Prostaglandins, Leukotrienes and Medicine (1987) 28, 277-284 !@I Longman Group UK Ltd 1987

A LOW PROTEIN-HIGH LINOLEATE DIET INCREASES GLOMERULAR PGE2 AND PROTECTS RENAL FUNCTION IN RATS WITH REDUCED RENAL MASS

Yasushi Ito, Uno Barcelli, Wataru Yamashita, Mark Weiss, James Deddens, and Victor E. Pollak

Division of Nephrology, University of Cincinnati Medical Center, Cincinnati, Ohio 45267-0585 (reprint requests to Uno Barcelli).

ABSTRACT _

Renal function deteriorates progressively in partially nephrec- tomized rats. This deterioration of renal function may be ameliorated by a diet either low in protein or high in linoleic acid. In the pre- sent experiment, partially nephrectomized rats were pair fed diets low in protein, high in linoleic acid or both low in protein and high in linoleic acid. Survival of renal function was most prolonged in rats fed a diet with both a low protein and high linoleic acid content; glomeruli from these animals demonstrated increased glomerular PGE2 production. This additive effect may be mediated hy increased produc- tion of the vasodilatory PGE2 by glomeruli.

INTRODUCTION

Dietary protein restriction preserves renal function in chronic renal failure in experimental animals (l-5) and in humans (6-8). Modifications of dietary lipid composition also have favorable effects. Feeding of linoleic acid (LA), a precursor of dienoic prostaglandins (PG) PGI2, PGE2, and thromboxane A2 (TXA2), ameliorates the deteriora- tion of renal function in immunologic (9,lO) and non-immunoloqic models of renal disease (11-18). This protective effect, associated with increased vasodilator PGs (10,111, is abolished by cyclooxygenase inhibition (12).

The present study was conducted in rats with reduced renal mass to investigate whether these two dietary modifications have additive pro- tective effects. The effects on renal function, and on blood pressure, lipid profile, and glomerular synthesis of eicosanoids, known important factors in progression of renal failure, were evaluated.

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MATERIAL AND METHODS

Four different diets (Table 1) were prepared by TEKLAD Test Diet (Madison, WI.). The normal protein-normal LA (NP-NLA) diet contained 22% (w/w) protein, 1% (w/w) LA; the low protein-normal LA diet (LP-NLA) 11% protein, 1% LA; the normal protein-high LA (NP-HLA) diet, 22% protein-15% LA and the low protein-high LA (LP-HLA) diet, 11% protein and 15% LA. The total fat content of the four diets was identical (20% w/w). In the low protein diet, casein (90% protein) was partially replaced by sucrose and fiber to make the diets isocaloric. There was a small difference in phosphorus content (0.58% and 0.49% w/w respectively in normal protein and low protein diets). Calcium content was identical (0.52%). The diets were pair between groups.

Composition

fed to avoid differences in food intake

Table 1

of the Experimental Diets

NP-NLA LP-NLA NP-HLA LP-HLA

Protein 240 120 240 120 Carbohydrate 242 350 242 350 Fat 200 200 200 200 Fiber 70 82 70 82 DL methionine 3 3 3 3 Corn starch 200 200 200 200 Mineral mix 35 35 35 35 Vitamin mix 10 10 10 10

Beef tallow Safflower oil Soybean oil

Fatty acids (%) Saturated Monosaturated Linoleic acid Other fatty acids

Major Constituents (g/Kg)

Source of Fats (g/Kg)

190 190 0 0 10 10

Composition of Fats (%)

0

200 0

0

200 0

48.9 48.9 10.8 10.8 44.2 44.2 11.0 11.0 4.4 4.4 76.6 76.6 2.5 2.5 1.6 1.6

Animals Female Sprague-Dawley rats, weighing 180-200 g underwent

3/4-nephrectomy in two-stages. Four weeks later, when their mean serum creatinine was 1.23 mg/dl, they were matched by serum creatinine and allocated to groups of 13 rats fed each of the 4 diets. Body weight,

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serum creatinine and urinary protein excretion were measured biweekly, systolic blood pressure by the tail cuff method at weeks 3 and 10. Blood was obtained by tail vein puncture, and urine by placing rats in metabolic cages for 23 hours, without food but with free access to water. The lipid profile was measured at week 12 after a 14 hour fast. At week 21, the aorta was cannulated under ketamine and xylazine anesthesia and the kidney remnant was perfused with 30 ml of Earle's balanced salt solution (EESS, Gibco). A small portion of the kidney was fixed with Mosmann's solution for light microscopy, and the rest was used for glomerular isolation.

Laboratory Methods Urinary protein was measured by Coomassie brilliant blue, serum

creatinine by the Jaffe reaction, total triglycerides and cholesterol by enzymatic methods. Glomeruli were isolated using a modification of the method reported by Hassid (14). Briefly, cortical strips were minced to paste-like consistency, passed initially through a 180 urn sieve, and then twice through a 140 urn sieve. Glomeruli were harvested over a 74 urn sieve. The purity was determined microscopically for each isolation, and was consistently 85-90%. Glomeruli were incubated in 1 ml modified Earle's balanced salt solution (EBSS), pH 7.4 containing BSA 1 mg/ml, 25 mM N-2-hydroxyethylpeperazine-N-2-ethanesulfonic acid (HEPES), at 37OC for one hour. The supernatant was removed after centrifugation, and frozen at -7OV until measurement. Glomerular protein content was measured by the Lowry method. PGE2, 6-keto-PGFtC1, and TXB2, the last two stable metabolites respectively of PG12 and TXA2, were measured by direct radioimmunoassay as reported elsewhere (15).

Microscopy: For light microscopy, 4 pm tissue sections were stained with periodic acid-Schiff (PAS). Histologic sections were coded and examined "blind." Glomerular and tubular changes were assessed. The percentage of focally and of globally sclerosed glomeruli was estimated. Tubular changes were graded'semiquantitatively from 0 to 4+; the degree of tubular dilatation, hypertrophy, hyperplasia and calcification and of intraluminal protein were measured.

Statistics: Two-way analysis of variance in normally distributed data allowed us to establish whether there was a significant effect of either manipulation, namely low protein or high LA diet, or if there was an effect of their interaction. Chi-square test was applied to data not normally distributed.

RESULTS

Weights, blood pressure and protein excretion The body weights were similar in all four groups throughout the

experimental period. The systolic blood pressures, measured at weeks 3 and 10, were also similar. All rats developed proteinuria of variable degrees, but there were no differences between groups.

Serum lipids Total serum triglycerides and cholesterol, measured at week 12 are

shown in Table 2. The serum triglycerides were lower in animals fed a high LA diet (p (0.00). No effect of either dietary manipulation was

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observed on serum cholesterol. The logarithm of the serum cholesterol, but not that of the serum triglycerides at week 12 correlated with the logarithm of the final serum creatinine value (r = 0.57, p ~0.001).

Table 2

Serum Lipids at Week 12

NP-NLA LP-NLA NP-HLA LP-HLA (n=7) (n=S) (n-7) (n=9)

Serum triglyceride 94 a7 45* 45f (mg/dl) (62, 141) (61, 123) (39, 51) (36, 56)

Serum cholestero 130 138 138 113 (mg/dl) (118, 143) (113, 168) (125, 153) (90, 142)

Geometric mean (-1 SD, +1 SD) *Effect of HLA diet, p C.005

Survival, Renal function, and renal histology At week 21: 5, 6, 5 and 7 rats were alive in groups NP-NLA, LP-NLA,

NP-HLA and LP-HLA respectively. In comparing the relationship of time to preservation of renal function we estimated the proportion of rats at each time point in which the serum creatinine level was less than double the serum creatinine at week 0.

100

60

60

40

20

: 1

UP - NLA (3---E,

LP - NLA &---Q

NP - HLA M

LP - HLA M

1 1 I I 1 I 1 1 I I 1

0 2 4 6 6 10 12 14 17 19 21

WEEKS

Figure 1: Survival of renal function in groups NP-NLA (O---O), LP-NLA (A ---A), NP-HLA (-1 and LP-HLA (M). The vertical axis repre- sents % of animals surviving with serum creatinine less than double the baseline value (n=13 for all groups).

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The survival curves of renal function for the four groups of rats are shown in Figure 1. The time at which 50% of the animals had a serum creatinine value equal to or less than double the control values was 15, 18 and 13 weeks for the NP-NLA, LP-NLA, NP-HLA groups. For the LP-HLA group, the estimated time was 22 weeks.

There were variable degrees of glomerular sclerosis whether global or focal. There were variable degrees of tubular dilatation and hyperplasia and of interstitial atrophy and fibrosis. The sclerosis score was defined as the sum of the global sclerosis plus l/2 of the focal sclerosis. Mean sclerosis scores were 13, 20, 21 and 24 for groups NP-NLA, LP-NLA, NP-HLA and LP-HLA respectively; the differences were not significant. There was a beneficial effect of low protein on interstitial fibrosis (p <0.05) and on tubular hyperplasia (p <O.Ol) Eicosanoid production

Production of PGE2, 6-keto-PGF,o and TXB2 by isolated glomeruli is shown in Table 3. There was additive stimulatory effect on PGE2 of low protein and high linoleic acid diet (p <0.05). No significant effects were observed on either 6-keto-PGFIa or TXB2 production.

Table 3

Glomerular Production of Eicosanoids at week 21

NP-NLA (n=S)

LP-NLA (n=6)

NP-HLA (n=4)

LP-HLA (n=7)

PGE2 (ng/mg protein)

9.8 8.1 9.1 15.5* (7.6, 12.5) (5.9, 11.2) (7.0, 11.9) (10.7, 22.4)

6-Keto-PGF,c (ng/mg protein)

7.1 7.4 7.7 8.4 (5.4, 9.4) 4.3, 12.5) (6.4, 9.2) (5.3, 13.3)

TXB2 (ng/mg protein)

3.5 3.0 1.3 3.3 (1.5, 7.9) (1.5, 5.5) (0.7, 2.4) 1.7, 6.4)

Geometric mean (-1 SD, +l SD) *Significant interaction effect of LP and HLA diets.

DISCUSSION

This experiment demonstrates a beneficial effect on renal function of a diet restricted in protein and enriched with linoleic acid. This effect is shown in a model of progressive renal failure, the rat with reduced renal mass. Each individual dietary manipulation was beneficial in prior reports, but no attempt to evaluate the effect of both dietary manipulations combined has been reported previously.

A beneficial effect of phosphate restriction in rats with reduced renal maas has been reported (16). The phosphorous content of the nor- ma1 and low protein diets in the present experiment were similar (0.58%

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and 0.49%), and lo-12 fold higher than in studies of the effect of a low phosphorous diet (16). This minor difference is unlikely to contribute to the result.

We observed an additive effect of low protein and high LA on the glomerular production of PGE2. Inhibition of cyclooxyqenase by indo- methacin (12) abolished the beneficial effects of LA feeding in rats with reduced renal mass. This finding and the demonstration of increased PGE2 excretion in urine of patients with chronic renal disease (17), Suggest a protective role for PGE2 in the adaptive mechanisms to loss of renal mass. Increased PGE2 has been reported in cortical strips (11) and urine (13) of rats with reduced renal mass fed hiqh LA diets, experiments in which concomitant protection of renal function was observed. PGE2 is a vasodilator, and may reduce glomerular hyperten- sion. The reduction of glomerular hypertension may be one of the mecha- nisms by which protein restriction prevents deterioration of renal function in the remnant nephron (4). The effect of both manipulations on glomerular hemodynamics may have been additive and contributory to the protection.

A lowering effect of LA on plasma triglycerides was also demonstrated. The effect of increased blood lipids in the development of glomerular sclerosis in the partially nephrectomized rat (18) and in NZB/W mice fed a diet high in saturated fats (19) has been demonstrated. The effects of plasma lipids on glomerular function is not well understood at the present time.

The present observations suggest that the combination of low protein with high linoleic acid or perhaps other polyunsaturated fats should be tested in patients with progressive chronic renal disease.

ACKNOWLEDGMENTS

This work was supported by NIH grant AM 17196, CLINFO RR 00068, and by grants from Research and Educational Funds of Dialysis Clinic and the Kidney Foundation of Greater Cincinnati.

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