephron seg- ToassesstheproximaltubuleresponsetotheHCdiet,westudied + ments to eliminate the metabolic acid load, the requirement of the regulation of key molecules involved in NH4 production and + – – Rhcg-mediated NH4 excretion, and effects on bone. To- excretion (Figure 2, A D, Supplemental Figures 2 4). The HC gether, these data provide a molecular explanation for the diet caused a transient increase in Rhcg mRNA, whereas Rhbg 2 2 stimulation and requirement of renal acid excretion. mRNA was not altered in Rhcg+/+ and Rhcg / kidneys (Supple- mental Figure 2). Four days of the HC diet induced a transient increase in system N/A transporter 3 (SNAT3) and phosphate- RESULTS dependent glutaminase (PDG) mRNA levels compared with normal diet (Figure 2, A and C). SNAT3 protein levels were also Diet High in Casein Protein Induces a Transient Acid higher after 4 days of the HC diet, while PDG showed a higher 2 2 Load That Rhcg / Mice Can Excrete protein expression at day 9 of the HC diet (Figure 2, B and D). Metabolic measures and acid-base status were assessed in Rhcg+/+, Cytosolic phosphoenolpyruvate carboxykinase (PEPCK) and 2 2 2 Rhcg+/ , and Rhcg / mice receiving a normal diet (basal sta- sodium-hydrogen exchanger 3 (NHE3) protein abundance re- tus with 20% protein), acid-loading high casein (HC) protein mained unchanged in Rhcg+/+ during HC treatment (Supple- diet, or non–acid-loading high soy control protein diet (HS) mental Figure 3, A and B). To test whether the delayed adaption 2 2 containing high or low SAA, respectively. Fifty percent of HC to HC diet acid load observed in Rhcg / mice could be partly + or HS diets were provided either as casein or soy protein, but explained by an adaptive enhanced NH4 production, we com- the diets were otherwise isocaloric and identical in their compared SNAT3, NHE3, PDG, and PEPCK mRNA and protein position. The HC but not the HS diet was associated with abundances in all three groups of mice after 4 and/or 9 days of slightly reduced food intake (Table 1, Supplemental Table 1). theHCdiet.NHE3andPEPCKmRNAandproteinexpression However, no difference was found in food and water intakes levels were similar among all three genotypes (Supplemental Fig- 2 among the three different genotypes at any time points mea- ure 3, C–H). However, at day 4 of the HC diet, both Rhcg+/ and 2 2 sured and under all three types of diet (normal, HC, and HS) Rhcg / increased mRNA and protein expression of SNAT3 (Table 1, Supplemental Table 1). Baseline blood and urine var- compared with Rhcg+/+ (Figure2,A,B,E,andF).PDG 2 2 iables (Table 1, Supplemental Tables 1–4) were similar among mRNA and protein were higher in Rhcg / than Rhcg+/+ (Fig- all three genotypes. A transient decrease in blood pH and ure 2, C, D, G, and H). After 9 days of the HC diet, SNAT3 and 2 2/2 HCO3 after 2 days of the HC diet (Figure 1, A and B), but PDG mRNA and protein were still higher in Rhcg than in 2 not the HS diet (Supplemental Figure 1, A and B), was identi- Rhcg+/+,whileinRhcg+/ mice only PDG protein levels were 2 2 2 2 2 cally observed in Rhcg+/+, Rhcg+/ , and Rhcg / , confirming elevated. Thus, Rhcg / mice increased expression of some the acid-loading effect of the HC diet. The HC diet did not alter proteins critical for proximal tubular ammoniagenesis. urinary pH but stimulated excretion of titratable acidity, most 2 2 2 likely in the form of phosphate (Table 1, Supplemental Table 3) Rhcg+/ and Rhcg / Mice Have Abnormal + to a similar extent in all three genotypes (Figure 1, C and D).

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