Molecular Phenotypes of Acute Rejection Predict Kidney Graft Prognosis

Abstract

Earlier detection of antibody-mediated rejection of kidney allografts may improve graft outcomes. Profiling of gene expression holds promise for the diagnosis and prognosis of antibody-mediated rejection. Here, we identified 730 patients who received kidney transplants during 2002–2005, including 21 patients (2.9%) who experienced early acute antibody-mediated rejection. We also identified a matched group of 43 patients with early acute T cell-mediated rejection to serve as controls. Compared with patients with T cell-mediated rejection, those with antibody-mediated rejection had significantly higher intrarenal mRNA expression of the cytoprotective heme oxygenase-1 but had lower expression of the regulatory T cell marker forkhead box P3 (FoxP3), the B cell marker CD20, and the chemokine regulated upon activation, normal T cell expressed and secreted (RANTES). T cell infiltration was similar in both groups of patients. Compared with grafts that had a favorable course, those that failed as a result of antibody-mediated rejection had expression profiles suggesting a lack of regulation (less FoxP3, TGF-β1, RANTES, and CD20). Grafts that failed as a result of T cell-mediated rejection only revealed lower expression of CD20 mRNA. In summary, these data suggest that severe antibody-mediated rejection and T cell-mediated rejection result in graft loss by distinct mechanisms. Molecular phenotypes of early acute rejection might help to identify grafts with poor prognosis, allowing earlier application of additional therapies.

Despite improvements in the first year renal allograft outcome, in the long term both acute and chronic rejections have been implicated to reduce renal allograft survival. Early antibody-mediated rejection (AMR) occurring within first 3 wk after kidney transplantation represents just a small part of all acute rejection episodes.^1–3 Although the fate of grafts developing early AMR likewise has been found to be superior to that of patients developing AMR later, the risk of graft loss is quite high despite aggressive immunosuppressive therapy.^4,5 Besides the well established therapy of acute T cell-mediated rejection (TCMR), the treatment schemes in early AMR still vary from center to center. Plasmapheresis and intravenous immunoglobulins have been used frequently, whereas rituximab, an anti-CD20 monoclonal antibody, or splenectomy is used mostly in resistant cases only.^6–8

Fitting with the diversity of therapeutical strategies, the AMR pathogenesis remains poorly understood. Whereas immunohistochemistry is well established,⁹ gene expression analyses of AMR just started. Surprisingly, AMR was shown to be associated with similar changes in the transcriptome like TCMR.¹⁰ Because microarray analyses are expensive, time-consuming, and often evaluated with improper statistics, the current update of the Banff classification recommends for future cytokine gene expression analysis using the reverse transcription (RT)-PCR method for the prediction of graft dysfunction.¹¹

Recently, we found that TGF-β1 and monocyte chemoattractant protein 1 (MCP-1) gene expression in case biopsies with allograft nephropathy help to identify those patients at risk for premature graft failure.^12,13 Early AMR is a rare complication of renal transplantation. Thus, there is limited information about gene expression profiles in early AMR in the literature. The aim of this study was thus to evaluate the patterns of intrarenal expression of several genes associated with B cell- and T cell-mediated inflammation, rejection, and tolerance^14,15 in relation to the renal allograft outcome in patients with early AMR and TCMR.

Results

Early AMR was diagnosed in 21 (2.9%) of 730 patients. Eight patients had revealed combined early AMR and TCMR (Banff IIa, IIB, and III in four, two, and two patients, respectively). Early AMR was diagnosed in average on the ninth postoperative day. As a control group, patients with early TCMR occurring within the same time window were enrolled in the study. Ten patients with AMR were treated using only plasmapheresis, and 11 patients were treated by combination of plasmapheresis and intravenous immunoglobulins (IVIGs). Despite trends toward better graft survival in the later group, Kaplan–Maier analysis showed no significant difference in graft survival. Grafts that failed had higher both glomerulitis and peritubular capillaries scores in the biopsy (data not shown).

First, we evaluated the gene expression differences of the selected genes between patients with biopsy-proven early acute AMR, TCMR, and combined AMR/TCMR. Biopsies from patients with early AMR and early TCMR showed comparable expression of CD3 and cytotoxic T lymphocyte (CTL) markers, suggesting similar levels of T cell infiltration. Remarkably, CD20 B cell expression was approximately five times higher in TCMR versus AMR (P < 0.05). Similarly, early TCMR was associated with higher intrarenal expression of CCL5, regulated upon activation, normal T cell expressed and secreted (RANTES) (P < 0.05), and forkhead box P3 (FoxP3) (P < 0.01), whereas the stress marker heme oxygenase-1 (HMOX1) was enhanced in AMR versus TCMR (P < 0.05) (Figure 1). All other markers were comparably expressed in all groups. Expression levels of all studied transcripts did not differ between acute tubular necrosis-like AMR and combined AMR/TCMR.

Figure 1.

Quantitative intrarenal mRNA expression within grafts that suffered from early acute AMR or TCMR. Filled boxes represent TCMR, blank boxes AMR, and stripped boxes combined AMR/TCMR. The results are expressed as the ratio of the gene of interest to the housekeeping gene HPRT. The box plots show 25th and 75th (boundaries of boxes), 50th (median), 10th and 90th (error bars) percentile values. *P < 0.05; **P < 0.01. Samples for gene expression analysis were obtained before the antirejection therapy.

Next, we wondered whether the gene expression patterns were associated with the outcome of AMR/TCMR. Nine out of 20 patients with AMR that resulted in graft failure during follow-up experienced approximately tenfold lower infiltration by B cells (CD20, P < 0.01) and plasma cells (IgJ P < 0.01) as well as the fivefold lower expression of the regulatory T cell (Treg) master transcription factor FoxP3 (P = 0.02). With Bonferroni correction for multiple testing, however, a critical P value was set at 0.01, and thus the FoxP3 expression achieved just borderline significance. The T cell (CD3) and CTL [perforin (PRF) and granzyme B (GZMB)] infiltration and the stress marker HMOX1 were not predictable for the outcome of AMR (Table 1).

Table 1.

Quantitative intrarenal mRNA expression in early acute AMR

Graft outcome	Failed grafts (n = 9)	Surviving grafts (n = 11)	P
TGF-β1	1.26 [0.03 to 6.95]	2.44 [1.03 to 3.60]	0.0367
TNF-α	5.12 [0.19 to 46.94]	4.91 [2.31 to 17.40]	0.4060
MCP-1	0.23 [0.02 to 1.73]	0.33 [0.09 to 0.61]	0.4938
RANTES	15.02 [0.83 to 66.49]	47.00 [8.96 to 159.65]	0.0118
HMOX1	1.18 [0.70 to 25.06]	1.35 [0.14 to 9.07]	0.2650
CD3	13.65 [0.57 to 17.93]	18.55 [7.65 to 28.54]	0.1091
CD20	4.17 [1.00 to 32.63]	53.65 [7.72 to 278.29]	0.0035
IgJ	0.85 [0.18 to 6.19]	7.27 [0.73 to 212.78]	0.0086
FoxP3	1.20 [0.29 to 5.00]	5.34 [1.73 to 25.51]	0.0200
GZMB	4.01 [0.15 to 6.74]	6.64 [1.72 to 20.31]	0.0955
PRF	10.04 [1.28 to 17.82]	17.65 [6.98 to 48.17]	0.0763
FoxP3/CD3	0.16 [0.11 to 2.13]	0.22 [0.09 to 1.82]	NS
GZMB/CD3	0.34 [0.20 to 0.60]	0.31 [0.11 to 1.10]	NS
PRF/CD3	1.08 [0.75 to 2.26]	0.82 [0.35 to 4.27]	NS

Relative quantities of specific gene expression relative to thatof the housekeeping gene HPRT and the calibrator sample are expressed asmedians [range]. Results reflect the gene expression patterns at thetime of diagnosis of early AMR and prior to therapy. One patient who died withfunctioning graft was not included in the analysis. The critical P fromBonferroni correction for multiple testing in Sankoh's modification is 0.01.Statistically significant values are represented in boldface type.

In contrast, grafts that failed after early TCMR during the follow-up showed before rejection therapy lower intrarenal mRNA expression of B cells (CD20, P < 0.01) and plasma cells (IgJ, P < 0.01) compared with grafts making a favorable course (Table 2). The CTL markers (PRF and GZMB) were comparable in both groups, and the GZMB-to-CD3 ratio was higher in the failing grafts; however, the low number of subjects who failed did not enable us to make a proper conclusion.

Table 2.

Quantitative intrarenal mRNA expression within grafts that suffered from early TCMR

Graft outcome	Failed grafts (n = 4)	Surviving grafts (n = 37)	P
TGF-β1	2.69 [0.04 to 5.46]	1.92 [0.01 to 7.03]	0.5762
TNF-α	3.70 [0.99 to 24.55]	6.07 [0.11 to 30.90]	0.5657
MCP-1	0.42 [0.22 to 1.62]	0.39 [0.12 to 1.66]	0.4394
RANTES	30.72 [9.39 to 180.12]	63.82 [4.13 to 184.98]	0.5730
HMOX1	0.82 [0.12 to 16.21]	0.79 [0.01 to 13.80]	0.5831
CD3	13.09 [2.00 to 17.00]	26.28 [0.29 to 199.68]	0.0809
CD20	20.63 [2.90 to 31.13]	108.46 [7.09 to 1519.93]	0.0033
IgJ	1.62 [1.18 to 2.73]	6.76 [0.37 to 220.48]	0.0003
FoxP3	4.38 [0.83 to 6.30]	9.99 [1.56 to 33.25]	0.0503
GZMB	9.26 [8.92 to 9.61]	8.16 [0.0005 to 69.20]	0.2732
PRF	22.24 [2.16 to 42.33]	14.96 [0.007 to 89.60]	0.9183
FoxP3/CD3	0.41 [0.26 to 0.48]	0.48 [0.12 to 1.19]	NS
GZMB/CD3	2.51 [0.57 to 4.46]	0.26 [0.00001 to 2.56]	NS
PRF/CD3	1.79 [1.08 to 2.49]	0.47 [0.0002 to 6.54]	NS

Relative quantities of specific gene expression relative to that of the housekeeping gene HPRT and the calibrator sample are expressed as median [range]. Samples for gene expression analyses were obtained prior to the antirejection therapy. The critical P from Bonferroni correction for multiple testing in Sankoh's modification is 0.01. Statistically significant values are represented in boldface type. Two patients who died with functioning graft were not included in the analysis.

Next, we analyzed the correlations between the expression of various genes. With Spearman rank analysis, the correlation of all measured genes with FoxP3 mRNA expression was performed. There were strong correlations of intrarenal expression of TGF-β1 (r_s = 0.709, P = 0.006), RANTES (r_s = 0.684, P = 0.004), and CD20 (r_s = 0.764, P = 0.004) with the intrarenal expression of FoxP3 mRNA during early AMR.

The Kaplan–Meier analysis of renal allograft survival showed patients with early AMR and low intrarenal expression of TGF-β1, RANTES, CD20, and FoxP3 at the time of biopsy [cutoff relative quantities (RQs) assessed from receiver operating characteristic analysis were 2.0, 16.7, 5.7, and 1.3, respectively] to have significantly shorter graft survival as compared with other patients (Figure 2). Similarly, patients with enhanced intrarenal CD20 mRNA expression had longer graft survival in TCMR (P < 0.01). Low mRNA expression of TGF-β1, RANTES, CD20, and FoxP3 in the biopsy revealing early AMR implied an increased risk for renal graft failure within next 12 mo [hazard ratio (HR) 11.96, P < 0.05; HR 5.61, P < 0.05; HR 11.46, P < 0.01; and HR 6.38, P < 0.05, respectively], and a similar effect was found in TCMR only for CD20 (HR 10.47, P < 0.05; Table 3). The analysis of multiway frequency tables confirmed the influence of low TGF-β1 intragraft mRNA expression during early AMR on renal graft failure (P < 0.01), independently of the treatment modality.

Figure 2.

Kaplan–Meier analysis of variables on renal graft outcome. (A) Intragraft TGF-β1 mRNA in AMR (log rank statistic 10.34; P < 0.01). (B) Intragraft RANTES mRNA in AMR (log rank statistic 7.32; P < 0.01). (C) Intragraft CD20 mRNA in AMR (log rank statistic 12.74; P < 0.001). (D) Intragraft FoxP3 mRNA in AMR (log rank statistic 7.56; P < 0.01).

Table 3.

Intrarenal gene expression during the early acute rejection and the risk of graft failure

	Variable	Hazard ratio	95% Confidence interval	P
AMR	TGF-β1 ≤ 2 (n = 11)	11.96	1.41 to 101.29	0.023
	RANTES ≤ 16.71 (n = 7)	5.61	1.38 to 22.73	0.016
	CD20 ≤ 5.7 (n = 5)	11.46	2.17 to 60.49	0.004
	FoxP3 ≤ 1.3 (n = 4)	6.38	1.41 to 28.89	0.016
TCMR	CD20 ≤ 31 (n = 8)	10.47	1.09 to 101.01	0.042

Low intragraft TGF-β1 (RQ ≤ 2), RANTES (RQ ≤ 16.71), CD20 (RQ ≤ 5.7), and FoxP3 (RQ ≤ 1.3) gene expressions in the biopsy with proved early AMR prior to the therapy and low intragraft CD20 (RQ ≤ 31) gene expression in the biopsy with proved early TCMR prior to the therapy were related with an increased risk of renal graft failure within the first 12 mo after kidney transplantation (univariate Cox regression model).

Discussion

The aim of this study was to evaluate the expression patterns of selected genes during early acute AMR and early acute TCMR. In contrast to some reports,¹⁰ we found significant differences between AMR and TCMR. Interestingly, T cell and CTL infiltrates were only slightly higher in TCMR, suggesting similar levels of T cell infiltration (total and CTL) in TCMR and AMR. The higher levels of FoxP3 in TCMR suggest a stronger counter-regulation by Tregs in TCMR compared with AMR. Because CCL5 (RANTES) seems to be involved in Treg recruitment, the higher RANTES expression in TCMR might explain the FoxP3 enrichment in cellular rejection. Remarkably, B cell infiltration (CD20) was also higher in TCMR versus AMR. Very recent data suggest an association of B cell expansion and allograft tolerance (RISET [Reprogramming the Immune System for Establishing of Tolerance]/Tolerance Indices Network, Hernandez et al., submitted). In summary, our data suggest a higher level of regulation in cellular versus humoral rejection that might explain the better long-term outcome of antirejection therapy in the former.

Therefore, we asked then whether a relation between gene expression and outcome is seen in early AMR and TCMR.

As suggested above, AMR shows fewer events of regulation (Tregs and B cells) compared with TCMR that have a better outcome in general, but looking at the failed grafts following AMR, the lack of regulation (low FoxP3, RANTES, TGF-β1, and CD20) was even more prominent. However, effector markers (T cells, CTL, macrophage inflammatory protein, etc.) do not predict the outcome of early AMR. The strongest prediction of graft failure within the next 12 mo is defined by low TGF-β1 and CD20 gene expression levels in the biopsy, revealing early AMR (HR 11.96, P = 0.023 and HR 11.46, P = 0.004, respectively). In fact, multiway frequency tables confirmed the key association of low TGF-β1 intragraft mRNA expression levels and renal graft failure after AMR, independently of the treatment modality. These data suggest that a lack of adequate counter-regulation might be a key element in the pathogenesis of severe AMR, resulting in graft loss.

In contrast, graft failure after early TCMR was associated with a higher effector T cell ratio (CTL/CD3). In contrast to AMR, markers of regulation (FoxP3 and TGF-β1) were not significantly different between the TCMR responders and nonresponders, suggesting a dominance of uncontrolled CTL in the pathogenesis of severe early TCMR, resulting in graft loss. Like in AMR, a lack of B cells is associated with graft failure, making a protective (“tolerogenic”) role for B cells possible.

TGF-β1 has been suggested as having both immunosuppressive and profibrogenic properties. Although in the long term the higher TGF-β1 expression within the renal allograft is associated with the poor kidney graft outcome,¹³ early after transplantation its higher expression might be related with improved tissue remodeling and repair after ischemic injury¹⁶ along with a more profound suppression of host immune response and thus be associated with better graft outcome.¹⁷ TGF-β1 has been shown to be critical in the generation of CD4⁺CD25⁺Foxp3⁺ Tregs.¹⁸ The increased frequencies of Tregs were described as an additional mechanism that induces alloimmune tolerance.¹⁹ The quantification of Tregs in allograft rejection is thus of considerable clinical interest. The presence of intragraft FoxP3-positive cells was not confined to tolerated grafts but was suggested to be a part of the normal immune response during rejection.²⁰ TGF-β1-driven Tregs may prevent an aberrant chronic T cell hyperactivation and were shown to inhibit inflammatory response to transplanted grafts and enhance its survival.²¹ Veronese et al.,⁹ using immunohistochemistry, analyzed 80 human renal transplant biopsies for the Treg transcription factor FoxP3. They found FoxP3-positive cells to be present within the interstitium in acute cellular rejection at a greater density than those in AMR. Similarly to the lower FoxP3 protein expression levels observed by Veronese et al., we find a lower level of FoxP3 mRNA expression in early AMR as compared with that in early TCMR. A recently published study by Bunnag et al. showed an association between FoxP3 mRNA expression in case biopsies and rejection.²² In this study, the authors observed no differences in FoxP3 mRNA expression between AMR and TCMR. They, however, evaluated just three early AMR cases; the other 11 AMRs were chronic AMR cases that occurred after the first year posttransplant. In multivariate analysis, higher FoxP3 mRNA levels did not correlate with favorable 6-mo graft outcomes, even when the analysis was restricted to biopsies with rejection. Contrary to this study²² and in agreement with the results of our study, Bestard et al.²³ suggest that immunostaining of FoxP3⁺ Tregs in protocol biopsies seems to be useful for the prediction of long-term kidney transplant outcome. Similarly, in the mouse renal transplant model, the presence of FoxP3 Tregs decreased during ongoing rejection.²⁴

Additionally, TGF-β1 might act as a potent inhibitor of complement C3 secretion under inflammatory conditions.²⁵ The locally synthesized complement component C3 was shown to contribute to both TCMR and AMR, with the highest expression levels found in C4d-positive indication biopsies with signs of cellular rejection.²⁶

In our study, there were also trends toward higher expression of markers related to B cell infiltration, such as CD20 within grafts with better prognosis. The B-lymphocyte infiltrate during acute rejection was shown to be associated with poor prognosis in some reports^27,28 although not in others.²⁹ B cells may be functioning as professional antigen-presenting cells in the graft and thus related to more severe rejection outcome. Recently, CD20 infiltrates during acute cellular rejection have been shown to possibly represent a heterogeneous population that might not help to identify a graft at risk for failure.³⁰ Moreover, very recent data suggest an association between enhanced B cell levels and operational tolerance in kidney transplant patients (unpublished observations).

In conclusion, we documented that gene expression profiles differ between early AMR and early TCMR of renal allografts. The differences are even more pronounced in grafts with poor outcome after rejection, supporting the view of different pathogenesis for AMR and TCMR. Aggressive therapeutical maneuvers in acute rejection might be associated with late complications, such as tumors, diabetes, and cardiovascular diseases. Phenotypical molecular traits of both AMR and TCMR identified in our study, in particular the lower distribution of CD20-positive cells associated with poor graft prognosis, are thus of potential clinical relevance. Rare occurrence of early AMR warrants larger prospective multicenter and biomarker-driven clinical trials to validate our results to include molecular phenotypes of rejection in management decisions.

Concise Methods

Patient Population

A total of 730 kidney transplant procedures were performed in our center in Prague from January 2002 through October 2005. Early acute AMR (within the first 3 wk posttransplant) was diagnosed in 21 patients (2.9%). A control group of 43 patients with TCMR out of these 730 patients were matched for posttransplant time, age, gender, and immunosuppression. Their basic demographic parameters are shown in Table 4. All patients were treated with maintenance immunosuppression using either tacrolimus or cyclosporin A (CsA) along with mycophenolate mofetil (MMF) and corticosteroids. In cases of increased panel reactive antibody (PRA) frequencies (>50%) before transplantation, patients received induction with antithymocyte globulin (ATG, Thymoglobuline, Genzyme) or anti-CD3 monoclonal antibody (OKT3-Muromonab, Janssen-Cillag). A survey of maintenance immunosuppression used is shown in Table 4. All patients were on follow-up in our outpatient clinic. Graft failure was defined as a return to dialysis treatment.

Table 4.

Basic patient characteristics

	AMR	TCMR
N	21	43
Age (yr)	47.0 ± 12.4	47 ± 10.7
First/second/third transplants	13/8/1	40/3/0
Donor gender (male %)	16/(76.2%)	22/(51.2%)
Number of A mismatches	1.1 ± 0.6	1.3 ± 0.6
Number of B mismatches	1.4 ± 0.6	1.3 ± 0.7
Number of DR mismatches	0.9 ± 0.7	1.0 ± 0.7
PRA (%)	44 ± 36	9 ± 16a
Hemodialysis duration (mo)	35.1 ± 26.5	27.2 ± 26.5
Immunosuppression: ATG or OKT3-Muromonab induction	10 (47.6%)	11 (25.6%)
Tacrolimus	19 (90.5%)	36 (83.7%)
CsA	2 (9.5%)	7 (16.3%)
MMF	20 (95.2%)	39 (90.7%)
SIR	1 (4.8%)	4 (9.3%)
Early AMR/TCMR:
Diagnosis (day)	9.0 ± 2.8	8.6 ± 4.0
Biopsy Cr (mg/dl)	5.1 ± 2.8	4.4 ± 3.0
Biopsy CCr (ml/min)	27.7 ± 16.4	33.5 ± 19.0
C4d+ (%)	100	0
CDCXM+ (%)	90.5	Not done
FXCM+ (%)	90.5	Not done
12 months Cr (mg/dl)	2.0 ± 0.8	1.7 ± 0.7
12 months CCr (ml/min)	43.5 ± 17.8	61.9 ± 20.1b

CCr, creatinine clearance; Cr, creatinine; SIR, sirolimus.

^aP < 0.001.

^bP < 0.05.

Histology

Biopsies were indicated either in the case of missing graft function within the first 5 to 7 postoperative days (delayed graft function) or stagnation of decreasing serum creatinine at pathologic levels or of rising serum creatinine. Biopsy procedures using 14-gauge biopsy needles were performed under ultrasound control. Diagnosis was established on the basis of histologic verification using the Banff 05 classification.³¹ Patients signed their informed consent before each biopsy to participate in the gene expression profile study, and the protocol was approved by the Ethical Committee of the Institute for Clinical and Experimental Medicine, Prague, Czech Republic.

The diagnosis of early AMR was characterized by C4d-positive immunohistology within the first 2 wk posttransplant along with evidence of donor-specific antibodies. C4d was documented using anti-C4d monoclonal antibody (Quidel Corp.) on frozen sections by indirect immunofluorescence method or immunoperoxidase methods on paraffin sections with a polyclonal anti-C4d antibody (Biomedika).

Small portions of renal tissue from the cortex were placed into tissue storage reagent RNAlater (Sigma) immediately after biopsy and stored at −20°C for further gene expression analysis, although most of the renal tissue taken by core biopsy was used for routine histology.

Evidence of Anti-HLA Antibody

Anti-donor-HLA antibody was demonstrated by either cytotoxic cross-match (CDCXM) or flow cytometry cross-match (FCXM). These tests were performed invariably if histology suspected early AMR. Non-HLA endothelial antibodies were not determined.

Treatment of Early Rejection

Ten patients with early AMR were treated using repeated sessions of plasmapheresis, and 11 patients were treated by a combination of repeated plasmapheresis followed by IVIGs (0.5 g/kg, Endobulin, Kiovig, Baxter-Immuno).

Acute TCMR BANFF I and IIa were treated with a methylprednisolone (MP) bolus, whereas ATG was administered in patients with rejections of BANFF IIb and III or in a case of steroid resistance. In eight cases that showed histologic signs of early AMR in combination with TCMR, a combined therapy of plasmapheresis/IVIG and MP/ATG was used as appropriate.

RNA Isolation and Real-Time Quantitative RT-PCR

The renal tissue was homogenized; total RNA was extracted using RNA Blue (Top-Bio) and reversely transcribed into cDNA, using the SuperScript II Reverse Transcriptase (Invitrogen). Complementary DNA was amplified by real-time quantitative PCR (TaqMan, 7300 Real Time PCR Systems, Applied Biosystems) using commercial primers and fluorogenic probes. Samples were tested for genomic DNA contamination and, if tested positive, were excluded from the study. On the basis of our preliminary data, we selected the following markers for gene expression analysis: cytokines such as TGF-ββ1, TNF-α, MCP-1, and RANTES (CCL5), for quantifying T, B, or plasma cell infiltration the markers CD3, CD20, and IgJ, respectively, FoxP3 as master transcription factor of Tregs, the cytotoxic markers GZMB and PRF for quantifying cytotoxic T cells/NK cells, and HMOX1 as a cytoprotective stress protein. Specific gene expression was calculated relative to that of the housekeeping gene hypoxanthine guanine phosphoribosyl transferase (HPRT), and the calibrator sample [comparative threshold cycle method (2^{−ΔΔ[supi]cT}). RQ software, Applied Biosystems) and was expressed as RQ.

Statistical Analysis

Basic statistical parameter data are given as absolute and relative frequency, average and SD, or median and range. Survival curves were estimated using the Kaplan–Meier method. Agreement between groups was tested using the log-rank test. The intergroup difference was analyzed by the chi-square test for discrete variables and t test for continuous variables. The differences in gene expression patterns were analyzed after logarithmic transformation, and their correlations were analyzed using the Spearman rank correlation test. Due to a strong correlation between the transcripts, Bonferroni correction modified by Sankoh was applied to correct for multiple testing.³²The univariate Cox regression model was used for analyzing the risk for renal graft failure. The influence of intrarenal gene expression in combination with the different treatment of AMR on graft survival was analyzed using the log-linear model in multiway frequency tables. The receiver operating characteristic curve analysis was used to calculate the cutoff values for low and high intrarenal gene expression. Unless stated otherwise, the P value <0.05 was considered to be statistically significant. Calculations were performed using the StatsDirect 2.6.6 program (StatsDirect).

Disclosures

None.