Low-dose carbon monoxide inhibits progressive chronic allograft nephropathy and restores renal allograft function.

Chronic allograft nephropathy (CAN) represents progressive deterioration of renal allograft function with fibroinflammatory changes. CAN, recently reclassified as interstitial fibrosis (IF) and tubular atrophy (TA) with no known specific etiology, is a major cause of late renal allograft loss and remains a significant deleterious factor of successful renal transplantation. Carbon monoxide (CO), an effector byproduct of heme oxygenase pathway, is known to have potent anti-inflammatory and antifibrotic functions. We hypothesized that inhaled CO would inhibit fibroinflammatory process of CAN and restore renal allograft function, even when the treatment was initiated after CAN was established. Lewis rat kidney grafts were orthotopically transplanted into binephrectomized allogenic Brown Norway rats under brief tacrolimus (0.5 mg/kg im, days 0-6). At day 60, CO (20 ppm) inhalation was initiated to recipients and continued until day 150 or animal death. Development of CAN was confirmed at day 60 with decreased creatinine clearance (CCr), significant proteinuria, and histopathological findings of TA, IF, and intimal arteritis. Air-treated control recipients continued to deteriorate with further declines of CCr and increases of urinary protein excretion and died with a median survival of 82 days. In contrast, progression of CAN was decelerated when recipients received CO on days 60-150, showing markedly improved graft histopathology, restored renal function, and improved recipient survival to a median of >150 days. CO significantly reduced intragraft mRNA levels for IFN-gamma and TNF-alpha at day 90. Expression of profibrotic TGF-beta/Smad was significantly suppressed with CO, together with downregulation of ERK-MAPK pathways. Continuous CO (20 ppm) treatment for days 0-30, days 30-60, or days 0-90, or daily 1-h CO (250 ppm) treatment for days 0-90, also showed efficacy in inhibiting CAN. The study demonstrates that CO is able to inhibit progression of fibroinflammatory process of CAN, restore renal allograft function, and improve survival even when the treatment is started after CAN is diagnosed.

Experimental orthotopic lung transplantation model in rats with cold storage.

This report describes a new experimental procedure, a rat unilateral, orthotopic lung transplantation with cold storage, and evaluates its relevancy and reliability to study the early events during cold ischemia/reperfusion (I/R) injury. This model, using the cuff technique, does not require extensive training and is relatively easy to be established. The model can induce reproducible degrees of pulmonary graft injury including impaired gas exchange, proinflammatory cytokine upregulation, or inflammatory infiltrates, depending on the preservation time. The results are consistent with the previous clinical evidence, thus suggesting that this model is a valid and reliable animal model of cold I/R injury.

Ex vivo carbon monoxide prevents cytochrome P450 degradation and ischemia/reperfusion injury of kidney grafts.

Renal ischemia/reperfusion injury is a major complication of kidney transplantation. We tested if ex vivo delivery of carbon monoxide (CO) to the kidney would ameliorate the renal injury of cold storage that can complicate renal transplantation. Orthotopic syngeneic kidney transplantation was performed in Lewis rats following 24 h of cold preservation in University of Wisconsin solution equilibrated without or with CO (soluble CO levels about 40 microM). Ischemia/reperfusion injury in control grafts resulted in an early upregulation of inflammatory mediator mRNAs and progressive deterioration of graft function. In contrast, the grafts preserved with CO had significantly less oxidative injury and this was associated with improved recipient survival compared to the control group. Renal injury in the control group showed considerable degradation of cytochrome P450 heme proteins, active heme metabolism and increased detrimental intracellular free heme levels. Kidney grafts preserved in CO-equilibrated solution maintained their cytochrome P450 protein levels, had normal intracellular heme levels and had less lipid peroxidation. Our results show that CO-mediated suppression of injurious heme-derived redox reactions offers protection of kidney grafts from cold ischemia/reperfusion injury.

Carbon monoxide ameliorates renal cold ischemia-reperfusion injury with an upregulation of vascular endothelial growth factor by activation of hypoxia-inducible factor.

BACKGROUND: We have previously shown that carbon monoxide (CO) inhalation at a low concentration provides protection against cold ischemia-reperfusion (I/R) injury after kidney transplantation. As vascular endothelial growth factor (VEGF) may promote the recovery process of impaired vascular endothelial cells during I/R injury, we examined whether protective effects of CO involved VEGF induction and its upstream hypoxia-inducible factor (HIF)-1 activation. METHODS: Lewis rat kidney graft, preserved in University of Wisconsin at 4 degrees C for 24 hr, was orthotopically transplanted into syngeneic recipient. Recipients were continuously maintained in air or exposed to CO (250 ppm) for 1 hr before and 24 hr after transplant. RESULTS: Prolonged cold preservation resulted in progressive impairment of kidney graft function with early inflammatory responses. Carbon monoxide significantly protected kidney grafts from cold I/R injury, improved renal function and enhanced recipient survival. Real-time reverse transcriptase-polymerase chain reaction revealed upregulation of HIF-1alpha and VEGF in the CO-treated kidney grafts as early as 1 hr after reperfusion. Western blot showed CO significantly upregulated VEGF expression 1 to 3 hr after kidney transplantation. Considerably more VEGF-positive cells were observed mainly in tubular epithelial cells in CO-treated, but not air-exposed, kidney grafts at 3 hr after reperfusion. YC-1, HIF-1alpha inhibitor, completely abrogated the actions of CO on VEGF induction and reversed the protective effects afforded by CO. Nitric oxide production in the grafts was increased by CO, however, abolished by YC-1. CONCLUSION: These results demonstrate that the protective effect of CO against renal cold I/R injury may involve VEGF upregulation through its upstream signal, HIF-1 activation.

Toll-like receptor 4 mediates the early inflammatory response after cold ischemia/reperfusion.

BACKGROUND: Ischemia/reperfusion (I/R) injury leads to graft dysfunction and may contribute to alloimmune responses posttransplantation. The molecular mechanisms of cold I/R injury are only partially characterized but may involve toll-like receptor (TLR)-4 activation by endogenous ligands. We tested the hypothesis that TLR4 mediates the early inflammatory response in the setting of cold I/R in a murine cardiac transplant model. METHODS: Syngeneic heart transplants were performed in mutant mice deficient in TLR4 signaling (C3H/HeJ) and wild-type mice (C3H/HeOuJ). Transplants were also performed between the strains (mutant hearts into wild-type recipients and the converse). Donor hearts were subjected to 2 hr of cold ischemia. The grafts were retrieved at 3 and 24 hr after reperfusion. Serum samples were collected for cytokine analysis. Reverse-transcription polymerase chain reaction and histologic analysis were used to assess intra-graft inflammation. RESULTS: After transplant, serum tumor necrosis factor (TNF), interleukin (IL)-6, JE/monocyte chemotractant protein (MCP)-1, IL-1beta, and troponin I levels, as well as intragraft TNF, IL-1beta, IL-6, early growth response (EGR)-1, intercellular adhesion molecule (ICAM)-1, and inducible nitric oxide synthase (iNOS) mRNA levels, were significantly lower in the mutant-->mutant group compared to the wild-type-->wild-type group (P< or =0.05). Intermediate levels of serum IL-6, JE/MCP-1, as well as intragraft TNF, IL-1beta, IL-6, and ICAM-1 mRNA were observed after transplants in the mutant-->wild-type and wild-type-->mutant groups. Immunohistochemistry revealed less myocardial nuclear factor-kappaB nuclear translocation at and less neutrophil infiltration in the mutant-->mutant group compared to the wild-type-->wild-type group. CONCLUSIONS: These findings demonstrate that TLR4 signaling is central to both the systemic and intragraft inflammatory responses that occur after cold I/R in the setting of organ transplantation and that TLR4 signaling on both donor and recipient cells contributes to this response.

Low-dose carbon monoxide inhalation prevents ischemia/reperfusion injury of transplanted rat lung grafts.

BACKGROUND: Carbon monoxide (CO), a byproduct of heme catalysis by heme oxygenases, has been shown to provide protection against ischemia/reperfusion (I/R) injury. We examined the cytoprotective effect of CO at a low concentration on cold I/R injury of transplanted lung grafts. METHODS: Orthotopic left lung transplantation was performed in syngenic Lewis to Lewis rat combination. Grafts were preserved in University of Wisconsin solution at 4 degrees C for 6 hours. Donors and/or recipients were exposed to CO (250 ppm) in air for 1 hour before surgery and then continuously post-transplantation. RESULTS: Blood oxygen partial pressure of graft pulmonary veins in the CO-treated group versus the air-treated group was significantly higher. The increase of messenger RNA of inflammatory mediators such as interleukin-6, tumor necrosis factor-alpha, inducible nitric oxide synthase, and cycloooxygenase-2 was markedly inhibited in the CO-treated group. The expression of phosphorylated-extracellular signal-regulated protein kinase 1/2 was significantly reduced in the CO-treated group. CO treatment reduced the number of infiltrating macrophages into the lung grafts. Vascular endothelial cells detected by CD31 stain were well preserved in CO-treated grafts, while those in air-treated grafts were faint and interrupted. CONCLUSIONS: These results demonstrate that exogenous low-dose CO treatment of donors and recipients can prevent lung I/R injury and significantly improve function of lung grafts after extended cold preservation and transplantation.

Mechanisms of Toll-like receptor 4 (TLR4)-mediated inflammation after cold ischemia/reperfusion in the heart.

BACKGROUND: Toll-Like Receptor 4 (TLR4) signaling mediates early inflammation after cold ischemia-reperfusion (I/R). We hypothesized that the TLR4 coreceptor CD14, the intracellular adaptor proteins myeloid differentiation factor 88 (MyD88) and TIR domain-containing-adaptor inducing IFNbeta (TRIF) would be required for cold I/R induced inflammation. High mobility group box 1 (HMGB1) is a putative endogenous activator of TLR4. Therefore, we also assessed the contribution of HMGB1 in cold I/R induced inflammation. METHODS: Syngeneic heart transplants were performed in mice deficient in CD14, MyD88, TRIF, or wild-type mice. In other experiments, anti-HMGB1 neutralizing antibody or control IgG was administered at reperfusion. Donor hearts were subjected to 2 hr of cold ischemia and retrieved after 3 hr of reperfusion. RESULTS: After cold I/R, grafts revealed striking translocation of HMGB1 out of the nucleus in cardiac myocytes. Administration of an anti-HMGB1 neutralizing antibody resulted in reduced systemic interleukin (IL)-6, tumor necrosis factor alpha (TNFalpha), and intercellular adhesion molecule-1 (ICAM-1) messenger RNA (mRNA) levels (P< or =0.05). Compared with controls, CD14 knock-out (KO) mice exhibited significantly lower (P< or =0.05) systemic IL-6 and JE/monocyte chemotractant protein-1 levels after cold I/R. Intragraft TNFalpha and IL-1beta mRNA levels were also significantly lower (P< or =0.05) in CD14 KO grafts. MyD88 KO mice exhibited significantly lower (P< or=0.05) systemic IL-6 levels compared with control mice after cold I/R. Intragraft TNFalpha, IL-6, and ICAM-1 mRNA levels were also significantly lower (P< or =0.05) in MyD88 KO grafts. Significantly lower levels (P< or =0.05) of serum IL-6, monocyte chemotractant protein-1 as well as intragraft TNFalpha, IL-6, IL-1beta, and ICAM-1 were observed after cold I/R in TRIF deficient animals compared with controls. CONCLUSIONS: CD14, MyD88, TRIF, and HMGB1 contribute to the inflammatory response that occurs after cold I/R. These results provide insight into the mechanisms of TLR4-mediated inflammation after cold I/R.

Carbon monoxide-saturated preservation solution protects lung grafts from ischemia-reperfusion injury.

OBJECTIVES: In previous work we have demonstrated that delivery of low concentrations (250 ppm) of carbon monoxide by means of inhalation to donors, recipients, or both protects transplanted lungs from ischemia-reperfusion injury (improved gas exchange, diminished intragraft and systemic inflammation, and retention of graft vascular endothelial cell ultrastructure). In this study we examined whether delivery of carbon monoxide to lung grafts in the preservation solution could protect against lung ischemia-reperfusion injury. METHODS: Orthotopic left lung transplantation was performed in syngeneic Lewis to Lewis rats. Grafts were preserved in University of Wisconsin solution with or without (control solution) carbon monoxide at 4 degrees C for 6 hours. Carbon monoxide gas (5% or 100%) was bubbled into University of Wisconsin solution at 4 degrees C for 5 minutes before use. RESULTS: In control animals, ischemia-reperfusion injury resulted in significant deterioration of graft function and was associated with a massive cellular infiltrate 2 hours after reperfusion. Grafts stored in University of Wisconsin solution with carbon monoxide (5%), however, demonstrated significantly better gas exchange and significantly reduced intragraft inflammation (reduced inflammatory mediators and cellular infiltrate). Experiments demonstrated that the protective effects afforded by 100% University of Wisconsin solution with carbon monoxide were not as potent as those of 5% University of Wisconsin solution with carbon monoxide. CONCLUSIONS: This study demonstrates that 5% carbon monoxide as an additive to the cold flush/preservation solution can impart potent anti-inflammatory and cytoprotective effects after cold preservation and transplantation of lung grafts. Such ex vivo treatment of lung grafts with carbon monoxide can minimize concerns associated with carbon monoxide inhalation and might offer the opportunity to significantly advance the application of carbon monoxide in the clinical setting.

A single intraperitoneal dose of carbon monoxide-saturated ringer's lactate solution ameliorates postoperative ileus in mice.

Treatment with inhaled carbon monoxide (CO) has been shown to ameliorate bowel dysmotility caused by surgical manipulation of the gut in experimental animals. We hypothesized that administration of CO dissolved in lactated Ringer's solution (CO-LR) might provide similar protection to that observed with the inhaled gas while obviating some of its inherent problems. Postoperative gut dysmotility (ileus) was induced in mice by surgical manipulation of the small intestine. Some mice were treated with a single intraperitoneal dose of CO-LR immediately after the surgical procedure, whereas other mice received only the LR vehicle. Twenty-four hours later, intestinal transit of a nonabsorbable marker (70-kDa fluorescein isothiocyanate-labeled dextran) was delayed in mice subjected to intestinal manipulation but not the sham procedure. Gut manipulation also was associated with increased expression within the muscularis propria of transcripts for interleukin-1beta, cyclooxygenase-2, inducible nitric-oxide synthase, intracellular adhesion molecule-1, and Toll-like receptor-4, as well as infiltration of the muscularis propria with polymorphonuclear leukocytes and activation of mitogen-activated protein kinases and nuclear factor-kappaB. All of these effects were attenuated by treatment with CO-LR. The salutary effect of CO-LR on gut motility, as well as many of the anti-inflammatory effects of CO-LR, was diminished by treatment with a soluble guanylyl cyclase (sGC) inhibitor, suggesting that the effects of CO are mediated via activation of sGC. These data support the view that a single intraperitoneal dose of CO-LR ameliorates postoperative ileus in mice by inhibiting the inflammatory response in the gut wall induced by surgical manipulation, possibly in a sGC-dependent fashion.

Donor graft adenoviral iNOS gene transfer ameliorates rat liver transplant preservation injury and improves survival.

The exact role of inducible NOS (iNOS) in liver ischemia/reperfusion (I/R) injury is controversial. This study was designed to investigate whether donor liver pretreatment with adenovirus encoding iNOS (AdiNOS) ameliorates I/R injury associated with liver transplantation. Orthotopic syngeneic LEW rat liver transplantation (OLT) was performed after 18 or 24 hours' preservation in cold UW. AdiNOS or control gene vector (AdLacZ) was delivered to the liver by donor intravenous pretreatment 4 days before graft harvesting. Uninfected grafts also served as control. Recipients were sacrificed 1 to 48 hours posttransplantation. An abundant hepatic iNOS protein expression and marked serum NO elevation was observed in the AdiNOS-treated group, without affecting endothelial nitric oxide synthase (eNOS) expression, before harvesting and after OLT. AdiNOS pretreatment markedly improved liver function assessed by serum aspartate aminotransferase/alanine aminotransferase levels and reduced liver necrosis formation. AdiNOS treatment also was associated with reduced ICAM-1 mRNA expression and neutrophil accumulation in the liver graft after OLT compared with untransfected or AdLacZ-treated group. Furthermore, AdiNOS delivery significantly improved transplant survival, compared with AdLacZ or saline controls. AdiNOS pretreatment did not attenuate I/R-induced apoptotic cell death in the liver graft. Administration of a selective inhibitor for iNOS abrogated the protection afforded by AdiNOS pretreatment. In conclusion, donor pretreatment with AdiNOS led to improved liver graft injury and posttransplantation survival. Downregulation of ICAM-1 mRNA and neutrophil infiltration may be associated with the mechanisms by which AdiNOS pretreatment confer the protection against transplant-associated hepatic I/R injury.