Background: Although high-mobility group box 1 protein (HMGB1) is an inflammatory mediator often associated with organ injury, little is known about its role in kidney transplantation. Pro-inflammatory activation of macrophages, epithelial, and endothelial cells is accompanied by translocation of HMGB1 from the nucleus to cytosol followed by extracellular release. Our objective was to determine whether HMGB1 release occurred in injured kidney transplants, and the etiology of release. Methods: Mouse kidney allografts were performed as previously described and studied at 8 weeks when there is development of fibrosis and impairment of renal function. Human and mouse proximal tubule epithelial cells (PTEC) were also studied. The extent of HMGB1 translocation from the nucleus to cytosol and HMGB1 released to culture media, sera, and urine were assessed by immunohistochemistry and western blot, respectively. To recapitulate cyclosporine (CsA) nephrotoxicity, some naïve mice were placed on a salt deprived diet for 4 weeks followed by treatment with CsA 30 mg/kg IP daily for 2 weeks. Results: In allografts, there was marked decrease of nuclear HMGB1 with translocation to the cytosol compared to naïve kidneys, particularly in the epithelium of the collecting ducts and vessels. Exposure of mPTEC to proinflammatory IFN-γ (5.2±2.1-fold; p<0.05) and IL-1α (9.4±2.1-fold; p<0.05), or the regulatory cytokine TGFβ (11.9±2.7-fold; p<0.05), resulted in significant HMGB1 release to the culture media compared to vehicle-treated cells. To investigate the implications of calcineurin inhibitor exposure in allografts, human PTECs were exposed to CsA and exhibited a dose-dependent and time-dependent accumulation of HMGB1 in the culture media compared to vehicle treated cells. Moreover, in kidney sections from CsA nephrotoxic mice, substantial HMGB1 translocation occurred particularly in outer medulla compared to vehicle treated controls. Serum and urine HMGB1 levels in CsA treated mice were significantly up-regulated at 1 week (5.9±0.2-fold and 3.2±0.2-fold; p<0.05) and 2 weeks (6.2±1.5-fold and 6.2±2.3-fold; p<0.05) respectively, compared to vehicle treated mice. Conclusion: These results demonstrate that HMGB1 release is associated with exposure to stress conditions and suggest that HMBG1 may be a novel marker and mediator of allograft injury. Further studies are focused on mechanisms of HMGB1 release and its contribution to allograft failure.

Hepatic damage occurs in males and ovariectomized (OVX), not in proestrus (PE), females following trauma-hemorrhage (T-H). The mechanism responsible for hepatoprotection remains unknown. We hypothesized protection in PE is a result of enhanced heme oxygenase-1 (HO-1)derived down-regulation of liver inflammatory responses. PE and OVX rats underwent T-H (midline laparotomy, 60% blood loss). PE rats received vehicle (Veh; saline), HO-1 inhibitor chromium mesoporphyrin IX chloride (CrMP; 2.5 mg/kg), zinc protoporphyrin IX (ZnPP; 25 mg/kg), or Akt/PI-3K inhibitor Wortmannin (Wort; 1 mg/kg) 30 min prior to resuscitation or sham operation i.p. OVX rats received Veh or 17 beta-estradiol (E2; 1 mg/kg) 30 min before hemorrhage. Rats were killed 2 h thereafter. Following T-H, left ventricular performance was maintained in PE and E2 OVX rats but was depressed in OVX and CrMP-, ZnPP-, and Wort-treated PE rats; liver damage was not evident in PE rats, and CrMP, ZnPP, and Wort abrogated protection; liver HO-1, p38 MAPK, Akt/PI3K, and Bcl-2 expression increased in PE and E2 OVX rats, which was abrogated by CrMP, ZnPP, and Wort, and liver ICAM-1, caspase-3, phospho-I kappa B-alpha, and NF-kappa B expression increased in OVX and CrMP-, ZnPP-, and Wort-PE rats; liver myeloperoxidase, NF-kappa B DNA-binding activity, TNF-alpha, IL-6, plasma proinflammatory cytokines, and cytokine-induced neutrophil chemoattractants increased in OVX and CrMP-, ZnPP-, and Wort-PE rats; and plasma estradiol levels and hepatic estrogen receptor-alpha and -beta expression decreased in OVX but were unaltered by CrMP, ZnPP, and Wort. Thus, enhanced HO-1 in PE and E2 OVX females modulates inflammatory responses and protects liver following T-H. J. Leukoc. Biol. 85: 1015-1026; 2009.

Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.