In the present study, we demonstrated that a selective c-Fos/AP-1 inhibitor, T-5224, suppressed LPS-induced TNF-α production dose-dependently. The inhibition of TNF-α is considered an effective treatment for septic AKI. The current study indicates that renal protection induced by T-5224 administration during endotoxemia occurs by controlling the expression of TNF-α and other downstream mediators.
Sepsis has been identified as the most common cause of AKI in intensive care units. Moreover, the combination of sepsis and AKI is associated with a very high mortality rate
. There is, therefore, an urgent need to identify novel therapeutic interventions with the potential to attenuate septic AKI. Numerous cytokines released from leukocytes and renal tubular cells in the injured kidney function as essential components of the initiation and extension of inflammation in AKI
. Hence, the pharmacological inhibition of these inflammatory mediators is a potential therapeutic target in the treatment of endotoxemia with bacterial infection. Recent studies indicated TNF-α is critical in mediating LPS-induced kidney injury
. Inhibition of TNF-α by genetic or antibody techniques inhibited renal injury following ischemia
 or LPS
Aikawa et al. designed and synthesized a selective c-Fos/AP-1 inhibitor termed T-5224 using 3D pharmacophore modeling based on the crystal structure of the AP-1–DNA complex
. c-Fos/AP-1 controls the expression of inflammatory cytokines including TNF-α by binding directly to AP-1 motifs in the promoter of the gene, and T-5224 treatment resolved arthritis in a mouse model. The transcription factor AP-1 is also activated by LPS, leading to enhanced TNF-α transcription
. T-5224 inhibited the expression of TNF-α and other cytokines in a mouse arthritis model
, suggesting it may also inhibit LPS-induced TNF-α production. Indeed, we demonstrated that T-5224 attenuated serum TNF-α levels dose-dependently following LPS administration and reduced serum BUN and Cr levels, indicating T-5224 can protect against LPS-induced AKI.
Apart from the inhibition of serum TNF-α during endotoxemia, T-5224 decreased other serum cytokines, namely IL-1β and IL-6. IL-1β is important in multiple organ failure and death during endotoxemia. IL-1β, a pro-inflammatory cytokine, evokes similar pathophysiological responses to TNF-α. Inhibition of IL-1β signaling by treatment with recombinant IL-1β receptor antagonist, resulted in decreased mortality in an animal model of endotoxic shock
. On the other hand, elevated plasma concentrations of IL-6 predict AKI in patients with severe sepsis
 and mortality in sepsis correlates with the degree of IL-6 up regulation
. T-5224 showed a remarkable inhibition of IL-1β and IL-6 production in mice challenged with LPS, suggesting that T-5224 can protect renal function against LPS, at least in part, via inhibiting both IL-1β and IL-6 production.
IL-10, an anti-inflammatory cytokine, plays an important role in improving survival in animals challenged with LPS and inhibits several macrophage functions including TNF-α production in vivo and in vitro. However, one study has reported that TNF-α signaling can promote IL-10 production
. Considering this, there was a possibility that T-5224 might inhibit IL-10 production. However, T-5224 did not suppress IL-10 production in our study. This observation is of interest since modulating the balance between pro-inflammatory and anti-inflammatory responses and restoring immune homeostasis of patients is important in the treatment of sepsis.
The effect of T-5224 on ICAM-1 mRNA expression in the kidney was also examined. ICAM-1 is a member of the immunoglobulin-like supergene family of adhesion molecules known to mediate adherence of polymorphonuclear neutrophils (PMNs) to endothelial cells
. Both LPS and TNF-α increase the cell surface expression of ICAM-1
, which is one of several downstream effectors induced by TNF-α. The present study demonstrated that mRNA expression of ICAM-1 was significantly increased in the kidney at 6 h after LPS injection and was markedly attenuated by treatment of T-5224. The role of ICAM-1 in PMN accumulation has been suggested in endotoxemic AKI and a deficiency of ICAM-1 may confer protection against LPS-induced AKI, by decreasing neutrophil adhesion and abrogating signaling events that occur in endothelium upon ligation of ICAM-1. Thus, our results suggested that T-5224 might also protect mice against LPS-induced AKI by decreasing ICAM-1 expression.
We also examined renal histological change at 48 h after LPS injection. As previous study reported, LPS induced severe renal tubular injury
. In this study, mice in LPS group also developed tubular injury. However, less tubular damage was observed in T-5224 group. Furthermore, pathological findings were well correlated with reduced expressions of TNF-α and other downstream effectors. These results indicated that T-5224 conferred protection against structural injury caused by LPS. Interestingly we observed few neutrophils in kidney, which suggests that ICAM-1 itself may cause renal injury through other as yet unknown neutrophil-independent mechanisms.
In the present study, T-5224 did not completely suppress all inflammatory mediators. One potential explanation for this could be that genes encoding TNF-α are also transcriptionally activated through nuclear factor (NF)-κB activation
. NF-κB, a transcription factor, is critically involved in the regulation of monocytic production of pro-inflammatory cytokines, such as TNF-α and IL-1β. T-5224 specifically inhibits the DNA binding activity of c-Fos/c-Jun, without affecting that of NF-κB/p65
. There are also several limitations in our study. First, we only examined BUN and Cr for kidney function, although these markers are insensitive and non-specific for acute changes to kidney function. Therefore, these biomarkers may not detect an ongoing injury to the kidney. Second, T-5224 was administrated immediately after LPS injection. Considering the clinical situation, the delayed administration of T-5224 should also be investigated in the future. Third, intravenous administration is generally more effective than oral administration. However, we demonstrated that oral treatment with T-5224 was effective enough to improve kidney injury in the acute phase of sepsis. The development of T-5224 for intravenous drug use is now underway.