研究背景: 药物肾毒性是急慢性肾功能不全的重要原因，其中肾脏近端小管主动分泌或重吸收药物（及其代谢产物），是肾损伤的主要部位。近端小管损伤可表现为功能障碍，即电解质和小分子物质重吸收障碍，临床表现为完全和不完全范可尼综合征，损伤严重时甚至发生细胞凋亡或坏死，是急性肾损伤(Acute kidney injury， AKI)最重要的原因。而肾小管损伤与修复过程中，不仅再现发育的过程，还和肾小球（管球反馈)、不同节段的小管之间发生相互协调和作用，决定肾脏最终的结局。研究药物近端小管损伤特点和机制，不仅有助于理解药物近端小管损伤修复的共同机制，也是探究小管之间，及其与小球之间相互作用的重要窗口，但因为临床样本量和研究手段的限制，国内外关于药物相关范可尼综合征的研究并不多。如最常见导致近端小管损伤的抗病毒药物富马酸替诺福韦醋（Tenofovir disoproxil fumarate，TDF)肾毒性，缺乏大样本中国人群临床资料，肾小管功能障碍与肾小球滤过率（Glomerularfiltrationrate，GFR)之间的关系并不明确；临床观察到长期口服噻嗪类利尿剂阻断远端小管钠氯共转运蛋白（Na+-Cl- co-transporter， NCC) 后，对近端小管功能的影响，但缺乏相应的机制研究。本研究首先观察TDF治疗人类免疫缺陷病毒(Human immunodeficiency virus， HIV)感染患者队列中近端小管损伤的临床特点，探索近端小管和肾小球滤过功能的相关性，进而在体外细胞实验中观察转运子损伤和内质网应激的关系；最后在噻嗪类利尿剂作用位点远端小管NCC功能障碍的Gitelman综合征（Gitelman syndrome，GS)患者队列中总结近端小管功能的临床特点，评估肾小球储备功能，分析可能的病理生理机制。研究目的:1.观察接受TDF治疗的HIV感染患者的近端小管损伤和肾小球滤过功能损伤的特点，分析小管功能损伤与GFR之间的关系；2.体外培养近端小管上皮细胞，观察替诺福韦（Tenofovir，TFV)损害不同转运子功能，及其与内质网应激的关系；3.分析GS队列中近端小管功能改变及肾小球储备功能特点，探讨远端小管功能障碍影响近端小管功能、肾小球储备功能的可能机制。研究方法:第一部分替诺福韦损伤肾脏近端小管的临床特点和可能机制1.替诺福韦治疗HIV感染患者的肾脏损伤特点纳入2001年9月1日至2019年8月31日在北京协和医院就诊、规律随访、接受TDF治疗的HIV感染患者。收集基线和随访的临床资料。定义近端小管损伤为满足低磷血症、低尿酸血症、低二氧化碳结合力、尿糖阳性、尿蛋白阳性中的2项及以上。定义肾功能快速恶化为估算肾小球滤过率（Evaluated glomerular filtration rate, eGFR)年下降率超过5ml/min/1.73m~2。总结该队列中近端小管损伤、肾功能损伤的发生比例和临床特点，分析二者之间的相关关系。2.替诺福韦损伤近端小管与内质网应激之间的关系:培养人近端小管上皮细胞系（HK2)，通过倒置相差显微镜观察细胞形态，免疫荧光染色鉴定HK2特异性标志物（细胞角蛋白CK18、Megalin)，以及特定转运子（钠糖共转运子SGLT2、钠磷转运子NaPi-IIa、尿酸盐阴离子转运体 URAT1)。使用CCK8细胞增殖实验检测不同TFV浓度和作用时间对HK2细胞存活率的影响，选择合适的TFV浓度和时间刺激HK2细胞，利用免疫荧光染色半定量方法评估TFV对转运子、内质网应激标志物GRP78/BiP的影响。第二部分Gitelman综合征患者肾脏近端小管功能改变纳入2005年8月1日至2019年12月31日在北京协和医院就诊、基因确诊为GS的患者，同时选取性别年龄匹配的健康对照。对见基因一代测 序为单杂合突变的患者，进一步行全外显子组测序明确基因突变情况。收集人口学信息、症状、血尿化验检查等临床资料。氢氯噻嗪试验通过计算口服氢氯噻嗪前后尿氯离子排泄分数的变化量，评估患者NCC功能。总结患者钙磷代谢、尿 酸代谢特点。同时建立蛋白负荷试验方法，通过计算口服高蛋白饮食前后GFR 的变化量，评估患者肾小球储备功能。研究结果:第一部分替诺福韦损伤肾脏近端小管的临床特点和可能机制1.共纳入接受TDF治疗的HIV感染患者375例，男性为主（占90.1%),平均 随访时长为37.9±26.1月，最长达116月。近端小管损伤的比例为6.7%，其中最常见临床表现是蛋白尿（20.3%)和低磷血症（12.3%)。近端小管损伤与低体重显著相关，而与年龄、TDF疗程、基线病毒载量、基线CD4+T淋巴细胞计数等无显著相关性。2.有完整血肌酐随访的患者共373例，随访结束时的eGFR较基线显著下降(104.6±15.2 比110.6±14.2ml/min/1.73m~2;P<0.001)，eGFR 年下降率平均为5.0±22.7ml/min/1.73m~2，其中23.6%的患者eGFR年下降率超过5ml/min/1.73m~2。女性与高eGFR年下降率显著相关。3.细胞实验：贴壁HK2细胞呈卵圆形或多边形，部分融合分布呈铺路石样。HK2细胞特异性标志物CK18、Megalin免疫荧光染色阳性，提示为近端小管上皮细胞。近端小管转运子SGLT2、URAT1、NaPi-IIa免疫荧光染色阳性，提示HK2细胞正常表达该转运子。细胞增殖毒性检测中，TFV刺激时间为24h时，所检测TFV浓度梯度范围内细胞存活率均大于95%。刺激时间为48h时，2.5pmol/L的TFV浓度下，细胞存活率小于95%。TFV(1pmol/L)干预肾小管上皮细胞48h后，转运子（SGLT2、URAT1、NaPi-IIa)表达均显著降低，而内质网应激标志物GRP78/BiP表达无显著性差异。第二部分：Gitelman综合征患者肾脏近端小管功能改变1. 共纳入基因确诊GS患者125例，33例（26.4%)患者见基因一代测序为单杂合突变。25例患者完成了全外显子组测序，9例（36%)患者最终确诊为复合杂合突变，全外测序新检出7个突变位点，包括3种错义突变、1种剪接位点突变、3种内含子突变。2. 与健康对照相比，GS患者血钙水平更高，尿钙、血镁、甲状旁腺素、25羟基维生素D水平更低，血磷无显著差异。血镁水平越低，甲状旁腺素水平也越低。离子钙水平低与代谢性碱中毒相关。3.GS患者血尿酸显著高于健康对照。男性、女性中高尿酸血症比例分别为19.4% 和34.5%。高尿酸血症组患者尿酸排泄分数显著低于血尿酸正常患者。4.共4例GS患者完成了蛋白负荷试验，肌酐清除率法计算平均肾小球储备功能为66.7±21.5ml/min/1.73m~2,肌酐联合胱抑素C法计算平均肾小球储备功能为12.1±4.4 ml/min/1.73m~2，与健康对照均无显著性差异。结论:在本研究条件下：1.TDF治疗的HIV感染患者中，6.7%出现近端小管损伤，23.6%肾功能年下降率超过5ml/min/1.73m~2。近端小管转运子功能损伤与eGFR年下降率无显著相关性。2.TFV下调近端小管上皮细胞转运子（SGLT2、URAT1、NaPi-IIa)表达。3.GS患者中，尿钙低、血总钙高、高尿酸高，与近端小管功能代偿相关。

Background: Drug nephrotoxicity is an important cause of acute and chronic renal insufficiency. The renal proximal tubule actively secretes or reabsorbs the drug (and its metabolites), which is the main site of kidney injury. The proximal tubule injury can be manifested as dysfunction, that is, reabsorption disorder for electrolytes and small molecule substances, leading to incomplete and complete Fanconi syndrome in clinical practice. In worse cases, even apoptosis or necrosis can occur, which is the most important reason of acute kidney injury (AKI). Tubule injury and repair can reproduce the development process, and it can also coordinate and interact with the glomerulus (tubuloglomerular feedback) and the tubules of different segments, which determines the outcome of the kidney. Investigation of the clinical characteristics and mechanism of proximal tubule injury related with drugs can not only help understand the common mechanisms of tubule injury and repairment, but also open an important window for exploring the interaction between tubules and their interaction with glomerulus. However, there are few studies on drug-related Fanconi syndrome all around the world, because of the limited sample size and clinical research methods. For example, tenofovir disoproxil fumarate (TDF), as the most common antiviral drug, can causes proximal tubule damage. The relationship between renal tubular dysfunction caused by TDF and decline of glomerular filtration rate (GFR) is not clear due to lack of clinical data from large cohorts, especially in the Chinese population. Long-term oral prescription of thiazide diuretics, which can block the sodium chloride co-transporter (NCC) of the distal tubule, which has been recognized to affect the function of the proximal tubule, but the underlying mechanism is largely unknown. In this study, we first observed the clinical characteristics of proximal tubule injury in the cohort of human immunodeficiency virus (HIV) infected patients treated with TDF, and explored the correlation between proximal tubule and GFR. Then we explored the relationship between transporter damage and endoplasmic reticulum (ER) stress in cell lines. Finally, we summarized the clinical characteristics of proximal tubule function in the cohort of patients with Gitelman syndrome (GS) with congenital NCC dysfunction, the same site of thiazide diuretic targeting, evaluated the glomerular renal functional reserve in GS patients, and analyzed the potential pathophysiological mechanisms. Aims: 1. To observe the characteristics of proximal tubule and GFR injury in HIV-infected patients receiving TDF and analyze the relationship between tubule dysfunction and GFR decline. 2. To culture human proximal tubule epithelial cells in vitro, and observe the relationship between transporter injury and ER stress induced by tenofovir (TFV). 3. To evaluate the function of proximal tubule and the glomerular renal functional reserve in the GS cohort, and explore the possible mechanisms of proximal tubule dysfunction and renal function secondary to distal tubule dysfunction. Methods: Part I: Clinical characteristics of proximal tubule injury caused by TDF and potential mechanism 1. Characteristics of kidney injury of HIV-infected patients treated with TDF HIV-infected patients treated with TDF who were regularly followed up in Peking Union Medical College from Sep 1, 2001 to August 31, 2019 were enrolled. Baseline and follow-up data were collected. Proximal tubule dysfunction was defined as meeting two or more of following criteria: hypophosphatemia, hypouricemia, low carbon dioxide binding capacity, positive urine glucose, and positive urine protein. Rapid deterioration of renal function was defined as the annual decline rate of estimated glomerular filtration rate (eGFR) exceeding 5ml/min/1.73m~2. The percentage and clinical characteristics of proximal tubule dysfunction and renal function impairment were analyzed, as well as the relationship between them. 2. The relationship between proximal tubule injury caused by TFV and ER stress Human renal proximal tubular epithelial cell line (HK2) was cultured. The morphology was observed by inverted phase contrast microscope, and immunofluorescence was used for detection of specific markers (CK18 and megalin) and transporters (SGLT2, NaPi-IIa, and URAT1). Cell proliferation and cytotoxicity assays was conducted to explore the effect of TFV to the survival rate of HK2 cells. Appropriate concentration of TFV was used to stimulate HK2 cells, and the impact on transporters and ER stress was evaluated. Part II: Clinical characteristics of renal proximal tubule function in GS patients Patients who were diagnosed with GS (confirmed by gene sequencing) in Peking Union Medical College Hospital from August 1, 2005 to December 31, 2019 were enrolled, as well as the selected gender- and age-matched healthy controls. For patients whose SLC12A3 gene was sequenced as a single heterozygous mutation, further whole exome sequencing was performed to clarify the gene mutation. Clinical data including demographic information, symptoms, and hematuria tests were collected. The hydrochlorothiazide test was performed to assess the function of NCC. Characteristics of calcium and phosphate metabolism and uric acid of patients were analyzed. The glomerular renal functional reserve in GS patients were evaluated by acute protein load test. Results: Part I: Clinical characteristics of proximal tubule injury caused by TDF and potential mechanism 1.A total of 375 HIV-infected patients receiving TDF were enrolled, mainly males (90.10%), with an average follow-up duration of 37.9±26.1 months, up to 116 months. The proportion of proximal tubule injury was 6.7%, and the most common clinical manifestations were proteinuria (20.3%) and hypophosphatemia (12.3%). Proximal tubule dysfunction was significantly associated with low body weight, but was not associated with age, TDF course, baseline viral load, baseline CD4+ T lymphocyte count. 2. A total of 373 patients had complete creatinine result during the follow-up. The eGFR at the end of the follow-up was significantly lower than the baseline (104.6±15.2 vs. 110.6±14.2 ml/min/1.73m~2,P<0.001). The average annual decline rate of eGFR was 5.0±22.7 ml/min/1.73m~2, of which 23.6% of patients had an annual decline rate of eGFR exceeding 5 ml/min/1.73m~2. Female was significantly related to the high annual decline rate of eGFR. 3. Cell experiments in vitro: HK2 cells were elliptical or polygonal, and looked like paving stone when fused together. The immunofluorescence detection was positive for CK18, megalin, SGLT2, NaPi-IIa, and URAT1. Cell proliferation and cytotoxicity assays showed that the survival rate of HK2 cells was more than 95% under different concentrations of TFV for 24h, while below 95% under 2.5μmol/L for 48h. When given TFV with a concentration of 1μmol/L for 48h, the expression level of transporters involving SGLT2, URAT1, and NaPi-IIa, were declined, while the expression level for GRP78/BiP showed no difference. Part II: Clinical characteristics of renal proximal tubule function in GS patients 1. A total of 125 patients with genetic diagnosed GS were enrolled, and 33 patients (26.4%) were with only a single heterozygous mutation. Twenty-five patients completed whole exome sequencing, and 9 patients (36%) were finally diagnosed with compound heterozygous mutations. Seven mutation sites were newly detected by whole exome sequencing, including 3 missense mutations, 1 splice-site point mutation and 3 intron mutations. 2.Compared with healthy controls, GS patients had higher blood calcium level. However, urinary calcium, serum magnesium, parathyroid hormone, and 25-hydroxyvitamin D were lower in GS patients. Blood phosphate was not significantly different between the two groups. Lower blood magnesium level was associated with lower parathyroid hormone level. Low levels of ionized calcium are associated with metabolic alkalosis. 3. Serum uric acid in GS patients was significantly higher than that in healthy controls. The proportion of men and women with hyperuricemia were 19.4% and 34.5%, respectively. Urinary fraction excretion of uric acid in the hyperuricemia group was significantly lower than that of patients with normal serum uric acid. 4. In 4 GS patients, the mean glomerular renal functional reserve was 66.7±21.5ml/min/1.73m~2 by creatinine clearance, and 12.1±4.4ml/min/1.73m~2bycystatin C-based eGFR, both of which showed no differences with health controls. Conclusion: Under the research conditions of this study: 1. Among the HIV-infected patients treated with TDF, 6.7% had proximal tubule dysfunction and 23.6% showed annual decline rate of eGFR exceeding 5ml/min/1.73m~2. Proximal tubule dysfunction wasn’t related to annual decline rate of eGFR. 2. The protein levels of proximal tubular transporters (SGLT2, URAT1, NaPi-IIa) were downregulated by TFV. 3. In GS patients, low urinary calcium, high serum calcium and high serum uric acid were related to the compensation of proximal tubular function.