The short-term effects of C-peptide on the early diabetes-related ultrastructural changes to the podocyte slit diaphragm of glomeruli in rats.

OBJECTIVE: This study aimed to investigate the structural changes in the slit diaphragm, caused by early diabetes, and the nephroprotective effect of C-peptide. METHODS: Streptozotocin-induced type 1 diabetic Wistar rats were divided into control, control plus C-peptide, early diabetes, and early diabetes plus C-peptide groups. C-peptide was infused into rats for one day. The slit diaphragm component proteins podocin, CD2AP, and nephrin, were stained immunofluorescently and their distribution quantitatively analyzed by determining the overlapping area ratio. The interfoot process gap length was measured from electron microscopic images. RESULTS: Diabetic duration correlated best with the area ratio of podocin and CD2AP (r = 0.626), followed by other protein combinations, showing progressive change in the slit diaphragm structure. C-peptide-treatment did not alter area ratios. The interfoot process gap length was wider in diabetic rats (p < 0.05) and did not narrow with C-peptide-treatment in either control or diabetic rats (both p < 0.05). CONCLUSIONS: Diabetes widened the interfoot process gap length and distorted the slit diaphragm structure progressively and heterogeneously in rats with early diabetes; this was not altered by C-peptide-treatment. The nephroprotective effect of C-peptide in decreasing the glomerular filtration rate appears to be functional rather than structural.

In vivo quantitative visualization analysis of the effect of C-peptide on glomerular hyperfiltration in diabetic rats by using multiphoton microscopy.

OBJECTIVE: The purpose of this study was to visualize glomerular hyperfiltration in rats at the early stage of diabetes under in vivo conditions and to quantitatively examine the effect of C-peptide on filtration. METHODS: Type 1 diabetes was induced by streptozotocin (50 mg/kg) in Wistar rats. The rats were divided into four groups: control, control plus C-peptide, early diabetes, and early diabetes plus C-peptide. C-peptide was continuously infused (50 pmol/kg/min). Filtration was visualized by a bolus shot of various sizes of dextrans (3 k to 70 k Da) conjugated with Texas Red and observed with a multiphoton microscope under inhaled anesthesia. Relative sieving coefficients were used to quantify filtration. RESULTS: Almost all smaller particles (3­10 k Da) were filtered both in control and diabetic rats. Filtration of larger particles (40­70 k Da) was seen in normal rats but was more apparent in diabetic rats, where it was progressive according to the duration of diabetes. C-peptide administration restored the leakage of larger particles to the level seen for the control. CONCLUSIONS: We visualized and analyzed hyperfiltration and confirmed that C-peptide has a nephroprotective effect. Furthermore, we found that the leakage of larger particles increased as the duration of diabetes increased.

Increased passive stiffness of cardiomyocytes in the transverse direction and residual actin and myosin cross-bridge formation in hypertrophied rat hearts induced by chronic ß-adrenergic stimulation.

BACKGROUND: Left ventricular (LV) hypertrophy is often present in patients with diastolic heart failure. However, stiffness of hypertrophied cardiomyocytes in the transverse direction has not been fully elucidated. The aim of this study was to assess passive cardiomyocyte stiffness of hypertrophied hearts in the transverse direction and the influence of actin-myosin cross-bridge formation on the stiffness. METHODS AND RESULTS: Wistar rats received a vehicle (control) or isoproterenol (ISO) subcutaneously. After 7 days, compared with the controls, ISO administration had significantly increased heart weight and LV wall thickness and had decreased peak early annular relaxation velocity (e') assessed by echocardiography. Elastic modulus of living cardiomyocytes in the transverse direction assessed by an atomic force microscope was significantly higher in the ISO group than in controls. We added butanedione monoxime (BDM), an inhibitor of actin-myosin interaction, and blebbistatin, a specific myosin II inhibitor, to the medium. BDM and blebbistatin significantly reduced the elastic modulus of cardiomyocytes in the ISO group. X-ray diffraction analysis showed that the reflection intensity ratio (I((1,0))/I((1,1))) at diastole was not different before and after treatment with BDM, which induces complete relaxation, in control hearts, but that I((1,0))/I((1,1)) was significantly increased after BDM treatment in the ISO group, indicating residual cross-bridge formation in hypertrophied hearts. CONCLUSIONS: Passive cardiomyocyte stiffness in the transverse direction is increased in hearts with ISO-induced hypertrophy and this is caused by residual actin-myosin cross-bridge formation.

Intermittent, moderate-intensity aerobic exercise for only eight weeks reduces arterial stiffness: evaluation by measurement of stiffness parameter and pressure-strain elastic modulus by use of ultrasonic echo tracking.

BACKGROUND AND PURPOSE: Aerobic exercise has been reported to be associated with reduced arterial stiffness. However, the intensity, duration, and frequency of aerobic exercise required to improve arterial stiffness have not been established. In addition, most reports base their conclusions on changes in pulse wave velocity, which is an indirect index of arterial stiffness. We studied the effects of short-term, intermittent, moderate-intensity exercise training on arterial stiffness based on measurements of the stiffness parameter (ß) and pressure-strain elastic modulus (E p), which are direct indices of regional arterial stiffness. METHODS: A total of 25 young healthy volunteers (18 men) were recruited. By use of ultrasonic diagnostic equipment we measured ß and E p of the carotid artery before and after 8 weeks of exercise training. RESULTS: After exercise training, systolic pressure (P s), diastolic pressure (P d), pulse pressure, systolic arterial diameter (D s), and diastolic arterial diameter (D d) did not change significantly. However, the pulsatile change in diameter ((D s - D d)/D d) increased significantly, and ß and E p decreased significantly. CONCLUSIONS: For healthy young subjects, ß and E p were reduced by intermittent, moderate-intensity exercise training for only 8 weeks.

Erythropoietin enhances hydrogen peroxide-mediated dilatation of canine coronary collateral arterioles during myocardial ischemia in dogs in vivo.

We have previously demonstrated that endothelium-derived hydrogen peroxide (H(2)O(2)) plays an important role in the canine coronary microcirculation as an endothelium-derived hyperpolarizing factor in vivo. However, it remains to be examined whether endogenous H(2)O(2) is involved in the dilatation of coronary collaterals during myocardial ischemia in vivo and, if so, whether erythropoietin (EPO) enhances the responses. Canine subepicardial native collateral small arteries (CSA; ≥ 100 µm) and arterioles (CA; <100 µm) were observed using an intravital microscope. Experiments were performed after left anterior descending coronary artery ischemia (90 min) under the following eight conditions (n = 5 each): control, EPO, EPO+catalase, EPO+N-monomethyl-l-arginine (l-NMMA), EPO+l-NMMA+catalase, EPO+l-NMMA+iberiotoxin [Ca(2+)-activated K(+) (K(Ca)) channel blocker], EPO+l-NMMA+apamin+charybdotoxin (K(Ca) channel blocker), and EPO+wortmannin (phosphatidylinositol 3-kinase inhibitor). Myocardial ischemia caused significant vasodilatation in CA but not in CSA under control conditions, which was significantly decreased by catalase in CA. After EPO, the vasodilatation was significantly increased in both sizes of arteries and was significantly decreased by catalase. The enhancing effect of EPO was reduced by l-NMMA but not by catalase in CSA and was reduced by l-NMMA+catalase in CA, where the greater inhibitory effects were noted with l-NMMA+catalase, l-NMMA+iberiotoxin, L-NMMA+apamin+charybdotoxin, or wortmannin. EPO significantly ameliorated ischemia-induced impairment of myocardial Akt phosphorylation, which was abolished by l-NMMA+catalase or wortmannin. EPO also ameliorated oxidative stress and myocardial injury, as assessed by plasma 8-hydroxydeoxyguanosine and troponin-T, respectively. These results indicate that EPO enhances H(2)O(2)-mediated dilatation of coronary collateral arterioles during myocardial ischemia in dogs in vivo.

Recent perspective on coronary bifurcation intervention: statement of the "Bifurcation Club in KOKURA".

The treatment of coronary bifurcation lesion remains a challenging issue even in the drug-eluting stent era. Frequent restenosis and stent thrombosis have been recently shown to be related not only to geometrical gap or stent structural deformation but also to rheological disturbance. Low wall shear stress at the lateral side of the bifurcation is likely to cause atherosclerotic changes due to easy access of the macrophages that induce chemical mediators. The turbulent flow over stent metal may facilitate accumulation of platelets, which results in thrombosis. The jailed strut and excess metal overlap may increase these risks. Since dramatic changes of the coronary flow pattern at the bifurcation are closely related to the genesis of atherosclerosis, future bifurcation intervention technique should be considered to restore the original physiological state as well as the anatomical structure. This article summarizes the global consensus of the members of the Asian Bifurcation Club and European Bifurcation Club at the KOKURA meeting. It also provides a perspective of basic sciences relating to bifurcation anatomy, physiology, and pathology, in the search for a best strategy for bifurcation intervention.

The alteration of a mechanical property of bone cells during the process of changing from osteoblasts to osteocytes.

Osteocytes acquire their stellate shape during the process of changing from osteoblasts in bone. Throughout this process, dynamic cytoskeletal changes occur. In general, changes of the cytoskeleton affect cellular mechanical properties. Mechanical properties of living cells are connected with their biological functions and physiological processes. In this study, we for the first time analyzed elastic modulus, a mechanical property of bone cells. Bone cells in embryonic chick calvariae and in isolated culture were identified using fluorescently labeled phalloidin and OB7.3, a chick osteocyte-specific monoclonal antibody, and then observed by confocal laser scanning microscopy. The elastic modulus of living cells was analyzed with atomic force microscopy. To examine the consequences of focal adhesion formation on the elastic modulus, cells were pretreated with GRGDS and GRGES, and then the elastic modulus of the cells was analyzed. Focal adhesions in the cells were visualized by immunofluorescence of vinculin. From fluorescence images, we could distinguish osteoblasts, osteoid osteocytes and mature osteocytes both in vivo and in vitro. The elastic modulus of peripheral regions of cells in all three populations was significantly higher than in their nuclear regions. The elastic modulus of the peripheral region of osteoblasts was 12053+/-934 Pa, that of osteoid osteocytes was 7971+/-422 Pa and that of mature osteocytes was 4471+/-198 Pa. These results suggest that the level of elastic modulus of bone cells was proportional to the stage of changing from osteoblasts to osteocytes. The focal adhesion area of osteoblasts was significantly higher than that of osteocytes. The focal adhesion area of osteoblasts was decreased after treatment with GRGDS, however, that of osteocytes was not. The elastic modulus of osteoblasts and osteoid osteocytes were decreased after treatment with GRGDS. However, that of mature osteocytes was not changed. There were dynamic changes in the mechanical property of elastic modulus and in focal adhesions of bone cells.

Visualisation of the effects of dilazep on rat afferent and efferent arterioles in vivo.

Although the effects of dilazep hydrochloride (dilazep), a nucleoside transport inhibitor, have been examined, there have been no visualisation studies on the physiological effects of dilazep on the glomerular arterioles. The purpose of this study was to visualise and evaluate the effects of dilazep and consequently the effects of adenosine, which dilazep augments by measuring glomelurar diameters, renal blood flow and resistance in rats in vivo. We time-sequentially examined afferent and efferent arteriolar diameter changes using an intravital videomicroscope and renal blood flow. We administered dilazep at a dose of 300 microg/kg intravenously. To further investigate the effects of dilazep, rats were pre-treated with 8-p-sulfophenyl theophylline (a nonselective adenosine receptor antagonist), 8-cyclopentyl-1,3-dipropylxanthine (an A1 receptor antagonist), or 3,7-dimethyl-1-propargylxanthine (an A2 receptor antagonist). Dilazep constricted the afferent and efferent arterioles at the early phase and dilated them at the later phase, with the same degree of vasoconstrictive and vasodilatory effect on both arterioles. A1 blockade abolished vasoconstriction and augmented vasodilatation at the later phase and A2 blockade abolished vasodilatation and augmented vasoconstriction at the early phase. Non-selective blockade abolished both early vasoconstriction and later vasodilatation. In conclusion, adenosine augmented by dilazep constricted the afferent and efferent arterioles of the cortical nephrons at the early phase and dilated both arterioles at the later phase via A1 and A2 adenosine receptor activation, respectively. That the ratio of afferent to efferent arteriolar diameter was fairly constant suggests that intraglomerular pressure is maintained in the acute phase by adenosine despite the biphasic flow change.

Coronary microcirculation in the beating heart.

The phase opposition of velocity waveforms between coronary arteries (predominantly diastolic) and veins (systolic) is the most prominent characteristic of coronary hemodynamics. This unique arterial and venous flow patterns indicate the importance of intramyocardial capacitance vessels and variable resistance vessels during a cardiac cycle. It was shown that during diastole the intramyocardial capacitance vessels have two functional components, unstressed volume and ordinary capacitance. Unstressed volume is defined as the volume of blood in a vessel at zero transmural pressure. In vivo observation of systolic narrowing of arterioles in mid-wall and in subendocardium indicates the increase in resistance by cardiac contraction.

Changes of asymmetric dimethylarginine, nitric oxide, tetrahydrobiopterin, and oxidative stress in patients with acute myocardial infarction by medical treatments.

The relationship among the nitric oxide synthase (NOS) inhibitor [asymmetric dimethylarginine (ADMA)], NOS cofactor [tetrahydrobiopterin (BH(4))], and superoxide anion in the patients with acute myocardial infarction (AMI) is still unknown. This study sought to assess the NOS inhibitor and cofactor with oxidative stress in AMI patients (n=9) during initial administration and 4 weeks after medical treatments. We measured plasma NOS inhibitor and cofactor (ADMA and BH(4)) by HPLC and plasma oxidized-LDL by ELISA. Blood samples from age-matched healthy volunteers (n=9) were taken for comparison. In AMI, plasma ADMA, oxidized-BH(4) (BH(2)+biopterin) and oxidized-LDL significantly increased (P<0.0001, P<0.01 and P<0.05 vs. healthy volunteers) and plasma BH(4), plasma nitrate and L-arginine/ADMA significantly decreased compared with healthy volunteers (P<0.0001, P<0.05 and P<0.005 vs. healthy volunteers). Medical treatments improved plasma ADMA, nitrate, BH(4) and oxidized-LDL. In conclusion, ADMA increased, and NO and BH(4) decreased with oxidative stress in AMI, and these mediators improved in AMI patients with medical treatments. These findings indicated that inhibition of NOS with oxidative stress plays a crucial role in endothelial dysfunction in patients with AMI.

Microheterogeneity of regional myocardial blood flows in low-perfused rat hearts evaluated by double-tracer digital radiography.

Using (3)H- and (125)I-labeled desmethylimipramine (DMI) for regional flow tracers, we established a two-time measurement method for the spatial pattern of myocardial perfusion in cross-circulated rat hearts. Myocardial extractions and retentions of these tracers were confirmed to be satisfactory; however, the latter were less than 90% after 3 min at a perfusion rate of 2.9 ml/min/g, limiting the present application to a short-time perfusion measurement. Distributions of myocardial depositions were separated by subtraction digital radiography with 400-microm pixel resolution. Its feasibility was examined by regression analysis between local deposition densities of (3)H- and (125)I-DMI injected simultaneously. The slope, y-intercept, and correlation coefficient (r) of the regression line were 0.98+/-0.04, 0.02+/-0.04, and 0.95+/-0.03, respectively, indicating the validity of the present image subtraction technique. The spatial pattern of myocardial perfusion in response to flow reduction was evaluated by the injections of (3)H- and (125)I-DMI, respectively, before and after a nearly 70% flow reduction. A significant correlation between normalized density distributions of these tracers was found in both subepicardium (r=0.77+/-0.12) and subendocardium (r=0.73+/-0.20), indicating the stable pattern of myocardial perfusion. However, the coefficient of variation of tracer densities showed a decrease of subendocardial flow heterogeneity from 35+/-15% to 31+/-16%. Thus, flow differences between originally high- and low-flow regions in subendocardium were reduced on a relative basis during low perfusion.

[Confocal microscopy].

Fluorescence-based molecular imaging has long and widely been used in the field of biomedical research, and now has become an indispensable tool. The development of confocal microscopy in the 1980s was a major breakthrough in the field. It can optically slice biological samples along the optical axis, which had been impossible with the conventional fluorescent microscopy. In this review, we first discuss the basic principle, and advantages of the confocal microscopy, and next describe the recent topics on the improved microscopy including high-speed confocal microscopy, FRET(fluorescence resonance energy transfer), FRAP (fluorescence recovery after photobleaching), and two photon excitation microscopy. Finally, we illustrate some examples of the biomedical applications of the confocal microscopy.

Beneficial effect of hydroxyfasudil, a specific Rho-kinase inhibitor, on ischemia/reperfusion injury in canine coronary microcirculation in vivo.

OBJECTIVES: We examined whether hydroxyfasudil, a specific Rho-kinase inhibitor, exerts cardioprotective effect on coronary ischemia/reperfusion (I/R) injury and, if so, whether nitric oxide (NO) is involved. BACKGROUND: Recent studies have demonstrated that Rho-kinase is substantially involved in the pathogenesis of cardiovascular diseases; however, it remains to be examined whether it is also involved in ischemia/reperfusion (I/R) injury. METHODS: Canine subepicardial small arteries (SA, >or=100 microm) and arterioles (A, <100 microm) were observed by a charge-coupled device intravital microscope during I/R. Coronary vascular responses to endothelium-dependent (acetylcholine, intracoronary [IC]) and -independent (papaverine, IC) vasodilators were examined after I/R under the following four conditions: control (n = 7), NO synthase inhibitor alone (N(G)-monomethl-L-arginine [L-NMMA], IC, n = 4), hydroxyfasudil alone (IC, n = 7), and hydroxyfasudil plus L-NMMA (n = 7). RESULTS: Hydroxyfasudil significantly attenuated serotonin (IC)-induced vasoconstriction of SA (-7 +/- 1% vs. 2 +/- 1%, p < 0.01). Coronary I/R significantly impaired coronary vasodilation to acetylcholine after I/R (SA, p < 0.05; and A, p < 0.01 vs. before I/R) and L-NMMA further reduced the vasodilation, whereas hydroxyfasudil completely preserved the responses. The vasoconstriction by L-NMMA after I/R was significantly improved by hydroxyfasudil in both-sized arteries (both p < 0.01). Expression of endothelial nitric oxide synthase (eNOS) protein in the ischemic endocardium of left anterior descending coronary artery area (as determined by Western blotting) significantly decreased (79 +/- 4%) compared with the nonischemic endocardium of LCX area (100 +/- 7%), which was improved by hydroxyfasudil (105 +/- 6%, p < 0.01). Hydroxyfasudil significantly reduced myocardial infarct size, and hydroxyfasudil with L-NMMA also reduced the infarct size compared with L-NMMA alone. CONCLUSIONS: Hydroxyfasudil exerts cardioprotective effects on coronary I/R injury in vivo, in which NO-mediated mechanism may be involved through preservation of eNOS expression.

Cardioprotective effect of hydroxyfasudil as a specific Rho-kinase inhibitor, on ischemia-reperfusion injury in canine coronary microvessels in vivo.

Rho-kinase modulates calcium sensitivity of the myosin light chain in smooth muscle cells and has been implicated as playing a pathogenetic role in cardiovascular disorders. This paper was aimed to determine whether hydroxyfasudil (a specific Rho-kinase inhibitor) exerts cardioprotective effect on coronary ischemia-reperfusion (I/R) injury, and if so, whether NO is involved. Canine subepicardial small arteries (diameter > or = 100 microm) and arterioles (diameter < 100 microm) were observed by a CCD intravital microscope during coronary I/R. Coronary vascular responses to endothelium-dependent (acetylcholine) and -independent (papaverine) vasodilators were examined after I/R under three conditions: control, preconditioning, and hydroxyfasudil. Coronary I/R significantly impaired coronary vasodilation to acetylcholine, whereas hydroxyfasudil completely preserved the responses, as did preconditioning. Hydroxyfasudil also significantly reduced myocardial infarct size. These results indicated that hydroxyfasudil exerts cardioprotective effects on coronary I/R injury in vivo, for which NO-mediated mechanism may be involved.

Hydrogen peroxide, an endogenous endothelium-derived hyperpolarizing factor, plays an important role in coronary autoregulation in vivo.

BACKGROUND: Recent studies in vitro have demonstrated that endothelium-derived hydrogen peroxide (H2O2) is an endothelium-derived hyperpolarizing factor (EDHF) in animals and humans. The aim of this study was to evaluate our hypothesis that endothelium-derived H2O2 is an EDHF in vivo and plays an important role in coronary autoregulation. METHODS AND RESULTS: To test this hypothesis, we evaluated vasodilator responses of canine (n=41) subepicardial small coronary arteries (> or =100 microm) and arterioles (<100 microm) with an intravital microscope in response to acetylcholine and to a stepwise reduction in coronary perfusion pressure (from 100 to 30 mm Hg) before and after inhibition of NO synthesis with N(G)-monomethyl-L-arginine (L-NMMA). After L-NMMA, the coronary vasodilator responses were attenuated primarily in small arteries, whereas combined infusion of L-NMMA plus catalase (an enzyme that selectively dismutates H2O2 into water and oxygen) or tetraethylammonium (TEA, an inhibitor of large-conductance K(Ca) channels) attenuated the vasodilator responses of coronary arteries of both sizes. Residual arteriolar dilation after L-NMMA plus catalase or TEA was largely attenuated by 8-sulfophenyltheophylline, an adenosine receptor inhibitor. CONCLUSIONS: These results suggest that H2O2 is an endogenous EDHF in vivo and plays an important role in coronary autoregulation in cooperation with NO and adenosine.

Dynamic changes in three-dimensional architecture and vascular volume of transmural coronary microvasculature between diastolic- and systolic-arrested rat hearts.

BACKGROUND: The phase difference of coronary arterial and venous flows indicates the importance of intramyocardial capacitance vessels in storing diastolic flow and in discharging volume in systole. However, the anatomic and functional characteristics of the capacitance vessels are unclear. We aimed to clarify those characteristics with their transmural difference by 3D visualization of transmural microvessels under diastole and systole. METHODS AND RESULTS: We performed complete intracoronary filling of a contrast medium into Langendorff's Wistar rat hearts under (1) St Thomas-perfused diastolic arrest (D-mode) and (2) BaCl(2)-induced systolic arrest (S-mode). Precise transmural 3D architectures of capillaries and of pre- and post-capillary microvessels (ie, microvessels larger than capillaries) were visualized clearly with a confocal laser scanning microscope and x-ray microcomputed tomography (microCT), respectively. Vascular volume fraction (VF) and systolic-induced VF reduction rate from D- to S-mode were analyzed. The net capillary VF in D-mode (20.4 +/- 0.9%) was 10 times that of larger microvessels and was reduced in S-mode by 32% without capillary collapse. Systolic-induced VF reduction rate was smaller in capillaries than in larger microvessels (48%; P<0.05). The larger microvessel VF in D-mode (2.2 +/-0.2%) was reduced in S-mode, accompanied by complicated 3D deformation. CONCLUSIONS: Capillaries were relatively resistant to the systolic extravascular compression compared with pre- and post-capillary microvessels, conveniently beneficial for the myocardial oxygen delivery throughout a cardiac cycle. Nevertheless, a larger change in the absolute volume of capillaries may function as effective capacitance. On one hand, the pre- and post-capillary microvessels showed a larger phasic change in resistance, which may function to maintain the capillary patency during systole.

Regression of capillary network in atrophied soleus muscle induced by hindlimb unweighting.

Little is known about the mechanisms responsible for the adaptation and changes in the capillary network of hindlimb unweighting (HU)-induced atrophied skeletal muscle, especially the coupling between functional and structural alterations of intercapillary anastomoses and tortuosity of capillaries. We hypothesized that muscle atrophy by HU leads to the apoptotic regression of the capillaries and intercapillary anastomoses with their functional alteration in hemodynamics. To clarify the three-dimensional architecture of the capillary network, contrast medium-injected rat soleus muscles were visualized clearly using a confocal laser scanning microscope, and sections were stained by terminal deoxynucleotidyltransferase-mediated dUTP nick-end labeling (TUNEL) and with anti-von Willebrand factor. In vivo, the red blood cell velocity of soleus muscle capillaries were determined with a pencil-lens intravital microscope brought into direct contact with the soleus surface. After HU, the total muscle mass, myofibril protein mass, and slow-type myosin heavy chain content were significantly lower. The number of capillaries paralleling muscle fiber and red blood cells velocity were higher in atrophied soleus. However, the mean capillary volume and capillary luminal diameter were significantly smaller after HU than in the age-matched control group. In addition, we found that the number of anastomoses and the tortuosity were significantly lower and TUNEL-positive endothelial cells were observed in atrophied soleus muscles, especially the anastomoses and/or tortuous capillaries. These results indicate that muscle atrophy by HU generates structural alterations in the capillary network, and apoptosis appears to occur in the endothelial cell of the muscle capillaries.

Role of asymmetrical dimethylarginine in renal microvascular endothelial dysfunction in chronic renal failure with hypertension.

We examined whether endothelial function of the renal microcirculation was impaired in a model of chronic renal failure (CRF), and further assessed the role of asymmetrical dimethylarginine (ADMA) and its degrading enzyme, dimethylarginine dimethylaminohydrolase (DDAH), in mediating the deranged nitric oxide (NO) synthesis in CRF. CRF was established in male mongrel dogs by subtotal nephrectomy, and the animals were used in experiments after a period of 4 weeks. The endothelial function of the renal afferent and efferent arterioles was evaluated according to the response to acetylcholine, using an intravital needle-lens charge-coupled device camera. Intrarenal arterial infusion of acetylcholine (0.01 microg/kg/min) elicited 22+/-2% and 20+/-2% dilation of the afferent and efferent arterioles in normal dogs. In dogs with CRF, this vasodilation was attenuated (afferent, 12+/-2%; efferent, 11+/-1%), and the attenuation paralleled the diminished increments in urinary nitrite+nitrate excretion. In the animals with CRF, plasma concentrations of homocysteine (12.2+/-0.7 vs. 6.8+/-0.4 micromol/l) and ADMA were elevated (2.60+/-0.13 vs. 1.50+/-0.08 micromol/l). The inhibition of S-adenosylmethionine-dependent protein arginine N-methyltransferase by adenosine dialdehyde decreased plasma ADMA levels, and improved the acetylcholine-induced changes in urinary nitrite+nitrate excretion and arteriolar vasodilation. Acute methionine loading impaired the acetylcholine-induced renal arteriolar vasodilation in CRF, but not normal dogs, and the impairment in CRF dogs coincided with the changes in plasma ADMA levels. Real-time polymerase chain reaction revealed downregulation of the mRNA expression of DDAH-II in the dogs with CRF. Collectively, these results provide direct in vivo evidence of endothelial dysfunction in canine CRF kidneys. The endothelial dysfunction was attributed to the inhibition of the NO production by elevated ADMA, which involved the downregulation of DDAH-II. The deranged NO metabolic pathway including ADMA and DDAH is a novel mechanism for the aggravation of renal function.

Increased pulmonary heme oxygenase-1 and delta-aminolevulinate synthase expression in monocrotaline-induced pulmonary hypertension.

Monocrotaline (MCT), a pyrrolizidine alkaloid plant toxin, is known to cause pulmonary hypertension (PH) in rats. Recent findings suggest that pulmonary inflammation may play a significant role in the pathogenesis in MCT-induced PH. Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme catabolism, is known to be induced by various oxidative stresses, including inflammation and free heme, and its induction is thought essential in the protection against oxidative tissue injuries. In this study, we examined expression of HO-1 as well as non-specific delta-aminolevulinate synthase (ALAS1), the rate-limiting enzyme in heme catabolism and biosynthesis, respectively, in a rat model of PH produced by subcutaneous injection of MCT (60 mg/kg). MCT treatment caused infiltration of inflammatory cells, fibrosis of the interstitium, and pulmonary arterial wall thickening with marked elevation of right ventricular (RV) pressure, which are characteristics of MCT-induced PH. Gene expression of tumor necrosis factor-alpha (TNF-alpha) as well as DNA binding activity of nuclear factor-kappaB (NF-kappaB) increased at 1 week after MCT treatment, reached a maximum at 2 weeks, and then decreased to the pretreatment level at 3 weeks. HO-1 expression was markedly increased at 1 week, and continued to increase by 3 weeks following MCT treatment, both at transcriptional and protein levels in the mononuclear cells in the lung. ALAS1 mRNA levels in the lung also significantly increased at 2 weeks after MCT treatment. These findings suggest that pulmonary HO-1 expression was presumably induced by proinflammatory cytokine(s) in MCT-treated rats, resulting in the derepression of heme-repressible ALAS1 expression, and that HO-1 induction plays a significant role as an inflammatory factor in this condition.