【摘要】：背景與目的：Gitelman綜合征(GS)由SLC12A3基因突變導致其編碼的腎臟遠曲小管鈉氯協(xié)同轉(zhuǎn)運蛋白(NCC)功能失活,是一種遺傳性失鹽性腎病,臨床表現(xiàn)為低血鉀、低血鎂、代謝性堿中毒、低尿鈣和腎素-血管緊張素Ⅱ-醛固酮系統(tǒng)(RAAS)活化,但血壓正常。目前臨床上尚缺乏簡單易行的診斷方法,限制了該病診治水平的提高。本研究擬建立GS患者臨床、基因和生理功能(氫氯噻嗪試驗)診斷方法,結(jié)合體外基因突變功能試驗,驗證中國人群GS患者的常見和部分新突變的致病性；觀察GS患者正常血鎂亞型和糖耐量低減的表型。觀察GS患者全身和局部RAAS活化及可能機制,探討維生素D及其類似物抑制腎素的作用。方法：1.制定臨床癥狀標準問卷、收集病例資料,提取外周血DNA,行SLC12A3基因外顯子直接測序,氫氯噻嗪實驗評估患者NCC蛋白功能狀態(tài)。總結(jié)中國人高頻和新發(fā)突變,構(gòu)建野生型和突變型NCC質(zhì)粒,體外轉(zhuǎn)錄成cRNA并進行顯微注射,用爪蟾卵母細胞表達體系定量驗證新突變功能。2.檢測GS患者血、尿電解質(zhì)水平(對照組為64位健康受試者)。行3h OGTT實驗結(jié)果評價GS患者的胰島素敏感性和胰島分泌功能,對照組為健康受試者(n=20)和糖尿病患者(n=20)。3.分析低鎂血癥和正常血鎂GS患者的臨床特點、NCC功能試驗差異,免疫組化法檢測腎活檢組織鎂離子轉(zhuǎn)運蛋白--瞬時受體電位陽離子通道亞家族M成員6(TRPM6)表達水平。4.RAAS臥立位實驗測定GS患者全身血漿腎素活性(PRA)、血管緊張素Ⅱ和醛固酮水平,免疫組化和熒光雙染觀察GS患者和胚胎腎組織中腎素產(chǎn)生細胞再募集現(xiàn)象。5.急性和慢性注射VDR激動劑,觀察血漿腎素活性、腎組織腎素mRNA和蛋白的變化,觀察VDR基因敲除小鼠腎素、環(huán)氧酶2(COX2)、NCC和TRPM6水平。體外實驗觀察VDR是否在球旁器顆粒細胞(JG)或致密斑(MD)細胞表達,并探究活性維生素D對低氯誘導的MD細胞COX2高表達的影響。結(jié)果：1.60位確診的GS患者常見的臨床表現(xiàn)為肌無力(79.7%)、乏力(55.9%)、手足痙攣或抽搐(45.8%)、心悸(40.7%)、感覺異常(35.6%)和夜尿增多(33.9%),且男性GS患者的臨床表現(xiàn)較女性患者重。2.54種突變中含14種新突變,中國GS患者突變頻率高于5%的突變?yōu)門60M.D486N和R913Q。體外功能實驗證明突變型NCC(T60M, L215F, D486N, N534K, Q617R和R928C)攝取22Na+的能力均顯著低于野生型,且突變型NCC轉(zhuǎn)運活性的下降是由轉(zhuǎn)運子特異性突變所致。3.建立了簡化氫氯噻嗪實驗診斷方法,確定了中國人群正常值范圍,用于臨床證實GS患者NCC蛋白功能受損。使用氫氯噻嗪前后氯排泄分數(shù)凈增加值來診斷GS的ROC曲線下面積為0.983,取2.86作為截點,以基因診斷為金標準,該實驗診斷低鉀、堿中毒GS患者的靈敏度為95%,特異度為95.2%。4. OGTT實驗結(jié)果提示GS患者存在糖代謝和胰島素功能異常,與健康對照相比,GS患者表現(xiàn)為血糖和胰島素分泌高峰延遲,血糖曲線下面積增加,胰島素分泌敏感性指數(shù)(ISSI)降低,而胰島素敏感性指標(ISOGTT、QUICKI 和 HOMA-IR)無顯著差異。5.正常血鎂患者臨床表現(xiàn)較低鎂血癥患者輕,對氫氯噻嗪的反應(yīng)好于低鎂血癥患者。NCC和TRPM6同時表達在腎臟遠曲小管。正常血鎂患者(n=2)遠曲小管TRPM6表達水平與輕微病變對照組(n=14)無顯著差異(7.82±5.23%vs.8.63±2.67%,p0.05),而低鎂血癥GS患者(n=10)TRPM6表達水平顯著低于對照組(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)腎組織球旁器增生,腎素表達水平顯著高于輕微病變對照組,球旁器和微動脈區(qū)域均發(fā)現(xiàn)了腎素與α-Actin共表達現(xiàn)象,重現(xiàn)了胚胎階段腎組織的情況。7.活性維生素D及其類似物均不能抑制急性腎素分泌,但腹腔注射兩周其類似物--RO化合物可抑制腎素mRNA表達,且不升高血鈣。VDR基因敲除小鼠血鈣降低,腎素mRNA表達增加2.9倍。MD細胞表達VDR,活性維生素D不能抑制低氯誘導的MD細胞COX2高表達。結(jié)論：建立了GS臨床、基因和生理功能試驗(氫氯噻嗪試驗)診斷模型。氫氯噻嗪實驗是一種簡單、經(jīng)濟、可靠的GS診斷和鑒別診斷方法。新發(fā)現(xiàn)了14種突變,明確了中國GS患者的高頻突變包括T60M、D486N和R913Q。男性GS臨床表現(xiàn)更重,部分患者存在糖耐量低減和胰島素分泌功能異常。正常血鎂是GS的一種亞型,血鎂水平與GS患者臨床嚴重程度及NCC功能相關(guān),TRPM6蛋白表達減少可能是導致GS患者低鎂血癥的重要原因。GS患者RAAS活化的結(jié)構(gòu)基礎(chǔ)是腎素產(chǎn)生細胞再募集,新型活性維生素D類似物—RO化合物可抑制腎素活性,有潛在治療GS的價值。
[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，

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