Pathogenesis and management of renal fibrosis induced by unilateral ureteral obstruction.

Regardless of the underlying etiology, renal fibrosis is the final histological outcome of progressive kidney disease. Unilateral ureteral obstruction (UUO) is an ideal and reproducible experimental rodent model of renal fibrosis, which is characterized by tubulointerstitial inflammatory responses, accumulation of extracellular matrix, tubular dilatation and atrophy, and fibrosis. The magnitude of UUO-induced renal fibrosis is experimentally manipulated by the species chosen, animal age, and the severity and duration of the obstruction, while relief of the obstruction allows the animal to recover from fibrosis. The pathogenesis of renal fibrosis is complex and multifactorial and is orchestrated by activation of renin-angiotensin system (RAS), oxidative stress, inflammatory response, transforming growth factor beta 1-Smad pathway, activated myofibroblasts, cell death (apoptosis, autophagy, ferroptosis, and necroptosis), destruction of intracellular organelles, and signaling pathway. The current therapeutic approaches have limited efficacy. Inhibition of RAS and use of antioxidants and antidiabetic drugs, such as inhibitors of sodium-glucose cotransporter 2 and dipeptidyl peptidase-4, have recently gained attention as therapeutic strategies to prevent renal scarring. This literature review highlights the state of the art regarding the molecular mechanisms relevant to the management of renal fibrosis caused by UUO.

The angiotensin receptor neprilysin inhibitor LCZ696 attenuates renal fibrosis via ASK1/JNK/p38 MAPK-mediated apoptosis in unilateral ureteral obstruction.

The angiotensin receptor neprilysin inhibitor LCZ696 affords superior cardioprotection and renoprotection compared with renin-angiotensin blockade monotherapy, but the underlying mechanisms remain elusive. Herein, we evaluated whether LCZ696 attenuates renal fibrosis by inhibiting ASK1/JNK/p38 mitogen-activated protein kinase (MAPK)-mediated apoptosis in a rat model of unilateral ureteral obstruction (UUO) and in vitro. Rats with UUO were treated daily for 7 days with LCZ696, valsartan, or the selective ATP competitive inhibitor of apoptosis signal-regulating kinase 1 (ASK1), GS-444217. The effects of LCZ696 on renal injury were examined by assessing the histopathology, oxidative stress, intracellular organelles, apoptotic cell death, and MAPK pathways. H2O2-exposed human kidney 2 (HK-2) cells were also examined. LCZ696 and valsartan treatment significantly attenuated renal fibrosis caused by UUO, and this was paralleled by downregulation of proinflammatory cytokines and decreased inflammatory cell influx. Intriguingly, LCZ696 had stronger effects on renal fibrosis and inflammation than valsartan. UUO-induced oxidative stress triggered mitochondrial destruction and endoplasmic reticulum stress, which resulted in apoptotic cell death; these effects were reversed by LCZ696. Both GS-444217 and LCZ696 hampered the expression of death-associated ASK1/JNK/p38 MAPKs. In H2O2-treated HK-2 cells, LCZ696 and GS-444217 increased cell viability but decreased the production of intracellular reactive oxygen species and MitoSOX and apoptotic cell death. Both agents also deactivated H2O2-stimulated activation of ASK1/JNK/p38 MAPKs. These findings suggest that LCZ696 protects against UUO-induced renal fibrosis by inhibiting ASK1/JNK/p38 MAPK-mediated apoptosis.

Inhibition of RIP1-RIP3-mediated necroptosis attenuates renal fibrosis via Wnt3α/ß-catenin/GSK-3ß signaling in unilateral ureteral obstruction.

Renal fibrosis represents the final common outcome of chronic kidney disease of virtually any etiology. However, the mechanism underlying the evolution of renal fibrosis remains to be addressed. This study sought to clarify whether RIP1-RIP3-mediated necroptosis is involved in renal fibrosis via Wnt3α/ß-catenin/GSK-3ß signaling in vitro and in a rat model of unilateral ureteral obstruction (UUO). Rats with UUO were administered RIP inhibitors (necrostatin-1 or GSK872) or ß-catenin/TCF inhibitor ICG-001 daily for 7 consecutive days. UUO caused significant renal tubular necrosis and overexpression of RIP1-RIP3-MLKL axis proteins, and was accompanied by activation of the NLRP3 inflammasome and renal fibrosis. Oxidative stress caused by UUO was closely associated with endoplasmic reticulum stress and mitochondrial dysfunction, which resulted in apoptotic cell death via Wnt3α/ß-catenin/GSK-3ß signaling. All of these effects were abolished by an RIP inhibitor (necrostatin-1 or GSK872) or ICG-001. In H2O2-treated HK-2 cells, both RIP inhibitor and ICG-001 decreased intracellular reactive oxygen species production and apoptotic cells, but increased cell viability. Activated Wnt3α/ß-catenin/GSK-3ß signaling was decreased by either RIP inhibitor or ICG-001. Our findings suggest that RIP1-RIP3-mediated necroptosis contributes to the development of renal fibrosis via Wnt3α/ß-catenin/GSK-3ß signaling in UUO and may be a therapeutic target for protection against renal scarring of other origins.

Coenzyme Q10 attenuates renal fibrosis by inhibiting RIP1-RIP3-MLKL-mediated necroinflammation via Wnt3α/ß-catenin/GSK-3ß signaling in unilateral ureteral obstruction.

OBJECTIVE: Coenzyme Q10 (CoQ10) protects against various types of injury, but its role in preventing renal scarring in chronic kidney disease remains an open question. Herein, we evaluated whether CoQ10 attenuates renal fibrosis by interfering with necroinflammation in a rat model of unilateral ureteral obstruction (UUO) and in vitro. METHODS: Rats with UUO were treated daily with CoQ10 or an RIP inhibitor (necrostatin-1 or GSK872) for 7 days. The influence of CoQ10 on renal injury caused by UUO was evaluated by histopathology and analysis of gene expression, oxidative stress, intracellular organelles, apoptosis, and Wnt3α/ß-catenin/GSK-3ß signaling·H2O2-exposed human kidney (HK-2) cells were also examined after treatment with CoQ10 or an RIP inhibitor. RESULTS: UUO induced marked renal tubular necrosis, upregulation of RIP1-RIP3-MLKL axis proteins, activation of the NLRP3 inflammasome, and evolution of renal fibrosis. UUO-induced oxidative stress evoked excessive endoplasmic reticulum stress and mitochondrial dysfunction, which triggered apoptotic cell death through Wnt3α/ß-catenin/GSK-3ß signaling. All of these effects were mitigated by CoQ10 or an RIP inhibitor. In H2O2-treated HK-2 cells, CoQ10 or an RIP inhibitor suppressed the expression of RIP1-RIP3-MLKL proteins and pyroptosis-related cytokines, and hindered the production of intracellular reactive oxygen species as shown by MitoSOX Red staining and apoptotic cell death but increased cell viability. The CoQ10 or Wnt/ß-catenin inhibitor ICG-001 deactivated H2O2-stimulated activation of Wnt3α/ß-catenin/GSK-3ß signaling. CONCLUSION: These findings suggest that CoQ10 attenuates renal fibrosis by inhibiting RIP1-RIP3-MLKL-mediated necroinflammation via Wnt3α/ß-catenin/GSK-3ß signaling in UUO.

Nicotine exacerbates tacrolimus-induced renal injury by programmed cell death.

BACKGROUND/AIMS: Cigarette smoking is an important modifiable risk factor in kidney disease progression. However, the underlying mechanisms for this are lacking. This study aimed to assess whether nicotine (NIC), a major toxic component of cigarette smoking, would exacerbates tacrolimus (TAC)-induced renal injury. METHODS: Sprague-Dawley rats were treated daily with NIC, TAC, or both drugs for 4 weeks. The influence of NIC on TAC-caused renal injury was examined via renal function, histopathology, oxidative stress, mitochondria, endoplasmic reticulum (ER) stress, and programmed cell death (apoptosis and autophagy). RESULTS: Both NIC and TAC significantly impaired renal function and histopathology, while combined NIC and TAC treatment aggravated these parameters beyond the effects of either alone. Increased oxidative stress, ER stress, mitochondrial dysfunction, proinf lammatory and profibrotic cytokine expressions, and programmed cell death from either NIC or TAC were also aggravated by the two combined. CONCLUSION: Our observations suggest that NIC exacerbates chronic TAC nephrotoxicity, implying that smoking cessation may be beneficial for transplant smokers taking TAC.

Dapagliflozin Alleviates Renal Fibrosis by Inhibiting RIP1-RIP3-MLKL-Mediated Necroinflammation in Unilateral Ureteral Obstruction.

Dapagliflozin, a sodium-glucose cotransporter-2 inhibitor, offers renoprotection in diabetes. However, potential for use in nondiabetic kidney disease remains unknown. Herein, we assessed whether dapagliflozin alleviates renal fibrosis by interfering with necroinflammation in a rat model of unilateral ureteral obstruction (UUO) and in vitro. After induction of UUO, rats were administered dapagliflozin daily for seven consecutive days. UUO induced significant renal tubular necrosis and overexpression of RIP1-RIP3-MLKL axis proteins; these coincided with NLRP3 inflammasome activation, and subsequent development of renal fibrosis. Oxidative stress caused by UUO is tightly associated with endoplasmic reticulum stress and mitochondrial dysfunction, leading to apoptotic cell death through Wnt3α/ß-catenin/GSK-3ß signaling; all of which were abolished by both dapagliflozin and specific RIP inhibitors (necrostatin-1 and GSK872). In H2O2-treated HK-2 cells, dapagliflozin and RIP inhibitors suppressed overexpression of RIP1-RIP3-MLKL proteins and pyroptosis-related cytokines, decreased intracellular reactive oxygen species production and apoptotic cell death, whereas cell viability was improved. Moreover, activated Wnt3α/ß-catenin/GSK-3ß signaling was inhibited by dapagliflozin and Wnt/ß-catenin inhibitor ICG-001. Our findings suggest that dapagliflozin ameliorates renal fibrosis by inhibiting RIP1-RIP3-MLKL-mediated necroinflammation via Wnt3α/ß-catenin/GSK-3ß signaling in UUO.

L-Carnitine protects against tacrolimus-induced renal injury by attenuating programmed cell death via PI3K/AKT/PTEN signaling.

Reducing immunosuppressant-related complications using conventional drugs is an efficient therapeutic strategy. L-carnitine (LC) has been shown to protect against various types of renal injury. In this study, we investigated the renoprotective effects of LC in a rat model of chronic tacrolimus (TAC) nephropathy. SD rats were injected with TAC (1.5 mg · kg-1 · d-1, sc) for 4 weeks. Renoprotective effects of LC were assessed in terms of renal function, histopathology, oxidative stress, expression of inflammatory and fibrotic cytokines, programmed cell death (pyroptosis, apoptosis, and autophagy), mitochondrial function, and PI3K/AKT/PTEN signaling. Chronic TAC nephropathy was characterized by severe renal dysfunction and typical histological features of chronic nephropathy. At a molecular level, TAC markedly increased the expression of inflammatory and fibrotic cytokines in the kidney, induced oxidative stress, and led to mitochondrial dysfunction and programmed cell death through activation of PI3K/AKT and inhibition of PTEN. Coadministration of LC (200 mg · kg-1 · d-1, ip) caused a prominent improvement in renal function and ameliorated histological changes of kidneys in TAC-treated rats. Furthermore, LC exerted anti-inflammatory and antioxidant effects, prevented mitochondrial dysfunction, and modulated the expression of a series of apoptosis- and autophagy-controlling genes to promote cell survival. Human kidney proximal tubular epithelial cells (HK-2 cells) were treated with TAC (50 µg/mL) in vitro, which induced production of intracellular reactive oxygen species and expression of an array of genes controlling programmed cell death (pyroptosis, apoptosis, and autophagy) through interfering with PI3K/AKT/PTEN signaling. The harmful responses of HK-2 cells to TAC were significantly attenuated by cotreatment with LC and the PI3K inhibitor LY294002 (25 µM). In conclusion, LC treatment protects against chronic TAC nephropathy through interfering the PI3K/AKT/PTEN signaling.

L-carnitine treatment attenuates renal tubulointerstitial fibrosis induced by unilateral ureteral obstruction.

BACKGROUND/AIMS: Accumulating evidence indicates that L-carnitine (LC) protects against multiorgan damage through its antioxidant properties and preservation of the mitochondria. Little information is available about the effects of LC on renal fibrosis. This study examined whether LC treatment would provide renoprotection in a rat model of unilateral ureteral obstruction (UUO) and in vitro. METHODS: Sprague-Dawley rats that underwent UUO were treated daily with LC for 7 or 14 days. The influence of LC on renal injury caused by UUO was evaluated by histopathology, and analysis of gene expression, oxidative stress, mitochondrial function, programmed cell death, and phosphatidylinositol 3-kinase (PI3K)/ AKT/forkhead box protein O 1a (FoxO1a) signaling. In addition, H2O2-exposed human kidney cells (HK-2) were treated with LC. RESULTS: LC treatment inhibited expression of proinflammatory and profibrotic cytokines, and was followed by a significant attenuation of tubulointerstitial inflammation and fibrosis. The increased oxidative stress caused by UUO was associated with mitochondrial dysfunction and excessive apoptosis and autophagy via PI3K/AKT/FoxO1a-dependent signaling, and this was abrogated by administration of LC. In H2O2-exposed HK-2 cells, LC decreased intracellular production of reactive oxygen species, and suppressed expression of profibrotic cytokines and reduced the number of apoptotic cells. CONCLUSION: LC protects against the progression of tubulointerstitial fibrosis in an obstructed kidney.

Exogenous pancreatic kininogenase protects against renal fibrosis in rat model of unilateral ureteral obstruction.

Tissue kallikrein has protective function against various types of injury. In this study, we investigated whether exogenous pancreatic kininogenase (PK) conferred renoprotection in a rat model of unilateral ureteral obstruction (UUO) and H2O2-treated HK-2 cells in vitro. SD rats were subjected to UUO surgery, then PK (7.2 U/g per day, ip) was administered for 7 or 14 days. After the treatment, rats were euthanized; the obstructed kidneys were harvested for further examination. We found that PK administration significantly attenuated interstitial inflammation and fibrosis, and downregulated the expression of proinflammatory (MCP-1, TLR-2, and OPN) and profibrotic (TGF-ß1 and CTGF) cytokines in obstructed kidney. UUO-induced oxidative stress, closely associated with excessive apoptotic cell death and autophagy via PI3K/AKT/FoxO1a signaling, which were abolished by PK administration. We further showed that PK administration increased the expression of bradykinin receptors 1 and 2 (B1R and B2R) mRNA and the production of NO and cAMP in kidney tissues. Coadministration with either B1R antagonist (des-Arg9-[Leu8]-bradykinin) or B2R antagonist (icatibant) abrogated the renoprotective effects of PK, and reduced the levels of NO and cAMP in obstructed kidney. In H2O2-treated HK-2 cells, addition of PK (6 pg/mL) significantly decreased ROS production, regulated the expression of oxidant and antioxidant enzymes, suppressed the expression of TGF-ß1 and MCP-1, and inhibited cell apoptosis. Our data demonstrate that PK treatment protects against the progression of renal fibrosis in obstructed kidneys.

Shen-Kang protects against tacrolimus-induced renal injury.

BACKGROUND/AIMS: Evidence suggests that Shen-Kang (SK), a traditional Chinese herbal medicine, protects against various types of renal injury. In this study, we evaluated whether SK treatment confers renoprotection in a rat model of chronic tacrolimus (TAC) nephropathy. METHODS: Rats were treated daily with TAC (1.5mg/kg, subcutaneously) and SK (450 mg/kg, intravenously) for 4 weeks. The effects of SK on TAC-induced renal injury were assessed by measuring renal function, urine albumin excretion, histopathology, inflammatory cell infiltration, expression of profibrotic (transforming growth factor ß1 [TGF-ß1] and TGF-ß inducible gene-h3 [ßig-h3]) and proinflammatory cytokines, oxidative stress, and apoptotic cell death. RESULTS: Administration of SK preserved glomerular integrity (fractional mesangial area and Wilms tumor 1-positive glomeruli), attenuated tubulointerstitial fibrosis, and reduced the number of ectodermal dysplasia 1-positive cells, and this was paralleled by improved urine albumin excretion and renal dysfunction. At the molecular level, SK treatment suppressed expression of TGF-ß1/Smad2/3, ßig-h3, and proinflammatory cytokines. Oxidative stress and apoptotic cell death were significantly decreased with SK treatment, and apoptosis-related genes were regulated toward cell survival (active caspase-3 and the B-cell lymphoma-2/Bcl2-associated X [Bcl-2/Bax] ratio). CONCLUSION: SK protects against TAC-induced renal injury.

Expression of brain-derived neurotrophic factor in kidneys from normal and cyclosporine-treated rats.

BACKGROUND: Accumulating evidence suggests that a decrease in brain-derived neurotrophic factor (BDNF) level induces a variety of psychiatric and neurological disorders. However, the expression and role of BDNF in the kidney have not been explored. The present study examined the expression of BDNF and tropomyosin-related kinase (Trk) receptors in an experimental model of chronic cyclosporine A (CsA) nephropathy. METHODS: Sprague-Dawley rats on a salt-deplete diet were treated daily for four weeks with vehicle or CsA. Urine profiles, apoptotic cell death, oxidative stress (8-hydroxy-2'-deoxyguanosine, 8-OHdG), and expression of BDNF and Trk receptors (TrkB and TrkC) were compared between groups. The impact of vasopressin infusion on the urine-concentrating ability was examined by measuring the expression of aquaporin-2 (AQP-2) and BDNF and urine profiles in normal and CsA-treated rats. RESULTS: Compared with the vehicle-treated rats, rats given CsA had enhanced urine volume and declined urine osmolality. Immunohistochemistry and immunoblotting showed that BDNF and Trk receptors were constitutively expressed in kidneys from vehicle-treated rats. This was confirmed by double immunofluorescent staining for Na-K-ATPase-α1, AQP-1, and AQP-2. By contrast, the expression of these factors decreased in kidneys from CsA-treated rats (BDNF: 51.1 ± 19.5% vs. 102.0 ± 30.3%, p < 0.01). Downregulation of BDNF was accompanied by impairment of urine osmolality, and this was reversed by exogenous infusion of vasopressin. Notably, the number of TUNEL-positive cells correlated negatively with BDNF expression and positively with urinary 8-OHdG excretion. CONCLUSIONS: BDNF is expressed in the collecting duct of the kidney and may be associated with urine-concentrating ability in an experimental model of chronic CsA-induced nephropathy. Our study provides a new avenue for further investigation of chronic CsA nephropathy.

Role of dipeptidyl peptidase-4 inhibitors in new-onset diabetes after transplantation.

Despite strict pre- and post-transplantation screening, the incidence of new-onset diabetes after transplantation (NODAT) remains as high as 60%. This complication affects the risk of cardiovascular events and patient and graft survival rates. Thus, reducing the impact of NODAT could improve overall transplant success. The pathogenesis of NODAT is multifactorial, and both modifiable and nonmodifiable risk factors have been implicated. Monitoring and controlling the blood glucose profile, implementing multidisciplinary care, performing lifestyle modifications, using a modified immunosuppressive regimen, administering anti-metabolite agents, and taking a conventional antidiabetic approach may diminish the incidence of NODAT. In addition to these preventive strategies, inhibition of dipeptidyl peptidase-4 (DPP4) by the gliptin family of drugs has recently gained considerable interest as therapy for type 2 diabetes mellitus and NODAT. This review focuses on the role of DPP4 inhibitors and discusses recent literature regarding management of NODAT.

Synergistic effects of leflunomide and benazepril in streptozotocin-induced diabetic nephropathy.

BACKGROUND: Leflunomide (LEF) and benazepril have renoprotective effects on diabetic nephropathy (DN) through their anti-inflammatory and anti-fibrotic activities. This study investigated whether combined treatment using LEF and benazepril affords superior protection compared with the respective monotherapies. METHODS: Diabetes was induced with streptozotocin (STZ, 65 mg/kg) by intraperitoneal injection in male Wistar rats. Two weeks after STZ injection, diabetic rats were treated daily for 12 weeks with LEF (10 mg/kg), benazepril (10 mg/kg), or a combination of both. Basic parameters (body weight, fasting blood glucose level, and 24 h urinary protein excretion), histopathology, inflammatory [inflammatory cell infiltration (ED-1), monocyte chemoattractant protein-1 (MCP-1), and Toll-like receptor-2 (TLR-2)] and glomerulosclerotic factors [transforming growth factor-ß1 (TGF-ß1) and connective tissue growth factor (CTGF)], and oxidative stress (8-hydroxy-2'-deoxyguanosine, 8-OHdG) were studied. RESULTS: Benazepril or LEF treatment significantly prevented body weight loss and 24 h urinary protein excretion induced by diabetes; combined treatment with LEF and benazepril further improved these parameters compared with giving each drug alone (all p < 0.01). Increased expression of inflammatory (MCP-1 and TLR-2) and glomerulosclerotic (TGF-ß1 and CTGF) factors in diabetic rat kidney was reduced by treatment with either LEF or benazepril and was further reduced by the combined administration of the two drugs (p < 0.01). These effects were accompanied by suppression of urinary 8-OHdG excretion. There was no significant between-group difference in blood glucose level. CONCLUSIONS: LEF treatment lessens DN, and combined treatment with LEF and benazepril provides synergistic effects in preventing DN.

The protective role of uteroglobin through the modulation of tissue transglutaminase in the experimental crescentic glomerulonephritis.

BACKGROUND AND METHODS: Tissue transglutaminase (tTG) may induce pro-inflammatory cytokines and produce irreversible end-products, thus promoting renal scarring. It has recently been confirmed that the crescent formation in murine experimental crescentic glomerulonephritis (ecGN) has been inhibited by the administration of recombinant uteroglobin (rUG). However, the ability of UG on tTG modulation has not been thoroughly assessed. In this study, we investigated the feasible protective role of UG in murine ecGN through the modulation of tTG and TGF-beta1 expressions. ecGN was induced by the administration of anti-GBM Ab into C57BL/6 mice. RESULTS: Both proteinuria and BUN levels were distinctively lower in rUG-treated mice compared to those of disease control mice. Glomerular injuries such as mesangial proliferation, matrix production and crescent formation were lessened with the rUG treatment, and these findings were parallel with the attenuated expression of tTG and TGF-beta1. tTG and TGF-beta1 were expressed mainly on mesangial areas by the induction of ecGN and rUG treatment markedly attenuated the expressions of these proteins in glomeruli without spatial changes. With the addition of LPS to mesangial cells, the expressions of tTG and TGF-beta1 were up-regulated, whilst the addition of cysteamine, tTG inhibitor, attenuated the expression of tTG and TGF-beta1 as well as the cellular proliferation which was further induced by LPS. CONCLUSION: We demonstrate for the first time that rUG is able to attenuate the renal injury through the modulation of expressions of tTG and TGF-beta1 in ecGN and further suggest a wide range of feasible molecular targets to reduce the severity of human glomerulonephritis.

Role of NKT cells in allogeneic islet graft survival.

Although NKT cells expressing CD1d-reactive TCR exerted protective role in autoimmune diseases, the regulatory function of CD1d-dependent NKT cells in alloimmune responses has not been investigated thoroughly. Here, we demonstrated the regulatory effects of NKT cells using a pancreas islet transplantation model. CD40/CD154 blocking induced long-term graft survival in most B6 recipients, but B6.CD1d(-/-) recipients showed co-stimulation blockade-resistant rejection. Adoptive transfer of NKT cells into B6.CD1d(-/-) restored tolerizing capacity of co-stimulatory blockade. Activation of NKT cells was effective for the prolongation of graft survival and up-regulated membrane-bound TGF-beta expression transiently on their cell surface. The activated CD1d-dependent NKT cells inhibited alloantigen-driven cell proliferation through cell contacts and the beneficial effect of CD154 blocking for allograft survival was related to TGF-beta pathway. Thus, we can conclude that NKT cells are essential for the stable allograft survival and the regulatory function is dependent on, at least in part, TGF-beta engagement.

The Differential Effects of Donor-Strain Immature Dendritic Cell Subtypes and the Blockade of CD40/CD154 Pathway on the Creation of Allograft Tolerance / 대한신장학회잡지

BACKGOUND: Dendritic cells (DC) are pivotal antigen presenting cells and serve a unique role in initiating immunity. To test the hypothesis that pre-immunization of recipient with certain DC subsets of donor origin can influence graft outcome. METHODS: We have studied the effects of immunization with allogeneic CD4+CD11c+(MDC) and CD8+CD11c+(LDC) DCs on the allograft response. RESULTS: Both immature MDC and LDC subsets from DBA/2 were able to prime naive allogeneic C57BL/6 (B6) T cells in MLR. In vitro allogeneic T cell responses were attenuated by the addition of anti-CD154 mAb into the culture. T cells from B6 mice that received DBA/2 MDC intravenously 4 weeks before testing mounted weaker MLR driven cell proliferation than T cells from LDC pretreated B6 mice. Consistent with the MLR results, combined pretreatment with MDC, but not LDC, plus anti-CD154 mAb produced donor-strain specific long-term graft survival and induced tolerance while treatment with LDC plus anti-CD154 mAb created minimal prolongation of allograft survival in a pancreas islet transplant model (DBA/2->B6). The beneficial effects exerted by MDC and anti-CD154 mAb pretreatment were correlated with TH1 to TH2 immune deviation and with the amplified donor-specific suppressive capacity by recipient CD4+CD25+T cells. CONCLUSION: These findings highlight the capacity of MDC to modulate alloimmune responses, and suggest therapeutic approaches for the induction of donor specific tolerance.