Immunological Mechanisms Underlying Allograft Rejection

与同种异体肾移植的短期生存相比，移植肾的长期生存并没有显著改善。慢性同种异体肾病（CAN）是活体受者晚期移植物衰竭的主要原因，占移植后6个月以上移植物衰竭的35-40%。CAN的确切病因尚不清楚，但免疫因素被认为是至关重要的：1)CAN与急性排斥反应有关；2)与尸体移植相比，活体同种异体移植物的存活率更高。另外，免疫抑制疗法对CAN缺乏效果表明非免疫因素也可能参与其中。经常提到的因素包括供体-受体尺寸差异、药物肾毒性、高血压和高脂血症。在CAN中，同种异体移植物的免疫和非免疫反应导致组织损伤、炎症细胞浸润和局部成纤维细胞增殖，最终导致间质基质沉积和纤维化。这一过程似乎受到细胞因子和生长因子的调控，如转化生长因子- β (tgf - β)、碱性成纤维细胞生长因子（bFGF）、血管内皮生长因子（VEGF）和血小板衍生生长因子（PDGF）。虽然研究人员已经使用了许多大鼠模型来研究CAN，但这些血管化移植模型使用了供体/受体菌株组合，只有很小的抗原差异，移植后4-6个月发生病理变化，使得模型劳动密集型且效率低下。我们最近观察到，虽然小鼠肾同种异体移植物在没有免疫抑制的情况下存活了很长时间，但移植物的功能在移植后6周逐渐恶化，并具有典型的人类CAN的组织学特征。此外，与CAN一致，我们发现1)与非排斥同种异体移植物相比，tgf - β在慢性排斥同种异体移植物中高表达，2)增强肾基质沉积。尽管减少了供体/受体MHC差异，并降低了移植物内tgf - β的表达，但CAN的严重程度没有改变。此外，tgf - β虽然本身是一种有效的纤维化细胞因子，但也具有免疫抑制特性，被认为有利于移植物的存活，这使得它不太适合作为预防CAN的靶点。这些研究表明，虽然MHC差异可能调节tgf - β和CAN的发育，但其他局部因素也会影响CAN。例如，我们发现同种异体抗体的发展对使用小鼠模型的CAN发展至关重要。我们提出减少细胞外基质沉积或组织可能会影响CAN的结果。我们的策略是直接使用必需基质合成酶的药物抑制剂来废除基质生产。脯氨酸-4-羟化酶是胶原形成所必需的酶，因为它在翻译后修饰前胶原α链。脯氨酸-4-羟化酶抑制阻止脯氨酸羟化前胶原链导致不稳定的前胶原折叠。新形成的异常前胶原被降解，从而减少间质胶原沉积。新型的phenanthrolinone化合物，在体内和体外都是脯氨酰4-羟化酶的竞争性抑制剂，似乎没有全身毒性。我们研究了一种新型的、专有的、口服活性的菲罗啉酮脯氨酸-4羟化酶（PHI）抑制剂在小鼠CAN模型中的作用。将来自C57BL/6 （H-2b）小鼠的肾脏移植到mhc不相容的CBY （H-2d）受体（同种异体移植物）中，或将其同窝同胞作为非排斥对照组。移植后3周，同种异体移植小鼠接受PHI（每天50mg/kg）或载药治疗3周，我们在移植后6周检测肾脏功能和与CAN相关的组织形态学变化。PHI治疗耐受性良好；接受治疗的小鼠体重稳定，没有明显的骨骼异常。最重要的是，异体移植PHI处理小鼠的肾小球滤过率（3.3 +/- 0.5 ml/min/kg）显著高于对照组（1.8 +/- 0.5 ml/min/kg, p<0.05），而同种异体移植小鼠的肾功能未受损害（6.45 +/- 0.53 ml/min/kg）。虽然我们发现各组间血管和肾小球损伤的严重程度没有差异，但同种异体移植物表现出CAN组织学改变，但与对照组（15.4 +/- 1.2;p<0.05）相比，接受PHI组的严重程度有所减轻（12.6 +/- 0.8），同种异体移植物间质炎症和纤维化减轻。与载体治疗组（1.0 +/- 0.3 mg蛋白/mg肌酐）相比，PHI治疗组尿蛋白（0.6 +/- 0.1 mg蛋白/mg肌酐）也显著降低。为了研究PHI如何介导这些影响，我们使用实时聚合酶链反应（RT-PCR）分析了移植体内的细胞因子谱，除了IL-4外，发现同种异体移植组之间Th1或Th2细胞因子以及tgf - β没有显著差异。然而，在pi处理的受体中，mRNA IL-4的相对表达量显著降低（相对于正常肾脏的6.12倍表达量），相比于载体处理的同种异体移植物（相对于正常肾脏的12.86倍，p=0.04）。因此，尽管PHI显著减少了同种异体移植物内的炎症细胞浸润，但我们只观察到移植物内细胞因子谱的适度变化。这些研究表明，PHI治疗可以减少can中的基质沉积，可能代表一种新的治疗方法。在其他研究中，我们研究了CTGF，这是一种38 Kd富含半胱氨酸的蛋白，是CCN生长因子家族的一员，由肾内皮细胞、成纤维细胞和系膜细胞产生。CTGF刺激胶原蛋白和纤维连接蛋白基质蛋白的产生。此外，虽然CTGF的表达受到tgf - β的调节，但它缺乏tgf - β的免疫调节特性。CTGF在许多啮齿动物肾脏疾病模型中上调，并与纤维化过程有关。在与纤维化相关的人类肾脏疾病中，CTGF的表达也增强。我们研究了CTGF作为tgf - β下游的靶标，但它显然参与基质积累，作为预防或治疗CAN的潜在治疗方法。我们使用实时RT-PCR测量了CTGF的表达（Northern证实），发现在移植后6周，与非排斥肾相比，CAN小鼠同种异体肾移植的CTGF mRNA表达显著增加（2.7倍）。在CTGF mRNA表达之前，移植后1周和2周同种异体移植的tgf - β mRNA诱导量分别是未移植肾脏的近12倍和6倍。因此，CTGF mRNA在具有CAN的同种异体移植物中表达，并与tgf - β mRNA的升高相关。我们还在NIH人类肾移植受者的试点研究中检测了CTGF。平均血清（50.1 +/- 3.9 ng/ml, n=25）和尿CTGF水平（32.2 +/- 6.0 ng/mg肌酐，n=20）与正常健康个体（n=10，分别为15.4 +/- 3.6 ng/ml和0.8 +/- 0.2 ng/mg肌酐）相比显著升高。虽然年龄、移植后时间、钙调磷酸酶抑制剂的使用和冷缺血时间等受体因素与CTGF水平无关，但活检显示CAN的患者平均尿CTGF水平最高，组织学正常的患者最低。这些研究的目的是为有慢性排斥风险的移植受者开发新的干预措施和筛查方法。

In contrast to short-term kidney allograft survival, long-term graft survival has not improved dramatically. The leading cause of late graft failure in living recipients is chronic allograft nephropathy (CAN), accounting for 35-40% of graft failure more than 6 months post-transplant. The precise etiology of CAN is not known, but immunologic factors are thought to be critical: 1) CAN is associated with acute rejection and , 2) living related allografts show improved graft survival compared to cadaveric grafts. Alternatively, immunosuppressive therapy's lack of effect on CAN suggests nonimmunologic factors may also be involved. Factors often mentioned include size donor-recipient size disparity, drug nephrotoxicity, hypertension, and hyperlipidemia. In CAN, combined immunologic and non-immunologic responses to the allograft result in tissue damage, inflammatory cell infiltration, and local fibroblast proliferation with the end result of interstitial matrix deposition and fibrosis. The process appears to be regulated by cytokines and growth factors such as transforming growth factor-beta (TGF-beta), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), and platelet derived growth factor (PDGF). While investigators have utilized a number of rat models to study CAN, these models of vascularized transplants have utilized donor/recipient strain combinations with only minor antigen differences and pathologic changes occur 4-6 months post-transplant making the models labor-intensive and inefficient. We recently observed that while mouse kidney allografts survive for prolonged periods without immunosuppression that the grafts' function deteriorates gradually and with histologic features typical of human CAN by 6 weeks post transplant. Further, consistent with CAN, we found that 1) TGF-beta was highly expressed in chronically rejecting allografts compared to nonrejecting isografts, and 2) enhanced kidney matrix deposition. Despite reducing the donor/recipient MHC disparity, with an associated reduction in intra-graft TGF-beta expression, CAN severity was unchanged. Further, TGF-beta, while itself a potent fibrogenic cytokine, also has immunosuppressive qualities believed to be beneficial to graft survival making it less desirable as a target to prevent CAN. These studies suggest that while MHC disparity may regulate TGF-beta and CAN development, other local factors influence CAN. For instance, we found that alloantibody development is critical for CAN development using the mouse model. We've proposed that reducing extra-cellular matrix deposition or organization may affect the CAN outcome. Our strategy is to abrogate matrix production directly using pharmacologic inhibitors of essential matrix synthesis enzymes. Prolyl-4-hydroxylase is an enzyme essential for collagen formation as it post-translationally modifies the procollagen alpha chains. Prolyl-4-hydroxylase inhibition prevents proline hydroxylation of procollagen chains leading to unstable procollagen folding. The newly formed abnormal procollagen is degraded thus decreasing interstitial collagen deposition. Novel phenanthrolinone compounds, competitive inhibitors of prolyl 4-hydroxylase both in vivo and in vitro, do not appear to have systemic toxicity. We studied the effects of a novel, proprietary, orally active phenanthrolinone prolyl-4 hydroxylase (PHI) inhibitor in our mouse CAN model. Kidneys from C57BL/6 (H-2b) mice were transplanted into MHC-incompatible CBY (H-2d) recipients (allografts) or, as a non-rejecting control group, their littermates. At 3 weeks post-transplant, allografted mice received PHI (50mg/kg per day) or vehicle for 3 weeks, and we assayed for renal function and histomorphologic changes associated with CAN 6 weeks post-transplant. PHI treatment was well tolerated; treated mice had stable body weights and displayed no apparent skeletal abnormalities. Most important, the glomerular filtration rate was significantly greater in PHI treated mice with allografts (3.3 +/- 0.5 ml/min/kg) compared to those receiving vehicle (1.8 +/- 0.5 ml/min/kg, p<0.05), while renal function was unimpaired in mice with isografts (6.45 +/- 0.53 ml/min/kg). While we saw no difference in vascular and glomerular injury severity between groups, the allografts displayed CAN histologic changes but the severity was reduced in those receiving PHI (12.6 +/- 0.8) compared to vehicle (15.4 +/- 1.2; p<0.05), with reduced allograft interstitial inflammation and fibrosis. PHI treatment was also associated with a substantial reduction in urinary protein (0.6 +/- 0.1 mg protein/mg creatinine) compared to vehicle treated recipients (1.0 +/- 0.3 mg protein/mg creatinine). To study how PHI might mediate these effects, we analyzed intragraft cytokine profiles using real-time polymerase chain reaction (RT-PCR) and, with the exception of IL-4, found no significant differences for Th1 or Th2 cytokines, nor for TGF-beta, between allograft groups. In PHI-treated recipients however, there was a significant reduction in relative mRNA IL-4 expression (6.12-fold expression relative to normal kidney) compared to vehicle treated allografts (12.86-fold relative to normal kidney; p=0.04). Thus, despite PHI significantly reducing the inflammatory cell infiltrate within allografts, we observed only modest intra-graft cytokine profile changes. These studies demonstrate that PHI treatment can reduce matrix deposition in CAN and may represent a novel therapy for humans. In other studies, we've pursued CTGF, a 38 Kd cysteine rich protein and a member of the CCN growth factor family, that is produced by kidney endothelial cells, fibroblasts, and mesangial cells. CTGF stimulates the production of collagen and fibronectin matrix proteins. Further, while CTGF expression is regulated by TGF-beta, it lacks TGF-beta's immunoregulatory properties. CTGF is upregulated in a number of rodent renal disease models and is implicated in the fibrotic process. In human renal diseases associated with fibrosis, CTGF expression is also enhanced. We studied CTGF as a target downstream of TGF-beta but one apparantly involved in matrix accumulation as a potential therapeutic approach to prevent or treat CAN. We measured CTGF expression using real time RT-PCR (confirmed by Northern) and found CTGF mRNA expression significantly increased (2.7-fold) in mouse kidney allografts with CAN compared to nonrejecting kidneys 6 weeks after transplant. CTGF mRNA expression was preceded by a nearly 12-fold and 6-fold TGF-beta mRNA induction in allografts at one week and two weeks post-transplant, respectively, compared to naive kidneys. Thus CTGF mRNA is expressed in allografts with CAN, and is associated with a rise in TGF-beta mRNA. We also examined CTGF in a pilot study of NIH human kidney transplant recipient. Mean serum (50.1 +/- 3.9 ng/ml; n=25) and urine CTGF levels (32.2 +/- 6.0 ng/mg creatinine; n=20) were significantly elevated compared to normal, healthy individuals (n=10; 15.4 +/- 3.6 ng/ml and 0.8 +/- 0.2 ng/mg creatinine, respectively). While recipient factors like age, time post transplant, calcineurin inhibitor use, and cold ischemic time did not correlate to CTGF levels, mean urinary CTGF levels were highest in patients with biopsies demonstrating CAN, and lowest in rerecipients with normal histology. The goal of these studies is to develop novel interventions and screening methods for transplant recipients at risk for developing chronic rejection.