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Facultyof Pharmacy Department of Biochemistry Thesis Submitted for the Degree of Philosophy Doctor in Pharmaceutical Sciences (Biochemistry) BY Nehal Mohsen Ahmed Elsherbiny M.Sc. Pharm. Sci., (2010) Under the supervision of Prof. Dr. Mamdouh M. El-Shishtawy Professor of Biochemistry and Dean of faculty of Pharmacy Mansoura University Prof. Dr. Prof. Dr. Gregory I. liou Laila A. Eissa Prof. of Biochemistry and Molecular Biology Department of Ophthalmology, School of Medicine Georgia Regents University, USA Professor of Biochemistry Faculty of Pharmacy-Mansoura University Dr. Mohammed M. H. Al-Gayyar Associate Professor of Biochemistry Faculty of Pharmacy- Mansoura University 2013 Page 196 Summary and Conclusion Summary and Conclusion Part I: diabetic nephropathy Diabetes is a major threat to global public that is associated with many complications threatening life. Diabetic nephropathy (DN) remains the most common cause for end stage renal disease. Adenosine is an endogenous purine nucleoside released from various tissues and organs. Adenosine controls the supply and demand of energy and modulates a variety of physiological responses by interacting with specific cell surface G-protein-coupled receptors Although, adenosine acts as local modulator with a cytoprotective function, extracellular adenosine usually disappears quickly due to rapid uptake into adjacent cells. Therefore, the current study investigated the effect of 5′-(NEthylcarboxamido)-adenosine (NECA), stable nonselective adenosine receptor agonist, on diabetes-induced increase in inflammatory cytokines and adhesion molecules. Enhancing adenosine receptor action by NECA was examined in renal tissues of streptozotocin-induced diabetic rats. Daily IP injection of 0.3mg/kg/day NECA was given for two weeks to six-weeks diabetic rats They were purchased from and kept in "Urology and Nephrology Center", Mansoura University, Egypt. Serum creatinine was determined by kinetic method. Serum Blood Urea Nitrogen (BUN) and urinary albumin were determined by colorimetric method. Oxidative stress was assessed by measuring tissue malondialdehyde by colorimetric method. Gene expression of interleukin (IL)-18, tumor necrosis factor alpha (TNF-α) and Intercellular Adhesion Molecule (ICAM)-1 was measured by real-time PCR. Activation of apoptotic pathway was demonstrated by measuring the Page 197 activation of c-Jun NH2-terminal kinases (JNK)–Mitogen-activated protein kinase (MAPK) using enzyme linked immuno-sorbent assay (ELISA). In addition, changes in kidney structure were examined to assess the severity of histopathological changes in kidney sections. This study found that diabetes-induced malondialdehyde formation activated the production of TNF-α, IL-18 and ICAM-1 which in turn activated the pro-apoptotic pathway in diabetic rats. Treatment with NECA protected diabetic rats by exerting hypoglycemic and antioxidant effects as well as reducing the gene expression of TNF-α, IL-18 and ICAM-1. These effects were associated with inhibition of JNK-MAPK apoptotic pathway. In addition, diabetic rats treated with NECA showed mild glomerular affection and vacuolation of tubular epithelium. These results demonstrated that the activation of adenosine receptors is a potential therapeutic target for DN. NECA acts via multiple mechanisms including: reducing diabetes-induced oxidative stress, inhibiting gene expression of IL-18 and ICAM-1 and blocking activation of JNK-MAPK pathway. Page 198 Summary and Conclusion Part II: Diabetic Retinopathy Diabetic retinopathy (DR) is the most feared complication of diabetes that is a leading cause of adult vision loss and blindness. As a result, DR is becoming an increasing burden to society that needs improved and preventive therapies. These therapies could be discovered by unraveling the pathophysiology of DR which in itself presents quite a unique challenge. Retinal inflammation is a vision-threatening complication in diabetic retinopathy. Amadori-glycated albumin (AGA), a risk factor for diabetes, induces microglia-mediated retinal inflammation, suggesting that microglial activation causes diabetic retinopathy, and may be a target for therapeutic intervention. Retinal inflammation is endogenously modulated by extracellular adenosine via adenosine A2AAR signaling. During inflammation, ATP released from stressed cells is converted to adenosine by ectonucleoside triphosphate diphosphohydrolase1 (CD39) and ecto-5’nucleotidase (CD73). Extracellular adenosine re-uptake by the equilibrative nucleoside transporter (ENT) allows for adenosine conversion to AMP by adenosine kinase (AK). This study aimed to evaluate the relative importance of CD73 and AK in regulating extracellular levels of adenosine in the retina. As AK is the key enzyme for the regulation of ambient levels of adenosine in the brain, this study hypothesizes that AK has a similar role in the retina. The hypothesis was tested by comparing retinal inflammation in wild type (WT) and CD73-/- (CD73KO) diabetic mice, and in diabetic mice treated and untreated with an AK inhibitor ABT 702 (of 1.5mg/kg IP twice Page 199 a week). This study also compared TNF-α release in AGA-treated retinal microglial cells ± AK or CD73 inhibitors. In diabetic mice, Western, real-time PCR and immunoflourescence analyses revealed up-regulation of retinal expression of A2AAR, ENT1, Iba1, TNF-α, ICAM1, caspase3, TUNEL positive cells and reactive oxygen species, but down-regulation of retinal AK expression. Treatment with AK inhibitor attenuated all the above-mentioned alterations in diabetic animals. In contrast, these retinal alterations remained unchanged in diabetic WT and CD73KO mice. Moreover, treatment with AK inhibitor, but not CD73 inhibitor, blocked TNF-α release in AGA-treated microglial cells. The data presented here provide experimental evidence that targeting adenosine kinase can inhibit diabetes-induced retinal abnormalities that are postulated in the development of diabetic retinopathy by potentially amplifying the endogenous therapeutic effects of accumulation of extracellular adenosine. Thus, ABT 702 appears to be a useful therapy to possibly inhibit the development/progression of retinopathy, the sight threatening complication faced by diabetic patients. In conclusion, this study demonstrated that enhancing adenosine signaling -by using adenosine receptors agonists or inhibiting endogenous adenosine metabolism- suppresses inflammation which may be a therapeutic option for the treatment of diabetic complications. Page 200