目的:Dent病1型(DD1)是由CLCN5突变引起的罕见X连锁遗传性病理,其特征主要是近端小管功能障碍,高钙尿症,肾结石/肾钙化病,进行性慢性肾病,和低重量的蛋白尿,这种疾病的分子标志。目前,没有具体的治疗方法,只有症状,不能阻止疾病的进展。在这项研究中,我们分离并表征了富含外泌体的尿细胞外囊泡(uEV),这将使我们能够鉴定与DD1进展相关的生物标志物,并更好地了解该疾病的病理生理基础。
方法:通过西班牙肾脏病学会(SEN)和西班牙小儿肾脏病学会(AENP)的全国性呼吁,尿液样本来自西班牙不同医院的患者和对照组,它们被处理以获得uEVS。这些患者的数据由各自的肾病学家提供和/或从RENALTUBE注册表中提取。通过超速离心分离uEV,形态学特征及其蛋白质和microRNA含量的提取。
结果:招募了25名患者和10名对照,从中处理尿液以分离uEV。我们的结果表明,与对照组相比,患者的uEVs/mL的相对浓度较低(0.26×106uEVs/mLvs1.19×106uEVs/mL,p<0.01)。此外,发现患者的uEV明显大于对照组(平均直径:187.8nmvs143.6nm,p<0.01)。最后,我们的数据表明,RNA已从患者和对照外泌体中正确提取.
结论:在这项工作中,我们描述了从患有Dent1疾病和健康对照的患者中分离和表征uEVs,这将有助于随后研究该病理学中差异表达的货物分子。
Dent\'s disease type 1 (DD1) is a rare X-linked hereditary pathology caused by CLCN5 mutations that is characterized mainly by proximal tubule dysfunction, hypercalciuria, nephrolithiasis/nephrocalcinosis, progressive chronic kidney disease, and low-weight proteinuria, the molecular hallmark of the disease. Currently, there is no specific curative treatment, only symptomatic and does not prevent the progression of the disease. In this study we have isolated and characterized urinary extracellular vesicles (uEVs) enriched in exosomes that will allow us to identify biomarkers associated with DD1 progression and a better understanding of the pathophysiological bases of the disease.
Through a national call from the Spanish Society of Nephrology (SEN) and the Spanish Society of Pediatric Nephrology (AENP), urine samples were obtained from patients and controls from different Spanish hospitals, which were processed to obtain the uEVS. The data of these patients were provided by the respective nephrologists and/or extracted from the RENALTUBE registry. The uEVs were isolated by ultracentrifugation, morphologically characterized and their protein and microRNA content extracted.
25 patients and 10 controls were recruited, from which the urine was processed to isolate the uEVs. Our results showed that the relative concentration of uEVs/mL is lower in patients compared to controls (0.26 × 106 uEVs/mL vs 1.19 × 106 uEVs/mL, p < 0.01). In addition, the uEVs of the patients were found to be significantly larger than those of the control subjects (mean diameter: 187.8 nm vs 143.6 nm, p < 0.01). Finally, our data demonstrated that RNA had been correctly extracted from both patient and control exosomes.
In this work we describe the isolation and characterization of uEVs from patients with Dent 1 disease and healthy controls, that shall be useful for the subsequent study of differentially expressed cargo molecules in this pathology.