Impact of dietary fatty acid supplementation on renal injury in obese Zucker rats

Kidney International, Vol. 39 (1991), pp. 1125—1134

Impact of dietary fatty acid supplementation on renal injury in obese Zucker rats BERTRAM L. KASISKE, MICHAEL P. O'DONNELL, HYUN LEE, YOUNGKI KIM, and WILLIAM F. KEANE Department of Medicine, Division of Nephrology, Hennepin County Medical Center, University of Minnesota, and Department of Pediatrics, University of Minnesota, Minneapolis, Minnesota, USA; and Department of Pathology, Seoul National University, Seoul, South Korea

Impact of dietary fatty acid supplementation on renal injury in obese Zucker rats. We previously reported that renal injury in hyperlipidemic, obese Zucker rats was associated with a relative deficiency of tissue polyunsaturated fatty acids (PUFA). In the present study 10-week-old obese Zucker rats were pair fed regular chow or chow containing either 20% sunflower oil rich in n-6 PUFA, fish oil rich in n-3 PUFA, coconut oil medium-chain saturated fatty acid, or beef tallow long-chain saturated fatty acid. At 34 weeks of age there were comparable reductions in albuminuria, mesangial matrix expansion, and glomeruloscierosis in the fish oil and sunflower oil groups. While both fish oil and sunflower

malities in FA profiles often accompany elevations in circulating lipoproteins, it is possible that alterations in plasma or tissue

FA could be important in the pathogenesis of renal injury

associated with hyperlipidemia. In the obese Zucker rat model of type II diabetes, hyperlipidemia, and progressive renal injury, we found that a relative deficiency of renal PUFA was associated with glomerular injury [17]. It was unclear, however, whether tissue FA abnormalities oil reduced serum triglycerides, and improved the composition of caused, or resulted from the renal injury. The present investitriglyceride-enriched lipoproteins, only fish oil decreased serum cho- gation was designed to further explore the role of abnormal FA lesterol. The effect of the dietary fatty acid supplementation on fatty metabolism by examining the effects of specific diet-induced, acid profiles were similar in isolated glomeruli and cortical tissue. In general, the amelioration in injury in the fish oil and sunflower oil fed FA alterations on the pathogenesis of glomerular injury in the rats was most closely linked to glomerular levels of PUFA, either n-6 or obese Zucker rat. Two PUFA diets, one enriched with n-6 and n-3. These data suggest that hyperlipidemia and abnormalities in tissue one with n-3 PUFA, were fed to obese Zucker rats. The effects FA are closely linked, and that dietary supplementation with PUFA of the PUFA diets were compared to standard chow (low fat) may ameliorate chronic, progressive renal injury. and to two saturated FA (SFA) enriched diets. Since SFA chain length may have important biologic effects [18], two different SFA diets were compared. Thus, these experiments examined Lipoprotein abnormalities are common in patients with the effects of dietary fat content (low vs. high fat), chain length chronic, progressive renal disease, and the presence of lipid (medium vs. long-chain SFA), the degree of saturation (SFA vs. deposits and foam cells has given rise to speculation that PUFA), and the position of the terminal double bond (n-6 vs. hyperlipidemia may be important in the pathogenesis of renal n-3), on circulating lipoproteins, blood pressure, renal tissue injury. Support for this hypothesis has come from a number of FA, and renal injury.

experiments in animal models of immune and non-immune mediated renal disease, where dietary and pharmacologic modifications of lipid metabolism have had a major impact on injury [1—16]. However, the specific lipid abnormalities that may be injurious have not been defined. Alterations in cholesterol, circulating lipoproteins, and fatty acid (FA) profiles have all been implicated in the pathogenesis of chronic progressive renal injury. Diet-induced hypercholesterolemia, for example, has been shown to cause renal injury in a number of different settings, and cholesterol-lowering agents reduced injury in several models of chronic, progressive renal disease [1—8]. In both immune and non-immune mediated renal disease models, dietary supplementation with n-3 or n-6 polyunsaturated FA (PUFA) ameliorated injury [9—14]. Since abnor-

Received for publication September 26, 1990 and in revised form January 3, 1991 Accepted for publication January 8, 1991

© 1991 by the International Society of Nephrology

Methods

Experimental design

Male, obese (fa/fa) Zucker rats (Charles River, Wilmington, Massachusetts, USA), were fed standard laboratory chow until 10 weeks of age. Rats were then randomly assigned to one of five experimental diets (described below). Since pilot studies demonstrated that obese Zucker rats fed the fish oil diet had slightly lower food intakes than obese rats fed the other diets, the rats in the present study were pair fed to rats in the fish oil group. All rats consumed the same amount of diet throughout the study period. Blood pressure, urine albumin excretion, and serum chemistries were measured at 16, 22, 28 and 34 weeks of age. At 34 weeks of age, rats were lightly anesthetized with ether and were killed by exsanguination. Plasma from each rat was used for lipoprotein studies. Renal tissue was processed for histology and FA analysis.

1125

1126

Kasiske et a!: Fatty acids and renal injury in Zucker rats

Table 2. Fatty acid composition of the experimental diets

Table 1. Major components of the diets Regular chow Protein (% wtlwt) Carbohydrate (% wtlwt) Fat (% wtlwt) Calories (KCaI/kg)

23 47 6

3.3

Oil diets 18

38 25

4.5

Fatty acid Lauric (12:0) Myristic (14:0) Palmitic (16:0)

Palmitoleic(l6:l) Stearic (18:0)

Diet composition One group of rats was fed standard laboratory chow (N = 12). The other four groups received standard chow supplemented with 20% (wt/wt) fat. The energy content of the standard chow was less than that of the fat-supplemented diets (Table 1). Since rats were pair fed, those in the standard chow group received

fewer calories than those in the other four groups. The food intake of the fat-supplemented rats was matched with that of

Oleic(18:l) Linoleic (18:2w6) Arachidonic (20:4w6)

E.P.A. (20:53) D.H.A. (22:6w3)

Experimental diet Fish Sunflower Coconut Beef oil tallow Standard oil oil — — — — 9.09 — 1.59 2.60 0.70 0.11 1.37 1.81 2.10 5.45 4.52 — — — 0.57 1.99 0.84 2.14 3.81 0.50 0.99 4.11 0.98 7.84 1.72 2.56 1.72 12.42 1.12 1.06 0.78 — — — — 0.13 — — — — 2.54 — — — — 2.13

Abbreviations are: E.P.A., eicosapentaenoic acid; D.H.A., docosahexaenoic acid. Values are g/100 g of diet. Components making up less than 0.10 g/lQO g diet are not shown.

standard chow rats on the basis of weight so that differences in protein intake would be minimized. Matching the groups by calorie intake would have avoided potential effects of caloric restriction, but would have increased the protein intake of the technique, as previously described [19]. Glomeruli were isostandard chow rats to a level (31%) that would have been much lated quickly using ice to prevent enzymatic degradation of FA. higher than the protein intake of the fat-supplemented rats To examine whether the glomerular isolation procedure altered (18%). All fat-supplemented diets contained, as a minimum, the FA profiles, FA were also determined on cortical tissue handled essential FA included in standard chow, reduced by 20%. Fat in the same way that tissue was handled for the isolation of supplements included: sunflower oil (N = 9), fish (menhaden) glomeruli. The FA profile of cortical tissue handled in this oil (N = 10), coconut oil (N = 15), and beef tallow (N = 12). manner was identical to that of cortical tissue that had been The FA composition of the diets was determined by gas processed immediately, suggesting that the glomerular isolation chromatography, using techniques described below (Table 2). procedure per se did not alter the FA profile. Lipids were extracted and separated using thin layer chroBlood pressure and urine albumin excretion matography and gas chromatography, as previously described Blood pressure was measured using the tail cuff method, as in detail [17]. Lipids were extracted using methanol and chlopreviously described in detail [8]. For 24-hour urine collections roform. The tissue extracts were separated into the major lipid rats were housed in metabolic cages and allowed free access to classes using 20 x 20 cm glass plates with 0.30 mm of silica gel water. Urine albumin was determined using a monospecific H, and a development system of petroleum ether:dimethyl antibody to rat albumin and a laser nephelometer, as previously ether:acetic acid (100:25:1, vol/vol/vol). The triglyceride and phospholipid zones were visualized using dichiorofluorescein described [81. and an ultraviolet lamp. Methyl esters of FAs were prepared by Serum cholesterol, triglycerides, and lipoproteins transesterification with 3 N HC1. The methyl esters were Tail vein blood samples for serum cholesterol and triglycer- analyzed on a Hewlett-Packard Model 5840A gas chromatoides were obtained, after an overnight fast, in rats subjected to graph (Hewlett-Packard Co. Avondale, Pennsylvania, USA) light ether anesthesia. Cholesterol and triglycerides were mea- equipped with a flame ionization detector and a fused silica sured using standard colorimetric techniques [7]. Lipoproteins capillary column (0.25 mm x 30 m) and 0.25 micron Durabondwere isolated from plasma using standard ultracentrifugation 225 (J & W Scientific Inc., Rancho Cordova, California, USA).

techniques. For the lipoprotein separation rat plasma was supplemented with 0.1 mrvi EDTA, 0.01 m butylated hydroxyHistology toluene, and 0.01% NaN3. Plasma was spun at 100,000 rpm for Coronal sections of the kidney were obtained for light mifour hours in a TL-100 ultracentrifuge with a TLA-100.3 rotor croscopy, and tissue was stained using periodic acid with (Beckman Instruments, Fullerton, California, USA) at 4°C. The Schiff's reagent (PAS). All glomeruli were examined in each

VLDL layer was then removed. The salt density of the in-

franate was sequentially adjusted using KBr and the lipoprotein fractions were isolated using centrifugation and density fractions: d 1.006-1.019 (IDL), d 1.1019-1.063 (LDL), and d 1.063-1.125 (HDL). Cholesterol and triglycerides were measured in each lipoprotein fraction.

Tissue fatty acids Renal cortical tissue was heated in 0.5 N acetic acid (95°C) for 30 minutes, frozen in liquid nitrogen, and stored at —70°C until analyzed. Glomeruli were isolated using a differential sieving

section to determine the incidence of focal, segmental glomeruloselerosis. Glomerulosclerosis was defined as global or segmental areas of capillary collapse with replacement by PASpositive material. The percent of glomeruli with any amount of sclerosis was determined in each tissue specimen. In addition, a glomeruloscierosis severity index was calculated using the formula:

[(S1+2x S +3 x S3 +4x S4)/ Total number of glomeruli] x 100.

1127

Kasiske et a!: Fatty acids and renal injury in Zucker rats Table 3. Body weight, organ weights, and glomerular areas

Diet

Body weight

Heart weight

g

Fish oil (N = 10) 736 Sunflower oil (N = 9) 791 Coconut oil (N = 15) 731 718 Beef tallow (N = 12) 595 Regular chow (N = 12) Shared superscripts indicate P >

86° 82° 107°

68° 47"

Liver weight

Kidney weight

Kidney weight

gllOOgbody wt 0.189 0.180 0.178 0.183 0.203

3.21

0.33a

0.040c

2.87 2.67

0.16" 041b 0.27" 038b

2.84 2.43

0.35°" 0.27"°

0.023'

2.81 3.60

072a.b 089d

0.457 0.373 0.383 0.399

3.19

0.56'"'

0.457± 0.066a

0.05 by analysis of variance. Values are means

Where SN was the number of glomeruli with sclerosis involving N of the four glomerular quadrants [7]. The amount of mesangial matrix expansion was determined using the formula:

[(M1+2xM2+3xM3+4xM4)/ Total number of glomeruli] x 100

2

g 0.053°"

0020a.b 0.017"

0.018"

Glomerular area

0.10lc 0050b,c

2.74 2.61

7930 7460 7280

7540

862° 944° 794° 751°

7690 593a

so.

The analysis was carried out using the Statistical Package for the Social Sciences (SPSS) microcomputer software [20]. The significance of differences between groups was assessed using oneway analysis of variance (ANOVA) with Duncan's multiple range comparison test. However, there were a number of similarities and differences (SFA vs. PUFA, high vs. low fat,

etc.) that made it arbitrary to designate individual diets as

'control' or 'experimental', and to compare differences using a one-way ANOVA. Moreover, repeated measures of the same variables at different times during the study period introduced the added complexity of multiple time-diet interactions. To camera (400x) and a computerized, digital image analysis simplify the analysis and avoid potential bias, multivariate, system (Southern Micro Instruments, Inc., Atlanta, Georgia, linear regression analysis was also carried out. The relative effects of the individual diets, or diet characterUSA). The integrated area within the perimeter was then istics, were examined by including each of these parameters as computed. Fifty glomeruli were measured to determine the mean area for each tissue specimen. The amount of tubuloin- an independent variable with a weight of 0 or 1. A stepwise terstitial damage was assessed by examining 10 to 15 micro- selection of the independent variables was then used to deterscopic fields (200x) and assigning a number 0 to 100 based on mine the diet groups that had statistically significant, indepenthe degree of tubular cast formation, epithelial damage, tubular dent effects on the dependent variable. The regression coeffiatrophy, and interstitial fibrosis. The mean of the individual cients for the diets and other independent variables indicated scores was used as a semiquantitative estimate of the degree of their relative contributions to the study parameter being examined. For variables that were measured repeatedly throughout tubulointerstitial damage for each tissue specimen. Immunocytological studies to determine the number of gb- the study period, such as, urine albumin excretion, blood merular macrophages were carried out using previously de- pressure, serum cholesterol, and serum triglycerides, the efscribed techniques [11. Briefly, tissue that had been snap frozen fects of the time of measurement (age of the rats) and interacin liquid nitrogen and stored at —70°C, was air dried, acetone- tions between the time of measurement and the diets were fixed, and incubated with appropriate dilutions of monoclonal included in the analysis. For the analysis of the effects of diet on antibodies for Ia-antigen positive macrophages (0X4, Pc! tissue FA, relative differences in cortical versus glomerular FA, Freeze Biologicals, Rogers, Arkansas, USA). The slides were were also taken into account. where MN was the number of glomeruli with matrix expansion involving N of the 4 glomerular quadrants [7]. Glomerular area was determined using planar morphometry. The perimeter of each glomerular tuft was traced using a video

then reacted to FITC conjugated rabbit antimouse IgG

F(Fab')2, followed by FITC goat antirabbit IgG F(Fab')2 (Pel Freeze). The number of positive cells in each glomerulus was determined with epifluorescence microscopy.

Results

Growth and organ weights

Statistical analysis Rats from all groups grew well throughout the study period. The frequency distribution of each study variable was exam- There were no differences in body weight among the fatined using indices of kurtosis and skewness, and the Kolmog- supplemented groups (Tables 3 and 4). Since rats were pair fed orov-Smirnov test to judge whether distributions were Gaus- to the fish oil group, rats in the standard chow group received sian. Variables requiring a logarithmic transformation to fewer calories and weighed less than rats in the high fat groups. achieve a normal distribution prior to analysis included: urine Interestingly, the kidneys from rats fed fish oil weighed more albumin excretion, serum cholesterol, serum triglycerides, as than the kidneys of rats in the other groups. This was true even well as lipoprotein cholesterol, triglyceride levels and the when kidney weight was factored by body weight. Although glomerulosclerosis severity index. Since glomerulosclerosis gbomerular areas correlated with kidney weights (r = 0.50, P < was also expressed as a fraction of the total number of glomer- 0.001), differences in the glomerular areas between the groups

uli, an arc sine transformation was used to normalize this were not statistically significant. The livers of rats fed beef distribution prior to analysis. All differences were considered significant for P < 0.05. Results are expressed as mean SD.

tallow weighed approximately 25% more than the livers of rats in the other diet groups.

1128

Kasiske er al: Fatty acids and renal injury in Zucker rats

Table 4. The relative effects of age and diet on body weight, blood pressure, fasting serum lipids, and albuminuria throughout the study period

Fraction of

Relative impact (coefficients) of age and diets Dependent variable

Body weight g Blood pressure mm Hg Triglycerides Ln mmol/liter Cholesterol Ln mmol/liter Albuminuria Ln mg124 hr

Age (per week)

Fish oil

Sunflower oil

14

Fish or

Coconut oil

sunflower

39

0.70

Beef tallow

Regular chow

—25

—107

0.03

0.74 0.14

—

6.6 —0.85

variability explained (r2)

0.62

—0.54

0.38 0.47

—0.82

0.15

0.64

—0.50

0.43

Values are multivariate, linear regression coefficients for age and diet effects(P < 0.05) on body weight, blood pressure, triglycerides, cholesterol and albuminuria. Shown in the last column is the fraction of the total variability for each dependent variable explained by the combination of independent variables. For example, body weight increased 14 g/week, sunflower oil fed rats weighed 39 g more, beef tallow fed rats weighed 25 g less, regular chOw fed rats weighed 107 g less, and 74% of the variability in body weight was explained by these variables. Values for triglycerides, cholesterol and urine albumin excretion were transformed using a natural logarithm prior to analysis.

Table 5. Serum chemistries, blood pressure, and albuminuria at 34 weeks of age Cholesterol

Triglycerides —________

Glucose

Blood pressure mm Hg

mmol/liter

Urine albumin mg/24 hr

1.9

l.0

2.75

2.3la

13.03

6.02a

112

l7

50

47

Sunflower oil

5.0

1.1"

2.98

l.77"

10.70

1.89""

117

12"

38

17"

Coconut oil

6.5 551 6,1 22b

15.5

34,31

10.88

187","

123

16"

86 76"

6.78

5,54tC

11.06

128a.b

118

13"

88

9.2

23.0

19.7"

9.33

3.10"

116

19"

164

Fish oil

(N =

10)

(N = 9) (N =

15)

Beef tallow (N=12) Regular chow

4.2c

119"

(N= 12) Shared superscripts indicate P > 0.05 by analysis of variance. Cholesterol, triglycerides, glucose, and urine albumin were logarithmically transformed before analysis. Values are means SD.

have had direct effects on lipoproteins that were not mediated Blood pressure and urine albumin excretion Blood pressure increased in all diet groups during the study by differences in albuminuria. Multivariate analysis was used to period, but never exceeded 125 mm Hg (Tables 4 and 5). determine the extent to which the effects of the diets on serum Overall, blood pressure was 6.6 mm Hg higher in rats fed beef lipoproteins were independent of, or mediated by, differences in tallow compared to the other diets. Urine albumin excretion urine protein excretion (Tables 6 and 7). The fish oil diet was associated with significant reductions in also increased during the study period (Tables 4 and 5). Albumin excretion was comparably reduced in rats fed fish oil and total, IDL, LDL, and FIDL cholesterol that did not appear to be sunflower oil. Indeed, the multivariate analysis indicated that the result of reduced albuminuria (Table 6). Rats fed regular the effects of fish oil and sunflower oil on urine albumin chow had higher levels of total, VLDL, and HDL cholesterol excretion were similar, and that both diets together substan- compared to rats fed oil-supplemented diets. Albuminuria, on tially reduced albuminuriá. Compared to the high fat diets, the other hand, was associated with higher levels of total, regular chow was associated with a greater amount of urine VLDL, IDL, LDL, and HDL cholesterol (Table 6). The effects of albuminuria and diet on cholesterol levels were relatively albumin excretion. strong, as indicated by the fact that up to 82% of the variability Serum lipids, plasma lipoproteins, and serum glucose in the total and lipoprotein cholesterol levels were accounted Only fish oil consistently reduced serum cholesterol levels for by these variables. In contrast, diet and albuminuria had (Tables 4 and 5). In contrast, both fish oil and sunflower oil little overall impact on the distribution of cholesterol between reduced serum triglycerides throughout the study period. Com- the individual lipoproteins. pared to the high fat diets, regular chow was associated with an Both the PUFA diets and albuminuria had major effects on increase in triglycerides. the triglyceride content of the lipoproteins (Table 7). The PUFA Albuminuria could influence serum lipoprotein levels, and, diets caused a relative shift in triglycerides from VLDL to therefore, diets may have indirectly affected lipoproteins by higher density lipoproteins. The opposite effect was seen for altering the amount of albuminuria. In addition, the diets may albuminuria, where there was a relative increase in VLDL

1129

Kasiske et a!: Fatty acids and renal injury in Zucker rats Table 6. The relative, independent effects of albuminuria and diet on lipoprotein cholesterol

Relative impact (coefficient s) of albuminuria and diets

Dependent variable Cholesterol levels mmollliter Total (5.2 0.5) VLDL (1.0 0.2) IDL (0.2 0.0) LDL (1.1 0.1) HDL (2.9 0.2) Cholesterol content (% of total) VLDL (16 1)

IDL(2 LDL (18

1)

Albumin excretion

Fish

ing/24 hr

oil

0.31

—

0.51

—1.80 —2.06

0.38 0.20 0.15

— —

1)

HDL (63± 2)

—1.00

0.54

—0.11

—0.88

—

— —0.93

—

Sunflower oil

Coconut oil

— — — — —

—

—0.80

— — —

— —

— — — —

—

Beef tallow

Fraction of Regular chow

variability explained (r2)

— — —

0.34

0.82

0.90

0.58

1.35

—.-

—

—

0.34

0.37 0.68 0.72

— —

1.37

— — —

0.39

—

—

0.32 0.10 0.39 0.27

Values are multivariate, linear regression coefficients for the effects of albüminuria and diet (P < 0.05) on lipoprotein cholesterol levels and composition. Shown in parentheses are the mean cholesterol levels and the fraction of total cholesterol in each lipoprotein fraction. Shown in the last column is the percent of the total variability for each dependent variable explained by the combination of independent variables. Values were transformed using a natural logarithm prior to analysis.

Table 7. The relative, independent effects of albuminuria and diet on lipoprotein triglycerides Relative impact (coefficients) of albuminuria and diets Albumin excretion

Dependent variable Triglyceride levels mmollliter Total (7.8 1.3) VLDL (7.5 1.3) IDL (0.1 0.1) LDL (0.1 0) HDL (0.1 0) Triglyceride content (% of total) VLDL (92 1) IDL (3 3) LDL (4 1) HDL (I 0)

Fish oil

Sunflower oil

— — — —

— — — — —

0.04

—0.17

—0.07

—0.37 —0.43 —0.37

0.90 1.78

1.24 1.02 1.27

mg124 hr

0.35

0.36 — — —

0.74

1.03

Coconut

Beef tallow

Regular chow

0.38

—

0.64

0.51

1.07 1.36

—0.50 —0.64 —0.71

—1.96

oil

— — — —

—1.75

—2.19

— —1.20 —0.94 —1.82

— — — — — — —

Fraction of variability explained (r2) 0.59 0.66 0.55 0.51

0.39 0.63 0.67 0.68 0.70

Values are multivariate, linear regression coefficients for the effects of albuminuria and diet (P < 0.05) on lipoprotein triglyceride levels and composition. Shown in parentheses are the mean triglyceride levels and the fraction of total triglyceride in each lipoprotein fraction. Shown in the last column is the percent of the total variability for each dependent variable explained by the combination of independent variables. Values were transformed using a natural logarithm before analysis.

triglycerides along with relative decreases in IDL, LDL, and tissue. There was relatively more triglyceride arachidonic acid HDL triglycerides. Fish oil and sunflower oil had little effect on in glomeruli than in cortex. Among phospholipid and triglycerabsolute lipoprotein triglyceride levels (Table 7). Coconut oil ide FA, the ratio of stearic/oleic acid was increased in glomeruli and beef tallow were associated with relatively lower levels of compared to cortical tissue (2.68 0.57 vs. 2.11 0.44, P < IDL, LDL, and HDL triglycerides, while total triglyceride 0.01, among phospholipids; 0.51 0.32 vs. 0.26 0.08, P < levels, and the fraction of total triglycerides found in VLDL, 0.01, among triglycerides). In general, the impact of the diets on tissue FA were similar were increased by regular chow, coconut oil, and albuminuria. Serum glucose levels were higher in rats fed fish oil compared in the cortex and in glomeruli, that is, very few dietary effects to rats fed regular chow (Table 5). Indeed, the highest glucose were seen in only cortical or glomerular FA profiles. Among the levels were from rats in the fish oil group. However, the phospholipid FA, the most striking effects were in the PUFA differences in glucose levels between the different fat-supple- (Figs. 1 and 2). Fish oil increased phospholipid n-3 PUFA while mented diet groups were not statistically significant by one-way causing a relative decrease in arachidonic acid. Sunflower oil ANOVA. increased linoleic acid and caused a relative decline in (n-3) eicosapentaenoic acid. Similar effects of fish oil and sunflower Tissue fatty acids oil on linoleic acid and n-3 PUFA were also seen in the There were major differences in the FA profiles of glomerular triglyceride FA profiles. However, in contrast to the pattern in and cortical tissues that were independent of the effects of diet phospholipids, the relative increases in triglyceride PUFA were (Figs. 1 and 2). Among phospholipid FA, glomeruli had rela- balanced by declines in oleic acid, rather than declines in the tively more linoleic, but less arachidonic acid than cortical other PUFA.

Kasiske et al. Fatty acids and renal injury in Zucker rats

1130 Cortex vs.

Standard

Fish

glomeruli

diet

oil

Sunflower Coconut oil

oil

Beef

tallow Fig. 1. The independent effects of diet and

Palmitic (16:0)

Palmitoleic (16:1)

Stearic (18:0) Oleic

(18:19) Linoleic

(18:26) DHGLA (20:3co6)

Arach idonic (20:4u6) EPA (20:5€o3)

OHA

•., •-

(22:6o3( •

5.0

, 5.0

-1

15.0 0.0 5.0 0.0 —5.0 5.0 0.0 —15.0 —5.0 5.0 0.0 —5.0 5.0 —10.0 0.0 5.0 10.0 —5.0 0.0 —5.0

Relative impact on the percent of total phospholipid fatty acids, %

Cortex vs. Standard glomeruli diet

Fish

oil

Sunflower

oil

Coconut oil

site (cortex vs. glomeruli) on phospholipid fatty acids. Values are multivariate, linear regression coefficients (P < 0.05) where each fatty acid was analyzed with the following independent variables: cortical vs. glomerular fatty acid with hatched bars indicating relatively more of the fatty acid in cortex (bars to the left of the vertical line) or in glomeruli (bars to the right of the vertical line); standard diet with a negative value indicating a relative reduction; fish oil; sunflower oil; coconut oil; and beef tallow. Interaction terms (diet effects found only in glomerular or cortical fatty acids) had minimal independent impact on fatty acids, and are not shown. Example: 2,05% more of the total phospholipid fatty acid was made up of palmitic acid in the renal cortex compared to the glonieruli. Sunflower oil and coconut oil caused relative reductions (—2.85% and —1.68%, respectively) in the proportions of total phospholipid fatty acids made up of palmitate in glomeruli and cortex.

Beef

tallow

Palm itic

(16:0) Palmitoleic (16:1) Stearic (18:0) Oleic (18:1w9(

Linoleic

(18:2w6) DHGLA (2O:3&o6(

Arach idon ic (20:4o)6)

DHA

(22:6w3)

Total polyenoic 5.0

r-r5.0

0.0 —5.0

0.0 —10.0

5.0 —5.0

-r rr

0.0

10.0 —5.0 5.0 15.0 0.0 —10.0 0.0 10.0 0.0 10.0 ---5.0 5.0 —10.0 5.0 —5.0 5.0

Relative impact on the percent of total triglyceride fatty acids, %

Fig. 2. The independent effects of diet and site (cortex vs. glomeruli) on triglyceride fatty acids. For an explanation of the results see the legend for Fig. 1.

compared to coconut oil (44 80), standard chow (24 24) and Histology The degree of mesangial matrix expansion was lowest in rats beef tallow (14 18) fed rats. Among the rats randomly selected for FA analysis, there fed sunflower oil (Figs. 3 and 4). Rats in the fish oil group also were correlations between tissue FA levels and glomerular had a reduced amount of mesangial matrix compared to regular chow, coconut oil and beef tallow fed rats. Both fish oil and injury. In particular, a decreased ratio of phospholipid linoleic/ sunflower oil caused comparable reductions in the incidence of arachidonic acid was associated with a greater amount of glomerulosclerosis compared to that seen in rats fed coconut oil mesangial matrix (Table 8). There was an even stronger, inverse correlation between total triglyceride PUFA levels and mesan(Fig. 5). By multivariate analysis, the two PUFA diets caused similar reductions in the incidence of focal glomerulosclerosis gial matrix expansion. Using multivariate, linear regression compared to the three other diet groups. The severity of the analysis, 84% of the variability in mesangial matrix could be glomerulosclerosis was similar in fish oil (severity index 6 explained by total glomerular triglyceride PUFA levels and 6) and sunflower oil (4 3) groups, but lower (P < 0.05) (independently) levels of triglyceride n-3 docosahexaenoic acid.

Kasiske et al. Fatty acids and renal injury in Zucker rats

1131

300

0

250

(0

• ••

CD

a)

200

•C

a)

E CD

150

CD

(4,

a)

100

50

—$—

I

•S

•

•a •• S •

Id

lcd —.— • • • $

S

•b -I-

S S

I 0—

S

.

FO

SO

CO

R

Fig. 3. The effects of diet fatty acid supplementation on mesangial

matrix expansion in obese Zucker rats. Abbreviations are: FO, fish oil; SO, sunflower oil; CO, coconut oil; R, regular/standard chow; BT, beef tallow. Shared superscripts indicate P> 0.05 by analysis of variance.

With the effects of these two glomerular PUFA parameters taken into account, diet had no additional, independent effect on mesangial matrix expansion. There was a fairly strong correlation between the amount of tubulointerstitial injury and mesangial matrix expansion (r = 0.61, P < 0.01). In general, however, there was less correlation

between tissue FA levels and tubulointerstitial injury than

4. A. Example of a normal glomerulus from a rat fed polyunsatbetween FA levels and mesangial expansion (Table 8). The best Fig. urated fatty acid. There is minimal mesangial matrix. B. Characteristic correlate to interstitial damage was the amount of oleic acid in glomerulus from an obese Zucker rat fed coconut oil or beef tallow showing mild to moderate mesangial matrix expansion. cortical phospholipids. When compared to lean Zucker rats fed standard chow, the

number of Ia+ cells per glomerulus was increased in all diet groups (Fig. 6). Rats in the beef tallow group had an increased number of glomerular Ia+ cells compared to the other four diet groups. In the latter four groups, the numbers of glomerular Ia+ cells were comparable. Discussion

Both hyperlipidemia and abnormalities in FA metabolism have been linked to the pathogenesis of chronic, progressive renal injury. In the present investigation, diets supplemented with either fish oil or sunflower caused comparable reductions in albuminuria, mesangial matrix expansion, and glomerulosclerosis in hyperlipidemic, obese Zucker rats. These results, combined with those of a previous investigation that correlated the degree of glomerular injury in obese Zucker rats with a relative deficiency in renal cortical PUFA [17], suggest that PUFA may be important in the pathogenesis of the renal injury in this model. Although reductions in serum cholesterol may have contributed to the amelioration of renal injury in the fish oil group, the reduction in injury in rats fed sunflower oil was not accompanied by lower levels of cholesterol. Moreover, neither fish oil nor sunflower oil altered the relative cholesterol content of the

individual lipoproteins. These observations suggest that the beneficial effects of the PUFA diets may not have been directly related to changes in cholesterol metabolism per se. However, these results do not necessarily negate the potential importance of cholesterol in the pathogenesis of glomerular injury. Indeed, a substantial amount of data suggest that dietary or pharmaco-

logic alterations in cholesterol metabolism can have a major effect on glomerular injury [1—81. It is possible that sunflower oil

ameliorated cholesterol-induced renal injury not by reducing serum cholesterol, but by altering lipoprotein composition, glomerular lipoprotein deposition, or tissue handling of cholesterol and cholesteryl esters.

Triglycerides were reduced in rats fed either fish oil or sunflower oil, and the PUFA enriched diets caused a relative reduction in the triglyceride content of VLDL. The multivariate analysis indicated that the effects of the PUFA diets on serum triglycerides were not mediated by alterations in urine protein excretion. Thus, changes in triglycerides and VLDL were most likely caused by PUFA diet-induced alterations in FA metabolism.

One of the major metabolic defects linked to obesity and hyperinsulinemia in the Zucker rat is an increase in hepatic FA

Kasiske et al: Fatty acids and renal injury in Zucker rats

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Fig. 5. The effects of diet fatty acid supplementation on glomerulosclerosis in obese Zucker rats. Abbreviations are: FO, fish oil; SO, sunflower oil; CO, coconut oil; R, regular/standard chow; BT, beef tallow. Shared superscripts indicate P> 0.05 by analysis of variance.

SO

CO

R

BT

Fig. 6. The effects of diet fatty acid supplementation on the number of

Ia-positive cells in the glomeruli of obese Zucker rats. Abbreviations

are: FO, fish oil; SO, sunflower oil; CO, coconut oil; R, regular/

standard chow; BT, beef tallow. Shared superscripts indicate F> 0.05 by analysis of variance. The hatched area represents the normal range for Ia-positive cells in lean Zucker rats.

Table 8. Relationship (r values) between tissue fatty acids and renal injury Association with Association with cortical fatty acid glomerular fatty acid Mesangial Interstitial Mesangial Interstitial expansion injury expansion - injury Phospholipid fatty acids Oleic Linoleic Oleic/linoleic Arachidonic Linoleic/arachidonic Total polyenoic Triglyceride fatty acids Oleic Linoleic Oleic/linoleic Arachidonic Linoleic/arachidonic Total polyenoic

0.36 —0.39

0.38 0.47 —0.04

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0.24

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synthesis [21]. The resulting increased FA pool leads to augrnented hepatic secretion of triglyceride rich lipoproteins, and hypertriglyceridemia [22]. The increase in FA synthesis is also associated with an increase in SFA, and a relative deficiency of PUFA in the liver, the kidney, and other tissues [17, 23—25].

Fish oil has been shown to decrease hepatic FA synthesis, reduce lipoprotein secretion, and enhance VLDL catabolism in normal rats [26—31]. Similar, but less striking effects have also been seen in normal rats fed diets supplemented with n-6 PUFA

[26—28, 311. Obese Zucker rats fed a diet enriched with n-6 PUPA had decreased hepatic PA synthesis and a relative

increase in PUPA levels [32], while obese rats fed a diet

enriched with n-3 PUFA had decreased activity of hepatic lipogenic enzymes and reduced serum triglyceride levels [331. Although reduced triglyceride levels and improved lipoprotein triglyceride composition were associated with an ameliora-

tion of renal injury in Zucker rats fed PUFA in the present study, whether these alterations explain the beneficial effects of

the PUPA diets is unclear. Recent investigations have found that a number of compositional alterations may make lipoproteins more atherogenic [34, 35]. Mechanisms may include increased monocyte chemotaxis, uninhibited uptake of lipoproteins by macrophages, and direct toxicity to endothelial cells. Although the specific effects of triglyceride lipoprotein enrich-

ment are unknown, it is interesting to speculate that the reduction in VLDL triglyceride in the present study may have played a role in reducing glomerular injury in rats fed PUFA. The effects of the diets on FA were similar in the cortex and glomeruli. Fish oil increased phospholipid and triglyceride n-3 PUFA, while sunflower oil increased n-6 PUPA, principally linoleic acid. Total triglyceride PUPA levels, were increased by both PUFA diets. Moreover, it was the alteration in triglyceride PUFA levels that was most closely associated with differences in mesangial matrix expansion. Whether the PUPA diet-induced renal tissue PA alterations in the present study caused the reduction in mesangial matrix expansion and glomerulosclerosis is unknown. However, there is a substantial amount of experimental data on the effects of PUFA that make it interesting to speculate on potential mechanisms for the observed reductions in renal injury. In general, fish oil and sunflower oil diets have very different effects on renal prostaglandins [36—38]. Moreover, the role of eicosanoids in PUFA diet-induced changes in renal injury has been controversial [14, 36—411. Although prostaglandins were not measured in the present study, it seems unlikely that the beneficial effects

1133

Kasiske et al. Fatty acids and renal injury in Zucker rats

of fish oil and sunflower oil can be explained by simple obese Zucker rats, or in other animal models of progressive renal disease, is unknown. The results of the present investigation add to a growing body of experimental data which suggest that PUFA may be imporlar injury [1, 42, 43]. Although the obese Zucker rats in the tant in the pathogenesis of chronic, progressive renal injury. present investigation had an increase in the number of glomer- Moreover, as shown in the present study, changes in FA are ular macrophages compared to lean littermates, the PUFA diets closely linked to abnormalities in triglycerides and in lipoproreduced injury without decreasing glomerular macrophages. tein composition that could possibly contribute to glomerular Nevertheless, it is possible that PUFA may have had major injury. The association between PUFA abnormalities and renal effects on macrophage function that could have reduced injury injury adds further support to the general hypothesis that the without reducing the number of macrophages [44]. Thus, the pathogenesis of chronic, progressive glomerulosclerosis may be results of this study do not preclude a role for macrophages in similar to that of systemic atherosclerosis. Ultimately, measures that effectively prevent the development and progression mediating lipid-induced renal injury. A number of investigations have shown that dietary restric- of systemic atherosclerosis may prove to be equally effective in tion of calories reduces renal injury independent of protein preventing end-stage renal disease. alterations in cyclooxygenase or lipoxygenase products. Previous studies have reported an association between the number of glomerular macrophages and lipid-induced glomeru-

intake [45, 46]. In the present study, rats fed regular chow received 25% fewer calories and weighed less than the rats in Acknowledgments the other diet groups. Despite this modest caloric restriction, Special thanks to Ms. Jan Lovick for helping to prepare this manurats fed standard chow did not have reduced renal injury. It is for publication and to the Zapata Haynie Corporation, Reedville, unlikely that the failure of calorie restriction to reduce injury script Virginia, for supplying the menhaden oil. The experiments were sup-

was solely the result of the slightly higher protein intake. However, potential alterations in the meal feeding pattern of the standard chow rats combined with a higher diet protein content could have offset any benefits observed with the calorie restriction seen in this group. If, for example, the rats in the standard

ported by United States Public Health Service grants ROl AM37396 and K08 DKO 1628 of the Department of Health and Human Services.

Reprint requests to Bertram L. Kasiske, M.D., Department of

Medicine, Hennepin County Medical Center, 701 Park Avenue, Minchow group ate the daily food allotment in a shorter period of neapolis, Minnesota 55415, USA.

time, potentially injurious glomerular hemodynamic alterations could have prevented a reduction in injury.

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