The Role of FGF23 on the Induction of Cardiovascular Damage in Anemia With an Without Chronic Kidney Disease

1 Study populations 1.1) Study in patients with iron deficiency anemia with normal renal function. The study will be carried out in the serum samples of patients. All patients will be provided with the corresponding informed consent.

Patients with an age ≥18 years, normal renal function (GFR> 60 ml / min / 1.73m2), hemoglobin levels <11 g / dl, serum ferritin levels <100ng / dL or saturation percentage of transferrin <30%. Patients who have a history of treated or active malignancy, proliferative hematological disorders or evidence of malnutrition (BMI <17), who are receiving oral iron treatment, or who have had intestinal bleeding or have received a blood transfusion in the last 90 days, will be excluded from the study.

1.1.2) Study in patients with anemia and CKD. This study will include patients with stage 3 and 4 of kidney disease with or without anemia that meet the criteria: age ≥18 years, hemoglobin levels <11 g / dl, serum ferritin levels <100ng / dL or percentage Transferrin saturation <30%. Patients who have severe proteinuria, symptoms of treated or active malignancy, inflammatory rheumatic diseases, HIV positivity, Hepatitis B or C virus, who are receiving anticoagulation with coumarin or any erythropoiesis stimulating agent, who have undergone a major surgery or had a cardiovascular event, intestinal bleeding or received a blood transfusion in the last 3 months prior to the start of the study will be excluded.

In relation to the sample size required for the development of the study, it is considered that a random sample of 183 individuals for the population of patients with iron deficiency anemia without CKD and 218 for the study to be carried out with patients with anemia and CKD, will be enough to estimate, with a 95% confidence and an accuracy of +/- 5 percentage units, a population percentage that is expected to be around 13% and 15%, respectively. As a percentage of necessary replacements, it is expected to be 5% and 10%, respectively. 1.2. Determinations A blood simple will be taken at the initial time (before receiving treatment), 12 months after the start of iron supplementation, which will be ferrous sulfate or ferric carboxymaltose for patients. The blood sample will be centrifuged immediately at 3500 r.p.m for 10 'and stored at -80 ° C until the analysis of the different study parameters. The levels of Ca, creatinine, urea, phosphorus, albumin, hemoglobin, ferritin and iron saturation levels will be determined, using standardized and automated commercial assays (Beckman AU), which will be processed. The determination of intact and C-terminal FGF23 levels will be carried out by E.L.I.S.A. using the Kainos Laboratories kit, Japan and Immutopics, CA, respectively. For the analysis of PTH levels, the intact PTH Kit (Immutopics, CA) will be used. Hepcidin will be determined by ELISA (Elabsciences, USA).The levels of 25 (OH) D will be determined by radioimmunoassay (RIA) (Immunodiagnostic Systems, UK). Renal function will be performed by calculating the glomerular filtration rate (eGFR) using the CKD-EPI (Chronic Kidney Disease Epidemiology Collaboration) formula, which includes serum creatinine, age, sex and race as variables. ELISA kits will be used for the quantitative determination of circulating levels of brain natriuretic peptide (BNP), myoglobin, and troponin, I, (Thermofisher scientific) and Bioplex to determine the levels of secreted cytokines. For the determination of oxidative stress in plasma, the activity of the enzyme glutathione peroxidase (GPX) will be analyzed, using the method of Flohé and Günzler.

Establishment, through the use of the latest technology of the non-coding transcriptomic profile (microRNAs) in the plasma of anemic patients without or with CKD and its association with the clinical and analytical profile of these pathologies. miRNAs profile study will be carried out with the Illumina SE50 platform (Novogene enterprise, Cambridge Science Park, UK). The study will be performed from plasma samples of patients with iron deficit and with or without renal failure. DEGseq R package will be used for identifying a differential expression. Gene Ontology (GO) enrichment analysis will be used on predicted target gene candidates of known and novel miRNAs. Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis will be used for identifying significantly enriched metabolic pathways or signal transduction pathways associated with differentially expressed miRNA target genes. A representative number of samples (n = 15 patients from each experimental situation including control subjects) will be considered. To validate the data obtained, real-time polymerase chain reaction (RT-PCR) of the selected miRNAS, differentially expressed, will be performed. The results obtained will be statistically analyzed to find possible correlations and associations with clinical and analytical parameters.