Decreased calcium and magnesium urinary excretion during prostaglandin synthesis inhibition in the rat

PROSTAGLANDINS DECREASEDCALcIuMAND~~IuMuRINARYM~IONDuRING PROSTAGLANDIN SYNTHESISINHIBITIONIN THE RAT. G. Friedlanderand C. Amiel Departmentof Physiology,FacultgXavier Bichat,Universit6Paris 7, and INSERMU.251,F-75018,Paris,France. ABSTRACT The effect of endogenousrensl prostaglandinson calcium and magnesium reabsorptionwes investigated.Renal tubular handling of calcium and magnesium wss studied by clearance methods in anesthetizedSprsgue-Dawleyand Brattlebororats, either intact or thyroparathyroidectomized (ATPTX),before and during prostaglandin synthesis inhibitionby meclofensmate,indomethacin,or piroxicam infusion.These three inhibitorshad similar effects on calcium and magnesium excretion : A significant decrease in absolute and fractional excretions of both cations wes observed in intact Sprague-Dawleyrats, and in ATPIX rats of both strains,but not in intact Brattlebororats. These results suggesten inhibitoryeffect of prosteglendinson vssopressin-,glucsgon-,but not PTH-mediated calciumand magnesiumreabsorption. This effectis likelyto occur in the thick ascendinglimb of Henle, which is both a target site for these polypeptidichormones,and a segmentwhere the bulk of calcium and magnesiumis reabsorbed. INTRODUCTION Prostsglandins(PGs)have been shown to inhibitthe vesopressin (AVP)-mediated water reabsorption(l-3), like1 througha decreasein AVP-sensitiveadenylatecyclsse stimulation94, 5). Recently, PGF2 has been demonstratedto inhibitAK&induced CAMP accumulationboth in the thick ascendinglimb (TAL) and the corticalcollectingtubule of the rat (6). Moreover PGE2 was demonstratedto inhibitnot only NaCl transportin mouse the AVP but also the ucagon (GLU)-mediated medullaryTAL (MALI (7.? On the other hand, a decreaseof calciumurinary excretionwas observedafter inhibitionof PG synthesisby indomethacinin rat, in monkey and in hypercelciuric patients (8). This effectwas reversed, in monkey,by intra-arterial FGE2 infusion(8). Finally, AVP, GLU, as well as parathyroidhormone (FTH) and calcitonin(Cl'),were shown to increasecalcium (Ca) and magnesium (Mg) tubular reabsorption (g-12), likely through stimulation of adenylatecyclsse (13, 14). The present study was thereforeundertakento further evaluate, in the rat, the effect of PG inhibitionon the renal transportof Ca and Mg in the presenceor absenceof the polypeptidichormoneswhich can be involved in an interactionwith prostaglandins.The data indicatethat the inhibitionof PG productionresultsin an increase in Ca and Mg reabsorption.These results suggest that the effect of JANUARY 1985VOL. 29 NO. 1

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FGs is likely to be exerted in the MAL, through an interaction polypeptidic hormones.

with

METHODS Preparation of the animals : Fifteen msle SpragueDawley (SD) rats (R. Janvier, Le Genest, France) and fourteen male homozygous Brattleboro (DI) rats (bred in the Departement de Biologie, C.E.N. Saclsy, France, by J.M. Juvsnon), of 196 f 3 g (SIN) aversge weight were used. Commercial chow of constant composition (mmoles per : Na : 130, K : 177 Ca : 230, Mg : 64, P : 297 ; vit.Dg : 4030 ; proteins : 200 & ; carbohydrates : 540 g/kg) was made available to all rats until 13-I 5 hours prior to the experiment. Free access to desionized water wss allowed. Anesthesia was achieved by the intraperitoneal administration of sodiuW+e l-5(I-methylpropyl)-2-thiobarbiturate (Inactine R), 8 mg x 100 g 4hsb.w. The animsls were placed on a heated table end the rectal temperature was maintained between 37 and 38’C. The arterial blood pressure wss monitored and blood samples were obtained through a catheter inserted into the right commoncarotid artery. A tracheotomy was performed. Both ureters were catheterized. Infusions were performed ss follows : for SD rats, isotonic saline 25 W--ib$$~ g b-w., via a tail vein, and Ringer’s bicarbonate (Na : : 115, HC03 : 30, K : 5 mF&iter) 25 pl/min/loO b.ti. via an &tern& jugular vein ; for DI rats, NaCl 75 mMand 1$2 diluted Ringer’s bicarbonate were infused via a tail vein end an external jugular vein respectively, at a total rate of 100 @/min/loO g b-w. In ATPTX rats, the parathyroids and the thyroid were removed surgically. The efficiency of thyroparathyroidectoq wss verified before any drug infusion by the disappearance of urinary phosphate (concentration lower t 0.025 mM). A priming dose of tritiated inulin (methoxy inulin- !?n H, New England Nuclear, Boston, MA, USA) was given via the tail vein, imediately followed by the sustaining dose. Experimental protocol

:

The experiments begsn after a 45 min equilibration period. Eight 30 min periods of urine collection were achieved : two control periods followed by six periods during continuous infusion Of meclof enamate, indomethacin or piroxicsm. At mid point of each period, a blood sample was obtained (Fig. 1). Meclofenemate (gift from Dr. Deluna, Substancia, Courbevoie, France) was infused in Ringer’s bicarbonate ss a loading dose Of 4 m$kg b.w. at the rate of 0.03 mg/min, then maintained as a sustaining infusion at 4 m&g per hour. Four experimental groups were studied. Group I : 4 intact SD rats ; Group II : 6 ATP’IXSD rats ; Group III : 7 intact DI rats ; Group IV : 7 ATITXDI rats.

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UJ

2,3 C

4

5

7

6

6

E

Pig. 1. Time-related graphshowingurine (U) collection periodsused for celculation. C : control; E : experimental. Arrowsdenoteplasma samples. Indomethacin (Signa, MO, USA)was administered to two addition&t ATPTXSD rats.Indomethacin was preparedby dissolving 50 mg in 0.5 ml of 0.5 M Na2 CD3, adding2 ml of H$ followed by 2.0 ml of 0.05M HCl and an appropriate amountof Ringer'sbicarbonate to yield a finalconcentration of 2.7 mg/ml.A loadinginfusionof 5 mg/kg at the rate of 0.047mg/minwas followedby a sustaining infusionof 6 and meclofenemate were m&g per hour.These dosesof indomethacin selectedbecause Higashihara et al. (13) have shown that urinary excretion of ICE2and FGP2awas suppressed by approximately 95 $ with eitherdrug, end becausethey inducedonly minor or no changesin glomerular filtration rate (15, 16). Piroxicam,a structurally differentinhibitorof PC synthesis for (17),was testedin a group of 3 ATITX SD rats. Preparation administration and doseswerethe sameas for meclofenamate. Analytical procedures : Bloodsampleswere collected on ried lithiumheparinate. Urine sampleswere weighted.l?oressayof %I inulinradioactivity, urine was appropriately diluted in water. Plasma end urine were then processed in an identical wey : a 20 ul samplewas addedto 20 ul of trichloracetic acid 10 $ w/v.Ten microliters of the supernatant were mixed with 5 ml of scintillation mixture (Aquesol,New England Nuclear,Boston,MA, USA). Countingswere performedin a liquid scintil.lator (Intertechnique, SL 4.000, Plaisir,Prance).Plasmaend urineconcentrations were determined as follows: phosphate according to Chen et al. (18),Na by flameemissionphotometry(Netheler end Hinz,G&H, Hamburg,Germany), Ca and Mg by flameatomicabsorption photometry(PyeUnicemSP 9 200, Cambridge, UK). Frectionel urinary excretions of calciumand magnesium were calculated assumingin all experimentsen ultrefilterability ratio of 0.705 and 0.807 for calciumand megnesium respectively, as determined in previousstudies (10). The right kidney was the experimentalone. Results were expressed as mean f SEM.l?orstatistical evaluation, in each rat the mean value of controlperiods@I end U2) ~&8 comparedto the mean JANUARY 1985VOL. 29 NO. 1

125

PROSTAGLANDINS value of experimentalperiods U5 to U8. Statisticalanalysis was performedby the paired and non-paired*tests.

Meclofenamateinfusioninduced a significantdecrease in urine flow rate in intact and ATFTX SD rats (GroupsI end II> and in ATFTX DI rats (GroupIV), whereas the decreasedid not reach significance in intact DI rats (Group III) (Table 1). The glomerularfiltration rate, however,was not affectedin either group, nor was the sodium fractionalexcretion,which did not vary significantlyduring the experiments. Table 1. Values for urinaryflow rate (V), glomerularfiltrationrate (Gm), sodium fractional excretion ( a), and Ca end Mg plasma concentrations during controlperiods and meclofenemateinfusion @Ia. Group I (n = 4)

Group II (n = 6)

C 7.7@3-78 lS.W.04 (@/m&kidney) M 2.39~kO.44~ 9.4w.30b

Group IV (n = 7)

58.5W.30

57.1W.86

49.7of8.20 39.m4.46b

1.19N.11

(ml/mt$idney) i 1 :f?‘g:%

Group III (n = 7)

1.05~.06 1 .W.43

0.88fo.03 O.SEN.05

0.93io.cB 0.89N.03

(37

C 0.6W.06 M 0.5oa0.03

PCs (ti)

C 2.31ti.23 M 2.22ti.25

1.43ti.OFe

2.24ti.09 1.9290.04f 2.2!3%.13 1.7W.C6bf

%(

C 0.61W.02 M 0.5B0.03

0.46a.02e 0.42a.02e

0.62SI.01 0.63+Q.O1

FE%

>

1.66ti.33 1.81iO.0@

0 KktO.21 0.77M.26 1.51ti.42 O.&&O.19

0.yio.01f 0.49a02f

a Values are expressedes mean f SEM. Statisticalanalysiswas performedas describedin methods. b,c Significant different from control (C) (p ( 0.05 and 0.01 respectively '3. d,e Significantlydifferentfrom the homologousvalue in Group I (~(0.05 and 0.01 respectively). f Significantlydifferentfrom the homologousvalue in Group III (p
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PROSTAGLANDINS

umoles/minvs 1.49 f 0.11 pmoles/min,p < 0.01). A slight but not significantdecrease was also observed in Group IV (1.31 f 0.12 umoles/minvs 1.03 f 0.06 pmoles min, NS, for the control and the meclofenamateperiod respectively I . Mg plasma concentrationand Mg filteredload were stablethroughoutthe experimentin each group. The effect of meclofenamateinfusionon Ca and Mg excretionsis summarizedin Table 2. Table 2. Effect of meclofenamate(M) on Ca end Mg absolute (U.V>and fractional(FE) excretionsa. Group II (n = 6)

Group III (n = 7)

Group IV (n = 7)

.

(nmolLJ$&

;

;:E:l;b

C 0.43ti.16 M o. l+o.o3b

FY (k a . (nmoli!$n)

27.6k9.00 80.1*16.10 18.1W.63 16.W7.45c

3.63*1.27

l.W.74

5.96*1.01

1.w0.57b

1.27ti.42

1.71iO.73b

C 162.1*35. 112.1%.57d 3zl 36.7i6.88d M 14.6Q.77 C 23.EW5.99

?Yg (

51.U17.18 15.8t7.57b

24.3W.93

77.1UO.96 75.3k7.52 56.6B.66 24.&7.67c 17.9Ok2.04 20.3Ok2.17 7.62k2.31c

M 3.19=W.48b 10.62%'.01d 13.10U.99

a Values are expresseda3 mean f SEPI.Statisticalanalysiswas performedas describedin methods. b,c,d Significantlydifferentfrom control (C) (p( 0.05, 0.01 and 0.001 respectively). Meclofenamsteinduced a decrease in absolute and fractional excretionsof Ca and Mg in intact and ATFTX SD rats. In intact DI rats (Group III), meclofenamateinduced only a slight and nonsignificantmodificationin Ca and Mg excretion.In contrast, a dramaticdecreasein Ca and Mg absoluteand fractionalexcretionswas observedin ATFTX DI rats (GroupIV) sfter meclofenamateinfusion. The effect of meclofenamateon Ca and Mg excretionin ATFTX SD rats was compared to that of two other structurallydifferent cyclooxygeneseinhibitors : Indomethacin end piroxicem. Indeed carboxylic acids (i.e. meclofenamate and indomethacin),unlike piroxicam,were previouslyreported to alter intracellularcelcium movements, and might therefore enhance the action of polypeptidic hormonesby a calcium-related mechanism,not necessarilyrelated to inhibitionof FG synthesis (19, 20). As shown in Fig. 2 ,piroxicam induced a significantdecrease of Ca end Mg fractionalexcretion, which was similar to that observed with meclofenamate and indomethacin.

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Ca

Mg

*

* O[

5a

*

I

P

Fi : Effect of meclofenamate(M) indomethacin(I) and piroxicam (PT'oiCa'endMg fractionalexcretion) (W) in ATETX SD-rats. Asterisk: Significantly differentfrom controlperiod (C). DISCUSSION The present study indicates that infusion of meclofenamate, indomethecinand piroxicam markedly reduced Ca and Mg urinary excretionsin two strainsof rats. The fact that three structur differentinhibitorsof renal prosteglentiin synthesis (15, 21, 22 had similar effects on Ca and Mg excretionssuggests a specific, prostaglandin-dependent effect, end supports a modulatingrole for endogenousprostsglandins in Ca and Mg tubularhandling. Since inhibitionof FG synthesiswas reportedto modify sodium trsnsportin the nephron,a possiblelink between calciumand sodium reabsorptionshouldbe examined.This phenomenoncould take place in the proximaltubule and/or in the loop of Henle. Althoughunlikely (15, 231, an enhanced sodium end calcium proximal reabsorption, consequenceof hemodynamicalterations,could not be ruled out. Such a phenomenon, however, would not account for the decreased Mg excretion,since the proximaltransportof this ion is quantitatively negligibleunder most circumstances(24). In the TAL, the question whether Ca and Mg reabsorptionis linked to a primaryactive sodium transportis still a matter of debate (25, 26). Anyhow, in our study the decreaseof Ca end Mg excretionwas not paralleledby a decreese of sodiumfractionalexcretion. Experimental groups were designed so that the interaction betweenPCs and polypeptidichormoneson Ca end Mg excretioncould be

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examined. This interaction is likelyto takeplacemainlyin the TAL, whichis both a targetsitefor FTH,GLU, CT and AVP (13, 141, and a segmentwhere the bulk of Mg end a substantial part of Ca are reabsorbed (24,27) underthe influence of theseShormones (9-12).The effectof meclofenamate in intactSD rats (GroupI> is therefore consistent with sn enhancedactionof one or severalamongthe four polypeptidic hormonesfollowinginhibitionof renal prostaglandin synthesis. That a decreasedCa end Mg excretionis also observed after meclofenamate infusionin ATFTX SD rats (GroupII) suggests that PGs couldinteractwith AVP and/orGLU ratherthan with FTH. Furthermore, since the effect of meclofenamate still persistsin ATFTX DI rats (GroupIV), this interaction is likelyto involve glucagon. In ATITX rats of both strains,the decreaseof Ca absolute excretioncould be partlyaccountedfor by a decreaseof calcium filteredload throughoutthe experimental period, related to a progressivedecrease of Ca plasma concentration which is a characteristic featureof acutethyroparathyroidectomy (28).However, the simultaneous decreaseof Ca fractionalexcretionsuggestsan enhancedtubularreabsorptive capaci afterPG inhibition. In intact DI rats (GroupIII B , the absenceof significant decreaseof Ca and Mg excretioncould be the consequence of two factors : first, a low basal reductionof renal prostaglendins relatedto the lackof AVP (29-32 P and thusa lessstrikingeffectof FG inhibition; second,a bluntedeffectof FGxinhibition by FTH : Indeed,FTH wes shownto stimulate adenylate cyclasein the cortical portionof TAG (13) only, whereasthe effectof FGs seems to be predominant in the medullsryportion (6). Thus,'in DI rats, the effectof FG inhibition couldhavebeen unmaskedonly in the absence of FTH (GroupIV). Anothereffectof FG inhibition by meclofensmate observedin groupsI, II end IV wes a decreaseof urinaryflow ratewhichcould have, per se, influenced the excretionof Ca, but not of Mg (9). Indeed, Ca, but not Mg excretion,was showed to be strongly correlated to urinaryoutpoutin Brattleboro DI rats treatedor not withAVP (9). In conclusion, the presentfindingsindicate thatmeclofenamate, indomethacin, 8.napiroxicam,most likely through prostaglendin inhibition, induce an increase in calcium and magnesium reabsorption. This couldbe accounted for by an inhibitory effectof prostsglsndins on the actionof polypeptidic hormoneson the thick ascendinglimb.The role of prostsglandins in calciumand magnesium homeostesis, however,remainsto be determined. AKN-EMENTS The authors eknowledgethe skilledtechnicalassistanceof Sylviene Couette and ChristianeCoureeu, and the secretarial assistance of Frencoise Carlier.

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PROSTAGLANDINS REFERENCE3

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mermnelien kidney.Kidney Int. -10:46-63.1976. 5. Stokes,J.B. Integratedactionsof renal medullaryprostaglsndins the control of water excretion. Am. J. Physiol. ;:O:F471-F@. 1981. 6. Torikai, S. end K. Kurokawa. Effect of PGE2 on vasopressindependentcell CAMP in isolatedsingle nephron segments.Am. J. Physiol.245:F58-F66.1983. 7. Npepper, R.M. end T.E. Andreoli.Site of PGE2 inhibitionof ADH-mediatedNaCl transportin mouse medullary thick ascending limb (mTAL).Kidney Int. 3:253(Abst). 1983. 8. Buck, A.C., C.J. I&e and W.F. Sampson.The influenceof renal prostsglandins on urinary calcium excretion in idiopathic urolithiasis. J. Ural. 129:421-426. 1983. 9. Rouffignac,C. de, B. Corman and N. Roinel. Stimulationby anti diuretic hormone of electrolyte tubular reabsorptionin rat kidney.Am. J. Physiol.a:Fl56-F164. 1983. 10. Bailly,C., N. Roinel and C. Amiel. m-like glucegonstimulation of Ca and Mg reabsorptionin Henle's loop of the rat. Am. J. Physiol.246:F205-F212. 1984. Il. Rouffignac,C. de, J.M. ElXLouf, N. Roinel, C. Bailly and C.

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PROSTAGLANDINS 13. Morel, F., M. Imbert-Tebouland D. Chabardbs.Distributionof hormone dependent edenylate-cyclasein 'the nephron and its physiologic&L significance. Ann. Rev. Physiol.43:569-581.1981. 14. Morel, F., D. Chabar&s, M. Imbert-Teboul., F. Lebouffant,A. Hus-Citharel'and M. Montegut. Multiple hormonal control of adenylatecyclase in distal segmentsof the rat kidney. Kidney Int. 21:S.55-S.62.1982. 15. Hzb,asFhara,E., J.B. Stokes,J.P. Kokko, W.B. Campbelland T.D. . Corticel and papillary micropunctureexamination of chloride transport in segments of the rat kidney during inhibition of prostsglandin production. J. Clin. Invest. 64: 1277-1287.1979. 16. Wilson, D.R., U. Honrath and H. Sonnenberg.Furosemideaction on collectingducts : effect of prostaglandinsynthesisinhibition. Am. J. Physiol.x:F666-F673. 1983. 17. Wisemen, E.H. Pharmacologic studies with a new class of nonsteroidal anti-inflammatoryagents - the oxicems - with special referenceto piroxicem (Feldene).Am. J. Med. 72:2-8. 1982. 18. Chen, P.S., Jr, T.Y. Toribaraand H. Warner. Microdetermination of phosphorus.Anal. Chem.28:1756-1758.1956. 19. Burch, R.M. and P.V. Halushka. 45Ca fluxes in isolated toad bladderepithelialcells : effectsof agentswhich alter water or sodiumtransport.J. Pharmacol.Exp. Ther. 224:108-117.1983. 20. Burch, R.M., W.C. Wise and P.V. Halushka. Prostaglandinindependent inhibition of calcium transport by nonsteroidel anti-inflammatorydrugs : differentialeffects of carboxylic acids and piroxicam.J. Pharmacol.Exp. Ther. 227:84-91. 1983. R.M. and H.R. Keiser. Prostaglandinbiosynthesisby 21. zusm, rabbit renomedullaryinterstitialcells in tissue culture. J. Clin. Invest.-60:215-223.1977. 22. Carty, T.J., J.D. Eskra, J.G. Lombardino and W.W. Hoffmen. Piroxicam,a potent inhibitorof prostaglendin productionin cell culture. Structure-activitystudy. Prosteglandins.-19:51-59. 1980. 23. Roman, R.J. and M.L. Kauker. Renal effects of prostaglandin synthetaseinhibitionin rats : micropuncturestudies. Am. J. Physiol.235:Flll-F118. 1978. 24. Qusmme, G.A. and J.H. Dirks. Magnesiumtransportin the nephron. Am. J. Physiol.239:F393-F401. 1980.

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25. Burg, M.B. Thick ascendinglimb of Henle'sloop.Kidney Int. 22:454-464. 1982. 26. Shareghi, G.R.and Z.S.Agus.Magnesium trensport in the cortical thick ascendinglimb of Henle'sloop af the rabbit.J. Clin. Invest. 1982. -69:759-769. 27. St&i,W.N. afid D. Rouse.Hormonalregulation of calciumtransport in thick ascending limb rensl tubules. Am. J. Physiol. 241:F171-F174. 1931. 28. Kuntziger,H., C. Amiel,N. Roinel end F. Morel.Effectsof parathyroidectomy and cyclicAMP on renaltransport of phosphate, calcium,and magnesium. Am. J. Physiol. 227:905-911. 1974. 29. Dunn,M.J.,H.P. Greely,H. Valtin,L.B. Kinterand R. Beewkes III. Renal excretionof prosteglendins E end F2 in diabetes insipidus rats.Am. J. Physiol. 235:E624-E 227. 1978. 30. Welker, LA., A.R. Whorton,M. Smigel,R. France end J.C. Frolich. Antidiuretichormone increasesrenal prosta@ndin synthesis in vivo.Am. J. Physiol. 235:F18&F185. 1978. 31.

Kirschenbaum, M.A., A.G. I.owe,W. Trizna and LG. Fine. Regulation of vasopressin actionby prosteglandins : evidence for prostaglendinsynthesis in the rabbit cortical collecting tubule.J. Clin.Invest.70:1193-1204. 1982.

32. Garcia-Perez, A. end W.L. Smith. Apical-basolateral m?brene asymmetry in canine cortice3 collectingtubule cells : bredykinin, arginine vasopressin, prosteglandin Q interrelationships.J. Clin.Invest.74:63-74. 1984. Editor: A. Nies

132

Received: 5-22-84

Accepted: 10-26-84

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