ORIGINAL ARTICLE

Malnutrition-Inflammation Syndrome in a Hemodialysis Population: The Influence of Polymorphic IL-6-174 and IL-10-1082 Genes
Suhardjono

ABSTRACT Aim: to find out inflammation prevalence in hemodialysis patients and whether polymorphic gene IL-6-174 and IL-101082 had prominent factors in malnutrition inflammation syndrome. Methods: a study on 81 patients who were on hemodialysis twice a week, 5 hours each session has been conducted. The subjects had no other co-morbidities and all of them used reprocessed diacetate cellulose dialyzers. Results: it was obtained that CRP blood level (6.235.57 mg/L), inflammation prevalence (23.5%), and malnutrition inflammation score (6.7) were lower compared with the data from Europe and the United States. Out of 64 patients examined, IL-6-174GG 95.31%, CC 3.13% and GC 1.56%, IL-1082AA 89.06%, GA 10.94%, but absence of GG genotype. Considering the scanty amount of allele C in IL-6-174 gene and G allele in IL-10-1082 gene, based on the statistic analysis performed, it did not reveal the influence of the difference in allele on the clinical manifestation. The proportion of these alleles were almost similar to that obtained in Korea, Japan and China, but it was different from that obtained in the US and in Europe. A very resolute impression was obtained in HD patients in Jakarta that IL-6174GG gene was protective in nature whereas IL-10-1082 AA gene had a less considerable role. Conclusion: the prevalence of inflammation and malnutrition-inflammation parameters (CRP, Malnutrition Inflammation Score) in our HD patients were lower than that reported in Western countries. This might be related to the low prevalence of IL-6-174C allele in our population. Key words: Hemodialysis, gene polymorphenism, IL-6, IL-10.

INTRODUCTION

Division of Nephrology, Department of Internal Medicine, Faculty of Medicine, University of Indonesia-dr. Cipto Mangunkusumo Hospital, Jakarta

The mortality rate in hemodialysis (HD) population is high. The majority of dialysis patients die from cardiovascular complications and far higher in patients with kidney failure compared to the general population.1 Among patients treated by dialysis, the risk ranges from 500-fold higher in individuals aged 2535 to 5-fold higher in individuals aged >85 years.2 Uremic patients were found to have increased risk factors for cardiovascular complications, hypertension, dyslipidemia, and inflammation. 3 Many efforts have been done to lower the mortality rate in dialysis population, which include the use of more advanced dialysis technology and the development of the National Kidney Foundation-Kidney Disease Outcomes Quality Initiative (NKF-K/DOQI) Clinical Practice Guidelines.4 Despite this improvement, the mortality rate in dialysis patients is still unacceptably high. Malnutrition is frequently found in HD patients. The prevalence varies between 18-75%. 5 Although malnutrition was rarely reported as a direct cause of death, many studies showed that patients with malnutrition were associated with higher mortality.5,6 There were some evidences, which showed that malnutrition in dialysis patients was different from those in general population.7,8 Many interventions such as administration of intradialytic parenteral nutrition, failed to improve malnutrition in dialysis patients.9 Decreased body weight, loss of fat, muscle mass, and appetite could be due to inflammation process, oxidative stress, and malnutrition.10 In dialysis patients, inflammation as measured by the levels of CRP, IL-6 or other inflammation markers, correlates well with mortality.11,12 Inflammation in HD population is associated with malnutrition-inflammationatherosclerosis syndrome (MIA) or malnutrition inflammation complex syndrome (MICS), a condition of accelerated atherosclerosis with high mortality.13,14 In end
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stage renal disease, IL-6, the major mediator of acute phase response, plays a key role as a central regulator of inflammation response.15 IL-6 is the most powerful CRP stimulator, and Bologa et al. reported that increased level of IL-6 to be the strongest predictor of mortality in hemodialysis patients.16 Some studies showed that polymorphism of IL-6-174, was associated with serum level of IL-6 and CRP.17 IL-6-174CC and IL-10AA genotypes were found to be associated with increased risk of inflammation.17,18 The biocompatibility of dialysis membrane and other dialysis systems, as well as the purity of dialysate, plays an important role as a stimulating factor for cytokines and inflammation process.19,20 Since dialysis in our unit was performed using reprocessed cellulose diacetate dialyzer with reverse osmosis water and without using ultrapure water filter, it was expected that we would have high prevalence of inflammation. The purpose of this study was to find out inflammation prevalence in hemodialysis patients and whether the proportion of polymorphic IL-6-174 and IL-10-1082 genes played a prominent factor in MICS.
METHODS Patients

Eighty one HD patients in stable condition and without clinical signs of infection were included in this study. Dialysis was performed using reprocessed cellulose diacetate dialyzers (FB-110T, FB130T, Nippro). Dialyzer was reprocessed using reverse osmosis water and formalin was used as disinfectant. Patients were included in the study, if they were on 5 hourly, twice weekly regular dialysis, and they had been on HD for more than 3 months. All patients gave informed consent. This study was approved by ethical committee of Faculty of Medicine, University of Indonesia, Jakarta.
Laboratory Methods

Genotyping of IL-6-174 and IL-10-182. Genotypes of IL-6-174 and IL-10-182 were examined in 64 out of 81 patients. Some patients moved to other cities, or stopped dialysis and they were not included in the study. Genotyping (IL-6, IL-10) was performed in GeneLab. Genotyping of IL-6-174 was performed using the methods as described by Fishman et al.21 and Ye et al.22 Genotyping of IL-10-1082 was done using the methods as described by Perrey.23 Blood samples from patients were collected in EDTA tube. DNA was extracted using QIAmp DNA mini kit (Qiagen, UK) extraction protocol. Isolated DNAs was stored until it was used for genotyping. The results of measurement IL-6-174G/C was confirmed with the help of Prof. Ian Day and Dr. Debbie Cumming, University of Southampton, UK. For IL-10-1082A/G confirmation, we got help from Dr.Vera Pravica, Manchester. Malnutrition inflammation score (MIS). Status of nutrition and inflammation was measured by malnutrition inflammation score (MIS) from KalantarZadeh.24 MIS is comprised of seven components of the conventional Subjective Global Assessment (SGA) of nutrition, which is a semi-quantitative scale with three severity levels. It combines three parameters, which includes body mass index (BMI), serum albumin, and TIBC to represent serum transferrin. Each MIS component has four levels of severity ranging from 0 (normal) to 3 (very severe).24
RESULTS Demographic and Malnutrition-Inflammation Parameters

C-Reactive Protein (CRP). Blood samples were collected from AV fistule just before the start of HD. We used the average CRP levels from two examinations. Determination of high sensitive C-Reactive Protein (hsCRP) was performed using immunometric assay by immulite analyzer and Immulite C-Reactive Protein reagens from DPC, Diagnostic Products Corporation, Los Angeles. Serum Albumin and Total Iron Binding Capacity (TIBC). Serum albumin was measured by bromocresol green method, using reagens from Roche. TIBC was determined by precipitation method with magnesium hydroxide carbonate.
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We examined 81 HD patients with an average age of 50.9 7.8 year-old. Sixty percent of the patients were males and 40% were females. The dialysis vintage was 39.2 7.2 months. The average MIS was 6.7, serum albumin 3.7 g/dL, and hsCRP 6.23 mg/L. Levels of CRP above 10 mg/L were found in 23.5% of patients. CRP levels above 5 mg/L was found in 44.4% of the patients and levels below 5 mg/L were 55.6%.
IL-6-174 and IL-10-1082 Gene Polymorphisms

Genotyping of IL-6-174 revealed that 95.31%, 1.56% and 3.13% of patients had IL-6-174 GG, GC and CC genes respectively. We also found that the majority of patients (89.06% ) had IL-10-1082AA genotype, and the rest (10.94%) had IL-10-1082GA genotype. None of the patients had IL-10-1082GG genotype. Considering the scanty amount of allele C in IL-6174 gene and G allele in IL-10-1082 gene, based on the statistic analysis performed it did not reveal the

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Table 1. Prevalence of Inflammation in Dialysis Patients Investigator Europe and North America - Zoccali et al - Owen et al - Qureshi et al - Zimmermann et al - Stenvinkel et al - Yeun et al Asia - Iseki et al - Noh et al - This study Year Patients CRP (mg/L) Prevalence (%)

1998 1998 1998 1999 1999 2000 1999 1998 2004

112 HD 1.054 HD 128 HD 280 HD 109 HD 91 HD 163 HD 106 PD 81 HD

>5 >8 >10 >8 >10 >5.2 >10 >8 >10 >5

65 35 53 46 32 51 21 14 23.5 44.4

Modified from Stenvinkel and Yeun25

influence of the difference in allele on the clinical manifestation (Fisher exact test, p=0.702, p=0.574, respectively).
DISCUSSION

In this study, we found that several parameters of inflammation were lower than those reported from dialysis centers in Europe and North America. We also found high levels of CRP (above 10 mg/L ), which was an indicator of inflammation, in 23.5% of patients. In table 1, we compared the result of our findings with the data that were collected by Stenvinkel and Yeun.25 If we used levels of CRP above 5 mg/L as a cut off of inflammation, 44.4% of our patients fulfilled the criteria of having an inflammation. These results were lower than those found by Zoccali et al (65%) and Yeun et al (51%). Data from Japan and Korea also reported lower rates of inflammation.25 Using the same cellulose diacetate dialyzer, we found that the average of CRP levels in this study (6.23 5.57 mg/L) were lower than those reported by Memoli et al. (12.9 2.8 mg/L ).19 Our results were almost similar to those using more biocompatible membrane (7.9 1.5 mg/L).19 In contrast, the study by Zimmerman et al, which used several biocompatible membranes (polysulfone, hemophane, polyamide) found CRP levels of 16.2 24.5 mg/L.26 The study by KalantarZadeh et al. reported lower levels of CRP (6.42 7.79 mg/L) using high flux biocompatible polysulfone membranes.27 It can be concluded in our study that using reprocessed and reused cellulose diacetate dialyzer did not increase the levels of CRP or inflammation process as high as those reported in Europe and North America.

MIS can be used as an indicator of Malnutrition Inflammation Complex Syndrome and as a predictor of dialysis outcome.24,27 It has a significant association with prospective hospitalization and mortality.24 Our study found an average of MIS was 6.5, which was lower than those reported in a survey among representative samples of HD facilities and patients participating in the DOPPS (Dialysis Outcomes and Practice Patterns Study) trial. For example, in DOPPS trial, MIS was reported at 10.7 in Germany and 9.0 to 10.1 in other European countries.28 Data from Japan showed an average MIS of 5.8, which was lower than the result in our study.28 The data support that inflammation is lower in Asian patients as compared to European patients. In DOPPS trial, it was found that for every 1 unit increase in MIS, there was an 8% increase in mortality (P<0.0001) and 6% increase in hospitalization (P<0.0001).28 The reason for the difference in prevalence of inflammations response between Asian and non-Asian patients is unkown. So far, there have been no data available in dialysis patients. There were some data about the prevalence of IL-6-174 and IL-10-1082 gene polymorphisms in general population. Pyo et al. reported the prevalence of IL-6-174 and IL-10-1082 genes in several countries.29 It was interesting that the prevalence of IL-6-174CC gene or C allele in Asian people was quite low. (Table 2 ) The proportion of these alleles in our dialysis population was almost similar to those found in Korean, Japanese and Chinese population, but different from those found in American and European population. The difference in the prevalence of inflammation was mainly influenced by IL-6-174. Although the prevalence of IL10-1082G in Asian population was low, it did not have any influence on inflammations response. (Table 3)
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Table 2. Prevalence of IL-6-174G/C Gene Polymorphisms in Several Countries/Races* This study N =128 (%) Allele G Allele C 96.1 3.9 China N= 192 (%) 99.8 0.2 Korea N= 622 (%) 100 0 Japan N = 192 (%) 100 0 AfricanAmerican N=116 (%) 89.7 10.3 White N = 430 (%) 61.7 38.3 Hispanic N= 50 (%) 80.0 20.0

*Data from Pyo et al. 29

Table 3. Prevalence Rate of IL10-1082A/G Gene in Several Countries/Races* This study N= 128 (%) Allele A Allele G 94.5 5.5
29

China N = 152 (%) 94.0 6.0

Korea N= 622 (%) 92.6 7.4

UK, Manchester N= 660 (%) 51.0 49.0

AfricanAmerican N=114 (%) 66.7 33.3

Caucasian N = 428 (%) 53.7 46.3

Hispanic N= 50 (%)

78.0 22.0

*Data from Pyo et al.

Inflammations response in our dialysis population was lower than that reported from developed countries. Due to variation in gene polymorphisms, it was necessary for us to pursue further study to determine the standard dose of dialysis, which is cost-effective for Indonesian population. Whether the recommended dialysis guidelines using more biocompatible membrane and ultrapure dialysate, as has been practised in developed countries so far, is applicable in our dialysis population needs to be determined. If the low prevalence of C allele IL-174 in our dialysis population is associated with inflammation responses, it probably we dont need to use more expensive biocompatible and ultrapure dialysate water. To lower mortality in dialysis patients, we can concentrate on other risk factors.

1082A/G. Professor Ian Day, Dr. Debbie Cumming, University of Southampton, Dr.Vera Pravica, Manchester, UK, for the gift of DNA and typing confirmation.
REFERENCES
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CONCLUSION

This study was performed in dialysis population using reprocessed cellulose diacetate dialyzer, without using ultrapure dialysate water, and 2 times a week. We found that the prevalence of inflammation and malnutrition-inflammation parameters (CRP, Malnutrition Inflammation Score) in our HD patients were lower than that reported in Western countries. This might be related to the low prevalence of IL-6-174C allele in our population.
ACKNOWLEDGMENTS

I thank Dr. Indika Pitono, from GeneLab, for his excellent effort genotyping IL-6-174G/C and IL-10148

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