Gitelman综合征诊断方法的建立及维生素D阻断其肾素活化机制初探
发布时间：2019-06-28 19:24
【摘要】：背景与目的：Gitelman综合征(GS)由SLC12A3基因突变导致其编码的肾脏远曲小管钠氯协同转运蛋白(NCC)功能失活,是一种遗传性失盐性肾病,临床表现为低血钾、低血镁、代谢性碱中毒、低尿钙和肾素-血管紧张素Ⅱ-醛固酮系统(RAAS)活化,但血压正常。目前临床上尚缺乏简单易行的诊断方法,限制了该病诊治水平的提高。本研究拟建立GS患者临床、基因和生理功能(氢氯噻嗪试验)诊断方法,结合体外基因突变功能试验,验证中国人群GS患者的常见和部分新突变的致病性；观察GS患者正常血镁亚型和糖耐量低减的表型。观察GS患者全身和局部RAAS活化及可能机制,探讨维生素D及其类似物抑制肾素的作用。方法：1.制定临床症状标准问卷、收集病例资料,提取外周血DNA,行SLC12A3基因外显子直接测序,氢氯噻嗪实验评估患者NCC蛋白功能状态。总结中国人高频和新发突变,构建野生型和突变型NCC质粒,体外转录成cRNA并进行显微注射,用爪蟾卵母细胞表达体系定量验证新突变功能。2.检测GS患者血、尿电解质水平(对照组为64位健康受试者)。行3h OGTT实验结果评价GS患者的胰岛素敏感性和胰岛分泌功能,对照组为健康受试者(n=20)和糖尿病患者(n=20)。3.分析低镁血症和正常血镁GS患者的临床特点、NCC功能试验差异,免疫组化法检测肾活检组织镁离子转运蛋白--瞬时受体电位阳离子通道亚家族M成员6(TRPM6)表达水平。4.RAAS卧立位实验测定GS患者全身血浆肾素活性(PRA)、血管紧张素Ⅱ和醛固酮水平,免疫组化和荧光双染观察GS患者和胚胎肾组织中肾素产生细胞再募集现象。5.急性和慢性注射VDR激动剂,观察血浆肾素活性、肾组织肾素mRNA和蛋白的变化,观察VDR基因敲除小鼠肾素、环氧酶2(COX2)、NCC和TRPM6水平。体外实验观察VDR是否在球旁器颗粒细胞(JG)或致密斑(MD)细胞表达,并探究活性维生素D对低氯诱导的MD细胞COX2高表达的影响。结果：1.60位确诊的GS患者常见的临床表现为肌无力(79.7%)、乏力(55.9%)、手足痉挛或抽搐(45.8%)、心悸(40.7%)、感觉异常(35.6%)和夜尿增多(33.9%),且男性GS患者的临床表现较女性患者重。2.54种突变中含14种新突变,中国GS患者突变频率高于5%的突变为T60M.D486N和R913Q。体外功能实验证明突变型NCC(T60M, L215F, D486N, N534K, Q617R和R928C)摄取22Na+的能力均显著低于野生型,且突变型NCC转运活性的下降是由转运子特异性突变所致。3.建立了简化氢氯噻嗪实验诊断方法,确定了中国人群正常值范围,用于临床证实GS患者NCC蛋白功能受损。使用氢氯噻嗪前后氯排泄分数净增加值来诊断GS的ROC曲线下面积为0.983,取2.86作为截点,以基因诊断为金标准,该实验诊断低钾、碱中毒GS患者的灵敏度为95%,特异度为95.2%。4. OGTT实验结果提示GS患者存在糖代谢和胰岛素功能异常,与健康对照相比,GS患者表现为血糖和胰岛素分泌高峰延迟,血糖曲线下面积增加,胰岛素分泌敏感性指数(ISSI)降低,而胰岛素敏感性指标(ISOGTT、QUICKI 和 HOMA-IR)无显著差异。5.正常血镁患者临床表现较低镁血症患者轻,对氢氯噻嗪的反应好于低镁血症患者。NCC和TRPM6同时表达在肾脏远曲小管。正常血镁患者(n=2)远曲小管TRPM6表达水平与轻微病变对照组(n=14)无显著差异(7.82±5.23%vs.8.63±2.67%,p0.05),而低镁血症GS患者(n=10)TRPM6表达水平显著低于对照组(4.96±1.88%vs.8.63±2.67%, p=0.001)。6.88%的GS患者存在全身RAAS活化,GS患者(n=18)肾组织球旁器增生,肾素表达水平显著高于轻微病变对照组,球旁器和微动脉区域均发现了肾素与α-Actin共表达现象,重现了胚胎阶段肾组织的情况。7.活性维生素D及其类似物均不能抑制急性肾素分泌,但腹腔注射两周其类似物--RO化合物可抑制肾素mRNA表达,且不升高血钙。VDR基因敲除小鼠血钙降低,肾素mRNA表达增加2.9倍。MD细胞表达VDR,活性维生素D不能抑制低氯诱导的MD细胞COX2高表达。结论：建立了GS临床、基因和生理功能试验(氢氯噻嗪试验)诊断模型。氢氯噻嗪实验是一种简单、经济、可靠的GS诊断和鉴别诊断方法。新发现了14种突变,明确了中国GS患者的高频突变包括T60M、D486N和R913Q。男性GS临床表现更重,部分患者存在糖耐量低减和胰岛素分泌功能异常。正常血镁是GS的一种亚型,血镁水平与GS患者临床严重程度及NCC功能相关,TRPM6蛋白表达减少可能是导致GS患者低镁血症的重要原因。GS患者RAAS活化的结构基础是肾素产生细胞再募集,新型活性维生素D类似物—RO化合物可抑制肾素活性,有潜在治疗GS的价值。
[Abstract]:BACKGROUND & OBJECTIVE: The mutation of Gittelman syndrome (GS) from the SLC12A3 gene results in the inactivation of the function of the co-transport protein (NCC) of the renal distal tubule, which is encoded by the SLC12A3 gene. It is a kind of hereditary loss of kidney and kidney disease. The clinical manifestation is hypokalemia, hypokalemia, metabolic alkalosis. The low urinary calcium and the renin-angiotensin II-aldosterone system (RAAS) were activated, but the blood pressure was normal. The diagnosis and treatment level of the disease is limited, and the diagnosis and treatment level of the disease is limited. In this study, the clinical, genetic and physiological functions of GS patients (hydrochlorofluorocarbon test) were established, and in combination with the in vitro gene mutation function test, the pathogenicity of common and partial new mutations in GS patients in Chinese population was verified, and the phenotype of normal blood magnesium and impaired glucose tolerance in GS patients was observed. The activation and possible mechanism of systemic and local RAAS in GS patients were observed, and the role of vitamin D and its analogues in the inhibition of renin was discussed. Method:1. A clinical symptom standard questionnaire was developed to collect the case data, to extract the peripheral blood DNA, to directly sequence the exon of the SLC12A3 gene, and to evaluate the functional status of the NCC protein in the patient. To sum up the high frequency and new mutation of Chinese human, the wild type and the mutant NCC plasmid were constructed, and the cRNA was transcribed into cRNA in vitro and microinjected, and the new mutation function was quantitatively verified by the expression system of Xenopus oocytes. The level of blood and urine electrolytes in GS (control group was 64 healthy subjects) was detected. The insulin sensitivity and the function of pancreatic islet secretion in GS patients were evaluated by the results of 3-h OGTT. The control group was healthy subjects (n = 20) and diabetic patients (n = 20). The clinical characteristics of patients with hypomagnesemia and normal blood and magnesium GS were analyzed. 4. The plasma renin activity (PRA), angiotensin 鈪，
本文编号：2507551
[Abstract]:BACKGROUND & OBJECTIVE: The mutation of Gittelman syndrome (GS) from the SLC12A3 gene results in the inactivation of the function of the co-transport protein (NCC) of the renal distal tubule, which is encoded by the SLC12A3 gene. It is a kind of hereditary loss of kidney and kidney disease. The clinical manifestation is hypokalemia, hypokalemia, metabolic alkalosis. The low urinary calcium and the renin-angiotensin II-aldosterone system (RAAS) were activated, but the blood pressure was normal. The diagnosis and treatment level of the disease is limited, and the diagnosis and treatment level of the disease is limited. In this study, the clinical, genetic and physiological functions of GS patients (hydrochlorofluorocarbon test) were established, and in combination with the in vitro gene mutation function test, the pathogenicity of common and partial new mutations in GS patients in Chinese population was verified, and the phenotype of normal blood magnesium and impaired glucose tolerance in GS patients was observed. The activation and possible mechanism of systemic and local RAAS in GS patients were observed, and the role of vitamin D and its analogues in the inhibition of renin was discussed. Method:1. A clinical symptom standard questionnaire was developed to collect the case data, to extract the peripheral blood DNA, to directly sequence the exon of the SLC12A3 gene, and to evaluate the functional status of the NCC protein in the patient. To sum up the high frequency and new mutation of Chinese human, the wild type and the mutant NCC plasmid were constructed, and the cRNA was transcribed into cRNA in vitro and microinjected, and the new mutation function was quantitatively verified by the expression system of Xenopus oocytes. The level of blood and urine electrolytes in GS (control group was 64 healthy subjects) was detected. The insulin sensitivity and the function of pancreatic islet secretion in GS patients were evaluated by the results of 3-h OGTT. The control group was healthy subjects (n = 20) and diabetic patients (n = 20). The clinical characteristics of patients with hypomagnesemia and normal blood and magnesium GS were analyzed. 4. The plasma renin activity (PRA), angiotensin 鈪，
本文编号：2507551
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