Volume 5, Number 6; 356-365, December 2014 http://dx.doi.org/10.14366/AD.2014.0500356
Original Article
Aging is Associated with Impaired Renal Function, INFgamma Induced Inflammation and with Alterations in Iron Regulatory Proteins Gene Expression Elísio Costa1,2*, João Fernandes2,3, Sandra Ribeiro1,2, JoséSereno3, Patrícia Garrido3, Petronila Rocha-Pereira2,4, Susana Coimbra2,5, Cristina Catarino1,2, Luís Belo1,2, Elsa Bronze-da-Rocha1,2, Helena Vala6,7, Rui Alves8,9, Flávio Reis3, Alice Santos-Silva1,2 1
Laboratório de Bioquímica, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, Porto, Portugal 2 Instituto de Biologia Molecular e Celular, Universidade do Porto, Porto, Portugal 3 Laboratório de Farmacologia e Terapêutica Experimental, Instituto de Imagem Biomédica e Ciências da Vida, Faculdade de Medicina, Universidade de Coimbra, Coimbra, Portugal 4 Centro Investigação Ciências da Saúde, Universidade Beira Interior, Covilhã, Portugal 5 CESPU, Institute for Research and Advanced Training in Health Sciences and Technologies, Gandhinagar-PRD, Portugal 6 Escola Superior Agrária de Viseu, Instituto Politécnico de Viseu, Viseu, Portugal 7 Centro de Estudos em Educação, Tecnologias e Saúde, Instituto Politécnico de Viseu, Viseu, Portugal 8 Departamento de Nefrologia, CHUC, Coimbra, Portugal 9 Faculdade de Medicina, Universidade de Coimbra, Coimbra, Portugal [Received October 18, 2013; Revised December 4, 2013; Accepted December 7, 2013]
ABSTRACT: 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.
Key words: Anemia, older population, elderly, renal failure, inflammation, erythropoietic disturbances
Epidemiological studies show an increased occurrence of anemia with aging, with a prevalence of about 5% at 65 years, to more than 20% at the age of 85 years [1,2].
Previous studies showed that anemia, per se, is associated with higher morbidity, risk of hospitalization, and mortality rates [2,3]. When the identifiable causes of
*Correspondence should be addressed to: Elísio Costa, Laboratório de Bioquímica, Departamento de Ciências Biológicas, Faculdade de Farmácia, Universidade do Porto, 4050-313 Porto, Portugal. Email:
[email protected] ISSN: 2152-5250
356
E. Costa et al
anemia are excluded, such as erythropoietic nutrient deficiencies, inflammatory conditions and chronic kidney disease (CKD), the prevalence of anemia is still high in the elderly population. In fact, several studies showed that as much as 1/3 of the anemia in older population is of unexplained cause [4-6]. Some possible explanations for the increased prevalence of anemia of unknown etiology in advancing age have been suggested [7-10]. Indeed, aging is characterized by a progressive mild proinflammatory status, as shown by the rising levels of proinflammatory markers [11]. This phenomenon can be partially explained by the increasing prevalence of chronic diseases; however, even when healthy older individuals were studied, they still had higher levels of pro-inflammatory cytokines and acute phase reactive proteins [11-14]. Additionally, a positive association between inflammation and erythropoietin (EPO) serum levels has been reported in old individuals without anemia [13]. These findings suggest that inflammation might hamper the response to EPO. Furthermore, it is known that increased levels of hepcidin (Hamp) are involved in the pathogenesis of the anemia of inflammation [10,11]. Nevertheless, it is still unclear whether the mild-proinflammatory state associated with aging, causes Hamp up-regulation and contributes to the development of anemia. More recently, it was reported that the anemia of chronic diseases is dependent on interferon-γ and that this cytokine reduces both the life span and the production of red blood cells [14]. This effect upon red blood cells has been attributed to interferon-γ capacity of inducing the expression of transcription factors of interferon regulatory factor-1 and PU.1 in human erythroid precursors, leading to inhibition of erythropoiesis. Aging is also associated with physiological, functional and morphological changes in kidneys, which are similar to those found in CKD, though to a lesser extent [15]. Inflammation is also associated to agingrelated diseases, and to progression of CKD [16]. The kidney is the major local of EPO synthesis, and one of the mechanisms proposed for the pathogenesis of the unexplained anemia in older people is an inadequate EPO production. In fact, it has been suggested that the ability of kidneys to secrete EPO in response to tissue hypoxia declines with aging. However, EPO levels in non-anemic older persons are conflicting, as some studies showed higher EPO levels, and other studies do not confirm that finding [17]. The aim of this work was therefore to contribute to a better understanding of the pathophysiology of anemia in elderly, by studying how aging affects renal function, iron metabolism and the inflammatory response, using for that purpose an experimental animal model; old male Wistar rats, aged 18 moths, were compared to young male Wistar rats, aged 2 months. The determinations included
Aging and erythropoietic disturbances
analytical blood studies (basic hematological study, reticulocyte count, glucose, urea and creatinine levels, Creactive protein, interleukin-6, interferon-γ, iron, ferritin, transferrin, soluble transferrin receptor and erythropoietin concentrations) kidney histopathological studies and gene expression analysis of transferrin receptor 2 (TfR2), hepcidin (Hamp), ferroportin (SLC40A1), hemojuvelin (HJV), transferrin (TF), hemochromatosis (Hfe) in liver tissue; divalent metal transporter 1 (DMT1) gene expression in duodenal tissue, as well EPO and EPO receptor (EPOR) gene expression in both liver and kidney. MATERIAL AND METHODS Animals and groups Two groups of male Wistar rats (Charles River Lab. Inc., Barcelona, Spain) were evaluated in this study: one of young rats, with only 2 months of age (n=8) and the other of natural aging rats, with 18 months of age (n=7). The rats were maintained in an air-conditioned room, subjected to 12 h dark/light cycles and given standard laboratory rat diet (IPM-R20, Letica, Barcelona, Spain) and free access to tap water. Animal experiments were conducted according to the European Communities Council Directives on Animal Care and to the National Authorities, and the study received approval from the Institutional Ethics Committee of the Faculty of Medicine from the University of Coimbra. Approval ID: FMUC/10/11. During the entire experimental protocol, the body weight and the amount of beverage consumption were monitored. Blood and tissue collection At the end of the experimental protocol the rats were submitted to intraperitoneal anesthesia with 2.0 mg/kg of a 2:1 (v:v) 50.0 mg/mL ketamine (Ketalar®, Parke-Davis, Pfizer Laboratories Lda, Seixal, Portugal) solution in 2.5% chlorpromazine (Largatil®, Rhône-Poulenc Rorer, Vitória Laboratories, Amadora, Portugal). Blood samples were immediately collected by venepuncture from the jugular vein into vacutainer tubes with anticoagulant (EDTA) and without anticoagulant, in order to obtain whole blood, plasma and serum. Under anesthesia the rats were sacrificed by cervical dislocation, and the liver, heart, kidneys and duodenum were immediately removed, weighed and placed in ice-cold Krebs buffer; the organs were carefully cleaned from adherent fat and connective tissue. Two small portions of the liver, kidney and duodenum from each rat were prepared for histopathological analysis (further placed in formaldehyde) and for the genetic studies; to isolate total
Aging and Disease • Volume 5, Number 6, December 2014
357
E. Costa et al
RNA, 0.2 g samples of liver, duodenum and kidney, from each rat, were immersed in RNA laterTM (Ambion, Austin, USA) and stored at 4 °C for 24h; afterwards, samples were frozen at -80oC. The body weight (BW) and the weight of kidneys (KW), heart weight (HW) and left ventricle
Aging and erythropoietic disturbances
weight (LVW) were used to calculate the tropism indexes (KW/BW, HW/BW, LVW/HW and LVW/BW).
Table 1. Body and tissue weight, trophism indexes, hematological, biochemical and inflammatory data, iron metabolism and EPO levels in young and old rats
*p
