The authors have declared that no competing interests exist.
Conceived and designed the experiments: LL JJTHR. Performed the experiments: LL. Analyzed the data: LL JK JJTHR. Contributed reagents/materials/analysis tools: JK NC IJMtB FJB. Wrote the paper: LL JK JJTHR. Corrected the manuscript: JK JJTHR SF.
The Endothelial Protein C Receptor (EPCR) is expressed on leukocytes, on endothelium of large blood vessels and to a lesser extent on capillaries. Membrane bound EPCR plays an important role in the activation of protein C which has anticoagulant, anti-inflammatory and cytoprotective effects. After cleavage by a protease EPCR is also found as a soluble protein. Acute rejection of kidney allografts can be divided in T-cell-mediated rejection (TCMR) and antibody-mediated (ABMR) rejection. The latter is characterized by strong activation of coagulation. Currently no reliable non-invasive biomarkers are available to monitor rejection.
Renal biopsies were available from 81 renal transplant patients (33 without rejection, 26 TCMR and 22 ABMR), we had access to mRNA material, matched plasma and urine samples for a portion of this cohort. Renal EPCR expression was assessed by RT-PCR and immunostaining. Plasma and urine sEPCR levels were measured by ELISA.
ABMR patients showed higher levels of EPCR mRNA than TCMR patients. EPCR expression on glomeruli was significantly elevated in ABMR patients than in TCMR or control patients. In the peritubular capillaries EPCR expression was higher in ABMR patients than in control patients. EPCR expression was higher in tubules and arteries of rejection patients than in control patients. Plasma sEPCR levels did not differ. Urine sEPCR levels were more elevated in the ABMR group than in patients with TCMR or without rejection. ROC analysis demonstrated that urinary sEPCR is appropriate to discriminate between ABMR patients and TCMR or control patients. We conclude that urinary sEPCR could be a novel non-invasive biomarker of antibody mediated rejection in renal transplantation.
The Endothelial Protein C Receptor (EPCR) is a type 1 transmembrane glycoprotein which belongs to the CD1 receptor family
The soluble form of EPCR (sEPCR), resulting from the cleavage of the extracellular domain of the membrane bound EPCR (mEPCR) by a metalloprotease
Currently the role of the EPCR/APC complex in renal transplantation is unknown; however APC has been extensively studied in inflammation settings and in sepsis. For example, Gupta
There are two types of acute allograft rejection that can occur either separately or together: T-cell-mediated rejection (TCMR) and acute antibody-mediated rejection (ABMR). TCMR is the most common form of acute allograft rejection, caused by effector T-cells that infiltrate and proliferate in the graft (-draining lymph nodes) leading to graft rejection
In the current study we investigate the EPCR expression pattern in kidney transplants on both mRNA and protein level; and correlate plasma and urine sEPCR levels upon acute renal allograft rejection. We describe how urinary sEPCR can distinguish between ABMR and TCMR.
Control Group (n = 33) | ABMR Group (n = 22) | TCMR Group (n = 26) | |
Gender (M/F) | 23/10 | 13/9 | 15/11 |
Age in years | 49 (19–75) a | 40 (11–63) a | 48.5 (16–68) |
Serum creatinine at the time of the biopsy ( µmol/L) | 130 (69–334) |
348 (84–881) |
335 (113–1341) * |
Time (in days) between transplantation and renal biopsy | 234 (0–771) | 399.5 (5–6187) | 47.5 (5–3675) |
GFR (mL/min/1.73 m2) | 48,78 (13,87–108.93) |
13.93 (5.12–122.23) |
16.88 (3.48–61.28) * |
Panel reactive antibody at the time of biopsy | 0 (0–24) * | 24 (0–100) *a | 1 (0–78) a |
Polycystic kidney disease | 7 | 7 | 3 |
Diabetes mellitus | 0 | 1 | 0 |
Focal glomerulosclerosis | 2 | 4 | 2 |
Hypertension | 4 | 4 | 5 |
Immune complex mediated diseases | 8 | 1 | 5 |
Vasculitis | 1 | 0 | 1 |
Urinary tract infection | 2 | 0 | 1 |
Other | 9 | 5 | 9 |
Mean age in years (SD) | 50,5 (16,2) | 45.6 (16.6) | 45.4 (11.9) |
No. Cadaveric/No. Living | 17/16 | 13/9 | 16/10 |
HLA mismatch | 3 (0–6) |
4 (3–6) |
3 (0–6) a |
Date are shown as median and range unless stated otherwise
a: p<0.05 (Mann-Whitney Test)
, *: p<0.001 (Mann-Whitney Test)
More HLA mismatches were present in patients with ABMR compared with control (p<0.001) and TCMR groups (p<0.05) (
PRA concentrations were measured before transplantation (before Tx), at the time of transplantation (Tx), at the time of biopsy and after the biopsy. PRA levels are shown for control (in black), antibody-mediated (ABMR, in light grey) and for T-cell-mediated rejection (TCMR, in dark grey) patients. PRA values within the groups did not differ significantly over time. However, at time of biopsy (i.e. during the acute rejection episode) PRA values in the ABMR group were significantly higher than in the TCMR group (P<0,05) and the control group (P<0,001). Results are shown as mean and range. * p<0.05 (Mann-Whitney Test)
All patients received immunosuppressive treatment consisting of CD25mAb (induction), corticosteroids, mycophenolate and a calcineurin inhibitor. Acute cellular rejections were treated with pulse doses of methylprednisolone 500 mg iv for 6 days. Antibody mediated rejections were treated with plasmapheresis for 7 days and rabbitATG (rATG). The starting dose of rATG was 5 mg/kg, 3 to 5 gifts were administered over 14 days. Dosages were titrated based on the total lymphocyte count after each administration (>300×109/L: dose 5 mg/kg;>200×109/L but <300×109/L: dose 3 mg/kg; >150×109/L but <200×109/L: dose 2 mg/kg; <150×109/L: no administration).
Control Group | ABMR Group | TCMR Group | |
Tubulitis | 0 (0 – 1) |
1 (0 – 3) |
1 (1 – 3)a § |
Mononuclear Cell Interstitial Inflammation | 0 (0 – 1) |
1 (0 – 3) |
2 (1 – 3)a § |
Glomerulitis | 0 (0 – 2) |
0,5 (0 – 3) |
1 (0 – 3)§ |
Arteriolar Hyaline Thickening | 0 (0 – 2) | 0 (0 – 2) | 0 (0 – 2) |
Intimal Arteritis | 0 (0 – 0) |
0 (0 – 3) |
0 (0 – 2)§ |
Glomerulopathy | 0 (0 – 2)a | 0 (0 – 3)a | 0 (0 – 1) |
Interstitial Fibrosis | 0 (0 – 2) | 0 (0 – 2) | 0 (0 – 3) |
Tubular Atrophy | 1 (0 – 2) | 1 (0 – 2) | 1 (0 – 3) |
Vascular Fibrous Intimal Thickening | 0 (0 – 1) | 1 (0 – 2) | 0 (0 – 3) |
Mesangial Matrix Increase | 0 (0 – 1) | 0 (0 – 3) | 0 (0 – 3) |
Capillaritis | 0 (0 – 2)a b | 0 (0 – 2)a | 0 (0 – 2)b |
No. C4d positive | 0 (0%) |
19 (86%) |
0 (0%)§ |
Data are shown as median and range.
a, b: p<0.05 (Mann-Whitney Test)
, §: p<0.001 (Mann-Whitney Test)
As shown in
EPCR mRNA levels were measured with qPCR on whole kidney biopsies. Results are shown as ratio between EPCR and HPRT Ct. Antibody-mediated (ABMR) rejection patients showed higher levels of EPCR mRNA than T-cell-mediated rejection (TCMR) patients. Results are shown as median, interquartile range and range. ** p<0.001 (Mann-Whitney Test)
In order to visualize the expression pattern of EPCR in transplant biopsies, we performed immunostainings.
Representative immunostainings of kidney biopsies for EPCR in glomeruli (×32 magnification), peritubular capillaries (×64), arteries (×64), veins (×64) and tubules (x64). Arteries were always positive; therefore no picture with a score of 0 is shown. For tubules no score of 3 was assigned.
EPCR expression was significantly higher in patients with ABMR compared to patients without rejection or with TCMR in glomeruli (p<0.001 and p<0.05, respectively,
Semi quantitative scores (on a scale from 0 to 5) of EPCR immunostainings. EPCR expression on glomeruli (A) and peritubular capillaries (B) are significantly elevated in ABMR patients than in TCMR or control patients. EPCR expression is higher in arteries (C) and in tubules (D) of rejection patients than in control patients. Results are shown as median, interquartile range and range. * p<0.05 (Mann-Whitney Test) ** p<0.001 (Mann-Whitney Test)
In arteries we observed higher EPCR expression in patients with ABMR and TCMR compared to patients without rejection (p<0.05,
EPCR scores of the venous endothelium did not show any differences between ABMR, TCMR and control (data not shown).
Plasma and urine levels of sEPCR were determined by ELISA. The urinary concentration of sEPCR was corrected for dilution. We confirmed the presence of intact sEPCR protein, and not degradation products by western blot analysis (data not shown).
Plasma sEPCR levels were not significantly different between ABMR patients (599 ng/mL [67 - 1355]), control patients (623 ng/mL [418 – 1102], p>0.05) or patients with TCMR (508 ng/mL [381 – 945], p>0.05) (
Plasma and urine sEPCR concentration were measured with ELISA, urinary sEPCR concentration is corrected for the urine dilution by dividing the concentration by the urinary creatinine concentration. Plasma sEPCR levels did not differ. Urine sEPCR levels are elevated in the ABMR group than in patients with TCMR or without rejection. Results are shown as median, interquartile range and range. * p<0.01 (Mann-Whitney Test)
We found no correlation between protein EPCR scores in the kidney and sEPCR concentration in plasma or urine (data not shown).
Recently, Sis et al.
Severe antibody-mediated inflammation of the microcirculation of the transplant, defined as the composite of Banff g and ptc scores [Sis et al. 2012 Am J Transplant], associated with higher EPCR scores in both the glomeruli (A) and the peritubular capillaries (B) as well as the composite EPCR score of glomeruli and peritubular capillaries (C). Severe microcirculatory inflammation associated with higher levels of soluble EPCR in the urine at time of biopsy. Data are represented as box-and-whisker plots with median and (interquartile) range. * p<0.05 (Mann-Whitney Test)
In order to evaluate the possible usefulness of urinary sEPCR as a non-invasive biomarker for ABMR, a ROC calculation was performed. The area under the ROC curve was used to summarize the discriminative ability of the test, the closer to 1 the better.
As shown in
Receiver operating characteristic (ROC) analysis of urine sEPCR concentration for the prediction of ABMR in kidney transplantation. ROC analysis demonstrated that urinary sEPCR is appropriate to discriminate between ABMR patients and TCMR or control patients. In grey: ABMR vs. Control, in black: ABMR vs. TCMR.
The cut-off value with the highest combined sensitivity and specificity for discriminating between ABMR patients and control patients was at 21.6 ng/mmol creatinine (75% sensitivity, 80% specificity). The optimum sEPCR concentration for discriminating between ABMR and TCMR patients was 22.1 ng/mmol creatinine (75% sensitivity, 82% specificity). The dotted line represents the line of equal sensitivity and specificity.
This study is the first to describe the levels of membrane bound and soluble EPCR during acute renal allograft rejection in a transplant patient cohort.
We observed higher EPCR mRNA levels in ABMR patients compared with TCMR patients. On the protein level, we observed in glomeruli more EPCR expression in ABMR patients compared with patients without rejection or with TCMR. In the peritubular capillaries EPCR expression was higher in ABMR patients than in control patients. Immunostaining also revealed a higher expression of EPCR in arteries and tubules from patients with ABMR and TCMR compared with patients without rejection. Finally we found increased concentrations of sEPCR in the urine of ABMR patients compared with patients with TCMR or without rejection. Although validation is needed, these findings together with the ROC analysis indicate that urinary sEPCR could be used as a non-invasive biomarker for antibody mediated kidney rejection.
There are some limitations to our study. Since it is a retrospective study some patients could not be studied for all the different tests. Furthermore the ABMR group contains a relatively small number of patients with urine sample available. The reason for this is the low incidence of ABMR within the renal transplant patient population in our institution. Nevertheless even with a population of 8 samples from the ABMR group and 21 from the TCMR group the ROC analysis achieves 80% power to detect a difference of 0.422 between the area under the curve under the null hypothesis of 0.5000 and an AUC under the alternative hypothesis of 0.875 using a two-sided z-test at a significance level of 0.05.
Another limitation of our study may be the lack of data on donor specific antibodies (DSA). ABMR diagnosis can be complex in the clinical setting, often requiring DSA. Indeed, C4d-negative ABMR is an increasingly recognized phenomenon
It is known from the literature that the expression of EPCR is not only limited on endothelial cells. On the mRNA level EPCR is transcribed in HUVEC
The roles of EPCR and protein C have been extensively studied in sepsis setting. EPCR and APC form a complex with PAR-1 and initiate biological effects such as anticoagulant, anti-inflammatory, anti-apoptotic and cytoprotective activities in vitro and in vivo
Importantly, Song
These findings clearly indicate that EPCR is part of a protective pathway. Considering this we assume that the higher EPCR expression during acute kidney rejection could be part of a protective mechanism for the graft. EPCR could exert its protective activities on several levels.
Firstly, higher EPCR expression could contribute to enhanced APC formation and therefore more activation of PAR-1 and its associated protective properties. This could suggest that an actively regulated protection mechanism involving the EPCR/APC/PAR-1 signaling cascade is taking place in the setting of acute allograft rejection.
Secondly, it is known that ABMR is characterized by activation of the coagulation cascade, resulting in elevated levels of FVIIa
EPCR not only exists as a membrane bound receptor but also as a soluble protein. sEPCR has a comparable affinity for APC as mEPCR
Considering this, it is not surprising to observe higher sEPCR production during rejection, knowing that allograft rejection is associated with endothelial damage and dysfunction especially in antibody mediated rejection
Renal transplantation is the most suitable therapy for end stage kidney disease. Despite recent progress in anti-rejection therapy, approximately 23% of transplanted patients undergo an episode of acute rejection within the first year post-transplant
Therefore we propose that urinary sEPCR could be of interest for diagnosing acute kidney antibody mediated rejection as proven by the ROC analysis which demonstrates that urinary sEPCR might be suitable to make the distinction between ABMR patients and non-rejecting patients (AUC = 0.875, p = 0.002) and importantly also between ABMR and TCMR patients (AUC = 0.875, p = 0.003). Although validation will be needed in a larger patient cohort, we conclude that urinary sEPCR could be a suitable candidate to diagnose antibody mediated renal rejection in the clinical setting, in a non-invasive way.
Eighty-one patients who underwent kidney transplantation between 1994 and 2008 were retrospectively selected from the patient population of the Academic Medical Center at the University of Amsterdam. Patients were selected based on pathological diagnosis. Renal biopsies that fulfilled the minimal criteria for diagnostic assessment (7 glomeruli and at least 1 artery) according to the Banff 1997 criteria were available from all patients
Patients were divided in 3 groups, according to the biopsy diagnosis. The TCMR group consisted of 26 patients with interstitial infiltration, tubulitis or intimal arteritis
Time matched mRNA, derived from frozen transplant biopsies, was available for 14 patients in the control group, 13 in the TCMR group and 11 in the ABMR group as well as matched serum samples for 21 patients in the control group, 22 in the TCMR group and 9 in the ABMR group. Matched urine samples (taken from 24 hours urine samples) were available for 22 patients in the control group, 21 in the TCMR group and 8 in the ABMR group.
Circulating donor specific antibodies are not systematically measured in kidney transplant patients in our institution. Consequently, according to the Banff criteria
Seven patients who showed signs of both TCMR and ABMR were included in the ABMR group.
All biological material was collected for previous studies
mRNA from complete kidney tissue was extracted from frozen renal biopsies cut into 25 µm thick sections using a Microm HM500 cryostat (Adamas Instruments BV) and collected in an Eppendorf tube containing TRIzol (Invitrogen, Breda, The Netherlands). After 5 minutes incubation at room temperature RNA was extracted using chloroform. cDNA was synthesised using a standard procedure.
Real-time reverse-transcriptase polymerase chain reaction (RT-PCR) was performed on a Lightcycler® 480 Real-Time PCR System using Lightcycler® 480 SYBR Green I Master (Roche Applied Science, Mijdrecht, the Netherlands). Specific primers were designed (synthesized by Eurogentec, Liège, Belgium) for human PROCR (EPCR) (forward
C4d staining was performed on frozen sections if available using a mouse anti-human C4d antibody (AbD Serotec, Dusseldorf, Germany, ref. 2222-8004). Alternatively, C4d staining was performed on paraffin sections using a rabbit anti-human C4d antibody (Cell Marque, Rocklin, USA, ref. 404A-14), as described previously
Paraffin sections of kidney biopsies were immunostained for EPCR. Antigen retrieval with Tris EDTA pH 9 (20 minutes at 121°C) was performed for optimal staining. 4 µm thick sections were incubated for 60 hours at 4°C with goat anti human EPCR monoclonal antibody (452 ng/mL, kind gift of Dr. C. Esmon, Oklahoma Medical Research Foundation). Primary antibody binding was detected with a peroxydase kit (30 minutes incubation at room temperature, Powervisoin poly HRP-Anti-mouse IgG, Immunologic, Duiven, Netherlands). Staining was developed with Ultra DAB (Immunologic, Duiven, Netherlands).
On EPCR stained sections, the intensity of immunostaining was scored semiquantitatively on a scale from 0–3 (respectively absent, weak, moderate or strong) following the method of Faust
Twenty µL aliquots of urine and plasma were pre-treated with 10 µL 1N HCl. After 10 minutes of incubation at room temperature acidified samples were neutralized with 9 µL 1N NaOH. Prior to the assay samples were diluted with Calibrator Diluent (RD5–24 provided with the ELISA kit) to reach a total dilution factor of 15.6 for urine samples and 39 for plasma samples. Soluble EPCR concentrations in urine and plasma were measured using Human EPCR Quantikine kit (R&D System, Abingdon, UK) according to the manufacturer's protocol. According to the data sheet the mean minimal detectable doses is 0.064 ng/mL. Optical densities were measured using a microplate reader set to 450 nm and were corrected with a wavelength of 570 nm. A standard curve was created using the trial version of MasterPlex® ReaderFit software (Hitachi Solution America Ltd.), capable of generating a four parameter logistic curve fit.
The urinary concentration of sEPCR was corrected for the urine dilution by dividing the urinary concentration of sEPCR by the urinary creatinine concentration (urine creatinine concentration did not differ between the groups, data not shown).
The GFR was estimated with use of the CKD-EPI formula
All data sets were tested for their distribution prior to analyses.
Data are expressed as median and range unless stated otherwise. Wilcoxon-Mann Whitney test, Kruskal Wallis, Spearman's correlation test, the multivariate analysis and Receiver Operating Characteristic (ROC) analyse were performed using SPSS 19 software (IBM Corporation, Stomer NY USA) and the R computing environment (