Novel Targets of Rituximab in FSGS

描述（由申请人提供）：移植后复发性局灶节段性肾小球硬化（FSGS）是一种非常普遍的疾病，利妥昔单抗（一种抗CD20的单克隆抗体）可能有潜在的适应症。除CD20外，利妥昔单抗已被证明可结合鞘磷脂二酯酶样3b前体（SMPDL-3b）并调节酸性鞘磷脂酶（ASMase）。我们验证了利妥昔单抗通过保存足细胞SMPDL-3b的表达和活性来预防移植后复发性FSGS的假设。我们对41例复发性FSGS高风险患者的初步研究表明，利妥昔单抗治疗与移植后蛋白尿发生率降低和eGFR降低相关。再灌注后活检中SMPDL-3b阳性细胞的数量在后来复发性疾病的患者中减少。利妥昔单抗部分阻止了患者血清治疗足细胞中SMPDL-3b的下调。利妥昔单抗和SMPDL-3b的过表达都能阻止肌动蛋白骨架的破坏和患者血清诱导的细胞凋亡，而在SMPDL-3b基因沉默的细胞中，这种作用减弱。体外应力纤维破坏的定量分析与移植后蛋白尿的程度有关，这表明有可能开发一种预测复发性FSGS的方法。我们的大学为招募大量FSGS患者提供了最好的临床基础之一。我们建议在所有接受肾移植的原发性FSGS患者中进行一项试点研究，作为实验研究的补充。我们将收集临床数据作为二次探索性分析。再灌注后肾活检和移植前血清将在随机接受利妥昔单抗或安慰剂的患者中收集。SMPDL-3b在肾脏活检和暴露于患者血清的培养足细胞中的表达将用于研究疾病机制并进行与临床结果的关联研究。将利用细胞骨架重塑、细胞活力、传统的裂隙膜蛋白表达和定位以及细胞脂质组成来研究利妥昔单抗是否以SMPDL-3b依赖的方式保护足细胞。我们的研究是非常重要的，因为它具有很强的临床意义，因为它可能导致利妥昔单抗治疗FSGS以及其他蛋白尿疾病的适应症的改变。这项研究具有创新性，因为它将为幼髓磷脂相关酶在足细胞功能中的作用提供新的见解，从而允许确定抗蛋白尿药物开发的新靶点。最后，我们用患者血清处理正常人类足细胞的体外试验可能成为一种移植前评估试验，用于识别高危患者。

DESCRIPTION (provided by applicant): Recurrent focal segmental glomerulosclerosis (FSGS) after transplantation is a highly prevalent condition where rituximab (a monoclonal antibody against CD20) may have a potential indication. Besides CD20, rituximab has been shown to bind sphingomyelin-phosphodiesterase-like-3b-precursor (SMPDL-3b) and to regulate acid-sphyngomyelinase (ASMase). We tested the hypothesis that rituximab prevents recurrent FSGS after transplantation via preservation of podocyte SMPDL-3b expression and activity. Our preliminary study in 41 patients at high risk for recurrent FSGS demonstrated that rituximab treatment was associated with lower incidence of post-transplant proteinuria and with decreased eGFR. The number of SMPDL-3b positive cells in post-reperfusion biopsies was reduced in patients that would later develop recurrent disease. Rituximab partially prevented the SMPDL-3b down-regulation observed in podocytes treated with the sera of affected patients. Both rituximab and SMPDL-3b overexpression prevented the disruption of the actin cytoskeleton and the apoptosis induced by patient sera, an effect that was diminished in cells where SMPDL- 3b gene was silenced. Quantitative analysis of the disruption of stress fibers in vitro was associated with the degree of post-transplant proteinuria, suggesting the possibility of developing a prediction assay for recurrent FSGS. Our University offers one of the best clinical grounds for the recruitment of a large population of patients with FSGS. We propose to utilize a pilot study in all patients with primary FSGS who undergo kidney transplantation as a feeder for the experimental studies. We will collect clinical data that will serve as secondary exploratory analyses. Post-reperfusion kidney biopsies and pre-transplant sera will be collected per protocol in patients randomized to receive rituximab or placebo. SMPDL-3b expression in kidney biopsies and in cultured podocytes exposed to patient sera will be utilized to study disease mechanisms and to perform association studies with clinical outcomes. Cell cytoskeleton remodeling, cell viability, traditional slit diaphragm proteins expression and localization and cellular lipid composition will be utilized to study if rituximab protects podocytes in a SMPDL-3b dependent manner. Our study is highly significant because it has strong clinical implications, since it may lead to a change in the approved indications for rituximab treatment of FSGS as well as other proteinuric diseases. The proposed study is innovative because it will offer new insights into the role of shyngomyelin related enzymes in podocyte function, thus allowing the identification of novel targets for antiproteinuric drug development. Finally, our in vitro assay treating normal human podocytes with patient sera may become a pre-transplant assessment test for the identifications of patients at high-risk.