Background

Renal transplantation is the ideal treatment for patients with end-stage renal disease [1]. It has been suggested that in human renal transplantation, pro-inflammatory Th1 lymphocytes and their cytokines mediate allograft rejection, whereas the Th2 lymphocytes and their cytokines are involved in the process of tolerance induction [2]. The inter-individual differences in cytokine production that influence allograft rejection might be impacted by the polymorphisms within the encoding genes that can regulate the various inflammatory responses within the graft [3].

Interleukin-1 (IL-1) plays a crucial role in the inflammatory response [4]. During allograft rejection, an increase in IL-1 production precedes allograft dysfunction and injury [5]. The genes of the IL-1 complex, which are located on chromosome 2q13, encode for three proteins: IL-1α, IL-1β, and IL-1 receptor antagonist (IL-1Ra). It has been reported that IL-1α is approximately 3000 times less active than IL-1β [6]. Each of the genes is polymorphic, and there is evidence that specific alleles are associated with increased susceptibility to inflammation [7]. A single nucleotide polymorphism (SNP) (rs16944) has been identified at bp position − 511 in the promoter region of the IL-1β gene with a substantial influence on its serum levels [8]. IL-1β plays an important role in the development and progression of acute kidney injury (AKI) [9]. In renal tubular cells, inflammasome-mediated caspase-1 activation and IL-1β generation are induced by several intra- and extra-cellular stimuli such as ischemic-reperfusion injury, hypotonic stress, adenosine triphosphate, mitochondrial dysfunction, uric acid crystals and lysosomal rupture [10].

The IL-1 receptor antagonist gene (IL-1RN) has a penta-allelic polymorphic site in intron 2 (rs2234663) containing variable numbers of an 86-bp tandem repeat (VNTR) sequence. The IL-1 complex is highly distinctive because the IL-1 receptor antagonist (IL-1Ra) acts as a natural inhibitor that binds to the IL-1 receptor inhibiting IL-1α and IL-1β binding [5].

The clinical outcome of renal transplantation is impacted by the recipient’s immune response to the transplanted kidney. The ability to manage a patient’s clinical course depends on the ability to control the immune response through immunosuppressive therapy [11]. Inhibition of IL-1 production is one of the main mechanisms by which corticosteroids suppress the immune response. IL-1Ra production is also enhanced in stable human kidney graft recipients and hence, could be a crucial factor in the early down-regulation of the allogeneic immune response. Drugs targeting IL-1 such as recombinant IL-1RN (anakinra), IL-1β traps (rilonacept), and neutralizing anti-IL-1β antibodies (canakinumab) are currently in clinical use; targeting IL-1 has shown promising results in renal transplantation patients [12].

This research work aimed to determine the IL-1β and IL-1RN gene polymorphisms among a group of renal transplant recipients and to study whether there is an association between these polymorphisms and their haplotypes with renal graft outcome.

Methods

Subjects

Our study included 31 patients (age range 18–48 years; mean age 32.68 ± 10.47; 23 males and 8 females) who had undergone living-donor renal transplantation at Kasr Al Ainy Hospital, Faculty of Medicine, Cairo University as well as 26 age- and gender-matched healthy controls.

Inclusion criteria included male and female adult (18–60 years old) patients undergoing renal transplantation. All patients had ABO- and HLA-matched living donors. Exclusion criteria included any patient with pre-renal or post-renal causes of renal allograft dysfunction.

Twenty-three patients experienced early renal allograft dysfunction [13], based on clinical diagnosis, including persistent elevation of the patient’s serum creatinine above their normal baseline, even after correction of hemodynamic status, urinary tract infections, and immunosuppressive drug level. Early dysfunction or failure occurring ≤ 6 months post-transplant can occur either immediately, during the initial hours and days post-transplant or within the first few weeks or months after transplant [13]. Eight patients had stable graft function (SGF).

Healthy controls for the study were recruited from the same geographical area, with no history of hypertension, diabetes, renal failure, vascular diseases, stroke, and/or cardiac diseases.

Laboratory methods

Blood samples and genotyping

Two milliliters of blood were collected in a tube containing EDTA as an anticoagulant for DNA extraction and stored at − 20 °C. Genomic DNA was isolated from peripheral blood leukocytes using a genomic DNA purification kit according to the manufacturer’s instructions (Thermo Scientific, USA).

Genotyping of IL-1β (− 511 C>T) (rs16944) by PCR–RFLP

IL-1β − 511 C/T genotyping was performed as previously described [14] with the primer pair (forward, 5′-TGG CAT TGATCT GGT TCATC-3′, and reverse, 5′-GTT TAG GAATCT TCCCAC TT-3′) (Bioneer, Korea) with initial denaturation at 95 °C for 1 min followed by 30 cycles of 95 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s with a final extension at 70 °C for 7 min using a PCR Thermal Cycler (ThermoHybaid, UK). PCR products were digested by restriction endonuclease Aval (ThermoScientific, USA) and visualized by electrophoresis on a 3% agarose gel stained with ethidium bromide. Alleles were coded as follows: T, 304 bp, and C, 190 and 114 bp.

Genotyping of IL-1RN VNTR (rs2234663) by PCR

IL-1RN genotyping was performed as previously described [15] with the primer pair (forward, 5′-CTC AGC AAC ACT CCT AT-3′, and reverse, 5′-TCC TGG TCT GCA GGT AA-3′) (Bioneer, Korea) with initial denaturation at 94 °C for 4 min followed by 32 cycles of 94 °C for 1 min, 50 °C for 1 min, and 72 °C for 1 min with a final extension at 72 °C for 10 min using a PCR Thermal Cycler. PCR products were analyzed by electrophoresis on a 1.5% agarose gel stained with ethidium bromide. Alleles 1–5 (IL-1RN 1–IL-1RN 5) were detected according to their sizes relative to a 100-bp DNA ladder: allele 1 (four repeats), 410 bp; allele 2 (two repeats), 240 bp; allele 3 (five repeats), 500 bp; allele 4 (three repeats), 325 bp; and allele 5 (six repeats), 595 bp.

Statistical analysis

Data were statistically described in terms of mean ± standard deviation (± SD), or frequencies (number of cases) and percentages when appropriate. A comparison of numerical variables between the study groups was done using the Mann–Whitney U test for independent samples. For comparing categorical data, the Chi-square (χ2) test was performed. An exact test was used instead when the expected frequency was less than 5. p values less than 0.05 were  considered statistically significant. All statistical calculations were done using the computer program SPSS (Statistical Package for the Social Science; SPSS Inc., Chicago, IL, USA) version 15 for Microsoft Windows.

Results

Characteristics of renal transplant cases and controls

The demographic and clinical data of the renal transplant cases and their age- and gender-matched healthy controls are shown in Table 1. Our renal transplant cases had a mean age of 32.68 ± 10.47 years with the majority (74.2%) being males. Around 45% of the cases developed end-stage renal disease as a result of hypertension. The mean time of pre-transplantation hemodialysis was 32.15 ± 25.73 months. The mean age of the donors was 35.29 ± 6.49 years, and the majority of donors (80.6%) were males (Table 1).

Table 1 Demographic and clinical data of renal transplant recipients and healthy controls
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Causes and clinical characteristics of the early allograft dysfunction group

The causes of early allograft dysfunction, as revealed by renal biopsy, were acute tubular injury, acute rejection, and thrombotic microangiopathy. The main clinical characteristics observed in the allograft dysfunction group were the rise of serum creatinine, elevated renal resistivity index, recent hypertension, proteinuria, and hematuria. None of these clinical features have been observed in the stable renal allograft group (Table 2).

Table 2 Clinical characteristics of the early renal allograft dysfunction group
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Frequency distribution of IL-1RN (VNTR) and IL-1β − 511C/T gene polymorphisms and IL-1RN/IL-1β haplotypes in renal transplant cases and healthy controls

The majority of our participants harbored the IL-1RN *1*1 genotype, while fewer cases displayed the less frequent genotype *1*2. However, the rare genotypes *2*2, *3*3, *1*3, and *2*4 were present in only four participants, so these genotypes were grouped as ‘others’ as shown in Table 3. Also, the less frequent haplotypes *1/T, *2/C, *2/T,*3/C, *3/T and*4/T were grouped as ‘others’. No statistically significant difference was encountered in the distribution of IL-1RN (p = 0.270) and IL-1β gene polymorphisms (p = 1.0) and their haplotypes (p = 0.259) between cases and controls (Table 3).

Table 3 Frequency distribution of IL-1RN (VNTR) and IL-1β − 511C/T gene polymorphisms and IL-1RN/IL-1β haplotypes in renal transplant recipients and healthy controls
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Association of IL-1RN (VNTR) and IL-1β − 511C/T gene polymorphisms and IL-1RN/IL-1β haplotypes with graft function in renal transplant recipients

Renal transplant recipients were further subdivided according to the graft function into stable allograft function (Group I) and early renal allograft dysfunction (Group II) as shown in Table 4. On comparing IL-1RN genotypes between the two groups, 87.5% of Group I were carriers of the IL-1RN *1*1 genotype versus 65.2% of Group II cases; however, the difference did not reach statistical significance, p = 0.379. As regards IL-1β genotype distribution, 95.7% of Group II were carriers of the IL-1β polymorphic genotypes CT + TT versus 25% of Group I cases; with a statistically significant difference, p < 0.001. Interestingly, on comparing the IL-1RN/IL-1β haplotype between the two groups, 68.75% of Group I were carriers of the wild-type haplotype *1/C versus 28.26% of Group II, with a statistically significant difference, p < 0.001 (Table 4).

Table 4 Association of IL-1RN (VNTR) and IL-1β − 511 C/T gene polymorphisms and IL-1RN/IL-1β haplotypes with graft function in Renal Transplant Recipients
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Discussion

Following renal transplantation, renal graft function is important for patient and graft survival. The immune response, to transplanted organs, is regulated by a network of cytokine interactions. However, the genes encoding these cytokines and their receptors are polymorphic. We attempted to study the impact of IL-1β and IL-1RN polymorphisms and their haplotypes on graft function. In the present study, we did not observe a statistically significant difference in the two studied gene polymorphisms between the healthy controls and renal transplant recipients. However, our results indicated that IL-1β − 511 C/T genotypes showed a statistically significant difference between stable allograft function and early renal allograft dysfunction, where 75% of the stable allograft function had the wild-type CC, compared to only 4.3% of the early renal allograft dysfunction. On comparing IL-1RN gene polymorphisms between the two groups, 87.5% of stable allograft function harbored the genotype *1*1 compared to 65.2% of early renal allograft dysfunction; however, the difference did not reach statistical significance. Interestingly, the IL-1RN/IL-1β haplotype showed a statistically significant difference when compared between the two groups, where 68.75% of those with stable allograft function were carriers of the haplotype IL-1RN *1/IL-1β *C versus 28.26% of those showing early renal allograft dysfunction.

Published results from previous studies were inconsistent as regards the role of IL-1RN and IL-1β polymorphisms and haplotypes on graft function and incidence of acute rejection (AR) in renal transplant recipients. Our findings contrasted those provided in a previous study from India by Manchanda et al. [16], where there was a significant difference in the distribution of IL-1β and IL-1RN genotypes between healthy controls and renal transplant groups. They stated that a statistically significant difference was also observed in IL-1RN genotypes when compared between stable graft function and delayed graft function groups whereas no significant difference in IL-1β − 511 promoter polymorphism was observed between these two transplant recipients’ groups [16].

Bhat et al. reported that the IL-1β − 511TT genotype was more prevalent in renal transplant cases versus controls and those experiencing rejection episodes versus those with stable graft function in their study conducted on participants from Kashmeer valley [3]. Ding et al. stated that there was no significant difference between recipients with an AR episode and those with an absence of AR regarding IL-1RN and IL-1β polymorphisms in the studied Chinese population [17]. These discordant findings might be due to differences in ethnic and geographical backgrounds, sample size, different clinical diagnostic processes, and immunosuppressive protocols [18, 19].

Interestingly, our study showed that the IL-1β − 511 TT genotype was present at higher frequencies in the early renal allograft dysfunction versus stable allograft function group, and this has been reported in other research work [3, 16], assuming it represents a “high secretor” phenotype leading to increased pro-inflammatory activity in autoimmune and infectious diseases [20].

Previous studies [21,22,23,24,25], analyzing the impact of the IL-1RN genotypes on levels of IL-1β and IL-1Ra, have provided discordant results. Studies on epithelial/endothelial cells have shown that the IL-1RN*1 allele is more anti-inflammatory with higher levels of IL-1Ra produced by cells harboring the IL-1RN*1 allele [21]. This is following the findings of Santtila et al. [22] who stated that the IL-1RN*2 allele is associated with increased IL-1 β release from peripheral blood mononuclear cells. Whereas Vamvakopoulos et al. [23] and Candiotti et al. [24] indicated that IL-1Ra concentrations were significantly higher in carriers of IL-1RN*2 than in IL-1RN*1 homozygotes. Also, they stated that IL-1RN*2 homozygotes showed a decreased IL-1 β release, in a dosage-dependent manner [23]. Other researchers [25] have indicated that they failed to demonstrate any IL-1RN allelic effect on IL1Ra expression manner.

The polymorphism of IL-1RN consists of perfect tandem repeats of a conserved 86–base pair sequence, which has been reported to contain putative protein-binding sites; an α-interferon silencer A, a β-interferon silencer B, and a short-term phase response element [24]. These binding sites may influence gene expression; however, the exact mechanism is under investigation.

It has been postulated that it is not only the IL-1β or IL-1RN genotypes but the haplotype of the IL-1RN and IL-1β that play a role in modulating the susceptibility to certain disease conditions [26]. In any individual, an SNP at a given locus may be a part of a haplotype that affects protein expression or function, whereas the same SNP in another individual may not form part of the functional haplotype. Haplotyping of IL-1 reflects that patients carrying high-producing alleles of IL-1β and low-producing alleles of IL-1RN are at higher risk of progression to rejection of allograft [26].

Our study has numerous strengths; we recruited 31 consecutive Egyptian live-donor renal transplant recipients and 26 age- and gender-matched healthy controls from the same geographical area. They represent an under-studied population, and there is a crucial need to shed light on the underlying gene polymorphisms which impact graft function in our studied groups. To the best of our knowledge, this is the first study investigating the impact of IL-1β and IL-1RN gene polymorphisms on graft function in Egyptian renal transplant recipients.

This study is not without limitations and our conclusion should be interpreted with caution due to the relatively small sample sizes. Although the sample size was relatively small, statistically significant results as regards the association of gene polymorphism with graft function were provided. However, this study should be replicated with a larger sample size before translating this information into clinical guidelines. Also, we did not measure the levels of IL 1 in renal transplant recipients and correlate it with the different IL-1β or IL-1RN genotypes and their haplotypes. Based on our results as well as others [3, 16, 20, 21], we assume that specific genotypes are ‘high’ or ‘low’ secretors.

Conclusions

This study sheds some light on the importance of detecting the underlying genetic polymorphisms that might impact graft function. Herein, we show that the IL-1β − 511 CT/TT polymorphic genotypes and IL-1RN/IL-1β polymorphic haplotypes were associated with early renal allograft dysfunction. To the best of our knowledge, this is the first report from Egypt suggesting that a high producer IL-1β genotype and a combination of low producer of IL-1RN with high producer IL-1β haplotype might act as risk factors for graft rejection. Thus, the measurement of IL-1 production in disease states might serve as a marker of the clinical course of the disease or can be used in the evaluation of therapeutic efficacy. Moreover, post-transplant down-regulation of IL-1 bioactivity, to reverse ongoing rejection, could provide an efficient therapeutic modality.

The results presented herein are observational data, and this is an exploratory research question that perhaps can be followed up prospectively and might open new avenues for personalized medicine. If the results of future studies were consistent with ours and in order to reduce the occurrence of graft dysfunction, clinicians can take necessary measures to identify the renal transplant recipients’ genotypes at risk of mounting an increased inflammatory response and hence administer the appropriate immunosuppressive therapy. Studies recruiting large numbers of renal transplant recipients, with prolonged periods of follow-up, are required before translating these findings to clinical guidelines for renal transplant recipients.