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Clinical |
Department of Internal Medicine,1 Eulji General Hospital; Department of Internal Medicine,2 Seoul National University Hospital, Seoul; Gachon University of Medicine and Science,3 Incheon; National Cancer Center,4 Gyeonggi-Do; Transplantation Research Institute,5 Cancer Research Institute,6 and Clinical Research Center,7 Seoul National University, Seoul, Korea
Correspondence to: K.H. Oh, Department of Internal Medicine, Seoul National University Hospital, 28 Yongon-Dong, Chongno-Gu, Seoul, 110–744, Republic of Korea. ohchris{at}hanmail.net
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONREFERENCES |
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Objective: To study the genetic effects of various
inflammatory cytokines on peritoneal solute transport rate (PSTR) in incident
Korean peritoneal dialysis (PD) patients. Design: Case-control association study. Methods: 132 patients with baseline peritoneal
equilibration test within 1 – 3 months after starting PD were enrolled.
We analyzed the influence of single nucleotide polymorphisms (SNPs) of
interleukin-6 (IL-6; -572G/C, T15A), tumor necrosis factor-alpha (TNF-
;
-1031C/T, -863C/A, -308G/A), and IL-10 (-1082A/G, -592A/C) on baseline PSTR.
Clinical parameters such as age, gender, presence of diabetes mellitus,
comorbidity, C-reactive protein, and residual renal function were also
included as covariates. Results: The T15A SNP of IL-6 (rs13306435) was
associated with PSTR. Patients with TA genotype (n = 18) had
significantly lower D4/P creatinine (0.65 ± 0.087 vs 0.73
± 0.110, p = 0.0046) and higher D4/D0
glucose (0.39 ± 0.174 vs 0.31 ± 0.119, p = 0.027) than
patients with TT genotype (n = 114). The log value of the dialysate
appearance rate of IL-6 had a strong positive correlation with D4/P
creatinine (r2 = 0.1294, p < 0.0001) and was
significantly lower in the TA genotype than the TT genotype (201.7 ±
14.42 vs 116.8 ± 88.91 pg/minute, p = 0.0358). By multiple
logistic regression, TA genotype was negatively associated with a higher PSTR
(high or high average; odds ratio 0.18; 95% confidence interval 0.048 –
0.666). Conclusions: In incident Korean PD patients, T15A
polymorphism of IL-6 is associated with dialysate IL-6 concentration and
baseline PSTR.
KEY WORDS: Interleukin-6; single nucleotide polymorphism.
Patients starting peritoneal dialysis (PD) vary significantly in peritoneal small solute transport rate (PSTR). PSTR determines a patient's dialysis prescription, as high transporters often need short dwells, high glucose-containing dialysis fluids, or icodextrin. In addition, many reports have shown an association between high transport status and poor patient or technique survival (1–3). A recent meta-analysis reported pooled summary relative risks for mortality and technique failure of 1.15 and 1.18, respectively, for every 0.1 increase in the dialysate over plasma ratio for creatinine (D/P Cr) (4). A worse clinical outcome associated with high transporters may be explained by (a) loss of ultrafiltration and consequent hypervolemia, (b) increased albumin loss and malnutrition, and (c) increased glucose absorption and worsening dyslipidemia (5).
To date, several clinical factors, including older age, diabetes, and
comorbidities, are known to possibly influence transport characteristics
(6). However, they are not
sufficient to predict PSTR because uremic patients starting dialysis often
have low serum albumin and high C-reactive protein, reflecting a state of
chronic inflammation. It has been suggested that chronic inflammation,
mediated by various inflammatory cytokines in the uremic milieu, may have an
effect on peritoneal transport. In particular, proinflammatory cytokines, such
as interleukin (IL)-6 and tumor necrosis factor-alpha (TNF-), and the
anti-inflammatory cytokine IL-10 may play important roles in chronic
inflammation in the uremic milieu
(7). Pecoits-Filho et
al. showed that plasma and dialysate concentrations of IL-6 were
associated with high PSTR, suggesting a role for chronic inflammation in
influencing peritoneal membrane transport
(8).
Therefore, it is possible that the genetic polymorphisms of inflammatory cytokines could predict the diversity seen in the peritoneal transport rate. A recent landmark study reported that the -174C/G single nucleotide polymorphism (SNP) of IL-6 (as well as serum albumin and comorbidity) is an independent predictor of high baseline PSTR in Caucasian PD patients (9).
In this context, we conducted clinico-genetic studies to elucidate the
determining factors of baseline PSTR in incident Korean PD patients. We
hypothesized that the chronic inflammation orchestrated by various pro- and
anti-inflammatory cytokines, in either the uremic milieu or local peritoneal
tissue, may influence baseline PSTR. Moreover, we also hypothesized that the
genetic polymorphisms of the pro- and anti-inflammatory cytokines might have
an association with PSTR. We selected the SNPs of IL-6, TNF-, and IL-10
with minor allele frequencies >0.05 in the East Asian population. We
genotyped the SNPs and correlated them with baseline PSTR as determined by
peritoneal equilibration test (PET). In our study, the IL-6 T15A SNP
(rs13306435) was found to be associated with dialysate IL-6 level and baseline
PSTR.
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METHODS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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CLINICAL PARAMETERS AND ASSESSMENT OF PSTR
Detailed demographic and clinical information and PD-associated parameters
were obtained. Cardiovascular disease was defined as coronary artery disease
including angina pectoris and acute myocardial infarction, cerebrovascular
accident, or peripheral artery disease.
A modified PET using 3.86% glucose dialysis solution was performed in all patients enrolled. In short, after an overnight dwell with 1.36% glucose PD fluid, patients were subjected to a 4-hour dwell with 3.86% glucose PD fluid. Dialysate samples were taken at 0, 1, 2, and 4 hours during the test. Blood samples were taken at 2 hours. The dialysate over plasma concentration ratio for creatinine at 4 hours (D4/P Cr) and dialysate over initial dialysate concentration ratio for glucose at 4 hours (D4/D0 glucose) were determined. Glucose concentration was measured by colorimetry using the glucose oxidase method and serum albumin by the bromcresol green method. Serum and dialysate creatinine were measured using a kinetic alkaline picrate (Jaffe) method with a sensitivity of 0.2 mg/dL (18 µmol/L). Dialysate creatinine concentration was corrected for glucose interference according to the correction factor of each local laboratory. Accuracy of creatinine measurements in plasma and dialysate was validated with control samples in the four participating centers. Patients were classified as high, high average, low average, and low transporters according to the criteria of Smit et al. based on D4/P Cr (10).
Urine and dialysate collections (24-hour) were performed for measurement of weekly urea and creatinine clearance, protein excretion through urine and dialysate, and normalized protein equivalent of nitrogen appearance.
Body surface area was calculated by the Du Bois and Du Bois equation (11). High-sensitivity C-reactive protein (hs-CRP) at the time of the PET was determined by immunoturbidometry (latex) with a sensitivity of 0.1 mg/L (normal value: <0.5 mg/L).
DNA EXTRACTION AND GENOTYPING
The SNPs of IL-6 [-572G/C (rs1800796), T15A (rs13306435)], IL-10 [-1082A/G
(rs1800896), -592A/C (rs1800872)] and TNF- [-1031C/T (rs1799964),
-863C/A (rs1800630), -308G/A (rs1800629)] were genotyped by single base primer
extension assay using the ABI PRISM SNaPShot Multiplex kit (Applied
Biosystems, Foster City, CA, USA) according to the manufacturer's
recommendation. Briefly, the genomic DNA flanking the SNP was amplified by
polymerase chain reaction (PCR) using forward and reverse primer pairs
(Table 1) and standard PCR
reagents in a 10-µL reaction volume containing 10 ng genomic DNA, 0.5
pmol/L of each oligonucleotide primer, 1 µL of 10X PCR gold buffer, 250
µmol/L dNTP, 3 mmol/L MgCl2, and 0.25 unit i-StarTaq DNA
Polymerase (iNtRON Biotechnology, Sungnam, Gyeonggi-Do, Korea). The PCRs were
carried out as follows: 10 minutes at 95°C for 1 cycle, 30 cycles at
95°C for 30 seconds, 55°C for 1 minute, 72°C for 1 minute,
followed by 1 cycle of 72°C for 7 minutes. After amplification, the PCR
products were treated with 1 unit each of shrimp alkaline phosphatase (Roche
Diagnostics, Mannheim, Germany) and exonuclease I (USB Corp., Cleveland, OH,
USA) at 37°C for 60 minutes and at 72°C for 15 minutes to purify the
amplified products. The purified amplification products (1 µL) were added
to a SNaPShot Multiplex Ready reaction mixture (Applied Biosystems) containing
0.15 pmol of genotyping primer for the primer extension reaction
(Table 1). The primer extension
reaction was carried out for 25 cycles of 96°C for 10 seconds, 50°C
for 5 seconds, and 60°C for 30 seconds. The reaction products were treated
with 1 unit shrimp alkaline phosphatase at 37°C for 1 hour and at 72°C
for 15 minutes to remove excess fluorescent dye terminators. The final
reaction samples (1 µL) containing the extension products were added to 9
µL Hi-Di formamide (Applied Biosystems). The mixture was incubated at
95°C for 5 minutes, followed by 5 minutes on ice, and then analyzed by
electrophoresis using the ABI Prism 3730xl DNA analyzer (Applied Biosystems).
Results were analyzed using GeneScan analysis software (Applied
Biosystems).
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DETERMINATION OF DIALYSATE IL-6 LEVELS
Samples for the determination of dialysate IL-6 concentration were obtained
from the dialysate effluent after overnight dwell 10 hours before the PET and
immediately frozen at –70°C until analysis. Dialysate IL-6 level was
determined by photometric enzyme-linked immunosorbent assay (ELISA)
(Boehringer Mannheim, Mannheim, Germany). Intra- and interassay coefficients
of variation were 2.6% and 7.1% respectively. Sensitivity was 7.5 pg/mL for
IL-6. For statistical analysis, dialysate appearance rate of IL-6 was
calculated as dialysate concentration times the drained volume divided by the
dwell time, and expressed as picograms per minute.
STATISTICAL ANALYSIS
Continuous variables are expressed as mean ± SD and categorical
variables are given as absolute counts and percentage. The differences in
clinical and peritoneal transport parameters between the transport groups were
analyzed by Kruskal–Wallis test. To compare genotype and allele
frequencies among transport groups, chi-square test was used. The peritoneal
transport parameters and dialysate appearance rate of IL-6 for each genotype
were compared using Mann–Whitney U test or Kruskal–Wallis test.
The hs-CRP and dialysate appearance rate of IL-6 were skewed and were
therefore transformed for regression analysis by taking the natural logarithm.
Multivariate analysis by logistic regression was performed using age, sex,
diabetes status, residual renal function, and the logarithm of hs-CRP as
covariates. The statistical tests were performed using SPSS version 12.0 (SPSS
Inc., Chicago, IL, USA) or GraphPad Prism version 4.0 (GraphPad Software, San
Diego, CA, USA). A p value < 0.05 was considered statistically
significant.
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RESULTS |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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DISTRIBUTION OF IL-6, TNF-, AND IL-10 SNPs ACCORDING TO TRANSPORT GROUP
Distribution of IL-6, TNF-, and IL-10 genotypes in peritoneal
transport groups are summarized in Table
3. The distributions of the 14 alleles (7 polymorphisms) were
within Hardy–Weinberg equilibrium. For the IL-6 polymorphism
(rs13306435), there was a significant correlation between T15A genotype and
peritoneal transport group (chi-square test, p = 0.025). Patients
with TA genotype (n = 18) had significantly lower D4/P Cr
(0.65 ± 0.087 vs 0.73 ± 0.110, p = 0.0046) and higher
D4/D0 glucose (0.39 ± 0.174 vs 0.31 ±
0.119, p = 0.0273) than patients with TT genotype (n = 114)
(Figure 1). No patient had an
AA genotype. In contrast with IL-6, SNPs of IL-10 and TNF- had no
influence on baseline PSTR.
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DIALYSATE IL-6 LEVEL, PSTR, AND IL-6 T15A POLYMORPHISM
To determine the functional significance of the IL-6 T15A polymorphism
(rs13306435), dialysate appearance rate of IL-6 was determined in the 115
patients available for samples. The log value of dialysate appearance rate of
IL-6 had a strong positive correlation with D4/P Cr
(r2 = 0.1294, p < 0.0001) and was lower in the
TA genotype than in the TT genotype (201.7 ± 14.42 vs 116.8 ±
88.91 pg/minute, p = 0.0358)
(Figure 2). In contrast to
dialysate IL-6, serum IL-6 levels were not different between the two genotypes
(data not shown). By multiple logistic regression using the clinical variables
gender, age, diabetes, cardiovascular disease, logarithm of hs-CRP, and
residual renal clearance as covariates, TA genotype was negatively associated
with a higher PSTR (high or high average), with an odds ratio of 0.18 (95%
confidence interval 0.048 – 0.666)
(Table 4).
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DISCUSSION |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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, and
IL-10 on baseline PSTR, as assessed by PET, in ethnic Korean PD patients. We
enrolled only incident patients with PET results within 1 – 3 months
after initiating PD. The T15A polymorphism in exon 5 of IL-6 (rs13306435) was
found to be an independent predictor of peritoneal transport rate in our
patient population. Moreover, IL-6 T15A polymorphism was significantly
associated with dialysate IL-6 level; that is, there were much lower dialysate
IL-6 levels in patients with TA genotype than in those with TT genotype. This
is the first Asian report on the association between genetic polymorphism of
IL-6 and baseline PSTR.
Many researchers have tried to find the major clinical factors that determine PSTR. For example, race (12), age (13), body mass index (6), presence of diabetes mellitus (14,15), uremia per se (16), and comorbidity (17) were reported by some but rejected by others. These conflicting results may be explained by differences in the timing of evaluation, ethnicity, or the possible effect of peritonitis prior to the evaluation. It is noteworthy that, in the large study on peritoneal solute transport by Davies (18), clinical factors as a whole could account for only 21% of the variability of PSTR. Recently, some investigators tried to elucidate the potential role of genetic polymorphism in the interindividual diversity of PSTR (9,18,19).
There have been only a few genetic studies in terms of peritoneal solute transport in PD patients. Researchers from Hong Kong studied the polymorphisms of possible candidate genes, such as plasminogen activator inhibitor-1 (20), endothelial nitric oxide synthase (21), and vascular endothelial growth factor (22); however, the results of those studies must be interpreted cautiously because of different methods of assessing PSTR and the admixture of incident and prevalent patients. Recently, Gillerot et al. showed that the promoter SNP of IL-6 (-174C/G, rs1800795) influenced baseline peritoneal permeability in Caucasian PD patients and exerted an influence on the mRNA level of IL-6 in the peritoneal membrane (9). However, in the case of IL-6 -174C/G polymorphism (rs1800795), the minor allele frequency was reported to be zero in the Asian population (accessed via http://www.ncbi.nlm.nih.gov/SNP). That is why the results of genetic studies with IL-6 -174C/G polymorphism cannot be directly applied to the Asian population.
It has been speculated that chronic inflammation plays a role in PSTR
(23,24).
The association between higher peritoneal membrane transport and lower serum
albumin is present before the initiation of dialysis. C-reactive protein, a
surrogate marker of inflammation, correlated well with D/P Cr. Also, plasma
and dialysate concentrations of IL-6 have been shown to be associated with
high PSTR, suggesting a role of chronic inflammation in influencing peritoneal
membrane transport
(8,25).
Proinflammatory cytokines (IL-6 or TNF-) and anti-inflammatory
cytokines (IL-10) may play important roles in chronic inflammation in the
uremic milieu (7). Therefore,
we focused on the inflammatory cytokines and carefully selected the SNPs with
minor allele frequency > 0.05 in the East Asian population.
In our study, there were no differences in clinical parameters among the
four transport groups. The SNPs of IL-10 and TNF- had no influence on
baseline PSTR. In contrast, we found that the T15A polymorphism of IL-6
(rs13306435) was an independent predictor of PSTR. This was still valid using
multiple logistic regression analysis after adjustments for possible
contributions by other clinical factors
(Table 4). Along with the other
report on IL-6 polymorphism
(9), our finding supports the
critical role of the IL-6 molecule and its genetic polymorphism in baseline
peritoneal transport.
Interestingly, dialysate IL-6 level, which strongly correlated with D4/P Cr [consistent with recent reports (8,25)], was significantly lower in the TA than in the TT genotype group (Figure 2). We measured serum IL-6 concentration from the same patient group (data not shown) and found that it was not correlated with dialysate IL-6 or with D4/P Cr (26). Intraperitoneal IL-6 is thought to be secreted mainly from mesothelial cells (27). The dialysate levels of IL-6 were more than fivefold higher than plasma concentrations of IL-6 in our study (data not shown). Therefore, the possibility of a leak from the systemic circulation was considered minimal. We also calculated the concentration of IL-6 attributed to local peritoneal production from the expected D/P ratio in order to avoid any chance of leakage from the circulation, and the result was not different (data not shown). Therefore, we can speculate that dialysate IL-6 mostly reflects local production of IL-6 in the peritoneum rather than diffusion from the circulation.
In contrast with the study by Gillerot et al. (9), there was no correlation between hs-CRP and dialysate IL-6 level (data not shown), or between hs-CRP and PSTR (Table 2). Recent studies that specifically investigated PSTR in incident PD patients also reported that high peritoneal transport is not consistently related to comorbidity, markers of systemic inflammation (hs-CRP or serum IL-6), or atherosclerosis in some populations (28–30).
Pecoits-Filho et al. (8) showed that dialysate and plasma levels of IL-6 do not correlate. In this context it could be said that systemic inflammation does not always parallel local inflammation. In our study, serum IL-6 — much lower concentration than that of dialysate — was not correlated with dialysate IL-6 or D4/P Cr. In addition, no difference was shown in serum IL-6 concentration between the two genotype groups (data not shown). These results imply that dialysate IL-6 concentration does not always reflect systemic IL-6 concentration, and that intraperitoneal IL-6 level, which is affected by IL-6 T15A SNP (rs13306435) through a yet unknown mechanism, could be one of the determinants of baseline PSTR.
The T15A variation in exon 5 changes the coding sequence of amino acid from Asp to Glu (D162E). The mechanism by which the T15A SNP of IL-6 (rs13306435) could affect the local concentration of IL-6 in the dialysate is not yet elucidated. The T15A polymorphism of IL-6 seems not to have a direct effect on cellular function of IL-6 because of the position of amino acid residue, similar physicochemical properties, and lack of minor allele homozygosity. Rather, the T15A SNP of IL-6 (rs13306435) may be a surrogate of unknown causal loci (another SNP, insertion–deletion polymorphism, or copy number variation) that affect the transcription level of IL-6 mRNA (31,32).
We cannot exclude bias due to enrollment from the different hospitals. We validated the measurements of creatinine, glucose, and other essential biochemical tests for the four participating centers before entering into the study. Although the patients (n = 20) recruited from one of the participating centers happened to have a tendency to lower D/P Cr compared to those from the other three centers, this might be due to the small number of patients enrolled from that center. When we analyzed our data with the remaining 112 patients, the result was not different from our conclusion. Also, our study population was entirely ethnic Korean. The duration of PD at the time of enrollment was within 3 months and without any previous history of peritonitis, which means a relatively homogenous population in terms of PD-associated factors and ethnicity. Finally, it could be said that although polymorphism has a significant effect on peritoneal transport, the TA genotype of T15A SNP (rs13306435) is present in only 15% of the total population and therefore is not the major determinant of peritoneal transport in most patients.
In conclusion, the T15A polymorphism of IL-6 (rs13306435) is associated with the dialysate IL-6 level and baseline PSTR in incident Korean PD patients. Our finding supports the role of IL-6 gene polymorphism in the interindividual diversity of the baseline PSTR and suggests a possible link between peritoneal inflammation and PSTR.
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ACKNOWLEDGMENTS |
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Received 30 January 2008; accepted 12 May 2008.
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REFERENCES |
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TOPABSTRACTMETHODSRESULTSDISCUSSIONREFERENCES |
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This article has been cited by other articles:
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  K.-H. Oh, J. Y. Jung, M. O. Yoon, A. Song, H. Lee, H. Ro, Y.-H. Hwang, D. K. Kim, P. Margetts, and C. Ahn Intra-peritoneal interleukin-6 system is a potent determinant of the baseline peritoneal solute transport in incident peritoneal dialysis patients Nephrol. Dial. Transplant., January 12, 2010; (2010) gfp670v2. [Abstract] [Full Text] [PDF]
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