Department of Immunology, IIS-Fundación Jiménez Díaz, UAM, REDINREN, Madrid, Spain.
Department of Nephrology/UAM/IRSIN, IIS-Fundación Jiménez Díaz, REDINREN, Madrid, Spain.
Transl Res. 2015 Nov;166(5):474-484.e4. doi: 10.1016/j.trsl.2015.05.007. Epub 2015 May 23.
Diabetic nephropathy (DN) is a major complication of diabetes mellitus and the most frequent cause of end-stage renal disease. DN progresses silently and without clinical symptoms at early stages. Current noninvasive available markers as albuminuria account with severe limitations (late response, unpredictable prognosis, and limited sensitivity). Thus, it urges the discovery of novel markers to help in diagnosis and outcome prediction. Tissue proteomics allows zooming-in where pathophysiological changes are taking place. We performed a differential analysis of renal tissue proteome in a rat model of early DN by 2-dimensional differential gel electrophoresis and mass spectrometry. Confirmation was performed by Western blot, immunohistochemistry (IHC), and selected reaction monitoring (SRM). Rat urine samples were collected and exosomes were isolated from urine to evaluate if these microvesicles reflect changes directly occurring at tissue level. The protein showing maximum altered expression in rat tissue in response to DN was further analyzed in human kidney tissue and urinary exosomes. Regucalcin protein or senescence marker protein-30 (SMP30) (Swiss-Prot Q03336) was found to be strongly downregulated in DN kidney tissue compared with healthy controls. The same trend was observed in exosomes isolated from urine of control and DN rats. These data were further confirmed in a pilot study with human samples. IHC revealed a significant decrease of regucalcin in human kidney disease tissue vs control kidney tissue, and regucalcin was detected in exosomes isolated from healthy donors' urine but not from kidney disease patients. In conclusion, regucalcin protein expression is reduced in DN kidney tissue and this significant change is reflected in exosomes isolated from urine. Urinary exosomal regucalcin represents a novel tool, which should be explored for early diagnosis and progression monitoring of diabetic kidney disease.
糖尿病肾病 (DN) 是糖尿病的主要并发症，也是终末期肾病的最常见原因。DN 在早期阶段无症状且无明显临床症状。目前，白蛋白尿等非侵入性可用标志物存在严重局限性（反应迟滞、预后不可预测、敏感性有限）。因此，迫切需要发现新的标志物来帮助诊断和预测预后。组织蛋白质组学可以深入研究发生病理生理变化的部位。我们通过二维差异凝胶电泳和质谱对早期 DN 大鼠模型的肾组织蛋白质组进行了差异分析。通过 Western blot、免疫组织化学 (IHC) 和选择反应监测 (SRM) 进行了验证。收集大鼠尿液样本并从尿液中分离外泌体，以评估这些微囊泡是否反映了组织水平直接发生的变化。在大鼠组织中对 DN 反应显示最大改变表达的蛋白质在人肾组织和尿外泌体中进一步进行了分析。与健康对照组相比，DN 肾脏组织中的 Regucalcin 蛋白或衰老标志物蛋白-30 (SMP30)（Swiss-Prot Q03336）表达明显下调。从对照和 DN 大鼠尿液中分离的外泌体中也观察到了相同的趋势。这些数据在一项人类样本的初步研究中得到了进一步证实。IHC 显示，Regucalcin 在人类肾脏疾病组织中的表达明显低于对照组，Regucalcin 在外泌体中检测到，而在健康供体的尿液中则没有。结论是，Regucalcin 蛋白在 DN 肾脏组织中的表达减少，这种显著变化反映在从尿液中分离的外泌体中。尿外泌体中的 Regucalcin 代表一种新的工具，应该探索用于糖尿病肾病的早期诊断和进展监测。
