Nehal M. Elsherbiny

Thymoquinone attenuates Doxorubicin-induced nephrotoxicity in rats: Role of Nrf2 and NOX4.

Doxorubicin (DOX) is a chemotherapeutic drug widely used for the treatment of various neoplastic conditions. However, its application is limited because of its toxic effects in many organs. Nephrotoxicity is a serious effect of DOX. The aim of this study was to determine the protective effect of thymoquinone (TQ), a predominant bioactive constituent of Nigella sativa oil, with well documented potent anti-oxidative and anti-inflammatory effects. Male Sprague Dawley rats received DOX (3.5mg/kg twice weekly) with or without TQ (50mg/kg/day, oral supplementation) for 3weeks. Elevated levels of serum urea, creatinine and urinary albumin excretion were observed in DOX-treated animals, indicating DOX-induced nephrotoxicity. Moreover, enhanced lipid peroxidation (LPO), as equivalents of malondialdehyde (MDA), in the renal tissue was accompanied by a significant decrease in the activities of superoxide dismutase (SOD) and glutathione-S-transferase (GST) in DOX-treated group. In addition, DOX treatment induced significant increase in renal levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and NADPH oxidase 4 (NOX-4), and marked decrease in interleukin-10 (IL-10) levels, nuclear factor erythroid 2-related factor 2 (Nrf2) mRNA levels and nuclear binding activity. Histopathological analysis showed severe damage in the renal tissue of DOX treated animals. Animals treated with TQ were found to have markedly reduced renal damage with restoration of all mentioned markers toward normal values. In conclusion, DOX-induced renal damage involved a redox imbalance in renal tissue, which could be reversed by TQ, suggesting a possible potential role for TQ in DOX-induced nephrotoxicity.

ABT-702, an adenosine kinase inhibitor, attenuates inflammation in diabetic retinopathy

AIMS This study was undertaken to determine the effect of an adenosine kinase inhibitor (AKI) in diabetic retinopathy (DR). We have shown previously that adenosine signaling via A2A receptors (A2AAR) is involved in retinal protection from diabetes-induced inflammation. Here we demonstrate that AKI-enhanced adenosine signaling provides protection from DR in mice. MAIN METHODS We targeted AK, the key enzyme in adenosine metabolism, using a treatment regime with the selective AKI, ABT-702 (1.5mg/kg intraperitoneally twice a week) commencing at the beginning of streptozotocin-induced diabetes at the age of eight weeks. This treatment, previously demonstrated to increase free adenosine levels in vivo, was maintained until the age of 16 weeks. Retinal inflammation was evaluated using Western blot, Real-Time PCR and immuno-staining analyses. Role of A2AAR signaling in the anti-inflammation effect of ABT-702 was analyzed in Amadori-glycated-albumin (AGA)-treated microglial cells. KEY FINDINGS At 16 weeks, when diabetic mice exhibit significant signs of retinal inflammation including up-regulation of oxidative/nitrosative stress, A2AAR, ENT1, Iba1, TNF-α, ICAM1, retinal cell death, and down-regulation of AK, the ABT-702 treated group showed lower signs of inflammation compared to control animals receiving the vehicle. The involvement of adenosine signaling in the anti-inflammation effect of ABT-702 was supported by the TNF-α release blocking effect of A2AAR antagonist in AGA-treated microglial cells. SIGNIFICANCE These results suggest a role for AK in regulating adenosine receptor signaling in the retina. Inhibition of AK potentially amplifies the therapeutic effects of site- and event-specific accumulation of extracellular adenosine, which is of highly translational impact.

Potential roles of adenosine deaminase-2 in diabetic retinopathy

The early activation of microglia that induces retinal inflammation in DR may serve as a target for therapeutic intervention of DR. Our demonstration that retinal inflammation is attenuated via adenosine receptor A(2A)AR supports the hypothesis that a mechanism to maintain extracellular concentrations of adenosine important in normal physiology is impaired in DR. Extracellular concentrations of adenosine are regulated by the interplay of equiliberative nucleoside transporter (ENT)s with enzymes of adenosine metabolism including adenosine deaminase-1 (ADA1), adenosine kinase (AK) and CD73. In the vertebrates but not rodents, a macrophage-associated ADA2 is identified. The role of ADA2 is, therefore, understudied as the sequencing probes or antibodies to mouse ADA2 are not available. We identified increased ADA2 expression and activity in human and porcine retinas with diabetes, and in Amadori glycated albumin (AGA)- or hyperglycemia-treated porcine and human microglia. In rodent as well as porcine cells, modulation of TNF-α release is mediated by A(2A)AR. Quantitative analysis of normal and diabetic porcine retinas reveals that while the expression levels of ADA2, A2AAR, ENT1, TNF-α and MMP9 are increased, the levels of AK are reduced during inflammation as an endogenous protective mechanism. To determine the role of ADA2, we found that AGA induces ADA2 expression, ADA2 activity and TNF-α release, and that TNF-α release is blocked by ADA2-neutralizing antibody or ADA2 siRNA, but not by scrambled siRNA. These results suggest that retinal inflammation in DR is mediated by ADA2, and that the anti-inflammatory activity of A(2A)AR signaling is impaired in diabetes due to increased ADA2 activity.

Cepharanthine and Piperine ameliorate diabetic nephropathy in rats: role of NF-κB and NLRP3 inflammasome.

AIMS Hyperglycemia leads to elevation of oxidative stress and proinflammatory cytokines which are the main causes of diabetic nephropathy (DN). NLRP3 inflammasome and thioredoxin-interacting protein (TXNIP) are recently assumed to participate in the development of DN. We aimed to investigate the effects of Cepharanthine (CEP), Piperine (Pip) and their combination in streptozotocin (STZ)-induced DN focusing on their role to modulate NLRP3 and TXNIP induced inflammation. MAIN METHODS Diabetic rats were treated with intraperitoneal (i.p.) injection of CEP (10mg/kg/day), Pip (30mg/kg/day) or their combination for 8weeks. Nuclear factor kappa B (NF-κB), tumor necrosis factor alpha (TNF-α) and interleukin-1β (IL-1β) were assessed by ELISA technique. TXNIP and NLRP3 genes expressions were evaluated by real time-PCR. KEY FINDINGS Diabetic rats showed significant increase in renal TXNIP and NLRP3 expression. CEP, Pip or their combination significantly decreased TXNIP and NLRP3 expression in diabetic kidneys. Hyperglycemia induced NF-κB activation leading to increased IL-1β and TNF-α levels. CEP, Pip or their combination showed significant inhibition of NF-κB together with decreased IL-1β and TNF-α levels in diabetic rats. Also, diabetic rats showed significant decrease in creatinine clearance and increase in blood glucose, serum creatinine, blood urea nitrogen, malondialdehyde, proteinuria, and kidney weight to body Weight ratio. All of these changes were reversed by CEP, Pip or their combination. SIGNIFICANCE The antioxidant and anti-inflammatory effects of CEP and Pip which were accompanied by inhibition of NF-κB and NLRP3 activation might be helpful mechanisms to halt the progression of DN.

Potential role of A2A adenosine receptor in traumatic optic neuropathy.

In traumatic optic neuropathy (TON), apoptosis of retinal ganglion cells is closely related to the local production of reactive oxygen species and inflammatory mediators from activated microglial cells. Adenosine receptor A2A (A2AAR) has been shown to possess anti-inflammatory properties that have not been studied in TON. In the present study, we examined the role of A2AAR in retinal complications associated with TON. Initial studies in wild-type mice revealed that treatment with the A2AAR agonist resulted in marked decreases in the TON-induced microglial activation, retinal cell death and releases of reactive oxygen species and pro-inflammatory cytokines TNF-α and IL-6. To further assess the role of A2AAR in TON, we studied the effects of A2AAR ablation on the TON-induced retinal abnormalities. A2AAR-/- mice with TON showed a significantly higher mRNA level of TNF-α, Iba1-1 in retinal tissue, and ICAM-1 expression in retinal sections compared with wild-type mice with TON. To explore a potential mechanism by which A2AAR-signaling regulates inflammation in TON, we performed additional studies using hypoxia- or LPS-treated microglial cells as an in vitro model for TON. Activation of A2AAR attenuates hypoxia or LPS-induced TNF-α release and significantly repressed the inflammatory signaling, ERK in the activated microglia. Collectively, this work provides pharmacological and genetic evidence for A2AAR signaling as a control point of cell death in TON and suggests that the retinal protective effect of A2AAR is mediated by attenuating the inflammatory response that occurs in microglia via interaction with MAPKinase pathway.

Evaluation of renal protective effects of inhibiting TGF-β type I receptor in a cisplatin-induced nephrotoxicity model

PURPOSE The use of cisplatin, the first of the platinum-containing anti-cancer drugs, is limited by the development of a myriad of adverse reactions, including nephrotoxicity. We conducted this study therefore to find out whether SB-431542, potent and specific inhibitor of type I transforming growth factor-beta receptor (TGF-βR1), could prevent or attenuate kidney damage in rats, and to elucidate its possible mechanism of action. METHODS Fifty rats were treated with cisplatin (10 mg/kg) in the presence (1 and 3 mg/kg) or absence of SB-431542. Morphological changes were assessed in kidney sections stained with H/E. Oxidative stress was evaluated in kidney homogenates by measuring malondialdehyde (MDA) and superoxide dismutase (SOD). Kidney samples were used for measurements of TGF-βR1, TGF-β1 and sCD93 by ELISA. Kidney tissue apoptosis was assessed by measuring caspase-3 activity. RESULTS The renal protective effect of SB-431542 was confirmed by the normal appearance of renal tissue and the relatively unaffected serum creatinine and urea levels. With SB-431542, there was significantly lower renal MDA and increased SOD compared with the cisplatin group. Furthermore, in the SB-431542 group, renal TGF-βR1, TGF-β1, sCD93 and caspase-3 levels were significantly lower. CONCLUSIONS Inhibition of TGF-βR1 provides protective effects against cisplatin-induced nephrotoxicity through several mechanisms, including attenuation of oxidative stress, inhibition of pro-inflammatory cytokines, blocking of renal fibrosis markers, and anti-apoptotic effects.

Sesamin attenuates neurotoxicity in mouse model of ischemic brain stroke

Stroke is a severe neurological disorder characterized by the abrupt loss of blood circulation into the brain resulting into wide ranging brain and behavior abnormalities. The present study was designed to evaluate molecular mechanism by which sesamin (SES) induces neuroprotection in mouse model of ischemic stroke. The results of this study demonstrate that SES treatment (30 mg/kg bwt) significantly reduced infarction volume, lipid per-oxidation, cleaved-caspase-3 activation, and increased GSH activity following MCAO in adult male mouse. SES treatment also diminished iNOS and COX-2 protein expression, and significantly restored SOD activity and protein expression level in the ischemic cortex of the MCAO animals. Furthermore, SES treatment also significantly reduced inflammatory and oxidative stress markers including Iba1, Nox-2, Cox-2, peroxynitrite compared to placebo MCAO animals. Superoxide radical production, as studied by DHE staining method, was also significantly reduced in the ischemic cortex of SES treated compared to placebo MCAO animals. Likewise, downstream effects of superoxide free radicals i.e. MAPK/ERK and P38 activation was also significantly attenuated in SES treated compared to placebo MCAO animals. In conclusion, these results suggest that SES induces significant neuroprotection, by ameliorating many signaling pathways activated/deactivated following cerebral ischemia in adult mouse.

Renal protective effects of arjunolic acid in a cisplatin-induced nephrotoxicity model

Cisplatin is the first platinum-containing anti-cancer drugs. Cisplatin notable side effect of nephrotoxicity limits its use in clinic. Meanwhile, arjunolic acid possesses anti-inflammatory properties and plays protective roles against chemically induced organ pathophysiology. This study was conducted to find out whether arjunolic acid could attenuate kidney damage in rats, and to elucidate its possible mechanism of action. Fifty rats were treated with cisplatin (10mg/kg) in the presence/absence of 100 or 250mg/kg arjunolic acid. Arjunolic acid is given 1h after cisplatin. Morphological changes were assessed in kidney sections stained with Hematoxylin/Eosin and Masson Trichrome. Kidney samples were used for measurements of transforming growth factor (TGF)-β1 and its type 1 receptor (TGF-βR1), tumor necrosis factor (TNF)-α and interleukin (IL)-1β by ELISA. Gene expression NFκB was determined by real time-PCR. Kidney tissue apoptosis was assessed by measuring the activities of caspase-3/8/9. The renal protective effect of arjunolic acid was confirmed by approximately normal appearance of renal tissue and the relatively unaffected serum creatinine and urea levels. Furthermore, arjunolic acid showed dose dependent reduction in cisplatin-induced elevation in renal levels of TGF-βR1, TGF-β1, TNF-α, IL-1β and caspases. These findings demonstrated that arjunolic acid attenuates cisplatin nephrotoxicity either indirectly by enhancing body antioxidant activity or directly through several mechanisms, including inhibition of pro-inflammatory cytokines, blocking activation of TGF-β1, and anti-apoptotic effects.

Adenosine kinase inhibition protects the kidney against streptozotocin-induced diabetes through anti-inflammatory and anti-oxidant mechanisms.

Adenosine provides anti-inflammatory effects in cardiovascular disease via the activation of adenosine A2A receptors; however, the physiological effect of adenosine could be limited due to its phosphorylation by adenosine kinase. We hypothesized that inhibition of adenosine kinase exacerbates extracellular adenosine levels to reduce renal inflammation and injury in streptozotocin-induced diabetes. Diabetes was induced in male C57BL/6 mice by daily injection of streptozotocin (50mg/kg/day, i.p. for 5 days). Control and diabetic mice were then treated with the adenosine kinase inhibitor ABT702 (1.5mg/kg, i.p. two times a week for 8 weeks, n=7-8/group) or the vehicle (5% DMSO). ABT702 treatment reduced blood glucose level in diabetic mice (∼20%; P<0.05). ABT702 also reduced albuminuria and markers of glomerular injury, nephrinuria and podocalyxin excretion levels, in diabetic mice. Renal NADPH oxidase activity and urinary thiobarbituric acid reactive substances (TBARS) excretion, indices of oxidative stress, were also elevated in diabetic mice and ABT702 significantly reduced these changes. ABT702 increased renal endothelial nitric oxide synthase expression (eNOS) and nitrate/nitrite excretion levels in diabetic mice. In addition, the diabetic mice displayed an increase in renal macrophage infiltration, in association with increased renal NFκB activation. Importantly, treatment with ABT702 significantly reduced all these inflammatory parameters (P<0.05). Furthermore, ABT702 decreased glomerular permeability and inflammation and restored the decrease in glomerular occludin expression in vitro in high glucose treated human glomerular endothelial cells. Collectively, the results suggest that the reno-protective effects of ABT702 could be attributed to the reduction in renal inflammation and oxidative stress in diabetic mice.

The role of IL-18 in type 1 diabetic nephropathy: The problem and future treatment

Diabetic vascular complication is a leading cause of diabetic nephropathy, a progressive increase in urinary albumin excretion coupled with elevated blood pressure leading to declined glomerular filtration and eventually end stage renal failure. There is growing evidence that activated inflammation is contributing factor to the pathogenesis of diabetic nephropathy. Meanwhile, IL-18, a member of the IL-1 family of inflammatory cytokines, is involved in the development and progression of diabetic nephropathy. However, the benefits derived from the current therapeutics for diabetic nephropathy strategies still provide imperfect protection against renal progression. This imperfection points to the need for newer therapeutic agents that have potential to affect primary mechanisms contributing to the pathogenesis of diabetic nephropathy. Therefore, the recognition of IL-18 as significant pathogenic mediators in diabetic nephropathy leaves open the possibility of new potential therapeutic targets.