Introduction
Chronic allograft failure (CAF) is characterized by typical histological changes that include arteriosclerosis, interstitial fibrosis, and tubular atrophy.1 Previous evidence indicates that T cells are the main population among the immune effectors that infiltrate a chronically rejected allograft.2,3 There are various subsets of CD4+ T cells, including the classical Th1 and Th2 cells, Th17 cells, follicular helper T cells, and regulatory T cells. Multiple evidence suggests a clear association between Th17 and acute allograft rejection.2,3
Owing to the genetic diversity of the host, the immune system’s ability to respond to alloantigens contributes to several events that together lead to tubular damage, interstitial fibrosis, and lead to graft failure. Cytokines are the chief mediators of inflammation and play an important role as regulators of inflammation. Polymorphisms located in the regulatory or coding region of cytokine genes influence transcription and production of cytokines either as a low or high production.4–6 We have previously shown that low interleukin (IL) 10 producing genotypes have a high correlation with graft inflammation and histology markers of CAF, however, no association was found with any of the production-affecting polymorphisms in the genes encoding pro-inflammatory cytokines.7IL17 is regarded as a crucial cytokine that bridges the gap between the innate and adaptive immune responses. It is chiefly produced by T cells known as Th17 cells, additionally by other cell types, including CD8+ T cells and γδ T cells. This cytokine promotes inflammation and recruits neutrophils to sites of infection or inflammation.8IL17 is linked to the development of autoimmune diseases such as rheumatoid arthritis, psoriasis, and multiple sclerosis. In these conditions, excessive IL17 production contributes to chronic inflammation and tissue damage. Human and experimental models have shown an elevated level of IL17 mRNA and proteins for renal allograft rejection.9–11
Indeed, the synthesis and regulation of cytokines like IL17 have a genetic component that can influence their production and function within the immune system. The genetic background of an individual can contribute to variations in cytokine levels, which in turn may affect susceptibility to various diseases, including autoimmune conditions and transplant rejection. These genetic associations known as single nucleotide polymorphisms (SNPs) can lead to changes in the structure or expression of cytokines, potentially impacting their biological activities and the immune responses they regulate. The IL17 genes are located at the 6p12 region of chromosome 6. Genetic polymorphisms in the IL17A and IL17F have been studied extensively in relation to autoimmune diseases and transplantation outcomes which may impact the immune response to the transplanted organ and have been linked with the association of chronic inflammatory diseases such as ulcerative colitis,12 Bahcet′s disease,13 asthma,14 or inflammatory bowel disease.15,16
To date, the impact of heritable differences in IL17 genes and CAF among kidney transplant patients from America and North America has not been reported. Many of the studies have evaluated IL17A and IL17F gene polymorphisms′ effects on long-term kidney allograft function, however, none of the studies has reported the association of IL17F gene polymorphism at the genotype level with delayed graft function and chronic kidney allograft failure. Recently, Romanowski et al.,17 evaluated the impact of the IL17 gene polymorphisms (IL17A and IL17F) in 269 Caucasian deceased donor renal transplant recipients on long-term kidney allograft function. They reported that IL17A gene promoter polymorphism was associated with significantly decreased long-term kidney transplant function. However, none of the genotypes of IL17F was directly associated with the delayed graft function. In the present study, we used various histological scores as clinical endpoints for chronic kidney allograft failure to assess the significance of IL17F polymorphisms. The objective of the present study was to examine the association of all known five SNPs of IL17F gene polymorphisms (-1507C/T rs1889570, -1165A/G rs1266828, -5045C/T rs7771511, -6328C/T rs766748 and -7488G/A rs763780) with transplant surveillance biopsies (TSBs) in 218 transplanted kidney recipients such as glomerular sclerosis (gs), interstitial fibrosis/tubular atrophy (IF/TA), tubular atrophy (ct), mesangial matrix increase (mm), interstitial fibrosis (ci), fibrous intimal thickening (cv), arteriolar hyaline thickening (ah), interstitial inflammation (i), tubulitis (t), glomerulitis (g), allograft glomerulopathy (cg) and intimal arteritis (v) for chronic kidney allograft failure. The five IL17 SNPs were selected because more than 90% association have been linked with kidney allograft function in previous studies.1–3,7,17 We expect that these SNPs may impact post-transplant immunological response against the transplanted kidney. We observed weak associations or no associations between the studied IL17F gene polymorphisms with glomerular sclerosis and IF/TA scores. We are in concordance with other studies and one of our previous studies, reported no association.7 A lack of association between IL17F SNPs and CAF following kidney transplantation was observed when more stringent criteria were applied.
Material and methods
Subjects
From a total of 536 renal transplants performed at the Southern Alberta Transplant Program between 1997 and 2006, a cohort of 218 renal patients was evaluated in this retrospective study. The availability of recipients′ DNA and the corresponding 6- to 12-month follow-up of TSB results were two criteria for inclusion. The control group consisted of 10 healthy individuals and was used for optimization purposes. Written informed consent from all the patients was obtained before transplantation for TSBs to be used. Recipient demographic data were collected using the ALTRA database and health record censoring. All procedures performed in studies involving human participants were in accordance with the Helsinki declaration or comparable ethical standards. Ethical clearance (ethics ID 24259) was obtained from University of Calgary, Calgary, Canada, and written informed consent was obtained from all individual participants included in the study.
Histologic quantitation of TSBs
To monitor the function of the graft, kidney transplant patients routinely undergo TSBs of the kidney performed at 6 to 12 months after renal transplants to enable early detection of acute and chronic histologic alterations as well as for subsequent intervention. TSBs were performed using 18-gauge needles under ultrasound guidance to obtain two core samples. Hematoxylin-eosin, Masson′s trichrome, periodic acid-Schiff, and periodic acid-Schiff-methenamine-silver stains were used for developing biopsy slides for the TSBs′ paraffin and plastic sections. A transplant pathologist blinded to the treatment and original case diagnosis, independently reviewed each biopsy under light microscopy. TSBs were assessed according to numerous histologic factors only if the biopsy specimen had at least one artery and seven glomeruli.18 Enrolled patients were categorized based on histological grades scored in surveillance biopsy. Histological grading was based on the following parameters: glomerular sclerosis (gs), interstitial fibrosis/tubular atrophy (IF/TA), tubular atrophy (ct), mesangial matrix increase (mm), interstitial fibrosis (ci), fibrous intimal thickening (cv), arteriolar hyaline thickening (ah), interstitial inflammation (i), tubulitis (t), glomerulitis (g), allograft glomerulopathy (cg) and intimal arteritis (v). Each parameter has four score categories ranging from 0 to 3 [0 (normal), 1 (mild), 2 (moderate), and 3 (severe)]; a score ≥ 1 was considered high and a score < 1 was considered low.18
DNA extraction
The genomic DNA from the 200 µL buffy coat was extracted using the Qiagen Kit (Qiagen QIAamp DNA Blood Mini Kit, Qiagen Inc., Mississauga, ON, Canada), according to the manufacturer’s recommendations.
Sanger sequencing
Genomic DNA samples from 10 healthy individuals were genotyped by direct sequencing for each of the studied polymorphisms. Five SNPs screening in the IL17F gene was performed with five pairs of primer sets by polymerase chain reaction (PCR) followed by Sanger sequencing (Table 1) using dideoxynucleotide chain terminators labeled with different fluorescent dyes (BigDye Terminator V1.1 cycle sequencing kit; Applied Biosystems Inc., Waltham, MA, USA). The thermal cycling conditions involved one cycle of 5 m at 95 °C, and then 30 cycles of 30 s at 96 °C, 10 s at 58 °C and 4 m at 72 °C. Fluorochrome-labeled DNA fragments were ethanol purified, heat denatured, and electrophoresed by capillary electrophoresis on an Applied Biosystems 3130 genetic analyzer (Applied Biosystems Inc.). The analysis of sequence was done by genetic analyzer software and representative results are reported in Figure 1.
Table 1Primers used for IL17 gene sequencing
| Primer name | Primer sequence |
|---|
| 1507 Sequencing primer forward | 5′-CCTTCCTTCCTCCTGGGTAG-3′ |
| 1507 Sequencing primer reverse | 5′-AACTTCTCCTGCCCACCTTT-3′ |
| 1165 Sequencing primer forward | 5′-CAGGTCTGCCTGACATCAAA-3′ |
| 1165 Sequencing primer reverse | 5′-CCCTGGATGGAAGAAATGAA-3′ |
| 5667 Sequencing primer forward | 5′-GTGTAATTCCAGGGGGAGGT-3′ |
| 5667 Sequencing primer reverse | 5′-GGCTTGCCTTTCTGAGTGAG-3′ |
| 6328 Sequencing primer forward | 5′-GCAAAGAGCCAGAAAATTCG-3′ |
| 6328 Sequencing primer reverse | 5′-CTTGGAAGACCAAGCACTCC-3′ |
| 7488 Sequencing primer forward | 5′-CCATCCGTGCAGGTCTTATT-3′ |
| 7488 Sequencing primer reverse | 5′-TGTACAGGCCCAGTGTAGGAA-3′ |
Genotyping of IL17F polymorphisms using PCR–SSP
PCR-sequence specific priming (SSP) was performed to detect the single nucleotide variations in IL17F gene for -1507C/T (rs1889570), -1165A/G (rs1266828), -5045C/T (rs7771511), -6328C/T (rs766748) and -7488 C/T(rs763780) using allele-specific primers (Table 2). PCR–SSP was optimized based on the sequencing results to ensure accurate results. The primers were designed with optimized length, %GC content, and Tm. Gene sequencing samples served as positive controls and optimized the conditions for PCR–SSP-based genotyping of IL17F variants. SNPs were optimized to a unique reaction condition and composition. The procedure involved the amplification of five fragments and subsequent use of the amplicons for each SNP as a DNA template for sequencing. The thermal cycling involved one step of 2 m at 96 °C, 40 cycles of 96 °C for 15 s with annealing at 58 °C for 30 s and extension of 72 °C for 45 s, and one step of extension for 3 m at 72 °C. Each reaction contained primers specific for nonpolymorphic HLA-DRA gene: FDRA360 (5′-GAGGTAACTGTGCTCACGAACAGC-3′) and RDRA595 (5′-GGTCCATACCCCAGTGCTTGAGAAG-3′). The 283 bp band produced by these primers serves as a quality control and works as an internal positive control. The PCR-amplification products were analyzed by ethidium bromide-stained agarose gel (Fig. 1).
Table 2Primers for allele-specific amplification of IL17 SNPs
| Primer name | Primer sequence |
|---|
| 1507 Primer forward C | 5′-GTAAATCAAAGAATTTCTTTATGGC-3′ |
| 1507 Primer forward T | 5′-GGTAAATCAAAGAATTTCTTTATGGT-3′ |
| 1507 Primer reverse | 5′-TCATCTAACATCACCCCCCAC-3′ |
| 1165 Primer forward | 5′-CCATTGCTATATGCCATGAACCT-3′ |
| 1165 Primer reverse T | 5′-GAAAACAGGGGTTAGGAAATCCT-3′ |
| 1165 Primer reverse C | 5′-GAAAACAGGGGTTAGGAAATCCC-3′ |
| 5046 Primer forward C | 5′-AATCTGTATAAGAAAAATAGAGGCTTAATAAAC-3′ |
| 5046 Primer forward T | 5′-AATCTGTATAAGAAAAATAGAGGCTTAATAAAT-3′ |
| 5046 Primer reverse | 5′-GGGTGGCTCCGAAGAAGG-3′ |
| 6328 Primer forward | 5′-GCCCCATAGTAAGTCTTAATAAACTCATCC-3′ |
| 6328 Primer reverse C | 5′-ATGAGAAAACCTTGGGACGGTACTG-3′ |
| 6328 Primer reverse T | 5′-ATGAGAAAACCTTGGGACGGTACTA-3′ |
| 7488 Primer forward T | 5′-GGATATGCACCTCTTACTGCACTT-3′ |
| 7488 Primer forward C | 5′-GGATATGCACCTCTTACTGCACTC-3′ |
| 7488 Primer reverse | 5′-CACCAAGGCTGCTCTGTTTCTT-3′ |
Statistical analysis
Fisher’s exact test was used to analyze the difference between categorical variables. A two-tailed p-value < 0.05 was regarded as statistically significant. Univariate and multivariate logistic regression analysis were used to test the influence of demographic and clinical parameters such as donor hypertension, acute rejection, and recipient age on the pathogenesis of CAF and mismatches at human leukocyte antigen (HLA) class I (HLA-A or HLA-B) and Class II (HLA-DR or HLA-DQ) loci, between donor and recipient pair with IF/TA sores was calculated using SPSS Statistics software (IBM Corp., Armonk, NY, USA) package.
Results
Demographic profile and clinical characteristics of subjects
All recruited patients in this study, received their kidney transplants between 1997 and 2006. TSB was collected at 224 ± 66 days post-transplantation. The patients′ mean age at the time of the TSB was 47 ± 13.1 years (12–79 years). Of the 218 patients, 57% were men and 43% were women. The majority of the recipients′ ethnicity was Caucasian (71%) followed by Asian (8%) (Table 3). The primary disease in the patients was diabetes or hypertension with a prevalence of 28%. The mean donor age was 36 ± 17 years, and the mean cold ischemia time was 10 ± 7 h. The majority of kidney patients (69%) received grafts from deceased donors. The number of HLA class I (HLA-A, HLA-B) mismatches was 59% and for HLA class II (HLA-DR or HLA-DQ) was 63% (Table 3).
Table 3Demographic profile and distribution of clinical characteristics of the studied population
| Characteristic | Distribution or range |
|---|
| Number | 218 |
| Period of transplantation | 1997–2006 |
| Time between transplant and collection of TSB | 224 ± 66 days |
| Recipient |
| Age | 47 ± 13 |
| Caucasian ethnicity | 72% |
| Male sex | 55% |
| Primary disease of diabetes or hypertension | 28% |
| Donor |
| Cadaver | 70% |
| Age | 36 ± 17 |
| Cold ischemia time in h | 10 ± 7 |
| Number of HLA class I mismatches > 0 | 59% |
| Number of HLA class II mismatches > 0 | 63% |
Histologic parameters of TSBs
Eleven histologic parameters were scored in 184 patients who were followed for 6 to 12 months for TSB results (Table 4). The majority of the patients had a histologic grade score ≥ 1. IF/TA, which was calculated based on individual scoring of interstitial fibrosis and tubular atrophy, was observed in 64% of the patients. Other histologic parameters that scored a grade of more than or equal to 1 were 64% tubular atrophy, 58% mesangial matrix, and 51% interstitial fibrosis. The remaining histologic parameters with a score of more than or equal to 1 were observed in 19–43% of patients except for chronic allograft cg and v. The last two parameters had a normal score of 0 in 99% of patients and therefore, were excluded from the analysis (Table 4). Univariate logistic regression analysis of the clinical variables, donor age, donor sex, donor hypertension, and mismatches at HLA class I and class II loci, between patient and donor was significantly associated with IF/TA (Table 5). A significant correlation was observed between an inflammatory grade ≥ 1 and the number of HLA mismatches between the recipient and donor, the episode of acute rejections, and the type of immunosuppressant (Table 5).
Table 4Histological parameters of post-transplant surveillance biopsies obtained at 6–12 months post transplantation
| Histological parameter | Patients with grade/score ≥ 1, % (n) | Patients with grades/scores of 0–3, n
|
|---|
| Normal (0) | Mild (1) | Moderate (2) | Severe (3) |
|---|
| Interstitial fibrosis/tubular atrophy (IF/TA) | 63.8 (139) | 79 | 122 | 14 | 3 |
| Tubular atrophy (ct) | 63.8 (139) | 79 | 122 | 14 | 3 |
| Mesangial matrix increase (mm) | 57.8 (126) | 92 | 77 | 34 | 15 |
| Interstitial fibrosis(ci) | 50.5 (110) | 108 | 88 | 18 | 4 |
| Fibrous intimal thickening (cv) | 42.9 (93) | 124 | 74 | 19 | 0 |
| Arteriolar hyaline thickening (ah) | 41.4 (90) | 128 | 61 | 23 | 6 |
| Glomerular sclerosis (gs) | 37.6 (82) | 136 | 47 | 20 | 15 |
| Interstitial inflammation (i) | 30.3 (66) | 152 | 56 | 7 | 3 |
| Tubulitis (t) | 21.1 (46) | 172 | 21 | 22 | 3 |
| Glomerulitis (g) | 19.3 (42) | 176 | 30 | 12 | 0 |
| Allograft glomerulopathy (cg) | 0.9 (2) | 216 | 2 | 0 | 0 |
| Intimal arteritis (v) | 0.5 (1) | 217 | 1 | 0 | 0 |
Table 5Significance of the demographical and clinical variables using univariate logistic regression analysis
| Clinical variable | Distribution | Code | Dependent variable, p-valuea
|
|---|
| IF/TA, yes, no | Inflammation, yes, no |
|---|
| Recipient age | ≤ 50 year = 133; 50 > year = 87 | ≥50 | 0.860 | 0.941 |
| Donor age | ≤ year = 209; > 50 year = 49 | ≥50 | 0.001b | 0.460 |
| Donor type | CAD = 152; living = 66 | CAD, living | 0.014b | 0.525 |
| Donor BMI | ≤ 30 = 186; > 30 = 25 | ≥30 | 0.403 | 0.465 |
| Donor hypertension | Yes = 23; no = 195 | Yes, no | 0.011b | 0.619 |
| Cold ischemia time | ≤ 18 h = 177; > 18 h = 34 | ≥18 h | 0.917 | 0.848 |
| Mismatch number | ≤ 2 = 35; > 2 = 183 | 2 and more | 0.092 | 0.044b |
| Mismatch class 1 (HLA-A or HLA-B) | 0 = 10, 1 = 16, 2 = 50, 3 = 60, 4 = 70 | 0,1,2,3,4 | 0.047b | 0.375 |
| Mismatch class 2 (HLA-Dr or HLA-DQ) | 0 = 19, 1 = 21, 2 = 72, 3 = 47, 4 = 47 | 1,2,3,4 | 0.034b | 0.242 |
| Acute rejection | Yes = 54; no = 164 | Yes, no | 0.090 | 0.000b |
| Delayed graft function | Yes = 13; no = 202 | Yes, no | 0.673 | 0.207 |
| CMV infection | Yes = 33; no = 185 | Yes, no | 0.987 | 0.684 |
| Immunosuppressant | CsA = 119; FK506 = 99 | CsA vs. FK506 | 0.972 | 0.000b |
| Induction therapy | Yes = 36; no = 182 | Yes, no | 0.251 | 0.253 |
| IL-2 therapy | Yes = 99; no = 119 | Yes, no | 0.750 | 0.993 |
Association of IL17F variants with changes in histopathological parameters
A total of five single nucleotide variants of IL17F gene were analyzed by SSP–PCR based genotyping in 218 renal transplant patients. To determine the accuracy of the PCR–SSP based typing, DNA from 10 healthy individuals was obtained and genotyped by direct sequencing (Fig. 1). Homozygotes and heterozygotes for each of the IL17F variants were then confirmed by PCR–SSP based genotyping.
All the renal transplant patients were scored for 12 histologic parameters for 6–12 months post-transplantation (Table 4). These scores were then tested for their association with the studied IL17F variants through univariate logistic regression analysis. Of the studied parameters, the incidence of glomerular sclerosis was found to be associated with IL17F 1165 G allele [p = 0.004, odds ratio (OR) = 0.39, 95% confidence interval (CI) = 0.21–0.74] and 1165 AA genotype (p = 0.017, OR = 2.7, 95% CI = 1.1–6.0). No association was observed between IL17F 1165 alleles and/or genotypes with any of the histologic parameters. Statistically significant associations of IF/TA, and tubular atrophy were observed with IL17F -1507C allele (p = 0.029, OR = 2.2, 95% CI = 1.09–4.47). Moreover, IL17F -1507T allele (rs1889570) was observed to be associated with better glomerulitis score (p = 0.041, OR = 0.3, 95% CI = 0.09–0.93) and allele C of IL17 -1165C/T (rs1266828) was associated with better glomerular sclerosis (p = 0.004, OR = 0.39) score (Table 6).
Table 6Association between IL17F gene polymorphisms and histologic parameters.
| Parameters | Values | TT | TC | CC | C | T |
|---|
| IL-17 -1165C/T (rs1266828) |
| gs | Frequency (with gs) | 33/65 (50.8%) | 20/65 (30.8%) | 12/65 (18.5%) | 32/65 (49.2%) | 53/65 (81.5%) |
| Frequency (without gs) | 34/117 (29.1%) | 50/117 (42.7%) | 33/117 (28.2%) | 83/117 (70.9%) | 84/117 (72%) |
| p-value | | 0.017 | 0.004 | NS |
| OR (95% CI) | | 2.7 (1.1–6.0) | 0.39 (0.21–0.74) | NS |
| IL17 -1507C/T (rs1889570) |
| IF/TA | Frequency (with IF/TA) | 20/119 (16.8%) | 90/119 (75.6%) | 9/119 (7.6%) | 99/119 (83.2%) | 110/119 (92.5%) |
| Frequency (without IF/TA) | 21/68 (30.9%) | 42/68 (61.7%) | 5/68 (7.3%) | 47/68 (69.1 %) | 63/68 (92.6%) |
| p-value | NS | NS | NS | 0.03 | NS |
| OR (95% CI) | NS | NS | NS | 2.2 (1.09–4.47) | |
| ct | Frequency (with ct) | 20/119 (16.8%) | 90/119 (75.6%) | 9/119 (7.6%) | 99/119 (83.2%) | 110/119 (92.5%) |
| Frequency (without ct) | 21/68 (30.9%) | 42/68 (61.7%) | 5/68 (7.3%) | 47/68 (69.1%) | 63/68 (92.6%) |
| p-value | NS | NS | NS | 0.029 | NS |
| OR (95% CI) | NS | NS | NS | 2.2 (1.09–4.47) | NS |
| g | Frequency (with g) | 10/38 | 22/38 | 6/38 | 28/38 (73.7%) | 32/38 (84.2%) |
| Frequency (without g) | 31/149 | 110/149 | 8/149 | 118/149 (83.7%) | 141/149 (94.6%) |
| p-value | NS | NS | NS | NS | 0.041 |
| OR (95% CI) | NS | NS | NS | NS | 0.30 (0.09–0.93) |
No other studied variant was found to be associated with any of the histological scores. The associations observed in the present univariate analysis were however, of weak significance and did not yield any significant relationship when corrected for multiple comparisons (data not shown).
Discussion
CAF is a complex inflammatory process that involves HLA, tumor necrosis factor α, transforming growth factor β, and other cytokines. Given that the biologic signature of CAF is an infiltration of inflammatory cells in the renal interstitium, the understanding of the causal agents of CAF is not fully understood. IL17 cytokine is chiefly produced by Th17 cells and plays an important role in the emergence of inflammatory disorders.19–21 The hypothetical framework of the present study was that, IL17, an important pro-inflammatory cytokine, known to have roles in genetic predisposition to a host of inflammatory diseases may be involved in the development of CAF. Five single nucleotide variants of exonic and regulatory regions of IL17F genes were investigated for their association with histological parameters related to CAF. The studied gene variants included IL17F -1507C/T (rs1889570), -1165A/G (rs1266828), -5045C/T (rs7771511), -6328C/T (rs766748) and -7488G/A (rs763780). As far as is known, this is the first study evaluating the roles of these variants in genetic predisposition to CAF in kidney transplant recipients from America and North America. Our current study did not rely solely on a single pathologist’s clinical diagnosis; rather, the association analysis was conducted utilizing specific histological scores that were accurately evaluated by a second pathologist. In the present study, univariate logistic regression analysis showed a weak association of alleles and genotypes of IL17F -1165 and alleles of -1507 with glomerular sclerosis as well as interstitial fibrosis/tubular atrophy and glomerulitis respectively. Although a trend was observed with these variants, however, upon correction for multiple comparisons, no association was observed.
IL17 is a proinflammatory cytokine that contributes significantly in tissue inflammation by inducing the release of mobilizing cytokines. Uncontrolled Th17 lineage is known to play a pathogenic role in the chronic inflammation process of autoimmune diseases, such as rheumatoid arthritis, inflammatory bowel disease, multiple sclerosis, and psoriasis. It has been demonstrated that ectopic expression of IL17F aggravates pulmonary neutrophilia and amplifies inflammatory responses in animal model.22 Genetic variants such as 6328G/A and 7488A/G, have been found to be associated with chronic inflammatory diseases, particularly atrophic gastritis, rheumatoid arthritis, and myocardial infarction.15,23,24
The results of the present study did not support the association of IL17 polymorphisms with the histopathology associated with CAF, suggesting that the role of these cytokines is more complicated than currently understood in this multifactorial anomaly. In an earlier study from our group,7 we reported that high IF/TA grade and interstitial inflammation scores, as well as a significant influx of inflammatory cells in the renal allograft interstitium, were all highly related to kidney transplant patients who were genetically predisposed to produce low levels of IL10 and no association of low production of IL10 was observed with other tested histologic parameters.
Recently, gene polymorphisms in IL17A and IL17F were associated with histopathological changes in kidney transplants.25 Authors reported a possible association between the IL17A gene polymorphisms and histopathological changes in biopsies. A weak association was reported with the C allele at the -7488 A/G IL17F gene polymorphism with IF/TA score, however, no significant association remained significant after multiple-level correction. Similar to the finding we also observed a weak association of C allele of IL17 -1507C/T (rs1889570) with IF/TA score (p = 0.03) (Table 6). Another paper from the group of Tunisia examines the effects of IL17F gene polymorphisms on kidney transplantation outcomes utilizing direct sequencing in 93 recipients of kidney transplants.26 The IL17F SNPs under study did not show any statistically significant correlation with the onset of acute rejection. They concluded that the IL17F 7489A/G A allele may be associated with a decreased risk of acute rejection and improved graft survival, as in the present study allele T of IL17 -1507C/T (rs1889570) was associated with the better glomerulitis score (p = 0.041, OR = 0.30) and allele C of IL17-1165C/T (rs1266828) was associated with better glomerular sclerosis (p = 0.004, OR = 0.39) (Table 6). Further, another study, published by Hejr et al.,27 on the association of IL17 gene polymorphisms with HBV-induced rejection in kidney transplants, is also in favor of our study. The studied gene polymorphism of IL17 (-197 A/G, rs2275913) did not associate with acute rejection. On the other hand, Romanowski et al. studied the impact of IL17A and IL17F gene polymorphisms on post-kidney transplant return to dialysis and long-term kidney allograft function in 269 Caucasian deceased donor renal transplant recipients.17 Genotyping was done on the IL17A and IL17F gene polymorphism and creatinine concentrations as a cutoff criterion for transplant rejection detection. None of the genotypes of IL17F was directly associated with the delayed graft function. In multivariate logistic regression analysis adjusted for recipients′ age and sex, they observed an association between the C allele and decreased probability of delayed graft function (OR = 0.44, p = 0.046). They further concluded that the GA genotype of the IL17F gene polymorphism (rs11465553) may be linked to a risk of graft function loss and a need for dialysis again following kidney transplantation.
Even though there is some experimental evidence directly associating Th17 cells with allograft rejection, the majority of clinical transplantation studies that have been published so far are concerned with IL17 expression and IL17 serum level.28,29 A study by Crispim et al. on kidney allograft outcome reported significantly increased levels of IL17 among samples derived from patients with rejection in contrast,30 to the nonrejection group. Studies have shown that IL17 is involved in experimental and human renal allograft rejection at an early stage, and human subclinical rejection allograft biopsy tissue has been reported to exhibit IL17 protein.11 Yuan et al. have demonstrated the importance of CD4+ Th17 cells for allograft rejection where an aggressive proinflammatory response leading to severe accelerated allograft rejection and vasculopathy was detected in CD4+ Th17 mediated cells.31 There is very little data or no study available on the expression profile of IL17 and delayed graft function compared to allograft rejection.
The absence of significant associations with chronic kidney allograft failure in this study could be the smaller sample size in genotype distribution and correlation with CAF which is one of the limitations of the study. Although more than 70% of the renal transplant recipients included in the study were Caucasians, a potential bias of demographic stratification may also be seen as a limiting factor. This study did not examine the role of IL17 polymorphism in donors and because of the lack of availability of serum at different time points, we were unable to detect the serological levels of IL17 in CAF which is another limiting factor. Because many patients were lost during follow-up, we were unable to do the survival analysis. Studies on hematopoietic stem transplant setting have shown that high-producing IL17 polymorphism in donors showed a trend of association with acute graft versus host disease.32
Conclusions
Our results do not support a major association of IL17F SNPs with predisposition to CAF. A lack of association of an important pro-inflammatory cytokine, IL17, in the present study, is consistent with our earlier observation and other studies published online. This emphasizes the nuanced nature of genetic associations and underscores the importance of carefully analyzing and interpreting genetic data while accounting for statistical considerations. Unfortunately, there are few studies on the effect of IL17 gene polymorphisms in chronic kidney allograft failure, which makes it challenging to compare the data. Therefore, more studies from different parts of the world and North America are needed to confirm these findings.
Abbreviations
- BMI:
body mass index
- CAF:
chronic allograft failure
- CAD:
Cadaver
- CI:
confidence interval
- CMV:
cytomegalovirus
- HLA:
human leukocyte antigen
- i:
interstitial inflammation
- IF/TA:
interstitial fibrosis/tubular atrophy
- IL:
interleukin
- OR:
odds ratio
- PCR:
polymerase chain reaction
- PCR:
polymerase chain reaction
- SSP:
sequence specific primer
- SNP:
single nucleotide polymorphism
- TSB:
transplant surveillance biopsy
Declarations
Acknowledgement
There is nothing to declare.
Ethical statement
All procedures performed in studies involving human participants were in accordance with the Helsinki declaration or comparable ethical standards. Ethical clearance (ethics ID 24259) was obtained from University of Calgary, Calgary, Canada, and written informed consent was obtained from all individual participants included in the study.
Data sharing statement
The data that support the findings of this study are available from the corresponding author upon reasonable request.
Funding
The authors would like to express their gratitude to the Calgary Laboratory Services, for the partial funding for the project (Total funds: $10,000).
Conflict of interest
The authors declare that they have no conflict of interests related to this study.
Authors’ contributions
Conception and study design (NB, FK and AL); data generation and analysis (GT, AL, FK and DIO); original manuscript draft and revision (GT and AL); scientific input (NB, AL, GT and FK); performed the study (DIO and AL); provided materials and reagents, and managed the study (AS, SY, NB, FK); revision of manuscript (GT, AL, DIO, RF, FK, AS, SY and NB). All authors have read and approved the manuscript.