It has been previously shown that biomarkers of inflammation are high even in the early stages of CKD [11, 12]. The increased inflammation could be caused by both genetic predisposition and environmental factors, being linked to the risk of CKD progression to ESRD. In our study, we have found strong suggestion of associations between ESRD and four SNPs located in IL4R (rs1801275), CCL2 (rs4586), NOS3 (rs7830), and an intergenic binding site for STAT4 (rs301640). All of these sites involve genes related to inflammation and immune response pathways.
On the one hand, two of the significant SNPs (rs1801275, and rs4586) reflected a certain protection against ESRD:
rs1801275 (A/G) is located at position g.54150A > G of the IL4R gene in chromosome 16. This position corresponds to exon 12, where it generates a missense change (p.Gln576Arg). IL-4 is a cytokine involved in Th2 immune response, and its effect depends on binding with IL4R. IL-4 has been found to be an important predictor of kidney injury. Modulation of the IL4 pathway during ESRD may be due to an intracellular regulation involving different pathways but also could be possible that polymorphisms within the IL4R gene could alter the signalling pathway of IL-4, leading to a progression or prevention of kidney damage . In our study, the presence of the AG genotype seems to indicate protection from ESRD development, which could be caused by a decrease in signalling of IL-4 through of its receptor. Although rs1801275 is a missense SNP which is located in a low complexity domain of the protein (SMART) and it has been predicted as tolerant (SIFT). To date, this SNP has shown associations with inflammatory and autoimmune diseases such as arthritis rheumatoid, asthma, allergic diseases and atopy [14, 15], but the entire role in CKD of IL-4 and its signalling pathway through IL4R still remains unknown and further studies are needed.
rs4586 (C/T) is a synonymous polymorphism located on exon 2 of the CCL2 gene in chromosome 17. The CCL2 gene encodes a key chemokine in recruiting mononuclear inflammatory cells to sites of inflammation. One example is the interstitium and glomerulus, where CCL2 causes renal interstitial and glomerular inflammation, leading to progressive renal injury . In our study, the presence of each additional copy of the C allele of rs4586 indicates protection against ESRD. Recent studies have suggested a beneficial effect of blocking the action of CCL2 on diabetic nephropathy and renal function through anti-fibrotic effects . By analyzing the CCL2 sequence via PATROCLES, we have found that the C allele of rs4586 generates a putative target site (TGCTGCTA) for six different microRNAs (hsa-miR-15a/15b/16/195/424/497), whereas the T allele disrupt this target site and consequently none of these microRNAs target this sequence. MicroRNAs are small RNA molecules (22 nucleotides) that have a great impact on posttranscriptional regulation and potentially large relevance to complex diseases. When a microRNA attaches to its target, it can silence expression via mRNA degradation or by preventing mRNA translation . Therefore we could hypothesize that C allele might exert its beneficial effect by binding a microRNA and blocking the transcription of CCL2 gene.
On the other hand, rs301640 and rs7830 polymorphisms indicated susceptibility to ESRD:
rs301640 (A/G) is a SNP located on chromosome 13, in an intergenic region between the eukaryotic translation initiation factor 4A1 pseudogene 6 (EIF4A1P6) and the gene encoding hsa-miR-3169. This SNP is located within a binding site for the transcription factor STAT4. In our study, the presence of each additional copy of A allele was associated with increased odds of ESRD. By using PROMO software [19, 20], we found that the A allele might disrupt the STAT4 binding site, which could potentially modify a distal enhancer. STAT4 plays an important role in Th1 differentiation by transmitting IL-12 signals to produce IFN-γ , which could induce pro-inflammatory cytokines leading to injury in target tissues.
rs7830 (G/T) is located in a region that belongs to two different genes. The sense strand corresponds to intron 26-27 of the NOS3 gene, whereas the antisense strand matches with the 3´-UTR of the autophagy related 9 homolog B (ATG9B) gene. On the one hand, NOS3 encodes for an enzyme that generates NO in endothelial cells and is involved in the regulation of vascular function . On the other hand, ATG9B encodes for a protein required for autophagy in several eukaryotic organisms, although its entire function is unknown . In our study, the T allele of rs7830 was associated with ESRD. We hypothesize that the effects of rs7830 on ESRD might be due to NOS3 rather than ATG9B. In fact, polymorphisms in NOS3 have been associated with atherosclerotic vascular diseases , renal dysfunction , and advanced diabetic nephropathy . Moreover, this SNP seems to be located within a silencer motif (TGGGGACT) , where the G to T allele change could disturb the splicing mechanism in NOS3 leading to different transcripts . Thus, this change might affect NOS3 expression and to be associated with the development of ESRD.
Although these findings have also been supported by previous studies where the SNPs described above have been associated with kidney disease or related (Additional file 1: Table S1 Content 1), we could not find any significant results after applying FDR correction. In regards to this, since p-value is depending on the sample size, it may be possible that we have not found any significant adjusted p-value because our sample size is not large enough to detect moderate effects. Thus only big effects would be detected in small populations. Moreover, it exists some controversy about adjusting the “p-value” after multiple tests on clinical-orientated studies [29, 30]. In addition, the weak association found for the studied polymorphisms could be due to an indirect involvement in ESRD. That is, it cannot be discarded that these SNPs might be in linkage disequilibrium with more powerful polymorphisms associated with ESRD. Another limitation of our study was that although we tried to select individuals for control-group with similar age to case-group, the comparison between means was significant (50.0 versus 52.8 years; p = 0.030, Table 1). We think that these differences are so slight that have a low clinical significance. However, logistic regression analysis was adjusted by age in order to avoid possible age interferences.
Therefore, this is a preliminary study which could be considered to generate hypothesis for future studies. In fact, the association of these SNPs with ESRD needs to be confirmed by replicating studies with a larger sample size, as well as functional studies should be performed in order to get further insights into ESRD susceptibility. Moreover, it will be interesting to include in the future some factors which may also influence ESRD development (diabetes, dyslipidemia, smoking, etc.) which could not be collected in this study. Besides, those patients with ESRD who are receiving dialysis and/or have not yet received kidney transplant should also be taken into account in further studies because they could carry on different genotypes.