Renal Allograft Rejection In Rats Research Articles

Prevention of chronic rejection by pravastatin in a rat kidney transplant model.

Pravastatin when administered with cyclosporine (CsA) has been shown to ameliorate transplant vasculopathy in the clinical setting. Previously we showed that pravastatin prevents chronic rejection in rat cardiac and liver transplant models. Here we determine whether pravastatin prevents chronic rejection in a rat renal allograft model. Orthotopic renal transplantations were performed using Fisher 344 rats as donors and Lewis rats as recipients. Recipients were treated with low-dose CsA for 10 days to prevent acute rejection. Recipients were divided into three groups: CsA, CsA + pravastatin, and syngeneic. Renal function was assessed by serum creatinine level at day 130. Allografts were evaluated by histology and immunohistochemistry. Serum levels of alloantibodies were measured by flow cytometry. Intragraft cytokine mRNA expression was determined by semiquantitative reverse transcriptase-polymerase chain reaction. Intragraft levels of the antiapoptotic Bag-1 gene were measured by Western blot. Unlike allografts from the pravastatin group, control allografts demonstrated glomerulosclerosis, vascular obliteration, tubular atrophy, and interstitial fibrosis. Serum creatinine levels and graft infiltration of T cells and macrophages in the pravastatin-treated animals were significantly lower. Intragraft cytokines showed a T helper 2 polarization and decreased transforming growth factor-beta in the pravastatin group. Intragraft expression of Bag-1 was increased in the pravastatin group. This study demonstrates the ability of pravastatin to inhibit chronic rejection in rat renal allografts. Pravastatin's pleiotropic effects of reducing intragraft inflammatory cytokines, inhibiting immune cell infiltration, and causing up-regulation of the antiapoptotic gene Bag-1 suggest that its ability to prevent transplant chronic rejection may be multifactorial.

In vivo detection of acute rat renal allograft rejection by MRI with USPIO particles

Magnetic resonance imaging (MRI) for non-invasively detecting renal rejection was developed by monitoring the accumulation of macrophages labeled with dextran-coated ultrasmall superparamagnetic iron oxide (USPIO) particles at the rat renal allografts during acute rejection. Five groups of male rats with DA-->BN renal allografts and one group with BN-->BN renal isografts were investigated by MRI before, immediately after, and 24 hr after intravenous infusion with different doses of USPIO particles. All infusions were done on post-operative day 4. MRI experiments were carried out in a 4.7-Tesla instrument using a gradient echo sequence. MR signal intensity (MRSI) of the cortex was found to decrease with higher dosages of USPIO particles. In the absence of USPIO infusion, a decrease in MRSI was seen in the medulla region, presumably due to hemorrhage associated with renal graft rejection, while no significant change was observed in the cortex. The optimal dose of USPIO particles for visualizing rejection-associated changes in our rat kidney model appears to be 6 mg Fe/kg body weight. Iron staining results correlated with the MRSI data, indicating that the signal reduction in the MR images was due to the presence of iron. Immunohistochemical results indicated that USPIO particles were mostly taken up by infiltrating macrophages in the rejecting grafts. Our results suggest that MRI with intravenous administration of dextran-coated USPIO particles appears to be a valuable and promising tool that can be used as a non-invasive and sensitive method to detect graft rejection in renal transplantation.

IMPACT OF FEBRILE INFECTIONS ON THE LONG-TERM FUNCTION OF KIDNEY ALLOGRAFTS

We prospectively determined the impact of febrile infectious disease on long-term renal graft function compared with a matched control group. Included in our study were 39 patients who presented with episodes of febrile infection with body temperature greater than 38C on 2 consecutive occasions, necessitating hospitalization. In addition, 39 controls without febrile infection requiring hospitalization within 2 months were chosen from the complete data pool of all renal transplant recipients followed at our transplant clinic using the matched pair technique. Renal function was estimated by serum creatinine and calculated creatinine clearance. Of the 39 patients with infection 15 had urinary tract infection and 24 had other, mostly bacterial infection, including pneumonia/severe bronchitis in 12, oral/dental infection in 2, gastroenteritis in 2, shunt sepsis in 1, herpes zoster in 1, cytomegalovirus in 1 and other in 5. Mean estimated creatinine clearance plus or minus standard deviation was similar in the infection and control groups at the beginning of the study (51 +/- 22 and 51 +/- 23 ml. per minute, respectively). During the infectious episode mean creatinine clearance significantly decreased to 38 +/- 17 ml. per minute in the infection group. After infection resolved creatinine clearance returned to an almost baseline mean value of 50 +/- 23 ml. per minute. However, after 2 years of followup there was a significant difference in mean creatinine clearance in the infection group versus controls (45 +/- 25 versus 52 +/- 25 ml. per minute, p = 0.022). To our knowledge we have shown for the first time in a prospective controlled study that febrile infectious episodes correlate with poor long-term renal graft function.

Recipient age and weight affect chronic renal allograft rejection in rats.

Nephron doses and immune responses change with age. Therefore, age is a potential risk factor for graft survival after kidney transplantation. The aim of this study was to determine whether age-related differences are of importance for long-term outcomes after renal transplantation. Kidneys from Fisher 344 rats were orthotopically transplanted into nephrectomized Lewis rats. Kidneys were transplanted using donors and recipients of three age levels, i.e., young (8 wk of age), adult (16 wk of age), and old (40 wk of age). Rats were killed 24 wk after transplantation, and functional, morphologic, and molecular evaluations were performed. Recipient age, rather than donor age, determined graft survival rates. No significant correlation was observed between donor kidney weight on the day of transplantation and morphologic results. Advanced recipient age was associated with reduced creatinine clearance, more severe histologic injuries, including extended glomerular sclerosis, interstitial fibrosis, and vascular lesions, more pronounced cellular infiltration, and greater expression of transforming growth factor-beta and platelet-derived growth factor A and B chains. Although no significant correlation between donor age or kidney weight on the day of transplantation and morphologic results was observed, there was a significant correlation between recipient body weight on the day of transplantation and allograft injury. It is concluded that recipient age and weight affect chronic renal rejection. Renal allografts may benefit from young recipient age but may deteriorate in old recipients, suggesting effects of recipient functional demand on long-term outcomes.

Treatment of chronic renal allograft rejection in rats with a low-molecular-weight heparin (reviparin).

Low-molecular-weight heparin (LMWH) has been shown to prolong survival of rat cardiac allografts independently from immunosuppressive treatment. Furthermore, long-term treatment reduces the development of chronic graft vascular disease after experimental heart transplantation. The aim of the present study was to determine whether treatment with the LMWH reviparin has a beneficial effect on chronic rejection in a rat renal allograft model. Kidneys of Fisher (F344) rats were transplanted into unilaterally nephrectomized Lewis (LEW) recipients. LEW-->LEW isografts served as controls. Animals were treated with cyclosporine (5 mg/kg/d) for the first 10 days. Nephrectomy of the remaining kidney was performed after 10 days. Allografted animals were treated either with reviparin (2 mg/kg/d subcutaneously) for 24 weeks (Allo-24), from week 12 to 24 (Allo-12), or with vehicle for 24 weeks. Proteinuria was determined at regular intervals. Kidneys were harvested after 24 weeks for histomorphological and immunohistochemical evaluation. No major bleeding complications were observed in reviparin-treated animals. Proteinuria was significantly reduced in allografted animals both by early as well as by late-onset treatment with reviparin. Transplant glomerulopathy was diminished in Allo-24 and in Allo-12 groups compared to vehicle-treated animals, whereas tubulointerstitial inflammation was influenced only in animals immediately treated with reviparin. Immunohistochemical studies demonstrated a marked reduction of renal monocyte and T-cell infiltration as well as expression of MHC II by treatment with reviparin. Treatment with the LMWH reviparin significantly improved chronic renal allograft rejection in the F344-to-LEW rat model, both after early and late start of therapy. Although the exact mechanisms of this beneficial effect remain unclear, our data offer a potential new therapeutical approach for prevention of chronic allograft nephropathy.

Expression of platelet-derived growth factor and its receptors in acute rat renal allograft rejection

Expression of platelet-derived growth factor and its receptors in acute rat renal allograft rejection

Effect of treatment with low-molecular-weight heparin on chronic renal allograft rejection in rats

Effect of treatment with low-molecular-weight heparin on chronic renal allograft rejection in rats

Treatment with a combined endothelin A/B-receptor antagonist does not prevent chronic renal allograft rejection in rats.

A markedly increased expression of endothelin (ET)-1 has been observed in renal allografts with chronic rejection, one of the most common causes of kidney graft loss. In this study we investigated the effect of treatment with a combined ET-A/B-receptor antagonist on the course of chronic renal allograft rejection. Experiments were performed in the Fisher-to-Lewis rat model of chronic rejection. Lewis-to-Lewis isografts and uninephrectomized Lewis rats served as controls. Animals were treated with either the oral combined ET-A/B-receptor antagonist LU224332 (20 mg/kg/day) or vehicle. Animal survival, blood pressure, creatinine clearance, proteinuria, and urinary ET excretion were investigated for 24 weeks. Kidneys were removed for light-microscopic evaluation and immunohistochemical assessment of cell-surface markers. Treatment with LU224332 did not improve survival after 24 weeks (0.47 vs. 0.38; p > 0.05 by log-rank test), nor did it have an influence on blood pressure, creatinine clearance, or proteinuria. Combined ET-A/B-receptor blockade was associated with a reduction of expression of cell-surface markers for macrophages (EDI), T-cells (R73), and major histocompatibility complex (MHC) II (F17-23-2), but did not lead to an improvement of histologic changes of chronic allograft rejection. Our data show that blocking both ET-A- and -B receptors, in opposition to a previously published beneficial effect of selective ET-A blockade, does not prevent the progression of chronic renal allograft rejection and does not prolong survival in this model. Functional integrity of the ET-B receptor therefore seems to play an important role in the nephroprotection provided by selective ET-A-receptor antagonists in chronic renal allograft nephropathy.

Effects of cytomegalovirus infection and prolonged cold ischemia on chronic rejection of rat renal allografts.

Previous studies have demonstrated that both cytomegalovirus (CMV) infection and prolonged cold ischemia of the allograft (CI) are associated with chronic rejection of renal transplants. The purpose of this study is to investigate the effect of CMV infection, of CI and of the combination of both, on the progression of chronic rejection, and to obtain a more detailed insight in their effects on the expression of adhesion molecules. Therefore, a rat transplantation model was used. Lewis recipients of renal allografts (with and without CI) from MHC-incompatible Brown Norway rats were inoculated with rat CMV or left uninfected. CMV infection alone resulted in an increased influx of CD4+ cells and macrophages early after infection, and in an increase in glomerular sclerosis and intima proliferation. CI caused an increase in infiltrating NK cells and an effect on intimal proliferation, glomerular sclerosis, and tubular atrophy. When CMV infection and CI were combined, an additive effect could be measured. This was however not the case for the function of the kidney. The creatinin showed a synergistic effect of the two influencing factors. Due to the CMV infection, an increase in CD49d cells was detected. CI resulted in an increase in CD18 cells and an increase in the expression of CD62P on vessels, and CD54 and CD44 on tubules. When CMV infection and CI were combined, all the effects caused by CMV and CI alone were present in an additional way. The results of the present study suggest that special attention should be paid to the recipient of an ischemically injured graft when either the donor or the recipient is CMV-infected. The patterns seen in histology, the infiltration of leukocytes and the expression of adhesion molecules, suggest that CI and CMV infection both have an effect on rejection, but act by different mechanisms.

SDZ-RAD prevents manifestation of chronic rejection in rat renal allografts.

Chronic rejection remains the most frequent cause of renal graft loss over the long term. However, effective treatment of this process is not yet available. SDZ-RAD (40-O-[2-hydroxyethyl]-rapamycin) is a new, orally active rapamycin derivative with potent immunosuppressive activity. We have examined the effects of SDZ-RAD in a well-established model of chronic renal allograft rejection in rats. Kidneys of Fisher (F334) rats were orthotopically transplanted into bilaterally nephrectomized Lewis recipients. To suppress an initial episode of acute rejection, rats were briefly treated with low doses of cyclosporine for the first 10 days. Thereafter they received either SDZ-RAD (0.5 mg/kg(day) or vehicle. At 24 weeks, functional evaluations were performed, kidneys were harvested, and histological, immunohistological, and reverse transcription-polymerase chain reaction evaluations were performed. Animals treated with SDZ-RAD developed lower proteinuria and less glomerulosclerosis as compared with controls. Additionally SDZ-RAD reduced the infiltration of macrophages and lymphocytes and the expression of intercellular adhesion molecule-1, laminin, and fibronectin. Furthermore, we observed a reduced expression of growth factor mRNA (transforming growth factor-beta and platelet-derived growth factor-AA) in these animals. Our results demonstrated that SDZ-RAD effectively ameliorates chronic renal allograft rejection in rats, probably mediated by suppression of growth factors.

Effects of cytomegalovirus infection and prolonged cold ischemia on chronic rejection of rat renal allografts

Abstract Previous studies have demonstrated that both cytomegalovirus (CMV) infection and prolonged cold ischemia of the allograft (CI) are associated with chronic rejection of renal transplants. The purpose of this study is to investigate the effect of CMV infection, of CI and of the combination of both, on the progression of chronic rejection, and to obtain a more detailed insight in their effects on the expression of adhesion molecules. Therefore, a rat transplantation model was used. Lewis recipients of renal allografts (with and without CI) from MHC-incompatible Brown Norway rats were inoculated with rat CMV or left uninfected. CMV infection alone resulted in an increased influx of CD4+ cells and macrophages early after infection, and in an increase in glomerular sclerosis and intima proliferation. CI caused an increase in infiltrating NK cells and an effect on intimal proliferation, glomerular sclerosis, and tubular atrophy. When CMV infection and CI were combined, an additive effect could be measured. This was however not the case for the function of the kidney. The creatinin showed a synergistic effect of the two influencing factors. Due to the CMV infection, an increase in CD49d cells was detected. CI resulted in an increase in CD18 cells and an increase in the expression of CD62P on vessels, and CD54 and CD44 on tubules. When CMV infection and CI were combined, all the effects caused by CMV and CI alone were present in an additional way. The results of the present study suggest that special attention should be paid to the recipient of an ischemically injured graft when either the donor or the recipient is CMV-infected. The patterns seen in histology, the infiltration of leukocytes and the expression of adhesion molecules, suggest that CI and CMV infection both have an effect on rejection, but act by different mechanisms.

Prevention of chronic renal allograft rejection in rats with an oral endothelin A receptor antagonist.

Chronic rejection is the most common cause of graft loss in renal transplantation. The pathomechanisms underlying chronic rejection are poorly understood, and no treatment has yet successfully been established. We hypothesized that, in analogy to models of reduced renal mass, the administration of a selective endothelin (ET) A receptor antagonist could improve the course of chronic rejection in renal allografts. Experiments were performed in the Fisher-to-Lewis rat model of chronic rejection. Lewis-->Lewis isografts served as controls. Animals were treated with either the oral selective ET-A receptor antagonist LU135252 (50 mg/kg/day) or vehicle. Animal survival, blood pressure, creatinine clearance, proteinuria, and urinary ET excretion were investigated for 24 weeks. Kidneys were removed for light microscopical evaluation, determination of ET mRNA expression and tissue protein concentration, and immunohistochemical assessment of cell surface markers. Rats with chronic rejection showed an increase in renal ET mRNA synthesis and ET protein content. Treatment with LU135252 resulted in a significant improvement in survival after 24 weeks (0.92 vs. 0.38, P<0.01 by log-rank test). Creatinine clearance was higher in animals treated with the selective ET-A receptor antagonist (P<0.05). LU135252 had no influence on blood pressure and proteinuria. Selective ET-A blockade was associated with significantly less morphological changes and a significant reduction of expression of cell surface markers for macrophages (ED1), T cells (R73), and MHC II (F17-23-2). The renal ET-A system plays an important role in the pathomechanisms underlying chronic renal allograft rejection, because the treatment with a selective ET-A receptor antagonist dramatically improves the course of chronic renal failure after allograft transplantation. These results offer a novel therapeutical option for treatment of chronic renal allograft rejection, for which so far no therapy is known.

Time course of chemokine expression in acutely rejecting rat kidneys

DURING acute rejection of rat renal allografts, the kidneys are heavily infiltrated by macrophages and T lymphocytes. Other leukocytes are quantitatively less important. Allograft infiltration is visible in perivascular regions on the first day posttransplantation, spreads to the renal interstitium and reaches its maximum on day 4. The number of allograft macrophages remains stable on days 5 to 6 after transplantation, and the number of T lymphocytes decreases during this time. The mechanisms leading to graft infiltration are not fully understood. Chemokines, a family of chemotactic cytokines, are probably involved.

Immunomodulatory functions of low-molecular weight hyaluronate in an acute rat renal allograft rejection model.

Low molecular weight hyaluronate (LMW-HA) blocks interactions between T lymphocyte CD44 and hyaluronate (HA), a heteropolysaccharide that is expressed on the surface of endothelial cells and ubiquitously in the extracellular matrix. This study was undertaken to assess the ability of LMW-HA to modify the course of experimental acute renal allograft rejection. Lewis (LEW) rats were bilaterally nephrectomized and received an orthotopic, fully MHC-mismatched, Wistar-Furth (WF) kidney transplant. Animals received either no treatment, low doses of cyclosporin A (CsA) on days 0 to 5, LMW-HA on days 0 to 5, or CsA plus LMW-HA on days 0 to 5 after transplantation. With no treatment, CsA monotherapy, or HA monotherapy, animals rejected their allografts at a median of 15, 13, and 7.5 d, respectively (P = NS). In contrast, combined CsA plus LMW-HA therapy prevented acute rejection and significantly prolonged graft survival (P = 0.008) to a median of 49.0 d. CsA/LMW-HA-treated grafts also demonstrated better preservation of renal function at day 30 (serum creatinine level, 1.38+/-0.8 mg/dl), compared with surviving animals treated with CsA alone (2.9+/-0.55 mg/dl, P<0.05). Histologic graft analysis of CsA/LMW-HA-treated animals at day 7 after transplantation showed minimal rejection and leukocyte infiltration, compared with all other groups. Intragraft gene expression analysis, using semiquantitative reverse transcription-PCR, at the same time point showed reductions of CD4, CD8, and interferon-gamma transcript levels in the combined-treatment group. This is the first study demonstrating the immunomodulatory functions of LMW-HA in vivo in the setting of organ transplantation. Defining the exact mechanisms that underlie this immunomodulation may provide the rationale to develop novel strategies for use in clinical transplantation.

Effects of cytomegalovirus infection and prolonged cold ischemia on chronic rejection of rat renal allografts

Effects of cytomegalovirus infection and prolonged cold ischemia on chronic rejection of rat renal allografts

Acute rejection of rat renal allografts is accelerated by donor brain death

Acute rejection of rat renal allografts is accelerated by donor brain death

Effect of hepatocyte growth factor on chronic rejection in rat renal allografts

Effect of hepatocyte growth factor on chronic rejection in rat renal allografts

IN VITRO AND IN VIVO IMMUNOMODULATORY EFFECTS OF RDP 1258, A NOVEL SYNTHETIC PEPTIDE

374 The synthetic MHC class I peptide HLA-B27 (residues 75-84) has been shown to have immunoinhibitory effects in animal and human transplant studies. There is evidence that these effects may be mediated by binding to intracellular heat shock proteins, in particular heme oxygenase-1 (HO-1). Immunosuppressive activities, physico-chemical properties and molecular dynamic studies of peptide 2702.75-84 and derivatives thereof were used for the rational design of a new agent, RDP 1258 (bc-1-nl). In vitro, this peptide was shown to be 50 times more potent in inhibiting HO-1. In vivo, administration of RDP 1258 resulted in systemic upregulation of HO-1 activity, a property consistent with observations made with other in vitro inhibitors of HO-1. RDP 1258 also inhibited the rat, mouse and human MLR in a dose dependent manner. This was associated with dose dependent inhibition of INF-γ production (Figure 1). Mitogen responses were not inhibited. The effects of RDP 1258 in an acute rat renal allograft rejection model were then tested. Bilateral nephrectomized LEW rats were transplanted with full MHC mismatched WF kidneys. Rejection was defined as death (see Figure 2). Control (n=6) animals received no treatment and survived for a median of 13.0 days. Treatment with peptide alone (1 mg i.p. on days -1, 0 and then twice weekly up to day 30; n=6) or low dose CsA alone (1 mg s.c on days 0-5; n=19) did not significantly prolong survival compared to controls: median survival 13.0 and 11.5 days, respectively. The combination of RDP 1258 + CsA (n=10), using the above regimens, significantly prolonged survival compared to all other groups(p<0.02, log rank test), to a median of 32.0 days.Fig. 1: Mean inhibition of proliferation and INF-γ production in rat MLRFig. 2: Kaplan-Meier survival plotIn conclusion, RDP 1258, a novel synthetic peptide, inhibited alloreactive responses in vitro and modified the course of acute renal allograft rejection. These effects may be related to in vivo upregulation of the immunomodulatory heat shock protein, HO-1.

EXPRESSION OF iNOS IN ACUTE HUMAN RENAL ALLOGRAFT REJECTION

136 Previous studies have demonstrated increased inducible nitric oxide synthase (iNOS) gene transcript expression in rat models of unmodified acute rejection. Furthermore, pulse corticosteroid therapy reduced iNOS mRNA expression and ameliorated rejection in these models. We studied the expression of iNOS in human kidney transplants with acute rejection. Our goal was to determine whether similar up-regulation of iNOS expression occurred in acute rejection of human kidney transplants. Methods. Twenty-four consecutive renal transplant recipients underwent allograft biopsies for acute deterioration of graft function, prior to initiation of pulse steroid therapy. Rejection was defined histologically, and by response to steroid therapy. Using competitive RT-PCR, the biopsies were examined for the presence of iNOS and glyceraldehyde phosphate dehydrogenase (GAPDH) mRNA. The expression of these genes was then compared between patients with acute rejection (AR) and those without. GAPDH expression served as a control for the efficacy of the reverse transcription. Results. Twelve biopsy specimens had AR and 12 had no rejection (NR). The non-rejection group included biopsies diagnosed as cyclosporine nephrotoxicity, preservation injury, and nonspecific changes. The biopsies with acute rejection were graded according to the Banff Classification Criteria, and were found to range in score from 1-3. GAPDH mRNA expression was present in all biopsy specimens. There was no difference in the iNOS/GAPDH mRNA expression between the two groups(7.92×10-5 in the rejection group vs. 5.62×10-5 in the NR group). Furthermore, iNOS mRNA expression was undetectable in 5/12 biopsies from the AR, and 7/12 biopsies from the NR group. There was no correlation between the prescence of iNOS transcripts and the histologic severity of rejection. Conclusions. There is no significant difference in iNOS mRNA expression between human renal transplant biopsies demonstrating acute rejection, and those without. In 5/12 of the AR biopsies iNOS mRNA expression could not be demonstrated. We find that the expression of iNOS transcripts in human renal allograft rejection is dissimilar to that found in unmodified rat renal allograft rejection.

De novo expression of MHC class II molecules by microglial cells during acute rat renal allograft rejection.

The expression of MHC class I and class II molecules in the cerebral cortex of rats was investigated at daily intervals from day 3 to day 6 after fully allogeneic (DA-->LEW) and isogeneic (LEW-->LEW) kidney transplantation. MHC class II molecules were temporarily induced on the previously negative microglial cells and on the endothelia of arterioles and venules during acute rejection. On the endothelia of all brain vessels MHC class I expression was enhanced. MHC class I+ cells with microglial cell morphology were discernible within the diffusely MHC class I+ brain parenchyma. In contrast, the brain parenchyma of isograft recipients and untreated control animals did not express detectable levels of MHC molecules. In conclusion, we demonstrate that a strong immune reaction in the periphery is able to activate microglial cells in the central nervous system.