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@#To establish a RP-HPLC method for the determination of content of sisomicin sulfate and sodium chloride injection. Thermo Aminoglycoside RP 18(4. 6 mm ×150 mm, 3 μm)column was used. The mobile phase consisted of Sodium heptane sulfonate solution(take 6 g of sodium heptane sulfonate, add 0. 1 mol/L potassium dihydrogen phosphate solution and dilute to 1 000 mL, adjust the pH to 1. 5 with phosphoric acid)- acetonitrile(77∶23). The detection wavelength was 205 nm, the flow rate was 1. 0 mL/min. and the column temperature was 35 °C. The separation of sisomicin peaks with related substances and the degradation products was good. The linear range of the peak area with sisomicin was 0. 010 024-1. 002 4 mg/mL(Y=4. 210 2×106 X+9. 107 0×103, r=0. 999 9, n=7), the detection limit was 0. 6 ng, the limit of quantification was 2 ng, and the recovery rate was at 99. 1%-100. 9%(RSD< 1. 0%, n=9). The method is sensitive, exclusive, accurate and suitable for the determination of sisomicin. Compared with the antibiotic microbiological test method, the specificity is better, the confidence interval of the result is narrowed, and the test time is saved.
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Objective To explore the molecular effect and interaction among nephrin, podocin, CD2AP and ?-actinin-4. Methods Firstly, the recombinant RNA interference (RNAi) plasmid-psiRNA-hH1GFPzeo, specifically targeting to the mRNA of nephrin, podocin, CD2AP or ?-actinin-4, was respectively tansfected into the mouse podocyte clone (MPC5) to each knockdown (KD) the expression of nephrin, podocin, CD2AP or ?-actinin-4. Molecular distributions were revealed by confocal microscopy, and the mRNA and protein expressions were detected with semi-quantitative RT-PCR and Western blotting. Results (1)In podocin KD group (siPod966 and siPod54), the mRNAs of podocin and nephrin were not detected, their protein decreased 92% and 79%, 82% and 67%, respectively. The mRNA and protein level of CD2AP increased 62% and 42%, 71% and 46%, respectively, whereas ?-actinin-4 did not change. In nephrin KD group (siNep492), the mRNA expression and protein level of nephrin were not detected, CD2AP increased 35% and 48%, respectively; and whereas podocin and ?-actinin-4 did not change. In CD2AP KD group (siCda744 and siCda21), the mRNA of expression CD2AP was not detected, and its protein level decreased 92% and 83%, the mRNA and protein of nephrin decreased 60% and 48%, 76% and 72%, respectively; podocin increased 38% and 22%, 56% and 44%, respectively; whereas ?-actinin-4 did not change. In ?-actinin-4 KD group (siAct1790 and siAct319), the mRNAs expression of ?-actinin-4 and nephrin decreased 69% and 58%, 64% and 49%, respectively; their protein level decreased 81% and 55%, 71% and 64%, respectively. However, the mRNAs of podocin and CD2AP increased 50% and 34%, 45% and 28%, respectively; and their protein level increased 64% and 46%, 65% and 42%, respectively. (2) With their expression change, the distributions of nephrin, podocin and CD2AP shifted evidently from the cell membrane surface to the nucleus circumference, whereas ?-actinin-4 showed no change, which was still localized in the cytoplasm and further extended to foot processes. Conclusion (1) Nephrin might more independently play a crucial role in the slit diaphragm complex. (2) Alpha-actinin-4 might interact direcdy or indirectly with nephrin, podocin and CD2AP. (3) The relationship among these podocyte molecules might not be spontaneous, either a single-directional or bi-directional reaction. (4) The normal localization of these podocyte molecules might depend on their normal expression quantity.
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Objective:To establish a podocyte cell injury model induced by puromycin aminonucleoside(PAN),an in vitro model for studying the role of podocytes,especial the slit diaphragm molecules in proteinuria at the cellular and molecular levels.Methods:MPC5 were treated for 24 and 48 hours by 15,45 and 75 mg/L PAN,respectively.The podocyte molecular behavior during podocyte injury was evaluated:the apoptotic podocyte cells were revealed with FITC-Annexin V and Propidium Iodide(PI) assay and the proliferative podocyte cells detected with MTT assay after PAN treatment.The distribution of Nephrin and Podocin was revealed with indirect-immnofluorescent staining under confocal microscope.The distribution of F-actin was revealed with direct-immnofluorescent staining under microscope.Results:The percentage of apoptotic podocyte cells was increased in a dose-and time-dependent manner after PAN treatment.In PAN-treated group,the apoptosis was obviously increased at hour 48,the PAN-45 treated group was 33.48%?14.55% and PAN-75 treated group 38.01%?12.13% vs the control group 6.38%?0.50%(P
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Objective To explore the molecular mechanisms underlying therapeutic responses of the anti-proteinuria drugs from the view of podocyte molecule. Methods Adriamycin (ADR) nephropathy was induced by a single tail intravenous injection of adriamycin. Lisinopril, prednisone and all-trans retinoic acid (ATRA) were administered once a day to the adriamycin-induced nephrotic rats at the first day after adriamycin injection respectively. Renal tissue samples were collected at day 3, 7, 14, and 28 after adriamycin injection respectively. The distribution, mRNA expression and protein expression of nephrin, podocin, CD2AP and ?-actinin-4 were examined by indirect immunofluorescence, real-time PCR and Western blotting, respectively. The interactions among nephrin and podocin, nephrin and CD2AP, as well as the nephrin phosphorylation were detected by immunoprecipitation, respectively. Results Compared to the control rats, 24 h urinary protein of the ADR rats increased significantly at day 14 (P
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Objective To dynamically observe the expression of slit diaphragm complex molecules, including nephrin, podocin, CD2AP, and cytoskeleton protein a-actinin-4, in adriamycin-induced nephrotic (ADN) rats, and to further explore the molecular behavior of podocyte proteins during the occurrence and development of proteinuria and their possible mechanisms. Methods Adriamycin nephropathy was induced by a single tail intravenous injection of adriamycin. Renal tissue samples were collected at day 3, 7, 14, and 28, respectively. The distribution, mRNA expression and protein expression of nephrin, podocin, CD2AP and a-actinin-4 were examined by indirect immunofluorescence, real-time PCR and Western blotting, respectively. Results (1) After the adriamycin injection, a significant increment of the 24-hour urinary protein was observed at day 14 and persisted up to day 28 (P