The Acute Effect of 25- Hydroxycholecalciferol on

Renal Handling of Phosphorus

EVIDENCEFORA PARATHYROID

HORMONE-DEPENDENTMECHANISM

MoRDEcAI M. PopovTzER, JoHN B. ROBINETrE, HECTORF. DELUCA, andMIcHAEL F. HoLIK

From the Department of Medicine, University of Colorado School of Medicineand Veterans Administration Hospital, Denver, Colorado 80220, and theDepartment of Biochemistry, University of Wisconsin,Madison, Wisconsin 53706

A B S T R A C T The acute effect of i.v. and direct intra-renal arterial infusion of 25-hydroxycholecalciferol(25HCC) and 1,25-dihydroxycholecalciferol (1,25-DHCC) on renal handling of phosphorus was evaluatedin the following groups of rats: (a) intact animals, (b)parathyroidectomized (PTX) hypocalcemic rats, (c)I'TX rats in which normocalcemia was maintained withcalcium supplements and (d) PTX animals in whichurinary phosphorus was augmented by (i) i.v. sodiumphosphate, (ii) expansion of the extracellular fluid vol-ume with normal saline, and (iii) i.v. parathyroid hor-mone (PTH). Clearances of inulin (Cin), phosphorus(CP), and fractional clearances of phosphorus (CP/Cin)of the experimental groups were compared with those ofthe corresponding control groups, and the clearances ofthe infused kidneys with those of the contralateralkidneys.

In intact animals, i.v. 25HCC decreased CP/Cix from0.29±0.04 (mean +SE) to 0.19+0.04, and i.v. 1,25-DHCCdecreased CP/Ci. from 0.25±0.04 to 0.15±0.02.The intrarenal infusion of both 25HCC and 1,25DHCCinto intact animals failed to produce a unilateral change;however, it decreased CP/Cin bilaterally. i.v. and intra-renal infusions of 25HCC or 1,25DHCC in PTX hy-pocalcemic and normocalcemic rats, and i.v. infusions of25HCC in PTX rats receiving either sodium phosphate

Presented in part at the 5th International Congress ofNephrology in Mexico City, Mexico, 11 October 1972(Abstract 463) and at the 26th Annual Meeting of theWestern Society for Clinical Research in Carmel, Calif.,1 February 1973. 1973. Clin. Res. 21: 203.

Received f or pulblication 21 May 1973 and in revisedform 30 August 1973.

or normal saline, all failed to produce significant changesin CP/Ci.. In contrast, 24HCC given i.v. to PTX ani-mals receiving exogenous PTH was associated with asignificant fall in CP/Cin from 0.34+0.08 to 0.13+0.02.These results indicate that 25HCC enhances tubular re-absorption of phosphorus in rats, only in the presenceof either endogenous or exogenous circulating PTH, butnot in its absence and thus imply a PTH-dependentmechanism of 25HCC action on the kidney. This effectdoes not appear to be related to the conversion of 25HCCinto 1,25DHCC, since the latter fails to affect tubularreabsorption of phosphorus in PTX rats.

INTRODUCTIONThe effect of vitamin D on renal handling of phos-phorus has been the subject of numerous investigations(1-7). The main difficulty encountered in interpretingchanges in urinary excretion of phosphorus has beenrelated to the calcemic action of the vitamin which, perse, by suppressing parathyroid hormone secretion,might, in an indirect fashion, alter renal handling ofphosphorus (8, 9). Thus, the en'hanced tubular reab-sorption of phosphorus associated with administrationof vitamin D to patients with osteomalacia (6, 7) andto rachitic animals with intact parathyroid glands (1)could be accounted for either by inhibition of para-thyroid hormone secretion or by a direct tubular ac-tion of the vitamin. In contrast, the phosphaturic actionof large doses of vitamin D observed in hypoparathy-roid humans (2) and in thyroparathyroidectomized dogs(4) has been either attributed to a direct effect of thesteroid on renal reabsorption of phosphorus (4) or to

The Journal of Clinical Investigation Volume 53 March 1974.913-921 913

an indirect effect mediated by the increase in serum cal-cium (10). However, the effect of the latter is ratlhercomplex, and an increase in serum calcium may leadeither to an increase or a decrease in the urinary ex-cretion of phosphorus indel)elldently of variations inparathyroid glands' activity (10, 11). hI addition, thelevel of calcium has beeil implicated as aan importantfactor, (letermininig the response of the bone to vita-miini D (12). The conceintration of calciumil may alsoplay an important role in other actions of vitamin D.In a recent study, a direct renal action of 25-hydroxy-cholecalciferol (25HCC)1 independent of parathyroidhormone secretion was suggested; however, the inter-dependence between the vitamin and the lhormone wasnot explored in great detail (5). The present study wasdesigned to investigate the effect of 25HCC on renalhandling of phosphorus with an attempt to characterizethe relationships between the action of the steroid andthe level of parathyroid hormone, and betweeni the se-rum concentration of calcium an(d the basal excretionrate of phosplhorus in the urine.

METHODSWhite female Sprague-Dawley rats (200-300 g) fed Purinapellet chow (Livestock and Poultry Feeds, Ralston PurinaCo., St. Louis, Mo.) diet with tap water ad lib. werestudied.

Clearance studiesThe clearance studies were performed in all animals at

the same part of the day between 8:00 a.m. and 4:00 p.m.After the induction of anesthesia with an intramuscularinjection of sodium pentobarbital (40 mg/kg body weight),the animals were placed on heated operating boards and atracheostomy tube was inserted. Rectal temperature wasmonitored by means of a thermistor and telethermometer(Yellow Springs Instrument Co., Yellow Springs, Ohio).The femoral artery and vein were exposed through anlinguinal incision and PE-20 tubings (Clay Adams, Div. ofBecton, Dickinson, and Co., Parsippany, N. J.) were in-serted into each vessel. The arterial line was used formonitoring mean arterial blood pressure with a Stathamstrain gauge, model 23AA (Statham Instruments, Inc.,Oxnard, Calif.) and for the collection of blood samples.The venous line was extended to a syringe mounted on avariable speed continiuous inftusion pump (Harvardl Ap-paratus Co., Inc., Millis, Mass.). Thle urinary bladder wasexposed and a funlnlel shaped enid of a PE-240 tubing wasintroduced through anl inlcisionl at the fuindus for urine col-lections. The incision was closed tightly with a purse strin-gsuture which excluded a major portion of the vesical lumeniand thus eliminated most of the dead space. The techniqueof renal artery catheterizationi which was employed in theexperiments with intrarenal arterial infusion was describedin detail in previous reports (13, 14). The teclhni(qlne of

.4qbbreviations uised int this paper: CiI, clearance of inn-lin; Cl., clearance of inulin phosphorus; CN., clearance ofsodium; 1,25DHCC, 1,25-dihydroxycholecalciferol; 25HCC,25-hydroxycholecalciferol; PTH, parathyroid hormone;PTX, parathyroidectomized.

measuring initlini clearances (CIn) and sodium clearances(CNa) in rats, and( the analytical procedures used in ourlaboratory have been described in detail in a recent report(14). All plasma and urine specimens w7ere analyzed forinulin (15) an(d phosphorus, and at least two plasma speci-mens obtainedl at tlle be-inningp ani(l the eill( of eachl exper'i-ment were analyzed for calcium. lilorganic phosl)horus wasmeasured utilizing- the methodology developed by the Amer-ican Monitor Co. (American '1Monitor Patenit N\o. 3-547-586). Calcium was (letermined with Perkin-Elmer AtomicAbsorption Spectrophotometer model 290 (Perkin-ElmerCorp., Norwalk, Connl.). The fractional phosphorus clear-ance was determined by factoring clearance of phosphorus(Cp) by CIn. Before administration both 25HCC and 1,25-dihydroxycholecalciferol (1,25DHCC) were dissolved inplasma obtained from rats similar to those that were studiedwith the vitamin. The infusioin rate of plasma in all ex-periments by each route of admiinistrationi was 0.3 ml/100

per h. In the control groups that did not receive 25HCCor 1,25DHCC, the corresponding vehicle was added to theilnfused plasma in a volume equal to that given to theexperimental groups.

Experimental groups

GroutP lto. In six rats with intact paratllyroi(l (glandsafter four control clearance periods, 0.1 ,sg/l010 per hof 25HCC was given intravenously over five collectiollperiods. Six additional rats treated in an idenitical nialnlerbut not receiving 25HCC served as control.

Group lb. In four rats with intact parathyroid glandsafter four control clearances, 0.02 jAg/100 g per h of25HCC were infused directly into the left renal artery dur-ing five collection periods. During the control collections,the intrarenal arterial infusion delivered plasma with thevehicle.

Group 2a. Parathyroidectomy was performed 2 daysbefore the clearance studies, using previously describedtechniques (16). In six parathyroidectomized (PTX) ratsafter four control clearances 0.15-0.30 ,ug/100 g per h of25HCC was given intravenously over five collection periods.Six additional PTX rats treated in a similar manner butnot receiving 25HCC served as control.

Grouip 2b. In four PTX rats after four control collec-tions, 0.02-0.10 ug/l00 g per h of 25HCC were infuseddirectly into the left renal artery over five collectionperiods. During control collection, plasma obtained fromsimilar PTX rats was infused directly into the left renalartery.

Group 3. Four PTX rats received oral suplplements ofcalcium as 50 mg/100 ml of elemental calcium in drinkingwater. In addition, they were receiving (laily 10 mg ofcalcium as calcium chloride by subcutaneous injection. D)ur-ing the clearance studies, the sustaining infusioni was sup-plemented withl calcium chloride, resulting in a concentra-tion of calcium of 20 mg/100 ml in the infusate. Afterfour control collections, 0.3 ug/100 g per h of 25HCC wasgiven intravenously over five clearance periods. Four addi-tional PTX control rats, receiving calcium supplements,were treated in a similar fashion, hut were not infusedwith 25HCC.

Group 4a. In six PTX rats after three control collec-tions, isotonic neutral sodium phosphate was infused in-travenously at the rate of 6 mg/100 g per h (0.2 mM/100 g per h) of elemental phosphorus. After three addi-tional clearance periods, as the phosphorus infusion con-tinued, i.v. infusion of 0.3 ug/100 g per h of 25HCC was

914 M. Al. Popovtzer, J. B. Robinette, H. F. DeLiuca, and M. F. Holick

started and was given for f our collection periods. Anadditional group of six PTX rats which were treated in anidentical f ashion but did not receive 25HCC served ascontrol.

Group 4b. Four PTX rats were treated in an identicalfashion as described in group 4a; however, sodium phos-phate was infused at a slower rate, 3 mg/100 g per h (0.1mM/100 g per h).

Group 5. In five PTX rats after three control collec-tions, normal saline was infused intravenously at the rateof 0.1 ml/100 g per min. After three additional clearanceperiods as the saline infusion continued, i.v. infusion of 0.3jug/100 g per h of 25HCC was started and was given forfour collection periods. An additional group of five PTXrats which were treated in an identical fashion but didnot receive 25HCC served as control.

Group 6. In six PTX rats after three control collec-tions, i.v. infusion of 1 U/100 g per h of parathyroid ex-tract (Eli Lilly and Co., Indianapolis, Ind.) was begun.After four additional periods, as the parathyroid extractinfusion continued, i.v. infusion of 0.1 ,ug/100 g per h25HCC was started and was given for four collectionperiods. Additional groups of six PTX rats which weretreated in an identical fashion but did not receive 25HCCserved as control.

Group 7a. In four rats with intact parathyroid glandsafter four control clearances, i.v. infusion of 0.1 ,ug/l00 gper h of 1,25DHCC was started and continued for fourperiods. An additional four rats which were treated in asimilar fashion but did not receive 1,25DHCC served ascontrol.

Group 7b. In four rats with intact parathyroid glandsafter four control clearances, intrarenal arterial infusion of0.1 ,ug/100 g per h of 1,25DHCC was started and continuedfor an additional four periods.

Group 8a. In six PTX rats after four control collec-tions, i.v. infusion of 1,25DHCC 0.1 ,tg/100 g per h wasstarted and continued over four periods. An additionalgroup of six PTX rats which were treated in a similarfashion but did not receive 1,25DHCC served as control.

Group 8b. In three PTX rats after four control col-lections, intrarenal arterial infusion of 0.1 ,ug/100 g per hof 1,25DHCC was begun and continued for an additionalfour periods.

In all studies the individual animals in the experimentaland in the corresponding control groups were littermates,matched by age, sex, weight, and feeding time; both theexperimental and the matched control rats were studied onthe same day and during the same hours, under identicalconditions.

The analysis of the results was based on the comparisonof the experimental with the respective control groups forthe corresponding clearanice periods, using the paired Stu-dent's t test. The results in the studies with direct intra-renal arterial infusion were evaluated by comparing valuesobtained on the ipsilateral side witli those on the contra-lateral side.

RESULTSThe effect of i.v. 25HCC on fractional clearance ofphosphorus (CP/Cln) in rats with intact paratlhyroidglands (group la) is depicted in Fig. 1. The resultsrepresent the mean+SE for both the control and the ex-perimental groups, during the control periods and dur-ing the infusion of plasma without or with 25HCC, in

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-0.30

P NS-0 20 - _

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O-----O CONTROLGROUPv-.* 25HCC GROUP

n-6P,O 01

-O 10

0 -80TIME (MINUTES)

80 -180

FIGURE 1 The effect of i.v. 25HCC on renal handling ofphosphorus in rats with intact parathyroid glands. P refersto the difference between the control and experimentalgroup.

the control and the experimental groups, respectively.The mean±SE of CP/Cin was calculated from the aver-ages of the control and the vitamin infusion periods.There was no dlifference in CP/C1. between the controland the experimental groups during the four controlperiods (0-80 nmin). During five periods (80-180 miii)in which 25HCC was given intravenously to the ex-perimental group, there was a marked decrease in CP/Cm.;whereas in the control group there was an increase,resulting in a ,highly significant discrepancy betweenthe two groups. The fall in CP/CI. became apparent 20-40 min after the beginning of 25HCC infusion. Thesechanges were not associated with significant variationsin CTn and serum concentrations of calcium and phos-

TABLE IRepresentative Experinient Showing the Response to iv.

25HCCin a Rat with Intac-t Parathyroid Glands

Time V* Cln Sc.1 Si, CP,./Cln

miti j.l/mnin jil/min mg/1OO ml mg/100 ml

0 Prime with inulin 5 mg/100 g and continue sustaininginfusion delivering inulin 0.17 mg/min per 100 g in5% dextrose in water at the rate of 0.040 ml/minper 100 g

0-60 Equilibration period60-80 107 3,990 9.2 2.4 0.2780-100 190 3,700 2.6 0.31

100-120 12(0 3,800 2.5 0.25120-140 135 3,960 9.0 2.7 0.26

Begin intravenous infusion of 25HCC 0.1 jg/100 g per h140-160 125 4,000 8.9 2.8 0.19160-18(1 120 3,560 2.8 0.14180-200 80 3,660 2.9 0.12200-22(0 85 3,650 3.0 0.11220-240 100 5,560 8.4 3.2 0.11

* Urine volume.t Concentration of calcium in serum.§ Concentration of inorganic phosphorus in the serum.

Effect of 25-Hydroxycholecalciferol on Renal Handling of Phosphorus 915

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FIGURE 2 The effect of 25HCC infusion directly into leftrenal artery (LT. Renal A.), with separate urine collectionfrom both the right (RT.) and the left (LT.) kidneys, onrenal handling of phosphorus in rats with intact parathyroidglands. P refers to the difference between the control andthe 25HCC infusion collection periods on both sides.

phorus. A representative experiment showing the re-sponse to i.v. 25HCC in a rat with intact parathyroidlglands is shown in Table I.

In group lb (Fig. 2) there was no (lifference inCE/Cm between both kidneys during the control pe-riods (0-80 min) and during the infusion of 25HCCinto the left renal artery (80-180 min). During the in-fusion periods, there was a marked but equal decreasein CP/Ci. on both sides. There were no significantchanges in Cin and serum calcium and phosphorus con-centrations during the whole study.

Although there were no changes in serum concen-trations of calcium during the administration of 25HCCsignificant enough to implicate secondary changes inparatlhyroid hormone secretion in the fall in CP/CI., thepossibility that a change in the rate of parathyroid hor-iinone secretion, in the presence of parathyroid glands,played a role in the decline could not be ruled out withcertainty. To further clarify this question, PTX ratswere studied. In each animal that entered the study, thesuccess of parathyroidectomy was ascertaine(l by a dropin serum calcium to a level of 6 mg/100 ml or less.

Lii L.ii

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-020 RT KIDNEY

CONTROL 4 n=P NS NS

LT. KIDNEY

- 0.10

0-80TIME (MINUTES)

80-180

FIGURE 4 The effect of 25HCC infusion directly into leftrenal artery on renal handling of phosphorus in PTX rats.

The effect of i.v. 25HCC on CP/Ci. in PTX hypo-calcemic rats (group 2a), compared with the effect onla control group of PTX animals who were not re-ceiving 25HCC is showni in Fig. 3. There was no sig-ni ficant difference in CP/CI. between the two groupsbefore (0-80 min) and during 25HCC infusion (80-180min), indicating a lack of effect of 25HCC on renalhandling of phosphorus in PTX rats. Similarly no re-sponse was noticeable when 25HCCwas infused directlyinto the left renal artery in group 2b (Fig. 4). In boththe 2a and b groups, there were no significant changesin CIn and serum concentrations of calcium and phos-phorus throughout the experiment.

In group 3 after a drop in serum calcium below 6 mg/100 ml after PTX, serum calcium was raised and main-tained at the range of 8.2-10.0 mg/100 ml in both thecontrol and the experimental groups, before and through-out the acute experiments. There was no difference inCl /Cdnduring the control periods (Fig. 5), between theconitrol and the exp)erimental grotups, nor during the in-

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4

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80-180

FIGUJRE 3 The effect of i.v. infusion of 25HCC on renalhandling of phosphorus in PTX rats with hypocalcemia.

FIGURE 5 The effect of i.v. infusion of 25H1CC on renalhandling of phosphorus in PTX rats with normocalcemia.

916 M. M. Popovtzer, J. B. Robinette, H. F. DeLuca, and M. F. Holick

fusion of 25HCC into the experimental group. Cin andserum concentrations of calcium and phosphorus did notchange significantly throughout the study.

In group 4a the constant infusion of sodium phosphatewas associated with a rise in serum phosphorus from5.20±0.16 (mean±SE) to 17.30+-1.10, and from 5.00±0.35 to 16.13+1.60 mg/10 ml in the control and the ex-

perimental groups, respectively. Fig. 6 depicts the vari-ations in CP/CIn in both groups, during the control pe-

riod (0-60 min), during sodium phosphate infusion(60-100 min), during phosphate with 25HCC infusionin the experimental group, and during phosphate infu-sion (without 25HCC) in the control grotup (100-180min). During the combined phosphate and 25HCC in-fusion periods in the experimental group, there was no

significant difference in CP/Ci compared to the controlgroup. Both groups exhibited a progressive increase in

phosphorus excretion reaching a mean maximum ofCP/Cin of about 50%. There were no significant changesin CIn throughout the study. There was a slight de-crease in serum calcium which was not significant.Similarly in group 4b, in which CP/Cln was increased bya smaller fraction, 25HCC had no effect on tubular re-

absorption of phosphorus (Table II). In group 5 salineinfusion was associated with considerable increases inboth CP/CIn and CNa/Cin; however 25HCC did not af-fect these changes (Table III).

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0.

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O-----O CONTROLGROUP

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P N NS

0-60 60-100TIME (MINUTES)

NS

loo-180

FIGURE 6 The effect of i.v. 25H1CC on renal handlingof phosphorus in PTX rats receiving sodium phosphate(NaP) infusion. NaP was started after 60 min, 25HCCwas infused in the experimental group after 100 min.

The sequential changes in CP/CIn in group 6, both inthe control and in the experimental animals, are de-picted in Fig. 7. There was no difference in CP/Cin be-tween the two groups during the control period (0-60min), nor during the infusion of parathyroid extract(60-140 minm); however there was a progressive in-crease in Ce/Cs5 in both groups. The addition of 25HCC

TABLE IIThe Effect of 25HCCon Renal Handling of Phosphorus in PTX Animals Receiving Sodium Phosphate Infusion (P-INF)

CIn UPv CP/Cln CN./CIn Sp

P-INF P-INF P-INF P-INF P-INFBase- +25 Base- +25 Base- +25 Base- +25 Base- +25line* P-INF$ HCC§ line P-INF HCC line P-INF HCC line P-INF HCC line P-INF HCC

,ul/min g/mmin mg/100 ml

Experimental rats

1 1,492 1,650 1,490 2.51 6.60 18.62 0.0280 0.0500 0.125 0.0010 0.0040 0.0080 6.0 8.0 10.02 940 1,060 1,015 0.16 4.81 17.54 0.0020 0.0420 0.135 0.0020 0.0050 0.0110 8.8 10.8 12.83 1,500 1,065 1,350 5.60 10.96 24.01 0.0450 0.1050 0.156 0.0050 0.0250 0.0210 8.3 9.8 11.44 1,277 1,010 1,160 3.79 6.44 12.06 0.0430 0.0750 0.105 0.0090 0.0040 0.0090 6.9 8.5 9.9

Mean 1,302 1,196 1,253 3.01 7.20 18.06 0.0295 0.0680 0.1302 0.0042 0.0095 0.0122 7.5 9.2 11.0±SE :1:31 ±150 ±104 1.14 1.30 2.45 40.0084 ±40.0141 i0.0106 40.0017 ±0.0050 ±0.0030 +0.2 ±0.6 i0.7

Control rats1 1,020 1,060 1,500 2.51 4.29 17.79 0.0390 0.0460 0.105 0.0090 0.0080 0.0090 6.3 8.8 11.32 1,470 1,060 1,060 6.77 16.31 17.84 0.0490 0.1620 0.165 0.0018 0.0015 'O.0130 9.4 9.5 10.23 1,028 944 1,360 0.65 1.00 19.99 0.0090 0.0100 0.105 0.0060 0.0080 0.0160 7.1 10.6 14.04 989 1,135 790 0.34 2.02 9.99 0.0050 0.0200 0.115 0.0020 0.0170 0.0200 6.9 8.9 11.0

Mean 1,126 1,049 1,175 2.57 6.05 16.40 0.0255 0.0595 0.1225 0.0047 0.0086 0.0145 7.4 9.5 11.6±SE ±114 ±40 158 1.48 3.53 2.20 ±0.0105 ±0.0309 ±0.0143 ±0.0016 40.0031 ±0.0023 +0.7 ±0.4 ±0.8

NSII NS NS NS NS NS NS NS NS NS NS NS NS NS NS

Average of three clearance periods before starting P-INF (0-60 min).t Average of three clearance periods after starting P-INF (60-120 min).

§ Average of four clearance periods 60 min after starting P-INF, during which 25HCCwas given to the experimental animals: the control animals received the vehicleonly (120-200 min).

11Denotes comparison between corresponding study periods of the experimental and control groups.

Effect of 25-Hydroxycholecalciferol on Renal Handling of Phosphorus 917

Tr

TABLE IIIThe Effect of 25HCCon Renal Handling of Phosphorus in PTX Aninmals Undergoing Extracellular Fluid

Volume Expansion with Saline Infusion (ECVE)

Cl,,

Base- +25linie* EC'VE$ H('('§

(N.i t 1!l

Base-liine

UPV

ECVEi }Q VEi+25 Base- +25

lJAIEV 11CC line li('VlE 1ICCBase-

linie

(''Cp;I7 Sp

+25 Base- 4-25ECV 11CC lilne EQVi 1E (CC

Experimental rats1 7002 8403 9724 7485 694

,Al/mill

1,0131,4231,2641,40)91,030

9121,6991,0351,2601,060

0.00(45(0.00120.00.320.0120(0.0)25(0

(.()8(1(1(1.115()(1.()81()().05(1()().()55()

0.1420)(1.19()01).1571)0. 1 3(0.1240

0.780.410.794.424.72

jsg/lmitn ing,/100 nil

15.553.664.46

24.477.5(0

17.468.82

12.2(025.17

9.23

(1.(112(1(0.0(045().01(1(1(0.1)650().105()

(0. 16(0(0(0.0221)0.035(0().1')3()(0. 14()()

(1.22(1(1(1.053(1(0.142(0(1.222(0(0.1300)

9.3 9.6 8.710.8 11.7 9.8

8. 1 1(.1 8.39. 1 9.(1 9.1)6.8 5.2 6.7

Mean 79(1 1,227 1,193 (1.0092+ESE +44 4+83 -+f153 40.0042

Control ratst2345

Mean4SE

81)() 80()741) 9)()(90() 1,481)50(1 1,59()70)0) 850

8(1()901(

1,46()1,2401, 1 (()

728 1,124 1,10()+63 +150 +111

NS11 NS NS

(0.()()40)0.01) 12(1.0(12(011.0112(00.002(0

((.1)762 (0.1404 2.22 11.12 14.584-0.011(11() 1.0129 0.96 3.94 3.06

0.068011.06.30)(0.0690)0.094(001.065(0

(1.138()(0.162110.0950)(1.12 8(00. 1 790

(.3()0.5t1.454.09(0.97

6.733.I02

17.7(017.69

1.91

15.366.0)5

17.7416.74

7.5(0

(1.1)062 11.1)716 (. 1384 1.46 9.41 12.67+11.0023 +11.11052 40.01 32 (1.69 3.47 2.45

NS NS NS NS NS NS

().1)393 (0.1)644 0.1534 8.8 9.1+410.0190) 40.11231 +0.0311 ±0.6 4-1.1

(0.0()35(0.012()(0.0 170(l.0900(1.1)1511

(1.0275+-0.0 150

NS

(1.11851)11.055(1(1.1300(0.1250)(0.0250)

(0.084()+0.0191

NS

(0.2()00(0.1120(0.135(0().145(0(0.075(0

(0.1334+41.0192

NS

8.S410.5

1(.9 9.9 9.65.8 6.1 6.119.5 9.2 9.()9.1 8.9 9.(19.2 9.11 9.1

8.9 8.6+(1.9 +11.6

NS NS

8.5+01.6

NS

* Average of three clearance periods before startinig _ECVF_ (0-60 min).I Average of three clearance periods after starting ECVE (60-120 min).§ Average of four clearance periods, 60 min after starting ECVE, during whiclh 25H(CC was giveni to the experimenital animals; the cotntrol aniimals received the vehlicleonly (120-200 min).11 Denotes comparison between corresponding study periods of the experimental and control groups.

to the parathyroid extract infusion in the experimentalgroup (140-220 min) led to a significant decrease inCP/Ci. in this group; whereas in the control groupwhiclh received only parathyroid hormone, CP/Cin con-tinued to rise, resulting in a large discrepancy in CP/CInbetween the two groups. These changes were not as-sociated with significant variations in Cin and serum

L

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z

0.50O-----O CONTROLGROUP

* 25HCC GROUP-0.40

-0.30 I"T

-020

4'--0.10

P NS NS NS <0.005 <0.0010.00 -

0-60 60-100 100-140 140-180 180 -220TIME (MINUTES)

FIGURE 7 The effect of i.v. 25HCC on renal handling ofphosphorus in PTX rats receiving constant infusion ofparathyroid hormone (PTH). PTH was started after 60min, 25HCC was started in the experimental group after140 min. P refers to the difference between correspondingcollection periods in the control and the experimentalgroup.

concentrations of calcium and phosphorus. This obser-vation demonstrated that 25HCC may decrease CP/Ci.in PTX animals, provided that exogenous hormone ispresent in the circulation.

The effect of 1,25DHCC on CP/Ci. in animals with in-tact parathyroid glands (group 7a) is shown in Fig. 8.There was no difference in CP/CI. between the control

cI-z

cr-

-J

U

()

0I0-In0a-

wUz

(I

C)

z

z

O-----O CONTROL GROUP*-0 *1,25DHCC GROUP

-030

T -- nI 6<

--PNS __- <0. 0

-0.201\

1,25ODHC

- o0io0 -80 80 -180

TIME (MINUTES)

FIGURE 8 The effect of i.v. 1,25,DHCC on renal handlingof phosphorus in rats with intact parathyroid glands. Prefers to the difference between corresponding collectionperiods in the control and the experimental groups.

918 M. M. Popovtzer, J. B. Robinette, H. F. DeLuca, and M. F. Holick

TABLE IVRepresentative Experiment Showing the Response to i.v.

1,25DHCC in a Rat with Intact Parathyroid Glands

Time V* Cln Scat SP§ CP/C,n

inini ,Al/min A4/min mg/100 ml mg/100 ml

0 Prime with inulin 5 mg/100 g and continue sustaininginfusion delivering inulin 0.17 mg/min per 100 g in 5%dextrose in water at the rate of 0.040 ml/min per 100 g

0-60 Equilibration period60-80 50 2,500 9.6 5.1 0.19080-100 S0 2,850 5.0 0.192

100-120 65 2,820 5.2 0.203120-140 100 2,130 9.5 5.0 0.392

Begin intravenous infusion of 1,25DHCC 0.1 Mg/'it00g per h

140-160) 135 2,790 5.2 0.10(160-18(0 163 2,80(0 5.3 0.095180-200 120 2,460 5.0 0.129200-22() 83 2,760 9.4 5.2 0.100

* Urine volume.t Concentration of calcium in serum.

§ Concentration of inorganic phosphorus in the serum.

and the experimental groups during the control periods(0-80 min). During four periods (80-160 min) in which1,25DHCC was given intravenously to the experimentalgroup, there was a marked decrease in CP/C1n in theexperimental animals; whereas in the control animalsthere was an increase resulting in a highly significantdiscrepancy in CP/CIn between the two groups. The fallin CP/CI. became apparent 20-60 min after the begin-ning of 1,25DHCC infusions. These changes were notassociated with significant changes in Cin and serumconcentrations of calcium and phosphorus. A representa-tive experiment showing the response to i.v. 1,25DHCCin a rat with intact parathyroid glands is shown inTable IV.

z

M

LUJ-J

U)

CE

cr0Ia-Uf)0

0-

z

-J

-J

-0.30 CONTROL

o LT. KIDNEY

1, 25DHCC-0.20 LT. RENAL A.

RT. KIDNEY --* \ \-

n0

4P<0005 <O0005

z

-J0

0Ia.

0Ia-

w

z4

-w-J

z.-J

z

O----O CONTROLGROUP

- 015 *- * 1,25DHCC GROUP

1,25DHCC- 0.10

n-6P NS

-0.05> --

0 -80 80-180

n = 6NS

TIME (MINUTES)

FIGURE 10 The effect of i.v. 1,25DHCC on renal handlingof phosphorus in PTX rats. P refers to the differencebetween the corresponding collection periods in both groups.

In group 7b (Fig. 9), there was no difference inCP/CI. between the two kidneys during the control pe-riods (0-80 min) and during the infusions of 1.25DHCCinto the left renal artery (80-160 min). During the in-fusion of 1,25DHCC there was a marked but equal de-crease in CP/Cin on both sides. There were no significantchanges in Cin and serum concentrations of calcium andphosphorus during the study. The effect of i.v. 1,25-DHCCon CP/CIn in PTX rats (group 8a) comparedwith the effect on a control group of rats not receiving1,25DHCC is shown in Fig. 10. There was no signifi-cant difference in CP/CI between the two groups be-fore (0-80 min) and -during 1,25DHCC infusion (80-160 min), suggesting a lack of effect of 1,25DHCC onrenal handling of phosphorus in PTX rats. Similarlyno response was elicited when 1,25DHCC was infuseddirectly into the left renal artery in group 8b (Fig. 11).In both the 8a and b groups there were no significantchanges in CIn and serum concentrations of calcium andphosphorus throughout the study.

DISCUSSIONThe results of the present study demonstrate that 25HCCenhances fractional reabsorption of phosphorus in the

LU

z

rsCEJLU

0

0-n0.I0.-O IO

0 -80

w

z4

w-J

z-J

z

80- 180

TIME (MINUTES)

FIGURE 9 The effect of 1,25DHCC infusion directly intoleft renal artery on renal handling of phosphorus in ratswith intact parathyroid glands. P refers to the differencebetween the control and, 1,25DHCC infusion periods inboth kidneys.

- 0.101,25DHCC

LT RENAL A.

- 0.05

0-80 80 -180TIME (MINUTES)

FIGURE 11 The effect of 1,25DHCC infusion directlyinto left renal artery on renal handling of phosphorus inPTX rats. P refers to the difference between the controland 1,25DHCC infusion periods in both kidneys.

Effect of 25-Hydroxycholecalciferol on Renal Handling of Phosphorus 919

presence of either endogenous or exogenous parathyroidhormone and imply a parathyroid hormone-dependentmechanism of action of 25HCC on the kidney. Since theobserved changes in CP/Ci. were not associated withsignificant variations in glomerular filtration rate or se-rum concentration of inorganic phosphorus, it may beconcluded that 25HCC produced a net increase in tu-bular reabsorption of phosphorus.

Our observations agree with a previous study whichdemonstrated a lack of acute effect of vitamin D on tu-bular reabsorption of phosphorus in PTX animals.The authors of the cited report considered the calcium-dependent secretory activity of parathyroid glands asthe underlying mechanism for the increase in tubularreabsorption of phosphorus after the administration ofvitamin D (4). In contrast, our study demonistrates anacute renal effect of vitamin D in the absence of para-thyroid glands, altlhouglh it required the presence ofparathyroid hormone. Our study provides detailed evi-dence regarding the effect of 25HCC, but not regardingthat of 1,25DHCC.

In keeping with our observations, previous studiesdemonstrated anl increase in tubular reabsorption ofphosphorus, after the administration of vitanmin D topatients with osteomalacia before a detectable rise inserum calcium became apparent (17). Similarly in thepresent study the increase in tubular reabsorption ofphosphorus during the administration of 25HCC (and1,25DHCC) to intact rats was not associated with sig-nificant changes in serum calcium concentration. Shouldthe renal response to vitamin D be secondarv to sup-pression of parathyroid hormone, a rise in seruni calciumwould be expected to precede or coincide witlh the in-crease in tubular reabsorption of phosphorus.

The nature of the association between 25HCC andparathyroid hormone with regar(d to the observed changesin CP/Cln is not clearly defined by the present study.Adequate parathyroid function maintains normal serumllcalcium. Under certain circumstances tlle metabolic ac-tion of 25HCC may be controlled by the level of calciumii(12). To examine the possibility that hypocalcemiacould be responsible for the lack of response to 25HCCin PTX rats, serum calcium was raised deliberately anidmaintained at normal range in group 3. Howvever, sus-tained normocalcemia failed to restore the renal responseto 25HCC in the PTX rats. Fuirthermore, subsequentexperiments demonstrated that administration of ex-ogenous parathyroid hormone (group 6) restored therenal response to 25HCC, even though it did not cor-rect the hypocalcemia.

The failure of 25HCC to produce a noticeable de-crease in CP/CiII in PTX rats could be due to the pre-existing low excretion rate of phosphorus. Parathvroidhormone could have restored the renal response to25HCC by increasing urinary excretion of phosplhorus.

Previous studies suggested that the phosphaturic re-sponse to extracellular fluid voluime expansion witlhnormal saline in rats was mainly mediated by changes inparathyroid gland activity and wvas minimal or absent inlacutely PTX rats (18). Wewere able to augmeint uri-nary excretion of phosphorus in rats subjected to PTXtwo days before, by expanding the extracellular fluidvolume with saline. Even though the amount of excretedphosphorus reached 15% or more of its filtered load,this increase failed to restore the renal response to25HCC. Similarly we were unable to see a response to25HCC in PTX rats in which CP/Ci Nwas increasedwith i.v. sodium phosplhate.

Recent studies demonstrated that the presence of para-thyroid hiormone is necessary for the conversioln of 25-HCCinto a 1,25DHCC in the kidney (19, 20). The lat-ter conmpounid lhas been shown to be the active fornm ofvitanmin D wlhiclh acts directlv on the gut and on the bonie(21-24). Should 1,25DHCC also be the form of vita-min D which acts directly on the kidney, parathyroidhormone could be necessary for the renal action of 25-HCC because of its role in coverting 25HCC into1,25DHCC. To test this possibility, 1,25DHCC wasgiven to PTX rats, botlh intraveniously and by a directinfusion into one renal artery, but had no apparent ef-fect on tubular reabsorption of phosphorus. However,the adnministration of 1,25DHCC to rats with intactparathyroid glands increased significantly fractional re-absorption of phosphorus.

The question whether 25HCC and/or 1,25DHCC di-rectly or indirectly affects tubular reabsorption of phos-phorus (in the presence of parathyroid hormone) is notanswered by the results of the present study. The lackof unilateral response during intrarenal arterial infusionof both 25HCC and 1,25DHCC does not support a di-rect action, but certainly (loes not exclude it. Witlh regardto the observations made during volume expanision, aword of cautioni is warranted: one cannot exclucle thepossibility that the phosphaturic effect of volume ex-pansion procedure coukl have influence(d the capacity of25HCC to reduce phosphorus excretion.

Several alternatives regarding the role of parathyroi(dhormonie in the renal response to vitamin D are worthcomment. First, parathyroid hormone may play a per-missive role in the renal response to vitamin D, similarto the role ascribed to vitamin D in facilitating the ac-tionl of parathyroid hormone on the bone (25, 26).Next, the preselnce of parathyroid hormone may beniecessary for converting 25HCCand/or 1,25DHCC intoan additional yet unknown conmpound(s) which act(s)lirectly oni the renal tubule. And filnally, vitaminl D may

act only on one componlent of inlorganic phosphoruistransport in kidiney whiclh has been described to be se-lectively sensitive to parathyroid hormone (27, 28). Inthe absence of parathyroid hormonie this transport sys-

920 M. M. Popovtzer, J. B. Robinette, H. F. DeLuca, and M. F. Holick

tern may be operating already at its maximal capacityprecluding any additional effect of vitamin D on phos-phorus reabsorption.

ACKNOWLEDGMENTSThis work was supported by a grant from the VeteransAdministration no. 7136-01 and 7136-02.

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7. Morgan, D. B., C. R. Paterson, C. G. Woods, C. N.Pulvertaft, and P. Fourman. 1965. Osteornalacia aftergastrectomy: a response to very small doses of vitaminD. Lancet. 2: 1089.

8. Baylor, C. H., H. E. Van Alestine, E. H. Keutmann,and S. H. Bassett. 1950. The fate of intravenouslyadministered calcium. Effect on urinary calcium andphosphorus, fecal calcium and calcium phosphorus bal-ance. J. Clin. Invest. 29: 1167.

9. Howard, J. E., T. R. Hopkins, and T. B. Connior.1953. On certain physiologic responses to intravenousinjection of calcium salts into normal, hyperparathyroidand hypoparathyroid persons. J. Clin. Enidocrinol. Me-tab. 13: 1.

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11. Lavender, A. R., and T. N. Pullman. 1963. Changes ininorganic phosphate excretion induced by renal arterialinfusion of calcium. Am. J. Physiol. 205: 1025.

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15. Galli, A. 1966. Colorimetric determination of inuliin inblood and in urine. Pathol. Biol. 14: 991.

16. Popovtzer, M. M., S. G. Massry, M. Villamil, and C.R. Kleeman. 1971. Renal handling of phosphorus inoliguric and nonoliguric mercury-induced acute renalfailure in rats. J. Clin. Invest. 50: 2347.

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20. Garabedian, M., M. F. Holick, H. F. DeLuca, and I.T. Boyle. 1972. Control of 25-hydroxycholecalciferolmetabolism by parathyroid glands. Proc. Natl. Acad.Sci. U. S. A. 69: 1673.

21. Frolik, C. A., and H. F. DeLuca. 1971. 1,25-dihydroxy-cholecalciferol: the metabolite of vitamin D responsiblefor increased intestintal calcium transport. Arch. Bio-chem. Biophys. 147: 143.

22. Wong, R. G., A. W. Norman, C. R. Reddy, and J. W.Coburn. 1972. Biologic affect of 1,25-dihydroxychole-calciferol (a highly active vitamin D metabolite) inacutely uremic rats. J. CliGt. Invest. 51: 1287.

23. Holick, M. F., M. Garabedian, and H. F. DeLuca.1972. 1,25-dihydroxycholecalciferol: metabolite of vita-min D3 active on bone in anephric rats. Science (Wash.D. C.). 176: 1146.

24. Boyle, I. T., L. Miravet, R. W. Gray, M. F. Holick,and H. F. DeLuca. 1972. The response of intestinalcalcium transport to 25-hydroxy and 1,25-dihydroxyvitamin D in nephrectomized rats. Endocrinology. 90:605.

25. Rasmussen, H., H. F. DeLuca, C. Arnaud, C. Hawker,and M. von Stedingk. 1963. The relationship betweenvitamin D and parathyroid hormone. J. Clin. Inivest. 42:1940.

26. Arnaud, C., H. Rasmussen, and C. Anast. 1966. Furtherstudies on the interrelationship between parathyroidhormone and vitamin D. J. Clint. Inivest. 45: 1955.

27. Glorieux, F., and C. R. Scriver. 1972. Loss of a para-thyroid hormone-sensitive component of phosphate trans-port in X-linked hypophosplhatemia. Science (Wash.D. C.). 997.

28. Glorieux, F. H., C. R. Scriver, T. M. Reade, H. Gold-man, and A. Roseborough. 1972. Use of phosphate andvitamin D to prevent dwarfism and rickets in X-linkedhypophosphatemia. N. En9l. J. Med. 287: 481.

Effect of 25-Hydroxycholecalciferol on Renal Handling of Phosphorus 921