Palmitic Acid-Induced Podocyte Apoptosis via the Reactive Oxygen Species-Dependent Mitochondrial Pathway.

BACKGROUND/AIMS: Chronic kidney disease (CKD) is often accompanied by hyperlipidemia, which accelerates progression of the disease. Podocyte injury can lead to dysfunction of the glomerular filtration barrier, which is associated with proteinuria, a risk marker for the progression of CKD. Our previous studies demonstrated that palmitic acid (PA) can induce podocyte apoptosis; however, the underlying mechanisms are unclear. In the present study, we investigated the specific molecular mechanisms of PA-induced apoptosis in cultured podocytes. METHODS: We cultured mouse podocytes and treated them with PA. Then, cell viability was measured using the Cell Counting Kit-8 colorimetric assay, lipid uptake was assessed by Oil Red O staining and boron-dipyrromethene staining, apoptosis was measured by flow cytometry, mitochondrial injury was assessed by JC-1 staining and transmission electron microscopy, and mitochondrial production of reactive oxygen species (ROS) was evaluated by fluorescence microscopy using the MitoSOX Red reagent. The effects of PA on the mitochondria-mediated caspase activation pathway were investigated by examining the expression of caspase-8, cleaved caspase-9, cleaved caspase-3, cleaved poly (ADP-ribose) polymerase (PARP), B-cell lymphoma 2 (Bcl-2), Bax, Bid, cytochrome c, and Fas-associated protein with death domain (FADD) using western blotting. The translocation of Bax and cytochrome c were detected by immunofluorescence. RESULTS: PA treatment significantly increased lipid accumulation and induced podocyte apoptosis. We investigated whether the two primary apoptosis signaling pathways (death receptor-mediated pathway and mitochondria-mediated pathway) were involved in the execution of PA-induced podocyte apoptosis, and found that the levels of FADD, caspase-8, and Bid did not significantly change during this process. Meanwhile, PA treatment induced an increase in Bax protein expression and a decrease in Bcl-2 protein expression, with Bax translocation to the mitochondria. Furthermore, PA treatment induced mitochondrial impairment, and triggered the release of cytochrome c from the mitochondria to cytosol, with a concomitant dose-dependent increase in the levels of cleaved caspase-9, cleaved caspase-3, and PARP. Meanwhile, PA treatment increased mitochondrial production of ROS, and the mitochondria-targeted antioxidant mitoTEMPO significantly ameliorated PA-induced podocyte apoptosis. CONCLUSION: Our findings indicated that PA induced caspase-dependent podocyte apoptosis through the mitochondrial pathway, and mitochondrial ROS production participated in this process, thus potentially contributing to podocyte injury.

[Study on High-yield Cultivation Measures for Arctii Fructus].

OBJECTIVE: To find out the high yield cultivation measures for Arctii Fructus. METHODS: Completely randomized block experiment design method was used in the field planting, to analyze the effect of different cultivation way on agronomic characters, phenological phase,quality and quantity of Arctii Fructus. RESULTS: Arctium lappa planted on August 28 had the best results of plant height, thousand seeds weight and yield. The highest yield of Arctii Fructus was got at the density of 1,482 plants/667 m2. Arctiin content was in an increase trend with the planting time delay and planting density increasing. The plant height, thousand seeds weight, yield and arctiin content by split application of fertilizer were significantly higher than that by one-time fertilization. Compared with open field Arctium lappa, plant height, yield, arctiin content and relative water content of plastic film mulching Arctium lappa was higher by 7.74%, 10.87%, 6.38% and 24.20%, respectively. In the topping Arctium lappa, the yield was increased by 11.09%, with 39. 89% less branching number. Early planting time and topping shortened the growth cycle of Arctium lappa plant. CONCLUSION: The high-yield cultivation measures of Arctii Fructus are: around August 28 to sowing, planting density of 1 482 plants/667 m2, split application of fertilizer for four times, covering film on surface of the soil and topping in bolting.

Optimization, validation and application of an assay for the activity of HMG-CoA reductase in vitro by LC-MS/MS.

A stable HMG-CoA reductase (HMGR) reaction in vitro was developed by a sensitive, selective and precise liquid chromatography-tandem mass spectrometry (LC-MS/MS) method. The optimized enzyme reaction condition contained 1.5 µg of HMGR, 20 nM of NADPH with 50 min of reaction time. The method was validated by several intra- and inter-day assays. The production transitions of m/z 147.0/59.1 and m/z 154.0/59.1 were used to detect and quantify mevalonolactone (MVAL) and MVAL-D7, respectively. The accuracy and precision of the method were evaluated over the concentration range of 0.005-1.000 µg/mL for MVAL and 0.010-0.500 µg/mL for lovastatin acid in three validation batch runs. The lower limit of quantitation was found to be 0.005 µg/mL for MVAL and 0.010 µg/mL for lovastatin acid. Intra-day and inter-day precision ranged from 0.95% to 2.39% and 2.26% to 3.38% for MVAL, 1.46% to 3.89% and 0.57% to 5.10% for lovastatin acid, respectively. The results showed that the active ingredients in Xuezhikang capsules were 12.2 and 14.5 mg/g, respectively. This assay method could be successfully applied to the quality control study of Xuezhikang capsule for the first time.

Ultra-high-performance liquid chromatography for the determination of exenatide in monkey plasma by tandem quadrupole mass spectrometry.

A highly sensitive ultra-high-performance liquid chromatographic-tandem mass spectrometry (UPLC/MS/MS) method was developed for the quantification of the synthetic peptide drug of exenatide in monkey plasma. Sample preparation was carried out by solid-phase extraction (SPE), and bivalirudin was used as the internal standard (IS). An excellent chromatographic separation was obtained on a reversed-phase C18 column with a gradient elution. Detection utilized a Qtrap 5500 system operated in the positive ion mode with multiple reaction monitoring (MRM). The proposed method was validated by assessing the specificity, linearity, intra- and inter-day precision and accuracy, recovery, and stability. The method resulted in a linear calibration range of 0.10-30 ng/mL, extracting with only 50 µL monkey plasma aliquots. The intra- and inter-day precisions (as relative standard deviation) were less than 7.5% and 9.6%, respectively. The method could be successfully utilized for the pharmacokinetic study of exenatide in monkeys following a single subcutaneous injection of Byetta.

GABAA receptors in VTA mediate the morphine-induced release of ascorbic acid in rat nucleus accumbens.

Local perfusion of morphine produces increased levels of extracellular ascorbic acid (AA) in the nucleus accumbens (NAc) of freely moving rats. However, the pathways that regulate morphine-induced AA release in the NAc are unclear. In the present study, we used high performance liquid chromatography with electrochemical detection (HPLC-ECD) to examine the effects of intra-ventral tegmental area (VTA) administration of a GABA(A) agonist and antagonist on morphine-induced increases in AA of the NAc. Also, using high performance liquid chromatography with fluorescent detection (HPLC-FD) and HPLC-ECD, the releases of γ-aminobutyric acid (GABA) and dopamine (DA) in the NAc induced by intra-VTA administration of a GABA(A) agonist and antagonist were also investigated. The results obtained showed that morphine (1 mM), locally perfused into the NAc, significantly increased AA release in the NAc and also GABA release. Intra-VTA infusion of bicuculline (150 ng/rat), a GABA receptor antagonist, not only abolished the enhanced extracellular AA and GABA levels produced by local perfusion of morphine but also decreased the basal release of extracellular GABA and increased the basal release of extracellular DA in the NAc. Muscimol (100 ng/rat), a GABA receptor agonist, affected the basal release of GABA and DA, but not the basal AA levels, or the morphine-induced changes in AA and GABA levels. These findings suggest that the GABA(A) receptors in the VTA play an important role in the modulation of morphine-induced AA release in the NAc, and the effect of morphine on AA release in the NAc is partially regulated by the GABA(A) receptor-mediated action of DA afferents from the VTA.