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: belma.turan@medicine.ankara.edu.tr

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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