Conjugated dienes: a critical trait of lipoprotein oxidizability in renal fibrosis
Bruno Poirier1,
Odile Michel1,
Raymond Bazin2,
Jean Bariéty1,
Jacques Chevalier1,,
Isaac Myara1,3 and
with the technical assistance of Anh-Thu Gaston,1
1 INSERM U 430, Broussais Hospital and Claude Bernard Association,
2 INSERM U 465, Institut des Cordeliers, Paris, and
3 Laboratory of Applied Biochemistry, Faculty of Pharmaceutical and Biological Sciences, Châtenay-Malabry, France
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Abstract
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Background. We assessed whether a differential oxidizability of apolipoprotein B (apo B)-containing lipoproteins (LDL and VLDL) may explain the oxidative stress that we had observed at the onset of renal fibrosis in Zucker obese (ZO) rats (Nephrol Dial Transplant 2000, 15: 467476).
Methods. Ex vivo copper-induced oxidation of lipoproteins was performed in 1-, 3-, and 9-month-old ZO and age-matched lean (ZL) rats. LDL/VLDL oxidizability was determined by spectrophotometry at 234 nm by monitoring the formation of conjugated diene hydroperoxides.
Results. A significant increase in lag time (reflecting the resistance to oxidation) was observed in ZO rats at 3 months while the maximal diene production (reflecting the amount of hydroperoxides formed during oxidation) was higher in ZO than in ZL rats as early as 1 month. Lipoproteins were larger in ZO than in ZL rats, as shown by their core to surface component ratio. Furthermore, ZO lipoproteins had increased vitamin E and polyunsaturated fatty acid (PUFA) content, with no change in vitamin E/PUFA ratio.
Conclusions. Rather than oxidizability of apo B-containing lipoproteins, the ability of these molecules to produce high levels of conjugated dienes, which can act as toxic tissue messengers, appears to be a critical trait in the development of renal fibrosis in this rat model of obesity and renal fibrosis.
Keywords: glomerulosclerosis; kidney lesion; LDL/ VLDL; obesity; oxidative stress; Zucker rat
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Introduction
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Although oxidative stress has been implicated in chronic renal failure (for review, see reference [1]), its role in the genesis of early renal injury is still poorly established, especially since oxidative stress is often accompanied by an inflammatory process and other factors such as hyperglycaemia [2]. Despite the frequent association between glomerulosclerosis and atherosclerosis [3], little attention has been paid to the oxidizability of the apolipoprotein B (apo B)-containing lipoproteins, including low-density lipoproteins (LDL) and very-low-density lipoproteins (VLDL), during renal glomerulosclerosis and interstitial fibrosis, when high susceptibility to oxidation of the apo B-containing lipoproteins seemed to be correlated with human coronary heart disease and atherosclerosis [4]. However, these observations are still a subject of debate [5].
Oxidation of lipoproteins involves the peroxidation of their polyunsaturated fatty acids (PUFA) and yields large amounts of lipid peroxidation products such as conjugated diene hydroperoxides. Cleavage of these products generates aldehydes, such as malondialdehyde, which act as toxic messengers in the processes of lesion formation [6]. Peroxidation starts only when antioxidant molecules, in particular vitamin E, present in the lipoprotein particle, have been consumed. This lag time represents the resistance of lipoprotein to lipid peroxidation. The susceptibility of lipoproteins to oxidation can readily be mimicked ex vivo by their exposure to cupric ions, and is commonly monitored by continuous spectrophotometric measurement of newly formed conjugated dienes [7]. The conventional action of vitamin E is illustrated by an increase in the resistance of lipoproteins to oxidation (reflected by a prolonged lag time) when vitamin E content increases. However, recent studies indicate that vitamin E can also act as a pro-oxidant (for review, see reference [8]).
A reduction in the lag time and/or a change in the composition and size of lipoproteins favours their oxidation and leads to an overproduction of lipid peroxidation products which, in turn, should promote the process of fibrosis in the kidney. Using the Zucker rat model of genetic obesity, hyperlipidaemia, and hyperinsulinaemia, we demonstrated that the initial steps of glomerular and interstitial fibrosis, which occur at 3 months and onwards, were associated with increases in lipid peroxidation products and oxidative stress in the absence of inflammatory cell infiltration, hypertension, or hyperglycaemia [9]. Lipid peroxidation products, indicated by the presence of thiobarbituric acid reactive substances (TBARS), have been found in kidney tissue and in the plasma LDL/VLDL fraction, and kidney antioxidant enzyme activity was modified in Zucker obese (ZO) rats compared with their Zucker lean (ZL) controls. However, the role of LDL/VLDL as a source of these peroxidation products remains unknown. To address this question, we examined the oxidizability of the apo B-containing lipoprotein (VLDL and LDL) fraction of ZO rats and ZL littermates at 1 month (before renal fibrosis is detectable), at 3 months (at the onset of renal lesions), and at 9 months of age (after a worsening of severe glomerular and tubulo-interstitial lesions [9]).
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Subjects and methods
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Animals
Male Zucker lean (Fa/fa, ZL) and obese (fa/fa, ZO) rats (INSERM U 465 husbandry, Paris, France, Dr Raymond Bazin) were identified and selected at 4 weeks of age by visual examination of inguinal fat deposits. They were raised in standard husbandry conditions, had free access to water and were fed ad libitum regular laboratory chow (M25, Extralabo, Provins, France) containing 38.3 mg/kg vitamin E, a standard amount for rat diets. At the time of sacrifice, at 1, 3, and 9 months, animals were anaesthetized with pentobarbital (i.p., 0.1 ml/100 g body weight) and blood was collected from the aorta into ethylene diamine tetra-acetic 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 8623, revised 1989, authorization 00577, 1989, Paris, France).
Isolation of lipoproteins
Blood obtained through aortic puncture was centrifuged at 4000 r.p.m. for 10 min at 4°C to remove chylomicrons, and plasma samples were supplemented with sucrose (6 g/l, final concentration) before freezing and storage at -80°C. Previous experiments indicated that sucrose is a good cryopreservative for LDL and does not modify lipid peroxidation parameters [10]. All samples were stored for less than 8 weeks. Three to five millilitres of potassium bromide (KBr) (Merck, Darmstadt, Germany) solution (d=1.063 g/ml) containing 1 mmol/l EDTA were 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 M ultracentrifuge using a 70.1 Ti rotor at 45 000 r.p.m. The top fraction (VLDL+LDL) was harvested. The VLDL/LDL fraction was isolated instead of the LDL fraction alone because the latter is present at low levels in rats [11] and is often contaminated by VLDL when separated from hypertriglyceridaemic plasma. The isolated lipoprotein fraction was dialysed at 4°C, twice for 2 h against 0.01 mol/l phosphate buffer (PBS), pH 7.4, supplemented with 0.16 M NaCl, and dialysed again overnight. The VLDL/LDL protein content was measured according to a Peterson assay with bovine serum albumin as a standard.
Susceptibility to copper oxidation
Susceptibility to copper oxidation was performed using a modification of the procedure described by Kleinveld et al. [12]. Lipoprotein oxidation was initiated by the addition, in capped quartz cuvettes, of 0.1 ml of copper chloride solution freshly prepared in water (final copper concentration 5 µmol/l) to 0.9 ml of dialysed lipoproteins, adjusted to a concentration of 50 µg of proteins/ml in PBS. This produces a ratio of cupric ions over lipoprotein particles higher than 50. The time-course of oxidation was followed by monitoring, every 2 min for 5 h at 37°C, the changes in absorbance at 234 nm using a Helios Unicam spectrophotometer (Unicam Spectrometry, Cambridge, U.K.), equipped with a thermostated 7-position automatic sample changer. This allowed the simultaneous analysis of six samples (three samples from ZO rats and three from age-matched ZL littermates) and a blank. Using experimental curves of oxidation kinetics (Figure 1
), the lag time, maximal conjugated diene (CD) production (CDmax), rate of diene production (Vmax), and time (Tmax) to reach maximal amount of CD formed were calculated. The lag time was defined from the absorbance curve as the time given by the intercept of the two straight lines from the lag and propagation phases on the time axis (A). CDmax was calculated using maximal absorbance value (B) and the BeerLambert law with an extinction coefficient for conjugated dienes being 29 500 L.mol/cm as given by Kleinveld et al. [12]. Vmax was determined as the slope of the absorbance curve during the propagation phase, and Tmax as the projection on the time axis of the intercept of the slope of the absorbance curve with the maximal absorbance value line (C). Previous studies showed that EDTA-treated plasma stored at -80°C in presence of sucrose (6 g/l) has no effect on oxidative susceptibility of LDL [12]. The intra-assay variation coefficients (n=6) remained at a low range: lag time 5%, CDmax 8%, Vmax 9%, and Tmax 4 %.

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Fig. 1. Typical time-course of the copper-induced oxidation of apo B-containing lipoproteins isolated from 3-month-old lean (ZL) and obese (ZO) rats. ZO rat lipoproteins showed a longer lag time (intercept of the lag and propagation phases on the time axis, A) and a higher maximal diene production (CDmax, B), a lower Vmax and a higher Tmax (C) than age-matched ZL littermate lipoproteins.
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Chemical characterization of lipoproteins
Total and free cholesterol, phospholipids, and triglycerides were determined with enzymatic test kits according to the manufacturer's procedures (Boehringer, Mannheim, Germany). Cholesterol ester content was calculated as total cholesterol minus free cholesterol. Total lipoprotein mass was calculated as the sum of the masses of the individual components (free cholesterol, cholesterol ester, triglycerides, phospholipids, and proteins). Protein was measured using the Peterson assay, as described above. Vitamin E was determined at 3 and 9 months in VLDL/LDL fraction as previously described [13]. Total fatty acids were determined according to Itaya et al. [14]. Fatty acids were analysed after esterification by gasliquid chromatography in a Sisons GC 8000 chromatograph (Thermoquest, Les Ullis, France) [15].
Statistical analysis
Results were expressed as means±SEM. Statistical analysis was carried out using a two-way analysis of variance (ANOVA) with age and group (genotype) as factors, followed by BonferroniDunn tests (Statview 5.0 software, Abacus Concept Inc, Berkeley, CA). Main effects and interactions were considered significant at P<0.05. In cases of a significant interaction between factors, a one-way ANOVA was used. Correlation coefficients between CDmax and PUFA were analysed using the Fisher P value test.
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Results
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Apo B-containing lipoprotein oxidation parameters
Using experimental curves of the oxidation kinetics, as illustrated in Figure 1
, parameters of oxidation were calculated (Table 1
). At 3 months, the lag-time was twofold longer in ZO than in ZL rats, and at 9 months, ZL lag time rose significantly. CDmax, constant in ZL rats at all ages, was higher in ZO than in ZL rats at 3 and 9 months. However, when expressed per ml of plasma, total CDmax was significantly increased in ZO rats as compared with ZL values as early as 1 month, and thereafter surpassed by two- and threefold the ZL values. Vmax was lower and Tmax higher in ZO than ZL rats at 1 and 3 months.
Chemical composition of apo B-containing lipoproteins
The lipid and protein contents of apo B-containing lipoproteins are shown in Table 2
. No significant change in lipid and protein contents was observed in 9-month-old ZO rats as compared with age-matched ZL littermates. At 1 month, cholesterol ester was decreased and phospholipid slightly increased in ZO rats. Free cholesterol to cholesterol ester and core to surface component ratios were not significantly different between ZO and ZL rats at 1 and 9 months of age. In contrast, marked alterations were observed at 3 months. The percentage of triglycerides in ZO rats rose to values twice as large as in ZL rats. Free and esterified cholesterol contents were 1.6- and 3.3-fold lower in ZO than in ZL rats, giving a ratio of free to esterified cholesterol twice that in ZO than in ZL rats. The low level of protein as well as the core to surface component ratio (triglyceride+cholesterol esters over phospholipids+free cholesterol+protein), index of lipoprotein size, indicate that the apo B-containing lipoproteins were larger in ZO than in ZL rats at 3 months.
As shown in Tables 3
and 4, apo B-containing lipoproteins were particularly rich in PUFA, the major substrate for lipid peroxidation, which comprised up to 50% of the whole fatty acids. While the total amount of fatty acids remained similar at 1 month in both ZO and ZL rats, the relative proportion of the different acids differed between ZO and ZL rats, with an increase in saturated fatty acids (SFA) and a decrease in PUFA percentages. At 3 months, this pattern became aggravated and involved a rise in the mono-unsaturated fatty acids (MUFA) percentage. Total fatty acid concentration per milligram of apo B was significantly increased in 3- and 9-month-old ZO rats as compared with age-matched ZL littermates: at 3 and 9 months, it was greater than the ZL values by threefold and twofold respectively (Table 3
). Thus, at 3 months of age, the concentrations per milligram of protein of each SFA and MUFA was at least fourfold higher than in ZL rats, while all PUFA were twofold more concentrated in ZO than in ZL rat lipoproteins (Table 4
). Linoleic (18:2 (n-6)), arachidonic (20:4 (n-6)), and docahexaenoic (22:6 (n-3)) acids were the most prominent PUFA in ZO and ZL lipoproteins. They increased 1.3- to 2.3-fold in ZO lipoproteins between 1 and 3 months, but decreased steadily with age in ZL lipoproteins. At 9 months, when the difference in percentage of SFA, MUFA, and PUFA was no longer observed between ZO and ZL rats, the concentrations of these fatty acids remained higher in ZO than in ZL animals, because of the higher level of whole fatty acids observed at that age. The (n-6)/(n-3) ratio was similar in both groups of animals at 1 and 3 months, but increased in ZL rats at 9 months (Table 3
).
Since the lag time strongly depends on the vitamin E content, we determined the level of this antioxidant in lipoproteins at the onset (3 months) and after the worsening (9 months) of the kidney lesions (Table 3
). At 3 and 9 months, vitamin E content was respectively 2.2- and 1.4-fold higher in ZO than in ZL rats. However, the level of vitamin E calculated per µmole of PUFA, which increased with age, remained identical in both groups (group effect, P=0.89).
The
5-desaturase index, lower in ZO than in ZL rats at all ages, decreased in both groups at 3 months, while the
6-desaturase index was higher in ZO than in ZL rats at all ages, despite a peak of this index in ZL rats at 3 months (Table 3
). Regression analysis showed a positive correlation between CDmax and whole PUFA except for 20:(3n-9), 22:(5n-6) and 20:(5n-3) fatty acids. Figure 2
illustrates such a correlation with linoleic and arachidonic acids and with all PUFA of ZL and ZO apo B-containing lipoproteins.

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Fig. 2. Correlations between maximal conjugated diene (CDmax) production and 18:(2n-6) linoleic acid (A), 20:( 4n-6) arachidonic acid (B) and all PUFA (C). CDmax was positively correlated to PUFA in ZL and ZO apo B-containing lipoproteins.
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Discussion
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We have showed here that apo B-containing lipoproteins isolated from obese Zucker rats displayed a significant increase in the lag time (reflecting resistance to oxidation) at 3 months of age, when renal lesions begin [9], compared with age-matched ZL littermates, while the maximal conjugated diene production (reflecting the total amount of hydroperoxides formed during oxidation) was higher in ZO than in ZL rats, even as early as 1 month, before renal fibrosis is initiated [9].
The lag time of apo B-containing lipoprotein oxidation depends on several factors. Exogenous factors such as hyperglycaemia, glycation processes, episodes of ketosis, and elevated levels of acetoacetate, known to favour oxidative stress and the development of vascular disease in diabetic patients [16,17] are unlikely in the ZO rat model, which maintains normal levels of glycaemia and blood indices of glycation [9] and does not develop ketosis [18]. Hydroperoxides bound to lipoproteins have also been reported to shorten the lag time [19]. Although the lipid hydroperoxide concentration in lipoproteins was not determined in this study, hydroperoxides and other products of peroxidation may play a minor role, since the lag time was increased in ZO rats. The antioxidant defence has a major role in the oxidizability of lipoproteins, and vitamin E is quantitatively the most important lipophilic antioxidant in lipoproteins [6]. The level of lipoprotein vitamin E was increased in 3- and 9-month-old ZO rats compared with age-matched ZL rats, and probably resulted from a greater consumption of the regular laboratory chow by obese, hyperphagic ZO rats. However, the increase in vitamin E level paralleled the PUFA increase in ZO rats, and the ratio of vitamin E content over the PUFA concentration remained identical in apo B-containing lipoproteins from both ZO and ZL rats at 3 and 9 months of age. Consequently, the lengthening of the lag time is unlikely to be the result of an increase in the level of vitamin E as an antioxidant associated with the lipoproteins.
Other antioxidants, not measured in this study, may be of importance [6]. The composition, size, or density of lipoproteins strongly determine their oxidizability. First, lag time depends on PUFA content: the higher the content, the shorter the lag time [20]. Despite the increase in total PUFA amount per mg of apo B determined in 3-month-old ZO rats, the lag time was longer in ZO than in ZL rats. This suggests that PUFA is not a relevant parameter in determining the duration of the lag time in ZO rats. Second, lag time strongly depends on the lipoprotein size and density: large-buoyant LDL have longer lag times than small-dense LDL [21]. This was the case in our experimental model: in 3-month-old ZO rats, lag time was 2.3-fold longer than in ZL littermates, while the lipoprotein size was larger, as shown by a 2.2-fold decrease of the protein content and a 2.3-fold increase of the core to surface component ratio of the lipoprotein.
This also applied to the lag time of ZL rat lipoproteins, which increased at 9 months in parallel with the protein content and the core to surface ratio of the particle. The reason that lipoprotein size peaked while protein content diminished specifically in 3-month-old obese rats remains to be explored. The lipid composition of lipoprotein is another major factor involved in the oxidation process. Increased free cholesterol lessens and increased cholesteryl esters aggravate the susceptibility to oxidation of LDL [21]. This was also the case in our study. As compared to age-matched ZL rats, free cholesterol/cholesteryl ester ratio in the VLDL/LDL fraction was significantly raised in 3-month-old ZO rats, while lag time lengthened in parallel. As suggested by Tribble et al. [21], a possible explanation for the association between lag time and free cholesterol/cholesteryl ester ratio is that a higher ratio leads to a higher lipoprotein rigidity, which results in reduced accessibility of oxidants.
The second parameter of lipid susceptibility towards oxidation involves the maximal capacity of the lipoprotein to produce conjugated dienes. The total amount of conjugated dienes (CDmax) that can be formed was higher in ZO than in ZL rats as early as 1 month. Because PUFA is the main substrate of lipid peroxidation [6,12] this explains the positive correlations between CDmax and almost every one of the PUFAs. The percentage of PUFA in apo B-containing lipoprotein was 30% lower in 3-month-old ZO rats than in age-matched ZL littermates. Since the experimental conditions used for isolation, handling, and storage were identical for both ZL and ZO rat lipoproteins, this precludes an artefactual peroxidation of ZO PUFA during the experimental procedure. In ZO rats, a relative deficiency of PUFA has been demonstrated in the liver and other tissues such as kidney (reviewed in ref. [22]). Thus, the decrease in lipoprotein PUFA percentage could be the result of an impairment of their hepatic synthesis, via an alteration in enzyme activities of fatty acid elongation and desaturation. The activity of desaturase enzymes can be assessed by the ratio of product to precursor molecules. For instance, the 20:(4n-6)/20:(3n-6) and 20:(3n-6)/18:(2n-6) ratios give an index of activity respectively of the
5- and
6-desaturases. We showed here that a decrease in
5-desaturase and an increase in
6-desaturase indices occurred in ZO rat lipoprotein, reflecting a possible alteration in hepatic synthesis of PUFA. The mechanisms explaining the increase in PUFA percentage in 9-month-old obese rats remain to be explored.
In ZO rats, the hepatic secretion of VLDL is increased, and secreted lipoproteins are both larger and enriched with triglycerides [23]. Large VLDL may directly damage the endothelium, principally via oxidative mechanisms. Although large VLDL cross the endothelial barrier more slowly than small VLDL particles, retention time of large VLDL within arterial tissue is greater [24]. Large VLDL therefore undergo an oxidative modification during a longer time period and may produce larger quantities of lipid peroxidation products. After formation, conjugated dienes are subsequently decomposed into toxic aldehydes (malondialdehyde, 4-hydroxynonenal) [6]. These aldehydes alter the lysine residues of apolipoprotein B. Modified apo B-containing lipoproteins may then be recognized by scavenger receptors expressed by macrophages and by other cells, including glomerular mesangial cells [25,26]. Aldehyde formation may also directly participate in the progression of glomerulosclerosis by enhancing the synthesis of extracellular matrix components by mesangial cells [27]. This hypothesis is supported by the present study, since an increase in MDA content was demonstrated in ZO rat kidney cortex at 3 months and thereafter [9]. In addition, mesangial cells may produce reactive oxygen species and oxidize LDL [28], thus providing an amplificatory loop of mesangial cell activation. Further experiments will be required to confirm these hypotheses.
In conclusion, our study demonstrated that, compared to age-matched lean littermates, apo B-containing lipoproteins from young ZO rats can produce larger amounts of conjugated dienes. This overproduction is probably related to an increase in the total amount of PUFA in lipoproteins. The ensuing increase in peroxidation products possibly activates mesangial cells and interstitial fibroblasts to initiate the fibrotic process seen at the 3rd month and onwards in this rat model of obesity and renal fibrosis.
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Acknowledgments
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Part of this work was presented as an Abstract at the 31st Annual Meeting of the American Society of Nephrology (Philadelphia, October 1998). We thank Drs Srinivas Kaveri and Antonino Nicoletti (INSERM U 430) for helpful discussions during the preparation of the manuscript.
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Notes
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Correspondence and offprint requests to: Dr Jacques Chevalier, Immunopathologie Rénale et Vasculaire, INSERM U 430, Hôpital Broussais, 96 Rue Didot, 75674 Paris Cedex 14, France. 
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Received for publication: 16. 8.00
Revision received 29. 1.01.