Activation of counter-regulatory mechanisms in a rat renal acute rejection model

Microarray analyses have been used to link changes at the level of gene expression to different kidney diseases to obtain markers for diagnosis and prognosis. Microarray analyses of human patients have been used to analyze post-transplant events [1–6]. A recent review of microarray-based studies and the search for biomarkers in organ transplantation is given by Kurian et al. [7]. Microarray analysis of kidney transplant biopsies with acute rejection identified gene expression patterns that distinguish three distinct subtypes of acute rejection that, although indistinguishable by histology, were marked by differences in mechanisms of immune activation and cell proliferation [8]. Interestingly, another study of acute rejection biopsies showed no evidence for up-regulation of cytotoxic T-cell effector molecules despite the fact that these have been considered markers for acute renal rejection [9]. In parallel, gene expression signatures of peripheral blood lymphocytes (PBLs) from transplant patients have also been shown to be capable of classifying patients with acute rejection [6]. Surprisingly, there was essentially no overlap in rejection-diagnostic genes up-regulated in PBL and genes up-regulated in the biopsies from the same patients, indicating that the blood must be considered a very different immune compartment in this setting. As a consequence of these initial studies, the hope of developing validated diagnostic biomarkers for transplantation is tempered by results that challenge an existing dogma on immune response mechanisms and demand additional studies to deconvolute.

There are a number of limitations inherent in clinical studies of gene expression in transplantation. Patients typically represent major differences in gender, age, co-morbidities, clinical histories, immunosuppressive regimes, race/ethnicities, and genetics and it is always difficult to collect samples at specific times relative to the procedures. These differences significantly influence gene expression and that operant variability compromises the results obtained by microarray analysis as well as the confidence to identify the principal underlying immune mechanisms. The use of experimental transplantation models in rats or mice is an opportunity to mitigate these variables and work in a controlled system. Several studies have made use of experimental transplantation to study changes in gene expression related to acute rejection after murine heart, kidney and lung transplantation [10–13]. While several studies have been performed using heart transplantation in rats [14, 15], to our knowledge, a study of acute kidney transplant rejection in rats has not been reported.

In previous studies [16, 17], we showed in well characterized rat renal transplantation models, no evidence of tissue necrosis at day 4 while histological changes consistent with acute rejection including activated lymphocyte infiltration were found consistently. In those studies we showed differences in gene expression for selected sodium and water transporters that were relevant to tissue injury and renal compensation. In the present study, we made use of these transplantation models and continued our studies using day 4 as the measuring point to represent a time at which there is full representation of acute rejection mechanisms but before the widespread renal tissue injury that can confuse the gene signatures. Thus, changes in gene expression related solely to acute rejection in allogeneic transplantation or linked to the surgical procedure in syngeneic transplantation can be evaluated. Native, untreated kidneys were used as the control group. No immunosuppression was used to specifically analyze the influence of acute rejection on gene expression independent of the effects of immunosuppressive regimens. Thereby, maximum homogeneity in procedures, sample preparation, timing, and handling was achieved. In contrast to the design of renal transplantation studies performed in mice [12, 13], we used bilaterally nephrectomized recipient rats, such that the animal's transplant was required to maintain the full metabolic and synthetic function of the organism as is the case in the clinical transplant situation.

The MHC antigens are the main barrier for an acceptance of the graft by the host organism and antigen presentation is essential for the activation of T-cells. T-cells are activated when the TCR recognizes allo-MHC class I molecules in the case of CD8+ and MHC class II molecules in the case of CD4+ T-cells [29]. Host T-cells can recognize MHC antigens by either indirect or direct presentation [30]. During indirect presentation, peptides derived from donor MHC class I or MHC class II molecules are processed and presented to CD4+ T-cells by host antigen presenting cells expressing the recipient's MHC class II molecules. The direct antigen presentation pathway is unique to transplantation. In this pathway the T-cell receptors directly recognize an intact MHC molecule expressed on donor cells. In this context, it is important that for our studies with acute rejection, nearly all the genes associated with the antigen processing and presentation pathways were affected (Fig. 3).

Our data also indicates that T-cell cytotoxicity is the main immunological pathway activated in response to acute rejection in allogeneic transplantation. The cytotoxic molecules are perforin/granzyme for CD8+ T-cells and the Fas/Fas-ligand system for CD4+ T-cells [31, 32]. In this context, the absolute changes in expression of genes involved in antigen processing and presentation by MHC class I molecules indicates that this part of the antigen presentation is intensely up-regulated. That is also consistent with the up-regulation of the immunoproteasome and TAP1/2 transcripts (Fig. 3) likely to be driven by the up-regulation of interferon γ [33, 34], which we have also documented. IFNγ can also induce the expression of the chemokines, CXCL9, CXCL10 and CXCL11 [35]. These data are consistent with other studies showing the changes in gene expression related to IFNγ or cytotoxic T lymphocytes in murine transplantation models [12, 13].

The induction of tolerance to the graft would be the treatment of choice after transplantation. Increased expression of protective genes like metallothionein-1 or α2β- crystallin were reported with a mouse cardiac transplant model [36, 37]. In this same context, we observed increased expression of a number of genes known to have protective or regulatory properties (Figs. 4, 5, 6, 7) creating a much more comprehensive list than reported in any previous studies. Our study also indicates the endogenous activation of counter regulatory mechanisms within several pathways that have been linked to acute rejection. For example, genes with anti- apoptotic functions include B-cell leukemia/lymphoma 2 related protein A1 (Bfl-1) (Fig. 5) and the Inhibitor of Apoptosis Proteins (IAP) [38, 39]. The expression of Heme Oxygenase 1 (HO1; 23-fold) was induced after allogeneic transplantation. The expression of HO1 can suppress graft rejection and leads to long term graft survival in some models [40]. The Cytotoxic T-Lymphocyte-Associated protein 4 (CTLA-4) is a negative regulator of T-cells (Fig. 4) [41]. The function of IL10 is to limit the action of the immune response. IL10 was initially characterized as a cytokine produced by Th2-cells, which inhibited the production of cytokines such as IL2, TNFα and IFNγ [42]. IL10 also modulates the expression of chemokines, chemokine receptors and MHC class II molecules [43]. Thus, the induction of IL10 after allogeneic transplantation indicates a possible feed back mechanism of the immune response (Table 1, Fig. 2).

The complement system is part of the innate immune response. The expression profile we demonstrate in allogeneic transplants for a number of complement components indicates the activation of the complement cascade by acute rejection (Fig. 7). Its major function is the elimination of pathogens and the stimulation of an inflammatory response [44, 45]. Local gene expression for complement components has been shown for C3 and C1 during acute rejection in kidney and heart transplantation models, respectively [36, 46]. One of the novel genes in our study with the strongest expression after allogeneic transplantation was the C4b binding protein alpha (C4bpa; 219 fold up-regulated). C4bpa is a cofactor for factor 1 in the degradation of C4b in the complement cascade [47]. The degradation of C4b inhibits the formation of the C4b2a complex, a key step in producing an inflammatory response [48].

Recently regulatory T-cells have been recognized as a promising tool for the induction of tolerance [49]. In these studies of our rat renal transplants we also find transcriptional evidence for the presence of regulatory T-cells within the graft. The function of regulatory T-cells is to regulate or suppress the activation, regulation and function of effector T-cells [50]. The development of regulatory T-cells appears to be controlled by FoxP3 [18], which we find is up-regulated after allogeneic transplantation (Fig. 2). The intragraft expression of FoxP3 has also been demonstrated in cardiac allograft patients [51]. The presence of regulatory T-cells within the grafts we studied is further supported by the concomitant increase in expression of CD25 [49] and tryptophan hydroxylase [52].

We have used a well-defined rat kidney transplantation model to demonstrate global changes in gene expression levels after syngeneic and allogeneic transplantation with the emphasis on early acute rejection in the allogeneic transplants. Using DNA microarrays and real time PCR analysis, we were able to identify major immune effector-related pathways that are activated after allogeneic transplantation and clearly linked by multiple lines of published evidence to acute rejection mechanisms of tissue injury. That we also found a list of genes that are equally highly differentially expressed in acute rejection but not presently linked in the literature represents opportunities for additional studies and discovery. In parallel, we have also identified a number of up-regulated genes that are linked to tissue protective and immune counter-regulation mechanisms. These observations fit with the evolving view of immunity as a balance between effector and regulatory mechanisms.