|Year : 2019 | Volume
| Issue : 1 | Page : 24-28
Overexpression of renal proapoptotic factors is attenuated subsequent to endurance exercise in Type I diabetes: An immunohistochemistry study
Nour S Erekat1, Rawan A Rababa'h2, Muhammed D Al-Jarrah3
1 Department of Anatomy, Faculty of Medicine, Jordan University of Science and Technology (JUST), Irbid, Jordan
2 Department of Legal Medicine, Toxicology and Forensic Sciences, Faculty of Medicine, JUST, Irbid, Jordan
3 Department of Rehabilitation Sciences, Faculty of Applied Medical Sciences, JUST, Irbid, Jordan
|Date of Web Publication||4-Feb-2019|
Nour S Erekat
Department of Anatomy, Faculty of Medicine, Jordan University of Science and Technology, P.O. Box 3030, Irbid 22110
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Upregulation of Apoptotic markers (p53 and active caspase-3) is reported in diabetic nephropathy. Exercise training is reported to exert renoprotective effects in diabetes. This study correlated the effects of endurance exercise training on the renal expression of p53 and active caspase-3 in a rat model of Type 1 diabetes. Materials and methods: Thirty healthy Sprague-Dawley rats were randomly divided into the following three groups: sedentary control (SC), sedentary diabetic (SD) rats, and exercised diabetic (ED) rats. The drug alloxan was administered to SD and ED groups of rats in order to induce diabetes mellitus. Expression of p53 and active caspase-3 in the renal tissue from each of the three different groups was investigated by immunohistochemistry. Increased blood levels compared to control group of rats validated the onset of diabetes in rats from SD and ED groups. Results: Significantly (P < 0.05) higher renal p53 and active caspase-3 expression was observed in SD versus SC group. Blood glucose levels and the expression of both p53 and active caspase-3 in the diabetic renal tissue were significantly reduced following endurance exercise training. Conclusion: This study attributes the renoprotective effects of endurance exercise training in diabetes to reduction in blood glucose levels and/or suppression of proapoptotic factors in the kidney.
Keywords: Active caspase-3, immunohistochemistry, kidney, p53, type 1 diabetes
|How to cite this article:|
Erekat NS, Rababa'h RA, Al-Jarrah MD. Overexpression of renal proapoptotic factors is attenuated subsequent to endurance exercise in Type I diabetes: An immunohistochemistry study. J Nat Sc Biol Med 2019;10:24-8
|How to cite this URL:|
Erekat NS, Rababa'h RA, Al-Jarrah MD. Overexpression of renal proapoptotic factors is attenuated subsequent to endurance exercise in Type I diabetes: An immunohistochemistry study. J Nat Sc Biol Med [serial online] 2019 [cited 2019 Dec 10];10:24-8. Available from: http://www.jnsbm.org/text.asp?2019/10/1/24/251506
| Introduction|| |
Diabetic nephropathy is a microvascular complication of diabetes mellitus, which leads to end-stage renal disease. Apoptosis potentially contributes to the progression of diabetic nephropathy. Tumor suppressor protein p53 and active caspase-3 are major biomarkers of apoptosis.,,, p53 is a nuclear transcription factor, which is expressed at low levels in adult nonstressed tissues., Stress can activate p53, leading to its nuclear accumulation, resulting in cell cycle arrest, apoptosis, or cell removal from the proliferative pool. p53 upregulation is reported in several different tissues in diabetes mellitus, including heart and kidney., p53 induces apoptosis through the activation of caspases. Caspases constitute a family of cysteine proteases, which exist in the cell as inactive zymogen. Caspase-3 is the executioner caspase of apoptosis that becomes activated upon cleavage in the apoptotic process, leading to the morphological features of apoptosis.,, Elevated levels of active caspase-3 is reported in many renal diseases.,, p53 and active caspase-3 are implicated in the pathogenesis of many renal diseases.,, Intermittent fasting is reported to attenuate p53 upregulation in diabetic kidney as a consequence of reduced hyperglycemia achieved by enhanced insulin-mediated glucose uptake. Furthermore, exercise training is reported to suppress caspase-3 activation and enhance phosphorylation of p53, which promotes mitochondrial biogenesis and the eventual downregulation of p53., The beneficial effects of exercise training in diabetes mellitus are also attributed to increase in insulin sensitivity and decrease in advanced glycation end products., However, the effect of endurance exercise training on expression of p53 and active caspase-3 has not be evaluated in the renal tissue from Type 1 diabetic experimental model. Using immunohistochemistry and light microscopy, this study evaluated the impact of endurance exercise training on the expression of p53 and active caspase-3, in kidneys from rats with alloxan-induced diabetes mellitus.
| Materials and Methods|| |
Thirty Sprague-Dawley rats were randomly selected and divided into the following three groups: sedentary control (SC, n = 10), sedentary diabetic (SD, n = 10), and exercised diabetic (ED, n = 10). The study was reviewed and approved by the university ethics committee. Animal care and experiments were performed according to the Research Committee Guidelines for Animal Experimentation at Jordan University of Science and Technology. Diabetes was induced according to the protocol described previously., Three days following intraperitoneal injection (120 mg/kg) of alloxan (SD and ED groups) or saline (SC group), diabetic rats were identified by evaluating fasting blood glucose levels above 250 mg/dL. Four weeks later, blood glucose levels were measured again. Finally, animals in all groups were sacrificed by cervical dislocation.
Exercise training at an aerobic level that provided adequate systemic and cellular adaptations was used to train the rats in the ED group. Briefly, a custom treadmill with eight separate lanes was used to make the diabetic rats run, at a speed of 18 m/min, 40 min/day for 5 days/week for 4 weeks.,,
Immunohistochemistry of p53 and active caspase-3 in the kidney
The immunohistochemistry protocol for p53 and active caspase-3 was performed as previously described.,,, 5-μm thick paraffin-embedded sections were prepared and subsequently deparaffinized and rehydrated. Further, antigen retrieval was performed. Subsequently, 3% hydrogen peroxide in methanol was used to block endogenous peroxidase activity. The sections were then washed in phosphate-buffered saline (PBS). A few of those sections were incubated with anti-p53 antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA), whereas others were incubated with anti-active caspase-3 antibody (Abcam, Cambridge, MA, USA). Anti-p53 antibody was diluted to 1:200, whereas anti-active caspase-3 antibody was diluted to 1:300. Then, sections were rinsed off with PBS before and after being incubated with biotinylated secondary antibody (LSAB kit, Dako, Carpinteria, CA, USA). Streptavidin horseradish peroxidase (LSAB kit, Dako, Carpinteria, CA, USA) was used to incubate the sections. Finally, after washing the sections with PBS, they were treated with 3, 3'-diaminobenzidine substrate until the desired color intensity was developed, and then the slides were washed with tap water to stop the reaction. The sections were counterstained with hematoxylin. The primary antibody was omitted in the negative control slides [Figure 2]g,[Figure 2]h,[Figure 2]i. Sections were viewed under the light microscopy.
|Figure 2: Immunohistochemical staining of p53 and active caspase-3 in the glomeruli and tubules in 5 μm thick paraffin-embedded kidney sections. (a, d, and g) From SC. (b, e, and h) From SD. (c, f, and i) From ED. (g, h, and i) are negative controls, SC: Sedentary control, SD: Sedentary diabetic, ED: Exercised diabetic. Scale bar shown in (a) applies to all images in the figure. P53 immunostaining is hardly observed in the sedentary control. However, p53 immunoreactivity is very strong in sedentary diabetic (at the tip of the arrows), but it is obviously decreased following endurance exercise training in the diabetic rats. Similarly, active caspase-3 immunostaining is barely detected in the sedentary control. However, active caspase-3 immunoreactivity is very strong in sedentary diabetic (at the tip of the arrows), but it is obviously decreased following endurance exercise training in the diabetic rats|
Click here to view
Data collection and analysis
Data collection and analysis were done as previously described.,,, A digital camera was used to photograph the sections. Ten slides from each animal of all ten animals in each of the three groups were analyzed by counting the total pixels of area occupied by positive staining using Adobe Photoshop software (Adobe Systems, San Jose, CA, USA). One-way analysis of variance followed by independent samples t-test was used to statistically compare P53 and active caspase-3 expression in kidney sections among the three different groups. All the data are represented as mean ± standard deviation. Differences in p53 expression and active caspase-3 expression were considered statistically significant at P < 0.05.
| Results|| |
At the completion of the study, blood glucose levels observed in the three different groups were 104 ± 6.2, 430 ± 13.1, and 237 ± 10 mg/dL in the SC, SD, and ED rats, respectively. The blood glucose levels were significantly elevated (P < 0.01, [Figure 1]) in the SD group when compared to those in the SC group. Endurance exercise training significantly reduced the blood glucose levels in the ED group (P < 0.01, [Figure 1]) when compared to the SD group.
|Figure 1: Blood glucose levels in the rats at the completion of the study. Blood glucose levels increased significantly in the SD group compared to SC groups (P < 0.001, *). Blood glucose levels significantly decreased (P < 0.001, **) in diabetic rats following endurance exercise training. SC: Sedentary control, SD: Sedentary diabetic, ED: Exercised diabetic|
Click here to view
Immunohistochemical staining revealed negligible expression of p53 and active caspase-3 in kidneys from SC group ([Figure 2]a and [Figure 2]d, respectively). However, p53 and active caspase-3 immunoreactivity was increased in the glomeruli and tubules of the diabetic kidneys ([Figure 2]b and [Figure 2]e, respectively), but it was hardly detected in kidneys from ED rats ([Figure 2]c and [Figure 2]f, respectively).
The induction of diabetes significantly upregulated p53 (P < 0.01, [Figure 3]) and active caspase-3 (P < 0.05, [Figure 4]) expression when compared to the control kidneys. Endurance exercise training significantly decreased both p53 (P < 0.01, [Figure 3]) and active caspase-3 (P < 0.05, [Figure 4]) expression in diabetic kidneys when compared to that in the SD group.
|Figure 3: Expression of p53 in the kidney. The level of P53 expression increased significantly in the SD group compared to SC groups (P < 0.01, *). P53 expression level significantly decreased (P < 0.01, **) in diabetic rats following endurance exercise training. SC: Sedentary control, SD: Sedentary diabetic, ED: Exercised diabetic|
Click here to view
|Figure 4: Expression of active caspase-3 in the kidney. The level of active caspase-3 expression increased significantly in the SD group compared to SC groups (P < 0.05, *). Active caspase-3 expression level significantly decreased (P < 0.05, **) in diabetic rats following endurance exercise training. SC: Sedentary control, SD: Sedentary diabetic, ED: Exercised diabetic|
Click here to view
| Discussion|| |
This study is the first of its kind to illustrate the impact of endurance exercise training on the expression of the apoptotic markers (p53 and active caspase-3) in kidney of experimental model of Type 1 diabetes. Endurance exercise training substantially suppressed hyperglycemia and reduced p53 and active caspase-3 expression in the renal tissues of diabetic rats.
Both alloxan and streptozotocin are cytotoxic chemicals that destroy insulin-secreting beta cells of the pancreas in rodents, inducing an experimental model of Type 1 diabetes. Markedly increased expression of active caspase-3 was reported in the kidney of streptozotocin-induced diabetic rats on the 3rd, 4th, and 5th days following the administration of streptozotocin. Hence, cellular damages in the kidney can occur at a very early stage of diabetes., Both p53 and active caspase-3 are abundant in the developing kidney, where they are involved in nephrogenesis and renal development., However, p53 and active caspase-3 levels are low in the adult nonstressed cells., This is in agreement with our finding of nearly undetectable p53 and active caspase-3 expression in kidneys of control rats ([Figure 2]a and [Figure 2]d, respectively).
The hyperglycemia and upregulation of p53 and active caspase-3 observed in our study are consistent with the previous studies showing significant increases in the expression of p53 and active caspase-3 in response to hyperglycemia. p53 and active caspase-3 have been identified as mediators of renal glomerular and tubular epithelial cell apoptosis in hyperglycemic conditions., Further previous studies have implicated hyperglycemia-induced p53 and active caspase-3 upregulation in the progression of diabetic nephropathy.
Exercise training is assumed to retard diabetic nephropathy by exerting a preventive effect on the development of microalbuminuria. Since the proapoptotic p53 and active caspase-3 have been implicated in the progression of diabetic nephropathy and the development of albuminuria, we sought to examine the impact of chronic exercise training on the renal p53 and active caspase-3 expression in diabetic rats in order to investigate the mechanism by which exercise training may beneficially influence diabetic nephropathy. Exercise training attenuated renal active caspase-3 upregulation in Type 2 diabetic kidneys. Similarly, our results illustrate significantly reduced renal p53 and active caspase-3 levels in rats with alloxan-induced diabetes mellitus following endurance exercise training. Exercise training has been shown to promote p53 phosphorylation, which enhances mitochondrial biogenesis that leads to reduced oxidative stress and enhanced antioxidant defense and thus decreasing p53 expression. In addition, exercise training is reported to increase Bcl-2/Bax ratio that consequently inhibits cytochrome c release and the subsequent activation of caspase-3, leading to attenuated apoptosis.
The suppressed hyperglycemia is concomitant with attenuated upregulation of p53 and active caspase-3 in the diabetic kidneys after exercise training, as shown in this study, and may explain the renoprotective effect of endurance exercise training. Exercise training is also reported to reduce advanced glycation end products in diabetic obese Zucker rats and enhance insulin sensitivity. Accordingly, it is likely that decreased hyperglycemia, following exercise training, may have reduced the expression of p53 and active caspase-3, indicative of apoptosis, in the renal glomeruli and tubules.
| Conclusions|| |
This is the first study to reveal the impact of endurance exercise training on proapoptotic p53 and active caspase-3 expression in renal tissue from an experimental model of Type 1 diabetes. Hence, chronic endurance exercise training can be advocated as preventive measure to avoid the development of diabetic nephropathy, which warrants clinical investigation.
Financial support and sponsorship
The Deanship of Research at Jordan University of Science and Technology, Irbid, Jordan. Research Grant #: 20140180.
Conflicts of interest
There are no conflicts of interest.
| References|| |
Rheinberger M, Böger CA. Diabetic nephropathy: New insights into diagnosis, prevention and treatment. Dtsch Med Wochenschr 2014;139:704-6.
Susztak K, Raff AC, Schiffer M, Böttinger EP. Glucose-induced reactive oxygen species cause apoptosis of podocytes and podocyte depletion at the onset of diabetic nephropathy. Diabetes 2006;55:225-33.
Erekat NS. Apoptotic mediators are upregulated in the skeletal muscle of chronic/progressive mouse model of Parkinson's disease. Anat Rec (Hoboken) 2015;298:1472-8.
Erekat NS. Cerebellar purkinje cells die by apoptosis in the shaker mutant rat. Brain Res 2017;1657:323-32.
Deshpande SD, Putta S, Wang M, Lai JY, Bitzer M, Nelson RG, et al.
Transforming growth factor-β-induced cross talk between p53 and a microRNA in the pathogenesis of diabetic nephropathy. Diabetes 2013;62:3151-62.
Küpper DS, Valera FC, Malinsky R, Milanezi CM, Silva JS, Tamashiro E, et al.
Expression of apoptosis mediators p53 and caspase 3, 7, and 9 in chronic rhinosinusitis with nasal polyposis. Am J Rhinol Allergy 2014;28:187-91.
Amundson SA, Myers TG, Fornace AJ Jr. Roles for p53 in growth arrest and apoptosis: Putting on the brakes after genotoxic stress. Oncogene 1998;17:3287-99.
Leigh-Brown S, Enriquez JA, Odom DT. Nuclear transcription factors in mammalian mitochondria. Genome Biol 2010;11:215.
Horn HF, Vousden KH. Coping with stress: Multiple ways to activate p53. Oncogene 2007;26:1306-16.
Nakamura H, Matoba S, Iwai-Kanai E, Kimata M, Hoshino A, Nakaoka M, et al.
P53 promotes cardiac dysfunction in diabetic mellitus caused by excessive mitochondrial respiration-mediated reactive oxygen species generation and lipid accumulation. Circ Heart Fail 2012;5:106-15.
Qiu LQ, Sinniah R, Hsu SI. Coupled induction of iNOS and p53 upregulation in renal resident cells may be linked with apoptotic activity in the pathogenesis of progressive IgA nephropathy. J Am Soc Nephrol 2004;15:2066-78.
Fan TJ, Han LH, Cong RS, Liang J. Caspase family proteases and apoptosis. Acta Biochim Biophys Sin (Shanghai) 2005;37:719-27.
Erekat NS. Autophagy precedes apoptosis among at risk cerebellar purkinje cells in the shaker mutant rat: An ultrastructural study. Ultrastruct Pathol 2018;42:162-9.
Slee EA, Adrain C, Martin SJ. Executioner caspase-3, -6, and -7 perform distinct, non-redundant roles during the demolition phase of apoptosis. J Biol Chem 2001;276:7320-6.
Bamri-Ezzine S, Ao ZJ, Londoño I, Gingras D, Bendayan M. Apoptosis of tubular epithelial cells in glycogen nephrosis during diabetes. Lab Invest 2003;83:1069-80.
Yano T, Itoh Y, Matsuo M, Kawashiri T, Egashira N, Oishi R, et al.
Involvement of both tumor necrosis factor-alpha-induced necrosis and p53-mediated caspase-dependent apoptosis in nephrotoxicity of cisplatin. Apoptosis 2007;12:1901-9.
Tikoo K, Tripathi DN, Kabra DG, Sharma V, Gaikwad AB. Intermittent fasting prevents the progression of type I diabetic nephropathy in rats and changes the expression of sir2 and p53. FEBS Lett 2007;581:1071-8.
Camera DM, Hawley JA, Coffey VG. Resistance exercise with low glycogen increases p53 phosphorylation and PGC-1α mRNA in skeletal muscle. Eur J Appl Physiol 2015;115:1185-94.
Zhao Y, Ma Z. Swimming training affects apoptosis-related microRNAs and reduces cardiac apoptosis in mice. Gen Physiol Biophys 2016;35:443-50.
Cuff DJ, Meneilly GS, Martin A, Ignaszewski A, Tildesley HD, Frohlich JJ, et al.
Effective exercise modality to reduce insulin resistance in women with type 2 diabetes. Diabetes Care 2003;26:2977-82.
Boor P, Celec P, Behuliak M, Grancic P, Kebis A, Kukan M, et al.
Regular moderate exercise reduces advanced glycation and ameliorates early diabetic nephropathy in obese zucker rats. Metabolism 2009;58:1669-77.
Rees DA, Alcolado JC. Animal models of diabetes mellitus. Diabet Med 2005;22:359-70.
Pourghasem M, Nasiri E, Shafi H. Early renal histological changes in alloxan-induced diabetic rats. Int J Mol Cell Med 2014;3:11-5.
Al-Jarrah M, Pothakos K, Novikova L, Smirnova IV, Kurz MJ, Stehno-Bittel L, et al.
Endurance exercise promotes cardiorespiratory rehabilitation without neurorestoration in the chronic mouse model of Parkinsonism with severe neurodegeneration. Neuroscience 2007;149:28-37.
Al-Jarrah MD, Erekat NS. Parkinson disease-induced upregulation of apoptotic mediators could be attenuated in the skeletal muscle following chronic exercise training. NeuroRehabilitation 2017;41:823-30.
Erekat N, Al-Khatib A, Al-Jarrah M. Heat shock protein 90 is a potential therapeutic target for ameliorating skeletal muscle abnormalities in Parkinson's disease. Neural Regen Res 2014;9:616-21.
] [Full text]
Erekat NS, Al-Jarrah AA, Shotar AM, Al-Hourani ZA. Hepatic upregulation of tumor necrosis factor alpha and activation of nuclear factor kappa B following methyl methacrylate administration in the rat. Int J Pharmacol 2018. [In press]. Doi: 10.3923/ijp. 2018.
Erekat NS, Al-Jarrah MD. Interleukin-1 beta and tumor necrosis factor-alpha upregulation and nuclear factor kappa B activation in the skeletal muscle from a mouse model of chronic/progressive Parkinson disease. Med Sci Monit 2018. [In Press]. DOI: 10.12659/MSM.909032.
Erekat N, Al Khatib A, Al-Jarrah M. Endurance exercise training attenuates the up regulation of iNOS in the skeletal muscles of chronic/progressive mouse model of Parkinson's disease. J Neurol Res 2013;3:108-13.
Erekat NS, Al-Jarrah MD, Al Khatib AJ. Treadmill exercise training improves vascular endothelial growth factor expression in the cardiac muscle of type I diabetic rats. Cardiol Res 2014;5:23-9.
Al-Jarrah M, Erekat N, Al Khatib A. Upregulation of vascular endothelial growth factor expression in the kidney could be reversed following treadmill exercise training in type I diabetic rats. World J Nephrol Urol 2014;3:25-9.
Lenzen S. The mechanisms of alloxan- and streptozotocin-induced diabetes. Diabetologia 2008;51:216-26.
Haligur M, Topsakal S, Ozmen O. Early degenerative effects of diabetes mellitus on pancreas, liver, and kidney in rats: An immunohistochemical study. Exp Diabetes Res 2012;2012:120645.
Saifudeen Z, Liu J, Dipp S, Yao X, Li Y, McLaughlin N, et al.
Ap53-pax2 pathway in kidney development: Implications for nephrogenesis. PLoS One 2012;7:e44869.
Araki T, Saruta T, Okano H, Miura M. Caspase activity is required for nephrogenesis in the developing mouse metanephros. Exp Cell Res 1999;248:423-9.
Ghosh S, Khazaei M, Moien-Afshari F, Ang LS, Granville DJ, Verchere CB, et al.
Moderate exercise attenuates caspase-3 activity, oxidative stress, and inhibits progression of diabetic renal disease in db/db mice. Am J Physiol Renal Physiol 2009;296:F700-8.
Kang BP, Frencher S, Reddy V, Kessler A, Malhotra A, Meggs LG, et al.
High glucose promotes mesangial cell apoptosis by oxidant-dependent mechanism. Am J Physiol Renal Physiol 2003;284:F455-66.
Mishra R, Emancipator SN, Kern T, Simonson MS. High glucose evokes an intrinsic proapoptotic signaling pathway in mesangial cells. Kidney Int 2005;67:82-93.
Kurdak H, Sandikci S, Ergen N, Dogan A, Kurdak SS. The effects of regular aerobic exercise on renal functions in streptozotocin induced diabetic rats. J Sports Sci Med 2010;9:294-9.
Brezniceanu ML, Liu F, Wei CC, Chénier I, Godin N, Zhang SL, et al.
Attenuation of interstitial fibrosis and tubular apoptosis in db/db transgenic mice overexpressing catalase in renal proximal tubular cells. Diabetes 2008;57:451-9.
Holten MK, Zacho M, Gaster M, Juel C, Wojtaszewski JF, Dela F, et al.
Strength training increases insulin-mediated glucose uptake, GLUT4 content, and insulin signaling in skeletal muscle in patients with type 2 diabetes. Diabetes 2004;53:294-305.
[Figure 1], [Figure 2], [Figure 3], [Figure 4]