EYDOCRINE RESEARCH COl\IMI'NICATIONS, 9 ( 2 ) , 135-143 ( 1 9 8 2 )

ACUTE I I F F E C T S OF A "PHYSIOI O G I C A L "

ON IIFNAL PHOSPHATE i R A N S P O R T DOSE 01' 1,25-DIHYDROXY VITAMIN D3

Helen Geirl-yaki a n d Jules B. PusLhett R t l n a 1 - F 1 PC t ro 1 y t e Sect i on

Dcpartinent of MrJdicine

P i t t sburgh , PA 15761 Universi ty o f P i t t c b u r g h School of Medicine

ABSTRACT

The renal phosphate t r a n s p o r t r rsponse o f t~h.~~.opdrdthyroid- ectomized, vitainin D--def ic imt r a t s to the i n f u s i o n of 1,25-di- h,ydroxy vitamin D3 ( 1 ,25 D3) was s tudied with and without the simultaneous adiiiinisi.rdtion o f a srilall ( o r "perlrii,i;sive") non- phosphaturic amoun t of bovine parathyroid hor-iiione (bPTli). A1 - though phosphate excret ion (UPV) was u n a l t e r e d by t h e infusion o f e i t h e r 0.1 U ( = .0025 11g o r 6 piiioles) o f '1,25 113 o r 0.2 U b,PTH per hour f o r 6 tiours, t h e i r combined adriiiriist,ration rc - didced UPV from 1 4 . 8 + 1.6 t o 10.3 ? 1 . 2 LIg/in-in. ( P .05) . There were no a l te ra t . ions in i n u l i n e x c r e t i o n . These data ver i - fy tha t : 1 ) 1,25 D3 i s an t iphosphatur ic i n t h i s c!xperiiiii-.ntal s c t - t i n g i n a very low dose which may r e p r e s e n t EI " l~hys io logica1" amount o f t h e inetabol i te ; and 2 ) t o enhance pho5phate t ranspor t , tlie 1 ,25 D3 requi res thi:? presence o f a small ("i:)ermissive") amount of PTH.

INrRODUCTIObJ

Several pub1 isht-tl ' , tud ies from t h i s and ot l ier l a b o r a t o r i e s

h<ive documpnted the acute an t iphosphatur ic i lctiori of the bio-

l o g i c a l l y a c t i v e metabol i tes of vitamin D, 25-h:idroxy d n d 1,25-

dihydroxy vitamin D3 ( 2 5 D3, 1 , 2 5 D3) i n both t h e thyroparathy-

r13idectoiiiized ( T P T X ) dog and rdt (1-5) . The qurs t ion has been

135

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136 GEORGAKI AND PUSCHETT

r a i s e d whether t h e dosages of t h e m e t a b o l i t e t h u s f a r employed

t o demonstrate t h i s e f f e c t on rena l phosphate t r a n s p o r t re -

present physiological o r pharmacological amounts. This s tudy

was performed t o analyze t h e e f f e c t s ( i f any) of t h e infus ion of

very small amounts of 1 ,25 D3 on phosphate excre t ion .

u t i l i z e d i s one-tenth t h a t p rev ious ly given.

The dose

MATERIALS AND METHODS

Weanling, 0-depleted r a t s were obta ined from the Holtzman

Company (Madison, WI) and fed a 0 - d e f i c i e n t d i e t containing

0.40% calcium (Ca) and 0.425% phosphorous (P) f o r two t o t h r e e

weeks. The animals were k e p t i n a room devoid of both na tura l

and f l u o r e s c e n t l i g h t .

For eva lua t ion of t h e r a c h i t i c effect of t h e d i e t , s i x t o

ten r a t s from each shipment of about 30 were f e d t h e D-deficient

d i e t b u t were a l s o given supplemental vitamin 0 (25 I.U./day).

A f t e r two t o t h r e e weeks on t h e d i e t , severa l r a t s from each

group were s a c r i f i c e d and blood obta ined by a o r t i c puncture f o r

t h e determinat ion o f Ca and P.

The D-def ic ien t , 2-3 week o l d r a t s were prepared f o r s tudy

according t o the technique o r i g i n a l l y descr ibed by Cotlove (6),

a s previously descr ibed from t h i s l a b o r a t o r y (4 ,5) .

were anes the t ized l i g h t l y with e i ther , fol lowing which TPTX was

performed by e lec t rocautery , and a bladder c a t h e t e r was inser ted .

An intravenous infusion was then begun via a t a i l vein with a so lu t ion

containing 4% glucose, 10 mM calcium, 20 mM sodium, 2.5 mM potassium

and 2 mM magnesium, each a s the ch lor ide s a l t . T h e r a t s a l s o received

The animals

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VITAMIN D AND THE KIDNEY 137

inul in in a dose of 5 mg./ml. The solut ion was irifused a t a r a t e of

1 . 1 4 ml/hr. so t h a t volume expansion would be avoided or minimized.

Following an equilibrium period of 14-16 hours, orie hour urine samples

were co l lec ted f o r a four-hour control period. The r a t s were then d i -

vided i n t o four experimental groups as follows: I ) Eight r a t s were

given only s a l i n e , a n d served a s a control g r o u p . 11) Eight r a t s re-

ceived 0.1 U ( = .0025 uq or 6 pmoles) of 1 ,25 D31 per hour f o r 6 hours.

111) Thirteen r a t s received b o t h the metabolite in the dose j u s t described

plus 0.2 U o f bovine PTH ( Inolex Inc . , Park Forest South, I L ) . I V ) Nine

r a t s were given the bPTH only.

A t the end of the six-hour experimental per iod, during which urine

specimens were obtained each hour, the animals were sacr i f iced and blood

was taken f o r the determination of Ca, P and inu l in by methodology pre-

viously described from t h i s laboratory (4,5).

we observed wide fluctiJations in the inu l in excret ion r a t e were discarded.

For each experiment, urinary excret ion values f o r control ( C ) a n d experi-

mental ( E ) phases of each study were compared u t i l i z i n g the mean values

from the l a s t two specimens of each of these two per iods. The mean f o r

a l l animals in each group f o r the C and E phases of the s tud ies were com-

pared u t i l i z i n g Student 's t t e s t f o r paired var iab les . Thus, each r a t

seirved as i t s own cont ro l . The data for serum ICa a n d P a n d f o r glomerular

f i l t r a t i o n r a t e ( G F R ) obtained f o r groups 1l'-IV was comp(3red t o t h a t of

the control ( g r o u p I ) r a t s u t i l i z i n g a t t e s t f o r independent var iab les .

Data from s tudies in which

1 . The 1,25 03 was a g i f t from Dr. Milan Uskokovic, Hoffman-La-Roche,

Inc . , Nutley, NJ.

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138

RESULTS ___-

GEORGAKI AND PUSCHETT

As d e p i c t e d i n f i g u r e 1, t h e r e was no a l t e r a t i o n i n phosphate excre-

t i o n i n t h e c o n t r o l group. Abso lu te phosohate e x c r e t i o n (UPV) was 11.86 i

1.43 and 10.93 t 1.42 i n t h e C and E per iods , r e s p e c t i v e l y (P > .60 ) .

Furthermore, v e r i f y i n g t h e s t a b i l i t y o f t h e p repara t i on , i n u l i n e x c r e t i o n

(UInV) was unchanged by t ime ( C : 9.59 _+ 0.70; E: 10.62 -t- 0.71 ug/min,

P > .25). The i n f u s i o n o f t h e m e t a b o l i t e a lone l i k e w i s e d i d n o t change

e i t h e r UPV (7.53 i- 1.65 + 6.93 +_ 2.16 ug/min, P > .60) o r UInV (8.69 i

0.87 + 8.58 i 0.76 ug/min, P > 90, group 11, f i g u r e 1 ) . However, when a

smal l dose o f bPTH was g i v e n s imu l taneous ly , u r i n a r y phosphate e x c r e t i o n

d e c l i n e d d r a m a t i c a l l y ( f rom 12.54 ? 1 .72 t o 8.61 i 1.27 ug/rnl, P < .02),

d e s p i t e t h e l a c k o f any c o n s i s t e n t v a r i a t i o n i n UInV (10.08 i 0.84 + 10.53

i- 0.37, P > .50; group 111, f i g u r e 1 ) . Furthermore, t h i s dose o f t h e

hormone, wh ich was r e q u i r e d f o r t h e d n t i p h o s p h a t u r i c e f f e c t o f t h e 1,25 D3

t o become m a n i f e s t d i d n o t i t s e l f cause any v a r i a t i o n i n t h e e x c r e t i o n

p a t t e r n o f e i t h e r P (8.12 i 1.17 -f 9.01 i ' 1 . 7 4 pg/rnin, P > . 50 )o r i n u l i n

(10.09 i 0.97 + 10.26 + 0.97 ug/min, P > .80; group I V , f i g u r e 1 ) .

The l e v e l o f abso lu te phosphate e x c r e t i o n d u r i n g t h e c o n t r o l phase

o f t h e s t u d i e s was somewhat h i g h e r i n t h e an imals t h a t r e c e i v e d 1,25 D3

and PTH (Group 111). T h i s v a r i a b i l i t y , wh ich has been no ted i n p rev ious

s tud ies , p robab ly r e l a t e s t o i n e q u a l i t y i n animal and/or r e n a l s i z e , b u t

o t h e r f a c t o r s c o u l d a l s o p l a y a r o l e .

i n t o two groups, those w i t h c o n t r o l phase phosphate e x c r e t i o n l e v e l s which

exceeded 13 ug/min, and those below t h i s l e v e l . I n t h e l a t t e r subgroup

(N = 7 ) , c o n t r o l phase phosphate e x c r e t i o n was 8.0 ?- 1.38 ug/min and f e l l

t o 5.73 * 1.18 ug/min ( P < .02). I n u l i n e x c r e t i o n was unchanged i n

these r a t s (8.87 5 1.30 + 10.50 i- 0.59 ug/min, P > .20). Thus, whatever

t h e l e v e l o f i n i t i a l phosphate e x c r e t i o n , t h e combina t ion o f 1,25(OH)2D3

and PTH was an t i phospha tu r i c .

We t h e r e f o r e subd iv ided t h e r a t s

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VITAMIN D AND THE K I D N E Y

C 0 NTR C) L 1,25 D, 1.25 D j

+ PTH

A

E .I

E 10 .. a, =L - I > n 5 3

0 C E C E C E

P NS NS P < .02

1-39

PTH

T

C E

NS

FIGURE- 1

t f f e c t s of 1,25-cIihydroxy vitamin D3 ( 1 , 2 5 D j ) and pa ra thy ro id hormone ( P T H ) , s i n g l y and i n combination, on a b s o l u t e phosphate e x c r e t i o n ( U p V ) . C , E = c o n t r o l , experimentdl pe r iods , respec- t i v e l y .

The serum phosphate concen t r a t ion (Pp) was iunalter-ed by 1 ,25 D3,

e i t h e r a lone o r i n combination w i t h bPTH ( P :> .Ot i and -> .20, r e s p e c t i v e l y ,

t a b l e I ) , bu t was e l e v a t e d from a con t ro l va lue of 6 . 6 : 0.4 mg'6 t o 10 .4 i

-1.6 mg% ( P .025) i n those r a t s t h a t rece ived t h e bPTH a lone . None o f

the levels o f GFR f o r any of t h e exper imenta l groups d i f f e r e d s i g n i f i c a n t l y

from the group I ( c o n t r o l ) r a t s ( t a b l e I ) .

A comparison of va lues f o r t h e D-de f i c i en t i ( - D ) animals and those

supplemented w i t h t h e v i tamin (+D) showed d i f f ' e rences f o r b o t h serum Ca

zind P.

5.2 mgZ ( P < .001) .

f o r - D ( P < .02).

For +D, mean serum Ca was 9.8 f 0.1 mg": and for ' -D r a t s i t was

Seruni P was 8.6 t 0.2 ma% f o r +D and 9.6 t 0 . 2 mg%

~~ DISCUSSION ______

In agreement wi th ou r prev ious s t u d i e s ( 5 ) , t h e s e da t a v e r i f y

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TABL

E I

MEAN

VAL

UES

FOR

SERU

M PH

OSPH

OROU

S AN

D CA

LCIU

M A

ND F

OR G

LOME

RULA

R FI

LTRA

TION

RA

TE I

N D-

DEFI

CIEN

T, TH

YROP

ARAT

HYRO

IDEC

TOMI

ZED

RATS

.

Gro

up*

N pp

**

P PC

a**

P GF

R**

P ml

/mi n

mg

%

mg%

I C

ontr

ol

8 6.

6 t

0.4

6.4 f 0

.5

1.59

f 0

.10

I1

1,25

03

8 8.

0 t

0.6

NS

5.4 f

0.3

NS

2.27

r 0

.70

NS'

111

1,25

D3

+ PT

H 10

7.

6 t

0.6

NS

6.2

f 0

.4

NS

1.27

f

0.13

NS

IV

PTH

9 10

.4 t

1.

2 <

.02

5.3 f 0

.5

NS

1.98

f 0

.?8

NS

* R

ats

wer

e gi

ven

eith

er 0

.1

U (.

0025

Fig

, 6

pmol

es)

1,25

-dih

ydro

xy

vita

min

D3

per

hour

fo

r 6

hour

s, o

r 0.

2 U

para

thyr

oid

horm

one

(PTH

, In

olex

, hi

ghly

pur

ifie

d bo

vine

hor

- m

one)

per

hou

r fo

r si

x ho

urs,

or

both

.

tion

rat

e.

** P

p, PC

a =

seru

m p

hosp

hate

and

cal

cium

val

ues,

res

pect

ivel

y; G

FR =

glo

mer

ular

fil

tra-

0

B

0 i2 H !z W

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V I T A M I N D AND THE K I D N E Y

an antiphosphaturic e f f e c t of 1,25 D3 - in the TPTX r a t on1.y when given

in c:ombination with a permissive dose of bPTH. However, the quant i ty

of the metabolite employed i s only one-tenth of the amount t h a t we have

previously u t i l i z e d , a n d represents the smallest dose of a vitamin D

metabolite thus f a r shown t o enhance reabsorption.

e f f e c t was not re la ted t o hemodynamic a l t e r a t i o n s s ince inul in excret ion

141

Furthermore, the 1,25 D3

was not changed.

The question as t o the so-called physiological dose of the vitamin D

metabolites in terms of the r renal tubular e f f e c t s i s a d i f f i c u l t one.

Bonjour and h i s colleagues ound t h a t i f they parathyroidectomized r a t s ,

they were able t o enhance the a b i l i t y of the i n t e s t i n e t o absorb phosphate

e i t h e r with a s ing le intravenous dose of 120 pmoles o f 1,25 D3 or by in-

j e c t i n g 13 pmoles twice da i ly f o r 11 days (a t o t a l of 286 pmoles) ( 7 ) . A

s imi la r dose (13 pmoles twiice da i ly f o r 8 days) was found t o res tore in-

t e s t i n a l calcium absorption i n TPTX rat.s (8) and was subsequently u t i l i z e d

f o r s tud ies of renal phosphate handling ( 9 ) . Hartenbower,et a1 (10) have

determined t h a t 12.5 nanograms (or approximately 30 pmoles,) ( 1 1 ) per day

administered t o D-deficient r a t s res tores serum calcium t o normal. There-

f o r e , the amount of the metabolite which we administered, selected because

i t represented one-tenth o f our or iginal dose, appears t o be in the same

range as those u t i l i z e d t o r e s t o r e the i n t e s t i n a l arid bone e f f e c t s of

vitamin D. Whether these ( l a t t e r ) act ions of the vitamin and those on

renal tubular t r a n s p o r t a re subserved by the same "physiological" dose i s ,

of course, problematical. Thus, whether even smaller amounts of 1,25 D 3

would have the same renal t ranspor t e f f e c t s as those we have seen with

the cur ren t a s well a s the higher dose ( 5 ) , i s n o t possible t o determine

a t present .

Only in t h a t group (of r a t s given the bPTH alone did there occur any

change i n the serum phosphate values, s imi la r t o our previous s tud ies ( 4 ) .

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142 GEORGAKI AND PUSCHETT

We presume, as before, t h a t t h i s represents a bone e f f e c t o f the hormone,

even i n the absence o f an a c t i o n on tubu la r t ranspor t .

were fas ted du r ing the i n f u s i o n , a gut e f f e c t seems u n l i k e l y .

Since these r a t s

I n summary, we have demonstrated t h a t as l i t t l e as 6 pmoles (o r

2.5 ng, o r 0.1 u n i t s ) o f 1,25 D3/hour f o r s i x hours reduces phosphate

e x c r e t i o n i n the TPTX, D -de f i c ien t r a t , when i t s admin i s t ra t i on i s com-

b ined w i t h the i n f u s i o n o f a permissive dose o f bPTH.

tempting t o c a l l t h i s amount o f the me tabo l i t e a phys io log i ca l dose,

f u r t h e r s tud ies w i l l be requ i red t o determine the accuracy o f t h i s

statement.

While i t i s

ACKNOWLEDGEMENT

These s tud ies were supported ( i n p a r t ) by the Veterans Admin is t ra t ion.

A p o r t i o n o f these data were presented t o the V I I I t h I n t e r n a t i o n a l Con-

gress o f Nephrology, Athens, Greece, June 7-12, 1981.

D r . Jules B. Puschett, 1191 Scai fe Ha l l , U n i v e r s i t y o f P i t t sbu rgh ,

P i t tsburgh, PA 15261.

Correspondence:

REFERENCES

1.

2.

3.

4.

Puschett, J.B., Moranz, J. and Kurnick, W.S., J. C l i n .

Invest . , z: 373, j972.

Puschett, J.B., Fernandez, P.D., Boyle, I.T., Gray, R.W.,

Omdahl, J.L. and DeLuca, H.F . , Proc. SOC. Exp. B i o l . Med.,

141: 379, 1972.

Popovtzer, M.M., Robinette, J.B., DeLuca, H.F. and Ho l i ck ,

J., Cl in. I n v e s t . 53: 913, 1974.

Puschett, J.B., Beck, W.S. and Jelonek, A . , Science 190: 473,

1975.

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VITAMIN D AND THE K I D N E Y 143

5. P u s c h e t t , J.B. and k'uhi-irian, M.S., J . Lab. C l i n . I4c.d. 92.: 895,

1978.

6. Co t love , E . , J . Appl. P h y s i o l . 16 : 764, 1961.

7. R i z r o l i , R . , F l e i s c h , H . and Ronjour , J . - P . , J . C l i n . I n L C s t .

60: 639, 1977.

8. R i z z o l i , R . , F l e i s c h H . and Bonjour , J . -P . , A m . J . P h y s i o l .

~ 233: E160, 1977.

9 . Bonjour , J .-P. , P r c c t o n , C . and F l c i s c h , H., ,I. C l i n . I l lvest .

- 60: 1419, 1977.

10. Har tenbower , D . L . , S t a n l e y , T.M., Coburn, J.W. ancl Norman,

A . W . , i n Vitamin D: Blochemi( a l , ChZiniciL and C l i n i c a l As- p e c t s R e l a t e d t o Calcium ~ Metabolism, ~~ ~ e d i t - d by 4.N. Noriiian,

e t a l , p. 587, Wal tc r de G r u y t e r , G e r l i n , 1977.

11 . Norman, A.W., J . Vutr. 102: ~- 1143, 1972.

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