Oxidative stress occurs in absence of hyperglycaemia and inflammation in the onset of kidney lesions in normotensive obese rats
Bruno Poirier1,
Marilyne Lannaud-Bournoville1,
Marc Conti2,
Raymond Bazin3,
Odile Michel1,
Jean Bariéty1,
Jacques Chevalier1 and
Isaac Myara1,4
1 INSERM U 430, Broussais Hospital, and Claude Bernard Association, Paris,
2 Laboratory of Biochemistry, Bicêtre Hospital,
3 INSERM U 465, Institut des Cordeliers, Paris and
4 Laboratory of Applied Biochemistry, Faculty of Pharmaceutical and Biological Sciences, Châtenay-Malabry, France
Correspondence and offprint requests to:
Dr Jacques Chevalier, Immunopathologie Rénale et Vasculaire, INSERM U 430, Hôpital Broussais, 96 Rue Didot, F-75674 Paris CEDEX 14, France.
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Abstract
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Background. Several factors favour the development of kidney lesions. We examined the role of oxidative stress in the onset of renal alterations that occur in Zucker obese (ZO) fa/fa rats.
Methods. Kidney structure, biological data, glycation parameters, advanced glycation end products (AGE), thiobarbituric acid-reactive substances (TBARS), circulating antibodies anti-malondialdehyde (MDA)-modified low-density lipoprotein (LDL), antioxidant defenses (Cu/Zn and Mn superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) activities, glutathione level), were determined in plasma and/or kidney of young and old ZO rats and lean (ZL) Fa/fa littermates.
Results. Renal lesions and functional decline appeared at 3 months in hyperlipidaemic, hyperinsulinaemic, normotensive ZO rats, independently of any macrophage-ED1+-cell infiltration. At 6 months and thereafter, kidney lesions and functional impairment worsened while numerous ED1+-cells invaded the interstitium. At 3 and 9 months, TBARS level in the LDL/very low-density lipoprotein fraction and in the kidney was higher in ZO than in ZL rats. Anti-MDA-LDL antibodies were increased in ZO rats. At 3 months, renal activity of Cu/Zn SOD was higher, and activities of catalase and GPx lower in ZO than in ZL rats, leading to an accumulation of hydrogen peroxide (H2O2). At 9 months, a decrease in Cu/Zn SOD activity and an increase in glutathione level were observed. Blood glucose and glycated proteins, as well as AGE in kidney, remained similar in both ZL and ZO rats, whatever their age.
Conclusion. These data suggest that oxidative stress triggers, at an early age, the onset of kidney lesions and functional impairment in ZO rats, in absence of hyperglycaemia, hypertension and inflammation.
Keywords: glomerulosclerosis; hyperinsulinaemia; lipid peroxidation; obesity; Zucker rat
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Introduction
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Several mechanisms may contribute to the onset and/or the progression of kidney lesions that occur in patients or animals suffering from metabolic disorders such as diabetes mellitus, obesity and/or hyperlipidaemia. Among them, lipid peroxidation and oxidative stress, high glucose levels and glycated products, have been frequently proposed.
Lipid peroxidation is initiated when polyunsaturated fatty acids (PUFA), principally located in cellular membranes and lipoproteins, interact with reactive oxygen species (ROS) which include hydrogen peroxide (H2O2), hypochlorous acid (HOCl), nitric oxide (NO) and free radicals such as superoxide anion (O2-), hydroxyl (OH), alkoxyl (RO) and peroxyl (ROO) radicals. In tissues, antioxidant defense, mainly made up of glutathione and antioxidant enzymes, opposes the toxic actions of ROS. Three major enzymes are involved in this defense: the Cu/Zn-dependent and Mn-dependent superoxide dismutases (Cu/Zn-SOD, Mn-SOD), catalase and glutathione peroxidase (GPx). Thus, oxidative stress is present in case of an imbalance between ROS production and antioxidant defense. Such oxidative stress has been demonstrated in obese [1], hyperlipidaemic [2] and diabetic patients and animals [3].
An abnormal glucose metabolism appears to be another key factor in the progression of kidney disease. A high glucose environment could intervene either directly, in stimulating kidney cells as suggested by in vitro experiments, or through the process of glycation, a non-enzymatic reaction of glucose with proteins, which produces Amadori compounds and, eventually, advanced glycation end products (AGE). Glycated proteins or AGE can directly activate glomerular cells. AGE receptors having been shown on rat mesangial cells. Glycated proteins can also favour lipid peroxidation: glucose and fructose lysine, the first Amadori rearrangement products, generate ROS in the presence of trace amounts of iron or copper which act as catalysts. Signal transduction by the AGE receptor appears to involve generation of free radicals [4].
Although hyperglycaemia, non-enzymatic glycation and oxidative stress could intervene in the development of kidney lesions seen in obesity-related disorders such as diabetic nephropathy, the respective roles of these metabolic factors in the genesis of early renal injury is not clearly established, especially as they are most often accompanied by an inflammatory process. Indeed, most of the data on glomerular sclerosis suggested that a release of cytokines by infiltrating inflammatory cells is the key factor which launches the abnormal accumulation of extracellular matrix components. However, recent observations using the Zucker rat model suggested that initial steps of the extracellular matrix remodelling occurred independently of an inflammatory process [5,6]. Thus, in order to distinguish between the factors involved in the genesis of renal structural changes from those driving their progression, we examined in Zucker obese (ZO) rats, the relationship between metabolic factors, oxidative stress, inflammation process and the time-course of renal morpho-functional changes leading to glomerulosclerosis and interstitial fibrosis. The Zucker rat is a useful model of genetic obesity presenting an autosomal recessive mutation of the fa gene encoding the leptin receptor. This strain shows abnormal glucose tolerance and peripheral insulin resistance similar to patients with type II non-insulin dependent diabetes mellitus (NIDDM). ZO rats express, already at weaning, pronounced hyperlipidaemia which worsens with age. They rapidly develop glomerulosclerosis, focal and segmental glomerular hyalinososis (FSGH) and interstitial fibrosis whose gravity increased with age [7]. These lesions occur in the absence of hypertension [7] or renal haemodynamic modification [8]. Male Zucker lean (Fa/fa) (ZL) littermates have normal serum lipids, glucose and insulin and normal renal structure and function [7], and serve as a useful internal control.
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Subjects and methods
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Animals
Male ZL (Fa/fa) and ZO (fa/fa) rats (Dr Raymond Bazin, INSERM U 465 husbandry, Paris, France) were identified and selected at 4 weeks of age by visual examination of inguinal fat deposits. They were raised in standard husbandry conditions, fed regular laboratory chow ad libitum (M25, Extralabo, Provins, France) and had free access to water. For determination of biological parameters, fasting animals were housed individually in metabolic cages with free access to water. Twenty-four-hour urine samples were collected and a blood sample was obtained by orbital sinus punction into tubes containing heparin. At the time of sacrifice at 1, 3, 6 and 9 months, animals were anaesthetized with pentobarbital (i.p., 0.1 ml/100 g body weight) and the kidneys were removed and weighed. At 3 and 9 months blood was also collected from the aorta into ethylene-diamine-tetraacetic-acid (EDTA) containing Vacutainers (Becton-Dickinson, Meylan, France) for lipid peroxidation assays. Animal care complied with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication 86-23, revised 1989, authorization 00577, 1989, Paris, France).
Kidney structure and immunohistochemistry
For histologic examination, kidneys were immediately placed on ice after removal and transverse sections were made at hilum and directly frozen in liquid nitrogen for immunohistochemistry study or fixed in alcoholic Bouin's solution, embedded in paraffin, sectioned (4 µm thick) and stained with Masson's trichrome, for routine histology. Overall tubulo-interstitial injury was defined as tubular dilation, degenerating tubular cells and cellular debris in the lumen, proteinasceous tubular casts, interstitial fibrosis and interstitial inflammatory cell infiltrates. It was graded on Masson's trichrome stained sections on a scale of 04 (0=normal; 0.5=small focal areas of damage; 1=involvement of less than 10% of the cortex; 2=involvement of 1025% of the cortex; 3=involvement of 2575% of the cortex; 4=extensive damage involving more than 75% of the cortex), to form a semi-quantitative index of cortical damage. Stained sections of at least 100 glomeruli were evaluated for the presence of FSGH lesions. The percentage of glomeruli with FSGH was determined for each tissue specimen. The differentiation and activation of interstitial mononuclear cells was assessed by incubating frozen kidney sections from rats aged 1, 3, 6 or 9 months with a mouse monoclonal antibody specific for a monocyte/macrophage cytoplasmic marker (ED1 antibody, Serotec, Oxford, UK), diluted 1:1000 in Tris-buffered saline pH 7.4, containing 0.1% bovine serum albumin (BSA, Sigma Chemical, St Louis, MO, USA) for 60 min at room temperature. The density of lymphocytes in the interstitium and in glomeruli was also estimated in frozen kidney sections of 3-month-old lean and obese rats, using a mouse monoclonal antibody directed against T cell receptors (1:100, 30 min, clone RT3, Serotec). Then, the sections were washed in Tris-buffered saline and incubated with rabbit anti-mouse immunoglobulin antibody (Dako Corporation, Carpinteria, CA, USA) and alkaline anti-phosphatase alkaline complexes (diluted 1:75) (Dako). The enzyme was revealed with freshly prepared Fast Red Substrate System (Dako) containing 0.33 mg/ml levamisole (Sigma) to reduce the staining background. Sections were counterstained with haematoxylin. The number of positive cells either in the glomerulus or in squares of 1.105 mm2 distributed over the interstitium area was counted, with a minimum of 50 glomeruli or 13 squares surveyed per kidney section as defined by convergent analysis. AGE deposits were detected by immunofluorescence on deparaffinized sections of 9-month-old ZL and ZO rats, using a rabbit anti-AGE antibody generously provided by Dr Bakala (Cell Biology Laboratory, University Paris 7, Paris, France), diluted 1:20 in phosphate buffer saline supplemented with 2% fish gelatin (Sigma), and revealed using a FITC-labelled goat anti-rabbit IgG (Cappel, Cachranville, PA, USA). For positive control, kidney sections of 30-month-old Wistar rats were used. Immunolabelled sections were observed under a Leica TCS SP confocal microscope (Leica, Heidelberg, Germany).
Biological parameters
Conscious systemic blood pressure was measured by a tail-cuff system (Ugo Basile Apelex, Varese, Italia). Orbital sinus blood samples were centrifuged at 2500 r.p.m. for 10 min at 4°C and aliquots of plasma were frozen and stored at -20°C. Triglycerides and total cholesterol were determined by the enzymatic and colorimetric GPO-PAP and CHOD-PAP detection kits respectively (Boerhinger, Mannheim, Germany). Glycaemia and plasma and urine creatinine concentrations were measured on a Synchron CX7 Beckman analyser (Beckman, Fullerton, CA, USA). Plasma insulin was measured by radioimmunoassay (CIS, Gif sur Yvette, France) with a rat insulin standard (Novo, Copenhagen, Denmark). Proteinuria was determined using the Coomassie Protein Assay Reagent (Pierce, Rockford, IL, USA) with bovine serum albumin as standard.
Sample preparation for lipid peroxidation parameters
Blood obtained through aortic stick was centrifuged at 4000 r.p.m. for 10 min at 4°C and aliquots of plasma for lipoprotein separation were frozen and stored at -80°C. Plasma, rather than serum was used in order to reduce the release of lipoperoxides during clotting and sucrose (6 g/l, final concentration), was added to plasma aliquots before freezing. Previous experiments indicated that sucrose was a good cryopreservative for low-density lipoprotein (LDL) and did not modify lipid peroxidation parameters. All samples were stored for less than 8 weeks.
For lipoprotein separation, we chose to investigate the combined LDL/very-low-density lipoprotein (VLDL) fraction since rats have a relatively small amount of LDL. KBr (Merck, Darmstadt, Germany) solution (d=1.063 g/ml) (35 ml) containing 1 mM EDTA was carefully added to 57 ml of plasma previously adjusted to a density of 1.063 g/ml with solid KBr in 10 ml centrifuge tubes (Beckman, Gagny, France). Samples were centrifuged for 18 h at 4°C in a Beckman L8 ultracentrifuge using a 70.1 Ti rotor at 45000 r.p.m. The top fraction (LDL+VLDL) was removed and the remaining plasma was adjusted to a density of 1.21 g/ml with solid KBr and, after having filled the centrifuge tube with KBr (d=1.21 g/ml)/EDTA as above, centrifuged again for 18 h to obtain high-density lipoproteins (HDL) in the upper phase and the remaining non-lipoprotein fraction in the pellet. Isolated lipoprotein and non-lipoprotein fractions were dialysed for 24 h at 4°C against four changes of 10 mM TrisHCl buffer, pH 7.4, containing 1 mM EDTA, and stored at 4°C in the dark for less than 30 days. Tris buffer scavenges reactive chemical species such as hydroxyl radical and EDTA prevents the artifactual elevation of lipid peroxidation products. Protein was measured according to a Peterson assay with bovine serum albumin as standard.
For kidney samples, small fragments (about 250 mg) were rinsed with 0.15 M NaCl containing 1 mM EDTA, frozen and stored in liquid nitrogen for less than 3 weeks. Tissues were homogenized in ice-bath for 30 s with 20 mM TrisHCl buffer pH 7.4 containing 1 mM EDTA (1 ml/100 mg tissue) using an Ultra Turax homogenizer (Janken Kunkel Ika-Werk, Staufen, Germany). The homogenate was centrifuged at 2300 g for 15 min at 4°C and the supernatant was used for TBARS and antioxidant enzyme activities. Tissue protein was determined using the Coomassie protein assay.
Lipid peroxidation parameters
Among indicators used to explore the lipid peroxidation process, thiobarbituric acid-reactive substances (TBARS) assay is the most popular owing to its sensitivity, although it is of relatively low specificity. TBARS content was assessed fluorometrically using 0.1 ml whole plasma, 0.1 mg plasma subfraction protein or 0.3 ml supernatant of tissue homogenate. In order to minimize within-run variations, each sample was performed in triplicate. Samples from each category were assayed at the same time.
Glutathione concentration was determined from frozen kidney samples homogenized in 3 volumes of 5% trichloroacetic acid (Merck), according to the technique of Ellman [9] with 5,5'-dithiobis (2-nitrobenzoic acid) (DTNB) (Sigma) as reagent. The activity of Cu/Zn- and Mn-SOD, catalase and GPx was measured as described by Thérond et al. [10].
Determination of antibodies against MDA-LDL was performed by an ELISA method in 96-well plates coated with MDA-modified human LDL, as previously described [11].
Glycated protein
Two methods based on boronate affinity chemistry were used for measuring the glycated haemoglobin: fully automated assay with the Abbott IMx analyser (Abbott, Laboratories, Abbott Park, IL, USA) and column chromatography (Glycogel II Boronate affinity gel, Pierce Inc). The percentage of glycated haemoglobin was determined according to the manufacturer. The Roche (Neuilly, France) reagent Unimate FRA was used for the colorimetric quantitative determination of plasma glycated protein (fructosamine). Total glycated proteins in plasma were also separated from nonglycated proteins by affinity chromatography on the Glycogel columns used above.
Statistical analysis
Results were expressed as mean±SEM. Statistical analysis was carried out using a two-way ANOVA analysis of variance with age and group (genotype) as factors, followed by Bonferroni-Dunn tests (Statview 5.0 software, Abaccus Concept Inc, Berkeley, CA). The overall age effect, group effect and interaction reached statistical significance if P<0.05. In cases of interaction between the factors, one-factor analysis of variance was used at one level of the other factor.
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Results
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Biological parameters
With age, no significant hypertrophy of the kidneys was observed as shown by kidney:body weight ratio which remained constant with age (ZO vs ZL rats: 3 months 0.59±0.01 vs 0.67±0.01, n=17; 9 months: 0.57±0.01 vs 0.70±0.01, n=17; group effect P<0.0001; age effect P=0.87; interaction P=0.01). However, while the kidney weight increased in proportion to the body in lean rats, it increased less than the body in obese rats. Mean systemic tail-cuff blood pressure remained at a normal range whatever the age and the genotype of the animals (ZO vs ZL rats: 3 months 103±6 vs 108±5 mm Hg, n=6; 9 months 129±3 vs 129±2 mm Hg, n=6; group effect P=0.54; age effect: P<0.0001; interaction P=0.54). As shown in Table 1
serum levels of triglycerides and cholesterol were higher in ZO than in ZL rats as early as 1 month. Triglycerides increased 3.5-fold and cholesterol 1.7-fold in ZO rats between 1 and 9 months. Plasma glucose concentration did not change with age and remained identical in both ZL and ZO groups, except at 3 months where ZO rats showed a weak hypoglycaemia. Plasma insulin rose dramatically in ZO rats between 1 and 6 months and stabilized thereafter. It remained roughly constant and low in ZL rats until 6 months, and doubled between 6 and 9 months. At 6 and 9 months, insulinaemia was respectively 9- and 4.2-fold higher in ZO than in ZL rats. Urinary volumes increased similarly with age in both groups. As compared to ZL rats, proteinuria developed markedly in ZO rats after 3 months. At 6 and 9 months, proteinuria was respectively 6.5 and 12.6 times greater in ZO rats than in lean littermates. Serum creatinine, which increased with age in both groups of animals up to 9 months, remained lower in ZO than in ZL rat. Creatinine clearance was lower in ZO than in ZL rats as early as 1 month. It increased slightly with age in both groups.
Kidney structure
At 9 months, in ZO rat kidneys, more than 20% of the glomeruli presented a FSGH, associated with severe tubular lesions, interstitial fibrosis and inflammatory cells, while age-matched lean littermates maintained a well preserved kidney structure (Figure 1
). As shown in Figure 2A
a few lesions, limited to some tubules showing proteinasceous casts, were detected as early as 3 months in ZO rats (damage incidence 0.35±0.22 arbitray units, n=6). Then, the lesions worsened to affect large areas of the tubulo-interstitial domain at 9 months. FSGH occurred at 6 months onwards in ZO rats (Figure 2B
), in parallel with diffuse glomerular sclerosis, characterized by an expansion of the extracellular matrices, as previously measured by automated image analysis [7]. In ZL rats, as well as in young ZO rats, rare monocytes/macrophages (ED1+ cells) were occasionally seen scattered in the interstitium. In ZO rats, ED1+-cell density increased at 6 months and onwards to overcome 5-fold the ZL value at 9 months (Figure 2C
). As previously shown [7], a few macrophages (12 cells per glomerulus) invaded the glomeruli of both ZO and ZL rats between 15 days and 1 month, slightly more in ZO than in ZL rats. The macrophage density did not increase afterwards. At 3 months, the density of lymphocytes in the interstitial area was similar in both groups of animals (ZO vs ZL rats: 120.5±21.8 vs 126.7±11.2 cells/mm2, P=0.81, n=5 rats/group) and was weak in the glomeruli (ZO vs ZL rats: 0.37±0.05 vs 0.28±0.02 cells/glomerulus, P=0.11, n=5 rats/group).

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Fig. 1. Histological aspect of 9-month-old lean (A) and obese (B) rat kidneys. FSGH as well as tubular and interstitial lesions were commonly seen in obese rats, but not in lean littermates. In close vicinity of sclerosed glomeruli (arrow) and damaged tubules (T), which present cellular debris, tubular cast and cystic formations, mononuclear cells invaded areas of interstitial fibrosis (open arrows). Bar=50 µm.
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Fig. 2. Time-course of histological changes in lean (filled circles) and obese rat kidneys (filled squares). In ZO rats, a few tubulo-interstitial lesions were detected at 3 months. They regularly worsened with age, to affect large areas of the kidney cortex at 9 months (A). FSGH was observed in ZO rats at 6 months and rapidly expanded to affect 25% of the glomeruli at 9 months (B). Interstitial ED1+ cells density markedly increased after 6 months in ZO rats as compared to ZL rats (C). When age/group interaction was significant, * indicates that ZO vs ZL rat values differed at a given age (P<0.05). n=6 rats per group.
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Lipid peroxidation parameters
Whole plasma TBARS were significantly higher in 6- and 9-month-old ZO rats than in ZL littermates, but were similar in both groups of animals at 1 and 3 months (Figure 3
). Thus, we examined TBARS in the plasma fractions (LDL/VLDL, HDL and non-lipoprotein fractions), at 3 and 9 months, that is before and after a difference in whole plasma TBARS between obese and lean rats has occurred. Regardless of the genotype of the animals, the total amount of TBARS was almost exclusively bound to lipoprotein fractions at 3 months, the non-lipoprotein fraction bearing only 0.1% of plasma TBARS. At 9 months, the total lipoprotein fraction accounted for 67 and 87% of whole plasma TBARS in ZL and ZO rats respectively. The concentration of TBARS in LDL/VLDL fraction was higher in ZO than in ZL rats at 3 and 9 months (group effect P=0.007) (Figure 4
). It increased between 3 and 9 months in both groups of animals, more in the ZO than in ZL littermates. In the HDL fraction (Fig. 4
), TBARS concentration was similar in 3-month-old ZL and ZO rats. It rose 2.4- and 4.3-fold thereafter in ZL and ZO rats respectively. In the non-lipoprotein fraction, TBARS concentration was significantly higher in 9-month-old ZO rats than in aged-matched ZL rats (ZO vs ZL rats: 3 months 4.3±0.4 vs 3.8±0.3 pmol/l, n=6; 9 months 8.2±0.6 vs 5.4±0.6 pmol/l, n=6) (Figure 4
). The increment in TBARS concentration in LDL/VLDL fraction was also reflected by the amount of circulating antibodies directed against MDA-modified LDL, which were detected in ZO rats as early as 1 month (Figure 5
). TBARS in kidney tissue were higher in ZO than in ZL rats at 3 and 9 months. They increased with age in both groups (Table 2
). The antioxidant defense status in the kidney is depicted in Table 2
. At 3 months, the kidney activity of Cu/Zn SOD was higher and the activities of catalase and GPx were lower in ZO than in ZL kidney. At 9 months, the Cu/Zn-SOD activity failed in both groups, more in ZO than in ZL rats. Glutathione concentration, low and similar in 3-month-old ZL and ZO rat kidneys, increased between 3 and 9 months to exceed in ZO rat kidney 1.6-fold the age-matched lean littermate value.

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Fig. 3. Age-dependant whole plasma TBARS concentration in lean and obese rats. After a decline during the first months of life, TBARS increased in ZO rats (filled circles) but remained roughly constant in ZL rats (filled squares) after 3 months. * indicates when age/group interaction was significant, ZO vs ZL rat values differed at a given age (P<0.05). n=9 rats per group.
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Fig. 4. TBARS in LDL/VLDL, HDL and non-lipoprotein plasma fractions of 3- and 9-month-old lean and obese rats. In the LDL/VLDL fraction, TBARS concentration was significantly higher in ZO rat (filled bars) as early as 3 months. It increased thereafter in both groups of animals, more in ZO than in ZL group (open bars). In the HDL fraction, TBARS concentration was similar in 3-month-old ZL and ZO rats and increased thereafter in both groups, more in ZO than in ZL rats. In the non-lipoprotein fraction, TBARS concentration was significantly increased in 9-month-old ZO rats. When age/group interaction was significant, * indicates that ZO vs ZL rat values differed at a given age (P<0.05). n=6 rats per group.
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Fig. 5. Circulating antibodies directed against MDA-modified LDL. The level of circulating anti-MDA-LDL antibodies, estimated as MDA-LDL/LDL binding ratio, were significantly increased at all ages in ZO (filled bars) as compared to ZL (open bars) rats. n=at least 10 rats per group (10<n>19).
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Glycated protein and AGE
Glycated haemoglobin, fructosamine and plasma glycated protein data are shown in Table 3
. No difference was observed between ZO and ZL rats except for fructosamine level which was lower in 3- and 9-month-old ZO rats. In 9-month-old rat kidney, immunofluorescence staining for AGE was low and limited to the Bowman's capsule and to the tubular basement membranes (TBM), with no difference between ZL and ZO rats (Figure 6
). Conversely, AGE were detected intensively in the glomerular extracellular matrices as well as in TBM of 30-month-old Wistar rat kidney.

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Fig. 6. Immunodetection of AGE in 9-month-old ZL (A) and ZO (B) rat kidney. In aged Zucker rat kidney, AGE were not detected in the glomerular tuft or in the interstitial area, the immunofluorescence being limited to the Bowman's capsule and the tubular basement membranes, with no difference between the two groups of animals. Conversely, the glomerular tuft of 30-month-old Wistar rat kidney, used as positive control, showed intensive fluorescence staining (C). Bar=50 µm.
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Discussion
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As shown in this study, ZO rats presented first signs of tubular alterations in absence of any macrophage or lymphocyte interstitial infiltration as early as 3 months. These alterations rapidly worsened into severe tubular lesions and interstitial fibrosis, while a 4-fold increase in macrophage density occurred between 6 and 9 months. This increase in interstial macrophage density is probably associated with an overexpression of osteopontin in the kidney cortex (Poirier et al., submitted paper). In glomeruli, although a small increase in macrophage density was detected at weaning in both lean and obese pups, this density remained low and did not increase after 1 month of age, whereas measurable expansion of mesangial matrix [7] and first FSGH (Figure 2
) were detected at 6 months onwards in ZO, but not in ZL, rat kidneys. Thus, the onset of glomerular and interstitial fibrosis appears to be independant of any inflammation process in ZO rat kidney. If macrophages do not trigger interstitial fibrosis, they most likely worsen the interstitial lesions once initiated, as already suggested in several experimental models (reviewed in reference [12]). Similarly, these renal changes have not been triggered by either high plasma glucose levels or systemic hypertension since glycaemia and systemic blood pressure remained normal throughout life in these ZO rats. By contrast, concomitant with the first appearance of kidney lesions and renal impairment, metabolic breakdown products of lipid peroxidation were found at 3 months in the LDL/VLDL fraction of ZO blood plasma as well as in ZO kidney homogenates. This peroxidation process worsened between 3 and 9 months and was accompanied by serum antibodies against MDA-modified LDL and a change in the kidney antioxidant defense.
The metabolic origin of plasma lipoproteins modified by lipid peroxidation products is a matter of speculation. One could suggest a direct oxidation of lipoproteins by ROS. This oxidation would take place in tissues rather than in plasma where high levels of antioxidants might prevent it. Lipid peroxidation products, such as MDA, could also be directly provided by tissues. They would be subsequently released into the blood circulation and distributed into plasma components, especially the LDL/VLDL fraction.
Several hypotheses may be put forward to explain the ROS accumulation in tissues: high glucose environment and subsequent glycation, hyperinsulinaemia, overconcentration of catalytically-active transition metals, development of an inflammatory process and overproduction of ROS and lipid peroxidation products from resident cells. First, in a high-glucose environment, proteins and lipoproteins trapped within tissues can undergo glycation to produce ROS and lipid peroxidation products [13]. However, the rats we used in this study remain normoglycaemic all life long, with no change in the levels of glycated proteins and AGE. Even more, fructosamines, that indicate the level of glycation of plasma proteins (especially albumin) were significantly lower in ZO than in ZL rats. This is probably the effect of a proteic hypercatabolism and/or a tendency to low glycaemia in obese rats. Thus, the glucose and associated product hypothesis has to be turned down. Second, insulin itself could induce the production of hydrogen peroxide in some tissues [14]. Since the ZO rat is characterized by early spontaneous development of hyperinsulinaemia, insulin could be a potential source of ROS production in this strain of rats. Third, transition metals (copper and iron) catalytically activate the oxidation of PUFA. An enhancement in plasma transition metal concentration has been noted in other diabetic animal models and in diabetic patients showing complications [15]. A 67 and 47% increase in copper and iron concentrations respectively, was observed in 1718-week-old ZO rats as compared with age-matched lean littermates [16]. However, it is unknown whether metals are present in a form able to catalyse the oxidation of lipids. Fourth, ROS can also be generated within the kidney by macrophages and polymorphonuclear leucocytes. In inflammatory cells, different sources of ROS have been suggested [17]. Among them, the eicosanoid synthetic pathway, which is involved in the formation of inflammatory mediators, also produces lipid peroxides. Another source is linked to the activity of the nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase. Upon activation of macrophages and polymorphonuclear leucocytes, plasma membrane NADPH oxidase leads to the formation of a superoxide radical that is converted into peroxide hydrogen and hydroxyl radical. Another possible source is lysosomal myeloperoxidase which oxidizes peroxide hydrogen to form hypochlorous acid, a strong oxidant capable of reacting with many biological molecules. Since inflammatory cells were rarely detected in the kidney cortex of young ZO rats, such a source of ROS is unlikely in young 3-month-old rats and other origins must be considered. However, as monocytes/macrophage density markedly increased in ZO rat kidney interstitium between 6 and 9 months, this source of ROS in kidney cortex probably occurs in ZO rats after 6 months, and might worsen the fibrosis process. The mechanism of activation of these infiltrating cells is still unclear and might be related, in particular, to hyperlipidaemia. In vitro incubation of triglyceride-rich emulsions with leucocytes enhances ROS release by these cells [18]. Fifth, it is highly possible that in the ZO rat kidney, the major source of ROS might be provided by resident cells, as a result of an imbalance between a ROS overproduction and the antioxident defense. The increase in kidney activity of Cu/Zn-SOD measured at 3 months, associated with the decrease in activities of catalase and GPx, brings on an accumulation in H2O2. The decrease in Cu/Zn-SOD activity which was observed in 9-month-old ZO rats is, at least in part, due to this H2O2 accumulation. As shown by Hodgson and Fridovich [19], the rate of Cu/Zn-SOD inactivation is directly dependent upon the concentration of both H2O2 and the enzyme. A decrease in SOD synthesis is also conceivable [20]. The increase in glutathione concentration, observed in 9-month-old ZO rats, could be an adaptative response to oxidative stress as it occurs in the lungs of rats exposed chronically to cigarette smoke [21]. Several lines of evidence support the concept that glomerular cells serve as source of ROS in non-inflammatory forms of kidney diseases, as shown by ex vivo experiments on freshly isolated glomeruli and in vitro studies on cultured mesangial cells and glomerular epithelial cells. Resident cells such as glomerular mesangial cells, either inherently or upon activation by LDL and/or insulin, oxidize native lipoproteins [22]. These newly oxidized LDL, as well as native LDL, would activate mesangial cells. For instance, they stimulate gene expression of collagen and other extracellular matrix components in mesangial cells, as recently observed by Lee et al. [23]. This leads to an autocrine amplification of the processus of sclerosis.
In conclusion, as shown in this study, the onset of renal lesions and impairment takes place in normotensive ZO rats very early, between 3 and 6 months. This is in the absence of hyperglycaemia and infiltrated inflammatory cells, but in the presence of an oxidative stress evidenced by modified antioxidant enzyme activities, lipid peroxidation product accumulation and plasma anti-MDA-LDL antibodies increase. This suggests that lipid peroxidation in the kidney tissue, as well as modification of the circulating LDL/VLDL fraction, are probably involved in the onset of kidney lesions in this normoglycaemic rodent model of obesity.
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Acknowledgments
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Parts of this work were presented in abstract forms at the XIth International Symposium on Atherosclerosis (Paris, October 1997) and at the 30th Annual Meeting of the American Society of Nephrology (San Antonio, November 1997). We acknowledge the technical assistance of Theano Irinopoulu in confocal microscopy, Michel Paing in photography, Morgane Galand (Ecole Nationale de Chimie, Physique Biologie, Paris) who participated in the detection of circulated antibodies directed against MDA-modified LDL and Anh-Thu Gaston and Marie-France Belair. We thank Dr Srinivas Kaveri (INSERM U 430) for helpful discussions during the preparation of the manuscript.
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Received for publication: 22. 3.99
Accepted in revised form: 16.11.99