The Effect of Mtor-Inhibition on Inflammation in Kidney Ischemia-Reperfusion Injury in Mice

Abstract

Introduction: Ischemia-reperfusion injury (IRI) plays an important role in acute renal transplant rejection and delayed allograft function and also long-term graft survival. Multiple stimuli initiating inflammation, including ROS and cytokines, participate in pathogenesis of IRI and are at some level controlled through activation of transcription factor NF-kappa B. We therefore determined the influence of mTOR-inhibition (Everolimus; eve) on inflammation and NF-kappa B activity after induction of IRI. Method: C57/BL6 mice were subjected to IRI by clamping both renal pedicles for 45 minutes. Application of eve started one day before IRI induction in a dose of 1.5 mg/kg b.w. subcutaneously daily. Both eve treated and non-treated mice were sacrificed 30 min, 1h, 2h, 6h, 12h, 1d, 2d, 3d and 7d after IRI. Kidneys were examined by morphology and standard molecular biology techniques including EMSA. Results: Eve treatment significantly increased at early (2d) (p< 0.05), however, significantly reduced kidney injury index at later (7d) time points (p< 0.004) compared with non-treated mice after IRI induction. Interstitial infiltration of F4/80-positive cells after IRI was significantly reduced at later time points in eve treated animals (p< 0.01), which is associated with a decreased expression in TGF-ß and iNOS. NF-kappa B activity was significantly increased in non-treated animals at early (6h) and also later (3d and 7d) time points, while eve treatment of mice significantly increased NF-kappa B binding activity at all time points, showing increased nuclear expression of phospho-p65 at all time points (p< 0.01) and Rel B at later time points (p< 0.01), when compared with non-treated animals. Biphasic increase in TNF-alpha and chemokine CCL2, CCL5 and CXCL10 expression, both in treated and non-treated animals, was observed. The second peak of TNF-alpha expression was significantly increased while CCL2 and CCL5 expression was significantly reduced in eve treated animals. The expression of the anti-inflammatory cytokine IL-10 was lower at later time points in eve treated mice. Conclusion: Expression profiles showed a biphasic expression of TNF-α and chemokines coinciding with the early inflammatory and late repair phase after IRI induction. The beneficial role of eve treatment in late repair phase could be partly reflected by decreased numbers of infiltrating macrophage. The pattern of NF-κB activation under m-TOR inhibition reflects activation via canonical and also non-canonical pathway in IRI which warrants further clarification.