OBJECTIVEDespite the beneficial effects of resveratrol (RSV) on cardiovascular disease and life span, its effects on type 2 diabetic nephropathy remain unknown. This study examined the renoprotective effects of RSV in db/db mice, a model of type 2 diabetes.RESEARCH DESIGN AND METHODSdb/db mice were treated with RSV (0.3% mixed in chow) for 8 weeks. We measured urinary albumin excretion (UAE), histological changes (including mesangial expansion, fibronectin accumulation, and macrophage infiltration), oxidative stress markers (urinary excretion and mitochondrial content of 8-hydroxy-2'-deoxyguanosine [8-OHdG], nitrotyrosine expression), and manganese-superoxide dismutase (Mn-SOD) activity together with its tyrosine-nitrated modification and mitochondrial biogenesis in the kidney. Blood glucose, glycated hemoglobin, and plasma lipid profiles were also measured. The phosphorylation of 5′-AMP–activated kinase (AMPK) and expression of silent information regulator 1 (SIRT1) in the kidney were assessed by immunoblotting.RESULTSRSV significantly reduced UAE and attenuated renal pathological changes in db/db mice. Mitochondrial oxidative stress and biogenesis were enhanced in db/db mice; however, Mn-SOD activity was reduced through increased tyrosine-nitrated modification. RSV ameliorated such alterations and partially improved blood glucose, glycated hemoglobin, and abnormal lipid profile in db/db mice. Activation of AMPK was decreased in the kidney of db/db mice compared with db/m mice. RSV neither modified AMPK activation nor SIRT1 expression in the kidney.CONCLUSIONSRSV ameliorates renal injury and enhanced mitochondrial biogenesis with Mn-SOD dysfunction in the kidney of db/db mice, through improvement of oxidative stress via normalization of Mn-SOD function and glucose-lipid metabolism. RSV has antioxidative activities via AMPK/SIRT1-independent pathway.

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A high performance liquid chromatographic (HPLC) method with chemiluminescence (CL) detection was developed for the sensitive determination of 1,3-diaminopyrene (1,3-DAP), 1,6-DAP, 1,8-DAP and 1-aminopyrene (1-AP). The HPLC conditions were as follows: column, Cosmosil 5C18 (4.6 mm i.d.X250 mm); mobile phase, 10 mM imidazoleperchloric acid buffer(pH 7.6)-acetonitrile (1:1, v/v); CL reagent, 0.02 mM bis(2,4,6-trichlorophenyl)oxalate (TCPO) and 15 mM hydrogen peroxide in acetonitrile. The oxidative degradation of DAPS and AP in the presence of metals was prevented by adding ascorbic acid to sample solutions. The calibration curves were straight over 2 orders of magnitude for all analytes, and their detection limits (as S/ N was 3) were in the sub-fmol range. Dinitro-and nitropyrenes in sooty emissions of diesel-and gasoline-engine cars could be determined by this HPLC after reductive conversion into DAPs and AP, respectively, by refluxing the samples in the presence of sodium hydrosulfide. Nitrated polycyclic aromatic hydrocarbons (NPAHs) have attracted much attention because of their potent mutagenic activity.' They are formed as undesirable by-products when various organic materials react with atmospheric nitrogen during combustion2,3 and are found in many environmental samples.4-6 Dinitropyrenes (DNPs) are the most mutagenic and carcinogenic of these NPAHs.'_9 Both gas chromatography (GC)10 and GC/ mass spectrometry6 have been reported as sensitive determination methods for DNPs. However, tedious procedures are required for cleaning and concentrating samples. On the other hand, a high performance liquid chromatographic (HPLC) method with fluorescence (FL) detection has also been reported' 1, with which DNPs were determined after a reductive conversion of DNPs into diaminopyrenes (DAPs). Although the FL detection was so selective that cleaning procedures became simpler, the sensitivity was not sufficiently high to detect them at trace levels in the environment.Recently, peroxyoxalate-chemiluminescence (P0-CL) detection has been developed as a highly sensitive and selective detection method for HPLC.'2,13 In our preliminary experiment, PO-CL detection was much more sensitive for DAPs and 1-AP than was FL detection. The observed peak heights, however, were significantly smaller than those from the extrapolated calibration curves when the injection amounts were less than pmol. This phenomenon disturbed the trace analysis. We found that DAPs and 1-AP were very unstable in the presence of metals and that this reaction was the main reason for the above phenomenon. The problem was solved by adding an antioxidant, such as ascorbic acid (AA), enabling the DAPs and 1-AP to be determined at trace levels. The purpose of this report is to propose an HPLC method with PO-CL detection for 1,3-, 1,6-, 1,8-DAPs and 1-AP whose sensitivities are much higher than those with FL detection. ExperimentalChemicals and solutions 1,3-, 1,6-and 1,8-DAPs and 1-AP were prepared according to a report by Hashimoto et a1.14 All other chemicals we...

The transient receptor potential vanilloid 1 (TRPV1) channel is highly expressed in a subset of sensory neurons in the dorsal root ganglia (DRG) and trigeminal ganglia of experimental animals, responsible for nociception. Many researches have revealed that some TRPV1-positive neurons co-express the transient receptor potential ankyrin 1 (TRPA1) channel whose activities are closely modulated by TRPV1 channel. However, it is less investigated whether the activities of TRPV1 channel are modulated by the presence of TRPA1 channel in primary sensory neurons. This study clarified the difference in electrophysiological responses induced by TRPV1 channel activation between TRPA1-positive and TRPA1-negative DRG. TRPV1 and TRPA1 channel activations were evoked by capsaicin (1 μM), a TRPV1 agonist, and allyl isothiocyanate (AITC; 500 μM), a TRPA1 agonist, respectively. Capsaicin perfusion for 15 s caused a large inward current without a desensitization phase at a membrane potential of −70 mV in AITC-insensitive DRG (current density; 29.6 ± 5.6 pA/pF, time constant of decay; 12.8 ± 1.8 s). The capsaicin-induced currents in AITC-sensitive DRG had a small current density (12.7 ± 2.9 pA/pF) with a large time constant of decay (24.3 ± 5.4 s). In calcium imaging with Fura-2, the peak response by capsaicin was small and duration reaching the peak response was long in AITC-sensitive neurons. These electrophysiological differences were completely eliminated by HC-030031, a TRPA1 antagonist, in an extracellular solution or 10 mM EGTA, a Ca2+ chelator, in an internal solution. Capsaicin perfusion for 120 s desensitized the inward currents after a transient peak. The decay during capsaicin perfusion was notably slow in AITC-sensitive DRG; ratio of capsaicin-induced current 60 s after the treatment per the peak current in AITC-sensitive neurons (78 ± 9%) was larger than that in AITC-insensitive neurons (48 ± 5%). The capsaicin-induced current in the desensitization phase was attenuated by HC-030031 in AITC-insensitive DRG. These results indicate that (1) TRPV1-mediated currents in TRPA1-positive neurons characterize small current densities with slow decay, which is caused by TRPA1 channel activities and intracellular Ca2+ mobilization and (2) desensitization of TRPV1-mediated current in TRPA1-positive neurons is apparently slow, due to appending TRPA1-mediated current.

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