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*
Department of Pediatrics, Erasmus Medical Center Rotterdam, Rotterdam, The
Netherlands
Department of Pediatric Surgery, Erasmus Medical Center Rotterdam,
Rotterdam, The Netherlands
Department of Cell Biology, Erasmus Medical Center Rotterdam, Rotterdam,
The Netherlands
§
Central Laboratory Animal Institute, Utrecht University, Utrecht, The
Netherlands
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Department of Cell Biology, University of Alabama at Birmingham,
Birmingham, Alabama.
¶
Department of Pediatrics, University of Alabama at Birmingham, Birmingham,
Alabama.
#
Department of Medicine, University of Alabama at Birmingham, Birmingham,
Alabama.
Correspondence to Dr. J. Nauta, Sophia Childrens Hospital, Molewaterplein 60, 3015 GJ Rotterdam, The Netherlands. Phone: 31-10-4636363; Fax: 31-10-4636801; E-mail: nauta{at}alkg.azr.nl
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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Of these conditions, the inherited polycystic kidney diseases (PKD) have been most extensively investigated. These disorders, which are transmitted as single Mendelian traits, cause significant morbidity and death among both adults and children (2). Autosomal dominant PKD (ADPKD) causes 6 to 8% of end-stage renal disease among adult patients. With autosomal recessive PKD (ARPKD), 30 to 50% of affected neonates die in the perinatal period. Surviving ARPKD patients, in combination with children with juvenile nephronophthisis, comprise 6 to 14% of all pediatric patients with end-stage renal disease (3,4).
Recent genetic studies have identified the principal genes involved in ADPKD (PKD1 and PKD2) (5,6). In addition, linkage studies have defined the predominant, if not exclusive, locus for ARPKD (PKHD1) (7,8). Although ADPKD is inherited as a dominant trait, multiple lines of evidence indicate that a second somatic mutation in this disorder may be necessary for disease expression (9,10). This suggests that, like ARPKD, ADPKD is initiated by recessive cellular mechanisms. However, the molecular pathogenic events involved in the initiation and progression of renal cystic diseases remain largely unknown.
In addition to human PKD, numerous mouse and rat models have been described (11,12,13). Most of these involve disruption of a single gene, and the mutant phenotypes closely resemble human PKD with respect to morphologic features, cyst localization, and disease progression. Several models are the result of spontaneous mutations, whereas others were engineered through either chemical mutagenesis or transgenic technologies. In addition, experimental models of PKD have been induced by chemical cystogens, primarily in rats.
Although the numerous mouse PKD mutations provide powerful models to characterize the genetic factors that regulate renal cyst initiation and disease progression, these models have limited utility for renal physiologic investigations. In contrast, rats represent a well established model system for investigating renal physiologic parameters such as renal blood flow, GFR, renal tubular transport, and BP regulation. In rats, PKD has been reported in the Han:SPRD-Cy model (14) and the Wistar-chi model (15). Han:SPRD-Cy rats have been well characterized and have been studied extensively as a model of ADPKD. The renal cystic disease in the Wistar-chi model resembles ARPKD in that lectin-binding studies localize the tubular cysts to collecting ducts. However, unlike in human ARPKD, the renal insufficiency progresses slowly and is associated with skeletal abnormalities.
In this report, we describe a new rat PKD model. Affected homozygotes develop rapidly progressive PKD that clinically and histologically resembles human ARPKD. This mutation occurred spontaneously in an outbred Wistar strain. We therefore designated the mutant locus wpk (Wistar polycystic kidneys). We have localized wpk to chromosome 5. This locus is distinct from the rat Han:SPRD-Cy locus, and its mouse and human orthologs are not allelic with any previously described mouse PKD model or human PKD gene. However, on the basis of the phenotypic similarities with human ARPKD, wpk rats provide a new experimental model for investigating the pathogenesis of recessive PKD. Because renal physiologic features have been well studied in rats, wpk rats should provide the first model system for evaluation of the abnormalities in renal tubular transport and systemic BP regulation that are associated with recessive PKD.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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The Han:SPRD-Cy rat model of PKD
(16) was used in
complementation experiments and as a phenotypic reference for our evaluation
of the homozygous wpk/wpk rats. In this model, +/Cy
heterozygotes develop slowly progressive PKD that leads to renal failure in
male animals only, at 
6 mo of age, whereas Cy/Cy homozygotes
develop a severe form of PKD within the first few weeks of life. Heterozygous
Han:SPRD-+/Cy breeding pairs were kindly provided in 1994 by F.
Deerberg (Central Institute for Laboratory Animal Breeding, Hannover,
Germany), and a breeding colony was established at the University of
Rotterdam. BB-DP rats, an inbred strain (F30) derived from biobreeding,
diabetes mellitus-prone rats, were kindly provided by H. A. Drexhage
(Department of Immunology, Erasmus University, Rotterdam, The
Netherlands).
To map the wpk locus, we used a (Wistar-+/wpk x BB-DP)F1 intercross, which was initially generated to test linkage to the PDK1 locus. The inbred BB-DP strain was selected on the basis of its genotype of Prm1, a polymorphic marker that is tightly linked to the rat PKD1 locus (17). Wistar-+/wpk heterozygotes of both genders were bred with BB-DP rats, and F1 progeny heterozygous for the wpk mutation were intercrossed to generate F2 rats. Affected homozygotes could be identified by abdominal palpation on postnatal day 12.
All animals were fed standard rat chow containing 24% protein and had free access to acidified tap water (pH 2.5 to 3.0). All experiments were conducted in accordance with the Dutch guidelines for the care and use of laboratory animals.
Clinical Parameters
Kidney weight, body weight, BP, and urine and blood chemistry values were
assessed for groups of six to eight rats at the age of 3 to 4 wk. Affected
wpk/wpk rats were compared with phenotypically unaffected
littermates. Affected Cy/Cy rats were compared with +/+ littermates.
In contrast to +/wpk heterozygotes, +/Cy rats could be
identified histologically, and these pups were excluded from further
analysis.
The weights of the left and right kidneys of wpk/wpk homozygotes did not differ. The kidney weights were therefore combined and expressed as a percentage of body weight. Data for male and female pups were pooled, because gender effects were never observed for prepubertal animals.
BP was measured in six pairs of 3- to 4-wk-old rats anesthetized with ketamine and thiopental, using an indwelling catheter in the femoral artery. Blood was obtained, by cardiac puncture, from six pairs of wpk/wpk homozygotes and unaffected littermates. Urea nitrogen, creatinine, and total bilirubin levels were measured in the plasma using a Kodak Ektachem 700 spectrophotometer (Kodak, Utrecht, The Netherlands). Urine was collected after cervical dislocation, after 2 h of fasting, from 4-wk-old affected and unaffected wpk rats. Urinary creatinine levels were measured by the Jaffé method and urinary protein levels by absorption spectroscopy.
For older animals, urinary protein excretion, BP, and blood chemistry values were compared between test-proven heterozygous +/wpk rats and wild-type control animals, at the age of 1.5 yr for female animals and 1 yr for male animals. Twenty-four-hour urine specimens were collected using metabolic cages. BP was determined by tail plethysmography. The animals were trained for this procedure before the measurements (18). Blood was obtained by aortic puncture, with ether anesthesia.
Tissue Preparation for Histologic Analysis and Electron Microscopy
(EM)
For histologic analysis, kidneys were obtained from wpk/wpk and
control animals on embryonic day 19 and postnatal days 0, 7, 14, and 21. The
cystic phenotype was established by histologic assessment. Multiple-organ
necropsies, including heart, lung, liver, pancreas, spleen, brain, and
intestine, were performed on 21-d-old homozygous mutants and adult
heterozygotes. Heterozygous female animals were studied at the age of 1.5 yr
and male animals were studied at the age of 1 yr.
For light microscopic and immunohistochemical analyses, the kidneys and livers were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) (pH 7.4) and embedded in paraffin. For liver histologic analyses, the left ventral lobe was dissected, fixed in 4% paraformaldehyde in PBS, and embedded in paraffin with the convexity toward the surface of the block.
For EM, representative kidney samples were fixed in 2.5% glutaraldehyde, postfixed in 1% osmium tetroxide, and dehydrated in a graded alcohol series. For scanning EM, the samples were then critical point-dried, mounted on stubs, coated with gold/palladium, and examined with a JEOL JSM 25 scanning electron microscope (JEOL, Tokyo, Japan). For transmission EM, the fixed and dehydrated samples were embedded in Epon. Thin sections were stained with uranyl acetate and lead citrate and examined with a Philips C100 electron microscope (Philips, Eindhoven, The Netherlands). Human kidney tissue from a neonate with ARPKD and end-stage renal failure was used as a comparative reference for the scanning EM analyses of wpk/wpk kidneys.
Evaluation of Biliary Ductal Plates
In addition to the renal cystic lesions, intrahepatic morphologic features
were evaluated for six affected wpk/wpk homozygotes and six
phenotypically unaffected animals (age, 21 d). The depth and angle of the
liver sections were standardized for histologic and morphometric analyses, as
described previously (19).
Bile ductules and recognizable portal areas were systematically examined in
each section, by one observer blinded to the phenotypes of the animals. A
recognizable portal area was defined as a venule accompanied by one or more
bile ductules.
Immunohistochemical Analyses
The localization of the renal cysts was evaluated by immunodetection of
nephron segment-specific proteins and lectin binding sites, on postnatal days
0, 7, 14, and 21. On the basis of preliminary studies, the following reagents
were selected. As a rat proximal tubule marker, we used polyclonal goat
anti-rat dipeptidyl peptidase 4 (dpp4) at dilutions of 1:500 (kindly provided
by E. de Heer, Leiden University Medical Center, Leiden, The Netherlands)
(20). As a marker of the thin
limb of Henle's loop (medulla) and proximal tubules (cortex), we used
polyclonal rabbit anti-rat aquaporin-1 (Aqp1) at 1:100 (generous gift of P.
Deen, University of Nijmegen, Nijmegen, The Netherlands). As a marker of the
thick ascending limb, we used polyclonal rabbit anti-human Tamm-Horsfall
glycoprotein at 1:200 (Biomedical Technologies, Stoughton, MA). As a marker of
cortical and medullary collecting tubules, we used polyclonal rabbit anti-rat
Aqp2 at 1:100 (generous gift of P. Deen, University of Nijmegen). All
antibodies were diluted in PBS with 0.5% dried milk and 0.15% glycine.
Schäfer et al. (16) reported increased expression of collagen type IV and laminin in the renal cysts of 2-mo-old Han:SPRD-+/Cy rats. Therefore, for comparative purposes, kidney sections from 21-d-old Wistar-wpk/wpk rats, 21-d-old Han:SPRD-Cy/Cy rats, and 2-mo-old+/Cy rats were analyzed with Jones' silver stain, as well as with a polyclonal rabbit anti-mouse collagen IV antibody at 1:200 (Collaborative Medical Products) (21) and a polyclonal rabbit anti-mouse laminin antibody at 1:50 (Eurodiagnostica, Arnhem, The Netherlands) (22).
The tissue sections were pretreated with 0.1% pronase in PBS for 5 to 10 min before the incubations with anti-Tamm-Horsfall glycoprotein, anti-dpp4, and anti-collagen IV and with 0.2% sodium dodecyl sulfate in PBS for 5 min before the incubations with anti-Aqp2. Anti-laminin and anti-Aqp1 were used without pretreatment of the tissue sections. Primary rabbit antibodies were detected using swine anti-rabbit peroxidase conjugate at 1:100 (Dako). Goat anti-dpp4 was detected using rabbit anti-goat peroxidase conjugate at 1:100 (Dako). The sections were stained with 0.05% diaminobenzidine/0.01% hydrogen peroxide and counterstained with hematoxylin.
Statistical Analyses
Clinical data are expressed as means and SD. Differences between
wpk and control animals were analyzed by t test.
PCR-Based Genotyping
To type progeny for the inheritance of alleles of anonymous DNA
microsatellite markers, spleen genomic DNA was prepared according to standard
protocols. Initial mapping was performed using interval haplotype analysis
(23). For these studies, we
selected microsatellite markers whose Wistar and BB-DP alleles differed in
size by at least 6 bp and mapped within approximately 10 cM of the proximal
and distal ends of each chromosome.
After chromosomal localization, further mapping was performed using markers spaced at 20-cM intervals along chromosome 5 (low-resolution linkage mapping study) and then within 4 cM of D5Rat73 (high-resolution linkage mapping study). All markers were chosen from the on-line Whitehead/Massachusetts of Technology database [accessible at http://www-genome.wi.mit.edu/; described by Szpirer et al. (24)]. PCR primer pairs for these markers were purchased from Research Genetics (Huntsville, AL).
Forward primers were end-labeled with [
-32P]ATP, and PCR
amplification was performed as described
(25). Amplified fragments were
analyzed on denaturing 6% polyacrylamide gels.
Analysis of Genetic Data
Genotype data, obtained by analyzing 35 affected F2 progeny for
microsatellite markers known to map to the ends of each autosome, were
subjected to interval haplotype analysis exactly as described by Neuhaus and
Beier (23). To construct low-
and high-resolution linkage maps, individual chromosomal haplotypes were
inferred from F2 genotypic data, as described previously
(25), and markers were ordered
to minimize the numbers of crossover events needed to account for the inferred
haplotypes.
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Results |
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The phenotype of homozygous mutants was characterized by progressive
nephromegaly and abdominal distension. Nephromegaly was first palpable on
postnatal day 12. By 4 wk of age, homozygous mutants were runted and had large
palpable kidneys. Affected rats died at 4 to 6 wk of age. The total kidney
weight was 10% of the body weight at 4 wk
(Table 1). In comparison, the
kidney weight was 1% of the body weight for unaffected littermates and 20% of
the body weight for Han:SPRD-Cy/Cy mutants at the same age. At 3 wk
of age, wpk/wpk homozygotes exhibited elevated plasma urea and
creatinine levels, proteinuria, and low urine osmolality after 2 h of fasting,
compared with unaffected littermates. The mean arterial BP, as assessed under
anesthesia by direct measurement in the femoral artery, was markedly elevated
(Table 1). The weight of the
liver relative to the kidney-free body weight and the plasma bilirubin levels
were not significantly different for homozygous mutants versus
unaffected littermates.
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Test-proven heterozygotes were clinically unaffected. There was no significant difference between 18-mo-old female +/wpk heterozygotes and age-matched, wild-type, control animals with respect to body weight (345 ± 24 versus 344 ± 29 g), systolic BP (135 ± 9 versus 128 ± 7 mmHg), plasma creatinine levels (41 ± 4 versus 46 ± 5 µM), or urinary protein excretion rate (20 ± 13 versus 27 ± 18 mg/d per 100 g body wt). Similarly, 1-yr-old male +/wpk heterozygotes did not differ from age-matched control animals with respect to body weight (547 ± 31 versus 517 ± 25 g), systolic BP (130 ± 7 versus 127 ± 8 mmHg), plasma creatinine levels (47 ± 5 versus 49 ± 7 µM), or urinary protein excretion rate (40 ± 26 versus 38 ± 21 mg/d per 100 g body wt).
Pathologic Features of wpk/wpk Kidneys
At a gross level, neonatal wpk/wpk kidneys exhibited normal
architecture, including normal lobulation, medullary rays, and well defined
corticomedullary demarcation. In affected pups, the kidneys progressively
enlarged but maintained a reniform shape, despite the progressive cystic
changes in the parenchyma. The capsular surface was smooth. The renal pelvis
and calices were not enlarged and maintained a normal relation to the
parenchyma (Figure 1). The
ureters were present and nondilated. This pattern is very similar to that
observed in the kidneys of human neonates with ARPKD.
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The histopathologic findings were characterized by progressive cystic dilation of the renal tubules. Early cysts were noted at 19 d of embryonic development (Figure 2, A and B). At that stage, as well as in neonatal kidneys, the lesions were predominantly localized in the renal cortex. The cysts were round or oval and were lined with either a single layer of cuboidal cells or flattened epithelia (Figure 2C). Some of the cysts were lined by a brush border-bearing cell type, suggesting proximal tubular epithelia. The cell density of the epithelial lining was high in most but not all cysts. Glomerular cysts were not observed.
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With subsequent disease progression, cysts developed throughout the entire kidney (Figure 2D); cortical cysts were arrayed in a radial orientation, whereas medullary cysts were generally round and of variable size. In kidneys with advanced cystic disease, there was an apparent reduction in the number of glomeruli and nondilated tubules, and those remaining appeared to be compressed between innumerable tubular cysts. Interstitial fibrosis was not observed, and the renal vasculature was unremarkable. In contrast to the early cystic kidneys, the cysts in more advanced disease were lined with a relatively homogeneous cell type. Within individual cysts, the cell densities varied considerably. Interestingly, areas of high cell density were often juxtaposed with similar areas in neighboring cysts (Figure 2E). This observation suggests that focal epithelial cell proliferation within cysts may be determined by the local biologic environment. However, true epithelial hyperplasia with associated polyps or microadenomas, as noted in human ADPKD (26), in Cy/Cy rats (16), and in the c-myc-overexpression model of PKD (27), was never observed.
Immunohistochemical Analyses
Cysts in neonatal kidneys were stained with either anti-dpp4,
anti-Tamm-Horsfall protein, or (from day 1 onward) anti-Aqp2 antibody,
indicating cellular characteristics typical of proximal tubules, thick
ascending limbs, or collecting tubules, respectively. Staining of medullary
cysts with anti-Aqp1, indicating a thin loop-derived cell type, was rarely
observed. Cortical cysts were occasionally stained with anti-Aqp1 in a weak
pattern. These cysts were lined by a brush border-bearing cell type and were
also stained with anti-dpp4, indicating proximal tubules. Individual cysts
were typically lined with only one cell type, and overlapping staining
patterns (except for anti-Aqp1 staining in proximal tubules) were not
observed. By 2 wk of age, the vast majority of cysts appeared to be derived
from collecting ducts, because the majority of cysts were stained with
anti-Aqp2. At 3 wk of age, >90% of the cysts expressed Aqp2
(Figure 2F). In comparison,
renal cysts in 3-wk-old Cy/Cy homozygotes appeared to arise from all
nephron segments (data not shown).
In wpk/wpk homozygotes, the renal tubular basement membrane morphologic features, as assessed with Jones' silver staining, were not significantly different between cystic tubules and normal tubules in either early or advanced disease. Moreover, there was no difference in the expression of the tubular basement membrane constituents collagen type IV and laminin when cystic and noncystic tubules were compared. In contrast, collagen type IV and laminin were overexpressed in the tubular basement membranes associated with some but not all renal tubular cysts in SRPD-+/Cy and SRPD-Cy/Cy kidneys (data not shown) (16).
Electron Microscopy
Scanning EM results were consistent with the light microscopic observation
that early and late cysts have different epithelial linings
(Figure 3). The cell surface
characteristics of early cystic tubules varied considerably among individual
cysts. Some tubules exhibited a homogeneous brush border, characteristic of
proximal tubules (Figure 3A).
Others exhibited more distal tubule or collecting duct characteristics,
including fewer microvilli and smoother apical surfaces. Some exhibited the
typical characteristics of collecting ducts, with a prominent single cilium,
as illustrated for 3-wk-old tissue (Figure
3E).
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Cysts in kidneys with advanced disease were radially oriented. In contrast to early cysts, these cysts were uniformly lined by a homogeneous cobblestone cell type, indicating a collecting duct origin (Figure 3, B to D). These cells exhibited the typical phenotype of principal cells, including relatively smooth apical surfaces and a well differentiated, conspicuous, single cilium (Figure 3E). These cilia were prominent for the vast majority of cortical cysts and less prominent or absent for medullary cysts. In addition, the epithelial lining of most cysts contained a small number of rough-surfaced cells with characteristics previously described for rat type B intercalated cells (Figure 3F) (28). This mixed pattern of principal and intercalated cells is characteristic of cortical and outer medullary collecting ducts in rats and is very similar to the pattern observed for the vast majority of the cysts in our human ARPKD reference sample (Figure 3, G and H) (28).
Transmission EM results were consistent with both the light microscopic and scanning EM findings. Early renal cysts in wpk/wpk rats were lined by well differentiated cells with the phenotypic characteristics of either proximal tubules, thick ascending limbs, distal tubules, or collecting ducts (Figure 4). Microvilli were well formed, cell junctions and basal laminae were intact, and the cellular organelles were normally distributed. At 3 wk of age, the vast majority of cortical and medullary cysts were lined by a homogeneous layer of cells with the characteristic phenotype of collecting duct cells (29). Abnormalities evident in human ADPKD and in the Han:SPRD-Cy/Cy model, e.g., basement membrane thickening, intercellular vacuoles, or intratubular micropolyps, were not observed (26). The basal laminae were intact, with normal architecture and thickness.
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Nonrenal Tissues
Multiorgan necropsies of 3-wk-old Wistar-wpk/wpk pups and adult
Wistar-+/wpk heterozygotes did not reveal any nonrenal structural
abnormalities. Of particular note, no evidence of ductal plate malformations
or biliary cysts was observed by light microscopy in multiple liver sections
from six 3-wk-old wpk/wpk rats. Morphometric analyses of standardized
liver sections revealed no evidence of portal triad abnormalities or bile
ductule proliferation in wpk/wpk homozygotes, compared with
unaffected control animals (Table
2). Similarly, pancreatic tissue exhibited normal ducts and normal
endocrine and exocrine structures in both mutant and control animals (data not
shown).
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(Wistar-+/wpk x BB-DP)F1 +/wpk Intercross
In our (Wistar-+/wpk x BB-DP)F1 intercross, F1
+/wpk hybrids were identified by progeny testing. Evaluation of aged
F1 male and female rats revealed no manifestations of renal cystic disease,
and F1 male and female animals bred in a robust manner.
Of the 225 F2 progeny generated to date, 55 (24.4%) exhibited recessive PKD. The number of F2 wpk/wpk pups is consistent with that expected for the Mendelian inheritance of a single recessive trait. Detailed histologic analysis of F2 mutants was not performed.
Genetic Mapping of wpk
To test whether wpk is allelic with the Cy locus on rat
chromosome 5, we crossed test-proven Wistar-+/wpk and
SPRD-+/Cy heterozygotes. None of the 35 F1 pups manifested the
severe, early-onset phenotype evident for either wpk/wpk or
Cy/Cy rats. As expected, 17 of the 35 pups (48.5%) expressed a
phenotype consistent with that described for SPRD-+/Cy heterozygotes.
These data exclude allelism between the wpk and Cy loci.
We then performed a whole-genome scan using interval haplotype analysis, as described by Neuhaus and Beier (23). In effect, by typing markers at the ends of each chromosome, we generated a series of 20 chromosomal intervals for the 35 affected F2 progeny of the (Wistar-+/wpk x BB-DP)F1 intercross.
Among the progeny of an intercross, a proportion of the F2 pups inherit chromosomes that are apparently nonrecombinant (NR), that is, the alleles of markers along these chromosomes correspond to a single parental strain (in this case, either Wistar or BB-DP). In the analysis of a recessive mutation such as wpk, a rapid genome scan can be performed by analyzing each chromosome for the distribution of NR intervals. The fewer NR chromosomes that correspond to the unaffected parental strain (BB-DP), the less likely it is that the loci along that chromosome are randomly distributed. Accordingly, this chromosomal interval is more likely to carry the mutation.
The distribution of chromosomal intervals is evaluated by 
2
analysis. The maximal inferred 2 value is calculated for each
chromosomal interval and expressed as a percentage of the maximal possible
2 value. Previous modeling experiments have established
>75% of the maximal possible 2 value as the threshold for
linkage (23). Therefore, this
strategy provides an efficient method to identify candidate chromosomes for
more detailed analyses, using standard recombinational mapping techniques.
Analysis of our data set revealed 88% of the maximal possible
2 value for chromosome 5 and <75% of the maximal possible
2 value for all other chromosomal intervals
(Table 3). These data provided
presumptive evidence for linkage to chromosome 5. We typed the initial cohort
of 35 affected F2 pups with a series of anonymous DNA microsatellite markers
spaced at approximately 20-cM intervals along this chromosome. These pups and
an additional 19 F2 pups (total of 54 pups and 108 meioses) were typed with
markers within 4 cM of D5Rat73. These data, which are summarized in
Figure 5, position wpk
within a 11.1-cM interval centered on D5Rat73.
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We screened 146 markers to identify the 40 informative markers required for interval haplotype analysis, indicating that the polymorphism rate between the outbred Wistar and BB-DP parental strains was only 27.5%. Similarly, for the low- and high-resolution mapping studies, we screened 16 markers to identify the six (37.5%) informative markers shown in Figure 5. This low polymorphism rate was not unexpected, given that the Wistar and BB-DP strains are phylogenetically related.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionsReferences |
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The majority of human patients with ARPKD are identified either in utero or at birth. The most severely affected fetuses exhibit enlarged echogenic kidneys and oligohydramnios because of poor fetal renal output. At birth, these neonates often exhibit a critical degree of pulmonary hypoplasia that is incompatible with survival. Renal function, although frequently compromised, is rarely a cause of neonatal death. For infants who survive the perinatal period, systemic hypertension and progressive renal failure usually evolve (4). In addition, patients with ARPKD have defects in both urine-diluting and urine-concentrating capacities. Hyponatremia, presumably resulting from defects in free water excretion, is often observed (4).
Similarly, renal cysts in wpk/wpk homozygotes develop in utero, and enlarged kidneys are palpable within the first few weeks of life. Disease progression is associated with continued renal enlargement, systemic hypertension, proteinuria, significant reductions in urine-concentrating capacity, progressive renal insufficiency, and death by 4 to 6 wk of age. As in human patients with ARPKD (30), early renal cysts in wpk/wpk homozygotes develop in both proximal and distal nephron segments. With disease progression, the cystic lesions in both the rat model and human ARPKD predominantly involve the cortical and medullary collecting ducts.
Although Wistar-wpk/wpk mutants do not express a biliary phenotype, we suspect that wpk-related disease expression may be modulated by the genetic background, as has been demonstrated for the cpk mouse model (31). Genetic backgrounds (32,33,34,35) as well as environmental factors, such as dietary protein restriction, use of soy proteins instead of casein proteins, use of dietary flaxseeds, use of nonacidified drinking water, and potassium citrate treatment, have been reported to alter disease progression in murine PKD models (36,37,38,39). Moreover, adequate treatment of high BP may have an additional beneficial effect on both renal and patient survival rates, as it does for human patients (40).
This wpk model complements two established rat models of PKD, i.e., the Cy model and the chi model. In contrast to both human ARPKD and the wpk model, the Cy mutation can be expressed as both a dominant and a recessive trait (14). We demonstrated that the renal phenotype in Cy/Cy rats is more severe than that currently described for the wpk model (Table 1). Histologic studies of the cysts in both Cy/Cy and +/Cy rats indicated that these may arise in any nephron segment. This is consistent with the pattern observed for human patients with ADPKD and is in contrast to the collecting tubular localization of the cysts in advanced stages of both human ARPKD and wpk/wpk disease. Although the chi model has not been as well characterized as either the Cy model or the wpk model, the chi model exhibits less progressive disease and includes skeletal abnormalities in addition to the renal lesions (15). In the original reports, there was no specific mention of whether biliary lesions are manifested in that model.
Systemic Hypertension
Hypertension is a common finding among human patients with PKD and
contributes to both morbidity and death. Longitudinal observations indicate
that hypertension is a major determinant of disease progression in ADPKD
(41). Among affected children,
hypertension occurs in both ADPKD and ARPKD but tends to be more severe in
ARPKD
(42,43,44,45,46).
Stimulation of the renal-angiotensin-aldosterone axis (RAAS) seems to be a major mechanism causing hypertension in ADPKD (41). Whether and to what degree increased activity of the RAAS contributes to hypertension in ARPKD remains unclear. The limited available data are conflicting. Histopathologic observations indicate that, with progressive disease, the glomeruli are compressed in the septa between expanding collecting duct cysts. Mechanical compression of the glomeruli and the intrarenal vasculature could, at least theoretically, stimulate RAAS activity. However, clinical data from affected neonates indicate that ARPKD is actually a low-renin state, with expansion of total body volume and occasional hyponatremia (40).
Given the extensive body of work on BP regulation in rats and the striking phenotypic similarities between the rat wpk model and human ARPKD, we propose that wpk rats may provide a new powerful model system for investigation of the physiologic and genetic mechanisms that contribute to hypertension in recessive PKD.
Absence of Biliary Abnormalities
In this study, we observed no biliary histopathologic features in
Wistar-wpk/wpk homozygotes. This is a shortcoming of the model,
because bile duct proliferation and periportal fibrosis are typically present
in all patients with ARPKD. However, because we did not examine the liver
histologic features of F2 wpk/wpk homozygotes, we cannot exclude the
possibility that the genetic background affects the expression of biliary
lesions in the wpk model, as has been demonstrated for the
cpk mouse model of ARPKD
(31,47).
Cellular Morphologic Features of wpk/wpk Renal Cysts
Characterization of the cystic epithelia in the wpk model has
yielded a number of interesting insights. First, the initial cysts seem to be
derived from functioning unobstructed nephrons, and the cystic epithelia
maintain their segment-specific phenotypes. These observations are consistent
with data for the mouse bpk and cpk models and suggest that
the disease-susceptibility genes in these models do not disrupt the early
stages of nephrogenesis, i.e., induction of mesenchyme-to-epithelium
transformation, acquisition of stem cell character, fate determination,
epitheliogenesis, and polarization
(48).
In more advanced disease, cysts in the Wistar-wpk/wpk kidneys are lined by well differentiated collecting duct cells, as assessed by segment-specific marker profiling and EM. Although the cystic epithelia seem to have escaped the normal mechanisms controlling tubular diameter, they retain specific epithelial phenotypes, with heterogeneous populations of principal cells and intercalated cells, and maintain their organization as monolayers. Interestingly, the cyst epithelium is not uniformly proliferative, with focal areas of increased proliferation adjacent to cysts. This observation suggests that cell proliferation is influenced by local environmental factors as well as by the defective gene. Interestingly, no abnormalities of the extracellular matrix or the basal lamina were associated with renal cyst initiation or progression in wpk/wpk kidneys.
The wpk Mutation as a New Model of Recessive PKD
The renal phenotype of the rat wpk model closely resembles those
of human ARPKD and the mouse cpk, bpk, and orpk models.
However, the wpk model is genetically distinct from all previously
described PKD loci. The wpk locus maps to proximal chromosome 5.
Although the rat Cy locus also maps to chromosome 5
(49), we have demonstrated
that wpk and Cy are not allelic.
In homology mapping, the human wpk ortholog maps to chromosome 8q11 (http://www.ncbi.nlm.nih.gov/Homology). These data exclude the wpk locus as a candidate for the human ARPKD gene, PKHD1, which maps to human chromosome 6p21-p12 (50). Similarly, the mouse wpk ortholog maps to proximal mouse chromosome 4 and thus is genetically distinct from mouse bpk on chromosome 10 (25), cpk on chromosome 12 (51), and orpk on chromosome 14 (52). It is interesting to note, however, that a principal modifying gene for both the mouse cpk and pcy models also maps to proximal chromosome 4 (35,53). This observation raises the interesting possibility that the mouse wpk ortholog may be a candidate PKD-modifying gene.
Although the (Wistar-+/wpk x BB-DP)F1 intercross was informative for localizing the rat wpk gene, the fact that the mutation arose in an outbred strain, coupled with the low polymorphism rate for the two parental strains, presents problems for further refinement of the genetic interval and application of positional cloning strategies to identify the wpk gene. Therefore, we are currently generating a congenic Wistar-+/wpk strain using a standard backcross strategy. By the 10th backcross generation, >99% of the genome in this congenic line will be derived from the inbred Wistar strain. At that point, we will generate a new F2 cohort by breeding the congenic Wistar-+/wpk line with a second inbred strain, e.g., Brown Norway, so that the polymorphism rate for the two parental strains is more robust. We will also monitor the available databases for potential candidate genes that map to the candidate wpk region on rat chromosome 5 or the homologous regions in the human and/or mouse genomes.
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