Carrie M. Elks

Brain nuclear factor-kappa B activation contributes to neurohumoral excitation in angiotensin II-induced hypertension.

AIMS Angiotensin II (ANG II)-induced inflammatory and oxidative stress responses contribute to the pathogenesis of hypertension. In this study, we determined whether nuclear factor-kappa B (NF-kappaB) activation in the hypothalamic paraventricular nucleus (PVN) increases oxidative stress and contributes to the ANG II-induced hypertensive response. METHODS AND RESULTS Rats were infused intravenously with ANG II (10 ng/kg per min) or saline for 4 weeks. These rats received either vehicle or losartan (LOS, 20 microg/h), an angiotensin II type 1 receptor (AT1-R) antagonist; pyrrolidine dithiocarbamate (PDTC, 5 microg/h), a NF-kappaB inhibitor; tempol (TEMP, 80 microg/h), a superoxide scavenger; LOS (20 microg/h), and PDTC (5 microg/h); or TEMP (80 microg/h) and PDTC (5 microg/h), given intracerebroventricularly (ICV) via osmotic minipump. ANG II infusion resulted in increased mean arterial pressure, renal sympathetic nerve activity, plasma proinflammatory cytokines (PIC), norepinephrine, and aldosterone. These rats also had higher levels of Fra-LI (an indicator of chronic neuronal activation), PIC, phosphorylated IKKbeta, NF-kappaB subunits, AT1-R, superoxide, and gp91phox (a subunit of NADP(H) oxidase) and lower levels of IkappaBalpha in the PVN than control animals. ICV treatment with LOS, PDTC, or TEMP attenuated these changes, and combined treatment with ICV LOS and PDTC, or ICV TEMP and PDTC prevented these ANG II-induced hypertensive responses. CONCLUSION These findings suggest that an ANG II-induced increase in the brain renin-angiotensin system activates NF-kappaB in the PVN and contributes to sympathoexcitation in hypertension. The increased superoxide in the PVN contributes to NF-kappaB activation and neurohumoral excitation in hypertension.

NF-κB-induced oxidative stress contributes to mitochondrial and cardiac dysfunction in type II diabetes

AIMS Inflammatory molecules and their transcription factor, nuclear factor kappa-B (NF-kappaB), are thought to play important roles in diabetes-induced cardiac dysfunction. Here, we investigated the effects of pyrrolidine dithiocarbamate (PDTC), a NF-kappaB inhibitor, in diabetic mice. METHODS AND RESULTS Obese db/db mice and heterozygous lean mice (n = 8) were allowed free access to drinking water (control) or water containing PDTC (100 mg/kg) for 20 weeks. Left ventricular (LV) function was measured using echocardiography at baseline and at study end. Mice were sacrificed and LV removed for gene expression, biochemical, immunofluorescence, and mitochondrial assays. LV and mitochondrial reactive oxygen species (ROS), superoxide and peroxynitrite were measured using electron spin resonance spectroscopy. Enhanced NF-kappaB activity in db/db mice was associated with increased oxidative stress as demonstrated by increased ROS, superoxide, and peroxynitrite production, and increased NF-kappaB, gp91phox, and Nox1 expression; PDTC ameliorated these effects. Mitochondrial free radical production and structural damage were higher in the db/db group than in the control, db/db PDTC, and PDTC-treated heterozygous animal groups. CONCLUSION This study demonstrates that NF-kappaB blockade with PDTC mitigates oxidative stress and improves mitochondrial structural integrity directly, through down-regulation of increased oxygen-free radicals, thereby increasing ATP synthesis and thus restoring cardiac function in type II diabetes.

HDAC Inhibition Attenuates Inflammatory, Hypertrophic, and Hypertensive Responses in Spontaneously Hypertensive Rats

Reactive oxygen species and proinflammatory cytokines contribute to cardiovascular diseases. Inhibition of downstream transcription factors and gene modifiers of these components are key mediators of hypertensive response. Histone acetylases/deacetylases can modulate the gene expression of these hypertrophic and hypertensive components. Therefore, we hypothesized that long-term inhibition of histone deacetylase with valproic acid might attenuate hypertrophic and hypertensive responses by modulating reactive oxygen species and proinflammatory cytokines in SHR rats. Seven-week-old SHR and WKY rats were used in this study. Following baseline blood pressure measurement, rats were administered valproic acid in drinking water (0.71% wt/vol) or vehicle, with pressure measured weekly thereafter. Another set of rats were treated with hydralazine (25 mg/kg per day orally) to determine the pressure-independent effects of HDAC inhibition on hypertension. Following 20 weeks of treatment, heart function was measured using echocardiography, rats were euthanized, and heart tissue was collected for measurement of total reactive oxygen species, as well as proinflammatory cytokine, cardiac hypertrophic, and oxidative stress gene and protein expressions. Blood pressure, proinflammatory cytokines, hypertrophic markers, and reactive oxygen species were increased in SHR versus WKY rats. These changes were decreased in valproic acid-treated SHR rats, whereas hydralazine treatment only reduced blood pressure. These data indicate that long-term histone deacetylase inhibition, independent of the blood pressure response, reduces hypertrophic, proinflammatory, and hypertensive responses by decreasing reactive oxygen species and angiotensin II type1 receptor expression in the heart, demonstrating the importance of uncontrolled histone deacetylase activity in hypertension.

Chronic NF-κB blockade reduces cytosolic and mitochondrial oxidative stress and attenuates renal injury and hypertension in SHR

Nuclear factor-kappaB (NF-kappaB) plays an important role in hypertensive renal injury; however, its roles in perpetuating mitochondrial oxidative stress and renal dysfunction remain unclear. In this study, we assessed the effects of chronic NF-kappaB blockade with pyrrolidine dithiocarbamate (PDTC) on renal dysfunction and mitochondrial redox status in spontaneously hypertensive rats (SHR). PDTC (150 mg.kg body wt(-1).day(-1)) or vehicle was administered orally to 8-wk-old SHR and their respective controls for 15 wk. Systolic blood pressure (SBP) was measured by tail-cuff plethysmography at the start of and at every third week throughout the study. After 15 wk of treatment, anesthetized rats underwent acute renal experiments to determine renal blood flow and glomerular filtration rate using PAH and inulin clearance techniques, respectively. Following renal experiments, kidneys were excised from killed rats, and cortical mitochondria were isolated for reactive oxygen species (ROS) measurements using electron paramagnetic resonance. Tissue mRNA and protein levels of NF-kappaB and oxidative stress genes were determined using real-time PCR and immunofluorescence or Western blotting, respectively. PDTC treatment partially attenuated the increase in SBP (196.4 +/- 9.76 vs. 151.4 +/- 2.12; P < 0.05) and normalized renal hemodynamic and excretory parameters and ATP production rates in SHR. PDTC treatment also attenuated the higher levels of cytosolic and mitochondrial ROS generation and tissue mRNA and protein expression levels of NF-kappaB and oxidative stress genes in SHR without any comparable responses in control rats. These findings suggest that NF-kappaB activation by ROS induces the cytosolic and mitochondrial oxidative stress and tissue injury that contribute to renal dysfunction observed in SHR.

Cross talk between cytokines and renin-angiotensin in hypothalamic paraventricular nucleus in heart failure: role of nuclear factor-κB

AIMS Nuclear factor-kappa B (NF-kappaB) is a potent inducer of pro-inflammatory cytokines (PIC) and oxidative stress in cardiovascular disease. In this study, we determined whether upregulation of NF-kappaB in the hypothalamic paraventricular nucleus (PVN) contributed to neurohumoral excitation either directly, or via interaction with the renin-angiotensin system (RAS), in heart failure (HF). METHODS AND RESULTS Rats were implanted with intracerebroventricular (ICV) cannulae and subjected to coronary artery ligation, or sham surgery (SHAM). Subsequently, animals were ICV treated with the angiotensin type 1 receptor (AT1-R) antagonist losartan (LOS, 20 microg/h), or SN50 (2 microg/h), which inhibits nuclear translocation of NF-kappaB, or tempol (TEMP, 80 microg/h), a membrane-permeable superoxide scavenger, or vehicle for 4 weeks. HF induced a significant increase in the expression of AT1-R, PIC, and NAD(P)H oxidase genes and NF-kappaB p50 in the PVN and in plasma norepinephrine (NE) levels when compared with SHAM rats. In contrast, ICV LOS, SN50, or TEMP attenuated PIC, NF-kappaB p50, AT1-R and NAD(P)H oxidase genes in the PVN compared with vehicle-treated HF rats. Treatment with LOS, SN50, or TEMP also reduced plasma levels of NE, angiotensin II, and PIC, and decreased left ventricular end diastolic pressure. CONCLUSION These findings indicate that NF-kappaB mediates the cross-talk between RAS and PIC in the PVN in HF, and that superoxide stimulates more NF-kappaB in the PVN and contributes to neurohumoral excitation.

Brain tumour necrosis factor-α modulates neurotransmitters in hypothalamic paraventricular nucleus in heart failure

AIMS Increased proinflammatory cytokines after myocardial infarction augment the progression of heart failure (HF) and are of prognostic significance. Recently, we demonstrated that increased proinflammatory cytokines in the brains of HF rats increased paraventricular nucleus (PVN) superoxide and down-regulated neuronal nitric oxide synthase (nNOS), contributing to sympathoexcitation. In this study, we explored the possible roles of brain proinflammatory cytokines and their effects on modulating PVN neurotransmitters in the exaggerated sympathetic activity in HF. METHODS AND RESULTS Sprague-Dawley rats with HF or sham-operated control (SHAM) rats were treated for 4 weeks with a continuous intracerebroventricular (ICV) infusion of the cytokine blockers-pentoxifylline (PTX, 10 microg/h and 40 microg/h), etanercept (ETN, 5 microg/h and 10 microg/h), or vehicle. Another set of HF and SHAM rats were treated with intraperitoneal (ip) infusion of a similar dose of PTX or ETN. HF rats had increased neuronal excitation accompanied by higher levels of glutamate, norepinephrine (NE), and tyrosine hydroxylase (TH), and lower levels of gamma-aminobutyric acid (GABA), nNOS, and 67-kDa isoform of glutamate decarboxylase (GAD67) in the PVN when compared with SHAM rats. Plasma cytokines, NE, epinephrine, angiotensin II, and renal sympathetic nerve activity (RSNA) were also increased in HF rats. ICV treatment with low doses of PTX or ETN attenuated, and high doses prevented, increases in levels of glutamate, NE, and TH, and decreases in levels of GABA, nNOS, and GAD67 in the PVN in HF rats. The same ICV treatments also attenuated the increased RSNA seen in HF rats. IP treatment with similar doses of PTX or ETN did not affect glutamate, NE, TH, GABA, nNOS, and GAD67 in the PVN and had no effect on RSNA of HF rats. CONCLUSION This study, for the first time, demonstrates that proinflammatory cytokines modulate neurotransmitters in the PVN and contribute to sympathoexcitation in HF.

TNF-induced mitochondrial damage: a link between mitochondrial complex I activity and left ventricular dysfunction

Mitochondrial damage is implicated in the progression of cardiac disease. Considerable evidence suggests that proinflammatory cytokines induce oxidative stress and contribute to cardiac dysfunction. This study was conducted to determine whether a TNF-induced increase in superoxide (O(2)(*)(-)) contributes to mitochondrial damage in the left ventricle (LV) by impairing respiratory complex I activity. We employed an electron paramagnetic resonance (EPR) method to measure O(2)(*)(-) and oxygen consumption in mitochondrial respiratory complexes, using an oxygen label. Adult male Sprague-Dawley rats were divided into four groups: control, TNF treatment (ip), TNF+ apocynin (APO; 200 micromol/kg bw, orally), and TNF+ Tempol (Temp; 300 micromol/kg bw, orally). TNF was injected daily for 5 days. Rats were sacrificed, LV tissue was collected, and mitochondria were isolated for EPR studies. Total LV ROS production was significantly higher in TNF animals than in controls; APO or Temp treatment ameliorated TNF-induced LV ROS production. Total mitochondrial ROS production was significantly higher in the TNF and TNF+ APO groups than in the control and TNF+ Temp groups. These findings suggest that TNF alters the cellular redox state, reduces the expression of four complex I subunits by increasing mitochondrial O(2)(*)(-) production and depleting ATP synthesis, and decreases oxygen consumption, thereby resulting in mitochondrial damage and leading to LV dysfunction.

A Blueberry-Enriched Diet Attenuates Nephropathy in a Rat Model of Hypertension via Reduction in Oxidative Stress

Objective and Background To assess renoprotective effects of a blueberry-enriched diet in a rat model of hypertension. Oxidative stress (OS) appears to be involved in the development of hypertension and related renal injury. Pharmacological antioxidants can attenuate hypertension and hypertension-induced renal injury; however, attention has shifted recently to the therapeutic potential of natural products as antioxidants. Blueberries (BB) have among the highest antioxidant capacities of fruits and vegetables. Methods and Results Male spontaneously hypertensive rats received a BB-enriched diet (2% w/w) or an isocaloric control diet for 6 or 12 weeks or 2 days. Compared to controls, rats fed BB-enriched diet for 6 or 12 weeks exhibited lower blood pressure, improved glomerular filtration rate, and decreased renovascular resistance. As measured by electron paramagnetic resonance spectroscopy, significant decreases in total reactive oxygen species (ROS), peroxynitrite, and superoxide production rates were observed in kidney tissues in rats on long-term dietary treatment, consistent with reduced pathology and improved function. Additionally, measures of antioxidant status improved; specifically, renal glutathione and catalase activities increased markedly. Contrasted to these observations indicating reduced OS in the BB group after long-term feeding, similar measurements made in rats fed the same diet for only 2 days yielded evidence of increased OS; specifically, significant increases in total ROS, peroxynitrite, and superoxide production rates in all tissues (kidney, brain, and liver) assayed in BB-fed rats. These results were evidence of “hormesis” during brief exposure, which dissipated with time as indicated by enhanced levels of catalase in heart and liver of BB group. Conclusion Long-term feeding of BB-enriched diet lowered blood pressure, preserved renal hemodynamics, and improved redox status in kidneys of hypertensive rats and concomitantly demonstrated the potential to delay or attenuate development of hypertension-induced renal injury, and these effects appear to be mediated by a short-term hormetic response.

Diet-induced renal changes in Zucker rats are ameliorated by the superoxide dismutase mimetic TEMPOL.

Diabetic nephropathy is the leading cause of renal failure in the United States. The obese Zucker rat (OZR; fa/fa) is a commonly used model of type 2 diabetes and metabolic syndrome (MetS), and of the nephropathy and renal oxidative stress commonly seen in these disorders. Heterozygous lean Zucker rats (LZRs; fa/+) are susceptible to high‐fat diet (HFD)‐induced obesity and MetS. The present study was designed to investigate whether 4‐hydroxy‐2,2,6,6‐tetramethylpiperidine‐N‐oxyl (TEMPOL), a membrane‐permeable radical scavenger, could alleviate the renal effects of MetS in OZR and LZR fed a HFD, which resembles the typical “Western” diet. OZR and LZR were fed a HFD (OZR‐HFD and LZR‐HFD) or regular diet (OZR‐RD and LZR‐RD) and allowed free access to drinking water or water containing 1 mmol/l TEMPOL for 10 weeks. When compared to OZR‐RD animals, OZR‐HFD animals exhibited significantly higher levels of total renal cortical reactive oxygen species (ROS) production, plasma lipids, insulin, C‐reactive protein, blood urea nitrogen (BUN), creatinine (Cr), and urinary albumin excretion (P < 0.05); these changes were accompanied by a significant decrease in plasma high‐density lipoprotein levels (P < 0.05). The mRNA expression levels of desmin, tumor necrosis factor‐α (TNF‐α), nuclear factor κB (NFκB), and NAD(P)H oxidase‐1 (NOX‐1) were significantly higher in the renal cortical tissues of OZR‐HFD animals; NFκB p65 DNA binding activity as determined by electrophoretic mobility shift assay was also significantly higher in these animals. The same trends were noted in LZR‐HFD animals. Our data demonstrate that TEMPOL may prove beneficial in treating the early stages of the nephropathy often associated with MetS.

Chronic Exercise Preserves Renal Structure and Hemodynamics in Spontaneously Hypertensive Rats

AIMS Exercise training (ExT) is a recommended adjunct to many pharmaceutical antihypertensive therapies. The effects of chronic ExT on the development of hypertension-induced renal injury remain unknown. We examined whether ExT would preserve renal hemodynamics and structure in the spontaneously hypertensive rat (SHR), and whether these effects were mediated by improved redox status and decreased inflammation. Normotensive WKY rats and SHR underwent moderate-intensity ExT for 16 weeks. One group of SHR animals was treated with hydralazine to investigate the pressure-dependent/independent effects of ExT. Acute renal clearance experiments were performed prior to sacrifice. Tissue free radical production rates were measured by electron paramagnetic resonance; gene and protein expression were measured by real time RT-PCR and Western blot or immunofluorescence, respectively. Plasma angiotensin II levels and kidney antioxidants were assessed. Training efficacy was assessed by citrate synthase activity assay in hind-limb muscle. RESULTS ExT delayed hypertension, prevented oxidative stress and inflammation, preserved antioxidant status, prevented an increase in circulating AngII levels, and preserved renal hemodynamics and structure in SHR. In addition, exercise-induced effects, at least, in part, were found to be pressure-independent. INNOVATION This study is the first to provide mechanistic evidence for the renoprotective benefits of ExT in a model of hypertension. Our results demonstrate that initiation of ExT in susceptible patients can delay the development of hypertension and provide renoprotection at the functional and ultrastructural level. CONCLUSION Chronic ExT preserves renal hemodynamics and structure in SHR; these effects are partially mediated by improved redox status and decreased inflammation.