Maria do Sameiro Faria

Inflammation, T-Cell Phenotype, and Inflammatory Cytokines in Chronic Kidney Disease Patients Under Hemodialysis and its Relationship to Resistance to Recombinant Human Erythropoietin Therapy

BackgroundResistance to recombinant human erythropoietin (rhEPO) occurs in some chronic kidney disease (CKD) patients, which may be due to enhanced systemic inflammatory response and to the erythropoiesis-suppressing effect of pro-inflammatory cytokines, some of which are produced by T cells.Aim of studyThe aim of this study was to investigate the relationship between resistance to rhEPO therapy in hemodialysis CKD patients and inflammatory markers [C-reactive protein (CRP), soluble interleukin (IL)-2 receptor (sIL2R), and serum albumin levels], blood cell counts, T-cell phenotype, cytokine production by T cells, and serum cytokine levels.Materials and MethodsWe studied 50 hemodialysis CKD patients, 25 responders and 25 nonresponders to rhEPO, and compared them to each other and with 25 healthy controls. When compared to controls, CKD patients showed increased serum levels of CRP, IL-6, and sIL2R and a T-cell lymphopenia, due to decreased numbers of both CD4+ and CD8+ T cells. T cells from CKD patients had an immunophenotype compatible with chronic T-cell stimulation as shown by the increased percentage of CD28−, CD57+, HLA-DR+, CD28−HLA-DR+, and CD57+ HLA-DR+ T cells and produce higher levels of IL-2, INF-γ, and TNF-α after short-term in vitro stimulation, although Th1 cytokines were not detectable in serum. Statistically significant differences were found between responders and nonresponders to rhEPO therapy for total lymphocyte and CD4+ T-lymphocyte counts, albumin (lower in nonresponders) and CRP (higher in nonresponders) levels.ConclusionCKD patients under hemodialysis present with raised inflammatory markers and decrease of total lymphocyte and CD4+ T-lymphocyte counts when compared with controls. Some of those markers are even further enhanced in nonresponders to rhEPO therapy patients, but resistance to this therapy cannot be justified by a Th1 polarized T-cell response.

Role of prohepcidin, inflammatory markers and iron status in resistance to rhEPO therapy in hemodialysis patients.

The aim of our study was to assess possible relations between prohepcidin, iron status and inflammatory markers in hemodialysis (HD) patients, as well as its association with resistance to recombinant human erythropoietin (rhEPO) therapy. Fifty HD patients and 25 healthy controls were enrolled in the study. Among HD patients, 25 were non-responders and 25 were responders to rhEPO therapy. Complete blood cell count, reticulocyte count, and circulating levels of ferritin, iron, transferrin saturation, C-reactive protein (CRP), soluble interleukin (IL)-2 receptor (s-IL2R), soluble transferrin receptor (s-TfR), IL-6 and prohepcidin were measured in all patients and controls. HD patients showed higher circulating levels of ferritin, s-TfR, CRP, IL-6, s-IL2R and prohepcidin, and lower levels of transferrin compared to healthy controls. Higher levels of s-TfR, CRP and lower levels prohepcidin were observed among non-responders compared to responders. Prohepcidin levels correlated negatively with s-TfR and reticulocyte count. The weekly rhEPO/kg dose was found to be positively correlated with CRP, hemoglobin and s-TfR. In conclusion, our data show that a close interaction exists between inflammation, iron status and prohepcidin serum levels that ultimately regulate intracellular iron availability. Prohepcidin and s-TfR, together with CRP, may prove to be good markers of resistance to rhEPO therapy in HD patients.

Hepcidin Serum Levels and Resistance to Recombinant Human Erythropoietin Therapy in Haemodialysis Patients

This title was also presented orally at Clinical Biochemistry workshop,Faro, 29 Janeiro, 2010

Neutrophil activation and resistance to recombinant human erythropoietin therapy in Hemodialysis Patients

AIM The aim of this work was to evaluate the neutrophil activation state in chronic kidney disease (CKD) patients under hemodialysis, and its linkage with resistance to recombinant human erythropoietin (rhEPO) therapy. METHODS We studied 63 CKD patients under hemodialysis and rhEPO treatment (32 responders and 31 non-responders to rhEPO therapy). In 20 of the CKD patients (10 responders and 10 non-responders to rhEPO therapy), blood samples were also collected immediately after dialysis. Twenty-six healthy volunteers were included in a control group. Hemoglobin levels, total and differential leukocyte counts, and circulating levels of C-reactive protein (CRP), elastase and lactoferrin were measured in all patients and controls. RESULTS Compared with controls, CKD patients presented with significantly higher CRP, neutrophil and elastase levels. When we compared the 2 groups of patients, we found that non-responders presented statistically significantly higher elastase plasma levels. A positive significant correlation was found between elastase levels and weekly rhEPO dose and CRP serum levels. After the hemodialysis procedure, a statistically significant rise in elastase, lactoferrin and, elastase/neutrophil and lactoferrin/neutrophil ratios were found. CONCLUSIONS Our data show that CKD patients under hemodialysis present higher elastase levels (particularly in non-responding patients), which could be related to the rise in neutrophils, and to be part of the enhanced inflammatory process found in these patients.

Altered Erythrocyte Membrane Protein Composition in Chronic Kidney Disease Stage 5 Patients under Haemodialysis and Recombinant Human Erythropoietin Therapy

Our aim was to evaluate red blood cell (RBC) membrane protein composition in chronic kidney disease (CKD) stage 5 patients under haemodialysis (HD) and recombinant human erythropoietin (rhEPO) therapy, and its linkage to rhEPO hyporesponsiveness. We evaluated in 63 CKD stage 5 patients (32 responders and 31 non-responders to rhEPO therapy) and in 26 healthy controls RBC count, haematocrit, haemoglobin concentration, haematimetric indices, reticulocyte count, reticulocyte production index, RBC osmotic fragility test and membrane protein analyses. CKD stage 5 patients presented significant changes in membrane protein composition, namely a reduction in spectrin, associated to altered protein 4.1/spectrin and spectrin/band 3 ratios. Non-responder CKD stage 5 patients were more anaemic, with more microcytic and anisocytic RBCs, than responders; significantly altered ankyrin/band 3 and spectrin/ankyrin ratios were also observed. CKD stage 5 patients under HD are associated with an altered protein membrane structure, which seems to the disease itself and/or to the interaction with HD membranes.

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).

Band 3 Profile as a Marker of Erythrocyte Changes in Chronic Kidney Disease Patients

Our aim was to study changes in red blood cell (RBC) membrane band 3 profile, as a cumulative marker of RBC changes, in chronic kidney disease (CKD) patients under haemodialysis and recombinant human erythropoietin (rhEPO) therapy and its linkage with resistance to this therapy. We studied 63 CKD patients, 32 responders and 31 non-responders to rhEPO therapy, and 26 healthy individuals. We evaluated the band 3 profile (% of band 3 monomer, high molecular weight aggregates (HMWAg), and proteolytic frag- ments (Pfrag)), membrane-bound haemoglobin (MBH), haematological data, total serum bilirubin, glutathione peroxidase (GPx) and superoxide dismutase activities, total antioxidant status (TAS) and plasma lipid peroxidation (TBA). Compared to controls, band 3 profile presented by CKD patients showed statistically significant lower HMWAg and Pfrag values and a significant higher value in band 3 monomer. GPx, TBA and TAS activities, and TBA/TAS ratio were also signifi- cantly higher in CKD patients. Comparing responders to non-responders CKD patients, significantly lower value in Pfrag and a trend for a higher value in MBH were found in non-responders. Our data suggest that CKD patients present younger RBC population, which could be related to the rhEPO therapy. The adverse plasma environment associated to CKD patients under hemodialysis imposes changes in band 3 profile, particu- larly in non-responders, suggesting that resistance to rhEPO therapy in CKD patients seems to be associated to an increase in RBC damage.

Changes in Red Blood Cells Membrane Protein Composition during Hemodialysis Procedure

Our aim was to evaluate the influence of the hemodialysis (HD) procedure in red blood cells (RBC) membrane protein composition. We evaluated hematological data (RBC count, hemoglobin concentration, and hematimetric indices) and RBC membrane protein composition (linear and exponential gradient polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate [SDS-PAGE] followed by densitometry analysis of RBC membrane proteins) before and immediately after the HD procedure in 20 patients (10 responders and 10 non-responders to recombinant human erythropoietin therapy [rhEPO]) and 26 healthy controls. Before HD, patients presented anaemia and significant changes in membrane protein composition, namely, a statistically significant reduction in spectrin associated with a significant increase in bands 6, as well as an altered membrane protein interaction (protein 4.1/spectrin, protein 4.1/band 3, protein 4.2/band 3 and spectrin/band 3). After HD, we found that patients showed a statistically significant increase in RBC count and hemoglobin, a further and statistically significant decrease in spectrin, an increase in band 3, and an altered spectrin/band 3 ratio. When comparing responders and non-responders patients after HD, we found that the non-responders presented a trend to a higher reduction in spectrin. Our data suggest that HD procedure seems to contribute to a reduction in spectrin, which is normally associated with a reduction in RBC deformability, being that reduction in spectrin is higher in non-responder patients.

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.

Partially Reversible Cardiomyopathy after Renal Transplant Associated with Anti-Troponin I Antibodies

Cardiomyopathy associated with dialysis has been shown to be reversible in some cases. A 58-year-old female patient with polycystic renal disease and end-stage renal disease had a renal transplant after 9 years on hemodialysis. Dilated cardiomyopathy with depressed left ventricular ejection fraction markedly improved after transplantation, with normalization of the ejection fraction. High titers for anti-troponin I antibodies were measured: IgG 1:640, IgM 1:80. In our case, the presence of anticardiac (anti-troponin I) antibodies may suggest that the syndrome of reversible cardiomyopathy seen after renal transplantation is associated with immunosuppression therapy acting on immunological mechanisms previously at play.