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NDT Advance Access originally published online on November 29, 2007
Nephrology Dialysis Transplantation 2008 23(4):1157-1165; doi:10.1093/ndt/gfm763
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© The Author [2007]. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org


Overexpression of PKD2 in the mouse is associated with renal tubulopathy

Stéphane Burtey1,*, Marta Riera1,*,**, Emilie Ribe1, Petra Pennekamp2, Edith Passage1, Roselyne Rance1, Bernd Dworniczak2 and Michel Fontés1

1 INSERM UMR 491, Medical Genetics and Development, IPHM, Faculté de Médecine de la Timone. 27 Bd. J. Moulin. 13385 Marseille cedex 5. France 2 Institute for Human Genetics, University Hospital Muenster, Vesaliusweg 12-14, 48149 Muenster, Germany

Michel Fontès, Génétique Médicale et Développement, INSERM UMR 491 Faculté de Médecine de la Timone, 27 Bd. J. Moulin 13385 Marseille cedex 5, France. Tel: +33-(0)4-91-25-71-59; E-mail: michel.fontes{at}medecine.univ-mrs.fr



   Abstract
Top
 Abstract
Introduction
 Subjects and methods
 Results
 Discussion
 Supplementary data
 References
 
Polycystin-2 (PC-2), a cation channel of the Trp family, is involved in autosomal dominant polycystic kidney disease (ADPKD) type 2 (ADPKD2). This protein has recently been localized to the primary cilium where its channel function seems to be involved in a mechanosensory phenomenon. However, its biological function is not totally understood, especially in tubule formation. In the present paper, we describe a mouse model for human PC-2 overexpression, obtained by inserting a human bacterial artificial chromosome (BAC) containing the PKD2 gene. Three lines were generated, expressing different levels of PKD2. One line, PKD2-Y, has been explored in more detail and we will present physiological and molecular exploration of these transgenic animals. Our data demonstrate that transgenic animals older than 12 months present tubulopathy with proteinuria and failure to concentrate urine. Moreover, the kidney cortex has been found disorganized. Finally, we observe that extracellular matrix protein expression is downregulated in these animals. In conclusion, overexpression of human PKD2 leads to anomalies in tubular function, probably due to abnormalities in tubule morphogenesis.

Keywords: abnormal extracellular matrix; fibronectin; PKD2; tubular dysfunction



   Introduction
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 Abstract
 Introduction
Subjects and methods
 Results
 Discussion
 Supplementary data
 References
 
Autosomal dominant polycystic kidney disease (ADPKD) is a common disorder primarily characterized by progressive renal enlargement and cyst formation, leading, in the majority of patients, to end-stage renal disease [1]. However, cyst formation has also been observed in the liver and pancreas and cerebral artery aneurysms have been reported [2] as well. The incidence of ADPKD in the general population is about 1 in 1000 individuals, making it one of the most common genetic disorders in humans [3].

This disorder is genetically heterogeneous. About 85% of cases are due to a mutation in the PKD1 gene [4], located on the short arm of chromosome 16 presenting early onset, while 15% present mutations in PKD2 [5], characterized by later onset of the disorder [6]. However, due to the increase in the lifespan of the general population in industrial countries, the prevalence of this form is tending to increase [7].

Polycystin-2 (PC-2), the protein encoded by PKD2, is a non-selective calcium channel of the Trp family [8]. Polycystin-1 (PC-1), encoded by PKD1, interacts with PC-2 and probably regulates the calcium channel activity of PC-2 [9]. The general assumption is that PC-2 is the channel and PC-1 the regulator of PC-2 channel activity, although we neither know the PC-1 ligands nor the concomitant cellular localization of the two proteins. PC-1 has been shown to modulate several downstream signalling pathways [10–13] although most of these reports rely on cell transfection with constructs representing only a small region of the protein (the total protein consists of 4302 amino acids). PC-2 can act in a signalling pathway without PC-1 [14,15]. Contrary to PC-1, PC-2 has been implicated in the mechanisms underlying vertebrate left–right axis specification [17].

Recently, PC-1 and PC-2 have been localized to cilia and have been demonstrated to play a role in the transduction of mechanosensation via calcium signalling [16]. Finally, it has been demonstrated that while PC-2 is present in the plasma membrane, most of the protein is located in the endoplasmic reticulum and acts as an intracellular Ca2+ release channel [18]. However, the reported data are quite puzzling, not answering questions concerning the function(s) of PC2, and moreover, the functions common to PC-1 and PC-2 in normal kidney organogenesis as well as during cystogenesis. In addition, while the primary cause of the disorder is clear (a mutation in PKD2), the cascade of events involved in cyst formation and growth has not been completely identified.

The phenotype of mice in which PKD2 has been knocked out has been well described [17,19]. However, while these models provide important information concerning the cystic disorder, they tell us little about the biological role of PC-2 in kidney development and tubule function.

In order to improve our understanding of the role of PC-2 in normal as well as in pathological conditions we constructed transgenic mice using a BAC encompassing the human PKD2 gene and its flanking elements. Here, we present the characterization of these three lines as well as data regarding kidney physiology and tubule structure. The transgenic mice displayed unexpected features: cortex disorganization and mild nephrogenic diabetes insipidus. In addition, data on gene expression in the kidney of transgenic animals will be presented and linked to the phenotype observed. These mice represent a new model of disease associated with PKD2.



   Subjects and methods
Top
 Abstract
 Introduction
 Subjects and methods
Results
 Discussion
 Supplementary data
 References
 
BAC isolation and construction of transgenic lines
DNA from the BAC containing human PKD2 (362G2, sequence accession number: AC084732 [GenBank] ) was purified and injected into fertilized mouse oocytes as described [20]. The genetic background of transgenic mice is C57BL/6. The data shown are obtained after more than 15 backcrosses to C57BL/6 mice. Mouse DNA was extracted from fibroblasts for quantitative Southern blot analysis. Pulse field gel electrophoresis was performed as described in [20] after digestion with BswI, blotting and hybridization with a PKD2 probe.

Molecular characterization of transgenic lines
RT-PCR was performed using primers (PKD2F: 5'-G AGA TGA AAT TAA AGA GTG C, PKD2R: 5'-G AAT TTG AAG AGC TTA ATC C) amplifying both the human and mouse PKD2 transcripts. Amplification products were cleaved with EcoRI allowing the human product (which is cleaved by the enzyme, generating two fragments of 398 bp and 318 bp) to be differentiated from the mouse product (which is not cleaved by the enzyme; amplicon size: 716 bp). In order to determine the level of expression of human PKD2 with respect to the endogenous mouse PKD2, we used primers amplifying both the human PKD2 and mouse PKD2 transcripts with the same efficiency and qPCR analysis as described below.

FISH analysis
Interphase and metaphase spreads from PKD2-Y fibroblasts were prepared according to standard cytogenetic protocols. The human PKD2 BAC clone used as a probe was labelled by random priming with biotin-14dCTP (Bioprime Labeling System, Life Technologies, Carlsbad, USA), hybridized to cytogenetic preparations and the signal revealed with avidin coupled to fluorescein. A commercial Y chromosome paint probe (Cambio cat: 1200-YMCy3) was used to label the entire mouse Y chromosome. Preparations were counterstained with 4,6-diamidino 2-phenyl indole (DAPI) at 100 ng/mL in Vectashield mounting media (Vector Laboratories, Burlingame, USA). Images were observed using an Axioplan-2 Zeiss fluorescent microscope and were captured with a CCD camera (Photometrics ‘Sensys’). Data were collected and merged using IPLab Spectrum software (Vysis).

Western blot analysis
Proteins were extracted from kidney samples using Ripa buffer. Fifty micrograms of protein were separated on a 7% denaturing acrylamid gel and blotted onto a PVD membrane. Membranes were blocked with PBS/Tween 0.1%/milk 5% for 1 h at room temperature and incubated overnight at 4°C with an affinity-purified rabbit antipeptide antibody raised against mouse Pc-2 aminoacid 941–956 (Pc-2-166) (B. Dworniczak, unpublished data). The secondary antibody was coupled with horseradish peroxidase (HRP) and incubated for 1 h at room temperature.

Histology and immunofluorescence
For immunofluorescence, four PKD2-Y males, four females and four C57/Bl6 non-transgenic males of the same age (1 month, 3 months, 5 months, 8 months and 12 months) were anaesthetized with isoflurane, and their kidneys were removed and gently frozen in liquid nitrogen. Twenty micrometre thick transverse kidney sections were fixed with paraformaldehyde 4% at room temperature for 15 min, permeabilized for 15 min with 0.5% Triton x100/PBS and blocked with 1% milk/0.5% Triton x100/PBS for 15 min. The sections were incubated overnight with a primary antibody against fibronectin (DakoCytomation A0245) (1:1000). Unbound primary antibodies were removed by washing three times for 10 min with milk/PBS; then, preparations were incubated with an FITC-conjugated IgG (SantaCruz SC-2090) (1:100) in milk/PBS for 1 h and washed with milk/PBS.

For histology, the kidneys were directly fixed in formalin and embedded in paraffin. Sections were stained with haematoxylin, eosin and safran.

Images were acquired with a Zeiss AxioImager M1 microscope equipped with a Zeiss CCD camera.

Evaluation of renal physiology
Mice were placed in metabolic cages to assess diuresis. The five mice studied in each group (transgenic and wild-type) were between 12 and 15 months of age. They were allowed to acclimatize to the metabolic cage for 2 days. After adaptation, a 24-h urine collection was performed. This set-up was used to study the mice's response to 24-h water deprivation, to injection of antidiuretic hormone (ADH; Minirin®, FERRING Pharmaceuticals) after 24-h water deprivation and to the replacement of drinking water by 5% glucose.

Equal volumes of mouse urine (5 µl) were analysed on SDS polyacrylamide gel that was stained with Coomassie blue (control: BSA).

Biochemical analysis was performed in a biochemical laboratory at Xavier Bichat Hospital, Paris, France.

RNA isolation and gene expression analysis
PKD2-Y males and females were anaesthetized with isoflurane; their kidneys were removed and snap frozen in liquid nitrogen.

After pulverization of the renal tissue in liquid nitrogen, total RNA was extracted using Trizol in accordance with the manufacturer's instructions. The quality and concentration of each sample were tested using an Agilent 2100 bioanalyzer.

Quantitative RT-PCR analysis
To analyse the expression of selected genes, RNA extracted from the frozen kidneys (see above) of PDK2-Y males and females was reverse transcribed. Then, cDNAs were quantified using a Nanodrop spectrophotometer. Specific primers (primers sequences can be obtained upon request) were designed according to the specifications of an on-line primer design protocol (Roche Diagnostics and Applied Sciences). The qPCR was performed using the LightCycler®480 (Roche Diagnostics and Applied Sciences), using the LightCycler®480 SYBR green I Master mix. Quantification of 18S rRNA was used as an internal control. The results were interpreted using the comparative CT method, where the amount of the target, normalized to the endogenous reference and relative to a calibrator, is given by 2–{Delta}{Delta} CT.

Statistical analysis
Data were analysed for statistical significance using the non-parametric Mann–Whitney test due to the small number of samples. We used the Prism v4 software to perform the statistical analysis.



   Results
Top
 Abstract
 Introduction
 Subjects and methods
 Results
Discussion
 Supplementary data
 References
 
Transgenic lines
A human BAC containing the PKD2 gene was used to construct transgenic mouse lines. The size of this BAC was 181 kb. Apart from PKD2, this BAC contains the human ABCG2 gene (the mammalian equivalent of the drosophila white gene). We checked that the ABCG2 gene is not expressed in kidney (data not shown) thus not affecting the renal phenotype. Transgenic mouse lines have been constructed using this BAC as previously described (20). Four transgenic founders were born, but only three showed germline transmission. These three lines were named PKD2-2, PKD2-4 and PKD2-Y. Southern blot analysis revealed that PKD2-2 has inserted two copies of the BAC into the genome, PKD2-Y five copies and PKD2-4 eight copies (data not shown).

FISH experiments located a unique insertion site for PKD2-2 (chromosome 2, Figure 1B) and PKD2-Y (Y chromosome, Figure 1A). PKD2-4 had two insertion sites and breeding was discontinued. In addition, the integrity of human inserted DNA was confirmed in PKD2-2 and PKD2-Y using pulse field electrophoresis (supplementary Figure 1).


Figure 1
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  		Fig. 1 Transgene localization and human PKD2 gene expression. (A) FISH analysis of PKD2-Y lineage. DNA from a human PKD2 BAC was biotin labelled, used as a probe and detected with avidin coupled to fluorescein. The same PKD2 BAC probe (green spots) was co-hybridized with a directly labelled mouse chromosome Y paint probe (red areas), showing that the transgene is integrated into the mouse Y chromosome. The nucleus is counterstained in blue with DAPI. (B) FISH analysis of PKD2-2 lineage. The transgene was integrated into mouse chromosome 2. The nucleus was counterstained in red by propidium iodide. (C) RNA was extracted from kidneys of PKD2-Y mice and used in RT-PCR experiments. Human and mouse PKD2 transcripts were simultaneously amplified (see the Methods section). The 2% agarose gel shows that the amplification product was cleaved in transgenic animals by EcoRI, but not in non-transgenic animals, demonstrating that the human PKD2 gene is expressed in the kidneys of transgenic animals. (D) Evaluation of human PKD2 expression versus endogenous expression in the mouse had been performed using qPCR. (E) Protein extracted from kidneys of wild-type animals and PKD2-Y had been analysed on polyacrylamide electrophoresis gels, blotted and stained with antibodies against polycystin 2 and against actin (as internal control).

 
Using a semi-quantitative RT-PCR test, we estimated the level of human PKD2 expression compared against the expression of the endogenous mouse gene to be 30% for PKD2-2 and 100% for PKD2-Y (Figure 1C). This was confirmed using qPCR (Figure 1D). Human PKD2 mRNA was overexpressed in all tissues tested (data not shown). Finally, we confirmed the overexpression of PC-2 using an antibody against PC-2 and found an overexpression of PC-2 of about 100% (Figure 1E and supplementary table).

Most results described below have been obtained analysing the PKD2-Y mouse line as being the line with the highest expression and a unique PKD2 insertion site.

General description and histology
The lifespan of transgenic animals is similar to non- transgenic littermates.

Histology of the kidneys, liver and pancreas of the PKD2-Y line was performed at different ages (1 month, 3 months, 5 months, 8 months and 12 months). The liver and pancreas were found to be normal.

Kidneys of transgenic animals did not present anomalies before 3 months of age. Between 3 and 8 months of age, they presented tubule dilation (Figure 2A) and mild inflammation (Figure 2B), but did not present cysts. After 8 months, a few microcysts were observed in the kidney (Figure 2C). No macroscopic cysts were observed. The presence and number of microscopic cysts were variable; in the majority of animals, there were no cysts. Fifteen percent of the transgenic mice (2/13 animals) had microcysts. We never observed cysts in the kidney of wild-type mice. In conclusion, mice younger than 8 months presented a very mild phenotype with regard to kidney cysts, similar to that observed in heterozygotes PKD2 invalid mice. In addition, no end-stage renal failure has been observed in PKD2Y animals.


Figure 2
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  		Fig. 2 Kidney histology I. Histological examination of the kidney from 5- to 8-month-old transgenic mice by light microscopy and haematoxylin, eosin and safran staining. (A) Kidney section showing tubule dilatation (arrow). (B) Kidney section showing mild inflammation (arrow). (C) Kidney section presenting small cysts (arrows).

 
In contrast, mice older than 1 year presented a general disorganization of the kidney cortex and anomalies in tubular structure (Figure 3A–C). These anomalies are constant in transgenic mice (PKD2-Y and PKD2-2) and never seen in wild-type mice. The phenotype was more severe in PKD2-Y mice (Figure 3C) than in PKD2-2 mice (Figure 3B). These figures are representative of the phenotype observed in six animals of PKD2-Y strain and wild-type strain, and two of PKD2-2 strain. The vacuoles are still observed in the kidney of transgenic mice and never in the kidney of wild-type mice. As seen in Figure 3D, almost all tubular cells embedded large vacuoles in their cytoplasms, whose origin is unknown. In addition, malformation of tubular structures was observed, with the presence of an abnormal number of bilayer epithelial structures (Figure 3E). Surprisingly, medullar structures seemed to be normal. In conclusion, histological examination revealed progressive disorganization in the cortex, with abnormal tubular organization and abnormal vacuolization of tubular cells. As these renal cortex anomalies could lead to anomalies of renal function, it was important to analyse the kidney physiology of these animals.


Figure 3
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  		Fig. 3 Kidney histology II. Histological examination of the kidney of transgenic and wild-type mice (12 months of age) using light microscopy and haematoxylin, eosin and safran staining. (A) Kidney section from normal mouse. (B) Kidney section of PKD2-2 animals. (C) Kidney section of PKD2-Y animals. (D) and (E) Kidney section from PKD2-Y mice. Note multiple vacuoles occupying a large part of cytoplasm (C). Arrow in (D): bilayer epithelial structure. Arrow heads in (B) and (C): vacuoles.

 
Renal function
Urine of PKD2-Y mice older than 1 year had been collected using metabolic cages and analysed. The pH of the urine was found to be close to normal. Proteinuria was significantly increased in PKD2-Y urine (Figure 4A) suggesting renal dysfunction. Urine of PKD2-2 line had been analysed as well and these mice presented a proteinuria intermediate between PKD2-Y and wild-type (Figure 4A). The electrophoretic protein patterns of PKD2-Y and PKD2-2 were qualitatively normal compared to urine from wild-type mice without microalbuminury, suggesting dysfunction of the proximal tubule (Figure 4B) without dysfunction in the glomeruli. In addition, normal and transgenic animals were submitted to hydric restriction. PKD2-Y continued to urinate, unlike the wild-type that stopped (Figure 4C), indicating that PKD2-Y mice were unable to concentrate the urine after physiological stress. The urine osmolality of PKD2-Y after water restriction was 2956 ± 816 mmol/kg H2O compared to 4280 ± 731 in wild-type mice (P < 0.013). Injection of ADH did not modify the volume of urine after water deprivation (data not shown). The inability to concentrate urine is therefore secondary to tubular dysfunction. The animals had no impairment of dilution capacity after water overload (data not shown). A summary of these data is given in Table 1.


Figure 4
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  		Fig. 4 Kidney physiology. (A) Protein concentration in the urine of PKD2-Y, PKD2-2 and wild-type animals (12 months of age) was determined in µg of protein per ml of urine. The difference was significant between all groups of mice P < 0.05 using the Mann–Whitney test. *P = 0.0058, **P = 0.0044, ***P = 0.0004. (B) 20 µl of urine from Tg (transgenic) and non-Tg 12-month-old animals were analysed on 15% denaturating polyacrylamide gels (lanes A and B). Lane C was loaded with a serum albumin solution. (C) PKD2-Y (five animals) and wild-type (five animals) mice were subjected to water deprivation. Urine was collected over 24 h and the urine volume is given in microlitre. At baseline, the Tg mice urinated the same volume as their controls. 24 h later (* P = NS), the amount of diuresis is significantly higher for PKD2-Y mice than for wild-type mice (**P < 0.0001).

 

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  		Table 1 Physiological data of PKD2Y mice compared to wild-type mice. There were five mice in each group. The mice are between 12 and 15 months old. Statistical analysis was done using the Mann–Whitney test

 
In conclusion, transgenic animals older than 12 months have a kidney dysfunction, probably due to anomalies in tubular function associated with abnormal kidney histology.

Gene expression in PKD2-Y mice
Most of the molecular data reported in ADPKD models are related to the PKD1 function (e.g. gene expression, activation of signalling pathways). In order to compare these models to our mouse line overexpressing PKD2, we chose to analyse expression of those genes described to be deregulated in ADPKD models.

As polycystins have been reported to activate cell proliferation [11] and apoptosis [21] (leading authors suggest a relationship between ADPKD and cancer [22,23]), we first focused our study on genes that are involved in these processes and whose expression is reported to be deregulated in PKD1 mouse models. Real-time PCR assays were performed using RNA extracted from the kidneys of normal and PKD2-Y mice. Results presented in Figure 5A show the level of gene expression in PKD2-Y versus wild-type animals at the 8-month age (the same results have been obtained with mice aged 1, 5 and 8 months). It is important to note that genes involved in apoptosis (p53, Bad and Bax and Bcl2) did not show significant variations. This was also the case for Ras pathway genes (H-, K- and N-Ras) that have been shown to be activated in cystic cells [24,25]. On the other hand, cMyc expression was upregulated as well as p21 expression. Identical results had been obtained analysing animals aged 1 month, 5 months and 8 months before the onset of tubulopathy.


Figure 5
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  		Fig. 5 Quantitative PCR. Analysis of RNA extracted from kidneys of transgenic and wild-type mice 8 months of age. After RT-PCR the relative amount of RNA was quantified using specific primers. Quantification of 18S rRNA was used as an internal control. Results from transgenic kidneys were always compared to data from wild-type kidneys which were taken as 1. (A) Expression of genes involved in apoptosis and cellular proliferation. Only the overexpression of p21 and cMyc was statistically significant. (B) Expression of extracellular matrix genes. The downregulation of all genes tested was statistically significant.

 
Expression of extracellular matrix genes
Several previous studies, using different cystic models, report anomalies in the gene and protein expression of extracellular matrix molecules [27]: laminins and integrins [28,29], fibronectin, syndecan and glypican-3 [30]. Moreover, the current working hypothesis says that the differentiation of the epithelium is highly dependent on interactions between epithelial cells and the extracellular matrix. Thus, one possibility is that tubular dysfunction observed in PKD2-Y animals could be due, at least partly, to anomalies in the extracellular matrix, a structure important for establishing and maintaining the normal architecture of kidney tubules. We therefore analysed the expression of genes coding for several proteins of the extracellular matrix using RNA prepared from PKD2-Y kidneys at different ages, and compared it with the expression in wild-type animals. In Figure 5B we only present the results of the 5-month-old mice because in younger mice (1 month), no difference between transgenic and normal animals could be detected and in older mice (8 months) transgenic animals presented the same difference with respect to gene expression as detected in 5-month-old transgenic animals.

It was obvious that fibronectin, decorin and glypican synthesis were strongly downregulated in young animals, although we did not observe kidney anomalies at this age. In contrast to this, expression of the connective tissue growth factor (CTGF) was normal. We therefore decided to analyse, in more detail, the expression of the gene coding for fibronectin, a protein clearly involved in epithelial differentiation. We could see (Figure 5B) that this gene was downregulated in both young and old PKD2-Y animals compared to wild-type animals.

In addition, we performed immunocytochemistry on kidney sections from PKD2-Y and wild-type mice using an antibody directed against fibronectin. As shown in Figure 6A, fibronectin was abundant in wild-type animals and mainly located around tubular structures. On the other hand, fibronectin was underexpressed or absent around tubules in the transgenic PKD2-Y line (Figure 6B). These data confirmed qPCR data and clearly showed that the extracellular matrix is abnormal in PDK2-Y animals, with a defect in extracellular matrix protein expression. This defect in ECM composition was associated with vacuolization of renal epithelium, abnormal structure of renal cortex and an inability to concentrate urine in response to water deprivation.


Figure 6
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  		Fig. 6 Fibronectin expression in the kidney. In view of the PCR results, kidneys from normal (A) and transgenic (B) 8-month-old mice have been sampled, fixed and processed as described in the Methods section. Tissue slices were stained using an antibody against fibronectin (see the Methods section). All slices were stained in the same time and duration of the photographs was exactly the same between normal and transgenic tissue slices.

 


   Discussion
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
Supplementary data
 References
 
Overexpression of human PKD2 in mice is associated with abnormal tubular function, disorganization of the renal cortex and abnormal expression of extracellular matrix components. Unlike the model overexpressing PKD1 [31,32], our mice did not exhibit polycystic kidney disease.

There is no valid explanation for this difference in phenotype. However in humans, ADPKD2 is characterized by later onset compared to ADPKD1 [6]. Therefore development of cysts in mice overexpressing PKD2 might be slower compared to mice overexpressing PKD1 and, in addition, the lifespan of mice might be too short to enable the development of detectable polycystic kidney disease symptoms.

Before 1 year of age we observed mild tubular dilatation, but after 1 year the phenotype was more striking. We observed two types of anomalies. First, numerous vacuoles could be observed in the cytoplasm of tubular cells. Interestingly this phenomenon was observed in a PKD animal model in which the gene coding for aquaporin 11 was disrupted [33]. In this model, vacuolization of proximal tubular cells appears before the development of cysts. The origin of these vacuoles is unknown as is their physiological significance.

The second anomaly concerns the general structure of the kidney cortex, presenting a general disorganization of this tissue. In addition, we observed that the tubular epithelium presented two epithelial cell layers with abnormal frequency. It should be noted that the same observation was reported in Hnf1β knock-out mice [34]. Whether these anomalies might be precursors of cyst formation is still an open question.

However, these anomalies prompted us to explore the renal physiology of these animals. The inability to concentrate urine under water deprivation suggested tubular dysfunction, and the insensitivity to ADH confirmed this hypothesis. To investigate the origin of these anomalies, we explored the expression of genes that could be involved and selected two types of genes: genes involved either in proliferation/apoptosis phenomena and genes coding for extracellular matrix proteins. A cMyc upregulation has been regularly observed in both human ADPKD [37] and animal models [38,39]. In addition, p21 overexpression has also been reported in cellular PC-1 overexpression models, as in our model, and defective p21 induction has previously been observed in a PKD1 knock-out model [11,40], eloquently reporting the deregulation of the Jak/STAT signalling pathway observed in vivo. Our data thus provide in vivo confirmation that not only PKD1 is involved in this deregulation, but probably PKD2 anomalies also result in the same phenomenon. One explanation for p21 overexpression could be the retention of Id2 in the cytoplasm associated with PC-2 overexpression [26]. We confirmed in vivo the importance of PC-2 in controlling the cell cycle, but on the other hand, we did not observe any significant perturbation of p53, Bad, Bax or Bcl2. We would like to point out that the same conclusion was drawn from a Myc transgenic model, suggesting that cMyc acts in cyst formation and in our model, in tubular disorganization through a p53- and Bcl-2-independent mechanism [41].

In conclusion, in kidney cells from PKD2-Y two genes (Myc and p21) controlling cell proliferation are overexpressed, but the expression of failsafe genes such as p53 or Bcl2 is not affected. This gene-expression pattern correlates to a status of the cells to be ‘gently’ stimulated to proliferate, but having intact failsafe signals preventing the cells from being precarcinogenic. These data could explain why ADPKD patients are not especially prone to kidney cancers, although their cells overexpress oncogenes.

The second set of genes we explored codes for extracellular matrix proteins. The authors have already described anomalies in the extracellular matrix in several models [42]. In a recent paper, using microarrays and RNA from the kidneys of Han:SPRD rats, we demonstrated that perturbation in the expression of genes coding for proteins of the extracellular matrix is an early event in PKD [30]. In contrast, inflammatory processes are a consequence of cyst formation, and are not associated with early pre-cystic phases. Data presented in this paper confirm these results, as a gene-like fibronectin is strongly downregulated in PKD2-Y kidneys from mice of 3 months of age, a stage where no histological anomalies could be detected. The same has been observed for syndecan and glypican-3. In contrast, CTGF expression in the kidneys of these animals is not affected. Finally, animals presenting tubular dysfunction also present downregulation of fibronectin, confirming that this structure is highly perturbed in old PKD2-Y animals. These observations could explain abnormal tubular differentiation in these animals. An early event could be tubule dilatation, involving an abnormal extracellular matrix followed by disruption of the normal structure of the cortex. A last point concerns a potential link between cMyc and p21 upregulation and downregulation of proteins of the extracellular matrix. This point should be explored in the future.

Finally, the present data were collected during the progression of cystic disorders. There are only a few data published concerning the potential disturbance of the renal physiology during presymptomatic stages. This holds true for animal models as well as for human patients. However, it has been reported that ADPKD patients who are not on dialysis and not presenting renal failure, do have abnormal tubular function [43,44]. Lack of solid data raises immediately a series of questions concerning processes in presymptomatic phases of ADPKD. Does tubulopathy, due to perturbation in tubule organization, occur before massive cystogenesis in ADPKD kidneys? Are anomalies of tubule organization a first step in cystogenesis? Answering these questions is important for understanding what happens during the early phases of the disorder and for defining therapeutic targets that will prevent cyst formation.



   Supplementary data
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Supplementary data
References
 
Supplementary data are available online at http://ndt.oxfordjournals.org/.



   Acknowledgments
 
This project has been supported by a Genzyme GRIP programme and a personal grant to MF by French AIRG. We thank Pr L. Daniel for helpful discussion about kidney pathology.

Conflict of interest statement. The results presented in this paper have not been published previously in whole or part, except in abstract format.



   Notes
 
* The first two authors have contributed equally to the work. Back

** Present address: Laboratory de fisiopatologia renal. Instituto de resercha val hebron. 08035 Barcelona. Back



   References
Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 Supplementary data
 References
 

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Received for publication: 7. 5.07
Accepted in revised form: 28. 9.07


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E. Y. Park, Y. H. Sung, M. H. Yang, J. Y. Noh, S. Y. Park, T. Y. Lee, Y. J. Yook, K. H. Yoo, K. J. Roh, I. Kim, et al.
Cyst Formation in Kidney via B-Raf Signaling in the PKD2 Transgenic Mice
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