Chunyan Hu

Methylglyoxal contributes to the development of insulin resistance and salt sensitivity in Sprague-Dawley rats.

Objectives Methylglyoxal, a metabolite of the glycolysis pathway, may play an important role in the development of diabetes and hypertension, but the exact mechanism has not been fully elucidated. The present study was designed to investigate whether methylglyoxal could directly induce insulin resistance and salt sensitivity in Sprague–Dawley rats. Methods Rats were allocated to four groups: control (normal drinking water), 1% methylglyoxal in drinking water, 1% methylglyoxal plus N-acetyl cysteine (NAC) (800 mg/kg per day), a methylglyoxal scavenger, or TM2002 (100 mg/kg per day), an advanced glycation endproducts (AGEs) inhibitor. After 4-week treatment insulin resistance was evaluated by an euglycemic hyperinsulinemic glucose clamp technique. In another set of rats, either a high-salt diet (4%) alone, standard rat chow with 1% methylglyoxal in drinking water or high-salt diet plus methylglyoxal was given for 4 weeks. Immunohistochemistry was performed to measure nitrotyrosine and methylglyoxal-induced AGEs, Nϵ-carboxyethyl-lysine (CEL) in the kidney. Results Four-week treatment with NAC or TM2002 completely improved methylglyoxal-induced insulin resistance. Co-administration of methylglyoxal and high-salt diet significantly increased systolic blood pressure, urinary albumin excretion, urinary thiobarbituric acid-reactive substances excretion and the renal nitrotyrosine expression in the kidney (markers of oxidative stress) compared with methylglyoxal or high-salt diet alone. Renal CEL was significantly increased in methylglyoxal-treated rats compared with nonmethylglyoxal-treated rats. Conclusion These results indicate that methylglyoxal-induced insulin resistance and salt sensitivity at least in part by increasing oxidative stress and/or AGEs formation in Sprague–Dawley rats. The present study provides further evidence for methylglyoxal as one of the causative factors in the pathogenesis of insulin resistance and salt-sensitive hypertension.

Effects of Renal Perfusion Pressure on Renal Medullary Hydrogen Peroxide and Nitric Oxide Production

Studies were designed to determine the effects of increases of renal perfusion pressure on the production of hydrogen peroxide (H2O2) and NO2−+NO3− within the renal outer medulla. Sprague-Dawley rats were studied with either the renal capsule intact or removed to ascertain the contribution of changes of medullary blood flow and renal interstitial hydrostatic pressure on H2O2 and NO2−+NO3− production. Responses to three 30-minute step changes of renal perfusion pressure (from ≈85 to ≈115 to ≈145 mm Hg) were studied using adjustable aortic occluders proximal and distal to the left renal artery. Medullary interstitial H2O2 determined by microdialysis increased at each level of renal perfusion pressure from 640 to 874 to 1593 nmol/L, as did H2O2 urinary excretion rates, and these responses were significantly attenuated by decapsulation. Medullary interstitial NO2−+NO3− increased from 9.2 to 13.8 to 16.1 &mgr;mol/L, with parallel changes in urine NO2−+NO3−, but decapsulation did not significantly blunt these responses. Over the range of renal perfusion pressure, medullary blood flow (laser-Doppler flowmetry) rose ≈30% and renal interstitial hydrostatic pressure rose from 7.8 to 19.7 cm H2O. Renal interstitial hydrostatic pressure and the natriuretic and diuretic responses were significantly attenuated with decapsulation, but medullary blood flow was not affected. The data indicate that pressure-induced increases of H2O2 emanated largely from increased tubular flow rates to the medullary thick-ascending limbs of Henle and NO largely from increased medullary blood flow to the vasa recta. The parallel pressure–induced increases of H2O2 and NO indicate a participation in shaping the “normal” pressure-natriuresis relationship and explain why an imbalance in either would affect the blood pressure salt sensitivity.

Carbonyl stress induces hypertension and cardio-renal vascular injury in Dahl salt-sensitive rats.

One major precursor of carbonyl stress, methylglyoxal (MG), is elevated in the plasma of chronic kidney disease (CKD) patients, and this precursor contributes to the progression of vascular injury, hypertension and renal injury in diabetic nephropathy patients. This molecule induces salt-sensitive hypertension via a reactive oxygen species-mediated pathway. We examined the role of MG in the pathogenesis of hypertension and cardio–renal injury in Dahl salt-sensitive (Dahl S) rats, which is a rat model of CKD. Nine-week-old Dahl S rats were fed a 1% NaCl diet, and 1% MG was added to their drinking water for up to 12 weeks. Blood pressure and cardio–renal injuries were compared with rats treated with tap water alone. The angiotensin II receptor blocker (ARB), candesartan (10 mg kg−1 day−1), was administered to MG Dahl S rats to determine the impact of this drug on the pathogenesis of MG-induced CKD. A progressive increase in systolic blood pressure was observed (123±1–148±5 mm Hg) after 12 weeks of MG administration. MG administration significantly increased urinary albumin excretion, glomerular sclerosis, tubular injury, myocardial collagen content and cardiac perivascular fibrosis. MG also enhanced the renal expression of Nɛ-carboxyethyl-lysine (an advanced glycation end product), 8-hydroxydeoxyguanosine (a marker of oxidative stress), macrophage (ED-1) positive cells (a marker of inflammation) and nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase activity. Candesartan treatment for 4 weeks significantly reduced these parameters. These results suggest that MG-induced hypertension and cardio–renal injury and increased inflammation and carbonyl and oxidative stress, which were partially preventable by an ARB.

Losartan modulates muscular capillary density and reverses thiazide diuretic-exacerbated insulin resistance in fructose-fed rats

The renin–angiotensin system (RAS) is involved in the pathogenesis of insulin sensitivity (IS). The role of RAS in insulin resistance and muscular circulation has yet to be elucidated. Therefore, this study sought to determine the mechanisms of angiotensin II receptor blockers (ARBs) and/or diuretics on IS and capillary density (CD) in fructose-fed rats (FFRs). Sprague-Dawley rats were fed either normal chow (control group) or fructose-rich chow for 8 weeks. For the last 4 weeks, FFRs were allocated to four groups: an FFR group and groups treated with the thiazide diuretic hydrochlorothiazide (HCTZ), with the ARB losartan, or both. IS was evaluated by the euglycemic hyperinsulinemic glucose clamp technique at week 8. In addition, CD in the extensor digitorum longus muscle was evaluated. Blood pressure was significantly higher in the FFRs than in the controls. HCTZ, losartan and their combination significantly lowered blood pressure. IS was significantly lower in the FFR group than in the controls and was even lower in the HCTZ group. Losartan alone or combined with HCTZ significantly increased IS. In all cases, IS was associated with muscular CD, but not with plasma adiponectin or lipids. These results indicate that losartan reverses HCTZ-exacerbated insulin resistance, which can be mediated through the modulation of muscular circulation in rats with impaired glucose metabolism.

Interference with PPARγ in endothelium accelerates angiotensin II-induced endothelial dysfunction

The ligand activated nuclear receptor peroxisome proliferator-activated receptor γ (PPARγ) in the endothelium regulates vascular function and blood pressure (BP). We previously reported that transgenic mice (E-V290M) with selectively targeted endothelial-specific expression of dominant negative PPARγ exhibited endothelial dysfunction when treated with a high-fat diet, and exhibited an augmented pressor response to angiotensin II (ANG II). We hypothesize that interference with endothelial PPARγ would exacerbate ANG II-induced endothelial dysfunction. Endothelial function was examined in E-V290M mice infused with a subpressor dose of ANG II (120 ng·kg(-1)·min(-1)) or saline for 2 wk. ANG II infusion significantly impaired the responses to the endothelium-dependent agonist acetylcholine both in basilar and carotid arteries from E-V290M but not NT mice. This impairment was not due to increased BP, which was not significantly different in ANG II-infused E-V290M compared with NT mice. Superoxide levels, and expression of the pro-oxidant Nox2 gene was elevated, whereas expression of the anti-oxidant genes Catalase and SOD3 decreased in carotid arteries from ANG II-infused E-V290M mice. Increased p65 and decreased Iκ-Bα suggesting increased NF-κB activity was also observed in aorta from ANG II-infused E-V290M mice. The responses to acetylcholine were significantly improved both in basilar and carotid arteries after treatment with Tempol (1 mmol/l), a scavenger of superoxide. These findings provide evidence that interference with endothelial PPARγ accelerates ANG II-mediated endothelial dysfunction both in cerebral and conduit arteries through an oxidative stress-dependent mechanism, suggesting a role for endothelial PPARγ in protecting against ANG II-induced endothelial dysfunction.

Cullin-3 mutation causes arterial stiffness and hypertension through a vascular smooth muscle mechanism

Cullin-3 (CUL3) mutations (CUL3Δ9) were previously identified in hypertensive patients with pseudohypoaldosteronism type-II (PHAII), but the mechanism causing hypertension and whether this is driven by renal tubular or extratubular mechanisms remains unknown. We report that selective expression of CUL3Δ9 in smooth muscle acts by interfering with expression and function of endogenous CUL3, resulting in impaired turnover of the CUL3 substrate RhoA, increased RhoA activity, and augmented RhoA/Rho kinase signaling. This caused vascular dysfunction and increased arterial pressure under baseline conditions and a marked increase in arterial pressure, collagen deposition, and vascular stiffness in response to a subpressor dose of angiotensin II, which did not cause hypertension in control mice. Inhibition of total cullin activity increased the level of CUL3 substrates cyclin E and RhoA, and expression of CUL3Δ9 decreased the level of the active form of endogenous CUL3 in human aortic smooth muscle cells. These data indicate that selective expression of the Cul3Δ9 mutation in vascular smooth muscle phenocopies the hypertension observed in Cul3Δ9 human subjects and suggest that mutations in CUL3 cause human hypertension in part through a mechanism involving smooth muscle dysfunction initiated by a loss of CUL3-mediated degradation of RhoA.

PPARγ Regulation in Hypertension and Metabolic Syndrome

Dysregulation of peroxisome proliferator-activated receptor gamma (PPARγ) activity leads to significant alterations in cardiovascular and metabolic regulation. This is most keenly observed by the metabolic syndrome-like phenotypes exhibited by patients carrying mutations in PPARγ. We will summarize recent findings regarding mechanisms of PPARγ regulation in the cardiovascular and nervous systems focusing largely on PPARγ in the smooth muscle, endothelium, and brain. Canonically, PPARγ exerts its effects by regulating the expression of target genes in these cells, and we will discuss mechanisms by which PPARγ targets in the vasculature regulate cardiovascular function. We will also discuss emerging evidence that PPARγ in the brain is a mediator of appetite and obesity. Finally, we will briefly review how novel PPARγ activators control posttranslational modifications of PPARγ and their prospects to offer new therapeutic options for treatment of metabolic diseases without the adverse side effects of thiazolidinediones which strongly activate transcriptional activity of PPARγ.

Endothelial PPAR-γ provides vascular protection from IL-1β-induced oxidative stress

Loss of peroxisome proliferator-activated receptor (PPAR)-γ function in the vascular endothelium enhances atherosclerosis and NF-κB target gene expression in high-fat diet-fed apolipoprotein E-deficient mice. The mechanisms by which endothelial PPAR-γ regulates inflammatory responses and protects against atherosclerosis remain unclear. To assess functional interactions between PPAR-γ and inflammation, we used a model of IL-1β-induced aortic dysfunction in transgenic mice with endothelium-specific overexpression of either wild-type (E-WT) or dominant negative PPAR-γ (E-V290M). IL-1β dose dependently decreased IκB-α, increased phospho-p65, and increased luciferase activity in the aorta of NF-κB-LUC transgenic mice. IL-1β also dose dependently reduced endothelial-dependent relaxation by ACh. The loss of ACh responsiveness was partially improved by pretreatment of the vessels with the PPAR-γ agonist rosiglitazone or in E-WT. Conversely, IL-1β-induced endothelial dysfunction was worsened in the aorta from E-V290M mice. Although IL-1β increased the expression of NF-κB target genes, NF-κB p65 inhibitor did not alleviate endothelial dysfunction induced by IL-1β. Tempol, a SOD mimetic, partially restored ACh responsiveness in the IL-1β-treated aorta. Notably, tempol only modestly improved protection in the E-WT aorta but had an increased protective effect in the E-V290M aorta compared with the aorta from nontransgenic mice, suggesting that PPAR-γ-mediated protection involves antioxidant effects. IL-1β increased ROS and decreased the phospho-endothelial nitric oxide synthase (Ser(1177))-to-endothelial nitric oxide synthase ratio in the nontransgenic aorta. These effects were completely abolished in the aorta with endothelial overexpression of WT PPAR-γ but were worsened in the aorta with E-V290M even in the absence of IL-1β. We conclude that PPAR-γ protects against IL-1β-mediated endothelial dysfunction through a reduction of oxidative stress responses but not by blunting IL-1β-mediated NF-κB activity.

Retinol-binding protein 7 is an endothelium-specific PPARγ cofactor mediating an antioxidant response through adiponectin

Impaired PPARγ activity in endothelial cells causes oxidative stress and endothelial dysfunction which causes a predisposition to hypertension, but the identity of key PPARγ target genes that protect the endothelium remain unclear. Retinol-binding protein 7 (RBP7) is a PPARγ target gene that is essentially endothelium specific. Whereas RBP7-deficient mice exhibit normal endothelial function at baseline, they exhibit severe endothelial dysfunction in response to cardiovascular stressors, including high-fat diet and subpressor angiotensin II. Endothelial dysfunction was not due to differences in weight gain, impaired glucose homeostasis, or hepatosteatosis, but occurred through an oxidative stress-dependent mechanism which can be rescued by scavengers of superoxide. RNA sequencing revealed that RBP7 was required to mediate induction of a subset of PPARγ target genes by rosiglitazone in the endothelium including adiponectin. Adiponectin was selectively induced in the endothelium of control mice by high-fat diet and rosiglitazone, whereas RBP7 deficiency abolished this induction. Adiponectin inhibition caused endothelial dysfunction in control vessels, whereas adiponectin treatment of RBP7-deficient vessels improved endothelium-dependent relaxation and reduced oxidative stress. We conclude that RBP7 is required to mediate the protective effects of PPARγ in the endothelium through adiponectin, and RBP7 is an endothelium-specific PPARγ target and regulator of PPARγ activity.

Role of specific T-type calcium channel blocker R(-) efonidipine in the regulation of renal medullary circulation.

Objectives: Blockade of the T-type calcium channel (TCC), which is expressed in the renal efferent arterioles of the juxtamedullary nephron and vasa recta, has been shown to protect against renal injury. Studies were designed to determine the effects of a specific TCC blocker, R(−) efonidipine [R(−)EFO], on the regulation of renal circulation. Methods and results: Renal medullary blood flux (MBF) and cortical blood flux (CBF) were simultaneously monitored using laser-Doppler flowmetry in Sprague-Dawley rats. Responses were also determined in rats with angiotensin II (AngII) induced renal ischemia. Intravenous (i.v.) or renal interstitial (r.i.) infusion of R(−)EFO (0.25 mg/h, i.v. or r.i.) significantly increased MBF by 24.0 ± 7.0 and 21.0 ± 4.4%, respectively, but without changing CBF or mean arterial pressure. The nitric oxide (NO) synthase inhibitor NG-nitro-L-argininemethylester (L-NAME, 1 &mgr;g/kg per min, i.v. or r.i.) significantly attenuated R(−)EFO-induced increase in MBF. R(−)EFO inhibited the AngII-mediated (50 ng/kg per min, i.v.) reduction of MBF (28.4 ± 1.7%), which was associated with increased urinary NO2− + NO3− excretion and decreased urinary hydrogen peroxide (H2O2) excretion. Intracellular H2O2 fluorescence (real-time fluorescence imaging) in the epithelial cells of isolated medullary thick ascending limb (mTAL) significantly increased following AngII stimulation (1 &mgr;mol/L, 235 ± 52 units), which was significantly inhibited by pre and coincubation with R(−)EFO. R(−)EFO stimulation also increased the intracellular NO concentration in the epithelial cells of mTAL (220 ± 62 units). Conclusion: These results suggest that TCC blockade with R(−)EFO selectively increases MBF, an effect that appears to be mediated by changes in renal NO and oxidative stress balance, which may protect against ischemic renal injury in the renal medullary region.