Novel peptide-based strategy to inhibit deleterious TNF signaling in nephrotoxic nephritis

 描述(申请人提供)：肾毒性血清(NTS)诱导的小鼠肾炎(NTN)的早期阶段以过度炎症和肾小球屏障功能障碍为特征，是人类急性肾炎(GN)的良好模型。肾小球内皮细胞和上皮室是早期治疗干预的重要靶点，因为它们在白细胞募集、蛋白尿和进行性肾功能衰竭中起着关键作用。目前旨在治疗或预防疾病进展的治疗方法是不够和有限的，强调了控制与GN相关的炎症的新的治疗方法的必要性。在这种情况下，细胞因子肿瘤坏死因子是一个理想的靶点，因为它在人类肾炎和小鼠肾炎中都是关键的参与。不幸的是，试图钝化肿瘤坏死因子与其受体的结合来治疗肾炎，却遇到了意想不到的副作用，包括增加了细菌感染的风险。因此，针对肿瘤坏死因子的有害作用，但保持其有益作用不变的治疗方法是可取的。我们的初步研究表明，TIP多肽模拟肿瘤坏死因子的凝集素样结构域，不干扰配体与其受体的结合，干扰至少两条途径，即足细胞和肾小球内皮细胞(GEC)在NTN中的死亡。一方面，该多肽可以钝化GEC中的p38致炎途径，对于促炎介质的产生、eNOS功能障碍的诱导和白细胞的渗透都是重要的。另一方面，TIP多肽可能通过诱导足细胞产生PGE2来保护足细胞屏障功能，在NTN中被证明具有保护作用。在NTN的早期阶段，该肽抑制全身产生的肿瘤坏死因子、IL-6和单核细胞趋化蛋白-1，减少巨噬细胞的渗透，减少水肿的形成，恢复肾小球滤过率和突触素的表达，并减少蛋白尿。我们的中心假设是，TIP多肽通过抑制肿瘤坏死因子诱导的GEC和足细胞中促炎症和屏障破坏途径的激活来保护肾脏免受NTN的影响，例如钝化白细胞渗透和肾小球损伤，而不干扰细胞因子的抗菌作用。在我们的三个具体目标中，我们 将结合体内概念验证NTN小鼠研究(目标1和2)足细胞和基于GEC的机制体外细胞研究(目标3)。我们将使用独特的模型和试剂，例如表达没有功能凝集素样结构域的肿瘤坏死因子突变体的敲入小鼠(目标2)。此外，我们将通过比较抗3(IV)抗体偶联多肽(Aim 1A)的有效性和在由跨膜肿瘤坏死因子内皮细胞过度表达(Aim 1B)引发的肾脏局部炎症模型中测试多肽来评估该多肽的局部和全身效应。了解TIP多肽对自身免疫性GN的保护作用机制，将促进我们对与肾病相关的炎症和抗炎途径的认识，并将有助于进行临床试验，以确定新型治疗性多肽对其他免疫性、炎症性疾病的疗效。

 DESCRIPTION (provided by applicant): The early stage of nephrotoxic serum (NTS)-induced nephritis (NTN) in mice is characterized by excessive inflammation and glomerular barrier dysfunction and represents a good model for human acute glomerulonephritis (GN). Glomerular endothelial and epithelial compartments represent attractive targets for early therapeutic intervention, due to their crucial roles in the recruitment of leukocytes, proteinuria and progressive renal failure. Current therapies aiming to treat or prevent progression of the disease are insufficient and limited, stressing the need for novel therapeutic approaches that control inflammation associated with GN. In this context, the cytokine TNF is an ideal target, since it is critically involved in both human GN and in murine NTN. Unfortunately, attempts to blunt TNF binding to its receptors for the treatment of GN were met with unexpected side effects, including increased risk for bacterial infection. Therefore, therapies that specifically target the deleterios effects of TNF, but leave its beneficial effects intact are desirable. Our preliminary studies indicate that the TIP peptide, which mimics TNF's lectin-like domain and which does not interfere with the ligand's binding to its receptors, interferes with at least two pathways importat in podocyte and glomerular endothelial cells (GEC) demise in NTN. On the one hand, the peptide blunts the p38 pro-inflammatory pathway in GEC, important for the generation of pro-inflammatory mediators, for the induction of eNOS dysfunction and for leukocyte infiltration. On the other hand, TIP peptide protects podocyte barrier function, possibly means by inducing PGE2 generation in podocytes, shown to be protective in NTN. In the early phase of NTN, the peptide blunts systemic generation of TNF, IL-6 and MCP-1, reduces macrophage infiltration, edema formation, restores glomerular filtration rate and synaptopodin expression and abrogates albuminuria. Our central hypothesis is that the TIP peptide protects the kidney against NTN by suppressing TNF-induced activation of pro-inflammatory and barrier-disruptive pathways in GEC and podocytes, respectively, as such blunting leukocyte infiltration and glomerular injury, without interfering with the anti-bacterial actions of the cytokine. In our three specific aims, we will combine in vivo proof-of-concept NTN mouse studies (aims 1 and 2) podocyte- and GEC-based with mechanistic in vitro cell studies (aim 3). We will use unique models and reagents, such as knock-in mice expressing a TNF mutant without functional lectin-like domain (aim 2). Moreover, we will assess local vs. systemic effects of the peptide upon comparing the efficacy of anti-3(IV) antibody-coupled vs. naked TIP peptide (aim 1A) and testing the peptide in a renal local inflammation model, initiated by endothelial overexpression of transmembrane TNF (aim 1B). Understanding the mechanism of TIP peptide- mediated protective effects against auto-immune GN will advance our knowledge on the inflammatory and anti- inflammatory pathways relevant for nephropathy and will help to conduct clinical trials to determine the efficacy of novel therapeutic peptides against other immune, inflammatory diseases.