Osteopontin level correlates with acute cellular renal allograft rejection

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Osteopontin level correlates with acute cellular renal allograft rejection Jina Wang, MD, PhD,a,b Qunye Tang, MD,a,b Yongyin Qiu, MD,a,b Ming Xu, MD,a,b Ruiming Rong, MD,a,b and Tongyu Zhu, MDa,b,* a b

Department of Urology, Zhongshan Hospital, Fudan University, Shanghai, China Shanghai Key Laboratory of Organ Transplantation, Shanghai, China

article info

abstract

Article history:

Background: Osteopontin (OPN) is a potent proinflammatory cytokine that is upregulated in

Received 8 July 2012

cell-mediated immunity and various inflammatory states of the kidney. However, the

Received in revised form

relationship between OPN levels plasma/urine and acute renal allograft rejection is still

31 July 2012

unknown. Therefore, we assessed the relationship between OPN levels in plasma/urine

Accepted 3 August 2012

and acute cellular rejection post-renal transplantation.

Available online 23 August 2012

Materials and methods: Clinical data and biologic samples of renal transplant recipients were analyzed retrospectively. Patients with biopsy-proved acute cellular rejection (ACR)

Keywords:

(n ¼ 22), protocol biopsy-proved non-rejection (non-R) (n ¼ 16), and living related donors as

Osteopontin

healthy control (HC) (n ¼ 10) were involved in this study. OPN level in plasma and urine was

Plasma

detected using the human OPN enzyme-linked immunosorbent assay kit. Type and grade

Urine

of ACR were diagnosed based on Banff’ 03 classification criteria of renal allograft pathology.

Renal transplantation

No prisoners or organs from prisoners were used in this study.

Acute cellular rejection

Results: Compared with non-R patients and HC, plasma and urine OPN levels in ACR patients were significantly increased (P < 0.05), whereas there was no significant difference between non-R patients and HC (P > 0.05). In ACR patients, plasma OPN level was positively correlated with Banff grading of acute rejection, and a cut-off value of 24.20 ng/mL was further demonstrated a good clinical value in receiver operation characteristic curve. Conclusions: The data obtained suggested that assessment of OPN levels in plasma and urine, especially in plasma, should be useful in predicting and evaluating the severity of ACR in renal transplant recipients. Crown Copyright ª 2013 Published by Elsevier Inc. All rights reserved.

1.

Introduction

Osteopontin (OPN), an arginine-glycine-aspartate domaincontaining extracellular matrix protein, can be produced by various tissues and cells in many conditions. As known as early T lymphocyte activation-1, it plays multifunctional roles

in immune response by influencing cell survival, migration, and differentiation, especially on T cells [1]. OPN is also identified as an amplifier of the Th-1 immune response and contributes to the development of Th-1 related disease [2,3]. In many inflammatory diseases, such as rheumatoid arthritis and multiple sclerosis, elevated OPN expressions were

Jina Wang and Qunye Tang contributed equally to this study. * Corresponding author. Shanghai Key Laboratory of Organ Transplantation, Zhongshan Hospital, Fudan University, Shanghai 200032, China. Tel.: þ86 21 64037287; fax: þ86 21 64038038. E-mail address: [email protected] (T. Zhu). 0022-4804/$ e see front matter Crown Copyright ª 2013 Published by Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.jss.2012.08.006

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reported and subsequently demonstrated to be facilitative for these diseases [4e8]. In a recent study of graft-versus-host disease (GVHD), a major immune rejection response after hematopoietic stem cell transplantation, elevated serum OPN level was found positively co-related with the pathogenesis of the disease. Furthermore, it was demonstrated to be critical for the disease initiation and persistence [4]. All these studies suggest a strong tie between OPN and immune-related diseases, and OPN level, especially in body fluids, may be predictable for these diseases. Acute renal allograft rejection is actually a kind of inflammatory response with significant accumulation and activation of monocytes, especially lymphocytes in the graft. Acute rejection can also be characterized as a Th1-directed immune response. In rats, OPN upregulation has been observed in several models of acute or chronic renal injury [9], with OPN displaying a peculiar expression pattern. One study suggested that inducible expression of OPN in the tubular epithelium may have a pathogenic role in acute renal allograft rejection by mediating interstitial monocyte infiltration and possibly tubular regeneration [10]. However, the direct relationship between OPN level and acute cellular rejection (ACR) of renal allografts is still not clear. Thus, we sought to characterize the predictive interaction between OPN and ACR by examining the level of OPN in body fluids (peripheral blood and urine) post-transplantation. In this study, we found that plasma OPN and urine OPN levels in patients with non-R were similar as in health control (HC) (P > 0.05), but significantly lower compared with ACR group (P < 0.01). The plasma OPN level was positively correlated with Banff grading of acute rejection (P < 0.05). Moreover, the receiver operating characteristic (ROC) curve demonstrated a plasma OPN cut-off value of 24.20 ng/mL, which was accepted as the discriminative level between patients with and without AR.

2.

Materials and methods

2.1.

Selection and description of participants

Twenty-two patients with biopsy-proved acute cellular rejection (ACR), 16 patients with stable renal function and protocol biopsy-proved non-rejection (non-R), and 10 living related donors as HC were involved in this study. Patients with fever of undetermined origin, pneumonia, and other causes of renal graft dysfunction (e.g., infection, pyelonephritis, CNI toxicity) were excluded from this study. Peripheral blood and first urine in the morning (urina sanguinis) were collected from these patients and living related donors. All these living related donor renal transplantations and this study were approved by the Ethics Committee of Zhongshan Hospital, Fudan University (Shanghai, China). Procedures in this study were in accordance with the Helsinki Declaration of 1975. No prisoners or organs from prisoners were used in this study. Informed consents were obtained from these patients and living related donors. All recipients started receiving combined immunosuppression from the first day after transplantation. Immunosuppressive agents in our center included mycophenolate

mofetil (MMF), prednisone (Pred), tacrolimus, or cyclosporine A.

2.2.

Samples and reagents

All peripheral blood and urina sanguinis samples tested in this study were collected before renal allograft biopsies or unilateral nephrectomy, so as to exclude the influence of biopsy and nephrectomy on the OPN levels. Plasma samples were separated from peripheral blood and frozen at e80 C until further analysis. Urina sanguinis samples were also frozen at e80 C until analysis. Type and grade of ACR were diagnosed based on Banff 03 classification criteria of renal allograft pathology. OPN was assayed in duplicate using a sandwich enzymelinked immunosorbent assay (Quantikine kit for Human Osteopontin Immunoassay; R&D Systems Inc., Minneapolis, MN, USA). The sample preparation and procedure were performed according to the manufacturer’s instructions.

2.3.

Statistical analysis

Student t-test was used to test the difference between two groups. ANOVA was used for statistics among multi-groups, and post hoc comparisons were then performed by Scheffe test. Spearman rank correlation coefficient and receiver operating characteristic (ROC) curve were also used to analyze results. Differences were considered statistically significant if the P value was less than 0.05.

3.

Results

3.1.

Characteristics of the transplant recipients

Table 1 shows the clinical profile of transplant recipients with ACR versus non-R group. There was no significant difference between the two groups with regards to age and the time of biopsy (P > 0.05). There was a significant difference of serum

Table 1 e Baseline characteristics of transplant recipients.

Age (y) Sex Male Female Biopsy time (mo) Scr at biopsy (mmol/L) Banff category IA IB IIA IIB Immunosuppression MMF þ CsA þ Pred MMF þ Tac þ Pred

ACR (n ¼ 22)

Non-R (n ¼ 16)

P value

44.00  14.36

45.06  17.35

>0.05

13 9 36.64  8.32 202.59  58.36

13 3 32.13  6.25 119.23  43.04

>0.05 <0.05

8 3 3 8

0 0 0 0

8 14

6 10

Tac ¼ tacrolimus; CsA ¼ cyclosporine A.

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creatinine (Scr) between these two groups (P < 0.05). As for the living related donors, there were 4 males and 6 females, aged 50.60  8.38 y.

3.2.

OPN level in plasma and urina sanguinis

All plasma and urine samples in the three groups were assayed in duplicate. Significant difference in plasma OPN levels were observed (P < 0.05) between the ACR and non-R patients, with mean OPN values of 41.84 ng/mL (41.84  18.51 ng/mL) and 19.38 ng/mL (19.38  8.23 ng/mL), respectively (Fig. 1A). There was no significant difference (P > 0.05) between the non-R and HC group, with mean OPN values of 19.38 ng/mL (19.38  8.23 ng/mL) and 12.23 ng/mL (12.23  5.95 ng/mL), respectively (Fig. 1A). We also assayed the OPN level in urina sanguinis (Fig. 1B) and got a similar conclusion as described above. The difference between the ACR and non-R patients was significant, with mean OPN values of 179.51 ng/mL (179.51  60.22 ng/mL) and 98.52 ng/mL (98.52  10.25 ng/mL), respectively (Fig. 1B). There was no significant difference between the non-R and HC group, with mean OPN values of 98.52 ng/mL (98.52  10.25 ng/ mL) and 76.32 ng/mL (76.32  9.86 ng/mL), respectively (Fig. 1B). These data demonstrated that OPN level in plasma and urine elevated significantly in patients who suffered from ACR.

3.3. Correlation between OPN level and other studied factors in ACR group We correlated the levels of OPN and other studied factors, such as serum creatinine (Scr) and histologic rejection grade, using Spearman rank correlation coefficient in ACR group (Table 2). We found that plasma OPN was positively correlated with acute rejection grade, but not with Scr level. The OPN level in urine was not found to correlate with acute rejection grade and Scr level. Student t-test was used to analyze plasma OPN level between grade IA and grade IIB patients (Fig. 2). Since the number of patients with grade IB and grade IIA were too small to do statistical analysis, those patients were not involved.

3.4. Clinical value of plasma OPN level in differentiating ACR from non-R The ROC curve was determined to differentiate ACR patients from non-R patients, and it demonstrated a plasma OPN value of 24.20 ng/mL as a cut-off value corresponding to the area under the ROC cure value of 0.923 [95% CI, 0.836e1.000], (P < 0.0001, Fig. 3). The ROC curve provided a sensitivity of 95.5% and specificity of 81.2% to identify ACR patients, suggesting a potential clinical value.

4.

Discussion

OPN is a secreted phosphoprotein that has a number of diverse biologic functions, including cell adhesion, migration, and signaling [11,12]. Although it was originally isolated from bone, OPN is also produced in the kidney, hence, also named as uropontin. In rodent and human kidney, OPN is constitutively expressed by distal tubular epithelium [13,14]. Its expression can also be induced and enhanced from proximal tubular epithelial associated with monocyte/macrophage infiltrates in numbers of rodent renal injury models [14e17] and human renal diseases [18e20]. However, the molecular mechanism of OPN expression regulation is still not completely understood. The hallmark of renal allograft acute rejection is tubulointerstitial inflammation; some researchers hypothesize that some intrarenal proinflammatory cytokines may act via autocrine/paracrine mechanism to stimulate OPN gene transcription and expression [10]. The consistently observed upregulation of OPN in areas of cellular infiltrate supports this

Table 2 e Correlation between OPN level and other studied factors in ACR group. Factor Fig. 1 e Quantitative analysis of OPN in plasma and urina sanguinis OPN levels in plasma and urina sanguinis samples from ACR group, non-R, and HC group were analyzed with human enzyme-linked immunosorbent assay kit. (A) OPN level in plasma. (B) OPN level in urine. NS means no significant difference.

Plasma OPN and Scr Plasma OPN and HRG Urine OPN and Scr Urine OPN and HRG

Number

Correlation coefficient

P value

22 22 22 22

0.043 0.739 0.035 0.014

0.77 <0.0001 0.82 0.87

HRG ¼ histologic rejection grade.

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Fig. 2 e Plasma OPN level in patients with different histologic rejection grade Statistical analysis of plasma OPN level between Banff grades IA and IIB. Grades were performed (P < 0.05). Patients with grade IB (n [ 3) and grade IIA (n [ 3) were not involved in statistical analysis because of the limited number.

hypothesis. Classic mediators of acute inflammation such as tumor necrosis factor-a and interleukin-1b strongly induce OPN expression [21]. Cytokine mRNA analysis on human renal allograft biopsies by a Polymerase Chain Reaction (PCR) based assay confirmed the presence of these two mediators in acute cellular rejection [22], while immunohistochemical studies demonstrate a marked tumor necrosis factor-a expression by infiltrating inflammatory cells and adjacent tubular cells in acute cellular rejection but not in normal kidney [1]. Another potential culprit that can induce OPN upregulation is endothelin-1 [23], which was shown to be upregulated on the tubular epithelial cells in acute rejection [15,24]. Taken together, these observations may explain the high level of

Fig. 3 e ROC curve of plasma OPN in patients The ROC curve was made so as to differentiate ACR patients from non-R patients. The open circle indicated the cut-off value of 24.20 ng/mL.

OPN expression in acute rejection. Nonetheless, other factors such as ischemia may also be involved. Is there correlation between the OPN level and the immunosuppressive regimen? In our center, all recipients started receiving combined immunosuppression from the first day after transplantation. Immunosuppressive agents included mycophenolate mofetil (MMF) þ prednisone (Pred) þ tacrolimus or MMF þ Pred þ cyclosporine A, and all patients involved in our study were with similar immunosuppressants plasma concentrations. We did not consider different combined immunosuppressive regimen as a variable factor for OPN level in this study. Since immunosuppressive regimen can prevent acute rejection episodes and the positive correlation we found between OPN level and acute rejection, theoretically, there may be a negative correlation between OPN level and immunosuppressive regimen. However, compared with healthy control who did not receive immunosuppressive regimen, patients without rejection had a little bit higher OPN level (no significant difference). Therefore, we think that there may be no obvious correlation between OPN level and immunosuppressive regimen. On the other side, the upregulated OPN may be the mediator and amplifier during AR. As a proinflammatory cytokine, OPN can not only directly induce the migration of inflammatory cells, but also trigger the production of some other chemokines such as Monocyte Chemoattractant Protein (MCP)-1 and Macrophage Inflammatory Protein (MIP)-1b. In the role of cellular immune response, OPN is regarded as a Th1 cytokine because it modulates cell-mediated immunity through promoting the Th1 response. In CD4þ T cells, OPN mRNA is expressed in Th1, but not Th2 cells [25], and soluble OPN can modulate the differentiation and proliferation of CD4þ and CD8þ T cells [17]. Alchi et al [10] reported that inducible expression of OPN in the tubular epithelium may have a pathogenic role in acute renal allograft rejection by mediating interstitial monocyte infiltration and possibly tubular regeneration. As a Th1-directed immune response, AR has significant monocyte accumulation and activation in the renal allograft, which is supposed to be initiated by chemoattractants, including OPN. Although previous studies revealed the possible relationship between OPN and inflammatory diseases or transplantation rejection, none of them stated OPN in peripheral fluids as a marker for noninvasive disease diagnosis. Zhao et al [4] recently found that in mice suffering from GVHD, the serum OPN level elevated significantly and persisted throughout the course of GVHD. Furthermore, blockade of OPN can attenuate GVHD with reduced accumulation of donor T cells in recipient organs [4]. One previous study [26] found that plasma osteopontin levels were significantly increased in non-rejecting patients compared with healthy volunteers. No differences could be detected in the rejecting patients compared either to non-rejecting patients or healthy volunteers. Our study found that in patients with acute rejection, the OPN level in plasma and urine elevated significantly. It is also interesting to note that the more severe of acute rejection, the higher concentration of OPN in plasma. However, the elevated plasma OPN level maybe the consequence and/or the promoter of acute rejection. To our knowledge, this is the first article, which states that plasma and urine OPN concentration correlate with acute

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cellular renal allograft rejection. In the present study, we demonstrated patients with ACR have higher levels of OPN in plasma and urine than patients without rejection. Besides these, we found that plasma OPN level was positively correlated with the severity of ACR. Plasma OPN may be a potential candidate for noninvasive diagnosis of ACR. It may also lead to the hypothesis that in acute rejection situation, treatment with an antibody against OPN may be clinically useful for reverting or attenuating acute rejection. Further prospective and multicenter studies to investigate the potential application of OPN would be worthwhile because of the limited number of patients in this study.

Acknowledgment This study was supported by grant from National Nature Science Foundation of China (81100533, 81100534, 81070595), Specialized Research Fund for the Doctoral Program of Higher Education of China (20110071120057), Science and Technology Commission of Shanghai Municipality (09411952000), Special Funds of 211 works of Fudan University (211Med-XZZD02), Shanghai Health Bureau (XBR2011019) and 11JC1402300.

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