we describe the case of a 1-year - old male persian cat diagnosed with congenital hepatic fibrosis ( chf ) associated with renal polycystic disease and , for the first time , we have shown that there was no c > a mutation in exon 29 of pkd1 ( polycystic kidney disease 1 ) . the cat presented with a history of chronic weight loss , anorexia , vomiting , depression and lethargy , with profuse salivation and ascites on clinical examination .
owing to the cat s deteriorating condition , it was euthanized . during necropsy , the liver was found to be enlarged , firm and reddish , and the kidney had multiple small cortical cysts .
immunohistochemistry revealed that bile duct cells and epithelial cells of renal cysts showed positive immunoreactivity to keratin 19 .
collagen fibers surrounding bile ducts within portal areas demonstrated reactivity to type iv collagen antibody , confirming the congenital nature of the process .
a diagnosis of ductal plate malformation consistent with chf associated with polycystic kidney in a young persian cat was made .
interestingly , genetic testing revealed a wild - type sequence at position 3284 in exon 29 of pkd1 .
the absence of the classic genetic mutation associated with the particular clinical presentation supports the hypothesis of a distinct etiopathogenesis among fibropolycystic diseases in domestic cats .
moreover , congenital hepatic fibrosis is a rare but important differential diagnosis for young persian cats and their crosses with clinical signs of chronic end - stage liver disease .
congenital hepatic fibrosis ( chf ) represents one of many fibrocystic hepatic diseases derived from biliary dysgenesis secondary to ductal plate malformation .
the condition is described in humans and some animal species , characterized by the persistence of embryonic bile ducts , abnormal branching of the intrahepatic portal veins and progressive fibrosis of the portal tracts . in humans ,
chf is often associated with other cyst - forming syndromes , such as autosomal recessive polycystic kidney disease ( arpkd ) , autosomal dominant polycystic kidney disease ( adpkd ) ( rarely ) , meckel  gruber syndrome , jeune syndrome and with many other genetic abnormalities . in veterinary medicine
, chf has been described in dogs , swiss freiberger foals , bovine fetuses and calves , and in an african green monkeys .
although only a single mutation in pkd1 has been identified in feline adpkd , the phenotypic spectrum is diverse , with most cats demonstrating renal rather than biliary malformations , with isolated cysts in the liver with or without involvement of the pancreas .
hepatic fibrosis has been reported in 2248% of cats with feline polycystic kidney disease ( pkd ) ; however , clinical signs associated with liver failure have rarely been reported , and the congenital nature of this process has not been established . moreover ,
these cases of hepatic fibrosis in cats with pkd were described before the discovery of the c > a transversion in exon 29 of feline pkd1 , so genetic testing had not been performed .
here we describe the clinical , morphological and genetic features of chf associated with pkd in a persian cat , in order to advise on a possible alternative genetic mutation for this specific clinical presentation .
a 1-year - old male persian cat weighing 3.1 kg was admitted to a veterinary teaching hospital ( faculty of veterinary medicine , methodist university of so paulo , sp , brazil ) , with a 4 week history of chronic weight loss , anorexia , vomiting and lethargy .
ascites fluid was classified as a modified transudate characterized by an albumin : globulin ratio of 0.89 ( ri > 1.0 ) and mild cellularity ( 1.500 cell/l ) composed of neutrophils and small lymphocytes .
serum biochemistry findings included mild increases in alanine aminotransferase ( 108 u / l ; ri
2070 u / l ) , -glutamyl transferase ( 7 u / l ; ri 15 u / l ) and aspartate aminotransferase ( 67 u / l ; ri 140 u / l ) .
albumin was low ( 1.4 g / dl ; ri 2.13.3 mg / dl ) .
a commercial immunoassay for feline leukemia virus antigen detection and feline immunodeficiency virus antibody ( idexx laboratories ) detection was negative .
owing to financial reasons and the cat s clinical status , the owner opted for euthanasia . at necropsy
, there was an increased amount of slightly turbid abdominal fluid and fibrin clots throughout the hepatic serosa .
the liver was enlarged , firm and red , with multifocal pale , white - to - tan circular areas measuring 0.51.0 cm in diameter restricted to the liver capsule .
tissue samples were obtained , formalin - fixed , paraffin - embedded , sectioned and stained with hematoxylin and eosin for microscopic examination .
microscopically , the liver was divided by portal - to - portal bridging bands of dense fibrous tissue , with numerous , small , irregular bile duct profiles that were often mildly to moderately ectatic .
these ducts were lined by oval - to - cuboidal epithelial cells with finely stippled chromatin and a variably prominent nucleolus ( figure 1 ) .
a few small , non - portal - associated bile ducts with spindle cells were dispersed throughout the liver section .
small amount of lymphocytes , plasma cells and macrophages infiltrated fibrous bands and portal areas .
masson s trichrome stain revealed dense collagenous tissue in the periportal area ( figure 2 ) .
islands of hepatocytes are separated by irregular bands of fibrous tissue ( portal  portal bridging fibrosis ) .
scale bar = 200 m the kidneys showed multiple , sometimes multi - loculated cysts that were 0.52.0 mm in diameter in the renal cortex , lined by cuboidal or squamous epithelium .
some cysts contained abundant mild eosinophilic material ( protein ) and multiple detached epithelial cells ( figure 3 ) .
scale bar = 500 m for immunohistochemical analysis , liver and kidney histological sections were deparaffinized , rehydrated and submitted to antigen retrieval in citrate buffer solution ( 10 mm , ph 6.0 ) in a pressure cooker for 3.5 mins . after blockade of endogenous peroxidase with 6% h2o2 solution ( merck ) for 30 mins , the slides were incubated in a humidified chamber overnight at 4c with the following primary antibodies : keratin 19 ( k19 ) ( novocastra ; leica microsystems ) for liver and kidney , and type iv collagen ( dako north america ) for liver .
the slides were then incubated with a goat anti - mouse horseradish peroxidase polymer from a commercial kit ( zymed laboratories ) for 30 mins .
kidney tissues were used as a positive control for k19 and type iv collagen expression .
bile duct cells and epithelial cells lining kidney cystic structures showed positive granular cytoplasmic immunoreactivity to k19 ( figure 4 ) .
collagen fibers surrounding bile ducts within portal areas demonstrated prominent reactivity to type iv collagen antibody ( figure 5 ) .
scale bar = 200 m for pcr , restriction fragment length polymorphism ( rflp ) analysis and sequencing , genomic dna was isolated from formalin - fixed , paraffin - embedded 5 m - thick tissue sections and immediately placed into 1.5 ml sterile dna / rna - free plastic microtubes with 1 ml of xylol to remove paraffin and 100% ethanol .
subsequently , a genomic dna mini kit was used accordingly to manufacturer s instructions ( qiaamp dna ffpe tissue kit ; qiagen ) .
pcr was performed to amplify exon 29 of pkd1 , as previously described . for the pcr - rflp analysis ,
approximately 5 l amplification product was digested with 10 u mly1 ( new england biolabs ) in a 10 l reaction that contained 1 ne buffer 4 at 37c for 3 h followed by inactivation of the enzyme at 65c for 10 mins .
residual amplification primers and deoxynucleotide triphosphates were removed from the pcr product using a qiaquick pcr purification kit ( qiagen ) .
amplicons were then subjected to nucleotide sequence determination and analyzed on an abi prism 377 sequencer ( applied biosystems ) . in both techniques ,
the result was negative for the c > a transversion in exon 29 of pkd1 in this cat ( figure 6 ) .
( a ) pcr fragment length polymorphism typing for feline polycystic kidney disease ( pkd ) mutation .
persian cat with congenital hepatic fibrosis and pkd without c > a mutation of exon 29 of pkd1 in lane 4 .
lane 1 = ladder ; lane 2 = blank ; lane 3 = positive control .
( b ) nucleotide sequences of the wild - type in exon 29 of feline pkd1 in a persian cat with congenital hepatic fibrosis .
( c ) nucleotide sequences of the c > a transversion at position 3284 in exon 29 of pkd1 in a persian cat with pkd ( positive control )
chf features a ductal plate malformation of interlobular bile ducts associated with fibrosis , and in humans often coexists with caroli s disease , von meyenburg complexes ( intrahepatic bile duct hamartomas ) , arpkd and adpkd .
portal bridging fibrosis , with marked increase in small or irregular bile duct profiles , minimal to absent inflammation , and lack of nodular regeneration or other histologic evidence of chronic hepatitis .
primitive bi - potential progenitor cells in the liver express keratins 8 , 18 and 19 . during gestation , progenitor cells that are committed to form hepatocytes
lose k19 expression , whereas those destined to form bile ducts retain it and gain k7 expression .
previous studies on ductal plate malformations in humans have documented the expression of keratins 7 , 8 and 19 in ductal plate cells . in the present case , a diffuse positive k19 immunoexpression was observed in all ductular structures , identifying them as bile ducts or progenitor cells rather than mature hepatocytes . regarding the immunoreactivity for type iv collagen , several studies have attempted to establish the pathophysiological mechanism behind the abnormal and excessive fibrotic response associated with chf .
degradation of the basement membrane and extracellular matrix ( ecm ) constituents , and the remodeling of the ecm are important processes of embryonic development .
type iv collagen , which is present in the mesenchymal tissue surrounding the ductal plate cells , may help in the migration of these cells , as well as in their differentiation and maturation into intrahepatic bile ducts . additionally , type iv collagen is present in mesenchyme surrounding ductal plates early in gestation . in this context ,
persistent type iv collagen immunoreactivity in the present report case supports the diagnosis of a ductal plate malformation .
some humans with chf have concurrent renal cystic disease , similar to this case , and this clinical presentation has been mainly associated with arpkd .
arpkd is a severe form of human pkd that primarily presents itself in infancy and childhood , being characterized by enlarged kidneys and dilated cystic bile ducts , extensive hepatic brosis and hepatic failure .
mutations at a single locus , pkhd1 ( polycystic kidney and hepatic disease 1 ) , are responsible for all typical forms of arpkd .
adpkd is the most common inherited renal cystic disease , occurring worldwide and across all ethnicities .
two main genes have been identified ( pkd1 [ chromosome region 16p13.3 ; 85% cases ] and pkd2 [ chromosome region 4q21 ; 15% cases ] ) .
clinically relevant aspects include both renal ( impaired urinary concentrating capacity , renal failure , hypertension and pain ) and extrarenal manifestations where involvement of the liver is the most common , owing to slow and variable expansion of the cysts . in cats , adpkd is a well - described disease , especially in persian cats .
congenital cysts originate from both the proximal and distal tubules , and can be found in both renal cortex and medulla .
the disease is progressive , and both the size and number of cysts can increase over time and may result in chronic renal failure later in life . also , it is fairly well described that adpkd in most cats is associated with congenital biliary cystic lesions , as multiple large cysts . however , the renal form is the more common variant in cats and is most commonly associated with clinical disease .
hepatic fibrosis is reported in 2248% of cats with pkd , with an age range of 114 years .
clinical signs of liver failure , such as emaciation , diarrhea and coma , secondary to severe chf have been reported only four times before in cats with pkd . in none of these cases was the genetic test for c > a transversion in exon 29 of feline pkd1 performed . in the present case ,
the cat exhibited chronic weight loss , anorexia , vomiting , lethargy and altered mental status , consistent with hepatic failure and hepatic secondary encephalopathy .
hepatic enzymes were slightly elevated , but in chf , the liver function tests may be normal with no regenerative hyperplasia present  in contrast to end - stage disease or cirrhosis .
this clinical presentation is uncommon in young cats , with the cat described herein only 12 months of age . owing to the low ratio of globulin to albumin in the ascitic fluid , feline infectious peritonitis was clinically suspected .
however , no gross and histological evidence was observed to support this assumption . because of the paucity of portal vein profiles , congenital portosystemic shunt or portal vein hypoplasia
might be considered in the differential diagnosis but were unlikely given the presumed portal hypertension in this case .
prominent biliary proliferation in these cases of chf might prompt the consideration of chronic extrahepatic biliary obstruction .
chf can be distinguished from chronic extrahepatic biliary obstruction by the typical lack of inflammation or evidence of cholestasis , but in some cases , the history and clinical and imaging data will need to be evaluated carefully .
feline adpkd can be diagnosed by ultrasound screening , which is recommended when cats are older than 10 months , in order to avoid a false - negative result , associated with the pkd1 genetic test or with kidney biopsy , which may show lesions suggestive of adpkd at an earlier stage .
the pathogenesis of adpkd in cats is unknown ; however , in humans , abnormalities in gene expression , cell polarity , fluid secretion , apoptosis and ecm are hypothesized . in cats , only a c
> a transversion in exon 29 of pkd1 was described as the genetic cause of adpkd .
although several studies showed that about 5% of animals observed with ultrasound or histopathological changes consistent with pkd are negative for this point mutation , none of them directly describes the relationship between renal cysts , chf and the absence of c > a transversion .
because information on littermates was not available in the current case , a hereditary basis could not be investigated .
therefore , this is the first case to show , through sequencing , chf in a cat with pkd not linked to c > a transversion in exon 29 of feline pkd1 .
it is likely that , as in humans , chf and pkd should be assigned to a specific gene and its mutation in juvenile persian and persian families of cats . although we did not find the c
> a transversion in pkd1 in this case , it is possible that other regions of this gene or even other genes , such as pkhd1 ( characteristic of the human arpkd form ) , which have not yet been described in cats , are involved in the pathogenesis of this particular phenotype .
linkage , candidate gene or genome - wide analyses should be considered in further genetic investigations .
the absence of the classic genetic mutation associated with the particular clinical presentation supports the hypothesis of a distinct etiopathogenesis among fibropolycystic diseases in domestic cats .
moreover , chf is a rare but important differential diagnosis for young persian cats and their crosses with clinical signs of chronic end - stage liver disease .
 case summarywe describe the case of a 1-year - old male persian cat diagnosed with congenital hepatic fibrosis ( chf ) associated with renal polycystic disease and , for the first time , we have shown that there was no c > a mutation in exon 29 of pkd1 ( polycystic kidney disease 1 ) . the cat presented with a history of chronic weight loss , anorexia , vomiting , depression and lethargy , with profuse salivation and ascites on clinical examination . 
 a mild elevation in liver - associated plasma enzymes suggested a hepatic disease . owing to the cat s deteriorating condition , 
 it was euthanized . during necropsy 
 , the liver was found to be enlarged , firm and reddish , and the kidney had multiple small cortical cysts . 
 immunohistochemistry revealed that bile duct cells and epithelial cells of renal cysts showed positive immunoreactivity to keratin 19 . 
 collagen fibers surrounding bile ducts within portal areas demonstrated reactivity to type iv collagen antibody , confirming the congenital nature of the process . 
 a diagnosis of ductal plate malformation consistent with chf associated with polycystic kidney in a young persian cat was made . 
 interestingly , genetic testing revealed a wild - type sequence at position 3284 in exon 29 of pkd1.relevance and novel informationthe absence of the classic genetic mutation associated with the particular clinical presentation supports the hypothesis of a distinct etiopathogenesis among fibropolycystic diseases in domestic cats . 
 moreover , congenital hepatic fibrosis is a rare but important differential diagnosis for young persian cats and their crosses with clinical signs of chronic end - stage liver disease . 
Case summary
Relevance and novel information
Introduction
Case description
Discussion
Conclusions