Sandra Ribeiro

Iron-Hepcidin Dysmetabolism, Anemia and Renal Hypoxia, Inflammation and Fibrosis in the Remnant Kidney Rat Model

Anemia is a common complication of chronic kidney disease (CKD) that develops early and its severity increases as renal function declines. It is mainly due to a reduced production of erythropoietin (EPO) by the kidneys; however, there are evidences that iron metabolism disturbances increase as CKD progresses. Our aim was to study the mechanisms underlying the development of anemia of CKD, as well as renal damage, in the remnant kidney rat model of CKD induced by 5/6 nephrectomy. This model of CKD presented a sustained degree of renal dysfunction, with mild and advanced glomerular and tubulointerstitial lesions. Anemia developed 3 weeks after nephrectomy and persisted throughout the protocol. The remnant kidney was still able to produce EPO and the liver showed an increased EPO gene expression. In spite of the increased EPO blood levels, anemia persisted and was linked to low serum iron and transferrin levels, while serum interleukin (IL)-6 and high sensitivity C-reactive protein (hs-CRP) levels showed the absence of systemic inflammation. The increased expression of duodenal ferroportin favours iron absorption; however, serum iron is reduced which might be due to iron leakage through advanced kidney lesions, as showed by tubular iron accumulation. Our data suggest that the persistence of anemia may result from disturbances in iron metabolism and by an altered activity/function of EPO as a result of kidney cell damage and a local inflammatory milieu, as showed by the increased gene expression of different inflammatory proteins in the remnant kidney. In addition, this anemia and the associated kidney hypoxia favour the development of fibrosis, angiogenesis and inflammation that may underlie a resistance to EPO stimuli and reduced iron availability. These findings might contribute to open new windows to identify putative therapeutic targets for this condition, as well as for recombinant human EPO (rHuEPO) resistance, which occurs in a considerable percentage of CKD patients.

Markers of Increased Cardiovascular Risk in Postmenopausal Women: Focus on Oxidized-LDL and HDL Subpopulations

Objective. To evaluate the effect of gender and menopause in cardiovascular risk (CVR) in a healthy population based on both classical and nontraditional markers. Methods. 56 men and 68 women (48 pre- and 20 postmenopause) were enrolled in the study. The following markers were analyzed: blood pressure (BP), body mass index (BMI), waist circumference (WC), glucose, total cholesterol (total-c), triglycerides (TGs), low-density lipoprotein cholesterol (LDL-c), oxidized-LDL (Ox-LDL), HDL-c and subpopulations, paraoxonase-1 activity, hsCRP, uric acid, tumor necrosis factor alpha (TNF-α), adiponectin, vascular endothelial growth factor (VEGF), and intercellular adhesion molecular 1 (ICAM1). Results. Relative to the women, men present significantly increased BMI, WC, BP, glucose, total-c, TGs, LDL-c, Ox-LDL, uric acid, and TNF-α and reduced adiponectin and total and large HDL-c. The protective profile of women is lost after menopause with a significantly increased BMI, WC, BP, glucose, LDL-c, Ox-LDL, hsCRP, and VEGF and decreased total and large HDL-c. Significant correlations were found in women population and in postmenopausal women between Ox-LDL and total, large, and small HDL-c and between TNF-α and total, large, and small HDL-c, LDL-c, and Ox-LDL. Conclusions. Men present higher CVR than women who lost protection after menopause, evidenced by nontraditional markers, including Ox-LDL and HDL subpopulations.

Risk Factors for Mortality in Hemodialysis Patients: Two-Year Follow-Up Study

Background. End-stage renal disease (ESRD) patients under hemodialysis (HD) have high mortality rate. Inflammation, dyslipidemia, disturbances in erythropoiesis, iron metabolism, endothelial function, and nutritional status have been reported in these patients. Our aim was to identify any significant association of death with these disturbances, by performing a two-year follow-up study. Methods and Results. A large set of data was obtained from 189 HD patients (55.0% male; 66.4 ± 13.9 years old), including hematological data, lipid profile, iron metabolism, nutritional, inflammatory, and endothelial (dys)function markers, and dialysis adequacy. Results. 35 patients (18.5%) died along the follow-up period. Our data showed that the type of vascular access, C-reactive protein (CRP), and triglycerides (TG) are significant predictors of death. The risk of death was higher in patients using central venous catheter (CVC) (Hazard ratio [HR] =3.03, 95% CI = 1.49–6.13), with higher CRP levels (fourth quartile), compared with those with lower levels (first quartile) (HR = 17.3, 95% CI = 2.40–124.9). Patients with higher TG levels (fourth quartile) presented a lower risk of death, compared with those with the lower TG levels (first quartile) (HR = 0.18, 95% CI = 0.05–0.58). Conclusions. The use of CVC, high CRP, and low TG values seem to be independent risk factors for mortality in HD patients.

Iron therapy in chronic kidney disease: Recent changes, benefits and risks

Anemia is a common complication in patients with chronic kidney disease (CKD), mainly due to inadequate renal production of erythropoietin. In hemodialysis (HD) patients this condition may be aggravated by iron deficiency (absolute or functional). The correction of this anemia is usually achieved by treatment with erythropoiesis stimulating agents (ESAs) and iron (oral or intravenous). Studies questioning the safety of ESAs (especially at higher doses) changed the pattern of anemia treatment in CKD patients. According to the new guidelines, when transferrin saturation is lower than 30% and ferritin lower than 500 ng/mL, a trial with iron should be started, to avoid therapy with ESAs or at least to reduce the doses needed to treat the anemia. Recent reports showed increasing ferritin levels, towards values above 800 ng/mL, in CKD patients treated according to the guidelines. In this review we focus on the risks of the increased iron use to treat CKD anemia, namely, iron overload and toxicity, increased risk of infections, as well as mortality.

Oxidized low-density lipoprotein and lipoprotein(a) levels in chronic kidney disease patients under hemodialysis: Influence of adiponectin and of a polymorphism in the apolipoprotein(a) gene

Chronic kidney disease (CKD) has been associated with an abnormal lipid profile. Our aim was to study the interplay between oxidized low‐density lipoprotein (ox‐LDL), adiponectin, and blood lipids and lipoproteins in Portuguese patients with CKD under hemodialysis (HD); the influence of the pentanucleotide repeat polymorphism in the apolipoprotein(a) (apo [a]) gene upon lipoprotein(a) (Lp[a]) levels in these patients. We studied 187 HD patients and 25 healthy individuals. ox‐LDL and adiponectin were measured using enzyme‐linked immunoassays. Apo(a) genotyping was performed by polymerase chain reaction, followed by electrophoresis in polyacrylamide gel. Compared with controls, patients presented with significantly higher levels of adiponectin, Lp(a), and ox‐LDL/low‐density lipoprotein cholesterol (LDLc) ratio; significantly lower levels of total cholesterol (TC), LDLc, apo A‐I, apo B, ox‐LDL, and TC/high‐density lipoprotein cholesterol (HDLc) ratio were also observed. Similar changes were observed for patients with or without statin therapy, as compared with controls, except for Lp(a). Multiple linear regression analysis showed that body mass index, HDLc, time on HD, and triglycerides (TG) were independent determinants of adiponectin levels, and that apo B, TG and LDLc were independent determinants of ox‐LDL concentration. Concerning the apo(a) genotype, the homozygous (TTTTA)8/8 repeats was the most prevalent (50.8%). A raised proportion of LDL particles that are oxidized was observed. Adiponectin almost doubled its values in patients and seems to be an important determinant in HDLc and TG levels, improving the lipid profile in these patients. Apo(a) alleles with a lower number of repetitions are more frequent in patients with higher Lp(a).

Resistance to Recombinant Human Erythropoietin Therapy in a Rat Model of Chronic Kidney Disease Associated Anemia

This study aimed to elucidate the mechanisms explaining the persistence of anemia and resistance to recombinant human erythropoietin (rHuEPO) therapy in a rat model of chronic kidney disease (CKD)-associated anemia with formation of anti-rHuEPO antibodies. The remnant kidney rat model of CKD induced by 5/6 nephrectomy was used to test a long-term (nine weeks) high dose of rHuEPO (200 UI/kg bw/week) treatment. Hematological and biochemical parameters were evaluated as well as serum and tissue (kidney, liver and/or duodenum) protein and/or gene expression of mediators of erythropoiesis, iron metabolism and tissue hypoxia, inflammation, and fibrosis. Long-term treatment with a high rHuEPO dose is associated with development of resistance to therapy as a result of antibodies formation. In this condition, serum EPO levels are not deficient and iron availability is recovered by increased duodenal absorption. However, erythropoiesis is not stimulated, and the resistance to endogenous EPO effect and to rHuEPO therapy results from the development of a hypoxic, inflammatory and fibrotic milieu in the kidney tissue. This study provides new insights that could be important to ameliorate the current therapeutic strategies used to treat patients with CKD-associated anemia, in particular those that become resistant to rHuEPO therapy.

Potential Cardiovascular Risk Protection of Bilirubin in End-Stage Renal Disease Patients under Hemodialysis

We evaluated the potential cardiovascular risk protection of bilirubin in hemodialysis (HD) patients. An enlarged set of studies were evaluated in 191 HD patients, including hematological study, lipid profile, iron metabolism, nutritional, inflammatory markers, and dialysis adequacy. The TA duplication screening in the UDP-glucuronosyltransferase 1 A1 (UGT1A1) promoter region was also performed. The UGT1A1 genotype frequencies in HD patients were 49.2%, 42.4%, and 8.4% for 6/6, 6/7, and 7/7 genotypes, respectively. Although no difference was found in UGT1A1 genotype distribution between the three tertiles of bilirubin, significant differences were found with increasing bilirubin levels, namely, a decrease in platelet, leukocyte, and lymphocyte counts, transferrin, oxidized low-density lipoprotein (ox-LDL), ox-LDL/low-density lipoprotein cholesterol ratio, apolipoprotein (Apo) A, Apo B, and interleukin-6 serum levels and a significant increased concentration of hemoglobin, hematocrit, erythrocyte count, iron, transferrin saturation, Apo A/Apo B ratio, adiponectin, and paraoxonase 1 serum levels. After adjustment for age these results remained significant. Our data suggest that higher bilirubin levels are associated with beneficial effects in HD patients, by improving lipid profile and reducing the inflammatory grade, which might contribute to increase in iron availability. These results suggest a potential cardiovascular risk protection of bilirubin in HD patients.

Aging is Associated with Impaired Renal Function, INF-gamma Induced Inflammation and with Alterations in Iron Regulatory Proteins Gene Expression

Our aim was to contribute to a better understanding of the pathophysiology of anemia in elderly, by studying how aging affects renal function, iron metabolism, erythropoiesis and the inflammatory response, using an experimental animal model. The study was performed in male Wistar, a group of young rats with 2 months age and an old one with 18 months age. Old rats presented a significant higher urea, creatinine, interferon (INF)-gamma, ferritin and soluble transferrin receptor serum levels, as well as increased counts of reticulocytes and RDW. In addition, these rats showed significant lower erythropoietin (EPO) and iron serum levels. Concerning gene expression of iron regulatory proteins, old rats presented significantly higher mRNA levels of hepcidin (Hamp), transferrin (TF), transferrin receptor 2 (TfR2) and hemojuvelin (HJV); divalent metal transporter 1 (DMT1) mRNA levels were significantly higher in duodenal tissue; EPO gene expression was significantly higher in liver and lower in kidney, and the expression of the EPOR was significantly higher in both liver and kidney. Our results showed that aging is associated with impaired renal function, which could be in turn related with the inflammatory process and with a decline in EPO renal production. Moreover, we also propose that aging may be associated with INF-gamma-induced inflammation and with alterations upon iron regulatory proteins gene expression.

Circulating cell-free DNA levels in hemodialysis patients and its association with inflammation, iron metabolism, and rhEPO doses.

To the Editor, Inflammation is a common feature in end-stage renal disease (ESRD) patients under hemodialysis (HD), which is enhanced in patients who develop resistance to recombinant human erythropoietin (rhEPO) therapy. The mechanisms/factors triggering the inflammatory process are still poorly clarified. It has been demonstrated, recently, a release of cell-free DNA (cfDNA) during dialysis procedure, which has been related to cellular necrosis and apoptosis, similar to that found in other clinical situations. The aims of this work were to evaluate the circulating cfDNA levels in ESRD patients and to search for relationships among hematological and inflammatory disturbances, as well as with iron metabolism, nutritional status, and dialysis adequacy. This transversal study included 153 ESRD patients under HD (89 males and 64 females, mean [±SD] age: 65.7 [14.4] years). Patients were under therapeutic HD three times per week for the duration of 3–5 hours, for a median time of 2.13 (0.82–5.24) years. For the HD procedure, high-flux polysulfone FX-class dialyzer of Fresenius (Bad Hamburg, Germany) was used. The main causes of renal failure in these patients were as follows: diabetic nephropathy (n = 55), hypertensive nephrosclerosis (n = 20), nephritic syndrome (n = 13), other diseases (n = 19), and of uncertain etiology (n = 46). Patients with autoimmune diseases, malignancy, and acute or chronic infection were excluded. All participants gave their written informed consent to participate in this study, which was previously approved by the Ethics Committee from the clinic of dialysis. A group of healthy volunteers (n = 20) with normal hematological and biochemical values, with no history of renal or inflammatory disease, and, as far as possible, ageand gender-matched with our patients, was also included. Blood was collected immediately before the second dialysis session of the week. Hematological data were accessed using an automatic blood cell counter (Sysmex K1000, Sysmex, Hamburg, Germany). Differential leukocyte and reticulocyte counts were performed by microscopy. Serum iron concentration was determined using a colorimetric method (Iron, Randox Laboratories Ltd, North Ireland, UK), whereas serum ferritin and transferrin were measured by immunoturbidimetry (Ferritin, Laboratories Ltd, North Ireland, UK; Transferrin, Laboratories Ltd). Enzyme-linked immunosorbent assays were used to measure soluble transferrin receptor (sTfR; human sTfR immunoassay, R&D Systems, Minneapolis, MN, USA). Plasma levels of hepcidin-25 were quantified using a peptide enzyme immunoassay (Bachem Group, Peninsula Laboratories LLC, San Carlos, CA, USA). Serum C-reactive protein (CRP) was determined by nephelometry (CRP [latex] High-Sensitivity, Roche Diagnostics, Basel, Switzerland); serum interleukin (IL)-6 was evaluated by enzyme immunoassays (Human IL-6 High Sensitivity ELISA, eBioscience, Vienna, Austria). The circulating cfDNA levels were assessed directly in serum samples, as recently described. Briefly, SYBR Gold Nucleic Acid Gel Stain (Invitrogen, Paisley, UK) was diluted in dimethyl sulfoxide and phosphate buffer; 10 μL of the serum sample was mixed 40 μL of SYBR Gold solution. Fluorescence was measured with a 96-well Correspondence to: E. Costa, PhD, Faculty of Pharmacy, University of Porto, Rua de Jorge Viterbo Ferreira no. 228, 4050-313 Porto, Portugal. E-mail: emcosta@ff.up.pt; elisio_costa@hotmail.com There is no conflict of interests. Hemodialysis International 2013; 17:664–670

Main Determinants of PON1 Activity in Hemodialysis Patients

Background/Aims: Cardiovascular diseases are the major cause of morbidity and mortality in hemodialysis (HD) patients. These patients present reduced paraoxonase 1 (PON1) activity that depends on genetic and non-genetic factors; however, how these factors influence PON1 activity in HD patients is poorly clarified. Our aim was to evaluate the influence of two polymorphisms and non-genetic factors on PON1 activity in HD patients. Methods: We evaluated 183 HD patients under recombinant human erythropoietin (rhEPO) treatment and 22 healthy individuals. The lipid profile [total cholesterol, triglycerides, HDL-c, LDL-c, apolipoprotein (Apo) A-I, Apo B, lipoprotein(a) and oxidized low-density lipoprotein (Ox-LDL)], inflammatory markers [adiponectin, interleukin-6 (IL-6) and C-reactive protein (CRP)], PON1 activity and PON1 gene polymorphisms (L55M and Q192R) were evaluated. Results: HD patients presented higher levels of IL-6, CRP and Ox-LDL/LDL-c, and lower PON1 activity, total cholesterol, HDL-c, LDL-c, Apo A and Apo B; the most frequent genotype was heterozygosity for L55M polymorphism and homozygosity for the Q allele, the more frequent genotype of Q192R polymorphism. Multiple regression analysis identified heterozygosity and homozygosity for L55M and Q192R polymorphisms, very low-density lipoproteins, LDL-c, Apo A and CRP levels, time on dialysis and rhEPO dose, as the independent variables significantly associated with PON1 activity. The associations with CRP, rhEPO and time on dialysis were negative. Conclusion: Our results show that the reduced PON1 activity in HD patients who are not under statin therapy is strongly associated with inflammation, longer time on dialysis and high rhEPO doses, suggesting that the reduction in PON1 activity may worsen the prognosis of these patients.