UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE ... · A minha orientadora, Prof a Dr a...
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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE CIÊNCIAS DA SAÚDE PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE EOSINÓFILOS E PROTEÍNA CATIÔNICA EOSINOFÍLICA NA URINA: UMA NOVA ABORDAGEM PARA O DIAGNÓSTICO DA INFLAMAÇÃO RENAL NO LÚPUS ERITEMATOSO SISTÊMICO TEREZA NEUMA DE SOUZA BRITO NATAL/RN 2011
Transcript of UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE CENTRO DE ... · A minha orientadora, Prof a Dr a...
UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
EOSINÓFILOS E PROTEÍNA CATIÔNICA EOSINOFÍLICA NA URINA: UMA NOVA
ABORDAGEM PARA O DIAGNÓSTICO DA INFLAMAÇÃO RENAL NO LÚPUS
ERITEMATOSO SISTÊMICO
TEREZA NEUMA DE SOUZA BRITO
NATAL/RN
2011
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TEREZA NEUMA DE SOUZA BRITO
EOSINÓFILOS E PROTEÍNA CATIÔNICA EOSINOFÍLICA NA URINA: UMA NOVA
ABORDAGEM PARA O DIAGNÓSTICO DA INFLAMAÇÃO RENAL NO LÚPUS
ERITEMATOSO SISTÊMICO.
ORIENTADORA: PROFA. DRA. VALÉRIA SORAYA DE FARIAS SALES
CO-ORIENTADORA: PROFA. DRA. MARIA JOSÉ PEREIRA VILAR
NATAL/RN
2011
Tese apresentada ao Programa de Pós-graduação em Ciências da Saúde, do Centro de Ciências da Saúde, da Universidade Federal do Rio Grande do Norte, como parte dos requisitos para obtenção do título de doutor em Ciências da Saúde
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Catalogação da publicação na fonte
B862e Brito, Tereza Neuma de Souza. Eosinófilos e proteína catiônica eosinofílica na urina: uma nova abordagem para o diagnóstico da inflamação renal no lúpus eritematoso sistêmico / Tereza Neuma de Souza Brito. – Natal/RN, 2011.
62f.: il. Orientadora: Profª Drª Valéria Soraya de Farias Sales. Co-orientadora: Profª Drª Maria José Pereira Vilar.
Tese (Doutorado em Ciências da Saúde) - Universidade Federal do Rio Grande do Norte. Centro de Ciências da Saúde.
1. Eosinófilos – Tese. 2. Proteína catiônica eosinofílica – Tese. 3. Lúpus eritematoso sistêmico – Tese. I. Sales, Valéria Soraya de Farias. II. Vilar, Maria José Pereira. III. Título.
UFRN/BSCCS CDU: 616.61-002
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UNIVERSIDADE FEDERAL DO RIO GRANDE DO NORTE
CENTRO DE CIÊNCIAS DA SAÚDE
PROGRAMA DE PÓS-GRADUAÇÃO EM CIÊNCIAS DA SAÚDE
COORDENADORA: PROFA. DRA. TÉCIA MARIA DE OLIVEIRA MARANHÃO
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TEREZA NEUMA DE SOUZA BRITO
EOSINÓFILOS E PROTEÍNA CATIÔNICA EOSINOFÍLICA NA URINA: UMA NOVA
ABORDAGEM PARA O DIAGNÓSTICO DA INFLAMAÇÃO RENAL NO LÚPUS
ERITEMATOSO SISTÊMICO
BANCA EXAMINADORA:
Presidente da Banca
Profa. Dra.Valéria Soraya de Farias Sales – UFRN
Membros da Banca
Profa. Dra. Valéria Soraya de Farias Sales - UFRN
Prof. Dr. Max Victor Carioca Freitas - Externo
Profa. Dra. Profa. Dra. Amália Cinthia Meneses do Rego - Externo
Prof. Dr. Maurício Galvão Pereira - UFRN
Prof. Dra. Elaine Lira Medeiros Bezerra - UFRN
Aprovada: 28 de novembro de 2011
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“Sei que meu trabalho é uma gota no oceano,
mas sem ele o oceano seria menor”.
(Madre Teresa de Calcutá)
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DEDICATÓRIA
Dedico este trabalho a...
Deus, por ter me dado força, fé, sabedoria e esperança para ser perseverante e
alcançar com as suas bênçãos mais uma vitória em minha vida. Pelo amor
incondicional, sem limites...
Meu marido Ribamar e a meus filhos Ana Luiza e André Luiz, razões maiores da
minha vida..., a meu genro João Luis e a nora Albirea Shinobu
Por toda paciência, amor, esforços desmedidos, orientações e força que me
fazem forte e pelo exemplo diário de como se deve constituir uma família.
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AGRADECIMENTOS
Aos pacientes, pela disponibilidade e pela lição de incentivo, apesar de toda a
sintomatologia da doença, não se negaram em participar da pesquisa.
Ao Programa de Pós-graduação em Ciências da Saúde pela oportunidade de realizar
esse trabalho e pelos conhecimentos adquiridos durante todo este periodo.
A minha orientadora, Profa Dra Valéria Soraya de Farias Sales primeiramente por ter
aceitado me orientar, pelos ensinamentos em imunologia, pela amizade,
companheirismo e pelo apoio.
A minha co-orientadora Profa Dra Maria José Pereira Vilar, obrigada pela disposição em
ajudar, discutir e ensinar. Sua participação foi fundamental para o desenvolvimento
desse trabalho.
A Dr José Bruno Almeida pelo apoio e incentivo constantes para que se faça uma
Uroanálise de qualidade e pelas correções e sugestões no trabalho.
A Maria do Carmo Cardoso de Medeiros (Carminha) pelos ensinamentos e ajuda na
imunofluorescência.
A Juliana Cavalcante e Dra. Luiza Karla P. Arruda (USP-RP) pela grande ajuda na
realização das dosagens da ECP.
A todas as pós-graduandas do laboratório de imunologia clínica pela grande ajuda nas
fosforilações da imunologia, pelas “cytokines” e também pela amizade.
Ao Departamento de Análises Clinicas e Toxicológicas, especialmente a chefia (Telma
Maria de Araújo Moura Lemos), a secretaria (Asinete, Maria José, Seu Josias e
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Mikelison) e ao pessoal da sala de coleta (Giliane Lemos, Lúcia Jacinto, Ana Júlia e
Alexandro) que não mediram esforços para ajudar no trabalho.
Aos funcionários: do HUOL, Virtolino Neto, Oscar e Elisângela, pela ajuda na coleta e
transporte das amostras até o laboratório.
A Dona Amélia Silva de Morais e Evanilda Lopes da Silva, do DACT, pelo cuidado em
manter todo o laboratório sempre pronto para realização das análises. Vocês foram
fundamentais para que as análises fossem corretas.
Aos residentes da enfermaria de reumatologia pela ajuda no preenchimento do
protocolo clínico e do Mex-SLEDAI.
Aos meus amigos e colegas farmacêuticos Zélia Maria de Sousa (Zelinha), Edna
Marques de Araújo Silva, Maria Goretti do Nascimento Santos, Geraldo Barroso C.
Júnior, Ana Claúdia Galvão Freire Gouveia, Sandra Resende de Andrade, Ivanaldo
Amâncio da Silveira, Maiza Rocha Abrantes, Elizabeth (Eliuza), Tatiana Xavier e
Ivonete Batista de Araújo pela torcida e pela ajuda intelectual e espiritual constantes e
também por dividir comigo as angústias dessa etapa da vida.
A Vivian Silbiger e André Luchesi pela ajuda na organização do artigo e nas dúvidas de
informática.
Aos professores e colegas da Bioquímica Clínica e da Uroanálise pelo incentivo e
apoio..
E, finalmente, a todos aqueles que direta ou indiretamente contribuíram para que eu
chegasse até aqui.
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LISTA DE ABREVIATURAS E SIGLAS
ACR - American College of Rheumatology
AIN - Acute interstitial nephritis
Anti-dsDNA - Anti-double-stranded DNA
AUC – Área sob a curva
CEP- Comitê de Ética em Pesquisa,
C3 e C4- complementos 3 e 4
Cr - Creatinina
ECP - Proteína Catiônica Eosinofílica
EDN - Neurotoxina Derivada do Eosinófilo
eGFR - Estimated glomerular filtration rate
EPO - Peroxidase do Eosinófilo
EPX - Proteína X do eosinófilo
FEIA - Fluoroenzymeimmunoassay
GM-CSF - Fator estimulador de colônias de granulócitos-macrófagos
HPF - High power field
HUOL – Hospital Universitário “Onofre Lopes”
IFN-γ - Interferon-γ
IL-5 – Interleucina 5
IQR – Range Interquartílico
kDA – Quilo Daltons
LES – Lúpus Eritematoso Sistêmico
LN – Nefrite lúpica
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LTC4 e LTD4 – Leucotrieno 4
MCP - Proteínas quimioatrentes de monócitos
MEX-SLEDAI - Mexican version of the SLE Disease Activity Index
Mg - Miligrama
MIP-1α - Proteína inflamatória do macrófago
NIA – Nefrite Intersticial Aguda
PAF - Fator ativador de plaquetas
Pg - Picograma
Pr - Proteína
RNase A – Ribonuclease A
ROC - Receiver operating characteristic
SD – Desvio Padrão
SLE – Systemic lupus erythematosus
TGF α/β - Fator de transformação do crescimento
TNF-α - Fator de necrose tumoral
uECP - Proteína Catiônica Eosinofílica Urinária
UFRN – Universidade Federal do Rio Grande do Norte
VIP - Peptídeo vasoativo intestinal
WHO - World Health Organization
µg - Micrograma
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LISTA DE TABELAS Artigo 1 Table 1: Baseline characteristics and laboratory parameters observed for select patients
per group, of all SLE patients included (n=74).
Table 2: Laboratory values for 74 SLE subjects with and without renal disease.
Table 3: Results of correlation between variables of the study and laboratory results that
evaluate the involvement of renal function and disease activity in SLE.
Table 4: Results of the ROC curve analyses using SLE patients with and without lupus
nephritis to detect cut-off levels of eosinophiluria, urine ECP and ECP/Cr ratio.
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LISTA DE FIGURAS
Artigo 1 Figure 1: Eosinophiluria by Hansel´s stain (400X) (arrow).
Figure 2: Curve of receiver operating characteristic (ROC) in SLE patients to detect cut-
off of eosinophiluria, uECP and uECP-Cr ratio, classified by state variables Pr/Cr ratio
≥2 (A) and eGFR ≤60 mL/min (B):
Artigo 2 Figure 1: Eosinophils in urine samples of the patients lupus nephritis, Hansel’s stain.
Urinalysis Discipline file of the Department of Clinical and Toxicological Analyses-UFRN,
Natal.
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SUMÁRIO
1 INTRODUÇÃO 16
2 REVISÃO DA LITERATURA 18
3 ANEXAÇÃO DE ARTIGOS 23
3.1 ARTIGO 1.Resumo expandido 24
3.1.1 Abstract 25
3.2 ARTIGO 2 26
3.2.1 Abstract 27
3.2.2 Introdution 28
3.2.3 Patients and methods 30
3.2.4 Results 34
3.2.5 Discussion 36
3.2.6 Key Messages 41
3.2.7 Acknowledgements 41
3.2.8 References 42
4 COMENTÁRIOS, CRÍTICAS E SUGESTÕES 51
5 APÊNDICES 56
6 ANEXOS 57
6.1 Anexo 1 – Protocolo clínico 57
6.2 Anexo 2 – Mex-SLEDAI 58
6.3 Anexo 3 - Parecer do Comitê de Ética 59
7. REFERÊNCIAS 60
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RESUMO
O objetivo desse trabalho foi investigar o eosinófilo e a proteína catiônica eosinofílica (ECP) na urina de pacientes com Lúpus Eritematoso Sistêmico (LES), com e sem nefrite lúpica, como possíveis marcadores de inflamação renal. Foram estudados 74 pacientes com LES � 20 com evidência clínica e laboratorial de nefrite lúpica (LN grupo) e 54 sem envolvimento renal (não-LN grupo)� quanto à eosinofilúria e ECP urinária (uECP). A eosinofilúria foi observada através da coloração de Hansel e as concentrações de ECP urinária foram obtidas por fluoroenzimaimunoensaio e em seguida corrigidas pela creatinina urinária (uECP/uCr). As variáveis do estudo foram comparadas com a hematúria glomerular, relação proteína/creatinina urinária (uPr/uCr), creatinina sérica, clearance de creatinina estimado, anti-dsDNA, níveis séricos dos complementos C3 e C4, relação IL-5 urinária/creatinina e com o Índice de atividade da doença LES (Mex-SLEDAI). A avaliação preditiva da eosinofilúria e uECP foi observada através da curva ROC e o nível de significância do estudo foi p valor<0,05. Os resultados mostraram que a eosinofilúria e as concentrações da uECP e uECP/uCr foram mais elevadas nos pacientes do LN grupo em relação ao não-LN grupo (p<0,001 para todos). Essas variáveis mostraram uma correlação estatisticamente significativa com a hematúria, dismorfismo eritrocitário glomerular, cilindrúria, relação uPr/uCr, creatinina sérica, clearance de creatinina estimado, anti-dsDNA, relação IL-5 urinária/creatinina e com o Mex-SLEDAI (p<0,05). Os resultados da curva ROC mostraram uma melhor performance (área sob a curva-AUC) para a uECP/uCr, usando como variável de classificação a uPr/uCr (AUC=0,94) e o clearance de creatinina estimado (AUC=0,84), p<0,0001. Conclui-se que dentre as variáveis do estudo, a uECP/uCr pode servir como um novo marcador de inflamação renal em pacientes com LES.
Palavras Chave: Eosinófilos. Proteína catiônica eosinofílica. Lúpus Eritematoso Sistêmico
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1 INTRODUÇÃO
O Lúpus Eritematoso Sistêmico (LES) é uma doença autoimune,
multifatorial caracterizada pela produção de auto-anticorpos, formação de
imunocomplexos e inflamação em diferentes órgãos, sendo o rim um dos órgãos
mais afetados. Sua patogênese é desencadeada tanto por fatores genéticos,
ambientais e hormônios sexuais quanto por anormalidades do sistema imune inato
e adaptativo. A ativação anormal de células T, B e células apresentadoras de
antígenos tem como resultado final a produção dos auto-anticorpos patogênicos
altamente específicos, a ativação do sistema complemento e a presença de
células inflamatórias em órgãos-alvo [1-4].
Dentre as células envolvidas na fisiopatologia de inúmeros processos
inflamatórios estão os eosinófilos, que são recrutadas para o local da inflamação
onde modulam a resposta imune inata e adaptativa através de uma série de
mecanismos, tais como secreção de proteínas catiônicas, citocinas pró-
inflamatórias, quimiocinas, mediadores lipídicos e expressão de receptores para
citocinas, imunoglobulinas, complemento, dentre outros [5-7].
A análise de células urinárias refletindo a inflamação renal no LES pode ser
uma ferramenta útil no monitoramento da doença renal [8]. A eosinofilúria pode
refletir uma resposta inflamatória não específica, mas a sua investigação tem sido
associada a várias condições clínicas que afetam os rins e o trato urinário,
especialmente os casos de nefrite intersticial aguda (NIA) induzida por fármacos
[9-11].
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A proteína catiônica eosinofílica (ECP) vem sendo estudada como
marcador em processos inflamatórios do trato respiratório, dermatite atópica e
outras doenças inflamatórias [12], mas no LES não foi encontrado relato na
literatura.
Com base nos achados de eosinófilos na urina de pacientes com LES,
doença autoimune de alta incidência aqui no Rio Grande do Norte [13] e no papel
importante dos eosinófilos nas doenças inflamatórias, foi proposto esse estudo
com o objetivo de avaliar a eosinofilúria e a concentração da ECP urinária em
pacientes com Lúpus Eritematoso Sistêmico, com e sem nefrite lúpica, como um
possível marcador de inflamação renal.
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2 REVISÃO DA LITERATURA
Estudos epidemiológicos de prevalência e incidência de LES em todo o
mundo apresentam resultados bem variáveis. A incidência está na faixa de 1 a
10/100.000 habitantes/ano e a prevalência de 16 a 70/100.000 habitantes/ano.
Acomete mais predominantemente as mulheres, entre 20 e 30 anos de idade [14].
No Brasil, particularmente no Nordeste, na cidade de Natal onde a exposição aos
raios ultravioletas é intensa, a incidência é de 8,7/100.000 habitantes [13].
O envolvimento renal no LES ocorre em aproximadamente 25 a 60% dos
pacientes e tem um espectro de severidade muito variado podendo evoluir, em
cerca de 10 a 15%, para uma doença renal terminal, a qual contribui
significativamente para morbidade e mortalidade desses pacientes. A biópsia renal
é o padrão ouro para classificar o tipo histológico de nefrite, no entanto, como é
um procedimento invasivo e com riscos de hemorragia e infecção, não é uma
metodologia satisfatória para monitorar o envolvimento renal, que exige um
monitoramento de longo prazo. O advento da precisão de biomarcadores para
prever, diagnosticar e monitorar a nefrite lúpica será uma grande conquista na
ajuda aos pacientes com LES [15-19].
Estudos sobre a patogênese da nefrite lúpica mostram uma interação entre
as células do parênquima renal e células inflamatórias recrutadas devido à
deposição glomerular de imunocomplexos e ao desequilíbrio na homeostase das
citocinas. Na literatura existe considerável evidência para o papel de células T
helper 1(Th1), T helper 17 (Th17) e células T reguladoras (treg) no LES, porém
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diversos estudos tem sugerido uma possível contribuição das células T helper
(Th2). Foi observado que os imunocomplexos são constituídos por
imunoglobulinas IgM, IgG e IgA bem como por IgE e já foi constadada a presença
de IgE auto-reativa no soro de alguns pacientes com LES sem associação à
atopia e alergia [20-22].
Dentre as células que desempenham importante papel na resposta Th2, o
eosinófilo, que é um leucócito multifuncional das doenças alérgicas, parasitárias e
que participa no reparo do tecido vem sendo apontado como uma célula envolvida
na modulação da resposta imune inata e adquirida [23].
O eosinófilo é também um componente do microambiente imune local, tem
atividade autócrina e quando ativado secreta para o local da inflamação, proteínas
citotóxicas, presentes em seus grânulos secretórios secundários ou específicos.
São elas, a Proteína Básica Principal (MPB), Proteína Catiônica Eosinofílica
(ECP), Neurotoxina Derivada do Eosinófilo/Proteína X do eosinófilo (EDN/EPX) e
a Peroxidase do Eosinófilo (EPO) [5-7, 23].
Além das proteínas catiônicas, o eosinófilo também secreta as
interleucinas, IL-2, IL-3, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, fator de
transformação do crescimento (TGF α/β), fator estimulador de colônias de
granulócitos-macrófagos (GM-CSF), fator de necrose tumoral (TNF-α) e interferon-
γ (IFN-γ)], mediadores lipídicos como o leucotrieno 4 (LTC4 e LTD4) e fator
ativador de plaquetas (PAF). Nos grânulos eosinofílicos, além da síntese e
armazenamento dessas citocinas há também a síntese e armazenamento de
quimiocinas como as pertencentes à família das eotaxinas 1, 2 e 3, a proteína
20
inflamatória do macrófago (MIP-1α), a RANTES, proteínas quimioatrentes de
monócitos MCP – 3 e 4, a substância P e o peptídeo vasoativo intestinal (VIP) [5,
6, 23].
A Proteína Catiônica Eosinofílica (ECP), é uma glicoproteína com peso
molecular entre 18 e 21 kDa, pertencente à superfamília das ribonucleases A
(RNase A). Corresponde a cerca de 30% das proteínas dos grânulos do eosinófilo
e está envolvida na resposta imune. As concentrações de ECP nos fluidos
biológicos indicam a ativação e degranulação do eosinófilo e são usadas para o
diagnóstico e monitoramento de doenças inflamatórias [12, 24-26].
A presença de eosinófilos na urina foi primeiramente sugerida por Galpin e
colaboradores (1978), que acharam essa célula na urina em nove pacientes que
tinham nefrite intersticial aguda induzida por meticilina e não encontraram em
outros pacientes com deficiência renal por outras causas. [27]
A eosinofilúria pode refletir uma resposta inflamatória não específica, mas a
sua investigação tem sido associada a várias condições clínicas que afetam os
rins e o trato urinário, especialmente os casos de nefrite intersticial aguda (NIA)
induzida por fármacos [9-11].
A análise das proteínas catiônicas do eosinófilo na urina, realizada em
pacientes com tumores na bexiga sugeriu que a ECP, por não ser filtrada no
glomérulo renal, quando encontrada na urina, poderia refletir atividade eosinofílica
local no trato urinário e mostrou sua importância no diagnóstico de neoplasia
urotelial, tanto como valor preditivo de prognóstico quanto no acompanhamento da
terapia [28].
21
A variação circadiana da ECP sérica e da EPX sérica e urinária foi
analisada e mostrou níveis máximos dessas proteínas durante a noite e no
período matinal. Isto sugere que estudos de marcadores inflamatórios no sangue e
na urina devem ser realizados para validação e que os marcadores inflamatórios
não exibem apenas uma variação circadiana, mas também podem sofrer
influências das variações sazonais e espontâneas do dia-a-dia [29]. A presença de
eosinofilúria e detecção da ECP, em extratos de urina foram evidenciadas em
crianças escolares da Tanzânia infectados pelo Schistosoma haematobium e que
apresentavam alterações patológicas na bexiga [24].
Em uma experiência com 183 pacientes com diversas condições clínicas,
sendo as mais comuns, infecção do trato urinário e nefrite intersticial aguda, que
tiveram suas urinas testadas para eosinofilúria, utilizando a coloração de Hansel,
foi constatado, que a mesma é um bom indicador de NIA [10]. Estudo em 148
pacientes, dos quais 44 apresentavam diagnóstico de comprometimento do trato
genitourinário com presença de leucócitos na urina foi encontrado eosinofilúria em
9 pacientes na proporção de 1/500 a 1/100 e em 2 pacientes valores superiores a
1/100. De 15 pacientes com NIA, 6 tinham eosinofilúria positiva acima de 1/100 e
em 36 pacientes com outras falhas renais, dez apresentavam positividade acima
de 1/100 [30].
Marcadores de comprometimento renal no LES como sedimento urinário
ativo, creatinina sérica, albumina sérica e excreção urinária de proteína (relação
proteína/creatinina, coleta de proteína na urina cronometrada, clearance de
creatinina estimado) são os mais utilizados rotineiramente. No entanto, sabe-se
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que essas medidas nem sempre podem distinguir com precisão entre a inflamação
ativa e os danos renais crônicos, por isso novos biomarcadores vem sendo
pesquisados na urina e dentre eles destacam-se as proteínas, citocinas,
moléculas de adesão e quimiocinas, que podem ser úteis e mostram ser uma
potencial ferramenta laboratorial para avaliar o rim. As medições urinárias podem
também refletir uma medida do grau de disfunção tubular, ao invés de refletir
apenas a patologia glomerular [31].
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3 ANEXAÇÃO DE ARTIGOS
Artigo 1: Resumo Expandido: Publicado: Clinical Chemistry and Laboratory
Medicine. Volume 46, Pages: S689–S772, ISSN (Online) 1437-4331, August 2008.
Published Online: August 2008. Impact Factor: 1,89
Artigo 2- Enviado para publicação em 07 de setembro de 2011 no periódico
Rheumatology e recebeu o número RHE-11-1135 .
Periódico: Rheumatology – ISSN 1462-0324 ISI Impact Factor: 4.236
24
Artigo 1
Title
EOSINOPHILURIA: FINDINGS FREQUENT OR SPORADIC?
Freire S.A.V.1, Oliveira T.V.C.1, Silva C.G.O.1, Pereira R.M.1, Xavier C.E.S.1,
Cavalcanti J.E.C.1, Sales V.S.F.1, Vilar M.J.P.2, Brito T.N.S.1
1Clinic Analysis and Toxicology Department, Federal University of Rio Grande do
Norte, Natal, 59012-570 Brazil
2University Hospital Onofre Lopes, Federal University of Rio Grande do Norte,
Natal, 59012-570 Brazil
H019 – Abstract
Introduction The eosinophiluria may reflect an inflammatory response is not
specific, but their investigation has been associated with several clinical conditions
that affect the kidneys and urinary tract, especially the cases of acute nephritis
interstitial (NIA) induced by drugs or immune complex.
Objective. This study aimed to analyze the occurrence of eosinophiluria in samples
of urine with leukocytes.
Methods We determined urine eosinophil count in first-morning samples by
Hansel’s stain, of 101 urine patients of both sexes, 1 to 88 years of age, with
several clinical indications, hospitalized in the University Hospital Onofre Lopes
and in the Pediatrics Hospital, both of the Federal University of Rio Grande do
Norte, from March 2007 to March 2008. The urinary sediment of these samples
showed 10 leukocytes high power field.
25
Results The results showed that about the clinical indication, 62.38% (n=64) of the
patients had some nephropathy and 37.62% (n=38) did not. Among the patients
who had kidney disease, 46.03% (n = 29) had lupus nephritis, 15.87% (n = 10)
ITU, 9.52% (n = 6) IRC and 7.93% (n = 5) GNDA. Among those who did not have
kidney disease, the systemic lupus erythematosus (SLE) was the most frequent
with 57.89% (n = 22) of cases. The eosinophiluria was positive in 36.63% (n = 37)
patients with an average 109 ± 2.98 eosinophils high power field.
Conclusions The findings of eosinophiluria were discrete and more frequent in
patients with lupus nephritis and systemic lupus erythematosus without clinical
indication of nephritis.
Keys words. eosinophiluria; systemic lupus erythematosus; Hansel’s stain;
leukocytes.
Figure 1 Eosinophils in urine samples of the
patients lupus nephritis, Hansel’s stain.
Urinalysis Discipline - Department of Clinical and
Toxicological Analysis - Federal University of Rio
Grande do Norte, Natal-RN, Brazil.
26
Artigo 2
Title
Eosinophil and eosinophil cationic protein in urine: a new approach to
diagnosis of renal inflammation in systemic lupus erythematosus.
Author names
Tereza N S Brito1, Maria J Vilar2, José B Almeida3, Juliana A Cavalcante4, Ana L S
B Faria4, Sarah D V Medeiros5, Maria C C Medeiros6, Telma M A M Lemos7, Edna
M A Silva1, Vanessa M A Silva5, Luanda B F C Souza7, Luisa K P Arruda8, Tatiana
X Costa5, Geraldo B Cavalcanti Júnior1, Vivian N Silbiger7, Valéria S F Sales1.
Author affiliation
1 Department of Clinical and Toxicological Analysis, Postgraduate program in
Health Sciences, Federal University of Rio Grande do Norte, Natal-RN, Brazil.
2 Division of Rheumatology, Department of Clinical Medicine, Postgraduate
program in Health Sciences, Federal University of Rio Grande do Norte, Natal-RN,
Brazil.
3 Division of Nephrology, Department of Integrated Medicine, Federal University of
Rio Grande do Norte, Natal-RN, Brazil
4 Clinical Hospital, University of São Paulo, Ribeirão Preto-SP, Brazil.
5.Postgraduate program in Health Sciences, Federal University of Rio Grande do
Norte, Natal-RN, Brazil
6 Hospital Universitario Onofre Lopes, Federal University of Rio Grande do Norte,
Natal-RN, Brazil.
27
7.Department of Clinical and Toxicological Analysis, Postgraduate in
Pharmaceutical Sciences, Federal University of Rio Grande do Norte, Natal-RN,
Brazil.
8 Department of Clinical Medicine, Faculty of Medicine of Ribeirão Preto, University
of São Paulo (FMRP-USP), Ribeirão Preto-SP, Brazil.
Corresponding author:
Dr. Valéria Soraya de Farias Sales, Rua General Cordeiro de Farias, Faculdade de
Farmácia, 2o andar, Disciplina Imunologia Clinica, Departamento de Analises
Clinicas e Toxicológicas, Centro de Ciências da Saúde, Petrópolis, Natal-RN,
Brazil. Email: [email protected]; Fax number: (84)3342-9797.
ABSTRACT
Objective: To investigate eosinophils and eosinophil cationic protein in urine
(uECP) in patients with systemic lupus erythematosus (SLE), either with or without
lupus nephritis (LN), as a possible urinary marker of renal inflammation.
Methods: Seventy-four patients with SLE—20 with clinical and laboratory evidence
of lupus nephritis (LN group) and 54 without evidence of renal involvement (non-LN
group)—were analyzed regarding eosinophiluria and uECP. Eosinophiluria was
observed by Hansel's stain and ECP by fluoroenzymeimmunoassay. Both uECP
and urinary IL-5 (uIL-5) were corrected by urinary creatinine. Eosinophiluria and
uECP were compared with glomerular erythrocyturia, protein/creatinine ratio (Pr/Cr
ratio), serum creatinine, estimated glomerular filtration rate (eGFR), anti-double-
28
stranded DNA (anti-dsDNA), serum levels of complement (C3 and C4), uIL-5/Cr
ratio, and SLE disease activity index. To assess the predictive performance of
study variables, receiver operating characteristic (ROC) curves were constructed.
A significant level was p value < 0.05.
Results: Patients of the LN group had higher eosinophiluria, uECP, and uECP/Cr
ratio levels than patients of the non-LN group (p<0.001 for all). These variables
showed a statistically significant correlation with erythrocyturia, glomerular
dysmorphic erythrocyte, casts, Pr/Cr ratio, serum creatinine, eGFR, anti-dsDNA,
uIL-5/Cr, and SLE disease activity index (all p<0.05). The ROC curve results
demonstrated the best performance (area under curve - AUC) of the ECP/Cr ratio
using a state variable Pr/Cr ratio (AUC=0.94) and eGFR (AUC=0.84), p<0.0001.
Conclusion: These findings indicate that among the study variables the uECP/Cr
ratio could serve as a novel urinary marker of renal inflammation in SLE patients.
Keywords
Eosinophils, Systemic Lupus Erythematosus, Eosinophil cationic protein.
INTRODUCTION
Systemic lupus erythematosus (SLE) [1], is a multifactorial autoimmune
disorder characterized by autoantibody production, immune complex formation,
and immunologically mediated tissue injury. Lupus nephritis (LN) is one of the most
serious manifestations of SLE, which affects 25–60 % of patients, and one of the
leading causes of morbidity and mortality of this disease [2-6].
29
Although the precise pathogenesis of LN has not been fully elucidated, it is
mostly attributable to the glomerular deposition of immune complexes and
imbalance of the cytokine homeostasis,[7] which leads to a cascade of
inflammatory events with recruitment of mononuclear cells, such as T cells,
macrophages, and dendritic cells [8]. There is considerable evidence of the role of
Th1 cells, Th17, and regulatory T (Treg) in SLE, and several studies have
suggested a possible contribution of Th2 cells [9].
Among the cells that can secrete cytokines capable of promoting T-cell
proliferation, activation of Th1, or Th2 polarization is the eosinophil. This is a
granulocyte that has been pointed out in modulating both innate and adaptive
immune responses. In response to the diverse stimuli, eosinophils are recruited
from the circulation to inflammatory foci where they modulate immune responses
through an array of mechanisms, such as secretion of cationic proteins and
expression of receptors for cytokines, immunoglobulins, complement, and mRNA
for a number of Toll-like receptors. They can initiate antigen-specific immune
responses by acting as antigen-presenting cells [10-14].
Once attracted to the site of inflammation, the eosinophil becomes activated
and are secreted four cationic protein preformed granules, highly cytotoxic, which
are eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), eosinophil
derived neurotoxin (EDN)/former eosinophil protein X (EPX), and major basic
protein (MBP) in addition to chemokines, cytokines, and growth factors. ECP is the
best known, assessed, and used extensively as a marker in asthma and other
inflammatory diseases and has been scrutinized in a number of functional studies.
30
Regarding cytokines, IL-5 is the most specific to the eosinophil lineage being
responsible for selective differentiation, regulating growth, activation, and survival
of eosinophils [10, 15].
Based on the findings of eosinophils in the urine of SLE patients, treated at
the Rheumatology Unit of University Hospital Onofre Lopes (HUOL), Federal
University of Rio Grande do Norte, Natal, Brazil, a region with high incidence of this
disease [16], and on the role of eosinophils in various inflammatory diseases, this
study was proposed with the aim of evaluating the eosinophiluria and urinary ECP
levels of SLE patients, either with or without LN, as a possible urinary marker to
renal inflammation of SLE patients.
PATIENTS AND METHODS
Study population
Seventy-four patients with SLE diagnosis according to the American College
of Rheumatology (ACR) [1] criteria, age ≥ 18 years, were selected in the
rheumatology unit (HUOL-UFRN). Informed consent was obtained from the
patients after approval by the local ethics committee, number 044/2006. The study
was conducted according to the ethical guidelines of our institution (UFRN) and the
Declaration of Helsinki. Disease activity was assessed by the Mexican version of
the SLE Disease Activity Index (MEX-SLEDAI) [17].
The patients were divided into two groups. The first group, (the LN group)
consisted of 20 patients with renal disease activity and clinical and laboratory
evidence suggestive of World Health Organization (WHO) class IV lupus
31
glomerulonephritis. The presence of renal disease activity was defined by MEX-
SLEDAI score ≥ 6 and by the presence of all of the following in the urine test:
haematuria ≥ 1+, proteinuria ≥ 2+, active urinary sediment with erythrocyturia
defined as ≥ 5 cells/ high power field (HPF or 40× magnification), casts (erythrocyte
and/or granular, fatty, waxy, and renal tubular epithelial cells), and glomerular
dysmorphic erythrocytes. In addition, the patients of the LN group also had to
present serum creatinina ≥ 1mg/dL, low eGFR ≤ 60 mL/min, high proteinuria 24-
hours ≥ 3g/L, high protein in the first morning urine (spot urine) corrected by
creatinine (Pr/Cr ratio) ≥ 3, positive titers of anti-dsDNA (≥1:40), and decreased
concentrations of serum C3 and C4 (Table 1).
Included in the LN group were only patients who received prednisone (≥ 1
mg/kg/day) for treatment of nephritis, up to a maximum of one week. Only three of
these patients also received one intravenous pulse cyclophosphamide (0.5 g/m2
body surface area) in the same period. All patients in group LN also received
antimalarial drugs, calcium channel blockers, angiotensin-converting enzyme
(ACE) inhibitors, proton pump inhibitors, and diuretics.
The second group (the non-LN group) consisted of 54 patients with MEX-
SLEDAI score zero and without evidence of renal involvement (Table 1). These
patients received prednisone at a dose maximum of 10 mg/day and antimalarial
drugs. Excluded in the present study were patients with immunodeficiency, history
of allergy, other autoimmune diseases, helminthiasis, prostate cancer, renal
cancer, bladder infections, nephrolithiasis, and urinary tract infections.
32
Laboratory measurement
The laboratory evaluation, including the stool analysis of all patients, was
performed twice, and 10 mL of venous blood, the midstream first morning urine,
and urine of 24 hours were collected from each patient. The venous blood to clot
for 60 min at 24oC, followed by centrifugation (10 min, 24oC, 1600g), and the
resulting serum samples were tested for anti-dsDNA, creatinine, eGFR, C3, and
C4.
Standard urinalysis for glomerular dysmorphic erythrocyte, eosinophiluria,
protein, and creatinine was performed in the first morning urine, midstream, and
the supernatant (followed by centrifugation 5 min, 24oC, 400 g) was stored at -80oC
until analysis of uECP and uIL-5. Following the standard protocol used in
laboratories, 24-h urine was collected and stored in the refrigerator. Next, this
sample was measured and centrifuged (5 min, 24oC, 400 g) for determination of
proteinuria and glomerular filtration rate (creatinine clearance). Urinalysis was
performed by experienced personnel following the good quality control procedures.
In this study, proteinuria was defined as Pr/Cr ratio, due to a strong
correlation between the results found by two methods and in accordance with the
recommendation of the Renal Disease Subcommittee of the American College of
Rheumatology Ad Hoc Committee on Systemic Lupus Erythematosus Response
Criteria [18].
Eosinophiluria research by Hansel's stain was conducted after concentrating
50 µL of urine sediment in a cytospin cytocentrifuge. The eosinophils were counted
per 10 fields (HPF) and the finding of only one was considered positive
33
eosinophiluria [19]. Importantly, this method is analyzer dependent and was
observed by three experienced analyzers.
The uECP measurements were performed by the Pharmacia CAP System®
ECP FEIA (fluoroenzymeimmunoassay) (Pharmacia, Uppsala, Sweden), with a
coefficient of variation of 2.5%, according to the instructions of the manufacturer.
The test is designed as a sandwich immunoassay. Inter- and intra-assay
coefficients of variation were less than 8% and the detection limit was 0.5 µg/L.
The uECP was determined following the same instructions used for serum ECP,
and uECP levels were corrected to urine creatinine (uECP/Cr ratio) with results
expressed as micrograms per milligram of creatinina (µg/mgCr).
The concentrations of uIL-5 were determined by quantitative sandwich
enzyme immunoassay (Quantikine® Minneapolis, United States of America),
according to the manufacturer’s instructions. This immunoassay is a solid phase
ELISA designed to measure IL-5 levels in cell culture supernates, serum, plasma,
and urine; the detection limit was <3,0 pg/mL. Concentrations of uIL-5 levels were
corrected to urine creatinine (uIL-5/Cr ratio) and results were expressed as
picrograms per milligram of creatinina (pg/mgCr).
Statistical analyses
Statistical analyses of results were performed by SPSS v.17 for Windows
(SPSS, Chicago, IL, USA). Continuous variables were tested for normal-
distribution using the Shapiro Wilk and Kolmogorov-Smirnov statistical tests.
Results are presented as mean±SD, median, interquartile range (IQR), and
minimum (min) and maximum (max) values. uECP correlation with disease activity
34
and renal function tests was assessed using Pearson and Spearman's rank
correlation coefficient.
Categorical variables were compared by chi-square test and continuous
variables were compared by Student's t-test and Mann-Whitney U test. A receiver
operating characteristic curve (ROC curve) was constructed using state variables,
Pr/Cr ratio ≥ 2, and estimated GFR≤ 60 mL/min for comparison with eosinophiluria,
uECP, and uECP/Cr ratio. Outcomes were determined by area under the ROC
curve (AUC), sensitivity, and specificity tables. Two-tailed p-values less than 0.05
were regarded as statistically significant. Assuming a significance level (α) of 0.05
and a power (β) of 80%, the sample size used was able to detect statistical
significance for differences between groups of 1.5 points in eosinophiluria, 3.9 units
in uECP, and 6.5 units in uECP/Cr ratio.
RESULTS
Baseline characteristics of patients
A total of 74 patients with SLE—20 patients (16 women and 4 men) with
active SLE and evidence of LN (MEX-SLEDAI score= 6.0-22.0) and 54 patients (all
women) with inactive disease and without LN (MEX-SLEDAI score =0)—were
evaluated for eosinophiluria, uECP, and uIL-5. The demographic characteristics of
the two groups are presented in Table 1. Of all the patients, 94.6% (n=70) were
women. The mean age was 29.5 ±8.5 years and the mean disease duration was
67.0±58.5 (1-228 months). There were no significant differences in ethnicity
between the two groups of patients.
35
Laboratory findings
Eosinophiluria (as shown in Figure 1) was observed in 45% (n=9) of the
patients in the LN group and 5.6% (n=3) in the non-LN group. The mean of
eosinophiluria was significantly higher in patients in the LN group than that for
patients in the non-LN group (p<0.001). The concentrations of uECP, uECP/ratio,
uIL-5, and uIL-5/Cr ratio were higher in the LN group than in the non-LN group (p
<0.05), (Table 2).
In addition, a statistically significant correlation was observed between study
variables and active renal disease parameters, such as haematuria, glomerular
dysmorphic erythrocyte, casts, Pr/Cr ratio, serum creatinine, eGFR, anti-dsDNA,
serum C4, and SLE disease activity index (all p<0.05). Moreover, the strongest
association was observed between uECP/Cr ratio and parameters: haematuria
(rs=0.76), Pr/Cr ratio (rs=0.75), serum creatinine (rs=0.70), and MEX-SLEDAI
(rs=0.72), p<0.001. Urinary IL-5 and uIL-5/Cr ratio showed a statistically significant
correlation with eosinophiluria, uECP, and uECP/Cr ratio (p<0.05) (Table 3). The
uIL-5/Cr also showed a statistically significant correlation with MEX-SLEDAI
(rs=0.41), p<0.01.
Cut-off values of 1/10 fields (HPF) for eosinophiluria, 2.25µg/L for uECP and
41.93 µg/mgCr for uECP/Cr ratio were chosen from the two ROC curves (Figure 2)
with data constructed with state variables by Pr/Cr ratio ≥ 3 and eGFR ≤60 mL/min
based on optimization of sensitivity and specificity (Table 4). The curves also
depict the specificity and sensitivity of the uECP/Cr ratio for renal inflammation in
SLE patients, providing a sensitivity of 90%, a specificity of 67.4%, and AUC 0.94
36
(defined by Pr/Cr), and a sensitivity of 94%, a specificity of 66.7%, and AUC of
0.84 (defined by eGFR), p<0.001 all (Table 4).
DISCUSSION
The present study was the first investigating eosinophiluria and uECP as a
possible urinary marker to evaluate renal inflammatory activity of SLE patients. The
first step to achieve this objective was selecting SLE patients with and without
renal inflammatory activity according to pre-established clinical and laboratorial
data. Lupus nephritis is an organ-specific autoimmune disease characterized by
intra-renal activation of inflammatory cells and the formation of glomerular, tubular,
and interstitial lesions [20]. Interestingly, this study revealed a significant increase
in eosinophiluria and uECP in patients with LN compared to patients without LN.
Lupus nephritis requires long-term monitoring over several years, as flares
may occur, as well as progressive deterioration of renal function. Renal biopsy
remains the gold standard to assess disease severity, while multiple biopsies to
gauge treatment efficacy are not feasible due to their invasive nature with risks of
bleeding and infection, thereby presenting a less satisfactory method for monitoring
renal involvement in SLE [21, 22].
Current laboratory markers for LN such as proteinuria, Pr/Cr ratio, urine
sediment analysis, eGFR, serum albumin, serum creatinine, anti-dsDNA, and
complement levels are unsatisfactory. They lack sensitivity and specificity for
differentiating renal activity and damage in LN [23-25].
37
Urinary biomarkers may also reflect to some extent the degree of tubular
dysfunction, rather than purely reflecting underlying glomerular pathology [26]. A
biomarker that can forecast lupus nephritis flare well before thresholds of
proteinuria, renal function, and urine sediment that signal clinical flare are reached
would be a valuable tool [27]. Thus, novel biomarkers that are able to discriminate
lupus renal activity and its severity, predict renal flares, and monitor treatment
response and disease progress are clearly necessary [24, 25].
The clinical and laboratory differences presented between the two groups of
patients selected for this study were consistent with literature data. Marks et al. [28]
found that LN patients had higher urine albumin/creatinine ratios versus non-
nephritis patients. Rubinstein et al. [29] found proteinuria (Pr/Cr ratio) greater than
2.0, decreased creatinine clearance, and SLE disease activity index (SLEDAI) ≥4
in SLE patients with biopsy-proven nephritis. A study by Guo et al. [30] found in
patients with LN class IV, values of 24-h proteinuria≥ 3.0 g/day, increased serum
creatinine, and a mean SLEDAI ≥9 in all patients, suggesting that most SLE
patients that merged with renal diseases were in the active stage.
Urine as a biological sample has the advantage that it is easily obtainable by
non-invasive means and thus allows investigators to avoid many regulatory hurdles
[31]. More significantly, this study showed that eosinophiluria, uECP, and uECP/Cr
levels were higher in the LN group than in the non-LN group. The eosinophiluria
had a higher frequency in the LN group and showed a regular and statistically
significant correlation with parameters of the renal inflammation and dysfunction
evaluated. Sensitivity (69.2%) and specificity (82%) to eosinophiluria, found in this
38
study, were similar to the findings of other authors in studies conducted in patients
with acute interstitial nephritis (AIN), as published by Corwin et al. [32] (a sensitivity
of 63%) and Ruffing et al. [33] (a sensitivity of 40% and specificity of 72%).
Eosinophils in urine may be associated with a variety of clinical conditions,
so some patients with indicator diseases were excluded in our study. There are no
systematic studies of kidney diseases which are associated with increased
amounts of eosinophils kidney [32, 34]. Therefore, no biopsies in this study could
evaluate whether the positive eosinophiluria was due exclusively to eosinophils
kidney.
The literature does not report the participation of eosinophils in the
inflammatory process of LN. However, several mechanisms have been shown to
contribute to the pathogenesis of LN, including the cell-mediated Th1 and Th17,
and, more recently as described by Charles et al. [9], mediated by basophils, IL-4,
and IgE, which promote a Th2 environment. Although eosinophils are generally
found in association with Th2 immunity, they express Th1, Th2, and regulatory
cytokines, as well as cytokines with strong pro- or anti-inflamatory properties,
chemokines, and lipid mediators [35]. Eosinophils also express receptors for a
wide range of cytokines, Th1, and Th2, and also chemokine receptors, which gives
them the ability to migrate to different tissues and to perform effector functions in
various types of inflammatory processes, both of the type Th1 or Th2 [36]. Thus,
based on these data we can assume that eosinophils are cells that may play a role
in kidney inflammation in LN.
39
Studies show that during inflammation whole eosinophil granules are
released from disrupted cells and those internal proteins are subsequently
released differentially through the process of piecemeal degranulation. Among the
components of these granules, ECP is RNAse A superfamily, protein rich in
arginine residues, which gives a high concentration of positive charges, promoting
a strong attraction for molecules negatively charged existing in cell membranes
[37]. This property may explain its cytotoxic power in the cell membranes causing
the formation of pores or channels on the surface of the membrane, disrupting its
lipid structure and possibly facilitating the entry of other cytotoxic molecules.
Sensitive assays have been developed for its measurement in biological fluids
which have contributed to the understanding of the role of the eosinophils in
disease [10, 15].
This study demonstrated increased uECP in patients of the LN group
compared to non-LN group patients and a statistically significant correlation, but
regular, between amounts of uECP and haematuria, glomerular dysmorphic
erythrocyte, casts, Pr/Cr ratio, serum creatinine, eGFR, uIL-5, and MEX-SLEDAI.
Interestingly, after correction of uECP by creatinine (ECP/Cr ratio), an increase
was noted in the correlation between this finding and the parameters haematuria,
Pr/Cr ratio, serum creatinine and MEX-SLEDAI. Additionally, using the ROC curve,
it was observed that the urinary ECP/Cr ratio had an excellent area-under-curve,
indicating that it has good predictive power of renal inflammatory activity in SLE
with a greater sensitivity and moderate specificity.
40
Inflammatory diseases tend to share common pathways and thus many a
potential biomarker will not be specific for a particular disease. Few biomarkers for
SLE have been validated and employed for making clinical decisions given the
complex etiopathogenesis, heterogeneous clinical manifestations, and varying
rates of disease progression among individual SLE patients [3, 27].
In the present study, the uIL-5 had higher concentration in the LN group and
after being corrected by creatinine (uIL-5/Cr ratio) showed better correlation with
ECP/Cr ratio and MEX-SLEDAI. No article has yet been published in the literature
showing the uIL-5/Cr in SLE. The increased levels of uIL-5 can justify the
appearance of eosinophils and ECP in urine of the SLE patients studied because
the cytokine IL-5, which is a key Th2 cytokine in eosinophil biology, is involved in
eosinophil differentiation, maturation, and migration of these cells [38].
This was the first study showing that the eosinophiluria and urinary ECP
may be useful as biomarkers of renal inflammation in SLE patients. The data are
encouraging and provide a basis for future research. We need to conduct a
longitudinal cohort study in which the number of patients will be increased and
follow-up will be done both for patients without LN and for patients with LN
undergoing immunosuppressive therapy. It is important to emphasize that renal
biopsies were not performed in this first phase of the research due to the absence
of a renal pathologist in our region. The longitudinal study of kidney biopsies can
provide data to assess whether the uECP and urinary eosinophils are capable of
discriminating the renal activity and severity of lupus, predicting kidney flares, and
monitoring response to treatment and disease progression.
41
Our findings not only suggest that uECP/Cr ratio may be a urinary biomarker
of renal inflammatory activity in SLE patients but also shows the need to
investigate the role of eosinophils in the inflammatory process of nephritis in
patients with systemic lupus erythematosus.
KEY MESSAGES
• Increased urinary eosinophils and uECP/Cr ratio levels in patients with
history of lupus nephritis.
• Urinary ECP/Cr ratio could serve as a novel marker of renal inflammation in
SLE.
Acknowledgements: We received a grant from Phadia Diagnostic Ltda for this
research and would like to express our gratitude to them, and thank our patients for
their participation.
Conflict of interest statement: All authors declare that they have no competing
interests.
Funding statement: This publication was made possible by Postgraduate
program in Health Sciences, Integrated Laboratory of Clinical Analysis and
University Hospital Onofre Lopes, all of the Federal University of Rio Grande do
Norte and Clinical Hospital, University of São Paulo. Its content is solely the
responsibility of the authors.
Authors' contributions: TNSB, MJV, JBM, ALSBF, GBCJ, VNS and VSFS
contributed to the study design, and to interpretation and analysis of the data. JAC,
LKPA performed the Pharmacia CAP System® ECP FEIA
42
(fluoroenzymeimmunoassay) assays. SDVM, MCCM, LBFCS and TNSB
performed the ELISA (IL-5), Immunofluorescence (Anti-dsDNA, ANA) and
Immunoturbidimetric (C3, C4) assays. TNSB, TMAML, EMAS, VMAS, TXC, MJV
and VSFS contributed to the sample collection and data acquisition.
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North Am 2010:6:131-43.
25. Mok CC. Biomarkers for lupus nephritis: a critical appraisal. J Biomed
Biotechnol 2010;2010:1-11 doi:10.1155/2010/638413 [published Online First: 22
March 2010.
26. Adhya Z, Borozdenkova S, Karim MY. The role of cytokines as biomarkers in
systemic lupus erythematosus and lupus nephritis. Nephrology Dialysis Transplant
2011;0:1-8 doi: 10.1093/ndt/gfq860 [published Online First: 03 March 2011.
27. Rovin BH, Zhang X. Biomarkers for lupus nephritis: the quest continues. Clin J
Am Soc Nephrol 2009;4:1858-65.
28. Marks SD, Shah V, Pilkington C, Tullus K. Urinary monocyte chemoattractant
protein-1 correlates with disease activity in lupus nephritis. Pediatr Nephrol
2010;25:2283-8.
29. Rubinstein T, Pitashny M, Levine B, et al. Urinary neutrophil gelatinase-
associated lipocalin as a novel biomarker for disease activity in lupus nephritis.
Rheumatology (Oxford) 2010;49:960-71.
30. Guo Q, Lu X, Miao L, Wu, M, Lu S, Luo P.. Analysis of clinical manifestations
and pathology of lupus nephritis: a retrospective review of 82 cases. Clin
Rheumatol 2010;29:1175-80.
31. Li Y, Tucci M, Narain S, et al. Urinary biomarkers in lupus nephritis.
Autoimmunity Reviews 2006;5:383-8.
32. Corwin HL, Bray RA, Haber MH. The detection and interpretation of urinary
eosinophils. Arch Pathol Lab Med 1989;113:1256-8.
46
33. Ruffing KA, Hoppes P, Blend D, Jarjoura D, Whittier FC. Eosinophils in urine
revisited. Clin Nephrol 1994;41:163-6.
34. Sutton JM. Urinary eosinophils. Arch Intern Med 1986;146:2243-4.
35. Tyagi P, Barclay D, Zamora R, et al. Urine cytokines suggest an inflammatory
response in the overactive bladder: a pilot study. International Urology and
Nephrology 2010;42:629-35.
36. Shamri R, Xenakis J, Spencer L. Eosinophils in innate immunity: an evolving
story. Cell and Tissue Research 2011;343:57-83.
37. Mallorquí-Fernández G, Pous J, Peracaula R, et al. Three-dimensional crystal
structure of human eosinophil cationic protein (RNase 3) at 1.75 Å resolution.
Journal of Molecular Biology 2000;300:1297-307.
38. Kim CK, Kita H, Callaway Z, et al. The roles of a Th2 cytokine and CC
chemokine in children with stable asthma: Potential implication in eosinophil
degranulation. Pediatric Allergy and Immunology 2010;21:e697-e704.
47
Figure 1: Eosinophiluria by Hansel´s stain (400X) (arrow).
Figure 2: Curve of receiver operating characteristic (ROC) in SLE patients to detect
cut-off of eosinophiluria, uECP and uECP-Cr ratio,classified by state variables
Pr/Cr ratio ≥2 (A) and eGFR ≤60 mL/min (B):
ROC Curve A: uECP/Cr ratio (AUC=0.94); uECP (AUC=0.62); Eosinophiluria
(AUC=0.55)
48
ROC curve B: uECP/Cr ratio (AUC=0.84); Eosinophiluria (AUC=0.72); uECP
(AUC=0.66);
Table 1. Baseline characteristics and laboratory parameters observed for select
patients per group, of all SLE patients included (n=74).
LN group (20) non-LN group
(54)
p-value
Age (y) 26.6±5.1 30.5±9.3 <0.01
Gender (men/women) % 20/80 0/100 <0.01
Ethnicity [W / N-W (pure Black /Pardo)] % 30/(20/50) 33/(4/63) 0.29
Disease duration of SLE (months) mean±SD 30±26.3 80.8±61.3 <0.01
Mex-SLEDAI (Min-Max) 6.0-22.0 0.0
Dipstick positive hematuria - (1+ to 4+), % (n) 100.0 (20.0) 0.0 (0.0) <0.001
Dysmorphic erythrocyte,% (n) 70.0 (14.0) 1.8 (1.0) <0.001
Erythrocyte, granular, fatty and waxy casts, % (n) 70.0 (14.0) 7.4 (4.0) <0.001
Oval fat bodies, % (n) 80.0 (16.0) 1.8 (1.0) <0.001
Erythrocytes sedimentation rate /HPF mean±SD 33.8±16.3 1.9±1.7 <0.001
Dipstick positive proteinuria (1+ to 4+), % (n) 100.0 (20.0) 5.6 (3.0) <0.001
Pr/Cr ratio (mg/mg) median (IQR) 3.5 (17.5) 0.3 (0.4) <0.001
24-h Proteinuria (g/L) median (IQR) 3.4 (11,0) 0.1 (0,3) <0.001
Serum creatinine (µmol/L) median (IQR) 114.0 (80.2) 70.7 (17.7) <0.001
eGFR (mL/min) median (IQR) 56.9 (48.1) 95.0 (33.0) <0.001
Creatinine clearance (mL/min/1.73 m2) median (IQR) 54.3 (36.8) 96.0 (32.0) <0.001
Serum C3 (mg/L) median (IQR) 466.5 (718.0) 1444.0 (802.5) <0.001
Serum C4 (mg/L) median (IQR) 231.0 (220.5) 438.0 (167.2) <0.001
Anti-dsDNA title median (IQR) 80.0 (110.0) 20.0 (10.0) <0.001
Number of individuals in parentheses. Continuous variables are presented as means
and standard deviation (±), medians, and interquartile range (IQR, in parentheses) and
were compared by Student's t tests and Mann-Whitney U tests.
Categorical variables were compared by chi-square test.
HPF,high power field or 400X; C3, complement 3; C4, complement 4.
Ethnicity: W=White; N-W= Not-White (pardo = Brazilian descendants of black, white and
indigenous people, according Brazilian Institute of Geography and Statistics).
49
Table 2. Laboratory values for 74 SLE subjects with and without renal disease.
LN group (20) non-LN group (54) p-value
Eosinophiluria, % (n) 45.0 (9.0) 5.6 (3.0) <0.001
Eosinophils sedimentation rate /HPF 1.7 (1.2) 0.17 (0.0) <0.001
Urinary ECP (µg/L), median (IQR) 4.1 (4.2) 2.0 (0.0) <0.001
Urinary ECP/Cr ratio (µg/mgCr) median (IQR)
93.5 (70.2) 33.1 (19.7) <0.001
Urinary IL-5 (pg/mL) median (IQR) 84.0 (185.2) 46.1 (63.43) <0.001
Urinary IL-5/Creatinina ratio (pg/mgCr) median (IQR)
231.9 (270.7) 81.3 (112.4) <0.001
Number of individuals in parenthesis. Continuous variables are presented as
means and standard deviation (±), medians and interquartile range (IQR, in
parenthesis) and were compared by Student's t-tests and Mann-Whitney-U tests.
Categorical variables were compared by chi-square test.
HPF, high power field or 400X; ECP, eosinophil cationic protein; ECP/Cr ratio,
eosinophil cationic protein-creatinine ratio; IL-5, Interleukin-5.
50
Table 3. Results of correlation between variables of the study and laboratory
results that evaluate the involvement of renal function and disease activity in SLE.
Correlation coefficient, rs (Spearman)
Eosinophiluria Urinary
ECP
ECP/Cr ratio p value
Haematuria 0.52 0.49 0.76 p<0.001
Glomerular dysmorphic erythrocyte 0.51 0.56 0.52 p<0.001
Casts 0.44 0.45 0.63 p<0.001
Pr/Cr ratio 0.46 0.55 0.75 p<0.001
Serum creatinine 0.59 0.57 0.70 p<0.001
eGFR -0.54 -0,47 -0.61 p<0.001
Anti-dsDNA 0.26 0.27 0.58 p<0.001
Serum C3 -0.21 -0.16 -0.30 p<0.05
Serum C4 -0.28 -0.44 -0.31 p<0.05
Urinary IL-5 0.31 0.32 0.27 p<0.05
Urinary IL-5/Cr (pg/mgCr) 0.36 0.28 0.50 p<0.001
Mex-SLEDAI 0.41 0.44 0.72 p<0.001
ECP, eosinophil cationic protein; Pr/Cr ratio, protein-creatinine ratio; C3,
complement 3; C4, complement 4; Mex-SLEDAI, Mexican version of the SLE
Disease Activity Index.
51
Table 4. Results of the ROC curve analyses using SLE patients with and without
lupus nephritis to detect cut-off levels of eosinophiluria, urine ECP and ECP/Cr
ratio.
The cut-off of Eosinophiluria, ECP/Cr and uECP (classified by state variables Pr/Cr
ratio ≥2 and eGFR ≤60 mL/min) were 1/ field (HPF), 42.2 µg/mgCr and 2.25 µg/L,
respectively. AUC, area under the ROC curve; IC, Confidence interval; Pr/Cr ratio,
protein-creatinine ratio; eGFR, estimated glomerular filtration rate; HPF,high power
field or 400X; ECP, eosinophil cationic protein; ECP/Cr ratio, eosinophil cationic
protein-creatinine ratio.
Criterion for renal inflammation in SLE- state variable Pr/Cr ratio
AUC Asymptotic 95% IC
Sensitivity Specificity p value
Eosinofiluria 0.55 0.35 - 0.75 69.2% 82.0% p>0.05
Urinary ECP 0.62 0.40 – 0.83 53.0% 87.0% p>0.05
ECP/Cr ratio 0.94 0.88 – 1.00 90.0% 67.4% p<0.0001
Criterion for renal inflammation in SLE- state variable eGFR
AUC Asymptotic 95% IC
Sensitivity Specificity p value
Eosinofiluria 0.72 0.50 - 0.82 69.2% 82.0% p<0.005
Urinary ECP 0.66 0.49 – 0.82 73,90% 94.0% p=0.05
ECP/Cr ratio 0.84 0.73 – 0.96 94.0% 66.7% p<0.0001
52
4 COMENTÁRIOS, CRÍTICAS E SUGESTÕES
O anteprojeto que deu origem a essa tese foi intitulado “Eosinófilos e
Proteína Catiônica Eosinofílica: achados em pacientes com Lúpus Eritematoso
Sistêmico”, aprovado sob o número 044/2006, CEP-UFRN. Todas as sugestões
do CEP-UFRN foram acatadas e o anteprojeto foi adequado para cumpri-las. Foi
elaborado a partir de um estudo piloto com o objetivo de identificar a freqüência de
eosinofilúria em pacientes que apresentavam comprometimento renal e
realizavam o exame de urina (sumário com sedimentoscopia) no Laboratório da
Disciplina de Uroanálise.
Esse estudo mostrou que dentre os pacientes que apresentavam
eosinofilúria, as que tinham indicação clínica de LES e nefrite lúpica apresentavam
uma maior positividade. Então foi decidido que seria analisado na urina desses
pacientes, não só o eosinófilo, mas também a proteína catiônica do eosinófilo, a
IL-5 que é uma das citocinas responsáveis pela ativação do eosinófilo e também a
IgE. Os artigos apresentando os resultados referentes a estas duas últimas
variáveis estão em fase de redação.
O presente estudo teve relevância pelo seu caráter inderdisciplinar, no qual
houve a integração de disciplinas da área médica como a reumatologia,
imunologia e nefrologia, auxiliados pelas Análises Clínicas, da área de Farmácia.
O mesmo propiciou também a integração entre as ciências básicas e clinicas que
pode ser refletida no auxilio ao diagnóstico da doença renal nesses pacientes
culminando em uma melhora para o tratamento. A inserção dos médicos
53
residentes e dos alunos de iniciação científica do Curso de Farmácia, no decorrer
da execução do projeto, foi de suma importância pela possibilidade de um contato
diário com os pacientes e entre os participantes do grupo de pesquisa e
acompanhamento desde o diálogo diário necessário para anuência do termo de
consentimento livre e esclarecido até a condução de soluções nas intercorrências.
As dificuldades com as quais nos deparamos foram relacionadas à
disponibilidade dos pacientes para coleta do sangue e da urina, devido a um
quadro clínico muito complexo, principalmente as pacientes do grupo com
evidências de nefrite lúpica. Para tanto, o preenchimento e assinatura do termo de
consentimento livre e esclarecido, em algumas vezes, teve que se adequar a
disponibilidade e condição clínica do paciente. Ainda relacionado às dificuldades,
pode-se citar a luta para conseguir doação de reagentes, pois o projeto não teve
financiamento.
A partir dos primeiros resultados da freqüência de eosinofilúria foi elaborado
um trabalho de conclusão de curso orientado pela doutoranda, intitulado
“Eosinofilúria: achados freqüentes ou esporádicos?” o qual foi apresentado no
20th International Congress of Clinical Chemistry and Laboratory Medicine- XXXV
Brazilian Congress of Clinical Analysis-VIII Brazilian Congress of Clinical Citology,
Fortaleza – Brasil. O resumo expandido “Eosinophiluria: findings frequent or
sporadic?” foi publicado em Clinical Chemistry and Laboratory Medicine, 2008.
Esse trabalho contribuiu com a informação da eosinofilúria em pacientes com LES.
Em seguida, as análises estatísticas permitiram observar as diferenças das
variáveis entre os dois grupos de pacientes com LES, calcular a sensibilidade e
54
especificidade das variáveis, determinadas na urina, que podem ser usadas como
marcador de inflamação renal no LES. Parte dos resultados está no manuscrito
que foi submetido ao periódico Rheumathology intitulado “Eosinophil and
eosinophil cationic protein in urine: a new approach to diagnosis of renal
inflammation in systemic lupus erythematosus”, 2011.
No momento encontra-se em fase de redação um artigo abordando além da
eosinofilúria e a ECP, a IL-5 e a IgE urinárias. Os aspectos trabalhados no
presente estudo estão em consonância com as pesquisas atuais na busca de
descoberta de biomarcadores urinários que proporcionem além do diagnóstico
precoce, o monitoramento do envolvimento renal em pacientes com LES.
A doutoranda é professora adjunto IV do Departamento de Análises
Clínicas e Toxicológicas, da UFRN, participa do grupo de pesquisa com atividades
registradas na base Bioanálises e pretende continuar ampliando a pesquisa sobre
marcadores urinários.
55
5 APÊNDICE
Participação em Congressos
DIAS, S. S. A., LOPES, Luciana Larissa Fonseca, BRITO, Ana Luiza de Souza,
SILVA, Leila Priscilla P, LIMA, Rosaninny Maria Moura, Walesca Cecília Costa de
Oliveira, LEMOS, Telma Maria de Araújo Moura, BRITO, T. N. S. Avaliação do
dismorfismo eritrocitário glomerular em pacientes com hematúria. In: 33
Congresso Brasileiro de Análises Clínicas e 6 Congresso Brasileiro de Citologia
Clínica, RBAC, v. 2, 2006.
BRITO, T. N. S., FONSECA, A. G., BATISTA, J. I., MOTA, G. R., PEREIRA, R. M.,
OLIVEIRA, F. K. J. Q. Perfil laboratorial da função renal em pacientes portadores
de Lupus Eritematoso Sistêmico In: 36 Congresso Brasileiro de Análises Clínicas -
9 Congresso Brasileiro de Citologia Clínica, RBAC, v. 41, p. 1B-66B, 2009
BRITO, T. N. S., MEDEIROS, S., ARAUJO, M., SILVA, E. M. A., LOPES, D. R.,
MELCHUNA, K., LIMA, A., VILAR, M. J. P., SALES, V. S. F. Immunological and
biochemical findings in patients with Systemic Lupus Erythematosus In: XXXIII
Meeting of the Brazilian Society for Immunology / II Extra Section of Clinical
Immunology, Intervention in the Immune System, 2008.
BARBOSA, K. C. C. S., NASCIMENTO, G. B., SILVA, E. M. A., SILVA, E. M. A.,
BRITO, T. N. S. Avaliação da taxa de filtração glomerular em pacientes com
doença renal: comparação entre dois métodos. In: 34o Congresso Brasileiro de
Análises Clínicas e 7o Congresso Brasileiro e Citologia Clínica, RBAC, v.2, 2007.
56
6 ANEXOS
6.1 Protocolo Clínico
Universidade Federal do Rio Grande do Norte
Centro de Ciências da Saúde
Programa de Pós-Graduação em Ciências da Saúde
EOSINÓFILOS E PROTEÍNA CATIÔNICA EOSINOFÍLICA: ACHADOS EM
PACIENTES COM LUPUS ERITEMATOSO SISTÊMICO
DATA: ____________________ RG HUOL: __
IDENTIFICAÇÃO:___________________________________________________
Data de nascimento:__________________ Idade: _____________
ENDEREÇO (referência):_____________________________________
Telefones: _________________________________________
Sexo: ( ) F ( ) M Cor(auto-denominada): ( ) Branca ( ) Pardo
( ) Preto ( ) Amarelo
Peso___________ Altura_______________
Início da doença (mês/ano): _______________________
Número de “pulsos” com ciclofosfamida: _________________
Antecedentes Alérgicos: Dermatite Atópica ( ) Asma ( ) Rinite ( ) Urticária ( )
Medicamentos em Uso: Corticóide ( ) Ciclofosfamida ( ) Antidepressivo ( )
Inibidor de ECA ( ) Inibidor de canal de cálcio ( ) Inibidor de Bomba de prótons ( )
Diurético ( ) Antibiótico ( )
57
6.2 MEX-SLEDAI
NOME:_________________________________________________________
DATA___/___/_____ NUM___________ SCORE TOTAL_______________
Pontuação se as alterações estão presentes na ocasião da visita ou nos 10 dias precedentes
PESO DESCRITOR DEFINIÇÃO
8 Alteração neurológica Psicose. Acidente cérebro-vascular. Convulsão. Síndrome mental-orgânica (alteração da função mental com alterações clínicas flutuantes da memória, função intelectual, além da incapacidade de concentrar atenção ao meio ambiente, insônia e aumento ou diminuição da atividade psicomotora). Mononeurite. Mielite.
6 Alteração renal Cilindrúria. Hematúria> 5 hem/campo. Proteinúria > 0,5 g/l urina simples. Creatinina > 5 mg/dL.
4 Vasculite Ulceração, gangrena, nódulos dolorosos nos dedos, infarto periungueal, splinter hemorrágico. Biópsia ou angiografia revelando vasculite.
3 Hemólise / Trombocitopenia
Hb < 12 g/dL e reticulocitose corrigida > 3%. Trombocitopenia < 100.000
3 Miosite Fraqueza muscular proximal com elevação da CPK
2 Artrite Mais de 2 articulações com edema e derrame
2 Alteração mucocutânea Rash malar. Ulceração naso-faríngea. Alopecia.
2 Serosite Pleurisia. Pericardite. Peritonite.
1 Febre / Fadiga Febre > 38 graus e fadiga inexplicada.
1 Leucopenia / Linfopenia Leuco < 4000. Linf < 1200
58
6.2 Parecer da aprovação do projeto
59
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