Long-term treatment with a beta-blocker timolol attenuatesrenal-damage in diabetic rats via enhancing kidneyantioxidant-defense system
Hilal Gokturk • N. Nuray Ulusu • Muslum Gok •
Erkan Tuncay • Belgin Can • Belma Turan
Received: 10 April 2014 / Accepted: 2 June 2014
� Springer Science+Business Media New York 2014
Abstract The factors with increasing diabetes-prevalence
lead to significant global increases in chronic kidney dis-
ease. Since hyperglycemia generates more ROS and
attenuates cellular antioxidant-defense mechanisms,
numerous studies demonstrated that hyperglycemia-
induced oxidative stress played a major role in the extra-
cellular matrix expansion in tissues. Although no direct
relation between activation of beta-adrenergic (b-AR)
system and kidney disease in diabetes and since b-blockers
demonstrate marked beneficial effects due to their scav-
enging free radicals and/or acting as an antioxidant in
diabetic animal studies, the eventual objective of the
present study was to determine whether timolol-treatment
of streptozotocin-induced diabetic rats (5 mg/kg, daily
following diabetes-induction, for 12-week) has advantage
to prevent hyperglycemia-induced renal-damage via
enhancing the depressed antioxidant defense in the kidney.
Light microscopy data and their quantification demon-
strated that timolol-treatment prevented basically glomer-
ular hypertrophy, expansion in mesangium cell size,
thickening and fibrosis in glomerular basement membrane,
and accumulation of glycogen into tubular epithelial cells.
Additionally, electron microscopy data demonstrated that
timolol-treatment could also prevent diabetes-induced
changes in the kidney tissue such as hypertrophy in
podocytes, lost of filtration gaps and slit-diaphragms, and
vacuolization in the distal tubular cells. Biochemical ana-
lysis basically on enzymes of antioxidant-defense system,
including glutathione-S-transferase, glutathione reductase,
and glucose-6-phosphate dehydrogenase, further supported
that diabetes-induced damage in the kidney is mostly
dependent on the increased oxidative stress and timolol,
having an antioxidant-like action, could protect the kidney
against hyperglycemia-induced damage without normali-
zation of high-blood glucose level. Consequently, it can be
suggested that although b-blockers are widely used for the
treatment of cardiovascular diseases, b-blocker therapy of
diabetics seems to be a new therapeutic approach against
hyperglycemia-induced kidney damage in diabetic patients.
Keywords Diabetes � Antioxidants � Beta-blockers �Kidney
Introduction
Diabetes Mellitus, as a metabolic disorder of multiple eti-
ologies, is characterized by chronic hyperglycemia with
disturbances of several intracellular metabolisms. The
increase in the incidence of diabetes is due to longevity of
life, changing lifestyle, obesity, sedentary work, and
changing dietary habits [1]. Experimental and clinical
H. Gokturk
Department of Histology-Embryology, Faculty of Medicine,
Yildirim Beyazit University, Ankara, Turkey
N. N. Ulusu
Department of Biochemistry, School of Medicine, Koc
University, Istanbul, Turkey
M. Gok
Departments of Medical Biochemistry, Faculty of Medicine,
Hacettepe University, Ankara, Turkey
E. Tuncay � B. Turan (&)
Department of Biophysics, Faculty of Medicine, Ankara
University, Ankara, Turkey
e-mail: [email protected]
B. Can
Department of Histology-Embryology, Faculty of Medicine,
Ankara University, Ankara, Turkey
123
Mol Cell Biochem
DOI 10.1007/s11010-014-2123-2
studies showed that hyperglycemia generates more reactive
oxygen species (ROS) and attenuates antioxidant mecha-
nisms in cells, and therefore, hyperglycemia-induced oxi-
dative stress can play important role in the extracellular
matrix expansion in tissues [2–4]. In addition, it is known
that diabetic nephropathy is the major cause of end-stage
renal disease in the industrialized world, of which result, in
part, from higher ROS concentrations [2–4]. Accordingly,
the concert of all pathogenetic changes in mammalian
system under hyperglycemia results in a particular
sequence of events such as marked structural and func-
tional changes including extracellular matrix protein
deposition and fibrosis in muscle cells with different
extents in different tissues.
Diabetes-associated complications are still an important
medical problem, in spite of the usage of many oral anti-
diabetic agents. Indeed, enhanced glucose uptake has been
identified in several different types of cell populations
within the diabetic kidney, including glomerular epithelial
cells, mesangial cells, and proximal tubular epithelial cells
[5]. Although intensive glycemic control is the most
desirable method to prevent progressive diabetic renal
disease, additional early interventions including limiting
cellular ROS generation and/or strengthening of antioxi-
dant-defense system in the diabetic kidney may enhance
the ability of specific susceptible cell populations to
decrease glucose uptake from hyperglycemic environ-
ments. Being relevant to this approach, a number of studies
have shown that different types of antioxidants and phy-
tochemicals, relatively very low-side effects and low-cost,
open new avenues for the treatment of various diseases
including diabetes, their therapeutic benefits in experi-
mental diabetic complications including nephropathy, via
attenuation of enzymes activities in antioxidant-defense
system have already afforded a promise for use of treat-
ment of diabetes [6–8].
Early experimental studies provide evidences that beta1-
adrenoceptor (b1-AR) activation mediates renin secretion
in the kidney tissue of spontaneously hypertensive rats, in
part, via their membrane stabilizing activities [9, 10].
Supporting these early studies, recent clinical study out-
comes pointed out the important role of b-blocker usage for
prevention of sudden cardiac death in patients on hemod-
ialysis [11, 12] as well as the importance of b-blocker
therapy on survival and major renal outcomes in patients
with diabetes [13, 14]. In line with these studies, we and
others have shown that some of non-selective b-blockers
including timolol could exert adrenoceptor-independent
effects, including scavenging of free radicals leading to
controlled cellular redox-status, besides their b-blockage
action [15–21]. In a cell-line study, Miyamoto et al. [16]
showed that both nipradilol and timolol possessed a novel
mechanism of their actions, and function as potent
protective agents against increased oxidative stress in the
cell types.
Our previous data showed that chronic treatment with
either timolol or propranolol possessed some important
beneficial effects on the heart function of the rats with
either diabetes or aging, without having anti-hyperglyce-
mic action [19, 20]. The eventual objective of the present
study was to determine whether timolol-treatment of
streptozotocin-induced diabetic rats has advantage to pre-
vent and/or to get under control the hyperglycemia-induced
renal-damage via enhancing the depressed antioxidant-
defense in the kidney tissue. Following timolol-treatment
of streptozotocin-induced diabetic rats (5 mg/kg, daily
following diabetes-induction for 12-week) prevented basi-
cally the changes observed in glomerulus, mesangium, and
tubular epithelial cells together with enhancement of some
antioxidant enzymes activities such glutathione reductase
(GR) and glucose-6-phosphate dehydrogenase. Therefore,
our present results suggest that chronic timolol-treatment
can protect the kidney tissue against hyperglycemia-
induced damages together with enhancing the tissue anti-
oxidant-defense system in patients with diabetic nephrop-
athy even if they live with a high-blood glucose level.
Materials and methods
Induction of diabetes
All animal care and experimental procedure were per-
formed by following Ankara University ethics guidelines
(No: 2011-108-403). The experimental procedure for dia-
betic animals, including timolol-treatment is performed as
described, previously [18–20].
For histological investigation, all kidney tissues were
prepared daily following the scarification of the animals.
For biochemical analysis, some kidney tissues were stored
at -80 �C for determination of enzymes activities.
Histological examination
For light microscopic evaluation, the kidney samples were
fixed in phosphate buffer 10 % formaldehyde for 2-day,
and then washed after fixation to remove the excess
material. The samples were dehydrated, using a serious of
alcohol solutions, and then as a clearing agent, an organic
solvent xylol was used to remove the alcohol. Following
the clearing process, the tissues were infiltrated with an
embedding-agent melted-paraffin. Following infiltration,
the paraffin was allowed to solidify, and thereby a firm
homogeneous mass containing the embedded-tissue was
obtained. The embedded-samples were sectioned to 3-lm
thickness by Leitz-1512 microtome. The sections were
Mol Cell Biochem
123
stained with hematoxylin-eosin (HE), Masson’s trichrome,
periodic acid schiff, periodic acid-silver methenamine (PA-
SM) or toluidine blue/azur II. All samples were photo-
graphed by Nikon-Eclipse E600 photomicroscope.
For electron microscopy investigation, tiny kidney sec-
tions were fixed in a solution of 2.5 % glutaraldehyde in a
phosphate buffer at pH 7.2 for 2–4 h and postfixed in 1 %
osmium tetroxide. Then, the materials were dehydrated in a
graded ethanol solutions and embedded in araldite 6005
(Ciba Geigy, Summit, NJ, USA). Sections were cut with an
ultramicrotome (Leica Ultracut R, Solms, Germany) with a
glass knife as semi-thin sections (700–1,000 nm) and then
these ultra-thin sections were stained with uranyl acetate-
lead citrate. All samples were photographed by a trans-
mission electron microscope (LEO 906-E, Oberkochen,
Germany).
Biochemical assays
All biochemical assays were performed as described, pre-
viously [22]. Shortly, the frozen kidney samples were first
homogenized with an ultra turrax homogenizer and then
the homogenate was centrifuged with an ultracentrifuge at
4 �C. The supernatant part was used to measure enzymes
activities and protein determinations. Protein contents of
the samples were determined by a micro-method using
bovine serum albumin as standard reference.
The activity of GR was measured in the supernatants
using a modified Staal method as described previously
[22]. Glutathione-S-transferase (GST) activity was also
assayed by measuring the conjugation of reduced gluta-
thione (GSH) with 1-chloro-2, 4-dinitrobenzene as descri-
bed previously [22]. The activities of glucose-6-phosphate
dehydrogenase (G-6-PD) and 6-phosphogluconate dehy-
drogenase (6-PGD) were also determined by a spectro-
photometer as described previously [22].
Data analysis and statistics
Groups were tested and compared using one-way ANOVA
and Tukey-post hoc test. Values of p \ 0.05 were taken as
statistically significant, and the data are presented as
mean ± SEM.
Results
General data for experimental animals
As we mentioned in our previous article, streptozotocin
(STZ)-injected rats displayed hyperglycemia as indicated
by significant increases in the blood glucose level com-
pared with age-matched controls following week-1 until
week-13 after STZ-injection (totally a 12-week treatment).
Timolol-treatment had no significant effect on high-blood
glucose levels; however, it improved some diabetic
symptoms including weight loss, as a small but a statisti-
cally significant level (p \ 0.05), by reducing weight loss
although the weight of the subjects remained less than
those of the controls [23].
In order to confirm whether STZ-injection of rats are
mimicking type 1 diabetes and the consequence of hyper-
glycemia in the renal-system, we monitored first some
diabetes-related renal-tissue damage associated markers
including the serum levels of urea, uric acid, and creati-
nine, similar to our previous study [24]. The serum levels
of urea and creatinine decreased in the diabetic group,
significantly compared to those of the controls, while their
serum uric acid levels were not significantly different
among these two groups, while these parameters were
normalized with timolol-treatment (data not presented).
Light microscopy analysis of kidney tissue
Marked morphological differences in the sections stained
with HE were observed basically in the distal and proximal
tubulus of the kidney tissue from 12-week diabetic rats
compared to those from the control group rats (Fig. 1a).
Our main observations included an expanded glomerulus
borders and prominent stasis in the glomerulus and inter-
stitium, mostly, due to the increased amount of erythro-
cytes in the investigated regions of the sections. The light
microscopy investigations also demonstrated marked evi-
dences for the epithelization in most of the glomeruli. In
addition, the observations related with distal convoluted
tubulus revealed that the cells are in pale-appearance with
loss of cytoplasmic constituents. Furthermore, timolol-
treatment of the diabetic rats significantly prevented these
above damages observed in the sections (Fig. 1b).
In order to understand further the effect of timolol-
treatment on diabetes-induced damaged in diabetic kidney
tissue, particularly developed in the glomerulus and Bow-
man’s capsule, we used sections from the kidney tissues
stained with different dyes such as either Masson’s tri-
chrome or periodic acid schiff (PAS). The sections stained
with either MT or PAS demonstrated that diabetes induced
marked thickening in the membrane of Bowman’s capsule
and the glomerulus, and marked dilation in the tubulus as
well as markedly increased amount of fibrosis in the
interstitial tissue, and erythrocyte stasis in the glomerulus
(Fig. 2a, c). These changes were not seen in the prepara-
tions from timolol-treated diabetic group (Fig. 2b, d).
In another set of examinations, we used sections from the
same experimental group rats stained with PA-SM. There
were also marked dilated tubulus and macula densa, thick-
ening in the basement membrane of both glomerulus and
Mol Cell Biochem
123
tubulus in the diabetic rats (Fig. 3a) while timolol-treatment
of the diabetic rats for a 12-week prevented these changes,
significantly (Fig. 3b). There were also normal sclerosing
areas in the kidney sections, marked hyalinisation at vessel
pole, normal appearance of proximal and dilated tubulus, and
less thickened basal membrane of tubulus and glomerulus in
the timolol-treated diabetic group, as well.
Furthermore, in order to provide quantitative data rela-
ted with the effects of timolol-treatment on glomerular
radius and thickness of glomerular basement membrane in
the kidney sections from diabetic rats, we also examined
semi-thin section of kidney tissue stained with toluidine
blue/azur II (Fig. 4a–c, respectively). Representative pho-
tomicrographs are showing the normal membrane appear-
ances of both glomerulus and Bowman’s capsule, and the
normal proximal and distal tubulus which were presenting
the main observations in the control group (Fig. 4a).
Marked degenerated corpuscule, fibrosis and increased
connective tissue around the blood vessels were detected in
the sections from the diabetic group, while these were not
detectable in timolol-treated diabetic group (Fig. 4b, c,
respectively). The percentage changes in the glomerular
radius as well as in the thickness of glomerular basement
membrane in diabetic and timolol-treated diabetic groups
compared to that of the control group were given as bar
graphs (Fig. 4d, e, respectively). Based on these results
from three groups, the untreated diabetic rats exhibited
larger glomerular radius, indicating glomerular hypertro-
phy with a marked thickening in the glomerular basement
membrane (p \ 0.05 vs. controls). Compared with
untreated diabetic rats, the hypertrophy and thickening in
the glomerular basement membrane were attenuated in the
timolol-treated diabetic group, significantly (p \ 0.05 vs.
untreated diabetics).
Electron microscopy analysis of kidney tissue
The results of electron microscopy evaluations, in here,
were similar to those of our previously published data [24].
Basically, we observed an increased number of mesangial
cells in the network of glomerular capillary walls as well as
an increased amount of lipid accumulation in the proximal
tubules in the sections from diabetic rats. In addition, there
were marked degenerative glomerulus and basement
membrane thickening in the glomerulus and tubules in
diabetic rats compared to those of the controls (Fig. 5b vs.
a). The cells in mesangial matrix had nucleus with differ-
entially degenerated, and heterochromatin and foci of
glomerular basement membrane thickening were seen in
the same sections, as well. Additionally, accumulation of
electron-dense materials into tubular mitochondria as well
as marked mitochondrial degenerations was appearing in
the sections from diabetic rats (Fig. 5b). The tubular
basement membranes in diabetic rats were thickened and
wavy in the same sections, as well.
The nucleus membrane of mesangial cells in diabetic
rats was infiltrated into the cell-matrix, which implies the
existence of some contractile filaments such as myosin in
the nucleus. In addition, in the same sections, thin filaments
inside the cytoplasm were concentrated near the nucleus
membrane. Compared to the controls, some modest glo-
merular lesions were noted in the sections from diabetic
rats. Furthermore, the glomerular capillaries were irregular,
enlarged, and attached to the Bowman’s capsule, although
its thickness with quantification was found not to be sig-
nificantly different among these groups (Fig. 5d; p [ 0.05).
In addition in diabetic rats, the degree of tubule-interstitial
damage was modest and there were some enlarged tubuli
with an indication of slight atrophy in the epithelial cells.
Fig. 1 Light microscopy examinations showing hematoxylin and
eosin-stained kidney sections. Micrographs showing expanded glo-
merulus borders (tailed arrow), dense erythrocytes stasis in glomer-
ulus and interstitium (vector spiral arrow), and clear crystalline cells
(diamond) in diabetic rats (a). Renal corpuscule sections showing less
crystalline cells of tubulus (diamond) and slight erythrocyte stasis in
glomerulus (vector spiral arrow) in timolol-treated diabetic rats (b).
In here, p proximal tubulus, d distal tubulus (bars = 50 lm)
Mol Cell Biochem
123
Moreover, a slight focal interstitial fibrosis was also
observed and the intra-renal arterial vessels showed modest
thickening of the walls in diabetic rats, as well.
The sections from timolol-treated diabetic rats were
almost in the normal appearances for all investigated sec-
tions, such as a normal appearance of podocytes on base-
ment membrane of glomerulus besides normal appearances
in the tubulus and epithelial cells (Fig. 5c).
Biochemical analysis of kidney tissue
The mean enzymes activities of antioxidant-defense system
in diabetic and timolol-treated diabetic rats compared to
those of the controls are given with bar graphs (Fig. 6). As
can be seen from this figure, the activity of GR, as one of
the most important enzyme in antioxidant-defense system
decreased (p \ 0.05 vs. controls) and most probably as a
cellular compensation mechanism, the GST activity
increased, significantly (p \ 0.05) in diabetic group com-
pared to those of the controls (Fig. 6a, b). In addition, the
glutathione-6-phosphate dehydrogenase (G-6-PD) activity
decreased in the diabetic group (p \ 0.05 vs. controls),
while the 6-phosphogluconate dehydrogenase (6-PGD)
activity of this group did not change, significantly
(p [ 0.05) (Fig. 6c, d). Timolol-treatment of diabetic rats,
for 12-week, induced almost a complete-protection against
the hyperglycemia-induced alterations in the antioxidant-
defense system of the kidney.
Discussion
The major importance of this study is to demonstrate
important beneficial effect of long-term treatment of
Fig. 2 Timolol-treatment of diabetic rats prevented the changes in
glomerulus and Bowman’s capsule. Representative sections stained
with either Masson’s trichrome (a, b) or periodic acid schiff, PAS (c,
d). Sections from diabetic rats in a showing thickened Bowman’s
capsule (tailed arrow), dense fibrosis at interstitial tissue (thick
arrow), and marked erythrocyte stasis in glomerulus (vector spiral
arrow) and in c showing thickened basement membrane of glomer-
ulus (thin arrow), dilated tubulus (clubs), crystalline cells in tubulus
(diamond). Sections from timolol-treated diabetic rats in b thickened
Bowman’s capsule (tailed arrow), erythrocyte stasis in glomerulus
(vector spiral arrow) and interstitial area (thick arrow) and crystalline
cells in tubulus (diamond), while in d hyalinosis at vessel pole
(spade), dilated tubulus (clubs), degenerated tubular cells (square)
and less crystalline cells in tubulus (diamond). In here, p proximal
tubulus, d distal tubulus (bars = 50 lm)
Mol Cell Biochem
123
Fig. 3 Light microscopy examinations showing periodic acid-silver
methenamine staining kidney sections. a Marked thickening in
basement membrane of glomerulus (arrow) and tubuli (arrowhead),
crystalline cells in tubulus (diamond), and macula densa (cursor) in
sections from diabetic rat kidneys. b Similar thickened basal
membrane of tubulus (tailed arrow) and less crystalline cells in
tubulus (diamond) and proximal tubulus (p) in sections from timolol-
treated diabetic rat kidneys (bars = 100 and 50 lm, respectively)
A B C
D
CON DM DM+TIM0
25
50
75
100
125 *
Glo
mer
ula
r ra
diu
s
E
Fig. 4 Timolol-treatment normalized the thickened glomerular base-
ment membrane and the enlarged glomerular radius. Representative
semi-thin sections stained with toluidin’s blue/azur II of control (a),
diabetic (b) and timolol-treated diabetic (c) rats. Normal appearances
of glomerulus and parietal membrane of Bowman’s capsule (flash),
proximal tubulus (p), distal tubulus (d) shown in a for control rats;
degenerated renal corpuscule (arrow) and normal appearances of
proximal tubulus (p) and distal tubulus (d) shown in b for diabetic
rats; fibrosis and increased connective tissue around blood vessels
(arrow), Bowman’s capsule (star), and normal appearances of
proximal tubulus (p) shown in c for timolol-treated diabetic rats
(bars = 50 lm). d Percentage changes given for the glomerular
radius of the sections from diabetic (DM) and timolol-treated diabetic
(DM?TIM) groups compared to that of the control (CON) group.
e Percentage changes in the thickness of glomerular basement
membrane of the sections from DM and DM?TIM groups compared
to that of the CON group. Values are given as mean (± SEM), and
* p \ 0.05 vs. CON group, � p \ 0.05 vs. DM group
Mol Cell Biochem
123
diabetic rats with a b-blocker, timolol such as significant
attenuation in the renal-damage via enhancement of kid-
ney antioxidant-defense system besides its systemic action
in diabetic rats. Therefore, a novel finding of our data is
that timolol-treatment induced important benefits in kid-
ney tissue via affecting some enzymes activities of anti-
oxidant-defense system from diabetic rats besides its
small but significant beneficial action on body-weight loss
seen in diabetic rats. Our histological examinations in
kidney tissue showed that timolol-treatment attenuated the
glomerulus sclerosis, irregular and enlarged glomerular
capillary structure via affecting their attachment to the
Bowman’s capsule besides marked increases in mesangial
cell numbers observed in the sections. Even though, a
long-period timolol-treatment could evoke marked
enhancement in the kidney antioxidant-defense system,
this present study strongly points out a possible role of
timolol to be a new candidate to induce a marked
attenuation in the renal-damages observed in the diabetic
patients.
In here, we demonstrated also a quantitative data related
with the glomerular radius and thickness of the glomerular
basement membrane in the sections from diabetic kidney
tissue, similar to ones published previously performed in
streptozotocin-induced hyperglycemic rat kidney using
different microscopic examinations [24]. These results are
also similar to the ones that published previously which
related with the walls of glomerular vessels in the early
stages of diabetes mellitus using light and transmission
electron microscopic examinations [25]. In addition, we, in
this study, showed that timolol-treatment has important
protective effect on the kidney tissue via enhancing the
tissue antioxidant-defense system. Indeed, supporting to
our present data, Toblli et al. [26] used nebivolol to treat
Zucker diabetic fatty rats for 6-month and evaluated its
effect on the control of blood pressure of the rats. Then,
Fig. 5 Transmission electron micrograph of kidney sections stained
with uranyl acetate-lead citrate and quantification of the changes in
the radius of Bowman’s capsule. Filtration slits between pedicels
(vector spiral arrow) and normal appearance of fenestra structure
(arrowhead) shown in a for control rats, CON group (9 12,930 and
bar = 1,000 nm); thickening in basement membrane of glomerulus
(tailed arrow) and increased mesangium matrix (star) shown in b for
diabetic rats, DM group (9 6,000 and bar = 1,000 nm) while e;
erythrocyte, n; neutrophile, crescent; blood platelet; regular podocytes
(arrow), irregular podocytes (thin arrow), regular fenestration
(arrowhead), and flattened fenestra structure (thick arrow) shown in
c for timolol-treated diabetic rats, DM?TIM group (9 10,000 and
bar = 1,000 nm). d Percentage changes in the radius of Bowman’s
capsule from DM and DM?TIM groups compared to that of the CON
group. Values are given as mean (± SEM)
Mol Cell Biochem
123
they demonstrated its beneficial effects on the kidney tissue
such as decreasing lipid peroxidation, preservation of
glomerular filtration rate, reducing proteinuria, inducing a
normal regulation of nephrin and podocin expressions, and
reduction of extracellular matrix proteins via control of
cellular redox-status. However, the exact underlying
mechanisms and the onset of alterations in kidney tissue at
the early phases of diabetes is unclear yet, although dif-
fused sclerosis in both glomerulus and mesangium, thick-
ening in the basal laminae, and proliferation of glomerular
cells have been already shown in the young diabetic mice,
previously by others [27].
It is known that nephropathy, globally renal-system
disorders, is one of the major problems in diabetic patients.
Although the current treatments of diabetic patients include
optimization in the control of both glycemic status and
blood pressure, it is more likely important to have more
innovative strategies such as a prevention of diabetes-
related pathologies such as nephropathy even if they have
high-blood glucose levels. Being parallel to the previous
statement, early and recent clinical studies imply the
importance of new therapy strategies, including the use
of b-blocker and angiotensin blocker together as therapies
and control of ROS generation and cellular redox-status are
also likely to feature in future treatment regimens for
diabetic patients [12, 26, 28–30]. Related with this subject,
Forbes et al. [31] reviewed widely the facts related with the
important role of oxidative stress as a major culprit in
kidney disease in diabetes. They proposed a unifying
hypothesis whereby mitochondrial production of ROS in
response to chronic hyperglycemia could be the key initi-
ator for the progression and development of diabetes
complications including nephropathy. In addition, they also
discussed the importance of antioxidants for reno-protec-
tion in diabetic patients. Therefore, right now, there is now
an increasing body of data on the role of strategies
involving a more targeted antioxidant approach being the
elusive additive therapy required to further optimize reno-
protection in diabetes.
The antioxidant-like action of timolol-treatment, pre-
sented previously, is in line also with previously published
data by others. Indeed, our recently published study
established that timolol-treatment of diabetic rats for
12-week led to a balanced oxidant/antioxidant level in the
circulation system of the rats such as normalization of
circulating plasma markers of oxidative stress, total oxi-
dant/total antioxidant status [23]. In the same study, to test
whether the observed timolol effects in diabetic rats are due
to its putative scavenging activity for ROS, we investigated
its direct antioxidant effect in H2O2-induced oxidant
medium and observed that timolol exerted a clear antiox-
idant effect in this fully oxidized medium in a concentra-
tion-dependent manner, besides it has an antioxidant-like
action with respect to trolox solution, although another b-
blocker propranolol, at any concentration did not show any
antioxidant effect. Those findings were also in line with an
earlier report, in that study, how an antioxidant role of
timolol as a scavenger was more effective on ROS than that
of propranolol because propanol as similar scavenger effect
on reactive nitrogen species (RNS) [32].
Accordingly, it seems highly recognizable to consider
the fact on the development of diabetic nephropathy is
associated with increased oxidative stress in the renal
system via both decreased NO production and increased
ROS generation [26, 28, 30, 33]. As mentioned in these
studies, it is well accepted that both types’ reactive mole-
cules can induce renal and tubular injury (mostly associ-
ated with proteinuria) via directly being associated with
oxidation of proteins and lipids. Indeed, the literature data
also confirm the above statement because proteinuria in the
renal system is shown to proceed by decreased NO syn-
thase (NOS), in most due to ROS-related suppression of
NOS [33]. As mentioned in previous paragraphs, the b-
CON DM
DM+TIM
0.0
2.5
5.0
7.5
10.0
12.5†
*
*
Glu
cose
-6-p
ho
sph
ate
deh
ydro
gen
ase
(U/m
g)
CON DM
DM+TIM
0.0
1.0
2.0
3.0
4.0
5.0 †
6-p
ho
sph
og
luco
nat
ed
ehyd
rog
enas
e (U
/mg
)
AB
C D
Fig. 6 Timolol-treatment of diabetic rats enhanced the depressed
activities of some antioxidant enzymes in the kidney tissues. Bar
graphs showing the effect of timolol-treatment of diabetic rats
(DM?TIM) on kidney tissue glutathione reductase (a), glutathione-S-
transferase (b), glucose-6-phosphate dehydrogenase (c), and 6-phos-
phogluconate-dehydrogenase (d) in diabetic rats (DM) compared to
that of the control (CON) group rats. Values are given as mean
(± SEM), and * p \ 0.05 vs. CON group, � p \ 0.05 vs. DM group
Mol Cell Biochem
123
blockers possess important ancillary properties besides
inhibiting b-adrenoceptors, while among them, nebivolol
activates NOS. Nebivolol and carvedilol preserve NOS
activity by reducing asymmetrical dimethylarginine and
enhance the bioavailability of NO because of their anti-
oxidant properties [14, 34, 35]. Even in the early studies
with experimental diabetic animals, the crucial roles of b-
blockers, due to their antioxidant-like actions in the pro-
tection against the renal-damage have been already dem-
onstrated [10, 36, 37]. Therefore, it can be summarized that
the data from the present and previous studies strongly
point out that oxidative stress is involved in the etiology of
diabetes-induced damage in the renal tissue, as most, via a
depressed endogenous antioxidant-defense mechanism.
As conclusion, diabetic nephropathy is a common
complication seen in diabetic patients, and a poor glycemic
control plays a significant role in its pathology. In addition,
early studies also showed that diabetic nephropathy is
morphologically characterized by the accumulation of
matrix proteins, in most, due to increased oxidative stress
and depressed antioxidant-defense system. Therefore, any
antioxidant-treatment of diabetic subjects can present
important benefits against hyperglycemia-induced renal-
damage. Thus, one can suggest that, owing to their ROS/
RNS-mediated actions (antioxidant-like actions), the new
generation b-blockers will find more clinical applications
in the treatment of renal diseases in diabetes mellitus.
Acknowledgments This work has been supported partially by grant
from TUBITAK SBAG-111S042.
Conflicts of interest No potential conflicts of interest relevant to
this article were reported.
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