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Increased intake of phosphorus induces depression of proximal tubular function in young rats
Nutrition Research, Vol. 17, No. 5, pp. 831-845, 1997 Copyright © 1997 Elsevier Science inc. Printed in the USA. All fights reserved 0271-5317D7 $17.00 + .00
ELSEVIER
PII S0271-5317(97)00051-1
I N C R E A S E D I N T A K E OF P H O S P H O R U S I N D U C E S D E P R E S S I O N OF P R O X I M A L T U B U L A R F U N C T I O N IN YOUNG R A T S Hiroshi Matsuzaki*l,M.S., M a r i k o Ueharal,Ph.D., K a z u h a r u Suzukil,Ph.D., Q i a o - L i n g Liu2,M.D., S h i g e r u SatoZ,Ph.D., Y u s u k e Kankel,Ph.D. and Shiro Gotol,M.D.,Ph.D. 1 D e p a r t m e n t of Nutrition, F a c u l t y of A g r i c u l t u r e , T o k y o U n i v e r s i t y of A g r i c u l t u r e , S e t a g a y a - k u , T o k y o , 156 J a p a n 2Central I n s t i t u t e for Electron M i c r o s c o p i c R e s e a r c h e s , N i p p o n Medical School, B u n k y o - k u , T o k y o , 113 J a p a n ABSTRACT The s t u d y investigated the effect of i n c r e a s i n g intake of p h o s p h o r u s on p r o x i m a l t u b u l a r f u n c t i o n . Male W i s t a r rats w e r e fed a p u r i f i e d diet c o n t a i n i n g 0.5%, 1.0% and 1.5% p h o s p h o r u s for 21 d a y s . K i d n e y d a m a g e was o b s e r v e d by light and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y . I n d i c a t o r s of k i d n e y f u n c t i o n w e r e e x a m i n e d as c r e a t i n i n e c l e a r a n c e , s e r u m u r e a n i t r o g e n c o n c e n t r a t i o n and u r i n a r y a l b u m i n e x c r e a t i o n . In a d d i t i o n , as i n d i c a t o r s of p r o x i m a l t u b u l a r f u n c t i o n , a c t i v i t y of N - a c e t y l - / 3 - D - g l u c o s a m i n i d a s e (NAG) in u r i n e and u r i n a r y / 3 2 - m i c r o g l o b u l i n e x c r e t i o n w e r e m e a s u r e d . The k i d n e y c a l c i u m c o n c e n t r a t i o n was i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet. K i d n e y calcification was o b s e r v e d in rats fed 1.0% and 1.5% p h o s p h o r u s diets. In rats fed 1.5% p h o s p h o r u s diet, d e p o s i t i o n of h y d r o x y a p a t i t e was o b s e r v e d in the collecting ducts. T h e r e was no u l t r a s t r u c t u r a l d a m a g e in the g l o m e r u l i and distal tubules. H o w e v e r , small v a c u o l e s , s w e l l i n g of the microvilli, l y s o s o m e and u n d e v e l o p e d r o u g h - e n d o p l a s m i c r e t i c u l u m w e r e f o u n d in the p r o x i m a l t u b u l e s . Creatinine clearance, serum urea nitrogen concentration and urinary albumin excretion were i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet. As the i n d i c a t o r s of p r b x i m a l t u b u l a r f u n c t i o n , NAG a c t i v i t y in u r i n e and u r i n a r y /32m i c r o g l o b u l i n e x c r e t i o n w e r e i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet. It was c o n c l u d e d that i n c r e a s e d i n t a k e of p h o s p h o r u s i m p a i r e d p r o x i m a l t u b u l a r function. 0 1997ElsevierSci~'tceInc.
KEY WORDS : P h o s p h o r u s , P r o x i m a l t u b u l a r f u n c t i o n , Rat
* A u t h o r to w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . 831
832
H. MATSUZAKI et al.
INTRODUCTION N e p h r o c a l c i n o s i s is a k i d n e y d i s o r d e r in rats, and r e f e r s to the d e p o s i t i o n of c a l c i u m p h o s p h a t e s m a i n l y in the c o r t i c o m e d u l l a r y j u n c t i o n of the k i d n e y (1). Etiological f a c t o r s of n e p h r o c a l c i n o s i s i n c l u d e sex (2), h o r m o n e s (3,4), s t r a i n (5,6) and age (7). The c o m p o s i t i o n of the diet also p l a y s a m a j o r role in the e t i o l o g y of n e p h r o c a l c i n o s i s , e s p e c i a l l y , calcium, m a g n e s i u m and p h o s p h o r u s are i m p o r t a n t n u t r i e n t s . High p h o s p h o r u s (810), low m a g n e s i u m ( 1 1 - 1 3 ) , and c a l c i u m : p h o s p h o r u s ratios b e l o w 1 ( 1 4 , 1 5 ) in the diet i n d u c e n e p h r o c a l c i n o s i s . Rats with nephrocalcinosis showed increased plasma urea c o n c e n t r a t i o n (16,17). In rats with n e p h r o c a l c i n o s i s , u r i n a r y a l b u m i n e x c r e t i o n was i n c r e a s e d , and was p o s i t i v e l y c o r r e l a t e d with the k i d n e y c a l c i u m c o n c e n t r a t i o n (18). T h e s e r e s u l t s i m p l y that n e p h r o c a l c i n o s i s m a y i m p a i r k i d n e y function. H o w e v e r , o t h e r w o r k e r s h a v e not s h o w n that p l a s m a u r e a c o n c e n t r a t i o n was e l e v a t e d in rats w i t h n e p h r o c a l c i n o s i s (19), and e f f e c t s of n e p h r o c a l c i n o s i s on u r i n a r y p r o t e i n e x c r e t i o n and inulin c l e a r a n c e (20). S c h o e n m a k e r s et al. (21) also r e p o r t e d that d e s p i t e k i d n e y c a l c i f i c a t i o n in rats fed a 0.4% p h o s p h o r u s diet, t h e r e w e r e no c h a n g e s on p l a s m a c r c a t i n i n e and u r e a levels and u r i n a r y c r e a t i n i n e , u r e a and a l b u m i n e x c r e t i o n . Thus, a l t h o u g h t h e r e h a v e b e e n m a n y s t u d i e s of k i d n e y f u n c t i o n in rats with n e p h r o c a l c i n o s i s , the details r e m a i n unclear. F u r t h e r m o r e , only a few a t t e m p t s h a v e b e e n m a d e so far to e x a m i n e p r o x i m a l tubuLar f u n c t i o n in k i d n e y s with high p h o s p h o r u s d i e t - i n d u c e d n e p h r o c a l c i n o s i s . In this s t u d y , rats w e r e fed a p u r i f i e d diet c o n t a i n i n g 0.5%, 1.0% and 115% phosphorus. The effect of i n c r e a s i n g i n t a k e of p h o s p h o r u s on p r o x i m a l t u b u l a r f u n c t i o n was e x a m i n e d using b i o c h e m i c a l i n d i c a t o r s and histological obscrvation. M A T E R I A L S AND METHODS
Experimental
d i e t s , a n i m a l s and h o u s i n g
The c o m p o s i t i o n of the e x p e r i m e n t a l diets is s h o w n in Table 1. M i n e r a l m i x t u r e was a m o d i f i c a t i o n of A I N - 7 6 m i n e r a l m i x t u r e (22) w i t h o u t calcium, m a g n e s i u m and p h o s p h o r u s , w h i l e the A I N - 7 6 A v i t a m i n m i x t u r e (23) was u s e d intact. P h o s p h o r u s was a d d e d to the p u r i f i e d diets at c o n c e n t r a t i o n s of 0.5%, 1.0% and 1.5% using p o t a s s i u m t r i p o l y p h o s p h a t e ( N A C A L A I TESQUE, INC., Kyoto, Japan). The c a l c i u m and m a g n e s i u m c o n c e n t r a t i o n in all p u r i f i e d diets w e r e also a d d e d 0.5% and 0.05% using c a l c i u m c a r b o n a t e (Kanto c h e m i c a l Co., INC., Tokyo, J a p a n ) and m a g n e s i u m oxide ( W a k o p u r e c h e m i c a l i n d u s t r i e s , Ltd., Osaka, J a p a n ) r e s p e c t i v e l y . The
PHOSPHORUS AND KIDNEY FUNCTION
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p u r i f i e d d i e t s w e r e s t o r e d at 4°C u n t i l use. TABLE 1 C o m p o s i t i o n of t h e E x p e r i m e n t a l
Diets
0.5% phosphorus
1.0% phosphorus
1.5% phosphorus
diet
diet
diet
Ingredient
g/100g
diet
Casein, milk
20.00
20.00
20.00
Starch, corn
15.00
15.00
15.00
Cellulose powder
5.00
5.00
5.00
Oil, c o r n
5.00
5.00
5.00
Mineral mixture1
1.66
1.66
1.66
Vitamin mixture 2
1.00
1.00
1.00
DL-Methionine
0.30
0.3(l
0.30
Choline bitartrate
0.20
0.2(l
0.20
K~P30]o
1.92
4.34
6.75
MgO
0.08
0.(I 8
0.08
CaCO3
1.25
1.25
1.25
48.59
46.17
43.76
Saccharose
Total 100.00 100.00 100.00 1 This m i n e r a l m i x t u r e is a m o d i f i c a t i o n of AIN-76 m i n e r a l m i x t u r e w i t h o u t c a l c i u m , m a g n e s i u m a n d p h o s p h o r u s sources. 2 AIN-76A v i t a m i n m i x t u r e . F i v e - w e e k - o l d m a l e W i s t a r r a t s (Clea J a p a n . , T o k y o , J a p a n ) w e r e u s e d for t h e e x p e r i m e n t . T h e r a t s w e r e h o u s e d in i n d i v i d u a l s t a i n l e s s s t e e l cages. During the e x p e r i m e n t , t h e r a t c a g e s w e r e l o c a t e d in a r o o m w i t h a c o n t r o l l e d 12h light : d a r k cycle (light, 0 8 0 0 - 2 0 0 0 h ) , temperaturc (22+1°C) a n d h u m i d i t y ( 6 0 - 6 5 % ) . The s t u d y was a p p r o v e d by the T o k y o U n i v e r s i t y of A g r i c u l t u r e A n i m a l Use C o m m i t t e e , a n d a n i m a l s w e r e m a i n t a i n e d in a c c o r d a n c e w i t h g u i d e l i n e s for t h e c a r e a n d u s e of l a b o r a t o r y a n i m a l s of T o k y o U n i v e r s i t y of A g r i c u l t u r e . All r a t s r e c e i v e d a p r e - e x p e r i m e n t a l diet c o n t a i n i n g 0.5% c a l c i u m , 0 . 0 5 % m a g n e s i u m a n d 0.5% p h o s p h o r u s ( T a b l e 1). Diet a n d d e m i n e r a l i z e d w a t e r w e r e f r e e l y a v a i l a b l e to r a t s . A f t e r a 7 d a y s pre-experimental p e r i o d , t h e r a t s w e r e d i v i d e d i n t o 3 g r o u p s of 6 r a t s e a c h on d a y 0 of t h e e x p e r i m e n t . M e a n b o d y w e i g h t s of t h e 3 g r o u p s w e r e
834
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similar. Each g r o u p was r a n d o m l y a s s i g n e d to one of the p u r i f i e d diets. fed 0.5% and 1.0% p h o s p h o r u s diet w e r e g i v e n a m o u n t s e q u i v a l e n t to c o n s u m e d by the rats fed 1.5% p h o s p h o r u s diet for 21 days. Rats had a c c e s s to d e m i n e r a l i z e d w a t e r t h r o u g h o u t the e x p e r i m e n t . Food i n t a k e b o d y w e i g h t w e r e r e c o r d e d daily.
Collection
of samples
and chemical
Rats that frec and
analyses
The rats h o u s e d i n d i v i d u a l l y in m e t a b o l i s m cages. Before d i s s e c t i o n , rats w e r e not fed and u r i n e was collected for 2 4 - h . At the end of the e x p e r i m e n t , the rats w e r e killed by d e c a p i t a t i o n . Blood was collected and c e n t r i f u g e d to s e p a r a t e the s e r u m . Both k i d n e y s w e r e r e m o v e d and w e i g h e d a f t e r the c a p s u l e s w e r e d i s c a r d e d . The right k i d n e y was used for chemical analysis. The left k i d n e y was used for h i s t o p a t h o l o g i c a L e x a m i n a t i o n . All s a m p l e s w e r e s t o r e d at - 4 0 ° C u n t i l a n a l y s i s . The r i g h t k i d n e y was d r i e d o v e r n i g h t at 100°C, and the d r y w e i g h t was m e a s u r e d . T h e k i d n e y was t h e n a s h e d at 5 5 0 ° C for 48h and the m i n e r a l s w e r e e x t r a c t e d for a n a l y s i s by 1 m o l / L HCI solution. Calcium in the k i d n e y was a n a l y z e d by a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y ( S h i m a d z u A A - 6 4 0 13) (24). P h o s p h o r u s in the k i d n e y was a n a l y z e d a c c o r d i n g to the m e t h o d of Gomori (25). C r e a t i n i n e in s e r u m and u r i n e was m e a s u r e d u s i n g the C r e a t i n i n e - T E S T Wako. Urea n i t r o g e n in s e r u m was m e a s u r e d using N y o s o t i s o - T E S T W a k o (all W a k o p u r e c h e m i c a l i n d u s t r i e s , Ltd., Osaka, Japan). The a c t i v i t y of N - a c e t y l - , 8 - D - g l u c o s a m i n i d a s e (NAG) in u r i n e was m e a s u r e d u s i n g the NAG TEST Shionogi (Shionogi & Co., Ltd., Osaka, Japan). U r i n a r y a l b u m i n and / 3 2 - m i c r o g l o b u l i n e x c r e t i o n w e r e m e a s u r e d u s i n g Rat a l b u m i n EIA s y s t e m and Rat / 3 2 - m i c r o g l o b u l i n EIA s y s t e m (all A m e r s h a m KK, T o k y o , Japan).
Histological
examination
Light m i c r o s c o p y The left k i d n e y was fixed in 10% n e u t r a l f o r m a l i n p h o s p h a t e b u f f e r . For h i s t o p a t h o l o g i c a l e x a m i n a t i o n , 5 /.zm thick l o n g i t u d i n a l s e c t i o n s of k i d n e y w e r e s t a i n e d w i t h h e m a t o x y l i n - e o s i n and Von Kossa's. Transmission electron microscopy In rats fed 1.5% p h o s p h o r u s diet, the left k i d n e y was fixed with 2.5% g l u t a r a l d e h y d e and p o s t - f i x e d with 1% o s m i u m t e t r o x i d e . The t i s s u e s a m p l e s w e r e d e h y d r a t e d t h r o u g h a g r a d e d alcohol series and e m b e d d e d in
PHOSPHORUS AND KIDNEY FUNCTION
835
Epok 812. U l t r a t h i n s e c t i o n s w e r e cut on a R e i h e r t - N i s s e i U l t r a c u t N u l t r a m i c r o t o m e w i t h a d i a m o n d knife, and s t a i n e d with u r a n y l a c e t a t e and lead citrate. The s e c t i o n s w e r e e x a m i n e d u n d e r an Hitachi H - 8 0 0 ( 7 5 k V ) electron microscope.
Statistical analysis The data was e x p r e s s e d as the m e a n s - + S E M . Statistical a n a l y s i s was p e r f o r m e d by o n e - w a y a n a l y s i s of v a r i a n c e (ANOVA) (26). D i f f e r e n c e s w e r e c o n s i d e r e d s i g n i f i c a n t w h e n the P v a l u e was <0.05. RESULTS
Body w e i g h t and w e i g h t gain Final b o d y w e i g h t and p h o s p h o r u s levels (Table 2).
weight
gain
were
not a f f e c t e d
by
dietary
TABLE 2 G r o w t h in Rats Fed Diets C o n t a i n i n g V a r i o u s L e v e l s of P h o s p h o r u s ] 0.5% phosphorus diet Body w e i g h t (g) Initial Final W e i g h t gain (g/day)
134 + 1 148 __+4 0.67 _+0.22
1.0% phosphorus diet
1.5% phosphorus diet
136 + 2 147 + 5
133 +1 149 _+6
0.51 __+0.23
0.77 + 0 . 2 4
1 Values are m e a n s _ S E M for six rats.
K i d n e y w e i g h t and c a l c i u m and p h o s p h o r u s c o n c e n t r a t i o n s Wet and d r y w e i g h t s of k i d n e y w e r e h i g h e r in rats fed 1.5% p h o s p h o r u s diet t h a n in rats fed 0.5% and 1.0% p h o s p h o r u s diets (Fig. 1A). Ki'dney c a l c i u m and p h o s p h o r u s c o n c e n t r a t i o n s w e r e also i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet (Fig. 1B).
Histological examination Light m i c r o s c o p y o b s e r v a t i o n
of k i d n e y
836
H. MATSUZAKI et al.
D e p o s i t i o n of c a l c i u m in the c o r t i c o m e d u l l a r y j u n c t i o n was o b s e r v e d in rats fed 1.0% and 1.5% p h o s p h o r u s diets, but rats fed 1.5% p h o s p h o r u s diet w a s s h o w n d e p o s i t i o n of c a l c i u m in the m e d u l l a as well. Deposits w e r e slight in rats fed 1.0% p h o s p h o r u s diet and s e v e r e in t h o s e fed 1.5% p h o s p h o r u s diet (Fig. 2). Electron m i c r o s c o p y o b s e r v a t i o n In rats fed 1.5% p h o s p h o r u s diet, the u l t r a s t r u c t u r a l d a m a g e was not f o u n d in g l o m e r u l i and distal t u b u l e s (Fig. 3, 5), but the m i c r o v i l l i d i s a p p e a r e d in s o m e of the distal t u b u l e s (Fig. 5). Small v a c u o l e s , s w e l l i n g of the microvilli, l y s o s o m e and u n d e v e l o p e d r o u g h - e n d o p l a s m i c r e t i c u l u m w e r c f o u n d in p r o x i m a l t u b u l e s (Fig. 4). D e p o s i t i o n of h y d r o x y a p a t i t e was f o u n d in the collecting ducts (Fig. 6).
200
1.2
~150 (1.8 ~O
100 0.4
E 50 E 0
0 Wet
[]
Dry
(I.5% p h o s p h o r u s diet
[]
Calcium 1.0% p h o s p h o r u s diet
[]
Phosphorus
1.5% p h o s p h o r u s diet
FIG. 1 K i d n e y w e i g h t (A) and c a l c i u m and p h o s p h o r u s c o n c e n t r a t i o n (B) in rats fed d i e t s c o n t a i n i n g v a r i o u s levels of p h o s p h o r u s . V a l u e s are m e a n s + _ S E M for six rats. M a t c h i n g s u p e r s c r i p t l e t t e r s d e n o t e s i g n i f i c a n t d i f f e r e n c e s , P<0.05. Indicators
of kidney
function
C r e a t i n i n e c l e a r a n c e was h i g h e r in rats fed 1.5% p h o s p h o r u s diet t h a n in rats fed 0.5% and 1.(1% p h o s p h o r u s diets (Fig. 7A). S e r u m u r e a n i t r o g e n c o n c e n t r a t i o n was i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet (Fig. 7B). U r i n a r y a l b u m i n e x c r e t i o n was h i g h e r in rats fed 1.5% p h o s p h o r u s diet c o m p a r e d with t h o s e in rats fed 0.5% and 1.0% p h o s p h o r u s diets (Fig. 7C).
PHOSPHORUS AND KIDNEY FUNCTION
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FIG. 2 Kidney s e c t i o n f r o m a rat fed diets c o n t a i n i n g v a r i o u s levels of p h o s p h o r u s s h o w n by Light microscopy. A : The I).5% p h o s p h o r u s dict did not s h o w d e p o s i t i o n of calcium. Von Kossa's s t a i n e d section, m a g n i f i c a t i o n x 1 8 . : The 1.0% p h o s p h o r u s diet showed slight d e p o s i t i o n of calcium (arrows). Von Kossa's s t a i n e d section, m a g n i f i c a t i o n x17. B
C : The 1.5% p h o s p h o r u s dict s h o w e d s e v e r e d e p o s i t i o n of calcium (arrows). Von Kossa's s t a i n e d section, m a g n i f i c a t i o n X17.
838
H. MATSUZAKI et al.
FIG. 3 G l o m e r u l a r c a p i l l a r i e s (Cap), m e s a n g i u m cells (Me) and p o d o c y t e (Po) in a rat fed 1.5% p h o s p h o r u s diet. T h e r e was no u l t r a s t r u c t u r a l d a m a g e .
FIG, 4 P r o x i m a l t u b u l e in a rat fed 1.5% p h o s p h o r u s diet s h o w e d small vacuoles (V), partial swelling of the microvilli (arrows), lysosome and a small amount of roughendoplasmic reticulum.
PHOSPHORUS AND KIDNEY FUNCTION
839
FIG. 5 Distal t u b u l e in a rat fed 1.5% phosphorus diet showed m i t o c h o n d r i a and w e l l developed rough-endoplasmic r e t i c u l u m ( a r r o w s ) . T h e r e was no u l t r a s t r u c t u r a [ d a m a g e , but the microvilli had partly d i s a p p e a r e d ( a r r o w heads).
FIG. 6 Collecting duct in a rat fed 1.5% phosphorus diet showed d e p o s i t i o n of h y d r o x y a p a t i t e ( a r r o w ) . I n s e r t was m a g n i f i e d deposition ofhydroxapatite.
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FIG. 7 Creatinine clearance (A), serum urea nitrogen concentration (B), u r i n a r y albumin cxcretion (C), a c t i v i t y o f N - a c e t y l - / 3 - D - g l u c o s a m i n i d a s e ( N A G ) in urine (D) a n d u r i n a r y /32-microglobulin excretion (El in r a t s f e d d i e t s containing various levels of phosphorus. Values are means Matching superscript letters denote significant differences,
+ S E M f o r six r a t s . P<0.05.
PHOSPHORUS AND KIDNEY FUNCTION
841
The NAG a c t i v i t y in u r i n e and u r i n a r y / f 2 - m i c r o g l o b u l i n e x c r e t i o n w e r e h i g h e r in rats fed 1.5% p h o s p h o r u s diet t h a n in rats fed 0.5% p h o s p h o r u s diet (Fig. 7D, E). DISCUSSION
A high p h o s p h o r u s d i e t - i n d u c e d n e p h r o c a l c i n o s i s o b s e r v e d m a i n l y at the c o r t i c o m e d u l l a r y j u n c t i o n (8). In this s t u d y , the s a m e r e s u l t was o b s e r v e d , n a m e l y , d e p o s i t i o n of c a l c i u m in rats fed 1.0% and 1.5% p h o s p h o r u s diets was f o u n d in the c o r t i c o m e d u l l a r y j u n c t i o n . H o w e v e r , thc d e p o s i t s of rats fed 1.5% p h o s p h o r u s diet w e r e not only f o u n d in the c o r t i c o m e d u l l a r y j u n c t i o n but also in the m e d u l l a . This r e s u l t s u g g e s t s that the distribution of c a l c i u m d e p o s i t i o n might differ by d e g r e e of n e p h r o c a l c i n o s i s . This d e p o s i t s c o n s t i t u t e d m a i n l y c a l c i u m and p h o s p h o r u s (20,27). The ratio of t h e s e e l e m e n t s in d e p o s i t s was s i m i l a r to the v a l u e f o u n d in h y d r o x y a p a t i t e (20). In this s t u d y , in rats fed 1.5% p h o s p h o r u s diet, d e p o s i t i o n of h y d r o x y a p a t i t e in the collecting d u c t s was o b s e r v e d by electron microscopy. It is i m p l i e d that high p h o s p h o r u s d i e t - i n d u c e d m i n e r a l d e p o s i t i o n is h y d r o x y a p a t i t e . I n c r e a s e of m i n e r a l d e p o s i t s in the k i d n e y m a y c a u s e i n c r e a s e of k i d n e y weight. Kidney w e i g h t was i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet, this is in a g r e e m e n t with o t h e r i n v e s t g a t o r s (18,28). I n c r e a s e of k i d n e y w e i g h t in rats with n e p h r o c a l c i n o s i s was m a i n l y r e s p o n s i b l e for i n c r e a s e of m i n e r a l c o n c e n t r a t i o n in the k i d n e y (18). I n c r e a s e of wet k i d n e y weight also r e l a t e to w a t e r and mineral c o n c e n t r a t i o n s in the k i d n e y (29). Rats fed 1.5% p h o s p h o r u s diet i n d u c e d i n c r e a s e d u r i n a r y a l b u m i n e x c r e t i o n . R i t s k e s - H o i t i n g a et al. (18) and Van Camp et al. (3(I) r e p o r t c d that u r i n a r y a l b u m i n e x c r e t i o n was i n c r e a s e d in rats with n e p h r o c a l c i n o s i s , and u r i n a r y a l b u m i n e x c r e t i o n was p o s i t i v e l y c o r r e l a t e d with the k i d n e y c a l c i u m c o n t e n t . I n c r e a s e of u r i n a r y a l b u m i n e x c r e t i o n was c a u s e d by a d e f e c t in the p e r m e a b i l i t y of the r e n a l g l o m e r u l a r b a s e m e n t m e m b r a n e , and the i m p o r t a n c e of the size b a r r i e r or c h a r g e b a r r i e r is k n o w n (31). In this s t u d y , g l o m e r u l a r b a s e m e n t m e m b r a n e in rats fed 1.5% p h o s p h o r u s diet did not s h o w u l t r a s t r u c t u r a l d a m a g e , s u g g e s t i n g that the i n c r e a s e in u r i n a r y a l b u m i n e x c r e t i o n m a y not be r e s p o n s i b l e for the d e f e c t in the p e r m e a b i l i t y of the r e n a l g l o m e r u l a r b a s e m e n t m e m b r a n e . A l b u m i n is m a i n l y r c a b s o r b e d in the p r o x i m a l t u b u l e (32). In o t h e r w o r d s , the i n c r e a s e d u r i n a r y a l b u m i n e x c r e t i o n m a y be c a u s e d by the o b s t r u c t i o n of a l b u m i n r e a b s o r p t i o n in the proximal tubule. In r e g a r d to p r o x i m a l t u b u l a r f u n c t i o n in rats with n e p h r o c a l c i n o s i s , rats with n e p h r o c a l c i n o s i s s h o w e d i n c r e a s e d NAG a c t i v i t y in u r i n e (33). In this s t u d y , NAG a c t i v i t y in u r i n e was i n c r e a s e d in rats fcd 1.5% p h o s p h o r u s diet. In a d d i t i o n , the d a m a g e s of p r o x i m a l t u b u l e s in rats
842
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fed 1.5% p h o s p h o r u s diet w e r e f o u n d by e l e c t r o n m i c r o s c o p y . T h e s e r e s u l t s i n d i c a t e d e p r e s s i o n of p r o x i m a l t u b u l a r f u n c t i o n . M o r e o v e r , u r i n a r y /32m i c r o g l o b u l i n e x c r e t i o n i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet. This r e s u l t i m p l i e s that r e a b s o r p t i o n in the p r o x i m a l t u b u l e is d e p r e s s e d . It is s u g g e s t e d that the i n c r e a s e in u r i n a r y a l b u m i n e x c r e t i o n in rats fed 1.5% p h o s p h o r u s diet m a y be due to the o b s t r u c t i o n of p r o x i m a l t u b u l a r a l b u m i n reabsorption. S e r u m u r e a n i t r o g e n c o n c e n t r a t i o n was i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet. P l a s m a u r e a was i n c r e a s e d in rats w i t h n e p h r o c a l c i n o s i s (16), and S c h a a f s m a et al. (17) r e p o r t e d that p l a s m a u r e a c o n c e n t r a t i o n s h o w e d p o s i t i v e c o r r e l a t i o n w i t h k i d n e y c a l c i u m c o n t e n t . R i t s k e s - H o i t i n g a ct al. (18) also c o n c l u d e d that the i n c r e a s e in p l a s m a u r e a in rats with nephrocalcinosis may c a u s e the enhanced reabsorption of u r e a by p r o l o n g a t i o n of t r a n s i t time in single n e p h r o n s . In c o n c l u s i o n , the u l t r a s t r u c t u r a l d a m a g e s w e r e o b s e r v e d in p r o x i m a l t u b u l e s of rats fed 1.5% p h o s p h o r u s diet by e l e c t r o n m i c r o s c o p y , and NAG a c t i v i t y in u r i n e and u r i n a r y / 3 2 - m i c r o g l o b u l i n e x c r e t i o n w e r e i n c r e a s e d . In rats fed 1.5% p h o s p h o r u s diet, u r i n a r y a l b u m i n e x c r e t i o n was i n c r e a s e d , w h e r e a s g l o m e r u l i did not s h o w u l t r a s t r u c t u r a l d a m a g e . It is s u g g e s t e d that i n c r e a s e d i n t a k e of p h o s p h o r u s i n d u c e s d e p r e s s i o n of p r o x i m a l t u b u l a r f u n c t i o n , and it m a y c a u s e an i n c r e a s e of the u r i n a r y a l b u m i n e x c r e t i o n . REFERENCES
1. S i l v e r m a n J, R i v e n s o n A. N e p h r o c a l c i n o s i s in 2 y o u n g rats: a case r e p o r t . Lab A n i m 1 9 8 0 ; 1 4 : 2 4 1 - 2 . 2. Cousins FB, Geary CPM. A s e x - d e t e r m i n e d r e n a l calcification in rats. Nature 1966;211:980-1. 3. E n g f e l d t B, Rhodin J, S t r a n d h J. S t u d i e s of the k i d n e y u l t r a s t r u c t u r e in h y p e r v i t a m i n o s i s D. Acta Chir Scand 1 9 6 2 ; 1 2 3 : 1 4 5 - 7 . 4. P a r k e r HM, F o r b e s RM. I n f l u e n c e of d i e t a r y calcium, m a g n e s i u m and p h o s p h o r u s on c y c l i c - A M P e x c r e t i o n and k i d n e y calcification in the rat. Proc Soc Exp Biol Med 1 9 7 6 ; 1 5 1 : 2 1 5 - 2 0 . 5. Du B r u y n DB.A c o m p a r i s o n of c e r t a i n rat s t r a i n s with r e s p e c t to s u s c e p t i b i l i t y to n e p h r o c a l c i n o s i s . S - A f t Med J 1 9 7 0 ; 4 4 : 1 4 1 7 - 8 .
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6. R i t s k e s - H o i t i n g a J, M a t h o t JNJJ, Van Z u t p h c n LFM, B c y n e n AC. I n b r e d s t r a i n s of r a t s h a v e d i f f e r e n t i a l s e n s i t i v i t y to d i e t a r y p h o s p h o r u s induced nephrocalcinosis. J Nutr 1992;122:1682-92. 7. Zalups RK. Age and the d e v e l o p m e n t of n e p h r o c a l c i n o s i s i n d u c e d by i n j e c t i o n s of n e u t r a l p h o s p h a t e in rats. A c t a M o r p h o l H u n g a r i c a 1984;32: 121-5. 8. M a c k a y EM, Oliver J. Renal d a m a g e f o l l o w i n g the i n g e s t i o n of a diet c o n t a i n i n g an e x c e s s of i n o r g a n i c p h o s p h a t e . J Exp Med 1 9 3 5 ; 6 1 : 3 1 9 - 3 3 . 9. H i t c h m a n AJ, H a s a n y SA, H i t c h m a n A, H a r r i s o n JE, T a m C. P h o s p h a t e i n d u c e d r e n a l c a l c i f i c a t i o n in the rat. Can J Physiol P h a r m a c o l 1 9 7 9 ; 5 7 : 92-7. 10. M a s u y a m a R, U e h a r a M, Suzuki K, Goto S. The action of m a g n e s i u m r e d u c i n g r e n a l c a l c i f i c a t i o n in r a t s r e c e i v i n g high p h o s p h o r u s s u p p l e m e n t e d diet. N u t r Res 1 9 9 5 ; 1 5 : 1 6 7 3 - 8 2 .
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11. Bunce GE, S a u b e r l i c h HE, R e e v e s PG, Oba TS. D i e t a r y p h o s p h o r u s and m a g n e s i u m d i f i c i e n c y in the rat. J N u t r 1 9 6 5 ; 8 6 : 4 0 6 - 1 4 . 12. H a r w o o d EJ. The i n f l u e n c e of d i e t a r y m a g n e s i u m on r e d u c t i o n of n e p h r o c a l c i n o s i s in r a t s fed p u r i f i e d diet. Lab A n i m 1 9 8 2 ; 1 6 : 3 1 4 - 1 8 . 13. P a r k e r HE. M a g n e s i u m a n d p h o s p h o r u s i n t e r r e l a t i o n s h i p s at d e f i c i e n t to n o r m a l d i e t a r y levels. N u t r Rep Int 1 9 8 5 ; 3 2 : 9 8 3 - 9 0 . 14. Clapp MJL. The e f f e c t of diet on s o m e p a r a m e t e r s m e a s u r e d t o x i c o l o g i c a l s t u d i e s in the rat. Lab A n i m 1 9 8 0 ; 1 4 : 2 5 3 - 2 6 1 .
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15. Clapp MJL, W a d e JD, S a m u e l s DM. Control of n e p h r o c a l c i n o s i s by m a n i p u l a t i n g the calcium: P h o s p h o r u s r a t i o in c o m m e r c i a l r o d e n t diets. Lab A n i m 1 9 8 2 ; 1 6 : 1 3 0 - 2 . 16. K a u n i t z H, J o h n s o n E. D i e t a r y p r o t e i n , fat, and m i n e r a l s in n e p h r o c a l c i n o s i s in f e m a l e rats. M e t a b o l i s m 1 9 7 6 ; 2 5 : 6 9 - 7 7 . 17. S c h a a f s m a G, D u u r s m a SA, V i s s e r WJ, D e k k e r PR. The i n f l u e n c e of d i e t a r y c a l c i u m on k i d n e y c a l c i f i c a t i o n and r e n a l f u n c t i o n in r a t s fed h i g h p h o s p h a t e diets. Bone 1 9 8 5 ; 6 : 1 5 5 - 6 3 .
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18. R i t s k c s - H o i t i n g a J, L e m m e n s AG, Danse LHJC, B e y n e n AC. P h o s p h o r u s i n d u c e d n e p h r o c a l c i n n s i s and k i d n e y f u n c t i o n in f e m a l e rats. J N u t r 1989;1 I 9 : 1 4 2 3 - 3 1 . 19 S t r u t h e r s BJ, D a h l g r e n RR, H o p k i n s DT. Biological e f f e c t s of f e e d i n g g r a d e d levels of alkali t r e a t e d s o y b e a n p r o t e i n c o n t a i n i n g l y s i n o a l a n i n e ( N - 2 - c a r b o x y e t h y l - L - l y s i n e ) in S D a n d W i s t a r rats. J Nutr 1 9 7 7 ; i 0 7 : 1 190-9. 20 S t o n a r d MD, S a m u e l s DM, L~ck EA. The p a t h o g e n e s i s and effect nn renal f u n c t i o n of n c p h r o c a l c i n o s i s i n d u c e d by d i f f e r e n t diets in f e m a l e rats. Fd Chem Toxic 1 9 8 4 ; 2 2 : 1 3 9 - 4 6 . 21
S c h o e n m a k e r s ACM, R i t s k e s - H o i t i n g a J, L e m m e n s AG, B e y n e n AC. l n f l u c n c e of d i e t a r y p h o s p h o r u s r e s t r i c t i o n on c a l c i u m and p h o s p h o r u s m e t a b o l i s m in rats. I n t e r n a t J Vit Nutr Res 1 9 8 9 ; 5 9 : 2 0 0 - 6 .
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29. M a t s u z a k i H, Arai K, U e h a r a M, Suzuki K, Sato S, K a n k e Y, Goto S. I n c r e a s i n g m a g n e s i u m i n t a k e p r e v e n t s high p h o s p h o r u s d i e t - i n d u c e d k i d n e y d a m a g e in y o u n g rats. N u t r Res (in p r e s s ) . 30. V a n C a m p I, R i t s k e s - H o i t i n g a J, L e m m e n s AG, B e y n e n AC. D i e t - i n d u c e d n e p h r o c a l c i n o s i s a n d u r i n a r y e x c r e t i o n of a l b u m i n in f e m a l e rats. Lab A n i m 1 9 9 0 ; 2 4 : 1 3 7 - 4 1 . 31. Olson JL, R e n n k e HG, V e n k a t a c h a l a m MA. A l t e r a t i o n s in the c h a r g e and size s e l e c t i v i t y b a r r i e r of the g l o m e r u l a r filter in a m i n o n u c l e o s i d e n e p h r o s i s in rats. Lab I n v e s t 1 9 8 1 ; 4 4 : 2 7 1 - 9 . 32. B o u r d e a u JE, C a r o n e FA, G a n o t e CE. S e r u m a l b u m i n u p t a k e in i s o l a t e d p e r f u s e d r e n a l t u b u l e s . J Cell Biol 1 9 7 2 ; 5 4 : 3 8 2 - 9 8 . 33. Kang SS, Price RG, Y u d k i n J, W o r c e s t e r NA, B r u c k d o r f e r KR. The i n f l u e n c e of d i e t a r y c a r b o h y d r a t e and fat on k i d n e y c a l c i f i c a t i o n and the u r i n a r y e x c r e t i o n of N - a c e t y l - /3- g l u c o s a m i n i d a s e . Br J N u t r 1 9 7 9 ; 4 1 : 6 5 - 7 1 .
Accepted for publication February 13, 1997.
ELSEVIER
PII S0271-5317(97)00051-1
I N C R E A S E D I N T A K E OF P H O S P H O R U S I N D U C E S D E P R E S S I O N OF P R O X I M A L T U B U L A R F U N C T I O N IN YOUNG R A T S Hiroshi Matsuzaki*l,M.S., M a r i k o Ueharal,Ph.D., K a z u h a r u Suzukil,Ph.D., Q i a o - L i n g Liu2,M.D., S h i g e r u SatoZ,Ph.D., Y u s u k e Kankel,Ph.D. and Shiro Gotol,M.D.,Ph.D. 1 D e p a r t m e n t of Nutrition, F a c u l t y of A g r i c u l t u r e , T o k y o U n i v e r s i t y of A g r i c u l t u r e , S e t a g a y a - k u , T o k y o , 156 J a p a n 2Central I n s t i t u t e for Electron M i c r o s c o p i c R e s e a r c h e s , N i p p o n Medical School, B u n k y o - k u , T o k y o , 113 J a p a n ABSTRACT The s t u d y investigated the effect of i n c r e a s i n g intake of p h o s p h o r u s on p r o x i m a l t u b u l a r f u n c t i o n . Male W i s t a r rats w e r e fed a p u r i f i e d diet c o n t a i n i n g 0.5%, 1.0% and 1.5% p h o s p h o r u s for 21 d a y s . K i d n e y d a m a g e was o b s e r v e d by light and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y . I n d i c a t o r s of k i d n e y f u n c t i o n w e r e e x a m i n e d as c r e a t i n i n e c l e a r a n c e , s e r u m u r e a n i t r o g e n c o n c e n t r a t i o n and u r i n a r y a l b u m i n e x c r e a t i o n . In a d d i t i o n , as i n d i c a t o r s of p r o x i m a l t u b u l a r f u n c t i o n , a c t i v i t y of N - a c e t y l - / 3 - D - g l u c o s a m i n i d a s e (NAG) in u r i n e and u r i n a r y / 3 2 - m i c r o g l o b u l i n e x c r e t i o n w e r e m e a s u r e d . The k i d n e y c a l c i u m c o n c e n t r a t i o n was i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet. K i d n e y calcification was o b s e r v e d in rats fed 1.0% and 1.5% p h o s p h o r u s diets. In rats fed 1.5% p h o s p h o r u s diet, d e p o s i t i o n of h y d r o x y a p a t i t e was o b s e r v e d in the collecting ducts. T h e r e was no u l t r a s t r u c t u r a l d a m a g e in the g l o m e r u l i and distal tubules. H o w e v e r , small v a c u o l e s , s w e l l i n g of the microvilli, l y s o s o m e and u n d e v e l o p e d r o u g h - e n d o p l a s m i c r e t i c u l u m w e r e f o u n d in the p r o x i m a l t u b u l e s . Creatinine clearance, serum urea nitrogen concentration and urinary albumin excretion were i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet. As the i n d i c a t o r s of p r b x i m a l t u b u l a r f u n c t i o n , NAG a c t i v i t y in u r i n e and u r i n a r y /32m i c r o g l o b u l i n e x c r e t i o n w e r e i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet. It was c o n c l u d e d that i n c r e a s e d i n t a k e of p h o s p h o r u s i m p a i r e d p r o x i m a l t u b u l a r function. 0 1997ElsevierSci~'tceInc.
KEY WORDS : P h o s p h o r u s , P r o x i m a l t u b u l a r f u n c t i o n , Rat
* A u t h o r to w h o m c o r r e s p o n d e n c e s h o u l d be a d d r e s s e d . 831
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INTRODUCTION N e p h r o c a l c i n o s i s is a k i d n e y d i s o r d e r in rats, and r e f e r s to the d e p o s i t i o n of c a l c i u m p h o s p h a t e s m a i n l y in the c o r t i c o m e d u l l a r y j u n c t i o n of the k i d n e y (1). Etiological f a c t o r s of n e p h r o c a l c i n o s i s i n c l u d e sex (2), h o r m o n e s (3,4), s t r a i n (5,6) and age (7). The c o m p o s i t i o n of the diet also p l a y s a m a j o r role in the e t i o l o g y of n e p h r o c a l c i n o s i s , e s p e c i a l l y , calcium, m a g n e s i u m and p h o s p h o r u s are i m p o r t a n t n u t r i e n t s . High p h o s p h o r u s (810), low m a g n e s i u m ( 1 1 - 1 3 ) , and c a l c i u m : p h o s p h o r u s ratios b e l o w 1 ( 1 4 , 1 5 ) in the diet i n d u c e n e p h r o c a l c i n o s i s . Rats with nephrocalcinosis showed increased plasma urea c o n c e n t r a t i o n (16,17). In rats with n e p h r o c a l c i n o s i s , u r i n a r y a l b u m i n e x c r e t i o n was i n c r e a s e d , and was p o s i t i v e l y c o r r e l a t e d with the k i d n e y c a l c i u m c o n c e n t r a t i o n (18). T h e s e r e s u l t s i m p l y that n e p h r o c a l c i n o s i s m a y i m p a i r k i d n e y function. H o w e v e r , o t h e r w o r k e r s h a v e not s h o w n that p l a s m a u r e a c o n c e n t r a t i o n was e l e v a t e d in rats w i t h n e p h r o c a l c i n o s i s (19), and e f f e c t s of n e p h r o c a l c i n o s i s on u r i n a r y p r o t e i n e x c r e t i o n and inulin c l e a r a n c e (20). S c h o e n m a k e r s et al. (21) also r e p o r t e d that d e s p i t e k i d n e y c a l c i f i c a t i o n in rats fed a 0.4% p h o s p h o r u s diet, t h e r e w e r e no c h a n g e s on p l a s m a c r c a t i n i n e and u r e a levels and u r i n a r y c r e a t i n i n e , u r e a and a l b u m i n e x c r e t i o n . Thus, a l t h o u g h t h e r e h a v e b e e n m a n y s t u d i e s of k i d n e y f u n c t i o n in rats with n e p h r o c a l c i n o s i s , the details r e m a i n unclear. F u r t h e r m o r e , only a few a t t e m p t s h a v e b e e n m a d e so far to e x a m i n e p r o x i m a l tubuLar f u n c t i o n in k i d n e y s with high p h o s p h o r u s d i e t - i n d u c e d n e p h r o c a l c i n o s i s . In this s t u d y , rats w e r e fed a p u r i f i e d diet c o n t a i n i n g 0.5%, 1.0% and 115% phosphorus. The effect of i n c r e a s i n g i n t a k e of p h o s p h o r u s on p r o x i m a l t u b u l a r f u n c t i o n was e x a m i n e d using b i o c h e m i c a l i n d i c a t o r s and histological obscrvation. M A T E R I A L S AND METHODS
Experimental
d i e t s , a n i m a l s and h o u s i n g
The c o m p o s i t i o n of the e x p e r i m e n t a l diets is s h o w n in Table 1. M i n e r a l m i x t u r e was a m o d i f i c a t i o n of A I N - 7 6 m i n e r a l m i x t u r e (22) w i t h o u t calcium, m a g n e s i u m and p h o s p h o r u s , w h i l e the A I N - 7 6 A v i t a m i n m i x t u r e (23) was u s e d intact. P h o s p h o r u s was a d d e d to the p u r i f i e d diets at c o n c e n t r a t i o n s of 0.5%, 1.0% and 1.5% using p o t a s s i u m t r i p o l y p h o s p h a t e ( N A C A L A I TESQUE, INC., Kyoto, Japan). The c a l c i u m and m a g n e s i u m c o n c e n t r a t i o n in all p u r i f i e d diets w e r e also a d d e d 0.5% and 0.05% using c a l c i u m c a r b o n a t e (Kanto c h e m i c a l Co., INC., Tokyo, J a p a n ) and m a g n e s i u m oxide ( W a k o p u r e c h e m i c a l i n d u s t r i e s , Ltd., Osaka, J a p a n ) r e s p e c t i v e l y . The
PHOSPHORUS AND KIDNEY FUNCTION
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p u r i f i e d d i e t s w e r e s t o r e d at 4°C u n t i l use. TABLE 1 C o m p o s i t i o n of t h e E x p e r i m e n t a l
Diets
0.5% phosphorus
1.0% phosphorus
1.5% phosphorus
diet
diet
diet
Ingredient
g/100g
diet
Casein, milk
20.00
20.00
20.00
Starch, corn
15.00
15.00
15.00
Cellulose powder
5.00
5.00
5.00
Oil, c o r n
5.00
5.00
5.00
Mineral mixture1
1.66
1.66
1.66
Vitamin mixture 2
1.00
1.00
1.00
DL-Methionine
0.30
0.3(l
0.30
Choline bitartrate
0.20
0.2(l
0.20
K~P30]o
1.92
4.34
6.75
MgO
0.08
0.(I 8
0.08
CaCO3
1.25
1.25
1.25
48.59
46.17
43.76
Saccharose
Total 100.00 100.00 100.00 1 This m i n e r a l m i x t u r e is a m o d i f i c a t i o n of AIN-76 m i n e r a l m i x t u r e w i t h o u t c a l c i u m , m a g n e s i u m a n d p h o s p h o r u s sources. 2 AIN-76A v i t a m i n m i x t u r e . F i v e - w e e k - o l d m a l e W i s t a r r a t s (Clea J a p a n . , T o k y o , J a p a n ) w e r e u s e d for t h e e x p e r i m e n t . T h e r a t s w e r e h o u s e d in i n d i v i d u a l s t a i n l e s s s t e e l cages. During the e x p e r i m e n t , t h e r a t c a g e s w e r e l o c a t e d in a r o o m w i t h a c o n t r o l l e d 12h light : d a r k cycle (light, 0 8 0 0 - 2 0 0 0 h ) , temperaturc (22+1°C) a n d h u m i d i t y ( 6 0 - 6 5 % ) . The s t u d y was a p p r o v e d by the T o k y o U n i v e r s i t y of A g r i c u l t u r e A n i m a l Use C o m m i t t e e , a n d a n i m a l s w e r e m a i n t a i n e d in a c c o r d a n c e w i t h g u i d e l i n e s for t h e c a r e a n d u s e of l a b o r a t o r y a n i m a l s of T o k y o U n i v e r s i t y of A g r i c u l t u r e . All r a t s r e c e i v e d a p r e - e x p e r i m e n t a l diet c o n t a i n i n g 0.5% c a l c i u m , 0 . 0 5 % m a g n e s i u m a n d 0.5% p h o s p h o r u s ( T a b l e 1). Diet a n d d e m i n e r a l i z e d w a t e r w e r e f r e e l y a v a i l a b l e to r a t s . A f t e r a 7 d a y s pre-experimental p e r i o d , t h e r a t s w e r e d i v i d e d i n t o 3 g r o u p s of 6 r a t s e a c h on d a y 0 of t h e e x p e r i m e n t . M e a n b o d y w e i g h t s of t h e 3 g r o u p s w e r e
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similar. Each g r o u p was r a n d o m l y a s s i g n e d to one of the p u r i f i e d diets. fed 0.5% and 1.0% p h o s p h o r u s diet w e r e g i v e n a m o u n t s e q u i v a l e n t to c o n s u m e d by the rats fed 1.5% p h o s p h o r u s diet for 21 days. Rats had a c c e s s to d e m i n e r a l i z e d w a t e r t h r o u g h o u t the e x p e r i m e n t . Food i n t a k e b o d y w e i g h t w e r e r e c o r d e d daily.
Collection
of samples
and chemical
Rats that frec and
analyses
The rats h o u s e d i n d i v i d u a l l y in m e t a b o l i s m cages. Before d i s s e c t i o n , rats w e r e not fed and u r i n e was collected for 2 4 - h . At the end of the e x p e r i m e n t , the rats w e r e killed by d e c a p i t a t i o n . Blood was collected and c e n t r i f u g e d to s e p a r a t e the s e r u m . Both k i d n e y s w e r e r e m o v e d and w e i g h e d a f t e r the c a p s u l e s w e r e d i s c a r d e d . The right k i d n e y was used for chemical analysis. The left k i d n e y was used for h i s t o p a t h o l o g i c a L e x a m i n a t i o n . All s a m p l e s w e r e s t o r e d at - 4 0 ° C u n t i l a n a l y s i s . The r i g h t k i d n e y was d r i e d o v e r n i g h t at 100°C, and the d r y w e i g h t was m e a s u r e d . T h e k i d n e y was t h e n a s h e d at 5 5 0 ° C for 48h and the m i n e r a l s w e r e e x t r a c t e d for a n a l y s i s by 1 m o l / L HCI solution. Calcium in the k i d n e y was a n a l y z e d by a t o m i c a b s o r p t i o n s p e c t r o p h o t o m e t r y ( S h i m a d z u A A - 6 4 0 13) (24). P h o s p h o r u s in the k i d n e y was a n a l y z e d a c c o r d i n g to the m e t h o d of Gomori (25). C r e a t i n i n e in s e r u m and u r i n e was m e a s u r e d u s i n g the C r e a t i n i n e - T E S T Wako. Urea n i t r o g e n in s e r u m was m e a s u r e d using N y o s o t i s o - T E S T W a k o (all W a k o p u r e c h e m i c a l i n d u s t r i e s , Ltd., Osaka, Japan). The a c t i v i t y of N - a c e t y l - , 8 - D - g l u c o s a m i n i d a s e (NAG) in u r i n e was m e a s u r e d u s i n g the NAG TEST Shionogi (Shionogi & Co., Ltd., Osaka, Japan). U r i n a r y a l b u m i n and / 3 2 - m i c r o g l o b u l i n e x c r e t i o n w e r e m e a s u r e d u s i n g Rat a l b u m i n EIA s y s t e m and Rat / 3 2 - m i c r o g l o b u l i n EIA s y s t e m (all A m e r s h a m KK, T o k y o , Japan).
Histological
examination
Light m i c r o s c o p y The left k i d n e y was fixed in 10% n e u t r a l f o r m a l i n p h o s p h a t e b u f f e r . For h i s t o p a t h o l o g i c a l e x a m i n a t i o n , 5 /.zm thick l o n g i t u d i n a l s e c t i o n s of k i d n e y w e r e s t a i n e d w i t h h e m a t o x y l i n - e o s i n and Von Kossa's. Transmission electron microscopy In rats fed 1.5% p h o s p h o r u s diet, the left k i d n e y was fixed with 2.5% g l u t a r a l d e h y d e and p o s t - f i x e d with 1% o s m i u m t e t r o x i d e . The t i s s u e s a m p l e s w e r e d e h y d r a t e d t h r o u g h a g r a d e d alcohol series and e m b e d d e d in
PHOSPHORUS AND KIDNEY FUNCTION
835
Epok 812. U l t r a t h i n s e c t i o n s w e r e cut on a R e i h e r t - N i s s e i U l t r a c u t N u l t r a m i c r o t o m e w i t h a d i a m o n d knife, and s t a i n e d with u r a n y l a c e t a t e and lead citrate. The s e c t i o n s w e r e e x a m i n e d u n d e r an Hitachi H - 8 0 0 ( 7 5 k V ) electron microscope.
Statistical analysis The data was e x p r e s s e d as the m e a n s - + S E M . Statistical a n a l y s i s was p e r f o r m e d by o n e - w a y a n a l y s i s of v a r i a n c e (ANOVA) (26). D i f f e r e n c e s w e r e c o n s i d e r e d s i g n i f i c a n t w h e n the P v a l u e was <0.05. RESULTS
Body w e i g h t and w e i g h t gain Final b o d y w e i g h t and p h o s p h o r u s levels (Table 2).
weight
gain
were
not a f f e c t e d
by
dietary
TABLE 2 G r o w t h in Rats Fed Diets C o n t a i n i n g V a r i o u s L e v e l s of P h o s p h o r u s ] 0.5% phosphorus diet Body w e i g h t (g) Initial Final W e i g h t gain (g/day)
134 + 1 148 __+4 0.67 _+0.22
1.0% phosphorus diet
1.5% phosphorus diet
136 + 2 147 + 5
133 +1 149 _+6
0.51 __+0.23
0.77 + 0 . 2 4
1 Values are m e a n s _ S E M for six rats.
K i d n e y w e i g h t and c a l c i u m and p h o s p h o r u s c o n c e n t r a t i o n s Wet and d r y w e i g h t s of k i d n e y w e r e h i g h e r in rats fed 1.5% p h o s p h o r u s diet t h a n in rats fed 0.5% and 1.0% p h o s p h o r u s diets (Fig. 1A). Ki'dney c a l c i u m and p h o s p h o r u s c o n c e n t r a t i o n s w e r e also i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet (Fig. 1B).
Histological examination Light m i c r o s c o p y o b s e r v a t i o n
of k i d n e y
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H. MATSUZAKI et al.
D e p o s i t i o n of c a l c i u m in the c o r t i c o m e d u l l a r y j u n c t i o n was o b s e r v e d in rats fed 1.0% and 1.5% p h o s p h o r u s diets, but rats fed 1.5% p h o s p h o r u s diet w a s s h o w n d e p o s i t i o n of c a l c i u m in the m e d u l l a as well. Deposits w e r e slight in rats fed 1.0% p h o s p h o r u s diet and s e v e r e in t h o s e fed 1.5% p h o s p h o r u s diet (Fig. 2). Electron m i c r o s c o p y o b s e r v a t i o n In rats fed 1.5% p h o s p h o r u s diet, the u l t r a s t r u c t u r a l d a m a g e was not f o u n d in g l o m e r u l i and distal t u b u l e s (Fig. 3, 5), but the m i c r o v i l l i d i s a p p e a r e d in s o m e of the distal t u b u l e s (Fig. 5). Small v a c u o l e s , s w e l l i n g of the microvilli, l y s o s o m e and u n d e v e l o p e d r o u g h - e n d o p l a s m i c r e t i c u l u m w e r c f o u n d in p r o x i m a l t u b u l e s (Fig. 4). D e p o s i t i o n of h y d r o x y a p a t i t e was f o u n d in the collecting ducts (Fig. 6).
200
1.2
~150 (1.8 ~O
100 0.4
E 50 E 0
0 Wet
[]
Dry
(I.5% p h o s p h o r u s diet
[]
Calcium 1.0% p h o s p h o r u s diet
[]
Phosphorus
1.5% p h o s p h o r u s diet
FIG. 1 K i d n e y w e i g h t (A) and c a l c i u m and p h o s p h o r u s c o n c e n t r a t i o n (B) in rats fed d i e t s c o n t a i n i n g v a r i o u s levels of p h o s p h o r u s . V a l u e s are m e a n s + _ S E M for six rats. M a t c h i n g s u p e r s c r i p t l e t t e r s d e n o t e s i g n i f i c a n t d i f f e r e n c e s , P<0.05. Indicators
of kidney
function
C r e a t i n i n e c l e a r a n c e was h i g h e r in rats fed 1.5% p h o s p h o r u s diet t h a n in rats fed 0.5% and 1.(1% p h o s p h o r u s diets (Fig. 7A). S e r u m u r e a n i t r o g e n c o n c e n t r a t i o n was i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet (Fig. 7B). U r i n a r y a l b u m i n e x c r e t i o n was h i g h e r in rats fed 1.5% p h o s p h o r u s diet c o m p a r e d with t h o s e in rats fed 0.5% and 1.0% p h o s p h o r u s diets (Fig. 7C).
PHOSPHORUS AND KIDNEY FUNCTION
837
FIG. 2 Kidney s e c t i o n f r o m a rat fed diets c o n t a i n i n g v a r i o u s levels of p h o s p h o r u s s h o w n by Light microscopy. A : The I).5% p h o s p h o r u s dict did not s h o w d e p o s i t i o n of calcium. Von Kossa's s t a i n e d section, m a g n i f i c a t i o n x 1 8 . : The 1.0% p h o s p h o r u s diet showed slight d e p o s i t i o n of calcium (arrows). Von Kossa's s t a i n e d section, m a g n i f i c a t i o n x17. B
C : The 1.5% p h o s p h o r u s dict s h o w e d s e v e r e d e p o s i t i o n of calcium (arrows). Von Kossa's s t a i n e d section, m a g n i f i c a t i o n X17.
838
H. MATSUZAKI et al.
FIG. 3 G l o m e r u l a r c a p i l l a r i e s (Cap), m e s a n g i u m cells (Me) and p o d o c y t e (Po) in a rat fed 1.5% p h o s p h o r u s diet. T h e r e was no u l t r a s t r u c t u r a l d a m a g e .
FIG, 4 P r o x i m a l t u b u l e in a rat fed 1.5% p h o s p h o r u s diet s h o w e d small vacuoles (V), partial swelling of the microvilli (arrows), lysosome and a small amount of roughendoplasmic reticulum.
PHOSPHORUS AND KIDNEY FUNCTION
839
FIG. 5 Distal t u b u l e in a rat fed 1.5% phosphorus diet showed m i t o c h o n d r i a and w e l l developed rough-endoplasmic r e t i c u l u m ( a r r o w s ) . T h e r e was no u l t r a s t r u c t u r a [ d a m a g e , but the microvilli had partly d i s a p p e a r e d ( a r r o w heads).
FIG. 6 Collecting duct in a rat fed 1.5% phosphorus diet showed d e p o s i t i o n of h y d r o x y a p a t i t e ( a r r o w ) . I n s e r t was m a g n i f i e d deposition ofhydroxapatite.
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H. MATSUZAKI et al. a
b
0.3 A
10
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FIG. 7 Creatinine clearance (A), serum urea nitrogen concentration (B), u r i n a r y albumin cxcretion (C), a c t i v i t y o f N - a c e t y l - / 3 - D - g l u c o s a m i n i d a s e ( N A G ) in urine (D) a n d u r i n a r y /32-microglobulin excretion (El in r a t s f e d d i e t s containing various levels of phosphorus. Values are means Matching superscript letters denote significant differences,
+ S E M f o r six r a t s . P<0.05.
PHOSPHORUS AND KIDNEY FUNCTION
841
The NAG a c t i v i t y in u r i n e and u r i n a r y / f 2 - m i c r o g l o b u l i n e x c r e t i o n w e r e h i g h e r in rats fed 1.5% p h o s p h o r u s diet t h a n in rats fed 0.5% p h o s p h o r u s diet (Fig. 7D, E). DISCUSSION
A high p h o s p h o r u s d i e t - i n d u c e d n e p h r o c a l c i n o s i s o b s e r v e d m a i n l y at the c o r t i c o m e d u l l a r y j u n c t i o n (8). In this s t u d y , the s a m e r e s u l t was o b s e r v e d , n a m e l y , d e p o s i t i o n of c a l c i u m in rats fed 1.0% and 1.5% p h o s p h o r u s diets was f o u n d in the c o r t i c o m e d u l l a r y j u n c t i o n . H o w e v e r , thc d e p o s i t s of rats fed 1.5% p h o s p h o r u s diet w e r e not only f o u n d in the c o r t i c o m e d u l l a r y j u n c t i o n but also in the m e d u l l a . This r e s u l t s u g g e s t s that the distribution of c a l c i u m d e p o s i t i o n might differ by d e g r e e of n e p h r o c a l c i n o s i s . This d e p o s i t s c o n s t i t u t e d m a i n l y c a l c i u m and p h o s p h o r u s (20,27). The ratio of t h e s e e l e m e n t s in d e p o s i t s was s i m i l a r to the v a l u e f o u n d in h y d r o x y a p a t i t e (20). In this s t u d y , in rats fed 1.5% p h o s p h o r u s diet, d e p o s i t i o n of h y d r o x y a p a t i t e in the collecting d u c t s was o b s e r v e d by electron microscopy. It is i m p l i e d that high p h o s p h o r u s d i e t - i n d u c e d m i n e r a l d e p o s i t i o n is h y d r o x y a p a t i t e . I n c r e a s e of m i n e r a l d e p o s i t s in the k i d n e y m a y c a u s e i n c r e a s e of k i d n e y weight. Kidney w e i g h t was i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet, this is in a g r e e m e n t with o t h e r i n v e s t g a t o r s (18,28). I n c r e a s e of k i d n e y w e i g h t in rats with n e p h r o c a l c i n o s i s was m a i n l y r e s p o n s i b l e for i n c r e a s e of m i n e r a l c o n c e n t r a t i o n in the k i d n e y (18). I n c r e a s e of wet k i d n e y weight also r e l a t e to w a t e r and mineral c o n c e n t r a t i o n s in the k i d n e y (29). Rats fed 1.5% p h o s p h o r u s diet i n d u c e d i n c r e a s e d u r i n a r y a l b u m i n e x c r e t i o n . R i t s k e s - H o i t i n g a et al. (18) and Van Camp et al. (3(I) r e p o r t c d that u r i n a r y a l b u m i n e x c r e t i o n was i n c r e a s e d in rats with n e p h r o c a l c i n o s i s , and u r i n a r y a l b u m i n e x c r e t i o n was p o s i t i v e l y c o r r e l a t e d with the k i d n e y c a l c i u m c o n t e n t . I n c r e a s e of u r i n a r y a l b u m i n e x c r e t i o n was c a u s e d by a d e f e c t in the p e r m e a b i l i t y of the r e n a l g l o m e r u l a r b a s e m e n t m e m b r a n e , and the i m p o r t a n c e of the size b a r r i e r or c h a r g e b a r r i e r is k n o w n (31). In this s t u d y , g l o m e r u l a r b a s e m e n t m e m b r a n e in rats fed 1.5% p h o s p h o r u s diet did not s h o w u l t r a s t r u c t u r a l d a m a g e , s u g g e s t i n g that the i n c r e a s e in u r i n a r y a l b u m i n e x c r e t i o n m a y not be r e s p o n s i b l e for the d e f e c t in the p e r m e a b i l i t y of the r e n a l g l o m e r u l a r b a s e m e n t m e m b r a n e . A l b u m i n is m a i n l y r c a b s o r b e d in the p r o x i m a l t u b u l e (32). In o t h e r w o r d s , the i n c r e a s e d u r i n a r y a l b u m i n e x c r e t i o n m a y be c a u s e d by the o b s t r u c t i o n of a l b u m i n r e a b s o r p t i o n in the proximal tubule. In r e g a r d to p r o x i m a l t u b u l a r f u n c t i o n in rats with n e p h r o c a l c i n o s i s , rats with n e p h r o c a l c i n o s i s s h o w e d i n c r e a s e d NAG a c t i v i t y in u r i n e (33). In this s t u d y , NAG a c t i v i t y in u r i n e was i n c r e a s e d in rats fcd 1.5% p h o s p h o r u s diet. In a d d i t i o n , the d a m a g e s of p r o x i m a l t u b u l e s in rats
842
H. MATSUZAKI et al.
fed 1.5% p h o s p h o r u s diet w e r e f o u n d by e l e c t r o n m i c r o s c o p y . T h e s e r e s u l t s i n d i c a t e d e p r e s s i o n of p r o x i m a l t u b u l a r f u n c t i o n . M o r e o v e r , u r i n a r y /32m i c r o g l o b u l i n e x c r e t i o n i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet. This r e s u l t i m p l i e s that r e a b s o r p t i o n in the p r o x i m a l t u b u l e is d e p r e s s e d . It is s u g g e s t e d that the i n c r e a s e in u r i n a r y a l b u m i n e x c r e t i o n in rats fed 1.5% p h o s p h o r u s diet m a y be due to the o b s t r u c t i o n of p r o x i m a l t u b u l a r a l b u m i n reabsorption. S e r u m u r e a n i t r o g e n c o n c e n t r a t i o n was i n c r e a s e d in rats fed 1.5% p h o s p h o r u s diet. P l a s m a u r e a was i n c r e a s e d in rats w i t h n e p h r o c a l c i n o s i s (16), and S c h a a f s m a et al. (17) r e p o r t e d that p l a s m a u r e a c o n c e n t r a t i o n s h o w e d p o s i t i v e c o r r e l a t i o n w i t h k i d n e y c a l c i u m c o n t e n t . R i t s k e s - H o i t i n g a ct al. (18) also c o n c l u d e d that the i n c r e a s e in p l a s m a u r e a in rats with nephrocalcinosis may c a u s e the enhanced reabsorption of u r e a by p r o l o n g a t i o n of t r a n s i t time in single n e p h r o n s . In c o n c l u s i o n , the u l t r a s t r u c t u r a l d a m a g e s w e r e o b s e r v e d in p r o x i m a l t u b u l e s of rats fed 1.5% p h o s p h o r u s diet by e l e c t r o n m i c r o s c o p y , and NAG a c t i v i t y in u r i n e and u r i n a r y / 3 2 - m i c r o g l o b u l i n e x c r e t i o n w e r e i n c r e a s e d . In rats fed 1.5% p h o s p h o r u s diet, u r i n a r y a l b u m i n e x c r e t i o n was i n c r e a s e d , w h e r e a s g l o m e r u l i did not s h o w u l t r a s t r u c t u r a l d a m a g e . It is s u g g e s t e d that i n c r e a s e d i n t a k e of p h o s p h o r u s i n d u c e s d e p r e s s i o n of p r o x i m a l t u b u l a r f u n c t i o n , and it m a y c a u s e an i n c r e a s e of the u r i n a r y a l b u m i n e x c r e t i o n . REFERENCES
1. S i l v e r m a n J, R i v e n s o n A. N e p h r o c a l c i n o s i s in 2 y o u n g rats: a case r e p o r t . Lab A n i m 1 9 8 0 ; 1 4 : 2 4 1 - 2 . 2. Cousins FB, Geary CPM. A s e x - d e t e r m i n e d r e n a l calcification in rats. Nature 1966;211:980-1. 3. E n g f e l d t B, Rhodin J, S t r a n d h J. S t u d i e s of the k i d n e y u l t r a s t r u c t u r e in h y p e r v i t a m i n o s i s D. Acta Chir Scand 1 9 6 2 ; 1 2 3 : 1 4 5 - 7 . 4. P a r k e r HM, F o r b e s RM. I n f l u e n c e of d i e t a r y calcium, m a g n e s i u m and p h o s p h o r u s on c y c l i c - A M P e x c r e t i o n and k i d n e y calcification in the rat. Proc Soc Exp Biol Med 1 9 7 6 ; 1 5 1 : 2 1 5 - 2 0 . 5. Du B r u y n DB.A c o m p a r i s o n of c e r t a i n rat s t r a i n s with r e s p e c t to s u s c e p t i b i l i t y to n e p h r o c a l c i n o s i s . S - A f t Med J 1 9 7 0 ; 4 4 : 1 4 1 7 - 8 .
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843
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in
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18. R i t s k c s - H o i t i n g a J, L e m m e n s AG, Danse LHJC, B e y n e n AC. P h o s p h o r u s i n d u c e d n e p h r o c a l c i n n s i s and k i d n e y f u n c t i o n in f e m a l e rats. J N u t r 1989;1 I 9 : 1 4 2 3 - 3 1 . 19 S t r u t h e r s BJ, D a h l g r e n RR, H o p k i n s DT. Biological e f f e c t s of f e e d i n g g r a d e d levels of alkali t r e a t e d s o y b e a n p r o t e i n c o n t a i n i n g l y s i n o a l a n i n e ( N - 2 - c a r b o x y e t h y l - L - l y s i n e ) in S D a n d W i s t a r rats. J Nutr 1 9 7 7 ; i 0 7 : 1 190-9. 20 S t o n a r d MD, S a m u e l s DM, L~ck EA. The p a t h o g e n e s i s and effect nn renal f u n c t i o n of n c p h r o c a l c i n o s i s i n d u c e d by d i f f e r e n t diets in f e m a l e rats. Fd Chem Toxic 1 9 8 4 ; 2 2 : 1 3 9 - 4 6 . 21
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845
29. M a t s u z a k i H, Arai K, U e h a r a M, Suzuki K, Sato S, K a n k e Y, Goto S. I n c r e a s i n g m a g n e s i u m i n t a k e p r e v e n t s high p h o s p h o r u s d i e t - i n d u c e d k i d n e y d a m a g e in y o u n g rats. N u t r Res (in p r e s s ) . 30. V a n C a m p I, R i t s k e s - H o i t i n g a J, L e m m e n s AG, B e y n e n AC. D i e t - i n d u c e d n e p h r o c a l c i n o s i s a n d u r i n a r y e x c r e t i o n of a l b u m i n in f e m a l e rats. Lab A n i m 1 9 9 0 ; 2 4 : 1 3 7 - 4 1 . 31. Olson JL, R e n n k e HG, V e n k a t a c h a l a m MA. A l t e r a t i o n s in the c h a r g e and size s e l e c t i v i t y b a r r i e r of the g l o m e r u l a r filter in a m i n o n u c l e o s i d e n e p h r o s i s in rats. Lab I n v e s t 1 9 8 1 ; 4 4 : 2 7 1 - 9 . 32. B o u r d e a u JE, C a r o n e FA, G a n o t e CE. S e r u m a l b u m i n u p t a k e in i s o l a t e d p e r f u s e d r e n a l t u b u l e s . J Cell Biol 1 9 7 2 ; 5 4 : 3 8 2 - 9 8 . 33. Kang SS, Price RG, Y u d k i n J, W o r c e s t e r NA, B r u c k d o r f e r KR. The i n f l u e n c e of d i e t a r y c a r b o h y d r a t e and fat on k i d n e y c a l c i f i c a t i o n and the u r i n a r y e x c r e t i o n of N - a c e t y l - /3- g l u c o s a m i n i d a s e . Br J N u t r 1 9 7 9 ; 4 1 : 6 5 - 7 1 .
Accepted for publication February 13, 1997.