TLR4 and murine ischemic acute renal failure

描述（由申请人提供）：我们最近发现 TLR4 的两种不同的功能丧失突变可减少缺血性急性肾衰竭（ARF）。使用骨髓嵌合体、免疫组织学和原位杂交，我们还发现最大缺血性 ARF 需要浸润白细胞（可能是巨噬细胞）上的 TLR4。位于直肠血管内皮细胞和外髓质小管上。后者位于损伤和炎症最严重的区域。基于上述，我们提出以下假设。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）。它们激活外髓质和肾小管的内皮细胞。激活的内皮细胞和肾小管有助于募集炎症反应。浸润的白细胞通过 TLR4 进一步激活，释放有毒分子，加剧损伤。三个具体目标检验了这一假设。目标 I) 通过使用免疫组织学、原位杂交和激光捕获解剖，然后进行微阵列分析，确定哪些基因受内皮和肾小管 TLR4 调节。我们将使用假设和微阵列驱动的方法。我们将比较 TLR4 充足和缺乏的小鼠中推定的 TLR4 调节基因的表达。目标 II) 使用骨髓嵌合体（经过致死辐射的小鼠，通过骨髓移植获救）研究最终导致最大缺血性 ARF 的炎症中间阶段。目标 III) 使用体外系统识别和研究内源性 TLR4 配体。识别 TLR4 配体。利用体外系统研究TLR4对内皮/肾小管表达的调节，并研究这些细胞中受TLR4调节的基因。这些体外实验补充了“具体目标 I 和 II”的体内实验。我们将采取严格的预防措施来防止这些实验中的内毒素污染。总而言之，这是一种治疗缺血性 ARF 的新方法，可能会产生新的见解和治疗方法，尽管有现代治疗，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.