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Downregulation of Protein Inhibitor of Activated STAT (PIAS) 1 Is Possibly Involved in the Process of Allograft Rejection
Downregulation of Protein Inhibitor of Activated STAT (PIAS) 1 Is Possibly Involved in the Process of Allograft Rejection Mohsen Nafara, Shiva Kalantaria, Shiva Samavata, Mir Davood Omranib, Shahram Arsang-Jangc, Mohammad Taherid,*, and Soudeh Ghafouri-Farde a Chronic Kidney Disease Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; bUrology and Nephrology Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; cClinical Research Development Center (CRDU), Qom University of Medical Sciences, Qom, Iran; dUrogenital Stem Cell Research Center, Shahid Beheshti University of Medical Sciences, Tehran, Iran; and eDepartment of Medical Genetics, Shahid Beheshti University of Medical Sciences, Tehran, Iran
ABSTRACT Background. Protein inhibitors of activated STAT (PIAS) proteins are regarded as negative regulators of cytokine-signaling and potent immunosuppressive proteins. However, their role in the process of organ transplant rejection has not been elucidated. Methods. In the current study, we compared transcript levels of PIAS1 to 4 in the peripheral blood of renal transplant recipients who experienced transplant rejection with those having normal transplant functions. Expression of PIAS1 was significantly higher in nonrejected group compared with the rejected group among male recipients; however, differences were insignificant among female recipients. Expressions of other PIAS genes were not different between study groups. Significant pairwise correlations were found between expression levels of PIAS genes in all study subgroups. The current investigation highlights the role of PIAS1 downregulation in the evolution of graft rejection and potentiates this gene as a predictive marker for transplant fate.
O
RGAN transplantation is regarded as a practical therapeutic option for patients suffering from end-stage renal disease. However, the efficacy of this treatment modality is hindered by acute allograft rejection as well as chronic rejection [1]. Although administration of powerful immunosuppressive agent has decreased the risk of acute rejection, chronic rejection rate has been remained high [2]. Because of the high mortality and morbidity rates of these complications, several studies have aimed at identification of the underlying causes of them. T cells and the inflammatory cytokines have central roles in the process of graft rejection [3]. Consequently, assessment of the regulatory mechanisms of cytokine production provides deeper insights and understanding of the transplant rejection mechanisms. Protein inhibitor of activated STAT (PIAS) family are a group of transcriptional coregulators that have small ubiquitin-like modifier (SUMO) E3 ligase function [4]. They suppress the function of signal transducer and activator of transcription (STAT) [5]. Suppression of the Janus kinaseSTAT pathway has been suggested as an immunosuppressive strategy in organ transplantation based on the observed specific effect of Janus kinase on ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, XX, 1e5 (2019)
the function of immune cells [6]. Other clues for the proposed immunosuppressive effects of PIAS proteins have been provided by the investigations that have shown the role of PIAS in diminishing the effects of cytokines [7]. Despite the suggested roles for PIAS in immunosuppression, their expressions have not been assessed in transplant recipients in association with transplant fate. However, the role of another family of STAT inhibitors, namely suppressor of cytokine signaling, has been highlighted in the process of transplant rejection. For example, expression of suppressor of cytokine signaling 1 in the peripheral blood mononuclear cells has been shown to be lower in patients who experienced organ rejection than those with normal
The first 2 authors contributed equally. This study was financially supported by the Urology and Nephrology Research Center. The authors declare they have no conflict of interest. *Address correspondence to Mohammad Taheri, Velenjak St, Tehran, Iran 1254698732. Tel/Fax: 00982123872572. E-mail: [email protected] 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.10.006
1
2
NAFAR, KALANTARI, SAMAVAT ET AL Table 1. Associations Between Relative Expression of Genes and Transplant Fate
Genes
Group
PIAS1
Total Male Female Total Male Female Total Male Female Total Male Female
PIAS2
PIAS3
PIAS4
Rejected
Nonrejected
Median (IQR)
Median (IQR)
0.68 0.632 0.814 0.722 0.697 0.815 0.924 0.908 0.955 0.804 0.777 0.934
(0.53-0.87) (0.51-0.78) (0.55-1.03) (0.59-0.91) (0.59-0.96) (0.48-0.91) (0.74-1.06) (0.74-1.07) (0.65-1.06) (0.62-1) (0.59-0.92) (0.67-1.11)
0.788 0.813 0.732 0.82 0.826 0.81 0.891 0.902 0.878 0.907 0.912 0.848
(0.64-0.87) (0.67-0.87) (0.58-0.78) (0.65-0.9) (0.68-0.91) (0.62-0.88) (0.81-0.98) (0.82-1.01) (0.81-0.91) (0.72-0.95) (0.76-0.96) (0.67-0.93)
Relative Expression Difference
Standard Error
P Value
95% Credible Interval
0.1605 0.1629 0.1075 0.0639 0.0675 0.0484 0.0176 0.004 0.044 0.0721 0.0867 0.0251
0.07 0.06 0.16 0.07 0.0791 0.1321 0.0176 0.07 0.13 0.08 0.08 0.1878
.821 .09 .292 .234 .182 .992 .797 .947 .729 .147 .121 .927
(0.28 to 0.04) (0.29 to 0.05) (0.22 to 0.41) (0.2 to 0.07) (0.23 to 0.09) (0.32 to 0.22) (0.14 to 0.1) (0.18 to 0.1) (0.22 to 0.3) (0.21 to 0.08) (0.24 to 0.07) (0.4 to 0.36)
P values were estimated from frequentist method. Abbreviation: IQR, interquartile range.
kidney function [8]. Based on the abovementioned evidence, we hypothesized that expression of PIAS genes might be different among transplant recipients in association with the fate of transplant. Consequently, we conducted the current study to appraise this hypothesis. MATERIAL AND METHODS Patients Sixty-one renal transplant recipients including both transplant-rejected (18 male and 11 female recipients) and transplant-nonrejected individuals (24 male and 8 female recipients) were enlisted in the present study. The latter group consisted of individuals with no creatinine rise in the previous 3 months and those without evidence of transplant rejection in protocol biopsy. The study groups were matched in age and sex of patients. Patients were recruited from Labbafi-Nejad Hospital, Tehran, Iran, during 2016-2017. Exclusion criteria were delayed
transplant function, urinary obstruction, and urinary tract infection. The transplant center has guidelines for accomplishment of protocol biopsies. These guidelines were executed. The function of renal graft was evaluated by measurement of creatinine clearance, protein excretion, and transplant ultrasonography and angiography. A total of 25% of creatinine rises through 2 consecutive evaluations were considered as warning for implementation of renal biopsy after excluding drug toxicity and obstructions. Acute rejection was documented as stated by the Banff criteria [9]. The study protocol was approved by ethical committee of Shahid Beheshti University of Medical Sciences. Written consent forms were obtained from all study participants.
Expression Assays Expression of PIAS genes were quantified in the peripheral blood of enrolled persons after extraction of total RNA and complementary DNA production using Hybrid-R Blood RNA purification kit
Fig 1. Relative expression of PIAS genes in study groups based on their sex.
DOWNREGULATION OF PROTEIN INHIBITOR
3
Table 2. Results of Quantile Regression Model for Assessment of Association Between Relative Expression of Genes and Independent Variables Gene
PIAS1
Variable
Group Sex Group*sex PIAS2 Group Sex Group*sex PIAS3 Group Sex Group*sex PIAS4 Group Sex Group*sex
Beta
SE
t
P Value
0.18 0.08 0.21 0.13 0.02 0.13 0.01 0.02 0.04 0.13 0.06 0.16
0.08 0.12 0.16 0.10 0.13 0.18 0.08 0.11 0.15 0.09 0.13 0.17
2.19 0.70 1.32 1.36 0.12 0.72 0.07 0.21 0.28 1.51 0.51 0.95
.032 .484 .193 .179 .908 .472 .948 .833 .781 .137 .611 .348
were carried out in in R 3.5.2 environment using pROC, qreg, and Stan with loo packages.
95% CI for Beta
(0.35 (0.31 (0.11 (0.32 (0.28 (0.23 (0.16 (0.25 (0.27 (0.31 (0.32 (0.18
to to to to to to to to to to to to
0.02) 0.15) 0.52) 0.06) 0.25) 0.49) 0.17) 0.2) 0.35) 0.04) 0.19) 0.5)
(GeneAll Biotech, Seoul, Korea) and PrimeScript 1st strand cDNA Synthesis kit (Takara Bio Inc, Tokyo, Japan), respectively. Expressions of PIAS genes were measured in Rotor Gene 6000 system (Corbett, Southport, Australia). The HPRT1 gene was considered as the reference gene. Reactions were prepared using TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Foster City, Calif, United States). The sequences of primers and probes and polymerase chain reaction conditions were the same as a recently published article [10]. All reactions were arranged in duplicate.
Statistical Methods The Bayesian regression model was used to test the difference in relative expression of PIAS genes between study groups by adjusting the effects of independent variables. The asymmetric Laplace prior distribution was assumed for parameterization of gene expression ratio with 4000 iteration and 1000 warm-up. The P values for regression model were estimated from quantile and interquartile regression model. The Spearman correlation was used for examination of the association between expressions of genes. Analyses
RESULTS General Data of Enrolled Patients
Rejected and nonrejected groups were matched in age (mean age [SD], 40.4 [15.6] years and 35.6 [16.1] years, respectively). The graft rejected group comprised 22 patients with signs of T cellemediated rejection and 7 patients with indications of antibody-mediated rejection. In the nonrejected group, 29 patients had creatinine rise while others did not. However, in those with creatinine rise, protocol biopsy specimen showed no sign of transplant rejection. Results of PIAS Genes Expression Analysis
The associations between relative expression of genes and transplant fate are shown in Table 1. Expression of PIAS1 was significantly higher in the nonrejected group than in the rejected group among male recipients; however, differences were insignificant among female recipients. Expressions of other genes were not different between study groups (Fig 1). The quantile regression model showed significant difference in PIAS1 expression between rejected and nonrejected groups (P ¼ .032) (Table 2). When classifying patients into 4 subgroups, namely antibody-mediated rejection, T cellemediated rejection, stable glomerular filtration rate, and normal biopsy with creatinine rise (protocol biopsy), Bayesian regression model showed no significant difference in expression of PIAS genes between study subgroups (Table 3). We also assessed correlations between expression levels of PIAS genes and detected significant pairwise correlations between all of them in all study subgroups (male, female,
Table 3. Results of Bayesian Regression Model to Compare Gene Expression Ratios Between Study Groups With Adjusting the Effects of Sex (Reference Category: Normal With Creatinine Rise) Genes
Group
Relative Expression Difference
SE
P Value
PIAS1
Antibody-mediated rejection T cellemediated rejection Stable GFR Protocol biopsy Antibody-mediated rejection T cellemediated rejection Stable GFR Protocol biopsy Antibody-mediated rejection T cellemediated rejection Stable GFR Protocol biopsy Antibody-mediated rejection T cellemediated rejection Stable GFR Protocol biopsy
0.093 0.0872 0.146 0.105 0.0235 0.1477 0.0921 0.0147 0.0363 0.0487 0.1716 0.0401 0.0413 0.1354 0.2388 0.0054
0.07 0.11 0.15 0.08 0.08 0.1 0.2 0.09 0.07 0.09 0.13 0.07 0.0804 0.0945 0.2388 0.0054
.419 .42 .123 .857 .847 .166 .762 .9 .619 .967 .3 .578 .681 .274 .316 .965
PIAS2
PIAS3
PIAS4
Abbreviation: GFR, glomerular filtration rate.
95% Credible Intervals
(0.23 (0.32 (0.44 (0.05 (0.19 (0.33 (0.45 (0.19 (0.11 (0.23 (0.41 (0.09 (0.22 (0.33 (0.56 (0.17
to to to to to to to to to to to to to to to to
0.05) 0.1) 0.15) 0.24) 0.12) 0.06) 0.28) 0.16) 0.17) 0.11) 0.11) 0.17) 0.11) 0.06) 0.15) 0.16)
4
NAFAR, KALANTARI, SAMAVAT ET AL
Fig 2. Results of pairwise correlation analysis between expression levels of PIAS genes.
rejected, and nonrejected subgroups). Figure 2 shows the results of pairwise correlation analysis. DISCUSSION
In the current investigation, we reported down-regulation of PIAS1 in male transplant recipients who rejected the graft compared with those with normal graft function. PIAS1 has been previously shown to bind with STAT1, hinder the DNA binding activity of STAT1, and suppress STAT1induced gene expression [5]. Downregulation of PIAS1 is expected to be associated with upregulation of STAT1. Previous reports have demonstrated links between STAT1 inhibition and immunosuppression. For instance, fludarabine-stimulated immunosuppression has been linked with suppression of STAT1 route [11]. STAT1 is a fundamental transcription factor facilitating interferon (IFN)-a/b signaling. It also modulates responses to IFN-g and other cytokines [12]. Meanwhile, IFN has been shown to be associated with renal transplants rejection [13], and several clinical observations propose inhibition of IFN as an effective strategy for improving renal transplant fate [13]. Moreover, IFN-a/b has sped up the process of graft rejection in a model of heart transplantation [14]. Consequently, upregulation of IFN responses might be a possible explanation for the observed lower levels of PIAS1 in patients who experienced transplant rejection. PIAS1 is also a crucial negative regulator of NF-kB. In the nucleus, PIAS1 binds to the p65 subunit of NF-kB and suppresses the NF-kBedependent gene activation in response to cytokines. In line with these observations, Pias1 null mice had higher levels of proinflammatory cytokines [15]. NF-kB has several important roles in many aspects of the allograft rejection process, such as ischemia/reperfusion
injury and oxidative organ damage. Not surprisingly, several immunosuppressive drugs alter function and expression of this transcription factor. Consequently, suppression of the NF-kB signaling pathway has been suggested as therapeutic modality for prevention of allograft rejection [16]. Based on these evidences, higher levels of NF-kB signaling activation is another explanation for the observed association between lower level of PIAS1 and transplant rejection. Finally, we assessed correlations between expression levels of PIAS genes and detected significant pairwise correlations between all of them in all study subgroups, which implies the existence of a single regulatory mechanism for PIAS genes that is not influenced by the process of graft rejection or sex of individuals. In conclusion, the current investigation highlights the role of PIAS1 downregulation in the evolution of graft rejection and potentiates this gene as a predictive marker for transplant fate.
REFERENCES [1] Matas AJ, Gillingham KJ, Payne WD, Najarian JS. The impact of an acute rejection episode on long-term renal allograft survival (t1/2). Transplantation 1994;57:857e9. [2] Meier-Kriesche HU, Schold JD, Kaplan B. Long-term renal allograft survival: have we made significant progress or is it time to rethink our analytic and therapeutic strategies? Am J Transplant 2004;4:1289e95. [3] Sanchez-Fueyo A, Strom TB. Immunologic basis of graft rejection and tolerance following transplantation of liver or other solid organs. Gastroenterology 2011;140:51e64. [4] Shuai K, Liu B. Regulation of gene-activation pathways by PIAS proteins in the immune system. Nat rev Immunol 2005;5: 593e605. [5] Liu B, Liao J, Rao X, Kushner SA, Chung CD, Chang DD, et al. Inhibition of Stat1-mediated gene activation by PIAS1. Proc Natl Acad Sci U S A 1998;95:10626e31.
DOWNREGULATION OF PROTEIN INHIBITOR [6] Moore CA, Iasella CJ, Venkataramanan R, Lakkis FG, Smith RB, McDyer JF, et al. Janus kinase inhibition for immunosuppression in solid organ transplantation: is there a role in complex immunologic challenges? Hum Immunol 2017;78:64e71. [7] O'Shea JJ. Targeting the Jak/STAT pathway for immunosuppression. Ann Rheum Dis 2004;63(Suppl. 2):67e71. [8] Wu TH, Lee HT, Lai CC, Yang AH, Loong CC, Wang HK, et al. Suppressor of cytokine signaling (SOCS) 1 is down-regulated in renal transplant recipients with rejection. Transpl Immunol 2016;38:54e9. [9] Racusen L, Rayner D, Trpkov K, Olsen S, Solez K. The Banff classification of renal allograft pathology: where do we go from here? Transplant Proc 1996;28:486e8. [10] Taheri M, Azimi G, Sayad A, Mazdeh M, Arsang-Jang S, Omrani MD, et al. Expression analysis of protein inhibitor of activated STAT (PIAS) genes in IFNbeta-treated multiple sclerosis patients. J Inflamm Res 2018;11:457e63.
5 [11] Frank DA, Mahajan S, Ritz J. Fludarabine-induced immunosuppression is associated with inhibition of STAT1 signaling. Nat Med 1999;5:444e7. [12] Reich NC. STATs get their move on. JAKSTAT 2013;2: e27080. [13] Magnone M, Holley JL, Shapiro R, Scantlebury V, McCauley J, Jordan M, et al. Interferon-alpha-induced acute renal allograft rejection. Transplantation 1995;59:1068e70. [14] Slater AD, Klein JB, Sonnenfeld G, Ogden LL 2nd, Gray LA Jr. The effects of interferon-alpha/beta in a model of rat heart transplantation. J Heart Lung Transplant 1992;11:975e8. [15] Liu B, Yang R, Wong KA, Getman C, Stein N, Teitell MA, et al. Negative regulation of NF-kappaB signaling by PIAS1. Mol Cell Biol 2005;25:1113e23. [16] Tsoulfas G, Geller DA. NF-kappaB in transplantation: friend or foe? Transpl Infect Dis 2001;3:212e9.
ABSTRACT Background. Protein inhibitors of activated STAT (PIAS) proteins are regarded as negative regulators of cytokine-signaling and potent immunosuppressive proteins. However, their role in the process of organ transplant rejection has not been elucidated. Methods. In the current study, we compared transcript levels of PIAS1 to 4 in the peripheral blood of renal transplant recipients who experienced transplant rejection with those having normal transplant functions. Expression of PIAS1 was significantly higher in nonrejected group compared with the rejected group among male recipients; however, differences were insignificant among female recipients. Expressions of other PIAS genes were not different between study groups. Significant pairwise correlations were found between expression levels of PIAS genes in all study subgroups. The current investigation highlights the role of PIAS1 downregulation in the evolution of graft rejection and potentiates this gene as a predictive marker for transplant fate.
O
RGAN transplantation is regarded as a practical therapeutic option for patients suffering from end-stage renal disease. However, the efficacy of this treatment modality is hindered by acute allograft rejection as well as chronic rejection [1]. Although administration of powerful immunosuppressive agent has decreased the risk of acute rejection, chronic rejection rate has been remained high [2]. Because of the high mortality and morbidity rates of these complications, several studies have aimed at identification of the underlying causes of them. T cells and the inflammatory cytokines have central roles in the process of graft rejection [3]. Consequently, assessment of the regulatory mechanisms of cytokine production provides deeper insights and understanding of the transplant rejection mechanisms. Protein inhibitor of activated STAT (PIAS) family are a group of transcriptional coregulators that have small ubiquitin-like modifier (SUMO) E3 ligase function [4]. They suppress the function of signal transducer and activator of transcription (STAT) [5]. Suppression of the Janus kinaseSTAT pathway has been suggested as an immunosuppressive strategy in organ transplantation based on the observed specific effect of Janus kinase on ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, XX, 1e5 (2019)
the function of immune cells [6]. Other clues for the proposed immunosuppressive effects of PIAS proteins have been provided by the investigations that have shown the role of PIAS in diminishing the effects of cytokines [7]. Despite the suggested roles for PIAS in immunosuppression, their expressions have not been assessed in transplant recipients in association with transplant fate. However, the role of another family of STAT inhibitors, namely suppressor of cytokine signaling, has been highlighted in the process of transplant rejection. For example, expression of suppressor of cytokine signaling 1 in the peripheral blood mononuclear cells has been shown to be lower in patients who experienced organ rejection than those with normal
The first 2 authors contributed equally. This study was financially supported by the Urology and Nephrology Research Center. The authors declare they have no conflict of interest. *Address correspondence to Mohammad Taheri, Velenjak St, Tehran, Iran 1254698732. Tel/Fax: 00982123872572. E-mail: [email protected] 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.10.006
1
2
NAFAR, KALANTARI, SAMAVAT ET AL Table 1. Associations Between Relative Expression of Genes and Transplant Fate
Genes
Group
PIAS1
Total Male Female Total Male Female Total Male Female Total Male Female
PIAS2
PIAS3
PIAS4
Rejected
Nonrejected
Median (IQR)
Median (IQR)
0.68 0.632 0.814 0.722 0.697 0.815 0.924 0.908 0.955 0.804 0.777 0.934
(0.53-0.87) (0.51-0.78) (0.55-1.03) (0.59-0.91) (0.59-0.96) (0.48-0.91) (0.74-1.06) (0.74-1.07) (0.65-1.06) (0.62-1) (0.59-0.92) (0.67-1.11)
0.788 0.813 0.732 0.82 0.826 0.81 0.891 0.902 0.878 0.907 0.912 0.848
(0.64-0.87) (0.67-0.87) (0.58-0.78) (0.65-0.9) (0.68-0.91) (0.62-0.88) (0.81-0.98) (0.82-1.01) (0.81-0.91) (0.72-0.95) (0.76-0.96) (0.67-0.93)
Relative Expression Difference
Standard Error
P Value
95% Credible Interval
0.1605 0.1629 0.1075 0.0639 0.0675 0.0484 0.0176 0.004 0.044 0.0721 0.0867 0.0251
0.07 0.06 0.16 0.07 0.0791 0.1321 0.0176 0.07 0.13 0.08 0.08 0.1878
.821 .09 .292 .234 .182 .992 .797 .947 .729 .147 .121 .927
(0.28 to 0.04) (0.29 to 0.05) (0.22 to 0.41) (0.2 to 0.07) (0.23 to 0.09) (0.32 to 0.22) (0.14 to 0.1) (0.18 to 0.1) (0.22 to 0.3) (0.21 to 0.08) (0.24 to 0.07) (0.4 to 0.36)
P values were estimated from frequentist method. Abbreviation: IQR, interquartile range.
kidney function [8]. Based on the abovementioned evidence, we hypothesized that expression of PIAS genes might be different among transplant recipients in association with the fate of transplant. Consequently, we conducted the current study to appraise this hypothesis. MATERIAL AND METHODS Patients Sixty-one renal transplant recipients including both transplant-rejected (18 male and 11 female recipients) and transplant-nonrejected individuals (24 male and 8 female recipients) were enlisted in the present study. The latter group consisted of individuals with no creatinine rise in the previous 3 months and those without evidence of transplant rejection in protocol biopsy. The study groups were matched in age and sex of patients. Patients were recruited from Labbafi-Nejad Hospital, Tehran, Iran, during 2016-2017. Exclusion criteria were delayed
transplant function, urinary obstruction, and urinary tract infection. The transplant center has guidelines for accomplishment of protocol biopsies. These guidelines were executed. The function of renal graft was evaluated by measurement of creatinine clearance, protein excretion, and transplant ultrasonography and angiography. A total of 25% of creatinine rises through 2 consecutive evaluations were considered as warning for implementation of renal biopsy after excluding drug toxicity and obstructions. Acute rejection was documented as stated by the Banff criteria [9]. The study protocol was approved by ethical committee of Shahid Beheshti University of Medical Sciences. Written consent forms were obtained from all study participants.
Expression Assays Expression of PIAS genes were quantified in the peripheral blood of enrolled persons after extraction of total RNA and complementary DNA production using Hybrid-R Blood RNA purification kit
Fig 1. Relative expression of PIAS genes in study groups based on their sex.
DOWNREGULATION OF PROTEIN INHIBITOR
3
Table 2. Results of Quantile Regression Model for Assessment of Association Between Relative Expression of Genes and Independent Variables Gene
PIAS1
Variable
Group Sex Group*sex PIAS2 Group Sex Group*sex PIAS3 Group Sex Group*sex PIAS4 Group Sex Group*sex
Beta
SE
t
P Value
0.18 0.08 0.21 0.13 0.02 0.13 0.01 0.02 0.04 0.13 0.06 0.16
0.08 0.12 0.16 0.10 0.13 0.18 0.08 0.11 0.15 0.09 0.13 0.17
2.19 0.70 1.32 1.36 0.12 0.72 0.07 0.21 0.28 1.51 0.51 0.95
.032 .484 .193 .179 .908 .472 .948 .833 .781 .137 .611 .348
were carried out in in R 3.5.2 environment using pROC, qreg, and Stan with loo packages.
95% CI for Beta
(0.35 (0.31 (0.11 (0.32 (0.28 (0.23 (0.16 (0.25 (0.27 (0.31 (0.32 (0.18
to to to to to to to to to to to to
0.02) 0.15) 0.52) 0.06) 0.25) 0.49) 0.17) 0.2) 0.35) 0.04) 0.19) 0.5)
(GeneAll Biotech, Seoul, Korea) and PrimeScript 1st strand cDNA Synthesis kit (Takara Bio Inc, Tokyo, Japan), respectively. Expressions of PIAS genes were measured in Rotor Gene 6000 system (Corbett, Southport, Australia). The HPRT1 gene was considered as the reference gene. Reactions were prepared using TaqMan Fast Universal PCR Master Mix (Applied Biosystems, Foster City, Calif, United States). The sequences of primers and probes and polymerase chain reaction conditions were the same as a recently published article [10]. All reactions were arranged in duplicate.
Statistical Methods The Bayesian regression model was used to test the difference in relative expression of PIAS genes between study groups by adjusting the effects of independent variables. The asymmetric Laplace prior distribution was assumed for parameterization of gene expression ratio with 4000 iteration and 1000 warm-up. The P values for regression model were estimated from quantile and interquartile regression model. The Spearman correlation was used for examination of the association between expressions of genes. Analyses
RESULTS General Data of Enrolled Patients
Rejected and nonrejected groups were matched in age (mean age [SD], 40.4 [15.6] years and 35.6 [16.1] years, respectively). The graft rejected group comprised 22 patients with signs of T cellemediated rejection and 7 patients with indications of antibody-mediated rejection. In the nonrejected group, 29 patients had creatinine rise while others did not. However, in those with creatinine rise, protocol biopsy specimen showed no sign of transplant rejection. Results of PIAS Genes Expression Analysis
The associations between relative expression of genes and transplant fate are shown in Table 1. Expression of PIAS1 was significantly higher in the nonrejected group than in the rejected group among male recipients; however, differences were insignificant among female recipients. Expressions of other genes were not different between study groups (Fig 1). The quantile regression model showed significant difference in PIAS1 expression between rejected and nonrejected groups (P ¼ .032) (Table 2). When classifying patients into 4 subgroups, namely antibody-mediated rejection, T cellemediated rejection, stable glomerular filtration rate, and normal biopsy with creatinine rise (protocol biopsy), Bayesian regression model showed no significant difference in expression of PIAS genes between study subgroups (Table 3). We also assessed correlations between expression levels of PIAS genes and detected significant pairwise correlations between all of them in all study subgroups (male, female,
Table 3. Results of Bayesian Regression Model to Compare Gene Expression Ratios Between Study Groups With Adjusting the Effects of Sex (Reference Category: Normal With Creatinine Rise) Genes
Group
Relative Expression Difference
SE
P Value
PIAS1
Antibody-mediated rejection T cellemediated rejection Stable GFR Protocol biopsy Antibody-mediated rejection T cellemediated rejection Stable GFR Protocol biopsy Antibody-mediated rejection T cellemediated rejection Stable GFR Protocol biopsy Antibody-mediated rejection T cellemediated rejection Stable GFR Protocol biopsy
0.093 0.0872 0.146 0.105 0.0235 0.1477 0.0921 0.0147 0.0363 0.0487 0.1716 0.0401 0.0413 0.1354 0.2388 0.0054
0.07 0.11 0.15 0.08 0.08 0.1 0.2 0.09 0.07 0.09 0.13 0.07 0.0804 0.0945 0.2388 0.0054
.419 .42 .123 .857 .847 .166 .762 .9 .619 .967 .3 .578 .681 .274 .316 .965
PIAS2
PIAS3
PIAS4
Abbreviation: GFR, glomerular filtration rate.
95% Credible Intervals
(0.23 (0.32 (0.44 (0.05 (0.19 (0.33 (0.45 (0.19 (0.11 (0.23 (0.41 (0.09 (0.22 (0.33 (0.56 (0.17
to to to to to to to to to to to to to to to to
0.05) 0.1) 0.15) 0.24) 0.12) 0.06) 0.28) 0.16) 0.17) 0.11) 0.11) 0.17) 0.11) 0.06) 0.15) 0.16)
4
NAFAR, KALANTARI, SAMAVAT ET AL
Fig 2. Results of pairwise correlation analysis between expression levels of PIAS genes.
rejected, and nonrejected subgroups). Figure 2 shows the results of pairwise correlation analysis. DISCUSSION
In the current investigation, we reported down-regulation of PIAS1 in male transplant recipients who rejected the graft compared with those with normal graft function. PIAS1 has been previously shown to bind with STAT1, hinder the DNA binding activity of STAT1, and suppress STAT1induced gene expression [5]. Downregulation of PIAS1 is expected to be associated with upregulation of STAT1. Previous reports have demonstrated links between STAT1 inhibition and immunosuppression. For instance, fludarabine-stimulated immunosuppression has been linked with suppression of STAT1 route [11]. STAT1 is a fundamental transcription factor facilitating interferon (IFN)-a/b signaling. It also modulates responses to IFN-g and other cytokines [12]. Meanwhile, IFN has been shown to be associated with renal transplants rejection [13], and several clinical observations propose inhibition of IFN as an effective strategy for improving renal transplant fate [13]. Moreover, IFN-a/b has sped up the process of graft rejection in a model of heart transplantation [14]. Consequently, upregulation of IFN responses might be a possible explanation for the observed lower levels of PIAS1 in patients who experienced transplant rejection. PIAS1 is also a crucial negative regulator of NF-kB. In the nucleus, PIAS1 binds to the p65 subunit of NF-kB and suppresses the NF-kBedependent gene activation in response to cytokines. In line with these observations, Pias1 null mice had higher levels of proinflammatory cytokines [15]. NF-kB has several important roles in many aspects of the allograft rejection process, such as ischemia/reperfusion
injury and oxidative organ damage. Not surprisingly, several immunosuppressive drugs alter function and expression of this transcription factor. Consequently, suppression of the NF-kB signaling pathway has been suggested as therapeutic modality for prevention of allograft rejection [16]. Based on these evidences, higher levels of NF-kB signaling activation is another explanation for the observed association between lower level of PIAS1 and transplant rejection. Finally, we assessed correlations between expression levels of PIAS genes and detected significant pairwise correlations between all of them in all study subgroups, which implies the existence of a single regulatory mechanism for PIAS genes that is not influenced by the process of graft rejection or sex of individuals. In conclusion, the current investigation highlights the role of PIAS1 downregulation in the evolution of graft rejection and potentiates this gene as a predictive marker for transplant fate.
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