Soy protein diet ameliorates renal nitrotyrosine formation and chronic nephropathy induced by...

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Life Sciences 74 (2004) 987–999

Soy protein diet ameliorates renal nitrotyrosine formation and

chronic nephropathy induced by puromycin aminonucleoside

Jose Pedraza-Chaverrıa,*, Diana Barreraa, Rogelio Hernandez-Pandob,Omar N. Medina-Camposa, Cristino Cruzc, Fernanda Murguıad, Cesar Juarez-Nicolasc,

Ricardo Correa-Rotterc, Nimbe Torresd, Armando R. Tovard

aDepartment of Biology, Faculty of Chemistry, Universidad Nacional Autonoma de Mexico (UNAM),

04510, Mexico, D.F., MexicobDepartment of Pathology, Instituto Nacional de Ciencias Medicas y Nutricion ‘‘Salvador Zubiran’’,

14000, Mexico, D.F., MexicocDepartment of Nephrology, Instituto Nacional de Ciencias Medicas y Nutricion ‘‘Salvador Zubiran’’,

14000, Mexico, D.F., MexicodDepartment of Physiology of Nutrition, Instituto Nacional de Ciencias Medicas y Nutricion ‘‘Salvador Zubiran’’,

14000, Mexico, D.F., Mexico

Received 10 April 2003; accepted 15 July 2003

Abstract

It has been shown that reactive oxygen species are involved in chronic puromycin aminonucleoside (PAN)

induced nephrotic syndrome (NS) and that a 20% soy protein diet reduces renal damage in this experimental

model. The purpose of the present work was to investigate if a 20% soy protein diet is able to modulate kidney

nitrotyrosine formation and the activity of renal antioxidant enzymes (catalase, glutathione peroxidase, Cu,Zn- or

Mn-superoxide dismutase) which could explain, at least in part, the protective effect of the soy protein diet in rats

with chronic NS induced by PAN. Four groups of rats were studied: (1) Control rats fed 20% casein diet, (2)

Nephrotic rats fed 20% casein diet, (3) Control rats fed 20% soy protein diet, and (4) Nephrotic rats fed 20% soy

protein diet. Chronic NS was induced by repeated injections of PAN and rats were sacrificed at week nine. The soy

protein diet ameliorated proteinuria, hypercholesterolemia, and the increase in serum creatinine and blood urea

nitrogen observed in nephrotic rats fed 20% casein diet. Kidney nitrotyrosine formation increased in nephrotic rats

fed 20% casein diet and this increase was ameliorated in nephrotic rats fed 20% soy protein diet. However, the soy

protein diet was unable to modulate the antioxidant enzymes activities in control and nephrotic rats fed 20% soy

protein diet. Food intake was similar in the two diet groups. The protective effect of a 20% soy protein diet on renal

0024-3205/$ - see front matter D 2003 Elsevier Inc. All rights reserved.

doi:10.1016/j.lfs.2003.07.045

* Corresponding author. Tel./fax: +52-55-5622-35-15.

E-mail address: [email protected] (J. Pedraza-Chaverrı).

J. Pedraza-Chaverrı et al. / Life Sciences 74 (2004) 987–999988

damage in chronic nephropathy induced by PAN was associated with the amelioration in the renal nitrotyrosine

formation but not with the modulation of antioxidant enzymes.

D 2003 Elsevier Inc. All rights reserved.

Keywords: Soy protein diet; Nephropathy; Nephrotic syndrome; Aminonucleoside; Nitrotyrosine; Antioxidant enzymes

Introduction

Reactive oxygen species (ROS) such as superoxide anion, hydroxyl radical and hydrogen peroxide

are involved in several renal diseases including experimental nephrotic syndrome (NS) induced by

puromycin aminonucleoside (PAN) (Baliga et al., 1999; Haugen and Nath, 1999; Nath and Norby,

2000; Shah, 1995; Ueda et al., 2001). Mammalian cells have a complex antioxidant system to detoxify

ROS produced as consequence of normal metabolism (Halliwell and Gutteridge, 1999; Nordberg and

Arner, 2001). An essential component of this system includes the antioxidant enzymes that work in

concert to catalytically scavenge free radicals. Highly reactive superoxide anions are rapidly converted

to hydrogen peroxide (H2O2) by superoxide dismutase (SOD, EC 1.15.1.1, superoxide:superoxide

oxidoreductase), whereas the antioxidant enzymes catalase (CAT, EC 1.11.1.6 H2O2:H2O2 oxidore-

ductase) and glutathione peroxidase (GPx, EC 1.11.1.9, glutathione:H2O2 oxidoreductase) protect cells

against the potentially harmful effects of H2O2 by converting it to H2O and O2 (Nordberg and Arner,

2001).

It has been shown that the overexpression or the exogenous administration of antioxidant enzymes

is useful to ameliorate or prevent the damage induced by ROS (Davis et al., 2001; Yin et al., 2001,

1990; Zhong et al., 2001) including experimental NS induced by PAN (Kawamura et al., 1991;

Nishimura et al., 1995; Ricardo et al., 1994). In contrast, the dietary deficiency of antioxidants or the

inhibition of antioxidant enzymes aggravates the renal damage induced by ROS (Nath and Paller,

1990; Pedraza-Chaverri et al., 1995, 1999). In this context, it has been observed that several

antioxidant agents such as probucol (Lee et al., 1997), vitamin E (Lee et al., 1997; Pedraza-Chaverri

et al., 1995; Trachtman et al., 1995), vitamin E/ascorbic acid (Ricardo et al., 1994), and dimethyl

thiourea (Ricardo et al., 1994) are able to ameliorate or prevent renal damage induced by PAN.

Additionally, it has been shown that the protective effect of some compounds against oxidative

damage is related to their capacity to increase the expression of antioxidant enzymes (Ahlbom et al.,

2001; de Cavanagh et al., 2000; Kawamura et al., 1991; Khaper et al., 1997; Lin et al., 1998; Seth et

al., 2000).

On the other hand, it has been shown that soy protein feeding has a protective effect on the

development of some diseases, some of them dealing with oxidative stress (Anderson et al., 1995; Aoki

et al., 2002; Messina et al., 1994; Velasquez and Bhathena, 2001). Several mechanisms have been

proposed to explain this protective effect (Barnes et al., 2000; Velasquez and Bhathena, 2001). For

example, it has been shown that soy feeding is associated with a decrease in the incidence of several

chronic inflammatory diseases (Barnes et al., 2000). This has been attributed to the presence of genistein

and daidzein, the principal isoflavones or phytoestrogens in the soybean, which have antioxidant

properties (Arora et al., 1998; Hwang et al., 2000; Kapiotis et al., 1997; Sadowska-Krowicka et al.,

1998; Wiseman et al., 2000). Genistein and daidzein can be nitrated by peroxynitrite (ONOO� )

(Boersma et al., 1999) which is a reactive nitrogen specie that is the product of the reaction between

J. Pedraza-Chaverrı et al. / Life Sciences 74 (2004) 987–999 989

superoxide anion and nitric oxide (Radi et al., 2001). Peroxynitrite promotes oxidative damage since it is

able to react with tyrosine residues in proteins forming nitrated proteins (Radi et al., 2001). The ability of

isoflavones to react with peroxynitrite is explained by the fact that they have structural similarities

(phenolic ring) to tyrosine (Boersma et al., 1999). Interestingly, it has been shown that genistein

decreases nitrotyrosine formation in gut inflammation induced by trinitrobenzene sulfonic acid (Sado-

wska-Krowicka et al., 1998). Additionally it has been found that isoflavones are able to modulate the

expression of antioxidant enzymes. For example, genistein is able to induce GPx expression in human

prostate cancer cells (Suzuki et al., 2002) and daidzein is able to increase CAT and GPx mRNAs and to

decrease Mn-SOD mRNA in hepatoma H4IIE cells (Rohrdanz et al., 2002). Furthermore, it has been

found that dietary flavonoids induce the expression of enzymes such as metallothionein (Kameoka et al.,

1999) or NADPH quinone reductase (Wang et al., 1998) which could be involved also in the protective

effects of these compounds.

We have recently shown that 20% soy protein diet improves renal damage and blood lipid

alterations in chronic nephropathy induced by PAN (Tovar et al., 2002). In this work we investigated

if a 20% soy protein diet is able to modulate nitrotyrosine formation and/or the activity of renal

antioxidant enzymes (CAT, GPx, Cu,Zn-SOD, Mn-SOD, and total SOD) which could explain, at least

in part, the protective effect of a 20% soy protein diet in rats with chronic nephropathy induced by

PAN.

Methods

Reagents and diets

Rabbit anti-nitrotyrosine polyclonal antibodies were from Upstate (Lake Placid, NY, USA). Anti-

rabbit Ig horseradish peroxidase antibody was purchased from Amersham Life Sciences (Bucking-

hamshire, England). Xanthine, xanthine oxidase, PAN, glutathione reductase, reduced glutathione

(GSH), nicotinamide adenine dinucleotide (NADPH), diethyldithiocarbamic acid (DDC), 3,3-diamino-

benzidine, and nitroblue tetrazolium (NBT), were from Sigma Chemical Co. (St. Louis, MO, USA).

Commercial kits to measure total cholesterol and triglycerides in serum were from Lakeside Diagnostics

(Mexico City). All other chemicals were reagent grade and commercially available. The composition of

both diets (20% soy protein and 20% casein diet) used has been previously reported (Tovar et al.,

2002). The only difference between both diets is the source of protein (soy or casein). This soy protein

diet contains 1.38, 0.71, and 0.19 mg/g protein of genistein, daidzein, and glycitein, respectively

(Tovar et al., 2002).

Experimental design

Male Wistar rats were obtained from the Experimental Research Department and Animal Care

Facilities at the Instituto Nacional de Ciencias Medicas y Nutricion (Mexico City). Animal studies

were approved by the Institutional Ethics Committee in Animal Experimentation and followed the

guidelines of Norma Oficial Mexicana (NOM-ECOL-087-1995). Four groups of rats were studied

(Tovar et al., 2002) (n = 10 rats per group): (1) control rats fed 20% casein diet (Casein); (2)

nephrotic rats fed 20% casein diet (Casein + NS), (3) control rats fed 20% soy diet (Soy), and (4)


nephrotic rats fed 20% soy diet (Soy + NS). All rats were maintained in individual metabolic cages

and had free access to water. Chronic experimental NS was induced by subcutaneous injections of

PAN: 50, 40, 40, and 25 mg/Kg body weight on weeks 0, 2, 4, and 6, respectively. Control rats were

injected with vehicle (0.9% saline solution). Blood from tail vein and 24-h urine were obtained every

week to perform biochemical determinations. Rats were sacrificed by decapitation 9 weeks after the

first injection of PAN and blood, kidneys, and urine were obtained. Blood serum was obtained and

stored at � 20 jC until the concentration of cholesterol, creatinine, and blood urea nitrogen (BUN)

were measured. One kidney was quickly removed for immunohistochemical localization of nitro-

tyrosine, a marker of endogenous production of peroxynitrite. The other kidney was removed and

stored at � 80jC until the measurement of antioxidant enzymes was performed. 24-h urine was

collected, centrifuged, and frozen at � 80 jC until the urinary excretion of total protein was

measured.

Analytical methods. Creatinine and BUN were measured using a creatinine analyzer model 2 and a

BUN analyzer 2 (Beckman Instruments, Fullerton, CA), respectively. Serum cholesterol was measured

enzymatically according to the instructions of the manufacturer. Total protein in urine was measured as

previously described (Pedraza-Chaverri et al., 1993).

Tissue homogenization. Kidney was homogenized in a Polytron (Model PT 2000, Brinkmann,

Westbury, NY, USA) for 10 seconds in cold 50 mM potassium phosphate, 0.1% Triton X-100, pH = 7.0

(Pedraza-Chaverri et al., 2000a). The homogenate was centrifuged at 19,000 � g and 4jC for 30 min

and the supernatant was separated to measure total protein and the activities of CAT, GPx, and SOD

(total SOD, Mn-SOD, and Cu,Zn-SOD).

Catalase. Renal CAT activity was assayed at 25 jC by a method based on the disappearance of H2O2

from a solution containing 30 mM H2O2 in 10 mM potassium phosphate at 240 nm (Pedraza-Chaverri et

al., 1999). Under the described conditions, the decomposition of H2O2 by CAT contained in the samples

follows a first-order kinetic as given by the equation k = 2.3/t log Ao/Awhere k is the first-order reaction

rate constant, t is the time over which the decrease of H2O2, due to CAT activity, was measured (15 s),

and Ao/A is the optical density at times 0 and 15 s, respectively. The results were expressed in k/mg

protein.

Superoxide dismutase. SOD activity in kidney homogenates was assayed by using a previously

reported method (Pedraza-Chaverri et al., 2000a). A competitive inhibition assay was performed using

xanthine-xanthine oxidase system to reduce NBT. Mixture reaction contains in a final concentration:

0.122 mM ethylenediaminetetraacetic acid (EDTA), 30.6 AM NBT, 0.122 mM xanthine, 0.006%

bovine serum albumin, and 49 mM sodium carbonate. Five hundred Al of tissue homogenate at the

appropriate dilution, were added to 2.45 ml of the mixture described above, then 50 Al xanthine

oxidase, in a final concentration of 2.8 U/l, were added and incubated in a water bath at 27 jC for 30

min. The reaction was stopped with 1 ml of 0.8 mM cupric chloride and the optical density was read at

560 nm. One hundred percent of NBT reduction was obtained in a tube in which the sample was

replaced by distilled water. The amount of protein that inhibited NBT reduction to 50% of maximum

was defined as one unit of SOD activity. Results were expressed as U/mg protein. To measure Mn-

SOD, Cu,Zn-SOD was inhibited with DDC (Pedraza-Chaverri et al., 2001). Therefore, to assay Mn-

SOD activity one sample was incubated with 50 mM DDC at 30 jC for 1 h and then was dialyzed for

three hours with three changes of 400 volumes of 5 mM potassium phosphate buffer (pH 7.8) � 0.1

mM EDTA. Cu,Zn-SOD activity was obtained by subtracting the activity of the DDC-treated samples

from that of total SOD activity.


Glutathione peroxidase. Renal GPx activity was assayed by a method previously described (Pedraza-

Chaverri et al., 1995). Reaction mixture consisted of 50 mM potassium phosphate pH = 7.0, 1 mM

EDTA, 1 mM sodium azide, 0.2 mM NADPH, 1 U/ml of glutathione reductase, and 1 mM GSH. One

hundred Al of the appropriate dilution tissue homogenates or serum were added to 0.8 ml of mixture

and allowed to incubate for 5 min at room temperature before initiation of the reaction by the addition

of 0.1 ml 0.18 mM H2O2 solution. Absorbance at 340 nm was recorded for 3 min and the activity was

calculated from the slope of these lines as Amoles of NADPH oxidized per min taking into account that

the millimolar absorption coefficient for NADPH is 6.22 mM� 1cm� 1. Blank reactions with

homogenates replaced by distilled water were subtracted from each assay. The results were expressed

as U/mg protein.

Histological analysis and immunohistochemical localization of nitrotyrosine

Rats were sacrificed and kidneys were immediately removed and longitudinally sectioned. Thin

slices of kidney tissue with cortex and medulla were fixed by immersion in buffered formalin (pH

7.4), dehydrated and embedded in paraffin. Sections (3 A) were stained with hematoxylin/eosin. For

immunohistochemistry, 3 A sections were deparaffined with xylol and rehydrated with ethanol.

Endogenous peroxidase was quenched/inhibited with 4.5% H2O2 in methanol by incubation for 1.5

h at room temperature. Nonspecific adsorption was minimized by leaving the sections in 3% bovine

serum albumin in phosphate buffer saline for 30 min. Sections were incubated overnight with a 1:700

dilution of anti-nitrotyrosine antibody (Barrera et al., 2003). After extensive washing with PBS, the

sections were incubated with a 1:500 dilution of a peroxidase conjugated anti-rabbit Ig antibody for 1

h, and finally incubated with hydrogen peroxide-diaminobenzidine for 1 min. Sections were

counterstained with hematoxilin and observed under light microscopy. In order to determine an

approximate concentration of nitrotyrosine, quantitative image analysis was performed with a Zeiss

KS 300 Imaging System, Release 3.0 (Hallbergmoos, Germany). This software determines densito-

metric means value of selected tissue regions. Thus, 50 tubular epithelial cells (all with nucleus and

same size), 20 glomeruli and 20 middle size arteries per rat were randomly chosen, and the intensity

of the nitrotyrosine staining was determined. All the sections from the four studied groups were

incubated under the same conditions with the same antibodies concentration, and in the same

running, so the immunostaining was comparable among the different experimental groups. We

normalized the data (arbitrary units) to 1.0 using the normal kidneys from Casein group. As positive

control, kidney sections from rats treated with potassium dichromate was used. We previously

showed that in this experimental model there are strong nitrotyrosine expression (Barrera et al.,

2003).

Table 1

Body weight (g) of the four groups of rats on weeks 0 and 9

Casein Casein + NS Soy Soy + NS

Week 0, n = 10 104 F 2a 95 F 6a 101 F 2a 103 F 2a

Week 9, n = 8–9 373 F 6a 279 F 22b 308 F 16c 211 F 15d

Data are mean F SEM. Groups with different letter are significantly different, p < 0.01.

Table 2

Food intake (g/day) in the four groups of rats studied on weeks 0 and 9

Casein Casein + NS Soy Soy + NS

Week 0, n = 10 15.3F 0.58 13.6F 0.90 14.6F 0.75 15.0F 0.78

Week 9, n = 8–9 20.3F 0.80 20.0F 1.37 19.5F 1.34 20.1F1.83


Statistics

Results are expressed as the mean F SEM. Data were analyzed by two way ANOVA and one

way ANOVA followed by Bonferroni’s multiple comparisons as appropriate using the software

Prism 3.02 (GraphPad, San Diego, CA, USA). A p value less than 0.05 was considered statistically

significant.

Results

Body weight was similar among the four groups of rats on week 0; however, on week 9, the body

weight of the four groups was significantly different (Table 1). Body weight was significantly lower in

Soy group compared to Casein group; however, food intake was similar in these two groups (Table 2), so

the decrease in body weigh was not consequence of changes in food intake.

In addition, chronic nephropathy induced a decrease in body weight in Casein + NS and Soy + NS

groups (Table 1). Food intake did not decrease in both nephrotic groups, except on week 5 where food

intake decreased slightly, but significantly in both nephrotic groups compared to their respective control

groups (Casein and Soy) (data not shown).

Biochemical markers. NS was evident by the proteinuria and hypercholesterolemia (Fig. 1). In

addition, nephrotic rats showed increased circulating levels of creatinine and BUN (Fig. 2). These renal

alterations were significantly diminished in Soy + NS group compared to Casein + NS group (Figs. 1

Fig. 1. Proteinuria (A) and serum cholesterol levels (B) in rats in the four groups of rats studied: (1) Casein, (2) Casein + NS, (3)

Soy, and (4) Soy + NS. Values are mean F SEM, n = 8–10. *p < 0.05 vs. Soy + NS group.

Fig. 2. Serum creatinine (A) and blood urea nitrogen (B) in rats in the four groups of rats studied: (1) Casein, (2) Casein + NS,

(3) Soy, and (4) Soy + NS. Values are mean F SEM, n = 8–10. *p < 0.05 vs. Soy + NS group.


and 2). Circulating levels of cholesterol, BUN, and creatinine were similar in Casein and Soy groups

(Figs. 1 and 2). These data confirmed the beneficial effect of soy on chronic nephropathy previously

published by our group (Tovar et al., 2002).

Kidney antioxidant enzymes in the four groups of rats studied. Total SOD, Cu,Zn-SOD, Mn-SOD,

and GPx activities were unchanged in the four groups of rats studied (Table 3). Catalase activity was

similar in both Casein and Soy groups and it decreased in both Casein + NS and Soy + NS groups

(Table 3).

Kidney histology and expression of renal nitrotyrosine. No histological alterations were observed in

rats fed 20% casein diet (Fig. 3A) or 20% soy protein diet (data not shown). Nephrotic rats fed 20%

casein diet developed segmental mesangial proliferation with matrix expansion, obliteration of capillary

lumens and fibrosis with adhesion between the glomerular tuft and Bowman’s capsule (Fig. 3B). Patches

of mononuclear inflammatory cells were seen in the interstitium, and many cortical tubules showed

epithelial damage (Fig. 3B). In contrast, nephrotic rats fed 20% soy protein had an evident lesser

damage, with small fibrotic or proliferative glomerular lesions in coexistence with normal tubules and

occasional interstitial inflammatory infiltrates (Fig. 3C).

The expression and cellular localization of nitrotyrosine in the kidney were determined by

immunohistochemistry. In Casein + NS group a high expression of nitrotyrosine was seen in proximal

Table 3

Kidney antioxidant enzymes in the four groups of rats studied on week 9

Enzyme Casein Casein + NS Soy Soy + NS

Total SOD, U/mg protein 18.3 F 0.7 17.6 F 0.3 20.4 F 0.7 18.8 F 0.8

Cu,Zn-SOD, U/mg protein 15.0 F 0.7 14.7 F 0.6 17.3 F 0.6 14.9 F 1.2

Mn-SOD, U/mg protein 3.3 F 0.6 2.6 F 0.3 3.1 F 0.2 3.9 F 0.5

Glutathione peroxidase, U/mg protein 0.06 F 0.003 0.07 F 0.004 0.07 F 0.005 0.06 F 0.003

Catalase, k/mg protein 0.3 F 0.01a 0.2 F 0.02b 0.3 F 0.02a 0.2 F 0.01b

Data are mean F SEM. n = 8–9. Cu, Zn-SOD, copper, zinc-superoxide dismutase; Mn-SOD, manganese-superoxide

dismutase. Groups with different letter are significantly different, p < 0.05.

Fig. 3. Representative micrographs of histopathology and nitrotyrosine immunohistochemistry in the kidney from the four

groups of rats studied. There are not histologic abnormalities in rats fed 20% casein diet (A). The kidney from casein + NS

group shows mesangial proliferation, interstitial inflammatory infiltrate and tubular damage (B). In contrast, small area of

proliferative lesions in the glomerulus, with normal interstitium and tubules are seen in nephrotic rats fed 20% soy protein

(C). There is no nitrotyrosine immunostaining in Casein (D) and Soy (E) groups. There is slight immunostaining in Soy +

NS group (F). In contrast, the kidney from Casein + NS group show high nitrotyrosine immunoreactivity in the epithelium of

the proximal tubules (G), capillaries and visceral epithelium from glomerulus (H), and muscular layer of middle size artery,

where the endothelial cells show the highest nitrotyrosine immunostaining (I).(A, B, C, D, E, F, and G = 200� , H and I =

400�).


convoluted tubules epithelium (Fig. 3G), endothelium and podocytes from glomeruli (Fig. 3H), in the

muscle layer of middle size arteries and arterioles, and particularly intense in the endothelium of these

vascular structures (Fig. 3I). Slight nitrotyrosine expression was observed in Soy + NS group (Fig. 3F).

Nitrotyrosine immunostaining was negative (basal staining) in the kidneys of Casein (Fig. 3D) and Soy

(Fig. 3E) groups.

Fig. 4 shows the automated morphometry determination of nitrotyrosine in proximal tubules (Fig.

4A), glomeruli (Fig. 4B), and vascular endothelium (Fig. 4C) from the four groups of rats studied.

Nitrotyrosine content increased 2.8-, 3.3-, and 2.0-fold in proximal tubules, vascular endothelium

and glomeruli, respectively, from Casein + NS group. This increase was partially prevented in

Fig. 4. Nitrotyrosine content in proximal tubules (A), glomeruli (B), and vascular endothelium (C) from the four groups of rats

studied on week 9: (1) Casein, (2) Casein + NS, (3) Soy, and (4) Soy + NS. Values are mean F SEM, n = 8–9. Groups with

different letter are significantly different, p < 0.001.


proximal tubules and vascular endothelium and completely prevented in glomeruli from Soy + NS

group (Fig. 4).

Discussion

We recently showed that a 20% soy protein diet improves renal damage and lipid alterations in

chronic aminonucleoside nephropathy (Tovar et al., 2002). The soy protein diet reduced proteinuria,

hypercholesterolemia, hypertriglyceridemia, VLDL-triglycerides, and LDL-cholesterol observed in


nephrotic rats fed 20% casein (Tovar et al., 2002). Furthermore, the 20% soy protein diet decreased the

incidence of glomerular sclerosis and proinflammatory cytokines (IL-1a, TNF-a, and TGF-h) (Tovar etal., 2002) and ameliorated the decrease in creatinine clearance observed in nephrotic rats fed 20% casein

indicating that the 20% soy protein diet improves not only structural damage but also renal function. The

improvement in lipid alterations and the decrease in proinflammatory cytokines may, undoubtedly,

contribute to the beneficial effect of 20% soy protein to the nephrotic kidney. Taking into account that

soy protein also contains isoflavones which have antioxidant properties (Arora et al., 1998; Hodgson et

al., 1996; Kapiotis et al., 1997; Sadowska-Krowicka et al., 1998; Wiseman et al., 2000), react with

peroxynitrite (Boersma et al., 1999), and are able to modulate some antioxidant enzymes in cell culture

(Rohrdanz et al., 2002; Suzuki et al., 2002) and that ROS have been involved in the pathogenesis of

aminonucleoside nephropathy (Gwinner et al., 1997; Pedraza-Chaverri et al., 1995, 1999; Posadas-

Sanchez et al., 2001; Shah, 1995), in this work we explored if the protective effect of 20% soy protein

diet may be related to the attenuation of nitrotyrosine formation and/or the modulation of the antioxidant

enzymes. According to our previous data (Tovar et al., 2002) we found that the 20% soy protein diet was

able to improve renal function (measured as proteinuria, circulating creatinine, and BUN) and to

ameliorate the high circulating cholesterol levels. Additionally, we found that the 20% soy protein diet

was unable to alter the activity of the antioxidant enzymes Cu,Zn-SOD, Mn-SOD, GPx, and CAT. This

is in contrast with other authors who have found that some isoflavones such as genistein (Suzuki et al.,

2002) and daidzein (Rohrdanz et al., 2002) are able to modulate antioxidant enzymes expression in cell

culture. However, the differences may be due to use of isolated isoflavones versus 20% soy protein diet

in this study and in vitro approaches in cell lines versus complete animal in this study. This may suggest

that the protective effect of 20% soy protein diet on chronic aminonucleoside nephropathy is not

secondary to the enhancement of the enzymatic antioxidant defenses. The increase in antioxidant

enzymes may explain why other compounds protect against oxidative stress; for example it has been

shown that testosterone increases CAT activity (Ahlbom et al., 2001), enalapril and captopril increase

GPx and glutathione reductase activities (de Cavanagh et al., 2000), glucocorticoid increases Cu,Zn-

SOD, Mn-SOD, GPx, and CAT activities (Kawamura et al., 1991), propranolol increases CAT and GPx

activities (Khaper et al., 1997), green tea increases SOD, CAT, and glutathione S-transferase activities

(Lin et al., 1998), and picroliv increases Cu,Zn-SOD content (Seth et al., 2000). Catalase activity

decreased in Casein + NS group and this decrease was not prevented by soy treatment in Soy + NS

group. The decrease in CAT activity in rats with aminonucleoside nephropathy is consistent with

previous reports (Gwinner et al., 1997; Pedraza-Chaverri et al., 1999, 2000b). Nitrotyrosine content

increased significantly in Casein + NS group which is consistent with the oxidative stress that has been

reported in these rats (Gwinner et al., 1997; Lee et al., 1997; Pedraza-Chaverri et al., 1995; Posadas-

Sanchez et al., 2001). To our knowledge, this is the first time that it has been reported the increase in

nitrotyrosine formation in chronic aminonucleoside nephropathy. It has been considered that nitro-

tyrosine is a marker of endogenous production of peroxynitrite, which is readily produced from

superoxide anion and nitric oxide. Interestingly, the 20% soy protein diet was able to ameliorate the

increase in nitrotyrosine formation. This may suggest that the protective effect of 20% soy protein diet is

associated with the amelioration of nitrotyrosine formation in aminonucleoside nephropathy. These data

are in close agreement with the data of Sadowska-Krowicka et al. (1998) who found that genistein

decreases nitrotyrosine formation in gut inflammation induced by trinitrobenzene sulfonic acid. We are

tempting to speculate that isoflavones present in the soy protein diet may be responsible for the decrease

in nitrotyrosine formation in kidney from rats with aminonucleoside nephropathy. Our data suggest that


a 20% soy protein diet may be a useful agent to prevent tissue damage associated with nitrotyrosine

formation.

Conclusion

In conclusion, our data suggest that the decrease in nitrotyrosine content, but not the increase in the

activities of the antioxidant enzymes Cu,Zn-SOD, Mn-SOD, GPx, and CAT, is associated with the

protective effect of 20% soy protein diet in aminonucleoside nephropathy. This may be an additional and

novel mechanism by which soy protein diet has a renoprotective effect in this experimental model.

Acknowledgements

This work was supported by CONACY (34687-M and 34338-M).

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