Polymorphisms of the ELANE Gene Promoter Region in End-Stage Chronic Kidney Disease Patients

End-stage renal disease (ESRD) patients have a high mortality rate that exceeds that of non-ESRD population. The hemodialysis procedure induces neutrophil activation and elastase release, which might have a role in the inflammatory process and in the development of oxidative stress. The ELANE gene encodes the neutrophil elastase. We analyzed the effect of ELANE promoter region polymorphisms and its relation with the circulating levels of elastase, as well as several clinical, biochemical and inflammatory markers in 123 ESRD patients. We found two duplications in heterozygosity in the promoter region and a new polymorphism, the c.-801G>A. ESRD patients heterozygous for the c.-903T>G polymorphism had no changes in the circulating levels of elastase or other evaluated variables, and those homozygous for the c.-741G>A polymorphism showed significant effects on neutrophils count, as well as in neutrophils/lymphocytes ratio, which might be associated with an increased inflammatory process.


Introduction
End-stage renal disease (ESRD) is a growing public health problem with an increasing worldwide prevalence. Inflammation is a common feature in ESRD patients under hemodialysis (HD) [1,2]; however, the mechanisms/factors triggering the inflammatory process are still poorly clarified. The HD procedure induces neutrophil activation and elastase release, which might be important in the inflammatory process and in the development of oxidative stress, two factors that increase the anemia and the risk of cardiovascular diseases [1][2][3]. Neutrophil elastase, a protease able to degrade several extracellular matrix proteins, is encoded by ELANE gene. Polymorphisms of ELANE gene have been associated with the development of several pathologies [4][5][6]. Indeed, the presence of mutations and single nucleotide polymorphisms (SNPs) in the codifying region and in the six repetitive tandem motifs of the promoter appear to influence the level of elastase expression, promoting proteolytic disturbances [5].
This study aimed to identify polymorphisms of the ELANE promoter region and assess their impact in the plasma levels of elastase, clinical and hematological data, iron metabolism, dialysis efficiency, inflammatory and nutritional markers.

Patients
We performed a cross-sectional study with 123 ESRD patients (69 males and 54 females, mean [˘SD] age: 65.3 [˘13.9] years) on regular HD. Patients were under therapeutic HD three times per week for 3-5 h, for a median time of 2.5 (1.2-5.2) years. High-flux polysulfone FX-class dialyzer of Fresenius (Bad Hamburg, Germany) was used for the HD procedure. The main causes of renal failure were diabetic nephropathy (n = 66), hypertensive nephrosclerosis (n = 21) and other causes (n = 36); patients with autoimmune diseases, malignancy, and acute or chronic infection were excluded. All participants gave their written informed consent to participate in this study, previously approved by the Ethics Committee from the dialysis clinic.

Methods
The ELANE promoter was screened in all patients using PCR-direct sequencing with forward (5'-GGAAGGACCAGAGAAGTGC-3') and reverse (5'-CTGCCAAACCTAGACCTGAG-3') primers, which amplify a 397bp DNA fragment. Sequencing was performed by automatized Sanger method (GATC Biotech ® facilities) and electropherograms were analyzed with Chromas lite 2.1.1™ software (Technelysium, Australia). Plasma elastase quantification was done by ELISA (Human PMN elastase platinum ELISA, eBioscience, Austria). Each polymorphism was statistically evaluated and correlated with clinical and dialysis adequacy markers (age, gender, type of vascular access, presence of diabetes, dialysis time, urea reduction ratio, Kt/V, creatinine, darbopoeitin dose), hematological data (hemoglobin, hematocrit, erythrocyte, MCV, MCH, MCHC, RPI, leukocyte, neutrophil, lymphocyte and reticulocyte counting, neutrophil/lymphocyte ratio), inflammatory (C-reactive protein, interleuquin-6 (IL-6), oxidized low-density lipoprotein (oxLDL), elastase and elastase/neutrophil ratio), nutritional (serum albumin and body mass index) and iron metabolism (iron, transferrin, transferrin saturation, ferritin and soluble transferrin receptor (sTfR) serum levels) markers, according to the methods previously described [7]. Statistical analysis was performed using the Statistical Package for Social Sciences (SPSS) version 21.0 for Windows (SPSS Inc., Armonk, NY, USA). The distribution of continuous variables was analyzed using the Kolmogorov-Smirnov test. The values of variables with normal distribution are presented as mean˘standard deviation, and those without normal distribution are expressed as median (interquartile range). According to the type of distribution, parametric or non-parametric for the comparisons between groups we used Student's t test or the Mann-Whitney test, respectively. A comparison of multiple variables between groups was performed using one-way ANOVA algorithm with the post hoc Tukey test. The association between categorical variables was analyzed using the test χ 2 or Fischer's exact test. Differences between groups were considered statistically significant at p < 0.05. Moreover, the adjustment of p value derived from multiple statistical tests was performed for the inflammatory associated variables (CRP, IL-6, OxLDL, elastase; elastase/neutrophil ratio, albumin, neutrophils, leucocytes, neutrophil/lymphocyte ratio) using the Bonferroni correction test and a p < 0.006 was considered as statistically significant.

Conclusions
ESRD patients under dialysis present high elastase plasma levels, which has been associated with the rise in neutrophils, common in inflammatory processes and also associated with the hemodialysis procedure. The c.-741G>A polymorphism in the ELANE promoter seems to be associated with higher neutrophil counts, and therefore, with an enhanced inflammatory process that is usually associated with a poor outcome. Further studies with a larger population are required to confirm the influence of the GG genotype for c.-741G>A polymorphism in the inflammatory process and to assess the impact of the new mutation and the extra blocks described herein for the first time.