Microarray gene expression profiling of chronic allograft nephropathy in the rat kidney transplant model.

Whole genome gene expression profiles were correlated with renal function and histology in a well-established animal model of chronic allograft nephropathy (CAN). Kidneys of F344 rats were transplanted into LEW recipients treated with a brief dose of FK506 (BFK). Blood and urine samples were collected weekly. Kidney grafts were harvested at an early (day 6) or late (days 30-90) phase after transplantation. BFK kidney grafts showed remarkable changes in function, histology, and gene expression profiles when compared to the isograft controls. In the early phase, renal function and histology were barely affected, yet the expression levels of 225 genes were significantly changed, reflecting both immune and non-immune pathways. In the late phase, however, 826 genes were affected in the BFK kidney grafts, including genes in the pathways of extracellular matrix and cell adhesion. Of these genes, 214 appear to be key factors for development of CAN, since they were affected at both early and late phases, including genes involved in the immune response, the inflammatory response, apoptosis, and metabolism. Kinetic studies with gene expression profiling can identify genes involved in the progressive development of chronic allograft rejection, leading to more detailed therapeutic approaches or useful biomarkers in clinical transplantation.

A blocking anti-CD28-specific antibody induces long-term heart allograft survival by suppression of the PKC theta-JNK signal pathway.

This study investigated the effects of a blocking anti-CD28 antibody (Anti-CD28-PV1-IgG3) in vitro and in vivo. Anti-CD28-PV1-IgG3, a hamster-mouse chimeric antibody against murine CD28, which does not provide CD28-positive signaling during TCR-driven T cell activation, enabled long-term survival of heart allografts across a complete mismatch of the MHC in rats. Among the T cell signaling proteins tested in the spleens from recipients, we found that recipients treated with anti-CD28-PV1-IgG3 exhibited suppression of alloantigen-initiated proximal TCR signaling events, including Lck, Zap70, Vav, and PI3K expression, and their PKC theta- and JNK-regulated expression/activation. This leads to attenuation of intragraft T cell infiltration and expression of T cell effector molecules. These results indicate that targeting the CD28 receptor with a blocking antibody leads to long-term allograft survival by reducing activation of alloantigen-mediated key signaling events in T cells that might be crucial for full T cell activation.

The use of unilateral PCR to identify prominent heteroduplexes formed during PCR of the mouse microsatellite locus D17Mit23.

Microsatellite markers are useful tools for understanding the evolutionary history of discrete segments of the mammalian genome. We used the microsatellite marker D17Mit23 to study the portion of the mouse genome known as the t complex, a naturally occurring variant of Chromosome 17. We identified an allelic variant of D17Mit23, which is shared by two forms of the t complex, the t haplotypes t(w2) and t(Lub2). Polymerase chain reaction (PCR) analysis of DNA samples from mice that were heterozygous for either t haplotype resulted in gel patterns with prominent bands of higher molecular weight in addition to the bona-fide D17Mit23 alleles. The appearance of these higher molecular weight bands, although consistent with heteroduplex formation, was not diminished through the use of reconditioning PCR. We used a modified form of asymmetric PCR, called "unilateral PCR," to show that the higher molecular weight bands are heteroduplexes and to identify their constituent strands. Certain microsatellite motifs may be especially prone to the production of prominent heteroduplex products, and this may lead to the erroneous genotyping of DNA samples.

Unique gene expression profiles of heart allograft rejection in the interferon regulatory factor-1-deficient mouse.

Interferon regulatory factor-1 (IRF1) is a transcription factor for many genes involved in innate and adaptive immune responses. By using DNA array technology, we have previously demonstrated that IRF1 is significantly upregulated during acute rejection in rat heart allografts and is restored to isograft levels when recipients are treated with the immunosuppressants tacrolimus or cyclosporin A (CsA). To understand the precise role of IRF1 in transplant rejection, we investigated the rejection responses of mice completely deficient of IRF1 protein. Heterotopic heart transplantations were performed using C57BL/6J wild-type (WT B6) and IRF1-deficient (IRF1-/-) mice as recipients, and C3H mice as donors. Graft survival was determined by abdominal palpation and rejection was confirmed by histology. On day 6 after transplantation, isografts and allografts were harvested and subjected to gene expression analysis by a commercial nylon array and by real-time RT-PCR. Median survival time of heart allografts was 8 days in the WT B6 mice and 10 days in the IRF1-/- mice. The gene expression profiles of allografts from the WT B6 and IRF1-/- recipients were nearly identical to each other and very different from the profile of the isograft control. Both WT B6 and IRF1-/- profiles showed 13 genes upregulated (IFN-gamma, MCP-2, MIP-1alpha, MIP-1beta, CCR5, MIG, IP-10 and others) and one gene downregulated (SDF2) among the 76 genes detectable on the array. In more detailed analyses, distinct cytokine and chemokine gene expression profiles were identified in the allografts from the WT B6 and IRF1-/- recipients. Whereas IL-4, IL-6, IL-13, MCP-1, MCP-3, and MPIF-2 were upregulated, RANTES, IL-2Rgamma and gp130 were downregulated in allografts from the IRF1-/- recipients when compared to the WT B6 control. Although the inactivation of the IRF1 gene did not sufficiently prevent acute allograft rejection in this model, a unique cytokine and chemokine gene expression profile was found in the absence of IRF1.

FK778, a powerful new immunosuppressant, effectively reduces functional and histologic changes of chronic rejection in rat renal allografts.

BACKGROUND: FK778 is a new derivative of the active leflunomide metabolite A77 1726. It effectively prevented acute allograft rejection in several experimental transplant models, and it is currently in phase II trials in human transplant recipients. In this study, we examined the effects of FK778 in a well-established model of chronic renal allograft rejection in the rat. METHODS: Kidneys of Lewis (LEW) and F344 rats were orthotopically transplanted into bilaterally nephrectomized LEW recipients as the isograft and allograft control, respectively. Allograft recipients were orally administered FK778 at doses of 3 mg/kg per day, 10 mg/kg per day, and 20 mg/kg per day for 10 days. Blood and 24-hr urine samples were collected once a week after grafting for plasma creatinine, allo-specific antibodies, and proteinuria determination. Kidney grafts were harvested on the 90th day after transplantation and subjected to histologic, immunohistologic, and reverse transcriptase-polymerase chain reaction analysis. Histologic sections were semiquantitatively scored using criteria adapted from the Banff' classification for transplant pathologic conditions. RESULTS: Recipients treated with FK778 for 10 days exhibited a dose-dependent decrease in proteinuria and plasma creatinine for the entire 90-day period after transplantation when compared with the allograft control. FK778, at doses of 10 mg/kg per day and 20 mg/kg per day, remarkably reduced chronic histologic changes, including tubular atrophy, glomerulosclerosis, fibrointimal hyperplasia, and transplant glomerulopathy. In addition, FK778 treatment was associated with decreased intragraft mononuclear cell infiltration, serum allo-specific immunoglobulin (Ig)M and IgG antibody production, and intragraft transforming growth factor beta messenger RNA expression in those recipients surviving 90 days after transplantation when compared with the allograft control. CONCLUSION: FK778 effectively reduces functional and histologic chronic kidney allograft rejection in the rat.