Effects of Cadmium Exposure on Rat Kidneys Leon C. King''~, Vernon Clark' and Edward J. Faeder2 IDepartment of Biology North Carolina Central University Durham, N.C. 27707 and =Biochemistry Branch Environmental Toxicology Division Environmental Protection Agency Research Triangle Park, N.C. 27711
There i s p r e s e n t l y a g r e a t deal of i n f o r m a t i o n s u g g e s t i n g t h a t exposure to cadmium both in the i n d u s trial and general environment r e s u l t s in human t o x i c i t y ( c f . F r i b e r g e__t_t a l . , 1971). However, the mechanism of cadmium induced renal damage and the development of b i o c h e m i c a l t e s t s r e f l e c t i n g the onset and s e v e r i t y of t h a t damage have not been worked o u t . Histochemical s t u d i e s on the d i s t r i b u t i o n of enzymes in the k i d n e y have shown t h a t s p e c i f i c s t r u c t u r e s of the renal nephtons are r i c h in a c i d phosphatase, a l k a l i n e phosphatase, g l u t a m i c o x o l o a c e t i c t r a n s a m i n a s e , c a r b o n i c anhydrase and l a c t i c dehydrogenase ( W a c h i s t e n , 1955; B o n t i n g e__tt a l . , 1960; P o l l a k and M a t t e n h e i m e r , 1962: M a t t e n h e i m e r , I~68). Cadmium-induced renal damage may cause the release of these enzymes i n t o the u r i n e (Nomiyama et a l . , 1973). The enzyme $ - g l u t a m y l t r a n s p e p t i d a s e (GT) is present in v a r i o u s t i s s u e s of the body, but the h i g h e s t activity i s found in the k i d n e y ( A l b e r t et a l . , 1961), Large changes in serum a c t i v i t y of GT have, however, been noted in c e r t a i n d i s e a s e s of the l i v e r ( S z c z e k l i k et a l . , 1961; Zein and Discombe, 1970), Evidence has shown t h a t acute and d e s t r u c t i v e d i s e a s e s of the u r o r e n a l system r e s u l t s in i n c r e a s e d activity of GT in u r i n e . Chronic d i s e a s e s produce s i g nificantly reduced a c t i v i t y (Levy and Dubach, 1972). I t has been p o s t u l a t e d t h a t when the k i d n e y b e c o m e s s a t u r a t e d w i t h cadmium, r e - a b s o r p t i o n decreases and t u b u l a r p r o t e i n u r i a appears. The o b j e c t i v e s of t h i s i n v e s t i g a t i o n were to s t u d y the e f f e c t s of cadmium t o x i c i t y on the k i d n e y at c h r o n i c and acute dosing and to determine whether t h e r e i s a r e l e a s e of the t u b u l a r enzyme GT i n t o the u r i n e .
572 Bulletin of Environmental Contamination & Toxicology, Vol. 16, No. 5 9 November 1976 by Springer,u New York Inc.
MATERIALS AND METHODS Male Sprague Dawley r a t s ( 2 2 5 - 2 5 0 g) were d i v i d e d into five groups, four rats per group. One c o n t r o l g r o u p was g i v e n 3 s u b c u t a n e o u s ( s c ) i n j e c t i o n s per week, f o r f o u r w e e k s , o f 200 ul s t e r i l e , isotonic saline. Two g r o u p s were a c u t e l y d o s e d : h i g h a c u t e dose was a s i n g l e sc i n j e c t i o n 9 mg Cd/kg body w e i g h t ; low a c u t e dose was 3 mg Cd/kg body w e i g h t . Two g r o u p s were chronically d o s e d , 3 t i m e s p e r week f o r f o u r weeks: h i g h c h r o n i c dose was 0 . 7 5 mg Cd/kg body w e i g h t , low c h r o n i c dose was 0 . 2 5 mg Cd/kg body w e i g h t . All animals were s a c r i f i c e d a t 4 w e e k s , and t h e k i d n e y s r e moved f o r m e t a l a n a l y s i s . The c o n d i t i o n o f t h e a n i m a l s was e v a l u a t e d d u r i n g t h e c o u r s e o f t r e a t m e n t by t h e f o l l o w i n g measurements; total body w e i g h t , 8 h o u r u r i n e c o l l e c t i o n every 3 days, GT a c t i v i t y i n u r i n e , and t o t a l urinary protein excretion. E v e r y t h i r d day u r i n e samples were c o l l e c t e d i n c o l d ( 2 - 4 ~ ) v i a l s f o r e i g h t h o u r s from r a t s i n d i vidually housed i n m e t a b o l i c c a g e s . The volume o f urine collected was r e c o r d e d . U r i n e samples were centrifuged a t l l O O x g f o r t e n m i n u t e s to remove c e l l u l a r d e b r i s and d i a l y z e d w i t h 3 changes o f c o l d d e i o n i z e d w a t e r f o r 16 h o u r s . Total acid insoluble protein excret e d was measured by a m o d i f i e d Lowry method ( L a y n e , 1 9 5 7 ) . GT was a s s a y e d u s i n g t h e method o f Szasz ( 1 9 6 9 ) . Cadmium analysis i n u r i n e , and cadmium and z i n c a n a l y s i s o f k i d ney were done a c c o r d i n g t o t h e method o f H i n n e r s e t a l . (1975).
RESULTS AND DISCUSSION The d o s i n g r e g i m e n was e s t a b l i s h e d i n such a way t h a t a f t e r f o u r w e e k s , t h e low c h r o n i c g r o u p had r e c e i v e d a b o u t t h e same t o t a l amount o f cadmium as t h e low a c u t e g r o u p , and t h e h i g h c h r o n i c g r o u p r e c e i v e d a b o u t t h e same t o t a l amount o f cadmium as t h e high acute group. Table 1 summarizes the metal accumulation data. K i d n e y cadmium c o n c e n t r a t i o n s in the a c u t e l y dosed a n i m a l s r e a c h e d h i g h e r l e v e l s t h a n i n the corresponding chronically dosed g r o u p s . Zinc levels remained essentially t h e same f o r a l l dosed groups. Cadmium l e v e l s i n u r i n e , f o r a l l g r o u p s , t h r o u g h o u t t h e e x p e r i m e n t , were l e s s t h a n 0 . 0 5 u g / m l .
573
TABLE 1
Metal Accumulation in Kidneys 1
Total Cd Injected mg
Group Control Low Chronic
. . .92
Total Cd Accumulated mg .
[Cd] ug/g
[Zn] ug/g
.07+.02
There i s p r e s e n t l y a g r e a t deal of i n f o r m a t i o n s u g g e s t i n g t h a t exposure to cadmium both in the i n d u s trial and general environment r e s u l t s in human t o x i c i t y ( c f . F r i b e r g e__t_t a l . , 1971). However, the mechanism of cadmium induced renal damage and the development of b i o c h e m i c a l t e s t s r e f l e c t i n g the onset and s e v e r i t y of t h a t damage have not been worked o u t . Histochemical s t u d i e s on the d i s t r i b u t i o n of enzymes in the k i d n e y have shown t h a t s p e c i f i c s t r u c t u r e s of the renal nephtons are r i c h in a c i d phosphatase, a l k a l i n e phosphatase, g l u t a m i c o x o l o a c e t i c t r a n s a m i n a s e , c a r b o n i c anhydrase and l a c t i c dehydrogenase ( W a c h i s t e n , 1955; B o n t i n g e__tt a l . , 1960; P o l l a k and M a t t e n h e i m e r , 1962: M a t t e n h e i m e r , I~68). Cadmium-induced renal damage may cause the release of these enzymes i n t o the u r i n e (Nomiyama et a l . , 1973). The enzyme $ - g l u t a m y l t r a n s p e p t i d a s e (GT) is present in v a r i o u s t i s s u e s of the body, but the h i g h e s t activity i s found in the k i d n e y ( A l b e r t et a l . , 1961), Large changes in serum a c t i v i t y of GT have, however, been noted in c e r t a i n d i s e a s e s of the l i v e r ( S z c z e k l i k et a l . , 1961; Zein and Discombe, 1970), Evidence has shown t h a t acute and d e s t r u c t i v e d i s e a s e s of the u r o r e n a l system r e s u l t s in i n c r e a s e d activity of GT in u r i n e . Chronic d i s e a s e s produce s i g nificantly reduced a c t i v i t y (Levy and Dubach, 1972). I t has been p o s t u l a t e d t h a t when the k i d n e y b e c o m e s s a t u r a t e d w i t h cadmium, r e - a b s o r p t i o n decreases and t u b u l a r p r o t e i n u r i a appears. The o b j e c t i v e s of t h i s i n v e s t i g a t i o n were to s t u d y the e f f e c t s of cadmium t o x i c i t y on the k i d n e y at c h r o n i c and acute dosing and to determine whether t h e r e i s a r e l e a s e of the t u b u l a r enzyme GT i n t o the u r i n e .
572 Bulletin of Environmental Contamination & Toxicology, Vol. 16, No. 5 9 November 1976 by Springer,u New York Inc.
MATERIALS AND METHODS Male Sprague Dawley r a t s ( 2 2 5 - 2 5 0 g) were d i v i d e d into five groups, four rats per group. One c o n t r o l g r o u p was g i v e n 3 s u b c u t a n e o u s ( s c ) i n j e c t i o n s per week, f o r f o u r w e e k s , o f 200 ul s t e r i l e , isotonic saline. Two g r o u p s were a c u t e l y d o s e d : h i g h a c u t e dose was a s i n g l e sc i n j e c t i o n 9 mg Cd/kg body w e i g h t ; low a c u t e dose was 3 mg Cd/kg body w e i g h t . Two g r o u p s were chronically d o s e d , 3 t i m e s p e r week f o r f o u r weeks: h i g h c h r o n i c dose was 0 . 7 5 mg Cd/kg body w e i g h t , low c h r o n i c dose was 0 . 2 5 mg Cd/kg body w e i g h t . All animals were s a c r i f i c e d a t 4 w e e k s , and t h e k i d n e y s r e moved f o r m e t a l a n a l y s i s . The c o n d i t i o n o f t h e a n i m a l s was e v a l u a t e d d u r i n g t h e c o u r s e o f t r e a t m e n t by t h e f o l l o w i n g measurements; total body w e i g h t , 8 h o u r u r i n e c o l l e c t i o n every 3 days, GT a c t i v i t y i n u r i n e , and t o t a l urinary protein excretion. E v e r y t h i r d day u r i n e samples were c o l l e c t e d i n c o l d ( 2 - 4 ~ ) v i a l s f o r e i g h t h o u r s from r a t s i n d i vidually housed i n m e t a b o l i c c a g e s . The volume o f urine collected was r e c o r d e d . U r i n e samples were centrifuged a t l l O O x g f o r t e n m i n u t e s to remove c e l l u l a r d e b r i s and d i a l y z e d w i t h 3 changes o f c o l d d e i o n i z e d w a t e r f o r 16 h o u r s . Total acid insoluble protein excret e d was measured by a m o d i f i e d Lowry method ( L a y n e , 1 9 5 7 ) . GT was a s s a y e d u s i n g t h e method o f Szasz ( 1 9 6 9 ) . Cadmium analysis i n u r i n e , and cadmium and z i n c a n a l y s i s o f k i d ney were done a c c o r d i n g t o t h e method o f H i n n e r s e t a l . (1975).
RESULTS AND DISCUSSION The d o s i n g r e g i m e n was e s t a b l i s h e d i n such a way t h a t a f t e r f o u r w e e k s , t h e low c h r o n i c g r o u p had r e c e i v e d a b o u t t h e same t o t a l amount o f cadmium as t h e low a c u t e g r o u p , and t h e h i g h c h r o n i c g r o u p r e c e i v e d a b o u t t h e same t o t a l amount o f cadmium as t h e high acute group. Table 1 summarizes the metal accumulation data. K i d n e y cadmium c o n c e n t r a t i o n s in the a c u t e l y dosed a n i m a l s r e a c h e d h i g h e r l e v e l s t h a n i n the corresponding chronically dosed g r o u p s . Zinc levels remained essentially t h e same f o r a l l dosed groups. Cadmium l e v e l s i n u r i n e , f o r a l l g r o u p s , t h r o u g h o u t t h e e x p e r i m e n t , were l e s s t h a n 0 . 0 5 u g / m l .
573
TABLE 1
Metal Accumulation in Kidneys 1
Total Cd Injected mg
Group Control Low Chronic
. . .92
Total Cd Accumulated mg .
[Cd] ug/g
[Zn] ug/g
.07+.02
