Original Paper
Nephron 1992;62:449-453
Nephrology Division and Pathology Department, Escola Paulista de Medicina, Sao Paulo, SP, Brazil
Effect of Eicosapentaenoic Acid on the Progression of Chronic Renal Failure in Rats
Key Words
Abstract
Eicosapentaenoic acid Chronic renal failure Glomerular hemodynamics Proteinuria Glomerular sclerosis
Eicosapentaenoic acid (EPA) can induce a shift in prostaglandin and leukotriene synthesis. The effects o f EPA supplementation of the diet on the progression of chronic renal failure (CRF) were evaluated in a model o f %renal mass ablation in rats. After 30 or 60 days o f CRF, elevation in single-nephron glomerular filtration rate due to an increase in glomerular plasma flow and hydraulic pressure was observed. These hemodynamic alterations were followed by a rise in proteinuria and glomerular sclerosis. EPA treatment for 30 or 60 days did not substantially modify the hemodynamic or morphological profiles induced by renal mass ablation. In the present non-immune model of CRF, preglomerular vasodilation with glomerular hyperperfusion and hypertension were responsible, at least in part, for the presence o f proteinuria and glomerular sclerosis. No additional vasodilation was observed in the present model of CRF, and, thus, hemodynam ic effects induced by EPA did not modify renal damage, in contrast to the EPA effects observed in immune-mediated models of CRF.
Introduction Fish oil is rich in polyunsaturated omega-3 fatty acids such as docosahexanoic acid and eicosapentaenoic acid (EPA) [1], These fatty acids compete with arachidonic acid by inhibiting its synthesis from linoleic acid and by compet ing as substrates for cyclooxygenase enzyme [2] as well as for the 2-position in membrane phospholipids. Arachi donic acid is the precursor o f 2-series prostaglandins (PGs), thromboxane (TxA2) and 4-series leukotrienes (LTB4), while the products o f EPA are the 3-series o f these eicosanoids, PGI3 and PGE3 (equipotents to PGT and PGE 2), TxA3 (physiologically inactive) and LTB5, which is less potent than LTB4 [3-6], Thus the net result is a change in the homeostatic balance toward a more vasodilatatory state with less platelet aggregation and inflammation [7], Because
Accepted: February 28.1992
of these effects, EPA has been implicated in the low in cidence of atherosclerosis among the Eskimos, who basi cally eat fish [8]. It has also been suggested that EPA plays a protector role in the progression of chronic renal failure (CRF) [9-12]. However, these findings are controversial since multifactor ial mechanisms appear to be involved in its pathogenesis. Besides inflammatory responses, altered PG synthesis, coa gulation abnormalities, and alterations in lipid metabolism observed in some models of CRF [13], the hemodynamic changes, such as increased glomerular pressure and flow, must be considered. In view o f the above considerations, the present study was undertaken to evaluate systemic parameters, renal hemodynamics and morphological re sponses to a diet supplemented with fish oil in a model of CRF induced by SA nephrectomy in rats.
Nestor Schor. M I). P h i) Escola Paulista de M edicina, Disciplina de Nefrologia R. Botucatu 740 04023 Säo Paulo. SP (Brazil)
© 1992 S. K arger AG, Basel 0028-2766/92/ 0624-O449S2.75/0
Downloaded by: Thomas Jefferson University Scott Library 147.140.20.32 - 10/16/2017 4:50:43 PM
V. E. R. Melhado, M. A. Boim C. Versolato, L. A. Mourn S. R. Stella, N. Schor
Induction o f CRF Male Munich-Wistar rats aged 3 months and weighing 250-320 g were subjected to % renal mass ablation by ligation of two or three branches o f the left renal artery and a right nephrectomy. Animals had free access to water and regular rat chow prior to the clearance and micropuncture studies. Simultaneously, fish oil contain ing 18% EPA (Lab. Fatec, Sao Paulo, Brazil) was given at a dose o f 150 m g/rat/day, per os. The fish oil composition employed was: EPA 18%; cholesterol 0.2%; docosahexanoic acid 12%; sardine oil 69.8%; vitamin A 50 IU /g; vitamin D 5 IU /g, and vitamin E I lU /g . Studies were performed 30 or 60 days after the induction o f chronic renal failure (CRF) along with an EPA-supplemented diet. Groups The animals were divided into six groups: control (C O N T; n = 8); control rats treated with EPA for 60 days (C'ONT + EPA: n = 7); 30 days o f CRF (CRF-30; n = 5); 30 days of CRF plus EPA (CRF30 + EPA; n = 7); 60 days o f CRF (CRF-60; n = 6) and 60 days o f CRF plus EPA (C R F-60+EPA : n = 6). Two days before the experiment, the rats were placed in individual metabolic cages, and 24-hour urine was collected for the determina tion o f urinary protein excretion measured by precipitation with 3% sulfosalicylic acid [14]. Micropuncture Studies The rats were anesthetized with Inactin (BYK, Konstanz, FRG), 100 mg/kg, i.p., placed on a temperature-regulated table and moni tored with an electronic rectal thermometer (Simpson Co., Chicago, 111., USA) to keep body temperature between 36 and 37 °C. After tracheotomy, the left femoral artery was catheterized with PE-50 tubing, and approximately 60 pi o f arterial blood was collected for baseline hematocrit and protein determinations [15]. The arterial ca theter was also used for subsequent periodic blood sampling for inulin measurement [16] and recording mean arterial pressure (MAP) with an electronic transducer (Model P23Db; Statham Instruments Div., Gould Inc., Hato Rey, P.R.), connected to a direct-writing recorder (Model 2.200; Gould Inc., Ohio, USA). PE-50 catheters were inserted into the right and left jugular veins infuse inulin (7.5%, 1.2 m l/h) and isoncotic rat serum to replace surgical losses and maintain euvolemia in the animals [17]. A laparotomy was then performed, and the left kidney was prepared for micropuncture studies as previously de scribed [18]. After a 45-min equilibration period, timed (1-2 min) samples of tubular fluid were collected from the surface proximal convolutions of at least three nephrons for the determination o f flow rate and inulin concentration [19], and the calculation o f the single-nephron glomeru lar filtration rate (SNGFR). Simultaneously, samples o f femoral arte rial blood were obtained for the determination o f systemic arterial pressure, hematocrit, total protein and inulin concentration in plasma. Colloid osmotic pressure o f plasma entering and leaving the glomerular capillaries was estimated from protein concentrations in femoral and efferent arterial blood plasma [20]. The protein level in the femoral arterial blood plasma was taken as a parameter o f protein concentration in the afferent arteriole. Direct measurements o f hydraulic pressure in single glomerular capillaries (Pcc), proximal tubules (PT), efferent arterioles and thirdorder peritubular capillaries were obtained in each rat using a continu ous-recording Servonull micropipette-transducer system (IPM Inc.,
450
San Diego, Calif.. USA). The transcapillary hydraulic pressure dif ference (AP) was calculated as A P = Pgc-P tColloid osmotic pressures, hydraulic pressures and SNGFR per mit the calculation o f the single-nephron filtration fraction, initial glomerular capillary plasma flow rate (Q,v), and the afferent (RA), efferent (R, )and total (R r= R,\+ Re) arteriolar resistances and glome rular capillary ultrafiltration coefficient (K,j, using equations given elsewhere [21]. Plasma cholesterol was measured by enzymatic oxidose and trigly cerides by enzymatic hydrolysis. Morphological Evaluation At the end o f the functional studies, the kidneys were perfused with a solution containing 2% glutaraldehyde buffered in phosphate, pH 1 2 -1 A, at a constant pressure of 100 mm Hg, for morphologic evalu ation. Glomerulosclerosis was defined as the absence o f cellular elements from the tuft, collapse of capillary lumens and folding o f the glomerular basement membrane with amorphous material, while me sangial matrix expansion was characterized by the presence o f in creased amounts o f periodic acid-Schiff positive material in the me sangial region. The severity o f the lesion was graded from 0 to 4 + , according to the percentage o f glomerular involvement. A I + lesion represented an involvement o f 25% o f the glomerulus, while a 2 + , 3 + or 4 + lesion indicated a glomerular involvement o f 25, 50 or 100%, respectively. The degree o f a lesion multiplied by the percentage o f the glomeruli with the same injury allows the calculation o f the sclerosis index, as performed by Raij et al. [22]. Statistical Analysis Data were compared by analysis o f variance followed by the Duncan test. Morphological data were compared using the KruskalWallis test followed by the Dunn test. Statistical significance was taken as p
Nephron 1992;62:449-453
Nephrology Division and Pathology Department, Escola Paulista de Medicina, Sao Paulo, SP, Brazil
Effect of Eicosapentaenoic Acid on the Progression of Chronic Renal Failure in Rats
Key Words
Abstract
Eicosapentaenoic acid Chronic renal failure Glomerular hemodynamics Proteinuria Glomerular sclerosis
Eicosapentaenoic acid (EPA) can induce a shift in prostaglandin and leukotriene synthesis. The effects o f EPA supplementation of the diet on the progression of chronic renal failure (CRF) were evaluated in a model o f %renal mass ablation in rats. After 30 or 60 days o f CRF, elevation in single-nephron glomerular filtration rate due to an increase in glomerular plasma flow and hydraulic pressure was observed. These hemodynamic alterations were followed by a rise in proteinuria and glomerular sclerosis. EPA treatment for 30 or 60 days did not substantially modify the hemodynamic or morphological profiles induced by renal mass ablation. In the present non-immune model of CRF, preglomerular vasodilation with glomerular hyperperfusion and hypertension were responsible, at least in part, for the presence o f proteinuria and glomerular sclerosis. No additional vasodilation was observed in the present model of CRF, and, thus, hemodynam ic effects induced by EPA did not modify renal damage, in contrast to the EPA effects observed in immune-mediated models of CRF.
Introduction Fish oil is rich in polyunsaturated omega-3 fatty acids such as docosahexanoic acid and eicosapentaenoic acid (EPA) [1], These fatty acids compete with arachidonic acid by inhibiting its synthesis from linoleic acid and by compet ing as substrates for cyclooxygenase enzyme [2] as well as for the 2-position in membrane phospholipids. Arachi donic acid is the precursor o f 2-series prostaglandins (PGs), thromboxane (TxA2) and 4-series leukotrienes (LTB4), while the products o f EPA are the 3-series o f these eicosanoids, PGI3 and PGE3 (equipotents to PGT and PGE 2), TxA3 (physiologically inactive) and LTB5, which is less potent than LTB4 [3-6], Thus the net result is a change in the homeostatic balance toward a more vasodilatatory state with less platelet aggregation and inflammation [7], Because
Accepted: February 28.1992
of these effects, EPA has been implicated in the low in cidence of atherosclerosis among the Eskimos, who basi cally eat fish [8]. It has also been suggested that EPA plays a protector role in the progression of chronic renal failure (CRF) [9-12]. However, these findings are controversial since multifactor ial mechanisms appear to be involved in its pathogenesis. Besides inflammatory responses, altered PG synthesis, coa gulation abnormalities, and alterations in lipid metabolism observed in some models of CRF [13], the hemodynamic changes, such as increased glomerular pressure and flow, must be considered. In view o f the above considerations, the present study was undertaken to evaluate systemic parameters, renal hemodynamics and morphological re sponses to a diet supplemented with fish oil in a model of CRF induced by SA nephrectomy in rats.
Nestor Schor. M I). P h i) Escola Paulista de M edicina, Disciplina de Nefrologia R. Botucatu 740 04023 Säo Paulo. SP (Brazil)
© 1992 S. K arger AG, Basel 0028-2766/92/ 0624-O449S2.75/0
Downloaded by: Thomas Jefferson University Scott Library 147.140.20.32 - 10/16/2017 4:50:43 PM
V. E. R. Melhado, M. A. Boim C. Versolato, L. A. Mourn S. R. Stella, N. Schor
Induction o f CRF Male Munich-Wistar rats aged 3 months and weighing 250-320 g were subjected to % renal mass ablation by ligation of two or three branches o f the left renal artery and a right nephrectomy. Animals had free access to water and regular rat chow prior to the clearance and micropuncture studies. Simultaneously, fish oil contain ing 18% EPA (Lab. Fatec, Sao Paulo, Brazil) was given at a dose o f 150 m g/rat/day, per os. The fish oil composition employed was: EPA 18%; cholesterol 0.2%; docosahexanoic acid 12%; sardine oil 69.8%; vitamin A 50 IU /g; vitamin D 5 IU /g, and vitamin E I lU /g . Studies were performed 30 or 60 days after the induction o f chronic renal failure (CRF) along with an EPA-supplemented diet. Groups The animals were divided into six groups: control (C O N T; n = 8); control rats treated with EPA for 60 days (C'ONT + EPA: n = 7); 30 days o f CRF (CRF-30; n = 5); 30 days of CRF plus EPA (CRF30 + EPA; n = 7); 60 days o f CRF (CRF-60; n = 6) and 60 days o f CRF plus EPA (C R F-60+EPA : n = 6). Two days before the experiment, the rats were placed in individual metabolic cages, and 24-hour urine was collected for the determina tion o f urinary protein excretion measured by precipitation with 3% sulfosalicylic acid [14]. Micropuncture Studies The rats were anesthetized with Inactin (BYK, Konstanz, FRG), 100 mg/kg, i.p., placed on a temperature-regulated table and moni tored with an electronic rectal thermometer (Simpson Co., Chicago, 111., USA) to keep body temperature between 36 and 37 °C. After tracheotomy, the left femoral artery was catheterized with PE-50 tubing, and approximately 60 pi o f arterial blood was collected for baseline hematocrit and protein determinations [15]. The arterial ca theter was also used for subsequent periodic blood sampling for inulin measurement [16] and recording mean arterial pressure (MAP) with an electronic transducer (Model P23Db; Statham Instruments Div., Gould Inc., Hato Rey, P.R.), connected to a direct-writing recorder (Model 2.200; Gould Inc., Ohio, USA). PE-50 catheters were inserted into the right and left jugular veins infuse inulin (7.5%, 1.2 m l/h) and isoncotic rat serum to replace surgical losses and maintain euvolemia in the animals [17]. A laparotomy was then performed, and the left kidney was prepared for micropuncture studies as previously de scribed [18]. After a 45-min equilibration period, timed (1-2 min) samples of tubular fluid were collected from the surface proximal convolutions of at least three nephrons for the determination o f flow rate and inulin concentration [19], and the calculation o f the single-nephron glomeru lar filtration rate (SNGFR). Simultaneously, samples o f femoral arte rial blood were obtained for the determination o f systemic arterial pressure, hematocrit, total protein and inulin concentration in plasma. Colloid osmotic pressure o f plasma entering and leaving the glomerular capillaries was estimated from protein concentrations in femoral and efferent arterial blood plasma [20]. The protein level in the femoral arterial blood plasma was taken as a parameter o f protein concentration in the afferent arteriole. Direct measurements o f hydraulic pressure in single glomerular capillaries (Pcc), proximal tubules (PT), efferent arterioles and thirdorder peritubular capillaries were obtained in each rat using a continu ous-recording Servonull micropipette-transducer system (IPM Inc.,
450
San Diego, Calif.. USA). The transcapillary hydraulic pressure dif ference (AP) was calculated as A P = Pgc-P tColloid osmotic pressures, hydraulic pressures and SNGFR per mit the calculation o f the single-nephron filtration fraction, initial glomerular capillary plasma flow rate (Q,v), and the afferent (RA), efferent (R, )and total (R r= R,\+ Re) arteriolar resistances and glome rular capillary ultrafiltration coefficient (K,j, using equations given elsewhere [21]. Plasma cholesterol was measured by enzymatic oxidose and trigly cerides by enzymatic hydrolysis. Morphological Evaluation At the end o f the functional studies, the kidneys were perfused with a solution containing 2% glutaraldehyde buffered in phosphate, pH 1 2 -1 A, at a constant pressure of 100 mm Hg, for morphologic evalu ation. Glomerulosclerosis was defined as the absence o f cellular elements from the tuft, collapse of capillary lumens and folding o f the glomerular basement membrane with amorphous material, while me sangial matrix expansion was characterized by the presence o f in creased amounts o f periodic acid-Schiff positive material in the me sangial region. The severity o f the lesion was graded from 0 to 4 + , according to the percentage o f glomerular involvement. A I + lesion represented an involvement o f 25% o f the glomerulus, while a 2 + , 3 + or 4 + lesion indicated a glomerular involvement o f 25, 50 or 100%, respectively. The degree o f a lesion multiplied by the percentage o f the glomeruli with the same injury allows the calculation o f the sclerosis index, as performed by Raij et al. [22]. Statistical Analysis Data were compared by analysis o f variance followed by the Duncan test. Morphological data were compared using the KruskalWallis test followed by the Dunn test. Statistical significance was taken as p
