In this manuscript, we further established the role of TLR4 in ischemic AKI by using mice with spontaneous mutations that inactivate TLR4. The C3H/HeJ and C57BL/10ScNJ strains used in our report are unrelated both by their genealogy32
and also by single-nucleotide polymorphism analysis.33
The fact that mutations in a single gene, TLR4
, should have such profound effects in such unrelated mice is an additional powerful genetic argument for the great importance of TLR4 in the pathogenesis of ischemic AKI. This work confirms and extends previous work that used a single transgenic knockout of TLR4 on the C57BL/6 genetic background.2,3
In this manuscript, we suggest that TLR4-mediated activation of leukocytes is one factor contributing to ischemic AKI extension. We provide several lines of data consistent with this hypothesis.
First, our radiation chimeras between two different pairs of TLR4 (+/+) and TLR4 (−/−) mice (C3H/HeJ and C3H/ HeOuJ, and C57BL/10ScNJ and C57BL/10SnJ) are strong arguments for the participation of TLR4 on both radioresistant and radiosensitive (presumable leukocyte) cell populations in the pathogenesis of ischemic AKI ( and ). Our data is consistent with Pulskens et al
but shows a much greater contribution of radiosensitive TLR4 (+/+) leukocytes compared with the report of Wu et al
The key chimera is the TLR4 (−/−) BM in the TLR4 (+/+)-irradiated recipient. The key question is whether all TLR4 (+/+) BMs are eliminated by the irradiation. We and Pulskens used two doses of 5 Gy for a total dose of 10 Gy, whereas Wu 2
used one dose of 9 Gy. Our higher dose of irradiation might have functionally eliminated all the TLR4 (+/+) leukocytes in the irradiated TLR4 (+/+) recipient. In contrast, the smaller dose of irradiation in the Wu study might have allowed a functionally significant number of TLR4 (+/+) leukocytes to survive in the irradiated recipient. These survivors may have proliferated while the mice were recovering from the irradiation and BM transplant, and these survivors and their progeny may have contributed to a TLR4 (+/+) inflammatory response to ischemia and the severe AKI seen in the Wu TLR4 (−/−) BM to irradiated TLR4 (+/+) recipient group.2
Second, we analyzed sham and ischemic kidneys from chimeric mice. We found that TLR4 (+/+) BM cells, presumably leukocytes, infiltrate the ischemic kidney of TLR4 (−/−) recipients (group C, ). This is consistent with TLR4 expression by leukocytes and infiltration of these TLR4-expressing leukocytes into the kidney in response to ischemia. To our knowledge, this is the first such analysis of TLR4 expression in ischemic kidneys from chimeric mice.
Despite the presence of TLR4 (+/+) BM cells, presumed leukocytes, in the ischemic kidneys of irradiated TLR4 (−/−) recipients, there was little ischemic AKI in these chimeras (). One would expect that any TLR4 (+/+) renal leukocyte would be stimulated by HMGB1 released by injured ischemic renal cells and would produce maladaptive IL6, as discussed in the next paragraphs, and injure the kidney. We believe that injury did not occur because although a sufficient number of TLR4 (+/+) leukocytes infiltrated the TLR4 (−/−) kidneys to be detected, there was an insufficient number to damage the kidney. We previously showed that endothelial TLR4 is required for the increased expression of adhesion molecules necessary for an efficient inflammatory response to renal ischemia.52
Radioresistant endothelial cells would be TLR4 (−/−) in the irradiated TLR4 (−/−) recipient. Absent this endothelial TLR4, there would be no increased adhesion molecule expression, and too few TLR4 (+/+) leukocytes would be able to infiltrate the ischemic kidney significantly damage it.66
However, in wild-type kidneys, endothelial TLR4 would be present, endothelial adhesion molecules would therefore increase during ischemic AKI, and there would be efficient inflammation by TLR4 (+/+) leukocytes. Thus, both the TLR4 on radioresistant endothelia and the TLR4 on radiosensitive leukocytes together cause the vigorous inflammatory response that exacerbates ischemic AKI.
Consistent with the above reasoning, we did not find statistically significant differences between IL6 mRNA when TLR4 (−/−) BM was transplanted into TLR4 (+/+) recipients versus when TLR4 (+/+) BM was transplanted into TLR4 −/− recipients. We believe that the similar low levels of IL6 occurred in these two groups because the TLR4 (+/+) leukocytes could not immigrate into the ischemic TLR4 (−/−) renal tissue, as discussed above.
Third, we developed a technique for isolating renal leukocytes from ischemic kidneys. We found that only leukocytes from TLR4 (+/+) kidneys produce maladaptive IL621
during ischemic AKI in vivo
Fourth, we also found that TLR4 (+/+), but not TLR4, (−/−) leukocytes produce IL6 in response to HMGB1 released by cells damaged during ischemia in vitro ().
Fifth, we demonstrate a role for TLR4 (+/+) macrophages in an in vitro
model of ischemic AKI. In this model, proximal tubule cells release HMGB1 after injury by ROS and this HMGB1 stimulates TLR4 (+/+), but not TLR4 (−/−), macrophages to produce IL6 (). This increase in IL6 from macrophages can be attenuated by glycyrrhizic acid, an inhibitor of HMGB161,62
The contribution of TLR4 (+/+) leukocytes to the pathogenesis of ischemic AKI, suggested by our data, is consistent with the literature because of the known expression of TLR4 on leukocytes6–9
and the known contribution of leukocytes to ischemic AKI.10–15
How leukocytes are activated during ischemic AKI remains to be fully understood. However, our data suggest that one pathway of activation is stimulation of leukocyte TLR4 by HMGB1, which are released by injured cells.
We acknowledge that other members of the family of damage-associated molecular pattern molecules and their ligands (for example, RAGE, TLR2, TLR9 and IL1) may also participate in ischemic AKI.16,67–74
We chose to focus on the HMGB1–TLR4 axis because direct molecular interactions between HMGB1 and TLR4 have recently been demonstrated,75
because antibodies against HMGB1 ameliorate ischemic AKI,76,77
and transgenic knockout2–5
and spontaneous mutagenesis ( and ) and antibodies to TLR4 () ameliorate ischemic AKI. Furthermore, shows that another TLR4 ligand HSP70 also activates IL6 production by macrophages, but much less effectively than HMGB1.
In conclusion, TLR4 was initially demonstrated on renal tubular cells after 24 h reperfusion.2,3,78,79
We have recently demonstrated endothelial expression of TLR4 at 4 h reperfusion and showed that renal endothelial TLR4 was required for expression of adhesion molecules. These adhesion molecules allow the maladaptive inflammatory response to ischemic injury at 4-h reperfusion.52
In this manuscript, we report that after leukocytes infiltrate the injured kidney, they produce maladaptive IL6 when their TLR4 receptors interact with HMGB1 released by injured renal cells. Thus, at least three cell types (epithelia, endothelia, and leukocytes) express TLR4 at various times during ischemic AKI; each may have different roles during ischemic AKI.