TLR4 and murine ischemic acute renal failure

描述(申请人提供)：我们最近发现两种不同的TLR4功能缺失突变可减少缺血性急性肾功能衰竭(ARF)的发生。利用骨髓嵌合体、免疫组织学和原位杂交，我们还发现，最大的缺血性ARF需要TLR4在浸润的白细胞(可能是巨噬细胞)上，以及。在外髓的直血管内皮细胞和小管上。后者位于损伤和炎症最严重的区域。在此基础上，我们提出了以下假设。缺血后受损的肾小管释放内源性TLR4配体(可能是热休克蛋白)。它们激活外髓和肾小管的内皮。激活的内皮和小管有助于招募炎症反应。渗透的白细胞通过它们的TLR4被进一步激活，释放出加剧损伤的有毒分子。有三个具体目标验证了这一假设。目的1)通过免疫组织学、原位杂交、激光捕获解剖和微阵列分析，确定内皮细胞和小管TLR4调控哪些基因。我们将使用假设和微阵列驱动的方法。我们将比较TLR4基因在TLR4充足和缺陷小鼠中的表达。目的ii)使用骨髓嵌合体(经致死性照射的小鼠，已被骨髓移植挽救)来研究最终导致最大缺血性ARF的炎症的中间阶段。目的利用体外系统鉴定和研究内源性TLR4配体。识别TLR4配体。利用体外系统研究TLR4在内皮/小管上的表达调控，并研究TLR4在这些细胞中的调控基因。这些体外实验是对“特异性靶标I和II”体内实验的补充。在这些实验中，我们将采取严格的预防措施来防止内毒素污染。总之，这是治疗缺血性ARF的一种新方法，可能会为这种疾病带来新的见解和治疗方法，尽管采用了现代疗法，但这种疾病的死亡率仍然很高。

DESCRIPTION (provided by applicant): We recently found that two different loss-of-function mutations of TLR4 decrease ischemic acute renal failure (ARF). Using bone marrow chimeras, immunohistology, and in situ hybridization, we also found that maximal ischemic ARF required TLR4 on infiltrating leukocytes (probably macrophages), and. on endothelia of the vasa rectae and tubules of the outer medulla. The latter is in the area where there is maximal injury and inflammation. Based on the above, we formulated the following hypothesis. Endogenous TLR4 ligands (possibly heat shock proteins) are released from injured renal tubules after ischemia. These activate the endothelia of the outer medulla and renal tubules. The activated endothelia and tubules help recruit an inflammatory response. The infiltrating leukocytes are further activated, via their TLR4, to release toxic molecules that exacerbate injury. Three Specific Aims test this hypothesis. Aim I) Determine what genes are regulated by endothelial and tubular TLR4 by using immunohistology, in situ hybridization, and laser capture dissection followed by microarray analysis. We will use hypothesis- and microarray- driven approaches. We will compare expression of the putative TLR4-regulated genes in TLR4-sufficient and -deficient mice. Aim II) Use bone- marrow chimeras (lethally irradiated mice, which have been rescued by bone-marrow transplants) to study intermediate stages of inflammation that culminate in maximal ischemic ARF. Aim III) Use in vitro systems to identify and study the endogenous TLR4 ligands. Identify the TLR4 ligands. Use in vitro systems to study the regulation of TLR4 expression on endothelia/ tubules, and to study the genes regulated by TLR4 in these cells. These in vitro experiments complement the in vivo experiments of "Specific Aims I & II". We will use stringent precautions to prevent endotoxin contamination in these experiments. Altogether, this is a novel approach to ischemic ARF and may yield new insights and therapies of a disease that continues, despite modern therapy, to have a high mortality.