Effect of Volume Expansion on Renal Citrate

and Ammonia Metabolism in KCl-Deficient Rats

SHELDONADLER, BARBARAZETr, BARBARAANDERSON,andDONALDS. FRALEY

From the Department of Medicine, University of Pittsburgh School of Medicineand Montefiore Hospital, Pittsburgh, Pennsylvania 15213

A B S T R A C T When rats with desoxycorticosteroneacetate (DOCA)-induced potassium chloride deficiencyare given sodium chloride there is simultaneously apartial correction of metabolic alkalosis and a markedreduction in urinary citrate excretion and renal citratecontent. To examine DOCA's role in this phenomenonand to determine how sodium chloride alters renalmetabolism, rats were made KC1 deficient using furose-mide and a KCl-deficient diet. Renal citrate and am-monia metabolism were then studied after chronic oralsodium chloride administration or acute volume ex-pansion with isotonic mannitol. Although both maneu-vers partially corrected metabolic alkalosis, sodiumchloride raised serum chloride concentration whilemannitol significantly decreased it. Urinary citrate ex-cretion decreased to 10% of control in rats givenNaCl and to 50% of control in rats infused with man-nitol. The filtered load of citrate was constant or in-creased indicating increased tubular citrate reabsorp-tion. Renal cortical citrate content also decreased ap-proximately 50%. Renal cortical slices from KCl-de-ficient rats incubated in low or normal chloride mediaproduced equal amounts of "CO2 from [1,5-"4C]citrate.In addition, urinary ammonia excretion increased byover 300% in both groups. This occurred in the man-nitol group despite increased urinary pH and flow rateindicating a rise in renal ammonia production.

It seems that neither DOCAnor an increase in serumchloride concentration explains the experimental results.Rather, it appears that volume expansion is responsiblefor increased renal tubular citrate reabsorption andrenal ammonia production. As these renal metabolic

This work was previously reported in abstract form:Adler, S., B. Zett, B. Anderson, and D. Fraley. 1974. Roleof chloride and volume expansion in regulating renal citratemetabolism. Clin. Res. 22: 654a.

Received for publication 31 December 1974 and in revisedform 14 April 1975.

responses ordinarily occur in response to acidosis, thedata are consistent with the hypothesis that volumeexpansion reduces renal cell pH in KCl-deficient rats.

INTRODUCTION

Recently, Adler, Zett, and Anderson (1) demonstratedthat when desoxycorticosterone acetate (DOCA)'-in-duced chronic potassium chloride-deficient rats aregiven sodium chloride, but no potassium salts, renalcortical citrate content and urinary citrate excretion pro-foundly decrease. Renal citrate metabolism was examinedbecause it has been shown that urinary citrate excretionand renal citrate content decrease in acidosis and in-crease in alkalosis (2, 3) presumably secondary to in-creased renal citrate oxidation in acidosis and a de-crease in alkalosis (4). However, Adler and hiscolleagues noted that despite the enormous reductionsin urinary and renal citrate seen after sodium chlorideadministration, metabolic alkalosis was still present (1).Inasmuch as changes in citrate metabolism in potas-sium-deficient diaphragm muscle have been shown tobe directly related to alterations in intracellular ratherthan extracellular pH (5), it was postulated that so-dium chloride administration in the potassium chloride-deficient rats may have caused a renal tubular cell acid-osis. Unfortunately, direct measurement of renal tubularcell pH is presently not possible in vivo (6), so thiswas not able to be confirmed directly.

To test the hypothesis that renal tubular cell pH islowered by sodium chloride ingestion in potassiumchloride deficiency and to determine the mechanismthrough which sodium chloride induces changes inrenal citrate metabolism, further experiments were per-formed. To eliminate the possible role of exogenously

'Abbreviation used in this paper: DOCA, desoxycorticos-terone acetate.

The Journal of Clinical Investigation Volume 56 August 1975 391-400 391

administered DOCA, rats were made potassium chlo-ride deficient by intraperitoneal furosemide administra-tion. The roles played by extracellular chloride concen-tration and volume expansion in altering renal cell pHwere studied by examining their effects on renal citrateand ammonia metabolism. The results indicate that theresponse of renal citrate metabolism to sodium chlorideingestion in the furosemide-induced potassium chloride-deficient rats was identical to the response of renalcitrate metabolism to sodium chloride ingestion in ratsmade deficient with DOCA. In addition to the changesin renal citrate metabolism, sodium chloride or mannitolinduced volume expansion increased ammonia excre-tion. These renal metabolic alterations appear to bedue to an expansion of intravascular volume ratherthan a change in plasma chloride concentration. Thedata are consistent with the hypothesis that expansionof intravascular volume in potassium chloride-deficientrats leads to a decrease in renal tubular cell pH.

METHODSPotassium chloride depletion protocol. Male Sprague-

Dawley rats weighing 350-375 g were used in all experi-ments. Each animal was placed on a zero potassium, zerochloride diet (obtained from Nutritional Biochemical Corpo-ration, Cleveland, Ohio). Animals were allowed water con-taining 75 meq/liter of sodium bicarbonate ad libitum duringthe entire study. Rats were maintained on the potassiumchloride-deficient diet for a 15-day period. On days 8-12each rat received a daily intraperitoneal dose of 2 mg offurosemide.

Repletion protocol. At the end of the 15-day period allfood was removed, and the rats were divided into fourequal groups: a KCI-repleted group tube fed 10 ml of a20% dextrose solution containing 200 meq/liter KCI twicedaily; a K-depleted group tube fed 10 ml of 20%o dextrosesolution containing no electrolytes twice daily; an NaCl-repleted group tube fed 10 ml of a 20%o dextrose solutioncontaining 200 meq/liter sodium chloride twice daily; and aKHCOa-repleted group tube fed 10 ml of a 20%o dextrosesolution containing 200 meq/liter KHCO. twice daily. Solu-tions were administered for 3 days during which the sodiumbicarbonate drinking water was continued ad libitum. Ratswere weighed each morning during this 3-day repletion.After 3 days of tube feeding rats were sacrificed the nextday as outlined below.

Citrate and ammonia excretion experiments in repletedrats. After receiving the sixth tube feeding each rat wasplaced in an individual metabolic cage and given only thesodium bicarbonate solution ad libitum. Urine was collectedfor the next 16 h. At the end of this time rats were removedfrom the cages which were then rinsed with distilled waterto insure complete urine collection. Each urine collectionwas filtered and analyzed for creatinine, chloride, citrate,and ammonia.

Tissue electrolyte and citrate experiments in repleted rats.Animals were anesthetized by the intraperitoneal injectionof inactin, 100 mg/kg of body weight. Each rat was anes-thetized separately to avoid any delay before killing therat by exsanguination from the abdominal aorta into a hepa-rinized syringe. Portions of renal cortical tissue, liver, andgluteal muscle were removed and rapidly frozen in liquid

nitrogen for citrate analysis. Other portions of these tissueswere placed in tared tubes for later determination of waterand potassium content. All the frozen tissues were weighedand homogenized in the frozen state. The homogenate wascentrifuged and the supernate saved for citrate analysis.

14CO0 production from [1,5-14C] citrate experiments. Aspreviously described (2) renal cortical slices were placedinto specially constructed 25-ml Erlenmeyer flasks havinga center well. Approximately 100 mg of renal cortex wasused in each flask. Each flask contained 4 ml of a carbondioxide-oxygen saturated Krebs-Ringer bicarbonate solutioncontaining 1.0 meq/liter of potassium. The substrate was 1mMacetate and 1 mMcitrate. In addition, each flask con-tained 0.5 ,hCi of [1,5-14C]citrate (obtained from New Eng-land Nuclear, Boston, Mass.). The solution in one groupof flasks contained 70 meq of sodium chloride, while thesolution in the other group contained 110 meq of sodiumchloride. Isoosmolarity was achieved by the addition ofmannitol to the first flask. Flasks were incubated for 90min in a metabolic shaker at 370C, and "CO2 was collectedand counted as previously described (2). Results are ex-pressed as disintegrations per minute per flask per 200 mg.Calculations of this value have also been previously de-scribed (2). Production of 'CO2 from labeled citrate inthe remainder of the paper will be termed citrate de-carboxylation.

Mannitol expansion experiments. Potassium chloride-de-pleted rats were employed in all experiments. After the15-day depletion protocol the potassium chloride-depletedrats were anesthetized by the intraperitoneal injection ofinactin, 100 mg/kg of body weight. A tracheostomy wasperformed in each animal to allow for removal of retainedsecretions. An indwelling jugular catheter was inserted forinfusion, and a femoral artery line was placed for bloodsampling. The infusion line was connected to a Harvardconstant infusion pump (Harvard Apparatus Co., Inc.,Millis, Mass.). A bladder catheter was inserted supra-pubically. After a 30-min stabilization period, animals weredivided into two groups. The dextrose-infused group re-ceived 8.3 ml/h of a 5% dextrose solution over a 210-minperiod. The mannitol-infused group was given 8.3 ml/h ofthe 5% dextrose solution for 90 min to act as a controlperiod. These animals were then infused with a 280 mMmannitol solution given at 37 ml/min for 150 min. In thedextrose group blood was obtained after the stabilizationperiod and at the conclusion of the experiment. In themannitol-infused group blood was obtained at the end ofthe stabilization period, at the end of the control period,and at the conclusion of the experimental period. Urine col-lected under oil in the dextrose-infused group was obtainedas a single sample over the entire 210 min. In the mannitol-infused rats a 90-min control period urine was obtained,and 30 min after mannitol infusion was started a 60-minand two 30-min urines were collected under oil. Urines inboth groups were analyzed for pH, creatinine, citrate, andammonia. Each group was infused with radioisotopes im-mediately after the stabilization period. Dextrose-infusedrats received 2 1ACi each of Na "Cl and ["C] DMOand 10,uCi of tritiated water. Double this quantity was given to themannitol-infused group to compensate for increased urinarylosses. Isotopes were injected early to allow sufficient tissueequilibration time to take place. At the conclusion of theexperiment animals in each group were killed by exsangui-nation from the abdominal aorta into a heparinized syringe.Portions of renal cortical tissue were removed and imme-diately frozen in liquid nitrogen, weighed, homogenized ina frozen state, and saved for citrate analysis as previously

392 S. Adler, B. Zett, A. Anderson, and D. S. Fraley

TABLE I

Arterial Acid-Base Status and Arterial and Tissue Potassium Levels in PotassiumChloride-Repleted, Potassium-Depleted, Sodium Chloride-Repleted,

and Potassium Bicarbonate-Repleted Rats*

Blood Tissue potassium

pH HCOS PCo2 K Muscle Kidney

meq/liter mmHg meq/liter meq/kg dry weight

KCl repleted, n = 5 7.424 20.8$ 324 3.9$ 4711 339±0.01 i1.7 ±-3 ±0.2 ±9 ±14

K depleted, n = 7 7.55§ 36.4§ 41§ 2.3§ 357§ 319±0.02 ±1.8 ±1 ±0.2 ±9 ±+9

NaCi repleted, n = 7 7.48 26.0t§ 35$ 2.6§ 365§ 31840.03 ±1.2 ±2 ±0.1 ±10 ±5

KHCO3repleted, n = 6 7.55§ 32.7§ 38§ 2.4§ 348§ 328±0O.01 ± 1.0 ±t1 ±0.2 ±12 46

* Values represent the mean±SE of the mean.$ Differ from K deplete P < 0.01, using Student t test.§ Differ from KC1 replete P < 0.01, using Student t test.

described (2). A portion of renal cortex, plus liver, glutealmuscle, and cardiac muscle tissue were placed in taredtubes for later determination of water and potassium con-tent. A portion of gluteal muscle, cardiac muscle, and livertissue was placed in weighed homogenizing tubes for deter-mination of intracellular pH, as previously described (7).

Analytic methods. Blood pH and PCo2 were measuredon a Radiometer BM3 MK2 blood micro system at 37'C.Urine pH was measured using a Radiometer PMS27 pHmeter. Bicarbonate concentration in the blood was calculatedfrom the Henderson-Hasselbalch equation employing a pKof 6.1. Sodium and potassium were measured on an ILflame photometer while chloride, creatinine, and blood ureanitrogen (BUN) were determined by autoanalyzer. Tissuewater and electrolytes were determined by drying tissue toconstant weight in a 700C oven for 16 h, as previously de-scribed (2). Citrate was determined enzymatically (8), andammonia was determined by microdiffusion (9). Tripleisotope determination of tissue pH employed the methodsdescribed by Schloerb and Grantham (10). Radioactivitywas measured in a three channel Packard liquid scintillationcounter (Packard Instrument Co., Inc., Downers Grove,Ill.) using a AES standardization and standard methods fordetermining efficiencies.

RESULTSEffect of sodium chloride repletion on renal citrate

metabolism and urinary ammonia excretion in KCl-depleted rats. Extracellular acid-base status and serumand tissue potassium levels in the four groups of ratsstudied are shown in Table I. With the exception of thepotassium chloride-repleted rats, metabolic alkalosiswas present in each group. Compared to potassium-depleted and potassium bicarbonate-repleted animals,however, the severity of the metabolic alkalosis wasless in the sodium chloride-repleted rats, P < 0.05.Thus, sodium chloride partially corrected the alkalosis,

a result previously shown by other investigators inDOCA-induced potassium-deficient animals (1, 11).The furosemide and dietary KC1 restriction regimeninduced a state of severe potassium depletion. Musclepotassium concentration was reduced 25% in each ofthe last three groups compared to values found bothin the potassium chloride-repleted animals and normalvalues obtained in this and other laboratories (P <0.001). Renal potassium concentration was also reducedbut the degree of reduction, as expected, was less thanin the muscle (1). It is important to note that despiteadministration of potassium bicarbonate serum and musclepotassium depletion was as severe as that found insodium chloride and potassium-depleted animals whoreceived no potassium. Administration of potassiumbicarbonate, unlike potassium chloride, was unable torestore extracellular acid-base conditions, serum po-tassium concentration, or tissue potassium concentra-tion to normal. The efficacy of sodium chloride admin-istration in repleting chloride stores is shown both bythe rise in serum chloride concentration from 80 to 94meq/liter and by the large amounts of chloride in theurine of the NaCl-repleted rats (Table II).

In addition to partially correcting the extracellularmetabolic alkalosis, administration of sodium chloridealso altered renal metabolism. As shown in Fig. 1 and2 urinary citrate excretion fell while urinary ammoniaexcretion rose in the sodium chloride-repleted rats.Citrate excretion in the sodium chloride-repleted group,compared to each of the three other groups, differedsignificantly (P < 0.001) as did ammonia excretion(P < 0.001 vs. K depleted and KHCOs and P < 0.02compared to KCl-repleted animals). The alteration in

Expansion, Renal Citrate, and Ammonia Metabolism in KCI Deficiency 393

TABLE ItArterial Blood Concentrations of Urea, Creatinine and Citrate, Urinary Chloride Excretion,

and Renal Cortical Citrate Content in the Four Groups of Rats*

BloodUrinary

Urea chloride Renal corticalnitrogen Creatinine Citrate excretion citrate content

g/100 ml g/100 ml pmol/ml meq/liter pmol/g wet weight

KCI replete, n = 5 11.2$ 0.48 0.059 121t 0.34640.6 =10.04 4O0.005 425 4:0.060

K deplete, n = 7 20.7§ 0.50 0.57 3.4§ 0.316±2.4 ±0.04 40.006 ±0.5 40.046

NaCl replete, n = 7 12.0t 0.46 0.060 147§ 0.158t§±t1.2 ±0.02 +0.008 ±28 ±0.025

KHCO3replete, n = 6 14.2 0.52 0.090t§ 4.2t 0.819t§±2.0 ±0.02 ±0.008 ±0.8 ±0.075

* Values represent the mean±SE of the mean.$ Differ from deplete P < 0.01, using Student t test.§ Differ from KCI replete P < 0.01, using Student t test.

citrate excretion in NaCl-repleted rats apparently wasnot secondary to changes in the filtered load of citrate.Table II shows that serum creatinine levels were notsignificantly different between groups, and only in thepotassium-depleted group was BUN elevated (P <0.01), indicating equal glomerular filtration rates inthe several groups. Blood citrate levels were elevatedonly in the potassium bicarbonate rats so filtered citratein each of the first three groups studied probably wasthe same. Increased citrate excretion in the KHCOs

group is easily accounted for by the elevated filteredload. Evidence that the observed change in renal citratemetabolism in the NaCl-repleted rats was not due tofiltered load is presented in the last column of Table II.The data show that in addition to the reduced citrateexcretion, renal cortical citrate content also decreasedsignificantly in the sodium chloride-repleted animals(P < 0.001 vs. each of the three other groups). Sodiumchloride given to KCl-deficient rats, therefore, induceschanges in both renal citrate and ammonia metabolism.

roF

IKCI K-DEPLETE NaCI

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C.)xw

0-

cr

>

cr-

4.01-

3.0 F

2.0 -

1.0 F

KHCO3

IKCI K-DEPLETE NaCI

FIGURE 1 Urinary citrate excretion in the four groups ofrepleted rats. The brackets represent ±1 SE of the mean.

FIGURE 2 Urinary ammonia excretion in the four groupsof replqtqd rats. The brackets represent ±1 SE of the mean.

394 S. Adler, B. Zett, A. Anderson, and D. S. Fraley

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TABLE IIIArterial Blood Values in KCO-Depleted Rats Infused with either Dextrose or Mannitol Solutions*

pH PCO2 HCO3 Na C1 K Creatinine Citrate

mmHg meq/lifer meq/liter g/100 ml Mmol/mlDextrose infused, n = 12

Initial 7.50 46 36.3 -±0.02 ±3 i±1.8

Final 7.49 46 35.1 134 79 2.4 0.56 0.073±40.02 ±3 ±1.6 ±2 ±2 ±0.1 ±t0.06 i0.007

Mannitol infused, n = 13Initial 7.47 52 37.3 140 2.4

±0.02 43 ±1.0 ±4 ±0.1Final 7.46 45: 31.81§ 113T§ < 70§ 2.6 0.57 0.080

±0.01 ±2 ± 1.3 ±-3 ±0.2 ±0.06 ±0.004

* Values represent the mean±SE of the mean.: Value differs significantly from initial value (P < 0.05).

§ Difference from final dextrose infused value statistically significant (P < 0.01).

In an attempt to delineate the mechanism involved,further experiments were performed.

14CO5 production from [1,5-14C]citrate in vitro. Theresults of the previous experiments might be due toalterations in the concentration of serum chloride bath-ing the kidney. It has been shown that urinary citrateexcretion is dependent on the ability of renal tubulesto reabsorb filtered citrate (12), and this resorptivecapacity is probably a function of the renal tubularcells ability to oxidize citrate accumulated intracellu-larly (4, 13). Measurement of citrate decarboxylationby renal cortical tissue at different external chlorideconcentrations should, therefore, partially describe chlo-ride's effect upon renal citrate metabolism. Renal corti-cal slices were obtained from potassium chloride-de-ficient rats whose extracellular acid-base status andserum and tissue potassium concentrations were sta-tistically identical to those of the potassium-depletedrats shown in Table I. The slices were incubated inlow (70 meq) and "normal" (110 meq) chloride solu-tions and 14CO2 production was measured. The resultsare shown in Fig. 3. As "CO2 production is equal inthe two groups it appears that chloride concentrationdoes not directly affect renal citrate metabolism. Tofurther examine the effect of chloride concentration onrenal metabolism and to determine whether changes inextracellular volume might account for the observedresults additional experiments were performed.

Expansion of intravascular volume by mannitol inpotassium-depleted rats: effect on renal citrate andammonia metabolism. Mannitol infusions were givento potassium chloride-depleted animals. The effect ofthis infusion was to expand intravascular volume andlower serum chloride concentration. Another group ofanimals was given low volume dextrose infusion over

a similar time period to serve as a control. Serum andtissue potassiuni concentrations in the animals beforeand after infusion were similar to that of the animalsshown in Table I. Muscle potassium concentration afterinfusion in the dextrose and mannitol groups was 304±30 and 312±36 meq/kg of dry weight, respectively,compared to a muscle potassium concentration in non-potassium chloride-depleted animals of 465 meq/kg dryweight (P < 0.001). Renal potassium content in thedextrose and mannitol rats was 267±26 and 252±32

, 500

3400

E

300-

0,

200-

C-)

Cr 1000_

C-)

NORMAL LOWCHLORIDE CHLORIDE

FIGURE 3 The effect of medium chloride concentration oncitrate decarboxylation by renal cortical slices. The num-bers within the bars represent medium chloride concentra-tions in each group while the brackets depict ±1 SE of themean.

Expansion, Renal Citrate, and Ammonia Metabolism in KCl Deficiency 395

TABLE IVCreatinine Clearance, Urinary pH, and Renal Citrate

Content in KCL-Depleted Rats Infused witheither Dextrose or Mannitol Solutions*

Urinary Renal corticalCcr pH citrate content

ml/min pmol/gwet weight

Dextrose infused, n = 12 1.35±0.29 6.8340.12 0.07540.008Mannitol infused, n = 13

Control 1.40 40.19 6.88 40.14Experiment 1 2.02 40.16t§ 7.064±0.05 0.049t14:0.005Experiment 2 1.6840.18 7.1040.05Experiment 3 1.8440.20t§ 7.08 40.05

* Each value represents the mean±SEM.Statistically significant differences from dextrose-infused animals

(P < 0.05).§ Statistically significant differences from control values in mannitol-infused rats (P < 0.05).

meq/kg dry weight, respectively, comlmeq/kg of dry weight in nondepletedMuscle and renal potassium contentcantly differ between dextrose andrats (P > 0.4).

The effect of the dextrose and mantextracellular conditions in potassiumrats is shown in Table III. Arterialafter infusion was unchanged in eitherinfusion had no effect on the serumbut did lower extracellular bicarbonasignificantly (P < 0.01). Neither serublood citrate levels were significantlynitol infusion. The result of acute m

1.2 F

1.0

0.6F

0.4F

0.2

DEXTRO

60 90 120 150

TIME (MINUTES)

FIGURE 4 The effect of mannitol-inducedon urinary citrate excretion. The closedobtained after 210 min of dextrose iopened circles are values obtained in tIrats. The dashed horizontal line representdextrose-infused group. The brackets demean.

pared to 312±18control animals.did not signifi-

zzI--

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E

0I--

cr-

-C

zZ-0

z-co60 90 120 150 180 210 240

TIME (MINUTES)

FIGURE 5 The effect of mannitol-induced volume expan-sion on urinary ammonia excretion. Conventions in thisfigure are the same as for Fig. 4.

mannitol-infused on extracellular conditions, therefore, was similar tothat previously shown in the rats chronically given so-

litol infusions on dium chloride. Serum bicarbonate concentration andchloride-depleted arterial PCo2 fell, metabolic alkalosis was maintained,pH before and and blood citrate levels were unaffected. A significant

group. Mannitol difference is that mannitol infusion decreased thepotassium level serum chloride concentration while sodium chloride ad-

ate concentration ministration raised serum chloride concentration tom creatinine nor normal levels.affected by man- As was seen with sodium chloride ingestion, manni-annitol infusions tol infusion lowered urinary citrate excretion. This is

shown in Fig. 4. Citrate excretion in each of the ex-perimental periods is significantly reduced comparedto control period values or to excretion in the dextrose-infused animals (P < 0.01). Fig. 5 shows the effect onammonia excretion. In this case, however, urinaryammonia was significantly elevated in each of the

)SE mannitol experimental periods compared to controlperiod values or to results obtained in dextrose-infusedanimals (P < 0.001). Citrate and ammonia excretionsduring the control period were similar in the twogroups (P > 0.5). The alteration in citrate excretionafter mannitol infusion is probably secondary to changesin the filtered load of citrate as experiments employingradioactive labeled citrate indicate that the citrate in

180 210 240 the urine comes from extrarenal citrate (13). Thus,urinary citrate essentially represents the net sum of

I volume expansion filtered citrate less reabsorbed citrate (14). Table IV,circle is the value however, shows that changes in filtered load do not

infusion while the account for the observed decrease in urinary citratehe mannitol-infused excretion. Glomerular filtration rate, calculated fromts the value for the the endogenous creatinine clearance, was increased dur-!pict ±1 SE of the t

ing mannitol infusion compared both to control period

396 S. Adler, B. Zett, A. Anderson, and D. S. Fraley

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TABLE V

Skeletal Muscle, Cardiac, and Liver Cell pH in KCI-Depleted1Rats Infusedwith either Dextrose or Mannitol Solutions*

Skeletal muscle Cardiac muscle Liver

K pHDxo K pHDmo K PHDmo

meq/kg dry weight meq/kg dry weight meq/kg dry weightDextrose infused, n = 12 304 6.88 273 7.10 336 7.14

49 40.03 4:8 4-0.03 418 40.03

Mannitol infused, n = 13 312 6.93 282 7.15 2821: 7.144111 410.03 :19 :1:0.02 4:8 ±0.02

* Values represent the mean±SE of the mean.t Differs significantly from dextrose-infused animals (P < 0.01).

values and to the filtration rate of the dextrose-infusedanimals. This increase was statistically significant onlyin periods two and four (P <0.05). This change infiltration rate combined with the slight increase inblood citrate (Table III) shows that filtered citrateincreased rather than decreased during mannitol in-fusion. Thus, decreased urinary citrate excretion isprobably due to increased tubular reabsorption of fil-tered citrate. Further proof that the alterations in uri-nary citrate excretion reflect altered renal citrate me-tabolism is given in Table IV. The data show thatrenal cortical citrate content in the mannitol-infusedrats was significantly reduced compared to that foundin the control rats (P < 0.001). Increased urinary am-monia excretion similarly appears to be secondary tochanges in renal metabolism rather than to a non-specific effect of the mannitol infusion. Urinary am-monia excretion ordinarily rises when urinary pHfalls (15). The data presented in Table IV show thaturinary pH rose rather than fell during mannitol in-fusion. The rise was not significant, however, com-pared to control period values or to values obtained inthe dextrose-infused rats (P < 0.1). Thus, chronicNaCl ingestion and acute mannitol infusion in KCl-depleted rats reduce renal cortical citrate content, lowerurinary citrate excretion, and increase urinary am-monia excretion.

Effect of mannitol-induced volume expansion onmuscle, liver, and cardiac pH. Although renal tubularcell pH cannot be directly determined in vivo, skeletalmuscle, liver, and cardiac muscle cell pH may be cal-culated from distribution of the weak acid 5,5-dimethyl-2,-4-oxazolidinedione (DMO) (16). Table V showsthe effect of mannitol infusion on the intracellular pHof these three tissues. Extracellular potassium con-centration was approximately 2.5 meq/liter (Table III)in these animals. The concentration of potassium ineach of the three tissues (Table V) demonstrates thata higher intracellular to extracellular gradient existed

at the conclusion of the infusion in each of the tissuesstudied. Membrane selectivity was thus maintained andthe DMOdistribution method for measuring cell pHmay be employed (16). It is apparent that mannitol in-fusion did not lower intracellular pH in any of thethree tissues. Indeed, there was a tendency in skeletaland cardiac muscle for intracellular pH to increase.This increase was not statistically significant (P >0.05). As shown in Table III, the slight elevation ofcell pH cannot be due to a change in extracellular pHfor final arterial blood pH in the mannitol-infused ratswas slightly lower than the final blood pH of the dex-trose-infused animals. The rise in cell pH might beexplained by the decreased C02 tension in the manni-tol-infused rats since carbon dioxide readily penetratescell membranes and might influence cell pH morethan bicarbonate (17). Citrate content was determinedin liver and gluteal muscle tissue. No differences werefound between dextrose and mannitol-infused animals.This is consistent with the absence of significant intra-cellular pH differences since tissue citrate content ap-parently is a function of intracellular pH (5).

DISCUSSION

These experiments extend previous work which dem-onstrated that administration of sodium chloride to po-tassium chloride-deficient rats alters renal citrate me-tabolism (1). Whether sodium chloride altered renalcitrate metabolism by changing extracellular chlorideconcentration or extracellular volume was not, however,examined in the earlier study. The present results showthat the alteration in renal citrate metabolism is notdue to changes in serum chloride concentration sincevolume expansion with mannitol had the same effect assodium chloride on extracellular acid base status andrenal citrate metabolism despite having an oppositeeffect on serum chloride concentration. Rather, it ap-pears that increased circulating volume is responsible

Expansion, Renal Citrate, and Ammonia Metabolism in KCI Deficiency 397

for the altered renal metabolism. Although circulatingvolume was not measured in the tube fed rats, weightloss in the NaCl-repleted animals over the 3 days offeeding was 10 g less than in K-depleted or KHCOarats. There was sufficient scatter in the data to makethis difference nonsignificant (P> 0.05, <0.1). Moststudies, however, show only small increases or de-creases in extracellular volume after chloride repletionor depletion (18), so the inability to obtain significantdata under the conditions of the present experiments isnot surprising. In addition, expansion of extracellularvolume in rats decreases renal bicarbonate reabsorptivecapacity (19). Thus, the increase in urinary pH duringmannitol infusion and the fall in serum bicarbonateafter NaCl mannitol administration is consistent withan increased circulating volume.

In addition to its effect on renal citrate metabolismvolume expansion increased ammonia excretion. Thelatter probably reflects increased renal ammonia pro-duction for two reasons. First, the increase was ac-companied by a simultaneous rise in urine pH in themannitol-infused rats. PNHI3 is in equilibrium betweenperitubular and luminal fluid (20), so the concentrationof total urinary ammonia (NH8 plus NH4+) variesdirectly with the pH gradient between peritubular andluminal fluid; the greater the gradient the higher theurinary ammonia excretion. As blood pH was constantthe gradient must have fallen. Less of the relativelynondiffusable NH4+ is then trapped in the tubular lu-men, and urinary ammonia excretion should fall if renalammonia production is constant (15, 21). Elevated uri-nary ammonia in these circumstances indicates in-creased renal ammonia production. This mechanismprobably explains the decreased ammonia excretionseen in the rats given potassium bicarbonate. A secondreason why increased production is probably responsiblefor the increased ammonia excretion is that renal bloodflow and glomerular filtration rate ordinarily rise aftervolume expansion (22). Endogenous creatinine clear-ance increased in the rats infused with mannitol so

renal blood flow probably also increased. As peritubularblood traps ammonia increased renal blood flow shouldincrease ammonia transport into renal venous bloodand decrease the amount of ammonia available for uri-nary excretion especially when the urine is only weaklyacid and urine flow rate is high. Increased renal bloodflow plus a rise in urinary pH indicate that elevatedurinary ammonia excretion after NaCl or mannitoladministration is due, therefore, to increased renalammonia production.

The volume expansion-induced alterations in renalcitrate and ammonia metabolism cannot, obviously, beexplained by changes in chloride concentration or bya nonspecific effect of mannitol. Three possibilities, not

mutually exclusive, may explain the results: the renalmetabolic changes could be due to the observed iso-hydric reduction in extracellular bicarbonate; theycould be secondary to a decrease in renal tubular cellpotassium concentration; or the changes could repre-sent a response to a reduction in renal tubular cell pH.Supporting the first possibility are the many studiesshowing that varying extracellular bicarbonate concen-tration at a constant extracellular pH alters intermedi-ary metabolism in a variety of tissues (23, 24). In-deed, Kamm, Fruisz, Goodman, and Cahill (25) haveshown that gluconeogenesis in rat renal cortical slicesvaries inversely with the bicarbonate concentration ofthe medium at a constant extracellular pH of 7.40.Since ammonia and glucose production by rat renalcortical slices obtained from metabolically acidotic ani-mals is greater than that seen in slices obtained fromalkalotic animals (26), it is possible the increased am-monia production in rats given NaCl or mannitol isdue to the decreased bicarbonate concentration. Citratemetabolism also appears to be bicarbonate dependent.Adler (27) has demonstrated that the citrate contentof rat diaphragm muscle is decreased in an iso-pHsystem when bicarbonate concentration is lowered.Furthermore, Simpson (4) has shown that under iso-hydric conditions in vitro citrate decarboxylation byrenal mitochondria proceeds more rapidly as bicar-bonate concentration is progressively lowered. Thisapparently is due to increased transport of citrate intothe mitochondria (28). Increased citrate decarboxyla-tion intramitochondrially would decrease renal citratecontent and increase reabsorption of citrate, the resultsobserved in the present study. Other work of Simpson(4), however, is not in accord with his observations inisolated mitochondria. Rabbit renal cortical slices incu-bated at a constant external pH of 7.39 and at bi-carbonate concentrations varying from 10 to 50 meq/liter showed identical citrate decarboxylation rates.These slices, however, were quite responsive to altera-tions in external pH as the citrate decarboxylation rate

varied inversely with the pH of the bathing medium.If the whole kidney responds like the slice then achange in bicarbonate concentration occurring iso-hydrically would not be expected to alter renal citratemetabolism. The exact role of the decreased bicarbonateconcentration, therefore, remains unclear.

A second possibility is that the altered renal metabo-lism is a consequence of altered intracellular potassiumconcentration. When normal animals are made alka-

lotic, urinary citrate excretion (29) and renal citratecontent (2) increase while renal ammonia productionand excretion decrease (30). In potassium deficiency,however, urinary citrate excretion falls (12), and uri-nary ammonia excretion rises (30) despite the presence

398 S. Adler, B. Zett, A. Anderson, and D. S. Fraley

of an extracellular alkalosis. This has been attributedto coexisting intracellular acidosis and extracellular al-kalosis in potassium depletion. Because the changes inpH are accompanied by a fall in tissue potassium con-centration, some investigators believe that the renalmetabolic alterations are due to decreased intracellularpotassium concentration rather than renal intracellularacidosis. In the present experiment, however, renal po-tassium content did not decrease further after sodiumchloride ingestion or mannitol infusion. In addition,Adler, Anderson, and Zett (5) have shown that thecitrate content of potassium-depleted rat diaphragmmuscle in vitro is a function of cell pH (calculatedfrom DMOdistribution) and not the tissue potassiumcontent. Thus, a fall in diaphragm muscle cell pH isaccompanied by a decrease in tissue citrate contentwithout any change in cellular potassium. It seems un-likely, therefore, that changes in intrarenal potassiumconcentration account for the renal metabolic altera-tions although changes in potassium concentrationwithin specific areas of the cell cannot be ruled out.

Finally, the alterations in renal citrate and am-monia metabolism may reflect reduced renal tubularcell pH. Acidosis apparently increases renal decarboxy-lation of exogenous citrate leading to a decrease inurinary citrate excretion and renal citrate content whilethe converse occurs in alkalosis (4). The observedchanges in renal citrate content and excretion thus arecompatible with renal cell acidosis. Indeed, Crawford,Milne, and Scribner (12), as well as other investigators(28), have postulated that renal citrate metabolism re-flects intracellular pH, using as supporting evidencefor this hypothesis the decreased citrate excretion andrenal cortical citrate content found in potassium de-ficiency alkalosis (12) and the known decrease inmuscle cell pH which occurs simultaneously (31, 32).In muscle there is direct evidence supporting the con-cept that citrate metabolism is a function of cell pHnot of extracellular acidity. Rats acutely administeredpotassium chloride intraperitoneally develop extracellu-lar metabolic acidosis, intracellular metabolic alkalosis,and elevated skeletal muscle citrate content (33). Ordi-narily, tissue citrate decreases in acidosis (2), so the

'change in citrate content presumably reflects the intra-cellular not the extracellular pH state. Also, as men-,tioned previously, Adler and coworkers (5) have beenable to relate citrate metabolism in diaphragm muscleto intracellular pH, not to cell potassium content orextracellular pH. Renal ammonia production is alsofelt to reflect intracellular rather than extracellular pHconditions. As is the case with renal citrate metabolismthis hypothesis is based on indirect evidence obtainedin abnormal metabolic states since renal cell pH hasonly been determined in an isolated renal tubular cell

preparation (34). Most investigators agree, neverthe-less, that both hydrogen ion secretion and renal am-monia production are regulated by intracellular pH.Both, for example, are increased in potassium de-ficiency despite a concomitant metabolic alkalosis (35).Also glutamine oxidation and presumably ammoniaproduction in renal cortical slices and mitochondria isdirectly related to changes in pH (36). Finally, Low-ance, Garfinkel, Mattern, and Schwartz (37) demon-strated that hypotonic volume expansion in dogs withchronic metabolic acidosis increases net acid excretionand restores plasma bicarbonate concentration to nor-mal. The increased net acid excretion probably wasdue in part to an increase in ammonia excretion. Al-though it is unclear how volume expansion might de-crease renal cell pH, the data are consistent with thehypothesis that volume expansion lowers renal tubularcell pH.

ACKNOWLEDGMENTSThe authors wish to thank Mrs. Elaine R. New for herassistance in manuscript preparation.

This work was supported in part by United States Pub-lic Health Service Grant HL 13810-04.

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