移植物转染反义细胞外信号调节激酶-2基因减轻慢性移植物血管病

目的 研究转染反义ERK2基因对慢性移植物血管病的抑制作用及其机制.方法 构建含有反义ERK2基因和带有LacZ基因的腺病毒载体.以BN大鼠为供者,Lewis大鼠为受者,制作大鼠腹主动脉移植模型.ERK2组接受转染反义ERK2基因的腹主动脉移植,LacZ组接受转染LacZ基因的腹主动脉移植,对照组接受不进行处理的腹主动脉移植.移植后60 d,取各组移植血管和血清样本,以2,3-二羟基丙醛-3-磷酸脱氢酶(GAPDH)为内参照,计算ERK2条带灰度与GAPDH条带灰度的比值(ERK2/GAPDH,即为ERK2的相对表达强度);测量移植血管内膜厚度(I)和中膜厚度(M),计算I/(I+M)之百分比;检测移植血管平滑肌细胞(VSMC)的数目和分布;检测血清血小板源性生长因子-BB(PDGF-BB)的浓度.结果 对照组、LacZ组和ERK2组ERK2/GAPDH的比值分别为1.21±0.15、1.02±0.06和0.47±0.09,后者显著低于前二者(P<0.05).对照组和LacZ组移植血管呈典型的移植物血管病表现,两组I/(I+M)比值分别为84.1%和79.9%;ERK2组移植血管呈移植动脉内膜炎表现,该组I/(I+M)比值为13.7%,显著低于前两组(P<0.05).对照组和LacZ组内膜增厚处散在分布大量VSMC,计数分别为(71.3±9.2)个和(76.4±11.3)个;ERK2组内膜处VSMC计数为(34.8±5.3)个,明显少于前两组(P<0.05).对照组和LacZ组受者血清PDGF-BB浓度分别为(1075±70)pg/ml和(1200±25)pg/ml,ERK2组为(626±27)pg/ml,ERK2组明显低于对照组和LacZ组(P<0.05).结论 转染反义ERK2基因可减轻慢性移植物血管病,并延缓其进展,其机制可能与VSMC增殖和迁移受到抑制以及PDGF-BB表达下调有关.     

大鼠供肾转染反义ERK2基因减缓肾移植后慢性移植肾肾病的作用及机制

目的 研究腺病毒介导的反义ERK2(A-dant-ERl(2)基因转染供肾对减缓肾移植后发生慢性移植肾肾病(CAN)的作用及机制.方法 建立大鼠间肾移植模型.按供肾移植前处理方式的不同分为对照组、空载病毒组和基因转染组,每组6只.对照组供肾灌注无菌HTK液0.5 ml,空载病毒组供肾灌注含5x109>pfu LaeZ基因腺病毒(Ad-LacZ)的HTK液0.5 ml,基因转染组供肾灌注含5x 109>pfu Adanti-ERK2的HTK液0.5 ml.肾移植术后24周行移植肾的病理学观察,免疫组织化学法观察肾小管上皮细胞表面标志蛋白E-Cadherin、Vimentin、TβRⅠ的表达以及CD4+、CD8+T淋巴细胞和ED-1+细胞的浸润情况;酶联免疫法检测受者血清中转化生长因子β1>(TGF-β1>)的含量.结果 肾移植术后24周,对照组和空载病毒组移植肾呈CAN表现;肾小球硬化,肾小管萎缩明显,伴严重间质纤维化以及明显的CD4+、CD8+T淋巴细胞和ED-1+细胞浸润;病变区肾小管上皮细胞E-Cadherin表达减少,Vimentin、TβRⅠ表达显著增多.基因转染组移植肾间质内仅有少量CD4+、CD8+T淋巴细胞和ED-1+细胞浸润;肾小管上皮细胞E-Cadherin表达正常.对照组和空载病毒组血清中TGF-β1>,含量明显高于基因转染组.结论 Adanti-ERK2基因转染移植肾可减缓CAN的发生,对移植肾具有保护作用.这种保护机制可能与减少炎症细胞的浸润、下调TGF-β1>,等致纤维化因子的表达以及抑制肾小管上皮细胞向间充质细胞的转化有关.     

Antisense ERK1/2 oligodeoxynucleotide gene therapy attenuates graft arteriosclerosis of aortic transplant in a rat model

Chronic rejection is a major cause of transplant loss that is effected by the extracellular signal-regulated kinases (ERK) pathway. This study investigated the effects of antisense ERK1/2 oligodeoxynucleotide(ODN) gene therapy on chronic rejection. METHODS: Lewis (RT1(1)) rats served as recipients of Brown-Norway (BN, RT1n) grafts. The BN rat abdominal aortas were harvested and orthotopically grafted into Lewis rats. The recipients were divided into three groups: (1) control group (n = 9), (2) random ODN transfer group (n = 10), and (3) antisense ODN transfer group (n = 10). At day 60 after transplantation, the recipients were sacrificed; the grafted aortas were evaluated histologically and immunohistochemically. ERK1/2 protein expression in the grafts was determined using Western Blot assays. Serum levels of slCAM-1 were detected by ELISA. RESULTS: In the control group and random ODN transfer group, we observed a remarkable degree of intimal hyperplasia and inflammatory cell infiltration, including macrophages and T cells. Compared with the control group, antisense ERK1/2 ODN gene therapy resulted in a significant reduction in neointimal proliferation (P < .01), inhibition of ERK1/2 protein expression (P < .01), decreased graft infiltration with CD4+ T lymphocytes (P < .01), CD8+ T lymphocytes(P < .05), and ED-1 macrophages (P < .01) with decreased serum levels of sICAM-1 (P < .05). We obtained a negative correlation between ERK1/2 expression and immune cell infiltration or ICAM-1 level. CONCLUSIONS: Antisense ERK1/2 gene therapy can attenuate graft arteriosclerosis so as to protect aortic allografts. The protection seemed to correlate with inhibition of inflammatory infiltration, implying that the ERK1/2 signal transduction pathway plays an important role in the process of chronic vascular rejection.     

Antisense extracellular signal-regulated kinase-2 gene therapy inhibits platelet-derived growth factor-induced proliferation, migration and transforming growth factor-beta(1) expression in vascular smooth muscle cells and attenuates transplant vasculopathy.

Platelet-derived growth factor-BB (PDGF-BB) enables vascular smooth muscle cells (VSMCs) to proliferate, migrate and secrete connective tissue matrix, which are critical events in transplant vasculopathy. However, little is known about the intracellular pathways that mediate these biologic responses of VSMCs. Extracellular signal-regulated kinase (ERK) pathway plays a major role in cellular responses and vascular diseases. In this study, we observed that the inhibition of ERK2 activity by recombinant adenovirus encoding antisense ERK2 (Adanti-ERK2) significantly suppressed the proliferation, converting of cell cycle from G(1) phase to S phase and directed migration, and partially abrogated transforming growth factor-beta(1) (TGF-beta(1)) expression in VSMCs stimulated with PDGF-BB. Ex vivo gene transfer of Adanti-ERK2 into rat aortic allograft attenuated chronic transplant vasculopathy by the inhibition of VSMC proliferation and migration. In conclusion, ERK2 is involved in PDGF-BB-induced VSMCs proliferation, migration and TGF-beta(1) expression and may be a potential therapeutic target for transplant vasculopathy.     

Reduction of ICAM-1 and LFA-1-positive leukocytes in the perivascular space of arteries under mycophenolate mofetil therapy reduces rat heart transplant vasculopathy

BACKGROUND: The interaction of lymphocyte function-associated antigen-1 (LFA-1)-positive host leukocytes with intercellular adhesion molecule-1 (ICAM-1) on graft endothelium may play a key role in allograft recognition, triggering the development of transplant vasculopathy (TVP). We investigated the correlation between TVP and ICAM-1 expression and accumulation of LFA-1-positive leukocytes in the perivascular space (PVS) of arteries under different immunosuppressive drugs. METHODS: After cardiac transplantation (Lewis to Fisher) animals were randomized 4 groups: cyclosporine (CsA), 3 mg/kg/day (n=74); mycophenolate mofetil (MMF), 40 mg/kg/day (n=96); FK 506, 0.3 mg/kg/day (n=96); and control, no therapy (n=74). Three or 4 animals from each group were harvested at intervals of 1 to 4 days within the study period of 60 days. Using immunohistochemistry, LFA-1-positive leukocytes were analyzed in intra- and epicardial arteries. ICAM-1 expression was scored histologically. TVP was assessed by digitizing morphometry and expressed as mean vascular occlusion. RESULTS: Accumulation of LFA-1-positive leukocytes in the PVS of arteries and the myocardium correlated with expression of ICAM-1 on graft endothelium. The severity of TVP in arteries correlated with the accumulation of LFA-1-positive leukocytes in PVS. All immunosuppressive drugs significantly reduced ICAM-1 expression, LFA-1 accumulation and extent of TVP, compared with controls. In MMF-treated animals, we also found a significant reduction of ICAM-1 expression, LFA-1 accumulation and extent of TVP compared with the groups treated with CsA and FK 506 (p <0.005). CONCLUSION: These data support an essential role of LFA-1/ICAM-1 interaction in the genesis of TVP that may be abrogated, especially by the use of MMF.     

Influence of aspirin on early allograft thrombosis and chronic allograft nephropathy following renal transplantation

BACKGROUND: Primary thrombosis and chronic allograft nephropathy are important causes of early and late graft loss, respectively, following renal transplantation. This study examined the potential for aspirin therapy to reduce these complications. METHODS: A consecutive series of 105 cadaveric renal transplants treated with aspirin 150 mg daily for the first 3 months after transplantation was compared with an untreated historical control group (n = 121). Protocol needle-core biopsies were performed on all transplants in both groups at 1 week and 12 months after transplantation. Needle-core allograft biopsies were performed at 3, 6 and 12 months after transplantation, and serum creatinine was measured at each outpatient attendance for the duration of follow-up. RESULTS: There was a significantly lower rate of primary allograft thrombosis in patients treated with aspirin (none of 105) compared with that in the control group (six (5 per cent) of 121; P = 0.03). There were no differences in renal function or 2-year allograft survival between the two groups. Aspirin-treated patients had a lower incidence of chronic allograft nephropathy at 1 year than controls although this did not reach statistical significance (16 versus 26 per cent; P = 0.075). There were no major bleeding complications in either group in association with peptic ulcer disease or following renal transplant biopsy. CONCLUSION: Aspirin reduced the rate of early graft thrombosis of renal transplants in this series but did not improve renal function or graft survival. A trend towards a lower rate of chronic allograft nephropathy was noted with aspirin treatment. These findings require confirmation in a prospective randomized trial.     

Targeting of macrophage activity by adenovirus-mediated intragraft overexpression of TNFRp55-Ig,IL-12p40, and vIL-10 ameliorates adenovirus-mediated chronic graft injury,whereas stimulation of macrophages by overexpression of IFN-gamma accelerates chronic graft injury in a rat renal allograft model

Adenovirus (Ad)-mediated gene transfer of immunoregulatory molecules prevents acute allograft rejection. It is here analyzed for the first time whether this approach may prevent the development of chronic renal allograft injury in rats. Renal allografts (F344-->Lewis rat) were ex vivo transduced in group I with control Ad-construct, group II with three different therapeutic Ad-constructs expressing the immunoregulatory molecules vIL-10, TNFRp55-Ig, and IL-12p40, and group III with AdIFN-gamma. Group IV served as untreated controls. Control grafts (IV) showed increasing proteinuria during the 24-wk follow-up. Chronic graft injury was accelerated by Ad-control (I) and even more by AdIFN-gamma (III). All rats carrying the AdIFN-gamma-transduced grafts died within 12 to 13 wk by advanced chronic renal failure associated with strong immune cell infiltration and immune gene expression. By contrast, the Ad-therapy group II showed less inflammation and improved graft histology and function if compared with the groups I and III. Moreover, significantly less infiltrating ED-1(+) macrophages and an improved histologic score even if compared with untreated controls (IV) was observed. However, after disappearance of therapeutic gene expression, group II showed increasing proteinuria probably as result of late T cell activation to the Ad-encoded proteins. Ex vivo transduction of allografts with Ad-control or even more AdIFN-gamma expression promotes intragraft inflammation and chronic graft injury. Targeting macrophage activation by a cocktail of therapeutic genes improved the results. These data support the pathogenetic role of cytokines in chronic graft injury; however, they also show the limitations of the Ad-mediated gene transfer.     

Use of FTY 720 and ICAM-1 antisense oligonucleotides for attenuating chronic renal damage secondary to ischemia-reperfusion injury

INTRODUCTION: Chronic allograft nephropathy is the main cause of graft loss. Although many factors are involved in its development, ischemia-reperfusion injury has received increasing attention as a risk determinant. In a previous study of syngeneic renal transplantation and ischemia, we demonstrated a protective effect of acute damage by FTY 720 and antisense oligonucleotides of ICAM-1 (Oligos). The purpose of the current study was to evaluate the impact of these agents on the development of chronic graft damage in the same model. METHODS: Lewis rat were used as donors and recipients. The harvested left kidney was kept in Collins solution for 2 hours. Recipient animals received treatment with FTY 720 or Oligos or saline. At 12 and 36 weeks after transplantation, creatinine clearance, GFR, proteinuria, and arterial blood pressure were recorded. Tissue from some animals were submitted for histological studies and quantification of mRNA TGF-beta1. RESULTS: All groups showed decreased levels of GFR and creatinine clearence, but only the untreated animals showed significant deterioration compared to the pretransplant values (0.53 +/- 0.24 versus 0.21 +/- 0.24 at 36 weeks respectively; P < .05). Proteinuria was also significant in control animals at 36 weeks. Blood pressure showed a moderate increase in all groups. Histological analysis showed that treated animals had fewer signs of chronic damage according to the Banff score. All groups displayed slight increases in TGF-beta1 without differences among them. CONCLUSIONS: In this model the use of FTY or antisense oligonucleotides of ICAM-1 were associated with less functional and morphological evidence of chronic graft damage secondary to an ischemia-reperfusion injury.     

Study of mRNA growth factors in urinary cells of kidney transplant recipients as predictors of chronic allograft nephropathy

BACKGROUND: Chronic allograft nephropathy (CAN) is responsible for a significant proportion of graft loss. Current diagnostic methods for CAN are inadequate, and noninvasive assays for detecting allograft dysfunction/rejection and predicting long-term outcomes are a priority in transplantation. METHODS: Urine samples were collected from 48 kidney transplant recipients (KTR): 18 recipients with stable graft function (creatinine levels<2.0 mg/dL) and proteinuria of less than 500 mg/24 hr (Group 1); 18 recipients with stable graft function and proteinuria of greater than 500 mg/24 hr (Group 2); and 12 recipients with biopsy confirmed CAN. Urinary cell levels of transforming growth factor-beta1 (TGF-beta1) mRNA or epidermal growth factor (EGF) mRNA were measured using real-time quantitative PCR assay, and levels were correlated with renal allograft status. The integrity of RNA isolated from urine sediments was also assessed using the Agilent 2100 Bioanalyzer. RESULTS: Urinary cell TGF-beta1 mRNA levels were higher in the CAN group compared to Group 1 (P<0.0001) or Group 2 (P<0.0001). Urinary cell EGF mRNA levels were higher in Group 1 compared to Group 2 (P<0.0001) or the CAN group (P<0.0001). There were no significant differences in the urinary cell levels of EGF mRNA between patients with greater than 500 mg/24 hr proteinuria (Group 2) and the CAN group (P=0.75). CONCLUSION: These results demonstrate that urinary cell TGF-beta1 mRNA levels distinguish CAN patients from long-term transplant patients with stable renal function and varied levels of proteinuria. Urinary cells may be a good resource for the noninvasive diagnosis of CAN.     

Methoxyethyl-modified intercellular adhesion molecule-1 antisense phosphorothiateoligonucleotides inhibit allograft rejection, ischemic-reperfusion injury, and cyclosporine-induced nephrotoxicity

BACKGROUND: The addition of phosphorothioate (PS) groups to natural phosphodiester (PD) antisense oligodeoxynucleotides (oligo) prevents their in vivo hydrolysis by nucleases allowing an RNase-dependent elimination of targeted mRNA. To further improve oligo function 2'-methoxyethyl (ME) groups were attached to selected nucleotides at the 3'-end because ME groups block RNase activity. METHODS/RESULTS: ME modification of PS- or PD/PS-oligo targeting human intracellular adhesion molecule (ICAM)-1 mRNA significantly increased the degree and duration of the in vitro inhibitory effects without compromising selectivity and specificity. A 7-day intravenous or oral therapy with rat ME/PS-modified ICAM-1 antisense oligo extended the survivals of kidney allografts. In addition, ME/PS-modified ICAM-1 antisense oligo reduced ischemic-reperfusion injury in kidneys, as measured by glomerular filtration rate, creatinine levels, and infiltration with leukocytes. Finally, a 14-day treatment with cyclosporine (CsA)-induced nephrotoxicity in syngeneic kidney transplants correlated with both increased ICAM-1 protein expression and infiltration with leukocytes. Graft perfusion and treatment of recipients with ICAM-1 antisense ME/PS-oligo alleviated the nephrotoxic effect and decreased ICAM-1 expression and leukocyte infiltration. CONCLUSIONS: ME/PS-modified ICAM-1 antisense oligo is very effective in inhibiting the ICAM-1-dependent mechanism of graft infiltration and tissue damage involved in allograft rejection, ischemic-reperfusion injury, and CsA-induced nephrotoxicity.     

Proteinuria after conversion to sirolimus in renal transplant recipients

Sirolimus (SRL) is a new, potent immunosuppressive agent. More recently, proteinuria has been reported as a consequence of sirolimus therapy, although the mechanism has remained unclear. We retrospectively examined the records of 25 renal transplant patients, who developed or displayed increased proteinuria after SRL conversion. The patient cohort (14 men, 11 women) was treated with SRL as conversion therapy, due to chronic allograft nephropathy (CAN) (n = 15) neoplasia (n = 8); Kaposi's sarcoma, Four skin cancers, One intestinal tumors, One renal cell carsinom) or BK virus nephropathy (n = 2). SRL was started at a mean of 78 +/- 42 (15 to 163) months after transplantation. Mean follow-up on SRL therapy was 20 +/- 12 (6 to 43) months. Proteinuria increased from 0.445 (0 to 1.5) g/d before conversion to 3.2 g/dL (0.2 to 12) after conversion (P = 0.001). Before conversion 8 (32%) patients had no proteinuria, whereas afterwards all patients had proteinuria. In 28% of patients proteinuria remained unchanged, whereas it increased in 68% of patients. In 40% it increased by more than 100%. Twenty-eight percent of patients showed increased proteinuria to the nephrotic range. Biopsies performed in five patients revealed new pathological changes: One membranoproliferative glomerulopathy and interstitial nephritis. These patients showed persistently good graft function. Serum creatinine values did not change significantly: 1.98 +/- 0.8 mg/dL before SRL therapy and 2.53 +/- 1.9 mg/dL at last follow-up (P = .14). Five grafts were lost and the patients returned to dialysis. Five patients displayed CAN and Kaposi's sarcoma. Mean urinary protein of patients who returned to dialysis was 1.26 (0.5 to 3.5) g/d before and 4.7 (3 to 12) g/d after conversion (P = .01). Mean serum creatinine level before conversion was 2.21 mg/dL and thereafter, 4.93 mg/dL (P = .02). Heavy proteinuria was common after the use of SRL as rescue therapy for renal transplantation. Therefore, conversion should be considered for patients who have not developed advanced CAN and proteinuria. The possibility of de novo glomerular pathology under SRL treatment requires further investigation by renal biopsy.     

Increased urinary excretion of transforming growth factor-beta(1) in renal transplant recipients during cytomegalovirus infection

AIMS: Cytomegalovirus (CMV) is a suggested risk factor for chronic allograft nephropathy, and transforming growth factor-beta (TGF-beta) is a key fibrogenic molecule in this process. CMV has been shown to induce the expression of TGF-beta and several cytokines. We analyzed the impact of CMV on urinary excretion of TGF-beta, ICAM-1, TNF-alpha and correlated findings with biopsy histology. MATERIAL: Urine samples from 46 renal transplant recipients were available for the study. Urine samples were taken when CMV infection was suspected, or for controlling of proteinuria or bacteriuria. METHOD: CMV was diagnosed by antigenemia and viral cultures. Patients with previous CMV infection were excluded from the analysis. Urine samples were analyzed by ELISA-method to detect the levels of TNF-alpha, ICAM-1 and TGF-beta(1). Banff '97 criteria were used for scoring of protocol biopsies taken 6 months after transplantation. RESULTS: At the time of the urine collection, 13/46 patients had CMV infection. Eight patients with no CMV infection were used as controls. TGF-beta(1) was significantly increased in the CMV group (samples taken mean 137+/-79 days post-transplantation) compared to controls (samples 139+/-64 days post-transplantation) (51.1+/-28.0 vs. 13.3+/-6.7 ng/mmol crea, p<0.001). No differences in the levels of other molecules were recorded. In the biopsies, interstitial fibrosis was significantly increased in the CMV group compared to controls. CONCLUSIONS: Urinary excretion of TGF-beta(1) was increased in patients during CMV infection. This was associated with increased fibrosis in the biopsies.     

Effect of histological damage on long-term kidney transplant outcome

BACKGROUND: Chronic renal allograft failure remains a major challenge to overcome. Factors such as donor quality, delayed graft function (DGF), acute rejection, and immunosuppression are known to affect long-term outcome, but their relationship to histological damage to graft outcome is unclear. METHODS: Protocol kidney biopsies (n=112) obtained at 3 months after transplantation yielded 102 with adequate tissue. Histology was scored by the Banff schema, and compared with implantation biopsies (n=91), repeat 12-month histology (n=39), decline in serum creatinine and serial isotopic glomerular filtration rate, onset of chronic allograft nephropathy (CAN), and actuarial graft survival censored for death with a functioning graft. RESULTS: At a median follow-up of 9.3 years, 20 patients had graft failure and 26 died with a functioning graft. Banff chronic nephropathy was present in 24% of 3-month biopsies, and was predicted by microvascular disease in the donor, cold ischemia, DGF, and acute vascular rejection (P<0.001). Acute glomerulitis at 3 months correlated with segmental glomerulosclerosis at 12 months, subsequent recurrent glomerulonephritis, and graft failure (P<0.01). Subclinical rejection at 3 months occurred in 29% of biopsies, correlated with prior acute rejection and HLA mismatch, and led to chronic histological damage by 12 months (r=0.25-0.67, P<0.05-0.001). Subclinical rejection, arteriolar hyalinosis, and tubulitis present at 3 months had resolved by 12 months. The 10-year survival rates for Banff chronic nephropathy were 90.4% for grade 0, 81.0% grade 1, and 57.9% for grades 2 or greater (P<0.01). Early tubulointerstitial damage at 3 months profoundly influenced graft survival beyond 10 years. CAN was predicted by kidney ischemia, 3-month chronic intimal vascular thickening, tubular injury, proteinuria, and late rejection. Chronic fibrointimal thickening of the small arteries and chronic interstitial fibrosis at 3 months independently predicted graft loss and decline in renal function (P<0.05-0.001). CONCLUSIONS: Early transplant damage occurs in the tubulointerstitial compartment from preexisting donor kidney injury and discrete events such as vascular rejection and DGF. Subsequent chronic damage and graft failure reflect accumulated previous injury and chronic interstitial fibrosis, vascular impairment, subclinical rejection, and injury from late rejection. CAN may be conceptualized as the sequelae of incremental and cumulative damage to the transplanted kidney. The duration of graft survival is dependent and predicted by the quality of the transplanted donor kidney combined with the intensity, frequency, and irreversibility of these damaging insults.     

Rat cytomegalovirus infection in kidney allograft recipients is associated with increased expression of intracellular adhesion molecule-1 vascular adhesion molecule-1, and their ligands leukocyte function antigen-1 and very late antigen-4 in the graft

BACKGROUND: Cytomegalovirus (CMV) infection is suggested to be a risk factor for chronic rejection. We have recently shown that rat CMV (RCMV) increases the inflammatory response and accelerates chronic rejection in a model of rat kidney allograft. In this study, the early inflammatory response and time-related expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) and their ligands, leukocyte function antigen-1 (LFA-1) and very late antigen-4 (VLA-4), in the grafts were investigated in RCMV-infected rats and compared to noninfected rats developing chronic rejection. METHODS: Transplantations were performed in a rat strain combination of DA (RT1a)->BN (RT1n) receiving triple drug immunosuppression. One group of rats was infected with RCMV, and the other was left uninfected. The grafts were harvested at different time points after transplantation. The adhesion molecules, their ligands and activation markers, MHC class II antigens and interleukin-2-receptors (IL-2-R), were demonstrated by monoclonal antibodies and immunoperoxidase staining from frozen sections of the grafts. Graft histology was evaluated according to the Banff criteria. RESULTS: RCMV caused a significant, prolonged increase of VCAM-1 and ICAM-1 expression in the vascular endothelium compared to the noninfected grafts. Also, the number of cells expressing activation markers, LFA-1 and VLA-4 was significantly enhanced in these animals. Significantly enhanced histological changes of chronic rejection were seen in the RCMV-infected group. CONCLUSIONS: Prolonged, increased expression of ICAM-1 and VCAM-1, and increased numbers of inflammatory cells expressing their ligands in the CMV infected grafts, were associated with accelerated chronic allograft nephropathy.     

阻断ERK2信号转导通路对血小板源生长因子-BB诱导血管平滑肌细胞增殖、迁移和表达转化生长因子-β1的影响

目的 观察ERK2信号转导通路在血小板源生长因子(PDGF-BB)诱导血管平滑肌细胞(VSMC)增殖、迁移和表达转化生长因子(TGF)-β1中的作用.方法 将原代培养的大鼠VSMC分为4组:(1)对照组;(2)PDGF刺激组;(3)Ad-LacZ组;(4)Adanti-ERK2组.用Westernblot检测细胞磷酸化ERK2蛋白水平;噻唑蓝(MTT)比色法测定细胞增殖率;流式细胞仪检测细胞细胞周期;Boyden小室测定细胞的跨膜迁移能力;酶联免疫吸附试验(ELISA)法检测细胞培养液上清中TGF-β1的浓度.结果 Adanti-ERK2组和对照组细胞增殖率、S期细胞百分比及跨膜迁移细胞数目明显低于PDGF刺激组和Ad-LacZ组(细胞增殖率:2.75%、0.00%比64.45%、61.88%;s期细胞百分比:14.18%、13.58%比38.14%、32.99%;跨膜迁移细胞数:8.2±3.2、6.3±2.6比24.8±6.1、23.3±5.8,均P<0.05);(2)Adanti-ERK2组和对照组细胞培养上清液中TGF-β1含量明显低于PDGF刺激组和Ad-LacZ组(P<0.05);而Adanti-ERK2组明显高于对照组(P<0.05).结论 ERK2信号转导通路参与调控PDGF-BB诱导的VSMC增殖、迁移和基因表达.反义ERK2基因预先转染阻断ERK信号转导能显著抑制PDGF-BB刺激的VSMC增殖、迁移,阻断细胞周期由G1期进入S期,并且部分下调TGF-β1的合成分泌.     

Chronic renal allograft nephropathy

Chronic rejection/chronic allograft nephropathy is the most prevalent cause of renal graft loss after the first year post-transplant. Chronic rejection/chronic allograft nephropathy is characterized by a slow progressive deterioration of graft function, often in combination with proteinuria and hypertension. Both immunologic and non-immunologic factors play key roles in the pathogenesis of chronic allograft nephropathy. Acute rejection episodes are the most prevalent risk factor for chronic rejection. Many risk factors for chronic allograft nephropathy have been identified, such as delayed graft function, nephron-dosing mismatch, repeated acute rejection episodes, and pathologically severe rejection. However, the precise pathogenesis of chronic allograft nephropathy remains elusive. The differential diagnosis of immunologically mediated chronic rejection and chronic rejection caused by non-immunologic factors is usually not possible using clinical parameters. The histopathologic findings of chronic allograft nephropathy are progressive interstitial fibrosis and remodelling of the vascular wall, and these findings are nonspecific. However, typical chronic transplant glomerulopathy, which affects glomerular tufts, as well as the multilayering of the peritubular capillary basement membrane, are characteristic of immunologic chronic rejection. Furthermore, in long-surviving patient with an allograft treated with a potent immunosuppressive agent, a calcineurin inhibitor, two or more concomitant independent lesions often develop. Therefore, the term "chronic allograft nephropathy" may be clinically preferable to "chronic rejection" to describe the gradual decline in graft function months or years after transplantation, in the absence of a well defined mechanism of graft dysfunction. The most effective way to prevent chronic allograft nephropathy is to avoid any kind of graft damage via either immunologic or non-immunologic mechanisms. Received: April 1, 2000 / Accepted: May 2, 2000     

Increased expression of intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 in rat cardiac allografts

This study was designed to investigate the role of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) during chronic cardiac allograft rejection. Wistar rats were used as donors, and SD rats as recipients heterotopic cardiac transplants. Recipients pretreated with inoculation of donor splenocytes (SPC) followed by cyclophosphamide (CP) were divided into 4 groups: (A) untreated group (n = 18) without immunosuppression; (B) SPC plus CP-treated group (n = 18) that were euthanized at 15-120 days posttransplantation; (C) CsA-treated group (n = 18) euthanized at 2-3 months posttransplantation; and (D) tolerance group (n = 18) treated with SPC plus CP and monitored for at least 1 year posttransplantation. Cardiac allografts were harvested at various times for immunohistochemical studies performed to evaluate the expression of ICAM-1 and VCAM-1. Pretreatment of animals with SPC and CP induced long-term cardiac allograft survival. Immunohistochemical staining demonstrated a low level of ICAM-1 and VCAM-1 expression in cardiac allograft muscle and coronary arteries among Groups B and D. In contrast, the expressions of ICAM-1 and VCAM-1 in cardiac allografts of Groups A and C were significantly higher than those in Groups B and D. Our results suggested that the expression of ICAM-1 and VCAM-1 plays an important role during the development of chronic cardiac allograft rejection.     

Adenovirus-mediated anti-sense ERK2 gene therapy inhibits tubular epithelial-mesenchymal transition and ameliorates renal allograft fibrosis

Purpose

Epithelial-mesenchymal transition (EMT) plays an important role in progress of renal allograft fibrosis. The adenovirus-mediated anti-sense extracellular signal-regulated kinase 2 (Adanti-ERK2) gene therapy was used to block ERK signaling pathway, and its effect on EMT and renal allograft fibrosis both in vivo and in vitro was explored.

Methods

We first generated an in vitro EMT model by connective tissue growth factor (CTGF) stimulation in a HK-2 cell culture system, and then applied Adanti-ERK2 gene therapy on it. The transition of epithelial marker (E-cadherin) to mesenchymal markers (??-SMA, Vimentin) and the cell mobility function alteration were monitored for the observation of EMT progress. In vivo, a rat renal transplant model with Fisher-Lewis combination was employed and the Adanti-ERK2 gene therapy was given. The tubular EMT changes and pathology of allograft fibrosis were examined.

Results

In vitro, Adanti-ERK2 gene therapy inhibited CTGF-induced tubular EMT and attenuated the cell motility function induced by CTGF. In vivo, Adanti-ERK2 gene therapy attenuated tubular EMT, modulated the infiltration of macrophages and CD8⁺, CD4⁺T lymphocytes, and ameliorated fibrosis effectively in the renal allografts 24 weeks after transplantation.

Conclusions

Adanti-ERK2 gene therapy inhibits tubular EMT and attenuates renal allograft fibrosis. It is possible to develop promising molecular drug(s) in the future based on ERK signaling pathway.     

Renoprotective effects of the AGE-inhibitor pyridoxamine in experimental chronic allograft nephropathy in rats.

BACKGROUND: Advanced glycation end products (AGEs) are involved in diabetic nephropathy (DN). The AGE formation inhibitor pyridoxamine (PM) is renoprotective in DN and in normoglycaemic obese Zucker rats. In chronic allograft nephropathy (CAN), renal AGE accumulation occurs as well. METHODS: To investigate whether inhibition of AGE formation is renoprotective in CAN, we studied the Fisher 344 to Lewis (F-L) allograft rat model of experimental CAN. Fisher to Fisher (F-F) isografts served as controls. Proteinuria, renal function and renal histology of untreated transplanted rats (F-L n = 8, F-F n = 8) were compared to rats receiving PM 2 g/l in drinking water for 20 weeks starting at transplantation (F-L n = 5, F-F n = 10). All rats received cyclosporin A (1.5 mg/kg/day) for 10 days after transplantation to prevent early acute rejection. RESULTS: Compared to untreated allografts, PM significantly decreased proteinuria (76 +/- 18 vs 29 +/- 3 mg/day), serum creatinine (130 +/- 12 vs 98 +/- 5 micromol/l), focal glomerulosclerosis (116 +/- 27 vs 16 +/- 5 AU), glomerular macrophage influx (5.6 +/- 0.6 vs 3.3 +/- 1.0), interstitial fibrosis (132 +/- 24 vs 76 +/- 2 AU) and interstitial macrophage influx (47.0 +/- 8.7 vs 15.4 +/- 5.0. Moreover, PM significantly ameliorated tubular accumulation of pentosidine, compared to untreated allografts (2.5 +/- 0.6 vs 0.3 +/- 0.3, all p < 0.05). In the isograft controls, these values did not differ between untreated and PM treated rats. CONCLUSION: PM exerts renoprotective effects and decreases renal pentosidine accumulation in experimental CAN, suggesting a detrimental role for renal AGE accumulation in the pathogenesis of renal damage in this non-diabetic model. These results indicate that inhibition of AGE formation might be a useful adjunct therapy to attenuate CAN.