ABSTRACT
OBJECTIVE: Diabetic nephropathy (DN) is a major diabetic micro-vascular complication, and podocyte apoptosis induced by high glucose (HG) is a typical early feature of DN. Studies have shown that microRNAs (miRNAs) play a crucial role in the pathogenesis of DN. The purpose of the current study was to explore the role and molecular mechanism of miR-770-5p in podocyte apoptosis in DN. PATIENTS AND MATERIALS AND METHODS: In vitro podocyte model of DN was conducted by treatment conditionally immortalized mouse podocytes with HG (30 mM D-glucose). The level of miR-770-5p in podocytes was detected using quantitative real-time PCR (qRT-PCR), and protein levels were measured using Western blot assay in our current study. The relationship between miR-770-5p and TP53 regulated inhibitor of apoptosis 1 (TRIAP1) was revealed by TargetScan and dual luciferase reporter assay. Cell proliferation ability and cell apoptosis were determined by using cell counting kit-8 (CCK-8) assay and flow cytometer (FCM), respectively. RESULTS: We found that miR-770-5p was significantly upregulated in podocytes under HG condition. TRIAP1 was a target gene of miR-770-5p and it was down-regulated in podocytes by HG treatment. Further analysis indicated that HG induced cell proliferation ability reduction, cell apoptosis enhancement and apoptotic peptidase activating factor 1(APAF1)/Caspase9 pathway exaltation in podocytes were prevented by miR-770-5p down-regulation. More importantly, the results showed that all the effects of miR-770-5p inhibitor on HG induced podocytes were eliminated by TRIAP1 silencing. S.-Z. Zhang, X.-J. Qiu, S.-S. Dong, L.-N. Zhou, Y. Zhu, M.-D. Wang, L.-W. Jin We showed that miR-770-5p was upregulated in the in vitro model of DN, and it might promote the development of DN through regulating podocyte apoptosis by targeting TRIAP1.
Subject(s)
Apoptosis , Diabetic Nephropathies/metabolism , Intracellular Signaling Peptides and Proteins/metabolism , MicroRNAs/metabolism , Models, Biological , Podocytes/metabolism , Animals , Cell Line , Diabetic Nephropathies/pathology , Glucose/pharmacology , Humans , Mice , Podocytes/pathology , Protein BindingABSTRACT
In this study, a simple, rapid and sensitive ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS) method is described for determination of domperidone in human plasma samples using oxcarbazepine as the internal standard (IS). Sample preparation was accomplished through protein precipitation with methanol, and chromatographic separation was performed on an Acquity BEH C18 column (2.1 mm×50 mm, 1.7 µm) with gradient profile at a flow of 0.45 mL/min. Mass spectrometric analysis was performed using a QTrap5500 mass spectrometer coupled with an electro-spray ionization (ESI) source in the positive ion mode. The MRM transition of m/z 426.3â175.2 was used to quantify for domperidone. The linearity of this method was found to be within the concentration range of 0.25-100.0 ng/mL for domperidone in human plasma. Only 1.5 min was needed for an analytical run. The method herein described was superior to previous methods and was successfully applied to the pharmacokinetic study of domperidone in healthy Chinese volunteers after oral administration.