Antioxidant effect of carnosine treatment on renal oxidative stress in streptozotocin-induced diabetic rats A Yay1, D Akkus1, H Yapıslar2, E Balcıoglu1, MF Sonmez1, S Ozdamar1 1Department of Histology and Embryology, University of Erciyes, Medical Faculty, Kayseri, Turkey, and 2Department of Physiology, Istanbul Bilim University, Medical Faculty Istanbul, Turkey
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Accepted January 30, 2014
Abstract Nitric oxide (NO) plays a significant role in the development of diabetic nephropathy. We investigated the effects of an antioxidant, carnosine, on streptozotocin (STZ)-induced renal injury in diabetic rats. We used four groups of eight rats: group 1, control; group 2, carnosine treated; group 3, untreated diabetic; group 4, carnosine treated diabetic. Kidneys were removed and processed, and sections were stained with periodic acid-Schiff (PAS) and subjected to eNOS immunohistochemistry. Examination by light microscopy revealed degenerated glomeruli, thickened basement membrane and glycogen accumulation in the tubules of diabetic kidneys. Carnosine treatment prevented the renal morphological damage caused by diabetes. Moreover, administration of carnosine decreased somewhat the oxidative damage of diabetic nephropathy. Appropriate doses of carnosine might be a useful therapeutic option to reduce oxidative stress and associated renal injury in diabetes mellitus. Key words: antioxidant therapy, carnosine, diabetes, eNOS, kidney, nephropathy
Diabetic nephropathy is a common microvascular complication of diabetes mellitus (Fioretto and Mauer 2007) that is characterized by hypertrophy of kidney structures including thickening of the basal membrane and progressive accumulation of extracellular matrix (Ibrahim and Hostetter 1997). Hyperglycemia, genetic factors, inflammatory cytokines and oxidative stress contribute to the development diabetic nephropathy (Nobrega et al. 2004). Nitric oxide (NO) must be considered in the pathogenesis of diabetic nephropathy, because NO plays a role in controlling renal and glomerular hemodynamics in the kidney (Dellamea et al. 2014). It has been reported that NO plays a significant role in the development of features of diabetic nephropathy (Eleftheriadis et al. 2013). NO is a gas that is
Correspondence: Arzu Yay, Ph.D., Department of Histology and Embryology, Medicine Faculty, University of Erciyes, Kayseri, Turkey 38039. Tel: 0 352 207 66 66- 207 66 60, Fax: 0 352 437 76 27. E-mail: [email protected] © 2014 The Biological Stain Commission Biotechnic & Histochemistry 2014, 89(8): 552–557.
DOI: 10.3109/10520295.2014.913811
generated by three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). Expression of eNOS is altered in many diseases including diabetes (Pitocco et al. 2010). Mensah-Brown et al. (2005) speculated that NO could be important in the development of tissue damage during later stages of experimental diabetes. Oxidative stress, which results from an imbalance between free radical production and antioxidant defenses, is associated with damage to a wide of range of molecules including proteins, lipids and nucleic acids; it also contributes to decreased NO bioavailability. The causal role of oxidative stress in diabetes suggests that NO probably is present in high concentration (Pall 2013). Oxidative damage is considered the main indicator of a loss of cellular function caused by oxidative stress (Mojica-Villegas et al. 2014). Oxidative stress occurs in diabetes as a result of excess free radicals and pro-inflammatory cytokines (Baynes et al. 1999, Aydogan et al. 2008). Carnosine is a naturally occurring water soluble dipeptide that has antioxidant properties. The antioxidant character of carnosine has been reported to 552
depend on its ability to inactivate reactive oxygen species and scavenge free radicals (Aydogan et al. 2008). We investigated earlier whether carnosine could reduce lipid oxidation, which increases in diabetic rats (Yapislar and Aydogan 2012). We report here our investigation of the effects of carnosine as an antioxidant for treatment of streptozotocin (STZ)-induced diabetic renal injury in rats.
Material and methods
Biotech Histochem Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
Animals and treatments All experiments conformed to the institutional guidelines for animal experimentation at Erciyes University Faculty of Medicine and we obtained approval from the research ethics board of the University of Erciyes. We used streptozotocin (STZ) to induce type 1 diabetes mellitus (Armstrong and Al-Awadi 1991). We used 410 36 g male Wistar albino rats obtained from the Hakan Cetinsaya Experimental and Clinic Research Center, Erciyes University, Kayseri, Turkey. Rats were assigned randomly to four groups of eight. The groups were: group 1, controls given 1 ml/kg normal saline; group 2, given 70 mg/ kg carnosine for 7 days; group 3, diabetics given 70 mg/kg STZ; group 4, diabetics given 70 mg/kg carnosine for 7 days before inducing diabetes. Carnosine (Sigma-Aldrich Chemie GmbH, Taufkirchen-Germany) and STZ (Sigma-Aldrich) were prepared in normal saline (200 mg STZ in 4 ml saline). Carnosine was given prior to STZ to determine its possible protective effect; all injections were intraperitoneal. Blood glucose levels of diabetic rats were checked by glucometer 3 days after the administration of STZ. Rats with blood glucose levels 250 mg/dl were considered diabetic. The rats were sacrificed using a lethal intramuscular injection of ketamine (120 mg/kg). A median abdominal incision was made and both kidneys were excised. Kidneys were fixed in 10% formalin for 24 h and dehydrated through a graded ethanol series. Tissues were embedded in paraffin, then cut at 5 μm. Sections were stained with periodic acidSchiff (PAS) to provide a morphological overview of the tissue and to show the brush border of the epithelial cells of the proximal tubule and the thickness of basal lamina. Immunohistochemistry
retrieval was performed by incubation in 10% citrate buffer (pH 6.0) at 95° C for 5 min, then cooling to room temperature for 20 min. The sections were incubated in 3% H2O2 for 10 min, then rinsed in phosphate-buffered saline (PBS). eNOS was detected in kidney sections using the alkaline phosphatase immunohistochemical technique and a rabbit polyclonal antibody (sc.654 Santa Cruz Biotechnology, Paso Robles, CA). Immunohistochemistry was performed using the alkaline phosphatase method (Ultravision Detection System, Thermo Scientific, San Jose, CA). The specimens were washed in PBS for 5 min, then incubated in Ultra V Block solution for 5 min. Sections then were incubated with polyclonal antibody for eNOS at a concentration of 5 μg/ml in Ultra V Block solution overnight at 4° C. PBS replaced the eNOS antibody for negative control sections. Sections were incubated with biotinylated goat anti-polyvalent for 20 min, then incubated with streptavidin alkaline phosphatase for 20 min and rinsed with PBS. Sections were incubated for 10 min with a fast red tablet (F4648-50SET; Sigma-Aldrich) dissolved in 5 ml naphthol phosphate substrate. After washing with de-ionized water, sections were counterstained with Gill’s hematoxylin and mounted with clear mounting medium (00-8010; Zymed Laboratories Inc. South San Francisco, CA). The stained sections were examined for eNOS immunoreactivity under an Olympus BX-51 light microscope (Olympus BX-51, Tokyo, Japan). Quantitative immunohistochemistry The mean immunoreactivity intensity was determined from five microscopic fields in the kidney sections for each animal (total of 30 microscopic fields) at an original magnification of 400. The mean immunoreactivity intensity of eNOS was calculated using Image J software. Statistical evaluation The data were analyzed using repeated measures of variance. To evaluate the statistical significance, data were analyzed by one-way analysis of variance (ANOVA). If statistically significant, the mean values obtained from each group were compared by Tukey’s post hoc test. The data are presented as means SD. Values for p 0.05 were considered significant.
Results
Sections were deparaffinized in xylene and rehydrated through a graded ethanol series. The sections were rinsed in de-ionized water and antigen
We measured body weights and blood glucose levels for all groups (Table 1). The weights at the
Carnosine and renal oxidative stress in diabetic rats
553
Table 1. Initial and final body weights of experimental rats Groups
Initial body weight Final body weight
C
D
DC
CC
420 12
417 22
409 12
418 15
426 16
338 17
341 19
421 19
Biotech Histochem Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
n 8 for all groups Data are means SD C, control; CC, carnosine treated; DC, diabetes carnosine; D, diabetes
beginning of the study were similar for all groups. Body weights of the diabetic rats decreased compared to the nondiabetic or untreated control rats. The initial and final body weights of untreated control and carnosine treated groups were not significantly different. The difference between initial and final body weights of diabetic rats and diabetic rats treated with carnosine was not significant (Table 1). The average blood glucose levels of untreated control rats (101.87 16.33) did not change during the experiment. Carnosine treatment did not produce significant changes (103.42 7.11) in otherwise normal rats. All diabetic rats exhibited high
blood glucose levels (483.27 80.89). Carnosine did not prevent development of diabetes induced by STZ; blood glucose levels remained elevated (452.43 78.62). Examples of kidney histopathology are shown in Fig. 1. The histologic examination of normal kidneys of the untreated control rats showed normal kidney morphology. The histologic appearance of carnosine-treated kidneys was similar to that of the untreated control group. The kidneys of diabetic rats showed degenerated glomeruli and thickened basement membranes. PAS staining indicated an increased number of glycogen-filled proximal tubules in the diabetic kidneys. The epithelium of proximal and distal convoluted tubules exhibited edematous changes. The carnosine diabetes group showed normal basement membranes, normal glomeruli, fewer glycogen-filled proximal tubules and absence of inflammatory cells. These observations suggest that carnosine alleviated STZ-induced histopathologic damage in kidneys (Fig. 1). We also tested, using eNOS immunohistochemistry, whether carnosine treatment can affect the intensity of eNOS immunoreactivity in diabetic rats; the results are summarized in Fig. 2. Cortical sections of carnosine treated and control kidneys showed similar eNOS staining. eNOS immunoreactivity was greater in the glomeruli and tubules
Fig. 1. Representative kidney sections. A) Control kidney section showing normal kidney histology. PAS. 400 . B) Diabetic kidney. The epithelia of proximal and distal convoluted tubules show edematous changes (arrowhead). Arrows indicate thickened basement membrane. PAS. 400 . C) Carnosine treated rats showing normal kidney histology. PAS. 200 . D) Diabetic kidney treated with carnosine. Number of glycogen-filled proximal tubules decreased significantly; normal glomerulus and basement membrane. PAS. 100 .
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Biotechnic & Histochemistry 2014, 89(8): 552–557
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Fig. 2. Representative images of immunohistochemical localizations of eNOS. Original magnifications 400. A) In control kidney section, eNOS staining does not appear in tubule or vascular structures, including the glomerular capillary. B) In diabetic kidney, eNOS immunoreactivity appears in the glomerulus and epithelium of proximal and distal convoluted tubules. C) In the carnosine treated rat, eNOS staining is absent (cf. Fig. 2A). D) In diabetic kidney treated with carnosine, eNOS appears faintly in the glomerulus.
of the kidneys of diabetic rats than in controls (Fig. 2). eNOS staining was apparent in the endothelial cells of all vascular structures including glomerular capillaries. The immunoreaction in glomeruli of eNOS in the carnosine diabetes group decreased slightly compared to diabetic rats and these differences were statistically significant (p 0.05) (Fig. 2, Table 2).
Discussion Diabetes and diabetic kidney disease continue to increase worldwide and present a serious public health concern. All diabetic patients are considered to be at risk for nephropathy (Ozbek 2012) and diabetic nephropathy is a leading cause of end-stage Table 2. Intensity of eNOS immunostaining Groups
Characteristics
Control Diabetes Diabetes carnosine Carnosine treated
72.40 0.61 80.51 1.62** 77.83 0.53* 72.45 0.78
Data are means SD *Compared to control, p 0.05 **Compared to control, p 0.001
kidney disease. A growing number of patients have diabetic kidney disease and these cases account for 35–40% of new dialysis patients worldwide (Ohkubo et al. 1995, Gang et al. 2008). Oxidative stress has been implicated in the development of complications diabetic nephropathy, because oxidative stress promotes the formation of advanced glycation end products (Browmlee 2001, Ha and Kim 1999). Oxidative stress occurs as a result of imbalance between the pro-oxidant and antioxidant systems, which damages cellular proteins, membrane lipids and nucleic acids, and eventually causes cell death. Oxidative stress is an important factor in many pathologies in addition to kidney diseases (Ozbek 2012). Oxidative stress causes lipid oxidation, which leads to propagation of free radicals (Yusong et al. 2003). We investigated whether carnosine exerts a protective effect against renal oxidative stress in STZ-induced diabetic rats using histopathological and immunohistochemical techniques. We found that oxidative stress was severe in diabetic rats. We investigated whether carnosine could protect the microstructure of the kidney from injury due to oxidative stress. We studied the histology of the kidneys of diabetic rats and found damage to the glomerulus, thickened basement membranes and edematous epithelial cells of the proximal
Carnosine and renal oxidative stress in diabetic rats
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convoluted tubule with increased glycogen accumulation. Our results are consistent with earlier reports (Sun et al. 2002, Sugano et al. 2006). Oxidative stress is a participant in the development and progression of diabetes and its complications (Ceriello 2000, Baynes and Thorpe 1999). High glucose levels increase directly hydrogen peroxide production in murine mesangial cells (Ruiz-Munoz et al. 1997) and lipid oxidation in the glomerulus (Ha et al. 1997). Oxidative stress may play a role in the pathophysiology of morphological and functional changes in the diabetic kidney. There also is evidence from animal studies that biomarkers of oxidative stress, such as advanced glycation end products (AGE) and mitogen-activated protein kinase (MAPK), are increased in diabetes (Suzuki et al. 1999, Kalousová et al. 2002, Shah et al. 2007). Our observations are consistent with these studies. We demonstrated that renal oxidative stress was greater in STZ-induced diabetic rats than in the untreated control animals. Our findings support the hypothesis of free radical-mediated injury to the kidney in diabetes. There is evidence that carnosine may play a beneficial role in diabetes and its complications (Janssen et al. 2005, Sauerhöfer et al. 2007, Rashid et al. 2007). Carnosine treatment prevents the up-regulation of pro-apoptotic molecules in the kidneys and loss of podocyte in diabetic rats (Isermann et al. 2007). Carnosine also diminished significantly apoptosis of glomerular cells and podocyte loss (Riedl et al. 2011). It has been shown that carnosine inhibits some of the deleterious effects of a high fructose diet (Hipkiss et al. 2001). Ansurudeen et al. (2012) demonstrated that daily injections and local application of carnosine enhanced significantly wound healing in diabetic db/db mice. Carnosine has been shown to suppress the secondary complications of diabetes in mice (Lee et al. 2005) including diabetic kidney disease in mice and humans (Sauerhöfer et al. 2007, Freedman et al. 2007). Carnosine inhibits NO-dependent activation of guanylate cyclase (Severina et al. 2000). The antioxidation function of carnosine is critical to its biological role. We evaluated the intensity of eNOS immunostaining in the kidney of STZ-induced diabetic rats and our findings suggest that increased oxidative stress plays a role in diabetes-induced nephropathy. Moreover, carnosine decreased significantly the intensity of eNOS immunostaining compared to diabetic rats. We demonstrated that STZ-induced diabetes causes damage to the kidney. Morphological assessments showed that the damage to the kidney caused by STZ was reduced by the administration of carnosine. Therefore, carnosine might be a useful 556
therapeutic option for reducing oxidative stress and associated renal injury in diabetes mellitus. To reach a definitive conclusion, however, further studies of doses, concentrations and routes of administration of carnosine on should be performed.
Acknowledgment This study was supported by Erciyes University Research Foundation. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.
References Ansurudeen I, Sunkari VG, Grünler J, Peters V, Schmitt CP, Catrina SB, Brismar K, Forsberg EA (2012) Carnosine enhances diabetic wound healing in the db/db mouse model of type 2 diabetes. Amino Acids 43: 127–134. Armstrong D, Al-Awadi F (1991) Lipid peroxidation and retinopathy in streptozotocin induced diabetes. Free Rad. Biol. Med. 11: 433–436. Aydogan S, Yapislar H, Artis S, Aydogan B (2008) Impaired erythrocytes deformability in H(2)O(2)-induced oxidative stress: protective effect of L-carnosine. Clin. Hemorheol. Microcirc. 39: 93–98. Baynes JW, Thorpe SR (1999) Role of oxidative stress in diabetic complications: a new perspective on an old paradigm. Diabetes 48: 1–9. Browmlee M (2001) Biochemistry and molecular cell biology of diabetic complications. Nature 414: 813–820. Ceriello A (2000) Oxidative stress and glycemic regulation. Metabolism 49: 27–29. Dellamea BS, Leitão CB, Friedman R, Canani LH (2014) Nitric oxide system and diabetic nephropathy. Diabetol. Metab. Syndrome 6: 17–22. Eleftheriadis T, Antoniadi G, Pissas G, Liakopoulos V, Stefanidis I (2013) The renal endothelium in diabetic nephropathy. Ren. Fail. 35: 592–599. Fioretto P, Mauer M (2007) Histopathology of diabetic nephropathy. Semin. Nephrol. 27: 195–207. Freedman BI, Hicks PJ, Sale MM, Pierson ED, Langefeld CD, Rich SS, Xu J, McDonough C, Janssen B, Yard BA, van der Woude FJ, Bowden DW (2007) A leucine repeat in the carnosinase gene CNDP1 is associated with diabetic end-stage renal disease in European Americans. Nephrol. Dial. Transplant. 22: 1131–1135. Gang JK, Young SK, Sang YH, Mi Hwa L, Hye K, Kum H, Dae R (2008) Lioglitazone attenuates diabetic nephropathy through an anti-inflammatory mechanism in type 2 diabetic rats. Nephrol. Dial. Transplant. 23: 2750–2760. Ha H, Kim KH (1999) Pathogenesis of diabetic nephropathy: the role of oxidative stress and protein kinase C. Diabetes Res. Clin. Pract. 45: 147–151.
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Ha H, Lee SH, Kim KH (1997) Effects of rebamipide in a model of experimental diabetes and on the synthesis of transforming growth factor-b and fibronectin, lipid peroxidation induced by high glucose in cultured mesangial cells. J. Pharmacol. Exp. Ther. 281: 1457–1462. Hipkiss AR, Brownson C, Carrier MJ (2001) Carnosine, the anti-aging, antioxidant dipeptide, may react with protein carbonyl groups. Mech. Ageing Dev. 122: 1431–1445. Ibrahim HN, Hostetter TH (1997) Diabetic nephropathy. J. Am. Soc. Nephrol. 8: 487–493. Isermann B, Vinnikov IA, Madhusudhan T, Herzog S, Kashif M, Blautzik J, Corat MA, Zeier M, Blessing E, Oh J, Gerlitz B, Berg DT, Grinnell BW, Chavakis T, Esmon CT, Weiler H, Bierhaus A, Nawroth PP (2007) Activated protein C protects against diabetic nephropathy by inhibiting endothelial and podocyte apoptosis. Nat. Med. 13: 1349–1358. Janssen B, Hohenadel D, Brinkkoetter P, Peters V, Rind N, Fischer C, Rychlik I, Cerna M, Romzova M, de Heer E, Baelde H, Bakker SJ, Zirie M, Rondeau E, Mathieson P, Saleem MA, Meyer J, Köppel H, Sauerhoefer S, Bartram CR, Nawroth P, Hammes HP, Yard BA, Zschocke J, van der Woude FJ (2005) Carnosine as a protective factor in diabetic nephropathy. Diabetes 54: 2320–2327. Kalousová M, Skrha J, Zima T (2002) Advanced glycation end products and advanced oxidation protein products in patients with diabetes mellitus. Physiol. Res. 51: 597–604. Lee YT, Hsu CC, Lin MH, Liu KS, Yin MC (2005) Histidine and carnosine delay diabetic deterioration in mice and protect human low density lipoprotein against oxidation and glycation. Eur. J. Pharmacol. 513: 145–150. Mensah-Brown EP, Obineche EN, Galadari S, Chandranath E, Shahin A, Ahmed I, Patel SM, Adem A (2005) Streptozotocin-induced diabetic nephropathy in rats: the role of inflammatory cytokines. Cytokine 31: 180–190. Mojica-Villegas MA, Izquierdo-Vega JA, ChamorroCevallos G, and Sánchez-Gutiérrez M (2014) Protective effect of resveratrol on biomarkers of oxidative stress induced by iron/ascorbate in mouse spermatozoa. Nutrients 6: 489–503. Nobrega MA, Fleming S, Roman RJ, Shiozawa M, Schlick N, Lazar J, Jacob HJ (2004) Initial characterization of a rat model of diabetic nephropathy. Diabetes 53: 735–742. Ohkubo Y, Kishikawa H, Araki E, Miyata T, Isami S, Motoyoshi S, Kojima Y, Furuyoshi N, Shichiri M (1995) Intensive insulin therapy prevents the progression of diabetic microvascular complications in Japanese patients with non-insulin-dependent diabetes mellitus: a randomized prospective 6-year study. Diabetes Res. Clin. Pract. 28: 103–117. Ozbek E (2012) Induction of oxidative stress in kidney. Int. J. Nephrol. 2012: 465897.
Pall M (2013) The NO/ONOO-cycle as the central cause of heart failure. Int. J. Mol. Sci. 14: 22274–22330. Pitocco D, Zaccardi F, Di Stasio E, Romitelli F, Santini SA, Zuppi C, Ghirlanda G (2010 ) Oxidative stress, nitric oxide, and diabetes. Rev. Diabet. Stud. 7: 15–25. Rashid I, van Reyk DM, Davies MJ (2007) Carnosine and its constituents inhibit glycation of low-density lipoproteins that promotes foam cell formation in vitro. FEBS Lett. 581: 1067–070. Riedl E, Pfister F, Braunagel M, Brinkkötter P, Sternik P, Deinzer M, Bakker SJ, Henning RH, van den Born J, Krämer BK, Navis G, Hammes HP, Yard B, Koeppel H (2011) Carnosine prevents apoptosis of glomerular cells and podocyte loss in STZ diabetic rats. Cell Physiol. Biochem. 28: 279–288. Ruiz-Munoz LM, Vidal VF, Lamreabe I (1997) Enaraplilat inhibits hydrogen peroxide production by murine mesangial cells exposed to high glucose concentrations. Nephrol. Dial. Transplant. 12: 456–464. Sauerhöfer S, Yuan G, Braun GS, Deinzer M, Neumaier M, Gretz N, Floege J, Kriz W, van der Woude F, Moeller MJ (2007) L-carnosine, a substrate of carnosinase-1, influences glucose metabolism. Diabetes 56: 2425–2432. Severina IS, Bussygina OG, Pyatakova NV (2000) Carnosine as a regulator of soluble guanylate cyclase. Biochemistry (Moscow) 65: 921–927. Shah SV, Baliga R, Rajapurkar M, Fonseca VA (2007) Oxidants in Chronic Kidney Disease. J. Am. Soc. Nephrol. 18: 16–28. Sugano M, Yamato H, Hayashi T, Ochiai H, Kakuchi J, Goto S, Nishijima F, Iino N, Kazama JJ, Takeuchi T, Mokuda O, Ishikawa T, Okazaki R (2006) High-fat diet in low dose streptozotocin-treated heminephrectomized rats induced all features of human type 2 diabetic nephropathy: a new rat model of diabetic nephropathy. Nutr. Metab. Cardiovasc. Dis. 16: 477–484. Sun L, Halaihel N, Zhang W, Rogers T, Levi M (2002) Role of sterol regulatory element-binding protein 1 in regulation of renal lipid metabolism and glomerulosclerosis in diabetes mellitus. J. Biol. Chem. 277: 18919–18927. Suzuki S, Hinokio Y, Komatu K, Ohtomo M, Onoda M, Hirai S, Hirai M, Hirai A, Chiba M, Kasuga S, Akai H, Toyota T (1999) Oxidative damage to mitochondrial DNA and its relationship to diabetic complications. Diabetes Res. Clin. Pract. 45: 161–168. Yapislar H, Aydogan S (2012) Effect of carnosine on erythrocyte deformability in diabetic rats. Arch. Physiol. Biochem. 118: 265–272. Yusong Y, Rajendra S, Abha S, Sanjay A, Yogesh CA (2003) Lipid peroxidation and cell cycle signaling: 4-hydroxynonenal, a key molecule in stress mediated signaling. Acta Biochim. Polon. 50: 319–336.
Carnosine and renal oxidative stress in diabetic rats
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Biotech Histochem Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
Accepted January 30, 2014
Abstract Nitric oxide (NO) plays a significant role in the development of diabetic nephropathy. We investigated the effects of an antioxidant, carnosine, on streptozotocin (STZ)-induced renal injury in diabetic rats. We used four groups of eight rats: group 1, control; group 2, carnosine treated; group 3, untreated diabetic; group 4, carnosine treated diabetic. Kidneys were removed and processed, and sections were stained with periodic acid-Schiff (PAS) and subjected to eNOS immunohistochemistry. Examination by light microscopy revealed degenerated glomeruli, thickened basement membrane and glycogen accumulation in the tubules of diabetic kidneys. Carnosine treatment prevented the renal morphological damage caused by diabetes. Moreover, administration of carnosine decreased somewhat the oxidative damage of diabetic nephropathy. Appropriate doses of carnosine might be a useful therapeutic option to reduce oxidative stress and associated renal injury in diabetes mellitus. Key words: antioxidant therapy, carnosine, diabetes, eNOS, kidney, nephropathy
Diabetic nephropathy is a common microvascular complication of diabetes mellitus (Fioretto and Mauer 2007) that is characterized by hypertrophy of kidney structures including thickening of the basal membrane and progressive accumulation of extracellular matrix (Ibrahim and Hostetter 1997). Hyperglycemia, genetic factors, inflammatory cytokines and oxidative stress contribute to the development diabetic nephropathy (Nobrega et al. 2004). Nitric oxide (NO) must be considered in the pathogenesis of diabetic nephropathy, because NO plays a role in controlling renal and glomerular hemodynamics in the kidney (Dellamea et al. 2014). It has been reported that NO plays a significant role in the development of features of diabetic nephropathy (Eleftheriadis et al. 2013). NO is a gas that is
Correspondence: Arzu Yay, Ph.D., Department of Histology and Embryology, Medicine Faculty, University of Erciyes, Kayseri, Turkey 38039. Tel: 0 352 207 66 66- 207 66 60, Fax: 0 352 437 76 27. E-mail: [email protected] © 2014 The Biological Stain Commission Biotechnic & Histochemistry 2014, 89(8): 552–557.
DOI: 10.3109/10520295.2014.913811
generated by three isoforms of nitric oxide synthase (NOS): neuronal NOS (nNOS), inducible NOS (iNOS) and endothelial NOS (eNOS). Expression of eNOS is altered in many diseases including diabetes (Pitocco et al. 2010). Mensah-Brown et al. (2005) speculated that NO could be important in the development of tissue damage during later stages of experimental diabetes. Oxidative stress, which results from an imbalance between free radical production and antioxidant defenses, is associated with damage to a wide of range of molecules including proteins, lipids and nucleic acids; it also contributes to decreased NO bioavailability. The causal role of oxidative stress in diabetes suggests that NO probably is present in high concentration (Pall 2013). Oxidative damage is considered the main indicator of a loss of cellular function caused by oxidative stress (Mojica-Villegas et al. 2014). Oxidative stress occurs in diabetes as a result of excess free radicals and pro-inflammatory cytokines (Baynes et al. 1999, Aydogan et al. 2008). Carnosine is a naturally occurring water soluble dipeptide that has antioxidant properties. The antioxidant character of carnosine has been reported to 552
depend on its ability to inactivate reactive oxygen species and scavenge free radicals (Aydogan et al. 2008). We investigated earlier whether carnosine could reduce lipid oxidation, which increases in diabetic rats (Yapislar and Aydogan 2012). We report here our investigation of the effects of carnosine as an antioxidant for treatment of streptozotocin (STZ)-induced diabetic renal injury in rats.
Material and methods
Biotech Histochem Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
Animals and treatments All experiments conformed to the institutional guidelines for animal experimentation at Erciyes University Faculty of Medicine and we obtained approval from the research ethics board of the University of Erciyes. We used streptozotocin (STZ) to induce type 1 diabetes mellitus (Armstrong and Al-Awadi 1991). We used 410 36 g male Wistar albino rats obtained from the Hakan Cetinsaya Experimental and Clinic Research Center, Erciyes University, Kayseri, Turkey. Rats were assigned randomly to four groups of eight. The groups were: group 1, controls given 1 ml/kg normal saline; group 2, given 70 mg/ kg carnosine for 7 days; group 3, diabetics given 70 mg/kg STZ; group 4, diabetics given 70 mg/kg carnosine for 7 days before inducing diabetes. Carnosine (Sigma-Aldrich Chemie GmbH, Taufkirchen-Germany) and STZ (Sigma-Aldrich) were prepared in normal saline (200 mg STZ in 4 ml saline). Carnosine was given prior to STZ to determine its possible protective effect; all injections were intraperitoneal. Blood glucose levels of diabetic rats were checked by glucometer 3 days after the administration of STZ. Rats with blood glucose levels 250 mg/dl were considered diabetic. The rats were sacrificed using a lethal intramuscular injection of ketamine (120 mg/kg). A median abdominal incision was made and both kidneys were excised. Kidneys were fixed in 10% formalin for 24 h and dehydrated through a graded ethanol series. Tissues were embedded in paraffin, then cut at 5 μm. Sections were stained with periodic acidSchiff (PAS) to provide a morphological overview of the tissue and to show the brush border of the epithelial cells of the proximal tubule and the thickness of basal lamina. Immunohistochemistry
retrieval was performed by incubation in 10% citrate buffer (pH 6.0) at 95° C for 5 min, then cooling to room temperature for 20 min. The sections were incubated in 3% H2O2 for 10 min, then rinsed in phosphate-buffered saline (PBS). eNOS was detected in kidney sections using the alkaline phosphatase immunohistochemical technique and a rabbit polyclonal antibody (sc.654 Santa Cruz Biotechnology, Paso Robles, CA). Immunohistochemistry was performed using the alkaline phosphatase method (Ultravision Detection System, Thermo Scientific, San Jose, CA). The specimens were washed in PBS for 5 min, then incubated in Ultra V Block solution for 5 min. Sections then were incubated with polyclonal antibody for eNOS at a concentration of 5 μg/ml in Ultra V Block solution overnight at 4° C. PBS replaced the eNOS antibody for negative control sections. Sections were incubated with biotinylated goat anti-polyvalent for 20 min, then incubated with streptavidin alkaline phosphatase for 20 min and rinsed with PBS. Sections were incubated for 10 min with a fast red tablet (F4648-50SET; Sigma-Aldrich) dissolved in 5 ml naphthol phosphate substrate. After washing with de-ionized water, sections were counterstained with Gill’s hematoxylin and mounted with clear mounting medium (00-8010; Zymed Laboratories Inc. South San Francisco, CA). The stained sections were examined for eNOS immunoreactivity under an Olympus BX-51 light microscope (Olympus BX-51, Tokyo, Japan). Quantitative immunohistochemistry The mean immunoreactivity intensity was determined from five microscopic fields in the kidney sections for each animal (total of 30 microscopic fields) at an original magnification of 400. The mean immunoreactivity intensity of eNOS was calculated using Image J software. Statistical evaluation The data were analyzed using repeated measures of variance. To evaluate the statistical significance, data were analyzed by one-way analysis of variance (ANOVA). If statistically significant, the mean values obtained from each group were compared by Tukey’s post hoc test. The data are presented as means SD. Values for p 0.05 were considered significant.
Results
Sections were deparaffinized in xylene and rehydrated through a graded ethanol series. The sections were rinsed in de-ionized water and antigen
We measured body weights and blood glucose levels for all groups (Table 1). The weights at the
Carnosine and renal oxidative stress in diabetic rats
553
Table 1. Initial and final body weights of experimental rats Groups
Initial body weight Final body weight
C
D
DC
CC
420 12
417 22
409 12
418 15
426 16
338 17
341 19
421 19
Biotech Histochem Downloaded from informahealthcare.com by McMaster University on 12/10/14 For personal use only.
n 8 for all groups Data are means SD C, control; CC, carnosine treated; DC, diabetes carnosine; D, diabetes
beginning of the study were similar for all groups. Body weights of the diabetic rats decreased compared to the nondiabetic or untreated control rats. The initial and final body weights of untreated control and carnosine treated groups were not significantly different. The difference between initial and final body weights of diabetic rats and diabetic rats treated with carnosine was not significant (Table 1). The average blood glucose levels of untreated control rats (101.87 16.33) did not change during the experiment. Carnosine treatment did not produce significant changes (103.42 7.11) in otherwise normal rats. All diabetic rats exhibited high
blood glucose levels (483.27 80.89). Carnosine did not prevent development of diabetes induced by STZ; blood glucose levels remained elevated (452.43 78.62). Examples of kidney histopathology are shown in Fig. 1. The histologic examination of normal kidneys of the untreated control rats showed normal kidney morphology. The histologic appearance of carnosine-treated kidneys was similar to that of the untreated control group. The kidneys of diabetic rats showed degenerated glomeruli and thickened basement membranes. PAS staining indicated an increased number of glycogen-filled proximal tubules in the diabetic kidneys. The epithelium of proximal and distal convoluted tubules exhibited edematous changes. The carnosine diabetes group showed normal basement membranes, normal glomeruli, fewer glycogen-filled proximal tubules and absence of inflammatory cells. These observations suggest that carnosine alleviated STZ-induced histopathologic damage in kidneys (Fig. 1). We also tested, using eNOS immunohistochemistry, whether carnosine treatment can affect the intensity of eNOS immunoreactivity in diabetic rats; the results are summarized in Fig. 2. Cortical sections of carnosine treated and control kidneys showed similar eNOS staining. eNOS immunoreactivity was greater in the glomeruli and tubules
Fig. 1. Representative kidney sections. A) Control kidney section showing normal kidney histology. PAS. 400 . B) Diabetic kidney. The epithelia of proximal and distal convoluted tubules show edematous changes (arrowhead). Arrows indicate thickened basement membrane. PAS. 400 . C) Carnosine treated rats showing normal kidney histology. PAS. 200 . D) Diabetic kidney treated with carnosine. Number of glycogen-filled proximal tubules decreased significantly; normal glomerulus and basement membrane. PAS. 100 .
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Fig. 2. Representative images of immunohistochemical localizations of eNOS. Original magnifications 400. A) In control kidney section, eNOS staining does not appear in tubule or vascular structures, including the glomerular capillary. B) In diabetic kidney, eNOS immunoreactivity appears in the glomerulus and epithelium of proximal and distal convoluted tubules. C) In the carnosine treated rat, eNOS staining is absent (cf. Fig. 2A). D) In diabetic kidney treated with carnosine, eNOS appears faintly in the glomerulus.
of the kidneys of diabetic rats than in controls (Fig. 2). eNOS staining was apparent in the endothelial cells of all vascular structures including glomerular capillaries. The immunoreaction in glomeruli of eNOS in the carnosine diabetes group decreased slightly compared to diabetic rats and these differences were statistically significant (p 0.05) (Fig. 2, Table 2).
Discussion Diabetes and diabetic kidney disease continue to increase worldwide and present a serious public health concern. All diabetic patients are considered to be at risk for nephropathy (Ozbek 2012) and diabetic nephropathy is a leading cause of end-stage Table 2. Intensity of eNOS immunostaining Groups
Characteristics
Control Diabetes Diabetes carnosine Carnosine treated
72.40 0.61 80.51 1.62** 77.83 0.53* 72.45 0.78
Data are means SD *Compared to control, p 0.05 **Compared to control, p 0.001
kidney disease. A growing number of patients have diabetic kidney disease and these cases account for 35–40% of new dialysis patients worldwide (Ohkubo et al. 1995, Gang et al. 2008). Oxidative stress has been implicated in the development of complications diabetic nephropathy, because oxidative stress promotes the formation of advanced glycation end products (Browmlee 2001, Ha and Kim 1999). Oxidative stress occurs as a result of imbalance between the pro-oxidant and antioxidant systems, which damages cellular proteins, membrane lipids and nucleic acids, and eventually causes cell death. Oxidative stress is an important factor in many pathologies in addition to kidney diseases (Ozbek 2012). Oxidative stress causes lipid oxidation, which leads to propagation of free radicals (Yusong et al. 2003). We investigated whether carnosine exerts a protective effect against renal oxidative stress in STZ-induced diabetic rats using histopathological and immunohistochemical techniques. We found that oxidative stress was severe in diabetic rats. We investigated whether carnosine could protect the microstructure of the kidney from injury due to oxidative stress. We studied the histology of the kidneys of diabetic rats and found damage to the glomerulus, thickened basement membranes and edematous epithelial cells of the proximal
Carnosine and renal oxidative stress in diabetic rats
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convoluted tubule with increased glycogen accumulation. Our results are consistent with earlier reports (Sun et al. 2002, Sugano et al. 2006). Oxidative stress is a participant in the development and progression of diabetes and its complications (Ceriello 2000, Baynes and Thorpe 1999). High glucose levels increase directly hydrogen peroxide production in murine mesangial cells (Ruiz-Munoz et al. 1997) and lipid oxidation in the glomerulus (Ha et al. 1997). Oxidative stress may play a role in the pathophysiology of morphological and functional changes in the diabetic kidney. There also is evidence from animal studies that biomarkers of oxidative stress, such as advanced glycation end products (AGE) and mitogen-activated protein kinase (MAPK), are increased in diabetes (Suzuki et al. 1999, Kalousová et al. 2002, Shah et al. 2007). Our observations are consistent with these studies. We demonstrated that renal oxidative stress was greater in STZ-induced diabetic rats than in the untreated control animals. Our findings support the hypothesis of free radical-mediated injury to the kidney in diabetes. There is evidence that carnosine may play a beneficial role in diabetes and its complications (Janssen et al. 2005, Sauerhöfer et al. 2007, Rashid et al. 2007). Carnosine treatment prevents the up-regulation of pro-apoptotic molecules in the kidneys and loss of podocyte in diabetic rats (Isermann et al. 2007). Carnosine also diminished significantly apoptosis of glomerular cells and podocyte loss (Riedl et al. 2011). It has been shown that carnosine inhibits some of the deleterious effects of a high fructose diet (Hipkiss et al. 2001). Ansurudeen et al. (2012) demonstrated that daily injections and local application of carnosine enhanced significantly wound healing in diabetic db/db mice. Carnosine has been shown to suppress the secondary complications of diabetes in mice (Lee et al. 2005) including diabetic kidney disease in mice and humans (Sauerhöfer et al. 2007, Freedman et al. 2007). Carnosine inhibits NO-dependent activation of guanylate cyclase (Severina et al. 2000). The antioxidation function of carnosine is critical to its biological role. We evaluated the intensity of eNOS immunostaining in the kidney of STZ-induced diabetic rats and our findings suggest that increased oxidative stress plays a role in diabetes-induced nephropathy. Moreover, carnosine decreased significantly the intensity of eNOS immunostaining compared to diabetic rats. We demonstrated that STZ-induced diabetes causes damage to the kidney. Morphological assessments showed that the damage to the kidney caused by STZ was reduced by the administration of carnosine. Therefore, carnosine might be a useful 556
therapeutic option for reducing oxidative stress and associated renal injury in diabetes mellitus. To reach a definitive conclusion, however, further studies of doses, concentrations and routes of administration of carnosine on should be performed.
Acknowledgment This study was supported by Erciyes University Research Foundation. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this paper.
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