Recent experimental data suggest that IRI rapidly activates innate immune responses. TLR4 has been shown to be upregulated in kidney IRI, particularly by TECs (29
). In this study, we found that expression of both TLR4 and a number of its endogenous ligands was increased following kidney ischemia and that the full development of kidney IRI was dependent upon signaling through the TLR4/MyD88 pathway. Mice genetically deficient in TLR4 or the adaptor molecule MyD88 were protected from kidney dysfunction and histological damage. Protection was associated with a reduction in proinflammatory cytokine and chemokine generation, a concomitant decrease in infiltration by macrophages and neutrophils, and a reduction in TEC apoptosis. We demonstrated that TLR4 signaling in kidney parenchymal cells made the more significant contribution to kidney damage although expression of TLR4 on leukocytes was clearly also important in IRI.
Within the kidney, both kidney parenchymal cells and BM-derived cells express pattern recognition molecules, including members of the TLR system. Interstitial and glomerular macrophages express TLR1, -2, -4, and -6, and DCs express TLR4, -7, -8, and -9, while TECs and mesangial cells express TLR1, -2, -3, -4, and -6 (15
). Expression by other types of kidney parenchymal cells such as endothelial cells or fibroblasts is possible; however, this has not been reported. Kidney DCs have recently been shown to form an interdigitating network extending throughout the interstitium and also present within the glomerulus in mesangial areas (35
). Thus, kidneys are well equipped to respond to TLR ligands. The classical ligand for TLR4 is bacterial LPS, and TLR4, present on both immune cells and kidney parenchymal cells, is involved in pathological kidney responses in experimental models of kidney infection and systemic bacterial sepsis (18
Endogenous ligands have been identified for many TLRs (19
), and several of these ligands have been implicated in the pathogenesis of IRI. HMGB1, recently identified as an inflammatory cytokine (38
), can be released from necrotic or damaged cells as a signal to trigger inflammation (40
). HMGB1 produced by ischemic hepatocytes binds to both TLR2 and TLR4, and this interaction is critical for the development of lethal hepatic IRI. Hyaluronan fragments have been shown to signal through TLR4 and/or TLR2 on DCs and endothelial cells (10
) and can function as an endogenous adjuvant in alloresponses, inducing expression of costimulatory molecules and production of TNF-α by DCs, thus enhancing T cell priming (10
). Chemokine and cytokine production by macrophages, kidney TECs, and lung epithelial cells mediated by the binding of hyaluronan to TLR4 has also been documented (42
). Biglycan, an ECM component, can promote inflammation by signaling through TLR2 and -4 on macrophages (24
). In the current study, we demonstrated upregulation of several ligands, including HMGB1, hyaluronan (and its synthetic enzyme HAS), and biglycan in kidney during IRI, suggesting that any or all of these may act as endogenous ligands for TLR4 in our model. In contrast, expression of HSP70 was not increased at either the gene or the protein level. Our studies provide circumstantial evidence that endogenous ligands are the source of TLR4 activation during IRI. Further studies will be necessary to determine the time course of expression of the different ligands in response to ischemic insults of varying severity and to evaluate their relative contributions to the development of kidney IRI.
TLR4 engagement by its ligands triggers multiple downstream effects including the activation and expression of proinflammatory cytokines (TNF-α, IL-1β, and IL-6) and chemokines responsible for neutrophil (IL-8 and MIP-2) and macrophage (MCP-1) accumulation, all features of IRI. We were able to confirm the proposed role of TLR4 in IRI by demonstrating that TLR4-deficient mice were protected against kidney IRI, with significantly lower serum creatinine, less tubular damage, and less interstitial neutrophil and macrophage accumulation versus WT controls. The major intracellular signaling pathway for TLR4 requires participation of the adaptor protein MyD88. Mice deficient in MyD88 were protected from kidney damage following IRI to an extent equal to TLR4-deficient mice, suggesting the MyD88-dependent TLR4 pathway is responsible for TLR4-mediated kidney IRI. The impact of TLR4 deficiency on cytokine and chemokine expression and macrophage and neutrophil accumulation in IRI was also consistent with involvement of the MyD88 pathway. TLR4 signaling may also proceed via a MyD88-independent pathway involving the adaptor molecule TRIF, stimulating IFN-inducible genes such as IP10. In contrast to the expression pattern of MyD88-dependent cytokines and chemokines, we found no significant difference in IP10 mRNA expression in TLR4–/–
, and WT kidney at day 1 after IRI. While maintained expression of IP10 in the MyD88–/–
group is consistent with an intact TLR4/TRIF pathway in these mice, it is perhaps surprising that no reduction of IP10 expression was observed in the TLR4–/–
group. These results do not exclude a role for the TRIF pathway but do suggest that signaling via MyD88 is the dominant pathway leading to kidney injury. MyD88 is also required for signaling through TLR2, another receptor involved in the pathogenesis of kidney IRI (31
). Genetic absence or knockdown of TLR2 resulted in reduced cytokine and chemokine production, reduced leukocyte infiltration, and protection from kidney dysfunction and tubular damage in the study of Leemans et al. (31
). Given that both TLR2 and -4 have similar downstream effects following engagement (10
) and signal through a common adaptor molecule, MyD88, it is somewhat surprising that more redundancy between their effects was not observed and that both TLR2- and TLR4-deficient mice were significantly protected against kidney IRI. Moreover, in our study, MyD88-deficient mice, in which signaling through both TLR2 and -4 is affected, did not enjoy greater protection from IRI than TLR4–/–
mice. One possible explanation for these observations is that the influence of an individual TLR on the inflammatory response to ischemia may depend upon the profile of endogenous ligands produced by ischemia and that this profile may vary according to the duration and severity of the insult as well as the tissue involved. Although our study and that of Leemans et al. employed very similar models, the duration of kidney ischemia in the latter was considerably longer than that in our study (45 versus 22 minutes), and endogenous ligand expression, consequently, could differ as well.
Both BM-derived cells and parenchymal cells are known to contribute to inflammation and injury within the kidney in various conditions. To determine the relative contribution of TLR4 on kidney parenchymal cells and leukocytes to kidney damage in IRI, BM chimeric mice were generated. We demonstrated that during IRI, mice with TLR4-deficient leukocytes but TLR4-competent parenchymal cells developed significantly more kidney dysfunction and damage compared with chimeras with TLR4-deficient parenchymal cells but competent leukocytes, suggesting functional TLR4 on kidney parenchymal cells makes the more significant contribution to IRI. Consistent with this, our in vitro results confirmed that kidney tubular cells required TLR4 to significantly upregulate proinflammatory cytokines and chemokines following ischemia and that in the absence of TLR4 or MyD88, they were protected against undergoing apoptosis. Similar consequences of TLR2 signaling in ischemic kidney tubular cells were also reported by Leemans et al. (31
). In addition to tubular cells, other TLR4-expressing intrinsic kidney cell types, such as endothelial cells, may be contributing to IRI. While we have not specifically addressed the role of endothelial cells, our in vitro data do suggest that TLR4 on kidney tubular cells plays a significant role in ischemia-mediated injury. The kidney is richly supplied with a network of resident DCs and macrophages (35
), and these, as well as infiltrating leukocytes, could promote IRI through the elaboration of soluble mediators and through cell-cell contact. Consistent with this, TLR4 expression by leukocytes also appears important in IRI, as chimeric mice lacking TLR4 on BM-derived cells alone were partially protected from kidney dysfunction and tubular injury as compared with WT controls. This was not seen in TLR2–/–
mice in IRI (31
), whereas TLR2 ligation on both BM-derived and parenchymal kidney cells plays a role in amplifying the inflammatory effects in immune-mediated glomerulonephritis (45
), another form of sterile tissue injury. In contrast, nonparenchymal cells are the critical cell type in the pathogenesis of TLR-mediated liver IRI (26
In summary, our results document the important role of TLR4- and MyD88-mediated signaling in the pathogenesis of kidney IRI and suggest that this pathway is central in the innate immune response that leads to kidney injury.