Schordan Sandra, Schordan Eric, Endlich Nicole, Lindenmeyer Maja T, Meyer-Schwesinger Catherine, Meyer Tobias N, Giebel Jürgen, Cohen Clemens D, Endlich Karlhans, Maurer Martin H
Department of Anatomy and Cell Biology, Ernst Moritz Arndt University, D-17487 Greifswald, Germany.
Proteomics. 2009 Oct;9(19):4519-28. doi: 10.1002/pmic.200800214.
Diabetic nephropathy is one of the most common complications of diabetes mellitus and the leading cause of end-stage renal disease. A reduction in podocyte number has been documented in the kidneys of these patients. To identify the molecular changes in podocytes that are primarily caused by high glucose (HG) concentrations and not by secondary alterations (e.g. glomerular hypertension), we investigated the protein expression profiles in a podocyte cell line under long-term HG exposure (30 versus 10 mM for 2 wk). Proteins were separated by 2-DE, and we identified 39 different proteins in 48 spots that were differentially regulated by more than twofold in response to HG concentrations using MALDI-TOF MS and MASCOT software. These proteins belong to several protein classes, including cytoskeletal proteins and specific annexins (annexins III and VI). Downregulation of annexins III and VI by HG concentrations was confirmed by qRT-PCR, Western blot, and immunostaining, and was also observed in glomeruli of kidney biopsies from patients with diabetic nephropathy. Our data demonstrate that HG concentrations per se are sufficient to strongly modify the protein expression profile of podocytes, the analysis of which contributes to the identification of novel targets involved in diabetic nephropathy.
糖尿病肾病是糖尿病最常见的并发症之一，也是终末期肾病的主要原因。这些患者的肾脏中已记录到足细胞数量减少。为了确定主要由高葡萄糖（HG）浓度而非继发性改变（如肾小球高血压）引起的足细胞分子变化，我们研究了长期暴露于HG（30 mM与10 mM，持续2周）的足细胞系中的蛋白质表达谱。通过二维电泳分离蛋白质，我们使用基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）和MASCOT软件在48个斑点中鉴定出39种不同的蛋白质，这些蛋白质在响应HG浓度时差异调节超过两倍。这些蛋白质属于几个蛋白质类别，包括细胞骨架蛋白和特定的膜联蛋白（膜联蛋白III和VI）。通过实时定量聚合酶链反应（qRT-PCR）、蛋白质印迹法和免疫染色证实了HG浓度导致膜联蛋白III和VI下调，并且在糖尿病肾病患者的肾活检肾小球中也观察到了这种下调。我们的数据表明，HG浓度本身足以强烈改变足细胞的蛋白质表达谱，对其分析有助于鉴定参与糖尿病肾病的新靶点。
