【作者】 刘艳；

【导师】 李晓；

【作者基本信息】 上海交通大学 ， 内科学（肾脏病学）， 2016， 硕士

【摘要】 系统性红斑狼疮(system lupus erythematosus,SLE)是一种可累及全身各器官系统,以慢性炎症为特征的自身免疫性疾病。狼疮性肾炎(lupus nephritis,LN)是SLE最为常见和严重的临床表现,部分患者可最终进展至终末期肾病(end-stage renal disease,ESRD)。近年来不少学者指出肾小管间质病变(tubulointerstitial lesion,TIL)的严重程度是影响LN患者预后的重要因素。研究表明免疫复合物在肾小管周围毛细血管、基底膜及间质沉积进而导致的炎性反应是LN肾损害,尤其肾小管间质损伤发生发展的关键环节,而肾小管间质缺氧性损伤是各种慢性肾脏病进展至ESRD的共同途径。P-选择素作为细胞粘附分子中选择素家族成员之一,可介导白细胞与血小板、内皮细胞的滚动及初始粘附聚集,是调节炎症和免疫反应等多种生物学过程的重要分子。本课题组前期工作发现P-选择素在LN患者血浆及肾组织均高度表达,P-选择素及其介导的树突状细胞可在肾间质浸润聚集,可能参与启动肾小管间质炎性反应和缺血缺氧性损伤。本研究旨在探讨LN患者TIL的特点及其与临床、其他病理指标和预后的相关性,并拟在狼疮模型小鼠中观察抗P-选择素单抗对LN肾小管间质病变的干预作用,探讨其可能存在的机制,期待为LN的治疗提供更多的思路。研究的第一部分,我们回顾性研究了2005年1月至2015年8月上海瑞金医院肾脏科350例经肾活检确诊的狼疮性肾炎患者的临床和病理资料,对肾小管间质病变,包括肾间质炎细胞浸润、肾小管萎缩、间质纤维化进行半定量评分,分析狼疮性肾炎患者肾小管间质病变情况,比较不同严重程度肾小管间质病变患者的临床、实验室及病理特点,并探讨影响狼疮性肾炎患者预后的因素。结果显示350例LN患者中298例(85.14%)存在TIL,其中中重度病变73例(20.86%),肾间质炎细胞浸润、肾小管萎缩、间质纤维化分别约81.71%、58.86%及60.29%。随着TIL程度加重,LN患者病情亦逐渐加重,表现为患者血压升高,肾功能减退,血尿酸水平、24h尿蛋白定量、肾小管性尿蛋白升高,血红蛋白下降等,而LN的疾病活动指标如抗ds-DNA抗体及补体C3、C4水平未随之改变。中重度TIL多见于IV型LN患者,其活动性及慢性指数均较高,肾间质炎细胞浸润、小管萎缩及间质纤维化之间相互影响,其中肾间质炎细胞浸润又与肾小球活动性病变,包括毛细血管内增生、白细胞浸润、核碎裂/坏死、细胞性新月体有关,而肾小管萎缩和间质纤维化与肾小球慢性病变,如纤维性新月体、肾小球硬化高度相关。但部分TIL可独立于肾小球病变发生,有14.3%的LN患者存在中重度肾小管间质病变而无或仅有轻度肾小球病变。Logistic回归分析显示,肾间质炎细胞浸润是狼疮性肾炎患者发生基线肾功能不全(OR=3.075;95%CI=2.112-4.479,P<0.001)及急性肾损伤(OR=2.954,95%CI,1.945-4.484;P<0.001)的危险因素。稳定随访的251例LN患者平均随访49.35±28.36个月,12例患者死亡,19例患者达肌酐翻倍或进入ESRD,中重度TIL狼疮患者较无或轻度病变者肾脏生存率降低,Cox多因素逐步回归分析显示男性(HR=3.272,95%CI=1.500-7.140,P=0.003)、随访期间无缓解(HR=19.631,95%CI=9.346-41.236,P<0.001)和肾小管间质慢性评分(HR=1.638,95%CI=1.328-2.021,P<0.001)是肾脏预后不良的独立危险因素。因此,在临床工作中,我们不仅要关注LN的肾小球病变情况,也要重视其小管间质病变,全面分析患者的病情,制定积极、有效的治疗方案,阻止病变的慢性化发展,改善患者预后。研究的第二部分,我们以雌性MRL/lpr自发性狼疮性肾炎模型小鼠为研究对象,拟在LN小鼠中观察抗P选择素单抗对肾脏损害,尤其是肾小管间质损害的干预作用,初步探讨LN肾脏病变的病理机制。我们发现MRL/lpr小鼠肾组织中P-选择素广泛表达,主要分布在肾小管间质,给予抗P-选择素单抗干预后,狼疮小鼠肾组织P-选择素表达下降,前期研究结果显示单抗干预组狼疮小鼠24h尿蛋白、尿白蛋白比肌酐比值(albumin to creatinine ratio,ACR)及血肌酐均显著低于同周龄生理盐水对照组,两组间血清抗ds-DNA抗体和C3水平无统计学差异。本研究进一步发现,与生理盐水对照组相比,单抗干预组小鼠肾间质及小血管周围炎细胞浸润明显减少,肾小管萎缩、间质纤维化少见,肾小管间质病变程度减轻。单抗干预组小鼠肾组织CD31标记的肾小管周围毛细血管数目增加,肾小管上皮细胞HIF-1α的表达及肾组织NADPH氧化酶亚单位gp91phox和p22phox的表达均减少。血氧水平依赖的磁共振成像显示,单抗干预后狼疮小鼠肾脏皮、髓质R2*值均较同周龄生理盐水对照组升高。上述实验结果提示抑制P选择素对狼疮性肾炎小鼠肾脏有保护作用,可在一定程度上减轻狼疮小鼠肾小管间质损害,肾脏缺氧状态及肾小管周围毛细血管丢失,但具体机制仍有待于进一步研究。综上所述,本研究通过回顾性分析LN患者的临床及病理学资料,发现LN患者普遍存在不同程度的肾小管间质病变,尤其肾小管萎缩是LN患者预后不良的危险因素。进一步采用抗P-选择素单抗干预MRL/lpr小鼠,发现抑制P选择素可减轻其肾小管间质损害,在一定程度上改善肾功能、减少尿蛋白,可能为LN治疗提供新思路。更多还原

【Abstract】 System lupus erythematosus(SLE)is a prototypic autoimmune disease that is characterized by systemic chronic inflammation that can affect multiple major organ systems.Lupus nephritis(LN)is the most common severe manifestation of SLE,many patients progress to end-stage renal disease(ESRD).Recently,it was found that the severity of tubulointerstitial lesion(TIL)is an important factor affecting the prognosis of LN patients.Previous studies indicated that immune deposits localizing in peritubular capillaries,tubular basement membranes and interstitium produce an inflammatory reaction which contributes to the development of TIL.Furthermore,hypoxia in tubulointerstitium is a final common pathway to ESRD.P-selectin is a selectin member of cell adhesion molecules that are widely accepted as critical modulator for various biological processes including inflammation and immunity,mediating initial leukocyte adhesion to activated platelet and endothelium.We found that P-selectin was highly expressed in both plasma and renal tissue of LN patients at early stage of our work,in addition,P-selectin and its mediated dendritic cell infiltrating in interstitium may trigger inflammatory reaction and hypoxia in tubulointerstitium damage.Our study aims to reveal the relationship between TIL,clinical-pathological characteristics and prognosis in patients with LN and explore the effect of anti-P-selectin mAb on tubulointerstitial damage in murine LN,provide a new insight into future treatment of LN.In the first part of the study,we retrospectively analysed 350 patients with biopsyproven lupus nephritis in our center from January 2005 to August 2015.Tubulointerstitial lesions including interstitial inflammation,tubular atrophy and interstitial fibrosis were assessed by semiquantitative score.Compared the clinical,laboratory and histological parameters of different severity of TIL.Follow-up data were obtained and renal survival analysis was conducted to determine the risk factors associated with the occurrence of the doubling of serum creatinine,end stage renal disease or death in patients with lupus nephritis.Among 350 patients,298 patients(85.14%)had TIL,and 73 patients(20.86%)had moderate to severe TIL.The prevalence of interstitial inflammation,tubular atrophy,and interstitial fibrosis was 81.71%,58.86% and 60.29% respectively.As the degree of TIL aggravated,blood pressure,the level of serum uric acid,24 h urinary protein and tubuloproteinuria of LN patients increased,meanwhile renal function and hemoglobin decreased.However,the activity indices,such as anti-ds-DNA antibody and complement C3,C4 did not change with TIL.Moderate to severe TIL was more common in patients with class IV,and activity and chronicity index were higher.Interstitial inflammation,tubular atrophy and interstitial fibrosis were associated with each other,in addition,interstitial inflammation also showed significant association with active glomerular lesions,including capillary hyperplasia,leukocyte infiltration,karyorrhexis or fibrinoid necrosis and cellular crescents.While,interstitial fibrosis and tubular atrophy obviously correlated with chronic glomerular lesions,such as glomerular sclerosis and fibrous crescents.But sometimes TIL can occur independent of glomerular lesions,with 14.3% of LN patients with more prominent TIL and nil or milder glomerular injury.In the logistic regression analysis,interstitial inflammation was risk factor for both renal sufficiency(OR=3.075;95% CI=2.112-4.479,P<0.001)and acute kidney injury(OR=2.954,95% CI=1.945-4.484;P<0.001).During a mean follow-up of 49.35±28.36 months,12 patients died,10 patients reached doubling of serum creatinine or end stage renal disease and renal survival decreased in those with moderate-to-severe TIL.Cox regression models showed that male(HR=3.272 95%CI=1.500-7.140,P=0.003),continuous without remission(HR=19.631,95%CI=9.346-41.236,P<0.001)and interstitial chronicity scores(HR=1.638,95%CI=1.328-2.021,P<0.001)were independent risk factors for poor renal outcomes.Thus,in the future clinical work,we should not only focus on glomerular lesions,but should also pay more attention to tubulointerstitial lesions,comprehensively analyzing patients’ conditions,taking aggressive and proper therapeutic measures to prevent chronic development and improve prognosis.In the second part of this work,female MRL/lpr mice which being the spontaneous model of lupus nephritis served as the research objects,to explore the effect of anti-Pselection mAb on renal injury,especially on tubulointerstitial lesions and the underlying mechanism.Our previous studies had showed widespread P-selectin expression in renal tissue of MRL/lpr mice,mainly distributing in tubulointerstitium.By anti-P-selectin monoclonal antibody(mAb)intervention,the expression of P-selectin was significantly decreased.Compared with saline-treated mice,urinary protein and urinary albumin to creatinine ratio(ACR)were much lower in anti-P-selectin mAb treated mice.In addition,the levels of serum creatinine(Scr)were obviously declined in anti-P-selectin mAb treated mice than those in saline-treated mice.There were no statistical differences in the levels of anti-dsDNA antibodies and complement C3 between two groups.In this study,we further found that anti-P-selectin mAb treated mice exhibited a significant improvement in tubulointerstitial lesions in comparison with saline-treated control.Moreover,the expression of hypoxia-inducible factor 1 alpha(HIF-1α),as well as gp91 phox and p22 phox,which being subunits of NAPDH oxidase,was actually down-regulated in renal tissues of MRL/lpr mice treated with anti-P-selectin mAb,however,CD31 marked peritubular capillary(PTC)density was elevated.Blood oxygen level–dependent magnetic resonance imaging(BOLD-MRI)showed that the mean R2* values of renal cortex and medulla were higher in anti-P-selectin mAb mice than those in saline-treated mice.These results indicated that intervention with anti-P-selectin mAb may attenuate tubulointerstitial injury,hypoxia and the loss of PTC to some extent in MRL/lpr mice,and might be an emerging therapeutic method on lupus nephritis.Further studies are still needed.In summary,we retrospectively analyzed the clinical and pathological data of lupus nephritis patients,and revealed that tubulointerstitial lesions were common in LN.Tubulointerstitial chronicity,particularly tubular atrophy,is a risk factor for poor prognosis of LN patients.We further investigated the effect of anti-P-selection mAb on renal damage in murine lupus nephritis and found inhibition of P-selectin may attenuate tubulointerstitial injury,which might be a therapeutic target in LN.更多还原