The identification and analysis of polymorphisms in genes that encode biochemical markers that are altered in CKD patients is important, since they might affect the outcome of the patients 1,21,22. The inflammatory biomarkers IL6 and PTX3 are particularly enhanced in ESRD patients and have been associated with a higher risk for CVD events, the major cause of death in these patients; still, how the polymorphisms of their encoding genes affect the inflammatory response and patient’s outcome, remains poorly clarified. It is important to identify and validate common genetic variants and genotypes, to know if and how they influence patients’ outcome; this knowledge may provide more adequate interventions in patients at higher risk. This study analyzed the allelic frequencies of two polymorphisms, namely IL6 (-174G > C) and PTX3 (+ 281A > G), in ESRD patients and in controls; evaluated their association with the degree of inflammation, shown by the blood levels of IL6, PTX3, CRP and GDF15, and with mortality risk, by recording events of death along one year.
For IL6 rs1800795, the frequency of CC, GG and CG genotypes for patients and controls was similar (9.7%, 47.4% and 42.9%, in ESRD patients; 9.4%, 53.1% and 37.5%, in controls) (Table 1). A study in a healthy Polish population, reported that the frequencies of IL6 (− 174 G/C) genotype were 21.9% for CC, 28.8%, for GG and 49.3% for CG, which were similar to those described for German and British populations; however the frequencies reported for Italian and American Caucasians, American Blacks and Asian Americans are similar to those observed in our study groups 38, in which GG is the most common genotype and CC genotype the less frequent 39. We found an allelic frequency of 0.28 and 0.31 for allele C and 0.72 and 0.69 for allele G, in controls and ESRD patients, respectively. According to the Reference SNP (rs) Report database for rs1800795, assessed on February, 25th 2021 (www.ncbi.nlm.nih.gov/snp/rs1800795), these frequencies are 0.44/0.56 for alleles C/G in the European population; the allelic frequencies in our studied population are closer to those reported for southern European 40 and North-American populations 41.
For PTX3 rs2305619, the genotype distribution was also similar between patients and controls (24.9%, 31.5% and 43.6% for AA, GG and AG, respectively, in ESRD patients; 15.6%, 31.3% and 53.2%, respectively, for controls). A study in healthy European subjects, reported that the distribution of AA, GG and AG genotypes was, respectively, 22.94%, 27.12% and 49.94% 33. Another study involving Taiwanese controls showed 12.2%, 41.5% and 46.3%, for AA, GG and AG genotypes, respectively 34. Our controls and ESRD patients presented similar genotype frequencies to those reported by Barbati et al.33. We also found that the allelic frequencies were 0.42 and 0.46 for allele A, and 0.58 and 0.54 for allele G, in controls and ESRD patients, respectively, which is in accordance with data obtained from Reference SNP (rs) Report database for rs2305619, assessed on February, 25th 2021 (www.ncbi.nlm.nih.gov/snp/rs2305619), for a European population (allele A: 0.48 and allele G: 0.52). Thus, the allelic frequencies for IL6 and PTX3 polymorphisms in our patient cohort were similar to those described in other European populations. Moreover, as the genotype distribution between controls and ESRD patients (Table 1) was similar, it seems that none of the different alleles were more prevalent in ESRD patients. Additionally, both IL6 and PTX3 polymorphisms genotype frequencies did not differ in ESRD patients, when sub-analyzed according to gender, etiology of CKD, type of vascular access for dialysis procedure, dialysis vintage, dialysis type, diabetes, hypertension and CVD history.
In our cohort of ESRD patients, IL6 (-174G > C) polymorphic genotypes showed that the GG and CC genotypes were associated with higher circulating levels of IL6, although without reaching statistical significance (Table 2). Actually, data on literature about the effect of IL6 (-174G > C) polymorphic genotypes on IL6 circulating levels is still controversial.
In a study with Indian ESRD patients with malnutrition inflammation complex syndrome, the C allele was associated with higher IL6 levels, and both CC and CG genotypes conferred a higher (about 3-fold) mortality risk than the GG genotype; moreover, the increased levels of IL6, when associated with higher TNF-α and low IL-10 levels, appeared to contribute for the activation of inflammatory pathways that lead to higher disease susceptibility, poorer nutritional status and lower survival rate 25. Another study, in a Southern Italian CKD cohort, also showed that patients with the CC genotype presented higher circulating levels of IL6 than those with CG or GG genotypes. Furthermore, CKD patients with CC genotype and high levels of IL6 showed a higher incidence rate (87%) of cardiovascular events, when compared to those with the CG or GG genotypes 28. In contrast, some studies in patients with other inflammatory clinical conditions, have suggested that the homozygous genotype for C allele confers protection, as it was associated with lower IL6 circulating levels 42–46. In a study in Korean patients on HD, the C allele of the IL6 (-174 G/C) was not detected, and this absence did not seem to interfere in IL6 circulating levels, in spite of the high circulating levels of IL6 (about three times higher than those found in our studied patients) 26. Different results were obtained in a study of Caucasian and African American ESRD patients on long-term dialysis, which showed the association of GG and CG genotypes with higher levels of IL6, as compared with those with the CC genotype 39.
Concerning the effect of IL6 (-174G > C) polymorphic genotypes on the other studied inflammatory biomarkers, we found that ESRD patients with CC genotype showed higher CRP levels (median: 0.52 mg/L), when compared to CG and GG genotypes (median: 0.42 mg/L and 0.30 mg/L, respectively), reaching statistical significance for CC versus CG (Table 2). Higher CRP values were also reported in CKD patients with CC genotype, compared with CG and GG genotypes, in other studies 28. Comparing PTX3 and GDF15 levels between IL6 polymorphic genotypes, we found that the CC genotype presented the highest median levels for both markers, reaching the increase in GDF15 a statistical significance. Thus, our data suggest that the CC genotype for IL6 (-174G > C) polymorphism contributes to an increased inflammatory response, by increasing CRP, PTX3 and GDF15 circulating levels in ESRD on dialysis.
Higher GDF15 concentrations have been associated with mortality and with heart failure events 47, as well as with CKD progression 48. Also, there is evidence that CRP stimulates GDF15 expression in endothelial cells 8,49. As already referred, our data show that in ESRD patients the CC genotype is associated to significantly increased GDF15 levels and a trend for higher IL6/CRP levels (Table 2); however, we found negative correlations between GDF15 and IL6/CRP for CC genotype patients, while for the other variants these correlations were positive (Table 3). This suggests that alternate transcription of IL6 gene might lead to the activation of different metabolic pathways, which could have a direct impact on GDF15 circulating levels.
The highest levels of hsCRP, PTX3 and GDF15 that we found in ESRD patients with the CC genotype, suggest that this genotype may increase the risk for CVD and for a poor outcome in ESRD patients. Epidemiologic studies have shown elevated levels of the inflammatory biomarkers, CRP, PTX3, and IL6 and GDF15 5,6,47 in ESRD patients, and their association with the risk for CVD events and mortality 12,28,47,48,50,51. Considering that the genotypic variations do not change over time, this highlights the importance of variants that although less frequent, may have biological value 51.
The evaluation of PTX3 levels has been proposed as an important sensitive tool to predict cardiovascular mortality risk in patients with advanced CKD, increasing before traditional systemic inflammation markers, such as CRP, commonly used in laboratorial/clinical practice 5,52. In our ESRD patient cohort, for the PTX3 polymorphism, a trend towards higher circulating PTX3 levels was found in the AG genotype, compared to the others. However, AA genotype presented the highest values of hsCRP and IL6, and GG genotype the lowest values (Table 2). As far as we know, no studies about the effect of this polymorphism have been performed in ESRD patients. Studies in patients at risk for myocardial infarction reported that this PTX3 polymorphism is associated with higher plasma PTX3 levels in individuals with AA genotype of rs2305619 33. Actually, several studies have proposed PTX3 as an inflammatory biomarker associated with inflammation and CVD, and as an early marker of CV mortality in ESRD patients 5,52. Moreover, elevated PTX3 levels have been also associated with lower eGFR and appear to independently predict incident CKD in the elderly, and, thus, appears to be a promising biomarker of kidney disease 15. Our data show that the AA genotype, not only presents the highest levels of hsCRP and IL6, but also presents a positive significant correlation between them (Table 3), suggesting a higher risk for a poorer outcome.
As referred, along one year we monitored patients, registering the events of death, in order to evaluate how the IL6 and PTX3 polymorphisms could affect their outcome. Along the one-year follow-up, 21 patients dropped from the study (e.g. transplant, recovering, abandoning KRT) and 28 patients died, showing a mortality rate of 10.4% (28 out of 268).
Evaluating mortality according to the PTX3 (+ 281A > G) polymorphism, we found that the genotype frequency between deceased and alive patients did not differ and the mortality rates were similar between genotypes (AA: 10.3%; GG: 8.2%; AG: 12.2%) (Table 4). Considering the IL6 (-174G > C) polymorphism, the genotype distribution in deceased patients was different from that observed for survivors, almost reaching statistical significance (p = 0.089); the mortality rates per genotype were 22.2% for CC genotype, 10.3% for GG and 7.8% for CG (Table 4). The survival cumulative curves (Fig. 1A) are also strongly suggestive of a poorer outcome for patients with the CC genotype; actually, the mortality rate in CC genotype carriers was more than 2-fold of that observed in the other two genotypes, which presented similar mortality rates, although a trend towards a lower value was observed in heterozygous individuals.
When comparing the circulating levels of the inflammatory markers in deceased and alive patients, we found that deceased patients with the CC genotype presented increased hsCRP (more than 3-fold), IL6 and PTX3 (both 1.5-fold) and decreased GDF15 levels. In the other two genotypes, the increase in hsCRP was lower for deceased patients, suggesting an enhancement in inflammation for CC genotype, and a less favorable outcome for their carriers (Table 5).
Curiously, the levels of GDF15 for alive patients with CC genotype, were significantly increased, when compared to the other two genotypes (Table 5). Considering that this genotype appears to be the less favorable (Table 4), we wonder about the importance of the influence of the IL6 (-174G > C) polymorphism on GDF15 levels, which most certainly deserves further investigation. Moreover, the cross-survival analysis (Cox regression) further suggests that the IL6 polymorphic CC genotype is associated with a higher mortality risk (approximately 3-fold higher than in CG genotype), and heterozygosity with the lowest mortality risk (Table 6 and Fig. 1A). Performing Cox regression analysis and adjusting for confounding factors, such as age, dialysis vintage, type of vascular access, diabetes and history of CVD, the CC genotype remained as an independent predictor of death (Table 6, adjusted models). Our data suggest that ESRD patients with CC genotype have almost three-fold higher risk for a poorer outcome, and the heterozygous, the most favorable outcome. In line with our findings, a meta-analysis of 74 studies with 86,229 subjects, evaluated the association of -174G > C IL6 (rs1800795) with the risk for CVD, and found that the C allele was associated with a higher risk for CVD 6,29.
When comparing the circulating levels of the inflammatory markers in deceased and alive patients, according to the PTX3 polymorphic genotypes, we found that deceased patients with the AA genotype presented the highest increase in IL6 (almost 3-fold than the survivors); the increase in the values of the other inflammatory markers in deceased patients were similar between genotypes. The mortality rates and survival cumulative curves for PTX3 polymorphism, were similar in all genotypes (Fig. 1B). Thus, in spite of the enhancement in the inflammatory markers, hsCRP and IL6 observed in AA genotype carriers and the striking increase in IL6 for AA genotype, in deceased patients, we did not find a significant impact of the PTX3 polymorphism in the outcome of these patients.