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Original Contribution Free Radical Biology & Medicine, Vol. 34, No. 2, pp. 186–195, 2003 Copyright

Original ContributionFree Radical Biology & Medicine, Vol. 34, No. 2, pp. 186–195, 2003 Copyright © 2003

Free Radical Biology & Medicine, Vol. 34, No. 2, pp. 186–195, 2003 Copyright © 2003 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/03/$–see front matter

PII S0891-5849(02)01195-4

EARLY OXIDATIVE STRESS IN THE DIABETIC KIDNEY: EFFECT OF DL- -LIPOIC ACID

IRINA G. OBROSOVA,* LAMIA FATHALLAH,* EDWIN LIU, and JAFFAR NOUROOZ-ZADEH

*Department of Internal Medicine, University of Michigan Medical Center, Ann Arbor, MI, USA; and Department of Medicine, University College London, Royal Free and University College Medical School, London, UK

(Received 14 August 2002; Revised 20 September 2002; Accepted 2 October 2002)

Abstract—Oxidative stress is implicated in the pathogenesis of diabetic nephropathy. The attempts to identify early markers of diabetes-induced renal oxidative injury resulted in contradictory findings. We characterized early oxidative stress in renal cortex of diabetic rats, and evaluated whether it can be prevented by the potent antioxidant, DL- -lipoic acid. The experiments were performed on control rats and streptozotocin-diabetic rats treated with/without DL- -lipoic acid (100 mg/kg i.p., for 3 weeks from induction of diabetes). Malondialdehyde plus 4-hydroxyalkenal concentration was increased in diabetic rats vs. controls (p .01) and this increase was partially prevented by DL- -lipoic acid. F 2 isoprostane concentrations (measured by GCMS) expressed per either mg protein or arachidonic acid content were not different in control and diabetic rats but were decreased several-fold with DL- -lipoic acid treatment. Both GSH and ascorbate (AA) levels were decreased and GSSG/GSH and dehydroascorbate/AA ratios increased in diabetic rats vs. controls (p .01 for all comparisons), and these changes were completely or partially (AA) prevented by DL- -lipoic acid. Superoxide dismutase, glutathione peroxidase, glutathione reductase, glutathione transferase, and NADH oxidase, but not catalase, were upregulated in diabetic rats vs. controls, and these activities, except glutathione peroxidase, were decreased by DL- -lipoic acid. In conclusion, enhanced oxidative stress is present in rat renal cortex in early diabetes, and is prevented by DL- -lipoic acid. © 2003 Elsevier Science Inc.

Keywords—Renal cortex, F 2 -isoprostanes, Malondialdehyde, 4-Hydroxyalkenals, Glutathione, Ascorbate, Antioxida- tive enzymes, Rat, Streptozotocin diabetes, Free radicals

INTRODUCTION

The epidemiological studies [1] and Diabetes Control and Complication Trial [2] point to a strong consistent relationship between hyperglycemia and the incidence and progression of diabetic nephropathy in patients with both Type 1 and Type 2 diabetes mellitus. One of the most important consequences of “glucose toxicity” in tissue-sites for diabetic complications is enhanced oxi- dative stress, i.e., increased production of reactive oxy- gen species combined with downregulation or insuffi- cient upregulation of antioxidant defense mechanisms. Enhanced oxidative stress has been documented in all three compartments of the renal cortex, i.e., glomeruli

Address correspondence to: Dr. Irina Obrosova, Division of Endo- crinology and Metabolism, Department of Internal Medicine, Univer- sity of Michigan Medical Center, 1150 West Medical Center Drive, MSRB II, Rm 5560C, Ann Arbor, MI 48109-0678, USA; Tel: (734) 763-3055; Fax: (734) 975-1849; E-Mail: iobrosso@umich.edu.

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[3,4], tubulo-interstitium [5], and vasculature [6], and has been implicated in mesangial expansion of extra- cellular matrix [7,8] and other early and late events of diabetic nephropathy, i.e., increased glomerular filtra- tion rate [9], urinary albumin excretion and proteinuria [7,8,10], glomerulosclerosis [7,11], and tubulo-inter- stitial fibrosis [11]. Studies in animal models of dia- betes revealed that some of these functional and mor- phological abnormalities can be prevented by antioxidants [7–11]. However, in general, the findings are inconclusive because several reports indicated the absence of improvement [12] and even worsening [13] of diabetic nephropathy with antioxidant treatment. The latter could result from insufficient understanding of the mechanisms underlying renal oxidative damage and, in particular, very contradictory information on the effects of diabetes on major components of free radical production and antioxidative defense, which hampers the selection of specific targets for antioxi-

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dant therapy as well as proper antioxidants and anti- oxidant combinations. Among all existing animal models of diabetes, the streptozotocin (STZ)-induced diabetic rat model was characterized best, and was found to develop all major nephropathic changes described in patients with diabetes mellitus. The attempts to identify early markers of renal cortex oxidative injury in STZ-diabetic rats resulted in contradictory ndings. Whereas most of the studies agree that enhanced oxidative stress and its major tissue man- ifestation, i.e., lipid peroxidation (LPO) are present in the kidney cortex shortly after induction of diabetes [14 16], no consensus has been achieved regarding the major mechanisms of free radical production as well as the extent of compromise of the key antioxidative defense mechanisms. One group suggested the importance of intramitochondrial superoxide production [17], whereas others point to the contribution of extramitochondrial, in addition to [18] or instead of [19] intramitochondrial mechanisms, to diabetes-induced kidney oxidative dam- age. The concentrations of the key biological nonenzy- matic antioxidant, reduced glutathione (GSH), were re- ported decreased [20,21] or unchanged [10,22] in the renal cortex of diabetic rats, and the major antioxidative defense enzyme activities, i.e., superoxide dismutase (SOD), catalase, glutathione peroxidase (GSHPx), gluta- thione reductase (GSSGRed), and glutathione transferase (GSHTrans) were found decreased, unchanged, or in- creased [16,19,2328]. The information on other com- ponents of free radical generation and antioxidative de- fense, e.g., the ascorbate system and NADH oxidase in the diabetic kidney, is either limited [29] or unavailable. The purpose of this study was to characterize early changes in LPO and antioxidative defense in the renal cortex of diabetic rats, and to evaluate whether these changes can be prevented by DL- -lipoic acid. DL- - lipoic acid is a universalantioxidant that combines free radical scavenging and metal chelating properties with an ability (after conversion to dihydrolipoic acid) to regenerate the levels of other nonenzymatic and enzy- matic antioxidants, i.e., GSH, ascorbate, -tocopherol, catalase, and GSH peroxidase [30,31]. DL- -lipoic acid effectively counteracts oxidative stress in the lens [32], retina [33,34], and peripheral nerve [35,36] of streptozo- tocin-diabetic rats, and prevents diabetes-induced endo- thelial dysfunction [37] and manifestations of diabetic neuropathy [35,36,38 40], retinopathy [34], and cataract [41]. In the same animal model, DL- -lipoic acid was found to prevent early glomerular injury manifested by increased urinary albumin excretion, fractional albumin clearance, glomerular volume, and glomerular content of immunoreactive transforming growth factor- and colla- gen 1(IV) [10]. Recent studies from the same group [8] revealed that chronic 7 month DL- -lipoic acid admin-

istration prevented renal insufciency, glomerulat mes- angial matrix expansion, and glomerulosclerosis. Fur- thermore, a pilot clinical study has demonstrated a benecial effect of DL- -lipoic acid on urinary albumin concentration in patients with diabetes mellitus [42].

MATERIALS AND METHODS

The experiments were performed in accordance with regulations specied by The Guiding Principles in the Care and Use of Animals (DHEW Publication, NIH 80-23) and the University of Michigan Protocol for An- imal Studies.

Animals

Male Wistar rats (Charles River, Wilmington, MA, USA), body weight 250 300 g, were fed a standard rat chow diet (ICN Biomedicals, Cleveland, OH, USA) and had ad libitum access to water. Diabetes was induced by a single injection of streptozotocin (Upjohn, Kalamazoo, MI, USA; 55 mg/kg body weight, i.p.). Blood samples for glucose measurements were taken from the tail vein ca. 48 h after streptozotocin injection and the day before the rats were killed. Rats with blood glucose 13.9 mmol/l were considered as diabetic. The experimental groups included control rats and diabetic rats treated with/without DL- -lipoic acid (Sigma, St. Louis, MO, USA; 100 mg · kg 1 · day 1 , i.p., for 3 weeks after induction of diabetes).

Experimental procedure

Rats were sedated with carbon dioxide and killed by cervical dislocation. Both kidneys were immediately dis- sected and frozen in liquid nitrogen. Four different sets of 30 mg renal cortex samples were either (i) extracted with 15.4 mol/l methanesulphonic acid and used for measurements of malondialdehyde (MDA) plus 4-hy- droxyalkenals (4-HA); (ii) extracted with 5% metaphos- phoric acid and used for measurements of dehydroascor- bate (DHAA) and total ascorbate (tAA); (iii) extracted with 6% perchloric acid, neutralized [32], and used for measurements of reduced (GSH) and oxidized (GSSG) glutathione; or (iv) homogenized in 1 ml 0.1 mol/l sodium-phosphate buffer, pH 6.5, and used for mea- surements of superoxide dismutase, catalase, GSH peroxidase, GSSG reductase, GSH transferase, and NADH oxidase activities and protein content. Renal cortex samples of 200 mg were homogenized in 2 ml 10 mmol/l phosphate buffer, pH 7.4, containing 100 mmol/l butylated hydroxytoluene (set 5). The homog- enates were used for measurements of total (i.e., free plus esteried) F 2 -isoprostane [4345] and arachi- donic acid [46] concentrations.

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Biochemical measurements

MDA plus 4-HA, GSH, and GSSG concentrations,

SOD, catalase, GSH peroxidase, GSSG reductase, GSH transferase, and NADH oxidase activities, and protein content were assayed as described [33]. For measurements of DHAA and AA, the renal cortex homogenates in 5% meta-phosphoric acid were centri- fuged at 4000 g for 10 min. In the DHAA assay, 0.1

ml of the supernatant was mixed with 0.9 ml 2 mol/l

Na-acetate buffer, pH 6.2. The reaction was started by addition of 0.02 ml 92.5 mM O-phenylenediamine (Sig- ma). The initial and nal readings were taken at exci- tation: 350 nm, emisssion: 430 nm. For measurements of tAA, 0.1 ml of the supernatant was mixed with 0.9 ml 2 M Na-acetate buffer, pH 6.2 and 10 U of ascorbate oxidase (Sigma) to convert ascorbate (AA) to DHAA.

After completion of the reaction, DHAA was quantied as described above. The differences in initial and nal readings in DHAA and tAA assays were compared with those of AA standards processed in the same run. AA levels were calculated as the difference between tAA and DHAA. For measurements of total F 2 -isoprostanes [4345], 1

ml aliquots of homogenate from step 5 were transferred

into glass tubes and 500 l of 4 mol/l KOH were added. After incubation at 45°C for 45 min, the pH was adjusted to 3 by adding 500 l of 4 mol/l HCI. The internal standard 3,3',4,4'-tetradeuterated 9 ,11 -prostaglandin F 2 (PGF 2 -d 4 , 5 ng in 100 l ethanol) was added and the samples were vortex mixed. Then 10 ml ethyl acetate was added, and the samples were vortexed for 20 s and centrifuged at 1000 g for 5 min. The upper organic layer was transferred into a new set of glass tubes. Total lipid extract was then applied onto an aminopropyl

(NH 2 ) cartridge. The cartridge was sequentially washed with 10 ml of hexane/ethyl acetate (30/70, v/v), acetoni- trile/water (90/10, v/v) and acetonitrile. Isoprostanes were eluted by washing the cartridge with 5 ml of ethyl acetate/methanol/acetic acid (85/10/5, v/v/v). The nal eluate from the NH 2 -cartridge was dried under nitrogen at 45°C and was then converted to pentauoro-benzyl ester/trimethylsylil ether derivatives. Gas-chromatogra- phy-mass spectrometry/negative ion chemical ionization (NICI) was carried out on a Hewlett Packard 5890 GC linked to a VG70SEQ using the NICI with ammonia as reagent gas as described elsewhere [44]. Briey, samples (2 l) were injected onto a SPB-1701 (30 m 0.25 mm I.D.; 0.25 m lm thickness; Supelco, Dorset, England). Separation was carried out using a temperature program:

initial temperature: 175°C; initial time: 2 min; rate:

30°C/min; nal temperature: 270°C; nal time: 30 min. Samples (2 l) were injected into a temperature pro- grammed Gerstel injector (Thames Chromatography,

Maidenhead, UK). Quantitative analysis was performed using selected ion monitoring (SIM) of the carboxylate anion [M-181]- at m/z 569 and 573 for F 2 -isoprostanes and PGF 2 -d 4 as the internal standard, respectively. For fatty acid analysis [46], 200 l aliquots of ho- mogenate (set 5) were mixed with 250 l ethanol con- taining 100 g of heptadecanoic acid as an internal standard. Five hundred microliters of ethyl acetate and 300 l water were then added to the homogenate, vor- texed, and centrifuged at 1000 g for 5 min. The upper organic layer was then transferred to a new set of glass vials. Five hundred microliters of ethyl acetate was added to the remaining aqueous phase and the mixture was vortexed and centrifuged as described above. The upper organic layers were pooled and dried under a stream of nitrogen. Fatty acid methyl esters were pre- pared by adding 500 l of 14% boron triuoride solution in methanol to the dried nal lipid extract and incubated at 60°C for 30 min. The samples were allowed to cool before water (1 ml) and hexane (1 ml) were added. The mixtures were vortexed and centrifuged. The hexane (upper) layer was collected and dried. The residue was redissolved in 100 l of hexane. Fatty acid analyses were carried out on a Fisons MFC 800 gas chromatograph equipped with a ame ionization detector and a splitless injector. Fatty acids methyl esters were separated on an Omegawax 320 column (0.32 m 30 m, lm thickness 0.25 m; Supelco) using a temperature gradient of 120 240°C at 6°C/min. One microliter samples were injected into the gas chromatograph.

Statistical analysis

The results are expressed as mean SEM. Data were subjected to equality of variance F test, and then to log transformation, if necessary, before one-way analysis of variance. When overall signicance (p .05) was at- tained, individual between group comparisons were made using the Student- Newman-Keuls multiple range test. Signicance was dened at p .05. When between- group variance differences could not be normalized by log transformation, the data were analyzed by the non- parametric Kruskal-Wallis one-way analysis of variance, followed by the Fishers PLSD test for multiple compar- isons.

RESULTS

The nal body weights were lower in diabetic rats than in the control group (Table 1). The initial body weights (not shown) were similar in control and diabetic groups. The nal body weights in DL- -lipoic acid- treated diabetic rats were slightly but signicantly lower than in the untreated diabetic group.

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Table 1. Final Body Weights and Blood Glucose Concentrations in Control Rats and Diabetic Rats Treated With/Without DL- -lipoic Acid (Mean SEM)

Body

Blood

glucose

concentration

 

weight (g)

(mmol/l)

Control (n 19)

352 4 293 7* 268 5* , **

3.61 0.11 20.6 0.61* 19.7 0.61*

Diabetic

(n

20)

Diabetic DL- -lipoic acid (n 16)

The number of observations is indicated in parentheses. * Signicantly different compared with controls (p .01). ** Signicantly different compared with untreated diabetic group (p .01).

Blood glucose concentration was increased 5.5-fold in diabetic rats compared with the control group, and was not affected by DL- -lipoic acid treatment. Renal cortex MDA plus 4-HA concentration was 2.4- fold higher in diabetic rats compared with the control group (p .01, Fig. 1). This increase was partially prevented by DL- -lipoic acid (p .05 vs. untreated diabetic group, and p .05 vs. controls). Renal cortex F 2 -isoprostane concentrations normal- ized per mg protein were similar in control and diabetic rats (Fig. 2A), but were 4-fold lower in DL- -lipoic acid-treated diabetic rats than in the corresponding un- treated group (p .01). Renal cortex arachidonic acid concentration was 1.4-fold lower in diabetic rats than in the control group (p .01, Fig. 2B), and this decrease was prevented by DL- -lipoic acid treatment (p .05 vs. untreated diabetic group). F 2 -isoprostane concentra-

untreated diabetic group). F 2 -isoprostane concentra- Fig. 1. Renal cortex MDA plus 4-HA concentrations in

Fig. 1. Renal cortex MDA plus 4-HA concentrations in control rats and diabetic rats treated with/without DL- -lipoic acid (Mean SEM, n 10). **Signicantly different compared with the control group (p .01); # Signicantly different compared with the untreated diabetic group (p .05).

tion, normalized per g of arachidonic acid (Fig. 2C), tended to increase in diabetic rats compared with the control group, but the difference between the groups did not achieve statistical signicance (p .12). It was 5.3 lower in DL- -lipoic acid-treated diabetic rats than in the untreated diabetic group (p .01). Renal cortex GSH concentration was slightly, but signicantly, decreased in diabetic rats compared with the control group (Fig. 3A, p .01), and this decrease was overcorrected by DL- -lipoic acid treatment (p .01 vs. untreated diabetic group). GSSG concentrations were similar among the experimental groups (Fig. 3B). GSSG/GSH ratio was 1.6-fold higher in diabetic rats than in the control group (p .01, Fig. 3C), and this increase was completely prevented by DL- -lipoic acid treatment (p .01 vs. untreated diabetic group). Renal cortex AA concentration was 2.2-fold lower in diabetic rats than in the control group (p .01, Fig. 4A), and this decrease was partially prevented by DL- -lipoic acid (p .01 vs. either control or untreated diabetic groups). DHAA concentrations were similar in control and diabetic groups, but was signicantly (p .05) lower in DL- -lipoic acid-treated diabetic group com- pared with the nondiabetic control group (Fig. 4B). DHAA/AA ratio (Fig. 4C) was 2.2-fold higher in dia- betic rats than in the control group (p .01), and this increase was completely prevented by DL- -lipoic acid (p .01 vs. untreated diabetic group). Renal cortex SOD activity was 1.4-fold higher in diabetic rats than in the control group, and this upregu- lation was completely prevented by DL- -lipoic acid (Table 2). Catalase activities were similar in control and diabetic groups but were lower in DL- -lipoic acid- treated diabetic group compared with nondiabetic con- trols. GSH peroxidase activity was increased 1.4-fold in diabetic rats compared with the control group, and this increase was not prevented by DL- -lipoic acid treat- ment. GSSG reductase and GSH transferase activities were 1.2- and 1.3-fold higher in diabetic rats than in the control group, and diabetes-associated upregulation of both enzymes was prevented by DL- -lipoic acid treat- ment. NADH oxidase activity was 1.6-fold higher in diabetic rats compared with the control group. This in- crease was completely arrested by DL- -lipoic acid; furthermore, NADH oxidase activity was 2.8-fold lower in DL- -lipoic acid-treated diabetic rats than in the non- diabetic control rats.

DISCUSSION

Our ndings of increased LPO, upregulated NADH oxidase, compromised concentrations of the key nonen- zymatic antioxidants GSH and AA, as well as the gluta- thione and ascorbate redox states, and increased SOD,

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190 I. G. O BROSOVA et al . Fig. 2. Renal cortex (A) F 2 -isoprostane

Fig. 2. Renal cortex (A) F 2 -isoprostane concentrations normalized per mg protein; (B) arachidonic acid concentrations; (C) F 2 - isoprostane concentrations normalized per g of arachidonic acid in control rats and diabetic rats treated with/without DL- -lipoic acid (Mean SEM, n 9 10). **Signicantly different compared with the control group (p .01); #,## Signicantly different compared with the untreated diabetic group (p .05 and .01, respectively).

GSH peroxidase, GSSG reductase, and GSH transferase activities clearly indicate that enhanced oxidative stress is present in the renal cortex at a very early stage of diabetes. Several lines of evidence suggest that renal oxidative stress in STZ-diabetic rats is a consequence of the diabetic state, and, primarily, hyperglycemia, rather than well-established [47] pro-oxidant effect of STZ itself. First, the pro-oxidant effect of STZ is limited to pancreatic -cells, whereas tissue targets for diabetic complications, e.g., retina and peripheral nerve, do not develop oxidative changes during the rst 10 d after induction of STZ-diabetes (Obrosova, unpublished). Second, the half-life of STZ is very short ( 15 min), whereas oxidative stress in the diabetic tissues, e.g. renal glomeruli, is more advanced in chronic than in short- term STZ-diabetes [48]. Third, impaired antioxidative defense [49], DNA oxidative damage [50], and other markers of oxidative stress (e.g., increased expression of heme oxygenase-1 [51]) in the diabetic kidney are cor- rected by insulin administered to animals with estab- lished STZ-induced diabetes. And fourth, oxidative

stress (i.e., lipid peroxidation [52,53] and GSH depletion [5355]) in diabetic tissues are corrected by aldose re- ductase inhibitors, i.e., agents that do not interfere with the STZ effect on -cells but arrest hyperglycemia-in- duced sorbitol pathway activation in other tissues, at the doses far below the effective doses of the most effective antioxidants, including DL- -lipoic acid. Diabetes-induced LPO in the kidney is manifested by

a 2.4-fold increased concentration of MDA plus 4-HA, and a trend towards an accumulation of F 2 -isoprostane, the specic product of arachidonic acid peroxidation. It

is important to realize that neither MDA plus 4-HA nor

F 2 -isoprostane are ideal markers of LPO, and that both variables are affected by changes in other metabolic pathways present in diabetes. In particular, MDA is the side product of prostaglandin metabolism [56,57], and tissue MDA concentrations are changed with inhibition of cyclooxygenase 1 [58], e.g., in diabetes [56]. In a similar fashion, tissue F 2 -isoprostane concentrations de- pend not only on the rate of LPO, but on arachidonic acid abundance, the rate of arachidonate metabolism to pros-

acid abundance, the rate of arachidonate metabolism to pros- Fig. 3. Renal cortex (A) GSH concentrations;

Fig. 3. Renal cortex (A) GSH concentrations; (B) GSSG concentrations; (C) GSSG/GSH ratios, in control rats and diabetic rats treated with/without DL- -lipoic acid (Mean SEM, n 710). *,**Signicantly different compared with the control group (p .05 and .01, respectively); ## Signicantly different compared with the untreated diabetic group (p .01).

Renal oxidative stress in diabetes

191

Renal oxidative stress in diabetes 191 Fig. 4. Renal cortex (A) AA concentrations; (B) DHAA concentrations;

Fig. 4. Renal cortex (A) AA concentrations; (B) DHAA concentrations; (C) DHAA/AA ratios, in control rats and diabetic rats treated with/without DL- -lipoic acid (Mean SEM, n 8). *,**Signicantly different compared with the control group (p .05 and .01, respectively); ## Signicantly different compared with the untreated diabetic group (p .01).

taglandin F 2 - and the prostaglandin clearance. Both arachidonate biosynthesis and metabolism [56,59], and prostaglandin F 2 - clearance [60] are compromised in diabetes. Thus, it is quite possible that both MDA and F 2 -isoprostane measurements in diabetic tissues, includ- ing the renal cortex, underestimate the rate of LPO. In any case, it is preferable to use several markers of LPO rather than MDA or F 2 -isoprostane alone. Our ndings of increased renal LPO in short-term diabetes are con- sistent with elevated lipid peroxide, malondialdehyde, and uorescent chromolipid concentrations [14 16,18,22] as well as increased rate of NADPH- dependent LPO [61] in other reports. One group has found LPO to depend on duration of diabetes with clearly manifested LPO 1 week after induction of diabe- tes, its drop to baseline at 24 weeks, and second pro- gressive increase after 5 and 6 weeks [19]. These obser- vations are different from ours because we found increased LPO in rats with 3 week duration of STZ- diabetes. Enhanced renal oxidative stress in short-term diabetes is associated with the loss of two major nonenzymatic antioxidants, GSH and AA, compromised glutathione and ascorbate redox states, upregulation of cytoplasmic

NADH oxidase but not downregulation of major antioxi- dative defense enzymes. The response of the glutathione and ascorbate systems of antioxidative defense in the diabetic kidney is similar to the one in two other tissue sites for diabetic complications, i.e., lens and peripheral nerve [32,35,36]. In all three tissues, GSH depletion parallels the loss of total glutathione, whereas accumu- lation of the oxidized form, GSSG, is either minor or absent. At the same time, a decrease in the renal cortex GSH concentration in diabetes of a 3 week duration is modest compared with the corresponding changes in peripheral nerve [62] and particularly, lens [63]. The latter is quite consistent with relatively low sorbitol path- way activity in the renal cortex compared with two other tissues [29,32,36,6264]. The inverse relationship be- tween the sorbitol pathway activity and GSH concentra- tions in tissue sites for diabetic complications has been revealed in a number of studies, performed not only with structurally diverse aldose reductase (AR) inhibitors [52,54,55,6567], but also in AR-overexpressing trans- genic [68] and AR-knockout [69] mice. The recent study of our group [70], however, indicates that the mecha- nisms underlying GSH depletion in diabetic tissues are complex and involve downstreampathways, i.e., poly-

Table 2. Antioxidative Defense Enzyme and NADH Oxidase Activities (nmol/min per mg Protein) in the Renal Cortex of Control Rats and Diabetic Rats Treated With/Without DL- -lipoic Acid (Mean SEM)

Control

Diabetic

Diabetic DL - -lipoic acid

SOD

719 52 (11)

974 93* (10)

663 49* ,## (10)

Catalase

3775 323 (8)

2906 488 (7)

2071 197* (8)

GSH peroxidase

558 30 (10)

754 29** (10)

724 32** (8)

GSSG reductase

110 10 (10)

135 6* (10)

113 5 # (10)

GSH tranferase

181 10 (10)

228 15** (9)

156 6 ## (7)

NADH oxidase

4.11 0.34 (11)

6.73 0.98** (7)

2.41 0.24 ## (9)

The number of observations is indicated in parentheses. * , ** Signicantly different compared with controls (p .05 and .01, respectively). # ,## Signicantly different compared with untreated diabetic group (p .05 and .01, respectively).

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I. G. OBROSOVA et al.

(ADP-ribosyl)ation. Renal AA depletion in streptozoto- cin-diabetic rats in our study is consistent with another report [29]. In contrast to the study [29], we found DHAA/AA ratio increased in the diabetic renal cortex. The latter is in agreement with compromised GSH con- centration and GSSG/GSH ratios considering that GSH and other cellular thiols, e.g., dihydrolipoic acid, play an important role in vitamin C homeostasis by regenerating AA from DHAA and semiascorbyl radicals [30]. AA concentrations in the lens have been found reduced by L-buthionine (S,R)-sulfoximine, an inhibitor of glutathi- one biosynthesis [31]. Diabetes-induced changes in antioxidative defense enzyme activities are known to be tissue-specic. All major antioxidative enzymes, i.e., SOD, GSH peroxi- dase, GSSG reductase, and GSH transferase are upregu- lated in the diabetic lens [54,71], but are downregulated in the diabetic retina [33]. SOD and catalase activities are decreased in the diabetic peripheral nerve [36,72], whereas the enzymes of glutathione metabolism remain unaffected [72]. Of interest, in the kidney, SOD, GSH peroxidase, GSSG reductase, and GSH transferase, but not catalase, appeared upregulated in short-term diabe- tes, which is consistent with a number of reports [19,24,25]. In contrast, in several other studies SOD activity was found decreased [4,16] or unchanged [26,7375]. Whereas the vast majority of studies in dia- betes models evaluated total SOD [4,16,23,24,26,7375], the report [19] describes different responses of ex- tamitochondrial Cu,Zn-SOD and intramitochondrial Mn- SOD. Of interest, only Cu,Zn-SOD activity was found upregulated in the kidney of diabetic rats, consistent with the increased Cu,Zn-SOD gene expression in the study [9]. The results [9,19] are in agreement with our ndings of a 1.6-increase in extramitochondrial NADH oxidase activity, which is one of the major extramitochondrial sources of superoxide anion radicals, and support the primary importance of extramitochondrial, rather than intramitochondrial, superoxide production in diabetes- induced renal oxidative damage. Most of the diabetes-induced changes observed in the present study were completely or partially prevented by the potent antioxidant DL- -lipoic acid, which is quite consistent with the effects of this compound in other tissues of diabetic animals [3236,40] as well as in other, nondiabetic models of oxidative stress [30,31]. DL- - lipoic acid caused a dramatic 5-fold decrease in renal cortex F 2 -isoprostane concentration, partially prevented accumulation of MDA plus 4-HA, arrested diabetes- induced GSH and AA depletion and increase in GSSG/ GSH and DHAA/AA ratios. Similar effects of DL- - lipoic acid on renal cortex MDA and GSH concentrations have been previously reported in the chronic STZ-diabetic rat model [8,11]. The antioxidant

effect of the compound in our study is also manifested by a 2.8-fold decrease in extramitochondrial NADH oxidase activity and a blunted upregulation of the key antioxida- tive defense enzymes, i.e., SOD, GSSG reductase, and GSH transferase in DL- -lipoic acid-treated diabetic group. The effect on NADH oxidase is probably related to the important role of DL- -lipoic acid in redox sig- naling [76,77]. In biological tissues, DL- -lipoic acid is rapidly reduced to dihydrolipoic acid by intramitochon- drial NADH-dependent dihydrolipoamide dehydroge- nase, thus shifting NAD /NADH ratios towards a more oxidized state in mitochondrial and secondary cytosolic compartments. An increase in NAD /NADH ratios with DL- -lipoic acid treatment has been described in several studies including those in the diabetic tissues [69,32,36,78]. Note that DL- -lipoic acid has a number of important metabolic effects, i.e., acts as a co-factor for pyruvate and 2-oxoglutarate complexes, increases glu- cose transport [79,80] and utilization [32,81]. Benecial effects on diabetic complications [3241] and, in partic- ular, on diabetic nephropathy [8,10,42], are likely to result from the whole complex of antioxidant and meta- bolic effects of the compound rather than from antioxi- dant properties alone. Several studies suggest that anti- oxidant properties of -lipoic acid can further be increased by the use of R- -lipoic acid instead of the racemic form [8284]. Furthermore, R-enantiomer is likely to have a better pharmacological prole than DL- -lipoic acid [82]. Thus, it is quite possible that R- enantiomer will be effective on diabetic complications at the doses far lower than those required for DL- -lipoic acid. Further preclinical studies are needed to evaluate R- -lipoic acid as a candidate for drug development in diabetic complications, and, in particular, diabetic ne- phropathy. In conclusion, enhanced oxidative stress is present in the renal cortex at a very early stage of diabetes, and is associated with the loss of two major nonenzymatic antioxidants, GSH and AA, and upregulation of extram- itochondrial NADH oxidase rather than downregulation of major antioxidative defense enzymes. The potent an- tioxidant DL- -lipoic acid effectively counteracts anti- oxidant loss and lipid peroxidation in the diabetic kidney. The ndings justify the rationale for evaluation of R- - lipoic acid as well as more narrow spectrum agents, i.e., stimulators of glutathione biosynthesis (N-acetyl-L-cys- teine) and inhibitors of NADH oxidase (apocynin), on diabetic nephropathy.

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