Meloxicam fails to augment the reno-protective effects of soluble epoxide hydrolase inhibition in streptozotocin-induced diabetic rats via increased 20-HETE levels.

The pro-inflammatory cyclooxygenase (COX)-derived prostaglandins and the anti-inflammatory cytochrome P450 epoxygenase-derived epoxyeicosatrienoic acids (EETs) play an important role in the regulation of renal injury. The current study examined whether COX inhibition augments the reno-protective effects of increased EETs levels via inhibiting EETs degradation by soluble epoxide hydrolase (sEH) in diabetic rats. Streptozotocin (50mg/kg, i.v) was used to induce diabetes in male Sprague Dawley rats. Rats were then divided into 5 groups (n=6-8); control non diabetic, diabetic, diabetic treated with the sEH inhibitor trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid (t-AUCB), diabetic treated with the COX inhibitor meloxicam and diabetic treated with meloxicam plus t-AUCB for 2 months. Glomerular albumin permeability and urinary albumin and nephrin excretion levels were significantly elevated in diabetic rats together with decreased glomerular α3 integrin and nephrin expression levels. Inhibition of sEH reduced glomerular albumin permeability, albumin and nephrin excretion levels and restored the decrease in glomerular α3 integrin and nephrin expression in diabetic rats. Meloxicam failed to reduce renal injury or even to synergize the reno-protective effects of sEH inhibition in diabetic rats. Furthermore, inhibition of sEH reduced the elevation in renal collagen deposition and urinary MCP-1 excretion levels together with a reduction in the number of renal TUNEL positive cells in diabetic vs. control rats (P<0.05). Meloxicam did not reduce renal inflammation or apoptosis in diabetic rats or even exacerbate the anti-inflammatory and anti-apoptotic effects of sEH inhibition. Renal 20-hydroxyeicosatetranoic acid (20-HETE) levels were elevated in diabetic rats and meloxicam further exacerbated this elevation. In conclusion, our study suggests that inhibition of COX failed to provide renal protection or to augment the reno-protective effects of sEH inhibition in diabetic rats, at least in part, via increased inflammatory 20-HETE levels.

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.

Role of haem oxygenase in the renoprotective effects of soluble epoxide hydrolase inhibition in diabetic spontaneously hypertensive rats.

We have shown previously that inhibition of sEH (soluble epoxide hydrolase) increased EETs (epoxyeicosatrienoic acids) levels and reduced renal injury in diabetic mice and these changes were associated with induction of HO (haem oxygenase)-1. The present study determines whether the inhibition of HO negates the renoprotective effect of sEH inhibition in diabetic SHR (spontaneously hypertensive rats). After 6 weeks of induction of diabetes with streptozotocin, SHR were divided into the following groups: untreated, treated with the sEH inhibitor t-AUCB {trans-4-[4-(3-adamantan-1-yl-ureido)-cyclohexyloxy]-benzoic acid}, treated with the HO inhibitor SnMP (stannous mesoporphyrin), and treated with both inhibitors for 4 more weeks; non-diabetic SHR served as a control group. Induction of diabetes significantly increased renal sEH expression and decreased the renal EETs/DHETEs (dihydroxyeicosatrienoic acid) ratio without affecting HO-1 activity or expression in SHR. Inhibition of sEH with t-AUCB increased the renal EETs/DHETEs ratio and HO-1 activity in diabetic SHR; however, it did not significantly alter systolic blood pressure. Treatment of diabetic SHR with t-AUCB significantly reduced the elevation in urinary albumin and nephrin excretion, whereas co-administration of the HO inhibitor SnMP with t-AUCB prevented these changes. Immunohistochemical analysis revealed elevations in renal fibrosis as indicated by increased renal TGF-ß (transforming growth factor ß) levels and fibronectin expression in diabetic SHR and these changes were reduced with sEH inhibition. Co-administration of SnMP with t-AUCB prevented its ability to reduce renal fibrosis in diabetic SHR. In addition, SnMP treatment also prevented t-AUCB-induced decreases in renal macrophage infiltration, IL-17 expression and MCP-1 levels in diabetic SHR. These findings suggest that HO-1 induction is involved in the protective effect of sEH inhibition against diabetic renal injury.

Novel molecular triggers underlie valproate-induced liver injury and its alleviation by the omega-3 fatty acid DHA: role of inflammation and apoptosis.

BACKGROUND/AIM: Hepatic injury is a hallmark adverse reaction to Valproate (VPA), a common used drug in the management of numerous CNS disorders, including epilepsy. DHA has a myriad of health benefits, including renal- and hepato-protective effects. Unfortunately, however, the underpinnings of such liver-pertinent VPA- and DHA-actions remain largely undefined. Accordingly, this study attempted to unveil the cellular and molecular triggers whereby VPA evokes, while DHA abates, hepatotoxicity. METHODS: We evaluated activity and/or expression of cellular markers of oxidative stress, inflammation, and apoptosis in rat liver, following treatment with VPA (500 mg/kg/day) with and without concurrent treatment with DHA (250 mg/kg/day) for two weeks. RESULTS AND CONCLUSION: VPA promoted hepatic oxidative stress as evidenced by enhancing activity/expression of NADPH-oxidase and its subunits, a ROS-generator, and by accumulation of lipid-peroxides. Moreover, VPA enhanced hepatic phosphorylation/activation of mitogen-activated protein kinase (MAPK), and expression of cyclooxygenase-2(COX-2), as proinflammatory signals. Besides, VPA promoted hepatocellular apoptosis, as attested by enhanced expression of cleaved caspase-9 and increased number of TUNEL-positive hepatocytes. Lastly, VPA upregulated levels of hypoxia-inducible factor-1-alpha (HIF-1α), a multifaceted modulator of hepatocytic biology, and activity of its downstream antioxidant enzyme heme-oxygenase-1(HO-1). These changes were significantly blunted by co-administration of DHA. Our findings demonstrate that VPA activated NADPH-oxidase and HIF-1α to induce oxidative-stress and hypoxia as initiators of hepatic injury. These changes were further aggravated by up-regulation of inflammatory (MAPK and COX-2) and apoptotic cascades, but could be partly lessened by HO-1 activation. Concurrent administration of DHA mitigated all VPA-induced anomalies.

Inhibition of 12/15-Lipoxygenase Reduces Renal Inflammation and Injury in Streptozotocin-Induced Diabetic Mice.

Previous studies suggest that 12/15 lipoxygenase (12/15-LO) is implicated in diabetic vascular complications. We hypothesize that 12/15-LO inhibition attenuates renal inflammation and injury in streptozotocin-induced diabetes. Diabetes was induced in wild-type C57BL/6J (WT) and 12/15-LO deficient mice using streptozotocin. Additionally, four groups of WT mice were also used; control non diabetic, diabetic, diabetic treated with the 12/15-LO inhibitor baicalein for 10 weeks and diabetic treated with baicalein only for the last 4 weeks of the experiment. After 10 weeks of induction of diabetes with streptozotocin, WT diabetic mice exhibited marked elevation in proteinuria together with elevation in the excretion levels of thiobarbituric acid reactive substance (TBARs), a marker of oxidative stress, and monocyte chemoattractant protein-1 (MCP-1), a marker of inflammation and these changes were significantly reduced in 12/15-LO deficient diabetic mice (P<0.05). Similarly, pharmacological inhibition of 12/15-LO with baicalein prevented the elevation in renal 12-HETE production, the major murine metabolic product of 12/15-LO, in diabetic mice, and this effect was associated with decreased proteinuria, TBARs excretion and renal collagen deposition compared to untreated diabetic mice. Interestingly, the protective effects of baicalein were not noticed when only administered in the last 4 weeks of diabetes compared to untreated diabetic mice. WT diabetic mice displayed elevation in renal interleukin-6 (IL-6) levels and these changes were only reduced in diabetic mice treated with baicalein for 10 weeks (P<0.05). The anti-inflammatory effects of baicalein or 12/15-LO deficiency were further confirmed in lipopolysaccharide (LPS)-induced acute renal inflammation as inhibition of 12/15-LO reduced the elevation in renal soluble epoxide hydrolase expression in LPS-injected mice. These results suggest that increased 12/15-LO activity and 12-HETE production contribute to the elevation of renal oxidative stress, inflammation and injury in streptozotocin-induced diabetic mice.

Valproate-induced liver injury: modulation by the omega-3 fatty acid DHA proposes a novel anticonvulsant regimen.

BACKGROUND: The polyunsaturated, ω-3 fatty acid, docosahexaenoic acid (DHA), claims diverse cytoprotective potentials, although via largely undefined triggers. Thus, we currently first tested the ability of DHA to ameliorate valproate (VPA)-evoked hepatotoxicity, to modulate its anticonvulsant effects, then sought the cellular and molecular basis of such actions. Lastly, we also verified whether DHA may kinetically alter plasma levels/clearance rate of VPA. METHODS AND RESULTS: VPA (500 mg/kg orally for 14 days in rats) evoked prominent hepatotoxicity that appeared as a marked rise (2- to 4-fold) in serum hepatic enzymes (γ-glutamyl transferase [γ-GT], alanine aminotransferase [ALT], and alkaline phosphatase [ALP]), increased hepatic lipid peroxide (LPO) and tumor necrosis factor-alpha (TNFα) levels, as well as myeloperoxidase (MPO) activity (3- to 5-fold), lowering of serum albumin (40 %), and depletion of liver reduced-glutathione (GSH, 35 %). Likewise, histopathologic examination revealed hepatocellular degeneration, replacement by inflammatory cells, focal pericentral necrosis, and micro/macrovesicular steatosis. Concurrent treatment with DHA (250 mg/kg) markedly blunted the elevated levels of liver enzymes, lipid peroxides, TNFα, and MPO activity, while raising serum albumin and hepatic GSH levels. DHA also alleviated most of the cytologic insults linked to VPA. Besides, in a pentylenetetrazole (PTZ) mouse convulsion model, DHA (250 mg/kg) markedly increased the latency in convulsion evoked by VPA, beyond their individual responses. Lastly, pharmacokinetic studies revealed that joint DHA administration did not alter serum VPA concentrations. CONCLUSIONS: DHA substantially ameliorated liver injury induced by VPA, while also markedly boosted its pharmacologic effects. DHA manipulated definite cellular machinery to curb liver oxidative stress and inflammation, without affecting VPA plasma levels. Collectively, these protective and synergy profiles for DHA propose a superior VPA-drug combination regimen.