ORAI channels are critical for receptor-mediated endocytosis of albumin
NATURE COMMUNICATIONS
Authors: Zeng, Bo; Chen, Gui-Lan; Garcia-Vaz, Eliana; Bhandari, Sunil; Daskoulidou, Nikoleta; Berglund, Lisa M.; Jiang, Hongni; Hallett, Thomas; Zhou, Lu-Ping; Huang, Li; Xu, Zi-Hao; Nair, Viji; Nelson, Robert G.; Ju, Wenjun; Kretzler, Matthias; Atkin, Stephen L.; Gomez, Maria F.; Xu, Shang-Zhong
Abstract
Impaired albumin reabsorption by proximal tubular epithelial cells (PTECs) has been highlighted in diabetic nephropathy (DN), but little is known about the underlying molecular mechanisms. Here we find that ORAI1-3, are preferentially expressed in PTECs and down-regulated in patients with DN. Hyperglycemia or blockade of insulin signaling reduces the expression of ORAI1-3. Inhibition of ORAI channels by BTP2 and diethylstilbestrol or silencing of ORAI expression impairs albumin uptake. Transgenic mice expressing a dominant-negative Orai1 mutant (E108Q) increases albuminuria, and in vivo injection of BTP2 exacerbates albuminuria in streptozotocin-induced and Akita diabetic mice. The albumin endocytosis is Ca2+-dependent and accompanied by ORAI1 internalization. Amnionless (AMN) associates with ORAIs and forms STIM/ORAI/AMN complexes after Ca2+ store depletion. STIM1/ORAI1 colocalizes with clathrin, but not with caveolin, at the apical membrane of PTECs, which determines clathrin-mediated endocytosis. These findings provide insights into the mechanisms of protein reabsorption and potential targets for treating diabetic proteinuria.
Hapten Synthesis and the Development of an Ultrasensitive Indirect Competitive ELISA for the Determination of Diethylstilbestrol in Food Samples
SCIENTIFIC REPORTS
Authors: Yang, Xingdong; Wang, Yinbiao; Song, Chunmei; Hu, Xiaofei; Wang, Fangyu; Zeng, Xianyin
Abstract
An ultrasensitive indirect competitive enzyme-linked immunosorbent assay (ic ELISA) using monoclonal antibodies (mAbs) was developed for the specific detection of diethylstilbestrol (DES) residues. To establish an ELISA based on mAbs, hapten diethylstilbestrol mono-carboxypropyl-ether (DES-MCPE) was chemically synthetized and then conjugated to bovine serum albumin (BSA) for immunization in mice. This ic ELISA was further optimized for DES determination. The sensitivity of the ic ELISA was found to be 0.49 mu g/kg and the limit of detection was 0.075 mu g/kg. DES residues in salmon meat and pork were tested with the recovery range from 74.0 to 85.2% and the coefficient of variation (CV) was less than 10%. Parallel analysis of DES samples from salmon meat showed comparable results from the ic ELISA with high-performance liquid chromatography. The ic ELISA provides a useful screening method for the quantitative detection of DES residues in animal-derived food.
COA
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