- Email: [email protected]
Dietary fat in experimental nephrotic syndrome: Beneficial effects of fish oil on serum lipids and, indirectly, on the kidney
L i f e Sciences, Vol. 40, P r i n t e d in the U.S.A.
BENEFICIAL Yasushl
pp. 2 3 1 7 - 2 3 2 4
Pergamon
D I E T A R Y FAT IN E X P E R I M E N T A L N E P H R O T I C SYNDROME: EFFECTS OF FISH OIL ON SERUM LIPIDS AND, INDIRECTLY, Ito,
Uno Barcelll, Moti Kashyap,
Divisions Blostatistics,
Journals
ON THE K I D N E Y
W a t a r u Yamashlta, Mark Weiss, Brian Thompson, James Deddens, and Victor E. Pollak
of N e p h r o l o g y , Lipid R e s e a r c h , and E p l d e m l o l o g y and D e p a r t m e n t of Medicine, and the D e p a r t m e n t of Pathology, U n i v e r s i t y of C i n c i n n a t i Medical Center.
(Recelved
in final form M a r c h
31, 1987)
Summary Three i s o c a l o r l c diets with d i f f e r e n t fat c o m p o s i t i o n were fed to rats for seven weeks after the p r o d u c t i o n of nephrotic syndrome by adriamycin. The effects of feeding 3% and 14% flsh oli were compared wlth those of feeding beef fat. At the fourth week of f e e d i n g the levels of t r i g l y c e r l d e s and c h o l e s t e r o l were lower in b o t h fish oli fed groups. At the seventh week these levels, and the LDL cholesterol, were lower only In the 14% fish oli group. In rats fed beef fat, but not ~n those fed flsh oli, there was a s t r i k i n g p o s i t i v e c o r r e l a t i o n of the f o u r - w e e k serum t r i g l y c e r l d e s and c h o l e s t e r o l wlth the s e v e n - w e e k serum c r e a t i n i n e level and wlth the degree of g l o m e r u l a r h y a l i n o s i s and e n d o t h e l i a l swelling. The f a v o r a b l e effects of flsh oil f e e d i n g on serum llplds may have a p r o t e c t i v e e f f e c t on the d e v e l o p m e n t of g l o m e r u l a r damage.
N e p h r o t l c syndrome is c h a r a c t e r i z e d by heavy proteinurla, h y p o a l b u m i n e m i a and h y p e r l i p i d e m l a . D e s p i t e an a p p a r e n t increased p r e v a l e n c e of a t h e r o s c l e r o s i s in nephrotlc patients (1,2,3), the need for and effects of t r e a t m e n t of h y p e r l l p l d e m l a are not well established. Increased toxicity of c l o f i b r a t e t r e a t m e n t has been reported in the presence of h y p o a l b u m i n e m l a (4). Dietary fish oil, rlch in o m e g a - 3 - f a t t y acids, reduces plasma t r l g l y c e r l d e levels In normal (5,6,7) and h y p e r l i p l d e m i c subjects (8,9,10), and has a b e n e f i c i a l e f f e c t in p r e s e r v i n g renal function in various forms of e x p e r i m e n t a l renal d i s e a s e (11,12,13). The p r e s e n t study was done to evaluate the effects of the c o m p o s i t i o n of the d i e t a r y fat on p l a s m a llplds, and on renal function and m o r p h o l o g y in rats with n e p h r o t l e syndrome induced by adrlamycln. It was d e s i g n e d p a r t i c u l a r l y to e v a l u a t e the effects of two levels (3% and 14%) of fish oil feeding, and to c o m p a r e them w i t h feeding beef fat. The beef tallow diet, c o n t a l n l n g 175 g/Kg fat, 44% of which was s a t u r a t e d fatty acid, m i m i c s the typical North A m e r i c a n diet. The 3% fish oil diet was d e s i g n e d to s i m u l a t e a dlet which could be r e a l i s t i c a l l y achieved in humans by d e c r e a s i n g some of the s a t u r a t e d fat and s u p p l e m e n t l n g the diets wlth s e m i - p u r i f l e d flsh oil. The 14% flsh oli diet was d e s i g n e d to a c h i e v e the m a x i m u m fish oll which S u p p o r t e d in part by NIH grants AM 34489, C L I N F O RR 00068, and a grant the R e s e a r c h and E d u c a t i o n a l Funds of D i a l y s i s Clinics, Inc.
Copyright
0 0 2 4 - 3 2 0 5 / 8 7 $3.00 + .00 (c) 1987 P e r g a m o n J o u r n a l s
Ltd.
from
2318
Fish Oil is B e n e f i c l a l
to N e p h r o t i c Rats
Vol.
40, No.
24,
1987
d o e s not i n t e r f e r e w i t h a c c e p t a b i l i t y . The fat c o m p o s i t i o n of thls d l e t s l m u l a tes that of an E s k i m o diet. A n i m a l s w e r e s t u d i e d at 4 w e e k s b e c a u s e renal d a m a g e is m i n i m a l at thls time (14), and w e r e s a c r i f i c e d at 7 w e e k s b e c a u s e term i n a l renal f a i l u r e was d e v e l o p l n g in some rats. M a t e r i a l and M e t h o d s Animals and Diets T h i r t y - f i v e S p r a g u e - D a w l e y f e m a l e rats (H~rlan, I n d i a n a p o l l s , Indiana) w e i g h i n g 2 2 0 - 2 7 0 g r e c e i v e d a s i n g l e dose of a d r l a m y c l n (Adria L a b o r a t o r i e s , C o l u m b u s , Ohio) 7.5 m g / K g i n t r a v e n o u s l y into the tall veln. S e v e n days later, m a t c h e d by u r i n a r y p r o t e i n e x c r e t l o n , the rats were a l l o c a t e d to three isoc a l o r i c d i e t s (Table i), c o n t a i n i n g beef t a l l o w (beef, n=ll), 3% f i s h oil (3% flsh, n=12) a n d 14% flsh oll (14% flsh, n=12). S i x rats, w h i c h had r e c e i v e d n o r m a l s a l i n e only, w e r e fed the beef d l e t and s e r v e d as n o n - n e p h r o t i c c o n t r o l s (C). This g r o u p was i n c l u d e d in o r d e r to p r o v i d e a p p r o p r i a t e r e f e r e n c e of v a l u e s for the three e x p e r i m e n t a l groups. The flsh oli was s u p p l l e d by R. P. S c h e r e r , Troy, M i c h i g a n as MaxEPA, a c o n c e n t r a t e d b l e n d of flsh oils from w h i c h v i t a m i n s A a n d D h a v e b e e n r e m o v e d and to w h i c h a l D h a - t o c o p h e r o l has b e e n added. The diets were p r e p a r e d by Teklad, Madison, W i s c o n s i n . P a l r f e e d i n g was e m p l o y e d t h r o u g h o u t , the rats in three groups b e i n g pair fed a g a i n s t rats in the g r o u p w i t h the l o w e s t food intake.
Table i The Experlmental Diets
Beef Tallow
3% Flsh 011
14% Fish O11
Ma3or Constltuents (g/Kg) Proteln DL Methlonlne Carbohydrate Flber Mineral mlxture Vltamln mlxture Total fat
180 30 500 70 35 i0 175
180 30 500 70 35 I0 175
180 30 500 70 35 i0 175
Source of Fats (g/Kg) Beef tallow Safflower oli MaxEPA
145 30 0
115 30 30
35 0 140
Composltlon of Fats (%) Fatty aclds (%) Saturated Monounsaturated Llnolelc acld (18:2 omega-6) Elcosapentaenolc acld (20:5 omega-3) Docosahexaenolc acld (22:6 omega-3) Other fatty aclds Cholesterol (g/Kg)
44.1 40.3 13.7
41.4 35.5 14.8
40.8 26.3 1.2
0
3.1
13.0
0
2.0
7.9
1.9
3.2
10.8
0.13
0.28
0.87
Vol.
40, No.
24,
1987
Fish Oil is B e n e f i c i a l
to N e p h r o t i c
Rats
2319
Rats were w e l g h e d weekly. Urine was c o l l e c t e d for 23 hours, w i t h o u t food b u t with free access to water, at w e e k 0 and 2 and w e e k l y thereafter. Blood p r e s s u r e was m e a s u r e d by the tail cuff method w i t h o u t a n e s t h e s i a at week 4. Serum a l b u m i n was m e a s u r e d at weeks 4 and 7, serum c r e a t l n i n e at week 7. Blood was drawn for serum llpids after a 15 hour fast at weeks 4 and 7. At sacrifice, k i d n e y tissue was o b t a i n e d under x y l a z l n e - k e t a m i n anesthesia, fixed in M o s s m a n ' s solutlon, stalned with periodic a c l d - S c h i f f (PAS), and exam i n e d "blinded." In the glomeruli, p a r t i c u l a r a t t e n t i o n was pald to e n l a r g e m e n t w i t h e n d o c a p i l l a r y swelling, to h y a l l n o s i s ( c a p i l l a r y l i p o p r o t e i n insudation), add to s e g m e n t a l and global sclerosis (mesanglal and GBM increase wlth capillary loop o b l l t e r a t l o n with and w i t h o u t collapse). In the k i d n e y s of each rat b e t w e e n 50 and 250 g l o m e r u l l were studied, and the number with each a b n o r m a l i t y counted. The tubular changes e v a l u a t e d s e m i q u a n t i t a t i v e l y (0-4+) w e r e d i l a t a tion, i n t r a l u m l n a l protein, p r o t e i n r e a b s o r p t i o n droplets, and n e p h r o c a l c l n o s l s . Laboratory Methods U r i n e p r o t e i n was m e a s u r e d with C o o m a s i e b r i l l i a n t blue, serum c r e a t i n i n e by the Jaffe reaction, and serum a l b u m i n with Bromcresol green. Fatty acid analysis was p e r f o r m e d by gas c h r o m a t o g r a p h y (15). For analysis of lipids, serum c o n t a i n i n g 0.2% E D T A was u l t r a c e n t r l f u g e d initlally, using a 50.3 TI Rotor in a L8-55 c e n t r i f u g e (Beckman I n s t r u m e n t Co., Palo Alto, Ca.) at 45000 rpm for 23 h o u r s after a d 3 u s t i n g the d e n s i t y to 1.020 g/ml wlth p o t a s s i u m bromide. The b o t t o m layer c o n t a i n e d LDL plus HDL c h o l e s t e r o l (tube A). This was further c e n t r l f u g e d at 45000 rpm for 23 hours at the d e n s i t y of 1.063 g/ml. The b o t t o m layer c o n t a i n e d HDL c h o l e s t e r o l . LDL was c a l c u l a t e d by s u b t r a c t i n g HDL f r o m total c h o l e s t e r o l in tube A. T r i g l y c e r i d e and c h o l e s t e r o l were m e a s u r e d by e n z y m a t i c m e t h o d s on a H l t a c h l 705 a u t o a n a l y z e r . Statistics Analysls of variance on single and repeated m e a s u r e m e n t s was used as appropriate. C o r r e l a t i o n was c a l c u l a t e d by linear regression. For n o r m a l l y d i s t r i b u t e d data, results are e x p r e s s e d as a r i t h m e t i c means + 1 SD. For data log normally d i s t r i b u t e d , the values were log t r a n s f o r m e d a n d results e x p r e s s e d as g e o m e t r i c means (-I SD, +I SD). D i f f e r e n c e s b e t w e e n groups were a n a l y z e d u s i n g the D u n c a n test. Results The Anlmals
lowest food intake was in the a d r i a m y c i n treated animals in the three other groups were pair fed a g a i n s t them.
fed beef
fat.
The results are s u m m a r i z e d in Table 2, the s t a t i s t i c a l s i g n i f i c a n c e of d i f f e r e n c e s b e t w e e n groups in Table 3. D u r i n g the 7 weeks of study i0/ii, ii/ii, and 12/13 rats s u r v i v e d r e s p e c t l v e l y in the beef, 3% fish and 14% fish groups. One rat each died in the beef and 14% flsh groups at week 4. Initially, rats had a normal food intake of a p p r o x i m a t e l y 15 g/day, which was m a i n t a i n e d c o n s t a n t t h r o u g h o u t the experiment. F e w rats had poor appetite, c o i n c i d i n g with the a p p e a r a n c e of the uremic syndrome. In the controls, the mean body w e i g h t was stable. In n e p h r o t l c rats fed beef tallow the weight, similar to the c o n t r o l s at week 0, d e c r e a s e d s i g n i f l c a n t l y by week 2, and remained lower thereafter. There was little c h a n g e in w e i g h t of rats fed 3% and 14% fish oil. At 4 and 7 weeks, the w e i g h t s of beth flsh oil fed groups were higher than those fed beef tallow. Blood p r e s s u r e was i d e n t i c a l in all groups at week 4. U r i n a r y p r o t e i n excretion, serum a l b u m i n and c r e a t l n i n e U r l n a r y p r o t e i n e x c r e t i o n ~n c o n t r o l s was minimal.
One week after
adriamy-
2320
Fish Oil is Beneflcial to Nephrotic Rats
Vol. 40, No. 24, 1987
Table 2 SummarM of Laboratory Data
Beef Tallow L- (n_--ll). . . . . n(= l O ) Week 4 Week 7
Control
(n=6) Week 4 Weight (g)*
248 + 5
Blood pressure*
118 + 7
P r o t e i n excretion
4.6 (2.4, 6.8)
(mg/23 h) Serum albumin (7/dl)
(n=6) Week 7 247 ~ I0
216 + 19
248 + i0
ii~ + I0 3.4 (1.6, 5.6)
3.27 3.01 (3.[0, 3.45)(2.86, 3.19)
Serum creatlnlne (mg/d[)
227 ~ 7
3% Flsh 01 1 [n=12) (n=12) Week 4 Week 7
_
14% Fish Oll (n=12) (n=ll) Week 4 Week 7
236 + i0
251 + 17
117 + 7
641 (547, 75[)
604 (456 799)
(607, 901)
800 (716, 895)
1.4g (1.4, [.6)
1.81 ([.56, 2.07)
1.59 ([.51, 1.68)
1.78 (1.62, 1.96)
0.63 (0°59, 0.68)
238 + i0
116 + 7
740
1.75 (i.16, 2.63)
574 (458, 719)
561 (455, 691)
1.66 (1.53, 1.81)
1.81 (1.62, 2.03)
1.56 (1.20, ?.02)
1.34 (0.79, 2.28)
rrlglycerx]e (mg/dl)
96 (60, 151)
27 (21, 36)
5L34 (2313, [1393)
3012 (@06, 11249)
1993 (827, 4801)
2790 (765, 18[71)
865 (405, 1645)
811 (306, 2147)
Total cholesterol
65 (56, 76)
59 (51, 69)
982 (571, [688)
g09 (463, [783)
681 (488, 951)
925 (530, 1617)
482 (365, 630)
412 (273, 619)
(mg/dl)
LDL (mg/dl)
9.5 (7.1, [2.7)
HDL (mg/dl)
46 (36, 58)
199 (147, 270)
223 (164, 303)
147 (i[8, 184)
73 (58, 91)
67 (63, 121)
82 (64, 105)
Table ~tatistlcal
$1gntf[cance
of
3
~le
Dlfferences
Adrlamycln
Beef
between
Tallow
Beef
vs 3% Week
Welght
Blood
pressure
Serum
albumin
Serum
creatlnxne
Fish 4
of
Tallow
3%
Fish
vs Oil Week
14% 7
Week
4
P
P
P
<0.01
<0.01
<0.01
NS <0.05
Groups
Rats
NS MS
NS
<0.01
-
Fxsh
OiL
vs Oxl week
14% 7
P <0.01
Week
4
Oil Week
7
P
P
NS
NS
-
NS
NS
N$
NS
Fish
-
NS
NS
Tryglycerxdes
<0.01
NS
<0.01
<0.05
<0.05
<0.05
Cholesterol Total
<0.05
NS
<0.01
<0.01
<0.05
<0.01
LDL
-
NS
-
<0.05
-
<0.01
HDL
-
NS
-
MS
-
NS
Vol.
40, No.
24,
1987
Fish Oil
is B e n e f i c i a l
to N e p h r o t i c
Rats
2321
cin a d m i n i s t r a t i o n all rats had d e v e l o p e d proteinuria, g r e a t e r than 10-fold that of controls. At this time they were d i v i d e d into the three d i e t a r y groups, m a t c h e d by u r i n a r y p r o t e i n excretion. At weeks 4 and 7, the p r o t e i n e x c r e t i o n in all three groups was 120-240 times g r e a t e r than in the controls. F r o m weeks 2 to 5 the p r o t e i n e x c r e t i o n of rats in the group fed 3% fish oil was not diff e r e n t from that of rats fed beef fat. The urine p r o t e i n e x c r e t i o n of rats fed 14% fish oil was lower than that of the groups fed beef (p <.04) and 3% fish (p <.03). Serum a l b u m i n values were d e c r e a s e d to about 45 to 60% of the controls in all three a d r i a m y c i n groups. S e r u m c r e a t i n i n e values were e l e v a t e d at week 7 in all animals r e c e i v i n g adriamycln. There was c o n s i d e r a b l e v a r i a t i o n b e t w e e n animals in each group. T h e y values were h i g h e r in those fed beef tallow than those fed 3% or 14% fish oil, b u t the d i f f e r e n c e s were not s i g n i f i c a n t . Renal h i s t o l o g y The k i d n e y s of the c o n t r o l rats were normal. In the a d r i a m y c i n treated animals, n o n - p r o l i f e r a t l v e g l o m e r u l a r lesions w i t h v a r y i n g degrees of c a p i l l a r y c o l l a p s e and s e g m e n t a l sclerosis were seen; there was no global g l o m e r u l a r sclerosis. In many animals there was s i g n i f i c a n t h y a l i n o s i s , g l o m e r u l a r e n l a r g e m e n t with e n d o c a p i l l a r y swelling, and visceral e p i t h e l i a l changes i n c l u d i n g s w e l l i n g and p r o l i f e r a t i o n which r e f l e c t proteinuria. In each group there was a s i g n i f i c a n t v a r i a t i o n in the changes o b s e r v e d from animal to animal; the changes, however, were most marked in the beef fed animals. In the beef fed a n i m a l s the p r o p o r t i o n of glomeruli with e n d o c a p i l l a r y swelling, with h y a l i n o sls, and wlth s e g m e n t a l sclerosis was r e s p e c t i v e l y 25%, 29% and 7%, in the 3% flsh Oli fed a n i m a l s 9%, 10%, and 9%, and in the 14% fish oli fed animals 16%, 10%, and 8% T u b u l a r d i l a t a t i o n ranged from 1 to 4+; i n t r a t u b u l a r p r o t e i n was a c o n s i s t e n t finding in a l m o s t all anlmals. N e p h r o c a l c l n o s i s (1-2+) was p r e s e n t in about 15% of animals. T u b u l a r d i l a t a t i o n in the animals fed beef tallow (3.0 + 0.2) and those fed 3% fish oil (2.8 + 0.3) was s i g n i f i c a n t l y g r e a t e r than in T h e anlmals fed 14% fish oil (2.1 + 0.~). Serum liplds In all animals in3ected with adrlamycin, serum llplds were m a r k e d l y higher than controls at week 4 and 7. Total serum triglycerides and c h o l e s t e r o l were lower in rats fed 3% fish oli than those fed beef tallow at week 4 but not at w e e k 7. Rats fed 14% flsh oli had lower c h o l e s t e r o l and t r i g l y c e r i d e levels than those fed beef tallow or 3% flsh oli at both week 4 and 7. There were no d i f f e r e n c e s in the levels of HDL c h o l e s t e r o l b e t w e e n groups at week 7. LDL chol e s t e r o l levels were lower in the 14% flsh oil fed rats than in the other two groups. R e l a t i o n s h i p b e t w e e n serum liplds and k i d n e y f u n c t i o n and histology. The r e l a t i o n s h i p s b e t w e e n the lipid values, urine protein, and serum a l b u m i n at week 4 and the serum c r e a t L n l n e and renal h i s t o l o g y at week 7 were also a s s e s s e d in the rats in3ected wlth adriamycin. In none of the three groups was there a s i g n i f i c a n t c o r r e l a t i o n b e t w e e n serum a l b u m i n or urine p r o t e i n at week 4 and the serum c r e a t l n i n e at week 7. W h e n the s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s were e x a m i n e d in all 33 animals w l t h nephrotic s y n d r o m e due to a d r l a m y c i n and fed the three d i f f e r e n t diets, there was a s t r i k i n g c o n s i s t e n c y . The r e l a t i o n s h i p s of log serum c r e a t i n i n e at w e e k 7 to the p e r c e n t of g l o m e r u l i wlth e n d o t h e l i a l s w e l l i n g and with h y a l i n o s i s were s i g n i f i c a n t in rats from all three groups c o n s i d e r e d together (r = 0.72, p <0.01; and r = 0.59, p <0.01 r e s p e c t i v e l y ) , and in each d i e t a r y group. By contrast, the c o r r e l a t i o n of log serum c r e a t i n l n e with the p e r c e n t of g l o m e r u l a r
2322
showing
Fish
Oil
segmental
is B e n e f i c i a l
to N e p h r o t i c
sclerosis
not
was
Rats
significant
(r =
Vol.
0.06,
40,
No.
24,
1987
p NS).
Both log triglycerides and log cholesterol measured at 4 weeks correlated positively w i t h log c r e a t i n i n e m e a s u r e d a t 7 w e e k s a n d w l t h the p e r c e n t of g l o meruli showing both endothelial swelling and hyalinosis ( T a b l e 4), a n d i n v e r s e l y w i t h b o d y w e i g h t a t w e e k 7. While these correlations were significant in a l l t h r e e g r o u p s of a n i m a l s c o n s i d e r e d as a w h o l e , the s t r i k i n g c o r r e l a t i o n s were s e e n i n t h e a n i m a l s f e d b e e f t a l l o w ; in t h e s e a n i m a l s b o t h the t r i g l y c e r i d e a n d cholesterol levels at 4 weeks were predictive of b o d y w e i g h t , s e r u m c r e a t i n i n e , glomerular endothelial swelling, and glomerular hyallnosis m e a s u r e d at 7 w e e k s .
Table 4 Relatlonshlp between L1pld Values at Week 4 and Serum Creatlnlne and Renal Hlstologlc flndlngs at Week 7
Renal Findings at 7 Weeks
Beef Tallow (n=10) r p
3% Fish O11 (n=12) r p
Log creatlnlne
0.74
<0.02
0.07
NS
0.38
NS
0.45
<0.01
Endothelzal swelllng (% glomerull)
0.67
<0.05
0.35
NS
0.07
NS
0.38
<0.05
Hyallnosls
0.71
<0.05
0.27
NS
0.I0
NS
0.56
<0.01
0.29
NS
0.42
NS
0.05
NS
Log Trlglycerlde
(% glomerull)
Segmental sclerosls (% glomerull)
0.27
NS
Log Cholesterol
14% Fish O11 (n=ll) r p
All An~mals (n=33) r p
at 4 Weeks
at 4 Weeks
Log creatlnlne
0.75
<0.02
0.22
NS
0.16
NS
0.46
<0.01
Endothellal swelllng (% glomerul~)
0.76
<0.01
0.50
NS
0.i0
NS
0.49
<0.01
Hyallnosls
0.79
<0.01
0.22
NS
0.05
NS
0.66
<0.01
0.39
NS
0.06
NS
0.08
NS
0.16
NS
(% glomerull)
Segmental sclerosls (% glomerull)
Discussion I n n o r m a l r a t s (16), n o r m a l h u m a n s ( 5 , 6 , 7 ) , a n d p a t i e n t s w i t h v a r l o u s d y s lipidemias ( 8 , 9 , 1 0 ) f e e d l n g f l s h o i l has b e e n s h o w n to e x e r t a f a v o r a b l e e f f e c t on triglycerlde and cholesterol levels. The present study showed an attenuation o f the e x t r e m e r i s e in t r i g l y c e r l d e and total cholesterol l e v e l s at 4 w e e k s in a n i m a l s f e d the l o w e r c o n c e n t r a t i o n of f l s h o ~ I a n d a n e v e n m o r e m a r k e d e f f e c t a t b o t h 4 a n d 7 w e e k s in t h o s e f e d the h l g h e r c o n c e n t r a t l o n of f l s h oli. The v e r y h i g h r i s e in L D L c h o l e s t e r o l , m e a s u r e d o n l y at 7 w e e k s , was s o m e w h a t attenuated o n l y in t h e h i g h f l s h o i l f e d a n l m a l s . T h i s e f f e c t w a s n o t d u e to differences in d i e t a r y f a c t o r s s u c h as the t o t a l c a l o r i c , l i p i d , or f i b e r c o n tent which were identical in a l l t h r e e g r o u p s ; nor was it d u e to t o t a l d i e t a r y cholesterol w h i c h w a s h i g h e s t in t h e h i g h f i s h o i l g r o u p . In nephrotic syndrome hyperllpidemia is t h o u g h t to r e s u l t f r o m a n o n selectlve hepatic overproduction of l l p o p r o t e i n in r e s p o n s e to h y p o a l b u m i n e m i a
Vol. 40, No. 24, 1987
and/or low oncotic of lipoprotein has adriamycin analog, of flsh oil may be (6).
Fish Oil is Beneficial
to Nephrotic
Rats
2323
pressure, or from decreased catabolism (3,18). Overproduction been shown in nephrotic syndrome induced in rats by an Daunorubicin (19). It Is possible that the favorable effects because of its action in decreasing hepatic VLDL production
Reducing the triglycerlde VLDL cholesterol is known to (17). Fish oil Is also known A 2 (6,11,20), which plays an adriamycln induced nephrosis
and LDL cholesterol, as in the present study, and protect against the development of atherosclerosis to suppress production of thromboxane important pathogenetic role in the proteinuria of (21).
The mechanlsms by which abnormalities of the lipids may produce progressive in3ury In the kidney have been discussed recently (22). In guinea pigs, high cholesterol feeding promotes the development of glomerular sclerosis (23); in the lupus nephritis of the NZB/NZW mouse, a dlet high in saturated fats accelerates progression of the renal disease (24). Using pharmacologic agents to lower lipid levels has had beneficial effects in partially nephrectomized rats (25), obese Zucker rats (26) and in rats with puromycin aminonucleoside nephrosis (27). Polyunsaturated fatty acids, beneficial ~n the renal disease of partlally nephrectomized rats, have also had favorable effects on the lipid profile (20). The present studies showed a marked effect of the lipid composition of the diet on both the plasma liplds and on the kidney. Feedlng beef fat to rats with adriamycin induced nephrotic syndrome resulted in extremely high levels of triglycerldes, cholesterol, and LDL cholesterol whlch were ameliorated by fish oil feeding. The lipid levels, measured relatively early, were strikingly predlctive of later adverse effects on body weight and on renal function and histology. Thls correlation was highly significant in animals fed beef fat, and was not significant in the high fish oil fed animals. Thls correlation suggests that elevated serum lipid levels early in this model of nephrotic syndrome may play a role in the pathogenesis of subsequent glomerular damage. The mechanisms might be similar to those operative in the development of atherosclerosls elsewhere in the vascular system; fish oil, rich in omega-3 fatty acids, reduces the serum llplds and may protect against the development of atherosclerosls. In the present experiment, of very short duratlon, glomerular sclerosis occurred only in minor degree. The development of glomerular sclerosis may have been prevented, at least in part, by the relatively low protein Intake due to progressive renal disease and as an effect of the pair feedings. The mean weight of the control group did not change. This might have been a consequence of the pair feedings. Other known effects of omega-3 fatty acids, such as changes In synthesis of products of the cylooxygenase and lipoxygenase pathways of fatty acid metabolism (7), reduction of platelet aggregabllity (7), decrease in systemic blood pressure (28), improvement of fibrlnolytlc activity (29) and effects on macrophage function (30,31), might have also played a protective role. References I. 2. 3. 4. 5. 6.
R.C. CURRY, JR., W.C. ROBERTS, Am. J. Med. 63 183-192 (1977) N.P. MALLICK, C.D. SHORT, Nephrology 27 54-57 (1981) D.B. BERNARD, In, Nephrotic Syndrome, (ed~) B.M. Brenner, J.H. Stein, chapter 4, Churchill Livingstone, New York, pp 85-120 (1982) J.F. BRIDGMAN, S.M. ROSEN, J.M. THORP, Lancet 2 506-509 (1972) W.S. HARRIS, W.E. CONNOR, M.P. MCMURRY, Metabolism 32 179-184 (1983) P.J. NESTEL, W.E. CONNOR, M.F. REARDON, S. CONNOR, S. WONG, R. BOSTON, J. Clin. Invest. 74 82-89 (1984)
2324
7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
31.
Fish oil is Beneflcial
to Nephrotic Rats
Vol. 40, No. 24, 1987
C. VON SCHACKY, S. FISCHER, P.C. WEBER, J. Clin. Invest. 76 1626-1631 (1985) B.E. PHILLIPSON, D.W. ROTHROCK, W.E. CONNOR, W.S. HARRIS, D.R. ILLINGWORTH, N. Engl. J. Med. 312 1210-1216 (1985) T. HAMAZAKI, R. NAKAZAWA, S. TATENO, H. SHISHIDO, K. ISODA, Y. HATTORI, T. YOSHIDA, T. FUJITA, S. YANO, A. KUMAGAI, Kidney Int. 26 81-84 (1984) W.S. HARRIS, W.E. CONNOR, S.B. INKELES, D.R. ILLINGWORTH, Metabolism 33 1016-1019 (1984) J.D. PRICKETT, D.R. ROBINSON, A.D. STEINBERG, J. Clin. Invest. 68 556-559 (1981) V.E. KELLEY, A. FERRETTI, S. IZUI, T.B. STROM, J. Immunol. 134 1914-1919 (1985) V. KHER, U. BARCELLI, M. WEISS, L. GALLON, P. PAJEL, P. LASKARZEWSKI, V.E. POLLAK, Prost. Leuk. Med. 22 323-334 (1986) T. BERTANI, A. POGGI, R. POZZONI, F. DELAINI, G. SACCHI, Y. THOUA, G. MECCA, G. REMUZZI, M.B. DONATI, Lab. Invest. 46 16-19 (1982) R. HITZEMANN, Neurochem. Res. 6 935 (1981) S. BALASUBRAMANIAM, L.A. SIMONS, S. CHANG, J.B. HICKIE, J. Lipid Res. 26 684-689 (1985) W.B. KANNEL, J.T. DOYLE, A.M. OSTFELD, C.D. JENKINS, L. KULLER, R.N. PODELL, J. STAMLER, Circulatlon 70(1) 155A-205A (1984) J.B. MARSH, J. Lipid Res. 25 1619-1623 (1984) N. MORISAKI, N. MATSUOKA, Y. SAITO, A. KUMAGAI, Metabolism 33 405-410 (1984) U. BARCELLI, J. MIYATA, Y. ITO, L. GALLON, P. LASKARZEWSKI, M. WEISS, R. HITZEMANN, V. POLLAK, Prostaglandins 32 211-219 (1986) G. REMUZZI, L. IMBERTI, M. ROSSINI, C. MORELLI, C. CARMINATI, G.M. CATTANEO, T. BERTANI, J. Clln. Invest. 75 94-101 (1985) J.F. MOORHEAD, M.K. CHAN, M. EL-NAHAS, Z. VARGHESE, Lancet 2 1309-1311 (1982) S.W. FRENCH, W. YAMANAKA, R. ~gTWALD, Arch. Path. 83 204-210 (1967) V.E. KELLEY, S. IZUI, Am. Assoc. Pathol. iii 288-297 (1983) B.L. KASISKE, M.P. O'DONNELL, F. DANIELS, W.F. KEANE, Clln. Res. 33 488A (1985) B.L. KASISKE, M.P. O'DONNELL, W.F. KEANE, Clin. Res. 34 600A (1986) F.G. SILVA, K.D.G. EDWARDS, C.L. PIRANI, Kldney Int. 16 789 (1979) P. SINGER, M. WIRTH, W. GODICKE, H. HEINE, Experlentia 41 462-464 (1985) U. BARCELLI, P. GLAS-GREENWALT, V.E. POLLAK, Thromb. Res. 39 307-312 (1985) T.H. LEE, R.L. HOOVER, J.D. WILLIAMS, R.I. SPERLING, J. RAVELESE, B.W. SPUR, D.R. ROBINSON, E.J. COREY, R.A. LEWIS, K.F. AUSTEN, N. Engl. J. Med. 1217-1224 (1985) K.S. KANT, J. SEXTON, U. BARCELLI, V.E. POLLAK, Clin. Res. 34 599A (1986)
BENEFICIAL Yasushl
pp. 2 3 1 7 - 2 3 2 4
Pergamon
D I E T A R Y FAT IN E X P E R I M E N T A L N E P H R O T I C SYNDROME: EFFECTS OF FISH OIL ON SERUM LIPIDS AND, INDIRECTLY, Ito,
Uno Barcelll, Moti Kashyap,
Divisions Blostatistics,
Journals
ON THE K I D N E Y
W a t a r u Yamashlta, Mark Weiss, Brian Thompson, James Deddens, and Victor E. Pollak
of N e p h r o l o g y , Lipid R e s e a r c h , and E p l d e m l o l o g y and D e p a r t m e n t of Medicine, and the D e p a r t m e n t of Pathology, U n i v e r s i t y of C i n c i n n a t i Medical Center.
(Recelved
in final form M a r c h
31, 1987)
Summary Three i s o c a l o r l c diets with d i f f e r e n t fat c o m p o s i t i o n were fed to rats for seven weeks after the p r o d u c t i o n of nephrotic syndrome by adriamycin. The effects of feeding 3% and 14% flsh oli were compared wlth those of feeding beef fat. At the fourth week of f e e d i n g the levels of t r i g l y c e r l d e s and c h o l e s t e r o l were lower in b o t h fish oli fed groups. At the seventh week these levels, and the LDL cholesterol, were lower only In the 14% fish oli group. In rats fed beef fat, but not ~n those fed flsh oli, there was a s t r i k i n g p o s i t i v e c o r r e l a t i o n of the f o u r - w e e k serum t r i g l y c e r l d e s and c h o l e s t e r o l wlth the s e v e n - w e e k serum c r e a t i n i n e level and wlth the degree of g l o m e r u l a r h y a l i n o s i s and e n d o t h e l i a l swelling. The f a v o r a b l e effects of flsh oil f e e d i n g on serum llplds may have a p r o t e c t i v e e f f e c t on the d e v e l o p m e n t of g l o m e r u l a r damage.
N e p h r o t l c syndrome is c h a r a c t e r i z e d by heavy proteinurla, h y p o a l b u m i n e m i a and h y p e r l i p i d e m l a . D e s p i t e an a p p a r e n t increased p r e v a l e n c e of a t h e r o s c l e r o s i s in nephrotlc patients (1,2,3), the need for and effects of t r e a t m e n t of h y p e r l l p l d e m l a are not well established. Increased toxicity of c l o f i b r a t e t r e a t m e n t has been reported in the presence of h y p o a l b u m i n e m l a (4). Dietary fish oil, rlch in o m e g a - 3 - f a t t y acids, reduces plasma t r l g l y c e r l d e levels In normal (5,6,7) and h y p e r l i p l d e m i c subjects (8,9,10), and has a b e n e f i c i a l e f f e c t in p r e s e r v i n g renal function in various forms of e x p e r i m e n t a l renal d i s e a s e (11,12,13). The p r e s e n t study was done to evaluate the effects of the c o m p o s i t i o n of the d i e t a r y fat on p l a s m a llplds, and on renal function and m o r p h o l o g y in rats with n e p h r o t l e syndrome induced by adrlamycln. It was d e s i g n e d p a r t i c u l a r l y to e v a l u a t e the effects of two levels (3% and 14%) of fish oil feeding, and to c o m p a r e them w i t h feeding beef fat. The beef tallow diet, c o n t a l n l n g 175 g/Kg fat, 44% of which was s a t u r a t e d fatty acid, m i m i c s the typical North A m e r i c a n diet. The 3% fish oil diet was d e s i g n e d to s i m u l a t e a dlet which could be r e a l i s t i c a l l y achieved in humans by d e c r e a s i n g some of the s a t u r a t e d fat and s u p p l e m e n t l n g the diets wlth s e m i - p u r i f l e d flsh oil. The 14% flsh oli diet was d e s i g n e d to a c h i e v e the m a x i m u m fish oll which S u p p o r t e d in part by NIH grants AM 34489, C L I N F O RR 00068, and a grant the R e s e a r c h and E d u c a t i o n a l Funds of D i a l y s i s Clinics, Inc.
Copyright
0 0 2 4 - 3 2 0 5 / 8 7 $3.00 + .00 (c) 1987 P e r g a m o n J o u r n a l s
Ltd.
from
2318
Fish Oil is B e n e f i c l a l
to N e p h r o t i c Rats
Vol.
40, No.
24,
1987
d o e s not i n t e r f e r e w i t h a c c e p t a b i l i t y . The fat c o m p o s i t i o n of thls d l e t s l m u l a tes that of an E s k i m o diet. A n i m a l s w e r e s t u d i e d at 4 w e e k s b e c a u s e renal d a m a g e is m i n i m a l at thls time (14), and w e r e s a c r i f i c e d at 7 w e e k s b e c a u s e term i n a l renal f a i l u r e was d e v e l o p l n g in some rats. M a t e r i a l and M e t h o d s Animals and Diets T h i r t y - f i v e S p r a g u e - D a w l e y f e m a l e rats (H~rlan, I n d i a n a p o l l s , Indiana) w e i g h i n g 2 2 0 - 2 7 0 g r e c e i v e d a s i n g l e dose of a d r l a m y c l n (Adria L a b o r a t o r i e s , C o l u m b u s , Ohio) 7.5 m g / K g i n t r a v e n o u s l y into the tall veln. S e v e n days later, m a t c h e d by u r i n a r y p r o t e i n e x c r e t l o n , the rats were a l l o c a t e d to three isoc a l o r i c d i e t s (Table i), c o n t a i n i n g beef t a l l o w (beef, n=ll), 3% f i s h oil (3% flsh, n=12) a n d 14% flsh oll (14% flsh, n=12). S i x rats, w h i c h had r e c e i v e d n o r m a l s a l i n e only, w e r e fed the beef d l e t and s e r v e d as n o n - n e p h r o t i c c o n t r o l s (C). This g r o u p was i n c l u d e d in o r d e r to p r o v i d e a p p r o p r i a t e r e f e r e n c e of v a l u e s for the three e x p e r i m e n t a l groups. The flsh oli was s u p p l l e d by R. P. S c h e r e r , Troy, M i c h i g a n as MaxEPA, a c o n c e n t r a t e d b l e n d of flsh oils from w h i c h v i t a m i n s A a n d D h a v e b e e n r e m o v e d and to w h i c h a l D h a - t o c o p h e r o l has b e e n added. The diets were p r e p a r e d by Teklad, Madison, W i s c o n s i n . P a l r f e e d i n g was e m p l o y e d t h r o u g h o u t , the rats in three groups b e i n g pair fed a g a i n s t rats in the g r o u p w i t h the l o w e s t food intake.
Table i The Experlmental Diets
Beef Tallow
3% Flsh 011
14% Fish O11
Ma3or Constltuents (g/Kg) Proteln DL Methlonlne Carbohydrate Flber Mineral mlxture Vltamln mlxture Total fat
180 30 500 70 35 i0 175
180 30 500 70 35 I0 175
180 30 500 70 35 i0 175
Source of Fats (g/Kg) Beef tallow Safflower oli MaxEPA
145 30 0
115 30 30
35 0 140
Composltlon of Fats (%) Fatty aclds (%) Saturated Monounsaturated Llnolelc acld (18:2 omega-6) Elcosapentaenolc acld (20:5 omega-3) Docosahexaenolc acld (22:6 omega-3) Other fatty aclds Cholesterol (g/Kg)
44.1 40.3 13.7
41.4 35.5 14.8
40.8 26.3 1.2
0
3.1
13.0
0
2.0
7.9
1.9
3.2
10.8
0.13
0.28
0.87
Vol.
40, No.
24,
1987
Fish Oil is B e n e f i c i a l
to N e p h r o t i c
Rats
2319
Rats were w e l g h e d weekly. Urine was c o l l e c t e d for 23 hours, w i t h o u t food b u t with free access to water, at w e e k 0 and 2 and w e e k l y thereafter. Blood p r e s s u r e was m e a s u r e d by the tail cuff method w i t h o u t a n e s t h e s i a at week 4. Serum a l b u m i n was m e a s u r e d at weeks 4 and 7, serum c r e a t l n i n e at week 7. Blood was drawn for serum llpids after a 15 hour fast at weeks 4 and 7. At sacrifice, k i d n e y tissue was o b t a i n e d under x y l a z l n e - k e t a m i n anesthesia, fixed in M o s s m a n ' s solutlon, stalned with periodic a c l d - S c h i f f (PAS), and exam i n e d "blinded." In the glomeruli, p a r t i c u l a r a t t e n t i o n was pald to e n l a r g e m e n t w i t h e n d o c a p i l l a r y swelling, to h y a l l n o s i s ( c a p i l l a r y l i p o p r o t e i n insudation), add to s e g m e n t a l and global sclerosis (mesanglal and GBM increase wlth capillary loop o b l l t e r a t l o n with and w i t h o u t collapse). In the k i d n e y s of each rat b e t w e e n 50 and 250 g l o m e r u l l were studied, and the number with each a b n o r m a l i t y counted. The tubular changes e v a l u a t e d s e m i q u a n t i t a t i v e l y (0-4+) w e r e d i l a t a tion, i n t r a l u m l n a l protein, p r o t e i n r e a b s o r p t i o n droplets, and n e p h r o c a l c l n o s l s . Laboratory Methods U r i n e p r o t e i n was m e a s u r e d with C o o m a s i e b r i l l i a n t blue, serum c r e a t i n i n e by the Jaffe reaction, and serum a l b u m i n with Bromcresol green. Fatty acid analysis was p e r f o r m e d by gas c h r o m a t o g r a p h y (15). For analysis of lipids, serum c o n t a i n i n g 0.2% E D T A was u l t r a c e n t r l f u g e d initlally, using a 50.3 TI Rotor in a L8-55 c e n t r i f u g e (Beckman I n s t r u m e n t Co., Palo Alto, Ca.) at 45000 rpm for 23 h o u r s after a d 3 u s t i n g the d e n s i t y to 1.020 g/ml wlth p o t a s s i u m bromide. The b o t t o m layer c o n t a i n e d LDL plus HDL c h o l e s t e r o l (tube A). This was further c e n t r l f u g e d at 45000 rpm for 23 hours at the d e n s i t y of 1.063 g/ml. The b o t t o m layer c o n t a i n e d HDL c h o l e s t e r o l . LDL was c a l c u l a t e d by s u b t r a c t i n g HDL f r o m total c h o l e s t e r o l in tube A. T r i g l y c e r i d e and c h o l e s t e r o l were m e a s u r e d by e n z y m a t i c m e t h o d s on a H l t a c h l 705 a u t o a n a l y z e r . Statistics Analysls of variance on single and repeated m e a s u r e m e n t s was used as appropriate. C o r r e l a t i o n was c a l c u l a t e d by linear regression. For n o r m a l l y d i s t r i b u t e d data, results are e x p r e s s e d as a r i t h m e t i c means + 1 SD. For data log normally d i s t r i b u t e d , the values were log t r a n s f o r m e d a n d results e x p r e s s e d as g e o m e t r i c means (-I SD, +I SD). D i f f e r e n c e s b e t w e e n groups were a n a l y z e d u s i n g the D u n c a n test. Results The Anlmals
lowest food intake was in the a d r i a m y c i n treated animals in the three other groups were pair fed a g a i n s t them.
fed beef
fat.
The results are s u m m a r i z e d in Table 2, the s t a t i s t i c a l s i g n i f i c a n c e of d i f f e r e n c e s b e t w e e n groups in Table 3. D u r i n g the 7 weeks of study i0/ii, ii/ii, and 12/13 rats s u r v i v e d r e s p e c t l v e l y in the beef, 3% fish and 14% fish groups. One rat each died in the beef and 14% flsh groups at week 4. Initially, rats had a normal food intake of a p p r o x i m a t e l y 15 g/day, which was m a i n t a i n e d c o n s t a n t t h r o u g h o u t the experiment. F e w rats had poor appetite, c o i n c i d i n g with the a p p e a r a n c e of the uremic syndrome. In the controls, the mean body w e i g h t was stable. In n e p h r o t l c rats fed beef tallow the weight, similar to the c o n t r o l s at week 0, d e c r e a s e d s i g n i f l c a n t l y by week 2, and remained lower thereafter. There was little c h a n g e in w e i g h t of rats fed 3% and 14% fish oil. At 4 and 7 weeks, the w e i g h t s of beth flsh oil fed groups were higher than those fed beef tallow. Blood p r e s s u r e was i d e n t i c a l in all groups at week 4. U r i n a r y p r o t e i n excretion, serum a l b u m i n and c r e a t l n i n e U r l n a r y p r o t e i n e x c r e t i o n ~n c o n t r o l s was minimal.
One week after
adriamy-
2320
Fish Oil is Beneflcial to Nephrotic Rats
Vol. 40, No. 24, 1987
Table 2 SummarM of Laboratory Data
Beef Tallow L- (n_--ll). . . . . n(= l O ) Week 4 Week 7
Control
(n=6) Week 4 Weight (g)*
248 + 5
Blood pressure*
118 + 7
P r o t e i n excretion
4.6 (2.4, 6.8)
(mg/23 h) Serum albumin (7/dl)
(n=6) Week 7 247 ~ I0
216 + 19
248 + i0
ii~ + I0 3.4 (1.6, 5.6)
3.27 3.01 (3.[0, 3.45)(2.86, 3.19)
Serum creatlnlne (mg/d[)
227 ~ 7
3% Flsh 01 1 [n=12) (n=12) Week 4 Week 7
_
14% Fish Oll (n=12) (n=ll) Week 4 Week 7
236 + i0
251 + 17
117 + 7
641 (547, 75[)
604 (456 799)
(607, 901)
800 (716, 895)
1.4g (1.4, [.6)
1.81 ([.56, 2.07)
1.59 ([.51, 1.68)
1.78 (1.62, 1.96)
0.63 (0°59, 0.68)
238 + i0
116 + 7
740
1.75 (i.16, 2.63)
574 (458, 719)
561 (455, 691)
1.66 (1.53, 1.81)
1.81 (1.62, 2.03)
1.56 (1.20, ?.02)
1.34 (0.79, 2.28)
rrlglycerx]e (mg/dl)
96 (60, 151)
27 (21, 36)
5L34 (2313, [1393)
3012 (@06, 11249)
1993 (827, 4801)
2790 (765, 18[71)
865 (405, 1645)
811 (306, 2147)
Total cholesterol
65 (56, 76)
59 (51, 69)
982 (571, [688)
g09 (463, [783)
681 (488, 951)
925 (530, 1617)
482 (365, 630)
412 (273, 619)
(mg/dl)
LDL (mg/dl)
9.5 (7.1, [2.7)
HDL (mg/dl)
46 (36, 58)
199 (147, 270)
223 (164, 303)
147 (i[8, 184)
73 (58, 91)
67 (63, 121)
82 (64, 105)
Table ~tatistlcal
$1gntf[cance
of
3
~le
Dlfferences
Adrlamycln
Beef
between
Tallow
Beef
vs 3% Week
Welght
Blood
pressure
Serum
albumin
Serum
creatlnxne
Fish 4
of
Tallow
3%
Fish
vs Oil Week
14% 7
Week
4
P
P
P
<0.01
<0.01
<0.01
NS <0.05
Groups
Rats
NS MS
NS
<0.01
-
Fxsh
OiL
vs Oxl week
14% 7
P <0.01
Week
4
Oil Week
7
P
P
NS
NS
-
NS
NS
N$
NS
Fish
-
NS
NS
Tryglycerxdes
<0.01
NS
<0.01
<0.05
<0.05
<0.05
Cholesterol Total
<0.05
NS
<0.01
<0.01
<0.05
<0.01
LDL
-
NS
-
<0.05
-
<0.01
HDL
-
NS
-
MS
-
NS
Vol.
40, No.
24,
1987
Fish Oil
is B e n e f i c i a l
to N e p h r o t i c
Rats
2321
cin a d m i n i s t r a t i o n all rats had d e v e l o p e d proteinuria, g r e a t e r than 10-fold that of controls. At this time they were d i v i d e d into the three d i e t a r y groups, m a t c h e d by u r i n a r y p r o t e i n excretion. At weeks 4 and 7, the p r o t e i n e x c r e t i o n in all three groups was 120-240 times g r e a t e r than in the controls. F r o m weeks 2 to 5 the p r o t e i n e x c r e t i o n of rats in the group fed 3% fish oil was not diff e r e n t from that of rats fed beef fat. The urine p r o t e i n e x c r e t i o n of rats fed 14% fish oil was lower than that of the groups fed beef (p <.04) and 3% fish (p <.03). Serum a l b u m i n values were d e c r e a s e d to about 45 to 60% of the controls in all three a d r i a m y c i n groups. S e r u m c r e a t i n i n e values were e l e v a t e d at week 7 in all animals r e c e i v i n g adriamycln. There was c o n s i d e r a b l e v a r i a t i o n b e t w e e n animals in each group. T h e y values were h i g h e r in those fed beef tallow than those fed 3% or 14% fish oil, b u t the d i f f e r e n c e s were not s i g n i f i c a n t . Renal h i s t o l o g y The k i d n e y s of the c o n t r o l rats were normal. In the a d r i a m y c i n treated animals, n o n - p r o l i f e r a t l v e g l o m e r u l a r lesions w i t h v a r y i n g degrees of c a p i l l a r y c o l l a p s e and s e g m e n t a l sclerosis were seen; there was no global g l o m e r u l a r sclerosis. In many animals there was s i g n i f i c a n t h y a l i n o s i s , g l o m e r u l a r e n l a r g e m e n t with e n d o c a p i l l a r y swelling, and visceral e p i t h e l i a l changes i n c l u d i n g s w e l l i n g and p r o l i f e r a t i o n which r e f l e c t proteinuria. In each group there was a s i g n i f i c a n t v a r i a t i o n in the changes o b s e r v e d from animal to animal; the changes, however, were most marked in the beef fed animals. In the beef fed a n i m a l s the p r o p o r t i o n of glomeruli with e n d o c a p i l l a r y swelling, with h y a l i n o sls, and wlth s e g m e n t a l sclerosis was r e s p e c t i v e l y 25%, 29% and 7%, in the 3% flsh Oli fed a n i m a l s 9%, 10%, and 9%, and in the 14% fish oli fed animals 16%, 10%, and 8% T u b u l a r d i l a t a t i o n ranged from 1 to 4+; i n t r a t u b u l a r p r o t e i n was a c o n s i s t e n t finding in a l m o s t all anlmals. N e p h r o c a l c l n o s i s (1-2+) was p r e s e n t in about 15% of animals. T u b u l a r d i l a t a t i o n in the animals fed beef tallow (3.0 + 0.2) and those fed 3% fish oil (2.8 + 0.3) was s i g n i f i c a n t l y g r e a t e r than in T h e anlmals fed 14% fish oil (2.1 + 0.~). Serum liplds In all animals in3ected with adrlamycin, serum llplds were m a r k e d l y higher than controls at week 4 and 7. Total serum triglycerides and c h o l e s t e r o l were lower in rats fed 3% fish oli than those fed beef tallow at week 4 but not at w e e k 7. Rats fed 14% flsh oli had lower c h o l e s t e r o l and t r i g l y c e r i d e levels than those fed beef tallow or 3% flsh oli at both week 4 and 7. There were no d i f f e r e n c e s in the levels of HDL c h o l e s t e r o l b e t w e e n groups at week 7. LDL chol e s t e r o l levels were lower in the 14% flsh oil fed rats than in the other two groups. R e l a t i o n s h i p b e t w e e n serum liplds and k i d n e y f u n c t i o n and histology. The r e l a t i o n s h i p s b e t w e e n the lipid values, urine protein, and serum a l b u m i n at week 4 and the serum c r e a t L n l n e and renal h i s t o l o g y at week 7 were also a s s e s s e d in the rats in3ected wlth adriamycin. In none of the three groups was there a s i g n i f i c a n t c o r r e l a t i o n b e t w e e n serum a l b u m i n or urine p r o t e i n at week 4 and the serum c r e a t l n i n e at week 7. W h e n the s t r u c t u r e - f u n c t i o n r e l a t i o n s h i p s were e x a m i n e d in all 33 animals w l t h nephrotic s y n d r o m e due to a d r l a m y c i n and fed the three d i f f e r e n t diets, there was a s t r i k i n g c o n s i s t e n c y . The r e l a t i o n s h i p s of log serum c r e a t i n i n e at w e e k 7 to the p e r c e n t of g l o m e r u l i wlth e n d o t h e l i a l s w e l l i n g and with h y a l i n o s i s were s i g n i f i c a n t in rats from all three groups c o n s i d e r e d together (r = 0.72, p <0.01; and r = 0.59, p <0.01 r e s p e c t i v e l y ) , and in each d i e t a r y group. By contrast, the c o r r e l a t i o n of log serum c r e a t i n l n e with the p e r c e n t of g l o m e r u l a r
2322
showing
Fish
Oil
segmental
is B e n e f i c i a l
to N e p h r o t i c
sclerosis
not
was
Rats
significant
(r =
Vol.
0.06,
40,
No.
24,
1987
p NS).
Both log triglycerides and log cholesterol measured at 4 weeks correlated positively w i t h log c r e a t i n i n e m e a s u r e d a t 7 w e e k s a n d w l t h the p e r c e n t of g l o meruli showing both endothelial swelling and hyalinosis ( T a b l e 4), a n d i n v e r s e l y w i t h b o d y w e i g h t a t w e e k 7. While these correlations were significant in a l l t h r e e g r o u p s of a n i m a l s c o n s i d e r e d as a w h o l e , the s t r i k i n g c o r r e l a t i o n s were s e e n i n t h e a n i m a l s f e d b e e f t a l l o w ; in t h e s e a n i m a l s b o t h the t r i g l y c e r i d e a n d cholesterol levels at 4 weeks were predictive of b o d y w e i g h t , s e r u m c r e a t i n i n e , glomerular endothelial swelling, and glomerular hyallnosis m e a s u r e d at 7 w e e k s .
Table 4 Relatlonshlp between L1pld Values at Week 4 and Serum Creatlnlne and Renal Hlstologlc flndlngs at Week 7
Renal Findings at 7 Weeks
Beef Tallow (n=10) r p
3% Fish O11 (n=12) r p
Log creatlnlne
0.74
<0.02
0.07
NS
0.38
NS
0.45
<0.01
Endothelzal swelllng (% glomerull)
0.67
<0.05
0.35
NS
0.07
NS
0.38
<0.05
Hyallnosls
0.71
<0.05
0.27
NS
0.I0
NS
0.56
<0.01
0.29
NS
0.42
NS
0.05
NS
Log Trlglycerlde
(% glomerull)
Segmental sclerosls (% glomerull)
0.27
NS
Log Cholesterol
14% Fish O11 (n=ll) r p
All An~mals (n=33) r p
at 4 Weeks
at 4 Weeks
Log creatlnlne
0.75
<0.02
0.22
NS
0.16
NS
0.46
<0.01
Endothellal swelllng (% glomerul~)
0.76
<0.01
0.50
NS
0.i0
NS
0.49
<0.01
Hyallnosls
0.79
<0.01
0.22
NS
0.05
NS
0.66
<0.01
0.39
NS
0.06
NS
0.08
NS
0.16
NS
(% glomerull)
Segmental sclerosls (% glomerull)
Discussion I n n o r m a l r a t s (16), n o r m a l h u m a n s ( 5 , 6 , 7 ) , a n d p a t i e n t s w i t h v a r l o u s d y s lipidemias ( 8 , 9 , 1 0 ) f e e d l n g f l s h o i l has b e e n s h o w n to e x e r t a f a v o r a b l e e f f e c t on triglycerlde and cholesterol levels. The present study showed an attenuation o f the e x t r e m e r i s e in t r i g l y c e r l d e and total cholesterol l e v e l s at 4 w e e k s in a n i m a l s f e d the l o w e r c o n c e n t r a t i o n of f l s h o ~ I a n d a n e v e n m o r e m a r k e d e f f e c t a t b o t h 4 a n d 7 w e e k s in t h o s e f e d the h l g h e r c o n c e n t r a t l o n of f l s h oli. The v e r y h i g h r i s e in L D L c h o l e s t e r o l , m e a s u r e d o n l y at 7 w e e k s , was s o m e w h a t attenuated o n l y in t h e h i g h f l s h o i l f e d a n l m a l s . T h i s e f f e c t w a s n o t d u e to differences in d i e t a r y f a c t o r s s u c h as the t o t a l c a l o r i c , l i p i d , or f i b e r c o n tent which were identical in a l l t h r e e g r o u p s ; nor was it d u e to t o t a l d i e t a r y cholesterol w h i c h w a s h i g h e s t in t h e h i g h f i s h o i l g r o u p . In nephrotic syndrome hyperllpidemia is t h o u g h t to r e s u l t f r o m a n o n selectlve hepatic overproduction of l l p o p r o t e i n in r e s p o n s e to h y p o a l b u m i n e m i a
Vol. 40, No. 24, 1987
and/or low oncotic of lipoprotein has adriamycin analog, of flsh oil may be (6).
Fish Oil is Beneficial
to Nephrotic
Rats
2323
pressure, or from decreased catabolism (3,18). Overproduction been shown in nephrotic syndrome induced in rats by an Daunorubicin (19). It Is possible that the favorable effects because of its action in decreasing hepatic VLDL production
Reducing the triglycerlde VLDL cholesterol is known to (17). Fish oil Is also known A 2 (6,11,20), which plays an adriamycln induced nephrosis
and LDL cholesterol, as in the present study, and protect against the development of atherosclerosis to suppress production of thromboxane important pathogenetic role in the proteinuria of (21).
The mechanlsms by which abnormalities of the lipids may produce progressive in3ury In the kidney have been discussed recently (22). In guinea pigs, high cholesterol feeding promotes the development of glomerular sclerosis (23); in the lupus nephritis of the NZB/NZW mouse, a dlet high in saturated fats accelerates progression of the renal disease (24). Using pharmacologic agents to lower lipid levels has had beneficial effects in partially nephrectomized rats (25), obese Zucker rats (26) and in rats with puromycin aminonucleoside nephrosis (27). Polyunsaturated fatty acids, beneficial ~n the renal disease of partlally nephrectomized rats, have also had favorable effects on the lipid profile (20). The present studies showed a marked effect of the lipid composition of the diet on both the plasma liplds and on the kidney. Feedlng beef fat to rats with adriamycin induced nephrotic syndrome resulted in extremely high levels of triglycerldes, cholesterol, and LDL cholesterol whlch were ameliorated by fish oil feeding. The lipid levels, measured relatively early, were strikingly predlctive of later adverse effects on body weight and on renal function and histology. Thls correlation was highly significant in animals fed beef fat, and was not significant in the high fish oil fed animals. Thls correlation suggests that elevated serum lipid levels early in this model of nephrotic syndrome may play a role in the pathogenesis of subsequent glomerular damage. The mechanisms might be similar to those operative in the development of atherosclerosls elsewhere in the vascular system; fish oil, rich in omega-3 fatty acids, reduces the serum llplds and may protect against the development of atherosclerosls. In the present experiment, of very short duratlon, glomerular sclerosis occurred only in minor degree. The development of glomerular sclerosis may have been prevented, at least in part, by the relatively low protein Intake due to progressive renal disease and as an effect of the pair feedings. The mean weight of the control group did not change. This might have been a consequence of the pair feedings. Other known effects of omega-3 fatty acids, such as changes In synthesis of products of the cylooxygenase and lipoxygenase pathways of fatty acid metabolism (7), reduction of platelet aggregabllity (7), decrease in systemic blood pressure (28), improvement of fibrlnolytlc activity (29) and effects on macrophage function (30,31), might have also played a protective role. References I. 2. 3. 4. 5. 6.
R.C. CURRY, JR., W.C. ROBERTS, Am. J. Med. 63 183-192 (1977) N.P. MALLICK, C.D. SHORT, Nephrology 27 54-57 (1981) D.B. BERNARD, In, Nephrotic Syndrome, (ed~) B.M. Brenner, J.H. Stein, chapter 4, Churchill Livingstone, New York, pp 85-120 (1982) J.F. BRIDGMAN, S.M. ROSEN, J.M. THORP, Lancet 2 506-509 (1972) W.S. HARRIS, W.E. CONNOR, M.P. MCMURRY, Metabolism 32 179-184 (1983) P.J. NESTEL, W.E. CONNOR, M.F. REARDON, S. CONNOR, S. WONG, R. BOSTON, J. Clin. Invest. 74 82-89 (1984)
2324
7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30.
31.
Fish oil is Beneflcial
to Nephrotic Rats
Vol. 40, No. 24, 1987
C. VON SCHACKY, S. FISCHER, P.C. WEBER, J. Clin. Invest. 76 1626-1631 (1985) B.E. PHILLIPSON, D.W. ROTHROCK, W.E. CONNOR, W.S. HARRIS, D.R. ILLINGWORTH, N. Engl. J. Med. 312 1210-1216 (1985) T. HAMAZAKI, R. NAKAZAWA, S. TATENO, H. SHISHIDO, K. ISODA, Y. HATTORI, T. YOSHIDA, T. FUJITA, S. YANO, A. KUMAGAI, Kidney Int. 26 81-84 (1984) W.S. HARRIS, W.E. CONNOR, S.B. INKELES, D.R. ILLINGWORTH, Metabolism 33 1016-1019 (1984) J.D. PRICKETT, D.R. ROBINSON, A.D. STEINBERG, J. Clin. Invest. 68 556-559 (1981) V.E. KELLEY, A. FERRETTI, S. IZUI, T.B. STROM, J. Immunol. 134 1914-1919 (1985) V. KHER, U. BARCELLI, M. WEISS, L. GALLON, P. PAJEL, P. LASKARZEWSKI, V.E. POLLAK, Prost. Leuk. Med. 22 323-334 (1986) T. BERTANI, A. POGGI, R. POZZONI, F. DELAINI, G. SACCHI, Y. THOUA, G. MECCA, G. REMUZZI, M.B. DONATI, Lab. Invest. 46 16-19 (1982) R. HITZEMANN, Neurochem. Res. 6 935 (1981) S. BALASUBRAMANIAM, L.A. SIMONS, S. CHANG, J.B. HICKIE, J. Lipid Res. 26 684-689 (1985) W.B. KANNEL, J.T. DOYLE, A.M. OSTFELD, C.D. JENKINS, L. KULLER, R.N. PODELL, J. STAMLER, Circulatlon 70(1) 155A-205A (1984) J.B. MARSH, J. Lipid Res. 25 1619-1623 (1984) N. MORISAKI, N. MATSUOKA, Y. SAITO, A. KUMAGAI, Metabolism 33 405-410 (1984) U. BARCELLI, J. MIYATA, Y. ITO, L. GALLON, P. LASKARZEWSKI, M. WEISS, R. HITZEMANN, V. POLLAK, Prostaglandins 32 211-219 (1986) G. REMUZZI, L. IMBERTI, M. ROSSINI, C. MORELLI, C. CARMINATI, G.M. CATTANEO, T. BERTANI, J. Clln. Invest. 75 94-101 (1985) J.F. MOORHEAD, M.K. CHAN, M. EL-NAHAS, Z. VARGHESE, Lancet 2 1309-1311 (1982) S.W. FRENCH, W. YAMANAKA, R. ~gTWALD, Arch. Path. 83 204-210 (1967) V.E. KELLEY, S. IZUI, Am. Assoc. Pathol. iii 288-297 (1983) B.L. KASISKE, M.P. O'DONNELL, F. DANIELS, W.F. KEANE, Clln. Res. 33 488A (1985) B.L. KASISKE, M.P. O'DONNELL, W.F. KEANE, Clin. Res. 34 600A (1986) F.G. SILVA, K.D.G. EDWARDS, C.L. PIRANI, Kldney Int. 16 789 (1979) P. SINGER, M. WIRTH, W. GODICKE, H. HEINE, Experlentia 41 462-464 (1985) U. BARCELLI, P. GLAS-GREENWALT, V.E. POLLAK, Thromb. Res. 39 307-312 (1985) T.H. LEE, R.L. HOOVER, J.D. WILLIAMS, R.I. SPERLING, J. RAVELESE, B.W. SPUR, D.R. ROBINSON, E.J. COREY, R.A. LEWIS, K.F. AUSTEN, N. Engl. J. Med. 1217-1224 (1985) K.S. KANT, J. SEXTON, U. BARCELLI, V.E. POLLAK, Clin. Res. 34 599A (1986)