EXPERIMENTAL AND CLINICAL TRANSPLANTATION

Objectives: It has been hypothesized that BK polyomavirus infection leads to nephropathy in kidney transplant patients via various plausible mechanisms, such as stimulation of chemokines. The CXCL11 gene may also play a role in BK polyomavirus-associated nephropathy. Our aim was to compare expression levels of CXCL11 in BK polyomavirus-infected versus noninfected kidney transplant patients with nephropathy and healthy controls.

Materials and Methods: We performed a cross-sectional study of 58 kidney transplant patients with the risk of BK polyomavirus infection; these patients were subgrouped as BK polyomavirus-infected (23 patients) and noninfected (35 patients). We also enrolled 30 healthy patients as controls in this study. The BK polyomavirus genome load was evaluated using a quantitative real-time polymerase chain reaction protocol in kidney transplant patients. We analyzed CXCL11 gene expression and protein levels using in-house SYBR green real-time polymerase chain reaction and enzyme-linked immunosorbent assay protocols.

Results: The expression level of the CXCL11 gene was increased 22.37 ± 23.1-fold in BK polyomavirus-infected kidney recipients and 12 ± 24-fold in noninfected patients versus that shown in controls.

Conclusion: From these results, we concluded that BK polyomavirus infection can induce CXCL11 gene expression in kidney transplant patients compared with that shown in patients without BK infection and healthy patients. However, further studies are needed to determine the accurate counteraction between BK polyomavirus infection and CXCL11 in kidney transplant patients.

Key words : Chemokine, BK polyomavirus, Renal transplant

BK polyomavirus, as a member of Polyomaviridae family, is a small nonenveloped virion that induces tumors and posttransplant nephropathy.¹ The virus is considered as a dangerous pathogen in immunocompromised individuals.² Although the main route of primary infection remains unknown, it appears that respiratory, oral, and body fluids can be considered as plausible transmission routes.³ The virus is prevalent in humans, with more than 80% of the population estimated to be seropositive up to early adolescence.^4-6

Under the pressures of immune responses, BK polyomavirus infection is suppressed but not eradicated, and latent forms of the disease can evolve. Therefore, BK polyomavirus may be reactivated when the immune system is suppressed.⁷ Virus reactivation is a main complication after renal transplant, inducing BK virus-associated nephropathy (BKVAN).^4-6,8 BK virus-associated nephropathy is characterized by inflammatory interstitial nephropathy.⁹ It appears that immunologic parameters may be involved in the pathogenesis of BKVAN. In addition, because transplant rejection is an immunologic phenomenon, it is important to understand the role of immunologic markers in the pathogenesis of BKVAN and also immune responses to the allograft.

Chemokines are important immune biomarkers that play key roles in either regulation of immune cell functions or migration via binding to their receptors.¹⁰ The CXCL chemokines are interferon-γ-inducible chemokines that mediate recruitment of T cells, natural killer cells, and monocytes and macrophages at the infection site, predominantly through their chemokine receptor belonging to the G protein-coupled cell surface receptor family CXCR3.^10,11 It has been reported that CXC (C-X-C motif) R3, an important chemokine receptor, induces intracellular signaling pathways after binding to its ligands such as CXCL11. Chemokine CXCL11, also known as I-TAC (interferon-inducible T-cell α), is a proinflammatory chemokine, and its high expression in serum has been established in several disorders such as multiple sclerosis,¹ viral infections,¹² Graves disease,¹³ and chronic allograft nephropathy.¹⁴ In addition, it has been reported that, during renal allograft rejection, the expression level of CXCL11 is increased in either blood circulation or kidney tissue.¹⁵ The CXCR3 ligands are excreted in the urine of kidney recipients during rejection and may therefore become useful for allograft monitoring.¹⁶ Animal studies have also confirmed the roles of CXCL11 in severe acute and chronic allograft rejection.¹⁷ However, the role of chemokines during BK virus infection compared with BK polyomavirus-negative allograft transplants has yet to be clarified. Here, our aim was to evaluate the mRNA expression and protein levels of CXCL11 in peripheral blood stem cells (PBMCs) and serum of BK polyomavirus-infected and noninfected renal transplant patients with nephropathy.

Materials and Methods

The study was approved by the Ethical Review Committee of the Institute. All of the protocols conformed to the ethical guidelines of the 1975 Declaration of Helsinki. Written informed consent was obtained from all participants.

Patients
This cross-sectional study was performed on 58 kidney transplant patients who were admitted to Namazi Hospital, Shiraz University of Medical Sciences (Shiraz, Iran) from 2012 to 2014. The patients had nephropathic features with the following criteria: clinical nephropathy symptoms, rising creatinine levels > 1.5 mg/dL, and glomerular filtration rate < 30 mL/min/1.73 m² body surface area. A standard immunosuppressive regimen for the transplant participants with nephropathy was applied as follows: initial therapy with 5 mg/kg cyclosporine followed by maintenance dose of 2 to 2.5 mg/kg, initial therapy with 120 mg/day prednisolone followed by 10 mg/day routinely, and 1000 mg/day mycophenolate mofetil at 2 times per day. In addition, to herpesvirus prophylaxis, intravenous acyclovir at 750 mg/day was administered from 3 days before transplant. After evaluation of BK virus infection, the participants were divided into BK virus infected (23 patients) and noninfected groups (35 patients). Thirty healthy sex- and age-matched nontransplanted individuals were also enrolled in the study as controls. One EDTA-treated blood sample was collected from each study patient and each control patient to evaluate CXCL11 gene expression and protein levels and also BK polyomavirus infectivity. Serum was also collected from each coagulated blood sample to evaluate biochemical indexes.

TaqMan polymerase chain reaction detection of BK polyomavirus
BK polyomavirus genomic DNA was extracted from plasma using the InvisorbSpin Virus DNA Blood Mini-kit (Invitek, Berlin, Germany) according to the manufacturer’s guidelines. BK polyomavirus DNA quantification was performed using Genesig BKV real-time polymerase chain reaction (PCR) kit (Primerdesign Ltd, Chandler’s Ford, UK) according to the manufacturer’s guidelines. Real-time PCR was performed in a final 20-μL volume containing 5 μL DNA, 10 μL Precision Master Mix (Applied Biosystems, Foster City, CA, USA), 1 μL specific primers and probe for BK-DNA, 1 μL specific primers and a probe targeting the internal control gene, and 3 μL diethylpyrocarbonate water. The following program was used in the Step One Plus Real-Time thermocycler (Applied Biosystems) for amplification of BK virus DNA and internal control: 1 cycle at 95°C (10 min) followed by 50 cycles at 95°C for 5 seconds and 60°C for 60 seconds. The quantitative PCR assay was able to detect >10 copies of BK virus DNA.

RNA extraction, cDNA synthesis, and real-time polymerase chain reaction
Total RNA was extracted from PBMCs using RNX Plus (CinnaGen, Tehran, Iran). The purity and integrity of RNA were determined by measuring the optical density at 260 nm/280 nm and by use of agarose gel (1%) electrophoresis. The samples were treated with DNase (1 μL/μg of RNA), and then 1 μg of each total RNA sample was reversely transcribed to cDNA using reverse transcriptase and random hexamer (Vivantis, Selangor, Malaysia). Briefly, mRNA (10 μg/10 μL), dNTPs (1 μL/10 mM), and random hexamer (1 μL/0.2 μg) were mixed and incubated at 65°C for 7 minutes and then on ice for 2 minutes. The following materials were then added to the prepared mRNAs: Moloney murine leukemia virus reverse transcriptase enzyme (1 μL/200 U), reverse transcriptase buffer (2 μL/10x), RNase inhibitor (1.3 μL/60 U), and nuclease-free water. The final mix was incubated at 42°C for 60 minutes and then at 85°C for 5 minutes to inactive reverse transcriptase enzyme. The synthesized cDNA were measured using appropriate primers (Table 1) and a commercial real-time SYBR green master mix from Takara Company (Tokyo, Japan). The amplification was performed in 96-well PCR plates (Exicycler 96 Quantitative Real-Time PCR System, Bioneer, Daejeon, Republic of Korea) with the following PCR program: 5 minutes at 95°C, 40 cycles of denaturation at 95°C for 60 seconds, 56 cycles for 55 seconds, and extension at 72°C for 60 seconds. Results were normalized to glyceraldehyde 3-phosphate dehydrogenase transcript levels, as the housekeeping gene, using the 2-ΔΔC(T) previously described method.¹⁸

Quantification of serum levels of CXCL11
Serum levels of CXCL11 were measured by enzyme-linked immunosorbent technique using a commercial reagent kit (Boster Biological Technology, Pleasanton, CA, USA) following the manufacturer’s instructions.

Statistical analyses
The raw data were analyzed using SPSS software (SPSS: An IBM Company, version 18, IBM Corporation, Armonk, NY, USA) and one-way analysis of variance and Kruskal-Wallis tests to analyze differences between groups regarding serum and mRNA levels of CXCL11. Pearson correlation and Spearman rho tests were used to analyze the relation between BK virus copy numbers and the serum and mRNA levels of CXCL11. P values < .05 were considered significant.

The expression level of the CXCL11 gene was increased 22.37 ± 23.1-fold in BK polyomavirus-infected kidney recipients and 12 ± 24-fold in noninfected patients compared with that shown in control participants. However, this difference was not significant (P = .515; 95% confidence interval, 0.509-0.529). Figure 1 illustrates the status of mean cycle threshold differences and expression levels of CXCL11 at mRNA levels among studied patients and controls.

The serum levels of CXCL11 in BK polyomavirus-infected renal transplant recipients, noninfected renal transplant recipients, and healthy controls were 864.1487 ± 37.1180, 37.1180 ± 31.9129, and 130.3503 ± 28.5669 pg/mL. The CXCL11 protein level was significantly higher in BK polyomavirus-infected patients compared with noninfected patients and healthy controls (P < .001; 95% confidence interval, 0.00-0.00) (Figure 2). Results showed that there was no significant association between BK polyomavirus copy numbers and CXCL11 protein level (r = -0.130; P = .404) and with mRNA level (r = 0.183; P = .555).

BK polyomavirus reactivation is a main complication after renal transplant, which results in BKVAN and subsequent renal loss.¹⁹ It has been hypothesized that chemokines play key roles in the recruitment of immune cells to the inflamed and infected tissues.^10,20 Here, we aimed to evaluate expression levels of CXCL11 in both mRNA and protein levels in PBMCs and plasma.

Our results demonstrated that the mRNA level of CXCL11 was not significantly increased in BK polyomavirus-infected kidney recipients compared with noninfected patients and healthy controls.

Our results did reveal that serum levels of CXCL11 were significantly increased in BK polyomavirus-infected compared with noninfected patients and healthy controls. Based on these results, postrenal transplant nephropathy may be associated with up-regulation of CXCL11 at protein levels.^15-17 Because mRNA levels of CXCL11 were not changed among the studied groups, it appears that BK virus infectivity may modulate translation-regulating mechanisms, resulting in up-regulation of protein levels of CXCL11.^15,21 Accordingly, it may hypothesized that increased translation of CXCL11 leads to increased secretion of the molecule and that its negative feedback results in decreased transcription from the CXCL11 gene.^17,21 In addition, another plausible reason for up-regulation of CXCL11 protein despite no changes in its mRNA level in PBMCs of BK polyomavirus-infected compared with noninfected recipients is that CXCL11 can be produced and secreted by other cell systems, including renal tubular epithelial cells.²¹ Collectively, based on the results, it appears that CXCL11 plays a key role in the pathogenesis of nephropathy-related complications following BK polyomavirus reactivation. It may be hypothesized that CXCL11 induced BKVAN by recruitment of immune cells and induction of inflammation.¹⁵ Previous investigations also confirmed the role played by CXCL11 in the pathogenesis of BKVAN. Panzer and associates reported that expression levels of CXCL11 and the number of CXCR3-positive T cells in renal transplant patients with nephropathy were elevated.¹⁵ Other investigations have also concurred regarding the role played by CXCR3, the receptor of CXCL11, in the induction of nephropathy after renal transplant.^22-25 Our results also confirmed the role of BK polyomavirus infection in deterioration of nephropathy via up-regulation of CXCL11. These results also demonstrated that there was no significant association between BK polyomavirus copy numbers and expression of CXCL11 in both mRNA and protein levels.

Our findings showed that BK polyomavirus may not only induce nephropathy directly but also uses some indirect mechanisms to induce graft loss during up-regulation of chemokine-like CXCL11. However, further studies are needed to determine the accurate association between BK polyomavirus infection and CXCL11 in kidney transplant patients.

Volume: 16 Issue: 1 February 2018

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