- Email: [email protected]
Protective effect of menhaden oil on renal necrosis occurring in weanling rats fed a methyl-deficient diet
Nutrition Research 22 (2002) 1077–1089 www.elsevier.com/locate/nutres
Protective effect of menhaden oil on renal necrosis occurring in weanling rats fed a methyl-deficient diet Marı´a C. Courre`gesa, Carla Carusoa, Jochen Kleinb, Alberto J. Monserrata,* a
Patologı´a Experimental, Departamento de Patologı´a, Facultad de Medicina, Universidad de Buenos Aires, J. E. Uriburu 950, 5° piso (1114) Buenos Aires, Argentina b Pharmakologisches Institut, Universitat Mainz, Obere Zahlbacher Str. 67 (D-55101), Mainz, Germany Received 26 July 2001; received in revised form 16 May 2002; accepted 18 May 2002
Abstract Weanling rats fed a lipotropic-deficient diet (LDD) may develop acute renal failure with morphological features that vary from focal tubular necrosis to widespread cortical necrosis and eventually reparative changes. The type of lipid in the diet influences the development of renal necrosis. The aim of this work was to study the effect of dietary menhaden oil on the development of acute renal failure induced in weanling rats fed a methyl-deficient diet. Experiment I: 40 weanling Sprague-Dawley male rats were allotted to 4 different groups and fed as follows: group 1, LDD with hydrogenated vegetable oil and corn oil as lipids; group 2, LDD with menhaden oil as lipid; group 3 and 4, similar to groups 1 and 2 plus choline. Rats were fed ad libitum until they died; surviving animals were killed on day 21. Mortality in the 4 groups was 60, 0, 0, and 10% respectively. Rats from groups 2, 3 and 4 did not show renal damage. Rats from group 1 showed tubular or cortical necrosis in dead rats and reparative changes in those killed on the 21st day. Experiment II was similar to experiment I, except that 45 weanling Wistar male rats were used and they were killed on the 7th day. All rats from group 1 showed renal necrosis; no renal damage was found in rats from groups 2, 3 and 4. Urea and creatinine alterations corroborated the renal changes. We conclude that menhaden oil displays a protective effect for renal necrosis induced by methyl-deficient diets in weanling rats. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Acute renal failure; Choline deficiency; Lipotropic factors deficiency; Methyl group deficiency; Menhaden oil
* Corresponding author. Tel.: ⫹54-11-4508-3602; fax: ⫹54-11-4508-3602. E-mail address: [email protected] (A.J. Monserrat). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 4 1 5 - 3
1078
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1. Introduction The important role of methyl metabolism in experimental animals as well as in human beings is widely and increasingly recognized. Nutrients involved in this metabolism are also known as lipotropic factors and are supplied in the diet mainly as methionine and choline, though folate and B12 are necessary for normal metabolism. Choline contributes to the structural integrity of cell membranes and signaling functions and it is involved in cholinergic neurotransmission, lipid transport and metabolism [1– 4]. Rats fed a diet deficient in lipotropic factors may develop liver pathology (steatosis, cellular death, cirrhosis and cancer) [1,2,5,6], heart changes (stetatosis and necrosis) [7,8] and renal pathology. In particular, weanling rats fed a lipotropic-deficient diet may develop acute renal failure with morphological features that vary from focal tubular necrosis to widespread cortical necrosis or eventual presence of reparative changes [2,9]. The type of lipid in the diet influences the development of the renal necrosis. For instance, coconut oil (rich in saturated fatty acids) has a protective effect [8,10,11], which would be due to its content of myristic acid [10,12]. Menhaden oil is rich in eicosapentanoic (20:5) and docosahexaenoic acid (22:6) and so may influence renal fatty acid composition and arachidonic acid metabolism which play an important role in renal physiology and pathology [13]. The purpose of this study, was to explore the effect of menhaden oil in this experimental model of acute renal failure. This could be important to a better knowledge of pathogenic mechanisms involved in renal tubular and cortical necrosis.
2. Materials and methods 2.1. Animals Forty Sprague-Dawley weanling male rats from the Veterinary School of the University of Buenos Aires (experiment I) and forty Wistar weanling male rats from the Department of Pathology of the School of Medicine of the University of Buenos Aires (experiment II) were employed. All animals had free access to one of four diets described below and drinking water. They were individually housed in suspended wire-bottomed cages in an air conditioned room and were exposed to light from 7.00 a.m. to 7.00 p.m. All conditions and handling of animals followed NIH Guidelines for the Care and Use of Laboratory Animals. Body weight and food intake were measured daily. 2.2. Diets Rats were given free access to semi-purified diets in powdered form as indicated in experimental design. The diets met the National Research Council Nutrition Requirements and varied in lipid composition and choline chloride content. The composition of each diet is described in Table 1.
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1079
Table 1 Composition of the diets (g/100 g) Diet composition a
Soy bean protein Sucrose Celluloseb Vitamin mixture (without B12 and choline)c Salts (W)d L-cystinee Hydrogenated vegetable oilf Corn oilg Menhaden oile
Group 1 and 3
2 and 4
20 48.5 4 4 2 0.5 14.3 5.7 0
20 48.5 4 4 2 0.5 0 0 20
Note. Diets 3 and 4 were supplemented with 0.35% of choline chloride. a Soybean protein grade II. U.S. Biochemical corp., USA. b Celufil. Non-nutritive Bulk, U.S. Biochemical Corp., USA. c Vitamin Diet Fortification Mixture. U.S. Biochemical Corp., USA. d Salt Mixture Wesson Modification, U.S. Biochemical Corp., USA. e U.S. Biochemical Corp., USA. f Flora Da´ nica, Buenos Aires, Argentina. g Mazola. Refinerı´as de Maı´z, Buenos Aires, Argentina.
2.3. Experimental design Experiment I: Forty Sprague-Dawley weanling male rats were distributed in 4 different groups. The rats from groups 1 and 2 were fed ad libitum the lipotropic-deficient diets shown in Table 1, while those in groups 3 and 4 were used as controls of the lipotropic-deficient animals and were fed the same diets of groups 1 and 2 respectively and supplemented with 0.35% choline chloride. The different lipids were given at a 20% concentration, the same concentration we have used in previous experiments evaluating the effects of coconut oil [11] and different fatty acids [12] in this experimental model. Groups 1 and 3 received a mixture of corn oil and hydrogenated vegetable oil to supply different fatty acids [9,11,12,14]. The rats were kept on the diets until they died or, if death did not occur, they were killed on the 21st day. Under light ether anesthesia, blood was drawn from the abdominal aorta and urea was determined in serum by standard procedures. The kidneys and liver were removed and weighed. Tissue samples were fixed in buffered formalin, embedded in paraffin, and sections were stained with hematoxylin-eosin and, in some animals, with PAS or Masson’s trichrome [15]. Frozen sections of the liver were stained with oil red O [16]. Fatty changes in the liver were evaluated in frozen sections stained with oil red O. Changes were classified as mild, moderate or severe according to the extent (from zonal to diffuse) of the fat deposition in the liver lobule. For topographical localization of different segments of the kidney, well known terminology was used [17]. A previous proposed classification was applied to evaluate the renal pathology [14]. (Table 2). Experiment II: Forty five Wistar male rats were distributed in 4 different groups, fed and
1080
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
Table 2 Histologic classificaiton of renal necrosis A B
C
D
Kidney without necrosis Acute tubular necrosis Grade 1: Isolated foci of cellular necrosis in some tubules Grade 2: Small groups of tubules with necrosis Grade 3: Zones of tubular necrosis Grade 4: Confluent zones of tubular necrosis Cortical necrosis Grade 5: Grade 4 plus isolated foci of cortical necrosis Grade 6: Grade 4 plus multiple foci of cortical necrosis Grade 7: Grade 4 plus confluent foci of cortical necrosis Grade 8: Massive cortical necrosis Repair: Repair is characterized by different degrees of interstitial fibrosis, tubular atrophy, tubular regeneration, glomerular fibrosis, etc. According to its extent it is divided into 4 grades.
kept as those rats in Experiment I. The rats were kept on the diets until day 7th. Blood was obtained as in Experiment I and in addition to urea, creatinine was also evaluated. The kidneys and liver were removed, weighed and studied as in Experiment I. 2.4. Determination of choline and choline metabolites The choline content of menhaden oil was determined essentially as previously described [18]. Briefly, menhaden oil was extracted with water. Aliquots of the water phase were evaporated to dryness, taken up in buffer and analyzed for content of free choline by high pressure liquid chromatography (HPLC). In addition, aliquots of the water phase were hydrolyzed by 6 N HCl (1 h, 80°C), a procedure which causes release of choline from glycerophosphocholine [18]. The oil phase was hydrolyzed with 6 N methanolic HCl (1 h, 80°C) to cleave phosphatidylcholine to choline. Subsequently, the methanolic phase was taken to dryness. The HPLC system for the analysis of choline consisted of a low-speed pump (BAS PM80), separation column (SepStik, 530 ⫻ 1 mm), enzyme reactor (50 ⫻ 1 mm) carrying immobilized choline oxidase and electrochemical detector (BAS LC-4C) with a platinum electrode operating at 0.5 V. The flow rate was 120 l/min. At an injection volume of 5 l, the detection limit of this system was 25 fmol/injection. 2.5. Statistical methods The Fisher’s exact test was employed to statistically evaluate mortality and the incidence of histopathologic renal lesions. Renal and body weights and length of survival as well as creatinine and urea in serum were expressed as mean ⫾ standard error and comparisons were made by two-way analysis of variance (ANOVA). In the case of significant differences ANOVA was followed by the Tukey-Kramer test [19]. Differences which resulted in p values lower than 0.05 were considered significant (S).
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1081
Table 3 Initial, fifth day and final body weights Body weight Initial
Experiment I Group 1 Group 2 Group 3 Group 4 Experiment II Group 1 Group 2 Group 3 Group 4
41.9 ⫾ 1.0* (10) 42.3 ⫾ 1.1 (10) 42.2 ⫾ 1.0 (10) 42.2 ⫾ 0.8 (10)
Final
5th day
Dead
Sacrificed
49.3 ⫾ 2.1 (6)
98.3 ⫾ 4.0⌿ (4) 96.0 ⫾ 3.6⌿ (10) 128.5 ⫾ 4.3 (10) 116.7 ⫾ 3.9 (9)
57.3 ⫾ 0.7 (10) 55.5 ⫾ 3.8 (10) 59.9 ⫾ 4.2 (10) 54.7 ⫾ 1.4 (9)
47.5 ⫾ 2.4⌿⌿ (11) 54.1 ⫾ 1.8 (12) 57.6 ⫾ 1.4 (12) 61.0 ⫾ 1.2 (10)
51.0 ⫾ 1.9 (11) 48.4 ⫾ 2.0 (12) 51.6 ⫾ 1.4 (12) 53.2 ⫾ 0.9 (10)
36.2 (1)
39.2 ⫾ 1.9 (11) 39.1 ⫾ 1.7 (12) 38.6 ⫾ 2.7 (12) 41.7 ⫾ 0.5 (10)
Values are expressed as Mean ⫾ SE. *: g ( ): number of rats. ⌿ 1 and 2 vs. 3 and 4 (p ⬍ 0.05); ⌿⌿ 1 vs. 2, 3 and 4 (p ⬍ 0.05).
3. Results The initial, final and fifth day body weights are shown in Table 3. As indicated by fifth day body weights, rats grew adequately well during the first days in both experiments. Later on, as a consequence of renal damage, rats belonging to group 1 lost weight (for example, see dead animals in group 1, experiment I). No differences between body weights of group 1 and group 2 were observed on the day of sacrifice. In experiment II, we obtained similar results, though in this case, we found significant differences between body weights of group 1 and 2 on the day of sacrifice. Mortality and length of survival in experiment I are shown in Table 4. As expected, in the choline-deficient rats mortality was high (6/10) in rats of group 1. No mortality was observed in choline-deficient rats receiving menhaden oil (group 2) and control groups 3 and 4, except one rat in group 4 that did not show renal damage in the histologic study. Length of survival was 9 days in rats of group 1. Renal weights, urea and creatinine values of both experiments are shown in Table 5. Renal weights were higher in choline deficient rats not supplemented with menhaden oil (group 1). Urea level in the serum was within normal ranges in choline-supplemented rats, while it was slightly elevated in those rats of group 2 (only in experiment I) and markedly increased in those of group 1. The acute renal changes were grossly characterized by an increase in size and weight and
1082
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
Table 4 Mortality and survival time Group
Total ratsn
Died ratsn
1 2 3 4
10 10 10 10
6 0 0 1
Length of survival 9.2 ⫾ 0.2 13.0
Values are expressed as Mean ⫾ SE. n : number of rats, *: Days.
by a purplish red discoloration. Necrosis involved mainly proximal convoluted tubules and was characterized by increased eosinophilia, changes in tinctorial affinities with Masson’s thricrome, pyknosis and mainly kariolysis; in more advanced stages glomerular and vascular necrosis were also observed. Concomitant with tubular necrosis hyalin casts were found. Repair was characterized by the progressive disappearance of necrotic tubules and casts and by different degrees of regeneration, tubular atrophy and fibrosis. The results of the histological study are shown in Table 6. In experiment I, dead rats of group 1 showed the typical renal lesions of tubular or cortical necrosis found in choline-deficiency. Rats sacrificed at the end of the experiment showed typical reparative changes in rats of group 1 (Fig. 1c), while no changes were observed in choline-deficient rats of group 2. As expected, choline-supplemented rats did not show renal damage. Rats from group 1 displayed a moderate or severe fatty liver, rats from group 2 a Table 5 Renal weights, urea and creatinine Dead rw Experiment I Group 1 Group 2 Group 3 Group 4 Experiment II Group 1 Group 2 Group 3 Group 4
Sacrificed rw/100 g bw
rw
Urea*
Creatinine*
rw/100 g bw
1.981 ⫾ 0.156 4.004 ⫾ 0.232 1.910 ⫾ 0.088⌿ (6) (6) (4) 1.344 ⫾ 0.059 (10) 1.555 ⫾ 0.050 (10) 0.598 1.857 1.550 ⫾ 0.059 (1) (1) (9)
1.957 ⫾ 0.145⌿ (4) 1.400 ⫾ 0.029 (10) 1.214 ⫾ 0.031 (10) 1.329 ⫾ 0.021 (9)
99 ⫾ 11⌿ (4) 35 ⫾ 5⌿⌿ (10) 18 ⫾ 4 (10) 26 ⫾ 2 (9)
1.525 ⫾ 0.041⌿ (11) 0.673 ⫾ 0.030 (12) 0.587 ⫾ 0.005 (12) 0.592 ⫾ 0.012 (10)
3.278 ⫾ 0.158⌿ (11) 1.270 ⫾ 0.082 (12) 1.025 ⫾ 0.018 (12) 0.971 ⫾ 0.014 (10)
136 ⫾ 13⌿ (11) 37 ⫾ 3 (12) 44 ⫾ 5 (12) 49 ⫾ 3 (10)
Values are expressed as Mean ⫾ SE. ( ): number of rats; rw: renal weight; *: mg/dL (only sacrificed rats). ⌿ 1 vs. 2,3 and 4 (p ⬍ 0.05); ⌿⌿ 2 vs. 3 and 4.
1.7 ⫾ 0.1⌿ (11) 0.6 ⫾ 0.1 (12) 0.7 ⫾ 0.1 (12) 0.7 ⫾ 0.1 (9)
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1083
Table 6 Renal histopthology Experment I
Group 1 n Grade Group 2 n Grade Group 3 n Grade Group 4 n Grade Experiment II
Dead rats
Sacrificed Rats
NN
TN
CN
4.0 ⫾ 0
2 5.5 ⫾ 0.3⌿
4
R
NN
CN
R 4 3.8 ⫾ 0.3⌿
10 10 1
9 NN
Group I n Grade Group 2 n Grade Group 3 n Grade Group 4 n Grade
TN
TN
CN
4 4.0 ⫾ 0⌿
7 5.6 ⫾ 0.2⌿
R
12 12 10
Note: In experiment II, there was no death and every rat was sacrificed. Values are expressed as Mean ⫾ SE. n: number of rats; grade: grade of renal necrosis; NN: no necrosis; TN; tubular necrosis; CN: corticl necrosis; R: repair. ⌿ 1 vs. 2, 3 and 4 (p ⬍ 0.05).
severe fatty liver, while those in group 3 showed a very mild stetatosis in 6/10 rats and those in group 4 did not show fatty changes. In experiment II, the gross and histopathological changes in the kidneys were morphologically similar to the acute changes observed in experiment I. Rats from group 1 showed the typical lesions of tubular (Fig. 1a) or cortical necrosis (Fig. 1b). No necrosis was found in the choline-deficient rats of group 2 (Fig. 1d) or in the choline-supplemented rats of group 3 and 4. Rats from group 1 displayed a moderate or severe fatty liver, rats from group 2 a severe fatty liver, while those in group 3 showed a mild stetatosis in 6/12 rats and those in group 4 showed mild fatty changes in 1/10 rats. In order to exclude that menhaden oil exerted protective actions because of its choline content, we performed an analysis of choline-containing compounds in this oil Neither choline nor glycerophosphocholine could be detected in aqueous extracts of the oil. The content of acid-hydrolyzable choline in the lipid components of the oil (probably phosphati-
1084
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
Fig. 1. Light microcopy of renal tissue from rats fed different choline-deficient diets. a) Experiment 2, group 1: tubular necrosis (H.E. ⫻ 350); b) Experiment 2, group 1: cortical necrosis, foci of calcification (H.E. ⫻ 350); c) Experiment 1, group 1 (day 21st): repair, foci of calcification (H.E. ⫻ 350); d) Experiment 2, group 2: no necrosis (H.E. ⫻ 350).
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1085
dylcholine) was determined as 12 pmol/mL oil. This minute amount of choline in menhaden oil cannot explain the protective effects of the diet on renal necrosis.
4. Discussion Weanling rats fed a diet deficient in lipotropic factors (choline, methionine, vitamin B12, folic acid) develop acute renal failure whose morphological features vary from focal tubular necrosis to widespread cortical necrosis [2,9]. The pathogenesis of this necrosis, however, is uncertain. The possible pathogenic role of changes in the renal lipids has been repeatedly studied in this experimental model, without clear evidence of a correlation between a particular lipid change and renal histology [20 –22]. Nevertheless, it is known that in rats fed a choline-deficient diet, the quantity as well as the quality of dietary lipids, can modulate the renal lesions [23,24]. Menhaden oil is rich in eicosapentanoic (20:5) and docosahexaenoic acid (22:6) and so may influence renal fatty acid composition and arachidonic acid metabolism which play an important role in renal physiology and pathology [13]. Thus, in the present study, we investigated the influence of menhaden oil on acute renal failure induced by choline-deficiency and our results clearly demonstrate a protective effect of the oil. Choline content of the oil can be excluded being the distribution of fatty acids in this diet responsible for this effect. Changes in lipid composition of cellular membranes could alter the generation of second messengers and cell signal transduction pathways making cells more resistant to the renal deleterious effects induced by choline deficiency. Another possibility is that dietary lipid content can cause changes in tubular lipids making cells more resistant to noxe. Specifically, we believe that menhaden oil diet caused a change of fatty acid composition which led to an alteration of eicosanoid formation and so to a modification of vasodilator and vasoconstrictor eicosanoids balance in favor of vasodilatation. This suggestion is based on previous findings that the type of lipid of the diet in experiments in vivo or in the culture medium in experiments in vitro can change the lipid composition of cellular structures and, thereby, modify cellular activities such as regulation of gene expression or cellular signaling and induce a different response to cellular injury [25–32]. Dietary fish oil can modulate different types of diseases in experimental animals and human beings [33], including different renal pathologies [34]. In a previous work, Barcelli et al. [35] found that rats fed fish oil showed a decrease of 18:2 and 20:4 fatty acids and an increase in 20:5 and 22:6 fatty acids in renal tissue. Logan et al. [29] demonstrated that dietary fish oil caused a diminution of the renal concentration of TXB2 and so an alteration of the relative amount of renal cortical PGI2 and TXB2, in favor of vasodilatation. Moreover, Croft et al. [36] studied the effects of a diet supplemented with 10% of either safflower oil, hydrogenated coconut oil containing 3% safflower oil or “max EPA” fish oil. They reported a reduction of arachidonic acid in serum fatty acids and in kidney phospholipids, a decrease of serum TXB2 and of urinary 6 keto-PGF1␣ and PGE2 in those rats fed fish oil compared to those rats receiving coconut oil or safflower oil. In a study by Schmitz et al. [37], intact glomeruli of rats fed a diet enriched with -3-fatty
1086
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
acids, or renal cortical fatty acids of rats with renal ablation and fed a diet rich in fish oil, showed a pattern of fatty acids reflective of the diet they received, with a decrease in linoleate and arachidonate and an increase in eicosapentaenoic and docosahexaenoic acid with a significant decrease in the urinary excretion of vasoconstrictor TXA2; in addition, angiotensin II-stimulated phospholipid turnover was attenuated in intact glomeruli pretreated with -3 fatty acids. In a model of acute renal failure induced in rats by endotoxin [38] the authors observed a rise in renal cortical generation rates of PGE2, 6-keto PGF1␣ and TXB2. Pretreatment with TXA2 synthetase inhibitor dazoxiben, abolished the rise in TXB2, prevented the fall in renal blood flow and allowed for preservation of glomerular filtration rate. Antagonism of endogenously produced leukotrienes with a receptor antagonist, while not preventing the rise in TXB2 induced by the lipopolysaccharide, ameliorated the fall in RBF. Finally, fish oil ameliorates acute renal failure induced in dogs by 120 minutes of ischemia. The animals that received fish oil during 6 weeks showed, in relation to the controls that received vegetable oil, a better renal function with significantly different glomerular filtration rate, creatinine clearance, plasma creatinine concentration and urine volume. The urinary excretion of TXB2 was lower in dogs fed fish oil [39]. In addition to already mentioned feeding studies, there is some pharmacological evidence to support our concept. Thus, in rats pretreated with ibuprofen to prevent prostanoid synthesis, the administration of PGE2 prevented the increase in kidney weight, the rise of creatinine in serum and the development of ischemic acute tubular necrosis [40]. Moreover, the PGE1 analog, misoprostol, protect against ischemia-induced renal dysfunction in rats [41] and against renal failure due to mercuric chloride. In primary cultures of proximal tubule epithelial cells, misoprostol diminished the damage induced by hypoxia and reoxygenation as prostacyclin and PGE2 do. Fish oil is not the only lipid that has a protective action on renal pathology induced by choline deficiency. Coconut oil and myristic acid can also have such an effect [10 –12]. Is there any relationship among these type of lipids? In a previous experiment, we have found that the protective effect of coconut oil is due to its content in myristic acid [12]. It is interesting to point out that though the concentration of myristic acid in coconut oil is around 19.5% (what mean a final concentration of 3.9% in the diet used by Zaki et al. [10] and by Monserrat et al. [11]), menhaden oil has a concentration of myristic acid of around 9.0% (so the final concentration in the experiment we are reporting now is only 1.8%, w/w). Coconut oil showed a protective effect when it was employed at a 20% concentration (with or without 1% safflower oil), but not at a 14% concentration (final concentration of myristic acid 2.7%) [11]. Though it remains to be studied which is the minimum concentration of myristic acid that afford renal protection against the renal lesions induced by choline-deficiency, it is clear that myristic content of menhaden oil is not enough to explain the protection exerted by the oil in our model of acute renal failure. In summary, alterations in renal hemodynamics have been postulated as the pathogenetic mechanism occurring in the acute renal failure induced by choline-deficiency [42,43]. There is an interrelationship among different vasoactive agents such as endothelin-1, nitric oxide, catecholamines, PAF and eicosanoids, that plays an important role in renal function in physiological as well as pathological conditions [34,44 –51]. Lelcuk et al. [52] suggested that the PGI2/TXA2 ratio is the critical moderator of the renal injury. There is evidence that
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1087
prostaglandins act locally in the kidney as regulatory hormones of vasoactive agents; thus, eicosanoids could play an important role in the pathogenesis of acute renal failure through the modulation of the tone of the renal vasculature. -3 fatty acids can modulate the relationship between vasodilatory and vasoconstrictor eicosanoids. In agreement with the previously mentioned facts, the present study shows that dietary lipid composition of menhaden oil drastically modulates renal necrosis in the animal model used by us. Further experiments are now in progress to probe the pathogenic mechanism proposed.
Acknowledgments This work was partially supported by grants from the National Council of Research of Argentina and from the University of Buenos Aires, Argentina. The technical assistance of Silvia Caram de Vicario and Silvia Farin˜ a is gratefully recognized.
References [1] Best C. The lipotropic agents in the protection of the liver, kidney, heart and other organs of experimental animals. Proc Royal Soc 1956;145:151– 69. [2] Griffith WH, Wade NJ. Effects of deficiency. In: Sebrell WH, Harris RS, editors. The vitamins: chemistry, physiology and pathology. Vol. 3. New York: Academic Press, 1971. p. 81–122. [3] Zeisel SH, Blusztajn JK. Choline and human nutrition. Annual Rev Nutr 1994;14:269 –96. [4] Zeisel SH, Szuhaj BF. Choline, phospholipids, and disease. Champaign, Illinois: AOCS Press, 1998. [5] Ghoshal AK, Farber E. Liver biochemical pathology of choline deficiency and of methyl group deficiency: a new orientation and assessment. Histol Histopathol 1995;10:457– 62. [6] Shin OH, Mar MH, Allbright CD, Citarella MT, da Costa KA, Zeisel SH. Methyl-group donors cannot prevent apoptotic death of rat hepatocytes induced by choline-deficiency. J Cell Biochem 1997;64:196 –208. [7] Arienti de Garcia I, Perazzo JC, Monserrrat AJ. Necrosis cardı´aca y deficiencia de factores lipotro´ picos. Medicina (Bs Aires) 1981;41:556 – 64. [8] Wilgram GF, Hartroft WS, Best CH. Dietary choline and the maintenance of the cardiovascular system in rats. Br Med J 1954;II:1–5. [9] Montes de Oca M, Perazzo JC, Monserrat AJ, Arrizurieta de Muchnik EE. Acute renal failure induced by choline deficiency: structural-functional correlations. Nephron 1980;26:41– 8. [10] Zaki FG, Hoffbauer FW, Grande F. Prevention of renal necrosis by coconut oil in choline-deficient rats. Arch Pathol (Chicago) 1966;81:85–9. [11] Monserrat AJ, Romero M, Lago N, Aristi C. Protective effect of coconut oil on renal necrosis occurring in rats fed a methyl-deficient diet. Renal Failure 1995;17:525–37. [12] Monserrat AJ, Cutrin JC, Coll C. Protective effect of myristic acid on renal necrosis occurring in rats fed a methyl-deficient diet. Research Exp Med 2000;199:195–206. [13] Remuzzi G, Fitzgerald GA, Patrono C. Thromboxane synthesis and action within the kidney. Kidney Int 1992;41:1483–93. [14] Monserrat AJ, Musso AM, Tartas N, Nicastro M, Konopka HF, Arienti de Garcı´a I, Sa´ nchez Avalos JC. Consumption coagulopathy in acute renal failure induced by hypolipotropic diets. Nephron 1981;28:276 – 84. [15] McManus JFA, Mowry RW. Staining methods: histologic and histochemical. New York: Hoeber, 1960. [16] Wilson W. Trichrome method for staining fat with oil red O in frozen sections. Bull Assoc Med Mus 1959;31:216 –20.
1088
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
[17] The Renal Commission of the International Union of Physiological Sciences. A standard nomenclature for structures of the kidney. Kidney Int 1988;33:1–7. [18] Klein J, Gonzalez R, Ko¨ ppen A, Lo¨ ffelholz K. Free choline, and choline metabolites in rat brain, and body fluids: sensitive determination, and implications for choline supply to the brain. Neurochem Int 1993;22: 293–300. [19] Systat 5.02 for Windows SYSTAT, Inc. Illinois, 1990. [20] Fewster ME, Hall MO. The renal phospholipid composition of choline-deficient rats. Lipids 1967;2:239 – 43. [21] Monserrat AJ, Porta EA, Ghoshal AK, Hartman SB. Sequential renal lipid changes in weanling rats fed a choline-deficient diet. J Nutr 1974;104:1496 –502. [22] Simon JB, Scheig R, Klatskin G. Relationship of early lipid changes in kidney and liver to the hemorrhagic renal necrosis of choline-deficient rats. Lab Invest 1968;19:503–9. [23] Griffith WH. Choline metabolism. III. The effect of cystine, fat and cholesterol on hemorrhagic degeneration in young rats. J Biol Chem 1940;132:639 – 44. [24] O’Neal RM, Still WJS, Hartroft WS. Increased lipotropic requirements with renal necrosis induced in rats by high-fat diets. J Nutr 1961;75:309 –18. [25] Alkhunazi AM, Yaqoob MM, Edelstein CL, Gengaro PE, Burke TJ, Nemenoff RA, Schrier RW. Araquidonic acid protects against hypoxic injury in rat proximal tubules. Kidney Int 1996;49:620 –5. [26] Bruckner G. Fatty acids, lipids and cellular signaling. In: Chow CK, editor. Fatty acids in foods and their health implications. New York: M Decker, 2000. p. 607–18. [27] Chapkin RS, Coble KJ. Remodeling of mouse kidney phospholipid classes and subclasses by diet. J Nutr Biochem 1991;2:158 – 64. [28] Iqbal M, Dingle JT, Moore T. Nutrition and lysosomal activity. The effect of dietary cod-liver oil on the distribution of polyunsaturated fatty acids in the kidney lysosomes of rats receiving deficient or adequate intakes of vitamin E. Br J Nutr 1969;23:31–9. [29] Logan JL, Michael UF, Benson B. Dietary fish oil interferes with renal arachidonic acid metabolism in rats: correlations with renal physiology. Metabolism 1992;41:382– 89. [30] Murphy MG. Dietary fatty acids and membrane protein function. J Nutr Biochem 1990:168 –79. [31] Sessler AM, Ntambi JK. Polyunsaturated fatty acid regulation of gene expression. J Nutr 1998;128:923– 6. [32] Sheridan AM, Fitzpatrick S, Wang C, Wheeler DC, Lieberthal W. Lipid peroxidation contributes to hydrogen peroxide induced cytotoxicity in renal epithelial cells. Kidney Int 1996;49:88 –93. [33] Nettleton JA. Omega-3 fatty acids, and health. New York: Chapman & Hall, 1995. [34] De Caterina R, Endres S, Kristensen SD, Schmidt EB. n-3 fatty acids, and renal diseases. Am J Kidney Dis 1994;24:397– 415. [35] Barcelli UO, Beach DC, Pollak VE. The influence of n-6 and n-3 fatty acids on kidney phospholipid composition and on eicosanoids production in aging rats. Lipids 1988;23:309 –12. [36] Croft KD, Codde JP, Barden A, Vandongen R, Beilin LJ. Onset of changes in phospholipid fatty acid composition and prostaglandin synthesis following dietary manipulation with n-6 and n-3 fatt acids in the rat. Biochem Biophys Acta 1985;834:316 –23. [37] Schmitz PG, Lane PL, Dalal R, Zhang K, Majidi BJ. -3 fatty acids attenuate glomerular capillary hydraulic pressure in rats with renal ablation. Kidney Int 1995;48:1792– 800. [38] Badr KF, Kelley VE, Rennke HG, Brenner BM. Roles for thromboxane A2 and leukotrienes in endotoxin induced acute renal failure. Kidney Int 1986;30:474 – 80. [39] Neumayer HH, Heinrich M, Schmissas M, Haller H, Wagner K, Luft FC. Amelioration of ischemic acute renal failure by dietary fish oil administration in conscious dogs. J Am Soc Nephrol 1992;3:1312–20. [40] Kaufman RP, Kobzik L, Shepro D, Anner H, Valeri CR, Hechtman HB. Vasodilator prostaglandins (PG) prevent renal damage after ischemia. Ann Surg 1987;205:195–98. [41] Paller MS, Manivel JC, Patten M, Barry M. Prostaglandins protect kidneys against ischemic and toxic injury by a cellular effect. Kidney Int 1992;42:1345–54. [42] Nagler AL, Dettbarn WD, Seifter E, Levenson SM. Tissue levels of acetylcholine and acetylcholinesterase in weaning rats subjected to acute choline deficiency. J Nutr 1968;94:13–19.
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1089
[43] Costa RS, Rossi MA, Oliveira JSM. Pathogenesis of the renal injury in choline deficiency: the role of catocholamines and acetylcholine. Br J Exp Path 1979;60:613–9. [44] Achard JM, de Cagny B, Pruna A, Fournier A. Pathophysiology of acute tubular necrosis. In Cantarovich F, Rangoonwala B, Verho M, editors. In: Progress in acute renal failure. New Jersey: Hoechst Marion Roussel, 1998. p. 23– 45. [45] Lin H, Smith MJ, Young DB. Roles of prostaglandins and nitric oxide in the effect of endothelin-1 on renal hemodynamics. Hypertension 1996;28:372–78. [46] Lo´ pez-Novoa JM. Potential role of platelet activating factor in acute renal failure. Kidney Int 1999;55: 1672– 82. [47] Maddox DA, Brenner BM. Glomerular ultrafiltration. In: Brrenner, Rector, editors. The kidney. 6th. ed. Philadelphia: WB Saunders, 2000. p. 319 –74. [48] Ratcliffe P. The pathopysiology of acute renal failure. In: Davison AM, Cameron JS, Gru¨ nfeld JP, Kerr DNS, Ritz E, Winearls C, editors. Oxford textbook of clinical nephrology. Oxford: Oxford University Press, 1998. p. 1531–56. [49] Robinette JB, Conger JD. Angiotensin and thromboxane in the enhanced renal adrenergic nerve sensitivity of acute renal failure. J Clin Invest 1990;86:1532–39. [50] Schramek H, Coroneos E, Dunn MJ. Interactions of the vasoconstrictor peptides, angiotensin II and endothelin-1, with vasodilatory prostaglandins. Seminars in Nephrology 1995;15:195–204. [51] Wilcox CS, Welch WJ, Snellen H. Thromboxane mediates renal hemodynamic response to infused angiotensin II. Kidney Int 1991;40:1090 –97. [52] Lelcuk S, Alexander F, Kobzik L, Valeri CR, Shepro D, Hechtman HB. Prostacyclin and thromboxane A2 moderate postischemic renal failure. Surgery 1985;98:207–12.
Protective effect of menhaden oil on renal necrosis occurring in weanling rats fed a methyl-deficient diet Marı´a C. Courre`gesa, Carla Carusoa, Jochen Kleinb, Alberto J. Monserrata,* a
Patologı´a Experimental, Departamento de Patologı´a, Facultad de Medicina, Universidad de Buenos Aires, J. E. Uriburu 950, 5° piso (1114) Buenos Aires, Argentina b Pharmakologisches Institut, Universitat Mainz, Obere Zahlbacher Str. 67 (D-55101), Mainz, Germany Received 26 July 2001; received in revised form 16 May 2002; accepted 18 May 2002
Abstract Weanling rats fed a lipotropic-deficient diet (LDD) may develop acute renal failure with morphological features that vary from focal tubular necrosis to widespread cortical necrosis and eventually reparative changes. The type of lipid in the diet influences the development of renal necrosis. The aim of this work was to study the effect of dietary menhaden oil on the development of acute renal failure induced in weanling rats fed a methyl-deficient diet. Experiment I: 40 weanling Sprague-Dawley male rats were allotted to 4 different groups and fed as follows: group 1, LDD with hydrogenated vegetable oil and corn oil as lipids; group 2, LDD with menhaden oil as lipid; group 3 and 4, similar to groups 1 and 2 plus choline. Rats were fed ad libitum until they died; surviving animals were killed on day 21. Mortality in the 4 groups was 60, 0, 0, and 10% respectively. Rats from groups 2, 3 and 4 did not show renal damage. Rats from group 1 showed tubular or cortical necrosis in dead rats and reparative changes in those killed on the 21st day. Experiment II was similar to experiment I, except that 45 weanling Wistar male rats were used and they were killed on the 7th day. All rats from group 1 showed renal necrosis; no renal damage was found in rats from groups 2, 3 and 4. Urea and creatinine alterations corroborated the renal changes. We conclude that menhaden oil displays a protective effect for renal necrosis induced by methyl-deficient diets in weanling rats. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Acute renal failure; Choline deficiency; Lipotropic factors deficiency; Methyl group deficiency; Menhaden oil
* Corresponding author. Tel.: ⫹54-11-4508-3602; fax: ⫹54-11-4508-3602. E-mail address: [email protected] (A.J. Monserrat). 0271-5317/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII: S 0 2 7 1 - 5 3 1 7 ( 0 2 ) 0 0 4 1 5 - 3
1078
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1. Introduction The important role of methyl metabolism in experimental animals as well as in human beings is widely and increasingly recognized. Nutrients involved in this metabolism are also known as lipotropic factors and are supplied in the diet mainly as methionine and choline, though folate and B12 are necessary for normal metabolism. Choline contributes to the structural integrity of cell membranes and signaling functions and it is involved in cholinergic neurotransmission, lipid transport and metabolism [1– 4]. Rats fed a diet deficient in lipotropic factors may develop liver pathology (steatosis, cellular death, cirrhosis and cancer) [1,2,5,6], heart changes (stetatosis and necrosis) [7,8] and renal pathology. In particular, weanling rats fed a lipotropic-deficient diet may develop acute renal failure with morphological features that vary from focal tubular necrosis to widespread cortical necrosis or eventual presence of reparative changes [2,9]. The type of lipid in the diet influences the development of the renal necrosis. For instance, coconut oil (rich in saturated fatty acids) has a protective effect [8,10,11], which would be due to its content of myristic acid [10,12]. Menhaden oil is rich in eicosapentanoic (20:5) and docosahexaenoic acid (22:6) and so may influence renal fatty acid composition and arachidonic acid metabolism which play an important role in renal physiology and pathology [13]. The purpose of this study, was to explore the effect of menhaden oil in this experimental model of acute renal failure. This could be important to a better knowledge of pathogenic mechanisms involved in renal tubular and cortical necrosis.
2. Materials and methods 2.1. Animals Forty Sprague-Dawley weanling male rats from the Veterinary School of the University of Buenos Aires (experiment I) and forty Wistar weanling male rats from the Department of Pathology of the School of Medicine of the University of Buenos Aires (experiment II) were employed. All animals had free access to one of four diets described below and drinking water. They were individually housed in suspended wire-bottomed cages in an air conditioned room and were exposed to light from 7.00 a.m. to 7.00 p.m. All conditions and handling of animals followed NIH Guidelines for the Care and Use of Laboratory Animals. Body weight and food intake were measured daily. 2.2. Diets Rats were given free access to semi-purified diets in powdered form as indicated in experimental design. The diets met the National Research Council Nutrition Requirements and varied in lipid composition and choline chloride content. The composition of each diet is described in Table 1.
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1079
Table 1 Composition of the diets (g/100 g) Diet composition a
Soy bean protein Sucrose Celluloseb Vitamin mixture (without B12 and choline)c Salts (W)d L-cystinee Hydrogenated vegetable oilf Corn oilg Menhaden oile
Group 1 and 3
2 and 4
20 48.5 4 4 2 0.5 14.3 5.7 0
20 48.5 4 4 2 0.5 0 0 20
Note. Diets 3 and 4 were supplemented with 0.35% of choline chloride. a Soybean protein grade II. U.S. Biochemical corp., USA. b Celufil. Non-nutritive Bulk, U.S. Biochemical Corp., USA. c Vitamin Diet Fortification Mixture. U.S. Biochemical Corp., USA. d Salt Mixture Wesson Modification, U.S. Biochemical Corp., USA. e U.S. Biochemical Corp., USA. f Flora Da´ nica, Buenos Aires, Argentina. g Mazola. Refinerı´as de Maı´z, Buenos Aires, Argentina.
2.3. Experimental design Experiment I: Forty Sprague-Dawley weanling male rats were distributed in 4 different groups. The rats from groups 1 and 2 were fed ad libitum the lipotropic-deficient diets shown in Table 1, while those in groups 3 and 4 were used as controls of the lipotropic-deficient animals and were fed the same diets of groups 1 and 2 respectively and supplemented with 0.35% choline chloride. The different lipids were given at a 20% concentration, the same concentration we have used in previous experiments evaluating the effects of coconut oil [11] and different fatty acids [12] in this experimental model. Groups 1 and 3 received a mixture of corn oil and hydrogenated vegetable oil to supply different fatty acids [9,11,12,14]. The rats were kept on the diets until they died or, if death did not occur, they were killed on the 21st day. Under light ether anesthesia, blood was drawn from the abdominal aorta and urea was determined in serum by standard procedures. The kidneys and liver were removed and weighed. Tissue samples were fixed in buffered formalin, embedded in paraffin, and sections were stained with hematoxylin-eosin and, in some animals, with PAS or Masson’s trichrome [15]. Frozen sections of the liver were stained with oil red O [16]. Fatty changes in the liver were evaluated in frozen sections stained with oil red O. Changes were classified as mild, moderate or severe according to the extent (from zonal to diffuse) of the fat deposition in the liver lobule. For topographical localization of different segments of the kidney, well known terminology was used [17]. A previous proposed classification was applied to evaluate the renal pathology [14]. (Table 2). Experiment II: Forty five Wistar male rats were distributed in 4 different groups, fed and
1080
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
Table 2 Histologic classificaiton of renal necrosis A B
C
D
Kidney without necrosis Acute tubular necrosis Grade 1: Isolated foci of cellular necrosis in some tubules Grade 2: Small groups of tubules with necrosis Grade 3: Zones of tubular necrosis Grade 4: Confluent zones of tubular necrosis Cortical necrosis Grade 5: Grade 4 plus isolated foci of cortical necrosis Grade 6: Grade 4 plus multiple foci of cortical necrosis Grade 7: Grade 4 plus confluent foci of cortical necrosis Grade 8: Massive cortical necrosis Repair: Repair is characterized by different degrees of interstitial fibrosis, tubular atrophy, tubular regeneration, glomerular fibrosis, etc. According to its extent it is divided into 4 grades.
kept as those rats in Experiment I. The rats were kept on the diets until day 7th. Blood was obtained as in Experiment I and in addition to urea, creatinine was also evaluated. The kidneys and liver were removed, weighed and studied as in Experiment I. 2.4. Determination of choline and choline metabolites The choline content of menhaden oil was determined essentially as previously described [18]. Briefly, menhaden oil was extracted with water. Aliquots of the water phase were evaporated to dryness, taken up in buffer and analyzed for content of free choline by high pressure liquid chromatography (HPLC). In addition, aliquots of the water phase were hydrolyzed by 6 N HCl (1 h, 80°C), a procedure which causes release of choline from glycerophosphocholine [18]. The oil phase was hydrolyzed with 6 N methanolic HCl (1 h, 80°C) to cleave phosphatidylcholine to choline. Subsequently, the methanolic phase was taken to dryness. The HPLC system for the analysis of choline consisted of a low-speed pump (BAS PM80), separation column (SepStik, 530 ⫻ 1 mm), enzyme reactor (50 ⫻ 1 mm) carrying immobilized choline oxidase and electrochemical detector (BAS LC-4C) with a platinum electrode operating at 0.5 V. The flow rate was 120 l/min. At an injection volume of 5 l, the detection limit of this system was 25 fmol/injection. 2.5. Statistical methods The Fisher’s exact test was employed to statistically evaluate mortality and the incidence of histopathologic renal lesions. Renal and body weights and length of survival as well as creatinine and urea in serum were expressed as mean ⫾ standard error and comparisons were made by two-way analysis of variance (ANOVA). In the case of significant differences ANOVA was followed by the Tukey-Kramer test [19]. Differences which resulted in p values lower than 0.05 were considered significant (S).
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1081
Table 3 Initial, fifth day and final body weights Body weight Initial
Experiment I Group 1 Group 2 Group 3 Group 4 Experiment II Group 1 Group 2 Group 3 Group 4
41.9 ⫾ 1.0* (10) 42.3 ⫾ 1.1 (10) 42.2 ⫾ 1.0 (10) 42.2 ⫾ 0.8 (10)
Final
5th day
Dead
Sacrificed
49.3 ⫾ 2.1 (6)
98.3 ⫾ 4.0⌿ (4) 96.0 ⫾ 3.6⌿ (10) 128.5 ⫾ 4.3 (10) 116.7 ⫾ 3.9 (9)
57.3 ⫾ 0.7 (10) 55.5 ⫾ 3.8 (10) 59.9 ⫾ 4.2 (10) 54.7 ⫾ 1.4 (9)
47.5 ⫾ 2.4⌿⌿ (11) 54.1 ⫾ 1.8 (12) 57.6 ⫾ 1.4 (12) 61.0 ⫾ 1.2 (10)
51.0 ⫾ 1.9 (11) 48.4 ⫾ 2.0 (12) 51.6 ⫾ 1.4 (12) 53.2 ⫾ 0.9 (10)
36.2 (1)
39.2 ⫾ 1.9 (11) 39.1 ⫾ 1.7 (12) 38.6 ⫾ 2.7 (12) 41.7 ⫾ 0.5 (10)
Values are expressed as Mean ⫾ SE. *: g ( ): number of rats. ⌿ 1 and 2 vs. 3 and 4 (p ⬍ 0.05); ⌿⌿ 1 vs. 2, 3 and 4 (p ⬍ 0.05).
3. Results The initial, final and fifth day body weights are shown in Table 3. As indicated by fifth day body weights, rats grew adequately well during the first days in both experiments. Later on, as a consequence of renal damage, rats belonging to group 1 lost weight (for example, see dead animals in group 1, experiment I). No differences between body weights of group 1 and group 2 were observed on the day of sacrifice. In experiment II, we obtained similar results, though in this case, we found significant differences between body weights of group 1 and 2 on the day of sacrifice. Mortality and length of survival in experiment I are shown in Table 4. As expected, in the choline-deficient rats mortality was high (6/10) in rats of group 1. No mortality was observed in choline-deficient rats receiving menhaden oil (group 2) and control groups 3 and 4, except one rat in group 4 that did not show renal damage in the histologic study. Length of survival was 9 days in rats of group 1. Renal weights, urea and creatinine values of both experiments are shown in Table 5. Renal weights were higher in choline deficient rats not supplemented with menhaden oil (group 1). Urea level in the serum was within normal ranges in choline-supplemented rats, while it was slightly elevated in those rats of group 2 (only in experiment I) and markedly increased in those of group 1. The acute renal changes were grossly characterized by an increase in size and weight and
1082
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
Table 4 Mortality and survival time Group
Total ratsn
Died ratsn
1 2 3 4
10 10 10 10
6 0 0 1
Length of survival 9.2 ⫾ 0.2 13.0
Values are expressed as Mean ⫾ SE. n : number of rats, *: Days.
by a purplish red discoloration. Necrosis involved mainly proximal convoluted tubules and was characterized by increased eosinophilia, changes in tinctorial affinities with Masson’s thricrome, pyknosis and mainly kariolysis; in more advanced stages glomerular and vascular necrosis were also observed. Concomitant with tubular necrosis hyalin casts were found. Repair was characterized by the progressive disappearance of necrotic tubules and casts and by different degrees of regeneration, tubular atrophy and fibrosis. The results of the histological study are shown in Table 6. In experiment I, dead rats of group 1 showed the typical renal lesions of tubular or cortical necrosis found in choline-deficiency. Rats sacrificed at the end of the experiment showed typical reparative changes in rats of group 1 (Fig. 1c), while no changes were observed in choline-deficient rats of group 2. As expected, choline-supplemented rats did not show renal damage. Rats from group 1 displayed a moderate or severe fatty liver, rats from group 2 a Table 5 Renal weights, urea and creatinine Dead rw Experiment I Group 1 Group 2 Group 3 Group 4 Experiment II Group 1 Group 2 Group 3 Group 4
Sacrificed rw/100 g bw
rw
Urea*
Creatinine*
rw/100 g bw
1.981 ⫾ 0.156 4.004 ⫾ 0.232 1.910 ⫾ 0.088⌿ (6) (6) (4) 1.344 ⫾ 0.059 (10) 1.555 ⫾ 0.050 (10) 0.598 1.857 1.550 ⫾ 0.059 (1) (1) (9)
1.957 ⫾ 0.145⌿ (4) 1.400 ⫾ 0.029 (10) 1.214 ⫾ 0.031 (10) 1.329 ⫾ 0.021 (9)
99 ⫾ 11⌿ (4) 35 ⫾ 5⌿⌿ (10) 18 ⫾ 4 (10) 26 ⫾ 2 (9)
1.525 ⫾ 0.041⌿ (11) 0.673 ⫾ 0.030 (12) 0.587 ⫾ 0.005 (12) 0.592 ⫾ 0.012 (10)
3.278 ⫾ 0.158⌿ (11) 1.270 ⫾ 0.082 (12) 1.025 ⫾ 0.018 (12) 0.971 ⫾ 0.014 (10)
136 ⫾ 13⌿ (11) 37 ⫾ 3 (12) 44 ⫾ 5 (12) 49 ⫾ 3 (10)
Values are expressed as Mean ⫾ SE. ( ): number of rats; rw: renal weight; *: mg/dL (only sacrificed rats). ⌿ 1 vs. 2,3 and 4 (p ⬍ 0.05); ⌿⌿ 2 vs. 3 and 4.
1.7 ⫾ 0.1⌿ (11) 0.6 ⫾ 0.1 (12) 0.7 ⫾ 0.1 (12) 0.7 ⫾ 0.1 (9)
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1083
Table 6 Renal histopthology Experment I
Group 1 n Grade Group 2 n Grade Group 3 n Grade Group 4 n Grade Experiment II
Dead rats
Sacrificed Rats
NN
TN
CN
4.0 ⫾ 0
2 5.5 ⫾ 0.3⌿
4
R
NN
CN
R 4 3.8 ⫾ 0.3⌿
10 10 1
9 NN
Group I n Grade Group 2 n Grade Group 3 n Grade Group 4 n Grade
TN
TN
CN
4 4.0 ⫾ 0⌿
7 5.6 ⫾ 0.2⌿
R
12 12 10
Note: In experiment II, there was no death and every rat was sacrificed. Values are expressed as Mean ⫾ SE. n: number of rats; grade: grade of renal necrosis; NN: no necrosis; TN; tubular necrosis; CN: corticl necrosis; R: repair. ⌿ 1 vs. 2, 3 and 4 (p ⬍ 0.05).
severe fatty liver, while those in group 3 showed a very mild stetatosis in 6/10 rats and those in group 4 did not show fatty changes. In experiment II, the gross and histopathological changes in the kidneys were morphologically similar to the acute changes observed in experiment I. Rats from group 1 showed the typical lesions of tubular (Fig. 1a) or cortical necrosis (Fig. 1b). No necrosis was found in the choline-deficient rats of group 2 (Fig. 1d) or in the choline-supplemented rats of group 3 and 4. Rats from group 1 displayed a moderate or severe fatty liver, rats from group 2 a severe fatty liver, while those in group 3 showed a mild stetatosis in 6/12 rats and those in group 4 showed mild fatty changes in 1/10 rats. In order to exclude that menhaden oil exerted protective actions because of its choline content, we performed an analysis of choline-containing compounds in this oil Neither choline nor glycerophosphocholine could be detected in aqueous extracts of the oil. The content of acid-hydrolyzable choline in the lipid components of the oil (probably phosphati-
1084
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
Fig. 1. Light microcopy of renal tissue from rats fed different choline-deficient diets. a) Experiment 2, group 1: tubular necrosis (H.E. ⫻ 350); b) Experiment 2, group 1: cortical necrosis, foci of calcification (H.E. ⫻ 350); c) Experiment 1, group 1 (day 21st): repair, foci of calcification (H.E. ⫻ 350); d) Experiment 2, group 2: no necrosis (H.E. ⫻ 350).
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1085
dylcholine) was determined as 12 pmol/mL oil. This minute amount of choline in menhaden oil cannot explain the protective effects of the diet on renal necrosis.
4. Discussion Weanling rats fed a diet deficient in lipotropic factors (choline, methionine, vitamin B12, folic acid) develop acute renal failure whose morphological features vary from focal tubular necrosis to widespread cortical necrosis [2,9]. The pathogenesis of this necrosis, however, is uncertain. The possible pathogenic role of changes in the renal lipids has been repeatedly studied in this experimental model, without clear evidence of a correlation between a particular lipid change and renal histology [20 –22]. Nevertheless, it is known that in rats fed a choline-deficient diet, the quantity as well as the quality of dietary lipids, can modulate the renal lesions [23,24]. Menhaden oil is rich in eicosapentanoic (20:5) and docosahexaenoic acid (22:6) and so may influence renal fatty acid composition and arachidonic acid metabolism which play an important role in renal physiology and pathology [13]. Thus, in the present study, we investigated the influence of menhaden oil on acute renal failure induced by choline-deficiency and our results clearly demonstrate a protective effect of the oil. Choline content of the oil can be excluded being the distribution of fatty acids in this diet responsible for this effect. Changes in lipid composition of cellular membranes could alter the generation of second messengers and cell signal transduction pathways making cells more resistant to the renal deleterious effects induced by choline deficiency. Another possibility is that dietary lipid content can cause changes in tubular lipids making cells more resistant to noxe. Specifically, we believe that menhaden oil diet caused a change of fatty acid composition which led to an alteration of eicosanoid formation and so to a modification of vasodilator and vasoconstrictor eicosanoids balance in favor of vasodilatation. This suggestion is based on previous findings that the type of lipid of the diet in experiments in vivo or in the culture medium in experiments in vitro can change the lipid composition of cellular structures and, thereby, modify cellular activities such as regulation of gene expression or cellular signaling and induce a different response to cellular injury [25–32]. Dietary fish oil can modulate different types of diseases in experimental animals and human beings [33], including different renal pathologies [34]. In a previous work, Barcelli et al. [35] found that rats fed fish oil showed a decrease of 18:2 and 20:4 fatty acids and an increase in 20:5 and 22:6 fatty acids in renal tissue. Logan et al. [29] demonstrated that dietary fish oil caused a diminution of the renal concentration of TXB2 and so an alteration of the relative amount of renal cortical PGI2 and TXB2, in favor of vasodilatation. Moreover, Croft et al. [36] studied the effects of a diet supplemented with 10% of either safflower oil, hydrogenated coconut oil containing 3% safflower oil or “max EPA” fish oil. They reported a reduction of arachidonic acid in serum fatty acids and in kidney phospholipids, a decrease of serum TXB2 and of urinary 6 keto-PGF1␣ and PGE2 in those rats fed fish oil compared to those rats receiving coconut oil or safflower oil. In a study by Schmitz et al. [37], intact glomeruli of rats fed a diet enriched with -3-fatty
1086
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
acids, or renal cortical fatty acids of rats with renal ablation and fed a diet rich in fish oil, showed a pattern of fatty acids reflective of the diet they received, with a decrease in linoleate and arachidonate and an increase in eicosapentaenoic and docosahexaenoic acid with a significant decrease in the urinary excretion of vasoconstrictor TXA2; in addition, angiotensin II-stimulated phospholipid turnover was attenuated in intact glomeruli pretreated with -3 fatty acids. In a model of acute renal failure induced in rats by endotoxin [38] the authors observed a rise in renal cortical generation rates of PGE2, 6-keto PGF1␣ and TXB2. Pretreatment with TXA2 synthetase inhibitor dazoxiben, abolished the rise in TXB2, prevented the fall in renal blood flow and allowed for preservation of glomerular filtration rate. Antagonism of endogenously produced leukotrienes with a receptor antagonist, while not preventing the rise in TXB2 induced by the lipopolysaccharide, ameliorated the fall in RBF. Finally, fish oil ameliorates acute renal failure induced in dogs by 120 minutes of ischemia. The animals that received fish oil during 6 weeks showed, in relation to the controls that received vegetable oil, a better renal function with significantly different glomerular filtration rate, creatinine clearance, plasma creatinine concentration and urine volume. The urinary excretion of TXB2 was lower in dogs fed fish oil [39]. In addition to already mentioned feeding studies, there is some pharmacological evidence to support our concept. Thus, in rats pretreated with ibuprofen to prevent prostanoid synthesis, the administration of PGE2 prevented the increase in kidney weight, the rise of creatinine in serum and the development of ischemic acute tubular necrosis [40]. Moreover, the PGE1 analog, misoprostol, protect against ischemia-induced renal dysfunction in rats [41] and against renal failure due to mercuric chloride. In primary cultures of proximal tubule epithelial cells, misoprostol diminished the damage induced by hypoxia and reoxygenation as prostacyclin and PGE2 do. Fish oil is not the only lipid that has a protective action on renal pathology induced by choline deficiency. Coconut oil and myristic acid can also have such an effect [10 –12]. Is there any relationship among these type of lipids? In a previous experiment, we have found that the protective effect of coconut oil is due to its content in myristic acid [12]. It is interesting to point out that though the concentration of myristic acid in coconut oil is around 19.5% (what mean a final concentration of 3.9% in the diet used by Zaki et al. [10] and by Monserrat et al. [11]), menhaden oil has a concentration of myristic acid of around 9.0% (so the final concentration in the experiment we are reporting now is only 1.8%, w/w). Coconut oil showed a protective effect when it was employed at a 20% concentration (with or without 1% safflower oil), but not at a 14% concentration (final concentration of myristic acid 2.7%) [11]. Though it remains to be studied which is the minimum concentration of myristic acid that afford renal protection against the renal lesions induced by choline-deficiency, it is clear that myristic content of menhaden oil is not enough to explain the protection exerted by the oil in our model of acute renal failure. In summary, alterations in renal hemodynamics have been postulated as the pathogenetic mechanism occurring in the acute renal failure induced by choline-deficiency [42,43]. There is an interrelationship among different vasoactive agents such as endothelin-1, nitric oxide, catecholamines, PAF and eicosanoids, that plays an important role in renal function in physiological as well as pathological conditions [34,44 –51]. Lelcuk et al. [52] suggested that the PGI2/TXA2 ratio is the critical moderator of the renal injury. There is evidence that
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1087
prostaglandins act locally in the kidney as regulatory hormones of vasoactive agents; thus, eicosanoids could play an important role in the pathogenesis of acute renal failure through the modulation of the tone of the renal vasculature. -3 fatty acids can modulate the relationship between vasodilatory and vasoconstrictor eicosanoids. In agreement with the previously mentioned facts, the present study shows that dietary lipid composition of menhaden oil drastically modulates renal necrosis in the animal model used by us. Further experiments are now in progress to probe the pathogenic mechanism proposed.
Acknowledgments This work was partially supported by grants from the National Council of Research of Argentina and from the University of Buenos Aires, Argentina. The technical assistance of Silvia Caram de Vicario and Silvia Farin˜ a is gratefully recognized.
References [1] Best C. The lipotropic agents in the protection of the liver, kidney, heart and other organs of experimental animals. Proc Royal Soc 1956;145:151– 69. [2] Griffith WH, Wade NJ. Effects of deficiency. In: Sebrell WH, Harris RS, editors. The vitamins: chemistry, physiology and pathology. Vol. 3. New York: Academic Press, 1971. p. 81–122. [3] Zeisel SH, Blusztajn JK. Choline and human nutrition. Annual Rev Nutr 1994;14:269 –96. [4] Zeisel SH, Szuhaj BF. Choline, phospholipids, and disease. Champaign, Illinois: AOCS Press, 1998. [5] Ghoshal AK, Farber E. Liver biochemical pathology of choline deficiency and of methyl group deficiency: a new orientation and assessment. Histol Histopathol 1995;10:457– 62. [6] Shin OH, Mar MH, Allbright CD, Citarella MT, da Costa KA, Zeisel SH. Methyl-group donors cannot prevent apoptotic death of rat hepatocytes induced by choline-deficiency. J Cell Biochem 1997;64:196 –208. [7] Arienti de Garcia I, Perazzo JC, Monserrrat AJ. Necrosis cardı´aca y deficiencia de factores lipotro´ picos. Medicina (Bs Aires) 1981;41:556 – 64. [8] Wilgram GF, Hartroft WS, Best CH. Dietary choline and the maintenance of the cardiovascular system in rats. Br Med J 1954;II:1–5. [9] Montes de Oca M, Perazzo JC, Monserrat AJ, Arrizurieta de Muchnik EE. Acute renal failure induced by choline deficiency: structural-functional correlations. Nephron 1980;26:41– 8. [10] Zaki FG, Hoffbauer FW, Grande F. Prevention of renal necrosis by coconut oil in choline-deficient rats. Arch Pathol (Chicago) 1966;81:85–9. [11] Monserrat AJ, Romero M, Lago N, Aristi C. Protective effect of coconut oil on renal necrosis occurring in rats fed a methyl-deficient diet. Renal Failure 1995;17:525–37. [12] Monserrat AJ, Cutrin JC, Coll C. Protective effect of myristic acid on renal necrosis occurring in rats fed a methyl-deficient diet. Research Exp Med 2000;199:195–206. [13] Remuzzi G, Fitzgerald GA, Patrono C. Thromboxane synthesis and action within the kidney. Kidney Int 1992;41:1483–93. [14] Monserrat AJ, Musso AM, Tartas N, Nicastro M, Konopka HF, Arienti de Garcı´a I, Sa´ nchez Avalos JC. Consumption coagulopathy in acute renal failure induced by hypolipotropic diets. Nephron 1981;28:276 – 84. [15] McManus JFA, Mowry RW. Staining methods: histologic and histochemical. New York: Hoeber, 1960. [16] Wilson W. Trichrome method for staining fat with oil red O in frozen sections. Bull Assoc Med Mus 1959;31:216 –20.
1088
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
[17] The Renal Commission of the International Union of Physiological Sciences. A standard nomenclature for structures of the kidney. Kidney Int 1988;33:1–7. [18] Klein J, Gonzalez R, Ko¨ ppen A, Lo¨ ffelholz K. Free choline, and choline metabolites in rat brain, and body fluids: sensitive determination, and implications for choline supply to the brain. Neurochem Int 1993;22: 293–300. [19] Systat 5.02 for Windows SYSTAT, Inc. Illinois, 1990. [20] Fewster ME, Hall MO. The renal phospholipid composition of choline-deficient rats. Lipids 1967;2:239 – 43. [21] Monserrat AJ, Porta EA, Ghoshal AK, Hartman SB. Sequential renal lipid changes in weanling rats fed a choline-deficient diet. J Nutr 1974;104:1496 –502. [22] Simon JB, Scheig R, Klatskin G. Relationship of early lipid changes in kidney and liver to the hemorrhagic renal necrosis of choline-deficient rats. Lab Invest 1968;19:503–9. [23] Griffith WH. Choline metabolism. III. The effect of cystine, fat and cholesterol on hemorrhagic degeneration in young rats. J Biol Chem 1940;132:639 – 44. [24] O’Neal RM, Still WJS, Hartroft WS. Increased lipotropic requirements with renal necrosis induced in rats by high-fat diets. J Nutr 1961;75:309 –18. [25] Alkhunazi AM, Yaqoob MM, Edelstein CL, Gengaro PE, Burke TJ, Nemenoff RA, Schrier RW. Araquidonic acid protects against hypoxic injury in rat proximal tubules. Kidney Int 1996;49:620 –5. [26] Bruckner G. Fatty acids, lipids and cellular signaling. In: Chow CK, editor. Fatty acids in foods and their health implications. New York: M Decker, 2000. p. 607–18. [27] Chapkin RS, Coble KJ. Remodeling of mouse kidney phospholipid classes and subclasses by diet. J Nutr Biochem 1991;2:158 – 64. [28] Iqbal M, Dingle JT, Moore T. Nutrition and lysosomal activity. The effect of dietary cod-liver oil on the distribution of polyunsaturated fatty acids in the kidney lysosomes of rats receiving deficient or adequate intakes of vitamin E. Br J Nutr 1969;23:31–9. [29] Logan JL, Michael UF, Benson B. Dietary fish oil interferes with renal arachidonic acid metabolism in rats: correlations with renal physiology. Metabolism 1992;41:382– 89. [30] Murphy MG. Dietary fatty acids and membrane protein function. J Nutr Biochem 1990:168 –79. [31] Sessler AM, Ntambi JK. Polyunsaturated fatty acid regulation of gene expression. J Nutr 1998;128:923– 6. [32] Sheridan AM, Fitzpatrick S, Wang C, Wheeler DC, Lieberthal W. Lipid peroxidation contributes to hydrogen peroxide induced cytotoxicity in renal epithelial cells. Kidney Int 1996;49:88 –93. [33] Nettleton JA. Omega-3 fatty acids, and health. New York: Chapman & Hall, 1995. [34] De Caterina R, Endres S, Kristensen SD, Schmidt EB. n-3 fatty acids, and renal diseases. Am J Kidney Dis 1994;24:397– 415. [35] Barcelli UO, Beach DC, Pollak VE. The influence of n-6 and n-3 fatty acids on kidney phospholipid composition and on eicosanoids production in aging rats. Lipids 1988;23:309 –12. [36] Croft KD, Codde JP, Barden A, Vandongen R, Beilin LJ. Onset of changes in phospholipid fatty acid composition and prostaglandin synthesis following dietary manipulation with n-6 and n-3 fatt acids in the rat. Biochem Biophys Acta 1985;834:316 –23. [37] Schmitz PG, Lane PL, Dalal R, Zhang K, Majidi BJ. -3 fatty acids attenuate glomerular capillary hydraulic pressure in rats with renal ablation. Kidney Int 1995;48:1792– 800. [38] Badr KF, Kelley VE, Rennke HG, Brenner BM. Roles for thromboxane A2 and leukotrienes in endotoxin induced acute renal failure. Kidney Int 1986;30:474 – 80. [39] Neumayer HH, Heinrich M, Schmissas M, Haller H, Wagner K, Luft FC. Amelioration of ischemic acute renal failure by dietary fish oil administration in conscious dogs. J Am Soc Nephrol 1992;3:1312–20. [40] Kaufman RP, Kobzik L, Shepro D, Anner H, Valeri CR, Hechtman HB. Vasodilator prostaglandins (PG) prevent renal damage after ischemia. Ann Surg 1987;205:195–98. [41] Paller MS, Manivel JC, Patten M, Barry M. Prostaglandins protect kidneys against ischemic and toxic injury by a cellular effect. Kidney Int 1992;42:1345–54. [42] Nagler AL, Dettbarn WD, Seifter E, Levenson SM. Tissue levels of acetylcholine and acetylcholinesterase in weaning rats subjected to acute choline deficiency. J Nutr 1968;94:13–19.
M.C. Courre`ges et al. / Nutrition Research 22 (2002) 1077–1089
1089
[43] Costa RS, Rossi MA, Oliveira JSM. Pathogenesis of the renal injury in choline deficiency: the role of catocholamines and acetylcholine. Br J Exp Path 1979;60:613–9. [44] Achard JM, de Cagny B, Pruna A, Fournier A. Pathophysiology of acute tubular necrosis. In Cantarovich F, Rangoonwala B, Verho M, editors. In: Progress in acute renal failure. New Jersey: Hoechst Marion Roussel, 1998. p. 23– 45. [45] Lin H, Smith MJ, Young DB. Roles of prostaglandins and nitric oxide in the effect of endothelin-1 on renal hemodynamics. Hypertension 1996;28:372–78. [46] Lo´ pez-Novoa JM. Potential role of platelet activating factor in acute renal failure. Kidney Int 1999;55: 1672– 82. [47] Maddox DA, Brenner BM. Glomerular ultrafiltration. In: Brrenner, Rector, editors. The kidney. 6th. ed. Philadelphia: WB Saunders, 2000. p. 319 –74. [48] Ratcliffe P. The pathopysiology of acute renal failure. In: Davison AM, Cameron JS, Gru¨ nfeld JP, Kerr DNS, Ritz E, Winearls C, editors. Oxford textbook of clinical nephrology. Oxford: Oxford University Press, 1998. p. 1531–56. [49] Robinette JB, Conger JD. Angiotensin and thromboxane in the enhanced renal adrenergic nerve sensitivity of acute renal failure. J Clin Invest 1990;86:1532–39. [50] Schramek H, Coroneos E, Dunn MJ. Interactions of the vasoconstrictor peptides, angiotensin II and endothelin-1, with vasodilatory prostaglandins. Seminars in Nephrology 1995;15:195–204. [51] Wilcox CS, Welch WJ, Snellen H. Thromboxane mediates renal hemodynamic response to infused angiotensin II. Kidney Int 1991;40:1090 –97. [52] Lelcuk S, Alexander F, Kobzik L, Valeri CR, Shepro D, Hechtman HB. Prostacyclin and thromboxane A2 moderate postischemic renal failure. Surgery 1985;98:207–12.