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- Materials and Methods
- Supporting Information
The occurrence of steroid-refractory acute rejection is a risk factor for adverse outcomes in renal allograft recipients [1-5]. First rejection episodes are most of the time treated by high-dose steroids, leading to reversal of the acute rejection. However, approximately 30% of patients have no or an inadequate response to corticosteroid therapy alone and require therapy with anti-thymocyte globulin (ATG) [2, 5-7]. Such steroid resistant patients may show progression of chronic damage to the graft, which has a detrimental effect on graft outcome [4, 5, 8].
Cellular and molecular markers in the graft tissue may complement clinical parameters and histomorphology when assessing steroid resistance of the acute rejection episode. Indeed, expression of markers, particularly those of inflammatory cell types, was found to be associated with therapy response (reviewed in Ref. ). Recently, we showed that the combination of T cell activation markers CD25:CD3 ratio and LAG-3 offers a superior prognostic value for assessing steroid response, compared with conventional parameters . We observed considerable molecular heterogeneity among the biopsy samples with acute rejection, underlining the complexity of the mechanisms involved in response to steroid therapy.
In the current study we aimed to gain further insight into the mechanisms underlying steroid resistance by identifying novel molecular markers of steroid-refractory acute rejection using genome wide expression profiling. Our results reveal that relatively high intragraft expression of metallothioneins (MT), a group of small cysteine-rich molecules that regulate intracellular zinc concentrations, during acute rejection is associated with resistance to steroid treatment. These proteins were further studied in biopsies and steroid-treated leukocyte cultures.
- Top of page
- Materials and Methods
- Supporting Information
We aimed to identify novel molecular markers associated with steroid-refractory acute rejection, and to gain insight into the mechanisms underlying steroid resistance. For this, we investigated intragraft gene expression profiles of renal allograft recipients with a first rejection episode. Our study reveals that the expression of metallothioneins (MT) within the renal allograft may distinguish patients with steroid resistant acute rejection from patients with steroid responsive acute rejection. The findings, which were generated in a discovery cohort of 36 patients, were validated in a validation cohort of 47 patients.
Until now, research in the field of steroid-refractory acute rejection in renal allografts has been focused on markers of inflammatory cells (reviewed in Ref. ). Recently, we found that the combination of T cell activation markers CD25:CD3 ratio and LAG-3 offers a superior prognostic value for assessing steroid response compared with conventional parameters . In the current study, we found that relatively high intragraft MT expression during acute rejection is associated with steroid resistance, and that MT-1 isoforms are expressed in activated macrophages and in tubular epithelial cells. These findings are in line with earlier findings in lung allograft recipients with steroid-refractory acute rejection. Yousem et al.  found increased percentages of MT-positive macrophages in transbronchial biopsy samples of lung allograft recipients who experienced steroid-refractory acute rejection. Interestingly, several studies in the oncological research field have also demonstrated that elevated expression of MT is related to treatment resistance [26-28].
In multivariate analysis, the combination of MT-1 with CYP4A11, TIMP1, and F2R represented the best predictive model. This multivariate MT-1 model has a slightly higher predictive value than the T cell activation model found in our previous study (see Figure 7) . Combination of the two models resulted in a predictive model with MT-1, TIMP1, F2R, CD25:CD3e ratio and LAG-3 as independent covariates, showing that our current findings strengthen the risk assessment of steroid resistance in patients having acute rejection. Patients with steroid-refractory acute rejection may benefit from immediate ATG treatment after the diagnosis of acute rejection.
Metallothioneins are a family of 11 proteins involved in the homeostasis of biologically essential metals [29-33]. Under physiological conditions MT are mainly expressed in the kidney and liver . MT can bind zinc ions, and by functioning as a zinc-donor or zinc-acceptor they can control cellular zinc distribution [29, 30]. A variety of DNA-binding proteins rely on zinc finger motifs to bind to their target sequences [29, 34]. Metallothioneins can influence the DNA-binding capacity of zinc-proteins by controlling the amount of zinc that is available for zinc finger domains [35, 36].
The glucocorticoid receptor (GR) is also a zinc-dependent protein . Glucocorticoid effects depend on GR-mediated transcriptional regulation of genes encoding for pro-inflammatory proteins. Binding of GR to glucocorticoid response elements (GREs) in the promoter region of target genes relies on two zinc finger motifs [29, 34, 37]. Increased expression of metallothioneins may lead to removal of zinc ions that are normally complexed in the zinc finger domains of GR, preventing its binding to GREs and inhibiting the immunomodulatory effects of the steroid-based anti-rejection treatment.
Another mechanism of action of the GR that may be affected by increased MT expression is its recruitment of histone deacetylase (HDAC)-2 . Upon ligand binding the GR becomes a target for HDAC-2, which in turn allows the GR to associate with and suppress the pro-inflammatory transcription factor NF-κB [38, 39]. In addition, the recruited HDAC-2 represses transcription from promoters through histone deacetylation, resulting in suppression of activated inflammatory genes within the nucleus [38, 40]. As the recruitment of HDAC-2 to the GR relies on zinc, anti-inflammatory effects of this process may also be inhibited by MT.
In vitro tests with PBMC have been used to correlate gene expression profiles with clinical disorders, including steroid responsiveness [41-43]. Following this strategy, we compared the RNA level of pro-inflammatory cytokine responses (TNFα, IFNγ) between stimulated PBMC and stimulated PBMC treated with methylprednisolone. This allowed distinction between responders and nonresponders to the steroid treatment. The concentration of 10−4 M methylprednisolone approximates the i.v. dosage of 1 g/day given in clinical practice. In all PBMC donors tested, treatment with methylprednisolone led to an increase in MT expression, which corresponds with earlier observations of MT as a steroid responsive gene [44, 45]. However, nonresponders displayed a significantly higher fold of MT upregulation upon methylprednisolone treatment than responders. These results are in line with the observation that patients who are steroid-resistant upon anti-rejection treatment have elevated MT expression. Further studies are needed to show that in vitro cultures of patient PBMC represent a useful indicator of the patient's response to steroid treatment in vivo.
Some comments need to be made with regard to the current findings. First, MT expression can be induced by the glucocorticoids . To prevent an influence of steroid-based anti-rejection treatment on the intragraft mRNA and protein expression levels, the biopsy samples investigated in our study were collected before the patients received high-dose methylprednisolone. All patients did receive low-dose steroids as part of the maintenance therapy. Second, due to high homology in the gene sequence of the MT-1 subtypes, it has proven difficult to distinguish the MT-1 subtypes from each other. As our microarray assays rely on 50-bp long probes to measure specific mRNA transcripts, we were able to measure the mRNA expression of the individual MT-1 isotypes. However, no distinction could be made between the MT-1 isoforms with qPCR or immunohistochemistry. The finding by microarray, that most MT-1 isoforms were upregulated in steroid resistant acute rejection, shows that the difficulty to distinguish between MT-1 isoforms likely will not have affected our findings.
In conclusion, resistance to steroid treatment is associated with a relatively high intragraft expression of zinc-regulating MT during acute rejection. MT are mainly expressed by activated macrophages and tubular epithelial cells within the kidney. The relationship between MT and steroid resistance was confirmed in vitro by treatment of activated PBMC with corticosteroids. An increased expression of MT may lead to regulation of intracellular zinc concentrations, and to inactivation of the DNA binding capacity of the GR. The current findings point to MT as potentially novel therapeutic targets.