Mol Cell Biochem DOI 10.1007/s11010-014-2123-2
Long-term treatment with a beta-blocker timolol attenuates renal-damage in diabetic rats via enhancing kidney antioxidant-defense system Hilal Gokturk • N. Nuray Ulusu • Muslum Gok Erkan Tuncay • Belgin Can • Belma Turan
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Received: 10 April 2014 / Accepted: 2 June 2014 Ó Springer Science+Business Media New York 2014
Abstract The factors with increasing diabetes-prevalence lead to significant global increases in chronic kidney disease. Since hyperglycemia generates more ROS and attenuates cellular antioxidant-defense mechanisms, numerous studies demonstrated that hyperglycemiainduced oxidative stress played a major role in the extracellular 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 scavenging 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.
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
Light microscopy data and their quantification demonstrated that timolol-treatment prevented basically glomerular 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 analysis 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 normalization 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 Kidney
Diabetes Antioxidants Beta-blockers
Introduction Diabetes Mellitus, as a metabolic disorder of multiple etiologies, 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
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studies showed that hyperglycemia generates more reactive oxygen species (ROS) and attenuates antioxidant mechanisms in cells, and therefore, hyperglycemia-induced oxidative 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 functional 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 antidiabetic 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 antioxidant-defense system in the diabetic kidney may enhance the ability of specific susceptible cell populations to decrease glucose uptake from hyperglycemic environments. Being relevant to this approach, a number of studies have shown that different types of antioxidants and phytochemicals, relatively very low-side effects and low-cost, open new avenues for the treatment of various diseases including diabetes, their therapeutic benefits in experimental diabetic complications including nephropathy, via attenuation of enzymes activities in antioxidant-defense system have already afforded a promise for use of treatment of diabetes [6–8]. Early experimental studies provide evidences that beta1adrenoceptor (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 outcomes pointed out the important role of b-blocker usage for prevention of sudden cardiac death in patients on hemodialysis [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
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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-hyperglycemic action [19, 20]. The eventual objective of the present study was to determine whether timolol-treatment of streptozotocin-induced diabetic rats has advantage to prevent and/or to get under control the hyperglycemia-induced renal-damage via enhancing the depressed antioxidantdefense in the kidney tissue. Following timolol-treatment of streptozotocin-induced diabetic rats (5 mg/kg, daily following diabetes-induction for 12-week) prevented basically 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 hyperglycemiainduced damages together with enhancing the tissue antioxidant-defense system in patients with diabetic nephropathy even if they live with a high-blood glucose level.
Materials and methods Induction of diabetes All animal care and experimental procedure were performed by following Ankara University ethics guidelines (No: 2011-108-403). The experimental procedure for diabetic 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
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stained with hematoxylin-eosin (HE), Masson’s trichrome, periodic acid schiff, periodic acid-silver methenamine (PASM) or toluidine blue/azur II. All samples were photographed by Nikon-Eclipse E600 photomicroscope. For electron microscopy investigation, tiny kidney sections 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 acetatelead citrate. All samples were photographed by a transmission electron microscope (LEO 906-E, Oberkochen, Germany). Biochemical assays All biochemical assays were performed as described, previously [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 glutathione (GSH) with 1-chloro-2, 4-dinitrobenzene as described previously [22]. The activities of glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD) were also determined by a spectrophotometer 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 compared 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 statistically 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 hyperglycemia in the renal-system, we monitored first some diabetes-related renal-tissue damage associated markers including the serum levels of urea, uric acid, and creatinine, 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 interstitium, mostly, due to the increased amount of erythrocytes in the investigated regions of the sections. The light microscopy investigations also demonstrated marked evidences 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, timololtreatment of the diabetic rats significantly prevented these above damages observed in the sections (Fig. 1b). In order to understand further the effect of timololtreatment on diabetes-induced damaged in diabetic kidney tissue, particularly developed in the glomerulus and Bowman’s capsule, we used sections from the kidney tissues stained with different dyes such as either Masson’s trichrome 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 preparations 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, thickening in the basement membrane of both glomerulus and
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Fig. 1 Light microscopy examinations showing hematoxylin and eosin-stained kidney sections. Micrographs showing expanded glomerulus borders (tailed arrow), dense erythrocytes stasis in glomerulus 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)
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 related 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 photomicrographs are showing the normal membrane appearances 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 hypertrophy 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
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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 differentially 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 glomerular 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 significantly 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.
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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 glomerulus (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)
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 sections, such as a normal appearance of podocytes on basement membrane of glomerulus besides normal appearances in the tubulus and epithelial cells (Fig. 5c).
cellular compensation mechanism, the GST activity increased, significantly (p \ 0.05) in diabetic group compared 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 antioxidantdefense system of the kidney.
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
Discussion The major importance of this study is to demonstrate important beneficial effect of long-term treatment of
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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
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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 timololtreated diabetic rat kidneys (bars = 100 and 50 lm, respectively)
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Fig. 4 Timolol-treatment normalized the thickened glomerular basement 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
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(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
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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)
diabetic rats with a b-blocker, timolol such as significant attenuation in the renal-damage via enhancement of kidney 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 kidney tissue via affecting some enzymes activities of antioxidant-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,
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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-Stransferase (b), glucose-6-phosphate dehydrogenase (c), and 6-phosphogluconate-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
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 diffused sclerosis in both glomerulus and mesangium, thickening 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 diabetesrelated pathologies such as nephropathy even if they have high-blood glucose levels. Being parallel to the previous
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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 initiator for the progression and development of diabetes complications including nephropathy. In addition, they also discussed the importance of antioxidants for reno-protection 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 renoprotection in diabetes. The antioxidant-like action of timolol-treatment, presented 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 oxidant/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 antioxidant effect in this fully oxidized medium in a concentration-dependent manner, besides it has an antioxidant-like action with respect to trolox solution, although another bblocker 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 molecules can induce renal and tubular injury (mostly associated 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 synthase (NOS), in most due to ROS-related suppression of NOS [33]. As mentioned in previous paragraphs, the b-
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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 antioxidant properties [14, 34, 35]. Even in the early studies with experimental diabetic animals, the crucial roles of bblockers, due to their antioxidant-like actions in the protection against the renal-damage have been already demonstrated [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 renaldamage. 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.
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Acknowledgments This work has been supported partially by grant from TUBITAK SBAG-111S042.
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Conflicts of interest No potential conflicts of interest relevant to this article were reported. 15.
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Long-term treatment with a beta-blocker timolol attenuates renal-damage in diabetic rats via enhancing kidney antioxidant-defense system Hilal Gokturk • N. Nuray Ulusu • Muslum Gok Erkan Tuncay • Belgin Can • Belma Turan
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Received: 10 April 2014 / Accepted: 2 June 2014 Ó Springer Science+Business Media New York 2014
Abstract The factors with increasing diabetes-prevalence lead to significant global increases in chronic kidney disease. Since hyperglycemia generates more ROS and attenuates cellular antioxidant-defense mechanisms, numerous studies demonstrated that hyperglycemiainduced oxidative stress played a major role in the extracellular 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 scavenging 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.
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
Light microscopy data and their quantification demonstrated that timolol-treatment prevented basically glomerular 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 analysis 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 normalization 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 Kidney
Diabetes Antioxidants Beta-blockers
Introduction Diabetes Mellitus, as a metabolic disorder of multiple etiologies, 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
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studies showed that hyperglycemia generates more reactive oxygen species (ROS) and attenuates antioxidant mechanisms in cells, and therefore, hyperglycemia-induced oxidative 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 functional 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 antidiabetic 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 antioxidant-defense system in the diabetic kidney may enhance the ability of specific susceptible cell populations to decrease glucose uptake from hyperglycemic environments. Being relevant to this approach, a number of studies have shown that different types of antioxidants and phytochemicals, relatively very low-side effects and low-cost, open new avenues for the treatment of various diseases including diabetes, their therapeutic benefits in experimental diabetic complications including nephropathy, via attenuation of enzymes activities in antioxidant-defense system have already afforded a promise for use of treatment of diabetes [6–8]. Early experimental studies provide evidences that beta1adrenoceptor (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 outcomes pointed out the important role of b-blocker usage for prevention of sudden cardiac death in patients on hemodialysis [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
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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-hyperglycemic action [19, 20]. The eventual objective of the present study was to determine whether timolol-treatment of streptozotocin-induced diabetic rats has advantage to prevent and/or to get under control the hyperglycemia-induced renal-damage via enhancing the depressed antioxidantdefense in the kidney tissue. Following timolol-treatment of streptozotocin-induced diabetic rats (5 mg/kg, daily following diabetes-induction for 12-week) prevented basically 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 hyperglycemiainduced damages together with enhancing the tissue antioxidant-defense system in patients with diabetic nephropathy even if they live with a high-blood glucose level.
Materials and methods Induction of diabetes All animal care and experimental procedure were performed by following Ankara University ethics guidelines (No: 2011-108-403). The experimental procedure for diabetic 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
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stained with hematoxylin-eosin (HE), Masson’s trichrome, periodic acid schiff, periodic acid-silver methenamine (PASM) or toluidine blue/azur II. All samples were photographed by Nikon-Eclipse E600 photomicroscope. For electron microscopy investigation, tiny kidney sections 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 acetatelead citrate. All samples were photographed by a transmission electron microscope (LEO 906-E, Oberkochen, Germany). Biochemical assays All biochemical assays were performed as described, previously [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 glutathione (GSH) with 1-chloro-2, 4-dinitrobenzene as described previously [22]. The activities of glucose-6-phosphate dehydrogenase (G-6-PD) and 6-phosphogluconate dehydrogenase (6-PGD) were also determined by a spectrophotometer 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 compared 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 statistically 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 hyperglycemia in the renal-system, we monitored first some diabetes-related renal-tissue damage associated markers including the serum levels of urea, uric acid, and creatinine, 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 interstitium, mostly, due to the increased amount of erythrocytes in the investigated regions of the sections. The light microscopy investigations also demonstrated marked evidences 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, timololtreatment of the diabetic rats significantly prevented these above damages observed in the sections (Fig. 1b). In order to understand further the effect of timololtreatment on diabetes-induced damaged in diabetic kidney tissue, particularly developed in the glomerulus and Bowman’s capsule, we used sections from the kidney tissues stained with different dyes such as either Masson’s trichrome 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 preparations 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, thickening in the basement membrane of both glomerulus and
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Fig. 1 Light microscopy examinations showing hematoxylin and eosin-stained kidney sections. Micrographs showing expanded glomerulus borders (tailed arrow), dense erythrocytes stasis in glomerulus 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)
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 related 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 photomicrographs are showing the normal membrane appearances 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 hypertrophy 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
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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 differentially 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 glomerular 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 significantly 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.
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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 glomerulus (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)
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 sections, such as a normal appearance of podocytes on basement membrane of glomerulus besides normal appearances in the tubulus and epithelial cells (Fig. 5c).
cellular compensation mechanism, the GST activity increased, significantly (p \ 0.05) in diabetic group compared 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 antioxidantdefense system of the kidney.
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
Discussion The major importance of this study is to demonstrate important beneficial effect of long-term treatment of
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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
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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 timololtreated diabetic rat kidneys (bars = 100 and 50 lm, respectively)
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Glomerular radius
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Fig. 4 Timolol-treatment normalized the thickened glomerular basement 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
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(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
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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)
diabetic rats with a b-blocker, timolol such as significant attenuation in the renal-damage via enhancement of kidney 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 kidney tissue via affecting some enzymes activities of antioxidant-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,
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Glucose-6-phosphate dehydrogenase (U/mg)
C
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-Stransferase (b), glucose-6-phosphate dehydrogenase (c), and 6-phosphogluconate-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
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 diffused sclerosis in both glomerulus and mesangium, thickening 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 diabetesrelated pathologies such as nephropathy even if they have high-blood glucose levels. Being parallel to the previous
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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 initiator for the progression and development of diabetes complications including nephropathy. In addition, they also discussed the importance of antioxidants for reno-protection 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 renoprotection in diabetes. The antioxidant-like action of timolol-treatment, presented 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 oxidant/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 antioxidant effect in this fully oxidized medium in a concentration-dependent manner, besides it has an antioxidant-like action with respect to trolox solution, although another bblocker 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 molecules can induce renal and tubular injury (mostly associated 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 synthase (NOS), in most due to ROS-related suppression of NOS [33]. As mentioned in previous paragraphs, the b-
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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 antioxidant properties [14, 34, 35]. Even in the early studies with experimental diabetic animals, the crucial roles of bblockers, due to their antioxidant-like actions in the protection against the renal-damage have been already demonstrated [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 renaldamage. 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.
10.
Acknowledgments This work has been supported partially by grant from TUBITAK SBAG-111S042.
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Conflicts of interest No potential conflicts of interest relevant to this article were reported. 15.
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