Background: Ischemia reperfusion injury is associated with a number of clinical conditions including renal transplantation. We recently showed that pretreatment of mice with leflunomide significantly reduces inflammatory cytokine production and cell death in the ischemic-reperfused kidney. The growth arrest- and DNA-damage-inducible protein 153 (GADD153) has emerged as a regulator of inflammation and cell death. Thus, the present study tested the hypothesis that leflunomide administration, at the time of reperfusion of the ischemic kidney, reduces GADD153 expression, DNA injury, inflammatory cytokine generation and cell death.

Methods: The right kidney of male C57BL/6 mice was subjected to 45 min of ischemia followed by administration of leflunomide (40 mg/kg; i.p.) at restoration of renal blood flow; the left kidney served as sham control. Untreated animals were given the vehicle, DMSO, but subjected to the same procedure. Four-hours after initiation of reperfusion, the kidneys were harvested for flow cytometry-based studies.

Results: The ischemic-reperfused kidneys showed significant elevations in cells positive for ΓH2AX (a marker of double strand DNA breaks) and GADD153 in association with increased pro-inflammatory cytokines, interleukin (IL)-17 and IL-23, positive cells but no change in the anti-inflammatory cytokine, IL-10, compared to those of their sham counterparts. These changes were associated with significant apoptotic and necrotic cell death in association with disruption of mitochondrial membrane potential (as revealed by the JC-1 assay). Leflunomide treatment resulted in significant reductions in ΓH2AX and GADD153 positive cells accompanied by reductions in IL-17 and IL-23 positive cells but increased IL-10 positive cells. Further, leflunomide treatment significantly reduced cell death and preserved mitochondrial membrane potential.

Conclusions: The results indicate that leflunomide treatment initiated at the time of reperfusion of the ischemic kidney exerts renoprotective effects through downregulation of GADD153 and inflammation. Since the timing of an ischemic insult is often not known, the demonstration of efficacy of leflunomide treatment at reperfusion may be of translational potential/value in conditions associated with renal ischemia reperfusion injury.

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