Beneficial effects of polyunsaturated fatty acids in partially nephrectomized rats

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BENEFICIAL EFFECTS OF POLYUNSATURATED FATTY ACIDS IN PARTIALLY NEPHRECTOMIZED RATS Barcelli, U-O., Miyata, J., Ito, Y., Gallon, L., Laskarzewski, P., Weiss, M., Hitzemann, R., Pollak, V.E. Departments of Internal Medicine and Pathology University of Cincinnati Medical Center, Cincinnati, Ohio, 45267-0585, USA

ABSTRACT

Evening primrose oil, safflower oil, and salmon oil, all with high polyunsaturated fatty acid content , were fed to partially nephrectomized rats; the effects were compared to those of feeding beef tallow. All three oils had favorable effects on progression of renal failure, salmon oil on kidney histology as well. The changes induced in platelet production of thromboxane A2, and in the renal production of various eicosanoids may explain the protective role of these oils. INTRODUCTION There is experimental evidence that some dietary polyunsaturated fatty acids (PUFA) have favorable effects on progression of renal disease in various animal models. Feeding linoleic acid (LA), an w-6 PUFA, was beneficial in progressive renal disease of the partially nephrectomized rat (1,2). This effect was abolished by indomethacin (21, suggesting a mechanism involving prostaglandins (PG) or thromboxanes (TX). Feeding the w-3 PUFAs eicosapentaenoic (EPA) and docosahexaenoic acids had beneficial effects in the NZB/NZW (3) and MRL/lpr (4) mouse models of lupus nephritis. Feeding both LA and EPA was beneficial in the glomerulonephritis induced in the mouse by repeated injections of apoferritin (5,6). Significant changes in production of PG and TX were demonstrated (6). In the partially nephrectomized rat model of chronic renal failure, we studied the effects of feeding three oils each with a high content of PUFA; they were compared with a control group fed a diet with predominantly saturated and monounsaturated fatty acids. The three PUFA diets were chosen because of their content of precursors of the monoenoic, dienoic, and trienoic PGs. METHODS Experimental design: Fifty-eight female Sprague-Dawley rats (initial weight 200-250 g) were partially nephrectomized in two stages. A left heminephrectomy and, one week later, a right total nephrectomy were performed under ketamine and xylazine anesthesia. Rats were matched by serum creatinine levels two weeks after the second surgical procedure and allocated to four groups, each fed dif-

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ferent diets prepared with beef tallow (BF), evening primrose oil (EVP), safflower oil (SAF), and salmon oil (SAL). Pair feedings were used throughout the experiment to minimize differences in weight gain. Body weight and serum creatinine were measured serially every two weeks, blood pressure by the tail method and urinary protein excretion every three weeks. Blood was obtained by tail vein puncture and urine by placing rats in metabolic cages for 16 hour periods, without food, but with free access to water. Urinary PG and TX excretion were measured at week 5 of pair feedings. Serum cholesterol, triglycerides, and high density lipoprotein cholesterol were measured at week 9 and at sacrifice. At week 21, rats were sacrificed by cardiac puncture and exsanguination under pentothal anesthesia. Blood was obtained to determine TXA2 generation by platelets. Kidney tissue was fixed in Mossman's solution for light microscopy. Diets. All diets had 20% (w/w) fat. The diet fed the animals that served as controls contained 19% beef tallow (BF) and, to provide essential fatty acids, 1% soybean oil. The EVP diet was prepared with 20% evening primrose oil (gift of Dr. D. Horrobin, Efamol, Inc.). Fatty acid analysis of the oils used to prepare the diets (Table 1) showed that the BF diet contained 4.4% LA; the EVP diet contained 7.6% GLA, but was also rich in LA (72.8%); the SAF diet contained 76.6% LA; and the SAL diet contained 1.6% LA, 11.9% arachidonic acid (AA), 8.5% EPA and 7.6% DHA. Table 1.

Composition of Fats in Experimental Diets

Fatty Acids

Saturated Monounsaturated Polyunsaturated GLA LA AA EPA DHA Other

BF (%I

EVP (%)

SAF (%I

SAL ($1

48.9

9.7

44.2

8.2

10.8 11.0

23.3 46.7

4.4

7.6 72.8

76.6

2.5

1.7

1.6

1.6 11.9 8.5 7.6 0.4

The remainder of the nutrients were identical in all four diets. Their constituents, by weight, were: casein 19%, DL methionine 0.3%, sucrose 2%, cornstarch 20%, cellulose 7%, a mineral mix 3.5%, and a vitamin mix 1%. These were in compliance with current nutritional standards (7).

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Laboratory Methods. Urinary protein was measured by using Coomassie Brilliant Blue; serum creatinine by the Jaffe reaction; total serum cholesterol, triglycerides and HDL were measured by enzymatic methods. Fatty acid analysis was performed by gas chromatography as previously reported (8).

PG and TX determinations. TXA2 generation by platelets: 1 ml of blood was incubated at 37OC for 30 minutes, centrifuged and serum TXB2 (the stable hydrolysis product of TXA2) measured by FUA. The cross reactivity of the antibody to TXB2 is reported elsewhere (9). Urinary PG: Five rats in each group were selected for this assay. They had stable serum creatinine levels, equal in all groups. At week 5, urine was collected in a metabolic cage for 16 hours in refrigerated thermos containers. Urine was extracted using Sep Paks. Urinary PGE,, PGE2, PGE3, 6-keto-PGFlo and TXB2 were separated by reverse phase HPLC and measured by BIA. To measure PGE3 we used an antibody to PGE2 with high cross reactivity, due to the lack of specific antibody to PGE3. The methods for eicosanoid measurement have been published in detail (9). Microscopy. For light microscopy, 4 micron 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 sclerosed glomeruli, whethex focally or globally, was estimated. Tubular changes were graded semiquantitatively from 0 to 4+ measuring the degree of tubular dilatation, degeneration and calcinosis. Statistics: Analysis of variance on single or repeated measurements, non-parametric tests, and survival analysis were used as appropriate. RESULTS Weight, blood pressure and survival: In the SAL group the diet was poorly accepted; some animals developed diarrhea and tended to gain less weight than those in the other three groups. Despite this, pair feeding successfully maintained similar weights in all four groups (Table 2). Systolic blood pressure increased equally in all four groups following the second operation, returned to normal levels by the ninth week (Table 21, and remained normal in all groups thereafter. There were no differences in survival. At 21 weeks lo/15 control rats (BF) were alive, and 13/14 fed EVP, 11/15 fed SAF, and lo/14 fed SAL. Serum lipids: The lipid profiles at week 9 and at sacrifice are summarized in Table 2. Compared with the controls, the triglyceride levels were significantly lower in all three PUFA fed groups at both time points. The HDL levels were significantly lower at sacrifice in rats fed EVP.

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Table 2.

Weight t Blood Pressure and Serum Lipids a BF

EVP

SAF

SAL

262 (244, 280) 134 (106, 162) iii (85, 145) 66 b (36, 130) 77 (60, 98)

276 (252, 300) 129 (112, 146) 113 (80, 160) 76 b (44, 138) 73 (55, 96)

263 (243, 283) 123 (108, 138) 119 (100, 142) 102 b (65, 160) 81 (65, 102)

Week 9 Weight

(g)

266 (240, 292) 128 (108, 148) Cholesterol (mg/dl) 130 (100, 170) Triglycerides (mg/dl) 184 (93, 365) HDL (mg/dl) 75 (58, 98) BP (mm Hg)

Week 21 Weight

(g}

275 269 275 (252, 298) (255, 283) (260, 290) 126 128 130 (105, 147) (107, 149) (iii, 149) Cholesterol (mg/dl) 134 116 123 (ii0, 166) (87, 156) (i00, 152) Triglycerides (mg/dl) 101 51 b 60 b (65, 156) (31, 84) i34, 98) HDL (mg/dl) 85 59b 81 (70, 104) (36, 98) (63¢ 103) aResults are expressed as arithmetic means (weight and blood eometric means (lipids) and, in parenthesis, 68% confidence Different from the control group BF, p <.05. BP (mm Hg)

g

263 (234, 292) 115 (98, 132) 141 (125, 160) 72 b (55, 98) 97 (86 t 108) pressure) or limits.

20°[

,Ootl 60 40

~

2o

llA

r/~

3

0

5

10

14

18

WEEKS

Fig.

i.

geometric

214

Serial means

urinary

protein

and

confidence

68%

excretion.

Results

are

expressed

as

limits.

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Urine protein: Serial urinary protein excretion is shown in Figure 1. Protein excretion in the EVP group did not differ from controls at all time periods. Protein excretion in the SAF group was less than in the controls only at week 5. In the SAL group proteinuria was less than in the controls at all time periods, the difference being significant at week 5, 14 and 18. Serum creatinine was elevated in all rats immediately after the second operation. At week 1, the mean serum creatinine was 0.86 mg/dl in all groups. Thereafter, it stabilized for several weeks. The serum creatinine rose progressively in most rats after week 8. The proportion of animals in each group with serum creatinine <1.3 mg/dl was analyzed by survival analysis (Fig. 2), as an index of preservation of renal function of about 30% of normal. All three experimental diets significantly prevented progressive deterioration of renal function, and to an equal degree.

80

60 %

&..._.

SF

A-.-

EVP

Q----SAF

40

+

SAL

20

0

5

10

15

20

25

WEEKS

Fig. 2.

Preservation of renal function. Results are expressed as number of animals with a serum creatinine <1.3 mg/dl.

x2 generation by platelets (Table 3): The production of TXA2 by platelets during blood clotting, measured as the stable metabolite TXB~, was markedly decreased by the high SAL diet; it was affected by neither the high EVP nor by the SAF diet.

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TXA~ Generation, Measured as E2 During Platelet Aggregation

Table 3.

Group

TXB2 (ng/mlja

BF (n=lO) EVP (n=13) SAF (n=12) SAL (n=9)

80 (58.2, 110.0) 81.3 (50.6, 130.7) 68.5 (44.7, 104.8) 30.3b (17.7, 51.7)

aResults are expressed as geometric means and, in parenthesis, 68% confidence limits. bDifferent from the control group BF, p <.05.

Urine eicosanoid excretion (Table 4): The high EVP diet induced a significant increment in the urinary excretion of 6-keto-PGFlc. High SAF feedings were associated with a decrease in PGE,, a four-fold increase in 6-keto-PGF,c and a 3.5-fold increase in PGE2 excretion. High SAL feedings induced a 16-fold increase in PGE3 and a significant reduction in TXB2 excretion.

Table

4.

Urinary

Eicosanoid

Excretion

(ng/16

h) at week

5a

Group

BF

(n=S) EVP

6K-PGF,

2.6 (1.8, 3.9)

4sb

PGE,

PGE2

18.8 (11.0, 32.0)

(3.5,

5.8)

11.1 (5.5, 22.0)

(n=5)

1o.G (9.2,

11.9)

(3.0,

SAL. (n=5)

3.7 (2.7, 5.2)

(n=5) SAF

5.5b 9.7)

11.5 (6.1.

21.2)

PGE3

TX82

21.3)

0.3 (0.2, 0.4)

3.4 (2.4, 4.9)

8.3 (5.4, 12.7)

0.5 (0.3, 0.7)

5.2 (4.1, 6.7)

0.5 (0.3, 0.7)

3.7 (2.8, 4.8)

4.9b

l.6b (0.2, 2.7)

13.6 (8.7,

47.9” (15.7,

146)

10.5 (9.8, 11.3)

aRe~ult~ are expressed as geometric means and, confidence limits. b. Different from the control group BF, p (.05.

(2.4,

10.1)

in parenthesis,

68%

Histology: In the control BF rats, the morphologic changes consisted mostly of glomerular sclerosis and tubular dilatation and degeneration: the percentage of sclerosed glomeruli was 24.8%. The percentage of sclerosed glomeruli in the EVP (25.2%) and SAF (19.8%) groups was not significantly different; that in the SAL (7.1%) group

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was significantly lower (p <.05). The mean tubular dilatation score in the control BF group was 3.1. The values in the EVP (2.5) and the SAF (2.3) groups were not different; that in the SAL (1.4) group was lower (p
The results of this experiment lend support to prior observations on the beneficial effects of some PUFA~ on the progression of renal disease. In a non-immunologic model of progressive renal disease, the rat with reduced renal mass, supplementation with three different oils rich in PUFAs had a favorable effect in ameliorating the deterioration of renal function that usually occurs. The salmon oil diet also had a favorable effect on the renal histologic findings. There were significant changes in renal production of PGs and TX, reflected in the urinary excretion of these eicosanoids. Evening primrose oil was fed because, although relatively rich in LA, the precursor of dienoic PGs, it also contains GLA, the precursor of monoenoic PGs. There was a significant increase in excretion of PGI2. The powerful vasodilator and platelet anti-aggregating effects of PG12 may at least in part explain the beneficial effect of the EVP diet. Safflower oil fed rats produced significantly larger amounts of urinary PGE2 and PG12. These PGs are vasodilators, and increase renal plasma flow and glomerular filtration rate. High LA feedings have been shown to preserve renal function in rats with reduced renal mass (1,2) and to increase creatinine clearance in normal humans (10). The fact that there was no positive correlation between serum creatinine at sacrifice and the degree of glomerular sclerosis in LA fed rats suggests the possibility that the beneficial effect of LA on renal function was exerted through functional mechanisms, possibly the production of vasodilatory prostaglandins and changes in transcapillary hydraulic pressure at the glomerular level. Salmon oil fed rats had a significant increase in urinary excretion of PGE3, as effective a vasodilator as PGE2, and a suppression of the urinary excretion of the vasoconstrictor TXA2. High EPA diets also very markedly inhibited production of TXA2 by platelets during aggregation. Pharmacologic inhibition of TXA2 formation is beneficial in the progressive renal deterioration of rats with reduced renal mass (111, and attenuates proteinuria in adriamycin-induced nephrosis (12).

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In contrast to our results, other authors have shown that a diet prepared with menhaden oil has deleterious effects in partially nephrectomized rats (13). In those experiments, suppression of the urinary excretion of PGE2 was demonstrated (13); in our experiments it was unaffected. EPA has also been shown to inhibit formation of PGI2 by vascular endothelium in rats (141, but not in humans (15). In the present experiment, urinary PGI2 excretion was not decreased. Cur rats fed salmon oil tended to develop diarrhea which may possibly have interfered with absorption of protein and other nutrients. Further studies will be needed to clarify these discrepancies. Other possible mechanisms may be invoked to explain the observations. Progression of renal failure in rats with decreased renal mass has been modified favorably by the anti-lipidemic drug clofibrate (16). In the present experiments, significant decreases in serum triglycerides, but not in cholesterol, were observed with all three experimental diets. The polyunsaturated fatty acids are also known to have effects on the clotting and fibrinolytic systems (17), membrane composition, and on leukotriene synthesis (18). Effects on these systems might have played a role, but were not studied. Modification of dietary lipids seems to be an effective and relatively simple maneuver which may have a preventive and therapeutic role in the progression of chronic renal disease. ACKNOWLEDGEMENTS This work was supported by National Institutes of Health grants AM 34489 and RR 00068 and by a grant from the Research and Education Fund of Dialysis Clinics, Inc. REFERENCES 1.

2.

3.

4.

5.

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Barcelli, U.O., M. Weiss, V.E. Pollak. Effects of a dietary prostaglandin precursor on the progression of experimentally induced chronic renal failure. J Lab Clin Med 100:786, 1982. Heifets, M., M. Purkerson, S. Klahr. A diet rich in linoleic acid improves renal function and decreases blood pressure in rats with a remnant kidney. Kidney Int 27:244, 1985 Prickett, J.D., D.R. Robinson, A.D. Steinberg. Effects of a dietary enrichment with eicosapentaenoic acid upon autoimmune nephritis in female NZB/N!ZW Fl mice. Arthritis Rbeum 26:133, 1983. Kelley, V.E., A. Ferretti, S. Izui, T.B. Strom. A fish oil diet rich in eicosapentaenoic acid reduces cyclooxygenase metabolites and suppresses lupus nephritis in MRL-lpr mice. J Immunol 134:1914, 1985. Kher, V., U. Barcelli, M. Weiss, V.E. Pollak. Effects of dietary linoleic acid enrichment on induction of immune complex nephritis in mice. Nephron 39:261, 1985.

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Kher, V., U. Barcelli, M. Weiss, L. Gallon, P. Pajel, P. Laskarzewski, V.E. Pollak. Protective effect of polyunsaturated fatty acid supplementation in apoferritin induced murine glomerulonephritis. Prostagl Leukotr Med (in press) Report of the American Institute of Nutrition ad hoc Committee on Standards for Nutritional Studies. J Nutr 107:1340, 1977. Hitzemann, R. Develomental changes in the fatty acids of synaptic membrane phospholipids: Effect of protein malnutrition. Neurochem Res 6:935, 1981. Gallon, L.S., U.O. Barcelli. Measurement of prostaglandin E3 and other eicosanoids in biologic samples using high pressure liquid chromatography and radioimmunoassay. Prostaglandins 31:217, 1986. Adam, 0, G. Wolfram. Effect of different linoleic acid intakes on prostaglandin biosynthesis and kidney function in man. Am J Clin Nutr 40:763, 1984. Purkerson, M.L., J.H. Joist, J. Yates, A. Valdes, A. Morrison, S. Klahr. Inhibition of thromboxane synthesis ameliorates the progressive kidney disease of rats with subtotal renal ablation. Proc Nat1 Acad Sci USA 82:193, 1985. Remuzzi, G., L. Imberti, M. Rossini, C. Morelli, C. Carminati, G.M. Cattaneo, T. Bertani. Increased glomerular thromboxane synthesis as a possible cause of proteinuria in experimental nephrosis. J Clin Invest 75:94, 1984. Scharschmidt, L., L. McGarry, P. Berger. The effects of fish oil eicosapentaenoic acid on PGE synthesis in mesangial cells and in Kid Int 27:264, 1985 (abstract) the 5/6 nephrectomized.rat. Scherhag, R., J.J. Kramer, R. Dusing. Dietary administration of eicosapentaenoic and linolenic acid increases arterial blood pressure and suppresses vascular prostacyclin synthesis in the rat. Prostaglandins 23:369, 1982. Fischer, S., P.C. Weber. Prostaglandin I3 is formed in-vivo in man after dietary eicosapentaenoic acid. Nature 307:165, 1984. Kasiske, B.L., M.P. O'Donnell, F. Daniels, W.F. Keane. The lipid lowering agent clofibric acid ameliorates renal injury in the 5/6 nephrectomy model of chronic renal failure. Clin Res 33:488A, 1985 (Abstract) Barcelli, U, P. Glas-Greenwalt, V.E. Pollak. Enhancing effect of dietary supplementation with omega-3 fatty acids on plasma fibrinolysis in normal subjects. Thromb Res 39:307, 1985. Lee, T.H., R.L. Hoover, J.D. Williams, R.I. Sperling, J. Ravalese,III, B.W. Spur, D.R. Robinson, E.J. Corey, R.A. Lewis, K.F. Austen. Effect of dietary enrichment with eicosapentaenoic and docosahexaenoic acids on in vitro neutrophil and monocyte leukotriene generation and neutrophil function. N Engl J Med 312:1217, 1985.

Editor: P.W. Ramwell

AUGUST

Received: 3-25-86

1986 VOL. 32 NO. 2

Accepted: 7-15-86

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