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Inducible NOS inhibition, eicosapentaenoic acid supplementation, and angiotensin II-induced renal damage
Abstract
Cytochrome P450(CYP)-dependent hydroxylation and epoxygenation metabolites of arachidonic acid (AA) influence renal vascular tone, salt excretion, and inflammation. Transgenic rats over expressing both human renin and angiotensinogen genes (dTGR) feature angiotensin II (Ang II)-induced organ damage, increased expression of inducible nitric oxide synthase (iNOS), decreased AA hydroxylation, and epoxygenation. As nitric oxide production via iNOS can inhibit CYP AA metabolism, we tested the hypothesis that by blocking iNOS or by supplementing eicosapentanoic acid (EPA), which can serve as an alternative CYP substrate, Ang II-induced vasculopathy could be ameliorated.
We treated dTGR with the iNOS inhibitor L-N(6)-(1-iminoethyl) lysine (L-NIL), EPA, and the combination of both treatments from week 4 to 7.
Immunohistochemistry showed that L-NIL and EPA reduced glomerular iNOS toward control levels. L-NIL-treated dTGR showed cardiac hypertrophy and albuminuria similar to untreated dTGR. EPA and the combination of EPA + L-NIL, ameliorated organ damage without lowering blood pressure. EPA and EPA + L-NIL reduced cardiac hypertrophy, albuminuria, renal fibronectin expression, and infiltration of monocytes/macrophages, compared to L-NIL and untreated dTGR. Reactive oxygen species were detected in glomeruli of untreated and L-NIL-treated dTGR, but was reduced in the EPA groups. EPA treatment reduced activator protein-1 (AP-1) activation and partially inhibited nuclear factor-kappaB (NF-kappaB) activity in kidneys of dTGR.
These results demonstrate that iNOS inhibition does not protect against Ang II-induced end-organ damage, while EPA treatment does. Our electromobility shift assay experiments revealed that EPA protection may involve inhibition of AP-1- and NF-kappaB-dependent pathways.
... 20 years ago, it was shown for the first time that hepatic and renal microsomes are able to metabolize EPA and DHA to ω-, and ω-1 hydroxy metabolites as well as to epoxides, analogue to CYP-dependent AA metabolism (Van Rollins et al., 1984;Van Rollins et al., 1989). There were a number of following studies, which confirmed that ω-3 PUFA are metabolized by CYPs in animals and human (Van Rollins et al., 1988;Theuer et al., 2005;Oliw and Sprecher, 1991;Knapp et al., 1991). ...
... Reactive oxygen species or reactive metabolic products can attack the P450 heme and therefore damage the enzyme, which is a signal for enzyme rapid degradation Correia et al., 2005). Reactive oxygen species were detected in glomeruli of dTGR animals (Theuer et al., 2005) but there is no evidence that it triggers proteasomal degradation of CYPs. Nitric oxide (NO) can bind to prostethic heme group of CYP and inhibit its enzymatic activity. ...
... • presumably also to accumulation of 20-HETE levels because of a lower rate of CYP2C-catalyzed 20-HETE epoxidation. , Oliw and Sprecher, 1991, Theuer et al., 2005. Knapp et al. (1991) found that humans ingesting ω-3 fatty acids excrete via urine substantial quantities of vicinal diols of EPA that result from CYP-catalyzed EPA epoxidation and subsequent hydrolysis by epoxide hydrolase. ...
... EPA has many effects, including antithrombotic, antihyperlipidemic (9), antiatherogenic (12), and anti-inflammatory actions (50). Recent studies have reported that EPA slows disease progression in human and experimental renal diseases including diabetic nephropathy (7,10,15,16,19,36,48,49). The mechanism by which EPA ameliorates glomerular lesions is thought to involve the inhibition of thromboxan A 2 (15), thromboxan B (49), activating protein-1 (48), monocyte chemoattractant protein-1 (19), mitogen-activated protein kinase/extracellular signal-regulated kinase signaling (19), prostaglandin E (36,49), and prostaglandin I (49). ...
... Recent studies have reported that EPA slows disease progression in human and experimental renal diseases including diabetic nephropathy (7,10,15,16,19,36,48,49). The mechanism by which EPA ameliorates glomerular lesions is thought to involve the inhibition of thromboxan A 2 (15), thromboxan B (49), activating protein-1 (48), monocyte chemoattractant protein-1 (19), mitogen-activated protein kinase/extracellular signal-regulated kinase signaling (19), prostaglandin E (36,49), and prostaglandin I (49). However, little is known about the ability of EPA to protect against diabetic tubular damage, especially its effects on the molecular mechanisms involved in oxidative stress and apoptosis. ...
The present study was designed to elucidate a possible mechanism of hyperglycemia-induced tubular injury and to examine a therapeutic potential of dietary eicosapentaenoic acid (EPA) for the prevention of diabetic kidney disease. Utilizing streptozotocin-induced diabetic mice, the extents of albuminuria and histological injuries were monitored at 2 wk after diabetic induction. Reactive oxygen species (ROS) production, apoptosis, and hypoxia in the kidney were evaluated by immunohistochemistry and Western blotting. An in vitro study was performed using rat proximal tubular cells (NRK-52E) to confirm the protective effect of EPA for methylglyoxal (MG)-induced ROS generation and staurosporine (STS)-induced mitochondrial apoptosis. The extents of albuminuria and histological tubular injuries were significantly lower in EPA-treated diabetic mice compared with untreated diabetic mice. The levels of lipid peroxidation product (4-hydroxy-2-nonenal), oxidative DNA damage (8-hydoxy-deoxyguanosine), and mitochondrial apoptosis (TUNEL, caspase-9, cleaved caspase-3, and cytochrome c release) in the tubular cells were also significantly lower in EPA-treated diabetic mice. Furthermore, hypoxia-inducible factor (HIF)-1α expression was significantly upregulated in the kidney tissues from EPA-treated mice compared with untreated diabetic mice. MG-induced ROS overproduction and STS-induced mitochondrial apoptosis in NRK-52E cells were significantly reduced by EPA treatment in vitro. These results indicated that the ROS generation and mitochondrial apoptosis were involved in hyperglycemia-induced tubular injury and EPA had a beneficial effect by suppressing ROS generation and mitochondrial apoptosis partly through augmentation of an HIF-1α response in diabetic kidney disease.
... 158 Renal arachidonate epoxygenase activity and protein levels of CYP2C11, CYP2C23, and CYP2J are reduced significantly in this animal model. [158][159][160] Importantly, treatment with fenofibrate increases epoxygenase expression, normalizes blood pressure and albuminuria, reduces nuclear factor B activity, and renal leukocyte infiltration in these rats, suggesting that hypertension and renal damage in this rat model is associated with down-regulation of P450 epoxygenase-dependent arachidonic acid metabolism. [158][159][160] Increased EET formation has been reported in the kidney of rats with liver cirrhosis. ...
... [158][159][160] Importantly, treatment with fenofibrate increases epoxygenase expression, normalizes blood pressure and albuminuria, reduces nuclear factor B activity, and renal leukocyte infiltration in these rats, suggesting that hypertension and renal damage in this rat model is associated with down-regulation of P450 epoxygenase-dependent arachidonic acid metabolism. [158][159][160] Increased EET formation has been reported in the kidney of rats with liver cirrhosis. 161 Although it is well documented that renal vasoconstriction leading to impaired renal function occurs during cirrhosis, this result suggests that increased EET synthesis may be a homeostatic response to preserve renal perfusion. ...
Small lipids such as eicosanoids exert diverse and complex functions. In addition to their role in regulating normal kidney function, these lipids also play important roles in the pathogenesis of kidney diseases. Increased glomerular cyclooxygenase (COX)1 or COX2 expression has been reported in patients with nephritis and in animal models of nephritis. COX inhibitors have shown beneficial effects on lupus nephritis and passive Heymann nephritis, but not anti-Thy1.1-induced nephritis. 5-Lipoxygenase-derived leukotrienes are involved in inflammatory glomerular injury. Lipoxygenase product 12-hydroxyeicosatetraenoic acid may mediate angiotensin II and transforming growth factor beta-induced mesangial cell abnormality in diabetic nephropathy. P450 arachidonic acid mono-oxygenase-derived 20-hydroxyeicosatetraenoic acid and epoxyeicosatrienoic acids are involved in several forms of kidney injury, including renal injury in metabolic syndrome. Ceramide also has been shown to be an important signaling molecule that is involved in the pathogenesis of acute kidney injury caused by ischemia/reperfusion and toxic insults. Those pathways should provide fruitful targets for intervention in the pharmacologic treatment of renal disease.
... ** P < 0.01 or *** P < 0.001 compared to cells treated with 5 mg/mL Alb. [21]. In colon cancer cells, EPA as Free Fatty Acid (EPA-FFA) treatment counteracted NOTCH1 activation stimulated by inflammation [22]. ...
Background:
It was reported that eicosapentaenoic acid (EPA) could prevent tubulointerstitial injury in kidney. EPA could also inhibit the epithelial-mesenchymal transition (EMT) of HK-2 cells stimulated by albumin (Alb) in vitro. However, the regulating molecular mechanism of EPA remains to be elucidated.
Methods:
An immortalized human proximal tubular cell line (human kidney-2 (HK-2) cells) was used in all experiments. MTT assay was employed to determine the effect of Alb or EPA on the cell viability of HK-2 cells. The miR-541 expression, the mRNA levels of EMT markers E-cadherin, α-smooth muscle actin (α-SMA), and fibrogenesis markers Collagen I and fibronectin (FN) were examined by RT-qPCR assay. The protein levels of E-cadherin, α-SMA and Collagen I, transforming growth factor β1 (TGF-β1)/Smad3/integrin-linked kinase (ILK) pathway-related protein TGF-β1, pSmad2/3, Smad7 and ILK were measured by western blot. Enzyme-linked immunosorbent assay (ELISA) was performed to detect FN expression. The target relationship between miR-541 and TGF-β1 was confirmed by bioinformatics, luciferase reporter assay and western blot.
Results:
Low doses of Alb had no effect on the cell viability of HK-2 cells, while EPA repressed the cell viability of HK-2 cells in a concentration-dependent manner. EPA could inhibit EMT and fibrosis and increase the miR-541 expression of HK-2 cells exposed to Alb. Interestingly, introduction of miR-541 effectively abolished the EMT and fibrosis of HK-2 cells stimulated by Alb. Bioinformatics analysis predicted TGF-β1 as a target gene of miR-541, and subsequent luciferase reporter assay and western blot further supported the prediction. miR-541 counter-regulated TGF-β1 expression, and inhibited the TGF-β1/Smad3/ILK pathway. Alb treatment activated the TGF-β1/Smad3/ILK pathway, while EPA inhibited the activation of the pathway. miR-541 inhibitors reversed the effects of EPA on EMT, fibrosis, and TGF-β1/Smad3/ILK pathway-related protein expression induced by Alb.
Conclusion:
EPA attenuates EMT and renal fibrosis through the TGF-β1/Smad3/ILK pathway in renal epithelial cells by targeting miR-541.
... Ovariectomy did not change NO levels in the present study; however, its levels were reduced by supplementation with LA and EPA. Corroborating these findings, previous studies reported that LA [52], EPA and DHA [53,54] have the ability to inhibit NO synthesis by nitric oxide synthase. ...
Background
Bilateral ovariectomy is an experimental model used to analyse the effects of menopause and develop strategies to mitigate the deleterious effects of this condition. Supplementation of the diet with antioxidants has been used to reduce potential oxidative stress caused by menopause. The purpose of the study was to analyse the effects of alpha-lipoic acid (LA), docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), dietary supplementation on oxidative stress in the livers of ovariectomized rats.
Methods
In this study, we evaluated the effect of dietary supplementation with LA, DHA and EPA for a period of 16 weeks on oestrogen levels and oxidative stress biomarkers in the livers of ovariectomized 25 three-month-old rats.
Results
Serum oestrogen levels were lower after ovariectomy but were not altered by dietary treatments. LA was capable of acting in the liver, recovering the activity of the antioxidant enzymes superoxide dismutase and glutathione peroxidase, and reducing protein oxidative damage. Moreover, LA supplementation reduced nitrite and nitrate levels. DHA and EPA recovered the antioxidant activity of cytosolic and mitochondrial superoxide dismutase, decreasing protein oxidation. Protection against lipid oxidation differed between treatments. The DHA-treated group showed increased levels of the lipid peroxidation biomarker malondialdehyde compared to the ovariectomized group. However, malondialdehyde levels were not altered by EPA treatment.
Conclusions
The results suggest that the antioxidant response varies among evaluated supplementations and all supplements were able to alter enzymatic and non-enzymatic antioxidants in the livers of ovariectomized rats. DHA presented the most evident antioxidant effect, decreasing protein and lipid damage.
... Although there is evidence of vitamin D deficiency affecting CVD, the mechanisms underlying how vitamin D protects the cardiovascular system are unclear. In vitro and clinical studies suggest that vitamin D receptor activation leads to downregulation of the renin-angiotensin system, inflammation inhibition, smooth muscle proliferation suppression, and vascular calcification [61][62][63]. Vitamin D receptor knockout mice develop hypertension and cardiac hypertrophy [64]. Epidemiologic studies have reported that vitamin D deficiency is associated with cardiovascular events in subjects with renal dysfunction and even in the general population [65,66]. ...
The prevalence rate of chronic kidney disease (CKD) is increasing worldwide, and cardiovascular disease (CVD) is a main cause of death in patients with CKD. The high incidence of CVD in CKD patients is related to chronic inflammation, dyslipidemia, malnutrition, atherosclerosis, and vascular calcification. Omega-3 polyunsaturated fatty acids ( ω -3 PUFAs) have been shown to reduce the risk of CVD. In this paper, we review the beneficial effects of ω -3 PUFAs on CVD and the possible cardioprotective mechanisms of ω -3 PUFAs in CKD patients by determining the effect of ω -3 PUFAs in the general population. ω -3 PUFAs have several cardioprotective benefits, such as reducing inflammation, decreasing oxidative stress, inhibiting platelet activity, exerting antiarrhythmic effects, and improving triglyceride levels, in the general population and patients with CKD. Modifications of erythrocyte membrane fatty acid content, including an increased ω -3 index and decreased oleic acid, after ω -3 PUFAs supplementation are important changes related to CVD risk reduction in the general population and patients with CKD. Further basic and clinical studies are essential to confirm the effects of ω -3 PUFAs on vitamin D activation, vascular calcification prevention, cardiovascular events, and mortality in CKD patients.
... Finally, Ng demonstrated a beneficial effect of fish oil therapy on human post-transplantation IgA nephropathy (53), while Tsipas reported a positive role of a balanced n-6/n-3 PUFA diet in cyclosporinemodel nephrotoxicity (54). Recently, it has been shown in transgenic rats that EPA treatment protected against angiotensin II (Ang II)-induced renal damage, by the inhibition of the activating protein-1 (AP-1)and NF-kB-dependent pathways (55). The mechanism through which PUFA exert their protective effects on nephropathy progression is unclear. ...
The role of polyunsaturated fatty acids in renal fibrosis. Several studies suggest a close relationship between polyunsaturated fatty acids (PUFA) and renal inflammation and fibrosis, which are crucial stages in chronic kidney disease (CKD). Beneficial effects of n-3 PUFA on the course of experimental and human nephropathies have been reported. PUFA can ameliorate chronic, progressive renal injury beyond the simple reduction of serum lipid levels. These pleiotropic effects of PUFA are due to their properties of interfering with the synthesis of a variety of inflammatory factors and events, through effects related both to the modulation of the balance of n-6 and n-3-derived eicosanoids and to direct action on the cellular production of the major cytokine mediators of inflammation and on endothelium function. The mechanisms by which PUFA can favorably interfere with some stages in renal fibrosis processes, such as mesangial cell activation and proliferation and extracellular matrix protein synthesis, include the regulation of some pro-inflammatory cytokine production, renin and nitric oxide (NO) systems and peroxisome proliferator-activated receptor gene expression. An optimal n-6/n-3 PUFA ratio dietary intake could offer new therapeutic strategies aimed at interrupting the irreversible process of renal fibrosis and ameliorating chronic renal injury. However, further experimental, epidemiological and clinical investigations are needed to confirm the role of PUFA in the renal fibrosis pathway and the natural history of chronic nephropathies.
... Several studies have shown a decrease in NF-jB activation by DHA and EPA [39][40][41][42][43][44]. However, Maziere et al. [45] observed an increase of NFjB activation in fibroblasts treated with DHA and EPA and Camandola et al. [46] stated that EPA (45 lM) exerts no effect on the nuclear translocation of NF-jB in the human promonocytic cell line U937. ...
Modulation of macrophage functions by fatty acids (FA) has been studied by several groups, but the effect of FA on nitric oxide production by macrophages has been poorly examined. In the present study the effect of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on NF-kappaB activity and NO production in J774 cells (a murine macrophage cell line) was investigated. All FA tested stimulated NO production at low doses (1-10 microM) and inhibited it at high doses (50-200 microM). An increase of iNOS expression and activity in J774 cells treated with a low concentration of FA (5 microM) was observed. The activity of NF-kappaB was time-dependently enhanced by the FA treatment. The inhibitory effect of FA on NO production may be due to their cytotoxicity, as observed by loss of membrane integrity and/or increase of DNA fragmentation in cells treated for 48 h with high concentrations. The results indicate that, at low concentrations FA increase NO production by J774 cells, whereas at high concentrations they cause cell death.
... 13 In our dTGR model, chronic n-3 PUFA treatment lowered blood pressure and improved renal and cardiac damage. 14 We now investigated the impact of n-3 PUFAs and DRI on cardiac electrophysiological remodeling in the dTGR, a model of high human renin hypertension. ...
We compared the effect n-3 polyunsaturated fatty acids (PUFAs) with direct renin inhibition on electrophysiological remodeling in angiotensin II-induced cardiac injury. We treated double-transgenic rats expressing the human renin and angiotensinogen genes (dTGRs) from week 4 to 7 with n-3 PUFA ethyl-esters (Omacor; 25-g/kg diet) or a direct renin inhibitor (aliskiren; 3 mg/kg per day). Sprague-Dawley rats were controls. We performed electrocardiographic, magnetocardiographic, and programmed electrical stimulation. Dietary n-3 PUFAs increased the cardiac content of eicosapentaenoic and docosahexaenoic acid. At week 7, mortality in dTGRs was 31%, whereas none of the n-3 PUFA- or aliskiren-treated dTGRs died. Systolic blood pressure was modestly reduced in n-3 PUFA-treated (180+/-3 mm Hg) compared with dTGRs (208+/-5 mm Hg). Aliskiren-treated dTGRs and Sprague-Dawley rats were normotensive (110+/-3 and 119+/-6 mm Hg, respectively). Both n-3 PUFA-treated and untreated dTGRs showed cardiac hypertrophy and increased atrial natriuretic peptide levels. Prolonged QRS and QT(c) intervals and increased T-wave dispersion in dTGRs were reduced by n-3 PUFAs or aliskiren. Both treatments reduced arrhythmia induction from 75% in dTGRs to 17% versus 0% in Sprague-Dawley rats. Macrophage infiltration and fibrosis were reduced by n-3 PUFAs and aliskiren. Connexin 43, a mediator of intermyocyte conduction, was redistributed to the lateral cell membranes in dTGRs. n-3 PUFAs and aliskiren restored normal localization to the intercalated disks. Thus, n-3 PUFAs and aliskiren improved electrical remodeling, arrhythmia induction, and connexin 43 expression, despite a 70-mm Hg difference in blood pressure and the development of cardiac hypertrophy.
Background:
Ulcerative colitis (UC) is a chronic mucosal inflammation of the large intestine that mostly affects the rectum and colon. The absence of safe and effective therapeutic agents encourages the discovery of novel therapeutic agents to effectively treat UC and its complications. The purpose of this research was to examine the protective impact of Eicosapentaenoic acid (EPA) in rats with UC induced by acetic acid (AA).
Method:
AA (2 ml, 3 % v/v) was injected intrarectally to cause UC. Before administering AA, EPA (300 and 1000 mg/kg) was given orally for 28 days.
Results:
EPA inhibited AA-induced UC by enhancing colonic histopathological changes like inflammation, goblet cell loss, glandular hyperplasia and mucosal ulceration, concomitant with a reduction in colon weight, colon weight/length ratio, C-reactive protein (CRP), and serum lactate dehydrogenase (LDH). EPA also effectively restored the imbalance between oxidants and antioxidants caused by AA. In addition, EPA increased the levels of trefoil factor-3 (TFF-3) and glucagon-like peptide-1 (GLP-1), while significantly reducing the expression of nuclear factor kappa B (NF-κB), interferon-γ (IFN-γ), and interleukin-6 (IL-6), transforming growth factor-1(TGF-β1), and phosphorylated epidermal growth factor receptor (P-EGFR), phosphatidylinositol-3-kinase (PI3K) and protein kinase B (AKT) expression in colonic tissues.
Conclusion:
EPA inhibited AA-induced UC in rats by modulating the TGF-β/P-EGFR and NF-κB inflammatory pathways, regulating the oxidant/antioxidant balance, and enhancing the colon barrier integrity.
Objective
Omega-3 fatty acids may reduce albuminuria and cardiovascular risk factors in patients with chronic kidney disease (CKD). We aimed to assess the effects of omega-3 fatty acid supplementation on albuminuria, blood pressure, pulse wave velocity, and inflammatory markers in patients with CKD.
Methods
Patients with CKD and a urine albumin excretion of at least 30 mg/g creatinine were supplemented for 3 months with 3,666 mg/day of docosahexaenoic and eicosapentaenoic acids or a corn oil supplement. The study was double blind. At baseline, 6 weeks, and 12 weeks, fasting blood and morning spot urine samples were obtained. Blood pressure, carotid intima media thickness, and pulse wave velocity were measured. The main outcome measure was a reduction of ≥20% in urine albumin.
Results
One hundred patients were randomized (50 received omega-3 fatty acids and 50 received corn oil). Four patients who received omega-3 fatty acids and 5 who received vegetable oil were lost to follow-up. In patients receiving omega-3 fatty acids, the omega-3 index increased from 3.08 (2.32-3.81) to 5.48 (3.045-7.04) percent. A 20% reduction in urine albumin excretion was observed in 13 participants of the control group and 19 participants of omega-3 group (Fisher’s exact P = .274). However, the supplement had a significant and positive effect on pulse wave velocity and triglyceride level.
Conclusion
An omega-3 fatty acid supplement of 3,666 mg/day did not modify urine albumin excretion in patients with CKD but did improve pulse wave velocity and serum triglyceride levels.
Toll‐like receptors (TLRs) induce an innate immune system. In general, there are two main pathways in TLRs: the myeloid differentiation primary response protein 88 (MyD88)‐dependent and toll‐interleukin‐1 receptor domain‐containing adapter‐inducing interferon‐β (TRIF)‐dependent pathways. In this study, it has been investigated whether eicosapentaenoic acid (EPA), a polyunsaturated fatty acid (PUFA), and arachidic acid (ACA), a saturated fatty acid (SFA), can modulate the TLR signaling pathways. EPA suppressed the activation of interferon regulatory factor 3 (IRF3) and the expression of Interferon gamma‐induced protein (IP‐10) induced by Toll‐like receptor 3 (TLR3) or TLR4 agonists by targeting TANK‐binding kinase 1 (TBK1); however, ACA did not. These results demonstrate that EPA inhibits the TRIF‐dependent signaling in the TLR3 and TLR4 pathways. The results raise the possibility that certain dietary PUFAs can modulate TLR‐derived signaling and inflammatory target gene expression and can alter the susceptibility to microbial infection and chronic inflammatory diseases.
Practical application
Eicosapentaenoic acid (EPA) is a bioactive lipid that modulates inflammation and immunity. TLRs play a central role as initiators of the innate immune responses. EPA regulates TRIF‐dependent pathways of TLRs by targeting TBK1. EPA may be a useful strategy to understand the mechanism of antiinflammatory activities.
Melatonin, a potent antioxidant molecule plays a role in blood pressure regulation. We hypothesized that melatonin may generate a protective effect in a high salt diet (HSD) rodent model mediated by decreasing renal oxidative stress. Dahl salt sensitive rats were divided into 3 groups according to diet: normal chow [control; HSD; HSD with melatonin (30/mg/kg/day) placed in their water (HSD+Mel) over an 8- week period. Blood pressure was measured by the tail cuff method. Kidney injury was evaluated by 24H urine protein excretion. Glomerular injury index (GII) (fibrotic glomeruli/100 glomeruli) was evaluated from a Masson's trichrom stained section. Kidney oxidative stress was determined by superoxide production via dihydroethidium staining. Expression of oxidative stress related genes was measured by RT-qPCR. Melatonin had no effect on blood pressure increase induced by HSD and attenuated proteinuria induced by HSD (HSD- 50.7±12, HSD+Mel- 22.3±4.3, controls- 6.5±1.0 gram protein/gram creatinine, p<0.001). HSD induced glomerular damage was significantly diminished by melatonin (GII in HSD -24±6, HSD+Mel-3.6±0.8, controls- 0.8±0.5. p<0.05). Superoxide production was significantly higher in kidneys of HSD fed rats than the controls (99±9 vs. 60±7 relative fluorescent units (RFU)/μm(2) , respectively, p<0.05). Melatonin also decreased superoxide production (74±5 RFU/μm(2) , p<0.05). The expression of kidney iNOS and p67(phox) mRNA was significantly higher in HSD than in the controls and HSD+Mel rats. Treatment with melatonin eliminated the deleterious effect of HSD in the kidneys of Dahl salt sensitive rats. The beneficial effect of melatonin is not mediated by lowering BP but by a direct anti-oxidative effect. This article is protected by copyright. All rights reserved.
Epidemiological and experimental studies have established the beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) on cancer. In particular, wide information is available regarding their effects on hormone responsive tumors, such as prostatic and mammary cancer, and tumors originating from colon. Recent studies have focused upon the improvement of chemotherapy effects when different drug treatments are accompanied by n-3 PUFA administration. The growth inhibitory action elicited by these fatty acids on tumors seems to be related to their ability to induce apoptosis and cell cycle arrest in cancer cells and to inhibit neo-angiogenesis. Different molecular mechanisms have been hypothesized to explain their anticancer effects, and this field is receiving increased attention. For a long time n-3 PUFAs have been considered to function only as prooxidant agents, competitors for arachidonic acid metabolism, or modifiers of membrane microenvironment and fluidity in cells. However, more recently, they have been shown to act as transcription regulators, being able to modulate the activity of different transcription factors, including NFkB, peroxisome proliferator-activated receptors, retinoid X receptors, and HIF-1. The expression of a wide array of genes involved in the regulation of cell proliferation, apoptosis, neo-angiogenesis and invasion of tumors have been shown to be modulated by n-3 PUFAs (proteins of BCL-2 family, cyclins and cyclin dependent kinase inhibitors, protein-kinases and phosphatases, COX-2, 5- LOX, VEGF and matrix-metalloproteinases). The modulating action brought about by n-3 PUFAs on the expression of lipid metabolism enzymes, such as fatty acids synthase and 3-hydroxy-3-methyl-glutaryl-CoA (HMG)-CoA reductase, have been recently involved in the anticarcinogenic action of these fatty acids. Telomerases, DNA topoisomerases and DNA polymerases are further molecular targets critical in the growth and survival of cancer cells recently observed to be modulated by n-3 PUFAs. The aim of this review is to examine the molecular mechanisms invoked so far in order to explain the anticancer effects of n-3 PUFAs. The definitive comprehension of the molecular mechanisms involved appears to be crucial to establish the appropriateness of n-3 PUFAs as chemopreventive or adjuvant therapeutic agents in human cancer.
Diabetic nephropathy is the leading cause of endstage renal disease, with both the incidence and prevalence continuing to increase worldwide. Current treatments include optimization of glycemic and blood pressure control by targeting the renin-angiotensin-aldosterone system (RAAS) with angiotensin-converting enzyme (ACE) inhibitors and/or angiotensin II receptor blockers. More innovative strategies are needed to prevent and treat this disease. New agents and approaches have recently been described that have the potential to delay the progression of diabetic kidney disease and minimize the growing burden of endstage renal disease. Possible targets include the formation of advanced glycation end products (AGEs) and the AGE receptor, increased oxidative stress and inflammation, protein kinase C, endothelin receptors, growth factors and cytokines, the vitamin D receptor, Rho-associated kinases, and the renal sympathetic system. This article reviews these recent developments as potential therapeutic interventions that may prevent this disease, with targets generally beyond the RAAS.
Fish oil containing n-3 polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) is well known to prevent the progression of IgA nephropathy. However, the mechanism through which fish oil prevents the progression of renal injury remains uncertain. We tried to clarify the effects of EPA on tubulointerstitial injury in the kidney both in vivo and in vitro. We examined the effects of EPA, especially to focus on nuclear factor kappa B (NF-κB), using Thy-1 nephritis models. Also the mechanism of EPA was investigated using small-interfering RNA (siRNA) in lipopolysaccharide (LPS)-stimulated proximal tubular epithelial cells (PTECs). In Thy-1 nephritis models, EPA significantly inhibited tubulointerstitial injury and the infiltration of macrophages into tubulointerstitial lesions except severe glomerular injury at early stage. Compared with control animals, NF-κB activation was significantly augmented in the Thy-1 nephritic kidney. However, treatment with EPA significantly reduced NF-κB activation, down-regulated the expressions of NF-κB-dependent molecules. Also in LPS-stimulated PTECs, LPS augmented NF-κB activation and the expression of NF-κB-dependent molecules. As in the case with the Thy-1 nephritis models, treatment with EPA inhibited them, prevented the degradation of IκBα in LPS-stimulated PTECs. Pre-treatment with siRNA for IκBα abolished the inhibitory effect of EPA on LPS-induced NF-κB activation, suggesting that EPA inhibited NF-κB activation by regulating IκBα. Our results indicate that EPA prevents the early progression of tubulointerstitial injury in Thy-1 nephritis models, and the inhibitory effect of EPA on the expression of inflammatory molecules via the regulation of IκBα in cultured cells may explain this mechanism.
The complex metabolic, vascular and inflammatory perturbations that characterize diabetes mellitus often lead to progressive albuminuria, renal injury and dysfunction (diabetic nephropathy [DN]), and diabetes is the leading cause of end-stage renal disease in the US and Europe. Diet has an important role in cardiometabolic disorders and its potential influence on DN is of interest. Fatty acids are a major source of energy, but in excess, fatty acids (particularly saturated fatty acids) can induce lipotoxicity. Omega-3 polyunsaturated fatty acids (PUFAs) confer protection against cardiovascular disease-the major cause of death in patients with DN-by virtue of their antihyperlipidemic, antihypertensive, anti-inflammatory and other properties. Omega-6 PUFAs are also cardioprotective. However, a significant proportion of adults consume insufficient quantities of these essential nutrients. This Review describes the role of omega-3 and omega-6 PUFAs in nutrition and metabolism, with a focus on experimental, epidemiologic and clinical studies that have investigated their renoprotective effect in patients with diabetes. Results from a number of studies suggest, but do not firmly establish, that long-chain omega-3 PUFAs (found in fish oil) reduce albuminuria in the setting of DN. Intake of omega-6 fatty acids is associated with reduced albuminuria in experimental settings and in epidemiologic studies of DN. Although PUFAs do not seem to attenuate glomerular dysfunction, insufficient evidence exists to rule out such an effect. We feel that further research is needed into the potential of PUFA consumption and supplementation in DN.
Long-chain polyunsaturated omega-3 fatty acids (n-3 PUFA), which are obtained primarily from dietary sources such as coldwater fish, have diverse and potent mediating effects on the immune, inflammatory, and metabolic pathways, signal transduction, and cell membrane physiology. N-3 PUFA are increasingly being studied for their clinical benefits in a variety of medical conditions, some of which are relevant to individuals with advanced chronic kidney disease (CKD). These include, among others, renoprotection in IgA nephropathy, cardioprotective effects via a variety of mechanisms including blood pressure and triglyceride reduction, maintenance of dialysis access patency, sparing of inflammation-associated muscle loss, and even mortality. However, further confirmatory work needs to be performed before establishing formal intake recommendations and dosing goals for advanced CKD patients. In the meantime, the current American Heart Association n-3 PUFA intake guidelines can be applied to CKD patients, especially given n-3 PUFA's potential benefits and negligible risk profile. Over time, it will be incumbent upon the nephrology community to more clearly define the utility and optimal dosing of n-3 PUFA in CKD patients with advanced disease via randomized clinical trials.
The therapeutic efficacy of individual components of fish oils (FOs) in various human inflammatory diseases still remains unresolved, possibly due to low levels of n-3 fatty acids docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) or lower ratio of DHA to EPA. Because FO enriched with DHA (FO-DHA) or EPA (FO-EPA) has become available recently, we investigated their efficacy on survival and inflammatory kidney disease in a well-established animal model of human systemic lupus erythematosus. Results show for the first time that FO-DHA dramatically extends both the median (658 d) and maximal (848 d) life span of (NZB x NZW)F1 (B x W) mice. In contrast, FO-EPA fed mice had a median and maximal life span of approximately 384 and 500 d, respectively. Investigations into possible survival mechanisms revealed that FO-DHA (versus FO-EPA) lowers serum anti-dsDNA Abs, IgG deposition in kidneys, and proteinuria. Further, FO-DHA lowered LPS-mediated increases in serum IL-18 levels and caspase-1-dependent cleavage of pro-IL-18 to mature IL-18 in kidneys. Moreover, FO-DHA suppressed LPS-mediated PI3K, Akt, and NF-kappaB activations in kidney. These data indicate that DHA, but not EPA, is the most potent n-3 fatty acid that suppresses glomerulonephritis and extends life span of systemic lupus erythematosus-prone short-lived B x W mice, possibly via inhibition of IL-18 induction and IL-18-dependent signaling.
We showed earlier that statin treatment ameliorates target-organ injury in a transgenic model of angiotensin (Ang) II-induced hypertension. We now test the hypothesis that rosuvastatin (1, 10, and 50 mg/kg/day) influences leukocyte adhesion and infiltration, prevents induction of inducible nitric oxide synthase (iNOS), and ameliorates target-organ damage in a dose-dependent fashion.
We treated rats harboring the human renin and human angiotensinogen genes (dTGR) from week 4 to 8 (n = 20 per group). Untreated dTGR developed severe hypertension, cardiac hypertrophy, and renal damage, with a 100-fold increased albuminuria and focal cortical necrosis. Mortality of untreated dTGR at age 8 weeks was 59%.
Rosuvastatin treatment decreased mortality dose-dependently. Blood pressure was not affected. Albuminuria was reduced dose-dependently. Interstitial adhesion molecule (ICAM)-1 expression was markedly reduced by rosuvastatin, as were neutrophil and monocyte infiltration. Immunohistochemistry showed an increased endothelial and medial iNOS expression in small vessels, infiltrating cells, afferent arterioles, and glomeruli of dTGR. Immunoreactivity was stronger in cortex than medulla. Rosuvastatin markedly reduced the iNOS expression in both cortex and medulla. Finally, matrix protein (type IV collagen, fibronectin) expression was also dose- dependently reduced by rosuvastatin.
Our findings indicate that rosuvastatin dose- dependently ameliorates angiotensin II-induced-organ damage and almost completely prevents inflammation at the highest dose. The data implicate 3-hydroxy-3-methylglutaryl coenzyme A function in signaling events leading to target-organ damage.
Inducible NO synthase (iNOS) has been implicated in the pathogenesis of hypertension and target organ damage. We hypothesized that induction of iNOS contributes to left ventricular (LV) hypertrophy and dysfunction in mice with 2-kidney, 1-clip hypertension. Deletion of iNOS diminishes oxidative stress, thereby attenuating LV hypertrophy and enhancing cardiac performance. 2-Kidney, 1-clip hypertension was induced in mice lacking iNOS and wild-type controls (C57BL/6J). Sham-clipped mice served as controls. Systolic blood pressure was measured weekly by tail cuff. Left ventricular ejection fraction (by echocardiography) and cardiac response (maximum and minimum dP/dt, as well as an indicator of isovolumic contraction) to isoproterenol (50 ng per mouse, i.v.) were studied at the end of the experiment. 4-Hydroxy-2-nonenal (a byproduct of lipid peroxidation and an indicator of oxidative stress) was measured by immunohistochemical staining. gp91(phox), endothelial NO synthase, and iNOS protein expression were determined by Western blot. We found that systolic blood pressure, LV weight, myocyte cross-sectional area, interstitial collagen fraction, ejection fraction, and cardiac response to isoproterenol did not differ between strains with sham clipping. 2-Kidney, 1-clip hypertension increased systolic blood pressure, LV weight, myocyte cross-sectional area, and interstitial collagen fraction similarly in both strains. However, in mice lacking iNOS, maximum and minimum dP/dt, as well as an indicator of isovolumic contraction, markedly increased in response to isoproterenol, associated with decreased cardiac 4-hydroxy-2-nonenal expression and urinary nitrate/nitrite. We concluded that deletion of iNOS does not seem to play a significant role in preventing 2-kidney, 1-clip hypertension-induced hypertension and cardiac hypertrophy; however, it does enhance preservation of cardiac function, probably because of a reduction of iNOS-induced oxidative stress.
CYP2C enzymes epoxidize arachidonic acid (AA) to metabolites involved in the regulation of vascular and renal function. We tested the hypothesis that eicosapentaenoic acid (EPA), a n-3 polyunsaturated fatty acid, may serve as an alternative substrate. Human CYP2C8 and CYP2C9, as well as rat CYP2C11 and CYP2C23, were co-expressed with NADPH-CYP reductase in a baculovirus/insect cell system. The recombinant enzymes showed high EPA and AA epoxygenase activities and the catalytic efficiencies were almost equal comparing the two substrates. The 17,18-double bond was the preferred site of EPA epoxidation by CYPs 2C8, 2C11, and 2C23. 17(R),18(S)-Epoxyeicosatetraenoic acid was produced with an optical purity of about 70% by CYPs 2C9, 2C11, and 2C23 whereas CYP2C8 showed the opposite enantioselectivity. These results demonstrate that EPA is an efficient substrate of CYP2C enzymes and suggest that n-3 PUFA-rich diets may shift the CYP2C-dependent generation of physiologically active eicosanoids from AA- to EPA-derived metabolites.
Cardiomyocytes release (or metabolize) several diffusible agents (e.g., nitric oxide [NO], endothelin-1 [ET-1], and angiotensin II) that exert direct effects on myocyte function under various pathologic conditions. Although cardiac hypertrophy is a compensatory mechanism in response to different cardiovascular diseases, there can be a pathologic transition in which the myocardium becomes dysfunctional. Recently, NO has been found to be an important regulator of cardiac remodeling. Specifically, NO has been recognized as a potent antihypertrophic and proapoptotic mediator in cultured cardiomyocytes. We demonstrated that ET-1-induced hypertrophic remodeling in neonatal cardiomyocytes was arrested by pretreatment with eicosapentaenoic acid (EPA), a major component of fish oil. In some recent studies, EPA has demonstrated cardioprotective effects by modulating NO. This study investigated the changes in NO synthase (NOS) in ET-1-induced hypertrophied cardiomyocytes and in total levels of nitrates and nitrites. Ventricular cardiomyocytes were isolated from 2-day-old Sprague-Dawley rats and were cultured in D-MEM/Ham F12 supplemented with 0.1% fatty acid-free bovine serum albumin for 3 days. At Day 4 of culture, the cardiomyocytes were divided into three groups: control group, ET-1 (0.1 nM) group, and ET-1 pretreated with EPA (10 microM) group. NOS gene expression was evaluated 24 hrs after treatment using real-time polymerase chain reaction. Endothelial NOS (eNOS) mRNA expression was decreased in the ET-1 group compared with controls and was unchanged by pretreatment with EPA. mRNA expression of inducible NOS (iNOS) was significantly increased in ET-1-treated cardiomyocytes and was suppressed by EPA pretreatment. Neuronal NOS gene expression and total NO level did not exhibit a statistically significant change in any of the groups. There may be some interaction between ET-1, eNOS, and iNOS in ET-1-induced and EPA-regressed hypertrophied cardiomyocytes that suppress iNOS expression without modulating total NO level or eNOS gene expression.
Accumulating evidence in both humans and animal models clearly indicates that a group of very-long-chain polyunsaturated fatty acids, the n-3 fatty acids (or omega-3), have distinct and important bioactive properties compared with other groups of fatty acids. n-3 Fatty acids are known to reduce many risk factors associated with several diseases, such as cardiovascular diseases, diabetes, and cancer. The mechanisms whereby n-3 fatty acids affect gene expression are complex and involve multiple processes. As examples, n-3 fatty acids regulate 2 groups of transcription factors, such as sterol-regulatory-element binding proteins and peroxisome proliferator-activated receptors, that are critical for modulating the expression of genes controlling both systemic and tissue-specific lipid homeostasis. Modulation of specific genes by n-3 fatty acids and cross-talk between these genes are responsible for many effects of n-3 fatty acids.
New Zealand green lipped mussel (NZGLM), abalone (AB), and shark cartilage (SC) are extensively used for treatment of and/or as preventatives for arthritis, despite a relative paucity of scientific evidence for efficacy. This research integrated a simulated digestion protocol with ultrafiltration and cartilage explants to generate new information on the anti-inflammatory and chondroprotective properties of NZGLM, SC, and AB. Each nutraceutical was artificially digested using simulated gastric and intestinal fluids, and the crude digest was ultrafiltered (50 kDa). Each filtrate was applied individually to cartilage explants before the explants were stimulated with IL-1 to induce an acute inflammatory response. Media were collected daily for 48 h and analyzed for prostaglandin E(2) (PGE(2)), glycosaminoglycan (GAG), and nitric oxide (NO), and cartilage tissue was differentially stained to determine the relative proportion of live and dead cells. SC and NZGLM significantly inhibited IL-1-induced PGE(2) synthesis and IL-1-induced GAG release, and AB was an effective inhibitor of IL-1-induced NO production. The three test nutraceuticals affect at least three major pathways involved in the catabolic cycle of arthritis and may prove important treatments and/or preventatives for the pain and degradation associated with this condition. The methodology and results describe a useful model for evaluating dietary nutraceuticals in vitro.
Angiotensin (Ang)-II induces vascular wall inflammation by activating NF- κB. Aspirin inhibits IKKβ in vitro; however, the in vivo relevance of the phenomenon is unclear. We tested the hypothesis that aspirin protects from Ang II-induced endorgan damage by inhibiting NF- κB activation in vivo. Rats harboring human renin and angiotensinogen genes received high- (600 mg/kg/day) or low- (25 mg/kg/day) dose aspirin. High-dose aspirin reduced mortality, cardiac hypertrophy, fibrosis, and albuminuria independent of blood pressure, whereas both doses reduced cyclooxygenase activity. High-dose aspirin inhibited NF- κB and AP-1 activation and inflammation in heart and kidney. These in vivo results serve to explain the clinical utility of high-dose aspirin in inflammatory disorders and suggest additional therapeutic avenues that may be relevant to cardiovascular disease.
Epidemiological and animal-based investigations have indicated that the development of skin cancer is in part associated with poor dietary practices. Lipid content and subsequently the derived fatty acid composition of the diet are believed to play a major role in the development of tumorigenesis. Omega 3 (ω3) fatty acids, including docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), can effectively reduce the risk of skin cancer whereas omega 6 (ω6) fatty acids such as arachidonic acid (AA) reportedly promote risk. To investigate the effects of fatty acids on tumorigenesis, we performed experiments to examine the effects of the ω3 fatty acids EPA and DHA and of the ω6 fatty acid AA on phorbol 12-tetradecanoate 13-acetate (TPA)-induced or epidermal growth factor (EGF)-induced transcription activator protein 1 (AP-1) transactivation and on the subsequent cellular transformation in a mouse epidermal JB6 cell model. DHA treatment resulted in marked inhibition of TPA- and EGF-induced cell transformation by inhibiting AP-1 transactivation. EPA treatment also inhibited TPA-induced AP-1 transactivation and cell transformation but had no effect on EGF-induced transformation. AA treatment had no effect on either TPA- or EGF-induced AP-1 transactivation or transformation, but did abrogate the inhibitory effects of DHA on TPA- or EGF-induced AP-1 transactivation and cell transformation in a dose-dependent manner. The results of this study demonstrate that the inhibitory effects of ω3 fatty acids on tumorigenesis are more significant for DHA than for EPA and are related to an inhibition of AP-1. Similarly, because AA abrogates the beneficial effects of DHA, the dietary ratio of ω6 to ω3 fatty acids may be a significant factor in mediating tumor development.
Excess dietary salt induces a cytochrome P450 arachidonic acid epoxygenase isoform in rat kidneys (Capdevila, J. H., S. Wei, J. Yang, A. Karara, H. R. Jacobson, J. R. Falck, F. P. Guengerich, and R. N. Dubois. 1992. J. Biol. Chem. 267:21720-21726). Treatment of rats on a high salt diet with the epoxygenase inhibitor, clotrimazole, produces significant increases in mean arterial blood pressure (122 +/- 2 and 145 +/- 4 mmHg for salt and salt- and clotrimazole-treated rats, respectively). The salt- and clotrimazole-dependent hypertension is accompanied by reductions in the urinary excretion of epoxygenase metabolites and by a selective inhibition of the renal microsomal epoxygenase reaction. The prohypertensive effects of clotrimazole are readily reversed when either the salt or clotrimazole treatment is discontinued. The indication that a salt-inducible renal epoxygenase protects against hypertension, are supported by studies with the Dahl rat model of genetic salt-sensitive hypertension. Dahl resistant animals responded to excess dietary salt by inducing the activity of their kidney microsomal epoxygenase(s) (0.102 +/- 0.01 and 0.240 +/- 0.04 nmol of products formed/min per mg of microsomal protein for control and salt-treated rats, respectively). Despite severe hypertension during excess dietary salt intake (200 +/- 20 mmHg), Dahl salt-sensitive rats demonstrated no increase in renal epoxygenase activity. These studies indicate that acquired or inherited abnormalities in renal epoxygenase activities and/or regulation can be related to salt-sensitive hypertension in rodents. Studies on the human renal epoxygenase and its relationship to salt hypertension may prove useful.
During the past few decades, intensive collaborative research in the fields of chronic and acute inflammatory disorders has resulted in a better understanding of the pathophysiology and diagnosis of these diseases. Modern therapeutic approaches are still not satisfactory and shock, sepsis and multiple organ failure remain the great challenge in intensive care medicine. However, the treatment of inflammatory diseases like rheumatoid arthritis, ulcerative colitis or psoriasis also represents an unresolved problem. Many factors contribute to the complex course of inflammatory reactions. Microbiological, immunological and toxic agents can initiate the inflammatory response by activating a variety of humoral and cellular mediators. In the early phase of inflammation, excessive amounts of interleukins and lipid-mediators are released and play a crucial role in the pathogenesis of organ dysfunction. Arachidonic acid (AA), the mother substance of the pro-inflammatory eicosanoids, is released from membrane phospholipids in the course of inflammatory activation and is metabolised to prostaglandins and leukotrienes. Various strategies have been evaluated to control the excessive production of lipid mediators on different levels of biochemical pathways, such as inhibition of phospholipase A2, the trigger enzyme for release of AA, blockade of cyclooxygenase and lipoxygenase pathways and the development of receptor antagonists against platelet activating factor and leukotrienes. Some of these agents exert protective effects in different inflammatory disorders such as septic organ failure, rheumatoid arthritis or asthma, whereas others fail to do so. Encouraging results have been obtained by dietary supplementation with long chain omega-3 fatty acids like eicosapentaenoic acid (EPA). In states of inflammation, EPA is released to compete with AA for enzymatic metabolism inducing the production of less inflammatory and chemotactic derivatives.
The mechanism-based cytochrome P-450 (CYP) inhibitor 1-aminobenzotriazole (ABT) was characterized as an inhibitor of renal arachidonic acid metabolism and administered to spontaneously hypertensive rats (SHRs) to determine the effect of reduced eicosanoid production on mean arterial pressure (MAP). A single intraperitoneal dose of ABT to Sprague-Dawley rats caused a dose-dependent loss of renal CYP content, arachidonic acid metabolism, and CYP4A protein. In the cortex and outer medulla, ABT showed a high degree of selectivity for the CYP4A enzymes, reflected by the potent inhibition of 19- and 20-hydroxyeicosatetraenoic acid (19- and 20-HETE) formation. A 50 mg/kg dose of ABT reduced cortical 20-HETE formation to 16.1 +/- 0.82% of control and outer medullary 20-HETE formation to 23.8 +/- 0.45% of control. In contrast, there was no inhibition of renal epoxygenase activity at this dose. Renal CYP content, arachidonic acid omega- and (omega-1)-hydroxylase activity, and CYP4A protein levels gradually return to control levels by 72 h after a single dose of ABT. Cortical 20-HETE formation recovered from 17.9 +/- 3.15% of control at 6 h to 84.8 +/- 4.67% of control at 72 h after ABT administration. A single injection of ABT to 7-wk-old SHRs caused an acute reduction in MAP, which remained suppressed for at least 12 h. The effect was maximal within 4 h and averaged 17-23 mmHg during the 4- to 12-h period after administration. 20-HETE formation was inhibited 85% in the cortex and 70-80% in the outer medulla during the period when MAP was reduced. A structurally related ABT analog 1-hydroxybenzotriazole had no effect on blood pressure or renal arachidonic acid metabolism. These results identify ABT as a selective inhibitor of renal CYP4A activity and provide further support for a role for 20-HETE in the regulation of blood pressure.
Inhibition of cytochrome P-450 (CYP450) enzymes with cobalt chloride (CoCl2) prevented hypertension, organ hypertrophy, and renal injury induced by DOCA and salt (1% NaCl) in uninephrectomized (UNx) rats. Systolic blood pressure (SBP) rose to 193 +/- 6 mmHg by day 21 from control levels of 150 +/- 7 mmHg in response to DOCA-salt treatment, a rise that was prevented by CoCl2 (24 mg. kg-1. 24 h-1). The effects of DOCA-salt treatment, which increased protein excretion to 88.3 +/- 6.9 mg/24 h on day 21 from 9.0 +/- 1.1 mg/24 h on day 3, were prevented by CoCl2. CoCl2 also attenuated the renal and left ventricular hypertrophy and the increase in media-to-lumen ratio in hypertensive rats. DOCA-salt treatment increased excretion of endothelin (ET)-1 from 81 +/- 17 to 277 +/- 104 pg. 100 g body wt-1. 24 h-1 associated with a fourfold increase in 20-hydroxyeicosatetraenoic acid (20-HETE) excretion from 3.0 +/- 1.1 to 12.2 +/- 1.9 ng. 100 g body wt-1. 24 h-1 (days 3 vs. 21). CoCl2 blunted these increases by 58 and 72%, respectively. In aortic rings pulsed with [3H]thymidine, ET-1 increased its incorporation. Dibromododec-11-enoic acid, an inhibitor of 20-HETE synthesis, attenuated ET-1-induced increases in [3H]thymidine incorporation. We distinguished effects of CoCl2 acting via CO generation vs. suppression of CYP450-arachidonic acid metabolism by treating UNx-salt-DOCA rats with 1-aminobenzotriazole (ABT), which suppresses CYP450 enzyme activity, and compared these results to those produced by CoCl2. ABT reduced hypertension, as did CoCl2. Unlike CoCl2, ABT did not prevent organ hypertrophy and proteinuria, suggesting that these effects were partially related to CO formation. Blockade of the ETA receptor with BMS-182874 reduced SBP, organ hypertrophy, and proteinuria, indicating the importance of ET-initiated abnormalities to the progression of lesions in UNx-salt-DOCA.
The epoxyeicosatrienoic acids (EETs) are products of cytochrome P450 epoxygenases that have vasodilatory properties similar to that of endothelium-derived hyperpolarizing factor. The cytochrome P450 isoform CYP2J2 was cloned and identified as a potential source of EETs in human endothelial cells. Physiological concentrations of EETs or overexpression of CYP2J2 decreased cytokine-induced endothelial cell adhesion molecule expression, and EETs prevented leukocyte adhesion to the vascular wall by a mechanism involving inhibition of transcription factor NF-kappaB and IkappaB kinase. The inhibitory effects of EETs were independent of their membrane-hyperpolarizing effects, suggesting that these molecules play an important nonvasodilatory role in vascular inflammation.
Human beings evolved consuming a diet that contained about equal amounts of n-3 and n-6 essential fatty acids. Over the past 100-150 y there has been an enormous increase in the consumption of n-6 fatty acids due to the increased intake of vegetable oils from corn, sunflower seeds, safflower seeds, cottonseed, and soybeans. Today, in Western diets, the ratio of n-6 to n-3 fatty acids ranges from approximately 20-30:1 instead of the traditional range of 1-2:1. Studies indicate that a high intake of n-6 fatty acids shifts the physiologic state to one that is prothrombotic and proaggregatory, characterized by increases in blood viscosity, vasospasm, and vasoconstriction and decreases in bleeding time. n-3 Fatty acids, however, have antiinflammatory, antithrombotic, antiarrhythmic, hypolipidemic, and vasodilatory properties. These beneficial effects of n-3 fatty acids have been shown in the secondary prevention of coronary heart disease, hypertension, type 2 diabetes, and, in some patients with renal disease, rheumatoid arthritis, ulcerative colitis, Crohn disease, and chronic obstructive pulmonary disease. Most of the studies were carried out with fish oils [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA)]. However, alpha-linolenic acid, found in green leafy vegetables, flaxseed, rapeseed, and walnuts, desaturates and elongates in the human body to EPA and DHA and by itself may have beneficial effects in health and in the control of chronic diseases.
Renal function is perturbed by inhibition of nitric oxide synthase (NOS). To probe the basis of this effect, we characterized the effects of nitric oxide (NO), a known suppressor of cytochrome P450 (CYP) enzymes, on metabolism of arachidonic acid (AA), the expression of omega-hydroxylase, and the efflux of 20-hydroxyeicosatetraenoic acid (20-HETE) from the isolated kidney. The capacity to convert [(14)C]AA to HETEs and epoxides (EETs) was greater in cortical microsomes than in medullary microsomes. Sodium nitroprusside (10-100 microM), an NO donor, inhibited renal microsomal conversion of [(14)C]AA to HETEs and EETs in a dose-dependent manner. 8-bromo cGMP (100 microM), the cell-permeable analogue of cGMP, did not affect conversion of [(14)C]AA. Inhibition of NOS with N(omega)-nitro-L-arginine-methyl ester (L-NAME) significantly increased conversion of [(14)C]AA to HETE and greatly increased the expression of omega-hydroxylase protein, but this treatment had only a modest effect on epoxygenase activity. L-NAME induced a 4-fold increase in renal efflux of 20-HETE, as did L-nitroarginine. Oral treatment with 2% sodium chloride (NaCl) for 7 days increased renal epoxygenase activity, both in the cortex and the medulla. In contrast, cortical omega-hydroxylase activity was reduced by treatment with 2% NaCl. Coadministration of L-NAME and 2% NaCl decreased conversion of [(14)C]AA to HETEs without affecting epoxygenase activity. Thus, inhibition of NOS increased omega-hydroxylase activity, CYP4A expression, and renal efflux of 20-HETE, whereas 2% NaCl stimulated epoxygenase activity.
The demonstration of in vivo arachidonic acid epoxidation and omega-hydroxylation established the cytochrome P450 epoxygenase and omega/omega-1 hydroxylase as formal metabolic pathways and as members of the arachidonate metabolic cascade. The characterization of the potent biological activities associated with several of the cytochrome P450-derived eicosanoids suggested new and important functional roles for these enzymes in cellular, organ, and body physiology, including the control of vascular reactivity and systemic blood pressures. Past and current advances in cytochrome P450 biochemistry and molecular biology facilitate the characterization of cytochrome P450 isoforms responsible for tissue/organ specific arachidonic acid epoxidation and omega/omega-1 hydroxylation, and thus, the analysis of cDNA and/or gene specific functional phenotypes. The combined application of physiological, biochemical, molecular, and genetic approaches is beginning to provide new insights into the physiological and/or pathophysiological significance of these enzymes, their endogenous substrates, and products.
gamma-Linolenic acid [GLA, 18:3(n-6)], eicosapentaenoic acid [EPA, 20:5(n-3)] and docosahexaenoic acid [DHA, 22:6(n-3)] have been reported to prevent cardiovascular diseases. However, they are highly unsaturated and therefore more sensitive to oxidation damage. We investigated the effects of a diet rich in these polyunsaturated fatty acids (PUFA) on blood pressure, plasma and lipoprotein lipid concentrations, total antioxidant status, lipid peroxidation and platelet function in spontaneously hypertensive rats (SHR) and normotensive Wistar Kyoto rats (WKY). Five-week-old SHR and WKY rats were fed for 10 wk either a diet containing Isio 4 oil or a diet rich in GLA, EPA and DHA (5.65, 6.39 and 4.94 g/kg dry diet, respectively). The total antioxidant status was assayed by monitoring the rate of free radical-induced hemolysis. VLDL-LDL sensitivity to copper-induced lipid peroxidation was determined as the production of thiobarbituric acid reactive substances. After dietary PUFA supplementation, a significant decrease in blood pressure of SHR rats (-20 mm Hg) was observed and the total antioxidant status was enhanced. VLDL-LDL resistance to copper-induced peroxidation was increased in both strains. The PUFA supplementation did not change platelet maximum aggregation in SHR rats, but it decreased the aggregation speed. In hypertensive rats, GLA + EPA + DHA supplementation lowers blood pressure, enhances total anti-oxidant status and resistance to lipid peroxidation, diminishes platelet aggregation speed and lowers plasma lipid concentrations. Thus, it enhances protection against cardiovascular diseases. Therefore, nutritional recommendations for cardiovascular disease prevention should take into account the pharmacologic properties of GLA, EPA and DHA.
Omega-3 fatty acids, which are abundant in fish oil, improve the prognosis of several chronic inflammatory diseases although the mechanism for such effects remains unclear. These fatty acids, such as eicosapentaenoic acid (EPA), are highly polyunsaturated and readily undergo oxidation. We show that oxidized, but not native unoxidized, EPA significantly inhibited human neutrophil and monocyte adhesion to endothelial cells in vitro by inhibiting endothelial adhesion receptor expression. In transcriptional coactivation assays, oxidized EPA potently activated the peroxisome proliferator-activated receptor alpha (PPAR alpha), a member of the nuclear receptor family. In vivo, oxidized, but not native, EPA markedly reduced leukocyte rolling and adhesion to venular endothelium of lipopolysaccharide (LPS)-treated mice. This occurred via a PPAR alpha-dependent mechanism because oxidized EPA had no such effect in LPS-treated PPAR alpha-deficient mice. Therefore, the beneficial effects of omega-3 fatty acids may be explained by a PPAR alpha-mediated anti-inflammatory effect of oxidized EPA.
The fatty acid composition of inflammatory and immune cells is sensitive to change according to the fatty acid composition of the diet. In particular, the proportion of different types of polyunsaturated fatty acids (PUFA) in these cells is readily changed, and this provides a link between dietary PUFA intake, inflammation, and immunity. The n-6 PUFA arachidonic acid (AA) is the precursor of prostaglandins, leukotrienes, and related compounds, which have important roles in inflammation and in the regulation of immunity. Fish oil contains the n-3 PUFA eicosapentaenoic acid (EPA). Feeding fish oil results in partial replacement of AA in cell membranes by EPA. This leads to decreased production of AA-derived mediators. In addition, EPA is a substrate for cyclooxygenase and lipoxygenase and gives rise to mediators that often have different biological actions or potencies than those formed from AA. Animal studies have shown that dietary fish oil results in altered lymphocyte function and in suppressed production of proinflammatory cytokines by macrophages. Supplementation of the diet of healthy human volunteers with fish oil-derived n-3 PUFA results in decreased monocyte and neutrophil chemotaxis and decreased production of proinflammatory cytokines. Fish oil feeding has been shown to ameliorate the symptoms of some animal models of autoimmune disease. Clinical studies have reported that fish oil supplementation has beneficial effects in rheumatoid arthritis, inflammatory bowel disease, and among some asthmatics, supporting the idea that the n-3 PUFA in fish oil are anti-inflammatory and immunomodulatory.
We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase–mediated Ca2+ sensitization of vascular smooth muscle (VSM) contraction. Subcellular localization and kinase activity of Src family protein kinases (SrcPTKs), except for c-Src, is controlled by a reversible S-palmitoylation, an event inhibited by eicosapentaenoic acid (EPA). We examined the possible involvement of SrcPTKs in SPC-induced Ca2+ sensitization and effects of EPA. We used porcine coronary VSM and rat aortic VSM cells (VSMCs) in primary culture. An SrcPTKs inhibitor, PP1, and EPA inhibited SPC-induced contraction, concentration-dependently, without affecting [Ca2+]i levels and the Ca2+-dependent contraction induced by high K+ depolarization. A digitized immunocytochemical analysis in VSMCs revealed that SPC induced translocation of Fyn, but not of c-Src, from the cytosol to the cell membrane, an event abolished by EPA. Translocation of Rho-kinase from the cytosol to the cell membrane by SPC was also inhibited by EPA and PP1. The SPC-induced activation of SrcPTKs was blocked by EPA and PP1, but not by Y27632, an Rho-kinase inhibitor. Rho-kinase–dependent phosphorylation of myosin phosphatase induced by SPC was inhibited by EPA, PP1, and Y27632. Translocation and activation of SrcPTKs, including Fyn, play an important role in Ca2+ sensitization of VSM contractions mediated by a SPC-Rho-kinase pathway.
Nitric oxide (NO) modulates transcription factors that bind specific cis-regulatory DNA responsible for coordinating the spatial and temporal patterns of gene expression that are initiated by a changing microenvironment. In this way NO helps to orchestrate gene transcription and forms the basis of functional cell responses to accommodate metabolic requirements and to coordinate endogenous defense mechanisms against a variety of stress and disease conditions. There is marked overlap between the signalling pathways triggered by NO, superoxide, and hypoxia. Understanding the redox-based regulation of signal transduction and gene expression will provide insights into how cell activities are constantly coordinated and how promising new therapies may be developed.
In a rat model of glomerular immune injury induced by administration of anti-glomerular basement membrane antibody and resembling human rapidly progressive glomerulonephritis, we explored whether activation of inducible nitric oxide synthase (iNOS) regulates synthesis of eicosanoids originating from cyclooxygenation or lipoxygenation of arachidonic acid. At early stages (24 hr) of injury, inhibition of iNOS using the selective inhibitor L-N6-(1-iminoethyl) lysine (L-NIL) at doses sufficient to reduce urinary excretion of nitrate/nitrite, reduced glomerular synthesis of the prostaglandins PGE2 and PGI2, but had no effect on that of thromboxane A2 (TxA2). The syntheses of 5-hydroxyeicosatetraenoic acid (HETE), 15-HETE and leukotriene B4 (LTB4) were also reduced. That of 12-HETE remained unchanged. We also explored the effect of arachidonate cyclooxygenation and lipoxygenation eicosanoids on iNOS expression. Administration of the cyclooxygenase (COX) inhibitor, indomethacin, at doses sufficient to inhibit glomerular prostaglandin synthesis, increased iNOS mRNA levels in glomeruli. Administration of the 5-lipoxygenase (5-LO) inhibitor, MK-0591, at doses sufficient to inhibit glomerular LTB4 synthesis also increased iNOS mRNA. The effect of 5-LO inhibition on iNOS expression was more pronounced than that of COX inhibition. In nephritic animals given the iNOS inhibitor, L-NIL, or indomethacin proteinuria worsened. In those given the 5-lipoxygenase inhibitor there was no change in urine protein excretion. These observations point to regulatory interactions between the arachidonic acid and the L-arginine: NO pathways in glomerulonephritis. These interactions are of importance in considering antiinflammatory strategies based on inhibition of iNOS or of specific eicosanoids.
It has been proposed that fish oil dietary supplementation in the chronic rat 5/6 renal ablation model may be either protective or toxic. These conflicting hypotheses were tested in rats who underwent renal ablation or sham surgery. Twenty rats received sham surgery, and 40 received 5/6 renal ablation. All rats were fed a regular laboratory diet up to 1 week postsurgery. At that time, one half of the renal ablation group was provided with an isocaloric diet supplemented with 24% MaxEPA (fish oil), 1% safflower oil, and antioxidants. The renal ablation rats developed hypertension, albuminuria, gammaglobulinuria, and a decline in glomerular filtration rate, which was less in the fish oil group compared with that in the regular laboratory diet group at 10 and 20 wk postsurgery. The fish oil renal ablation rats had significantly less glomerulosclerosis than did the regular laboratory diet renal ablation animals, and no more glomerular fibrin deposition than did the sham controls. The renal ablation regular laboratory diet rats had a significant dyslipidemia at 20 wk which was prevented in the fish oil renal ablation cohort. The fish oil renal ablation rats also demonstrated a significant decline in renal tissue arachidonic acid incorporation and a concomitant increase in eicosapentaenoic acid and docosahexaenoic acid incorporation. The mortality of the renal ablation group was greater than that of the sham controls but not significantly different for the fish oil or the regular laboratory diet groups. These results support the hypothesis that the fish oil diet containing specific antioxidant, vitamin E, and essential fatty acid supplementation is protective in the rat remnant nephron model and prevents the evolution of glomerulosclerosis with associated renal functional impairment, while preserving glomerular filtration.
Liver and kidney microsomes were isolated from rats raised on high-fat diets. In terms of energy, the high-fat diets contained 4% vegetable and 40% fish, vegetable or coconut oils. Each microsomal preparation was fortified with 1 mM NADPH and incubated with 5,8,11,14,17-eicosapentaenoic acid (20:5(n-3]. The number of metabolites formed was assessed by reverse-phase high-performance liquid chromatography (HPLC). To identify the major metabolites, large-scale incubations were done with 20:5(n-3) and microsomes from phenobarbital-treated rats. After extracts from the phenobarbital and dietary studies were combined, individual products were isolated by reverse- and normal-phase HPLC. The metabolites were identified by mass spectrometry, by chromatographic properties, and by comparing their retention times and mass spectra with those of chemically synthesized standards. For liver microsomes, the major metabolites were: 17,18-, 14,15-, 11,12- and 8,9-dihydroxyeicosatetraenoic acids, 20-hydroxyeicosapentaenoic acid, and 19-hydroxyeicosatetraenoic acid. For renal microsomes, the major metabolites were 20-hydroxyeicosapentaenoic and 19-hydroxypentaenoic acids. Because formation of these metabolites required NADPH and was enhanced by phenobarbital pretreatment, 20:5(n-3) appears to be oxidized by cytochrome P-450 monooxygenases. Based on reverse-phase high performance liquid chromatograms, all three high-fat diets may produce the same types of monooxygenase metabolites from 20:5(n-3). It remains unknown whether fish-oil diets induce the synthesis of monooxygenases to oxidize n-3 fatty acids, because these preliminary studies involved only two animals per dietary group.
To determine whether purified omega-3 and omega-6 fatty acids influence the progression of hypertensive renal failure in salt-loaded stroke-prone spontaneously hypertensive rats (SHRSP) with established hypertension or during the developmental stage of their hypertension.
Groups of eight SHRSP aged 1 or 4 months were fed, for 12 weeks, synthetic diets containing 2% sodium (wt:wt) and either 5% olive oil or 4.5% gamma-linolenic acid (omega-6), eicosapentaenoic acid (omega-3) or docosahexaenoic acid (omega-3).
Urinary protein excretion and blood pressure were measured after 6, 9 and 12 weeks. The rats were killed and their tissues were collected for fatty acid and eicosanoid analysis.
Young rats (aged 1 month) fed diets containing gamma-linolenic acid or olive oil developed marked proteinuria by 9 weeks, whereas no change was observed after 12 weeks in rats fed docosahexaenoic acid or eicosapentaenoic acid. Blood pressure was lower in those fed docosahexaenoic acid or eicosapentaenoic acid than in the gamma-linolenic acid or olive oil groups. Adult rats (aged 4 months) fed the docosahexaenoic acid diet had significantly lower proteinuria than those fed gamma-linolenic acid, eicosapentaenoic acid or olive oil, but there were no differences in blood pressure among the groups. Kidneys from rats fed omega-3 fatty acids had increased levels of docosahexaenoic acid or eicosapentaenoic acid, or both, whereas those from rats fed gamma-linolenic acid and olive oil contained virtually no omega-3 fatty acids. Thromboxane B2 and 12-hydroxyeicosatetraenoic acid production in renal cortex extracts was lowest in rats fed docosahexaenoic acid and eicosapentaenoic acid.
Dietary omega-3 fatty acids retard the development of hypertension-induced proteinuria. This may be caused by a favourable influence on fatty acid and eicosanoid metabolism and reduction of blood pressure.
Wistar rats (4-week-old) were administered with streptozotocin (45 mg/kg) through tail veins. After 3 months, diabetic rats were divided into 2 groups. One group (EPA group, n = 16) was fed a lipid-free diet (90%, w/w) plus lard (8%) and 90% pure eicosapentaenoic acid ethyl ester (2%) for 6 months. The other group (control group, n = 16) was fed in the same way except that eicosapentaenoic acid ethyl ester was replaced by safflower oil. Twenty-four-hour urine was collected just before starting the experimental diets and during the 6-month experimental period at monthly intervals. There were no differences in food intake and body weight between the two groups throughout the experiment. The mean microalbuminuria of the EPA group became significantly lower than that of the control group after 4 months on the diets through the end of the study (6 months). The mean microalbuminuria levels at the end of the study were 1.38 mg/day in the EPA group (n = 9) and 5.19 mg/day in the control group (n = 6) (p < 0.01). Eicosapentaenoic acid administration might retard the progression of diabetic nephropathy by reducing microalbuminuria.
Renal transplantation studies indicate that some form of renal dysfunction underlies the development of hypertension in Dahl salt-sensitive (S) rats; however, the factors responsible for altering kidney function remain to be determined. Previous studies have indicated that Dahl S rats require a higher renal perfusion pressure to excrete the same amount of sodium and water as normotensive rats and that this is due largely to an elevation in Cl- transport in the thick ascending limb of the loop of Henle. There are now five lines of evidence that suggest an abnormality in the renal metabolism of arachidonic acid by enzymes of the P4504A family may contribute to the increase in loop Cl- transport and the development of hypertension in Dahl S rats. In this regard, the formation of 20-HETE and the levels of P4504A protein are reduced in the outer medulla of Dahl S rats. Perfusion of the loop of Henle of Dahl S rats with exogenous 20-HETE normalizes the elevated loop Cl- transport. In addition, a genetic marker in the P4504A2 gene, which encodes for the enzyme that makes 20-HETE, cosegregates with the development of hypertension in an F2 cross of Dahl S and Lewis rats. Finally, induction of renal production of 20-HETE with clofibrate prevents the development of hypertension in Dahl S rats and inhibition of renal 20-HETE formation produces hypertension in Lewis rats fed a high salt diet. These results implicate the CYP4A2 locus as a candidate gene that contributes to the alterations in renal function and the development of hypertension in Dahl S rats.
Carbohydrate antigen 19-9 (CA19-9) is a tumor-associated antigen defined by a murine monoclonal antibody, NS 19-9, which was established from the colon cancer cell line SW1116 in 1979 by Koprowski et al. [1]. We concentrated on the fact that signet ring cell carcinoma of the pancreas in two cases manifested very high levels of carcinoembryonic antigen (CEA) despite the absence of hepatic metastasis [2].
Hypertension and kidney damage in the double transgenic rat (dTGR) harboring both human renin and human angiotensinogen genes are dependent on the human components of the renin angiotensin system. We tested the hypothesis that monocyte infiltration and increased adhesion molecule expression are involved in the pathogenesis of kidney damage in dTGR. We also evaluated the effects of long-term angiotensin-converting enzyme (ACE) inhibition, AT1 blockade, and human renin inhibition on monocyte recruitment and inflammatory response in dTGR. Systolic blood pressure and 24-hour albuminuria were markedly increased in 7-week-old dTGR as compared with age-matched normotensive Sprague Dawley rats. We found a significant monocyte/macrophage infiltration in the renal perivascular space and increased expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) in the interstitium, intima, and adventitia of the small renal vessels. alphaLbeta2 integrin and alpha4beta1 integrin, the corresponding ligands for ICAM-1 and VCAM-1, were also found on infiltrating monocytes/macrophages. The expression of plasminogen activator inhibitor-1 and fibronectin in the kidneys of dTGR were increased and distributed similarly to ICAM-1. In 4-week-old dTGR, long-term treatment with ACE inhibition (cilazapril), AT1 receptor blockade (valsartan), and human renin inhibition (RO 65-7219) (each drug 10 mg/kg by gavage once a day for 3 weeks) completely prevented the development of albuminuria. However, only cilazapril and valsartan were able to decrease blood pressure to normotensive levels. Interestingly, the drugs were all equally effective in preventing monocyte/macrophage infiltration and the overexpression of adhesion molecules, plasminogen activator inhibitor-1, and fibronectin in the kidney. Our findings indicate that angiotensin II causes monocyte recruitment and vascular inflammatory response in the kidney by blood pressure-dependent and blood pressure-independent mechanisms. ACE inhibition, AT1 receptor blockade, and human renin inhibition all prevent monocyte/macrophage infiltration and increased adhesion molecule expression in the kidneys of dTGR.
Angiotensin (Ang) II-induced organ damage has fascinated students of hypertension since the work of Wilson and Byrom. We are investigating a double transgenic rat (dTGR) model, in which rats transgenic for the human angiotensinogen and renin genes are crossed. These rats develop moderately severe hypertension but die of end-organ cardiac and renal damage by week 7. The heart shows necrosis and fibrosis, whereas the kidneys resemble the hemolytic-uremic syndrome vasculopathy. Surface adhesion molecules (ICAM-1 and VCAM-1) are expressed early on the endothelium, while the corresponding ligands are found on circulating leukocytes. Leukocyte infiltration in the vascular wall accompanies PAI-1, MCP-1, and VEGF expression. The expression of TGF-beta and deposition of extracellular matrix proteins follows, which is accompanied by fibrinoid vasculitis in small vessels of the heart and kidneys. Angiotensin-converting enzyme inhibitors and AT1 receptor blockers each lowered blood pressure and shifted pressure natriuresis partially leftward by different mechanisms. When combined, they normalized blood pressure, pressure natriuresis, and protected from vasculopathy completely. Renin inhibition lowered blood pressure partially, but protected from vasculopathy completely. Endothelin receptor blockade had no influence on blood pressure but protected from vasculopathy and improved survival. We show evidence that Ang II stimulates oxidative stress directly or indirectly via endothelin 1 and that NFkappaB is upregulated in this model. We speculate that the transcription factors NFkappaB and AP-1 are involved with initiating chemokine and cytokine expression, leading to the above cascade. The unique model and our pharmacological probes will enable us to test these hypotheses.
The protean properties of 20-hydroxyeicosatetraenoic acid (HETE), vasoactivity, mitogenicity, and modulation of transport in key nephron segments, serve as the basis for the essential roles of 20-HETE in the regulation of the renal circulation and electrolyte excretion and as a second messenger for endothelin-1 and mediator of selective renal effects of ANG II. Renal autoregulation and tubular glomerular feedback are mediated by 20-HETE through constriction of preglomerular arterioles, responses that are maintained by 20-HETE inhibition of calcium-activated potassium channels. 20-HETE modulates ion transport in the proximal tubules and the thick ascending limb by affecting the activities of Na+-K+-ATPase and the Na+-K+-2Cl- cotransporter, respectively. The range and diversity of activity of 20-HETE derives in large measure from COX-dependent transformation of 20-HETE to products affecting vasomotion and salt and water excretion. Nitric oxide (NO) exerts a negative modulatory effect on 20-HETE formation; inhibition of NO synthesis produces marked perturbation of renal function resulting from increased 20-HETE production. 20-HETE is an essential component of interactions involving several hormonal systems that have central roles in blood pressure homeostasis, including angiotensins, endothelins, NO, and cytokines. 20-HETE is the preeminent renal eicosanoid, overshadowing PGE2 and PGI2. This review is intended to provide evidence for the physiological roles for cytochrome P-450-derived eicosanoids, particularly 20-HETE, and seeks to extend this knowledge to a conceptual framework for overall cardiovascular function.
In most arterial beds a significant endothelium-dependent dilation to various stimuli persists even after inhibition of nitric oxide synthase and cyclo-oxygenase. This dilator response is preceded by an endothelium-dependent hyperpolarization of vascular smooth muscle cells, which is sensitive to a combination of the calcium-dependent potassium-channel inhibitors charybdotoxin and apamin, and is assumed to be mediated by an unidentified endothelium-derived hyperpolarizing factor (EDHF). Here we show that the induction of cytochrome P450 (CYP) 2C8/34 in native porcine coronary artery endothelial cells by beta-naphthoflavone enhances the formation of 11,12-epoxyeicosatrienoic acid, as well as EDHF-mediated hyperpolarization and relaxation. Transfection of coronary arteries with CYP 2C8/34 antisense oligonucleotides results in decreased levels of CYP 2C and attenuates EDHF-mediated vascular responses. Thus, a CYP-epoxygenase product is an essential component of EDHF-mediated relaxation in the porcine coronary artery, and CYP 2C8/34 fulfils the criteria for the coronary EDHF synthase.
Mesangial cell proliferation is a characteristic feature of IgA nephropathy and many other forms of glomerulonephritis. Recent clinical studies have shown that dietary fish oil supplementation retards renal disease progression in patients with IgA nephropathy. The mechanism by which this effect occurs is unknown.
The anti-Thy 1.1 (ATS) model of mesangial proliferative glomerulonephritis was employed to test the hypothesis that dietary fish oil supplementation reduces mesangial cell proliferation following acute injury. Subcultured rat mesangial cells were used to determine the in vitro effects of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), the primary components of fish oil, on proliferation.
Following antithymocyte serum (ATS) administration, proteinuria was significantly decreased in animals treated with fish oil compared with sesame oil-treated controls. In ATS rats given fish oil, there was less mesangial cell and matrix expansion, mesangiolysis, or basement membrane disruption (delta% = -40%). ATS rats receiving fish oil had less glomerular cell proliferation (PCNA-delta% = -50%) and a reduction of alpha-smooth muscle actin expression (delta% = -27%) by mesangial cells. In subcultured rat mesangial cells, DHA, but not EPA, significantly inhibited proliferation.
Fish oil inhibits mesangial cell activation and proliferation in ATS glomerulonephritis, reduces proteinuria, and decreases histologic evidence of glomerular damage. In vitro, the antiproliferative effects of fish oil are more likely related to the action of DHA. We suggest that orally administered fish oil, or purified DHA, may have a suppressive effect in acute phases or relapses of glomerulopathies by inhibiting activation and proliferation of mesangial cells.
We recently described autoantibodies (angiotensin-1 receptor autoantibodies, AT(1)-AA) directed at the AT(1) receptor in the serum of preeclamptic patients, whose placentas are commonly infarcted and express tissue factor (TF). Mechanisms of how AT(1)-AA might contribute to preeclampsia are unknown. We tested the hypothesis that AT(1)-AA cause vascular smooth muscle cells (VSMC) to express TF.
IgG from preeclamptic patients containing AT(1)-AA was purified with anti-human IgG columns. AT(1)-AA were separated from the IgG by ammonium sulfate precipitation. We transfected Chinese hamster ovary cells overexpressing the AT(1) receptor with TF promoter constructs coupled to a luciferase reporter gene. VSMC were obtained from human coronary arteries. Extracellular signal-related kinase activation was detected by an in-gel kinase assay. AP-1 activation was determined by electromobility shift assay. TF was measured by ELISA and detected by immunohistochemistry. Placentas from preeclamptic women stained strongly for TF, whereas control placentas showed far less staining. We proved AT(1)-AA specificity by coimmunoprecipitating the AT(1) receptor with AT(1)-AA but not with nonspecific IgG. Angiotensin (Ang) II and AT(1)-AA both activated extracellular signal-related kinase, AP-1, and the TF promoter transfected VSMC and Chinese hamster ovary cells, but only when the AP-1 binding site was present. We then demonstrated TF expression in VSMC exposed to either Ang II or AT(1)-AA. All these effects were blocked by losartan. Nonspecific IgG or IgG from nonpreeclamptic pregnant women had a negligible effect.
We conclude that AT(1)-AA and Ang II both stimulate the AT(1) receptor and initiate a signaling cascade resulting in TF expression. These results show an action of AT(1)-AA on human cells that could contribute to the pathogenesis of preeclampsia.
Low rates of coronary heart disease was found in Greenland Eskimos and Japanese who are exposed to a diet rich in fish oil. Suggested mechanisms for this cardio-protective effect focused on the effects of n-3 fatty acids on eicosanoid metabolism, inflammation, beta oxidation, endothelial dysfunction, cytokine growth factors, and gene expression of adhesion molecules; But, none of these mechanisms could adequately explain the beneficial actions of n-3 fatty acids. One attractive suggestion is a direct cardiac effect of n-3 fatty acids on arrhythmogenesis. N-3 fatty acids can modify Na+ channels by directly binding to the channel proteins and thus, prevent ischemia-induced ventricular fibrillation and sudden cardiac death. Though this is an attractive explanation, there could be other actions as well. N-3 fatty acids can inhibit the synthesis and release of pro-inflammatory cytokines such as tumor necrosis factoralpha (TNFalpha) and interleukin-1 (IL-1) and IL-2 that are released during the early course of ischemic heart disease. These cytokines decrease myocardial contractility and induce myocardial damage, enhance the production of free radicals, which can also suppress myocardial function. Further, n-3 fatty acids can increase parasympathetic tone leading to an increase in heart rate variability and thus, protect the myocardium against ventricular arrhythmias. Increased parasympathetic tone and acetylcholine, the principle vagal neurotransmitter, significantly attenuate the release of TNF, IL-1beta, IL-6 and IL-18. Exercise enhances parasympathetic tone, and the production of anti-inflammatory cytokine IL-10 which may explain the beneficial action of exercise in the prevention of cardiovascular diseases and diabetes mellitus. TNFalpha has neurotoxic actions, where as n-3 fatty acids are potent neuroprotectors and brain is rich in these fatty acids. Based on this, it is suggested that the principle mechanism of cardioprotective and neuroprotective action(s) of n-3 fatty acids can be due to the suppression of TNFalpha and IL synthesis and release, modulation of hypothalamic-pituitary-adrenal anti-inflammatory responses, and an increase in acetylcholine release, the vagal neurotransmitter. Thus, there appears to be a close interaction between the central nervous system, endocrine organs, cytokines, exercise, and dietary n-3 fatty acids. This may explain why these fatty acids could be of benefit in the management of conditions such as septicemia and septic shock, Alzheimer's disease, Parkinson's disease, inflammatory bowel diseases, diabetes mellitus, essential hypertension and atherosclerosis.
In the porcine coronary artery, a cytochrome P450 (CYP) isozyme homologous to CYP 2C8/9 has been identified as an endothelium-derived hyperpolarizing factor (EDHF) synthase. As some CYP enzymes are reported to generate reactive oxygen species (ROS), we hypothesized that the coronary EDHF synthase may modulate vascular homeostasis by the simultaneous production of ROS and epoxyeicosatrienoic acids. In bradykinin-stimulated coronary arteries, antisense oligonucleotides against CYP 2C almost abolished EDHF-mediated responses but potentiated nitric oxide (NO)-mediated relaxation. The selective CYP 2C9 inhibitor sulfaphenazole and the superoxide anion (O(2-)) scavengers Tiron and nordihydroguaretic acid also induced a leftward shift in the NO-mediated concentration-relaxation curve to bradykinin. CYP activity and O(2-) production, determined in microsomes prepared from cells overexpressing CYP 2C9, were almost completely inhibited by sulfaphenazole. Sulfaphenazole did not alter the activity of either CYP 2C8, the leukocyte NADPH oxidase, or xanthine oxidase. ROS generation in coronary artery rings, visualized using either ethidium or dichlorofluorescein fluorescence, was detected under basal conditions. The endothelial signal was attenuated by CYP 2C antisense treatment as well as by sulfaphenazole. In isolated coronary endothelial cells, bradykinin elicited a sulfaphenazole-sensitive increase in ROS production. Although 11,12 epoxyeicosatrienoic acid attenuated the activity of nuclear factor-kappaB in cultured human endothelial cells, nuclear factor-kappaB activity was enhanced after the induction or overexpression of CYP 2C9, as was the expression of vascular cell adhesion molecule-1. These results suggest that a CYP isozyme homologous to CYP 2C9 is a physiologically relevant generator of ROS in coronary endothelial cells and modulates both vascular tone and homeostasis.
NO is an essential cytotoxic agent in host defense, yet can be autotoxic if overproduced, as evidenced in inflammatory lesions and tissue destruction in experimental arthritis models. Treatment of streptococcal cell wal1-induced arthritis in rats with N:(G)-monomethyl-L-arginine (L-NMMA), a competitive nonspecific inhibitor of both constitutive and inducible isoforms of NO synthase (NOS), prevents intraarticular accumulation of leukocytes, joint swelling, and bone erosion. Because increased inducible NOS (iNOS) expression and NO generation are associated with pathogenesis of chronic inflammation, we investigated whether a selective inhibitor of iNOS, N:-iminoethyl-L-lysine (L-NIL), would have more directed anti-arthritic properties. Whereas both L-NMMA and L-NIL inhibited nitrite production by streptococcal cell wall-stimulated rat mononuclear cells in vitro and systemic treatment of arthritic rats with L-NMMA ablated synovitis, surprisingly L-NIL did not mediate resolution of inflammatory joint lesions. On the contrary, daily administration of L-NIL failed to reduce the acute response and exacerbated the chronic inflammatory response, as reflected by profound tissue destruction and loss of bone and cartilage. Although the number of iNOS-positive cells within the synovium decreased after treatment with L-NIL, immunohistochemical analyses revealed a distinct pattern of endothelial and neuronal NOS expression in the arthritic synovium that was unaffected by the isoform-specific L-NIL treatment. These studies uncover a contribution of the constitutive isoforms of NOS to the evolution of acute and chronic inflammation pathology which may be important in the design of therapeutic agents.
During the past 15 years, nitric oxide (NO) and NO synthases have become an important research topic in cellular and molecular biology. NO is produced by many if not all mammalian cells and fulfils a broad spectrum of signaling functions in physiological and pathophysiological processes. In this review, recent advances in our understanding of the mechanisms by which NO regulates the expression of eukaryotic genes will be summarized. The currently available data illustrate that NO has multiple molecular targets: it can not only directly influence the activity of transcription factors but also modulates upstream signaling cascades, mRNA stability and translation, as well as the processing of the primary gene products.
Epoxyeicosatrienoic acids (EETs) are released from endothelial cells and potently dilate small arteries by hyperpolarizing vascular myocytes. In the present study, we investigated the structural specificity of EETs in dilating canine and porcine coronary microvessels (50-140 microm ID) and activating large-conductance Ca2+-activated K+ (BK(Ca)) channels. The potencies and efficacies of EET regioisomers and enantiomers were compared with those of two EET homologs: epoxyeicosaquatraenoic acids (EEQs), which are made from eicosapentaenoic acid by the same cytochrome P-450 epoxygenase that generates EETs from arachidonic acid, and epoxydocosatetraenoic acids (EDTs), which are EETs that are two carbons longer. With EC50 values of 3-120 pM but without regio- or stereoselectivity, EETs potently dilated canine and porcine microvessels. Surprisingly, the EEQs and EDTs had comparable potencies and efficacies in dilating microvessels. Moreover, 50 nM 13,14-EDT activated the BK(Ca) channels with the same efficacy as either 11,12-EET enantiomer at 50 nM. We conclude that coronary microvessels and BK(Ca) channels possess low structural specificity for EETs and suggest that EEQs and EDTs may thereby also be endothelium-derived hyperpolarizing factors.
Nitric oxide (NO) regulates inflammatory responses partly by cell-specific inhibition of the transcription factor nuclear factor kappaB (NF-kappaB). This study investigated the effect of continuous oral administration of an NO donor (molsidomine [Mol]), NO precursor (L-arginine [L-arg]), or selective inhibitors of inducible NO synthase (iNOS; aminoguanidine [AG], L-N(6)-(1-iminoethyl)lysine [L-NIL]) on the progression of tubulointerstitial inflammation and NF-kappaB activation in a non-immune model of chronic glomerular disease (Adriamycin nephropathy [AN]), from day 8 until day 30 after disease induction. On day 30, rats with AN had heavy proteinuria, reduced creatinine clearance, and tubulointerstitial disease. Treatment with both AG and L-NIL exacerbated the progression of AN as evidenced by (1) increased renal cortical malondialdehyde; (2) reduced creatinine clearance; and (3) increased tubular atrophy, interstitial volume, and monocyte infiltration. Unexpectedly, Mol also increased renal malondialdehyde and worsened tubular injury, whereas L-arg had no effect. The increase in renal cortical NF-kappaB activation in AN was not altered by AG, L-NIL, or Mol, but the mRNA expression of monocyte chemoattractant protein-1, interleukin-10, and osteopontin were elevated in these groups. Nitrite release from kidney slices reduced in AN. Treatment with Mol restored renal nitrite release to normal, whereas neither L-arg nor the NOS inhibitors had an effect. It is concluded that endogenous iNOS-derived NO has a protective role against tubulointerstitial injury and cytokine production in AN. However, the pro-oxidant activity of NO donors may limit their potential benefit in proteinuric renal disease.
This article is a report on a symposium sponsored by the American Society for Pharmacology and Experimental Therapeutics and held at the Experimental Biology 01 meeting in Orlando, FL. The presentations addressed the mechanisms of inhibition and regulation of cytochrome P450 and flavin monooxygenase enzymes by nitric oxide. They also highlighted the consequences of these effects on metabolism of drugs and volatile amines as well as on important physiological parameters, such as control of blood pressure, renal ion transport, and steroidogenesis. This is achieved via regulation of P450-dependent prostacyclin, hydroxyeicosatetraenoic acid, and epoxyeicosatrienoic acid formation. Conversely, the mechanisms and relative importance of nitric oxide synthases and P450 enzymes in NO production from endogenous and synthetic substrates were also addressed.
Since the initial reports that renal cytochrome P450 (CYP) enzymes can metabolize arachidonic acid to substances which affect arterial tone, it has become increasingly clear that CYP enzymes expressed within the cardiovascular system play a crucial role in the modulation of vascular homeostasis. There is strong evidence suggesting that the activation of a CYP epoxygenase in endothelial cells is an essential step in nitric oxide and prostacyclin-independent vasodilatation of several vascular beds, particularly in the heart and kidney. A smooth muscle CYP omega-hydroxylase, on the other hand, generates a vasoconstrictor eicosanoid that is central to the myogenic response. Moreover, CYP epoxygenase and omega-hydroxylase products, as well as CYP-derived reactive oxygen species, are intracellular signal transduction molecules involved in several signaling cascades affecting numerous cellular processes, including vascular cell proliferation and angiogenesis. This review summarizes the vascular effects of epoxyeicosatrienoic acids and 20-hydroxyeicosatetraenoic acid, both of which are CYP-derived metabolites of arachidonic acid, endogenously generated within endothelial and vascular smooth muscle cells. Although the link between CYP expression/activity and cardiovascular disease is currently tentative, the evidence being accumulated to suggest that CYP pathways are altered in animal models of hypertension and atherosclerosis can no longer be ignored. The development of selective pharmacological tools is, however, a prerequisite for the analysis of the involvement of specific CYP isoforms in the regulation of vascular homeostasis in human subjects.
A number of laboratories have sought to elucidate the role of nitric oxide (NO) in both acute and chronic inflammatory diseases. It is now well appreciated that NO can influence many aspects of the inflammatory cascade ranging from its own expression to recruitment of leucocytes to the effected tissue. With the advent of mice selectively deficient in the various isoforms of nitric oxide synthase (NOS), the role that NO may play in various disease states can now be examined in vivo. One such syndrome that has gained much attention in recent years is ischaemia and reperfusion-induced tissue injury. Ischaemia-reperfusion (I/R) injury is an important clinical consideration in situations such as transplantation, trauma, liver or bowel resection and haemorrhagic shock. A hallmark of I/R is the production of reactive oxygen species (ROS) during the reperfusion phase and it is thought that the production of ROS mediate much of the post-ischaemic tissue injury. This review will examine the current state of knowledge regarding the regulatory mechanisms by which NO can influence various aspects of the inflammatory cascade as well as its role in a model of I/R injury in vivo.
The fatty acid composition of inflammatory and immune cells is sensitive to change according to the fatty acid composition of the diet. In particular, the proportion of different types of polyunsaturated fatty acids (PUFA) in these cells is readily changed, and this provides a link between dietary PUFA intake, inflammation, and immunity. The n-6 PUFA arachidonic acid (AA) is the precursor of prostaglandins, leukotrienes, and related compounds, which have important roles in inflammation and in the regulation of immunity. Fish oil contains the n-3 PUFA eicosapentaenoic acid (EPA). Feeding fish oil results in partial replacement of AA in cell membranes by EPA. This leads to decreased production of AA-derived mediators. In addition, EPA is a substrate for cyclooxygenase and lipoxygenase and gives rise to mediators that often have different biological actions or potencies than those formed from AA. Animal studies have shown that dietary fish oil results in altered lymphocyte function and in suppressed production of proinflammatory cytokines by macrophages. Supplementation of the diet of healthy human volunteers with fish oil-derived n-3 PUFA results in decreased monocyte and neutrophil chemotaxis and decreased production of proinflammatory cytokines. Fish oil feeding has been shown to ameliorate the symptoms of some animal models of autoimmune disease. Clinical studies have reported that fish oil supplementation has beneficial effects in rheumatoid arthritis, inflammatory bowel disease, and among some asthmatics, supporting the idea that the n-3 PUFA in fish oil are anti-inflammatory and immunomodulatory.
Nitric oxide (NO) released under inflammatory and infectious conditions has been implicated in the down-regulation of many cytochrome P450 genes, but its mechanism of action remains unknown. We showed that the expression of the CYP2D6 gene is down-regulated at the transcriptional level by NO in HepG2 cells. The NO donor (+/-)-N-[(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexene-1-yl]-3-pyridine carboxamide (NOR4) decreased the expression of CYP2D6 mRNA in a concentration-dependent manner. Using a CYP2D6 promoter-luciferase construct, we found that NOR4 and another NO donor, S-nitrosoglutathione (GSNO), reduced the luciferase activity in a concentration-dependent manner. A guanylate-cyclase inhibitor failed to prevent suppression of CYP2D6 promoter activity by GSNO, indicating that the activity of the CYP2D6 promoter is suppressed via an NO-guanylate cyclase-independent pathway. Deletion analysis of the CYP2D6 promoter revealed that the -80 to +65 region, which contains the nuclear receptor hepatocyte nuclear factor-4 (HNF4) binding site, was responsible for the suppression of CYP2D6 promoter activity by NO. Therefore, we examined NO responsiveness of the HNF4 binding site by electrophoretic mobility-shift assays and site-direct mutagenesis. The DNA-binding activity of HNF4 was directly inhibited by NO donors, GSNO, and S-nitroso-N-acetyl-penicillamine in a concentration-dependent manner. Mutation of the HNF4 binding site in the CYP2D6 promoter partially restored the suppression of the promoter activity by NO donors. These results demonstrated that NO down-regulates CYP2D6 gene expression, at least in part, by directly inhibiting HNF4 binding to the CYP2D6 promoter.
Recent studies have indicated that arachidonic acid is primarily metabolized by cytochrome P-450 (CYP) enzymes in the brain, lung, kidney, and peripheral vasculature to 20-hydroxyeicosatetraenoic acid (20-HETE) and epoxyeicosatrienoic acids (EETs) and that these compounds play critical roles in the regulation of renal, pulmonary, and cardiac function and vascular tone. EETs are endothelium-derived vasodilators that hyperpolarize vascular smooth muscle (VSM) cells by activating K(+) channels. 20-HETE is a vasoconstrictor produced in VSM cells that reduces the open-state probability of Ca(2+)-activated K(+) channels. Inhibitors of the formation of 20-HETE block the myogenic response of renal, cerebral, and skeletal muscle arterioles in vitro and autoregulation of renal and cerebral blood flow in vivo. They also block tubuloglomerular feedback responses in vivo and the vasoconstrictor response to elevations in tissue PO(2) both in vivo and in vitro. The formation of 20-HETE in VSM is stimulated by angiotensin II and endothelin and is inhibited by nitric oxide (NO) and carbon monoxide (CO). Blockade of the formation of 20-HETE attenuates the vascular responses to angiotensin II, endothelin, norepinephrine, NO, and CO. In the kidney, EETs and 20-HETE are produced in the proximal tubule and the thick ascending loop of Henle. They regulate Na(+) transport in these nephron segments. 20-HETE also contributes to the mitogenic effects of a variety of growth factors in VSM, renal epithelial, and mesangial cells. The production of EETs and 20-HETE is altered in experimental and genetic models of hypertension, diabetes, uremia, toxemia of pregnancy, and hepatorenal syndrome. Given the importance of this pathway in the control of cardiovascular function, it is likely that CYP metabolites of arachidonic acid contribute to the changes in renal function and vascular tone associated with some of these conditions and that drugs that modify the formation and/or actions of EETs and 20-HETE may have therapeutic benefits.
A particular Lewis rat substrain (LEW/Maa) develops chronic glomerulonephritis in the anti-Thy 1 model (aThy 1-GN) characterized by increased microaneurysm formation, chronic glomerular sclerosis and persistent albuminuria. This phenotype is accompanied by increased and prolonged glomerular induction of inducible nitric oxide synthase (iNOS) when compared to the LEW/Moe substrain, in which aThy 1-GN resolves quickly. We investigated the effect of selective iNOS inhibition by l-N6-(1-iminoethyl)-lysine (L-NIL) administration on aThy 1-GN in LEW/Maa rats.
Nephritic rats were studied over a period of 7 days. L-NIL-treated animals received 20 mg/day L-NIL in the drinking water starting two days prior to disease induction. iNOS activity was determined in cultured glomeruli and in urine samples, respectively. Severity of aThy 1-GN was determined by scoring glomerular matrix expansion and microaneurysm formation, and by albuminuria measurements (ELISA). Immunohistochemical evaluation was performed including staining for macrophages (ED-1), platelets (PL-1) and fibrin deposition.
L-NIL treated rats (+NIL) showed a significant decrease in peak nitrate production by ex vivo cultured glomeruli, and in urinary nitrate excretion versus untreated nephritic rats (-NIL). Mean arterial pressure remained unchanged in both +NIL and -NIL rats. +NIL rats developed significantly increased albuminuria (+44%) associated with a significant increase in glomerular platelet (+45%) and fibrin deposition (+48%).
Selective inhibition of iNOS aggravated albuminuria in chronic aThy 1-GN in LEW/Maa rats. Induction of iNOS during the inflammatory phase of this model may be a partially protective mechanism by interfering with intraglomerular coagulation processes.
The glomerulus is a unique vascular network with the potential to express several isoforms of nitric oxide synthase (NOS). Induction of inducible NOS (iNOS) occurs as part of a rapid initial response to immune injury in glomerulonephritis (GN). Studies on rodent models suggest that this is due to activation of transcription factors by reactive oxygen species (ROS), generated in responses to Fcgamma and CR engagement. iNOS operates in a complex milieu among multiple other inflammatory mediators, changing expression of constitutive NOS (endothelial NOS, eNOS), a critical regulator of glomerular function, and auto-regulating its own expression. As yet there is no consensus as to the role of high output NO generated by iNOS in the glomerulus, although many studies have demonstrated that NO inhibition can alter the level of proteinuria and leukocyte infiltration, and other manifestations of injury such as thrombosis, proliferation, and matrix production. This article reviews the evidence accumulated from experimental studies over the past decade, and discusses how these conflicting data can be reconciled to form a working hypothesis on the role of NO in GN.
P450-dependent arachidonic acid (AA) metabolites regulate arterial tone by modulating calcium-activated (BK) potassium channels in vascular smooth muscle cells (VSMC). Because eicosapentaenoic acid (EPA) has been reported to improve vascular function, we tested the hypothesis that P450-dependent epoxygenation of EPA produces alternative vasoactive compounds. We synthesized the 5 regioisomeric epoxyeicosattrienoic acids (EETeTr) and examined them for effects on K(+) currents in rat cerebral artery VSMCs with the patch-clamp technique. 11(R),12(S)-epoxyeicosatrienoic acid (50 nmol/L) was used for comparison and stimulated K(+) currents 6-fold at +60 mV. However, 17(R),18(S)-EETeTr elicited a more than 14-fold increase. 17(S),18(R)-EET and the remaining four regioisomers were inactive. The effect of 17(R),18(S)-EETeTr was blocked by tetraethylammonium but not by 4-aminopyridine. VSMCs expressed P450s 4A1 and 4A3. Recombinant P450 4A1 hydroxylated EPA at C-19 and C-20 and epoxygenated the 17,18-double bond, yielding the R, S- and S, R-enantiomers in a ratio of 64:36. We conclude that 17(R),18(S)-EETeTr represents a novel, potent activator of BK potassium channels. Furthermore, this metabolite can be directly produced in VSMCs. We suggest that 17(R),18(S)-EETeTr may function as an important hyperpolarizing factor, particularly with EPA-rich diets.
Nitric oxide (NO) plays an important regulatory/modulatory role in a variety of inflammatory conditions. NO is a small, short-lived molecule that is released from a variety of cells in response to homeostatic and pathologic stimuli. It may act as a vasodilator and a platelet inhibitor and may interfere with adhesion molecules to prevent neutrophil adhesion. NO release may also lead to the formation of highly reactive species such as peroxynitrite and stable nitrosothiols and may cause mitochondrial damage and nitration of protein tyrosine residues. In addition, NO inhibits cell proliferation via inhibition of polyamine synthesis and cell uptake and may well act as a 'brake' on the proliferative response following cytokine exposure. All three isoforms of nitric oxide synthases are found in the kidney during inflammation. The site of NO release impacts significantly on its net function and structural impact. NO plays a protective role in many forms of immune injury, such as nephrotoxic serum-induced glomerulonephritis, autoimmune tubular interstitital nephritis, and experimental allergic encephalomyelitis. NO overproduction in sepsis, after cytokine exposure, inducible NO synthase transcription, and local inflammation can autoinhibit endothelial NO synthase, leading to selective renal and mesenteric vasoconstriction.
Transgenic rats overexpressing both human renin and angiotensinogen genes (dTGR) develop hypertension, inflammation, and renal failure. We tested the hypothesis that these pathological features are associated with changes in renal P450-dependent arachidonic acid (AA) metabolism. Samples were prepared from 5- and 7-week-old dTGR and from normotensive Sprague-Dawley (SD) rats, ie, before and after the dTGR developed severe hypertension and albuminuria. At both stages, dTGR showed significantly lower renal microsomal AA epoxygenase and hydroxylase activities that reached 63% and 76% of the control values at week 7. Furthermore, the protein levels of several potential AA epoxygenases (CYP2C11, CYP2C23, and CYP2J) were significantly reduced. Immunoinhibition studies identified CYP2C23 as the major AA epoxygenase, both in dTGR and SD rats. Immunohistochemistry showed that CYP2C23 was localized in cortical and outer medullary tubules that progressively lost this enzyme from week 5 to week 7 in dTGR. CYP2C11 expression occurred only in the outer medullary tubules and was markedly reduced in dTGR compared with age-matched SD rats. These findings indicate site-specific decreases in the availability of AA epoxygenase products in the kidney of dTGR. In contrast to renal microsomes, liver microsomes of dTGR and SD rats showed no change in the expression and activity of AA epoxygenases and hydroxylases. We conclude that hypertension and end-organ damage in dTGR is associated with kidney-specific downregulation of P450-dependent AA metabolism. Because the products of AA epoxygenation have anti-inflammatory properties, this alteration may contribute to uncontrolled renal inflammation, which is a major cause of renal damage in dTGR.
The heart is richly endowed with K(ATP) channels, which function as biological sensors, regulating membrane potentials and electrical excitability in response to metabolic alterations. We recently reported that the cytochrome P450 metabolites of arachidonic acid, epoxyeicosatrienoic acids (EETs), potently activate cardiac K(ATP) channels by reducing channel sensitivity to ATP. In the present study, we further demonstrate that 11(S),12(R)-EET activated the cardiac K(ATP) channels with an EC(50) of 39.5 nM, whereas 11(R),12(S)-EET was totally inactive. In addition, 11(S),12(R)-EET but not 11(R),12(S)-EET hyperpolarized the resting membrane potentials and shortened the duration of cardiomyocyte action potentials. By studying homologs and analogs of 11,12-EET, we also found that all four EET regioisomers are equipotent activators of the K(ATP) channels, reducing the ATP sensitivity by more than 10-fold; however, neither altered chain length, double bond number, epoxide position, nor methylation of the carboxyl group affected channel inhibitions by ATP. All the fatty epoxides studied are potent K(ATP) channel activators, but the omega-3 homolog was particularly potent, reducing ATP sensitivity 27-fold. Together, the results indicate that the presence of an epoxide group in a particular three-dimensional configuration is a critical determinant for K(ATP) channel activation, and its effect is augmented by a double bond at omega-3 position. The results also suggest that fatty epoxides are important modulators of cardiac electrical excitability.
We recently reported that sphingosylphosphorylcholine (SPC) is a novel messenger for Rho-kinase-mediated Ca(2+) sensitization of vascular smooth muscle (VSM) contraction. Subcellular localization and kinase activity of Src family protein kinases (SrcPTKs), except for c-Src, is controlled by a reversible S-palmitoylation, an event inhibited by eicosapentaenoic acid (EPA). We examined the possible involvement of SrcPTKs in SPC-induced Ca(2+) sensitization and effects of EPA. We used porcine coronary VSM and rat aortic VSM cells (VSMCs) in primary culture. An SrcPTKs inhibitor, PP1, and EPA inhibited SPC-induced contraction, concentration-dependently, without affecting [Ca(2+)](i) levels and the Ca(2+)-dependent contraction induced by high K(+) depolarization. A digitized immunocytochemical analysis in VSMCs revealed that SPC induced translocation of Fyn, but not of c-Src, from the cytosol to the cell membrane, an event abolished by EPA. Translocation of Rho-kinase from the cytosol to the cell membrane by SPC was also inhibited by EPA and PP1. The SPC-induced activation of SrcPTKs was blocked by EPA and PP1, but not by Y27632, an Rho-kinase inhibitor. Rho-kinase-dependent phosphorylation of myosin phosphatase induced by SPC was inhibited by EPA, PP1, and Y27632. Translocation and activation of SrcPTKs, including Fyn, play an important role in Ca(2+) sensitization of VSM contractions mediated by a SPC-Rho-kinase pathway.