Crosstransplantation of kidneys in normal and Hyp mice Evidence that the Hyp mouse phenotype is unrelated to an intrinsic renal defect

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Crosstransplantation of kidneys in normal and

Hyp mice. Evidence that the Hyp mouse

phenotype is unrelated to an intrinsic renal

defect.

T Nesbitt, … , R Griffiths, M K Drezner

J Clin Invest. 1992;89(5):1453-1459. https://doi.org/10.1172/JCI115735.

Although deranged phosphate transport is the fundamental abnormality in X-linked

hypophosphatemic (XLH) rickets, it remains unknown if this defect is the consequence of an intrinsic kidney abnormality or aberrant production of a humoral factor. To discriminate between these possibilities, we examined phosphate homeostasis in normal and Hyp mice, subjected to renal crosstransplantation. We initially evaluated the effects of uninephrectomy on the indices of phosphate metabolism that identify the mutant biochemical phenotype. No differences were found in the serum phosphorus concentration, fractional excretion of phosphate (FEP), or tubular reabsorption of phosphate per milliliter of glomerular filtrate (TRP) in uninephrectomized normal and Hyp mice, compared with sham-operated controls. Subsequently, single kidneys from normal or Hyp mice were transplanted into normal and Hyp mouse recipients. Normal mice transplanted with normal kidneys and Hyp mice

engrafted with mutant kidneys exhibited serum phosphorus, FEP, and TRP no different from those of uninephrectomized normal and Hyp mice, respectively. However, engraftment of normal kidneys in Hyp mice and mutant kidneys in normal mice affected neither serum phosphorus (4.69 +/- 0.31 and 8.25 +/- 0.52 mg/dl, respectively) nor FEP and TRP of the recipients. These data indicate that the Hyp mouse phenotype is neither corrected nor transferred by renal transplantation. Further, they suggest that the phosphate transport defect in Hyp mice, and likely X-linked hypophosphatemia, is the result […]

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Crosstransplantation

of

Kidneys

in

Normal and

Hyp

Mice

EvidenceThat theHypMouse

Phenotype

Is Unrelatedtoan Intrinsic Renal Defect

Teresa Nesbitt, Thomas M.Coffman, RobertGriffiths,and MarcK. Drezner

DepartmentsofMedicine and CellBiology,and The SarahW.Stedman CenterForNutritional Studies DukeUniversityMedical Center

and Veterans Administration MedicalCenter, Durham,North Carolina 27710

Abstract

Although deranged phosphate transport is the fundamental ab-normality in X-linked hypophosphatemic(XLH) rickets,it re-mainsunknown ifthis defect is the consequence ofanintrinsic kidneyabnormality or aberrant production of a humoral factor. Todiscriminate between thesepossibilities,weexamined phos-phatehomeostasis in normal and Hypmice, subjectedtorenal crosstransplantation. Weinitiallyevaluated the effects of uni-nephrectomy on the indices ofphosphatemetabolism that

iden-tify the mutant biochemical phenotype. No differences were

found in theserumphosphorusconcentration,fractional

excre-tionof phosphate(FEP),ortubularreabsorptionofphosphate per milliliter ofglomerular filtrate(TRP)inuninephrectomized normal and Hyp mice, compared with sham-operated controls. Subsequently, single kidneys from normalorHypmicewere

transplanted intonormal and Hypmouse recipients.Normal mice transplantedwith normalkidneysand Hyp miceengrafted with mutant kidneys exhibited serum phosphorus, FEP, and

TRPnodifferentfrom those ofuninephrectomizednormaland Hypmice, respectively. However, engraftment of normal kid-neys in Hyp mice and mutantkidneysin normal miceaffected neither serum phosphorus(4.69±031 and8.25±0.52 mg/dl, respectively) norFEPand TRPof therecipients.These data indicate that the Hyp mouse phenotype is neither correctednor

transferredbyrenaltransplantation.Further,theysuggest that the phosphate transport defect in Hyp mice, and likely X-linkedhypophosphatemia,is theresult of a humoral factor, and is not an intrinsic renal abnormality. (J. Clin. Invest. 1992. 89:1453-1459.) Key words: hyp mice * rickets * phosphate . kidney

Introduction

X-linked hypophosphatemia

(XLH)'

is the most common form of inherited vitamin D-resistant rickets/osteomalaciain humans (1). The X-linked hypophosphatemic (Hyp) mouse

Portionsofthis work have appeared in abstract form in 1990. (J. Bone Miner. Res. 5(2):S205.)Address correspondence and reprint requests

toDr. Teresa Nesbitt, Box 3285, Duke University Medical Center, Durham, NC 27710.

Receivedfor publication30July 1991 and in revisedform 05

De-cember1991.

1.Abbreviations usedinthis paper: FEP, fractional excretion of phos-phate;Pi, phosphate;TRP, tubularreabsorptionof phosphate per mil-liliter of glomerularfiltrate,XLH, X-linkedhypophosphatemia.

serves as amodel for thedisease, sharingmanyof theclinical and biochemical abnormalities observed in patients with XLH (2). Although many investigators have identified deranged phosphate (Pi)transport in the renal proximal tubule of the murinehomologueastheprimary abnormality underlying this disease (3, 4), it remains unknown whether this defect is the

consequence ofan intrinsic kidney abnormality, or results

fromproduction ofahumoral factor that alters proximal tu-bule brush border membrane function. Indeed, these possibili-ties have been inadequately explored, and previous studies have produced conflicting data. Such investigations include those in which in vivo evaluation of phosphatehomeostasis, following parabiotic union of normal and Hyp mice, favored anhormonalcause(5-7), and those in which comparison of phosphatetransportin cell cultures derived from proximal

tu-bules of these animal models indicated thepresenceofan in-trinsic renal abnormality (8-10). Each of these experimental approaches, however, is fraught with significant limitations, and neither permits simultaneous investigation ofthe

indepen-dent influences of renal and humoral factors onphosphate

me-tabolism. Incontrast,renal crosstransplantation innormaland Hyp mice, and subsequent assessment of phosphatetransport

in normal kidneys, engrafted in agenetically abnormal hor-monal/metabolic domain, and kidneys frommutantanimals, transplanted intoanormalmetabolicmilieu, permit

examina-tion ofboth possibilities. Therefore, inthe present study,we

used the technique of crosstransplantation to comprehensively

examineif the disorderedphosphate transportin XLHresults

fromaprimaryorsecondary disturbance of kidney function.

Methods

Animals

NormalC57BL/6Jmicewerematedwith C57BL/6J heterozygous

female Hyp mice,aspreviouslydescribed (11). Male Hyp and normal

miceobtained fromthe resultant litters were selected for study at 6-8 wkofage.Allanimals received anappropriatediet (asdescribed below) anddeionizedwateradlib. from weaning until the time of study.

Experimental protocols

CHARACTERIZATION OF THE HYP MOUSE BIOCHEMICAL PHENOTYPE

Initiallywedeveloped thebiochemical indices that would permit

identification of the Hyp mouse phenotype in subsequent studies in which animals were subjected to renal transplantation. From weaning untilsacrifice,normaland Hypmice received a diet containing 0.6% of bothcalcium and phosphorus (Teklad Premier Laboratory Diets,

Ma-dison, WI). Identical numbers of normal and Hyp mice mice were

sham operated,leavingbothkidneys undisturbed,and atl-wk inter-valsthereafter blood obtained via retroorbital sinus puncture for deter-mination of the plasma phosphorus concentration. 4wk

postopera-tively,theanimalswere anesthetized (65 mg/kg phenobarbital i.p.),

andthefractional excretion of phosphate (FEP) and tubular reabsorp-tion of phosphate per milliliter of glomerular filtrate (TRP)

deter-Transplantation ofKidneysinNormal and HypMice 1453

J.Clin. Invest.

0021-9738/92/05/1453/07 $2.00

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mined. At the end of the clearance procedure the animals were euthan-ized byexsanguination. In addition, phosphate-depleted (normal) mice wereevaluated in order to establish the criteria to differentiate hypo-phosphatemiaof differingorigins. These normal mice were fed a

phos-phorus-deficient diet(0.2%inorganic phosphorus, 0.6% calcium;

Tek-lad Premier Laboratory Diets) for 1 wk before phosphate clearance

studies.

Evaluationofphosphate clearance. Phosphate clearance was

mea-sured bypreviously established methods (12). After animals were anes-thetizedwithpentobarbital,asilasticcatheter (0.012 in. id, 0.025 in. od; DowCorningCorp.,Midland,MI) was placed in theleftjugular veinfor infusion of

['4C]Inulin

(New England Nuclear, Boston, MA). The right carotid artery was catheterized with tubing (PE-10; Clay Adams, Parsippany,NJ) forelectronic monitoring ofblood pressure

(Comfort Electronics, Durham, NC)andarterialbloodcollection. In

addition,acatheter(PE-50; Clay Adams) wasinsertedthrough a supra-pubic incisionintoanick in the bladder wallfor urine collection.The catheterwassecuredwithasingle ligature,and theabdominalwall and skin closedto preventdehydration. Aftervolume replacement with normalsaline(2% body wt over 20 min), a priming dose of['4C]Inulin

wasgiven, followed by acontinuous infusion to maintain constant

blood levels.Aftera30-minequilibrium period,theclearanceof [4C]-Inulin was measuredduringtwo successive 30-minperiods. Arterial bloodwasobtainedatthemidpointof each collectionperiod,and urine

attheconclusion ofeach time period. Subsequently,theglomerular

filtration rate(GFR)wascalculated fromtheconcentrationof

['4C]-Inulinin theurineandserumsamples(["4C]InulinunneX

_Volume.n/J

['4CJInulin,,,m

per min perkg).Inaddition,theserumandurine

con-centrationsofphosphorus were measured in preinfusion samplesin

ordertocalculate FEP and TRPaccordingtothefollowing formulas:

FEP= UpjXUvoume/FilteredVolumeX

_Spwhere

Filtered Volume=GFR(milliliter/minuteperkidneyperkilogram)

XBW(kg)XminutesX(no.kidneys)

TRP/mlGFR =FilteredLoadPi-_Upi/GFRwhere

FilteredLoad SF>XGFR(milliliter/minuteperkidneyper

kilogram)XBW(kilogram) Xminutes X(no. kidneys)

EVALUATION OF RENAL TRANSPLANTATION AND CROSSTRANSPLANTATION

NormalandHypmicewereengraftedwithasingle kidneyfromeither

animalmodel.Appropriatecontrols forthese studieswereobtainedby

subjectingmice ofboth genotypesto ashamsurgical procedure,leaving bothkidneys undisturbed,andaright uninephrectomy,tomimic the

singlekidneymodel used in thetransplantationstudies.Throughout

the study,all animals received a dietcontaining 0.6% calcium and

phosphorus.Bloodwasobtained fromtheretroorbitalsinusat1-2 wk

intervalsaftersurgeryforthedeterminationofserumphosphorus lev-els. Inaddition,4 wkpostoperatively,the micewereanesthetized(65

mg/kgpentobarbital,i.p.),and the FEP andTRP,aswellastheGFR,

weredetermined,asdescribedabove.

Renaltransplantation technique. In renal transplantationstudies

we engrafted asingle kidneyinto recipients thatwere subsequently

nephrectomized.Weusedanendtoside,aortato aortaanastomosisfor the renaltransplantation, using techniques previously established in

rats (13). Donormice wereanesthesized with Nembutal(50 mg/kg

i.p.),and theaortaandvena cavaseparatedanddissected fromapoint distaltotheleft renal vesselsto an areaproximalto therightrenal

vessels. Threeligatures were preplaced around (a) theaortaandvena

cava, distaltotheleftkidney,and(b)thevena cavaandaorta individu-ally, distaltotherightrenalvessels. The leftureter wasdissectedtoits insertion in the bladderwall, therightureterwascauterized and

sev-ered, and aligature was preplaced around the urethra. The donor mouse was then set aside.

Recipient micewereanesthetized with4% Halothane and

main-tained withaconcentrationof 1.5%.Heparin (1,000U/ml;Elkins-Sinn

Inc., CherryHill, NJ) was administered to both recipient and donor mice.Afteraleftnephrectomy, theabdominal aorta and vena cava weredissected and separated, and the recipient mouse was set aside.

The donorkidneywasobtainedbyligatingthe preplaced ligatures andseveringthevessels/urethradistaltotheties.Thekidney, urethra, and bladder were then placed in anice-cold solution of0.1mlheparin/ 20 ml stock solution (4.5 g sodium chloride, 100 g mannitol, and 1 g

Chloromycetin Sodium/liter sterilewater).

Intherecipients,vascular clamps wereplaced proximally and

dis-tallyinpreparation foraorotomy and venotomy. Thefree proximal

endsofthe donor aorta and vena cavawere sutured to the respective

recipientaorta andvena cavawith 11-0Dermalon, and the vascular

clamps removed. Theproximal 1/5oftherecipient bladder and the

proximal 2/3 ofthe donor bladder wereexcised in preparation for

creatinga newurinarybladder in the transplanted mouse. The

remain-ing 1/3 ofthe donor bladderwith itsattatched ureter was sutured to the remaining4/5oftherecipientbladder,using10-0 Novafil.

Theabdominalwall andskin were closed separately with

continu-oussuturesof4-0silk.4dafter transplantation, theengraftedmouse

wasanesthetizedwith Halothane and a nephrectomy performed on the

remainingnativekidney.

Analytical methodology

Urinevolumewasdetermined by weighing.Plasma(14)andurine

(15) inorganicphosphorus were determined by acolorimetric

tech-nique.

Statistical

methods

All results areexpressedasthe mean±SE.Significance ofthe differ-encebetween groupswasevaluatedusing pairedttestingandanalysis

ofvariance (with appropriateaposteriori multiplerangecomparison

procedures) (16). 4-10animals comprisedeach experimental group.

Results

Characterization

of

the Hyp mouse biochemical phenotype

Theserumphosphorus concentration inHypand

phosphate-depletedmice, although characteristicallydecreasedto alevel significantlyless than thatinnormals,didnotdistinguish these animalmodels(Fig. 1). However, Hypmice exhibitedan FEP

far inexcessof that manifested byphosphate-depleted mice, andaTRPthatwastwofold less than that ineither normalsor phosphate-depleted normals(Fig. 1).Collectively,these obser-vations establishedthat the decreasedcirculatinglevelof phos-phorus in Hyp mice isuniquelyduetorenalphosphate

wast-ing. Therefore, identification of the Hypmousephenotype in transplantedoruninephrectomizedanimalsnotonlyrequires documentation ofpersistent hypophosphatemia,butthe pres-enceofanormalFEPandadecreasedTRP,evidence ofabnor-mal renalphosphate handling.

Effects

ofuninephrectomy, renal

transplantation,

and

renal

crosstransplantation

on

phosphate

homeostasis

Uninephrectomy. Initially,weperformed uninephrectomy in normal and Hyp miceto determine theeffects ofa single kidneyonthe indices ofphosphate homeostasis that identified

the mutant biochemical phenotype. 4 wk

postoperatively,

bodyweightand GFR(perkidney)in both

uninephrectomized

normal andHyp mice were no different than those of their respective sham operatedcontrols(Fig.2A).Similarly, unine-phrectomyhadno effecton theplasmaphosphorusvalues in eitheranimalmodel (Fig. 3 A), and the presence ofa

single

kidney didnot significantly alterthemeasures ofrenal

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FRACTIONAL EXCRETION OFPHOSPHORUS

0

NS

20-I0

O

0t

NORMAL HYP Pi-OEPLETE TUBULAR REABSORPTION

OFPHOSPHORUS

3F NS

NORMAL HYP Pi-DEPLETE

Figure 1.Serum phosphorus concentration, fractional excretion of phosphorus, and tubular reabsorption of phosphorus in sham-oper-ated normal, Hyp, and phosphate-depleted mice. Serumphosphorus levels inHyp micewerenotdifferent from those in Pi-depleted mice, although theyweresignificantlylower thanthosein normal mice(P

<0.001). The fractionalexcretion ofphosphoruswas nodifferentin

normal and Hyp mice but markedly reduced in Pi-depleted mice (P

<0.001). The tubular reabsorptionofphosphoruswassignificantly reducedin Hypmice (P<0.01), compared with normal mice, while Pi-depleted animal models displayed TRP levelsnodifferent from those in normals. Dataareexpressed as mean±SEM of 6-10

indi-vidual determinationspergroup.

whichweused in subsequent studies hadnoeffectonPi

homeo-stasis.

Renal transplantation. Likewise,renaltransplantation

be-tweenadonor and recipientof thesamegenotypedid not

influ-encebody weightorGFR (Fig.2B). Moreover,thisprocedure

didnotaffectphosphatehomeostasis. 4 wk aftersurgery,

nor-malmice engrafted withanormalkidneymaintainedaplasma

phosphorus level significantly greater than Hyp mice

trans-planted withamutantkidney (Fig. 3 B),arelationship

identi-caltothat observedinbothsham-operatedand

uninephrecto-mized animals. Indeed, throughoutthepostoperative interval,

the engrafted normalmice did notvarytheirplasma

phospho-rus (9.6-10.8 mg/dl) from the normal range, while

trans-planted Hyp mice exhibited a persistently decreased plasma

phosphorus, 5.4-6.4 mg/dl, (Fig. 5 A).Renaltransplantation,

likewise, did notalter the dynamics of renal phosphate

han-dling in normal or Hyp mice. Normal mice engrafted with

normal kidneys and Hyp mice transplanted withmutant

kid-neysmaintainedFEPandTRPvalues comparabletothose in

their respective sham-operated controls (Fig. 4B). Thus, the

FEP inthe transplantedmutantswasnodifferent from that of

engrafted normals,inspite ofaremarkably diminished filtered

phosphate load, and the TRPwastwofold reduced (Fig. 4 B).

These observations indicate that a single normal and Hyp

mousekidney transplant functions in normal andmutant

recip-ients, respectively, to maintain phosphorus homeostasis and

thedistinctive biochemical profiles of the host animal models.

Renal crosstransplantation. Insubsequent studieswe

inves-tigatedtheeffects ofrenalcrosstransplantationin normal and

Hyp mice with donors and recipients of differentgenotypes. 4

wkaftersurgery, normal mice transplanted with Hypmouse

kidneys and Hyp mice engrafted with normal kidneys

exhib-itedbody weights and GFRsnodifferent than their

sham-oper-A

7-

Figure

2.

(A)

GFR and

6

body weights

in sham

c ^ IT. _ anduninephrectomized

normal and Hypmice

n 4_ _ _ 4 wk after thesurgical

3- procedure. The GFR

. 2 was nodifferentin each

Io

I., of theseanimal models.

SHAM

Nd

N SHAM_In contrast, body

SHAM Nx SHAM Nx weights of normal mice

NORMAL HYP were significantly

BODY 28.4 28.6 20.9 19.7 _greater_than_{Hyp mice,}

WEIGHT ±0.8 ±1.3 ±0.7 ±0O7 grae nHymi,

whether sham operated

B @ 7 or uninephrectomized

(r 6 I _{(Nx) (P}<_0.001).

How-X

5m

ever,uninephrectomy

4 4 hadnoeffect on this

U- variablein normalor

Cd *Er_

_Hyp

mice. Dataare

ex-C- 2 pressedasmean ± SEM

E - - of fourtosix individual

o _ determinationsper

DONOR: NORMAL HYP HYP NORMAL _group._(B)_{GFR and}

RECIPIENT: NORMAL HYP NORMAL HYP

BODY 26.8 21.0 27.7 22.0 body weights in normal

WEIGHT 1.4 ±0.9 ±0.9 *0.8 andHypmice 4 wk after

renaltransplantation

orrenal crosstransplan-tation. Nosignificantdifferences in GFR were present in any of the groups ofmice. Body weights of normal mice were significantly greaterthan those of Hyp mice (P<0.01),regardless of the trans-plantedkidneytype. Data areexpressedas mean± SEMof fourto

six individual determinationspergroup.

A

N

E

NORMAL

B

10

8

6

4

IIu DONOR: NORMAL

RECIPIENT: NORMAL

HYP HYP

HYP NORM

Figure 3. (A) Serum phosphorus

concentra-tions insham and unin-ephrectomized (Nx) normaland Hyp mice 4 wkafter the surgical

i i procedure. Uninephrec-tomyhadnosignificant effecton serum phos-phorus levels in either

M Nx normalorHypmice.

HYP However, both

sham-operated and unine-phrectomized Hyp mice

weresignificantly hypo-phosphatemic relative

totheir respective

nor-mals_(P<_0.001).Data

areexpressedas mean

± SEMof 6-10

individ-ualdeterminationsper group.(B)Serum

phos-_LL phorusconcentrations

NORMAL in normal and hypmice

4AL HYP 4wkafterrenal

trans-plantationorrenal

crosstransplantation. Serum phosphorus levelswerereflectiveof

whether the recipientwas anormalorHypmouse,andwereunrelated

tothe donormousemodel. Thus,Hypmouserecipients(black bars)

displayedasignificant hypophosphatemia(P<0.001), compared with

normalmouserecipients (white bars),regardlessof whetherthe

transplantedkidneywasdonated byanormalorHypmouse.Data areexpressedas mean±SEM offourtosixindividualdeterminations

pergroup.

Transplantation ofKidneysinNormal andHypMice 1455

SERUM PHOSPHORUS

E

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FRACTIONALEXCRETION TUBULARREABSORPTION tionAl

A ePHOSPHORUSOFP S ig .(A) Fractional

40 3 excretionofphosphorus

30-

l

alland

7

tubular

ciE ; 2

reabsorp-20O[rE

tion ofphosphorusin

jl lE

_

shamand

uninephrec-HAM

ISA N. SHAM N- SHAM N. SHAM N. _{tomized normal and}

Hypmice4 wkafterthe

FRACTIONAL EXCRETION TUBUR REABSORPTION

B OFPHOSPHORUS OFPHOSP _surgicalprocedure.

40- _^ ^ Uninephrectomy had

2 | nox l weffectoneither

pa-4J lIwtE,31

rameterin both normal

0-_

j

_{and Hyp}

J

_

^_0

_mouse_models.

HONOR. NORMAL _HOP HYP NORMAL HONORNRl. HOP HOPNOML

RtECMNT NORMA HYPNRA HYP RECRPENT ORVA HHYPp N A mYP Incontrast, while

sham-operatedand

unine-phrectomized normaland Hypmicedisplayednodifference inthe

fractional excretion of phosphorus,theseHyp mousemodelshad a

significantlyreduced(P<0.001)tubularreabsorption ofphosphorus

comparedwith normals.Dataare expressedas mean±SEM of6-10

individual determinationspergroup.(B)Fractionalexcretionof

phosphorusandtubularreabsorption of phosphorusinnormaland

Hypmice4 wkafter renal transplantation orrenal crosstransplanta-tion. Thefractional excretion of phosphoruswasnotsignificantly

differentin anygroup. However,thetubular reabsorption of

phos-phorus wasreflective ofwhether therecipientwas a normalorHyp

mouse, andwasunrelatedtothedonormouse model.Thus,Hyp

mouse recipients(black bars) displayeda TRP thatwas significantly

lessthan normalmouserecipients (whitebars), regardless of whether

thetransplantedkidneywasdonatedbya normal orHypmouse (P

<0.001).Data areexpressedas mean±SEMof fourtosix individual

determinationspergroup.

12

10 8 I6 4 2

DONOR RECIPIENT:

12

io

:B81 Ca6

4

12r

10 8 6 4 2

0 1 2 3 4 0 1

Weeks AfterTransplant

NORMAL NORMAL

r I12

- i f

810

F

~~~~6-F

~~~~4-

i-F ah~~~~2

0

DONOR: RECIPIENT:

2 3 4 0 1

Weeks After Transplant

HYP NORMAL

N(

Figure 5. (A) Weeklyserumphosphoruslevels innor

mice after renaltransplantation.Serumphosphorus 1e

changesignificantlyin the 4-wkpostsurgicalintervalii

orHypmouserecipients.Dataareexpressedasmean

to five individual determinations pergroup.(B)Weel

phoruslevelsin normal andHypmice afterrenalcros

tion.Normal miceengraftedwith_Hypmouse_kidney normalserumphosphoruslevelsthroughouttheposto Likewise, Hypmicecrosstransplantedwith normal ki

tained hypophosphatemiacharacteristicof the mutan wkafter thesurgical procedure.Dataareexpressedas

offive to sixindividualdeterminationspergroup.

atedoruninephrectomizedcounterparts(Fig.2B). Moreover, the normal mice withkidneys fromHyp mice maintaineda plasma phosphorusconcentrationwithin the normalrangeand did notdevelopthe characteristichypophosphatemia of

Hyp

mice(Fig. 3B).In contrast, Hypmice engraftedwith normal kidneys displayedadecreasedplasma phosphorus concentra-tion.Furthermore,theinabilityof crosstransplantation to alter theplasma phosphoruslevels ofgraft recipientswaspersistent andunwavering.Within 1 wk of surgery, Hyp mice

crosstrans-plantedwith normalkidneys continuedtoexhibit hypophos-phatemiawhich remained steadystatethroughoutthe 4 wkof observation (Fig. 5B). Likewise, normalmiceengrafted with

mutant kidneys maintained normal plasma phosphorus

throughout the post-crosstransplantationperiod.

The normophosphatemia evident in normal mice

cross-transplantedwithkidneysfrom Hypmicewasassociated with evidence of normal renalphosphate handling. Both FEP and TRP in these animalswere nodifferent than those of normal mice bearing normal transplants (Fig.4 B). Indeed, these

val-uesindicated that the kidney from Hyp mice, resident in the normalhost,wasfully capable ofnormalphosphatetransport.

In contrast, the normal kidney functioning in the mutant

milieu exhibited evidence of markedly abnormal renal phos-phate transport. Consequently, Hypmice, crosstransplanted with normal kidneys, displayed an FEP and TRP no different thanthose of Hyp mice engrafted with kidneys from mutant animals (Fig. 4 B). Moreover, the TRP of these animalswas significantly less than those of the normal mice

crosstrans-planted with kidneys from Hypmice.

Discussion

Renalphosphate reabsorption occurs intheproximal

convo-luted tubule via a sodium-dependent, phosphate cotransporter thatmediates apicaltransportofphosphate from the tubular lumen into the cell

_(17).

The

_activity

_{of this system is} deter-minedby the integrated effects of multiple intrarenalevents,

HYP and the modifying influence of a variety of hormones, and

HYP metabolic anddietaryfactors.Therefore,decreasedphosphate

transportin XLHmay be dueto ageneticdefect that results in

aprimaryorextrarenalabnormality(s) which alters the activity

ofthisapical transport system. Such defects may respectively include: (a) a primary modification of the plasma membrane protein(s) that constitutes the Na+/Pi cotransporter and/or the _-I----j intracellularenzymes/cofactorsor renal paracellular hormones

thatregulateitsactivity; and (b)ahormonal/metabolic

imbal-ance thatsecondarily disturbsacomponent of the transport

2 3 ₄ _mechanism_and_{results in}_aberrant_{function. Until}_{recently, the}

majority of available evidence suggested that a primary renal ORMAL abnormality, such as the absence ordiminished activity ofa

HYP

transport-specific protein,underlies thisdisease.Several

obser-mal andHyp vationssupport this possibility: (a) the expressed abnormality

Evels did not inphosphate transport is confined to the kidney ( 18-20) andis

neithernormal _{restricted to the proximal convoluted tubule (21, 22); and}_(b)

FSEM of four known hormones that regulate this process, such as

parathy-klyserumphos- roid hormone, circulate within the normal range (23-25).

ss

maintained

These

data, however, provide only inferential evidence in favor perative perod ofa primary renal defect, and considerable controversy persists

pdneys

main- regarding the target organ for the genetic defect in XLH. Thus,

It

phenotype 4 in the present studies we used renal crosstransplantation of

mean±SEM kidneysbetween normal andHypmicetodirectlyexamine the

independent influencesofrenal and humoral factorson

phos-A

E

B

-1

I

-j

(6)

phatemetabolism in this genetically determined

hypophospha-temicstate.This experimentalapproachprovidedthe

opportu-nityto examine the function of kidneys fromHyp mice in a normal hormonal/metabolic milieu and the function of

nor-malkidneys in the abnormal domain of the mutant.

Thetechnique oforgancrosstransplantation has been used previouslytodistinguishwhether thephenotypic and/or bio-chemical characteristics ofavariety ofgenetic diseasesresult frominnatedefectsin end-organfunctionand/orsystemic hor-monal/metabolic influences. Suchinvestigations have identi-fiedanintrarenalabnormalityas aprimarydefect inagenetic form ofhypertensionin therat(26),andanintrahepaticdefect

ascausalof deranged uricacid metabolisminDalmationdogs

(27). In contrast, alterations intheaffected host environment

have been determined as the primary cause ofresistance to

arteriosclerosisinpigswithvonWillebrand'sdisease(28),and

asprimaryorcoexistentabnormalitiesinavian muscular dys-trophy(29). Centraltoeachoftheseinvestigationsis elucida-tion ofdefinitive criteriato identifythe abnormal phenotype under study, and documentation that surgical intervention doesnotaltertheirexpression. In ourstudies,weobserved that

measurement of plasma phosphorus and TRP reproducibly

discriminates the Hyp mouse from both normal and phos-phate-depleted normal mice(Fig. 1).Moreover, thesingle

kid-neymodel thatwe employed didnotinfluenceexpression of theseabnormalities inspite ofcontraryobservations in other animal species (30). Indeed, we found that uninephrectomy and/or renaltransplantation between matched murine models didnotnegativelyinfluence the GFRoralterphosphate

clear-ance in the resultantsingle kidney. Consequently, theserum

phosphorus concentrationwasunchanged in each treated ani-mal model (Fig. 3). The surgical intervention that we used, therefore, did not confound interpretation ofdata from the crosstransplantation studies.

Thus, in contrast to prevailingopinion, ourobservations afterrenalcrosstransplantation indicatethat theexpressed ab-normality ofphosphatereabsorption inXLHdoesnotresult from anintrinsic renal defect, but morelikely froman hor-monal/metabolic abnormality. In this regard, normal mice transplanted with kidneysfrom Hypmice maintaineda

nor-malplasmaphosphorusconcentration throughoutthe

postop-erativeperiod,andsurprisingly,didnotdevelop hypophospha-temia,inspite of functioning grafts fromthemutants(Fig. 3). Furthermore, thenormophosphatemiawasassociated with evi-dence of normal renalphosphate

reabsorption,

whichincluded

FEPandTRPvalues well within the normalrange. In concert,

crosstransplantation ofa normal kidney into a Hyp mouse

failedtoalterthecharacteristichypophosphatemiaof thegraft recipient. Moreover, the Hyp miceengraftedwith normal

kid-neysdisplayedFEPandTRPvaluescharacteristicof the mu-tantbiochemical phenotype (Fig.4). Allof these changes

oc-curred without variation in thebodyweightof the hostor di-minished GFR in thesingle kidney (Fig.2). Therefore, these dataillustrate that theHyp mousephenotype is neither

trans-ferrednorcorrectedbyrenaltransplantation. Rather, the

po-tential influenceof crosstransplanted kidneys from normalor Hyp mice is negated, and their function determined bythe hormonal/metabolic milieu ofthe hostanimal.

Whether aberrant regulation ofrenal 25-hydroxyvitamin

D-1_{a-hydroxylase activity,}anadditional kidney abnormality

in XLH, similarly occurs asa consequence ofan abnormal hormonal/metabolic milieu remains unknown.Nevertheless,

sincestudies in theHypmousehavedocumented that the

de-rangedrenalenzymeactivity is likely localizedtothe proximal convoluted tubule, the site of defective phosphate transport,

thecausesof these abnormalities areundoubtedly linked.

In-deed,currently available evidence (31-33)suggeststhat the en-zymedysfunction isanacquired defectsecondarytothis abnor-mality. Thus, theapparenthormonal/metabolic defect in Hyp mice mostlikely indirectlyinfluencesthe expresseddefect in calcitriol production.

Our dataagreewith previous observations(5, 6), in which thetechnique ofparabiotic unionwasusedtodemonstratethat

normal mice paired withHypmice,beforeandafter

parathy-roidectomydeveloped hypophosphatemia secondary to a

re-versibleincrease in renalphosphateexcretion. However,

sev-eral salientpoints limit interpretation of these parabiotic

stud-ies.Inthisregard, the serumphosphorus level andtheurinary

phosphate excretion in the normal parabiont were

interme-diate between those evident in the complementary Hypmouse

and shamoperated normals.Further, normal mice parabiosed

tonormalmice also exhibitedadecrease inserumphosphorus andanincrease inurinary phosphate excretion, compared with sham operated normals. Althoughsomeof these observations

maybe the resultofaninabilitytocontrol thecrosscirculation

rate in a parabiotic union, such confounding alterations in phosphate homeostasis in parabionts maybe due to muscle wasting and/or transfer ofphosphorus to the

phosphate-de-pleted bonesof themutant. Inany case, nodatacanbe

gener-atedfrom study of parabionts regarding the exclusive abilityof thekidneyfromaHypmouse tomaintainorcontributetothe abnormalbiochemical profile.Thus, thepossibility ofa

com-bineddefect in the endorganandthe hostenvironmentcannot

beruledout.Indeed,previousexperimentsindicate that sucha

dualabnormalitymayproduce the characteristicphenotype in avian myotonic dystrophy (29). These limitations have

pre-cludeddefinitiveconclusions about the hormone dependence of XLH.

In any case, our observations provide noinformation

re-garding the identityofthe abnormalhormonal/metabolic

fac-torthatunderliesXLH.Although elevatedserumparathyroid hormone levels have beenreported inHypmice (34),this alter-ation of themetabolic milieuunlikelyunderlies the abnormal Pi homeostasis inthe mutants. Inthis regard, parathyroidec-tomy ofHypmice doesnotaltertheserum phosphorus

con-centration (35). Bycontrast,therecentdiscoveryand biochemi-calcharacterizationoftheparaneoplasticsyndrome, tumor-in-duced osteomalacia, provides substantial evidence thatfavors theexistence ofauniquehumoralagent. Inthisacquired

dis-ease, hypophosphatemic rickets/osteomalacia occurs

second-ary todiminishedrenal tubularreabsorption of phosphate and

abnormallyregulated calcitriolproduction (36). Moreover,

re-movalof smalltumorsleadstopromptrestoration ofthe bio-chemical abnormalities and healing ofthe bone disease(37,

38). Consequently, overproductionofahormone(s),nativeor

ectopicto thetissue oforigin, almost certainly underlies this syndrome. Thegeneticerror in XLH,therefore, may resultin altered production(or turnover)ofsuch a naturallyoccurring

humoralfactor(s).

Moreover, other evidencedoesexistthat supports the possi-bilityofahumoralphosphaturic factorinX-linked hypophos-phatemia.Morgan et al.(39) reported thattransplantation ofa

normalkidneyintoapatientwithXLHresultedincontinued hypophosphatemiaand decreased renalreabsorptionof

(7)

phorus. This evidence is tempered by the tendencyof trans-planted kidneys in humans tofunction abnormally and exhibit lowrenal retention ofphosphate (40). Further, atthetimeof study in the posttransplantation period, an abnormal hor-monal milieu secondary to renal failurelikely persisted, and may haveinfluenced phosphate homeostasis.

Regardless, there are additional data that are difficult to reconcile with a humoraloriginforXLH.Renal cellsfromHyp mice displaydiminishedphosphate uptake(8-10)and abnor-malvitamin Dmetabolism (8, 41) after several days in culture, albeittheabnormalitiesare not asprofoundasthoseobserved in vivo.Additionally,osteoblasts from Hypmice,when

trans-planted to normal mice, produce abnormally mineralizing bone,consistentwithexpression ofthe geneeffectinthis cell type (42). Although several investigators have suggested that the presence ofcellular heterogeneity,potential dedifferentia-tion,ormemoryretention ofin vivo hormonal influences may explain someof thesediscrepantinvitroobservations, insuffi-cient informationis availabletoascertain the influence of any ofthesefactors.

Alternatively,

these

disparate

findings

may be the consequenceofauniquehormonaleffectthatresults ina

blockadeof,orfailuretoexpress,anessential gene function ina varietyofcell types. The absenceofthe geneeffectmay induce the well known end-organeffects ofXLH, which include ab-normal renalphosphate transport andimpairedbone mineral-ization. While culture ofsuch impaired cells, or their

trans-plantationtoaforeignenvironment,wouldremovethealleged hormonal influence,thesetechniquesmaynotresult in

expo-sureofthe cellsto essential factorsnecessaryto reinitiate ex-pression ofthe gene.As aconsequence, theabnormalities

pres-entin these cells invivomay be

perpetuated

in

culture,

orafter transplantation.Furtherstudiesarenecessary,

however,

to de-terminethereasonforthese

apparently contradictory

observa-tions.

Nevertheless,the presentfindings providethefirst

unequiv-ocal evidence that the

kidney

is not the target organ for the genetic

abnormality

thatunderliesXLH.

Indeed,

our observa-tions indicatethatthe Hypmouse

phenotype

is neither trans-ferred nor corrected by renal

transplantation. Therefore,

we postulatethat the XLHsyndrome

likely

resultsfrom hormon-ally induced alterationsin the function of renal

proximal

tu-bule brush border membranes.

Acknowledgments

The authorsthankChrisBest for his expert technical assistance.

Thisworkwas supported byNational Institutes of Healthgrants

AR-27032,DK-38015,and DK-38 108. Dr. CoffmanisaResearch

As-sociateatthe VeteransAdministrationHospital.

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