The physiological effect of adenosine is

The physiological effect of adenosine is now considered a new direction in halting the progression of organ damage in cardiovascular disease [24], [25], [26]. Adenosine has diverse functions, depending on its interaction with different receptor subtypes: A1, A2A, A2B, and A3[24], [25], [26]. Stimulation of adenosine A2A receptor (A2A) has been shown to provide potent anti-inflammatory effects and A2A receptor are highly expressed in the glomeruli and immune cells such as monocyte/macrophages, neutrophils and T cells [24], [27], [28], [29]. In the kidney, A2A receptor activation has been shown to attenuate inflammation and renal injury associated with diabetic nephropathy [24]. Agonists of A2A receptor also preserved the normal structure of podocyte foot processes, slit diaphragms, and the full report cytoskeleton in puromycin-induced podocyte injury in mice [30]. Given the crucial role of inflammation in the development of diabetic renal injury and the fact that adenosine kinase could limit the extracellular adenosine levels by its conversion to adenosine mono-phosphate, we target adenosine kinase, the key enzyme in adenosine metabolism, using the selective adenosine kinase inhibitor ABT702 to increase free adenosine levels. We hypothesize that adenosine kinase inhibition attenuates diabetes-induced renal injury as the potential effects of ABT702 in diabetes-induced renal complications have not been clearly addressed. Thus, we evaluated the efficacy of ABT702 in alleviating diabetes-induced renal injury in the current study in the context of its potential effects on oxidative stress and inflammation.
Materials and methods All procedures with animals were performed in accordance with the Public Health Service Guide for the Care and Use of Laboratory Animals (Department of Health, Education, and Welfare publication, NIH 80-23) and Georgia Regents University guidelines. Ten-week-old male C57BL/6 mice were given daily injection of streptozotocin (50mg/kg; i.p.) for 5 days after a 4h fast; control animals received the vehicle (citrate buffer; 0.01mol/L, pH: 4.5). Diabetes was confirmed by measurement of fasting blood glucose levels of >250mg/dl three days after streptozotocin injection. Thereafter, diabetic mice were randomly subdivided to receive the adenosine kinase inhibitor 4-amino-5-(3-bromophenyl)-7-(6-morpholinopyridin-3-yl)pyrido[2,3-d]pyrimidine ABT702 (CAS214697-26-4), 1.5mg/kg, i.p., Santa Cruz Biotechnology, Santa Cruz, CA two times a week or injections of the vehicle, 5% DMSO (n=7–8/group) for eight weeks. This dose has previously shown to lower oxidative stress, inflammation and apoptosis in diabetic retina [25]. Eight weeks after initiation of ABT702 treatment, mice were individually placed in metabolic cages for collection of 24-h urine samples for determination of total protein, albumin, nephrin, podocalyxin, collagen, creatinine, thiobarbituric acid reactive substances (TBARS), nitrate/nitrite and monocyte chemoattractant protein-1 (MCP-1) excretions. Mice were then anesthetized with sodium pentobarbital (50mg/kg i.p.) and blood samples obtained for glucose and creatinine determination. Kidney were isolated.
Results
Discussion Targeting early features of renal injury in diabetes could prevent the progression of ESRD. Adenosine signaling activation is known to provide anti-inflammatory effects [36]; however, the physiological effects of exogenous adenosine intake are limited by its hemodynamic effects (hypotension and bradycardia), rapid reuptake and subsequent intracellular metabolism by adenosine kinase [24], [25], [26], [30]. Accordingly, the use of adenosine kinase inhibitors is considered a new avenue to amplify the endogenous physiological effects of adenosine while minimizing unwanted hemodynamic side effects of exogenous adenosine. The current study demonstrates that 8-week diabetic mice showed a marked albuminuria and nephrinuria together with early histological changes of diabetic renal injury such as collagen deposition and increased collagen excretion. Furthermore, diabetic renal injury was associated with activation of NADPH oxidase and oxidative stress in the kidney together with a marked degree of renal inflammation as manifested by increased macrophage infiltration, activation of NFκB and increased MCP-1 excretion. Administration of the adenosine kinase inhibitor, ABT702, reduced blood glucose levels and ameliorated streptozotocin-induced albuminuria and collagen deposition together with decreased oxidative stress and renal inflammation in mice. In vitro, ABT702 also reduced the elevation in glomerular permeability and MAPK phosphorylation and restored the decrease in glomerular occludin expression in HGECs treated with high glucose condition for 5 days.