背景与目的：

机体维持水盐电解质和血压的平衡依赖于肾脏小球和小管结构的完整性和正常的管球反馈（tubuloglomerular feedback，TGF）。远端小管上的钠氯共转运子（NCC）是由SLC12A3基因编码的跨膜蛋白，它与近端小管髓攀升支粗段和致密斑的钠钾氯共转运子（NKCC2）、集合管的上皮钠通道（ENaC）共同组成了肾脏的水盐电解质平衡的重要防线，其中NKCC2和ENaC是致密斑和集合管管球反馈的重要感应子，而NCC与TGF的关系并不明确。SLC12A3基因失活突变导致NCC功能障碍即Gitelman综合征（GS），是一种罕见的失盐性肾病，是研究NCC功能的天然疾病模型，但相关病理生理机制的研究不多，缺乏评价临床严重程度、可指导治疗的生物标志物和功能影像学方法。

本研究拟观察较大样本量的GS患者临床病理特点、生活质量和性别差异，比较NCC生理功能试验对GS的临床诊断价值；观察尿液PGE2及其代谢产物水平与临床的相关性，初步探索外泌体及功能核磁在GS中的应用价值。建立两种不同的盐敏感高血压小鼠模型：高盐-高醛固酮-低肾素模型（DS）和轻中度高尿酸导致的早期高血压模型（HUA），探索NCC在高血压发生和肾损害中可能的作用，并在腺苷1型受体基因敲除（A1AR^-/-）的模型中探讨管球反馈与NCC的相关性。

方法：

第一部分：先天NCC功能障碍的GS患者临床特点及生物影像标志物探索

1.    纳入临床疑诊为失盐性肾病的患者，提取外周血DNA、PCR扩增后行SLC12A3基因测序进行基因诊断，作为诊断的金标准，采用氢氯噻嗪试验阻断NCC以评价其体内功能，分析比较该功能试验与经典的GS临床指标在诊断试验中的敏感性、特异性差异；

2.    搜集GS患者临床资料，观察患者血压和水盐代谢的特点；留取患者血浆和24h尿标本ELISA法测定PGE2及其代谢产物PGEM的水平，分析其与临床资料及NCC功能的相关性；采用SF-36量表分析其生活质量，并与性别年龄匹配的健康受试者比较。比较GS患者以上资料的性别差异。收集女性患者的妊娠资料，观察妊娠期间血电解质水平的变化及妊娠相关并发症。

3.    总结本中心肾活检的GS患者肾脏病理资料，免疫组化半定量分析肾脏皮质COX-2的表达与对照（肾小球轻微病变患者）的差异。提取尿液外泌体并进行形态、粒径和标志物的鉴定，免疫印迹法检测患者NCC、COX-2、COX-1蛋白表达水平。采用轴位IVIM（体内不相干运动成像）和DKI（弥散峰度成功能核磁）显像的方法，探索GS患者肾脏潜在的功能改变，分析其功能影像学参数与临床资料的相关性。

第二部分：盐敏感高血压小鼠模型的建立及NCC相关生理的改变

1.    分别建立C57bl背景下的单肾切除-高盐-高醛固酮的盐敏感高血压（DS）小鼠模型（肾素抑制）和轻度高尿酸血症(HUA)诱导的早期盐敏感高血压（高肾素型）小鼠模型，尾夹法监测血压变化，观察血压及水盐代谢的变化；评价血压与饮食中盐含量的关系；病理染色观察肾脏病变特点、评价小管间质损害。

2.    在两种模型中分别检测肾组织3种钠离子转运通道（NCC/NKCC-2/γENaC）蛋白和mRNA水平。定量上游的调控激酶SGK-1的蛋白表达，检测炎症焦亡信号caspase-1/IL18/IL1β及相关指标的异常。

3.    放免法观察调节血压和出球小动脉收缩的肾素、血管紧张素II和醛固酮水平，免疫组化进行球旁器肾素定位，免疫印迹法观察与管球反馈和入球动脉舒缩功能相关的腺苷受体表达水平。

第三部分：NCC在管球反馈缺失后对血压和水盐代谢的可能作用

1.    在GS患者和非GS患者中进行速尿试验，计算试验前后的肌酐清除率以推测管球反馈的状态，比较GS患者和非GS患者肌酐清除率的变化，初步推测体内管球反馈的活化程度。

2.    在管球反馈缺失的A1AR^-/-小鼠中，分别观察DS模型和HUA模型的血压及水盐代谢变化，评价肾脏病变特点；免疫印迹法检测3种钠离子转运蛋白（NCC/NKCC-2/γENaC）和炎症焦亡信号caspase-1/IL18/IL1β的水平；比较A1AR敲除后其他类型腺苷受体的变化。

3.    分离野生型和A1AR^-/-小鼠肾小球的入球动脉，显微灌注观察不同尿酸水平对入球动脉的急性刺激作用；免疫组化半定量评估尿酸对野生型和A1AR^-/-小鼠入球动脉的慢性重塑作用。

结果：

第一部分：先天NCC功能障碍GS患者临床特点及生物影像标志物

1.    105例低血钾患者检测到SLC12A3基因突变，共携带69种突变，平均血压为110.0±12.4/72.1±9.8mmHg，并伴有不同程度的肾素、血管紧张素II、醛固酮活化；氢氯噻嗪实验提示患者NCC不同程度受损。以基因诊断为金标准，氢氯噻嗪功能试验的敏感性和特异性分别为93.1%和88.9%，曲线下面积为0.989(95% CI 0.961-1.000)，均优于低血镁和低尿钙单个指标或联用的临床诊断价值；

2.    男性患者较女性发病早、血钾低、血压高、多尿更显著，但生活质量无差异。5例妊娠患者孕期前3月血钾水平较妊娠前显著下降，对症处理后，妊娠过程平稳、结局良好。男性尿PGE2及其代谢产物（PGEM）水平显著高于女性，且GS患者显著高于同性别的对照，并观察到升高的PGE2与更严重的NCC功能障碍（氢氯噻嗪试验）、代谢性碱中毒和电解质紊乱相关。肾皮质的COX-2蛋白表达显著低于对照。尿液外泌体蛋白定量检测到GS患者NCC表达显著减低，COX-2和COX-1的表达均增加。

3.    GS患者肾脏病理以球旁器增生为主，可伴有肾小球肾炎和间质小管损伤。常规核磁显示GS患者肾脏无异常，功能成像中，弥散峰度成像的K值和D值均与血Cl和HCO₃^-相关性良好，而IVIM的D值和DP值则与NCC体内功能损害程度相关。

第二部分：盐敏感高血压小鼠模型的建立及NCC相关生理的改变

1.    成功建立高盐高醛固酮的DOCA盐敏感高血压小鼠，表现为收缩压和舒张压持续升高，并伴有尿量增加、尿钠排出量增加和尿渗透压下降；肾脏显著增大，较早(4w)出现小管间质损害和不同程度的间质纤维化，伴有NCC/NKCC-2的表达上调和Casepase-1/IL-1β的激活、COX2 高表达。

2.    氧嗪酸钾（250mg/kg/d）灌胃及0.1%尿酸水饲养成功诱导小鼠轻中度高尿酸血症，并引起血压轻度升高，不伴有尿酸肾病的间质小管改变。小鼠血压与饮食中盐浓度相关，符合盐敏感型高血压的特点，即血压随饮食中盐浓度升高而升高，但其肾组织NCC/NKCC-2/ENaC和炎症焦亡分子的表达量均无显著变化。

3.    高盐高醛固酮小鼠肾素水平降低，管球反馈相关的A1AR受体无显著差异，A2aR显著降低；高尿酸盐敏感高血压模型中，球旁器肾素表达增加，肾素管球反馈相关的A1AR表达显著升高，而A2aR的表达降低，同时存在RAS系统和管球反馈活化。

第三部分：NCC在管球反馈缺失后对血压和水盐代谢的可能作用

1.    速尿阻断致密斑NKCC2后，GS患者Ccr显著降低，降低的幅度超过NCC 功能正常的非GS患者。

2.    管球反馈缺失的A1AR^-/-小鼠，建立DOCA高血压模型后，肾脏纤维化提前发生，伴随A1AR^-/-时上调的A2bR恢复到野生型水平；单肾切除后，A1AR^-/-小鼠血压轻度升高，NCC表达上调；高盐高醛固酮刺激不能进一步上调NCC，但可以通过上调NKCC-2促进水和盐的排泄，维持血压与WT-DS小鼠相似。

3.    管球反馈缺失的高尿酸模型中血压恢复正常，入球动脉慢性重塑消失，伴有A2bR上调；急性尿酸直接刺激入球动脉收缩，并呈剂量依赖性。敲除A1AR后该效应消失，且扩张入球动脉。

结论：

1.    先天性NCC功能障碍的GS患者表现为正常或低血压、低血钾，尿钾、钠、氯排泄增加，以基因诊断为金标准，采用NCC功能试验（氢氯噻嗪试验）临床诊断GS的敏感性和特异性均优于传统的临床诊断标准（低血镁和低尿钙）。

2.    尿液PGE2水平升高有助于评价临床表现严重程度和NCC功能缺失程度，是潜在的治疗位点；外泌体COX-1和COX-2表达的增加可能是PGE2升高的机制，也可能与活化的管球反馈相关。功能核磁DKI和IVIM参数与NCC功能相关，需要进一步的研究证实。

3.    低肾素的盐敏感高血压DS模型存在NCC和NKCC2表达上调，炎症可能为高滤过晚期的继发表现；管球反馈缺失后NCC可能与维持正常状态下的血压和电解质平衡相关，在醛固酮和高盐的进一步刺激下，NKCC-2表达增加。

4.    在高尿酸诱导的盐敏感高血压中，NCC改变和炎症活化不突出，入球动脉的急性收缩和慢性重塑则是这一时期的高血压的重要发病机制，A1AR可能参与其中。

Background and objectives:

Water and electrolytes balance in body is dependent on the functional integrity of glomerulus and renal tubules, and the tubuloglomerular feedback (TGF) between them. The sodium-chloride symporter (also known as Na⁺-Cl⁻ cotransporter, NCC) is a transmembrane protein encoded by the gene SLC12A3. It contributes to the balance of water and electrolytes as same as the the Na-K-Cl cotransporter 2 (NKCC2) in thick ascending limb of the loop of Henle and the macula densa in nephrons, and the Epithelial Sodium Channel (ENaC) in the collecting tubule. NKCC2 and ENaC are proved to be an important inductor for TGF, while the role of NCC in TGF is still unclear. Gitelman syndrome (GS), a rare inherited salt-losing tubulopathy mainly caused by loss-of-function mutations in the SLC12A3 gene, is a natural disease modal to study the function of NCC. However, there is no related research about clinical and pathophysiology of GS, and no practical biomarker and functional imageology methods for evaluating the severity and guiding the treatment of GS.

This study aimed to observe the clinical and pathological features, quality of life and gender difference in a GS cohort with a fairly large sample size. Compare the diagnostic value of funcitonal test of NCC. Evaluate the correlation between PGE2 level or its metabolin in urine and the clinical manifestations. Explore the the diagnosing value of exosome and functional MRI for GS patients. Establish two different salt- sensitive hypertension mice model, i.e. the deoxycorticosterone acetate (DOCA)-salt hypertensive model and hypeluricemia induced hypertensive model, and evaluate the role of NCC in the development of hypertension and kidney damage. Furthermore, investigate the relationship of tubuloglomerular feedback and NCC in the Adenosine receptor type 1 knockout (A1AR^-/-) model.

Methods:

Part 1. Study about the characteristics of clinical manifestations, biomarkers and imageology in the congenital NCC dysfuncion patients with GS

1.  Collect the clinical data of the suspicious GS patients, extract the DNA of peripheral blood for exome sequencing, taking the gene test as gold standard, evaluate the function of NCC in vivo by using hydrochlorothiazide. Compare the diagnostic value of hydrochlorothiazide test to hypomagnesemia and hypocalcinuria for GS patients.

2.  Observe the feature of blood pressue and fluid-electrolyte metabolism in GS patient. Gather the plasma and 24h urine for testing PGE2 and its metabolin levels by ELISA， analysis the correlation between PGE2 with clinical manifestations and NCC function. Compare the gender difference in the manifestations of GS, summarize the clinical data of pregnancy and the SF-36 scale for the quality of life in GS patients. Measure the PGE2 and its metabolites both in blood and urine, to test the gender difference and the correlation of clinical manifestation. Collect the data of female patients with pregnancy, observe the elctrolytes level and complications during pregnancy.

3.  Collect the renal pathological data of GS patients, compare the expression of COX-2. Extract the exosome of urine, measure the expression of NCC, COX-2 and COX-1. Collect the medical imagology data of GS patients using the functional-MRI (DKI and IVIM) of kidney.

Part 2. The establishment of salt sensitive hypertension mice model and the change of NCC associated water electrolyte balance.

1.  Establish the uninephrectomized and deoxycorticosterone acetate (DOCA)-salt hypertensive mice model, and establish the early-stage hypertensive model induced by mild hyperuricemia. Monitor the variation of blood pressure, together with the change of uric acid and electrolytes levels. Evaluate the relation between salt-intake with BP. Evaluate the renal tubulointerstitial damage by PAS and Masson staining.

2.  In two mice model, measure the expression of NKCC/NKCC2/ENaC including protein and mRNA, quantify the expression of SGK-1, and test the caspase-1/IL-18/IL1β in inflamation-pyroptosis pathway and adenosine receptors.

3.  Measure the levels of renin, angiotensin, aldosterone by radio-immunity assay. Locate the expression of renin in juxtaglomerular complex by immunohistochemistry. Observe the expression of adenosine by immunoblotting

Part 3. The possible effect of NCC on blood pressure and water electrolyte metabolism when lacking tubuloglomerular feedback.

1.  By furosemide test, calculate teh clearance rate of creatinine (CCR), speculate the state of tubuloglomerular feedback. And compare the CCR of GS and non-GS patients to evaluate the activity of tubuloglomerular feedback.

2.  In A1AR knockout mice model, observe the difference of BP, water, adn electrolytes in DS model and HUA model. measure the expression of NKCC/NKCC2/ENaC including protein and mRNA and test the caspase-1/IL-18/IL1β in inflamation-pyroptosis pathway and adenosine receptors.

3.  Transect the afferent arterioles of wild or A1AR knockout mice, observe the effect of different UA levels on afferent arterioles by mircoperfusion. And evalutae the chronic remodelling effect of UA to afferent arterioles by semi-quantitative immunohistochemistry analysis

Results:

Part 1. study about the characteristics of clinical manifestations, biomarkers and imageology in the congenital NCC dysfuncion patients with GS

1.    Totally 105 hypokalemia patients detected SLC12A3 gene mutation and diagnosed as Gitelman syndrome, and totally 69 types of mutation were found. The mean blood pressure of GS patients was 110.0±12.4/72.1±9.8mmHg combined with Renin-AngiotensionII-Aldosterone system activation. Hydrochlorothiazide test(HCT) indicated different degree of NCC depression in GS patients. Compared to the gold standard of gene diagnose, the sensitivity and specificity of hydrochlorothiazide test was 93.1% and 88.9% respectively, the AUC was 0.989(95% CI 0.961-1.000), all were better than those of the common used criteria of hypomagnesemia and hypocalcinuria.

2.    Male GS patients seemed to have an early onset, with lower serum potassium levels, higher blood pressure and more severe polyuria, but no difference in the test of quality of life. There were 5 female patients got pregnant after disease onset, they performed a decrease of serum potassium in the first trimester, after symptomatic treatment they all had stable process to good outcome. The average PGE2 levels of GS patients were higher than the normal control group of the same gender, and male patients had a higher level than female patients. The elevated PGE2 levels might be associated with more severe NCC damage, metabolic alkalosis and electrolyte disturbance. of GS patients was worse than normal control, and there was no difference between male and female. COX-2 expression was decreased in the cortex of kidney, while a decrease of NCC expression and increase of COX-2 and COX-1 expression were detected in urine exosome.

3.    The renal pathological results of GS patients showed the proliferation of juxtaglomerular apparatus. While the conventional MRI showed no abnormal of kidney for GS patients, the mean kurtosis and mean diffusivity level of diffusion kurtosis imaging (DKI) had a good correlation with serum chlorine and bicarbonate, and the D value and DP value of the inteavoxel incoherent motion imaging (IVIM) had a correlation with the NCC function.

Part 2. The establishment of salt sensitive hypertension mice model and the change of NCC associated water electrolyte balance.

1.    The DOCA-salt hypertensive mice model was established, with had a significant continuous elevation of systolic and diastolic blood pressure，together with increased urine volume and more electrolytes excretion in urine. The kidneys were magnified and significant tubulointerstitial injury and fibrosis. The expression of NCC/NKCC-2/ENaC at 4th week and the expression of caspase-1/IL1β at 8th week were both increased.

2.    Oxonic acid gavage（250mg/kg/d）together with 0.1% uric acid drinking water stably induced a mild hyperuricemia and the blood pressure was upregulated, without the injury of urate nephropathy. The hypertension was regulated with the change of diet salt， consistent with the feature of salt sensitive hypertension. There was no significan change in the amout of NCC/NKCC-2/ENaC and caspase-1/IL1β of inflamation-pyroptosis.

3.    The renin activety in the DOCA-salt hypertensive mice was suppressed and the tubuloglomerular feed back associated adenosine receptors，A2AR was decreased，while no change of A1AR. As to the hyperuricemia hypertension mice, A1AR was upregulated while A2AR was downregulated，RAS and tubuloglomerular feed back was activated.

Part 3. The possible effect of NCC on blood pressure and water electrolyte metabolism when lacking tubuloglomerular feedback.

1.    When the macula densa NKCC2 was blocked by furosemide, patients with normal NCC function（non GS） showed a weak decrease of creatinine clearance rate，while GS patients showed a significant decreasion.

2.    When lacking tubuloglomerular feedback (A1AR-/- mice), after the model was established，the fibrosis happened earlier，wihle the upregulated A2bR returned to normal; the difference of NCC expression disappeared. In the latter model, the expression of NKCC-2 was up-regulated, the excretion of urine volume and urine sodium increased, and. The blood pressure descended in the first week, and ascended thereafter, but not higher than the wild type, nor was the kidney damage.

3.    When A1AR was knockout, the hyperuricemia at same level would not induce hypertension, nor was the lesion of afferent arterioles, with the up-regulated of A2bR. The microperfusion of afferent arterioles showed that uric acid directly stimulated the contraction of afferent arterioles, which showed to be dose dependent. Furthermore, the effect disappeared after the A1AR knockout.

Conclusions:

1.    The congenital NCC dysfuncion patients with GS showed normal tension、hypokalemia，the excretion of potassium, sodium and chloride were increased. Compared to the gold standard of gene diagnose, the sensitivity and specificity of hydrochlorothiazide test was better than those of the common used criteria of hypomagnesemia and hypocalcinuria.

2.   The elevated PGE2 levels might be helpful to evaluate the degree of clinically severity and dysfunction of NCC. The exosome COX-2 expression was elevated and may be the mechanism of the elevated PGE2, also maybe the reason of activated tubuloglomerular feedback. Functional MRI of DKI and IVIM may be associated with the NCC function but further research was needed.

3.  The DOCA salt-sensitive hypertensive model with low renin showed an increase of NCC and NKCC2 expression. The inflammation might be secondary to the high filtration. At the absence of tubuloglomerular feedback, NCC might contribute to the balance of normal blood pressure and electrolytes. Stimulated by aldosterone and high salt-intake, the expression of NKCC-2 increased.

4.  In the hyperurecima-induced hypertension, the change of NCC and activation of inflammation were not obvious. The acute contraction and chronic damage of afferent arterioles played an important role of the mechanism for hypertension.