Pure red cell aplasia and anti-erythropoietin antibodies in patients on hemodialysis: a report of two cases and a literature review / Aplasia pura de células vermelhas e anticorpo antieritropoietina em pacientes de hemodiálise: relato de dois casos e revisão da literatura

ABSTRACT Introduction: Anemia is a frequent multifactorial complication of CKD seen in patients on dialysis derived mainly from impaired erythropoietin (EPO) production. A less common cause of anemia in individuals with CKD is pure red cell aplasia (PRCA) secondary to the production of anti-EPO antibodies. Objective: This paper aimed two describe two cases of PRCA secondary to the production of anti-EPO antibodies including choice of treatment, patient progression, and a literature review. Materials: This study included the cases of two patients with CKD on hemodialysis with severe anemia in need of specific investigation and management. Results: Patient 1 with CKD secondary to hypertension treated with EPO for 7 months showed persistent decreases in hemoglobin (Hb) levels despite the subcutaneous administration of increasing doses of EPO; the patient required recurring blood transfusions. Workup and imaging tests were negative for the main causes of anemia in individuals with CKD on dialysis. Patient 2 with CKD secondary to adult polycystic kidney disease had been taking EPO for 2 years. The patient developed severe abrupt anemia the month he was started on HD, and required recurring transfusions to treat the symptoms of anemia. Workup and imaging findings were inconclusive. Specific laboratory tests confirmed the patients had anti-EPO antibodies. After six months of immunosuppressant therapy (corticosteroids + cyclosporine) the patients were stable with Hb > 9.0 g/dl. Conclusion: PRCA is a rare condition among patients on dialysis treated with rhEPO and should be considered as a possible cause of refractory anemia. Treating patients with PRCA may be challenging, since the specific management and diagnostic procedures needed in this condition are not always readily available.

RESUMO Introdução: Anemia é complicação frequente da Doença Renal Crônica (DRC) em pacientes dialíticos. Apresenta caráter multifatorial principalmente pela insuficiente produção de eritropoietina (EPO). Situação rara causadora de anemia na DRC é Aplasia Pura de Células Vermelhas (APCV), em decorrência da produção de anticorpos anti-EPO. Objetivo: Descrever 2 casos de APCV com formação de anticorpos anti-EPO, sua abordagem clínica, evolução e revisão de literatura. Métodos: Dois pacientes em hemodiálise que desenvolveram anemia grave, necessitando investigação e manejo específico. Resultados: Paciente nº 1: feminina, 75 anos, DRC secundária à hipertensão arterial. Após 7 meses com EPO desenvolveu queda persistente em valores de hemoglobina (Hb) mesmo com incremento em doses EPO SC, necessitando transfusões de sangue recorrentes. Extensa investigação laboratorial e de imagem resultou negativa para principais causas de anemia. Paciente nº 2: masculino, 66 anos, DRC secundária à DRPA, há 2 anos em uso de EPO. No mês de entrada em HD desenvolveu anemia severa, também exigindo transfusões recorrentes para tratamento da anemia sintomática. Extensa investigação laboratorial e por imagem, sem chegar a uma conclusão definitiva. Em ambos os casos a presença de anticorpos anti-EPO foi confirmada por exames laboratoriais específicos. Terapia imunossupressora resultou em estabilização do quadro e Hb > 9,0 g/dl em ambos os pacientes, 6 meses após início do tratamento. Conclusão: APCV é condição rara entre pacientes dialíticos que recebem EPOHuR e deve ser lembrada como causa de anemia refratária. Seu manejo específico e diagnóstico laboratorial nem sempre acessível, tornando desafiadora a condução dos casos para o nefrologista.

Long-term and stable correction of uremic anemia by intramuscular injection of plasmids containing hypoxia-regulated system of erythropoietin expression

Relative deficiency in production of glycoprotein hormone erythropoietin (Epo) is a major cause of renal anemia. This study planned to investigate whether the hypoxia-regulated system of Epo expression, constructed by fusing Epo gene to the chimeric phosphoglycerate kinase (PGK) hypoxia response elements (HRE) in combination with cytomegalovirus immediate-early (CMV IE) basal gene promoter and delivered by plasmid intramuscular injection, might provide a long-term physiologically regulated Epo secretion expression to correct the anemia in adenine-induced uremic rats. Plasmid vectors (pHRE-Epo) were synthesized by fusing human Epo cDNA to the HRE/CMV promoter. Hypoxia-inducible activity of this promoter was evaluated first in vitro and then in vivo in healthy and uremic rats (n = 30 per group). The vectors (pCMV-Epo) in which Epo expression was directed by a constitutive CMV gene promoter served as control. ANOVA and Student's t-test were used to analyze between-group differences. A high-level expression of Epo was induced by hypoxia in vitro and in vivo. Though both pHRE-Epo and pCMV-Epo corrected anemia, the hematocrit of the pCMV-Epo-treated rats exceeded the normal (P < 0.05), but that of the pHRE-Epo-treated rats didn't. Hypoxia-regulated system of Epo gene expression constructed by fusing Epo to the HRE/CMV promoter and delivered by plasmid intramuscular injection may provide a long-term and stable Epo expression and secretion in vivo to correct the anemia in adenine-induced uremic rats.

Expression of erythropoietin and its receptor increases in colonic neoplastic progression: The role of hypoxia in tumorigenesis.

Background: Tissue hypoxia is a characteristic patho-physiologic property of colorectal cancer. This process may also add to a therapeutic problem of solid tumor resistance to chemo- and radiation therapy. Erythropoietin (Epo) expression is induced by tissue hypoxia. Acting via its receptor (EpoR), Epo inhibits apoptosis of erythroid cells and has been shown to rescue neurons from hypoxic damage. Increased Epo and EpoR expression has been recently described in human breast, renal and cervical carcinoma. Given the characteristic tumor diathesis present in majority of colorectal cancers, we examined whether Epo signaling may play a role in colonic neoplastic progression. Materials and Methods: Expression of Epo and EpoR was examined using immunohistochemistry in 24 cases of primary colorectal and metastatic adenocarcinomas versus adenomas and normal colonic mucosa. Immunohistochemical stains were evaluated semiquantitatively based on a four-tiered scale. Based on the combination of extent and intensity of immunoreactivity, an immunostaining score (0-300) was determined for each sample. Expression of Epo and EpoR protein and mRNA was examined using Western blot and reverse transcriptase-polymerase chain reaction (RT-PCR), respectively, in both normal colonic tissue and carcinoma specimens in five cases. Results: Epo expression was sequentially increased in normal colonic mucosa (8.3 ± 5.6, mean ± SEM), adenoma (26.4 ± 9.1), primary carcinoma (96.1 ± 12.8) and metastatic carcinoma (122 ± 51.3). EpoR expression was also sequentially increased in normal colonic mucosa (22.3 ± 11.8), adenoma (108.7 ± 24.2), primary carcinoma (178.7 ± 16.6) and metastatic carcinoma (220 ± 58.3) (P< 0.05 for all results). Epo and EpoR showed enhanced expression in the areas adjacent to ischemia/necrosis. Western blot and RT-PCR analysis revealed increased EpoR protein and mRNA levels in carcinoma compared to normal mucosal colon specimens. Focal stromal Epo and EpoR immunoreactivity was present in 10 and 12 cases, respectively. Conclusions: The uniform increase in the expression of Epo and EpoR along the colonic neoplastic sequence and further increase in ischemic/necrotic areas indicates that the Epo signaling pathway is an important component in colon carcinogenesis including possible epithelial-stromal interactions.

Defective erythropoietin production and reticulocyte response in acute Plasmodium falciparum malaria-associated anemia.

To elucidate the relationship between falciparum malaria-associated anemia and serum erythropoietin (Epo) levels and reticulocyte response during acute malaria infection, 87 adults aged 18-65 years presenting with acute, uncomplicated malaria were examined on enrollment and for 28 days of follow-up. The 87 patients were divided into 2 groups: those with anemia (n = 45) and those without (n = 42). Serum samples were taken on admission (Day 0), then on Days 7, 21, and 28, to measure the reticulocyte count, absolute reticulocyte count, reticulocyte hemoglobin content, and erythropoietin level (Epo). The absolute reticulocyte counts for the anemic patients were significantly higher than for those without anemia on Days 0, 7, 21, and 28. The serum Epo levels for the anemic patients were significantly higher than the non-anemic group only on Day 0 (44.39 +/- 4.06 vs 25.91 +/- 4.86 mlU/ml, p < 0.001). Inadequate Epo production was found in 31.03% (27/87) of patients on Day 0, 37.93% (33/87) on Day 7, 43.67% (38/87) on Day 21, and 39.08% (34/87) on Day 28. These results indicate defective Epo production and reticulocyte response in adult patients suffering from acute P. falciparum malaria, which differs from pediatric patients. Our findings may provide the basis for further study into the choice of therapeutic strategies to treat acute P. falciparum malaria-associated anemia with recombinant human Epo to correct refractory anemia due to malaria.

Erythropoietin & erythroid progenitors in rats exposed to chronic hypoxia.

In order to better understand the mechanisms affecting erythropoietin (Epo) synthesis and red cell mass increase under chronic hypoxia, we examined Epo production and erythroid progenitors (CFU-E) in rats exposed to normobaric hypoxia for four weeks. Hypoxia induced the rise of hematocrit (Htc), hemoglobin (Hb) concentration and the red blood cell (RBC) number with a plateau in hematocrit values after two weeks. After 24 h of hypoxia, Epo levels were increased 20 fold, followed by a significant decrease. After the first week of hypoxia, the values were still higher than in the controls, but after two weeks Epo levels did not differ significantly from the normal values. The fall of Epo levels coincided with the plateau values of hematocrit. The changes in the CFU-E number followed the changes in Epo concentration: a two fold increase after 24 h of hypoxia; a further increase during the next two weeks reaching a peak on day 14, and then a progressive decrease at the time when Epo concentration was at a normal level. Although decreased, but still higher than normal, the CFU-E number during the last two weeks of hypoxia could be necessary for the maintenance of an achieved steady state under persistent hypoxic conditions with normal Epo concentration sufficient to maintain the existing rate of erythropoiesis.

Expression of active human erythropoietin in the mammary gland of lactating transgenic mice and rabbits

Transgenic mice and rabbits were generated using a chimeric gene comprising the human erythropoietin (hEPO) cDNA under the 5' and 3' regulatory sequences of the rabbit whey acidic protein gene. Transgenic mice expressed hEPO at levels of 0.01 mg/l in the milk of lactating females showing that the genetic construct was functional. Reverse transcriptase polymerase chain reaction with RNA from various tissues showed that this transgene was expressed mainly in the ovary and mammary gland. In rabbits, we demonstrated the germ line transmission of the transgene. The hEPO was obtained in the milk of lactating females at levels of up to 0.0003 mg/l. Although the expression levels were low, biologically active hEPO was obtained in the milk of transgenic rabbits without any apparent detrimental effect for the animals. In vitro, the specific activity of the rabbit-derived hEPO was higher than that reported for the natural hEPO, thus suggesting differences in the glycosylation pattern in at least part of the molecules secreted by the mammary gland of transgenic rabbits

Eritropoyetina recombinante humana (r-HuEPO) en pacientes con insuficiencia renal crónica

Se presentan 43 pacientes con insufiencia renal crónica (IRC), en hemodíalisis, con hemoglibina (HB) menor de 8 gr/dL. Se excluyeron quienes presentaron anemias por deficiencia de ácido fólico, B12, HTA de difícil control, drogadicción, hiperparatiroidismo e intoxicación por aluminio. Se administraron por vía venenosa 50 U/kg tres veces por semana de eritropoyetina recombinante humana (r-HuEPO). La edad promedio fue 40+- 17 años, con predominio del sexo masculino (57 por ciento). Después de 12 semanas de tratamiento, la Hb aumentó de 7.1+-1 a 10.5+- 1.6 gr/dL (p<0.001). Se observó una discreta leucocitosis, los niveles séricos de Na, K, Ca, Po4, Cr, aminotransferars y bilirrubinas no presentaron cambios significativos. El hierro y la ferritna sérica disminuyeron en repuesta al incremento del número de globulos rojos. Hubo aumento de la presión arterial en 17 por ciento y un paciente presentó trombosis de la fistula A-V. Conclusión: la r-HuEPO es segura y efectiva en el tratamiento de la anemia secundaria a IRC.

Depressed plasma erythropoietin levels in rats with hemodynamically-mediated acute renal failure

Ratas adultas hembra de la cepa Wistar fueron inyectadas con metahemoglobina (M-Hb), (125 mg/Kg de peso corporal) con el propósito de inducir un primer episodio de insuficiencia renal aguda mediada hemodinámicamente (HMARF). Luego de once días de este tratamiento, un grupo de ratas fue inyectado nevamente con M-Hb con el fin de provocar un segundo episodio de HMARF. La evolución de la función exocrina renal, los estudios plasmáticos y renalaes de inmunoeritropoyetina (iEpo), en condiciones de normoxia e hipoxia, fueron determinados en los días 1, 2, 3, 5 y 10 post-M-Hb. La inyección de M-Hb provocó: a) aumentos transitorios en la concentración plasmática de urea, excreción fracción de sodio y volumen urinario, y b) una significativa disminución de las osmolalidad urinaria. En relación con las determinaciones citadas, los mayores efectos ocurrieron luego de la primera inyección de M-Hb y en el día 5§ post pigmento. El examen histológico mostró infiltración celular intersticial, descamación del epitelio tubular proximal y colapso o dilatación de la luz tubular. El tratamiento con M-Hb disminuyó significativamente los niveles plasmáticos y renales de iEpo en condiciones de normoxia e hipoxia. Las disminuciones observadas fueron más pronunciadas luego de la primera inyección de M-Hb en relación con los valores obtenidos luego del segundo intento de inducir HMARF. Los resultados obtenidos indican la existencia de correlación entre el daño tubular y la producción de Epo en condiciones de normoxia e hipoxia. Estos hallazgos apoyan el concepto de que la producción de Epo está relacionada con la función del tubo proximal del nefron