Polycystic kidney disease: A review and occurrence in Slovenia with comparison between ultrasound and genetic testing

Abstract

Polycystic kidney disease (PKD) is an inherited autosomal kidney disease which is most commonly identified in Persian and Persian related cats. Positive cats have multiple cysts of various sizes that occur in the renal cortex and medulla and occasionally in other abdominal organs. PKD often leads to renal failure which occurs from mid to late in life. Renal cysts can be diagnosed ultrasonographically after 7 weeks of age by an experienced ultrasonographer and a high resolution machine. However, ultrasonography is now being replaced by genetic screening. A total of 340 cats of variable breeds aged from 5 months to 18 years were ultrasonographically examined in the past 7 years at the University Veterinary Small Animal Clinic. Of these, 13.8% were PKD positive with very high prevalence in Persian cats (36%). There was no sex predilection identified. The C>A transversion at position 3284 on exon 29 of PKD1 gene, resulting in a stop mutation has been identified in the heterozygous state in eight affected cats examined (Persian breed). All heterozygous cats were also ultrasonographically positive.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common genetic disorder in humans, occurring in 1 in 200 to 1 in 100 people (Kimberling et al 1991). The disease occurs in all races, affecting as many as five million people worldwide, and accounts for approximately 10% of renal dialysis patients. A form of ADPKD similar in clinical features to human ADPKD occurs in Persian and Persian crosses (Biller et al 1996a, Eaton et al 1997, Barrs et al 2001, Beck and Lavelle 2001).

In approximately 85% of human patients with ADPKD the disease is caused by a mutation in the PKD1 gene located on chromosome 16p13.3. The remaining 15% of affected ADPKD patients were discovered to be a result of a defective PKD2 gene on chromosome 4q21–23 or an unidentified, but suspected third genetic locus. Similar to PKD1, PKD2 encodes for ‘an integral membrane protein’, and the homology between the two proteins exists. There are additional genes that are known to cause different types of polycystic kidney condition in humans (Biller et al 1996a, Barrs et al 2001, Beck and Lavelle 2001, Young et al 2005). The clinical signs of this disease often overlap, therefore in humans DNA testing is required to determine which gene is causing the clinical presentation. Diseases that have similar clinical presentation are called heterogeneous diseases. Because of the disease heterogeneity in humans it was difficult to find out which gene caused polycystic kidney disease (PKD) in cats (Eaton et al 1997).

Study of the pathogenesis of cyst formation and growth in ADPKD has been limited because of the lack of availability of animal models that closely mimic the human disease. Several models of autosomal recessive polycystic kidney disease (ARPKD) that closely mimic human ADPKD have been described. In addition, the pcy mouse model of ARPKD shows some similarities in clinical features to human ADPKD but differs in morphology and inheritance. The clinical and morphological similarity of ADPKD in Persian cats to human disease and the autosomal dominant pattern suggest that this disease represents a good model for ADPKD in humans (Eaton et al 1997, Beck and Lavelle 2001).

Feline PKD is an inherited disease that has been recognised mostly in Persian and Persian related cats.

The disease is characterised by cysts of various sizes that occur in the renal cortex and medulla and occasionally in the liver, pancreas and uterus. Affected cats remain clinically normal for most of their lives, and renal failure only occurs later in life (usually after 7 years of age). Although the dysfunction of the kidney does not occur until mid to late in life, the cysts are present from birth, although are smaller in younger animals. The size of cysts can vary from less than 1 mm to greater than 1 cm in size, with older animals having larger and more numerous cysts. The number of cysts also varies from one to more than 200 per kidney. There is a marked difference in when and how quickly cats succumb, with the possibility of a long enough life that a PKD cat can die of other causes before kidney failure. However, it is never certain when the cysts will grow and cause problems (Biller et al 1996a,b).

Feline renal cystic disease can be diagnosed by ultrasound with a high level of confidence. In affected cats, cysts can be detected as early as 7 weeks of age (Biller et al 1996a). The sonographic features of renal cysts include: a round-to-ovoid contour; anechoic contents; smooth, sharply demarcated thin walls with a distinct far-wall border, and strong distal acoustic enhancement (through transmission). Cysts may deform the kidney outline if they are large. High frequency transducers are required to give sufficient resolution and a 7.0 MHz transducer, or higher should be used. The medulla is hypoechoic to the renal cortex; therefore, it is easier to detect cysts in the cortex in comparison to the medulla. Ultrasonography had sensitivity of 75% when performed at 16 weeks of age and a sensitivity of 91% when performed at 36 weeks of age (Biller et al 1996a,b). Research showed that the absence of cysts on ultrasound examination at 6 months of age was correlated with absence of PKD at necropsy (Biller et al 1996a). Actually only one cat had just one cyst on one kidney, but because it was a young Persian it was diagnosed as PKD. All other cats had more than one cyst.

In 2004 Lyons et al published identification of a mutation C>A at position 3284 resulting in feline PKD. The C>A transversion was found in heterozygous state in Persians and other breeds out-crossed with Persians. A presently available DNA test can identify cats that will develop PKD in the future. Using polymerase chain reaction (PCR) amplification product size 559 on exon 29 is amplified. The mutation causes a restriction enzyme site alteration for MLY1, producing two fragments of 316 and 243 bp. Cats with wild type of gene PKD1 do not have any stop mutation, resulting in only non-digested amplification product in size 559 bp after restriction fragment length polymorphism (RFLP). However, the heterozygous cats with mutation have digested amplification product and non-digested amplification product. A different mutation could still possibly cause feline PKD but would have to be in strong linkage disequilibrium with this stop codon (Lyons et al 2004).

Materials and methods

A total of 340 cats have been examined ultrasonographically over the last 7 years (January 1999–October 2006). Of these, 104 were Persian cats, five Siamese, two Burmese, two Norwegian Forest cats, one Angora, one Himalayan, one British Shorthair, one European and 223 domestic shorthair cats. The age of the cats ranged from 5 months to 18 years. Cats were referred to the clinic either at their owner's request, specifically to be screened for the presence of PKD, or were referred for the investigation of medical problems. Both kidneys were examined ultrasonographically using a Vingmed System Five ultrasound machine with 10 MHz linear transducer. PKD was confirmed if at least one cystic structure was found on at least one kidney. All examinations were performed by one ultrasonographer [Aleksandra Domanjko Petrič (ADP)] with 16 years of experience.

DNA from 24 Persian cats was isolated from whole blood using Wizard Genomic DNA purification kit (Promega) following manufacture protocol. Ethylenediamine tetraacetic acid (EDTA) anticoagulated blood was collected by venepucture. PKD exon 29 product was amplified by PCR from genomic DNA using the primers:

• PKD1 F CAG GTA GAC GGG ATA GAC GA

• PKD2 R TTC TTC CGT GTC AAC GAC TG.

Approximately 12.5 ng of DNA was used per PCR reaction. Reaction condition for each samples were as follows: 22.5 μl platinum super mix (Invitrogen) and 1 pmol of each primers in 25 μl reaction volumes. Cycling parameters included 10 min of denaturation at 94°C followed by 35 cycles of 1 min denaturation at 94°C, annealing for 1 min at 58°C, and a 72°C extension for 1 min. RFLP analysis of amplificate products followed. Approximately 5 μl of amplification product was digested with 10 U of Mly1 (New England Biolabs) in a 10 μl reaction that contained 1× NE Buffer 4 at 37°C for 3 h. After incubation followed inactivation of the enzyme at 65°C for 10 min. Digestion products were analysed on 2% agarose gels.

Statistics

Examined cats were divided into two groups – Persians (because the rest of breeds were represented solitarily) and non-Persians (including domestic shorthairs), representing two big groups in order to calculate the prevalence.

Prevalence was calculated for predisposed examined cats (Persian) with the formula: (predisposed positive/total predisposed) and for non-predisposed breeds (non-predisposed positive/total non-predisposed).

Results

The study was partially retrospective and partially prospective; in the retrospective population were 104 Persians and 223 non-Persians. Eighty Persians were examined by ultrasound only, and among them were 29 PKD positive (36.3%). The remaining 24 were examined by both ultrasound and genetic testing, and among those eight Persians were positive on both tests (33.3%). Although further comparison between ultrasound and genetic testing is needed, these results confirm previous observations that ultrasound is very accurate when performed by an experienced ultrasonographer with a high resolution transducer.

Of the 340 cats examined 47 (13.8%) were positive for feline PKD, with one or more cystic structures of various sizes identified within the renal parenchyma of one or both kidneys ( Fig 1). Actually only one cat had just one cyst on one kidney, but because it was a young Persian it was diagnosed as PKD. All other cats had more than one cyst on each kidney.

Fig 1.

Cystic structures of various sizes within the renal parenchyma.

In the population of positive cats were 37 Persian cats, one European cat and nine domestic shorthairs. These cats ranged in age from 5 months to 18 years (an average age of 7.78 and median 7 years) ( Fig 2). Of the 104 Persian cats, 37 (36%) had the disease, with prevalence of 0.36. Of non-predisposed cats 10 (4.2%) were affected with PKD and the calculated prevalence was 0.042.

Fig 2.

Age distribution of cats positive for feline PKD examined in our study at the time of diagnosis.

Amongst the positive cats there were 24 males and 23 females. In two Persian cats we found numerous cysts in the liver as well ( Fig 3).

Fig 3.

Numerous cysts in the liver in a cat with PKD.

In 10 affected cats with clinical signs (anorexia, polyuria, polydipsia, vomiting and excessive salivation) the renal biochemical parameters urea and creatinine were measured. The results showed that almost all cats had higher values of creatinine (average of 202.3 μmol/l or 2.29 mg/dl), and normal or higher values of urea (Table 1). The mean age of these cats was 12.2 years, which correlates with the fact that most of the PKD affected cats remain clinically normal until late in life when renal failure occurs (Fig 2).

Table 1.

Table of urea and creatinine values in PKD affected cats in this study (values above normal references are written in bold numbers)

Urea mmol/l (reference: 5.9–12.5 mmol/l)	Urea mg/dl (reference: 16.5–35.0 mg/dl)	Creatinine μmol/l (reference: 71–108 μmol/l)	Creatinine mg/dl (reference: 0.80–1.22 mg/dl)	Age (years)
11.49	32.18	224.7	2.54	20
4.95	13.87	101.5	1.15	9
8.1	22.69	129.4	1.46	4
6.8	19.05	132.1	1.49	12
88.5	247.90	409.2	4.62	13
19.29	54.03	210.5	2.38	14
35.14	98.43	381.5	4.32	8
21.2	59.38	152.4	1.72	15
10.27	28.77	147.9	1.67	13
11.62	32.55	133.7	1.51	14

Genetic tests in 24 cats showed eight heterozygous cats with stop mutation on PKD1 gene as causative for feline PKD1. All heterozygous cats were PKD positive also by ultrasound examination (Fig 4). A DNA test of feline PKD showed complete co-segregation of the stop mutation with the disease phenotype. The remaining 16 cats were negative for PKD both by genetic test and ultrasonography. Homozygous cats for the mutation on PKD1 gene were not found.

Fig 4.

Amplification product of exon 29 was digested with the restriction enzyme Mly1. All cats affected with PKD produced the wild type 559 bp fragment and the digested 316 and 243 bp fragments. Lanes 1, 2, 13, 19, 20, 55 are unaffected cats, and lanes 3 and 4 are affected cats. Table shows ultrasound (US) examination's results and genotype (G).

Discussion

Feline PKD has been reported in the literature as primarily affecting Persian and Persian-cross cats and was first described as a familial disease in cats. The similarity between PKD in Persian cats and human ADPKD was found to provide a valuable animal model to research the deadly human kidney disease. Both diseases are inherited in an autosomal dominant manner and are characterised by renal cysts that are present from birth and that progressively grow in size as the individual ages, eventually causing renal failure. The age of onset is variable, but in the average occurs late in life (Eaton et al 1997).

The latest studies revealed the prevalence of PKD in Persian cats to be very high (37–49%). Our study shows a prevalence of 4.2% in domestic shorthairs and 36% in Persian cats in Slovenia. This prevalence is slightly lower in comparison to other studies (Table 2) which maybe is a consequence of more cats being screened in the last years (Cooper 2000, Beck and Lavelle 2001, Barrs et al 2001, Cannon et al 2001, Barthez et al 2003).

Table 2.

The comparison of prevalence of PKD in some countries worldwide

Country/region	Prevalence of PKD in Persian and other long-haired breeds (%)
United Kingdom	49.2 (Cannon et al 2001)
Sydney	45.0 (Barrs et al 2001)
University of Melbourne	45.0 (Beck and Lavelle 2001)
Brisbane	42.0 (Barrs et al 2001)
France	41.8 (Barthez et al 2003)
United States	37.0 (Cooper 2000)
Slovenia	36.0 (this study)

The number of screened cats of other breeds was too small to calculate a reasonable prevalence. There was an almost equal number of positive male (51%) and females (49%) found, suggesting that the disease has no sex predilections found by other authors (Barrs et al 2001). It is important to diagnose the disease before breeding in order to avoid passing on the condition. Ultrasound examination proved to be a very sensitive tool in experienced hands with a high definition transducer and can give accurate results before cats are bred. The results of the ultrasound examination were in agreement with the genetic test results in the 24 cats in which both tests were performed. This comparison between examinations shows that ultrasound examination of PKD in cats is reliable. Detection of feline PKD in cats younger than 3 months is better using genotyping of PKD1 gene than ultrasound examination. In future genotyping of PKD1 gene will be used for detection of PKD1 gene in very young cats where very small cysts, especially in medulla, might not be seen. All affected cats are heterozygous for mutation on PKD1 gene (Pp), therefore, 36% of Persian breed cats born in Slovenia are heterozygous (Pp) and 64% of cats are homozygous normal (pp). Frequency of wild type PKD1 gene is 0.76 thus frequency of PKD1 gene with mutation is 0.24. We can conclude that 6% of cat fetuses are homozygous for PKD1 gene with mutation.

Those fetuses homozygous for PKD1 gene probably die in the embryonic period or are stillborn (Lyons et al 2004).