Fish oil has protective and therapeutic effects on proteinuria in passive Heymann nephritis

Kidney International, Vol. 43 (1993), pp. 359—368

Fish oil has protective and therapeutic effects on proteinuria in passive Heymann nephritis WOLFGANG J. WEISE, YASUHIRO NATORI, JERROLD S. LEVINE, YVONNE M. O'MEARA, ANDREW W. MIN-ro, ERIC C. MANNING, D. J0RDI GOLDSTEIN, DALE R. ABRAHAMSON, and DAVID J. SALANT Evans Memorial Department of Clinical Research at The University Hospital, Boston University Medical Center, Boston, Massachusetts, and Department of Cell Biology and Anatomy, University of Alabama at Birmingham, Birmingham, Alabama, USA

Fish oil has protective and therapeutic effects on proteinuria in passive Heymann nephritis. Passive Heymann nephritis (PHN) is a rat model of membranous nephropathy induced by injecting anti-Fx1A. The onset of proteinuria in PHN is caused by complement-mediated injury to gbmerular epithelial cells (GEC) accompanied by enhanced glomerular eicosanoid production. In addition, sublethal injury by complement of

rat GECs in culture leads to phospholipase activation, phospholipid hydrolysis and release of arachidonic acid and dienoic prostanoids. Based on these findings, we undertook to determine if substituting arachidonic acid (omega-6) in GEC membrane phospholipids with omega-3 fatty acids derived from fish oil would alter the development

hypertension of advanced renal failure may predispose patients

to accelerated atherosclerosis. To date, no therapy has been uniformly effective in reducing proteinuria without adversely affecting renal function. Studies on the pathogenesis of experimental membranous nephropathy suggest a logical therapeutic strategy that can be tested in a rat model closely resembling human membranous nephropathy. Passive Heymann nephritis (PHN) is induced by injection of an antiserum (anti-Fx1A) that

identifies antigens on the glomerular epithelial cell (GEC)

and course of proteinuria in PHN. We found that rats fed a diet plasma membrane and is characterized by subepithelial immune containing 10% fish oil for four weeks prior to antibody injection developed 50 to 60% less proteinuria between two and six weeks after anti-Fx1A than rats fed an equivalent diet containing 10% safflower oil, and had substantial enrichment of glomerular phospholipids with omega-3 fatty acids and displacement of arachidonic acid. This outcome was associated with a 50% reduction in release of glomerular thromboxane B2 (stable metabolite of thromboxane A2) in the fish oil group. More importantly, when PHN rats with well established proteinuria while on regular chow were randomized to three dietary groups, those fed fish oil had a 25 to 50% decline in proteinuria as compared to those fed lard or safflower oil. This difference was evident within two weeks of randomization and persisted until the end of the study after eight weeks. In neither study could the differences in urine protein excretion be accounted for by protein or calorie deprivation, or by differences in

blood pressure, renal function, immune response to sheep IgG, or glomerular deposition of IgG or complement. Thus, our results indicate that dietary fish oil has protective and therapeutic effects with regard to proteinuria in PHN. These benefits may relate to alterations in membrane phospholipid composition in favor of omega-3 fatty acids and release of less reactive trienoic eicosanoids.

deposits. The onset of proteinuria is mediated by the C5b-9 membrane attack complex of complement which assembles in the GEC plasma membrane and produces alterations in GEC morphology and enhanced glomerular eicosanoid production [reviewed in 1]. In addition, antibody-directed insertion of C5b-9 into rat GECs in culture leads to phospholipase C and A2 activation, membrane phospholipid hydrolysis and prostanoid release [2]. These findings suggest that GEC membrane phos-

pholipid hydrolysis might contribute to the development of proteinuria, perhaps through the intermediary action of thromboxane A2 (TXA2) or other lipid mediators [4—61. Many human and animal studies have shown that dietary

supplementation with fish oil, a major source of omega-3 fatty acids, alters membrane phospholipid fatty acid composition and eicosanoid production [6—8]. Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the most abundant omega-3 fatty acids, accumulate in the plasma membrane and partially replace arachidonic acid (AA) as substrates for cyclooxygenase and The cyclooxygenase and lipoxygenase products Membranous nephropathy is the most common cause of lipoxygenase. of EPA (especially TXA3 and LTB5) have much less vasoconidiopathic nephrotic syndrome in adults and an important cause strictive and inflammatory action than those of AA (TXA2 and of chronic renal failure. Even in the absence of renal failure, LTB4) [9, 10]. These alterations may well be responsible for the many patients with membranous nephropathy suffer considerbeneficial effects of fish oil in murine lupus nephritis [11] and able morbidity from intractable edema. In addition, the hyperother models of experimental nephritis [12]. Fish oil also lipidemia associated with severe nephrotic syndrome and the favorably alters various factors that may contribute to progres-

sive glomerular injury and atherosclerosis, including blood Received for publication May 21, 1992 and in revised form September 8, 1992 Accepted for publication September 10, 1992

© 1993 by the International Society of Nephrology

rheology [13], platelet aggregation [8] and plasma lipid composition [14, 15].

We undertook this study to determine if fish oil affects the development and course of proteinuria in PHN and, if so, to define possible mechanisms by analyzing the influence of fish oil

359

360

Weise

et a!: N-3 fatty acids in membranous nephritis

on the primary immune response, antibody deposition, complement-induced glomerular prostanoid production and renal func-

tion during and after active immune injury. In addition to

immune response and plasma lipids. In addition, a subgroup of

rats fed either fish oil or safflower oil for four weeks was

sacrificed to measure the glomerular phospholipid fatty acid demonstrating substantial protection against the development composition prior to antibody injection. of proteinuria in PHN, our results are the first to document a Experimental procedures therapeutic effect of dietary fish oil in rats with established membranous nephropathy. Possible mechanisms for this outInduction of passive Heymann nephritis. PHN was induced come are discussed. in anesthetized (3% chloral hydrate, 1 ml/100 g body wt i.p.) rats (200 to 250 g) by two intravenous injections of 1.0 and 075 Methods ml of sheep anti-Fx1A 24 hours apart. Preparation and characterization of this antiserum has been described [16]. Rats fed the Animals and materials fish oil diet had a higher mortality rate from anesthesia and Female Sprague-Dawley rats were obtained from Charles antibody injection than the other animals (15% vs. 10%). Urine River Laboratories (Wilmington, Massachusetts, USA). 3H- protein excretion was measured biweekly by the sulfosalicylic and 3H-inulin were purchased from acid method using a commercial serum standard [16]. At the POE2, 3H-TXB,, 1251 Du Pont-New England Nuclear Research Products (Boston, end of both studies all animals were sacrificed and kidneys were Massachusetts, USA). PGE2, TXB, and albumin standards removed for morphologic studies. Renal clearance studies were were purchased from Sigma Chemicals (St. Louis, Missouri, performed on selected rats on day 10. Other animals were USA), and antisera to PGE7 and TXB, were obtained from sacrificed on day 6 and kidneys were removed for isolation of Advanced Magnetics (Cambridge, Massachusetts, USA). Para- glomeruli to measure glomerular prostanoid production and aminohippuric acid (PAR) was obtained from Aldrich Chemical antibody deposition. Co. (Milwaukee, Wisconsin, USA). Glomerular antibody deposition. A dual isotope method was used to assess the potential effect of various dietary lipids on Diets glomerular antibody deposition [16]. Rats were injected with 15 Experimental and control diets consisted of an isocaloric rat mg '251-labeled Yi anti-Fx1A (2 x 106 cpm/rat), prepared and chow (Ralston Purina, Richmond, Indiana, USA) containing characterized as previously described [16], On day 6 after 21% protein (casein), 59% carbohydrate (sucrose and dextrin), anti-FxlA, all animals were injected with 131j BSA to account 10% lipid and supplements (mineral and vitamin mix). The lipid for nonspecific glomerular binding and were sacrificed one hour in the experimental diet was derived from fish oil (MaxEPA oil, later. Glomeruli were isolated from the kidneys of individual provided by R.P. Scherer, Troy, Michigan, USA), a source of rats by differential sieving and were enumerated in a Fuchsomega-3 fatty acids. Vitamin E, 50 lU/kg, was added to the diet Rosenthal hemocytometer. 125J and 1311 radioactivity of glomerand the fish oil and diets were stored at —20°C to prevent uli and serum were measured in a gamma counter and specific peroxidation. The lipid content of the control diets was made up glomerular deposition of 125j labeled anti-Fx1A was calculated of safflower oil, a source of omega-6 fatty acids, or lard, a as described [11. Results are expressed as micrograms of IgG source of saturated fat. The diets were prepared before each bound/38,000 glomeruli. experiment and rats were pair-fed as follows. Based on their Fatty acid composition of glomerular phospholipids. Glomeraverage daily food consumption prior to dietary modification, uli were isolated, washed with ice-cold phosphate-buffered rats were given sufficient food to last about two days. Since the saline, pH 7.4 (PBS), and stored at —70°C until assayed. Tissue safflower and lard fed rats ate their food more quickly than the lipids were extracted using a procedure modified from that of fish oil group, the food hoppers were not refilled until the fish oil Foich et al [171. A 2:1 mixture of chloroform/methanol (C/M) group had finished their ration. All rats were weighed every two was used to extract total lipids from isolated glomeruli. The weeks to ensure equal food intake and weight gain. organic layer was evaporated under N2, resolubilized in C/M (2:1) and separated by thin layer chromatography using LK6D Experimental protocols silica gel plates (Whatman Inc., Clifton, New Jersey, USA) and Two experimental protocols were established to study the a solvent system of hexane:ethylether:glacial acetic acid (70: effect of dietary lipids on the development of proteinuria 30:1). The phospholipid region was identified from co-chro(Preventive study) and on already established proteinuria matographed standards, scraped, and the lipids eluted from the (Therapeutic study). In the preventive study, PHN was induced gel with C/M (1:1) and concentrated. Transmethylation was by injection of anti-Fx1A after both groups of rats had been fed performed using BF3/CH3OH (Supelco, Bellafonte, Pennsylvatheir respective diets (fish oil or safflower oil) for four weeks. nia, USA) [18]. Fatty acid methylesters were separated on a 6 The animals continued on their diets and were followed for six foot glass gas-chromatography column (Hewlett Packard Co., to eight weeks after anti-FxlA injection, that is, well into the Avondale, Pennsylvania, USA) packed with GP 5% DEGS-PS autologous phase of proteinuria. In the therapeutic study, all on 100/200 Supelcoport (Supelco) and equipped with a flame animals were injected with anti-Fx1A and maintained on regular ionization detector. The carrier gas N2 flow rate was 40 mllmin. rat chow (Rodent Laboratory Chow 5001, Ralston Purina) until The oven, injector and detector temperatures were 170, 250 and the autologous phase of proteinuria was established (3 weeks). 250°C, respectively. Peaks were identified according to authenAt this point, the animals were randomly assigned to receive tic standards (Supelco) and quantification was performed using diets containing either fish oil, safflower oil or lard and were an auxiliary integrator. followed for eight weeks. In both studies, urine and blood were Measurement of glomerular prostanoid production. All kidcollected biweekly to assess proteinuria, renal function, host neys were perfused with 200 ml of ice-cold PBS in vivo prior to

Weise et al: N-3 fatty acids in membranous nephritis

361

removal. Glomeruli were then isolated by sieving and incubated

were placed in the right jugular vein and femoral artery to infuse

in plastic tubes containing 2 ml of Krebs-Ringer-bicarbonate buffer, pH 7.2, for 30 minutes at 37°C according to the method of Stahl et a! [4]. Glomerular PGE2 and TXB2 production were determined by direct radioimmunoassay of the indomethacintreated supernatants without prior extraction or chromatographic separation. The specific assay procedures have been described in detail elsewhere [5]. The cross reactivity of other arachidonate metabolites with the utilized antisera at 50% displacement were as follows: anti-PGE2: PGE1 50%, PGA2 6%, PGA1 3%, PGF2 1.3%, 6-keto-PGE1 1%, l5-keto-PGF2 1%, 15-keto-PGE2 1%, all other AA metabolites <1%; antiTXB2: all measured AA metabolites <0.1%. All results are expressed as picogram of prostanoid per milligram of glomerutar protein per minute incubation time. Measurement of host immune response. Rat anti-sheep IgG serum titers were measured by ELISA to determine the host immune response to injected sheep anti-Fx1A. Microtiter plates (Falcon, Becton Dickinson Co., Oxnard, California, USA) were coated with normal sheep IgG (10 pg/ml in PBS) and incubated for 16 hours at 4°C in an air-tight moist chamber. The antigen was discarded and blocking buffer (1% BSA-0.15 M Tris-HC1,

plasma, 3H-inulin and PAH and to monitor mean arterial pressure and to collect blood. Mean arterial pressure was measured by an electronic transducer (model 1280 C, Hewlett

Packard) connected to a recorder (model 7282 A, Hewlett Packard). To maintain euvolemia during the experiment, each rat received a continuous intravenous infusion of isooncotic rat plasma (1 ml/100 g body wt) for the first 30 minutes. Glomerular filtration rate and whole kidney effective renal plasma flow rate

were measured by inulin and PAH clearance, respectively. 3H-inulin and 0.9% PAH in 0.9% NaCl were infused at a constant rate of 0.028 mllmin. After a 40-minute equilibration period, three collection periods of 20 minutes each were performed. 3H-inulin in blood and urine samples was measured in a lipid scintillation counter. PAH was determined in proteinfree filtrates of plasma and urine by a colorimetric method [20]. The clearances were calculated from standard equations. Morphologic studies. At the end of both studies kidney tissue from representative rats was examined by light microscopy (LM), immunofluorescence (IF) and electron microscopy (EM).

Tissue for LM was fixed in 10% neutral buffered formalin, sectioned at 4 microns, and stained with hematoxylin and eosin

pH 7.6) was added and incubated for one hour at room stain. Tissue for IF was snap frozen in dry-ice-isopentane, and temperature. All wells were washed three times with washing buffer (0.05% Tween 20, 15 mi Tris-HC1, pH 7.6, 135 mM NaCl). Serially diluted (1/50-1/800) test sera were added and incubated for one hour at room temperature. The serum samples were then removed and wells washed again with washing buffer. Biotinylated goat anti-rat IgG (Cappel Laboratories, Cochranville, Pennsylvania, USA) at a 1/1000 dilution was added and incubated for one hour at room temperature. After washing, wells were incubated with avidin-peroxidase (Cappel, 2.5 pg/ml) for 30 minutes at room temperature. After a final wash, freshly prepared developing buffer containing 50 mM phosphate-citrate (pH 5), 0.04% (wt/vol) ortho-phenylenediamine and 0.014% (vol/vol) H202 was added to the welts and incubated for 5 to 10 minutes. The reaction was stopped with

sectioned and stained as previously described [16, 21]. Antisera utilized in the staining procedure included fluorescein-conjugated IgG fractions of monospecific polyclonal antibodies against sheep IgG, rat IgG and rat C3 (Cappel Laboratories) and monoclonal anti-rat CSb-9 (from WG Couser, University of Washington). Cortical kidney tissue was fixed in situ with

Karnovsky's fixative and processed further for EM as described before [22].

Statistical analysis The values given in tables and text are expressed as mean SD unless indicated otherwise. All analyses were done with StatViewTM 512 + (BrainPower, Inc., Calabasas, California, USA). Two-way analysis of variance (ANOVA) was used to 2.5 M H2S04 to each well and absorbance was read at 490 nm in test changes within groups and between groups over time. A a Minireader II (Dynatech Laboratories, Inc., Alexandria, two-tailed unpaired t-test was used for comparisons between Virginia, USA). Nonspecific reactivity was determined from two groups, and a paired (-test was used where indicated for control reactions in which serum samples were deleted. All before and after comparisons within groups. Mann-Whitney U measurements were performed in duplicate and results are test and Kruskal-Wallis analysis of variance were used for expressed in arbitrary ELISA units as described in detail by nonparametric data. A P value of <0.05 was considered signifButler, Cantarero and McGivern [19]. icant, Measurement of serum albumin. Serum albumin was measured by ELISA in which plates were coated with sheep anti-rat Results albumin (Cappel Laboratories) dissolved in PBS at a concentration of 20 g/ml. Mter coating and appropriate blocking and Effect of dietary fatty acid substitution on fatty acid content of glomerular phospholipids and on growth rate washing steps, albumin standards and test sera, at a dilution of 1 x 10, were added, and subsequently assayed and developed Since glomerular epithelial cells are the primary targets of with biotinylated sheep anti-rat albumin followed by avidin- injury in this model, we analyzed glomerular phospholipid fatty peroxidase. All measurements were performed in duplicate and acid composition to determine the degree of omega-3 fatty acid test results were read off a standard curve relating albumin substitution of plasma membranes. The results are shown in concentration to OD 490 nm. Table 1. Total glomerular omega-3 fatty acid content in the fish Measurement of serum creatinine, triglyceride and choles- oil group was 10-fold and 20-fold greater than that in the lard terol. All measurements were performed by the University and safflower oil groups, respectively. Arachidonate (w-6 20:4) Hospital clinical laboratory using standard automated methods. content in the fish oil group was reduced by about 50% Clearance studies. Rats were anesthetized with 3% chloral compared to the control diets indicating a substantial reduction hydrate (1 ml/l00 g body wt), After tracheostomy, polyethylene of substrate for the cyclooxygenase and lipoxygenase enzyme catheters (PE 50, Clay Adams, Parsippany, New Jersey, USA) systems. The safflower oil group had the highest omega-6 fatty

362

Weise

et at: N-3 fatty acids in membranous nephritis

300

Table 1. Glomerular phospholipid fatty acid content of glomeruli from

rats fed lard, safflower oil or fish oil for four weeks

w-9

Fatty acid

Lard

14:0 16:0

1.2

15.9

16:1

1.3

17:0 18:0

5.7 24.9 9.0 7.5 0.0

o-9

18:1

o-6

18:2 18:3

w-3 o-6

o.3

20:4 20:5

(0-9

24:1

a'6

22:4 22:5 22:6

(0-3 (0-3

25.1

0.8 4.4 3.0 0.0

total o,.3

1.0 1.8

total o-6

35.6

Safflower oil

Fish

C

oil

0

0.8

0.0

C.)

16.9

16.4

ci,

0.0 5.4 24.3 8.7

2.2 7.2 25.3

0

12.8

6.2 0.0

0,2 22.0 0.3 3.7 4.2 0.0 0.4 0.9 39.0

10.5

200

Ci 0.

100

ci,

C

11.3

9.2 0.0 2.5 4.8 4.5 18.5 20.0

Values are expressed in mol % of total glomerular phospholipid fatty acid content and represent the mean of three animals in each dietary group.

0

4

14

28

35

42

Time after anti-Fx1A, days Fig. 1. Effect of dietary lipids on the development of proteinuria in

passive Heymann nephritis (preventive study). Rats were fed safflower oil (, 10) or fish oil (U, 7) for four weeks and were then injected with anti-FxIA on day 0 and day I. Values are mean SE. Numbers in parentheses represent the number of animals in each group. * ANOVA P < 0.01 (FO vs. SO).

acid content reflecting the high omega-6 fatty acid content of this diet (76.9%). In a cohort of rats following the preventive protocol, animals were weighed eight times during the four-week pre-immunization period of feeding with fish oil (FO) or safflower oil (SO). Growth rates, as determined by body weight gain, were similar

300 C

0 w

in the two groups and the weights after four weeks were not significantly different (FO: 210.4

15.6 g, N

16; SO: 229.2

12.4g;N= 11;P=rNS).

.

a)'1 ci)

Effect of dietary fatty acid substitution on proteinuria, body weight and serum albumin Preventive study. Animals were fed their respective diets for

200

100-

C

four weeks and then were injected with anti-Fx1A. Urine protein excretion was significantly reduced in the fish oil group at 14 days after antibody injection (FO: 74.5 12.5; SO: 226.5 25.6 mg124 hr) and this difference persisted until the end of the study at 42 days (FO: 49.6 10.7; SO: 91.6 14.4 mg124 hr; Fig. 1). Five days after antibody injection only about half the animals in each group was proteinuric and, therefore, statistical

Fig. 2. Effect of dietary lipids on established proteinuria in passive Heymann nephritis (therapeutic study). Three weeks after anti-FxlA

analysis was not performed at this time point. Rats in the

weeks. Numbers in parentheses represent the number of animals in

Time after initiation of diets, days when proteinuria was well established (day 0), rats were randomized to (8), safflower oil, PA (15), or fish oil, U three dietary groups [lard, (15)1 and the effect on urine protein excretion was measured every two

safflower group gained slightly but significantly more weight each group. Values are mean SE. * ANOVA P < 0.05 (FO vs. SO + after disease induction than the fish oil group (after 6 weeks FO: 21.1 7.7%, N 7; SO: 30.1 11.1%, N = 9; P < 0.05). Serum albumin concentrations declined markedly 14 days after

lard).

antibody injection as compared to predisease levels and re- added as a second control diet in this study to determine if the turned towards normal levels after seven weeks (Fig. 3). The apparent benefit observed in initial experiments with fish oil difference between the levels of the fish oil and safflower oil might instead have been due to an adverse effect of safflower groups at 14 days was not statistically significant although it was oil. Fourteen days after randomization urine protein excretion in accord with the milder proteinuria in the fish oil group. Therapeutic study. All animals in this study were injected with anti-Fx1A and maintained on regular rat chow until the autologous phase was established and rats were severely proteinuric (3 weeks). At this point the animals were randomized to three different diets. Lard, a diet that closely resembles regular

was significantly reduced in the fish oil group compared to the two control groups (Fig. 2). This difference remained significant until the end of the study period (Fig. 2) despite wide variations in urinary protein excretion in both control groups during this

time. There was no difference in urinary protein excretion between the lard and the safflower oil groups at any time during

rat chow in terms of total lipid fatty acid composition, was the study period, which allows us to draw two conclusions.

363

U,

0

I I

Table 3. Effect of dietary lipids on the host antibody response to anti-Fx1A as measured by the serum titer of rat anti-sheep IgG Weeks after anti-FxlA

4

2

8

S0(9)

I

Lard (7)

4.75

10.00

4.00

3.95—33.35

2.55—20.85

0.65—9.30

3

FO (8)

Dietary groups Fig. 3. Serum albumin before (pre, U) and after immunization (PA 2 weeks; D 4 weeks; 7 weeks) in rats fed fish oil (FO) or safflower oil (SO) from four weeks prior to anti-FxIA injection. Values are mean s. * P < 0.001 (paired 1-test before and after immunization), # P < NS (FO vs. SO).

Table 2. Effect of dietary lipids on glomerular antibody binding six days after administration of anti-FxIA Glomerular antibody binding, ugIkidney

(mean SD) 22.8 23.9

4.5 6.0

Number of animals is in parentheses. P = NS (unpaired 1-test).

First, there is no independent effect of the degree of fatty acid saturation on proteinuria in this model. Second, the difference in protein excretion between fish oil and safflower oil is due to a beneficial effect of the marine oil rather than a harmful effect of safflower oil. Two weeks after randomization of heavily proteinuric rats to the various diets, weight gain was less in the fish oil group (FO: 9.9 6.1%, N = 15; SO: 16.1 7.6%, N = 15; lard: 16.5 4.8, N 8; P < 0.02) but by four weeks weights in the three groups had equalized. We did not attempt to determine body water content to account for potential differences in fluid retention. Serum albumin levels were mildly reduced in all groups at the

time of randomization to the different diets and remained

7

3.00

5.30

0.40—9.80

0.30—20.90

9.60 2.05—23.40

I

o

SO (7)

Dietary lipid

1.90 0.35—13.60

Weeks after anti-FxIA Therapeutic

SO (9)

4.70 0.95—15.85

I

I')

0

•

FO (5)

FO (9)

5.35 0.30--8.05

I

o 0

•

Preventive

o-.

Serum albumin, gAiter

•

0a

Weise et al: N-3 fatty acids in membranous nephritis

3.00 0.00—8.95

3.85

6.30

1.20—23.45

0.40—22,85

Values are median ELISA units and range. Number of samples tested is in parentheses. Nonparametric statistical analyses showed no significant differences between groups at any time point.

different between the fish oil and safflower oil groups six days after injection of 15 mg '251-anti-Fx1A (Table 2), and was within a range similar to that previously reported [23].

The effect of the various diets on the humoral immune response to injected sheep anti-Fx1A was measured two, four and six weeks after antibody injection in the preventive study and after three and seven weeks in the therapeutic study. There was no significant difference between the groups at any time (Table 3) to explain the decline in proteinuria seen in the fish oil group. Glomerular prostaglandin synthesis The results of TxB2 and PGE2 synthesis by isolated glomeruli from PHN and control rats (injected with normal sheep IgG) in the heterologous phase (6 days after antibody injection) are

shown in Figures 4 and 5. In rats fed safflower oil, TXB2 synthesis by nephritic glomeruli was significantly elevated compared to that of normal controls. Fish oil lowered basal glomerular TXB2 production in controls and prevented the increase in PHN (Fig. 4). There was no difference in PGE2 synthesis between nephritic and control glomeruli on either diet, although PGE2 synthesis was significantly reduced in glomeruli from fish oil fed rats when compared with those fed safflower oil (Fig. 5), reflecting the reduced diene-prostaglandin synthesis in animals fed a diet rich in fish oil.

Effect of dietary lipids on renal function To examine the possibility that the reduction in proteinuria with fish oil in the preventive study may reflect alterations in renal function, we measured renal hemodynamics in animals 10 to 14 days after anti-FxlA. The safflower oil and the fish oil Effect of dietary fatty acid composition on glomerular groups with PHN exhibited a decline in inulin clearance of antibody deposition and host immune response to anti-FxIA about 25% and 6% and effective renal plasma flow rate of about Potentially, the observed benefits of fish oil on proteinuria 47% and 21%, respectively, when compared to normal noncould be due to decreased glomerular deposition of antibody or nephrotic animals [241. As shown in Table 4, the fish oil group a blunted humoral response by the host to injected sheep of PHN rats had significantly higher effective renal plasma flow anti-Fx1A. This is not supported by the results shown in Tables rate, and a trend towards higher inulin clearance and decreased 2 and 3. Glomerular antibody deposition was not significantly filtration fraction than the safflower oil group. Mean arterial

similarly low throughout the study (after 8 weeks FO: 40.0 5.8 g/dl, N = 15; SO: 38.9 4.9 g/dl, N = 14; lard: 36.2 4.8 gIdl, N = 7; P = NS).

Weise et I

al: N-3 fatty acids in membranous nephritis

*

Table 5. Effect of dietary lipids on renal function as measured by serum creatinine in rats injected with anti-Fx1A

•

a 0 0

364

0

0 0

SO (8) FO (5)

Therapeutic Lard (7)

Serum creatinine mg/dl (mean

*

SD)

3 weeks after anti-FxIA Before anti-FxIA 0.40 0.07 0.54 o.05 0.56 020 0.39 0.15 8 weeks after randomization to diet 0.74 0.60

SO (7) FO (8)

0 0

Glomerular TXB2 pg/mg/rain

00

Group Preventive

0.56

0.15c 0.12" 0.14

a p < 0.05 vs. before anti-Fx1A

bp NS vs. SO; ANOVA P < 0.05 vs. SO and FO

0

"P = NS vs. FO; ANOVA

Fig. 4. Glomerular thromboxane B2 production in the heterologous phase (preventive study). Symbols are: (fl) PHN group (7); () control group (3). Numbers in parenthesis represent number of animals. Values are mean SE. * P < 0.05 (FO PHN vs. SO PHN) ** P < 0.05 (SO PHN vs. SO control).

Table 6. Effect of dietary lipids on plasma cholesterol and triglyceride in rats injected with anti-Fx1A Cholesterol Group

Triglyceride mg/dl

a'

a 0 0

Preventive, 3 weeks after anti-FxIA 134.4 40.4 SO (9) 96.4 l5.3a FO (5)

Glomerular POE2 pg/mglmln (0

00

Therapeutic, 8 weeks after randomization to diet 192.6 106.5 Lard (7) SO (7) FO (8)

180.1 114.1

94.0c 55,7J

99.9 64.0

58.6

219.9 151.6 50.5

146.0 46.6c

113b

17.4e

H

0 0

Number studied is in parentheses. Data are means SD. a p < 0.1; b P < 0.5; Student's 1-test P = NS vs. lard;" P NS vs. lard and SO; C p < 0.05 vs. lard and

0 0

SO; ANOVA

0

nine were seen in the fish oil and safflower oil groups in the therapeutic study, but the level was significantly higher in the group on a lard diet (Table 5).

Fig. 5. Glomerular prostaglandin E2 production in the early heterologous phase (preventive study). Symbols are: (EJ) PHN group (7); () control group (3). Numbers in parenthesis represent number of animals. Values are mean SE. * P < 0.05 (FO vs. SO; both PHN and control). Table 4. Effect of dietary lipids on renal hemodynamics Inulin clearance

Effective RPF mi/mm

SO (4) 0.724 FO (4) 0.911

0.115 1.896

0.411 39,93

4.59

MAP mm Hg 98.4

354a 101.8

6.0

35a SE. Numbers of animals are in parentheses.

0.l39a 2.833

Values are mean

Filtration fraction %

0.406" 32.55

Abbreviation is MAP, mean arterial pressure. a Not significant P < 0.05 (FO vs. SO)

,

Effect of dietary lipids on plasma lipids and blood pressure The effect of dietary lipids on the plasma lipid profile is shown in Table 6. Plasma cholesterol and triglycerides were consistently lower in the fish oil group in both studies, but only the triglyceride level in the therapeutic study reached statistical significance. In the preventive study, mean arterial pressure of anesthetized rats in the early autologous phase was not different in the safflower and fish oil groups (Table 4). Similarly, systolic blood pressure was not significantly different in awake rats at

any time point after randomization in the therapeutic study 14.9, N = 8; SO: 140 14.2 mm Hg, N = 7; P = NS).

(after 8 weeks FO: 134 lard:

143

14.7, N

7;

Morphologic studies

Immunofluorescence at the end of both the preventive and therapeutic studies showed moderate granular staining of capillary loops for rat IgG and sheep IgG and trace staining for rat pressure was similar in the two groups. Renal function was also C3. There were no apparent differences between the fish oil evaluated from serial measurements of serum creatinine and group and the two control groups. Staining for CSb-9 was representative results are shown in Table 5. In the preventive present and of similar intensity and distribution in all animals study, the serum creatinine was elevated by about 35% in both from both studies (Fig. 6). Similar light microscopic changes the fish oil and safflower oil groups reflecting the reduced inulin

were seen in the glomeruli of all nephritic groups in both

clearance observed earlier. Similar increases in serum creati- studies. The glomeruli were uniformly enlarged, the capillary

Weise et al: N-3 fatty acids in membranous nephritis

365

deposited in glomeruli. However, because immunofluorescence is not a quantitative technique, we cannot exclude the possibility that omega-3 substitution of target cells reduces the assembly of C5b-9 in the plasma membrane or accelerates its removal. These findings contrast with those of Scharschmidt et al [28], who found that the benefits of fish oil on proteinuria and GFR in

an accelerated autologous phase model of rat nephrotoxic nephntis were accompanied by reduced humoral immunity to, and slower immune elimination of, the injected antiserum. On maintained on fish oil (a) and safflower oil (b)for four weeks before and the other hand our studies agree closely with those by Rahman, seven days after anti-FxIA injection (x 400). Sauter and Young [29], who showed that pretreatment with fish oil reduced the development of proteinuria, inhibited glomerular eicosanoid production, and preserved renal function without loops were dilated and there was no hypercellularity. A mod- qualitatively altering glomerular immune deposits or host imerate interstitial infiltrate was present in all nephritic groups and mune response in a planted antigen model of membranous was not affected by dietary lipid content. nephropathy. Together these two studies of membranous neTo determine if there was an ultrastructural basis for the phropathy show that despite an equivalent degree of sublethal differences in proteinuria, four glomeruli from three separate glomerular epithelial cell injury, fish oil therapy ameliorates rats from each group were examined by electron microscopy in proteinuria, the major functional manifestation of this lesion. the preventive study. All animals displayed the same changes in Our results differ from those of Stahl et al [4] and Rahman et al glomerular capillary wall architecture. The most prominent [29] in one respect: we found no increase in glomerular POE2 in abnormal features were the presence of numerous subepithelial PHN rats on the omega-6 diet. While there is no obvious electron dense deposits and extensive foot process effacement explanation for this difference, it may have to do with differ(Fig. 7 A and B). These changes were found in each glomerulus ences in the type and content of lipids in the control diets. and appeared to occur with the same general frequency and The most original and important result was obtained in the severity in both groups of animals (FO and SO). In addition, therapeutic study. When rats with severe, well-established blebbing of the surface membrane and intracellular absorption proteinuria were randomized to receive saturated fat (lard) or droplets were seen in the podocytes in both groups. In all cases, two unsaturated fat diets (omega-3 or omega-6), urine protein the endothelial cells appeared normal and there were no inflam- excretion in those fed omega-3 fell by about 50% within two matory cell infiltrates or evidence of fibrin deposition within any weeks and remained significantly lower than in those fed lard or of the glomeruli examined. safflower oil for the duration of the study. Again the differences in urine protein excretion could not be accounted for by protein Discussion or calorie deprivation, or by differences in blood pressure, renal The principal findings of our study are that the development function, immune response to sheep IgG or glomerular IgO or of proteinuria is substantially inhibited and established protein- complement deposition. As in other studies of PHN in rats fed uria is remarkably lowered when rats with PHN are fed a diet standard rat chow [24, 30], we found that GFR was reduced by enriched with fish oil. To the best of our knowledge, this is the about 30 to 40% during the autologous phase in rats on fish oil first report of a therapeutic effect of fish oil in experimental or safflower. Interestingly, rats fed lard in our study experiglomerulonephritis. The effects of fish oil are likely due to the enced an even larger increase in serum creatinine than those fed incorporation of omega-3 fatty acids into the membrane phos- safflower or fish oil. The explanation of this finding is presently pholipid fraction of glomerular cells as shown by a 10- to 20-fold unknown. increase in the total glomerular omega-3 fatty acid content The role of lipid mediators in the pathogenesis of proteinuria compared to control diets and a reduction in glomerular AA in this model is incompletely understood. Antibody-induced content of about 50%. These and similar findings by others C5b-9 assembly stimulates glomerular eicosanoid synthesis, [25—27] indicate that four weeks of dietary fish oil is sufficient to especially TxA2 [4, 5], but pharmacologic inhibition of cyclooxinduce near maximal glomerular membrane phospholipid fatty ygenase and thromboxane synthetase in PHN has had variable acid substitution. effects on proteinuria. In an isolated perfused rat kidney model Dietary omega-3 fatty acid supplementation provided marked of PHN in which proteinuria was shown to be due to C5b-9but incomplete protection against the development of protein- induced GEC injury [31], Cybulsky et al showed that high-grade uria. The beneficial effect was first evident during the heterol- inhibition of thromboxane synthetase with OKY-046 reduced ogous phase and persisted for at least six weeks, that is, well proteinuria by about 60% [5]. On the other hand, Stahl and into the autologous phase. The effects of fish oil feeding during colleagues reported that 80 to 90% inhibition of C-induced the most proteinuric phase (2 weeks after antibody) appeared glomerular TxA2 production with UK-38485 in vivo was not also to be reflected in a less severe fall in serum albumin and in accompanied by a decrease in proteinuria [4]. Not surprisingly, Fig. 6. Representative glomeruli stained for C5b-9 from PHN rats

a more modest weight gain. The protection could not be Zoja, et a! also found that lesser degrees of thromboxane explained by group differences in glomerular antibody binding,

synthetase inhibition (40%) were without effect on proteinuria,

blood pressure or renal function, or by protein or calorie but they showed a substantial reduction in proteinuria after deprivation. Moreover, dietary omega-3 fatty acid supplemen- cyclooxygenase inhibition with indomethacin [32]. Since ultratation did not inhibit the autologous immune response to sheep structural studies in our current study showed equivalent GEC

IgG or limit the amount of immunoreactive C3 or C5b-9 injury in all dietary groups, it appears that the effect of

366

Weise et

a!: N-3 fatty acids in membranous nephritis 1.

us

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—

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'.•. .':.,,'?-.--. i-:-

----'(S. tcr ,

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r Fig. 7. Representative electron micrographs of glomerular capillary walls from PHN rats maintained on fish oil (a) and safflower oil (b) for four weeks before and seven days after anti-FxIA injection. Prominent subepithelial electron-dense deposits (arrows) are present in both groups of animals. Broad foot processes (*), containing dense accumulation of cytoskeletal elements, are also seen in the epithelial podocytes (Ep). The endothelial layer (En) is normal and no inflammatory cells are present in the capillary lumens (CL). Abbreviations are: RBC, erythrocyte; US, urinary space. a and b x 21,000. Scale bar = 1 m.

thromboxane (or other diene-lipid mediators) on glomerular TxA2 that is generated [33]. Thus, fish oil blocks both the permselectivity in PHN may be to exaggerate the sieving defect generation and action of TxA2. [241 caused by antibody and complement [5, 30].

Since omega-3 fatty acid substitution has multiple effects, it is

The present study supports this view. Fish oil completely appropriate to consider other mechanisms that might account inhibited the increase in glomerular TxA2 production seen after six days in PHN rats on safflower oil and substantially attenuated proteinuria. To understand why fish oil is more effective than thromboxane synthetase inhibitors in vivo it is important to

for the beneficial action of fish oil in PHN. Marine oils also alter

the production of peptide (including interleukin 1 and tumor necrosis factor) and other lipid mediators (LTB4) of inflamma-

tion [10, 29, 34]. As yet there is no evidence that peptide

appreciate that EPA and DHA have at least two effects with mediators have any role in human or experimental membranous regard to thromboxane. First, they divert thromboxane down nephropathy. Although Lianos et al noted that LTB4-producing the less inflammatory triene pathway leading to TxA3 instead of leukocytes populate the glomeruli of PHN rats during the first TxA2. Second, TxA is an effective competitive inhibitor of any few hours after antibody injection, neither he nor Rahman et al

Weise et al: N-3 fatty acids in membranous nephritis

found evidence that LTB4 contributes to proteinuria in experimental membranous nephropathy [29, 35, 36]. On the other hand, it was recently reported that LTD4, presumably derived

367

lary pressure is instrumental in modulating the magnitude of proteinuria in PHN [24] and is a major factor in the pathogenesis of glomerubosclerosis in other disease models, there is good

from infiltrating monocytes, contributes to proteinuria and

reason to believe that lowering plasma lipids in PHN may be alters glomerular hemodynamics in the early autologous phase favorable. of PHN [37]. These findings correspond to a report by Hara et In summary, it appears likely that the beneficial preventive al who found that monocytes contribute to proteinuria at a and therapeutic effects of dietary omega-3 supplementation on similar stage in PHN [38]. Thus, although we have never found proteinuria in PHN are the result of a complex interplay evidence of leukocyte involvement in early PHN [1, 2, 21], it is between altered inflammatory, hemodynamic and metabolic possible that fish oil might act in the autologous phase by factors. Whatever the mechanism, dietary omega-3 supplemeninhibiting the production of 4-series leukotrienes by monocytes. tation may offer a safe therapeutic strategy to diminish proteinIt is also worth relating our observations to those of other uria in patients with membranous nephropathy who have severe investigators who have used a diet deficient in essential fatty and intractable edema. Additional studies are necessary, howacids (EFAD) to study the role of inflammatory cells in various ever, to assess the long-term efficacy and safety of omega-3 forms of renal injury [39, 40]. Although both omega-3 substitu- fatty acids on renal function and hyperlipidemia in nephrotic tion and EFAD deplete cellular phospholipids of omega-6 fatty subjects, and to determine if this dietary manipulation is able to acids and reduce TxA2 and LTB4 production, the benefits of ameliorate or delay the progressive nature of this disease and its EFAD, at least in nephrotoxic nephritis, appear to be due to consequences in humans. decreased production of a novel lipid chemoattractant and depletion of resident or infiltrating macrophages and not to an Acknowledgments effect of eicosanoids [41].

Proteinuria in PHN results from GEC injury caused by formation of the C5b-9 complex. Effacement of foot processes,

villous transformation and vesiculation of the plasma membranes are the major features of this sublytic injury. We have described the in vitro correlates of these morphologic alterations of GECs [42] and shown them to be associated with changes in levels of intracellular messengers. In cultured rat GECs, antibody-directed formation of C5b-9 results in Ca2 influx and mobilization of Ca2 from intracellular stores as a consequence of phospholipase C (PLC) activation and inositol triphosphate (1P3) production [2]. Furthermore, free AA is released by PLA2, and TxB2 and PGFSa are generated and feed

This study was presented at the 22nd annual meeting of the American

Society of Nephrology, December 3, 1989 and published in abstract form in Kidney mt 37:437, 1990. This work was supported by research grants DK30932 (DJS) and DK34972 (DRA), an institutional training grant DK07053 (WJW, JSL and ECM) from the National Institute of Diabetes, Digestive and Kidney Diseases, an institutional BGRS award from The University Hospital (WJW). Dr. Abrahamson is a recipient of an Established Investigator Award from the American Heart Association. The authors are grateful for the assistance of Ann Tercyak of the

Boston University Biophysics Department. Dr. Weise's present address is: Lehrstuhl fur Innere Medizin-Nephrologie, Universität Erlangen-Nurnberg, Kontumazgarten 14-18, 8500 Nurnberg 80, Germany.

Reprint requests to Dr. David J. Salant, Renal Section (E428), The

back to further enhance PLC activity [2]. These events may University Hospital, 88 E. Newton Street, Boston, Massachusetts lead to altered GEC function and contribute to impaired gb- 02118, USA. merular permselectivity in PHN, however, our recent work References suggests that they contribute to the survival of sublethally injured GECs as well [43]. Thus, incorporation of omega-3 fatty

acids into GEC plasma membrane phospholipids might alter these cell signaling mechanisms and facilitate more efficient C5b-9 shedding from the cell surface, thereby providing enhanced protection to the injured GEC. Alternatively, altered membrane fluidity [44, 45], impaired CSb-9 insertion into the plasma membrane, or improved ability of GEC to repair C5b9-induced injury may provide additional mechanisms by which omega-3 incorporation exerts its favorable effect on the impaired gbomerular filtration barrier.

An added benefit of fish oil in this study was its ability to lower plasma lipids. Whether this effect was due to a direct action on hepatic lipoprotein synthesis or an indirect consequence of reduced proteinuria cannot be determined from this work. Nevertheless, this outcome could have long range advan-

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