Immunohistochemical distribution of leptin in kidney tissues of melatonin treated diabetic rats S Elis Yildiz1, T Deprem2, E Karadag Sari2, SA Bingol1, S Koral Tasci2, S Aslan2, G Nur3, M Sozmen4 of Health Sciences, Kafkas University, Kars, 2Department of Histology and Embryology, Faculty of Veterinary Medicine, Kafkas University, Kars, 3Directorate of Food Control Laboratory, Hatay, and 4Department of Pathology, Faculty of Veterinary Medicine, Ondokuz Mayıs University, Samsun, Turkey Biotech Histochem Downloaded from informahealthcare.com by University of Hong Kong Libraries on 02/02/15 For personal use only.
1School
Accepted July 29, 2014
Abstract We examined using immunohistochemistry the distribution of leptin in kidney tissues of melatonin treated, streptozotocin (STZ) diabetic rats. The animals were divided into five groups: control, sham, melatonin-treated, diabetic and melatonin-treated diabetic. Kidney sections were prepared and stained with hematoxylin and eosin, and Crossman’s triple staining for histological examination. The immunohistochemical localization of leptin in the kidney tissue was determined using the streptavidin-biotin-peroxidase method. We determined that on days 7 and 14, the leptin immunoreactivity of the diabetic and melatonin-treated diabetic groups was weaker than for the other groups. Weak immunoreactivity was found in the proximal and distal tubules of the kidney in the diabetic and melatonin-treated diabetic groups on days 7 and 14, and strong immunoreactivity was found in the control, sham and melatonin groups. Melatonin application had no significant effect on leptin production in the kidney tissues of diabetic rats. Key words: diabetes mellitus, immunohistochemistry, kidney, leptin, melatonin Diabetes mellitus is a state of chronic hyperglycemia that is characterized by disorders of insulin secretion, insulin action or both (Solomon et al. 2002) (Catalanov Marshall 1992). Oxidative stress, caused by increased free oxygen radicals and decreased protective antioxidant capacity due to diabetes, has been thought to affect kidneys adversely (Vural et al. 2001, Baydas et al. 2002). Melatonin is secreted by the pineal gland and plays a role in regulating sleep, reproduction, immunity and limitation of tumor formation (Cam et al. 2003, Guerrero-Romerov et al. 2005). Melatonin has a strong antioxidant effect that can stimulate the endogenous antioxidant system and oxygen scavenger activity (Vijayalaxmive et al. 2002). Melatonin reduces oxidative stress in diabetes; therefore, it has been suggested that it might provide protection
against radical-mediated renal injury in diabetes (Vijayalaxmive et al. 2002, Cam et al. 2003, GuerreroRomero and Rodriguez Morana 2005). Leptin is a hormone produced by the ob gene in fat cells, heart, placenta, lung, liver, kidney, pancreas, spleen, testes and colon (Wallace 2000). Leptin in the circulation reaches the central nervous system by crossing the blood-brain barrier using specific receptors; in the central nervous system, it reduces food consumption and increases energy consumption (Caro et al. 1997). Leptin also decreases basal and glucose-induced insulin secretion (Tallman and Taylor 1999, Wauters et al. 2000). We investigated the immunohistochemical distribution of leptin in the renal tissues of melatonintreated streptozotocin (STZ) induced diabetic rats.
Material and methods Correspondence: Sevda Elis Yildiz, Department of Histology and Embryology, School of Health Science, University of Kafkas, Kars, Turkey. Tel: ⫹ 90 0474 225 15 67. E-mail: sevdaelis36@ hotmail.com © 2014 The Biological Stain Commission Biotechnic & Histochemistry 2014, Early Online: 1–8.
DOI: 10.3109/10520295.2014.983548
Animals Our experimental protocol was approved by the Kafkas University Animal Experiments Local Ethical Committee. Fifty 250 g male Sprague-Dawley 1
rats, 8–12 weeks old, were kept in standard cages at 22 ⫾ 2° C and a 12 h light and 12 h dark cycle. Animals were provided normal rat food and tap water ad libitum. Rats were divided randomly into five groups: group 1, control; group 2, ethanol and normal saline treated sham group; group 3, melatonin treated; group 4, diabetic; group 5, melatonin treated diabetic.
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Experimental diabetes Experimental diabetes was induced by intraperitoneal injection of 50 mg/kg STZ dissolved in 0.1 M citrate buffer, pH 4.5 (Kanitkar and Bhonde 2000). Prior to induction of experimental diabetes, the animals were fasted for 12 h, then fed standard rat food and water 4 h after STZ administration (Ozturk et al. 2005). At 72 h after STZ administration, blood glucose levels of rats fasted for 6 h were measured using a glucometer in blood taken from the tail. The animals with blood glucose levels ⬎ 200 mg/dl were considered diabetic (Kanitkar and Bhonde 2000). Melatonin treatment We administered a 10 mg/kg dose of melatonin to male Sprague-Dawley rats between 19:00 and 21:00 during the winter; this rate of melatonin was used by Bharti et al. (2011). Rats in the melatonin treated diabetic group were given melatonin 72 h after STZ injection, i.e., immediately after detection of diabetes. We injected the diabetic, melatonin treated diabetic and melatonin groups intraperitoneally with 10 mg/kg/day melatonin diluted with normal saline and dissolved in ethanol in a total volume of 0.4 ml for 14 days. An equal volume of ethanol and normal saline were injected into the sham group. Animals in the group control received no treatment. On days 7 and 14 after treatments, the rats were weighed, blood glucose levels were measured and kidney tissue samples were excised after cervical dislocation under ether anesthesia.
multiple comparison test. Values for p ⬍ 0.05 were considered significant. Histopathology For light microscopy, the left kidney was fixed in phosphate-buffered 10% formalin, processed and embedded in paraffin, then 5 μm sections were cut using conventional techniques. Sections were stained with hematoxylin and eosin (H & E) and Crossman’s triple stain (Luna 1968). The severity and prevalence of injury to the kidneys of the experimental and control groups were assessed semiquantitatively as: 0, no injury; 1, minimal; 2, moderate; 3, severe. Immunohistochemistry The streptavidin-biotin-peroxidase technique (Tru 1990) was used to investigate the immunohistochemical distribuition of leptin in the kidney tissues. After deparaffinization and rehydration, the sections were incubated in 3% H202 for 10 min to block endogenous peroxide activity. All tissues were washed with PBS and processed in 0.1 M citrate buffer solution, pH 6.0, in an 800 watt microwave oven for 10 min to expose antigenic sites. Sections were washed again with PBS, then incubated in blocking buffer (Histostain Plus Broad Spectrum (AEC) Ref. 85.9943; Invitrogen, Carlsbad, CA) for 10 min to prevent nonspecific binding. Primary leptin antibody was applied to the sections (rabbit polyclonal IgG antibody A-20, sc482; Santa Cruz Laboratories, Santa Cruz, CA) diluted 1:500 in a humid chamber at room temperature for 1 h. After incubation, sections were counterstained with hematoxylin. The sections were examined using a microscope (BX-051 Olympus, Tokyo, Japan) and photos were taken. Leptin immunoreactivity in cells was assessed using a semiquantitative method: 0, no reaction; 1, slight staining; 2, moderate staining; 3, intense staining (Zhu 1989, Seidal et al. 2001). Negative controls consisted of sections that were treated with PBS without primary antibodies.
Live weights and kidney weights Live weights and kidney weights of all rats in all groups were measured on days 7 and 14 of our study. The weights of the kidneys were normalized to the body weights (kidney weight/body weight ⫻ 100) (Sechi et al. 1997). The results were compared statistically using one-way analysis of variance (one-way ANOVA). The statistical significance of differences between the groups was assessed using Duncan 2
Results Live and tissue weight On days 7 and 14, a significant increase in live weights was observed in the control, sham and melatonin groups; the live body weights of the diabetic and melatonin treated diabetic groups were not significantly different (Table 1).
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Table 1. Mean weights (g) of rats in all groups on days 7 and 14
Days day 7 (n ⫽ 10) day 14 (n ⫽ 5) a,b,cValues
Sham
Control
Melatonin
Diabetic group
Melatonin-treated diabetic group
238.70 ⫾ 14.63a 224.54 ⫾ 13.01c
238.83 ⫾ 24.23a 220.48 ⫾ 18.13bc
191.56 ⫾ 60.82b 189.51 ⫾ 51.70a,b,c
185.09 ⫾ 36.45b 184.88 ⫾ 9.78a,b
193.93 ⫾ 27.08b 173.52 ⫾ 23.75a
with different letters in the same line were significantly different from each other. p ⬍ 0.05.
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Table 2. Mean kidney weights (g) of rats in all groups on days 7 and 14
Days day 7 (n ⫽ 5) day 14 (n ⫽ 5) a,b,cValues
Sham
Control
Melatonin
Diabetic group
Melatonin-treated diabetic group
4.81 ⫾ 0.29 4.48 ⫾ 0.30a
4.96 ⫾ 0.31 4.96 ⫾ 0.31a,b
4.45 ⫾ 0.50 4.79 ⫾ 0.68a,b
5.27 ⫾ 0.34 5.53 ⫾ 0.70b
4.99 ⫾ 0.69 5.29 ⫾ 0.57b
with different letters in the same line were significantly different from each other. (p ⬍ 0.05).
On day 7, there were no significant differences in kidney weights among the groups. On day 14, differences in kidney weights among the control, sham, diabetic and melatonin treated diabetic groups were statistically significant (p ⬍ 0.05) (Table 2). Histology Injuries to kidney tissues in all groups were evaluated semiquantitatively. The control, sham and melatonin groups showed normal histological structure (Fig. 1). In the diabetic groups, necrosis of the epithelial cells of proximal and distal tubules and collecting tubules of diabetic rats was observed (Figs. 2–4); the tubule lumens were enlarged owing to the necrosis of the epithelial cells (Figs. 2–4). Vacuol and hydropic degeneration was observed in tubule epithelial cells that were not necrosed and pyknosis was observed in desquamated epithelial cell nuclei. We observed that glomeruli were partially contracted or glomerular spaces were expanded as a result of glomerular atrophy (Figs. 2–4). The Pathologic disorders in the kidney were significantly less severe in the melatonin treated diabetic group than in the diabetic group.
Fig. 1. Control rat kidney. Renal tubules and glomeruli show normal structure. H & E. Bar ⫽ 100 μm.
Immunohistochemistry The location of leptin immunoreactivity was similar for all groups, but it was weaker in the diabetic and melatonin treated diabetic groups than in the other groups. Leptin immunoreactivity was observed in renal tissues of rats in all groups on days 7 and 14. Strong leptin immunoreactivity was found in the renal cortex except for the diabetic and melatonin
Fig. 2. Renal lesion in kidney of diabetic group rat on day 7. Severe and widespread necrosis in proximal and distal tubule epithelium (long arrows). Tubule cavities are dilated owing to glomerular atrophy (short arrows). H & E. Bar ⫽ 200 μm.
The effect of melatonin on leptin in diabetic kidney 3
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Fig. 3. Renal lesion in the kidney of diabetic group rat on day 7. Severe and widespread necrosis in proximal and tubule distalis epithelium (long arrows). Tubule cavities are dilated owing to glomerular atrophy (short arrows). H & E. Bar ⫽ 100 μm.
treated diabetic groups. Leptin immunoreactivity was weak in renal medullas of all groups, however. Although weak immunoreactivity was detetected in proximal, distal tubule cells and collecting tubules of rats in the diabetic and melatonin treated diabetic groups on days 7 and 14 (Figs. 5, 6, 10, 11), strong immunoreactivity in proximal and distal tubule cells, and collecting tubules was observed in rats in the melatonin, sham and control groups (Figs. 7– 9, 12–14). These were differences statistically significant. No leptin immunoreactivity was observed in the glomeruli or vascular endothelium of renal tissues of any group (Table 3).
Fig. 4. Renal lesion in the kidney of diabetic group rat on day 7. Severe and widespread necrosis in proximal and distal tubule epithelium (long arrows). Dilation of tubule cavities owing to glomerular atrophy (short arrow). H & E. Bar ⫽ 50 μm.
4
Fig. 5. Renal section of diabetic group rat on day 7. Weak to intense leptin immunoreactivity in proximal and tubule distal tubules is shown. Bar ⫽ 100 μm.
Discussion We investigated changes in body and kidney weights, histopathologic structural changes and immunohistochemical distribution of leptin in the kidney in melatonin treated rats with STZ-induced diabetes. Andallu (2003) reported that weight loss in diabetic rats is due to destruction of body tissue proteins. We found decreased live body weights in both the diabetic and melatonin treated diabetic groups compared to the other groups. These findings are consistent with literature reports that the body weights of diabetic rats are significantly lower than control groups (Hayashi et al. 2001, Fujita et al. 2005, Vardı et al. 2005, German et al. 2010, Bingol and Kocamis 2010). Cam et al. (2003) and Aksoy et al. (2003) reported that melatonin treatment had no significant effect on body weights of diabetic
Fig. 6. Renal section of melatonin diabetic group rat on day 7. Weak to intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 100 μm.
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Fig. 7. Renal section of melatonin group rat on day 7. Intense leptin immunoreactivity in proximal and distal tubules is shown. Bar ⫽ 100 μm.
Fig. 10. Renal section of diabetic group rat on day 14. Weak to intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 50 μm.
Fig. 8. Renal section of sham group rat on day 7. Intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 100 μm.
Fig.11. Renal section of melatonin diabetic group rat on day 14. Weak to intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 50 μm.
Fig. 9. Renal section of control group rat on day 7. Intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 100 μm.
Fig. 12. Renal section of melatonin group rat on day 14. Intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 50 μm.
The effect of melatonin on leptin in diabetic kidney 5
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Fig. 13. Renal section of sham group rat on day 7. Intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 50 μm.
Fig. 14. Renal section of control group rat on day 14. Intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 50 μm.
patients; however, Bartnes et al. (1985) reported that while melatonin did not affect body weight during the first 8 weeks, it caused decreased body weight during the following 6 weeks. We found no significant differences between the body weights of the diabetic and melatonin treated diabetic groups on day 14 of melatonin administration. Sechi et al. (1997), Wade et al. (2001) and Bingöl and Kocamıs (2010) reported that kidney weights
of mice with experimental diabetes increased compared to their control groups. We also found that kidney weights increased more in the diabetic and melatonin treated diabetic groups than in the control, sham and melatonin groups. We suggest that the increased kidney weight is caused by hypertrophy due to diabetes. Lee et al. (2007) reported that diabetes causes glomerular enlargement, sclerosis and tubulointerstitial fibrosis in rat kidney. Basement membrane thickening of the glomerulus and tubule caused by accumulation of extracellular matrix in experimental diabetic and cases of nephropathy has been described by others (Lehman and Schleicher 2000, Singh and Crook 2000, Reeves and Andreoli 2000, Ozturk et al. 2005). We observed necrosis of the epithelial cells of proximal, distal and collecting tubules in the diabetic group. A lesser degree of necrosis was observed in the melatonin treated diabetic group. Although Cam et al. (2003) and Vardı et al. (2005) detected hydropic changes in the diabetic group in their study, they also stated that melatonin administration limits hydropic changes. We showed that melatonin administration reduces tissue damage caused by diabetes and these results are comparable to those reported by Cam et al. (2003) and Vardı et al. (2005). It has been proposed melatonin prevents pathological changes that occur in diabetes by stabilizing cell membranes (Vijayalaxmi et al. 2002). It has been reported that leptin suppresses insulin secretion by activating ATP-sensitive K⫹ channels in pancreatic β cells; thus, β cells become hyperpolarized prior to depolarization for insulin secretion (Harvey et al. 1997). It has been reported also that leptin decreases basal and glucose-stimulated insulin secretion by exerting negative feedback on insulin secretion (Wauters et al. 2000, German et al. 2010). Serradeil-Le Gal et al. (1997) and Hama et al. (2004) detected leptin receptors in the renal cortex, and proximal and distal tubule cells. We observed weak immunoreactivity in the renal cortex, proximal and distal tubules of diabetic and melatonin treated diabetic groups at days 7 and 14. Also, strong
Table 3. Comparison of leptin immunoreactivity among groups
Diabetic group Renal structures Proximal tubule Distal tubule Medulla
6
Melatonin-treated diabetic group
Melatonin, sham and control groups
7 days
14 days
7 days
14 days
7 days
14 days
weak weak weak
weak weak weak
weak weak weak
weak weak weak
strong strong weak
strong strong weak
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immunoreactivity was detected in the proximal and distal tubules of the renal cortex in the melatonin, sham and control groups. Immunoreactivity of leptin in all groups showed similar characteristics, but it was weaker in the diabetic and melatonin treated diabetic groups compared to the other groups. Previous studies have shown that leptin levels were high in the blood of obese people; high leptin levels develop in proportion to fat mass (Montague et al. 1997). Application of melatonin was observed to reduce renal injury in STZ-induced diabetic rats; however, no effect on leptin immunoreactivity was observed. Further studies of long-term use of melatonin are required to demonstrate the effects of melatonin on diabetic complications.
Acknowledgment Our research was supported by a grant from the Kafkas University, BAP (Project No: 2012-KSYO-18) and presented at the 21st National Electron Microscope Congress, P: 123, 28–31 May, 2013, Mersin, Turkey.
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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Reeves BR, Andreoli TE (2000) Transforming growth factor β contributes to progressive diabetic nephropathy. Proc. Natl. Acad. Sci. 97: 7667–7669. Sechi LA, Ceriello A, Griffin CA, Catena C, Amstad P, Schambelan M, Bartoli E (1997) Renal antioxidant enzyme mRNA levels are increased in rats with experimental diabetes mellitus. Diabetologia 40: 23–29. Seidal T, Balaton AJ, Battifora H (2001) Interpretation and quantification of immunostains. Am. J. Surg. Pathol. 25: 1204–1207. Serradeil-Le Gal C, Raufaste D, Brossard G, Pouzet B, Marty E, Maffrand JP, Le Fur G (1997) Characterization and localization of leptin receptors in the rat kidney. FEBS Lett. 10: 185–191. Singh LP, Crook ED (2000) Hexosamine regulation of glucose-mediated laminin synthesis in mesangial cells involves proetin kinases A and C. Am. J. Physiol. Kidney Physiol. 279: 646–654. Solomon D, Davey D, Kurman R (2002) The 2001 Bethesda system. Terminology for reporting cervical cytology. J. Am. Med. Assoc. 287: 2114–2119. Tallman DL, Taylor CG (1999) Potential interactions of zinc in the neuroendocrine-endocrine disturbances of diabetes mellitus type 2. J. Physiol. Pharmacol. 77: 919–933.
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Tru LD (1990) Principles of immunohistochemistry. In: Atlas of Diagnostic Immunohistopathology, Tru LD, Ed., New York Press. New York. pp. 1–31. Vardi N, Iraz M, Ozturk F, Ucar M, Gul M, Esrefoglu M, Otlu A (2005) Improving effects of melatonin on the histologic alterations of rat kidneys induced by experimental diabetes. Inonu Univ. J. Med. 12: 145–152. Vijayalaxmi TCR, Reiter RJ, Herman TS (2002) Melatonin: from basic research to cancer treatment clinics. J. Clin. Oncol. 20: 2575–2601. Vural S, Sabuncu T, Arslan SO (2001) Melatonin inhibits lipid peroxidation and stimulates the antioxidant status of diabetic rats. J. Pineal Res. 31: 193–198. Wada J, Zhang H, Tsuchiyama Y, Hiragushi K, Hida K, Shikata K, Kanwar YS, Makino H (2001) Gene expression profile in streptozotocin-induced diabetic mice kidneys undergoing glomerulosclerosis. Kidney Int. 59: 1363–1373. Wallace AM (2000) Measurement of leptin binding in the human circulation. Ann. Clin. Biochem. 37: 244–252. Wauters M, Considine RV, Van Gaal LF (2000) Human leptin: from an adipocyte hormone to an endocrine mediator. Eur. J. Endocrinol. 143: 293–311. Zhu QY (1989) Analysis of blood vessel invasion by cells of thyroid follicular carcinoma using image processing combined with immunohistochemistry. Zhonghua Yi Xue Za Zhi 69: 573–575.
Biotechnic & Histochemistry 2014, Early Online: 1–8
1School
Accepted July 29, 2014
Abstract We examined using immunohistochemistry the distribution of leptin in kidney tissues of melatonin treated, streptozotocin (STZ) diabetic rats. The animals were divided into five groups: control, sham, melatonin-treated, diabetic and melatonin-treated diabetic. Kidney sections were prepared and stained with hematoxylin and eosin, and Crossman’s triple staining for histological examination. The immunohistochemical localization of leptin in the kidney tissue was determined using the streptavidin-biotin-peroxidase method. We determined that on days 7 and 14, the leptin immunoreactivity of the diabetic and melatonin-treated diabetic groups was weaker than for the other groups. Weak immunoreactivity was found in the proximal and distal tubules of the kidney in the diabetic and melatonin-treated diabetic groups on days 7 and 14, and strong immunoreactivity was found in the control, sham and melatonin groups. Melatonin application had no significant effect on leptin production in the kidney tissues of diabetic rats. Key words: diabetes mellitus, immunohistochemistry, kidney, leptin, melatonin Diabetes mellitus is a state of chronic hyperglycemia that is characterized by disorders of insulin secretion, insulin action or both (Solomon et al. 2002) (Catalanov Marshall 1992). Oxidative stress, caused by increased free oxygen radicals and decreased protective antioxidant capacity due to diabetes, has been thought to affect kidneys adversely (Vural et al. 2001, Baydas et al. 2002). Melatonin is secreted by the pineal gland and plays a role in regulating sleep, reproduction, immunity and limitation of tumor formation (Cam et al. 2003, Guerrero-Romerov et al. 2005). Melatonin has a strong antioxidant effect that can stimulate the endogenous antioxidant system and oxygen scavenger activity (Vijayalaxmive et al. 2002). Melatonin reduces oxidative stress in diabetes; therefore, it has been suggested that it might provide protection
against radical-mediated renal injury in diabetes (Vijayalaxmive et al. 2002, Cam et al. 2003, GuerreroRomero and Rodriguez Morana 2005). Leptin is a hormone produced by the ob gene in fat cells, heart, placenta, lung, liver, kidney, pancreas, spleen, testes and colon (Wallace 2000). Leptin in the circulation reaches the central nervous system by crossing the blood-brain barrier using specific receptors; in the central nervous system, it reduces food consumption and increases energy consumption (Caro et al. 1997). Leptin also decreases basal and glucose-induced insulin secretion (Tallman and Taylor 1999, Wauters et al. 2000). We investigated the immunohistochemical distribution of leptin in the renal tissues of melatonintreated streptozotocin (STZ) induced diabetic rats.
Material and methods Correspondence: Sevda Elis Yildiz, Department of Histology and Embryology, School of Health Science, University of Kafkas, Kars, Turkey. Tel: ⫹ 90 0474 225 15 67. E-mail: sevdaelis36@ hotmail.com © 2014 The Biological Stain Commission Biotechnic & Histochemistry 2014, Early Online: 1–8.
DOI: 10.3109/10520295.2014.983548
Animals Our experimental protocol was approved by the Kafkas University Animal Experiments Local Ethical Committee. Fifty 250 g male Sprague-Dawley 1
rats, 8–12 weeks old, were kept in standard cages at 22 ⫾ 2° C and a 12 h light and 12 h dark cycle. Animals were provided normal rat food and tap water ad libitum. Rats were divided randomly into five groups: group 1, control; group 2, ethanol and normal saline treated sham group; group 3, melatonin treated; group 4, diabetic; group 5, melatonin treated diabetic.
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Experimental diabetes Experimental diabetes was induced by intraperitoneal injection of 50 mg/kg STZ dissolved in 0.1 M citrate buffer, pH 4.5 (Kanitkar and Bhonde 2000). Prior to induction of experimental diabetes, the animals were fasted for 12 h, then fed standard rat food and water 4 h after STZ administration (Ozturk et al. 2005). At 72 h after STZ administration, blood glucose levels of rats fasted for 6 h were measured using a glucometer in blood taken from the tail. The animals with blood glucose levels ⬎ 200 mg/dl were considered diabetic (Kanitkar and Bhonde 2000). Melatonin treatment We administered a 10 mg/kg dose of melatonin to male Sprague-Dawley rats between 19:00 and 21:00 during the winter; this rate of melatonin was used by Bharti et al. (2011). Rats in the melatonin treated diabetic group were given melatonin 72 h after STZ injection, i.e., immediately after detection of diabetes. We injected the diabetic, melatonin treated diabetic and melatonin groups intraperitoneally with 10 mg/kg/day melatonin diluted with normal saline and dissolved in ethanol in a total volume of 0.4 ml for 14 days. An equal volume of ethanol and normal saline were injected into the sham group. Animals in the group control received no treatment. On days 7 and 14 after treatments, the rats were weighed, blood glucose levels were measured and kidney tissue samples were excised after cervical dislocation under ether anesthesia.
multiple comparison test. Values for p ⬍ 0.05 were considered significant. Histopathology For light microscopy, the left kidney was fixed in phosphate-buffered 10% formalin, processed and embedded in paraffin, then 5 μm sections were cut using conventional techniques. Sections were stained with hematoxylin and eosin (H & E) and Crossman’s triple stain (Luna 1968). The severity and prevalence of injury to the kidneys of the experimental and control groups were assessed semiquantitatively as: 0, no injury; 1, minimal; 2, moderate; 3, severe. Immunohistochemistry The streptavidin-biotin-peroxidase technique (Tru 1990) was used to investigate the immunohistochemical distribuition of leptin in the kidney tissues. After deparaffinization and rehydration, the sections were incubated in 3% H202 for 10 min to block endogenous peroxide activity. All tissues were washed with PBS and processed in 0.1 M citrate buffer solution, pH 6.0, in an 800 watt microwave oven for 10 min to expose antigenic sites. Sections were washed again with PBS, then incubated in blocking buffer (Histostain Plus Broad Spectrum (AEC) Ref. 85.9943; Invitrogen, Carlsbad, CA) for 10 min to prevent nonspecific binding. Primary leptin antibody was applied to the sections (rabbit polyclonal IgG antibody A-20, sc482; Santa Cruz Laboratories, Santa Cruz, CA) diluted 1:500 in a humid chamber at room temperature for 1 h. After incubation, sections were counterstained with hematoxylin. The sections were examined using a microscope (BX-051 Olympus, Tokyo, Japan) and photos were taken. Leptin immunoreactivity in cells was assessed using a semiquantitative method: 0, no reaction; 1, slight staining; 2, moderate staining; 3, intense staining (Zhu 1989, Seidal et al. 2001). Negative controls consisted of sections that were treated with PBS without primary antibodies.
Live weights and kidney weights Live weights and kidney weights of all rats in all groups were measured on days 7 and 14 of our study. The weights of the kidneys were normalized to the body weights (kidney weight/body weight ⫻ 100) (Sechi et al. 1997). The results were compared statistically using one-way analysis of variance (one-way ANOVA). The statistical significance of differences between the groups was assessed using Duncan 2
Results Live and tissue weight On days 7 and 14, a significant increase in live weights was observed in the control, sham and melatonin groups; the live body weights of the diabetic and melatonin treated diabetic groups were not significantly different (Table 1).
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Table 1. Mean weights (g) of rats in all groups on days 7 and 14
Days day 7 (n ⫽ 10) day 14 (n ⫽ 5) a,b,cValues
Sham
Control
Melatonin
Diabetic group
Melatonin-treated diabetic group
238.70 ⫾ 14.63a 224.54 ⫾ 13.01c
238.83 ⫾ 24.23a 220.48 ⫾ 18.13bc
191.56 ⫾ 60.82b 189.51 ⫾ 51.70a,b,c
185.09 ⫾ 36.45b 184.88 ⫾ 9.78a,b
193.93 ⫾ 27.08b 173.52 ⫾ 23.75a
with different letters in the same line were significantly different from each other. p ⬍ 0.05.
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Table 2. Mean kidney weights (g) of rats in all groups on days 7 and 14
Days day 7 (n ⫽ 5) day 14 (n ⫽ 5) a,b,cValues
Sham
Control
Melatonin
Diabetic group
Melatonin-treated diabetic group
4.81 ⫾ 0.29 4.48 ⫾ 0.30a
4.96 ⫾ 0.31 4.96 ⫾ 0.31a,b
4.45 ⫾ 0.50 4.79 ⫾ 0.68a,b
5.27 ⫾ 0.34 5.53 ⫾ 0.70b
4.99 ⫾ 0.69 5.29 ⫾ 0.57b
with different letters in the same line were significantly different from each other. (p ⬍ 0.05).
On day 7, there were no significant differences in kidney weights among the groups. On day 14, differences in kidney weights among the control, sham, diabetic and melatonin treated diabetic groups were statistically significant (p ⬍ 0.05) (Table 2). Histology Injuries to kidney tissues in all groups were evaluated semiquantitatively. The control, sham and melatonin groups showed normal histological structure (Fig. 1). In the diabetic groups, necrosis of the epithelial cells of proximal and distal tubules and collecting tubules of diabetic rats was observed (Figs. 2–4); the tubule lumens were enlarged owing to the necrosis of the epithelial cells (Figs. 2–4). Vacuol and hydropic degeneration was observed in tubule epithelial cells that were not necrosed and pyknosis was observed in desquamated epithelial cell nuclei. We observed that glomeruli were partially contracted or glomerular spaces were expanded as a result of glomerular atrophy (Figs. 2–4). The Pathologic disorders in the kidney were significantly less severe in the melatonin treated diabetic group than in the diabetic group.
Fig. 1. Control rat kidney. Renal tubules and glomeruli show normal structure. H & E. Bar ⫽ 100 μm.
Immunohistochemistry The location of leptin immunoreactivity was similar for all groups, but it was weaker in the diabetic and melatonin treated diabetic groups than in the other groups. Leptin immunoreactivity was observed in renal tissues of rats in all groups on days 7 and 14. Strong leptin immunoreactivity was found in the renal cortex except for the diabetic and melatonin
Fig. 2. Renal lesion in kidney of diabetic group rat on day 7. Severe and widespread necrosis in proximal and distal tubule epithelium (long arrows). Tubule cavities are dilated owing to glomerular atrophy (short arrows). H & E. Bar ⫽ 200 μm.
The effect of melatonin on leptin in diabetic kidney 3
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Fig. 3. Renal lesion in the kidney of diabetic group rat on day 7. Severe and widespread necrosis in proximal and tubule distalis epithelium (long arrows). Tubule cavities are dilated owing to glomerular atrophy (short arrows). H & E. Bar ⫽ 100 μm.
treated diabetic groups. Leptin immunoreactivity was weak in renal medullas of all groups, however. Although weak immunoreactivity was detetected in proximal, distal tubule cells and collecting tubules of rats in the diabetic and melatonin treated diabetic groups on days 7 and 14 (Figs. 5, 6, 10, 11), strong immunoreactivity in proximal and distal tubule cells, and collecting tubules was observed in rats in the melatonin, sham and control groups (Figs. 7– 9, 12–14). These were differences statistically significant. No leptin immunoreactivity was observed in the glomeruli or vascular endothelium of renal tissues of any group (Table 3).
Fig. 4. Renal lesion in the kidney of diabetic group rat on day 7. Severe and widespread necrosis in proximal and distal tubule epithelium (long arrows). Dilation of tubule cavities owing to glomerular atrophy (short arrow). H & E. Bar ⫽ 50 μm.
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Fig. 5. Renal section of diabetic group rat on day 7. Weak to intense leptin immunoreactivity in proximal and tubule distal tubules is shown. Bar ⫽ 100 μm.
Discussion We investigated changes in body and kidney weights, histopathologic structural changes and immunohistochemical distribution of leptin in the kidney in melatonin treated rats with STZ-induced diabetes. Andallu (2003) reported that weight loss in diabetic rats is due to destruction of body tissue proteins. We found decreased live body weights in both the diabetic and melatonin treated diabetic groups compared to the other groups. These findings are consistent with literature reports that the body weights of diabetic rats are significantly lower than control groups (Hayashi et al. 2001, Fujita et al. 2005, Vardı et al. 2005, German et al. 2010, Bingol and Kocamis 2010). Cam et al. (2003) and Aksoy et al. (2003) reported that melatonin treatment had no significant effect on body weights of diabetic
Fig. 6. Renal section of melatonin diabetic group rat on day 7. Weak to intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 100 μm.
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Fig. 7. Renal section of melatonin group rat on day 7. Intense leptin immunoreactivity in proximal and distal tubules is shown. Bar ⫽ 100 μm.
Fig. 10. Renal section of diabetic group rat on day 14. Weak to intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 50 μm.
Fig. 8. Renal section of sham group rat on day 7. Intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 100 μm.
Fig.11. Renal section of melatonin diabetic group rat on day 14. Weak to intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 50 μm.
Fig. 9. Renal section of control group rat on day 7. Intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 100 μm.
Fig. 12. Renal section of melatonin group rat on day 14. Intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 50 μm.
The effect of melatonin on leptin in diabetic kidney 5
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Fig. 13. Renal section of sham group rat on day 7. Intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 50 μm.
Fig. 14. Renal section of control group rat on day 14. Intense leptin immunoreactivity in proximal and tubule distal tubles is shown. Bar ⫽ 50 μm.
patients; however, Bartnes et al. (1985) reported that while melatonin did not affect body weight during the first 8 weeks, it caused decreased body weight during the following 6 weeks. We found no significant differences between the body weights of the diabetic and melatonin treated diabetic groups on day 14 of melatonin administration. Sechi et al. (1997), Wade et al. (2001) and Bingöl and Kocamıs (2010) reported that kidney weights
of mice with experimental diabetes increased compared to their control groups. We also found that kidney weights increased more in the diabetic and melatonin treated diabetic groups than in the control, sham and melatonin groups. We suggest that the increased kidney weight is caused by hypertrophy due to diabetes. Lee et al. (2007) reported that diabetes causes glomerular enlargement, sclerosis and tubulointerstitial fibrosis in rat kidney. Basement membrane thickening of the glomerulus and tubule caused by accumulation of extracellular matrix in experimental diabetic and cases of nephropathy has been described by others (Lehman and Schleicher 2000, Singh and Crook 2000, Reeves and Andreoli 2000, Ozturk et al. 2005). We observed necrosis of the epithelial cells of proximal, distal and collecting tubules in the diabetic group. A lesser degree of necrosis was observed in the melatonin treated diabetic group. Although Cam et al. (2003) and Vardı et al. (2005) detected hydropic changes in the diabetic group in their study, they also stated that melatonin administration limits hydropic changes. We showed that melatonin administration reduces tissue damage caused by diabetes and these results are comparable to those reported by Cam et al. (2003) and Vardı et al. (2005). It has been proposed melatonin prevents pathological changes that occur in diabetes by stabilizing cell membranes (Vijayalaxmi et al. 2002). It has been reported that leptin suppresses insulin secretion by activating ATP-sensitive K⫹ channels in pancreatic β cells; thus, β cells become hyperpolarized prior to depolarization for insulin secretion (Harvey et al. 1997). It has been reported also that leptin decreases basal and glucose-stimulated insulin secretion by exerting negative feedback on insulin secretion (Wauters et al. 2000, German et al. 2010). Serradeil-Le Gal et al. (1997) and Hama et al. (2004) detected leptin receptors in the renal cortex, and proximal and distal tubule cells. We observed weak immunoreactivity in the renal cortex, proximal and distal tubules of diabetic and melatonin treated diabetic groups at days 7 and 14. Also, strong
Table 3. Comparison of leptin immunoreactivity among groups
Diabetic group Renal structures Proximal tubule Distal tubule Medulla
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Melatonin-treated diabetic group
Melatonin, sham and control groups
7 days
14 days
7 days
14 days
7 days
14 days
weak weak weak
weak weak weak
weak weak weak
weak weak weak
strong strong weak
strong strong weak
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immunoreactivity was detected in the proximal and distal tubules of the renal cortex in the melatonin, sham and control groups. Immunoreactivity of leptin in all groups showed similar characteristics, but it was weaker in the diabetic and melatonin treated diabetic groups compared to the other groups. Previous studies have shown that leptin levels were high in the blood of obese people; high leptin levels develop in proportion to fat mass (Montague et al. 1997). Application of melatonin was observed to reduce renal injury in STZ-induced diabetic rats; however, no effect on leptin immunoreactivity was observed. Further studies of long-term use of melatonin are required to demonstrate the effects of melatonin on diabetic complications.
Acknowledgment Our research was supported by a grant from the Kafkas University, BAP (Project No: 2012-KSYO-18) and presented at the 21st National Electron Microscope Congress, P: 123, 28–31 May, 2013, Mersin, Turkey.
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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