original paper cellular...

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Original Paper

Cell Physiol Biochem 2010;26:1059-1072 Accepted: November 04, 2010Cellular PhysiologyCellular PhysiologyCellular PhysiologyCellular PhysiologyCellular Physiologyand Biochemistrand Biochemistrand Biochemistrand Biochemistrand Biochemistryyyyy

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Induction of Metabolic Acidosis with AmmoniumChloride (NH4Cl) in Mice and Rats – SpeciesDifferences and Technical Considerations

Marta Nowik1*, Nicole B. Kampik1*, Marija Mihailova1, DominiqueEladari2 and Carsten A. Wagner1

1Institute of Physiology and Zurich Center for Human Integrative Physiology (ZHIP), University of Zurich,Zurich, 2UMRS 872 (CRC) INSERM, Equipe 3; Université Paris Descartes; Departement de Physiologie,HEGP, AP-HP, Paris, *these authors contributd equally to this study and share first authorship

Carsten A. WagnerInstitute of Physiology and Zurich Center for Integrative Human PhysiologyUniversity of Zurich, Winterthurerstrasse 190, 8057 Zurich (Switzerland)Tel. +41-44-63 55023, Fax +41-44-63 56814E-Mail [email protected]

Key WordsAmmonium chloride • Metabolic acidosis • Rodents •Aquaporins pendrin • Ammoniagenesis

AbstractAmmonium chloride addition to drinking water isoften used to induce metabolic acidosis (MA) inrodents but may also cause mild dehydration.Previous microarray screening of acidotic mousekidneys showed upregulation of genes involved inrenal water handling. Thus, we compared twoprotocols to induce metabolic acidosis in mice andrats: standard 0.28M NH4Cl in drinking water or anequivalent amount of NH4Cl in food. Both treatmentsinduced MA in mice and rats. In rats, NH4Cl in watercaused signs of dehydration, reduced mRNAabundance of the vasopression receptor 2 (V2R),increased protein abundance of the aquaporin waterchannels AQP2 and AQP3 and stimulated phosphor-ylation of AQP2 at residues Ser256 and Ser261.In contrast, NH4Cl in food induced massive diuresis,decreased mRNA levels of V2R, AQP2, and AQP3,did not affect protein abundance of AQP2 and AQP3,

and stimulated phosphorylation at Ser261 but notpSer256 of AQP2. In mice, NH4Cl in drinkingwater stimulated urine concentration, increasedAQP2 and V2R mRNA levels, and enhanced AQP2and AQP3 protein expression with higher levels ofAQP2 pSer256 and pSer261. Addition of NH4Cl tofood, stimulated diuresis, had no effect on mRNAlevels of AQP2, AQP3, and V2R, and enhancedonly AQP3 protein abundance whereas pSer256-AQP2 and pSer261-AQP2 remained unaltered.Similarly, AQP2 staining was more intense andluminal in kidney from mice with NH4Cl in water butnot in food. Pendrin, SNAT3 and PEPCK mRNAexpression in mouse kidney were not affected bythe route of NH4Cl application. Thus, addition of NH4Clto water or food causes MA but has differentialeffects on diuresis and expression of mRNAs andproteins related to renal water handling. Moreover,mice and rats respond differently to NH4Cl loading,and increased water intake and diuresis may be acompensatory mechanism during MA. It may be nec-essary to consider these effects in planning and in-terpreting experiments of NH4Cl supplementation toanimals.

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Introduction

The kidney plays a central role in maintaining thecomposition of body fluids by regulating water, NaCl, acid-base, and solute reabsorption and excretion, respectively.The role of specific genes in these processes have beenstudied extensively over the last decade using geneticallymodified animals, often requiring provocation tests touncover defects. The standard protocol to induce meta-bolic acidosis in experimental animals involves applica-tion of ammonium chloride (NH4Cl), which is suppliedmost frequently in drinking water [1-11], but also in foodor fixed amounts of gelled diet [6, 12-16]. Administrationof NH4Cl in both, drinking water and food is also used[17-18].The first report describing the effect of ammo-nium chloride ingestion on acid-base balance dates backto 1921 [19].

Metabolic acidosis induced by NH4Cl administeredin food and drinking solution has been associated withdecreased reabsorption of NaCl and water in the proxi-mal tubule of dog and rat [17, 20-21]. In the collectingduct in rats, MA (NH4Cl provided in gelled diet or stand-ard 0.28M NH4Cl solution) has been linked to alteredexpression and activity of the epithelial sodium channelENaC [13, 22]. Moreover, altered expression of the va-sopressin-regulated water channel AQP2 and a concomi-tant increase of circulating levels of vasopressin in re-sponse to 0.28M NH4Cl in water loading have been de-scribed in rats [9]. In contrast, Nanogutchi and colleaguessuggested that elevated expression of AQP2 in rats wasdue to dehydration and not MA itself [16]. Similarly, astudy on the aquaporin 6 water channel revealed elevatedabundance of AQP6 only in rats receiving NH4Cl in wa-ter but not in food [6]. The authors suggested that changesin water intake and not disturbed acid-base status of ani-mals would be responsible. Recently, microarray screen-ing of kidneys from 2 and 7 days acidotic mice revealedthe upregulation of a number of genes involved in waterhomeostasis (AQP1, AQP2, AQP3, AQP4, the vaso-pressin receptor V2R, and the urea transporter Slc14a2)[23]. Acidotic mice, however, consumed around 20% lesswater than control animals.

Thus, the effects of acidosis and particularly the ap-plication of NH4Cl have remained controversial with re-spect to its effects on renal water handling. Most authorsused the standard protocol, adding 0.28M NH4Cl to drink-ing water [9, 21, 24-27]. Nonoguchi et al. added NH4Clto food [16]. Therefore discrepancies observed in thesestudies may be explained by different routes of NH4Clapplication, variable degrees of dehydration in studies

adding NH4Cl to drinking water, and species differencesbetween rats and mice.

Therefore, we aimed to clarify if the route of NH4Clapplication affects the physiologic response of the kid-ney and examined the induction of metabolic acidosis inresponse to standard 0.28M NH4Cl in drinking water andcompared it with NH4Cl addition to food. Both protocolswere tested in mice and rats to test also for species spe-cific responses. Our data indicate that NH4Cl in drinkingwater induces dehydration in addition to MA and thatsome of the effects ascribed previously to MA may berather the consequences of dehydration. Moreover, dis-tinct differences between mice and rats exist in their re-sponse to NH4Cl-loading.

Materials and Methods

AnimalsAll experiments were performed on 12 weeks old C57BL/

6J male mice (25-30 g) and male Wistar rats (160-200 g). Allexperiments were performed according to Swiss Animal Wel-fare laws and approved by the local veterinary authority(Veterinäramt Zürich). All animals received standard rodentchow GLP 3433 (PROVIMI KLIBA, Kaiseraugst, Switzerland).

Induction of metabolic acidosisTwo experimental protocols were usedProtocol 1: Experimental protocol for acid load in drink-

ing water. C57BL/6J male mice and Wistar rats were given 0.28M NH4Cl + 0.5% sucrose in drinking water (tap water) for 3days. Control animals received tap water + 0.5% sucrose adlibitum. All animals had free access to standard rodent chow.This acid load averaged 6.74 ± 0.49 mmoles NH4Cl/day/rat and1.06 ± 0.06 mmoles NH4Cl/day/mouse.

Protocol 2: Experimental protocol for acid load in food.C57BL/6J male mice and Wistar rats were given NH4Cl mixedwith standard powdered rodent chow for 3 days in the follow-ing proportion: 2 g (mice) or 3 g (rats) of NH4Cl / 100 g ofstandard rodent chow. All animals received tap water + 0.5%sucrose ad libitum and had free access to the chow. Controlanimals received the same diet but without NH4Cl. This acidload averaged 9.41 ± 0.24 mmoles NH4Cl/day/rat and 1.38 ± 0.04mmoles NH4Cl/day/mice.

Metabolic cages studiesAll animals were adapted to metabolic cages for 3 days

before data collection. In total, 4 rats and 6 mice were acid-loaded using Protocol 1, 4 rats and 6 mice were acid-loadedusing Protocol 2, and 4 rats and 6 mice served as controls.Daily chow, water intake and body weights were measured andurine was collected under mineral oil. At the end of experi-ments, animals were anesthetized with isoflurane/pressurizedair and heparinised arterial blood was collected from the tailartery in rats and heparinised mixed venous blood was col-

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lected in mice and analyzed immediately for pH, blood gases,and electrolytes on a Radiometer ABL 800 (Radiometer,Copenhagen, Denmark) blood gas analyzer. Serum was col-lected and frozen until further analysis. Both kidneys wereharvested, rapidly frozen in liquid nitrogen, and stored at-80°C until mRNA extraction.

Urine and serum analysis was performed as describedpreviously [23]. Aldosterone in urine was measured witha radioimmunoassay using a commercially available kit(DPC Dade Behring, La Défense, France). Briefly, 24 hoururines were collected in metabolic cages and kept at -20°C untiluse. For each sample, 50 μl urine were hydrolized overnightwith 3.2 N HCl, treated with ethyl acetate to extract aldosteroneand evaporated. Dried extracts were resuspended in theappropriate solvent following the manufacturer’srecommendations and aldosterone was then measured by

radioimmunoassay.

RNA extraction, reverse transcription, and real-timequantitative PCRKidneys were harvested and rapidly frozen in liquid ni-

trogen. Total snap-frozen kidneys were homogenized in RLT-Buffer/1% -mercaptoethanol (Qiagen, Basel, Switzerland).Total RNA was extracted using the RNeasy Mini Kit(Qiagen) according to the manufacturer’s instructions. Qualityand concentration of the isolated RNA preparations wereanalyzed by the ND-1000 spectrophotometer (NanoDropTechnologies). 300 ng of RNA was used as a template forreverse transcription using the TaqMan Reverse Transcrip-tion Kit (Applied Biosystems, Forster City, CA, USA). Realtime RT-PCR was performed as described previously [23].Primers used are listed in Table 1.

Induction of Acidosis in Rodents

Table 1. Primers and probes used for quantitative real-time PCR in mice (M) and rats (R). All primers and probes were designedusing Primer Express software (Applied Biosystems). Either primers or probes are spanning intron-exon boundaries to excludegenomic DNA contamination. The nucleotide numbers of the primers and probes are given in brackets.

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Western Blot AnalysisPreparation of crude membrane proteins from the total

kidneys was performed as described elsewhere [26]. 50 μg ofcrude membrane proteins were solubilised in Laemmli samplebuffer and SDS-PAGE was performed on 10 % polyacrylamidegels. Immunoblotting and image analysis was performed asdescribed previously [23, 28]. The primary antibodies used were:rabbit anti aquaporin 2 (1:5000) (kindly provided by J. Loffing,Institute of Anatomy, University of Zurich, Switzerland [29],rabbit anti aquaporin 3 (1:200) (Alomone Labs, Jerusalem, Is-rael), rabbit anti pSer256-AQP2 1:3000 (kindly provided by Dr.Soren Nielsen, University of Aarhus, Denmark), rabbit antipSer261-AQP2, 1:2000 (Lifespan Biosciences, Seattle, WA,USA), and mouse monoclonal anti- -actin antibody (42 kD;Sigma, St. Louis, MO; 1:5000); secondary antibodies used were:donkey anti-rabbit or sheep anti-mouse antibodies linked tohorseradish peroxidase (1:10000) (GE Healthcare, Little ChalfontBuckinghamshire, UK) or goat anti-rabbit antibody (1:5000)linked to alkaline phosphatase (Promega, USA).

ImmunohistochemistryMouse kidneys were perfusion fixed through the left

ventricle with a PFA fixative solution and subsequent immuno-histochemistry was performed as described previously [25, 30].Sections were incubated with polyclonal goat anti-human

Table 2. NH4Cl-loading of rats. Bloodgas status and electrolytes in blood andurine data from Wistar rats under controlconditions and after 3 days acid-loading(0.28 M NH4Cl + 0.5% sucrose in drink-ing water or 3g NH4Cl / 100g food + 0.5%sucrose in drinking water). Control ratsreceived only 0.5% sucrose in their drink-ing water. Values are means ± SEM, n = 4per group. */# p 0.05, **/## p 0.01,***/### p 0.001, * significantly differentbetween control and acidotic group,# significantly different between bothacidotic groups.

aquaporin 2 (1:1000) (Santa Cruz Biotechnology) followed byAlexa 488-conjugated donkey anti-goat antibody (1:400)(Invitrogen, Basel, Switzerland).

Statistical analysisResults are expressed as mean ± SEM. All data were tested

for significance using the one way ANOVA and Tukey posttest or unpaired student’s t-test where appropriate.

Results

NH4Cl in water induces metabolic acidosis anddehydration in ratsIn rats acid-loading with NH4Cl either in drinking

water or food resulted in induction of metabolic acidosis,as evident from the reduction of blood pH and bicarbonatelevels (Table 2). However, the degree of metabolicacidosis was less pronounced in rats ingesting NH4Cl withfood despite ingesting even a slightly higher load (6.74 ±0.49 mmoles NH4Cl/day/rat. for Protocol 1; 9.41 ± 0.24mmoles NH4Cl/day/rat for Protocol 2). As expectedserum chloride was elevated in both acidotic rat groups,


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however, animals ingesting NH4Cl in food had significantlylower chloride levels than animals with NH4Cl in water.Plasma sodium concentration and hematocrit were notchanged in both experimental rat groups (Table 2). Plasmaurea concentration and osmolality were elevated only inrats NH4Cl-loaded with water (Table 2). During acid-loading rats gained less weight when receiving NH4Cl indrinking water and their food intake was lower comparedto control (Table 2). Water consumption increasedsignificantly in the group receiving NH4Cl in foodparalleled by higher urinary output.

Fig. 1. AQP2, AQP3,V2R, and ENaC mRNA abun-dance in rat kidney. Real-time RT-PCR was used toassess AQP2, AQP3, V2R and ENaC mRNA levelsin kidneys from control and NH4Cl-loaded animals(n = 4 rats/group). In rat kidney, AQP2, AQP3, V2Rand ENaC mRNA was reduced in animals receivingNH4Cl in food. NH4Cl in drinking water decreasedonly V2R mRNA. * p 0.05, ** p 0.01.

Fig. 2. Expression of AQP2and AQP3 proteins duringacid-loading in rat kidney. (A)Immunoblotting for glyco-sylated and non-glycosylatedvariants of AQP2 and AQP3water channels in totalmembrane fractions fromcontrol and NH4Cl-loaded rats(n = 4 animals/group). Allmembranes were reprobed for

-actin. (B) Bar graphssummarizing data fromimmunoblotting. All data werenormalized against -actin. * p

0.05, *** p 0.001.

Urine analysis revealed that rats from both acidoticgroups acidified their urine to a similar extend andammonium excretion as well as titratable acid (TA) andnet acid excretion (NAE) was increased accordingly.NH4Cl-loading in water did not influence water intakeand urine output but increased urine osmolality. NH4Cl-loading in food, however increased urine volume withoutchanging urine osmolality. Urinary urea and aldosteronewere elevated in both acidotic rat groups, with a 3-foldhigher aldosterone in animals receiving NH4Cl in waterthan in food (Table 2).

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Changes in AQP2, AQP3, V2R, and ENaCmRNA and protein expression in rat kidneydepend on the route of NH4Cl applicationInduction of MA for 3 days with 0.28M NH4Cl in

the drinking water in rats had no effect on mRNAexpression of both AQP2 and AQP3, and subunit ofepithelial sodium channel ENaC but decreased V2RmRNA expression (Fig. 1). In contrast, addition of NH4Clto food decreased mRNA expression of both aquaporins,V2R, and ENaC (Fig. 1).

Induction of metabolic acidosis using NH4Cl-containing drinking solution resulted in elevated abundanceof both, glycosylated and non-glycosylated AQP2 speciesand AQP3 in rat kidney (Fig. 2A, B). Application of NH4Clin food had no apparent effect on AQP2 and AQP3protein abundance (Fig. 2A, B). To estimate the impactof NH4Cl application on AQP2 activation, we assessedphosphorylation of AQP2 at positions Ser256 and Ser261

(Fig. 3A, B). Phosphorylation of Ser256 is associated withactivation of AQP2 [31-32], whereas phosphorylation ofSer261 may be associated with increased internalizationand degradation [33-34]. Addition of NH4Cl to drinkingwater stimulated phosphorylation at serines 256 and 261in native rat kidney. In contrast, NH4Cl in food, stimulatedonly phosphorylation at serine 261 (Fig. 3A, B).

NH4Cl-loading alters distribution of AQP2 waterchannelImmunohistochemistry was used to examine

whether NH4Cl-loading alters segmental and/orsubcellular distribution of the aquaporin 2 water channelin rat kidney. AQP2 is predominantly expressed on theapical side of principal cells along the entire connectingtubule and collecting duct system [35]. Low magnificationoverviews showed a higher intensity for AQP2 stainingin kidneys from animals receiving NH4Cl in water but

Fig. 3. Abundance of phosphorylatedpSer256 and pSer261 AQP2 protein duringacid-loading in rat kidney. (A)Immunoblotting for glycosylated and non-glycosylated variants of AQP2 water chan-nel phosphorylated at serine residues 256(pSer256-AQP2) or 261 (pSer261-AQP2) intotal membrane fractions from control andNH4Cl-loaded rats (n = 4 animals/group). Allmembranes were reprobed for -actin. (B)Bar graphs summarizing data fromimmunoblotting. All data were normalizedagainst -actin. * p 0.05, ** p 0.01.


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not in food (Fig. 4 A). At higher magnification a morepronounced membrane associated staining in CNT, CCD,OMCD, and IMCD was seen in all NH4Cl treated ratswhich appeared to be even more intense in rats receivingNH4Cl in water.

NH4Cl in water induces more pronouncedmetabolic acidosis and decreases diuresis in miceIn mice acid-loading with both protocols resulted

in a fall of blood pH and HCO3- and in increases in

Cl- and Ca2+ levels. Neither serum urea nor osmolalitywere affected by acid-loading, also hematocritwas unchanged in both acidotic groups. Mice withNH4Cl in food lost body weight, whereas mice on normaldiet or receiving NH4Cl in water did not (Table 3);however food intake was not changed by either way ofacid-loading. Water consumption was elevated in mice

receiving NH4Cl in food paralleled by higher urinaryoutput.

Acid loading with both protocols resulted instimulated urinary acidification,elevated ammonium,titratable acids, and net acid excretion. Interestingly, miceingesting NH4Cl in food showed much higher urinaryammonium excretion than NH4Cl-loaded in water,although NH4Cl intake was not significantly differentbetween protocols (1.19 ± 0.08 mmoles NH4Cl/day/mouse for NH4Cl in water; 1.38 ± 0.04 mmoles NH4Cl/day/mice for NH4Cl in food). Elevated urine osmolalityin mice acid-loaded in water was paralleled by lower urineoutput despite normal water intake. NH4Cl-loading in food,however, increased water consumption and urine volumeand reduced urine osmolality. Both NH4Cl loadingprotocols increased urinary aldosterone excretion to asimilar extent.

Fig. 4. Immunolocalization of AQP2water channel in rat kidney duringacid-loading. (A) Low magnification(100 x) overview over cortex and innermedulla of rat kidneys from untreatedanimals (control) or animals receivingNH4Cl in water or food, respectively. (B)Higher magnifications (400 x) of connect-ing tubule (CNT), cortical collecting duct(CCD) and inner medullary collectingducts (IMCD).


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NH4Cl in water increases AQP2 and AQP3 mRNAand protein abundanceIn mice acid-loading with NH4Cl in water resulted

in increased AQP2 and V2R mRNA abundance but had

Table 3. NH4Cl-loading of mice. Blood gasstatus and electrolytes in blood and urinedata from C57BL/6 mice under controlconditions and after 3 days acid-loading(0.28 M NH4Cl + 0.5% sucrose in drinkingwater or 2g NH4Cl/ 100g food + 0.5 %sucrose in drinking water). Control micereceived only 0.5% sucrose in their drinkingwater. Values are means ± SEM, n = 6per group. */# p 0.05, **/## p 0.01, ***/###

p 0.001, * significantly different betweencontrol and acidotic group, # significantlydifferent between both acidotic groups.

Fig. 5. AQP2, AQP3, V2R, and ENaCmRNA abundance in mouse kidney. Real-timeRT-PCR was used to assess AQP2, AQP3,V2R, and ENaC mRNA levels in kidneysfrom control and NH4Cl-loaded animals(n = 6 mice/group). In mouse kidney, expres-sion of AQP2 water channel and V2R mRNAwas increased only in mice receiving NH4Clin water, but not in food. In contrast, the

subunit of the epithelial sodium channelENaC was elevated only in mice receivingNH4Cl in food. AQP3 expression remainedunchanged in both groups. * p 0.05,** p 0.01.

no effect on expression of AQP3 and ENaC mRNA(Fig. 5). In contrast to rats, mice ingesting NH4Cl in fooddid not show any changes in mRNA abundance of AQP2,AQP3, or V2R, but expression of ENaC mRNA was


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Fig. 6. Expression of AQP2 and AQP3 proteins during acid-loading in mouse kidney. (A) Immunoblotting for glycosylated andnon-glycosylated variants of AQP2 and AQP3 water channels in total membrane fractions from control and NH4Cl-loaded mice.All membranes were reprobed for -actin, 4 representative animals/ group are shown. (B) Bar graphs summarizing data fromimmunoblotting. All data were normalized against -actin. (C) Immunolocalization of AQP2 water channel in kidneys from controland acid-loaded mice shows higher intensity in kidneys from mice loaded with NH4Cl in water. Pictures were taken from the innermedulla at an original magnification of 200 x. * p 0.05, ** p 0.01.

elevated (Fig. 5). Furthermore, only mice ingesting NH4Clin water but not in food had elevated abundance ofglycosylated and non-glycosylated AQP2 (Fig. 6A, B).Moreover, both acidotic mice groups exhibited elevatedlevels of the aldosterone regulated AQP3 water channel(Fig. 6A, B). Addition of NH4Cl to drinking waterstimulated AQP2 phosphorylation at serines 256 and 261in native mouse kidney. In contrast, NH4Cl in food hadno significant effect on AQP2 phosphorylation at serineresidues 256 and 261 (Fig. 7A, B).

NH4Cl-loading alters distribution of AQP2 waterchannelImmunohistochemistry of mouse kidneys showed

similar findings as in rat kidney. AQP2 staining in CNT,CCD, OMCD and IMCD appeared to be more membraneassociated in mice treated with NH4Cl in water but alsomice with NH4Cl in water had some AQP2 at the luminalmembrane (Fig. 8A, B).

NH4Cl in food and water alters abundance ofacidosis regulated genesWe and others have previously shown that the anion

exchanger pendrin, the glutamine transporter SNAT3, andphospho-enol pyruvate carboxy kinase (PEPCK) areregulated on mRNA levels during metabolic acidosisinduced by NH4Cl [7, 10, 23, 36, 37]. qPCR demonstratedthat pendrin mRNA abundance was similarly reduced inmice receiving NH4Cl in food or drinking water (Fig. 5).Also, SNAT3 and PEPCK mRNA levels were increasedin both mouse groups irrespective of the route of NH4Cladministration (Fig. 5).

Discussion

The purpose of this study was to compare the in-duction of metabolic acidosis with NH4Cl added either todrinking water or food. Application of NH4Cl with drink-


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ing water is a well established and extensively usedmethod to induce metabolic acidosis [1-4, 7-8, 10, 27]. Inour hands, both protocols induced MA in rats and miceas evident from lower blood pH, increased urinary acidi-fication, and acid excretion. Both acidotic mice and ratgroups were also able to acidify their urine and excreteacids to a similar extend regardless of the diet (Tables 2-3). It seems, however, that animals receiving NH4Cl infood were less acidotic compared to the group receivingNH4Cl in drinking water. Total NH4Cl intake was similarin both groups suggesting that the route of applicationaffects compensatory mechanisms.

The major findings of our comparison of differentNH4Cl-loading protocols are: 1) both protocols inducedmetabolic acidosis in mice and rats, 2) differences be-tween rats and mice exist with respect to their renal com-

pensatory responses as evident from different expres-sion levels of AQP2, AQP3, ENaC, and V2R mRNAand protein, 3) NH4Cl in drinking water induces signs ofa mild dehydration in rats, as demonstrated by higher al-dosterone levels and higher urine osmolality, 4) NH4Cl inwater stimulated urinary concentration in mice withoutother signs of dehydration, 5) NH4Cl in water increasedphosphorylation of residues in AQP2 associated withhigher AQP2 activity and insertion into the membraneparalleled by enhanced staining of AQP2 at the luminalmembrane, and 6) dehydration and MA affect independ-ently expression patterns of genes and proteins involvedin renal water handling (Table 4).

We found higher urea excretion in rats and mice inthe food group as compared to the water group. In con-trast, ammonium excretion was higher only in mice re-

Fig. 7. Abundance of phosphorylatedpSer256 and pSer261 AQP2 protein during acid-loading in mouse kidney. (A) Immunoblottingfor glycosylated and non-glycosylated vari-ants of AQP2 water channel phosphorylatedat serine residues 256 (pSer256-AQP2) or 261(pSer261-AQP2) in total membrane fractionsfrom control and NH4Cl-loaded mice (n = 4-5animals/group). All membranes were reprobedfor -actin. (B) Bar graphs summarizing datafrom immunoblotting. All data were normal-ized against -actin. ** p 0.01, *** p 0.001.


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ceiving NH4Cl in food compared to mice with NH4Cl inwater. Mice and rats on the acid diet tended to receive ahigher NH4Cl load than the respective water groups andtherefore an overall increased nitrogen load that mightexplain the observation. Moreover, the higher urinary ureaexcretion might also reflect, at least in mice, increasedcatabolism of proteins as suggested by a higher weightloss.

The excretion of the chloride load (given by way ofthe chloride contained in food plus the NH4Cl added ei-ther to drinking water or food) was lower in rats receiv-ing NH4Cl in food. Since urinary chloride excretion mightreflect net chloride intake and ammonium absorption, itmight indicate a lower absorbed NH4Cl load in rats re-ceiving NH4Cl in food. However, interpretation of uri-nary chloride excretion is difficult since it may not di-rectly correlate with intestinal chloride or even ammo-nium absorption. Recent experiments investigating sodiumbalance in rats and mice demonstrated that intake and

Fig. 8. Immunolocalization of AQP2 waterchannel in mouse kidney during acid-loading.(A) Low magnification (100 x) overview overcortex and inner medulla of mouse kidneys fromuntreated animals (control) or animals receiv-ing NH4Cl in water or food, respectively. (B)Higher magnifications (400 x) of connectingtubule (CNT), cortical collecting duct (CCD)and inner medullary collecting ducts (IMCD).

excretion did not equal and that substantial amounts ofsodium were stored under the skin [38]. Thus, more de-tailed studies on the net absorptive rate of ammoniumintake are needed to estimate the true acid load providedby the two different application routes.

Dehydration and extracellular volume contraction ac-tivates the renin-angiotensin-aldosterone system andstimulates synthesis of angiotensin II and secretion ofaldosterone [39-42]. Induction of MA resulted in eleva-tion of urine aldosterone excretion in both acidotic ratand mice groups; however, aldosterone levels were 3-fold lower in rats receiving NH4Cl in food compared tothe NH4Cl in water group. Surprisingly, elevated aldos-terone levels were not reflected by changes in urinarysodium and potassium excretion. This might be explainedby direct effects of acidosis on aldosterone targets suchas the ROMK potassium channel or the ENaC sodiumchannel. The function of ROMK is modulated by intrac-ellular pH where a fall in intracellular pH reduces chan-


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nel function and potassium secretion [43-44]. Water rea-bsorption by the luminal AQP2 water channel in the col-lecting duct is a major factor in water homeostasis andurinary concentration. The subcellular localization andactivity of this channel is regulated on the molecular levelby phosphorylation of several residues including serines256 and 261[31-34] integrating various signals includingstimulation by vasopressin or angiotensin II [45]. Our re-sults indicate that AQP2 is stimulated as indicated by in-creased phosphorylation of Ser256 and by enhanced stain-ing at the luminal membrane in animals receiving NH4Clin water. Hematocrit and plasma sodium concentrationwere unchanged in both rat groups. Additionally NH4Clingestion in food resulted in increased water intake andurine output and decreased urine osmolality in both miceand rats which is in agreement with observations of Moriet al. [15]. Collectively, these data are consistent withmild dehydration in animals receiving NH4Cl in drinkingwater resulting in elevated aldosterone levels andupregulation and stimulation of several components ofthe urinary concentration machinery. The regulation ofAQP3 observed also in the NH4Cl in food group may beat least in part due to the slightly increased levels of al-dosterone which has been shown to increase AQP3 ex-pression [46].

All animals receiving NH4Cl in food or water devel-oped increased urinary calcium excretion which was moreprofound in the food groups. Hypercalciuria is thought todevelop at least in part due to inhibition of the TRPV5calcium channel in the distal convoluted tubule and con-necting tubule [11]. Elevated urinary calcium may thenaffect localization and expression of the AQP2 waterchannel along the collecting duct via a calcium-sensingreceptor [47-48] promoting increased diuresis. However,

Table 4. Short summary of findings in mice and rats usingtwo different acid-loading protocols. - higher than in controlgroup, - lower than in control group, - not changed.

Fig. 9. Regulation of SNAT3, PEPCK, and pendrin in kidneys from NH4Cl-loaded mice. Real-time RT-PCR was used to assessSNAT3, PEPCK, and pendrin mRNA levels in kidneys from control and NH4Cl-loaded animals (n = 6 mice/group). All threemRNAs were regulated irrespective of the route of NH4Cl application. *** p 0.001.

whether this mechanism contributes to the higher urinaryexcretion in the food group remains to be examined.

Our results of NH4Cl-loading in drinking water aresimilar to those of Amlal et al. [9] suggesting that the


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6 Promeneur D, Kwon TH, Yasui M, KimGH, Frokiaer J, Knepper MA, Agre P,Nielsen S: Regulation of AQP6 mRNAand protein expression in rats in responseto altered acid-base or water balance.Am J Physiol Renal Physiol2000;279:F1014-1026.

7 Frische S, Kwon, TH, Frokiaer J, MadsenKM, Nielsen S: Regulated expression ofpendrin in rat kidney in response tochronic NH4Cl or NaHCO3 loading. Am JPhysiol Renal Physiol 2003;284:F584-593.

8 Stehberger PA, Shmukler BE, Stuart-Tilley AK, Peters LL, Alper SL, WagnerCA: Distal renal tubular acidosis in micelacking the AE1 (band3) Cl-/HCO3

-

exchanger (slc4a1). J Am Soc Nephrol2007;18:1408-1418.

9 Amlal H, Sheriff S, Soleimani M:Upregulation of collecting ductaquaporin-2 by metabolic acidosis: Roleof vasopressin. Am J Physiol Cell Physiol2004;286:C1019-1030.

10 Curthoys NP, Taylor L, Hoffert JD,Knepper MA: Proteomic analysis of theadaptive response of rat renal proximaltubules to metabolic acidosis. Am JPhysiol Renal Physiol 2007;292:F140-147.

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- exchanger pendrin in the ratkidney is regulated in response to chronicalterations in chloride balance. Am JPhysiol Renal Physiol 2004;287:F1179-1188.

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and Mg2+ transport proteins. J Am SocNephrol 2006;17:617-626.

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results reported there may be mainly due to dehydrationbut not MA in rats. Ingestion of equivalent amounts ofNH4Cl in food had no effect on AQP2 protein levels inour hands. Also immunohistochemistry analysis of mouseand rat kidneys showed more intense AQP2 staining withenhanced accumulation at the luminal membrane morepronounced in the NH4Cl in water group. Concomitantwith increased protein abundance was mRNA expres-sion of AQP2 in mice loaded with NH4Cl in water. Inter-estingly, there was no change in AQP2 mRNA expres-sion in rats subjected to NH4Cl in water. Moreover, ex-pression of AQP2 was even decreased in animals in-gesting NH4Cl in food. Thus, not urinary concentrationbut increased diuresis may actually be part of the renalcompensatory response to metabolic acidosis.

Taken together, our study demonstrates that the routeof NH4Cl application – drinking water versus food – canaffect the regulation of renal genes and proteins, particu-larly those involved in water handling. NH4Cl in drinkingwater causes mild dehydration and upregulation andstimulation of several components of the urinaryconcentrative mechanism. Moreover, our study showsthat distinct species differences exist between mice andrats in their response to NH4Cl-loading in drinking water.

Therefore, future studies should consider these differen-tial effects of NH4Cl addition in planning and interpretinganimal experiments. In addition, species differences ex-ist between rats and mice and may be more pronouncedthan previously anticipated. Finally, it may be of interestto consider similar studies in humans assessing urinaryconcentration and water balance.

Acknowledgements

This study was supported by grants to C A Wagnerfrom the Swiss National Science Foundation (31-109677and 3100A0-122217)), the 6th EU Frame work projectEuReGene, and a ZIHP PhD student fellowship to MNowik. D. Eladari was supported by the TNH from theFondation Leducq and by a grant PHYSIO 2007-RPV07084 from L’Agence Nationale de la Recherche(ANR). The use of the ZIHP Core Facility for RodentPhysiology is gratefully acknowledged. The authors thanksFrançoise Leviel and the technicians from the Depart-ment of Physiology of Hopital Européen GeorgesPompidou for their skillful technical assistance in aldos-terone measurements.


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