Free Radical Biology & Medicine, Vol. 34, No. 2, pp.

186 195, 2003

Copyright 2003 Elsevier Science Inc.
Printed in the USA. All rights reserved
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PII S0891-5849(02)01195-4

Original Contribution
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)

AbstractOxidative 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. F2
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.

KeywordsRenal cortex, F2-isoprostanes, Malondialdehyde, 4-Hydroxyalkenals, Glutathione, Ascorbate, Antioxida-

tive enzymes, Rat, Streptozotocin diabetes, Free radicals

INTRODUCTION [3,4], tubulo-interstitium [5], and vasculature [6], and

has been implicated in mesangial expansion of extra-
The epidemiological studies [1] and Diabetes Control
cellular matrix [7,8] and other early and late events of
and Complication Trial [2] point to a strong consistent
diabetic nephropathy, i.e., increased glomerular filtra-
relationship between hyperglycemia and the incidence
tion rate [9], urinary albumin excretion and proteinuria
and progression of diabetic nephropathy in patients with
[7,8,10], glomerulosclerosis [7,11], and tubulo-inter-
both Type 1 and Type 2 diabetes mellitus. One of the
stitial fibrosis [11]. Studies in animal models of dia-
most important consequences of glucose toxicity in
betes revealed that some of these functional and mor-
tissue-sites for diabetic complications is enhanced oxi-
phological abnormalities can be prevented by
dative stress, i.e., increased production of reactive oxy-
antioxidants [711]. However, in general, the findings
gen species combined with downregulation or insuffi-
are inconclusive because several reports indicated the
cient upregulation of antioxidant defense mechanisms.
absence of improvement [12] and even worsening [13]
Enhanced oxidative stress has been documented in all
of diabetic nephropathy with antioxidant treatment.
three compartments of the renal cortex, i.e., glomeruli
The latter could result from insufficient understanding
of the mechanisms underlying renal oxidative damage
Address correspondence to: Dr. Irina Obrosova, Division of Endo- and, in particular, very contradictory information on
crinology and Metabolism, Department of Internal Medicine, Univer- the effects of diabetes on major components of free
sity of Michigan Medical Center, 1150 West Medical Center Drive,
MSRB II, Rm 5560C, Ann Arbor, MI 48109-0678, USA; Tel: (734) radical production and antioxidative defense, which
763-3055; Fax: (734) 975-1849; E-Mail: iobrosso@umich.edu. hampers the selection of specific targets for antioxi-

186
 Renal oxidative stress in diabetes
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 findings. 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-
187
istration prevented renal insufficiency, glomerulat mes-
angial matrix expansion, and glomerulosclerosis. Fur-
thermore, a pilot clinical study has demonstrated a
beneficial 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 specified 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 kg1 day1, 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 esterified) F2-isoprostane [43 45] and arachi-
donic acid [46] concentrations.
188 I. G. OBROSOVA et al.

Biochemical measurements Maidenhead, UK). Quantitative analysis was performed

using selected ion monitoring (SIM) of the carboxylate
MDA plus 4-HA, GSH, and GSSG concentrations,
anion [M-181]- at m/z 569 and 573 for F2-isoprostanes
SOD, catalase, GSH peroxidase, GSSG reductase, GSH
and PGF2-d4 as the internal standard, respectively.
transferase, and NADH oxidase activities, and protein
For fatty acid analysis [46], 200 l aliquots of ho-
content were assayed as described [33].
mogenate (set 5) were mixed with 250 l ethanol con-
For measurements of DHAA and AA, the renal cortex
taining 100 g of heptadecanoic acid as an internal
homogenates in 5% meta-phosphoric acid were centri-
standard. Five hundred microliters of ethyl acetate and
fuged at 4000 g for 10 min. In the DHAA assay, 0.1
300 l water were then added to the homogenate, vor-
ml of the supernatant was mixed with 0.9 ml 2 mol/l
texed, and centrifuged at 1000 g for 5 min. The upper
Na-acetate buffer, pH 6.2. The reaction was started by
organic layer was then transferred to a new set of glass
addition of 0.02 ml 92.5 mM O-phenylenediamine (Sig-
vials. Five hundred microliters of ethyl acetate was
ma). The initial and final readings were taken at exci-
added to the remaining aqueous phase and the mixture
tation: 350 nm, emisssion: 430 nm. For measurements
was vortexed and centrifuged as described above. The
of tAA, 0.1 ml of the supernatant was mixed with 0.9 ml
upper organic layers were pooled and dried under a
2 M Na-acetate buffer, pH 6.2 and 10 U of ascorbate
stream of nitrogen. Fatty acid methyl esters were pre-
oxidase (Sigma) to convert ascorbate (AA) to DHAA.
pared by adding 500 l of 14% boron trifluoride solution
After completion of the reaction, DHAA was quantified
in methanol to the dried final lipid extract and incubated
as described above. The differences in initial and final
at 60C for 30 min. The samples were allowed to cool
readings in DHAA and tAA assays were compared with
before water (1 ml) and hexane (1 ml) were added. The
those of AA standards processed in the same run. AA
mixtures were vortexed and centrifuged. The hexane
levels were calculated as the difference between tAA and
(upper) layer was collected and dried. The residue was
DHAA.
redissolved in 100 l of hexane. Fatty acid analyses were
For measurements of total F2-isoprostanes [43 45], 1
carried out on a Fisons MFC 800 gas chromatograph
ml aliquots of homogenate from step 5 were transferred
equipped with a flame ionization detector and a splitless
into glass tubes and 500 l of 4 mol/l KOH were added.
injector. Fatty acids methyl esters were separated on an
After incubation at 45C for 45 min, the pH was adjusted
Omegawax 320 column (0.32 m 30 m, film thickness
to 3 by adding 500 l of 4 mol/l HCI. The internal
0.25 m; Supelco) using a temperature gradient of 120
standard 3,3',4,4'-tetradeuterated 9,11-prostaglandin
240C at 6C/min. One microliter samples were injected
F2 (PGF2-d4, 5 ng in 100 l ethanol) was added and the
into the gas chromatograph.
samples were vortex mixed. Then 10 ml ethyl acetate
was added, and the samples were vortexed for 20 s and Statistical analysis
centrifuged at 1000 g for 5 min. The upper organic
The results are expressed as mean SEM. Data were
layer was transferred into a new set of glass tubes. Total
subjected to equality of variance F test, and then to log
lipid extract was then applied onto an aminopropyl
transformation, if necessary, before one-way analysis of
(NH2) cartridge. The cartridge was sequentially washed
variance. When overall significance (p .05) was at-
with 10 ml of hexane/ethyl acetate (30/70, v/v), acetoni-
tained, individual between group comparisons were
trile/water (90/10, v/v) and acetonitrile. Isoprostanes
made using the Student- Newman-Keuls multiple range
were eluted by washing the cartridge with 5 ml of ethyl
test. Significance was defined at p .05. When between-
acetate/methanol/acetic acid (85/10/5, v/v/v). The final
group variance differences could not be normalized by
eluate from the NH2-cartridge was dried under nitrogen
log transformation, the data were analyzed by the non-
at 45C and was then converted to pentafluoro-benzyl
parametric Kruskal-Wallis one-way analysis of variance,
ester/trimethylsylil ether derivatives. Gas-chromatogra-
followed by the Fishers PLSD test for multiple compar-
phy-mass spectrometry/negative ion chemical ionization
isons.
(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]. Briefly, samples RESULTS
(2 l) were injected onto a SPB-1701 (30 m 0.25 mm
I.D.; 0.25 m film thickness; Supelco, Dorset, England). The final body weights were lower in diabetic rats
Separation was carried out using a temperature program: than in the control group (Table 1). The initial body
initial temperature: 175C; initial time: 2 min; rate: weights (not shown) were similar in control and diabetic
30C/min; final temperature: 270C; final time: 30 min. groups. The final body weights in DL--lipoic acid-
Samples (2 l) were injected into a temperature pro- treated diabetic rats were slightly but significantly lower
grammed Gerstel injector (Thames Chromatography, than in the untreated diabetic group.
 Renal oxidative stress in diabetes
Table 1. Final Body Weights and Blood Glucose Concentrations in
Control Rats and Diabetic Rats Treated With/Without DL--lipoic
Acid (Mean SEM)
Blood
glucose
Body concentration
weight (g) (mmol/l)
Control (n 19) 352 4 3.61 0.11
Diabetic (n 20) 293 7* 20.6 0.61*
Diabetic DL--lipoic acid (n 16) 268 5*,** 19.7 0.61*
The number of observations is indicated in parentheses.
* Significantly different compared with controls (p .01).
** Significantly 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 F2-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). F2-isoprostane concentra-
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). **Significantly different compared with the control group (p
.01); #Significantly different compared with the untreated diabetic
group (p .05).
189
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 significance (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
significantly, 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 significantly (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 findings 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,
190 I. G. OBROSOVA et al.

Fig. 2. Renal cortex (A) F2-isoprostane concentrations normalized per mg protein; (B) arachidonic acid concentrations; (C) F2-
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). **Significantly different compared with the control group (p .01); #,##Significantly 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 first 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 F2-isoprostane,
the specific product of arachidonic acid peroxidation. It
is important to realize that neither MDA plus 4-HA nor
F2-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 F2-isoprostane concentrations de-
pend not only on the rate of LPO, but on arachidonic acid
abundance, the rate of arachidonate metabolism to pros-

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). *,**Significantly different compared with the control group (p .05 and
.01, respectively); ##Significantly different compared with the untreated diabetic group (p .01).
 Renal oxidative stress in diabetes 191

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). *,**Significantly different compared with the control group (p .05 and .01,
respectively); ##Significantly different compared with the untreated diabetic group (p .01).

taglandin F2- and the prostaglandin clearance. Both
arachidonate biosynthesis and metabolism [56,59], and
prostaglandin F2- clearance [60] are compromised in
diabetes. Thus, it is quite possible that both MDA and
F2-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 F2-isoprostane alone. Our findings
of increased renal LPO in short-term diabetes are con-
sistent with elevated lipid peroxide, malondialdehyde,
and fluorescent 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 2 4 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 [29,32,36,62 64]. 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
[52,54,55,65 67], 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 downstream pathways, 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)

Diabetic DL --lipoic
Control Diabetic 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.

* ,** Significantly different compared with controls (p .05 and .01, respectively).
# ,##
Significantly different compared with untreated diabetic group (p .05 and .01, respectively).
192 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-specific. 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 [4,16,23,24,26,73
75], 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 findings
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 F2-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]. Beneficial
effects on diabetic complications [32 41] 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 [82 84]. Furthermore, R-enantiomer is
likely to have a better pharmacological profile 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 findings 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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