Acute renal ischemia elicits an inflammatory response that exacerbates injury; this inflammation requires the increased expression of endothelial adhesion molecules that facilitate diapedesis of leukocytes into renal tissues. 1–10
However, the regulation of these endothelial adhesion molecules is not well understood.
On the basis of observations made in this study, we now suggest that endothelial TLR4 contributes to this regulation. First, we show increased expression of endothelial TLR4 of the ISOM at 4 h reperfusion. Because TLR4 had previously been reported only on tubules at 24 h and later, we used four different techniques to confirm our new findings: analysis of dissected renal tissues, in situ
hybridization, immunohistology, and analysis of isolated renal endothelial cells. Second, we developed a technique for isolating renal endothelia and quantitatively assaying their expression of adhesion molecules. We find increased expression of adhesion molecules during ischemic AKI only in wild-type, but not TLR4 (−/−), endothelia. Third, we find that HMGB1, released by injured renal cells during ischemic AKI, interacts with endothelial TLR4 to activate adhesion molecule expression only by wild-type, but not TLR4 (−/−), endothelia in vitro
. Finally, transgenic knockout of TLR4 ameliorates ischemic injury and inflammation;16,17
this shows a role for TLR4 in ischemic AKI. We propose that one role is the regulation of endothelial adhesion molecule expression. All together, these observations are consistent with the idea that endothelial TLR4 regulates the expression of the endothelial adhesion molecules required for the inflammatory response to injury early after ischemia/reperfusion.
Although our proposed role for endothelial TLR4 has not previously been demonstrated in ischemic AKI, such a role has been shown during ischemic injury of the cremaster muscle,42
and during endotoxin injury of the lung.43
Further support for our hypothesis is the localization of TLR4 to endothelia of the ISOM. The medullary thick ascending limb and the S3 straight proximal tubule that reside in this area are the tubules most vulnerable to ischemic injury in both rodents and humans.23,44–48
During ischemic AKI, injury to these cells should result in the release of HMGB1, and this HMGB1 should stimulate TLR4 on the adjacent endothelium. Because of endothelial activation in the ISOM, this is the area of greatest inflammation during ischemic AKI.5
At 24 h, but not 4 h, reperfusion, we found TLR4 protein on proximal tubules. To our knowledge, these are the first studies to use double immunofluorescence for TLR4 and tubule-specific markers to localize TLR4. At even later reperfusion times (days 3–5), TLR4 has also been reported on distal tubules and medullary thick ascending limb cells by using anti-Tamm Horsfall antibodies and in situ
hybridization for TLR418
and on proximal tubules and distal nephrons by using anti-TLR4 antibodies; tubules were identified by morphology alone.19
Although we see strong, consistent expression of TLR4 on proximal tubules, we did occasionally see inconsistent staining of more distal segments. This study is focused on endothelial expression of TLR4 and events during the first 4 h of reperfusion; ongoing studies in our laboratory pursue the localization and functions of TLR4 on tubules and other cells after 24 h of reperfusion.
Although tubular TLR4 would regulate injury after 24 h, it would not affect earlier injury and inflammation. We suggest that this earlier injury results from TLR4 expression on renal endothelia, on the TLR4-dependent expression of endothelial adhesion molecules and on the ensuing inflammation. The different timing of TLR4 expression by tubules and endothelia reflects the different regulation of this molecule on these two cell types. During ischemic AKI, reactive oxygen species is released early during ischemia/reperfusion.26–28
We found that this reactive oxygen species increased endothelial TLR4 expression in vitro
. This is consistent with the early expression of TRL4 on endothelia. On the other hand, tubular TLR4 is stimulated by interferon-γ and tumor necrosis factorα.18
This is consistent with the late expression of these cytokines.49–51
All together, our observations support the maladaptive role of endothelial activation in ischemic AKI,5,6,35,52–55
and also support the following hypothesis: at 4 h reperfusion, acute ischemic injury results in the release of HMGB1; HMGB1 interacts with endothelial TLR4; this activates endothelial cells to express adhesion molecules; these endothelial adhesion molecules allow the maladaptive inflammation that exacerbates ischemic AKI.
In conclusion, the contribution of TLR4 to ischemic AKI is complex, and just beginning to be understood. At least two different cell types—renal tubules, as previously reported,16,18,19
and renal endothelia, as now reported—express TLR4. Each cell type contributes to renal injury in a different way and at a different time after reperfusion.