UNIVERSIDADE FEDERAL DE SANTA MARIA

CENTRO DE CIÊNCIAS RURAIS

PROGRAMA DE PÓS-GRADUAÇÃO EM MEDICINA VETERINÁRIA

Marcella Barrella Ambrosio

DOENÇA RENAL CRÔNICA E UREMIA EM GATOS DOMÉSTICOS

Santa Maria, RS
2020
 Marcella Barrella Ambrosio

DOENÇA RENAL CRÔNICA E UREMIA EM GATOS DOMÉSTICOS

Dissertação apresentada ao Curso de Pós-

Graduação em Medicina Veterinária na
Universidade Federal de Santa Maria (UFSM,
RS), como requisito para a obtenção do título de
Mestre em Medicina Veterinária.

Orientadora: Profª Drª. Glaucia Denise Kommers

Santa Maria, RS
2020
 Marcella Barrella Ambrosio

DOENÇA RENAL CRÔNICA E UREMIA EM GATOS DOMÉSTICOS

Dissertação apresentada ao Curso de Pós-

Graduação em Medicina Veterinária na
Universidade Federal de Santa Maria (UFSM,
RS), como requisito para a obtenção do título de
Mestre em Medicina Veterinária.

Aprovado em 18 de fevereiro de 2020:

_____________________________________
Glaucia Denise Kommers, PhD (UFSM)
(Presidente/Orientador)

_____________________________________
Maria Andréia Inkelmann, Dra. (UNIJUÍ)

_____________________________________
Mariana Matins Flores, Dra. (UFSM)

Santa Maria, RS
2020
 AGRADECIMENTOS

Agradeço em primeiro lugar minha família por estar comigo em todos os momentos,
nem sempre fisicamente, mas com certeza em pensamento. Desejando sempre o melhor e
torcendo por mim, apoiando meus sonhos, mesmo quando o mundo todo me fazia pensar em
desistir.
Aos amigos, tanto do LPV-UFSM, quanto os de Santa Maria, São Paulo, do mundo e
de outras galáxias, pelo companheirismo e por colocarem sal e açúcar na minha vida. Da vida
nada se leva, apenas as memórias dos bons momentos que passamos juntos.
Agradeço à minha orientadora Glaucia Kommers pelas oportunidades que me
proporcionou, pela confiança, apoio e pelo aprendizado. Por me fazer me sentir um ser-
humano único, e não apenas um número, com compreensão das minhas virtudes e defeitos.
Agradeço à professora Mariana Flores, pela igual oportunidade de aprendizado, pela
paciência e confiança. A convivência com ela permitiu que todo o caminho fosse menos
árduo.
Agradeço ao Programa de Pós-Graduação e a CAPES pelo apoio estrutural e
financeiro.
Aos médicos-veterinários, professores, pós-graduandos, residentes e estagiários que
pude compartilhar experiências desde que iniciei meu trajeto na Patologia Veterinária.
Deixo uma singela homenagem às famílias brasileiras que não puderam estudar em
uma universidade federal, nem ver seus filhos, nem ninguém da sua família com um diploma.
Não por falta de merecimento ou esforço, mas por viverem em uma sociedade não igualitária,
em que a educação não é oferecida de forma igual a todos. Que tudo que está sendo produzido
nessa universidade retorne de alguma forma a essas pessoas e que votemos nos nossos
representantes com sabedoria. No momento que vendermos nossa educação, venderemos,
portanto, nossa alma ao capital.
 “Que nada nos defina. Que nada nos sujeite.
Que a liberdade seja a nossa própria substância.”

(Simone de Beauvoir)
 RESUMO

DOENÇA RENAL CRÔNICA E UREMIA EM GATOS DOMÉSTICOS

AUTORA: Marcella Barrella Ambrosio

ORIENTADORA: Glaucia D. Kommers

Esta dissertação de mestrado foi dividida em duas partes, resultando em dois artigos
científicos. O primeiro artigo consistiu em um estudo comparativo entre as alterações
macroscópicas, histológicas e características imuno-histoquímicas de 25 gatos com doença
renal crônica (DRC) com fibrose intersticial. O diagnóstico morfológico de nefrite túbulo
intersticial crônica (NTIC) foi o mais comumente observado (20/25) e cinco gatos (5/25)
apresentaram doença glomerular primária. Foi observada redução do tamanho dos rins em 22
dos 25 casos e os rins diminuídos de tamanho apresentaram graus mais elevados de fibrose
intersticial (p=0.021) e a redução do tamanho renal foi correlacionada à severidade da
inflamação crônica (p=0.0039) e da fibrose intersticial (p<0,001). O aspecto macroscópico
arredondado dos rins, presente apenas na NTIC foi atribuído à fibrose intersticial, atrofia
tubular, obsolescência glomerular e redução da espessura da camada cortical lateralmente ao
hilo renal, região compatível com os polos renais. A imunomarcação celular para actina-alfa
de músculo liso (α-SMA) foi observada em todos os estágios da DRC, demonstrando a
importância dos miofibroblastos nos diferentes processos e graus de intensidade da DRC com
desenvolvimento de fibrose intersticial. O segundo estudo teve como objetivo determinar a
prevalência e os aspectos clínicos da uremia em gatos, além dos aspectos patológicos e a
distribuição anatômica das lesões extrarrenais da uremia. No período estudado (janeiro de
2000 a outubro de 2019) foram necropsiados 1.330 gatos, dentre os quais 78 apresentaram
lesões extrarrenais de uremia (5,8%). Em 75% dos casos, a azotemia prolongada e uremia
foram consequência de doenças renais. A DRC na forma de NTIC foi o processo renal mais
comumente observado. As principais lesões extrarrenais de uremia observadas nos gatos
foram o edema pulmonar e a gastrite hemorrágica e/ou ulcerativa. Mineralização de tecidos
moles e hiperplasia das paratireoides foram achados incomuns, e osteodistrofia fibrosa não foi
observada. A apresentação multissitêmica de lesões extrarrenais de uremia foi observada em
apenas 24% dos casos, e algumas lesões normalmente encontradas em cães urêmicos não
foram observadas nos gatos deste estudo.

Palavras-chave: Gato. Doença renal crônica. Uremia. Histoquímica. Imuno-histoquímica

 ABSTRACT

CHRONIC KIDNEY DISEASE AND UREMIA IN DOMESTIC CATS

AUTHOR: Marcella Barrella Ambrosio

ADVISOR: Glaucia Denise Kommers

This masters dissertation was composed of two different parts, resulting in two scientific
articles. The first article consisted of a comparative study between macroscopic, histological
and immunohistochemical features changes in 25 cats with chronic kidney disease (CKD)
with interstitial fibrosis. The morphological diagnosis of chronic tubular interstitial nephritis
(CTIN) was the most commonly observed (20/25) and five cats (5/25) had primary glomerular
disease. Reduction of kidney size was observed in 22 of the 25 cases and the reduced kidneys
had higher degrees of interstitial fibrosis (p = 0.021) and the reduction in kidney size was
correlated with the severity of chronic inflammation (p = 0.0039) and interstitial fibrosis (p
<0.001). The rounded gross appearance of the kidneys, observed only in CTIN, was attributed
to interstitial fibrosis, tubular atrophy, glomerular obsolescence and reduced thickness of the
cortical layer laterally to the renal hilum, a region compatible with the renal poles. Cellular
immunostaining for α-smooth muscle actin (α-SMA) was observed in all stages of CKD,
demonstrating the role of myofibroblasts in the different processes and degrees of severity of
CKD with the development of interstitial fibrosis. The second study aimed to determine the
prevalence and clinical aspects of uremia in cats, in addition to the pathological aspects and
the anatomical distribution of nonrenal uremic lesions. During the studied period (from
January 2000 to October 2019), 1,330 cats were necropsied in LPV-UFSM, of which 78 had
nonrenal uremic lesions (6%). In 75% of the cases, prolonged azotemia and uremia were the
result of kidney disease. CKD in the form of NTIC was the most commonly observed renal
process. The most frequente nonrenal uremic lesions observed in cats were pulmonary edema
and hemorrhagic and/or ulcerative gastritis. Soft tissue mineralization and parathyroid
hyperplasia were uncommon findings and fibrous osteodystrophy was not observed. The
multisystemic presentation of nonrenal uremic lesions was observed only in 24% of the cases,
and some uremic lesions usually observed in uremic dogs were not observed in the cats of this
study.

Keywords: Cat. Chronic kidney disease. Uremia. Histochemistry. Immunohistochemistry.

 SUMÁRIO

1 INTRODUÇÃO ................................................................................................................. 8
2 REVISÃO BIBLIOGRÁFICA ......................................................................................... 9
2.1 DOENÇA RENAL CRÔNICA ..................................................................................... 9
2.2 FIBROSE RENAL E MIOFIBROBLASTOS ............................................................. 12
2.3 INSUFICIÊNCIA RENAL, AZOTEMIA E UREMIA ................................................ 14
3 ARTIGO 1 ...................................................................................................................... 51
4 ARTIGO 2 ...................................................................................................................... 81
5 DISCUSSÃO ................................................................................................................... 72
6 CONCLUSÃO ................................................................................................................. 74
7 REFERÊNCIAS .............................................................................................................. 75
 8

1 INTRODUÇÃO

A doença renal crônica (DRC) é a forma mais comum de doença renal em gatos
domésticos (O’NEILL et al., 2014; POLZIN, 2017; SOSNAR et al., 2003) e sua prevalência
varia entre os estudos, podendo chegar até 50% (MARINO et al., 2014). Apesar de acometer
gatos de todas as idades, a maior parte dos animais com DRC consiste em adultos e idosos
(BARTGES, 2012; BROWN et al., 2016; DIBARTOLA et al., 1987). A DRC como causa de
morte de gatos é mais expressiva em animais idosos, quando comparada a animais jovens
(HAMILTON; HAMILTON; MESTLER, 1969; TOGNI et al., 2018).
Apesar de a DRC ser frequentemente observada em gatos na rotina diagnóstica do
Laboratório de Patologia Veterinária da Universidade Federal de Santa Maria (LPV-UFSM),
há características morfológicas (macroscópicas e microscópicas) nas lesões renais crônicas de
gatos que chamam a atenção, principalmente quando comparadas à espécie canina.
Exemplificando, durante a necropsia de gatos com DRC, é frequentemente observada a
acentuada redução do tamanho, com perda do formato normal, observando-se rins encolhidos
e arredondados (uni ou bilateralmente). O aspecto histológico observado na DRC da espécie
felina conta com comprometimento predominante do compartimento túbulo-intersticial.
De fato, as DRCs de origem glomerular não são tidas como a principal etiologia da
DRC nos gatos, diferentemente do que ocorre em humanos (SUMNU et al., 2015) e no cão
(CIANCIOLO et al., 2016). A forma de DRC mais comumente observada em gatos é a nefrite
túbulo-intersticial crônica (NTIC) sem causa específica, com evolução para fibrose intersticial
acentuada, na qual a etiologia, na maior parte dos casos, ainda é pouco compreendida
(BROWN, et al., 2016; CHAKRABARTI et al., 2013; DIBARTOLA, 1987; MCLELAND et
al., 2015; SCHERK, 2015). Assim, os objetivos deste estudo foram caracterizar
detalhadamente os aspectos macroscópicos, histológicos e imuno-histoquímicos de rins de
gatos em diferentes estágios de DRC, com ênfase no estudo da fibrose intersticial como a
alteração comum predominante na DRC.
Além da DRC, outras doenças do trato urinário, tanto inferior quanto superior, são
importantes na medicina felina. Visto que há uma escassez de informações sobre as lesões
extrarrenais de uremia em gatos na rotina diagnóstica do LPV-UFSM e a literatura utiliza o
cão como o principal modelo das lesões de uremia, objetivou-se também estudar a
prevalência, epidemiologia, manifestações clínicas e distribuição anatômica das lesões
extrarrenais de uremia em gatos.
 9

2 REVISÃO BIBLIOGRÁFICA

Nesta revisão de literatura será abordada, de maneira sucinta, a DRC em gatos, o

desenvolvimento da fibrose renal e a origem dos miofibroblastos, e por fim, aspectos da
insuficiência renal, azotemia e uremia.

2.1 DOENÇA RENAL CRÔNICA

A DRC é definida por anormalidades estruturais e funcionais envolvendo um ou os

dois rins, com período de desenvolvimento de pelo menos três meses. (POLZIN, 2017).
Apresenta um amplo espectro de abrangência de lesões histológicas, podendo variar de uma
lesão leve e unilateral, até comprometer parte significativa do parênquima de ambos os rins
(POLZIN, 2011).
A DRC é a forma mais comum de doença renal observada em gatos (O’NEILL et al.,
2014; POLZIN, 2017; SOSNAR et al., 2003). A prevalência foi estimada em estudo realizado
por Lund et al. (1999), sendo de 1,9 % considerando todos os gatos atendidos por um ano em
clínicas particulares do Estados Unidos. Segundo estudo de Sosnar et al. (2003), a prevalência
foi de 16,4% em 164 felinos analisados, podendo chegar até 50% (considerando população
de 100 animais) de acordo com Marino et al. (2013).
A DRC pode afetar animais de todas as faixas etárias (BARTGES, 2012;
DIBARTOLA et al., 1987), entretanto, é mais comum em gatos idosos, afetando 49% dos
gatos com mais de 15 anos (CHAKRABARTI et al., 2013). Em estudo realizado com 74
gatos, o intervalo etário de animais com DRC foi de nove meses a 22 anos de idade,
demonstrando que gatos de todas as idades podem manifestar a doença (DIBARTOLA et al.,
1987). Mesmo assim, a prevalência é demonstrada como maior em animais mais velhos,
sendo de 28% em gatos com mais de 12 anos e de 31% em gatos com mais de 15 anos
(LAWSON et al., 2015).
Em estudo realizado entre 1964 e 2013 acerca da causa de morte ou razão para
eutanásia de gatos necropsiados no LPV-UFSM, a DRC estava entre as principais causas de
morte em gatos, totalizando 2,64%. Considerando apenas os indivíduos idosos (com idade
superior a 10 anos), esse valor subiu para 53,5% (TOGNI et al., 2018).
Apenas em uma minoria dos casos é possível atribuir as lesões renais a doenças
específicas (CHAKRABARTI et al., 2013; MINKUS et al., 1994). A principal forma
 10

histológica da DRC observada em gatos é a NTIC (CHAKRABARTI et al., 2013;

DIBARTOLA, 1987; MCLELAND et al., 2015; SCHERK, 2015). Nessa doença os rins
normalmente se apresentam diminuídos de tamanho, firmes e com a superfície irregular
(BROWN et al., 2016; SCHERK, 2015). A lesão histológica consiste em infiltrado
inflamatório principalmente linfocítico no ambiente túbulo intersticial, acompanhado de
fibrose intersticial, degeneração, necrose e perda do epitélio tubular e obsolescência
glomerular (MCLELAND et al., 2015). A distribuição da lesão varia de multifocal a
segmentar (BROWN et al., 2016). O grau de comprometimento do tecido renal está
relacionado ao estágio clínico da doença (MCLELAND et al., 2015).
A causa da NTIC observada em gatos com DRC ainda não foi completamente
estabelecida pela literatura, é possível que fatores genéticos, individuais e ambientais possam
estar envolvidos no seu desenvolvimento (JEPSON, 2016). A idade avançada, exposição à
nefrotoxinas, hipotensão, desidratação e a ocorrência de eventos isquêmicos ao longo da vida
do animal, que possibilitem episódios brandos e recorrentes de insuficiência renal aguda
(IRA), são considerados como os principais fatores envolvidos na patogênese da DRC na
forma de NTIC (BROWN et al., 2016; JEPSON, 2016). Adicionalmente, períodos de
obstrução do trato urinário por nefrólitos ou ureterólitos são potencialmente capazes de causar
lesão aguda ao parênquima renal, o que pode evoluir para NTIC (JEPSON, 2016).
A glomerulonefrite crônica (GNC), considerada a principal causa de DRC em cães
(MACDOUGALL et al., 1986; MULLER-PEDDINGHAUS; TRAUTWEIN, 1977; VADEN,
2017), é menos frequentemente observada em gatos (DIBARTOLA et al., 1987, VADEN,
2017). De acordo com estudo realizado com biópsias renais de gatos com DRC, 70,4% dos
120 animais apresentavam NTIC, com médio a alto grau de severidade, com quantidade
variável de fibrose renal. Em segundo lugar, em termos de prevalência, estavam as GNCs,
incluindo todas as suas variedades, representando 14,8% dos casos (MINKUS et al., 1994).
Há dois possíveis mecanismos de desenvolvimento da GNC. O primeiro envolve a
formação de anticorpos contra antígenos da membrana basal glomerular e o segundo consiste
na formação de imunocomplexos na corrente sanguínea e deposição nos glomérulos. Nesse
caso, os antígenos que compõem os imunocomplexos podem ser endógenos, no caso de
doenças autoimunes, ou exógenos quando originados de infecções sistêmicas por bactérias,
vírus ou parasitas (ALPERS; FOGO, 2013, BRESHEARS; CONFER, 2017, SLAUSON;
LEWIS, 1979). Qualquer estímulo antigênico persistente é potencialmente capaz de causar
uma GNC (CIANCIOLO; MOHR, 2016). No caso da não identificação da doença primária, a
 11

GNC é considerada idiopática (GRANT; FORRESTER, 2001). Na Tabela 1 estão listadas as

doenças que podem estar associadas às GNCs nos gatos.

Tabela 1 – Doenças sistêmicas associadas às GNCs em gatos

Doenças sistêmicas
Neoplasias Distúrbios mieloproliferativos
Linfoma
Infecciosas Infecções bacterianas crônicas
Peritonite infecciosa felina
Imunodeficiência felina
Vírus da leucemia felina
Poliartrite por Mycoplasma gatae
Inflamatórias Pancreatite
Colângio-hepatite
Poliartrite crônica progressiva
Lupus eritematoso sistêmico
Outros Idiopática
Toxicidade por mercúrio
Glomerulopatia familiar (gatos da raça Abissínio)

Fonte: (VADEN; GRAUER, 2011; SCHERK, 2015; WHITE et al., 2008).

Gatos com GNC podem desenvolver síndrome nefrótica, caracterizada por

hipoproteinemia, proteinúria, hiperlipidemia, edema subcutâneo ou generalizado, perda de
peso e atrofia muscular (CIANCIOLO; MOHR, 2016; PRESSLER, 2011; WHITE et al.;
2008) ou diferentes graus de severidade de insuficiência renal crônica (IRC) (SCHERK,
2015).
Embora menos frequentes, existem outras possíveis etiologias envolvidas no
desenvolvimento da DRC na espécie felina. Dentre as doenças congênitas, a mais comumente
observada é a doença dos rins policísticos, que afeta principalmente gatos da raça Persa.
Outras doenças congênitas já identificadas são a amiloidose renal dos gatos Abissínios e
Siameses, doença glomerular dos gatos Abissínios e a displasia renal (REYNOLDS;
LEFEBVRE, 2013). As doenças adquiridas que podem levar à DRC são as nefrolitíases,
 12

pielonefrites bacterianas, infecções pelos vírus da leucemia felina (FelV) e da

imunodeficiência felina (FIV), recuperação incompleta do tecido renal após necrose tubular
aguda de origem tóxica, além das causas neoplásicas, que compreendem os linfomas e
carcinomas renais (CHAKRABARTI et al., 2013; REYNOLDS; LEFEBVRE, 2013).

2.2 FIBROSE RENAL E MIOFIBROBLASTOS

A fibrose se caracteriza pelo depósito excessivo de colágeno e outros componentes da

matriz extracelular (MEC) em um determinado tecido ou órgão (KUMAR; ABBAS; ASTER,
2013). A fibrose renal é a substituição do parênquima renal por tecido conjuntivo fibroso
maduro (BRESHEARS; CONFER, 2017).
Os rins são potencialmente capazes de se regenerar (LAWSON et al., 2015) e a fibrose
ocorre como mecanismo alternativo à não regeneração dos componentes do néfron, quando
as nefrotoxinas não são completamente removidas, quando não há manutenção da
integridade da membrana basal tubular após lesões isquêmicas, ou quando o epitélio tubular
não resiste às agressões dos agentes agressores. Pode envolver os glomérulos, túbulos e
interstício, e infrequentemente trata-se de um evento primário (BRESHEARS; CONFER,
2017).
A fibrose é o evento que marca o estabelecimento da DRC (MACK; YANAGITA,
2014). Quando a fibrose é acentuada e extensa há destruição do parênquima renal, levando à
perda estrutural e prejuízos funcionais ao órgão, e é comum o desenvolvimento de
insuficiência renal crônica e uremia (BRESHEARS; CONFER, 2017; SUN et al., 2016). O
mecanismo da progressão da fibrose não é completamente elucidado, mas acredita-se que
proteinúria, inflamação crônica, hipóxia, idade avançada dos animais e hiperfosfatemia
estejam envolvidas no estabelecimento e aceleração desse processo (LIU, 2006).
Os fibroblastos são o principal constituinte celular do interstício renal. São células
mesenquimais quiescentes, fusiformes e alongadas, conhecidas pela expressão de marcadores
celulares como receptor β do fator de crescimento derivado de plaquetas (PDGFR- β) e CD73
(MACK; YANAGITA, 2014; STRUTZ; ZEISBERG, 2006). Promovem sustentação do tecido
renal e são responsáveis pela homeostase do ambiente renal mediante a produção e
degradação equilibrada de MEC (STRUTZ; ZEISBERG, 2006; SUN et al., 2016). Atuam
também na organização do ambiente intersticial, promovendo comunicação celular com o
epitélio tubular, endotélio vascular e células hematopoiéticas circulantes (SUN et al., 2016).
 13

Miofibroblastos são células responsáveis pela produção de MEC nos rins com fibrose
intersticial, e compartilham características estruturais com fibroblastos, porém expressam a
proteína citoplasmática actina-alfa de músculo liso (α-SMA) e possuem alta atividade
proliferativa. São células contráteis, alongadas, com projeções citoplasmáticas constituídas de
microfilamentos, retículo endoplasmático rugoso bem desenvolvido e contam com
hemidesmossomos (STRUTZ; ZEISBERG, 2006). Foram identificadas em humanos e em
diversos modelos animais de fibrose renal, principalmente no interstício e em menor
quantidade nos glomérulos (SUN et al., 2016). São consideradas as células responsáveis pela
deposição excessiva de MEC nos processos de fibrose renal e podem ser detectadas pela
expressão de α-SMA (STRUTZ; ZEISBERG, 2007).
Apesar de muito estudada, ainda é controversa a origem dos miofibroblastos no tecido
renal, pois há marcada variedade entre os resultados de diferentes grupos de pesquisa. A
dificuldade se dá principalmente pela da heterogeneidade das células identificadas nos estudos
e pelo fato dessas pesquisas utilizarem diferentes modelos experimentais. De maneira sucinta,
esses estudos consistem em rastreamento de linhagem celular in vivo, e o principal modelo
utilizado é a obstrução ureteral unilateral em roedores, que reproduz a fibrose intersticial e
lesão tubular em relativamente pouco tempo (MACK; YANAGITA, 2014). O consenso
existente mais atual é que os miofibroblastos renais possuam múltiplas origens. As teorias
mais aceitas são que a transdiferenciação de fibroblastos residentes e a migração de células
precursoras hematopoiéticas para os rins sejam a fonte mais expressiva. Outra possibilidade é
que uma menor parcela seja limitada aos pericitos, e aos processos de transição epitelial-
mesenquimal das células epiteliais tubulares, e endotelial-mesenquimal das células endoteliais
vasculares (MACK; YANAGITA, 2014, STRUTZ; ZEISBERG, 2006, SUN et al., 2016).
Em humanos, a expressão de α-SMA em biópsias de rins de pacientes com DRC está
diretamente relacionada à intensidade de fibrose intersticial e, portanto, à gravidade da DRC
(BOUKHALFA et al., 1996). O aumento da expressão de α-SMA em rins de felinos e caninos
com DRC também já foi reportado (ARESU et al., 2007, SAWASHIMA et al., 2000;
YABUKI et al., 2010).
Yabuki et al. (2010) reportou imunomarcação positiva para α-SMA no interstício de
rins de gatos com DRC e a expressão foi correlacionada estatisticamente com o aumento dos
valores da creatinina plasmática e ao grau de fibrose intersticial, indicando que as células α-
SMA positivas, interpretadas como miofibroblastos, desempenham um papel fundamental no
processo fibrogênico e na progressão da DRC. Considerando a origem dos miofibroblastos
nesse estudo, a expressão de vimentina, marcador celular expresso por células mesenquimais,
 14

foi observada nos túbulos renais e foi correlacionada com a fibrose intersticial e a expressão
de α-SMA, sugerindo que a transição epitelial-mesenquimal seja um possível mecanismo
envolvido no surgimento dos miofibroblastos na DRC do gato.
Em estudo imuno-histoquímico em gatos com nefrite túbulo-intersticial crônica
realizado por Sawashima et al. (2000), foi observada imunomarcação para α-SMA no
interstício peritubular e periglomerular nas áreas de fibrose intersticial. Além disso, a
expressão de α-SMA foi correlacionada positivamente com o aumento de ureia e creatinina
plasmáticas. Torna-se interessante ressaltar que nesse estudo a expressão de α-SMA foi
observada em estágios iniciais da lesão renal, antes mesmo da colagenização tecidual,
indicando que α-SMA pode ter utilidade na detecção precoce da fibrose renal em gatos.

2.3 INSUFICIÊNCIA RENAL, AZOTEMIA E UREMIA

Insuficiência renal ocorre quando aproximadamente 75% da capacidade funcional dos

rins está comprometida (BRESHEARS; CONFER, 2017; BROWN et al., 1997). IRA é
caracterizada por redução súbita da função renal (KHAN; KHAN, 2015) com oligúria ou
anúria repentinas e azotemia (CIANCIOLO; MOHR, 2016).
As principais causas de IRA são a necrose tubular aguda de origem infecciosa
(bacteriana ou viral), tóxica (medicamentosa, plantas tóxicas ou metais pesados), isquemia
renal, eventos obstrutivos, processos neoplásicos do trato urinário inferior ou descompensação
de uma lesão renal crônica. A IRA costuma ser um processo reversível (ALPERS; FOGO,
2013; BREASHEARS; CONFERS, 2017; CIANCIOLO; MOHR, 2016; SCHERK, 2015;
SERAKIDES; SILVA, 2016). As manifestações clínicas e alterações bioquímicas costumam
ser mais severas na IRA, pois mecanismos compensatórios sistêmicos ainda não foram
estabelecidos (DIBARTOLA; WESTROPP, 2015).
A IRC é o resultado comum das doenças renais crônicas, normalmente de caráter
irreversível, caracterizada por poliúria, polidipsia, perda de peso, anemia arregenerativa, e
sinais prolongados de uremia (CIANCIOLO; MOHR, 2016; DIBARTOLA;WESTROPP,
2015). Algumas doenças renais crônicas com curso clínico silencioso e insidioso podem
resultar em manifestações clínicas de IRA, pela ocorrência repentina (CIANCIOLO; MOHR,
2016).
Azotemia é a elevação do nível plasmático de pequenas moléculas hidrossolúveis,
compostos ligados a proteínas e moléculas de peso molecular intermediário (BRESHEARS;
 15

CONFER, 2017; DIBARTOLA; WESTROPP, 2015; ROSS, 2011), devido à retenção

causada por doenças renais ou causas extrarrenais (CIANCIOLO; MOHR, 2016). Dentre
essas moléculas a ureia e a creatinina são os compostos mais estudados e melhor
caracterizados, e identificados rotineiramente nos exames bioquímicos dos animais
(DIBARTOLA; WESTROPP, 2015; ROSS, 2011).
A azotemia pré-renal ocorre devido à menor perfusão dos rins por desidratação severa,
perda de volemia por hemorragias graves ou insuficiência cardíaca congestiva. A azotemia
pós-renal ocorre quando há obstrução do trato urinário inferior (ureteres, vesícula urinária ou
uretra) (ALPERS; FOGO, 2013; CIANCIOLO; MOHR, 2016).
A uremia consiste em uma síndrome clínica resultante do acúmulo de toxinas
urêmicas na circulação, e envolve manifestações clínicas e lesões multissistêmicas
(BRESHEARS; CONFER, 2017; ROSS, 2011). Alterações bioquímicas, metabólicas e
endócrinas estão envolvidas no seu desenvolvimento (BRESHEARS; CONFER, 2017;
CIANCIOLO; MOHR, 2016). As doenças renais crônicas são as principais causadoras dessa
síndrome, devido ao caráter progressivo e irreversível. No entanto, injúrias agudas ou eventos
obstrutivos do trato urinário inferior também podem causar uremia (CLARKSON; THOMAS,
2011).
Os sinais clínicos observados em animais com uremia são múltiplos e variam de
acordo com o indivíduo, grau e velocidade da perda da função renal, e tempo que o animal
permaneceu vivo em estado urêmico (BRESHEARS; CONFER, 2017; ROSS, 2011). Dentre
os principais estão anorexia, perda de peso, letargia, náusea, vômito, halitose urêmica,
estomatite e glossite ulcerativas, diarreia, melena ou hematoquezia, dificuldade respiratória
(associada ao edema pulmonar), alterações neurológicas e de estado de consciência
(encefalopatia urêmica), fragilidade e fraturas ósseas (associadas à osteodistrofia fibrosa) e
hemorragias (DIBARTOLA; WESTROPP, 2015; PETERS et al., 2005; POLZIN, 2017).
Os mecanismos envolvidos nas lesões da uremia são a necrose fibrinoide vascular
resultando em aumento da permeabilidade vascular, vasculite, trombose, infarto e necrose
tecidual; injúria cáustica dos epitélios devido à conversão de ureia salivar ou gástrica em
amônia pela atividade bacteriana (BRESHEARS; CONFER, 2017); redução da resistência
dos eritrócitos na circulação, redução da hematopoese (CHALHOUB, LANGSTON,
EATROFF, 2011); e desbalanço entre cálcio e fósforo (CIANCIOLO; MOHR, 2016).
As lesões de uremia são mais comumente observadas na espécie canina
(CIANCIOLO; MOHR, 2016). As principais lesões extrarrenais de uremia são a estomatite e
glossite ulcerativas e necrosantes, mais observadas em cães e gatos; as lesões gástricas, que
 16

ocorrem em cães e menos comumente em gatos urêmicos, e consistem em edema da mucosa

gástrica, com necrose e ulcerações, além de mineralização das camadas médias e profundas da
parede gástrica; colite ulcerativa e hemorrágica, mais comumente observada em bovinos e
equinos; pericardite fibrinosa, e arterite envolvendo átrios, aorta e tronco pulmonar, também
são lesões observadas na espécie canina (GOLDSTEIN et al., 1998; BOEDEC et al., 2012;
BRESHEARS; CONFER, 2017; PETERS et al., 2005; POLZIN, 2017). A maior parte dos
animais urêmicos apresenta edema pulmonar, e a pneumopatia urêmica, caracterizada por
edema, mineralização dos septos alveolares com deposição de fibrina e inflamação
granulomatosa, é geralmente observada na espécie canina (BRESHEARS; CONFER, 2017;
CIANCIOLO; MOHR, 2016). Calcificação dos tecidos moles ocorre principalmente em cães,
e o sítio anatômico mais afetado é o tecido conjuntivo subpleural dos espaços intercostais
craniais; locais menos frequentes de mineralização são coração, aorta, laringe, diafragma,
intestino, língua e traqueia (SILVEIRA et al., 2015). Adicionalmente, animais urêmicos
podem apresentar anemia não regenerativa, hiperplasia das paratireoides e osteodistrofia
fibrosa (BRESHEARS; CONFER, 2017; CIANCIOLO; MOHR, 2016; GEDDES et al., 2013;
KING et al., 1992; POLZIN, 2017).
 51

1 3 ARTIGO 1 –

2 Chronic Kidney Disease with Interstitial Fibrosis in Cats: a Comparative Study between

3 Macroscopic and Histological Changes and the Presence of Myofibroblasts

4

5 M. B. Ambrosio*, E. C. Lamego*, L. A. S. Tondo*, S. M. P. de Melo*, L. M.

6 Eisenhardt*, L. C. Binder°, M. M. Flores*, R. A. Fighera*, G. D. Kommers*.

8 *Laboratório de Patologia Veterinária, Departamento de Patologia, Universidade Federal

9 de Santa Maria, Av. Roraima 1000, Camobi, Santa Maria-RS, Brasil.

10 °Laboratório de Doenças Parasitárias, Departamento de Medicina Veterinária Preventiva e

11 Saúde Animal, Universidade de São Paulo, Av. Prof. Dr. Orlando Marques de Paiva, 87,

12 Cidade Universitária, São Paulo-SP, Brasil.

13

14

15

16

17

18

19 Corresponding author: G. D. Kommers (e-mail: [email protected]).

20

21

22
 52

1 Abstract

3 Chronic kidney disease (CKD) is frequently seen in domestic cats, and chronic

4 tubulointerstitial nephritis (CTIN) is the most common morphological form of the disease.

5 Interstitial fibrosis is the main histological component in the kidneys of cats with CKD, and

6 its role in the progression of the disease has been a recent focus of attention. Although the

7 etiology behind the majority of the cases of CKD in cats is not completely understood, it is

8 already well established that tubulointerstitial damage is the main event leading to kidney

9 failure and to end-stage kidney disease. Thus, the early diagnosis of tubulointerstitial

10 histological lesion would contribute to the development of therapies aiming to retard or even

11 to prevent the progression of CKD in cats. The main objective of this study was to perform a

12 gross, histological and immunohistochemical characterization of the kidneys of 25 cats with

13 CKD, considering tissue loss and the development of interstitial fibrosis, aiming to contribute

14 to the understanding of morphological changes in renal tissue of cats with CKD. The

15 morphological diagnosis of CTIN was the most commonly observed (20/25), followed by

16 chronic glomerulonephritis (CG) (5/25). The kidneys reduced in size had higher degrees of

17 interstitial fibrosis (p = 0.021) and the reduction in renal size was correlated with the severity

18 of chronic inflammation (p = 0.0039) and interstitial fibrosis (p <0.001). The rounded gross

19 appearance of the kidneys, present only in CTIN, was attributed to interstitial fibrosis, tubular

20 atrophy, glomerular obsolescence and reduced thickness of the cortical layer laterally to the

21 renal hilum, a region compatible with the renal poles. Cellular immunostaining for alpha

22 smooth muscle actin (α-SMA) was observed in all stages of CKD, demonstrating the

23 importance of myofibroblasts in the development of interstitial fibrosis in the kidneys of cats

24 with CKD.

25 Keywords: chronic kidney disease; cat; myofibroblasts; interstitial fibrosis

 53

1 Introduction

3 Chronic kidney disease (CKD) is considered nowadays the most important metabolic

4 disease in feline medicine (Brown et al., 2016; Jepson, 2016) and the prevalence can reach up

5 to 50%, depending on the population of cats studied (Marino et al., 2014). Further, there is an

6 increase in the prevalence of the disease considering aged cats (DiBartola et al., 1987; Minkus

7 et al., 1994; Bartges, 2012). The disease presents a wide variety in the intensity of histological

8 impairment, ranging from a mild and unilateral lesion to extensive damage affecting both

9 kidneys (Polzin, 2011).

10 Although congenital and acquired diseases can lead to CKD in cats

11 (Chakrabarti et al., 2013; Reynolds and Lefebvre, 2013), in most of the cases it is not possible

12 to define the specific cause of the disease at the time of diagnosis (DiBartola et al., 1987,

13 Minkus et al., 1994; Chakrabarti et al., 2013). The morphological diagnosis most commonly

14 attributed to cats with CKD is chronic tubulointerstitial nephritis (CTIN) with interstitial

15 fibrosis (DiBartola et al., 1987; Minkus et al., 1994; Chakrabarti et al., 2013; McLeland et al.,

16 2015).

17 Renal fibrosis is considered the final common pathway of CKD (Liu, 2006; Lawson et

18 al., 2015; Cianciolo and Mohr, 2016) and it is one of the main histological elements present in

19 feline CKD (Chakrabarti et al., 2013; McLeland et al., 2015). Many studies have dedicated

20 efforts to understand the relationship between interstitial fibrosis and the disease progression.

21 It is already well established in human medicine that, regardless of the primary cause of CKD,

22 changes in the tubulointerstitial environment are related to the reduction of renal function

23 (Schainuck et al., 1970; Nath, 1992).

24 Studies point out to interstitial fibrosis as the histological component best related to

25 the degree of severity of CKD in cats and to the worsening of clinical and laboratory findings
 54

1 (Chakrabarti et al., 2013; McLeland et al., 2015). The development of renal fibrosis in CKD

2 has been associated with the proliferation of myofibroblasts, cells of the renal interstice

3 responsible for the excessive deposition of extracellular matrix (ECM) in chronic processes

4 (Strutz and Zeisberg, 2007). These cells have already been detected in human CKD

5 (Boukhalfa et al., 1996; Novakovic et al., 2012), and in the CKD of dogs (Aresu et al., 2007;

6 Yabuki et al., 2010; Kutlu and Alcigir, 2019) and cats (Sawashima et al., 2000; Yabuki et al.,

7 2010; Kutlu and Alcigir, 2019) through the expression of the alpha isoform of smooth muscle

8 actin protein (α-SMA).

9 Kidneys with advanced CKD usually become small and fibrous, and the marked loss

10 of renal mass is usually associated with reduction of functional capacity of the organ

11 (Cianciolo and Mohr, 2016). In human medicine, studies looking for correlations between

12 macroscopic renal changes (assessed by imaging exams) and histological lesions revealed that

13 most patients (69%) with reduced kidney size had severe chronic kidney damage in

14 histopathological analysis (Moghazy et al., 2005).

15 Although the specific etiology for many cases of CKD in cats is not already

16 completely understood, the knowledge of histological events and cellular components

17 involved in renal damage in cats CKD becomes useful to enable an accurate diagnosis of the

18 disease and also to contribute to the development of therapies aiming to prevent or to slow

19 down the progression of the injury. Thus, this study was designed to perform a macroscopic,

20 histological and immunohistochemical characterization of kidneys of cats with different

21 degrees of severity of CKD, aiming to contribute to the understanding of morphological

22 changes in the renal tissue, taking into account tissue loss, the development of interstitial

23 fibrosis, and its consequences on tissue morphology.

24

25
 55

1 Materials and Methods

2 For the morphological study of CKD, 25 necropsied cats were selected at the LPV-

3 UFSM necropsy records between the years 2006 and 2019. The inclusion criteria of the study

4 were the presence of chronic histological lesion, no evidence of “acute-on-chronic” disease,

5 absence of obstructive episodes (urolithiasis) before the development of renal injury, and

6 sufficient availability of tissue included in paraffin blocks to perform serial histological

7 sections. Kidneys from ten cats also necropsied in the LPV-UFSM between the years 2018

8 and 2019, without gross and histological kidney changes and without clinical history of

9 kidney disease were used as normal controls.

10 Information regarding the age, sex and breed of each case was obtained from the

11 necropsy records. Kidney shrinkage (unilateral or bilateral) and changes in the kidney normal

12 conformation (shrinking and rounding) were assessed, based on the anatomical characteristics

13 and the normal size of the kidneys for the feline specie. In addition, other macroscopic

14 changes such as increased consistency (firm kidneys), colour change, irregularity of the

15 capsular surface; reduction of the cortical layer, presence of white streaks and areas of

16 depression of the renal parenchyma (infarcts / scars) were also recorded.

17 The kidney samples of all cats had been collected during post-mortem examination,

18 fixed in 10% neutral buffered formalin, and embedded in paraffin according to the standard

19 histological procedure. Paraffin blocks were cut into serial and alternating 3mm sections, and

20 subsequently stained with hematoxylin-eosin (HE) for histological evaluation of the kidney

21 injury. Periodic Acid Schiff (PAS) and Masson's Trichrome stain (TM) were performed in all

22 cases in order to confirm thickening of the glomerular basement membranes (of the

23 glomerular capillaries and Bowman's capsule) and to better demonstrate the interstitial

24 collagenized connective tissue, respectively.

 56

1 In all cases the kidneys were assessed bilaterally, but in those where only one kidney

2 showed histological lesion, only the affected kidney was considered in subsequent

3 evaluations. In cases where the lesion was distinct between the two kidneys, both were

4 considered.

5 Tissues stained with MT and HE were scored for interstitial fibrosis and interstitial

6 inflammation, respectively, on a scale from 0 to 4, adapted from Chakrabarti et al. (2013) as

7 follows: 1 = no fibrosis/inflammation to 25% of the section compromised; 2 = 26% to 50% of

8 the section compromised; 3 = 51% to 75% of the section compromised; 4 = more than 75% of

9 the section compromised by fibrosis/inflammation.

10 The thickness of the cortical layer was obtained considering the average of four

11 consecutive measurements of renal cortex in a histological section of each kidney, obtained

12 using a microscope Olympus BX5 with digital camera DP21, controlled by the Olympus

13 CellScens Standard program. In cases where the cortical thickness was uniform, four random

14 measurements were taken, and in cases of irregular reduction, the largest and the shortest

15 distance were used.

16 The differences in the fibrosis and inflammation scores between kidneys with

17 macroscopic reduction and those without macroscopic reduction were obtained using Mann-

18 Whitney statistical test. Spearman's Correlation test was performed to verify the existence of a

19 correlation between the fibrosis and inflammation scores and the thickness of cortical layer.

20 Statistical tests were performed using the software R (R Development Core Team 2019).

21 Immunohistochemistry (IHC) as performed according to the following steps:

22 deparaffinization and rehydration of silanized slides with 2μm to 3μm sections; blocking

23 endogenous peroxidases with two treatments with 3% hydrogen peroxide (H202) for 10

24 minutes each; antigen retrieval by heating the sample for ten minutes in a citrate buffer (pH

25 6.0) in a microwave; blocking unspecific reactions in 5% casein in PBST at 25 ºC for 30 min;

 57

1 incubation with human anti-α-SMA antibody (clone 1 A4; α isoform of human smooth muscle

2 actin, EasyPath [EP-12-52833]) diluted at 1:200, at 25ºC for 60 min; incubation with one step

3 polymer (Kit EasyLink One [EP-12-20504]) at 25ºC for 30 min; washing was performed with

4 PBTS; immunoreactivity was detected with 3, 3’diaminobenzidine (DAB; Dako, Carpinteria,

5 CA) as chromogen for 3 to 5 min; and counterstaining was performed with Harris

6 haematoxylin. For negative control sections, PBST was used instead of the primary antibody.

7 For positive controls, sections of uterus of cats were used and smooth muscle cells from blood

8 vessels of the kidneys were used as internal positive controls.

9
10 Results

11

12 Renal histologic data were obtained from 25 cats with CKD of which age, sex and

13 breed information are in Table 1. The renal lesion of twenty cats (20/25; 80%) was

14 compatible with CTIN, and in five cases (5/25; 20%) the morphological diagnosis was of

15 chronic glomerulonephritis (CG).

16

17 Morphological aspects

18 Chronic tubulointerstitial nephritis (n=20)

19 The most common gross finding was the marked shrinkage and rounding of the

20 kidneys (Fig. 1). In most of the cases, capsular surface had a smooth and regular appearance,

21 usually with a focal area of parenchyma retraction (scarring), often located bilaterally to the

22 renal hilum (equivalent to the renal poles) (Fig. 2). The reduction in the size of rounded

23 kidneys varied from bilateral (10/20) (Fig. 3), to bilateral asymmetric (3/20) and unilateral

24 (5/20), with reduction of the right kidney in three cases, and of the left one in two cases. In

25 two cases (2/20), there was no reduction in kidney size. In three cases of unilateral kidney
 58

1 shrinkage, a less severe histological lesion was observed in the contralateral kidney, and in

2 three cases of asymmetric bilateral kidney shrinkage the histological lesion was distinct

3 between the two kidneys. In this way, 26 different histological lesions were observed in the

4 cases of CTIN.

5 On histology, there was variation in the intensity of interstitial fibrosis and

6 inflammation between cases and also in the cortical layer thickness (Table. 2). In general,

7 CTIN was characterized by predominantly lymphocytic interstitial inflammation with

8 interstitial fibrosis organized in bands, with radial orientation, and segmental multifocal

9 distribution (Fig. 4). These bands consisting of inflammation and fibrosis coincided with the

10 areas of reduced cortical thickness. All, or almost all of the glomeruli located within these

11 bands showed marked shrinkage, hyalinization (PAS-positive) and fibrosis of the glomerular

12 tuft (light blue colour on the TM) (Fig. 5) with capillaries loss, characterizing glomerular

13 obsolescence.

14 Tubules in these areas showed atrophy and loss of tubular lumens. Tubular loss with

15 tubular basement membrane disruption was frequently observed, with interstitial lipid present

16 in the interstice. The lipid content was sometimes observed inside the cytoplasm of foamy

17 macrophages. The areas of tissue retraction observed in the gross examination coincided with

18 the areas of greatest histological lesion (accentuated cortical thinning, glomerular

19 obsolescence, interstitial fibrosis and inflammation) (Fig. 6). In the bands consisting of

20 inflammation and interstitial fibrosis, it was possible to observe a marked proliferation of

21 small arteries usually surrounding the glomeruli with obsolescence. The morphologically

22 viable glomeruli near to the segmental areas of lesion showed moderate to severe

23 periglomerular fibrosis and Bowman's capsule thickening. Additional histological

24 components observed were intratubular hyaline casts (20 cases), multifocal mineralization (18

25 cases), tubular epithelial necrosis (11 cases) and intratubular birefringent crystals (7 cases).
 59

1
2

3 Chronic glomerulonephritis (n=5)

5 In four cases (4/5) the kidneys were reduced in size on gross examination, with

6 maintenance of the natural shape of the kidneys in all cases. Delicate granulation of the

7 capsular surface was observed, and at longitudinal section, the parenchyma showed variable

8 amount of whitish streaks. On histology, two cases were diagnosed as membranous CG, two

9 cases were diagnosed as membranoproliferative CG, and mesangioproliferative CG was

10 observed in one case. The latter was characterized by expansion of the mesangial matrix

11 associated with mesangial hypercellularity. Glomerular sclerosis was observed, characterized

12 by obliteration of glomerular capillaries by extracellular matrix in areas of the glomerulus

13 (segmental sclerosis), or of the entire glomerular tuft (global sclerosis). Sclerotic glomeruli

14 were observed randomly and with multifocal distribution.

15 Cases of CG presented variable intensity of interstitial inflammation and fibrosis;

16 cortical thinning was usually uniform (Table. 3). Multifocal lymphoplasmacytic inflammatory

17 infiltrate was observed, mainly in the periglomerular region and there was a predominance of

18 interstitial fibrosis in the corticomedullary junction, with uniform distribution throughout the

19 histological section. Intratubular hyaline casts (5 cases), mineralization foci (3 cases),

20 intratubular birefringent crystals (2 cases) and tubular epithelial necrosis (1 case) were

21 observed.

22

23 Statistic

24

25 There was a significant difference of interstitial fibrosis scores between kidneys with

26 macroscopic reduction than those kidneys without macroscopic diminution (p = 0,021) with
 60

1 significance set at the 5% level. The kidneys reduced in size showed greater interstitial

2 fibrosis scores than those without changes in size. Considering interstitial inflammation, there

3 was no significant difference between both groups. The decrease of cortical layer thickness

4 was found to be significantly correlated with interstitial inflammation score (p = 0.0039) and

5 also strongly correlated with interstitial fibrosis (p<0,001).

6 Immunohistochemistry

7 Control cats (n=10)

8 Smooth muscle cells from blood vessels demonstrated strong intracytoplasmic

9 immunostaining for α-SMA. In three cases, there were no other cells with positive

10 immunostaining for α-SMA. In the other cases, few glomerular cells showed weak

11 immunostaining for α-SMA, and a variable amount of strongly α-SMA positive spindle cells

12 were observed in the interstice of the corticomedullary junction (Fig. 8).

13

14 Chronic tubulointerstitial nephritis (n=20)

15

16 In the cortical region corresponding to the radial bands of chronic inflammation, α-

17 SMA immunostaining was markedly enhanced. The positive α-SMA cells ranged from

18 spindle shape to slightly stellate (Fig. 9). In the interstice of the corticomedullary junction

19 there was a marked α-SMA immunostaining and in the areas where interstitial fibrosis was

20 more pronounced, the positive cells were in close contact with the atrophic tubules. The

21 number of positive α-SMA cells was moderate in the external medulla, usually organized in

22 radial orientation throughout the tissue (Fig. 10), and mild to sparse in the internal medulla. In

23 fields free of histological lesion, immunostaining for α-SMA was similar to that observed in

24 the control kidneys.

 61

1 The morphologically healthy glomeruli, detected in the regions adjacent to the areas

2 of glomerular obsolescence showed a moderate amount of cells strongly positive for α-SMA.

3 In periglomerular spaces with deposition of fibrous tissue there were fusiform and delicate

4 positive cells. The number of positive cells inside the glomeruli decreased as the glomeruli

5 loss its cellularity and showed expansion of fibrous tissue (Fig. 11), until immunostaining

6 became absent in glomerular obsolescence.

7 In the areas of marked tissue retraction, located mainly laterally to the renal hilum, it

8 was possible to recognize a greater abundance of α-SMA-positive cells, with similar intensity

9 in the cortical, corticomedullary junction and medullary regions. These regions showed

10 proliferation of small arteries, which were evidenced by α-SMA positive smooth muscle cells.

11
12 Glomerulonefrite crônica (n=5):

13 Cells with positive immunostaining for α-SMA were located around the glomeruli,

14 in the middle of the inflammatory infiltrate and in the regions of periglomerular fibrosis

15 (Fig.12). In the corticomedullary junction positive α-SMA spindle cells were observed

16 throughout the histological section. No positive immunostaining was observed in the inner

17 medulla.
18

19 Discussion

20

21 The systematic and comparative analysis carried out in this study between gross and

22 histological aspects of the kidneys of cats with CKD allowed to the identification of a distinct

23 pattern of lesion present in the kidneys with CTIN. Shrinkage and rounding aspect, observed

24 in most cases of CTIN, especially in cases with more than 50% of the renal parenchyma

25 compromised by chronic inflammation and interstitial fibrosis (scores III and IV), was not
 62

1 observed in CG. The explanation for the round conformation of the kidneys with CTIN was

2 the accentuation of the histological lesion bilaterally to the renal hilum, with greater

3 narrowing of the cortical layer, greater intensity of inflammation and fibrosis and a higher

4 number of α-SMA positive cell, compatible with myofibroblasts, in that specific region. Such

5 changes resulted in flattening of renal poles, an alteration possibly similar to that previously

6 described as "scars" in the renal poles (Lucke, 1968). Interestingly, in the study conducted by

7 Lucke (1968), round kidneys had more marked histological lesion, while the cats had a

8 greater decline in kidney function, suggesting that this is the presentation of the final stages of

9 CKD in cats.

10 These areas of tissue loss, which led to the flattening of the renal poles, were

11 compatible with areas of greater intensity of interstitial inflammation and fibrosis. It is known

12 that CTIN, the most common presentation of CKD in cats (DiBartola et al., 1987; Chakrabarti

13 et al., 2013; McLeland et al., 2019), has as its main components chronic inflammation and

14 interstitial fibrosis, with multifocal to segmental distribution (Brown et al., 2016). These two

15 histological elements were observed in all cases of CTIN of this study, reaching up to 75% of

16 the renal parenchyma in some cases. The main recognized mechanism in the development of

17 interstitial fibrosis is chronic inflammation (Lawson, 2015), in which the recruitment of

18 inflammatory cells after tissue injury results in the production of pro-fibrotic cytokines, which

19 lead to the activation of myofibroblasts, and to the production of ECM (Meng, 2019). This is

20 one of the mechanisms recognized in the development of interstitial fibrosis in feline CTIN

21 (Lawson et al., 2015) and a correlation between chronic inflammation and interstitial fibrosis

22 has already been observed in the kidneys of cats with CKD (Chakrabarti et al., 2013).

23 Lymphocytes were the main cells present in the cases of CTIN in this study.

24 Lymphocytic inflammation has been described in the early stages of CKD in cats, becoming

25 more pronounced and accompanied by interstitial fibrosis in cases of greater impairment of

 63

1 the renal parenchyma (McLeland et al., 2015). Granulomatous inflammation, observed in

2 cases of more severe interstitial inflammation (score III and IV) in this study, was also

3 observed by McLeland et al. (2015), especially in cats with more than 50% of renal tissue

4 affected. In addition, a significant correlation has recently been established between the

5 infiltration of macrophages in renal tissue, interstitial fibrosis and the progression of CKD in

6 cats, indicating that macrophages also act in the development of interstitial fibrosis (Ohara et

7 al., 2019). Granulomatous inflammation is these cases is considered to be secondary to

8 ischemic tubular injury, with tubular rupture, and leakage of lipids from tubular epithelial

9 cells to the renal interstice (Brown et al., 2016; Schmiedt et al., 2016).

10 Glomerular obsolescence is characterized by thickening and contraction of the

11 glomerular basement membrane towards the vascular pole, and as the glomerulus undergoes

12 retraction, there is deposition of acellular fibrous connective tissue between the glomerular

13 tuft and the Bowman capsule, resulting in reduced size glomeruli (Hughson et al., 2002). This

14 type of lesion, frequently observed in CTIN in this study, has been described as the

15 predominant glomerular lesion in cats CKD (Chakrabarti et al., 2013). It is considered a

16 lesion secondary to the primary tubulointerstitial process, and it is morphologically

17 compatible with ischemic glomerular obsolescence (Brown et al., 2016). In humans,

18 glomerular obsolescence is attributed to reduced glomerular perfusion by narrowing of

19 interlobular arched artery secondary to vascular lesions (Hughson et al., 2012). In the case of

20 cats, glomeruli with obsolescence are considered part of atubular nephrons, and have been

21 associated with failure in the regeneration of atrophic tubules secondary to the expansion of

22 interstitial fibrous connective tissue or secondary to tubules that undergo ischemic

23 tubulorrhexis (Brown et al., 2019).

24 Current experimental studies have collaborated with the understanding of the

25 pathogenesis involved in feline CTIN (Schmiedt et al., 2016; Brown et al., 2019). Changes
 64

1 observed in the kidneys of cats six months after induced ischemic lesion (90 minutes of renal

2 ischemia) consisted of inflammation and interstitial fibrosis, tubular atrophy and glomerular

3 obsolescence with markedly compromised fields, interspersed with healthy areas (Brown et

4 al., 2019). This set of alterations described is very similar to the histological pattern observed

5 in spontaneous cases of CTIN in this study. The experimental findings have been compared to

6 the histological lesion frequently observed in cats naturally affected by CKD (Brown et al.,

7 2016; Brown et al., 2019). They support the hypothesis that multiple mild ischemic insults, or

8 one single severe ischemic insult, followed by failures in tissue repair and propagation

9 processes inherent to the feline specie, may be involved in the development of CKD in cats in

10 the form of CTIN (Jepson, 2016).

11 In most of the cases of CKD in this study, it was notable the reduction of kidneys’

12 size. The kidneys reduced in size had statistically higher scores of interstitial fibrosis than

13 normal sized kidneys. It was also notable that, with the reduction in renal size, the degrees of

14 inflammation were higher, but with no statistical difference for this parameter (p = 0.054).

15 Additionally, the strong correlation between the increase in the scores of interstitial fibrosis

16 and the reduction in renal cortex point to fibrosis as the main histological component present

17 in the loss of renal tissue. Chronic inflammation was also correlated with renal cortex

18 thinning, but there was a tendency for this parameter to show lower scores when compared to

19 fibrosis, which probably reflects a chronological characteristic of chronic kidney injury.

20 Inflammation is the initial response to tissue injury, and through the production of pro-fibrotic

21 factors and activation of ECM-producing cells (myofibroblasts) there is a predominance of

22 interstitial fibrosis in the most advanced terminal stages of CKD (Liu, 2006; Meng, 2019).

23 The detection of α-SMA in kidneys of cats with CKD allows the identification of

24 interstitial cells with positive immunostaining for this antibody and these cells are considered

25 to be myofibroblasts (Swashima et al., 2000; Yabuki et al., 2010). In fact, the anti-α-SMA
 65

1 antibody is a reliable tool and is considered the most efficient in the identification of renal

2 myofibroblasts, which by conventional histology cannot be differentiated from fibroblasts

3 (Strutz and Zeisberg, 2006).

4 In the present study, it was possible to identify spindle cells positive for α-SMA in

5 the renal interstice, both in CTIN and CG. Myofibroblasts were more evident in the interstice

6 of kidneys with CTIN, which showed more severe histological lesion. In CTIN, the

7 distribution of α-SMA-positive cells was compatible with the areas affected by chronic

8 inflammation and interstitial fibrosis and, therefore, areas with greater tissue loss. It is

9 possible that myofibroblasts are involved in the process of tissue loss in the kidneys of cats

10 with CKD, as these cells have a contractile cellular apparatus (contractile proteins) in the

11 composition of the cytoskeleton, which allows them to exert mechanical forces in the

12 contraction of wounds and in the pathological conditions that culminate in tissue contraction

13 and loss of delicate renal tissue architecture (Tomasek et al., 2002).

14 Glomerular mesangial cells act in the production of cytokines and growth factors

15 involved in fibrosis, and are an expressive source of cell differentiation in myofibroblasts in

16 the processes of glomerular sclerosis (Liu, 2016; Meng, 2019). Positive immunostaining for

17 α-SMA in these cells has already been reported in human glomerular diseases (Boukhalfa et

18 al., 1996). In the CTIN of the cats in this study, positivity for α-SMA was observed in

19 morphologically healthy glomeruli located near to damaged areas, with subsequent reduction

20 of the immunostaining in glomeruli undergoing obsolescence. This characteristic was

21 observed by Sawashima et al. (2000) in cats with CKD and these findings may indicate a

22 probable involvement of myofibroblasts originating from mesangial cells in the continuous

23 process of glomerular obsolescence. The loss of mesangial cells with the progression of the

24 lesion resulted in the absence of positive immunostaining in completely fibrous glomeruli.

 66

1 Cases of CG demonstrated less involvement of the parenchyma by chronic

2 inflammation and fibrosis (scores I and II) when compared to cases of CTIN. Even so,

3 positive α-SMA cells were also observed, especially in areas of interstitial fibrosis such as

4 periglomerular space corticomedullary junction. This finding demonstrates that, although

5 primary glomerular processes are infrequent in cats (DiBartola et al., 1987; Minkus et al.,

6 1994), they also culminate into tubulointerstitial injury. These consequences must be

7 considered, since the progression of CKD with renal failure is related to the extent of

8 tubulointerstitial involvement regardless of the primary cause (Nangaku, 2010).

9 Likewise, the observation of positive α-SMA cells in the renal interstice of cats with

10 initial CTIN and CG (with less than 25% of the parenchyma compromised by the

11 tubulointerstitial lesion) corroborates the findings of Sawashima et al. (2000), who reported

12 increased expression of α-SMA in kidneys of cats in early stages of CKD, with absent or mild

13 deposition of collagenous connective tissue in the renal interstice. The short interval between

14 tissue damage and myofibroblast proliferation in renal tissue was also demonstrated by the

15 presence of immunostaining for α-SMA in the initial repair phase after experimental ischemic

16 injury (Schmiedt et al., 2016). These evidences are very relevant, considering that the

17 expression of α-SMA may represent an early marker of tubulointerstitial damage in kidneys

18 of cats with CKD.

19 The reduction in kidney size was a common aspect observed in the CKD in this

20 study and it is suggestive that chronic inflammation with progression to interstitial fibrosis,

21 together with proliferation of myofibroblasts would be involved in this process. The reduction

22 in kidney size in CTIN occurred in a singular way, with frequent shrinkage and rounding of

23 the kidneys. This unique characteristic was attributed not only to higher degrees of intensity

24 of renal fibrosis, but also to the marked impairment of the glomeruli and tubules located in the
 67

1 renal perihilar region. The results of this study reinforce the participation of myofibroblasts in

2 the development of interstitial fibrosis in CKD in the feline species.

3

4 Acknowledgements

6 This research was financially supported by the Coordenação de Aperfeiçoamento de

7 Pessoal de Nível Superior (CAPES), Brazil. The student M. B. Ambrosio has a Master’s

8 scholarship from CAPES.

10 Conflict of Interest Statement

11

12 The authors declare no conflicts of interest with respect to the research, authorship or

13 publication of this article.

14

15

16

17
 68

1 References

3 Aresu L, Rastaldi MP, Scanziani E, Baily J, Radaelli E et al. (2007) Epithelial-mesenchymal

4 transition (EMT) of renal tubular cells in canine glomerulonephritis. Virchows Archiv:

5 European Journal of Pathology, 451, 937–942.

6 Bartges JW (2012) Chronic kidney disease in dogs and cats. Veterinary Clinics of North

7 America - Small Animal Practice, 42, 669–692.

8 Boukhalfa G, Desmouliere A, Rondeau E, Gabbiani G, Sraer JD (1996) Relationship between

9 alpha-smooth muscle actin expression and fibrotic changes in human kidney. Experimental

10 Nephrology¸ 4, 241–247.

11 Brown AB, Rissi DR, Dickerson VM, Davis AM, Brown SA (2019) Chronic Renal Changes

12 After a Single Ischemic Event in an Experimental Model of Feline Chronic Kidney

13 Disease. Veterinary Pathology, 56, 536-543.

14 Brown CA, Elliot J, Schmiedt CW, Brown SA (2016) Chronic kidney disease in aged cats:

15 clinical features, morphology, and proposed pathogeneses. Veterinary Pathology, 53, 309-

16 326.

17 Chakrabarti S, Syme HM, Brown CA, Elliot J (2013) Histomorphometry of feline chronic

18 kidney disease and correlation with markers of renal dysfunction. Veterinary Pathology,

19 50, 147–155.

20 Cianciolo RE, Mohr FC (2016) Urinary sistem. In: Jubb, Kennedy and Palmer’s Pathology of

21 Domestic Animals, 6th Edit., MG Maxie, Elsevier, St Louis, pp. 377-463.

22 DiBartola SP, Rutgers HC, Zack PM (1987) Clinicopathologic findings associated with

23 chronic renal disease in cats: 74 cases (1973 -1984). Journal of American Veterinary

24 Medical Association, 190, 1196–202.

 69

1 Hughson MD, Johnson K, Young RJ, Hoy WE, Bertram JF (2002) Glomerular size and

2 glomerulosclerosis: relationships to disease categories, glomerular solidification, and

3 ischemic obsolescence. American Journal of Kidney Diseases, 39, 679–688.

4 Jepson RE (2016) Current understanding of the pathogenesis of progressive chronic kidney

5 disease in cats. Veterinary Clinics of North America: Small Animal Practice, 46, 1015-

6 1048.

7 Kutlu T, Alcigir G (2019) Comparison of renal lesions in cats and dogs using

8 pathomorphological and ımmunohistochemical methods. Biotechnic & Histochemistry, 94,

9 126-133.

10 Lawson J, Elliot J, Wheeler-Jones C, Symed H, Jepson R (2015) Renal fibrosis in feline

11 chronic kidney disease: known mediators and mechanisms of injury. Veterinary Journal,

12 203, 18–26.

13 Liu Y (2006) Renal fibrosis: new insights into the pathogenesis and therapeutics. Kidney

14 International, 69, 213–217.

15 Lucke VM (1968) Renal disease in the domestic cat. Journal of Pathology and Bacteriology,

16 95, 67–91.

17 Marino CL, Lascelles BDX, Vaden SL, Gruen ME, Marks SL (2014) Prevalence and

18 classification of chronic kidney disease in cats randomly selected from four age groups and

19 in cats recruited for degenerative joint disease studies. Journal of Feline Medicine and

20 Surgery, 16, 1 –8.

21 McLeland SM, Cianciolo RE, Duncan CG, Quimby JM (2015) A comparison of biochemical

22 and histopathologic staging in cats with chronic kidney disease. Veterinary Pathology, 52,

23 524-534.
 70

1 McLeland S, Quimby J, Lappin MR (2019) Alpha-enolase staining patterns in the renal

2 tissues of cats with and without chronic kidney disease. Veterinary Immunology and

3 Immunopathology, 212, 23-26.

4 Meng XM (2019) Inflammatory mediators and renal fibrosis. Advances in Experimental

5 Medicine and Biology, 1165, p. 381-406.

6 Minkus G, Reusch C, Horauf P, Breuer W, Darbhs J et al. (1994) Evaluation of renal biopsies

7 in cats and dogs — histopathology in comparison with clinical data. Journal of Small

8 Animal Practice, 35, 465–472.

9 Moghazi S, Jones E, Schroepple J, Arya K, McClellan W et al. (2005) Correlation of renal

10 histopathology with sonographic findings. Kidney International, 67, 1515–1520.

11 Nangaku M (2004) Mechanisms of tubulointerstitial injury in the kidney: final common

12 pathways to end-stage renal failure. Internal Medicine, 43, 9–17.

13 Nath KA (1992) Tubulointerstitial changes as a major determinant in the progression of renal

14 damage. American Journal of Kidney Diseases, 20, 1-17.

15 Novakovic ZS, Durvov MG, Puljak L, Saraga M, Ljutic D (2012) The interstitial expression

16 of alpha-smooth muscle actin in glomerulonephritis is associated with renal function.

17 Medical Science Monitor, 18, 235-240.

18 Ohara Y, Yabuki A, Nakamura R, Ichii O, Mizukawa et al. (2019) Renal infiltration of

19 macrophages in canine and feline chronic kidney disease. Journal of Comparative

20 Pathology, 170, 53-59.

21 Polzin, DJ (2011) Chronic kidney diseases in small animals. Veterinary Clinics: Small Animal

22 practice, 41, 15–30.

23 Reynolds BS, Lefebvre HP (2013) Feline CKD: pathophysiology and risk factors-what do we

24 know. Journal of Feline Medicine and Surgery, 15, 3–14.

 71

1 Sawashima KO, Mizuno S, Mizuno-Horikawa Y, Shimada A, Kudo T et al. (2000).

2 Expression of α-smooth muscle actin and fibronectin in tubulointerstitial lesions of cats

3 with chronic renal failure. American Journal of Veterinary Research, 61, 1080–1086.

4 Schainuck LI, Striker GE, Cutler RE, Benditt EP (1970) Structural-functional correlations in

5 renal disease. II. The correlations. Human pathology, 4, 631-641.

6 Schmiedt CW, Brainard BM, Hinson W, Brown SA, Brown CA (2016) Unilateral renal

7 ischemia as a model of acute kidney injury and renal fibrosis in cats. Veterinary pathology,

8 53, 87-101.

9 Strutz F. Zeisberg M (2006) Renal fibroblasts and myofibroblasts in chronic kidney disease.

10 Journal of the American Society of Nephrology, 17, 2992–2998.

11 Tomasek JJ, Gabbiani G, Hinz B, Chaponnier C, Brown RA (2002) Myofibroblasts and

12 mechanoregulation of connective tissue remodelling. Nature Reviews Molecular Cell

13 Biology, 3, 349-363.

14 Yabuki A, Mitani S, Fujiki M, Misumi K, Endo Y, et al. (2010) Comparative study of chronic

15 kidney disease in dogs and cats: induction of myofibroblasts. Research in Veterinary

16 Science, 88, 294-299.

17
 72

10

11

12

13

14

15

16 Fig. 1. CTIN, kidney, cat. Kidneys bilaterally reduced in size and markedly rounded,

17 positioned dorsally in the retroperitoneum. In detail, the gross appearance of a control kidney.

18

19
 73

9 Fig 2. CTIN, kidney, cat. Left kidney rounded by flattening of the renal poles. Right kidney

10 with focal area of tissue retraction (scar), located laterally to the renal hilum.

11

12

13

14

15

16

17

18

19

20

21

22 Fig 3. CTIN, kidney, cat. Bilaterally reduced in size and rounding kidneys. There is a greater

23 narrowing of renal cortex bilaterally to the renal hilum, what turned into rounded gross

24 appearence.

25
 74

9 Fig 4. Light micrograph of a kidney of a cat with CTIN. Cortical lymphocytic inflammation is

10 accompanied by interstitial fibrosis organized in bands with radial orientation. PAS. Bar,

11 50mm.

12

13

14

15

16

17

18

19

20

21

22 Fig 5. Light micrograph of a kidney of a cat with CTIN. The glomerulus is diminished in size

23 and exhibits marked wrinkling of glomerular basement membrane, with capillary loss and

24 deposition of fibrous connective tissue (ischemic obsolescence). Masson’s trichrome stain.

25 Bar, 20mm.
 75

9 Fig 6. Light micrograph of a kidney of a cat with TICN. More than 75% of the renal

10 parenchyma is affected by interstitial fibrosis (collagenous matrix stained in blue). There is

11 collapse of cortical layer and marked tubular atrophy and loss. Masson’s trichrome stain. Bar,

12 100mm.

13

14

15

16

17

18

19

20 Fig 7. Light micrograph of a kidney of a cat with CG. There is periglomerular

21 lymphoplasmocytic inflammation and segmental to global glomerular sclerosis with

22 multifocal distribution. HE. Bar, 50mm.

23
 76

10 Fig 8. Immunohistochemical labelling for α-SMA in a control cat.

11 Few myofibroblasts in the interstice surrounding the distal convoluted tubules. In detail,

12 positive smooth muscle cells of the glomerular arteriole. Bar, 20mm.

13

14

15

16

17

18

19
*
20

21

22 Fig 9. Immunohistochemical labelling for α-SMA, CTIN, cat. Positive spindle cells within the

23 lymphocytic inflammatory infiltrate. In detail, myofibroblasts are visible surrounding a

24 glomerular obsolescence. No positive cells inside glomerular corpuscle. Bar, 20mm.

 77

9 Fig 10. Immunohistochemical labelling for α-SMA. NTIC, cat. Positive spindle cells with

10 radial orientation in outer medulla. Bar, 20mm.

11

12

13

14

15

16

17

18

19 Fig 11. Immunohistochemical labelling for α-SMA. CTIN, cat. Glomerular α-SMA

20 expression. Positive spindle cells inside glomeruli undergoing obsolescence. Bar, 20mm.

21
 78

9 Fig. 12. Immunohistochemical labelling for α-SMA. CG, cat. Fusiform to slightly stellate

10 positive cells in periglomerular lymphoplasmacytic inflammation. Bar, 20mm.

11

12

13

14

15

16

17
 79

1 Table 1

2 Age, sex and breed of 25 cats with CKD

Morphological Number of Cases Average ages Sex Breed

diagnosis (interval)

CTIN 20 11.4 (3 to 20 11 female 15 MB*

years) 9 male 5 Persian
1 Siamese

CG 5 11 (8 to 14 3 female 4 MB*
years) 2 male 1 Siamese

3 *mixed-breed cats

4 Table 2
5 Interstitial fibrosis and inflammation scores, gross changes and thickness of cortical
6 layer from 25 cats with CTIN.

Fibrosis Inflammation(*) Macroscopic Rounding Cortical layer

(*) shrinkage (*) thickness average in
(*) mm (interval)
VI (5) VI (1) 207.7
III (3) (5) (5) (171.03-265.04)
II (1)
I (0)

III (7) VI (0) 290.6

III (5) (7) (202.7-555.7)
II (2) (7)
I (0)

II (10) VI 361.3
III (7) (4) (505.04-276.9)
II (5)
I (5)

I (4) IV 403.3
III (2) (1) (330.3-541.6)
II
I (4)
7 *number of occurences

10
 80

1 Table 3

2 Interstitial fibrosis and inflammation scores, gross changes and thickness of cortical
3 layer from 5 cats with CG

Fibrosis Inflammation Macroscopic Rounding Cortical layer

(*) (*) shrinkage (*) thickness average in
(*) mm (interval)
II (3) VI (0) 303.9
III (0) (3) (0) (127.4-401.5)
II (3)
I (0)
I (2) IV (0) 537.5
III (0) (1) (0) (345.3-729.7)
II (0)
I (2)
4 *number of occurences

5
 81

1 4 ARTIGO 2 –

3 Nonrenal lesions of uraemia in 78 domestic cats

4 M. B. Ambrosio*, M. M. Hennig*, H. H. L. Nascimento, A. dos Santos, M. M. Flores*,

5 R. A. Fighera*, L. F. Irigoyen*, , G. D. Kommers*

7 *Laboratório de Patologia Veterinária, Departamento de Patologia, Universidade Federal

8 de Santa Maria, Av. Roraima 1000, Camobi, Santa Maria-RS, Brasil.

10

11

12

13

14

15 Autor para correspondência: G. D. Kommers (e-mail: [email protected]).

16

17

18

19 Artigo a ser submetido para a revista Jounal of Comparative Pathology,

20

21

22

23

24

25
 82

1 Summary

3 Uraemia is a clinical syndrome caused by the increase of uraemic toxins in blood

4 stream as a consequence of intrinsic kidney or lower urinary tract diseases. Cats seem to be

5 more affected by urinary diseases than dogs, especially considering chronic kidney disease

6 (CKD) as one of the most important illness for the specie. Considering the lack of information

7 regarding the systemic consequences of uraemia in cats, this study aims to investigate the

8 prevalence, clinical and pathologic aspects of nonrenal lesions of uraemia in cats with urinary

9 tract diseases, with special attention to the differences between cats and what is known for

10 dogs. Cats necropsied between 2000 and 2019 were investigated for urinary tract diseases and

11 nonrenal lesions of uraemia. The prevalence of cats with nonrenal uremic lesions was 5,8%,

12 and there was a higher number of adults and elderly animals when compared to young cats,

13 and more male cats were affected than female. Anorexia, apathy and vomiting were the most

14 common clinical signs reported, and CKD was observed in the majority of cats with uraemia.

15 Pulmonary oedema was the most frequent lesion observed, and there was a notable variation

16 between the gastric lesions observed in this study and what has been reported in the literature.

17 Haemorrhagic and ulcerative gastritis was frequently observed. Soft tissue mineralization and

18 parathyroid hyperplasia were uncommon features of cats with uraemia and fibrous

19 osteodystrophy was not observed. Cats with urinary tracts diseases of this study did not show

20 the same variety of nonrenal uremic lesions usually present in dogs with uremic syndrome

21 and multsisystemic presentation of uraemia was only observed in about 24% of the cases.

22

23 Keywords: cat, chronic kidney disease, uraemia.

 83

1 Introduction

3 Uraemia is a clinical syndrome associated with renal failure and lower urinary tract

4 diseases (Cianciolo and Mohr, 2016), caused by the increase of metabolic compounds, which

5 under normal conditions should be excreted by the healthy kidneys and involves clinical signs

6 and systemic lesions (Vanholder et al., 2003; Serakides and Silva, 2016). It is a complex event

7 that, besides the retention of toxins in the blood, involves derangements on hormonal balance

8 and enzymatic processes (Cowgill, 2003).

9 Clinical manifestations and gross findings of uraemia are important evidences of renal

10 failure. There are differences in the severity of uremic lesions between species, and they

11 depend mostly upon the period of time that the animal was exposed to uremic toxins. Thus, in

12 acute renal failure (ARF) these lesions can be less severe when compared to long term kidney

13 diseases (Cianciolo and Mohr, 2016; Breshears and Confer, 2017).

14 According to previous studies developed in this same laboratory (LPV-UFSM) about

15 uraemia in dogs, the syndrome prevalence ranged from 3,8% (Silveira et al., 2015) to 4,43%

16 (Dantas and Kommers, 1997), considering the total number of dogs necropsied during a

17 determined period of time. In another research aiming to determine the prevalence of renal

18 failure in dogs in a veterinary routine, it was found to be around 11% from a universe of 935

19 dogs. From these patients, 76.6% died of renal failure (Sosnar et al., 2003).

20 The literature often uses the dog as the main model to describe nonrenal uremic

21 lesions in domestic animals (Cianciolo and Mohr, 2016; Breshears and Confer, 2017), but the

22 urinary tract diseases also play an important role in morbidity and mortality of cats. The

23 prevalence of renal failure in cats according to Sosnar et al. (2003) has exceeded the

24 prevalence in dogs. Togni et al. (2018) reported that urinary tract diseases are among the main

25 causes of death and euthanasia in cats. Furthermore, CKD is the most common metabolic
 84

1 disease in cats, and the prevalence is considered higher than in dogs (Polzin 2011; Brown et

2 al., 2016). There are few researches specifically focused on the systemic consequences of

3 urinary tract diseases in the feline specie, and the more accurate knowledge of the type and

4 frequency of development of uremic lesions may lead to advances in the management of

5 uremic patients.

6 Therefore, the aim of this study was to characterize the prevalence, clinical and

7 pathological aspects of uraemia in cats, considering the anatomical distribution of lesions,

8 attempting to the main differences between the development of the syndrome in dogs and

9 cats.

10 Material and Methods

11

12 Cases of cats with nonrenal lesions of uraemia submitted to necropsy were

13 investigated in the archives of the LPV-UFSM between January 2000 and October 2019.

14 Cases were included in the study when the necropsy reports showed urinary tract diseases as

15 the cause of death concomitant with lesions considered nonrenal lesions of uraemia according

16 the specific literature (Breshears and Confer, 2017). The total number of cats necropsied in

17 the laboratory in this period was also recorded.

18 Information about age, sex and breed, clinical signs, laboratory findings (blood urea

19 nitrogen, serum creatinine concentration and haematocrit value, according to [Fielder, 2020]),

20 gross and histologic findings were taken from necropsy reports. Considering the age category,

21 cats were divided in three groups, according to Togni et al. (2018): young (less than one year

22 old), adults (from one to ten years old) and elderly (more than ten years).

23 The type of primary injury that led to azotaemia and, consequently, to the uremic

24 syndrome was analysed and divided into prerenal, renal and postrenal causes. Nonrenal

25 uremic lesions were described according to gross and histologic aspects and the anatomical
 85

1 distribution for each case, according to specific literature (Breshears and Confer,

2 2017). Paraffin blocks of selected cases were sectioned in 3.0 µm thick and submitted to

3 haematoxylin and eosin (HE) technique in order to illustrate this study.

4 Results

6 During the studied period, 1.330 cats were necropsied in the LPV-UFSM and 78 cats

7 (5.8%) presented nonrenal uremic lesions. Table 1 shows information referent to age, sex and

8 breed of the studied cats. In 72 cases (72/78, 92%) cats had clinical signs related to uraemia

9 (Table 2). Additional clinical signs reported were dehydration, hypothermia, weight loss,

10 polyuria, dysuria, haematuria, anuria, polydipsia, salivation, abdominal pain, diarrhoea and

11 dyspnoea.

12 In 27 cases, there was available data about the increase of urea and creatinine values,

13 which characterized azotaemia, and reduction of haematocrit value was only mentioned in

14 nine cases. Creatinine values, when reported, ranged from 3.2 mg/dl to 26.0 mg/dl (reference

15 ranges 0.9-2.2 mg/dL) and urea values ranged from 300.0 mg/dl to 767.0 mg/dl (reference

16 ranges 19-34 mg/dL). The minimum haematocrit value observed was 9% and the maximum,

17 21% (reference ranges 30-45%)

18 Considering the origin of azotaemia, and consequently the origin of nonrenal uraemic

19 lesions (Table 3), 58 cases (58/78; 74.3%) were attributed to renal causes. In two cases,

20 azotaemia was caused by concomitant renal and post renal lesions (acute tubular injury with

21 necrohemorrhagic cystitis and chronic interstitial nephritis with traumatic ureteral rupture). In

22 18 (18/78; 23%) it was a consequence of a postrenal injury. The frequency and distribution of

23 uremic lesions are demonstrated in Table 4 and 5.

24 The most prevalent uremic lesion was pulmonary oedema which affected 40 cats

25 (40/78; 51.2%). Usually, the lungs were wet, heavy and slightly reddened. The cut surface of
 86

1 the parenchyma drained variable amounts of fluid and the trachea contained a foamy fluid.

2 Histology showed light eosinophilic amorphous material inside alveolar lumens (Fig. 1). In

3 five cases, beyond pulmonary oedema, lesion of uremic pneumopathy was also observed.

4 They were characterized by a firm lung parenchyma with multiple slightly white and firm

5 areas in the pleural surface. On the histology, there were granular basophilic deposits in

6 alveolar septa, interpreted as multifocal mineralization, with variable amounts of

7 inflammatory infiltrate of neutrophils and foamy alveolar macrophages with deposition of

8 strongly eosinophilic fibrillar material (fibrin).

9 The second most common uremic lesion observed was ulcerative or erosive and

10 haemorrhagic gastritis (28/78; 35.8%). Gastric mucosa showed multiple ulcers and/or

11 erosions with haemorrhagic content in most of the cases (Fig. 2). In 22/28 cases the stomach

12 had a strong ammoniac odour when opened, in three cases there was mineralization of gastric

13 mucosa and in two cases there was also oedema of the submucosal layer. In two cases gastritis

14 was associated with thrombosis and fibrinoid necrosis of the vascular wall.

15 Ulcerative glossitis and stomatitis occurred in 22 cases (22/78; 28.2%) and was

16 characterized by bilateral focal ulceration in the tongue ventral surface (Fig. 3) or multiple

17 small ulcers and erosions in different sites of oral cavity mucosa and lips. Histologically, there

18 was epithelial necrosis and ulceration with marked neutrophilic and histiocytic inflammatory

19 infiltrate. There was necrosis and haemorrhage of the tongue muscle. In some cases, there was

20 secondary bacterial infection.

21 Soft tissue mineralization was present in 11 cases (11/78; 14.1%). The organs

22 involved in this condition were, in descending order of frequency, aorta (6), lungs (5),

23 stomach (3), intercostal muscles (3), heart (endocardium; 3), spleen (1), tongue (1) and

24 adrenal glands (1). Grossly, it was observed white and firm focal areas with irregular cut

25 surface with crumbly texture with variable intensity and distribution between different organs.
 87

1 In the histology, mineralization was identified as depositions of coarse and strongly

2 basophilic material on the tissue surface.

3 Anaemia was observed in nine cats (9/78; 11.5%) in which haematocrit values were

4 below the reference value for the specie. These cats also presented marked pale visible

5 mucous membranes, watery blood, pale aspect of blood marrow on gross examination,

6 rarefaction of erythroid lineage cells on bone marrow microscopic examination.

7 Haemorrhagic enteritis was observed in four cases (4/78; 5.1%) in which the serosa

8 presented numerous petechiae and the mucosa surface was markedly red, filled with

9 haemorrhagic content. In one of these cases there were multiple ulcers in enteric mucosa. In

10 one case it was observed a focally extensive necrotic area in the mucosal layer. On histology,

11 the epithelium was multifocally ulcerated; the remaining epithelium was necrotic and there

12 was an adjacent neutrophilic inflammatory infiltrate with fibrin deposition.

13 In only three cases (3/78; 3.8%) bilateral parathyroid hyperplasia was observed. There

14 was a notable increased in size of these endocrine glands. In one case (1/78; 1.8%) an

15 ulcerative oesophagitis was present. There was evident necrosis of the oesophageal mucosa

16 and multiple focus of erosion of the lining epithelium with fibrin deposition.

17 Cavities effusions were a common gross finding in this study. They were observed in

18 23 (23/78; 29.4%) cases affecting pleural cavity, in 13 cases (13/78; 16.6%) in the abdominal

19 (peritoneal) cavity and in five cases (5/78; 6.4 %) there was fluid accumulation in the

20 pericardial sac. Three cats with chronic glomerulonephritis had a combination of pleural

21 effusion, ascites, pulmonary oedema and subcutaneous oedema, findings that were considered

22 suggestive of nephrotic syndrome in the necropsy reports.

23
 88

1 Discussion

3 This study made possible to picture the profile of 78 cats with urinary tract diseases

4 that had developed systemic lesions considered nonrenal uraemic lesions. There were more

5 adults and elderly cats with uraemia in comparison to young cats, and about two thirds of the

6 cats were male. This data was considered noteworthy, although it does not allow deducing age

7 or sexing predispositions, since the total population of necropsied cats was not analysed

8 regarding these parameters. The main disease that leads to uraemia is chronic kidney disease

9 (CKD), mainly because of the prolonged exposure to uremic toxins (Ross, 2011; Bartges,

10 2012). Moreover, many studies point out a higher prevalence of CKD considering a geriatric

11 population of cats (DiBartola et al., 1987; Lawler et al., 2006; Chakrabarti et al., 2013;

12 Sparkes et al., 2016; Togni et al., 2018). Furthermore, among urinary tract diseases, feline

13 lower urinary tract disease (FLUTD) is the name attributed to a group of conditions related to

14 disturb of urine elimination in cats (Little, 2012) and castrated males have increased risk for

15 all the specific causes of FLUTD (Lekcharoensuk et al, 2001).

16 As observed in dogs (Dantas and Kommers, 1997; Silveira et al., 2015), the majority

17 of cats developed uraemic syndrome as a consequence of primary kidney diseases. Intrinsic

18 renal failure in cats can be originated from many types of diseases, such as polycystic kidney

19 disease (PKD), obstructive urolithiasis and pyelonephritis (Syme et al., 2006), as observed in

20 the present study. However, the most frequent histopathological diagnosis attributed to cats

21 with renal failure was chronic tubulointerstitial nephritis in this study, which is the most

22 common recognized form of CKD in cats (Lucke, 1968; Dibartola et al., 1987; Chakrabarti et

23 al., 2013; Brown et al., 2016).

24 The most frequent clinical sign observed in this study was anorexia. Anorexia is an

25 unspecific sign present in both ARF and CRF (Elliot, 2011, Polzin, 2011; DiBartola and
 89

1 Westropp, 2015; Quimby, 2016). It can be attributed to changes in taste and olfaction, to the

2 nausea and vomiting caused by increased uremic toxins in the blood stream (Elliot, 2011), to

3 ulcerative lesions in the oral cavity, and to gastritis (DiBartola and Westropp, 2015).

4 Vomiting was observed in 30% of the cases of uremic cats and this value represented a half of

5 the results obtained in studies with dogs (Dantas and Kommers, 1997; Silveira et al. 2015).

6 Indeed, vomiting is considered a more common clinical manifestation in uremic dogs than in

7 cats (DiBartola and Westropp, 2015). In uremic dogs, vomiting is a consequence of

8 stimulation of chemoreceptors in a trigger zone in the stomach by uremic toxins (Borison and

9 Hebertson, 1959) and it is possible that the same mechanism would be involved in vomiting

10 in uremic cats (Quimby, 2016; Sparkes et al., 2016).

11 Pulmonary oedema was the most prevalent uraemic lesion observed in this study. The

12 percentage of affected cats (51,2%) was similar to what was previously observed in dogs

13 (Silveira et al., 2015). In these uremic dogs pulmonary oedema was also among the most

14 common uremic lesions, being only overcome by ulcerative gastritis and mineralization of

15 soft tissues. Uraemic pulmonary oedema is a consequence of the increased vascular

16 permeability caused by the caustic effect of uremic toxins (Bass, 1952; Rackow et al., 1978;

17 Cianciolo and Mohr, 2016; Breshears and Confer, 2017). It is important to emphasize that

18 pulmonary oedema was the only uraemic lesion observed in 11 cats without available blood

19 urea and creatinine values available in the necropsy reports. In these cats, it was not observed

20 any other pathological process rather than primary urinary disease that could explain the

21 pulmonary oedema or justify the cat’s natural death (ten cases) or euthanasia (one case).

22 However, it is not possible to determine certainly if the oedema was caused by uraemia in

23 these cases.

24 The combination of pulmonary oedema with transudation of fibrin, mineralization and

25 inflammation, characterizing uremic pneumopathy (Breshears and Confer, 2017) was rarely
 90

1 observed in the cats of this study and it is commonly observed in uremic dogs (Cianciolo and

2 Mohr, 2016; Dantas and Kommers, 1997; Boedec et al, 2012, Silveira et al., 2015).

3 Uremic gastropathy is a well described condition in uremic dogs, characterized by

4 gastric oedema and congestion, haemorrhagic gastric content, mineralization and vascular

5 lesions (Uzal et al, 2016). Among the available studies, gastric histopathologic findings in

6 uremic dogs are variable. According to Peters et al. (2005), the histopathologic changes

7 observed in the stomach of 22 out of 28 dogs (78.5%) with renal failure was oedema,

8 mineralization and vasculopathy, whereas ulceration and necrosis of gastric wall were

9 uncommon features. Recently, Cardoso et al. (2019) found uremic gastropathy in 15 of the 16

10 necropsied uremic dogs (93.15%). Erosive and/or ulcerative gastritis, oedema, hyperaemia,

11 ammoniac odour and necrosis of gastric mucosa were commonly observed, while

12 mineralization was only observed in four cases. According to Dantas and Kommers (1997)

13 and Silveira el al. (2015), uremic gastropathy was the most prevalent lesion in uremic dogs,

14 comprising 79,1 % and 56.5% of the cases, respectively. Haemorrhagic and ulcerative

15 gastritis with oedema, vascular lesions and mineralization were frequently observed in these

16 studies.

17 The most common gastric lesions observed in the cats of this study were ulcerative/

18 erosive and haemorrhagic gastritis. These changes were different from the results obtained in

19 a study of uremic gastropathy in 37 cats with CKD. Gastric ulceration, oedema and vascular

20 fibrinoid changes were not observed in gastric histopathologic analysis. Otherwise, the most

21 important gastric lesions observed were fibrosis and mineralization (McLeland et al., 2014).

22 This result can suggest that gastric lesion in uremic cats may show individual variations.

23 Ulcerative stomatitis and glossitis are common features of canine uraemia,

24 characterized by dark and fetid ulcers in the tongue, lips and cheeks (Uzal et al, 2016). It was

25 a frequent gross finding in the present study and also reported by Lucke (1968) in uremic cats.
 91

1 The development of ulcerative lesions in oral cavity probably is a combination of ammonia

2 caustic effect on oral mucous membranes and vascular ischemic injury. Ammonia is

3 originated from the degradation of urea in salivary content by local urease-producing bacteria

4 (DiBartola and Westropp, 2015; Uzal et al, 2016).

5 Anaemia ranges between 32% and 65% of cats with CKD (Dibartola et al., 1987;

6 Elliot and Barber, 1998). Considering only cats with CKD (48 cases) in this study, the

7 percentage of anaemia was around 18%. Impairment of renal endocrine function, results in

8 reduced haematopoiesis by reducing erythropoietin production (Chalhoub et al.,2011) and it

9 is considered the main source of anaemia in CKD, but a multifactorial character has been

10 attributed to the pathogenesis of this type of anaemia (Chalhoub et al, 2011; Cianciolo and

11 Mohr, 2016). Factors like inflammation (Stenvinkel, 2001), uremic toxins causing reduction

12 in red blood cells survival and blood loss in gastrointestinal tract are believed to be involved

13 (Chalhoub et al., 2011).

14 There was a notable difference between the findings related to calcium metabolism

15 between the cats of this study and what is reported in uremic dogs. Multiple soft tissue

16 mineralization were frequently observed in uremic dogs, ranging from 55.9% (Silveira et al.,

17 2015) to 93.7% (Cardoso et al., 2019) of the cases. Parathyroid hyperplasia was observed in

18 9,3% of uremic dogs (Silveira et al., 2015) and fibrous osteodystrophy ranged from 6.2%

19 (Cardoso et al., 2019) to 8% of the cases (Silveira et al., 2015). In the present study, soft

20 tissue mineralization rate was about 13%.

21 Similarly, parathyroid hyperplasia was an uncommon feature of this study and fibrous

22 osteodystrophy was not reported in clinical history or observed during necropsies. In a

23 previous study that investigated some aspects of systemic consequences of CKD in cats,

24 parathyroid hyperplasia was observed only in six cats with long term kidney injury. In these

25 cases, there was no evidence of pathological fractures or softening of the bones but a detailed
 92

1 microscopic analysis of the bones revealed mild osteoclastic bone resorption, with deposition

2 of fibrous tissue in the resorption site. These mild bone lesions in cats did not show a

3 predilection for the skull bones (mandible and maxillae), which are the most severally

4 affected bones in the canine renal secondary hyperparathyroidism (Lucke, 1968).

5 Additional lesions, frequently observed in uraemic syndrome in dogs, such as mural

6 endocarditis, fibrinous pericarditis, ulcerative laryngitis and multiple soft tissue mineralization

7 affecting heart, aorta, larynx, diaphragm and intestines (Dantas and Kommers, 1997; Silveira

8 et al. 2015) were not observed in the uraemic cats analysed herein. In addition, the present

9 study showed multisystemic presentation of uraemia in only 24% of the cases, suggesting that

10 the consequences of urinary tract disease are less severe in cats in comparison to dogs.

11 The classic concept about the progression of CKD is based on a slow and progressive

12 decline in kidney function, with continuous functional loss during the animal's life, until the

13 development of renal failure and uraemia. However, many cats with CKD show stable renal

14 function for a long period of time, sometimes dying from other diseases rather than renal

15 processes (Polzin, 2011). There is a hypothesis that the feline CKD caused by

16 tubulointerstitial lesion might not be merely a degenerative disease that causes mortality. One

17 study trying to determine the relationship between kidney injury, cause of death, and age in a

18 population of 676 adult cats detected longer mean life span in cats with kidney disease in

19 comparison to animals with different causes of death. The authors therefore suggested that

20 CKD in cats could represent an adaptive survival process of the feline specie (Lawler et al.,

21 2006). Additionally, it is important to emphasize that the most common feature usually

22 observed in the CKD of cats in the form of a tubulointerstitial lesion is a multifocal to

23 segmental distribution, with remaining areas of preserved parenchyma (Brown et al., 2016)

24 what could provide maintenance of the renal function. Such facts may help to understand, at
 93

1 least considering the cases of CKD in this study, the smallest variety of nonrenal lesions of

2 uraemia and the lower occurrence of multiple organs injury in uraemic cats.

5 Acknowledgements

6 This research was financially supported by the Coordenação de Aperfeiçoamento de

7 Pessoal de Nível Superior (CAPES), Brazil. The student M. B. Ambrosio has a Master’s

8 scholarship from CAPES.

9 Conflict of Interest Statement

10 The authors declare no conflicts of interest with respect to the research, authorship or

11 publication of this article.

12
 94

1 References

2 Bass HE, Greenberg MD, Singer E, Miller MA (1952) Pulmonary changes in uremia. Journal

3 of the American Medical Association, 148, 724-726.

4 Boedec LK, Heng HG, Snyder PW, Pressler BM (2012) Pulmonary Abnormalities in Dogs

5 with Renal Azotemia. Journal of Veterinary Internal Medicine, 26, 1099-106.

6 Borison HL, Hebertson LM. Role of medullary emetic chemoreceptor trigger zone (CT zone)

7 in postnephrectomy vomiting in dogs. American Journal of Physiology, 197, 850-852.

8 Breshears, MA, Confer AW (2017) The urinary system. In: Pathologic Basis of Veterinary

9 Disease. 6th. Edit, JF Zachary, Elsevier, St. Louis, pp. 617-681.

10 Brown CA, Elliot J, Schmiedt CW, Brown SA (2016) Chronic kidney disease in aged cats:

11 clinical features, morphology, and proposed pathogeneses. Veterinary Pathology, 53,309-

12 326.

13 Cardoso PGS, Pinto MPR, Moroz LR, Fontes TN, Santos RS et al. (2019) Dystrophic

14 mineralization in uremic dogs: an update. Pesquisa Veterinária Brasileira, 39, 889–899.

15 Chakrabarti S, Syme HM, Brown CA, Elliot J (2013) Histomorphometry of feline chronic

16 kidney disease and correlation with markers of renal dysfunction. Veterinary Pathology,

17 50, 147–155.

18 Chalhoub S, Langston C, Eatroff A (2011) Anemia of renal disease: What it is, what to do and

19 what’s new. Journal of Feline Medicine and Surgery, 13, 629–640.

20 Cianciolo RE, Mohr FC (2016) Urinary sistem. In: Jubb, Kennedy and Palmer’s Pathology of

21 Domestic Animals, 6th Edit, MG Maxie, Elsevier Ltd, St Louis, pp. 377-463.

22 Cowgill LD (2003) Advanced therapeutic approaches for the management of uraemia – the

23 met and unmet needs. Journal of Feline Medicine and Surgery, 5, 57–67.

24 Dantas AF, Kommers GD (1997) Lesões extrarenais de uremia em 72 cães. Ciência Rural,

25 27, 301-306.
 95

1 DiBartola SP, Rutgers HC, Zack PM (1987) Clinicopathologic findings associated with

2 chronic renal disease in cats: 74 cases (1973 -1984). Journal of the American Veterinary

3 Medical Association, 190, 1196–202.

4 Dibartola SP, Westropp JL (2015) Doenças do Trato Urinário. In: Medicina Interna de

5 Pequenos Animais.5th Edit. RW Nelson, CG Couto, Elsevier, Rio de Janeiro, pp. 698-703.

6 Elliott DA (2011) Nutritional considerations for the dialytic patient. Veterinary Clinics of

7 North America: Small Animal Practice, 41, 239–250.

8 Elliot J, Barber PJ (1998) Feline chronic renal failure: clinical findings in 80 cases diagnosed

9 between 1992 and 1995. Journal of Small Animal Practice, 39, 78-85.

10 Lawler DF, Evans RH, Chase K, Ellersiek M, Qinghong L et al. (2006). The aging feline

11 kidney: a model mortality antagonist? Journal of Feline Medicine and Surgery, 8, 363-371.

12 Lekcharoensuk C, Osborne CA, Lulich JP. Epidemiologic study of rick factor for lower

13 urinary tract diseases in cats. JAVMA, 218, 219-239.

14 Little SE (2015) Distúrbios do trato urinário. In: O gato: medicina interna. 1st. Edit. SE

15 Little, Roca, Rio de Janeiro, pp. 1406-1913.

16 Lucke VM (1968) Renal disease in the domestic cat. The Journal of Pathology and

17 Bacteriology, 95 ,67–91.

18 Maxie MG, Newman SJ (2007) The urinary system. In: Jubb, Kennedy, and Palmer’s

19 Pathology of Domestic Animals. 5th. Edit. MG Maxie, Elsevier, Saint Louis, pp.425-522.

20 McLeland SM, Lunn KF, Duncan CG, Refsal, KF, Quimby JM (2014) Relationship among

21 Serum Creatinine, Serum Gastrin, Calciumphosphorus Product, and Uremic Gastropathy in

22 Cats with Chronic Kidney Disease. Journal of Veterinary Internal Medicine, 28, 827-837.

23 Peters RM, Goldstein RE, Erb HN, Njaa BL (2005) Histopathologic Features of Canine

24 Uremic Gastropathy: A Retrospective Study. Journal of Veterinary Internal Medicine, 19,

25 315-320.
 96

1 Polzin, DJ (2011) Chronic kidney diseases in small animals. Veterinary Clinics: Small Animal

2 practice, 41, 15–30.

3 Polzin DJ (2011) Chronic kidney disease. In: Nephrology and urology of small animals. J

4 Bartges, DJ Polzin, 1st Edit, Blackwell Publishing Ltd, Iowa, pp. 433-469.

5 Quimby JM (2016) Update on medical management of clinical manifestations of chronic

6 Kidney disease. Veterinary Clinics: Small Animal Practice, 46, 1163–1181.

7 Rackow EC, Fein A. Sprung C. Grodmsn RS (1978) Uremic pulmonary edema. The

8 American Journal of Medicine, 64, 1978.

9 Ross SJ (2011) Azotemia and uremia. In: Nephrology and urology of small animals. 8. Edit. J

10 BARTGES, DJ POLZIN, Elsevier, Saint Louis, pp. 393-398.

11 Serakides R, Silva JF (2016) Sistema urinário. In: Patologia Veterinária, 2. Edit, R Santos,

12 AC Alessi, Roca, Rio de Janeiro, pp. 518-527.

13 Silveira IP, Inkelmann MA, Tochetto C, Rosa FB, Fighera RA (2015) Epidemiologia e

14 distribuição de lesões extrarenais e uremia em 161 cães. Pesquisa Veterinária Brasileira,

15 35, 562-568.

16 Sosnar M, Kohout P, Ruzicka M, Vrbasová L (2003) Retrospective study of renal failure in

17 dogs and cats admitted to university of veterinary and pharmaceutical sciences brno during

18 1999-2001. Acta Veterinaria Brno, 72, 593–598.

19 Sparkes AH, Caney S, Chalhoub S, Elliot J, Finch N (2016) ISFM Consensus Guidelines on

20 the Diagnosis and Management of Feline Chronic Kidney Disease. Journal of Feline

21 Medicine and Surgery, 18, 219–239.

22 Stenvinkel P. The role of inflammation in the anemia of end-stage kidney disease.

23 Nephrology Dialysis Transplantation, 16, 36-40.

 97

1 Syme H, Markwell P, Pfeiffer D, Elliott J (2006) Survival of cats with naturally occurring

2 chronic renal failure is related to severity of proteinuria. Journal of Small Animal Practice,

3 20, 528-535.

4 Togni M, Curtis A, Vargas DP, Kommers GD, Irigoyen LF (2018) Causas de morte e razões

5 para eutanásia em gatos na Região Central do Rio Grande do Sul (1964-2013). Pesquisa

6 Veterinária Brasileira, 38, 741–750.

7 Uzal FA, Plattner, BL, Hostetter JM (2016) Alimentary system. In:. Jubb, Kennedy, and

8 Palmer’s Pathology of Domestic Animals. 6th Edit, MG Maxie, Elsevier Ltd, St Louis, pp.

9 51-53.

10 Vanholder R, DeSmet R, Glorieux G, Argilés A, Baurmeister U. et al. (2003) Review on

11 uremic toxins: classification, concentration, and interindividual variability. Kidney

12 International, 63, 1934-1943.

13
 98

8 Fig. 1. Pulmonary oedema. (A) Presence of a considerable amount of foamy fluid in the

9 trachea and uncollapsed lungs with wet appearance. (B) Alveoli filled with protein-rich

10 amorphous eosinophilic fluid.

11
 99

3 Fig. 2. Uraemic gastritis. (A) Multiple and variable size visible ulcers (from 0.5 to 2.0 cm

4 diameter) in the gastric mucosal. (B) There is epithelium loss and parietal and principal

5 mucosal cells necrosis. Throughout the mucosa, there are multiple areas of haemorrhage (C)

6 Haemorrhagic gastric content. (D) Deposition of a bright eosinofilic material in the walls of

7 small arteries of gastric submucosa layer (uremic vascular fibrinoid necrosis).

8
 100

7 Fig. 3. Uraemic ulcerative glossitis. (A) Extensive bilateral symmetric ulcers on the ventral

8 surface of the tongue. (B) Histologically, there is focally extensive ulceration of lining

9 stratified squamous keratinized epithelium of the tongue, with erythrocyte and fibrin

10 accumulation in the adjacent connective tissue.

11
 101

2 Fig. 4. Soft tissue mineralization. (A) Thoracic cavity (parietal pleura), streaks of white

3 material in the subpleural surface, characterizing intercostal mineralization. (B) Deposition of

4 basophilic mineral material between intercostal muscle fibers. (C) Multifocal mineralization

5 of tongue skeletal muscle. (D) Focally extensive mineralization of the cortical zona

6 fasciculata of the adrenal gland.

7
 102

1 Table 1

2 Epidemiological information about of the 78 cats with nonrenal uremic lesions

Breed Sex Age category

Mixed-breed 51 Male 56 Adult 54
Siamese 13 Female 22 Elderly 17
Persian 10 Young 7
Burmese Sacred 2
Exotic Shorthair 1
Himalayan 1
3

4 Table 2

5 Clinical manifestations of uraemia in 78 cats.

Clinical signs Number of cases Percentage (%)

Anorexia 39 48.75%
Apathy 32 40%
Vomiting 24 30%
Neurological signs 18 22.5%
Oral ulceration 8 10%
6

7 Table 3

8 Prevalence of primary urinary tract diseases affecting cats with uraemia

Origin of azotaemia Total

number of
cases
Lesion Occurrence*
Renal 58
Chronic tubulointerstitial nephritis 32
Chronic glomerulonephritis 16
Acute tubular necrosis 14
Bacterial pyelonephritis 4
Polycystic kidney disease (PKD) 3
Hydronephrosis 3
Nephrolithiasis 2
Granulomatous interstitial nephritis a 1
Renal papillary necrosis 1
Ischemic acute infarctions 1
Post renal 18
Necrotic/haemorrhagic/fibrinous cystitis 16
Urethral obstruction 8
Traumatic urinary bladder rupture 1
Traumatic ureteral rupture 1
9
10 a: associated to feline infectious peritonitis
11 * total number of occurrences considering multiple lesions in the same animal
 103

1 Table 4

2 Type and frequency of nonrenal uremic lesions in 78 cats.

Uremic lesion Number Percentage (%)

of cases
Pulmonary oedema 40 51.2%
Gastritis 28 35.8%
Ulcerative 16
Ulcerative and haemorrhagic 7
Erosive 2
With oedema of gastric wall 2
Thrombosis and vascular fibrinoid necrosis 2
With ammonia odour 22
Oral cavity lesions 22 28.2%
Ulcerative glossitis 14
Ulcerative stomatitis 8
Anaemia 12 11.5%
Soft tissue mineralization 11 14.1%
Aorta 6
Lungs 5
Stomach 3
Intercostal muscles 3
Heart 3
Spleen 1
Tongue 1
Adrenal gland 1
Enteritis 4 5.1%
Haemorrhagic 4
Ulcerative 1
Parathyroid hyperplasia 3 3.8%
Necrotic and erosive esophagitis 1 1.3%
3

4 Table 5

5 Distribution of nonrenal lesions in 78 cats

6 Number of affected Number of cases Percentage (%)

organs/anatomical sites
7
One 37 47.4%
8 Two 22 28.2%
Three or more 19 24.4%
9

10
11
 72

5 DISCUSSÃO

Esta dissertação de mestrado foi dividida em duas partes, o que resultou em dois
artigos científicos. A fonte de inspiração para os temas centrais de ambos os artigos foi o
próprio cotidiano da rotina diagnóstica do LPV-UFSM. A observação de características
únicas da DRC na espécie felina, tanto macroscópicas (redução do tamanho e marcado
arredondamento dos rins), quanto histológicas, possibilitaram a realização de um estudo de
caracterização macroscópica, histológica e imuno-histoquímica de 25 gatos com DRC.
Apesar da relação entre a perda funcional e estrutural dos rins não ser necessariamente
constante, na DRC há perda funcional dos néfrons (POLZIN, 2011b), resultando, geralmente,
em redução do tamanho dos rins (CIANCIOLO; MOHR, 2016). A redução do tamanho dos
rins foi observada em 22 dos 25 casos do estudo, variando de bilateral, bilateral assimétrica e
unilateral. Os rins reduzidos de tamanho apresentaram maiores graus histológicos de fibrose
intersticial (p=0.021) e a redução do tamanho renal foi correlacionada à severidade da
inflamação crônica (p=0.0039) e da fibrose intersticial (p<0,001).
Os resultados da observação criteriosa da macroscopia dos rins dos 25 gatos deste
estudo demonstraram que a diminuição do tamanho dos rins com arredondamento, foi uma
apresentação exclusiva da NTIC. A causa para o arredondamento renal foi a combinação de
redução da espessura da camada cortical, inflamação crônica, fibrose intersticial, atrofia
tubular e esclerose glomerular. Todos esses caracteres histológicos apresentaram-se mais
acentuados lateralmente ao hilo renal, se perpetuando em direção aos polos renais, levando ao
achatamento, e posterior arredondamento dos rins.
Além da característica macroscópica única, a NTIC caracterizou-se por inflamação
linfocítica com fibrose intersticial organizada em faixas, com orientação radial, com
distribuição multifocal segmentar, com redução da espessura da cortical e obsolescência
glomerular, muito semelhante ao descrito em estudos anteriores (CHAKRABARTI et al.,
2013; DIBARTOLA et al., 1987; MCLELAND et al., 2019). Os componentes histológicos
descritos sugerem que a etiologia da NTIC, principal forma de DRC observada na espécie
felina, ainda não completamente compreendida, envolva eventos isquêmicos do tecido renal
(BROWN et al., 2016)
A presença de miofibroblastos, células imunomarcadas positivamente para α-SMA,
observada tanto na NTIC, quanto na GNC dos gatos deste estudo, sugere que essas células
estejam envolvidas nos processo de fibrose intersticial e na obsolescência glomerular nas
 73

DRCs, mesmo nos casos de lesão menos acentuada. A perda tecidual, observada
principalmente nas NTIC, fortemente correlacionada à severidade da fibrose intersticial, pode
ter envolvimento dos miofibroblastos tanto como produtores de MEC, quanto pela função
contrátil destas células (STRUTZ; ZEISBERG, 2006, SUN et al., 2016).
Dada a importância das doenças do trato urinário em felinos (SOSNAR et al., 2003;
TOGNI et al., 2018) e a escassez de informações sobre uremia na espécie felina, tanto nos
trabalhos realizados no LPV-UFSM, quanto na literatura, foi realizado um estudo visando
determinar a prevalência, e caracterizar os aspectos clínicos, patológicos e a distribuição
anatômica das lesões extrarrenais de uremia em gatos.
Em um universo de 1330 gatos necropsiados no LPV-UFSM entre os anos de 2000 e
outubro de 2019, 78 gatos apresentaram lesões extrarrenais de uremia (5,8%). Este estudo
permitiu traçar o perfil dos gatos com uremia, além das principais manifestações clínicas
observada nos animais. Lesões intrínsecas renais foram as principais doenças atribuídas à
azotemia, e consequentemente à uremia, e as lesões crônicas foram as mais prevalentes,
especialmente a NTIC. Esse predomínio de lesões renais crônicas pode ser explicado pelo seu
caráter progressivo, resultando em exposição prolongada do animal às toxinas urêmicas
(CLARKSON; THOMAS, 2011).
As lesões extrarenais de uremia comumente observadas nos gatos deste estudo, em
ordem decrescente de frequência, foram: edema pulmonar, gastropatia urêmica, glossite e
estomatite ulcerativas. Menos frequentemente, os animais apresentaram mineralização de
tecidos moles, anemia, enterite hemorrágica ou ulcerativa e hiperplasia das paratireoides. Vale
ressaltar, que o caráter multissistêmico da uremia normalmente relatado para a espécie canina
(CARDOSO et al., 2019; DANTAS; KOMMERS, 2007; SILVEIRA et al., 2015;) foi
observado em apenas 24% dos gatos deste estudo, os quais apresentaram ainda uma menor
variedade de lesões relacionadas à uremia.
 74

6 CONCLUSÃO

O estudo de caracterização dos aspectos morfológicos da DRC em gatos revelou que a

NTIC foi o principal diagnóstico morfológico observado nos casos estudados. O formato
arredondado dos rins, observado somente na NTIC, foi atribuído a maior severidade da
fibrose intersticial e da inflamação crônica, junto à redução da espessura da camada cortical
renal, atrofia tubular e esclerose glomerular, observadas de maneira mais acentuada
principalmente nas regiões laterais ao hilo renal. A técnica de imuno-histoquímica utilizando
o anticorpo anti-α-SMA foi uma ferramenta útil para a detecção de miofibroblastos no tecido
renal de gatos com diferentes graus de intensidade de DRC. Houve maior intensidade da
imunomarcação para α-SMA nos casos em que o comprometimento tecidual pela lesão
histológica era mais acentuado, reforçando o papel dos miofibroblastos no desenvolvimento
da fibrose intersticial presente na DRC dos felinos.
Lesões consideradas extrarrenais de uremia foram observadas em 78 gatos com lesões
do trato urinário, entre os anos de 2000 e 2019. As principais causas de uremia nesses gatos
foram doenças renais crônicas, principalmente na forma da NTIC. Edema pulmonar e gastrite
ulcerativa e/ou hemorrágica e foram as lesões mais frequentemente observadas, em 51,2% e
35,8% dos casos, respectivamente. Lesões relacionadas às falhas no metabolismo do cálcio e
fósforo (mineralização de tecidos moles, hiperplasia das paratireoides e osteodistrofia fibrosa)
foram achados incomuns. A apresentação multissistêmica não foi a forma mais comum da
uremia na espécie felina e os gatos apresentaram menor variedade de lesões extrarrenais de
uremia. Isso pode ser reflexo da estabilidade da função renal comumente observada em gatos
com DRC e ao comprometimento segmentar do tecido renal no caso das NTIC, que pode
conferir manutenção prolongada da função renal ao longo da vida do animal.
 75

7 REFERÊNCIAS

ALPERS, C. E.; FOGO, A. B. O Rim e seu sistema coletor. In: KUMAR, V.; ABBAS, A. K.;
ASTER, J. C. Robbins Patologia Básica. 9. ed. São Paulo: Elsevier, 2013. cap. 13, p. 517-
550.

ARESU, L. et al. Epithelial-mesenchymal transition (EMT) of renal tubular cells in canine

glomerulonephritis. Virchows Archiv, v. 451, n. 5, p. 937–942, 2007.

BARTGES, J. W. Chronic kidney disease in dogs and cats. Veterinary Clinics of

North America - Small Animal Practice, v. 42, p. 669–692, 2012.

BOEDEK, L. K. Pulmonary Abnormalities in Dogs with Renal Azotemia. Journal of

Veterinary Internal Medicine, v. 26, n. 5, sep-oct. 2012.

BRESHEARS, M. A.; CONFER, A. W. The Urinary System. In: ZACHARY. J. F.

Pathologic Basis of Veterinary Disease. 6. ed. St. Louis: Elsevier, 2017. cap. 11, p. 617-681.

BROWN, S. A. Pathophysiology and Management of Progressive Renal Disease. The

veterinary journal, v. 154, p. 93-109, 1997.

BROWN, C. A. et al. Chronic kidney disease in aged cats: clinical features, morphology, and
proposed pathogenesis. Veterinary Pathology, v. 53, n. 2, p. 309-326, 2016.

CARDOSO, P. G. S. et al. Dystrophic mineralization in uremic dogs: an update. Pesquisa

Veterinária Brasileira, v. 39, n. 11, p. 889–899, nov. 2019.

CHALHOUB, S.; LANGSTON, C.; EATROFF, A. Anemia of renal disease: what it is, what
to do and what’s new. Journal of Feline Medicine and Surgery, v. 13, p. 629-640, 2011.

CHAKRABARTI, S. et al. Histomorphometry of feline chronic kidney disease and

correlation with markers of renal dysfunction. Veterinary Pathology, v. 50, n. 1, p. 147–155,
2013.

CIANCIOLO, R. E., et al. World small animal veterinary association renal pathology
initiative: classification of glomerular diseases in dogs. Veterinary Pathology, v. 53, n. 1, p.
113-135, 2016.

CIANCIOLO, R. E.; MOHR, F. C. Urinary System. In: MAXIE, M. G. Jubb, Kennedy, and
Palmer’s Pathology of Domestic Animals. 6. ed. St. Louis: Elsevier, 2016. v. 2, cap. 4, p.
377-463.

CLARKSON, C. E.; FLETCHER, T. F. Anatomy of the kidney and proximal ureter. In:
BARTGES, J.; POLZIN, D. J. Nephrology and urology of small animals. 1st ed. Iowa:
Blackwell Publishing Ltd, 2011. cap. 1, p. 21-27.
 76

DANTAS, A. F. M.; KOMMERS, G. D. Lesões extrarenais de uremia em 72 cães. Ciência

Rural, v. 27, n. 2, p. 301-306, 1997.

DIBARTOLA, S. P.; RUTGERS, H. C.; ZACK, P. M. Clinicopathologic findings associated

with chronic renal disease in cats: 74 cases (1973-1984). Journal American Veterinary
Medical Association, v. 190, p. 1196–202, 1987.

DIBARTOLA, S. P.; WESTROPP, J. L. Manifestações Clínicas das Doenças do Trato

Urinário. In: NELSON, R. W.; COUTO, C. G. Medicina interna de pequenos animais. 5
ed. [S.l.]: Elsevier, 2015. Cap. 41, p. 1838-1857.

GEDDES, R. F. et al. The role of phosphorus in the pathophysiology of chronic kidney

disease. Journal of Veterinary Emergency and Critical Care, v. 23, n. 2, p. 122-133, 2013.

GOLDSTEIN, R.E. et al., Gastrin concentrations in plasma of cats with chronic renal failure.
Journal American Veterinary Medical Association, v. 213, p. 826–828, 1998.

GRANT, D. C.; FORRESTER, S. D. Glomerulonephritis in dogs and cats: diagnosis and

treatment. Compendium in continuing education for the practising veterinarian: north
American edition, v. 23, n. 9, p. 798-804, 2011.

HAMILTON, J. B.; HAMILTON, R. S.; MESTLER, G. E. Duration of life and causes of

death in domestic cats: influence of sex, gonadectomy, and inbreeding. Journal of
Gerontology, v. 24, n. 4, p. 427-437, 1969.

JEPSON, R. E. Current understanding of the pathogenesis of progressive chronic kidney

disease in cats. Veterinary Clinics of North America: Small Animal Practice, v. 46, n. 6,
p. 1015-1048, 2016.

KHAN, T. M.; KHAN, K. N. Acute Kidney Injury and Chronic Kidney Disease. Veterinary
Pathology, v. 52, n. 3, p. 441-444, 2015.

KING, L. G. et al. Anemia of chronic renal failure in dogs. Journal of Veterinary Internal
Medicine, v. 6, n. 5, p. 264–70, 1992.

KUMAR, V.; ABBAS, A. K.; ASTER, J. C. Inflamação e Reparo. In: ___. Robbins
Patologia Básica. 9. ed. São Paulo: Elsevier, 2013. cap. 2, p. 29-72.

LAWSON, J. et al. Renal fibrosis in feline chronic kidney disease: Known mediators and
mechanisms of injury. Veterinary Journal, v. 203, n. 1, p. 18–26, 2015.

LIU, Y. Renal fibrosis: new insights into the pathogenesis and therapeutics. Kidney
International, vol. 69, n. 2, p. 213- 217, jan. 2006.

LUND, E. M. et al. Health status and population characteristics of dogs and cats examined at
private veterinary practices in the United States. Journal American Veterinary Medical
Association, v. 214, n. 9, p.1336–1341, 1999.
 77

MACDOUGALL, D. F. et al. Canine chronic renal disease: Prevalence and types of

glomerulonephritis in the dog. Kidney International, v. 29, n. 6, p. 1144–1151, 1986.

MACK, M. YANAGITA, M. Origin of myofibroblasts and cellular events triggering fibrosis.

Kidney international, v. 87, n. 2, p. 297-307, aug. 2014.

MARINO, C. L. et al. Prevalence and classification of chronic kidney disease in cats

randomly selected from four age groups and in cats recruited for degenerative joint disease
studies. Journal of Feline Medicine and Surgery, v.16, n. 6, p. 1-8, 2014.

MCLELAND, S. M. et al. A comparison of biochemical and histopathologic staging in cats

with chronic kidney disease. Veterinary Pathology, v. 52, n. 3, p. 524-534, 2015.

MINKUS G. et al. Evaluation of renal biopsies in cats and dogs — histopathology in

comparison with clinical data. Journal of Small Animal Practice, v. 35, n. 9, p. 465–472,
1994.

MULLER-PEDDINGHAUS, R.; TRAUTWEIN, G. Spontaneous Glomerulonephritis in

Dogs: I. Classification and Immunopathology. Veterinary Pathology, v. 14, n. 1, p. 1–13,
1977.

O’NEILL, D. G. et al. Prevalence of disorders recorded in cats attending primary-care

veterinary practices in England. The veterinary journal, v. 202, n. 2, p. 286-291, nov. 2014.

PETERS, R. M. et al. Histopathologic Features of Canine Uremic Gastropathy: A

Retrospective Study. Journal of Veterinary Internal Medicine, v. 19, p. 315-320, 2005.

PRESSLER, N. Nephrotic syndrome. In: BARTGES, J.; POLZIN, D. J. Nephrology and

urology of small animals. 1st ed. Iowa: Blackwell Publishing Ltd, 2011. cap 44, p. 415-438.

POLZIN, D. J. Chronic kidney diseases in small animals. Veterinary Clinics of North

America: Small Animal Practice, v. 41, n. 1, p. 15–30, 2011a.

POLZIN, D. J. Chronic kidney disease. In: BARTGES, J.; POLZIN, D. J. Nephrology and
urology of small animals. 1st ed. Iowa: Blackwell Publishing Ltd, 2011b. cap.48, p. 433-469.

POLZIN, D. J. Chronic kidney disease. In: ETTINGER, S. J.; FELDMAN, E. C.; CÔTÉ, E.
Textbook of Veterinary Internal Medicine. 8th.ed. Saint. Louis: Elsevier, 2017. cap. 324, p.
4693-4734.

ROSS, S. J. Azotemia and uremia. In: BARTGES, J.; POLZIN, D. J. Nephrology and
urology of small animals. 1st ed. Iowa: Blackwell Publishing Ltd, 2011. cap. 41, p. 393-398.

SAWASHIMA, K. et al. Expression of α-smooth muscle actin and fibronectin in

tubulointerstitial lesions of cats with chronic renal failure. American Journal of Veterinary
Research, v. 61, n. 9, p. 1080–1086, 2000.

SILVEIRA, I. P. et al. Epidemiologia e distribuição de lesões extrarenais e uremia em

161 cães. Pesquisa Veterinária Brasileira, vol. 35, n. 4, p. 562-568, jun. 2015.
 78

SERAKIDES, R.; SILVA, J. F. Sistema Urinário. In: SANTOS, R. de L.; ALESSI, A.C.
(Org.). Patologia veterinária. 2. ed. Rio de Janeiro: Roca, 2016. cap. 5, p. 460-532.

SHERK, M. Trato urinário superior. In: LITTLE, S. E. O gato: medicina interna. 1. ed. Rio
de Janeiro: Roca, 2015. cap. 32, p. 1378-1386.

SLAUSON, D. O.; LEWIS, R. M. Comparative pathology of glomerulonephritis in animals.

Veterinary Pathology, v. 16, n. 2, p. 135–164, 1979.

SOSNAR, M. et al. Retrospective study of renal failure in dogs and cats admitted to
university of veterinary and pharmaceutical sciences brno during 1999-2001. Acta
Veterinaria Brno, v. 72, n. 4, p. 593–598, 2003.

STRUTZ, F.; ZEISBERG, M. Renal Fibroblasts and Myofibroblasts in Chronic Kidney Disease.
Journal of the american society of nephrology, v. 17, n. 11, p. 2992-2998, nov, 2006.

SUMNU, A.; GURSU, M.; OZTURK, S. Primary glomerular diseases in the elderly. World
Journal Nephrology, v. 4, n. 2, p. 263–270, 2015.

SUN, Y. B. et al. The origin of renal fibroblasts/myofibroblasts and the signals that trigger
fibrosis. Differentiation, v. 92, n. 3, p. 102-107, sep. 2016.

TOGNI, M. et al. Causas de morte e razões para eutanásia em gatos na Região Central do Rio
Grande do Sul (1964-2013). Pesquisa Veterinária Brasileira, v. 38, n. 4, p. 741–750, abr.
2018.

VADEN, S. L.; GRAUER, G. F. Glomerular disease. In: BARTGES, J.; POLZIN, D. J.

Nephrology and urology of small animals. 1st ed. Iowa: Blackwell Publishing Ltd, 2011.
cap. 53, p. 556-563.

VADEN, S. L. Glomerular Diseases. In: ETTINGER, S. J.; FELDMAN, E. C.; CÔTÉ, E.

Textbook of Veterinary Internal Medicine. 8. ed. St. Louis: Elsevier, 2017. cap. 325, p.
4735- 4764.

WHITE, J. D. Persistent haematuria and proteinuria due to glomerular disease in related

Abyssinian cats. Journal of Feline Medicine and Surgery, v. 10, n. 3, p. 219-229, 2008.

YABUKI, A. et al. Comparative study of chronic kidney disease in dogs and cats: induction
of myofibroblasts. Research in Veterinary Science, v. 88, n. 2, p. 294-299, 2010.