【作者】 翟亦晖；

【导师】 徐虹； Franz Schaefer； Claus Peter Schmitt；

【作者基本信息】 复旦大学 ， 儿科学， 2010， 博士

【摘要】 慢性肾脏病(chronic kidney disease, CKD)是严重威胁人类健康和生命的常见病之一,其发病率高、进展至终末期肾病(end stage renal disease, ESRD)死亡率高,给患者和家庭带来巨大的经济和精神负担,并消耗大量的社会卫生资源,已成为全球面临的严重公共卫生问题。流行病学调查显示,CKD发病呈现年轻化趋势,不少尿毒症患者年龄仅有二三十岁。由此预测,儿童中的CKD不仅是儿童时期ESRD的重要病因,这部分患儿还将成为成年期CKD乃至ESRD的高危患病群体。因此,儿童CKD同样需引起重视。面对CKD的严峻挑战,如何早期发现CKD高危人群,并及时有效干预,是延缓CKD进展、减少ESRD发生、改善预后的重要途径。另外,对部分进展至ESRD的儿童进行安全有效的肾替代治疗、延长生命、提高生活质量,同样至关重要。因此,本研究就儿童CKD的早期发现和终末期腹透治疗透析液生物相容性两方面,展开进一步研究。为探讨儿童CKD早期发现的策略,本研究首先通过中小学生学校尿液筛查,分析上海市部分地区儿童血尿／蛋白尿的检出率,评价不同的尿液筛查方法和筛查的合适年龄段,并根据随访资料及病例回顾性分析,进一步探讨学校尿液筛查对儿童CKD早期发现的意义。结果发现一次尿检血尿(隐血)和／或蛋白尿者超过5%,两次均阳性者占1%；进一步通过四种不同筛查方案的比较,发现二次尿液试纸目测法既简便经济,又具备一定的敏感性和特异性,是值得推广的筛查方案；而筛查的适宜年龄段仍需更大样本量的研究而进一步明确。本研究筛查尿检异常者的随访率较低(24%),但3年间67名长期随访的儿童中确诊了3例IgA肾病；其中一例虽然临床仅表现为无症状蛋白尿伴镜下血尿,但肾活检病理提示近40%肾小球硬化；经积极治疗,随访3年后尿检正常,肾功能正常。进一步对我科1979-2009年肾活检确诊的158例儿童原发IgA肾病回顾性分析发现,无症状尿检异常者为33例(20.9%),其中有10%(3／33)患儿肾脏病理损害严重,该比例与有症状组中病理损害严重者占的比例(12／125)相似,提示学校尿液筛查对于早期发现这部分临床无典型症状、隐匿起病但病理改变严重的重症患儿具有重要的作用。儿童CKD虽经早期发现并合理干预,仍有少部分会进展至ESRD。对这部分重症患儿的精心治疗同样至关重要,目前由于肾源的紧缺,腹膜透析为儿童首选的肾替代治疗方法。然而长期暴露于传统的非生物相容性的腹透液(peritonealdialysis fluid, PDF)会导致腹膜间皮细胞(human peritoneal mesothelial cells, HPMC)损伤、脱落,腹膜纤维化和新生血管形成,最终导致超滤衰竭,腹透治疗失败。因此,新型腹透液相继问世,以pH中性、葡萄糖降解产物水平低为特点的“新型腹透液”具有较好的生物相容性,能保护HPMC的生物活性和功能,维持腹膜稳定性,改善腹膜超滤功能,延长ESRD患者的存活率。本课题着重于目前已应用于临床的两种新型腹透液,研究其缓冲体系的差异,即碳酸氢盐缓冲腹透液(bicarbonate-buffered PDF, B-PDF)和乳酸盐缓冲腹透液(lactate-buffered PDF, L-PDF)对一个腹膜功能关键蛋白——水通道蛋白-1 (aquaporin-1, AQP1)表达和相关功能的影响,探讨临床慢性腹透患者透析液选择的依据。为研究不同腹透液对HPMC AQP1表达的影响,首先通过实时荧光定量PCR和western blot结果发现,B-PDF能够以时间依赖的方式上调HPMC AQP1的表达；而L-PDF则以时间、剂量依赖的方式下调AQP1的表达。另外,细胞免疫荧光实验也证实B-PDF刺激的间皮细胞膜上以及胞浆内有更强的AQP1荧光信号。为进一步研究B-PDF和L-PDF pH值的微小差异(7.4 vs 7.0)对AQP1表达的影响,分别用NaOH或HCl调节腹透液pH值,结果显示,随pH值上升,B-PDF进行性上调HPMC AQP1 mRNA的表达水平；而L-PDF对AQP1表达的调节则无pH相关性,提示pH值不单独发挥调节效应。为研究不同腹透液对HPMC迁移能力的影响,分别采用Transwell迁移实验、细胞划痕实验以及实时显微镜三种方法检测不同腹透液对HPMC迁移能力的影响,结果发现,与L-PDF相比,B-PDF能显著增加HPMC的迁移能力和损伤修复能力。并进一步通过siRNA干扰实验发现,AQP1而非AQP3参与调节HPMC的迁移和损伤修复能力,并且不同腹透液对HPMC迁移能力的影响是AQP1所依赖的。此外,为研究不同腹透液对腹膜钠过筛的影响,进一步通过大鼠腹膜平衡实验观察大鼠腹膜的钠过筛效应,结果显示,与L-PDF相比,B-PDF刺激的腹膜有较快较强的钠过筛效应,提示其可能实现更好的超滤作用。综上所述,本研究通过对上海市部分中小学生的尿液筛查以及回顾性病例资料分析,发现“健康”儿童具有一定的尿检阳性率,而二次尿液试纸目测法是值得推广的筛查方案,病例随访和回顾性资料分析进一步肯定了尿液筛查在早期发现儿童CKD的意义；并关注儿童ESRD腹透治疗透析液的生物相容性,通过体外原代细胞培养,研究不同腹透液对腹膜间皮细胞AQP1表达和功能的影响,发现与L-PDF相比,B-PDF能上调HPMC AQP1的表达水平,并通过AQP1的作用增加细胞的迁移和损伤修复能力；进一步通过动物实验研究不同腹透液对腹膜钠过筛的影响,发现B-PDF较L-PDF能增加腹膜的钠过筛效应。比较两种新型腹透液发现,B-PDF可能更好的保护腹膜细胞功能,维持腹膜稳定性和超滤作用,具有更好的生物相容性。对于新型腹透液生物相容性的深入研究有助于增进新型腹透液与腹膜之间相互作用的了解,还将为临床长期慢性腹透患儿透析液的选择提供实验和理论依据。更多还原

【Abstract】 Chronic kidney disease (CKD) is a worldwide health and life threatening disease. With high morbidity and mortality, CKD has become a severe public health issue, bringing heavy burden to the patients and their families, and meanwhile, resulting in large expenditure of social health resource. Epidemic studies show that more and more young people develop CKD and some uremic patients are only at their twenties or thirties, thus it can be expected that CKD in children is not only a cause of end stage renal disease (ESRD) in childhood, but may also a major cause of CKD and ESRD in adults. Therefore, great attention should also be paid to CKD in children.Facing severe challenge of CKD, early detection of people at high risk and effective therapies are of great significance in slowing progression of CKD and reducing the incidence of ESRD. Simultaneously, it’s also very important to safely and effectively treat the children with ESRD, improve the survival rate and their quality of life. Therefore, this study on one hand focuses on the strategy of early detection of CKD in children and on the other hand, on biocompatibility of peritoneal dialysis (PD) fluids used for PD, the major renal replacement therapy (RRT) modality for children with ESRD.To study the strategy on early detection of CKD in children, we performed school urine screening with various screening methods in four districts of Shanghai. We analyzed the positive rate of urine abnormality, in terms of hematuria and/or proteinura, compared various screening methods and investigated appropriate age for screening. Then based on the follow-up data and retrospective case analysis, we evaluated the significance of school urine screening in early detection of CKD, especially IgA nephropathy in children.Data showed positive rate over 5% at the first screening and 1% at second screening. Two times of dipstick screening was found to be a superior method due to its low direct cost and appropriate sensitivity and specificity, whereas the appropriate age for urine screening required further study with larger samples.Although the follow-up rate was rather low, only 24%,3 cases of IgA nephropathy were diagnosed among 67 children within 3 years of follow-up. Of note, one case was with minor clinical symptoms but severe renal pathologic lesion, and after appropriate treatment, satisfied outcome was achieved with normal urinalysis and renal function. Further study was based on a retrospective case analysis of 158 cases of primary IgA nephropathy in children diagnosed in our hospital between the year 1979 and 2009. Data showed that about one-fifth of patients had the disease onset without obvious clinical symptoms and were not aware of kidney disease until a random urine test. Among them, nearly half only had minor histological lesion manifested by renal biopsy, however,-10% showed severe renal lesion, and the ratio of severe case in these asymptomatic children was similar as that in symptomatic children. All these suggested that school urine screening was of great significance in early detection of CKD in children, especially children who presented minor in clinical symptoms but severe in renal pathology.Even with early detection and careful treatment, a few of patients with CKD still inevitably progress to ESRD. It’s of no doubt nephrologists’ duty to make great efforts on treating these patients as well. Because of lacking kidney allografts, PD is the first choice of RRT modality for children with ESRD. However, long-term exposure of peritoneum in the traditional bioincompatible PD fluids leads to damage and denudation of human peritoneal mesothelial cells (HPMC) and peritoneal fibrosis and neoangiogenesis, which finally results in loss of ultrafiltration and technical failure of PD. Therefore, new generation of PD fluids have been introduced and these pH neutral, low glucose degradation products (GDP) PD fluids are considered to be more biocompatible, as manifested by protected bioactivity and function of HPMC, preserved stability of peritoneum, improved ultrafiltration, and prolonged survival of ESRD patients. In this study, we focused on the regulation of expression and relevant function of aquaporin-1 (AQP1), a key protein in the peritoneum during PD, by two new PD fluids with different buffer, bicarbonate (B-PDF) or lactate (L-PDF). To study the regulation of AQP1 by different PD fluids, realtime RT-PCR and western blot were carried out and results showed that B-PDF time-dependently up-regulated expression of AQP1 in HPMC, while L-PDF time-and dose-dependently down-regulated expression of AQP1. Immunofluorence further confirmed stronger signals of AQP1 on cell membrane and in cell plasma with respect to HPMC treated with B-PDF. To further study the pH effect on regulation of expression of AQP1, pH of PD fluids was adjusted by NaOH or HC1 to various value as 6,7, and 8. Data showed that by increasing pH value, B-PDF up-regulated mRNA expression of AQP1 in HPMC, while no regulatory effect on expression of AQP1 with respect to L-PDF, suggesting pH only had partial effect on regulation of expression of AQP1.To study the migration capacity of cells treated with different PD fluids, Transwell migration assay, wound healing assay and time-laps microscopy were performed and data showed that B-PDF significantly increased cell migration and wound healing compared to L-PDF. Further siRNA experiments demonstrated that AQP1, not AQP3, participated in HPMC migration and wound healing, and B-PDF increased cell migration in an AQP1-dependent manner.Furthermore, to study the regulation of peritoneal sodium sieving with various PD fluids,2h peritoneal equilibrium test (PET) in a rat model was performed and data showed that B-PDF increased peritoneal sodium sieving compared to L-PDF, suggested that B-PDF might better preserve the ultrafiltration capacity of peritoneum.In conclusion, based on the school urine screening, we reported the positive rate of urine abnormality in well-children in Shanghai, and recommended two times dipstick screening for further big scale screening. The follow-up cases and retrospective case analysis showed the importance of urine screening in early detection of CKD in children. Further in vitro and in vivo studies on two new PD fluids showed B-PDF up-regulated expression of AQP1 in HPMC and in an AQP1-dependent manner increase HPMC migration and wound healing capacity, and as well increased peritoneal sodium sieving. By comparison of two new PD fluids, B-PDF might better protect peritoneal cell function and preserve peritoneal stability and ultrafiltration, thus with better biocompatibility. More studies on new PD fluids will extend our understanding of biocompatibility and will provide experimental and theoretical evidence for clinical option of new PD fluids for patients.更多还原