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.
