# T-Cell Cytokine Gene Polymorphisms and Vitamin D Pathway Gene Polymorphisms in End-Stage Renal Disease due to Type 2 Diabetes Mellitus Nephropathy: Comparisons with Health Status and Other Main Causes of End-Stage Renal Disease

Authors: Alicja E. Grzegorzewska; Grzegorz Ostromecki; Paulina Zielińska; Adrianna Mostowska; Paweł P. Jagodziński
Journal: Journal of Diabetes Research
 (2014)

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## Abstract

Background. T-cell cytokine gene polymorphisms and vitamin D pathway gene polymorphisms were evaluated as possibly associated with end-stage renal disease (ESRD) resulting from type 2 diabetes mellitus (DM) nephropathy. Methods. Studies were conducted among hemodialysis (HD) patients with ESRD due to type 2 DM nephropathy, chronic glomerulonephritis, chronic infective tubulointerstitial nephritis, and hypertensive nephropathy as well as in healthy subjects. A frequency distribution of T-cell-related interleukin (IL) genes (IL18 rs360719, IL12A rs568408, IL12B rs3212227, IL4R rs1805015, IL13 rs20541, IL28B rs8099917, IL28B, and rs12979860) and vitamin D pathway genes (GC genes: rs2298849, rs7041, and rs1155563; VDR genes: rs2228570, rs1544410; and RXRA genes: rs10776909, rs10881578, and rs749759) was compared between groups. Results. No significant differences in a frequency distribution of tested polymorphisms were shown between type 2 DM nephropathy patients and controls. A difference was found in IL18 rs360719 polymorphic distribution between the former group and chronic infective tubulointerstitial nephritic patients (Ptrend=0.033), which also differed in this polymorphism from controls (Ptrend=0.005). Conclusion. T-cell cytokine and vitamin D pathway gene polymorphisms are not associated with ESRD due to type 2 DM nephropathy in Polish HD patients. IL18 rs360719 is probably associated with the pathogenesis of chronic infective tubulointerstitial nephritis.


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## Body

## 1. Introduction

Diabetes mellitus (DM) is the most common cause of end-stage renal disease (ESRD) in many hemodialysis (HD) centers. In Australia and New Zealand, the incident ESRD population (1991–2005) who began renal replacement therapy (RRT) included 30.0% type 2 DM and 4.5% type 1 DM subjects [1]. In the HEMODIALYSIS (HEMO) study, the group of HD patients comprised approximately 45% of DM subjects [2].Diabetic ESRD patients compared to nondiabetic ESRD subjects show higher both mortality rate [3] and prevalence of coronary artery disease (CAD) [4], are more prone to severe infections [5] and worse response to hepatitis B vaccination [6], and more often suffer from adynamic bone disease associated with low serum parathyroid hormone (PTH) levels [7]. In this paper we will focus on ESRD due to type 2 DM nephropathy. Together with altered glucose metabolism and insulin resistance, deficiency of vitamin D [8] and aberrant T-cell cytokine balance [9] were found to be associated with this severe complication of type 2 DM. There is a link between vitamin D and T-cell functional balance: active form of vitamin D [1,25(OH)2D] has the inhibitory effect on the T helper (Th) 17 and Th1 response [10].Abnormalities in T-cell cytokine equilibrium [11–13] and plasma vitamin D concentrations [14–16] are related to cardiovascular events [13, 16] and immunononcompetence during infections [11, 14] and vaccinations [12, 15]. Serum PTH levels are dependent on serum vitamin D concentrations [17], and T cells are implicated in the mechanism of PTH action in bone [18].Vitamin D activity may be adequately expressed if vitamin D pathway components (vitamin D binding protein, also referred to as group-specific component (GC), vitamin D receptor (VDR), and retinoid X receptors (RXRs)) are properly structured and regulated. The recent study by Zhang et al. [19] has shown thatVDRBsmI polymorphism correlates with type 2 DM nephropathy and may be susceptible for early onset of this nephropathy. Among T-cell-related cytokine gene polymorphisms, promoter polymorphic variants ofIL10 [20, 21] andIL6[22] were already associated with the risk of type 2 DM nephropathy. Monocyte chemoattractant protein 1 (MCP-1) has been reported to participate in the pathogenesis of early type 2 DM nephropathy [23], but MCP1 polymorphism in the promoter region was not differentially distributed between ESRD patients with type 2 DM nephropathy and healthy controls [24, 25].To our knowledge, there are scarce data, if any, on ESRD due to type 2 DM nephropathy showing a frequency distribution of single nucleotide polymorphisms (SNPs) of T-cell-related IL genes:IL18rs360719,IL12Ars568408,IL12Brs3212227,IL4Rrs1805015,IL13rs20541,IL28Brs8099917, andIL28Brs12979860 as well as vitamin D pathway genes: GC genes (GCrs2298849, rs7041, and rs1155563), VDR genes (VDRrs2228570, rs1544410), and RXR α genes (RXRArs10776909, rs10881578, and rs749759). The aim of our study was to determine the potential association between aforementioned polymorphisms of T-cell-related cytokine genes and vitamin D pathway genes and ESRD due to type 2 DM nephropathy. For comparisons, aforementioned genotype frequencies of healthy controls as well as ESRD patients with other main causes of ESRD were used. Polymorphism related associations, if exist, could contribute to explanation of susceptibility to ESRD due to type 2 DM nephropathy and phenotype differences between ESRD patients with type 2 DM nephropathy and other causes of ESRD.


## 2. Material and Methods

### 2.1. Patients and Controls

Blood samples for genotype analyses are collected since 2009 from ESRD patients (estimated glomerular filtration rate (eGFR) category G5 in accordance with KDIGO recommendations [26]). All subjects were treated with HD on enrolment. Controls were recruited from blood donors and healthy volunteers unrelated to patients. All enrolled individuals live/lived in the Greater Poland region of Poland.Genotyping ofIL18rs360719,IL12Ars568408,IL12Brs3212227,IL4Rrs1805015, andIL13rs20541 polymorphisms was performed in 2009–2012 using currently available material. Results had been analyzed in our previous studies in the context of responsiveness to the surface antigen of hepatitis B virus (HBsAg) using data of all (not segregated) patients [27–30]. For this study, we used results of controls and patients with type 2 DM nephropathy, chronic glomerulonephritis, chronic infective tubulointerstitial nephritis, and hypertensive nephropathy.IL28Brs8099917,IL28Brs12979860,GCrs2298849,GCrs7041,GCrs1155563,VDRrs2228570,VDRrs1544410,RXRArs10776909,RXRArs10881578, andRXRArs749759 polymorphisms were analyzed in winter 2013/2014 among HD patients with ESRD (n=893) due to type 2 DM nephropathy (n=366), chronic glomerulonephritis (n=178), chronic infective tubulointerstitial nephritis (n=118), and hypertensive nephropathy (n=231) as well as healthy controls (n=378).DM was not diagnosed in patients having renal diseases other than type 2 DM nephropathy.Healthy individuals and HD patients with other renal diseases as cause of ESRD served as reference groups for a frequency distribution of tested polymorphic variants. All examined subjects were of Caucasian race.Basic clinical and laboratory data were collected on enrolment and they are updated every year.


### 2.2. Genotyping

Genomic DNA for genotype analysis was isolated from peripheral blood lymphocytes by salt-out extraction procedure.Genotyping ofIL18rs360719,IL12Ars568408,IL12Brs3212227,IL4Rrs1805015, andIL13rs20541 polymorphisms was performed as previously described [27–30].IL28Brs8099917 andIL28Brs12979860 polymorphisms were genotyped using high-resolution melting curve analysis (HRM) on the LightCycler 480 system (Roche Diagnostics, Mannheim, Germany) with the use of 5x HOT FIREPol EvaGreen HRM Mix (Solis BioDyne, Tartu, Estonia). The PCR program consisted of an initial step at 95°C for 15 min to activate HOT FIREPol DNA polymerase, followed by 50 amplification cycles of denaturation at 95°C for 10 s, annealing at 61°C for 10 s, and elongation at 72°C for 15 s. Amplified DNA fragments were then subjected to HRM with 0.1°C increments in temperatures ranging from 76 to 96°C. Primers used for PCR with subsequent HRM analysis were as follows: rs8099917F 5′TTTGTCACTGTTCCTCCTTTTG3′, rs8099917R 5′AAGACATAAAAAGCCAGCTACCA3′, rs12979860F 5′CGTGCCTGTCGTGTACTGAA3′, and rs12979860R 5′AGGCTCAGGGTCAATCACAG3′.Genotyping of theGCrs1155563,GC rs2298849,RXRArs10881578, andRXRArs10776909 polymorphisms was carried out by HRM on the Bio-Rad CFX96 Real Time PCR system (Bio-Rad, Hercules, CA). DNA fragments amplified with the use of specific primers were subjected to HRM with 0.1°C increments in temperatures ranging from 71 to 92°C. Genotyping of theGCrs7041,RXRA rs749759,VDRrs1544410, andVDRrs2228570 was performed using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method according to the manufacturer’s instructions (Fermentas, Vilnius, Lithuania). Primer sequences and conditions for HRM and PCR-RFLP analyses are presented in Table 1.Table 1
HRM and RFLP conditions for the identification of polymorphisms genotyped in the vitamin D pathway related genes.
Gene symbol
rs number
Alleles
Primers for PCR amplification (5′-3′)
Annealing temp. (°C)
PCR product length (bp)
HRMa analysis
RFLPb analysis
Melting temp. range (°C)
Restriction enzyme
Restriction fragment length (bp)
GC
rs7041
G/T
F: GGAGGTGAGTTTATGGAACAGC
66.3
493
HaeIII
T = 493
R: GGCATTAAGCTGGTATGAGGTC
G = 414 + 79
rs1155563
C/T
F: GGTTATTCTAAGACTGTGCTCTTGC
63.0
116
71–78
R: ATGTGTTCTCACTGTTCGACTCC
rs2298849
C/T
F: TCCACTGGCAAAACACATTAC
60.6
118
73–83
R: GGGACATCTGCATTTATCCTG
RXRA
rs10881578
A/G
F: TCTTGAGCAATGCCAGCAG
60.6
75
80–90
R: CCACAGCTCACACATCCAATC
rs10776909
C/T
F: CAGCCTGTGGCCTGCTCA
60.6
95
82–92
R: AACCTCCGGCCCTTGGAG
rs749759
A/G
F: ATAGGGCTTGCCTGCCTAGA
62.6
382
BstXI
A = 382
R: CTCCACCATAGCCCAAGTGA
G = 243 + 139
VDR
rs1544410
A/G
F: GGAGACACAGATAAGGAAATAC
60.6
248
FspI
A (B) = 248
R: CCGCAAGAAACCTCAAATAACA
G (b) = 175 + 73
rs2228570
C/T
F: GCACTGACTCTGGCTCTGAC
72.5
341
FokI
C (F) = 341
R: ACCCTCCTGCTCCTGTGGCT
T (f) = 282 + 59
aHRM analysis: high resolution melt analysis.
bRFLP analysis: restriction fragment length polymorphism analysis.For quality control, the genotyping analysis was blinded to the subject’s case-control status. In addition, approximately 10% of the randomly chosen samples were regenotyped. Samples that failed the genotyping were excluded from further statistical analyses.


### 2.3. 25(OH)D Testing

Plasma 25(OH)D was determined in blindly selected 162 HD patients in the winter season of the year to avoid differences in sunlight exposure between patients who used to sunbathe and those who did not. Plasma 25(OH)D concentration was measured in HD patients who had not been treated with vitamin D or had stopped such a treatment for at least 3 weeks to obtain the so-called basic vitamin D concentrations. Under these conditions, there were no patients showing optimal plasma 25(OH)D levels (35–80 ng/mL for adults). To examine plasma 25(OH)D levels, a chemiluminescent microparticle immunoassay (CMIA) was used according to the manufacturer’s instructions (Abbott Diagnostics ARCHITECT 25-OH VITAMIN D CMIA).


### 2.4. Statistical Methods

Results are presented as percentage for categorical variables, as mean with one standard deviation for normally distributed continuous variables or as median with range for not normally distributed continuous variables as tested by the Shapiro-Wilk test. Statistical tests used for comparison of data obtained in selected groups are indicated atP values.Hardy-Weinberg equilibrium (HWE) was tested to compare the observed genotype frequencies to the expected ones using Chi-square test. Distributions of tested polymorphisms were consistent with HWE with three exceptions which are indicated in tables showing analysis of genotype and allele distributions. The Fisher exact probability test or Chi-square test was used to evaluate differences in genotype and allele prevalence between the examined groups. Homozygotes for the major allele were the reference group. The odds ratio (OR) withP value and 95% confidence intervals (95% CI) value were calculated. All probabilities were two-tailed. Polymorphisms were tested for association using the Chi-square test for trend (Ptrend). Power analysis was performed by Fisher’s exact test.Values ofP<0.05 were judged to be significant. However, associations were reported only if the following conditions were fulfilled.(1)
A genotype distribution was consistent with HWE in a tested group and a referent group.(2)
Ptrend was below 0.05.(3)
Odds ratio remained significant after the Bonferroni correction applied for multiple testing, if appropriate.Aforementioned statistical calculations were performed using GraphPad InStat 3.10, 32 bit for Windows, created on July 9, 2009 (GraphPad Software, Inc., La Jolla, USA), CytelStudio version 10.0, created on January 16, 2013 (CytelStudio Software Corporation, Cambridge, USA), and Statistica version 10, 2011 (StatSoft, Inc., Tulsa, USA).


## 2.1. Patients and Controls

Blood samples for genotype analyses are collected since 2009 from ESRD patients (estimated glomerular filtration rate (eGFR) category G5 in accordance with KDIGO recommendations [26]). All subjects were treated with HD on enrolment. Controls were recruited from blood donors and healthy volunteers unrelated to patients. All enrolled individuals live/lived in the Greater Poland region of Poland.Genotyping ofIL18rs360719,IL12Ars568408,IL12Brs3212227,IL4Rrs1805015, andIL13rs20541 polymorphisms was performed in 2009–2012 using currently available material. Results had been analyzed in our previous studies in the context of responsiveness to the surface antigen of hepatitis B virus (HBsAg) using data of all (not segregated) patients [27–30]. For this study, we used results of controls and patients with type 2 DM nephropathy, chronic glomerulonephritis, chronic infective tubulointerstitial nephritis, and hypertensive nephropathy.IL28Brs8099917,IL28Brs12979860,GCrs2298849,GCrs7041,GCrs1155563,VDRrs2228570,VDRrs1544410,RXRArs10776909,RXRArs10881578, andRXRArs749759 polymorphisms were analyzed in winter 2013/2014 among HD patients with ESRD (n=893) due to type 2 DM nephropathy (n=366), chronic glomerulonephritis (n=178), chronic infective tubulointerstitial nephritis (n=118), and hypertensive nephropathy (n=231) as well as healthy controls (n=378).DM was not diagnosed in patients having renal diseases other than type 2 DM nephropathy.Healthy individuals and HD patients with other renal diseases as cause of ESRD served as reference groups for a frequency distribution of tested polymorphic variants. All examined subjects were of Caucasian race.Basic clinical and laboratory data were collected on enrolment and they are updated every year.


## 2.2. Genotyping

Genomic DNA for genotype analysis was isolated from peripheral blood lymphocytes by salt-out extraction procedure.Genotyping ofIL18rs360719,IL12Ars568408,IL12Brs3212227,IL4Rrs1805015, andIL13rs20541 polymorphisms was performed as previously described [27–30].IL28Brs8099917 andIL28Brs12979860 polymorphisms were genotyped using high-resolution melting curve analysis (HRM) on the LightCycler 480 system (Roche Diagnostics, Mannheim, Germany) with the use of 5x HOT FIREPol EvaGreen HRM Mix (Solis BioDyne, Tartu, Estonia). The PCR program consisted of an initial step at 95°C for 15 min to activate HOT FIREPol DNA polymerase, followed by 50 amplification cycles of denaturation at 95°C for 10 s, annealing at 61°C for 10 s, and elongation at 72°C for 15 s. Amplified DNA fragments were then subjected to HRM with 0.1°C increments in temperatures ranging from 76 to 96°C. Primers used for PCR with subsequent HRM analysis were as follows: rs8099917F 5′TTTGTCACTGTTCCTCCTTTTG3′, rs8099917R 5′AAGACATAAAAAGCCAGCTACCA3′, rs12979860F 5′CGTGCCTGTCGTGTACTGAA3′, and rs12979860R 5′AGGCTCAGGGTCAATCACAG3′.Genotyping of theGCrs1155563,GC rs2298849,RXRArs10881578, andRXRArs10776909 polymorphisms was carried out by HRM on the Bio-Rad CFX96 Real Time PCR system (Bio-Rad, Hercules, CA). DNA fragments amplified with the use of specific primers were subjected to HRM with 0.1°C increments in temperatures ranging from 71 to 92°C. Genotyping of theGCrs7041,RXRA rs749759,VDRrs1544410, andVDRrs2228570 was performed using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) method according to the manufacturer’s instructions (Fermentas, Vilnius, Lithuania). Primer sequences and conditions for HRM and PCR-RFLP analyses are presented in Table 1.Table 1
HRM and RFLP conditions for the identification of polymorphisms genotyped in the vitamin D pathway related genes.
Gene symbol
rs number
Alleles
Primers for PCR amplification (5′-3′)
Annealing temp. (°C)
PCR product length (bp)
HRMa analysis
RFLPb analysis
Melting temp. range (°C)
Restriction enzyme
Restriction fragment length (bp)
GC
rs7041
G/T
F: GGAGGTGAGTTTATGGAACAGC
66.3
493
HaeIII
T = 493
R: GGCATTAAGCTGGTATGAGGTC
G = 414 + 79
rs1155563
C/T
F: GGTTATTCTAAGACTGTGCTCTTGC
63.0
116
71–78
R: ATGTGTTCTCACTGTTCGACTCC
rs2298849
C/T
F: TCCACTGGCAAAACACATTAC
60.6
118
73–83
R: GGGACATCTGCATTTATCCTG
RXRA
rs10881578
A/G
F: TCTTGAGCAATGCCAGCAG
60.6
75
80–90
R: CCACAGCTCACACATCCAATC
rs10776909
C/T
F: CAGCCTGTGGCCTGCTCA
60.6
95
82–92
R: AACCTCCGGCCCTTGGAG
rs749759
A/G
F: ATAGGGCTTGCCTGCCTAGA
62.6
382
BstXI
A = 382
R: CTCCACCATAGCCCAAGTGA
G = 243 + 139
VDR
rs1544410
A/G
F: GGAGACACAGATAAGGAAATAC
60.6
248
FspI
A (B) = 248
R: CCGCAAGAAACCTCAAATAACA
G (b) = 175 + 73
rs2228570
C/T
F: GCACTGACTCTGGCTCTGAC
72.5
341
FokI
C (F) = 341
R: ACCCTCCTGCTCCTGTGGCT
T (f) = 282 + 59
aHRM analysis: high resolution melt analysis.
bRFLP analysis: restriction fragment length polymorphism analysis.For quality control, the genotyping analysis was blinded to the subject’s case-control status. In addition, approximately 10% of the randomly chosen samples were regenotyped. Samples that failed the genotyping were excluded from further statistical analyses.


## 2.3. 25(OH)D Testing

Plasma 25(OH)D was determined in blindly selected 162 HD patients in the winter season of the year to avoid differences in sunlight exposure between patients who used to sunbathe and those who did not. Plasma 25(OH)D concentration was measured in HD patients who had not been treated with vitamin D or had stopped such a treatment for at least 3 weeks to obtain the so-called basic vitamin D concentrations. Under these conditions, there were no patients showing optimal plasma 25(OH)D levels (35–80 ng/mL for adults). To examine plasma 25(OH)D levels, a chemiluminescent microparticle immunoassay (CMIA) was used according to the manufacturer’s instructions (Abbott Diagnostics ARCHITECT 25-OH VITAMIN D CMIA).


## 2.4. Statistical Methods

Results are presented as percentage for categorical variables, as mean with one standard deviation for normally distributed continuous variables or as median with range for not normally distributed continuous variables as tested by the Shapiro-Wilk test. Statistical tests used for comparison of data obtained in selected groups are indicated atP values.Hardy-Weinberg equilibrium (HWE) was tested to compare the observed genotype frequencies to the expected ones using Chi-square test. Distributions of tested polymorphisms were consistent with HWE with three exceptions which are indicated in tables showing analysis of genotype and allele distributions. The Fisher exact probability test or Chi-square test was used to evaluate differences in genotype and allele prevalence between the examined groups. Homozygotes for the major allele were the reference group. The odds ratio (OR) withP value and 95% confidence intervals (95% CI) value were calculated. All probabilities were two-tailed. Polymorphisms were tested for association using the Chi-square test for trend (Ptrend). Power analysis was performed by Fisher’s exact test.Values ofP<0.05 were judged to be significant. However, associations were reported only if the following conditions were fulfilled.(1)
A genotype distribution was consistent with HWE in a tested group and a referent group.(2)
Ptrend was below 0.05.(3)
Odds ratio remained significant after the Bonferroni correction applied for multiple testing, if appropriate.Aforementioned statistical calculations were performed using GraphPad InStat 3.10, 32 bit for Windows, created on July 9, 2009 (GraphPad Software, Inc., La Jolla, USA), CytelStudio version 10.0, created on January 16, 2013 (CytelStudio Software Corporation, Cambridge, USA), and Statistica version 10, 2011 (StatSoft, Inc., Tulsa, USA).


## 3. Results

Characteristics of the examined HD patients are presented in Tables2 and 3. ESRD patients due to type 2 DM nephropathy compared to non-DM ESRD patients showed older age at RRT onset, shorter treatment with RRT, higher death rate on RRT, higher prevalence of CAD and myocardial infarction, lower serum PTH level, and lower frequency of parathyroidectomy and treatment with cinacalcet.Table 2
Characteristics of hemodialysis patients (n = 893).
Parameter
Type 2 DM nephropathy
Other causes of ESRD
P value
Demographic data
n = 366
n = 527
Male sex,n (% of all)
201  (54.9)
307  (58.3)
0.337b
Age at RRT beginning, years
62.9 ± 14.1
57.2 ± 17.2
<
0.000
1
c
RRT duration, years
3.29  (0.06–28.0)
4.42  (0.12–28.2)
<
0.000
1
c
Death rate, cases per 100 patient-years
0.48
0.42
Death rate, cases per 100 RRT-years
7.97
4.63
Clinical data
n = 332
n = 527
Coronary artery disease,n (% of all)
174  (52.4)
168  (31.9)
<
0.000
1
b
Myocardial infarction,n (% of all)
98  (29.5)
101  (19.2)
0.00
9
b
Parathyroidectomy,n (% of all)
2  (0.60)
21  (3.98)
0.000
9
b
Treatment with cinacalcet hydrochloride
24  (7.2)
98  (18.6)
<
0.000
1
b
Laboratory data
n = 366
n = 527
Anti-HBc positive,n (% of all)
95  (26.0)
126  (23.9)
0.528b
HBsAg positive,n (% of all  anti-HBc positive)
7  (7.4)
11  (8.7)
0.807b
Anti-HCV positive,n (% of all)
26  (7.1)
57  (10.8)
0.062b
HCV RNA positive,n (% of all anti-HCV  positive)
14  (53.8)
39  (68.4)
0.225b
Responders to hepatitis B vaccine,n (% of all)
202  (55.2)
315  (59.8)
0.191b
25(OH)D (ng/mL)a
13.3 ± 3.9
14.5 ± 5.6
0.182a,d
Total calcium (mg/dL)
8.83 ± 0.67
8.91 ± 0.82
0.264d
Phosphates (mg/dL)
5.03 ± 1.44
5.25 ± 1.49
0.054d
PTH (pg/mL)
296  (12.9–3,757)
463  (12.7–3,741)
<
0.000
1
c
Total alkaline phosphatase (U/L)
98.2  (25.8–1,353)
97.1  (40.5–1,684)
0.528c
25(OH)D: 25-hydroxycholecalciferol, anti-HBc: antibodies to core antigen of hepatitis B virus, anti-HCV: antibodies to hepatitis C virus, HBsAg: surface antigen of hepatitis B virus, DM: diabetes mellitus, ESRD: end-stage renal disease, HCV RNA: ribonucleic acid of hepatitis C virus, PTH: parathyroid hormone, and RRT: renal replacement therapy.
A significant difference is indicated using bold font.
a
n = 66 for type 2 DM nephropathy; n = 96 for other renal diseases.
bFisher’s exact test.
cMann-Whitney test.
dUnpaired t-test, Welch corrected.Table 3
Characteristics of hemodialysis patients grouped by a cause of ESRD.
Parameter
Type 2 DM nephropathy (1)
Chronic  glomerulonephritis (2)
Chronic  tubulointerstitial  nephritis (3)
Hypertensive nephropathy (4)
P value
Demographic data
n = 366
n = 178
n = 118
n = 231
Male sex,n (% of all)
201  (54.9)
110  (61.8)
63  (53.4)
134  (58.0)
0.386b
Age at RRT beginning, years
62.9 ± 14.1
47.4 ±  17.6
59.9 ± 16.6
63.3 ± 13.6
<
0.000
1
c 1  versus  2: <0.001c 2  versus  3: <0.001c 2  versus  4: <0.001c
RRT duration, years
3.29  (0.06–28.0)
5.73  (0.16–28.2)
4.82  (0.33–26.5)
3.82  (0.12–20.4)
<
0.000
1
c 1  versus  2: <0.001c 1  versus  3: <0.01c 2  versus  4: <0.001c
Death rate, cases per 100 patient-years
0.48
0.41
0.44
0.42
Death rate, cases per 100 dialysis-years
7.97
2.87
5.28
6.70
Clinical data
n = 332
n = 178
n = 118
n = 231
Coronary artery disease,n (% of all)
174  (52.4)
43  (24.2)
29  (24.6)
96  (41.5)
<
0.000
1
b  1  versus  2: <0.0001e 1  versus  3: <0.0001e 1  versus  4: 0.013e 2  versus  4: 0.0002e 3  versus  4: 0.002e
Myocardial infarction,n (% of all)
98  (29.5)
25  (14.0)
17  (14.4)
59  (25.5)
<
0.000
1
b 1  versus  2: <0.0001e 1  versus  3: <0.0001e 1  versus  4: <0.0001e 2  versus  4: 0.005e 3  versus  4: 0.02e
PTX,n (% of all)
2  (0.60)
14  (7.9)
5  (4.2)
2  (0.87)
<
0.000
1
b 1  versus  2: <0.0001e 1  versus  3: 0.015e 2  versus  4: 0.0004e 3  versus  4: 0.046e
Treatment with cinacalcet hydrochloride
24  (7.2)
48  (27.0)
21  (17.8)
29  (12.6)
<
0.000
1
b 1  versus  2: <0.0001e 1  versus  3: 0.0008e 1  versus  4: 0.017e 2  versus  4: 0.0003e
Laboratory data
n = 366
n = 178
n = 118
n = 231
Anti-HBc positive,n (% of all)
95  (26.0)
53  (29.8)
25  (21.2)
48  (20.8)
0.233b
HBsAg positive,n (% of all  anti-HBc positive)
7  (7.4)
10  (18.9)
0  (0.0)
1  (2.08)
0.000
7
b 1  versus  2: 0.032e 2  versus  3: 0.007e 2  versus  4: 0.001e
Anti-HCV positive,n (% of all)
26  (7.1)
33  (18.5)
11  (9.3)
13  (5.6)
<
0.000
1
b 1  versus  2: 0.0004e 2  versus  3: 0.031e 2  versus  4: <0.0001e
HCV RNA positive,n (% of all anti-HCV  positive)
14  (53.8)
27  (81.8)
4  (36.4)
8  (61.5)
<
0.000
1
b 1  versus  2: <0.0001e 2  versus  3: 0.0004e 2  versus  4: <0.0001e
Responders to hepatitis B vaccine,n (% of all)
202  (55.2)
107  (60.1)
70  (59.3)
138  (59.7)
0.598b
25(OH)D (ng/mL)a
13.3 ± 3.9
14.2 ± 7.3
15.7 ± 4.3
14.1 ± 3.9
0.453d
Total calcium (mg/dL)
8.83 ± 0.67
8.85 ± 0.85
9.04 ± 0.61
8.88 ± 0.87
0.239d
Phosphates (mg/dL)
5.03 ± 1.44
5.63 ± 1.59
4.92 ± 1.29
5.15 ± 1.47
0.000
7
d 1  versus  2: <0.001c 2  versus  3: <0.01c 2  versus  4: <0.05c
PTH (pg/mL)
296  (12.9–3,757)
632  (12.7–3,118)
426  (45.8–3,741)
364  (19.5–2,351)
<
0.000
1
c 1  versus  2: <0.001c 1  versus  3: <0.05c 1  versus  4: <0.05c 2  versus  4: <0.001c
Total ALP (U/L)
98.2  (25.8–1,353)
113  (44.5–860)
89.0  (40.5–1,684)
90.9  (41.0–1,110)
0.01
0
c 2  versus  4: <0.05c
25(OH)D: 25-hydroxycholecalciferol, anti-HBc: antibodies to core antigen of hepatitis B virus, anti-HCV: antibodies to hepatitis C virus, HBsAg: surface antigen of hepatitis B virus, DM: diabetes mellitus, ESRD: end-stage renal disease, HCV RNA: ribonucleic acid of hepatitis C virus, PTH: parathyroid hormone, and RRT: renal replacement therapy.
a
n = 66 for type 2 DM nephropathy, n = 40 for chronic glomerulonephritis, n = 13 for chronic interstitial nephritis, and n = 43 for hypertensive nephropathy.
bChi squared test.
cKruskal-Wallis test.
dANOVA test.
eFisher’s exact test.In respect of the examined parameters, type 2 DM nephropathy patients differed the most significantly from chronic glomerulonephritic subjects, the least significantly from hypertensive nephropathy patients.There were no differences in frequency distributions of tested genotypes between type 2 DM nephropathy patients and healthy subjects (Table4) as well as other ESRD patients analyzed together (Table 5) which could be judged as significant associations.Table 4
Comparison of the distribution of polymorphic variants of tested genes between ESRD patients treated with hemodialysis due to type 2 DM nephropathy and healthy subjects.
Parameter
Type 2 DM nephropathy (frequency)
Healthy subjects (frequency)
Odds ratio (95% CI)
Two-tailedP
P
trend
IL18 rs360719
n = 248
n = 240
TT
133  (0.54)
121  (0.50)
Referent
0.233
CT
102  (0.41)
98  (0.41)
0.947  (0.654–1.372)
0.777
CC
13  (0.05)
21  (0.09)
0.563  (0.270–1.174)
0.145
CT + CC
115  (0.46)
119  (0.50)
0.879  (0.616–1.254)
0.526
MAF
128  (0.26)
140  (0.29)
0.845  (0.638–1.119)
0.268
IL12A rs568408
n = 234
n = 240
GG
173  (0.74)
171  (0.71)
Referent
0.782
AG
52  (0.22)
63  (0.26)
0.816  (0.534–1.246)
0.389
AA
9  (0.04)
6  (0.03)
1.483  (0.517–4.256)
0.600
AG + AA
61  (0.26)
69  (0.29)
0.874  (0.583–1.309)
0.538
MAF
70  (0.15)
75  (0.16)
0.976  (0.684–1.393)
0.965
IL12B rs3212227
n = 247
n = 240
AA
156  (0.63)
151  (0.63)
Referent
0.639
AC
84  (0.34)
77  (0.32)
1.056  (0.721–1.547)
0.846
CC
7  (0.03)
12  (0.05)
0.563  (0.217–1.473)
0.345
AC + CC
91  (0.37)
89  (0.37)
0.990  (0.685–1.430)
1.000
MAF
98  (0.20)
101  (0.21)
0.927  (0.680–1.268)
0.699
IL4Rrs1805015
n = 303
n = 225
TT
205  (0.68)
162  (0.72)
Referent
0.304
CT
82  (0.27)
53  (0.24)
1.223  (0.818–1.828)
0.360
CC
16  (0.05)
10  (0.04)
1.264  (0.559–2.861)
0.684
CT + CC
98  (0.32)
63  (0.28)
1.229  (0.843–1.793)
0.295
MAF
114  (0.19)
73  (0.16)
1.197  (0.866–1.653)
0.313
IL13rs20541
n = 303
n = 230
CC
168  (0.55)
124  (0.54)
Referent
0.457
CT
114  (0.38)
84  (0.36)
1.002  (0.695–1.443)
1.000
TT
21  (0.07)
22  (0.10)
0.705  (0.371–1.338)
0.324
CT + TT
135  (0.45)
106  (0.46)
0.940  (0.666–1.326)
0.726
MAF
156  (0.26)
128  (0.28)
0.899  (0.684–1.182)
0.489
IL28B rs8099917
n = 339
n = 375
TT
219  (0.65)
245  (0.65)
Referent
0.504
GT
107  (0.31)
123  (0.33)
0.973  (0.709–1.336)
0.872
GG
13  (0.04)
7  (0.02)
2.078  (0.814–5.302)
0.169
GT + GG
120  (0.35)
130  (0.35)
1.033  (0.759–1.405)
0.875
MAF
133  (0.20)
137  (0.18)
1.092  (0.837–1.423)
0.560
IL28B rs12979860
n = 336
n = 372
CC
141  (0.42)
164  (0.44)
Referent
0.669
CT
157  (0.47)
166  (0.45)
1.100  (0.804–1.505)
0.576
TT
38  (0.11)
42  (0.11)
1.052  (0.643–1.723)
0.900
CT + TT
195  (0.56)
208  (0.56)
1.090  (0.809–1.469)
0.595
MAF
116  (0.29)
250  (0.34)
1.049  (0.842–1.307)
0.713
GCrs2298849
n = 364a
n = 375
TT
226  (0.62)
237  (0.63)
Referent
0.250
CT
110  (0.30)
124  (0.33)
0.930  (0.679–1.274)
0.688
CC
28  (0.08)
14  (0.04)
2.097  (1.077–4.086)
0.035
CT + CC
138  (0.38)
138  (0.37)
1.049  (0.778–1.413)
0.762
MAF
166  (0.23)
152  (0.20)
1.162  (0.907–1.490)
0.262
GCrs7041
n = 343
n = 361
GG
112  (0.33)
116  (0.32)
Referent
0.572
GT
163  (0.47)
186  (0.52)
0.908  (0.650–1.268)
0.609
TT
68  (0.20)
59  (0.16)
1.194  (0.773–1.844)
0.440
GT + TT
231  (0.67)
245  (0.68)
0.977  (0.712–1.339)
0.936
MAF
299  (0.44)
304  (0.42)
1.062  (0.860–1.312)
0.612
GCrs1155563
n = 362
n = 377
TT
180  (0.50)
189  (0.50)
Referent
0.541
CT
141  (0.39)
155  (0.41)
0.955  (0.703–1.297)
0.815
CC
41  (0.11)
33  (0.09)
1.305  (0.789–2.155)
0.311
CT + CC
182  (0.50)
188  (0.50)
1.017  (0.762–1.356)
0.941
MAF
223  (0.31)
221  (0.29)
1.074  (0.859–1.341)
0.567
VDRrs2228570
n = 345
n = 371
CC
101  (0.29)
103  (0.28)
Referent
0.401
CT
175  (0.51)
183  (0.49)
0.975  (0.691–1.376)
0.930
TT
69  (0.20)
85  (0.23)
0.828  (0.544–1.260)
0.394
CT + TT
244  (0.71)
268  (0.72)
0.929  (0.671–1.285)
0.679
MAF
313  (0.45)
353  (0.48)
0.915  (0.743–1.126)
0.432
VDRrs1544410
n = 359
n = 372
GG
137  (0.38)
148  (0.40)
Referent
0.753
AG
165  (0.46)
165  (0.44)
1.080  (0.787–1.483)
0.686
AA
57  (0.16)
59  (0.16)
1.044  (0.678–1.607)
0.912
AG + AA
222  (0.62)
224  (0.60)
1.071  (0.795–1.442)
0.705
MAF
279  (0.39)
283  (0.38)
1.035  (0.839–1.278)
0.788
RXRArs10776909
n = 364
n = 378
CC
233  (0.64)
250  (0.66)
Referent
0.426
CT
111  (0.30)
112  (0.30)
1.063  (0.774–1.461)
0.746
TT
20  (0.05)
16  (0.04)
1.341  (0.679–2.651)
0.490
CT + TT
131  (0.36)
128  (0.34)
1.098  (0.812–1.485)
0.590
MAF
151  (0.21)
144  (0.19)
1.112  (0.862–1.435)
0.452
RXRArs10881578
n = 365
n = 377
AA
197  (0.54)
183  (0.48)
Referent
0.168
AG
134  (0.37)
154  (0.41)
0.808  (0.775–1.046)
0.185
GG
34  (0.09)
40  (0.11)
0.790  (0.479–1.301)
0.376
AG + GG
168  (0.46)
194  (0.51)
0.804  (0.603–1.073)
0.143
MAF
202  (0.28)
234  (0.31)
0.850  (0.680–1.063)
0.172
RXRArs749759
n = 355
n = 370
GG
207  (0.58)
221  (0.60)
Referent
0.850
AG
125  (0.35)
123  (0.33)
1.085  (0.794–1.216)
0.632
AA
23  (0.06)
26  (0.07)
0.944  (0.522–1.708)
0.881
AG + AA
148  (0.42)
149  (0.40)
1.061  (0.789–1.426)
0.706
MAF
171  (0.24)
175  (0.24)
1.024  (0.804–1.304)
0.894
ESRD: end-stage renal disease, DM: diabetes mellitus, and MAF: minor allele frequency.
aNot consistent with Hardy-Weinberg equilibrium.Table 5
Comparison of the distribution of polymorphic variants of tested genes between ESRD patients treated with hemodialysis due to type 2 DM nephropathy and the most common causes of ESRD other than type 2 DM nephropathy (chronic glomerulonephritis, chronic tubulointerstitial nephritis, and hypertensive nephritis).
Genotype
Type 2 DM nephropathy (frequency)
Other causes of ESRD (frequency)
Odds ratio (95% CI)
Two-tailedP
P
trend
IL18 rs360719
n = 248
n = 353
TT
133  (0.54)
186  (0.53)
Referent
—
0.362
CT
102  (0.41)
135  (0.38)
1.057  (0.752–1.485)
0.795
CC
13  (0.05)
32  (0.09)
0.568  (0.287–1.124)
0.107
CT + CC
115  (0.46)
167  (0.47)
0.963  (0.696–1.334)
0.868
MAF
128  (0.26)
199  (0.28)
0.886  (0.684–1.149)
0.370
IL12A rs568408
n = 234
n = 337
GG
173  (0.74)
234  (0.69)
Referent
—
0.303
AG
52  (0.22)
89  (0.26)
0.790  (0.533–1.060)
0.275
AA
9  (0.04)
14  (0.04)
0.870  (0.368–2.055)
0.831
AG + AA
61  (0.26)
103  (0.31)
0.801  (0.552–1.163)
0.260
MAF
70  (0.15)
117  (0.17)
0.837  (0.606–1.157)
0.319
IL12B rs3212227
n = 247
n = 352
AA
156  (0.63)
205  (0.58)
Referent
—
0.176
AC
84  (0.34)
132  (0.38)
0.836  (0.593–1.068)
0.337
CC
7  (0.03)
15  (0.04)
0.613  (0.244–1.540)
0.376
AC + CC
91  (0.37)
147  (0.42)
0.814  (0.582–1.136)
0.236
MAF
98  (0.20)
162  (0.23)
0.828  (0.624–1.098)
0.215
IL4Rrs1805015
n = 303
n = 436
TT
205  (0.68)
295  (0.68)
Referent
—
0.871
CT
82  (0.27)
121  (0.28)
0.975   (0.700–2.360)
0.933
CC
16  (0.05)
20  (0.05)
1.151  (0.583–2.275)
0.728
CT + CC
98  (0.32)
141  (0.32)
1.000  (0.731–1.368)
1.000
MAF
114  (0.19)
161  (0.18)
1.023  (0.784–1.335)
0.919
IL13rs20541
n = 303
n = 436
CC
168  (0.55)
242  (0.56)
Referent
—
0.902
CT
114  (0.38)
166  (0.38)
0.989  (0.726–1.348)
1.000
TT
21  (0.07)
28  (0.06)
1.080  (0.594–1.967)
0.878
CT + TT
135  (0.45)
194  (0.44)
1.002  (0.746–1.346)
1.000
MAF
156  (0.26)
222  (0.25)
1.015  (0.800–1.287)
0.950
IL28B rs8099917
n = 339
n = 493
TT
219  (0.65)
317  (0.64)
Referent
—
0.858
GT
107  (0.31)
162  (0.33)
0.956  (0.709–1.289)
0.820
GG
13  (0.04)
14  (0.03)
1.344  (0.620–2.916)
0.549
GT + GG
120  (0.35)
176  (0.36)
0.987  (0.739–1.318)
0.941
MAF
133  (0.20)
190  (0.19)
1.022  (0.799–1.309)
0.910
IL28Brs12979860
n = 336
n = 488
CC
141  (0.42)
209  (0.43)
Referent
—
0.952
CT
157  (0.47)
221  (0.45)
1.053  (0.783–1.415)
0.763
TT
38  (0.11)
58  (0.12)
0.971  (0.612–1.541)
0.907
CT + TT
195  (0.56)
279  (0.57)
1.036  (0.782–1.373)
0.830
MAF
116  (0.29)
337  (0.35)
1.006  (0.819–1.237)
0.994
GCrs2298849
n = 364a
n = 524
TT
226  (0.62)
339  (0.65)
Referent
—
0.109
CT
110  (0.30)
165  (0.31)
1.000  (0.745–1.342)
1.000
CC
28  (0.08)
20  (0.04)
2.100  (1.155–3.819)
0.014
CT + CC
138  (0.38)
185  (0.35)
1.119  (0.848–1.477)
0.436
MAF
166  (0.23)
205  (0.20)
1.215  (0.964–1.530)
0.111
GCrs7041
n = 343
n = 506
GG
112  (0.33)
182  (0.36)
Referent
—
0.247
GT
163  (0.47)
236  (0.47)
1.122  (0.824–1.528)
0.480
TT
68  (0.20)
88  (0.17)
1.256  (0.846–1.863)
0.267
GT + TT
231  (0.67)
324  (0.64)
1.159  (0.867–1.548)
0.340
MAF
299  (0.44)
412  (0.41)
1.125  (0.925–1.369)
0.259
GCrs1155563
n = 362
n = 527
TT
180  (0.50)
252  (0.48)
Referent
—
0.614
CT
141  (0.39)
213  (0.40)
0.927  (0.696–1.234)
0.610
CC
41  (0.11)
62  (0.12)
0.926  (0.597–1.435)
0.740
CT + CC
182  (0.50)
275  (0.52)
0.927  (0.709–1.211)
0.585
MAF
223  (0.31)
337  (0.32)
0.947  (0.772–1.161)
0.638
VDRrs2228570
n = 345
n = 503
CC
101  (0.29)
130  (0.26)
Referent
—
0.541
CT
175  (0.51)
275  (0.55)
0.819  (0.594–1.130)
0.249
TT
69  (0.20)
98  (0.19)
0.906  (0.606–1.356)
0.682
CT +  TT
244  (0.71)
373  (0.74)
0.842  (0.620–1.143)
0.273
MAF
313  (0.45)
471  (0.47)
0.943  (0.776–1.145)
0.588
VDRrs1544410
n = 359
n = 512
GG
137  (0.38)
189  (0.37)
Referent
—
0.598
AG
165  (0.46)
235  (0.46)
0.969  (0.720–1.303)
0.880
AA
57  (0.16)
88  (0.17)
0.894  (0.599–1.332)
0.613
AG + AA
222  (0.62)
323  (0.63)
0.948  (0.718–1.253)
0.722
MAF
279  (0.39)
411  (0.40)
0.948  (0.778–1.152)
0.626
RXRArs10776909
n = 364
n = 526
CC
233  (0.64)
308  (0.59)
Referent
—
0.298
CT
111  (0.30)
196  (0.37)
0.749  (0.561–0.999)
0.050
TT
20  (0.05)
22  (0.04)
1.202  (0.641–2.254)
0.629
CT + TT
131  (0.36)
218  (0.41)
0.794  (0.603–1.046)
0.108
MAF
151  (0.21)
240  (0.23)
0.883  (0.702–1.112)
0.317
RXRA rs10881578
n = 365
n = 525
AA
197  (0.54)
252  (0.48)
Referent
—
0.134
AG
134  (0.37)
220  (0.42)
0.779  (0.586–1.035)
0.096
GG
34  (0.09)
53  (0.10)
0.821  (0.513–1.312)
0.478
AG + GG
168  (0.46)
273  (0.52)
0.787  (0.602–1.029)
0.088
MAF
202  (0.28)
326  (0.31)
0.850  (0.690–1.046)
0.139
RXRArs749759
n = 355
n = 514
GG
207  (0.58)
265  (0.52)
Referent
—
0.082
AG
125  (0.35)
212  (0.41)
0.755  (0.567–1.005)
0.059
AA
23  (0.06)
37  (0.07)
0.796  (0.459–1.381)
0.490
AG + AA
148  (0.42)
249  (0.48)
0.761  (0.579–1.000)
0.053
MAF
171  (0.24)
286  (0.28)
0.823  (0.661–1.025)
0.092
ESRD: end-stage renal disease, DM: diabetes mellitus, and MAF: minor allele frequency.
aNot consistent with Hardy-Weinberg equilibrium.Comparisons of genotype and allele frequencies between type 2 DM nephropathy patients and other ESRD groups revealed associations only with chronic infective tubulointerstitial nephritic patients in respect ofIL18rs360719 (Table 6, no significant results are shown). Frequency ofIL18rs360719 allele C carriers was higher in type 2 DM nephropathy patients than in those with chronic infective tubulointerstitial nephritis. The latter group showed lower frequency ofIL18rs360719 allele C carriers compared to healthy controls (Table 6).Table 6
Selected comparisons of the polymorphic variants distribution of tested genes between type 2 DM nephropathy patients, chronic infective tubulointerstitial nephritic patients, and healthy subjects.
Genotype
Genotype frequencies
Odds ratio (95% CI)
Two-tailedP
P
trend
Type 2 DM nephropathy  versus  chronic infective tubulointerstitial nephritis
IL18 rs360719
n = 248
n = 77
TT
133  (0.54)
54  (0.70)
Referent
0.033
CT
102  (0.41)
19  (0.25)
2.180  (1.217–3.905)
0.00
9
a
CC
13  (0.05)
4  (0.05)
1.320  (0.412–4.228)
0.783
CT + CC
115  (0.46)
23  (0.30)
2.030  (1.173–3.512)
0.01
2
a
MAF
128  (0.26)
27  (0.18)
1.636  (1.031–2.596)
0.046
Chronic infective tubulointerstitial nephritis versus healthy controls
IL18 rs360719
n = 77
n = 240
TT
54  (0.70)
121  (0.50)
Referent
0.005
CT
19  (0.25)
98  (0.41)
0.434  (0.242–0.781)
0.00
6
a
CC
4  (0.05)
21  (0.09)
0.427  (0.140–1.303)
0.160
CT + CC
23  (0.30)
119  (0.50)
0.433  (0.250–0.750)
0.00
4
a
MAF
27  (0.18)
140  (0.29)
0.516  (0.326–0.818)
0.006
DM: diabetes mellitus; MAF: minor allele frequency.
Significant differences are indicated using bold font.
aSignificant after the Bonferroni correction (P<0.017).Type 2 DM nephropathy patients with diagnosed CAD differed in tested genotype frequencies neither from type 2 DM nephropathy subjects without CAD (Table7) nor from healthy controls (Table 8).Table 7
Comparison of the distribution of polymorphic variants of tested genes between ESRD patients treated with hemodialysis due to type 2 DM nephropathy grouped by diagnosis of CAD.
Parameter
Type 2 DM nephropathy with CAD (frequency)
Type 2 DM nephropathy without CAD (frequency)
Odds ratio (95% CI)
Two-tailedP
P
trend
IL18 rs360719
n = 124
n = 109
TT
68  (0.55)
53  (0.49)
Referent
0.269
CT
51  (0.41)
49  (0.45)
1.128  (0.725–1.754)
0.653
CC
5  (0.04)
7  (0.06)
0.628  (0.194–2.036)
0.557
CT + CC
56  (0.45)
56  (0.51)
0.879  (0.560–1.380)
0.645
MAF
61  (0.25)
63  (0.29)
0.803  (0.532–1.211)
0.345
IL12A rs568408
n = 117
n = 102
GG
83  (0.71)
77  (0.63)
Referent
0.361
AG
28  (0.24)
22  (0.22)
1.181  (0.623–2.236)
0.630
AA
6  (0.05)
3  (0.03)
1.855  (0.448–7.678)
0.502
AG + AA
34  (0.29)
25  (0.25)
1.262  (0.691–2.304)
0.542
MAF
40  (0.17)
28  (0.14)
1.311  (0.776–2.214)
0.378
IL12B rs3212227
n = 124
n = 109
AA
78  (0.63)
69  (0.63)
Referent
0.906
AC
43  (0.35)
36  (0.33)
1.057  (0.611–1.829)
0.889
CC
3  (0.02)
4  (0.04)
0.664  (0.143–3.069)
0.708
AC + CC
46  (0.37)
40  (0.37)
1.017  (0.597–1.734)
1.000
MAF
49  (0.20)
44  (0.20)
0.974  (0.618–1.535)
0.909
IL4Rrs1805015
n = 144
n = 127
TT
95  (0.66)
86  (0.68)
Referent
0.947
CT
42  (0.29)
32  (0.25)
1.188  (0.689–2.048)
0.581
CC
7  (0.05)
9  (0.07)
0.704  (0.251–1.972)
0.605
CT + CC
49  (0.34)
41  (0.32)
1.082  (0.652–1.797)
0.797
MAF
56  (0.19)
50  (0.20)
0.985  (0.644–1.504)
0.944
IL13rs20541
n = 144
n = 127
CC
80  (0.56)
71  (0.56)
Referent
0.867
CT
55  (0.38)
46  (0.36)
1.061  (0.640–1.759)
0.898
TT
9  (0.06)
10  (0.08)
0.799  (0.307–2.077)
0.808
CT + TT
64  (0.44)
56  (0.44)
1.014  (0.627–1.640)
1.000
MAF
73  (0.25)
92  (0.26)
0.967  (0.657–1.423)
0.944
IL28B rs8099917
n = 163
n = 145
TT
105  (0.64)
97  (0.67)
Referent
0.752
GT
52  (0.32)
42  (0.29)
1.144  (0.700–1.870)
0.618
GG
6  (0.04)
6  (0.04)
0.924  (0.288–2.961)
1.000
GT + GG
58  (0.36)
48  (0.33)
1.116  (0.697–1.189)
0.719
MAF
64  (0.20)
54  (0.19)
1.068  (0.714–1.597)
0.829
IL28B rs12979860
n = 163
n = 142
CC
69  (0.42)
66  (0.46)
Referent
0.352
CT
73  (0.45)
62  (0.44)
1.126  (0.698–1.816)
0.715
TT
21  (0.13)
14  (0.10)
1.435  (0.674–3.055)
0.448
CT + TT
94  (0.58)
76  (0.54)
1.183  (0.752–1.861)
0.490
MAF
115  (0.35)
90  (0.32)
1.175  (0.838–1.647)
0.396
GC  rs2298849
n = 172
n = 158a
TT
99  (0.58)
106  (0.67)
Referent
0.173
CT
60  (0.35)
40  (0.25)
1.606  (0.989–2.608)
0.067
CC
13  (0.07)
12  (0.08)
1.160  (0.505–2.663)
0.833
CT + CC
73  (0.42)
52  (0.33)
1.503  (0.959–2.355)
0.088
MAF
166  (0.25)
64  (0.20)
1.313  (0.909–1.895)
0.174
GC  rs7041
n = 161
n = 151
GG
57  (0.35)
46  (0.30)
Referent
0.844
GT
69  (0.43)
82  (0.54)
1.327  (0.825–2.134)
0.277
TT
35  (0.22)
23  (0.15)
1.629  (0.900–2.949)
0.136
GT + TT
104  (0.65)
105 (0.70)
1.061  (0.721–1.559)
0.769
MAF
139  (0.43)
128  (0.42)
1.025  (0.746–1.409)
0.943
GC  rs1155563
n = 172
n = 157
TT
82  (0.48)
79  (0.50)
Referent
0.645
CT
70  (0.41)
61  (0.39)
1.106  (0.697–1.755)
0.724
CC
20  (0.12)
17  (0.11)
1.133  (0.554–2.321)
0.856
CT + CC
90  (0.52)
78  (0.50)
1.112  (0.721–1.714)
0.660
MAF
110  (0.32)
95  (0.30)
1.084  (0.779–1.508)
0.695
VDRrs2228570
n = 162
n = 152
CC
43  (0.27)
44  (0.29)
Referent
0.316
CT
93 (0.57)
68  (0.45)
1.400  (0.829–2.363)
0.230
TT
26  (0.16)
40  (0.26)
0.665  (0.348–1.272)
0.252
CT + TT
119  (0.73)
108  (0.71)
1.128  (0.688–1.849)
0.705
MAF
145  (0.45)
148  (0.49)
0.854  (0.624–1.169)
0.365
VDRrs1544410
n = 170
n = 155
GG
65  (0.38)
61  (0.39)
Referent
0.772
AG
79  (0.46)
72  (0.46)
1.030  (0.641–1.653)
0.905
AA
26  (0.15)
22  (0.14)
1.109  (0.569–2.160)
0.865
AG + AA
105  (0.62)
94  (0.61)
1.048  (0.671–1.639)
0.909
MAF
131  (0.39)
116  (0.37)
1.048  (0.763–1.440)
0.833
RXRArs10776909
n = 172
n = 158
CC
112  (0.65)
104  (0.66)
Referent
0.621
CT
48  (0.28)
47  (0.30)
0.948  (0.585–1.537)
0.902
TT
12  (0.07)
7  (0.04)
1.592  (0.604–4.198)
0.473
CT + TT
60  (0.35)
54  (0.34)
1.032  (0.655–1.625)
0.908
MAF
72  (0.21)
61  (0.19)
1.107  (0.756–1.621)
0.672
RXRA rs10881578
n = 173
n = 158
AA
89  (0.51)
92  (0.58)
Referent
0.192
AG
65  (0.38)
53  (0.34)
1.268  (0.796–2.019)
0.345
GG
19  (0.11)
13  (0.08)
1.511  (0.704–3.241)
0.340
AG + GG
84  (0.49)
66  (0.42)
1.316  (0.852–2.032)
0.226
MAF
103  (0.30)
79  (0.25)
1.272  (0.902–1.793)
0.199
RXRA rs749759
n = 169
n = 153
GG
100  (0.59)
89  (0.58)
Referent
0.812
AG
59  (0.35)
54  (0.35)
0.972  (0.610–1.551)
1.000
AA
10  (0.06)
10  (0.07)
0.890  (0.354–2.238)
0.818
AG + AA
69  (0.41)
64  (0.42)
0.960  (0.615–1.496)
0.910
MAF
79  (0.23)
74  (0.24)
0.956  (0.665–1.375)
0.882
CAD: coronary artery disease, ESRD: end-stage renal disease, DM: diabetes mellitus, and MAF: minor allele frequency.
aNot consistent with Hardy-Weinberg equilibrium.Table 8
Comparison of the distribution of polymorphic variants of tested genes between type 2 DM nephropathy patients with diagnosis of CAD and healthy controls.
Parameter
Type 2 DM nephropathy with CAD (frequency)
Healthy controls (frequency)
Odds ratio (95% CI)
Two-tailedP
P
trend
IL18 rs360719
n = 124
n = 240
TT
68  (0.55)
121  (0.50)
Referent
0.186
CT
51  (0.41)
98  (0.41)
0.926  (0.590–1.453)
0.819
CC
5  (0.04)
21  (0.09)
0.424  (0.153–1.174)
0.122
CT + CC
56  (0.45)
119  (0.50)
0.837  (0.542–1.294)
0.440
MAF
61  (0.25)
140  (0.29)
0.792  (0.558–1.124)
0.223
IL12A rs568408
n = 117
n = 240
GG
83  (0.71)
171  (0.71)
Referent
0.626
AG
28  (0.24)
63  (0.26)
0.916  (0.546–1.535)
0.794
AA
6  (0.05)
6  (0.03)
2.060  (0.645–6.583)
0.348
AG + AA
34  (0.29)
69  (0.29)
1.015  (0.624–1.653)
1.000
MAF
40  (0.17)
75  (0.16)
1.113  (0.731–1.695)
0.695
IL12B rs3212227
n = 124
n = 240
AA
78  (0.63)
151  (0.63)
Referent
0.475
AC
43  (0.35)
77  (0.32)
1.081  (0.681–1.717)
0.813
CC
3  (0.02)
12  (0.05)
0.484  (0.133–1.766)
0.397
AC + CC
46  (0.37)
89  (0.37)
1.001  (0.639–1.567)
1.000
MAF
49  (0.20)
101  (0.21)
0.924  (0.631–1.354)
0.757
IL4Rrs1805015
n = 144
n = 225
TT
95  (0.66)
162  (0.72)
Referent
0.285
CT
42  (0.29)
53  (0.24)
1.351  (0.838–2.179)
0.221
CC
7  (0.05)
10  (0.04)
1.194  (0.440–3.240)
0.798
CT + CC
49  (0.34)
63  (0.28)
1.326  (0.845–2.083)
0.246
MAF
56  (0.19)
73  (0.16)
1.247  (0.848–1.832)
0.305
IL13rs20541
n = 144
n = 230
CC
80  (0.56)
124  (0.54)
Referent
0.469
CT
55  (0.38)
84  (0.36)
1.015  (0.653–1.578)
1.000
TT
9  (0.06)
22  (0.10)
0.634  (0.278–1.447)
0.324
CT + TT
64  (0.44)
106  (0.46)
0.936  (0.616–1.422)
0.831
MAF
73  (0.25)
128  (0.28)
0.881  (0.630–1.231)
0.510
IL28B rs8099917
n = 163
n = 375
TT
105  (0.64)
245  (0.65)
Referent
0.584
GT
52  (0.32)
123  (0.33)
0.986  (0.663–1.467)
1.000
GG
6  (0.04)
7  (0.02)
2.000  (0.656–6.094)
0.229
GT + GG
58  (0.36)
130  (0.35)
1.041  (0.709–1.530)
0.845
MAF
64  (0.20)
137  (0.18)
1.093  (0.786–1.521)
0.658
IL28B rs12979860
n = 163
n = 372
CC
69  (0.42)
164  (0.44)
Referent
0.281
CT
73  (0.45)
166  (0.45)
1.045  (0.705–1.549)
0.841
TT
21  (0.13)
42  (0.11)
1.188  (0.656–2.154)
0.644
CT + TT
94  (0.58)
208  (0.56)
1.074  (0.740–1.558)
0.776
MAF
115  (0.35)
250  (0.34)
1.077  (0.819–1.416)
0.644
GC  rs2298849
n = 172
n = 375
TT
99  (0.58)
237  (0.63)
Referent
0.080
CT
60  (0.35)
124  (0.33)
1.158  (0.786–1.706)
0.486
CC
13 (0.07)
14  (0.04)
2.223  (1.008–4.901)
0.052
CT + CC
73  (0.42)
138  (0.37)
1.266  (0.876–1.830)
0.220
MAF
166  (0.25)
152  (0.20)
1.311  (0.969–1.774)
0.092
GC  rs7041
n = 161
n = 361
GG
57  (0.35)
116  (0.32)
Referent
0.748
GT
69  (0.43)
186  (0.52)
0.755  (0.496–1.150)
0.196
TT
35  (0.22)
59  (0.16)
1.207  (0.714–2.040)
0.502
GT + TT
104  (0.65)
245  (0.68)
0.864  (0.584–1.278)
0.482
MAF
139  (0.43)
304  (0.42)
1.044  (0.801–1.362)
0.800
GC  rs1155563
n = 172
n = 377
TT
82  (0.48)
189  (0.50)
Referent
0.378
CT
70  (0.41)
155  (0.41)
1.041  (0.710–1.527)
0.845
CC
20  (0.12)
33  (0.09)
1.397  (0.757–2.578)
0.332
CT + CC
90  (0.52)
188  (0.50)
1.103  (0.769–1.583)
0.646
MAF
110  (0.32)
221  (0.29)
1.134  (0.861–1.494)
0.411
VDRrs2228570
n = 162
n = 371
CC
43  (0.27)
103  (0.28)
Referent
0.386
CT
93 (0.57)
183  (0.49)
1.217  (0.788–1.880)
0.384
TT
26  (0.16)
85  (0.23)
0.733  (0.416–1.290)
0.321
CT + TT
119  (0.73)
268  (0.72)
1.064  (0.702–1.613)
0.833
MAF
145  (0.45)
353  (0.48)
0.893  (0.687–1.160)
0.434
VDRrs1544410
n = 170
n = 372
GG
65  (0.38)
148  (0.40)
Referent
0.880
AG
79  (0.46)
165  (0.44)
1.090  (0.734–1.620)
0.687
AA
26  (0.15)
59  (0.16)
1.003  (0.581–1.732)
1.000
AG + AA
105  (0.62)
224  (0.60)
1.067  (0.735–1.549)
0.776
MAF
131  (0.39)
283  (0.38)
1.021  (0.784–1.329)
0.931
RXRArs10776909
n = 172
n = 378
CC
112  (0.65)
250  (0.66)
Referent
0.483
CT
48  (0.28)
112  (0.30)
0.957  (0.638–1.434)
0.838
TT
12  (0.07)
16  (0.04)
1.674  (0.767–3.656)
0.209
CT + TT
60  (0.35)
128  (0.34)
1.046  (0.716–1.529)
0.846
MAF
72  (0.21)
144  (0.19)
1.125  (0.819–1.545)
0.518
RXRA rs10881578
n = 173
n = 377
AA
89  (0.51)
183  (0.48)
Referent
0.682
AG
65  (0.38)
154  (0.41)
0.868  (0.591–1.275)
0.494
GG
19  (0.11)
40  (0.11)
0.977  (0.535–1.783)
1.000
AG + GG
84  (0.49)
194  (0.51)
0.890  (0.621–1.276)
0.582
MAF
103  (0.30)
234  (0.31)
0.942  (0.714–1.243)
0.725
RXRA rs749759
n = 169
n = 370
GG
100  (0.59)
221  (0.60)
Referent
0.924
AG
59  (0.35)
123  (0.33)
1.060  (0.718–1.566)
0.842
AA
10  (0.06)
26  (0.07)
0.850  (0.395–1.830)
0.710
AG + AA
69  (0.41)
149  (0.40)
1.023  (0.707–1.482)
0.925
MAF
79  (0.23)
175  (0.24)
0.985  (0.727–1.334)
0.983
CAD: coronary artery disease, DM: diabetes mellitus, and MAF: minor allele frequency.


## 4. Discussion

Genetic studies involving DM nephropathy and related complications are not consistent in many aspects [31–34]. Some polymorphisms tested in this study were reported as being associated with type 1 DM (IL12Brs3212227 [35],IL4R[36, 37],IL13[37],VDR rs1544410 [38, 39], andVDRrs2228570 [38]), type 2 DM susceptibility (VDRrs2228570 [40],VDR rs1544410 [41]), and phenotype of type 2 DM (VDRrs2228570 [42],VDR rs1544410 [41, 43]).VDRrs2228570 andIL4polymorphisms were also related to the risk of chronic kidney disease [44, 45]. On the other hand, there are also data indicating no major effect ofIL12Bon type 1 DM susceptibility in the entire study group [46], no association ofIL4Rwith type 1 DM [47], no evident causal relationship between vitamin D pathway genes and type 2 DM, myocardial infarction or mortality [48], similar distribution of genotypes, allele and haplotypes ofVDRrs2228570 andVDR rs731236 between type 2 DM patients and controls [49], no contribution ofVDR rs1544410 to type 1 DM susceptibility [50], and no association ofVDR rs1544410 with chronic kidney disease susceptibility [51].In this study we were not able to show significant differences in the frequency distribution of tested polymorphic variants of T-cell-related cytokine genes or vitamin D pathway genes between HD patients with ESRD due to type 2 DM nephropathy and controls as well as HD patients with other causes of ESRD analyzed together. This lack of association was present although the examined type 2 DM nephropathy patients showed clinical complications more frequently than HD patients with other renal diseases: higher dialysis related mortality rate [3], higher prevalence of CAD including myocardial infarction [4], lower serum PTH, and lower frequency of parathyroidectomy and treatment with cinacalcet, all of them predictive for higher tendency to adynamic bone disease [7]. Type 2 DM nephropathy patients with or without diagnosis of CAD also did not differ in tested genotype distributions.Development of ESRD substantially ameliorates interpatient clinical variability related to underlying renal impairment and exposes uremia-related signs and symptoms. Comparisons of type 2 DM nephropathy patients in respect of tested genotype frequencies with subjects showing other common causes of ESRD revealed that the former group has a higherIL18rs360719 minor allele frequency than chronic infective tubulointerstitial nephritic group. In this case, lowerIL18rs360719 minor allele frequency in tubulointerstitial nephritic patients was observed also when their results were compared to those of healthy subjects. Sánchez et al. [52] have found a significant increase in the relative expression of IL-18 mRNA in individuals carrying the rs360719 minor allele. IL-18 is IFN-γ inducing factor. Infective tubulointerstitial nephritic patients are known to have diminished ability of blood leukocytes to produce IFN-γ [53]. Our study indicates that this may be related to lower frequency ofIL18rs360719 minor allele in this group compared to controls and type 2 DM nephropathy patients. In type 2 DM patients with overt nephropathy, positive correlations between plasma IFN-γ, proteinuria, and eGFR were found [54].Due to limited financial support, we did not perform any functional studies regarding T-cell-related interleukin and vitamin D pathway genes, especially that multiple influences independent or dependent on genetic profile need to be taken into account in such studies conducted in the uremic milieu. Although the examined patients showing ESRD due to type 2 DM nephropathy were well-defined group, they obviously were not consistent in HLA DRB1 alleles. The latter could be important in modulating susceptibility to advanced type 2 DM nephropathy and related complications, like it was shown for type 1 DM [55] or type 2 DM [41], regardless of their complications.


## 5. Summary

Distributions of tested T-cell cytokine gene polymorphisms or vitamin D pathway gene polymorphisms are not significantly different among patients with ESRD due to type 2 DM nephropathy and healthy individuals. Subjects with ESRD due to type 2 DM nephropathy differ in clinical manifestation from patients with other nephropathies leading to dialysis dependency, but differences in tested genotype distributions were found only inIL18rs360719 compared with chronic tubulointerstitial nephritic patients. This difference probably arose from the fact that pathology of chronic infective tubulointerstitial nephritis might have been associated with this specific polymorphism.


## 6. Conclusions

In Polish HD patients, T-cell cytokine gene polymorphisms and vitamin D pathway gene polymorphisms are not associated with ESRD due to type 2 DM nephropathy.IL18polymorphism is worthy to be further investigated in chronic infective tubulointerstitial nephritic patients as being possibly associated with this disease.



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*Source: 120317-2014-12-22.xml*