第一部分：

研究背景及目的：IgA肾病（IgA nephropathy, IgAN）是亚洲目前最常见的原发性肾小球疾病之一。IgAN具有异质性、进展性等特点，其临床表现多样，确诊IgA肾病的患者，20%-40%的患者会在20年内进展至终末期肾病。降低持续性蛋白尿是IgA肾病治疗的主要目标之一，目前治疗方式主要包括ACEI/ARB类足量使用和免疫抑制治疗。目前，IgA肾病患者免疫抑制治疗是否获益仍存在争议，指南中对免疫抑制治疗的推荐级别整体较低，特别是对那些肾病理损伤较严重和（或）肾功能下降的患者。与目前现有大规模IgAN队列相比，我院IgAN队列具有病情较重、免疫抑制治疗较积极且随访时间较长等特点。本研究通过回顾性分析CKD 3-4期IgAN且随访超过5年的患者，探究免疫抑制治疗对病情较重的患者治疗的有效性，并了解哪些患者接受免疫抑制治疗可能获益，以期为IgAN患者提供新的治疗策略。

   研究方法：为一项单中心回顾性研究，收集2012年1月至2014年12月在我院首次诊断IgA肾病的496例患者的基线临床数据并进行为期5年以上的随访。根据患者的肾功能水平及免疫抑制治疗情况将患者分成4组：①CKD 3期免疫抑制治疗（96例），②CKD 3期无免疫抑制治疗组（30例），③CKD 4期免疫抑制治疗（26例），④CKD 4期无免疫抑制治疗组（12例）。以死亡、进展至ESRD或透析、血肌酐倍增作为主要研究终点，以eGFR进行性下降（下降＞1ml/min/1.73m²/年）作为次要研究终点。并对CKD3期接受免疫抑制治疗的96例患者进行亚组分析，探究免疫抑制治疗的IgAN患者预后影响的因素，以及是否存在长期预后的短期预测指标。使用IBM SPSS Statistics 22.0软件进行数据分析，定义p＜0.05 具有统计学显著性。

研究结果：研究共纳入符合入排标准的164例患者，其中男性98例、女性66例，男女比例为1.5:1；平均年龄42.75±13.04岁（18-69岁），平均随访时间为5.51±1.13年。CKD 3期患者126例、CKD 4期患者38例，不同分期CKD患者根据是否接受免疫抑制治疗分组后，基线年龄、性别、肾脏病病程、既往高血压及糖尿病等代谢性疾病病史方面均无明显差异。Cox单因素及多因素回归分析表明，免疫抑制治疗可显著改善CKD 3期IgAN患者的预后（HR 0.435, 95％CI 0.200-0.944, p= 0.035），但对CKD 4期IgAN患者的预后无明显改善作用（p=0.364）。亚组分析显示，基线eGFR（OR 0.909, 95％CI 0.834-0.991, p= 0.031）与主要研究终点和尿蛋白缓解相关，但CKD3a期及3b期接受免疫抑制治疗的肾脏预后无明显差异（p=0.197）。毛细血管襻坏死（OR 0.189[95% CI 0.050-0.709], p=0.014）、新月体小球比例（OR 0.200[95% CI 0.100-0.521], p=0.003）、牛津分型T2相对T0评分（OR 5.490, 95% CI 1.323-22.727,p=0.019）、C2相对C0评分（OR 0.580, 95% CI 0.070-0.910, p=0.009）与患者次要研究终点相关。接受免疫抑制治疗的患者1年内部分或完全缓解，可提示较好的长期肾脏预后（HR 0.555 [95％CI 0.296-0.759；p= 0.035]），且治疗后达到缓解的时间越短，其预后可能更好。

研究结论：CKD 3期的IgAN患者接受免疫抑制治疗可能获益，不仅改善蛋白尿缓解情况，还可以延缓肾功能恶化。但对于CKD4期的患者，积极的免疫抑制治疗需要谨慎。基线肾功能较好或者肾脏病理襻坏死、新月体等急性病变较多或间质纤维化、小管萎缩等慢性病变较少的患者接受免疫治疗后预后较好，在确诊后可给予积极的治疗。此外，在接受免疫抑制治疗1年内可达完全或部分缓解的患者长期预后较好。

第二部分：

研究背景及目的：IgA肾病患者可存在足细胞肥大、足突融合、足细胞内吞形成囊泡等病理表现，且超过70%的患者存在局灶节段硬化（S1），是肾脏预后不良的独立危险因素。此外，足细胞损伤程度也可作为提示疾病严重程度的指标和疾病进展的预测指标。探讨如何减轻足细胞损伤也许可成为IgA肾病潜在的药物治疗靶点及研究方向，可减轻患者蛋白尿及慢性肾脏病进展。而Hippo信号通路及其中最重要的转录激活因子Yes相关蛋白（YAP）在足细胞的细胞凋亡及损伤后修复的过程中发挥着重要作用。本研究关注IgA肾病患者的足细胞损伤及YAP表达情况，推测在IgAN中YAP对足细胞可能起到一定的保护作用，且YAP表达水平与患者疾病严重程度及肾脏预后具有相关性，从而为减轻IgAN患者尿蛋白水平，减缓肾小球硬化及改善患者预后提供新的治疗思路。

研究方法：筛选2012年1月至2014年12月于北京协和医院住院行肾穿刺首次诊断IgAN的患者共496例，随机抽取Lee氏3级及4级患者，并根据性别、年龄匹配得到蛋白尿≥3.5g/d的Lee 3-4级患者16例，尿蛋白1-3.5g/d的Lee 3-4级患者16例，匹配Lee 2级患者3例作为疾病对照；同时获取健康对照5例，均为肾脏肿瘤行单侧肾切除后获得其远离肾脏肿瘤的相对健康的肾脏皮质。免疫组化及免疫荧光染色确定YAP在肾脏的表达情况，免疫荧光WT1及YAP共染确定核内表达YAP的足细胞，利用YAP核（+）的足细胞数与该小球足细胞总数的比例代表YAP表达水平。并对不同病理损伤等级的IgAN患者及不同YAP表达水平的IgAN患者进行亚组分析。

研究结果：（1）IgAN Lee氏3-4级患者与健康对照相比，足细胞计数明显减少（26.18[23.96, 28.39] vs 35.37[29.40，41.75], p=0.043;16.37[13.42, 19.31] vs 35.37[29.40，41.75], p=0.001），并且随着病理级别增加，足细胞计数减少及损伤加重。

（2）IgAN Lee 2-4级患者细胞核内YAP阳性的足细胞比例均较健康对照组明显下降，且随着病理级别增加，YAP核（+）的足细胞比例逐渐减少。根据散点图及Pearson相关性检验，YAP的表达水平与足细胞计数具有正相线性关系（r=0.876，p<0.001），YAP核阳性的足细胞比例增加，肾小球的足细胞数越多。

    （3）IgAN患者YAP的表达水平与疾病严重程度具有相关性，高YAP表达组具有较低的24hUP [2.60（1.18，4.02）vs 3.86（3.38，3.34），p=0.038]及更高的eGFR [106.68±20.01 vs 64.62±19.64, p<0.001]，组织病理检查还具有较低的球性硬化比例[3.77%（1.30%，6.26%）vs 30.40%（21.13%，39.68%），p<0.001]、节段性硬化比例[2.33%（0.94%，3.73%）vs 13.16%（7.25%，19.08%），p<0.001] ]及新月体比例[6.21%（2.94%，9.48%）vs 16.59%（9.60%，23.57%）,p=0.011]，且小管间质纤维化程度更轻（p<0.001）。根据MEST-C分组显示，存在肾小球硬化（S1）、肾小管萎缩或间质纤维化（T1-2）、重度细胞/纤维细胞性新月体（C2）病变的患者足细胞YAP表达水平更低。

    （4）在中位随访6年后，8例IgAN患者到达结局终点，根据COX单因素及多因素回归分析表明，足细胞高YAP表达可显著改善患者预后（HR 0.106[95% CI 0.013-0.869]; p=0.037）。

    研究结论：本研究发现IgA肾病患者存在足细胞损伤，而YAP可能对足细胞起到保护作用。足细胞YAP表达水平与患者临床实验室指标、组织病理评分等具有相关性，且可对患者肾脏预后具有预测价值。阻断Hippo通路使YAP核内表达增加可能成为IgAN新的治疗手段。

PART I:

Background and objective:IgA nephropathy (IgAN) was first proposed by Berger and Hinglais in 1968, and it is currently one of the most common primary glomerular diseases in Asia, has the characteristics of heterogeneity and progression. In patients diagnosed with IgA nephropathy, 20%-40% of patients will progress to end-stage renal disease (ESRD) within 20 years, and the clinical manifestations of patients are diverse. Reducing persistent proteinuria is one of the main goals of the treatment in IgAN mainly by including ACEI/ARB and immunosuppressive therapy. The benefits of immunosuppressive therapy in patients with IgAN remain controversial, especially for those with more severe renal pathology findings and reduced renal function.In comparisonto previous research, the IgAN cohort in our hospital was more severe at the time of diagnosis and more active in immunosuppressive therapy.Therefore, we conducted a real-world study to observe the effect of immunosuppressive therapy on long-term prognosis in IgAN patients in CKD stage 3 and stage 4, and to explore the predictive role of whether the treatment of immunosuppressants is effect or not on long-term prognosis. We aimed to provide a new treatment strategy for IgAN patients especially for those with more sever conditions.

Methods: This was a single center retrospective cohort study. A total of 496 biopsy-confirmed IgAN patients aged ³18 years oldwere screened for eligibilitybetween January 2012 and December 2014. Patients were divided into four groups according to CKD stages and the treatment of immunosuppressants therapy. The patients were divided into four groups: (1) CKD stage 3 with immunosuppressive therapy (96 subjects) ; (2) CKD stage 3 without immunosuppressive therapy (30 subjects)；(3) CKD stage 4 with immunosuppressive therapy (26 subjects); and (4) CKD stage 4 without immunosuppressive therapy (12 subjects). The primary end points were doubling of serum creatinine，progression to ESRD, or death for all causes. The secondary end point was rapid decrease in eGFR (＞1ml /min/1.73m²/ year). Subgroup analysisof CKD3 immunosuppressant treatment group was conducted to explore the factors affecting the prognosis of IgAN patients after treatment and whether there are short-term alternative indicators. 

Results：Atotal of 164 patients were enrolled including 98 males and 66 females, with a male-to-female ratio of 1.5:1. The median age was 43 years, and the mean follow-up time was 5.51 years. There were 126 patients in CKD 3 stage and 38 patients in CKD 4 stage and no significant differences in baseline age, gender, duration of kidney disease, history of metabolic diseases such as hypertension and diabetes mellitus among CKD patients with different stages according to whether or not they received immunosuppressive therapy. Cox single-cause and multi-cause regression analysis showed that immunosuppressive therapy significantly improved prognosis in patients with CKD stage 3 IgAN (HR 0.435[95%CI 0.200-0.944]; P=0.035)，but no difference for CKD stage 4 IgAN (p=0.364＞0.05). Subgroup analysis showed that baseline eGFR (OR 0.909[95%CI 0.834-0.991]; p=0.031), serum IgG level (OR 0.809 [95%CI 0.658-0.995]; p=0.045) were associated with primary outcome and loop necrosis (OR 0.189[95% CI 0.050-0.709], p=0.014), proportion of crescents (OR 0.200[95% CI 0.100-0.521], p=0.003), MEST-C score T2 relative to T0 (OR 5.490[95%CI 1.323-22.727], p =0.019), C2 relative to C1(OR 0.741[95%CI 0.090-0.915], p =0.016), C2 relative to C0 (OR 0.580[95%CI 0.070-0.910], p =0.009) were associated with secondary outcome.Remission within 1 year could be used as an indicator of good long-term prognosis (HR 0.555[95%CI 0.296-0.759; p =0.035]).

Conclusions: For IgAN patients with CKD stage 3, immunosuppressive therapy should be actively applied under the general treatment, but for patients with CKD stage 4, immunosuppressive therapy should be used carefully. Patients with good baseline renal function, more acute lesions of renal pathology such as loop necrosis, crescents or fewer chronic lesions such as interstitial fibrosis and tubule atrophy, would have a better prognosis after immunosuppressive therapy, and can be given active treatment after diagnosis. Besides, patients who achieved complete or partial remission within 1 year would have better long-term prognosis.

PART II:

Background and objective:IgA nephropathy patients may have pathological manifestations such as podocyte hypertrophy, tip lesion and resorption droplets within podocytes. More than 70% of patients have focal segmental sclerosis (S1) lesion, which is an independent risk factor for poor renal prognosis. In addition, the degree of podocyte damage can also be used as an indicator of disease severity and a predictive indicator of disease progression. Reducing podocyte damage may become a potential drug treatment target and research direction for IgA nephropathy, which can reduce the degree of proteinuria and glomerular sclerosis in patients. The Hippo signaling pathway and its most important transcriptional activator Yes- associated protein (YAP) play an important role in the podocyte apoptosis and the podocyte repair after injury. In this study, we focus on podocyte damage and YAP expression in patients with IgA nephropathy. We supposed that YAP can protect podocytes in IgAN, and the level of YAP expression is related to the severity and the prognosis. We aimed to explore whether YAP could slow down glomerular sclerosis and improve the prognosis of patients in IgA nephropathy.

Methods: Screening patients who were hospitalized in Peking Union Medical College Hospital for the first diagnosis of IgAN by renal biopsy from January 2012 to December 2014. According to propensity score matching of gender and age，16 patients of Lee’s grade 3-4 with proteinuria ≥3.5g/d, 16 patients of Lee’s grade 3-4 with proteinuria 1-3.5g/d and 3 patients with Lee grade 2 were matched as disease control. Besides, 5 healthy control were obtained, all of which were treated with unilateral nephrectomy to obtain relatively healthy renal cortex away from the renal tumor. Immunohistochemistry and immunofluorescence staining to determine the expression of YAP in the kidney, co-staining of WT1 and YAP to determine the expressing of YAP in podocytes nucleus. The ratio of the number of podocytes positive for YAP nucleus to the total number of podocytes in the globule represents the YAP expression level. Subgroup analysis of IgAN patients with different pathological damage grades and IgAN patients with different YAP expression levels was carried out.

Results：(1)The podocyte count in patients with IgAN Lee grade 3-4 was significantly lower than that in healthy controls (26.18[23.96, 28.39] vs 35.37[29.40, 41.75], p=0.043; 16.37[13.42, 19.31] vs 35.37[29.40, 41.75], p=0.001). The podocyte count decreased and the injury aggravated with the increase of pathological grade.

(2) The proportion of podocytes positive for YAP nucleus decreased significantly in IgAN. And as the pathological grade increases, the proportion gradually decreases. According to the scatter plot and Pearson correlation test, the expression level of YAP has a positive linear relationship with the count of podocytes (r=0.876, p<0.001). The proportion of podocytes positive for YAP nucleus increases, then the number of podocytes in the glomerulus increases. 

   (3) The expression level of YAP in IgAN patients is correlated with the severity of the disease. The high YAP expression group has lower 24hUP [2.60 (1.18, 4.02) vs 3.86 (3.38, 3.34), p=0.038], higher eGFR [106.68±20.01 vs 64.62±19.64, p<0.001], lower proportion of spherical sclerosis [3.77% (1.30%, 6.26%) vs 30.40% (21.13%, 39.68%), p<0.001] and segmental sclerosis [2.33% (0.94%, 3.73%) vs 13.16% (7.25%, 19.08%), p<0.001]], lower proportion of crescent [6.21% (2.94%, 9.48%) vs 16.59 %(9.60%, 23.57%), p=0.011], and the degree of tubulointerstitial fibrosis is lighter (p<0.001). According to MEST-C grouping, YAP expression level of podocytes in patients with glomerulosclerosis (S1), renal tubular atrophy or interstitial fibrosis (T1-2), severe crescent (C2) was higher.

   (4) After a median follow-up of 6 years, 8 patients with IgAN reached the end point. According to COX univariate and multivariate regression analysis, high podocyte YAP expression can significantly improve the prognosis of patients (HR 0.106[95% CI 0.013-0.869]; p=0.037).

    Conclusions: In this study, we found the presence of podocyte injury with IgA nephropathy patients, and YAP may have a protective effect on podocytes. The expression level of YAP in podocytes is correlated with patients' clinical laboratory indexes, histopathological scores and has predictive value for the prognosis. Blocking the Hippo pathway to increase the expression of YAP in the nucleus may become a new treatment for IgAN.