Kidney International, Vol. 57 (2000), pp. 891–897

Renal citrate metabolism and urinary citrate excretionin the infant rat

JOEL Z. MELNICK, PATRICIA A. PREISIG, ROBERT J. ALPERN, and MICHEL BAUM

Department of Pediatrics, Northwestern University Medical School, Chicago, Illinois, and Departments of Pediatrics andInternal Medicine, University of Texas Southwestern Medical School, Dallas, Texas, USA

Renal citrate metabolism and urinary citrate excretion in the reported that from infancy to adolescence, urinary citrateinfant rat. excretion decreases with a concomitant increase in uri-

Background. Although hypercalciuria has the same preva- nary Ca-oxalate saturation [5]. However, the mechanismlence in children as adults, children rarely develop renal stones.for this effect is unexplained.This may be explained by a greater urinary citrate excretion

Urinary citrate plays an important role in inhibitingin infants compared with adults. The present study examinesthe renal excretion of citrate and renal cortical citrate metabo- the formation of calcium-containing kidney stones bylism in infant and adult rats. chelating calcium, directly preventing the crystallization

Methods. Adult male and newly weaned infant rats were and precipitation of calcium [6] and interacting withacclimated to metabolic cages and fed synthetic diets. UrineTamm-Horsfall proteins to inhibit Ca-oxalate crystalliza-was collected after two days, and renal cortical citrate metabo-tion [7]. Low urinary citrate occurs in approximatelylism was assayed.

Results. Infant rats had a lower plasma [HCO32] and higher half of adult patients with renal stones [8]. Because the

plasma [K1] and had a fourfold higher urinary citrate:creatinine filtered load of citrate remains relatively constant, proxi-ratio and a twofold higher concentration of citrate in their

mal tubular citrate reabsorption regulates urinary citrateurine compared with adult rats. This higher urinary citrateexcretion [9]. Chronic regulation of proximal tubularexcretion was not due to a difference in renal proximal tubular

Na/citrate cotransporter activity, nor renal cortical citrate syn- citrate reabsorption has been shown to be associated withthase or ATP citrate lyase activities in infants as compared changes in the apical membrane Na/citrate cotransporterwith adults. However, infant rat kidneys had significantly lower (abstract; Aruga et al, J Am Soc Nephrol 8:2A, 1997)mitochondrial aconitase (m-aconitase) activity. Renal cortical

[10, 11], cytosolic ATP citrate lyase [12], and mitochon-citrate concentrations were comparable in infant and adult rats.drial aconitase (m-aconitase) [13].Manipulation of plasma [K1] to adult levels did not affect the

higher urinary citrate excretion in infant rats. The purpose of this study was to characterize the me-Conclusions. Urinary citrate excretion in infant rats is tabolism of citrate in the renal proximal tubule of infant

greater than in adults but does not parallel tissue [citrate].rats. The results demonstrate that infant rats excreteThus, this higher urinary citrate is likely due to maturationalurine with a higher total concentration of citrate and adifferences in the proximal tubule, other than Na/citrate co-

transport, that directly affect citrate transport. higher citrate:creatinine ratio than adult rats. This higherurinary citrate excretion is not a result of the higherplasma potassium seen in infant rats and is associated

The incidence of renal stones in children with hyper- with a decrease in renal m-aconitase activity.calciuria is approximately 5 to 15% [1, 2], and is muchlower than the 60 to 90% that is observed in adults with

METHODShypercalciuria [3]. This lower incidence of urolithiasis inExperimental designchildren with hypercalciuria may be due to higher uri-

nary concentrations of inhibitors that prevent stone for- Male Sprague-Dawley rats (Harlan, 180 to 200 g) weremation [4]. In support of this hypothesis, Hoppe et al used as adults. Infant Sprague-Dawley rats were received

as 18-day-old litters and were weaned two days later.Adult rats and 22-day-old rats were placed in metabolicKey words: urolithiasis, potassium, renal development, ATP citrate

lyase, aconitase, hypercalciuria cages for two days on ad libitum water and a syntheticdiet [(in g) 180 casein, 200 cornstarch, 500 sucrose, 35Received for publication August 7, 1998corn oil, 35 peanut oil, 10 CaHPO4, 6 MgSO4, 6 NaCl,and in revised form August 19, 1999

Accepted for publication October 11, 1999 8.3 K2HPO4, and 10 vitamin fortification mixture; ICNPharmaceutical, Cleveland, OH, USA]. During the third 2000 by the International Society of Nephrology

891

Melnick et al: Urinary citrate excretion in infant rats892

day, 24-hour urine collections were obtained in the pres- 7.4] extravesicular solutions [15]. We measured Na-dependent citrate uptake during the linear period overence of thymol [14], and the animals were sacrificed after

intraperitoneal anesthesia (100 mg/kg body wt of Inactin; 10 seconds using the Millipore filtration technique with0.65 mm pore size cellulose nitrate filters (DAWP 2500;Promonta, Germany). Blood was obtained first by aortic

puncture through an abdominal incision in adults and Millipore, Bedford, MA, USA) at room temperature.Enrichment of the vesicle preparation was determinedby decapitation in infants. Subsequently, kidneys were

removed through an abdominal incision, decapsulated using g-glutamyltransferase activity [15, 16]. Vesicle vol-umes were calculated from 14C-citrate uptake at twoand freeze clamped, or placed in ice-cold phosphate buf-

fered saline (PBS; pH 7.4). hours using the known citrate concentration in the extra-vesicular solution.For studies comparing infant rats receiving normal

and low-potassium diets, 22-day-old rats were allowedMitochondrial preparation and enzyme assaysto acclimate in metabolic cages for two days on a syn-

thetic diet that contained a similar amount of K1 [(in g) Mitochondria were harvested using differential cen-trifugation as described previously by our laboratory180 casein, 200 cornstarch, 500 sucrose, 35 corn oil, 35

peanut oil, 10 CaHPO4, 6 MgSO4, 6 NaCl, 6.8 Na2HPO4, [13]. One gram of renal cortical tissue was homogenizedin 10 mL ice-cold homogenization solution [300 mmol/L7.1 KCl, and 10 vitamin-fortification mixture]. Subse-

quently, rats were randomly divided either to continue sucrose, 5 mmol/L KH2PO4, 1 mmol/L EGTA, 0.1% bo-vine serum albumin, 10 mmol/L Na3citrate, 5 mmol/L 3-receiving the control potassium diet or to receive a low-

potassium diet wherein one half of the KCl was replaced [N-morpholino] propanesulfonic acid (MOPS), pH 7.4;50 mg/mL PMSF, and 4 mg/mL aprotinin] with fourwith NaCl. All animals were pair-fed and 24-hour urines

were collected during day 3; subsequently, the rats were strokes of a Potter-Elvehjem. Large pieces of cellulardebris were pelleted at 700 3 g for 10 minutes, and thesacrificed, and blood was obtained.

Blood gas determinations were performed using a remaining supernatant was centrifuged at 7500 3 g for10 minutes. The resultant supernatant was decanted andCMS System 1304 blood gas analyzer (Instrumentation

Laboratory, Lexington, MA, USA); electrolytes and cre- used for the cytosolic preparation to measure ATP ci-trate lyase activity, as described in the following section.atinine were measured on an Astra-7 electrolyte analyzer

(Beckman, Palo Alto, CA, USA). Citrate measurements The remaining mitochondrial pellet was resuspended toa protein concentration of approximately 10 mg/mL inwere performed using a citric acid enzymatic kit (Boeh-

ringer Mannheim, Indianapolis, IN, USA) on the Cobas 150 mmol/L KCl and 5 mmol/L MOPS, pH 7.4 withKOH, and frozen at 2708C.Fara II chemistry system (Roche Diagnostic Systems,

Somerville, NJ, USA). Urinary calcium was measured Mitochondrial suspensions were freeze thawed twiceat 2708C to disrupt the mitochondrial membranes [17].using the Beckman Synchron CX3 autoanalyzer.For the citrate synthase assay, 5 mL of the freeze-thawed

Measurement of renal cortical apical membrane mitochondrial suspension were added to 470 mL of 5Na/citrate cotransporter mmol/L Tris-HCl, pH 8.1, containing 0.17 mmol/L 5,59-

dithio-bis-(2-nitrobenzoic) acid and 0.40 mmol/L acetylThe renal cortex was dissected and minced on an icedPetri dish and was homogenized in 15 mL of buffer CoA. Twenty-five microliters of 10 mmol/L Na2oxaloace-

tate (in 1 mol/L Tris-HCl, pH 8.1) started the reaction.[300 mmol/L mannitol, 16 mmol/L HEPES/20 mmol/LTris, pH 7.4, 5 mmol/L ethyleneglycoltetraacetic acid The generation of CoA was determined spectrophoto-

metrically at 412 nm with an extinction coefficient of(EGTA), and 100 mg/mL phenylmethylsulfonyl fluoride(PMSF)] with a Polytron (Brinkman Industries, West- 13.6/mmol [17].

Mitochondrial aconitase activity was determined spec-bury, NY, USA). Brush border membrane vesicles wereisolated by Mg21 precipitation and differential centrifu- trophotometrically on freeze-thawed mitochondria by

measuring aconitate generation at 240 nm [18]. Fiftygation as previously described by our laboratory [12].Membranes were pelleted and resuspended to a protein microliters of the mitochondrial suspension were added

to 950 mL of 100 mmol/L Tris-HCl, pH 8.0, that containedconcentration of approximately 5 mg/mL in intravesicu-lar buffer that contained valinomycin (50 to 60 mg/mg 50 mmol of D,L Na3isocitrate. An extinction coefficient of

3.6/mmol was used to calculate the aconitate produced.protein).Na-dependent citrate transport was determined in

ATP citrate lyase assaybrush border membrane vesicles as previously describedusing the difference between 1,5-14C-citrate (New En- The supernatant obtained from differential centrifuga-

tion described in the preceding section was centrifugedgland Nuclear, Boston, MA, USA) uptake in Na-free[(in mmol/L) 100 cholineCl, 50 KCl, 0.1 K3citrate, 16 at 48,000 3 g 3 20 minutes at 48C. The resultant superna-

tant was then placed at 2208C overnight in the presenceHEPES/12 Tris, pH 7.4] and Na-containing [(in mmol/L)100 NaCl, 50 KCl, 0.1 K3citrate, 16 HEPES/12 Tris, pH of 6.5 mmol/L dithiothreitol to reduce the sulfhydryl

Melnick et al: Urinary citrate excretion in infant rats 893

Table 1. Plasma and urine values for adult and infant rats

Adults Infants

PlasmapH 7.4260.01 7.3960.01a

pCO2 mm Hg 40.061.3 39.561.6HCO3 mEq/L 26.260.5 23.760.9a

Na mEq/L 14261 14061K mEq/L 3.760.1 4.960.2a

Cl mEq/L 10761 10761Citrate mg/L 40.662.5 47.662.1

UrineCitrate mg/day 2.7660.47 1.6060.21a

Citrate mg/L 158624 318636a

Creatinine mg/day 11.1960.62 1.4560.08b

Citrate : creatinine 0.2560.05 1.1260.13b

%FE citrate 4.160.5 19.162.1b

Fig. 1. Sodium (Na)/citrate cotransporter activity was determined inN 5 7 for both groups.adult (h) and infant ( ) rats. Na-dependent (left bars) and Na-indepen-aP , 0.05

bP , 0.001 dent (right bars) 14C-citrate uptake was determined in renal corticalbrush border membrane vesicles (N 5 6).

groups of the enzyme [19]. ATP citrate lyase activityexcreted less total urinary citrate compared with adults.was measured using the hydroxamate assay as describedThis is not an unexpected observation because of thepreviously [12].lower glomerular filtration rate in infants. However, Ta-

Measurement of renal citrate ble 1 also shows that infant rats excreted urine witha higher citrate concentration. This would be of moreTissue citrate concentration was determined using thephysiologic significance because the concentration of in-freeze-clamp method [20]. In anesthetized adult rats, thehibiting and promoting compounds determines the satu-kidneys were exposed through an abdominal incision.ration characteristics of the urine. Furthermore, infantThe cortex of the kidney was pinched, in situ, and wasrats had a higher citrate:creatinine ratio and, notwith-immediately frozen using brass clamps cooled in liquidstanding the higher plasma citrate, a greater percentagenitrogen. Because of the size of the infant kidneys, thefractional excretion of citrate. The citrate:creatinine ratioentire organ was removed and frozen in the clamps.and the fractional excretion citrate correct for changesApproximately 100 mg of frozen tissue was placed in 2in the glomerular filtration rate and give a more accuratemL of 1 mol/L perchloric acid to inactivate tissue en-measure of tubular citrate reabsorption. Thus, these stud-zymes. After homogenization with a Polytron, the super-ies demonstrate that infant rats have higher urinary citratenatant was neutralized with NaOH to pH 7.0. The citrateexcretion and a lower tubular citrate reabsorption.concentration was determined as for urine samples. Lac-

tate concentration, as an index of tissue hypoxia, wasNa/citrate cotransporter activity in infants and adultsmeasured using a commercially available kit from Sigma

To determine if a difference in proximal tubular apical(St. Louis, MO, USA).membrane Na/citrate cotransporter activity accounted

Materials for the higher urinary citrate excretion in infants, wemeasured 14C-citrate uptake in brush border membraneSigma supplied all chemicals unless otherwise noted.vesicles. As can be seen in Figure 1, there was no differ-All protein concentrations were determined using theence in Na-dependent citrate uptake (Na/citrate cotrans-bicinchoninic acid assay method (Pierce, Rockford, IL,porter activity; in pmol citrate/mg protein/10 s) betweenUSA).adults (28.2 6 1.0) and infants (31.2 6 1.7). There was

Statistical analysis also no difference in brush border membrane vesicleenrichments (adults 11.5 6 2.1, infants 8.1 6 1.2) orData are expressed as mean 6 SE and are comparedvolumes (in mL/mg protein; adults 1.18 6 0.20, infantsusing the unpaired t-test.0.99 6 0.11).

RESULTS Citrate metabolism in infant and adult rat kidneysPlasma and urine composition Because renal citrate metabolism has been shown to

play an important role in regulating urinary citrate excre-Table 1 shows that infant rats had a significantly lowertion [12, 13], we examined whether a difference in renalplasma [HCO3

2], and pH, and a higher plasma [K1]compared with adults. Table 1 also shows that infant rats cortical enzyme activities was associated with the in-

Melnick et al: Urinary citrate excretion in infant rats894

Fig. 2. Citrate synthase activity in adult and infant rats. Citrate synthase Fig. 4. Mitochondrial-aconitase (m-aconitase) activity in adult and in-activity was determined spectrophotometrically as the appearance of fant rats. m-Aconitase activity was determined spectrophotometricallyCoA from acetyl CoA and oxaloacetate in mitochondrial suspensions as aconitate generation from isocitrate in mitochondrial suspensions(N 5 6). (N 5 6). *P , 0.05.

Fig. 5. Renal cortical citrate concentration in adult and infant rats.Fig. 3. Adenosine 59-triphosphate (ATP) citrate lyase activity in adultKidneys were freeze clamped in vivo, and pulverized under liquid N2.and infant rats. ATP citrate lyase activity was measured spectrophoto-Citrate levels were measured spectrophotometrically (N 5 9).metrically as the generation of acetylhydroxamate (N 5 6). *P , 0.05.

creased urinary citrate excretion in infants. As shown in cortical mitochondria citrate metabolism. We have shownpreviously that regulation of m-aconitase partially medi-Figure 2, there was no difference in renal cortical citrate

synthase activity (in nmol citrate/mg protein/minute; ates acidosis-induced hypocitraturia and alkalosis-inducedhypercitraturia [13]. As shown in Figure 4, infant rat kid-adults 332.8 6 28.8, infants 301.0 6 15.0).

Adenosine 59-triphosphate (ATP) citrate lyase activity neys had a significantly lower renal cortical m-aconitaseactivity compared with adults (in nmol aconitate/mg mitoserves an important role in the hypocitraturia of meta-

bolic acidosis [12]. This cytosolic enzyme cleaves citrate protein/min; 163.7 6 12.6, 51.5 6 4.7, P , 0.001). Thislower renal m-aconitase activity in infants may be re-to oxaloacetate and acetyl CoA and is prevalent in kid-

ney tissue [21]. We used the hydroxamate assay because sponsible for hypercitraturia in these animals.the kidney contains high native NADH dehydrogenase

Renal citrate concentration in infant and adult ratsactivity that would interfere with the malate dehydroge-nase-coupled assay [12]. Figure 3 shows that infant rat As a result of the difference in renal citrate metabo-

lism, in animals with hypocitraturia or hypercitraturia,kidneys had a significantly higher ATP citrate lyase activ-ity as compared with adult kidneys (in nmol acetylhy- there is a decrease or increase, respectively, in renal citrate

concentration [22]. We reasoned that infant rats woulddroxamate/mg mito protein/30 min; 41.2 6 9.0 vs. 81.6 69.6, P , 0.05). have a higher renal citrate compared with adults. How-

ever, Figure 5 shows that there is no difference in renalBecause an increase in ATP citrate lyase activity wouldnot be expected to increase urinary citrate excretion, we citrate concentration (in mmol/g kidney wet weight;

adults 0.54 6 0.04 vs. infants 0.58 6 0.06). This findingmeasured m-aconitase activity, a regulated step in renal

Melnick et al: Urinary citrate excretion in infant rats 895

Table 2. Plasma and urine values for control and infant rats fed a they also showed progressively higher urinary calciumlow-K diet

oxalate saturation that peaks in prepubescence [5].Control Low-K The proximal tubule serves as the principal segment

Plasma for renal tubular citrate reabsorption and provides thepH 7.3560.02 7.3560.02 major site for the regulation of renal citrate absorptionpCO2 mm Hg 44.462.6 45.561.7

and excretion [9]. The first step in proximal tubular ci-HCO3 mEq/L 24.260.9 25.361.1Na mEq/L 13761 14061a trate absorption is citrate transport across the apicalK mEq/L 4.660.2 3.960.1b membrane on the Na/citrate cotransporter. Chronic met-Cl mEq/L 10761 10761

abolic acidosis causes hypocitraturia and an adaptiveUrineVolume mL/day 4.560.3 8.960.7b increase in Na/citrate cotransporter activity [11], protein,Citrate mg/day 1.4060.14 3.0060.82 and mRNA abundance (abstract; ibid). In our study,Creatinine mg/day 0.9460.04 1.1160.18

Na/citrate cotransporter activity in infant rats was noCitrate:creatinine 1.5060.14 2.5860.56Calcium mg/day 0.7560.13 0.1860.09b different than in adults. A similar observation occurs

associated with the increased urinary citrate excretionN 5 6 for both groups.aP , 0.05 of chronic alkali feeding [11].bP , 0.01

Although proximal tubule Na/citrate cotransporter ac-tivity increases in chronic metabolic acidosis, the tissuecitrate concentration decreases [22]. This would suggestthat citrate metabolism plays an important role in thewas not a result of a difference in specimen handlingregulation of urinary citrate excretion. Cytosolic citratebecause renal lactate concentrations were similar (inmetabolism through the enzyme ATP citrate lyase playsmmol/g kidney wet weight; adults 0.71 6 0.05 vs. infantsan important role in decreasing urinary citrate excretion0.62 6 0.09).in metabolic acidosis and hypokalemia [12]. However,

Urinary citrate excretion in infant rats on a low- in this study, infant rats have higher renal cortical ATPpotassium diet citrate lyase activity when compared with adults, a change

in the wrong direction to explain the hypercitraturia.Because hypokalemia can cause hypocitraturia, weBecause rat kidneys continue to grow and to developreasoned that the higher plasma potassium in infant ratsfrom birth to four weeks of age [23], the higher activitymight cause the higher urinary citrate excretion. To re-of ATP citrate lyase may be explained by the fact thatduce the plasma [K1], we lowered the potassium contentsynthesis of lipids for growing tissue requires this enzymeof the diet. Between the time of weaning and completion[24]. Additionally, to supply the high energy demandedof the study, animals that received the low-potassiumby proximal tubular transport processes, infant rat kid-diet gained 17 6 3 g, which was not different from controlney transitions from anaerobic glycolysis to fatty acidanimals that gained 15 6 2 g, or from animals that re-oxidation during the first postnatal month [25, 26]. Be-mained on the commercial diet, which were not placedcause ATP citrate lyase is a crucial step in the formationin metabolic cages and gained 16 6 2 g. As can beof fatty acids, its activity may also reflect these changesseen in Table 2, infant rats fed the low-K diet had ain energy substrate demand.significantly lower plasma [K1], which was not different

The mitochondrial tricarboxylic acid cycle serves as anfrom the adults, and had a higher plasma [HCO32]. Addi-

important pathway for renal citrate metabolism. Chronictionally, decreasing plasma [K1] was associated with ametabolic acidosis and chronic alkali feeding regulatehigher urine output and total urinary citrate excretionthe activity and protein abundance of m-aconitase, thewithout affecting urinary citrate concentration. Further-first step for mitochondrial citrate metabolism [13]. Inmore, the low-K infant rats had a higher urinary citrate:addition, the prostate synthesizes and secretes citrate, ancreatinine ratio, but was not statistically significant. How-effect mediated by the inhibition of m-aconitase [27]. Weever, these rats had a significant decrease in both urinaryshow here that similar to observations in chronic alkalicalcium excretion and urinary calcium:creatinine ratiofeeding and prostate, infant rat kidneys excrete more ci-compared with controls. Thus, the higher urinary citratetrate and have a lower m-aconitase activity than adults.excretion in infant rats, compared with adult rats, was

There was no difference in renal cortical citrate con-not due to hyperkalemia.centration between infant and adult rats. A decrease incitrate catabolism, as seen in animals with metabolic

DISCUSSION alkalosis, results in an increase in renal citrate concentra-This study demonstrates that infant rats have a signifi- tion [22]. This may decrease the driving force for citrate

cantly higher urinary citrate:creatinine ratio, as well as transport across the apical membrane and result in hyper-urinary citrate concentration, compared with adult rats. citraturia. However, proximal tubule citrate uptake de-

pends on a sodium gradient as well, and may be affectedHoppe et al found similar results in children in whom

Melnick et al: Urinary citrate excretion in infant rats896

by other factors such as basolateral Na1,K1-ATPase ac- REFERENCEStivity. Although we demonstrate that the Na/citrate co- 1. Bennett AH, Colodny AH: Urinary tract calculi in children.transport activity was not different in infant and adult J Urol 109:318–320, 1973

2. Stapleton FB, Southwest Pediatric Nephrology Study Group:brush border membrane vesicles, the Na1,K1-ATPaseIdiopathic hypercalciuria: Association with isolated hematuria andis lower in infant kidneys [28, 29]. Thus, the increase in risk for urolithiasis in children. Kidney Int 37:807–811, 1990

urinary citrate excretion in infant rats likely reflects a 3. Coe FL: Treated and untreated recurrent calcium nephrolithiasisin patients with idiopathic hypercalciuria, hyperuricosuria, or norelative immaturity of proximal tubular transport possi-metabolic disorder. Arch Intern Med 87:404–410, 1977bly through a change in the sodium gradient [30]. 4. Elliot JS: Calcium oxalate urinary calculi: Clinical and chemical

The lower plasma [HCO32] observed in infant rats is aspects. Medicine 62:36–43, 1983

5. Hoppe B, Jahnen A, Bach D, Hesse A: Urinary calcium oxalatesimilarly observed in children [31] and would be expectedsaturation in healthy infants and children. J Urol 158:557–559, 1997to result in lower urinary citrate excretion [12]. However, 6. Pak CY: Citrate and renal calculi. Miner Electrolyte Metab 13:257–

human infants, compared with adolescents, excrete twice 266, 19877. Hess B, Zipperle L, Jaeger P: Citrate and calcium effects onas much urinary citrate when corrected for weight [5].

Tamm-Horsfall glycoprotein as a modifier of calcium oxalate crys-Similarly, in our study, despite the lower plasma tal aggregation. Am J Physiol 265:F784–F791, 1994[HCO3

2], infant rats excrete fourfold more citrate when 8. Nicar MJ, Skurla C, Sakhaee K, Pak CY: Low urinary citrateexcretion in nephrolithiasis. Urology 21:8–14, 1983corrected for creatinine in their urine compared with

9. Hamm LL: Renal handling of citrate. Kidney Int 38:728–735, 1990adult rats. However, this study examined only a single 10. Brennan TS, Hering-Smith KS, Hamm LL: Effect of pH on citratetime point in renal development. The mechanism for the reabsorption in the proximal convoluted tubule. Am J Physiol

255:F301–F306, 1988increased urinary citrate in humans may differ from that11. Jenkins AD, Dousa TP, Smith LH: Transport of citrate acrosspresented in infant rats. renal brush border membrane: Effects of dietary acid and alkali

Because hypokalemia decreases urinary citrate excre- loading. Am J Physiol 249:F590–F595, 198512. Melnick JZ, Srere PA, Elshourbagy NA, Moe OW, Preisigtion [15], the relative hyperkalemia in infants may in-

PA, Alpern RJ: Adenosine triphosphate citrate lyase mediatescrease urinary citrate excretion even in the presencehypocitraturia in rats. J Clin Invest 98:2381–2387, 1996

of low plasma [HCO3]. However, decreasing infant rat 13. Melnick JZ, Preisig PA, Moe OW, Srere PA, Alpern RJ: Renalcortical mitochondrial aconitase is regulated in hypo- and hyperci-plasma [K1] to that seen in adults resulted in a slighttraturia. Kidney Int 54:160–165, 1998increase in plasma [HCO3

2] and a further increase in14. Nicar MJ, Hsu MC, Johnson T, Pak CY: The preservation of

urinary citrate:creatinine. Thus, the higher urinary ci- urine samples for determination of renal stone risk factors. ClinChem 18:382–384, 1987trate excretion in infant rats does not result from their

15. Levi M, McDonald LA, Preisig PA, Alpern RJ: Chronic K deple-higher plasma [K1]. The infant rats fed a low-K diet alsotion stimulates rat renal brush-border membrane Na-citrate co-

had a lower rate of urinary calcium excretion than the transporter. Am J Physiol 261:F767–F773, 1991rats on the control diet. This is in contrast to results in 16. Szasz G: A kinetic photometric method for serum gamma-

glutamyl transpeptidase. Clin Chem 15:124–136, 1996adult humans [32]. The cause for the lower rate of cal-17. Robinson JB Jr, Brent LG, Sumegi B, Srere PA: An enzymaticcium excretion in rats fed the low-K diet is unclear. approach to the study of the Krebs tricarboxylic acid cycle, in

In summary, this study has shown that infant rats, Mitochondria: A Practical Approach, edited by Darley-UsmarVM, Rickwood D, Wilson MT, Washington D.C., IRL Press,despite mild metabolic acidosis, excrete more citrate in1987, p 153their urine than adult rats. Furthermore, we have shown 18. Plank DW, Howard JB: Identification of the reactive sulfhydryl

that this difference in urinary citrate excretion is associ- and sequences of cysteinyl-tryptic peptides from beef heart aconi-tase. J Biol Chem 263:8184–8189, 1988ated with lower renal cortical m-aconitase activity but

19. Cottam GL, Srere PA: The sulfhydryl groups of citrate cleavagenot renal cortical citrate concentration. Our results sug- enzyme. Arch Biochem Biophys 130:304–311, 1969gest that there is a maturational difference in renal citrate 20. Wollenberger A, Ristau O, Schoffa G: Eine einfache technik

der extrem schnellen abkuhlung groberer gewebestucke. Pflugersmetabolism and transport in infants that results in aArch 270:399–412, 1960higher urinary citrate excretion, which likely accounts

21. Srere PA: The citrate cleavage enzyme. J Biol Chem 234:2544–for the paucity of urolithiasis in the pediatric population. 2547, 1959

22. Simpson DP: Citrate excretion: A window on renal metabolism.Am J Physiol 244:F223–F234, 1983ACKNOWLEDGMENTS

23. Arataki M: On the postnatal growth of the kidney with specialreference to the number and size of the glomeruli (albino rat).These studies were supported by National Institutes of Health grantsAm J Anat 36:399–436, 1926R01-DK41612 (M.B.) and P01-DK20543 (R.J.A.). J.Z.M. was sup-

24. Abraham S, Madvig P: Enzyme activities during development ofported by a National Institutes of Health institutional training grantsome organs of the rat. J Nutr 110:100–104, 1980T32-DK07659 and a grant from the Northwestern University. The

25. Djouadi F, Bastin J, Kelly DP, Merlet-Benichou C: Transcrip-authors gratefully acknowledge the advice and assistance from Drs.tional regulation by glucocorticoids of mitochondrial oxidative en-Paul Srere and Moshe Levi, and the technical help from Ms. Paulettezyme genes in the developing rat kidney. Biochem J 315:555–562,Padalino and Ms. Virginia Ho.1996

26. Bastin J, Delaval E, Freund N, Razanoelina M, Djouadi F,Reprint requests to Dr. Joel Z. Melnick, Northwestern UniversityBismuth J, Geloso JP: Effects of birth on energy metabolism inMedical School, Children’s Memorial Hospital, 2300 Children’s Plaza,the rat kidney. Biochem J 252:337–341, 1988Box #37, Chicago, Illinois 60614, USA.

E-mail: j-melnick@nwu.edu 27. Costello LC, Franklin RB: Concepts of citrate production and

Melnick et al: Urinary citrate excretion in infant rats 897

secretion by prostate 1: Metabolic relationships. Prostate 18:25–46, 30. Baum M, Quigley R: Ontogeny of proximal tubule acidification.Kidney Int 48:1697–1704, 19951991

28. Aperia A, Larsson L: Induced development of proximal tubular 31. Edelmann CM Jr, Soriano JR, Boichis H, Gruskin AB, AcostaMI: Renal bicarbonate reabsorption and hydrogen ion excretionNaKATPase, basolateral cell membranes and fluid reabsorption.

Acta Physiol Scand 121:133–141, 1984 in normal infants. J Clin Invest 46:1309–1317, 196732. Lemann J Jr, Pleuss JA, Gray RW, Hoffman RG: Potassium29. Schwartz GJ, Evan AP: Development of solute transport in rabbit

proximal tubule. III. Na-K-ATPase activity. Am J Physiol 246: administration increases and potassium deprivation reduces uri-nary calcium excretion in healthy adults. Kidney Int 39:973–983, 1991F845–F852, 1984