Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on October 20, 2004
Human Molecular Genetics 2004 13(24):3069-3077; doi:10.1093/hmg/ddh336

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 13/24/3069    most recent ddh336v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Google Scholar Articles by Leeuwen, I. S. L.-v. Articles by Peters, D. J.M. Search for Related Content PubMed PubMed Citation Articles by Leeuwen, I. S. L.-v. Articles by Peters, D. J.M. Social Bookmarking          What's this?

Human Molecular Genetics, Vol. 13, No. 24 © Oxford University Press 2004; all rights reserved

Lowering of Pkd1 expression is sufficient to cause polycystic kidney disease

Irma S. Lantinga-van Leeuwen¹, Johannes G. Dauwerse¹, Hans J. Baelde³, Wouter N. Leonhard¹, Annemieke van de Wal³, Christopher J. Ward⁴, Sjef Verbeek¹, Marco C. DeRuiter², Martijn H. Breuning¹, Emile de Heer³ and Dorien J.M. Peters^1,*

¹Center for Human and Clinical Genetics and ²Department of Anatomy and Embryology, Leiden University Medical Center, 2333 AL Leiden, The Netherlands, ³Department of Pathology, Leiden University Medical Center, 2300 RC Leiden, The Netherlands and ⁴Division of Nephrology, Mayo Clinic, Rochester, MN 55905, USA

Received July 9, 2004; Revised October 5, 2004; Accepted October 12, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Autosomal dominant polycystic kidney disease (ADPKD) is a major cause of renal failure and is characterized by the formation of many fluid-filled cysts in the kidneys. It is a systemic disorder that is caused by mutations in PKD1 or PKD2. Homozygous inactivation of these genes at the cellular level, by a ‘two-hit’ mechanism, has been implicated in cyst formation but does not seem to be the sole mechanism for cystogenesis. We have generated a novel mouse model with a hypomorphic Pkd1 allele, Pkd1^nl, harbouring an intronic neomycin-selectable marker. This selection cassette causes aberrant splicing of intron 1, yielding only 13–20% normally spliced Pkd1 transcripts in the majority of homozygous Pkd1^nl mice. Homozygous Pkd1^nl mice are viable, showing bilaterally enlarged polycystic kidneys. This is in contrast to homozygous knock-out mice, which are embryonic lethal, and heterozygous knock-out mice that show only a very mild cystic phenotype. In addition, homozygous Pkd1^nl mice showed dilatations of pancreatic and liver bile ducts, and the mice had cardiovascular abnormalities, pathogenic features similar to the human ADPKD phenotype. Removal of the neomycin selection-cassette restored the phenotype of wild-type mice. These results show that a reduced dosage of Pkd1 is sufficient to initiate cystogenesis and vascular defects and indicate that low Pkd1 gene expression levels can overcome the embryonic lethality seen in Pkd1 knock-out mice. We propose that in patients reduced PKD1 expression of the normal allele below a critical level, due to genetic, environmental or stochastic factors, may lead to cyst formation in the kidneys and other clinical features of ADPKD.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the formation of multiple fluid-filled cysts in the kidneys, ultimately leading to renal failure and a need for renal replacement therapy in most patients. The kidney is the most severely affected organ, but the disease is systemic and extra-renal manifestations such as cyst formation in liver and pancreas, hypertension and cerebral aneurysms are frequently observed (1).

ADPKD has a prevalence of 1 : 1000, and the majority of patients, 85%, have a mutation in the PKD1 gene (2,3). This gene encodes a large protein, polycystin-1, which forms multiprotein complexes at the cell membrane and is thought to function in cell–cell/cell–matrix interactions, signal transduction and in mechanosensation (4). The C-terminal region of polycystin-1 has been shown to interact with the PKD2 gene product, polycystin-2, which is mutated in 15% of the ADPKD patients, and functions as a cation channel (5–7).

Renal cysts in ADPKD patients arise from the nephrons and collecting ducts owing to alterations in the epithelium. Although all cells carry the same germ-line mutation, only a minority of nephrons form cysts. As an explanation for focal cyst development, a ‘two-hit’ hypothesis was proposed analogous to hereditary types of cancer (8). Indeed, somatic mutations resulting in homozygous inactivation of PKD1 or PKD2 were identified in a subset of renal and hepatic cysts, as well as in clonal expansion of cyst-lining cells (9,10). Additional support was provided by Pkd2 mice carrying one null allele and one unstable Pkd2 allele, which are prone to develop cysts (11). However, the presence of polycystin-positive cysts in ADPKD patients and cyst formation in mice overexpressing Pkd1 suggest that cystogenesis is due to a more complex mechanism than recessive inactivation at the cellular level (12,13).

In this study, we investigated the effect of a reduced dosage rather than a lack of Pkd1 expression in a novel mouse model. Mice with only 13–20% of correctly spliced Pkd1-transcripts are viable showing polycystic kidneys and a variety of extra-renal manifestations observed in ADPKD-patients.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Polycystic kidneys in Pkd1^nl/nl mice
By gene targeting we generated a mutant Pkd1 allele, Pkd1^nl, harbouring a selection cassette containing the neomycin resistance gene (neo) flanked by loxP sites in intron 1 of Pkd1. A third loxP site was inserted into intron 11 to allow deletion of exons 2–11 (Fig. 1). Since it is known that the presence of a selection cassette may interfere with RNA processing (14), we intercrossed heterozygous mutant mice and analyzed the offspring. Mice homozygous for the Pkd1^nl allele were born at the expected frequency (Table 1). Homozygous Pkd1^nl/nl mice, however, showed growth retardation and early mortality. The severity of the symptoms varied among these mice. The majority of the Pkd1^nl/nl mice had distended abdomens and a 40% reduction in body weight at weaning compared with their littermates. These mice died within 1–2 months after birth. Sacrificed 1-month-old mice had pale, massively enlarged cystic kidneys (Fig. 2A). Notably, 25% of the homozygous mice manifested a less severe phenotype and became fertile adults. Their body weights at age of weaning were >10 g and these mice showed less enlarged kidneys (Table 2). Three animals (10%) stayed alive for at least 1 year, the eldest mouse was sacrificed at 15 months of age.

View larger version (12K): [in this window] [in a new window]   Figure 1. Generation of the Pkd1 hypomorphic allele. A fragment of the genomic wild-type Pkd1 locus is shown. The Pkd1 targeting vector was constructed by inserting a PGK promoter-driven neomycin-resistance gene flanked by loxP sites into intron 1 and a third loxP site into intron 11. A 5'-probe covering exon 1, and a 3'-probe covering exons 24–26 from the Pkd1 gene were used for genotyping by Southern blotting. PGK, phosphoglycerate kinase gene; Neo, neomycin phosphotransferase gene; TK, thymidine kinase gene; Ex, exon.

 

View this table: [in this window] [in a new window]   Table 1. Offspring of Pkd1 mutant mice

 

View larger version (100K): [in this window] [in a new window]   Figure 2. Polycystic kidneys and structure of Pkd1 transcripts in Pkd1nl/nl mice. (A) Kidney from a 1-month-old Pkd1nl/nl mouse is enlarged, pale, and cystic, compared with kidneys from heterozygous and wild-type littermates. (B) Schematic representation of Pkd1 transcripts in Pkd1nl/nl mice. RT-PCR on renal RNA with primers in Pkd1 exons 1 and 2 showing the expected 81 bp fragment in normal mice and an additional 579 bp fragment, containing 498 bp of the selection cassette sequence, in heterozygous and homozygous Pkd1nl mice. M, marker. (C) Alternative splicing of Pkd1 results in a mixture of wild-type and mutant transcripts. Ex, exon (only three of the 46 Pkd1 exons are shown). (D) Nucleotide and predicted amino acid sequences of the mutant Pkd1 transcripts that result in an early protein translation stop (asterisk). (E) Northern blot analysis using a Pkd1 exon 15 probe demonstrated the 14 kb Pkd1 transcript in 7-weeks-old wild-type (lane 1), heterozygous (lane 2) and homozygous (lane 3) littermates. Hybridization of the same filter with a neo cDNA probe showed the inclusion of neo-cassette-derived sequences in the Pkd1 transcript of mutant mice. Glyceraldehyde 3-phospate dehydrogenase (GAPDH) was used as a loading control.

 

View this table: [in this window] [in a new window]   Table 2. Phenotypes of Pkd1nl/nl mice

 
Histological analysis of 1-month-old Pkd1^nl/nl mice showed kidneys with dilated and cystic tubules and some small areas of mild fibrosis and mild inflammation. In the renal cortex of the most severely affected mice large cysts replaced most of the normal renal parenchyma (Fig. 3A and B). The 15-month-old homozygous mouse also exhibited a bilaterally cystic phenotype, although the majority of the cysts were rather small. Kidneys of age-matched heterozygous and wild-type littermates showed no abnormalities. In addition, we observed no cysts in old heterozygous Pkd1^nl/+ mice (n=7, age >13 months) (Table 3).

View larger version (82K): [in this window] [in a new window]   Figure 3. Renal and extrarenal pathology in Pkd1nl/nl mice. (A–F) Kidney sections from homozygous 4–7-weeks-old Pkd1nl/nl mice. Sections stained with haematoxylin and eosin (HE) show many cysts. (A) 4-weeks-old mouse with a less severe phenotype. (B) 4-weeks-old mouse with a severe phenotype. (C–E) Segment identity was characterized by staining with lectin Dolichos biflorus (C), as a marker for collecting tubules, and by using antisera directed against Tamm-Horsfall protein (D), as a marker for thick ascending limb and distal convoluted tubules, and megalin (E), a proximal tubule marker. The majority of the cysts are derived from loops of Henle, distal-tubules, and collecting ducts. (F) Expression of polycystin-1 was detected in a subset of cysts using the monoclonal antibody PKS-A (12). (G) Pancreas and (H) liver sections stained with HE show the presence of bile duct dilatations and small cysts (cy) in Pkd1nl/nl mice. (I and J) Consecutive transverse sections of the descending aorta showed an intramural bleeding. Sections are stained with HE (I) and resorchin fuchsin to detect elastic lamels (J). The elastic lamels diverge by the intramural bleeding (asteriks).

 

View this table: [in this window] [in a new window]   Table 3. Cystic phenotype of Pkd1 mutant mice

 
Most cysts arise from the loops of Henle, distal tubules and collecting ducts, as defined by the segment-specific markers Tamm-Horsfall protein and Dolichos biflorus, respectively. Less than 10% of the cysts stained with proximal tubule marker megalin, and glomerular cysts were rarely identified (Fig. 3C–E).

Aberrant splicing in Pkd1^nl/nl mice
To assess whether the Pkd1 allele is a hypomorphic allele due to interference with normal splicing, we carried out reverse transcriptase–PCR analyses on RNA isolated from renal tissues, with primers specific for exons 1 and 2. A larger amplification product of 579 bp was observed in homozygous and heterozygous mice, whereas the normal littermates revealed only the expected 81 bp-sized PCR product (Fig. 2B). cDNA sequencing revealed that a 498 bp fragment was included by the use of cryptic splice sites from the neo cassette (Fig. 2C). We also identified two additional splice donor sites. All splice-variants use the same cryptic acceptor site, resulting in a frameshift and predicted to result in a premature translational stop (Fig. 2D).

To confirm that the phenotype of Pkd1^nl/nl mice is due to aberrant splicing of Pkd1, we removed the neo cassette by crossbreeding the mice with EIIa-Cre mice (15). These mice mosaically express Cre-recombinase in the germ-line, such that partial or complete excision of loxP-flanked DNA sequences occurs. In this way, we obtained mice in which the neo cassette was removed but leaving Pkd1 unchanged. Homozygous offspring showed normal growth and no histological abnormalities up to 15 months of age (n=5), thus eliminating the possibility that the pathogenic properties of the Pkd1^nl/nl mice are due to mutations introduced during the ES cell-targeting. Intercrossing mice with a deletion of both the selection-cassette and the floxed part of the Pkd1 gene (exons 2–11) produced no viable offspring homozygous for the deletion, as expected from previously described mutant Pkd1-mice (16–19).

Residual wild-type expression in Pkd1^nl/nl mice
Wild-type transcript was also present in Pkd1^nl/nl mice, showing that natural splice sites are also used. We quantified the proportion of wild-type Pkd1 transcripts with a real-time PCR assay. We designed a PCR primer pair in the 5' part of the Pkd1 gene (5'PCR) that specifically amplified wild-type transcripts and a second PCR primer pair in the 3' part of the Pkd1 gene (3'PCR) that amplified both wild-type and splice-variant transcripts. The 3'PCR versus 5'PCR ratio's showed that in the majority of Pkd1^nl/nl mice 13–20% of the Pkd1 transcripts are spliced as in wild-type mice (Fig. 4A; Table 2). Consistently, heterozygous mice have an average of 55% correctly spliced Pkd1 transcripts. In general, there was no apparent correlation between the phenotypic features and the relative levels of normal Pkd1 transcript in the severe and less severely affected mice (Table 2). However, the few homozygous mice that stayed alive for more than 1 year showed distinct higher levels of normal Pkd1 expression (38 and 48%, Table 2), consistent with their mild cystic phenotype and extended lifespan.

View larger version (9K): [in this window] [in a new window]   Figure 4. Real-time PCR analysis of renal RNA samples of 4–8-weeks-old Pkd1nl/nl mice. Means of duplicate measurements are shown. (A) Wild-type Pkd1 transcripts relative to total Pkd1 mRNA. The ratio of Pkd1 3'PCR versus Pkd1 5'PCR is normalized to the mean ratio of 10 wild-type littermates, which has been set to 1. Homozygous Pkd1nl/nl mice show an 6-fold increase in ratio, indicating that one-sixth (17%) of the transcripts are correctly spliced. (B) Pkd1 (Pkd1 3'PCR) and (C) Pkd2 expression normalized to expression of the HPRT gene that served as reference. Error bars represent standard error. The data represent the mean ± SE of 7–10 animals. Asteriks (*) indicate significant difference from normal, P<0.05 (Student's t-test).

 
Notably, real-time RT–PCR detected a 2–3-fold increase in the total amount of Pkd1 transcripts in the 4–8-weeks-old Pkd1^nl/nl mice compared with Pkd1^+/+ controls (2.69±0.67 versus 1.00±0.40; P<0.001), whereas no significant differences were found in heterozygous mice (1.08±0.17) (Fig. 4B). Northern blot analysis confirmed the elevated expression of total Pkd1 in homozygous mice Pkd1^nl/nl, and the inclusion of neo-cassette-derived sequences in the Pkd1 transcripts (Fig. 2E). The upregulation may represent a continued immaturity of the epithelium or loss of terminal differentiation of the epithelium in the cystic kidneys in Pkd1^nl/nl mice. The latter seems more likely, as expression of Pkd2 was not increased in the cystic kidneys (1.13±0.22; P=0.21), while both Pkd1 and Pkd2 expression are highest in embryonic and neonatal kidneys (20). Notably, in human PKD1 kidneys upregulation of the normal transcript has also been reported (12).

Polycystin-1 expression in normal and cystic tubules
Consistent with the residual wild-type expression of the Pkd1^nl allele, we observed staining for polycystin-1 in kidneys from Pkd1^nl/nl mice, using the monoclonal antibody PKS-A (12). Although the majority of the cysts in Pkd1^nl/nl mice were virtually negative for polycystin-1 staining, small and large polycystin-1 positive cysts were detected as well (Fig. 3F). A few cysts exhibited a heterogeneous staining pattern indicating variation in polycystin-1 expression. This patchy staining has been observed in human cystic kidneys as well and suggested to be dependent on the stage of cell division or other intracellular factors (21).

Extrarenal abnormalities in Pkd1^nl/nl mice
Only mild cystogenesis was observed in liver and pancreatic sections of Pkd1^nl/nl mice, concordant to human ADPKD. In the livers, either no cysts (6 of 10) or small cysts and bile duct dilatations (4 of 10) were detected, and in the pancreas dilatation of ducts and occasionally a cyst (8 of 10) were detected (Fig. 3G and H) (Table 3).

Cardiovascular abnormalities, such as cardiac valve abnormalities, saccular intracranial aneurysms and dissecting thoracic aortic aneurysms are also associated with human ADPKD (22). Accordingly, cardiac septation defects and vascular fragility have been reported for Pkd1 and Pkd2 knockout embryos (17,18,23). Therefore, we analyzed the abdominal and thoracic aorta from six Pkd1^nl/nl animals, two of them presented in Table 2. All mice showed dissecting aneurysms, often accompanied with large intramural bleedings (Fig. 3I and J).

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Our findings provide the first evidence that decreased levels of wild-type Pkd1 could lead to the clinical features of ADPKD. We showed that the intronic neomycin-insertion into Pkd1 had a profound effect on splicing efficiency, resulting in a mixture of mutant and wild-type sequences. Approximately 15–20% of the Pkd1 transcripts were correctly spliced in the majority of the homozygous Pkd1^nl/nl mice. These hypomorphic mice show that low levels of wild-type Pkd1 expression can overcome the embryonic lethality seen in knock-out mice where Pkd1 protein is absent. Although the hypomorphic Pkd1^nl/nl mice survive, they develop severe cystic kidneys as well as extrarenal features of ADPKD. Although the severity of the symptoms varied among the Pkd1^nl/nl mice, there was no apparent correlation between the phenotypic features and the relative levels of the normally spliced Pkd1 transcript except for the few animals with a much milder phenotype. It may be, however, that at the time of cyst-initiation the relative expression levels showed more variation due to stochastic fluctuations.

During life-time, ADPKD patients acquire 100–1000 of renal cysts, leading to chronic renal failure in 50% of patients by the age of 60 years (24). Two proposed mechanisms for focal cyst development are the occurrence of somatic mutations in the normal alleles (second hits) and gene-dosage effects. The second-hit hypothesis is supported by studies showing loss of, or mutations in, the normal allele in the epithelial cells of a subset of cysts (9,10). In all those cysts somatic mutations occurred via a separate event. The model, however, has some shortcomings, such as failure to identify somatic mutations in a significant number of cysts and expression of polycystin-1 in a subset of cysts in kidneys of PKD1-patients. These findings may be explained in part by acquired missense mutations resulting in mutant proteins that are still expressed (12). A trans-heterozygous two-hit model has also been suggested with somatic mutations in the PKD2 gene in a PKD1 kidney, and vice versa (25,26). However, no hot-spot for missense mutations has been identified and trans-heterozygous mutations have been identified in only a small fraction of cysts. In addition, severely affected cystic kidneys in a group of early-onset ADPKD patients would require a large number of second hits within the first few months of life (21,27).

The alternative hypothesis for cystogenesis implies that mutations in one allele may be sufficient to cause cyst formation. This gene-dosage-effect is called haploinsufficiency. In general, gene expression levels show considerably more variation in the presence of only one functional allele (28). Therefore, stochastic fluctuations in Pkd1-gene dosage, below a tissue-specific threshold, may interfere with the control of processes involved in maintenance of the renal epithelial architecture. Interestingly, a growing number of reports are supporting the notion of haploinsufficiency in tumorigenesis for a variety of tumors, in addition to Knudson's classical two-hit model. Like cystogenesis in ADPKD, these tumours have a relatively late age of onset (29–31).

Consistent with human ADPKD, most cysts arise from the loops of Henle, distal tubules and collecting ducts, as defined by segment specific markers, and glomerular cysts were only rarely observed. Mice also showed mild cyst formation in the pancreas and liver. In contrast, massive cystic dilatation of pancreatic ducts has been reported for homozygous Pkd1 knock-out embryos, and these pancreatic cysts occurred well before the renal cysts appeared (16,19).

Pkd1^nl/nl mice also develop aortic aneurysms, frequently accompanied by intramural bleedings. In ADPKD patients, aortic aneurysms have also been reported (32). However, the prevalence of cerebral aneurysms has been studied more extensively and is increased 10-fold compared with the general population (33–35). It seems that reduced gene expression results in structural weakness of the arterial wall that, influenced by hemodynamic factors, cause aneurysm formation. Homozygous Pkd1 and Pkd2 knock-out mice die in utero having massive oedema and focal haemorrhages (16,23), and a heterozygous Pkd2 mouse model develop aneurysms only upon the induction of systemic hypertension (36). In the Pkd1^nl/nl mice, aortic aneurysms are spontaneously formed, strongly suggesting that Pkd1-gene expression indeed is crucial for development and maintenance of the structural integrity of the blood vessel wall.

Phenotypic severity in homozygous mice was variable, with kidney/body weight ratios ranging from 2 to 33% and death occurring as early as day 25 and as late as 15 months. Death may be caused by renal failure. However, as we found aortic aneurysms in mice sacrificed at 4–8 weeks of age, rupture of aneurysms could also be the cause of early mortality. The mixed genetic background, 129Ola and C57Bl/6, is likely to contribute to the phenotypic variations, as genetic background has been reported to modulate cystic pathology in PKD mouse models (37). Stochastic fluctuations in gene expression, however, may also contribute to the phenotypic variation. Interestingly, patients with ADPKD also show diversity in disease progression and in extra-renal manifestations. Although the factors that affect disease progression are not clear, modifying genes, environmental factors as well as stochastic effects on gene expression are probably involved (38).

Given the similarities to human polycystic kidney disease, Pkd1^nl/nl mice may serve as an attractive model to study cyst and aneurysm formation and the effects of therapy. In particular, as Pkd1 knock-out mice are embryonic lethal and heterozygous knock-out mice show only a mild cystic phenotype at old age. Diets or drugs tested as potential therapeutic interventions sometimes show opposing effects, depending on the animal model used. For instance EGF-R antagonists have opposing effects in the HAN:SPRD-rat and in the PCK-rat models for PKD, emphasizing the need for models that mimic human disease (39,40). Promising therapeutic results have been obtained with a vasopressin V2 receptor antagonist. This drug, OPC31260, has now been evaluated in animal models of the recessive form of PKD, and most recently in a Pkd2 mouse model but not yet in a Pkd1 mouse model (41,42).

Transgenic mice overexpressing Pkd1 also show cyst formation, although mainly of glomerular origin (13). Hence, a balanced Pkd1-dosage seems critical to establish and maintain intact renal epithelial architecture. Our findings suggest that in addition to somatic mutations, reduced Pkd1 expression of the normal allele due to genetic, environmental or stochastic factors may lead to clinical features of ADPKD in patients. Because steady-state expression levels of Pkd1 need to be retained within predetermined boundaries, we doubt whether modulation of PKD1 gene expression may ever become sufficiently precise to be successful as a treatment modality for ADPKD. Manipulation of downstream effects of aberrant PKD1 expression is likely to be more promising to slow down or prevent cyst growth.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 
Generation of Pkd1^nl/nl mice
A Pkd1 genomic clone was isolated from a mouse 129/SV PAC library (RPCI-21) and a 30 kb SacII fragment containing exon 1 was subcloned. A loxP-flanked neomycin resistance gene (neo) under the control of the phosphoglycerate kinase (PGK) promoter was cloned in antisense orientation into the Eco105I site in intron 1 and a synthetic double-stranded oligonucleotide containing a loxP site was cloned into the BglII site in intron 11. As a result, a 7 kb region containing exons 2–11 of the Pkd1 gene is flanked by loxP sites (Fig. 1). The targeting vector contained 8.5 kb of homologous DNA sequence on the short arm and 10 kb on the long arm. A thymidine kinase gene under the control of the PGK promoter was inserted at the end of the 3'-arm for counter-selection. The linearized targeting construct was electroporated into 129/Ola embryonic stem (ES) cells, which were then subjected to selection with G418. Homologous recombination was identified by Southern blot hybridizations in 10% of the G418-resistant clones using 5'- and 3'-external Pkd1 probes and a neo probe. Chimeric mice were obtained by injection of Pkd1^nl/+ ES cells into C57BL/6 blastocysts, and two independently targeted ES clones (#4 and #18) were found to transmit the Pkd1^nl allele through the germline. The F1 and F2 progenies in mixed C57BL/6x129/Ola background were analyzed in this study. The genotypes were assessed by PCR analysis of tail genomic DNA.

All experiments using mice were approved by the local animal experimental committee of the Leiden University Medical Center and by the Commission Biotechnology in Animals of the Dutch Ministry of Agriculture.

Histological analysis
Pkd1^nl/nl mice and their heterozygous and wild-type littermates were sacrificed and their tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 4 or 5 µm and stained with haematoxylin and eosin, periodic acid-Schiff or resorchin fuchsin.

Kidney-segment identity was characterized using lectin Dolichos biflorus (Sigma-Aldrich, Zwijndrecht, The Netherlands), and antibodies against Tamm Horsfall protein (Cappel-Organon Teknika, Durham, NC, USA) and megalin (43). To detect polycystin-1, sections were stained with PKS-A, a monoclonal antibody directed to the C-terminal region of human polycystin-1 (12), in combination with the DAKO ARK animal research Kit (DAKO, Glostrup, Denmark).

RNA analysis
Tissues were snap-frozen in liquid nitrogen and stored at –80°C. We isolated total RNA from kidneys using TRIzol reagent (Invitrogen Life Technologies, Breda, The Netherlands) and cDNA was synthesized with SuperScript II (RNase H) reverse transcriptase (GibcoBRL, Breda, The Netherlands). An aliquot of 1 µg total RNA was reverse transcribed and amplified by PCR using Pkd1-specific and neo-cassette-specific primers. Each mutant transcript was sequenced.

Real-time PCR analysis of renal RNA samples was performed using TaqMan technology (PE Applied Biosystems, Nieuwerkerk a/d/ IJssel, The Netherlands) as published previously using oligo-dT and hexamer primers (44). We developed two Pkd1 PCR primer pairs: the 5'Pkd1 PCR specifically amplifies wild-type Pkd1 mRNA, and the 3'Pkd1 PCR amplifies both mutant and wild-type Pkd1 transcripts. The 5' Pkd1 5'PCR forward primer was designed to span the exon 1–exon 2 boundary, and the reverse primer is situated in exon 3. The Pkd1 3'PCR forward primer spans the exon 45–exon 46 boundary, whereas the reverse primer is located in exon 46. Primer and probe sequences are presented in Table 4. The expression of the housekeeping gene HPRT (hypoxanthine guanine phosphoribosyl transferase) served as reference for gene expression. We performed all reactions in duplicate. For northern blot analysis, RNA (10 µg/lane) was separated on 1% agarose–formaldehyde gels, transferred to nylon membranes and hybridized to Pkd1, neo and glyceraldehyde 3-phospate dehydrogenase probes.

View this table: [in this window] [in a new window]   Table 4. Primer and probe sequences

 

	ACKNOWLEDGEMENTS

 
We thank Yvonne de Jong, Jos van der Kaa and Jill Claassen for their technical assistance, and Stefan White for reading the manuscript. This research was supported by grants from the Dutch Kidney Foundation (98.1745) and the Netherlands Organisation for Scientific Research (NWO) (VIDI 016.036.353), and the Center of Medical System Biology (CMSB) established by the Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research (NGI/NWO).

	FOOTNOTES

 
^* To whom correspondence should be addressed at: Center for Human and Clinical Genetics, Leiden University Medical Center, Sylvius Laboratory, Wassenaarseweg 72, 2333 AL Leiden, The Netherlands. Tel: +31 715276048; Fax: +31 715276075; Email: d.j.m.peters{at}lumc.nl

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS REFERENCES

 

1. Gabow, P.A. (1990) Autosomal dominant polycystic kidney disease: more than a renal disease. Am. J. Kidney Dis., 16, 403–413.[Web of Science][Medline]

2. Peters, D.J.M. and Sandkuijl, L.A. (1992) Genetic heterogeneity of polycystic kidney disease in Europe. In Breuning, M.H., Devoto, M., Romeo, G. (eds). Contributions to Nephrology: Polycystic Kidney Disease Karger, Basel, Vol. 97, pp. 128–139.

3. The European Polycystic Kidney Disease Consortium (1994) The polycystic kidney disease 1 gene encodes a 14 kb transcript and lies within a duplicated region on chromosome 16. Cell, 77, 881–894.[CrossRef][Web of Science][Medline]

4. Wilson, P.D. (2004) Polycystic kidney disease. N. Engl. J. Med., 350, 151–164.[Free Full Text]

5. Tsiokas, L., Kim, E., Arnould, T., Sukhatme, V.P. and Walz, G. (1997) Homo- and heterodimeric interactions between the gene products of PKD1 and PKD2. Proc. Natl Acad. Sci. USA, 94, 6965–6970.[Abstract/Free Full Text]

6. Gonzalez-Perrett, S., Kim, K., Ibarra, C., Damiano, A.E., Zotta, E., Batelli, M., Harris, P.C., Reisin, I.L., Arnaout, M.A. and Cantiello, H.F. (2000) Polycystin-2, the protein mutated in autosomal dominant polycystic kidney disease (ADPKD), is a Ca2+-permeable nonselective cation channel. Proc. Natl Acad. Sci. USA, 98, 1182–1187.

7. Hanaoka, K., Qian, F., Boletta, A., Bhunia, A.K., Piontek, K., Tsiokas, L., Sukhatme, V.P., Guggino, W.B. and Germino, G.G. (2000) Co-assembly of polycystin-1 and -2 produces unique cation-permeable currents. Nature, 408, 990–994.[CrossRef][Medline]

8. Reeders, S.T. (1992) Multilocus polycystic disease. Nature Genet., 1, 235–237.[CrossRef][Web of Science][Medline]

9. Qian, F.J., Watnick, T.J., Onuchic, L.F. and Germino, G.G. (1996) The molecular basis of focal cyst formation in human autosomal dominant polycystic kidney disease. Cell, 87, 979–987.[CrossRef][Web of Science][Medline]

10. Pei, Y., Watnick, T., He, N., Wang, K., Liang, Y., Parfrey, P., Germino, G. and St George-Hyslop, P. (1999) Somatic PKD2 mutations in individual kidney and liver cysts support a ‘two-hit’ model of cystogenesis in type 2 autosomal dominant polycystic kidney disease. J. Am. Soc. Nephrol., 10, 1524–1529.[Abstract/Free Full Text]

11. Wu, G., D'Agati, V., Cai, Y., Markowitz, G., Park, J.H., Reynolds, D.M., Maeda, Y., Le, T.C., Hou, H., Jr, Kucherlapati, R. et al. (1998) Somatic inactivation of Pkd2 results in polycystic kidney disease. Cell, 93, 177–188.[CrossRef][Web of Science][Medline]

12. Ward, C.J., Turley, H., Ong, A.C.M., Comley, M., Biddolph, S., Chettty, R., Ratcliffe, P.J., Gattner, K. and Harris, P.C. (1996) Polycystin, the polycystic kidney disease 1 protein, is expressed by epithelial cells in fetal, adult and polycystic kidney. Proc. Natl Acad. Sci. USA, 93, 1524–1528.[Abstract/Free Full Text]

13. Pritchard, L., Sloane-Stanley, J.A., Sharpe, J.A., Aspinwall, R., Lu, W., Buckle, V., Strmecki, L., Walker, D., Ward, C.J., Alpers, C.E. et al. (2000) A human PKD1 transgene generates functional polycystin-1 in mice and is associated with a cystic phenotype. Hum. Mol. Genet., 9, 2617–2627.[Abstract/Free Full Text]

14. Lewandoski, M. (2001) Conditional control of gene expression in the mouse. Nat. Rev. Genet., 2, 743–755.[CrossRef][Web of Science][Medline]

15. Lakso, M., Pichel, J.G., Gorman, J.R., Sauer, B., Okamoto, Y., Lee, E., Alt, F.W. and Westphal, H. (1996) Efficient in vivo manipulation of mouse genomic sequences at the zygote stage. Proc. Natl Acad. Sci. USA, 93, 5860–5865.[Abstract/Free Full Text]

16. Lu, W., Peissel, B., Babakhanlou, H., Pavlova, A., Geng, L., Fan, X., Larson, C., Brent, G. and Zhou, J. (1997) Perinatal lethality with kidney and pancreas defects, in mice with a targetted Pkd1 mutation. Nature Genet., 17, 179–181.[CrossRef][Web of Science][Medline]

17. Kim, K., Drummond, I., Ibraghimov-Beskrovnaya, O., Klinger, K. and Arnaout, M.A. (2000) Polycystin 1 is required for the structural integrity of blood vessels. Proc. Natl Acad. Sci. USA, 97, 1731–1736.[Abstract/Free Full Text]

18. Boulter, C., Mulroy, S., Webb, S., Fleming, S., Brindle, K. and Sandford, R. (2001) Cardiovascular, skeletal, and renal defects in mice with a targeted disruption of the Pkd1 gene. Proc. Natl Acad. Sci. USA, 98, 12174–12179.[Abstract/Free Full Text]

19. Wu, G., Tian, X., Nishimura, S., Markowitz, G.S., D'Agati, V., Park, J.H., Yao, L., Li, L., Geng, L., Zhao, H. et al. (2002) trans-Heterozygous Pkd1 and Pkd2 mutations modify expression of polycystic kidney disease. Hum. Mol. Genet., 11, 1845–1854.[Abstract/Free Full Text]

20. Guillaume, R. and Trudel, M. (2000) Distinct and common developmental expression patterns of the murine Pkd2 and Pkd1 genes. Mech. Dev., 93, 179–183.[CrossRef][Web of Science][Medline]

21. Ong, A.C., Harris, P.C., Davies, D.R., Pritchard, L., Rossetti, S., Biddolph, S., Vaux, D.J., Migone, N. and Ward, C.J. (1999) Polycystin-1 expression in PKD1, early-onset PKD1, and TSC2/PKD1 cystic tissue. Kidney Int., 56, 1324–1333.[CrossRef][Web of Science][Medline]

22. Perrone, R.D. (1997) Extrarenal manifestations of ADPKD. Kidney Int., 51, 2022–2036.[Web of Science][Medline]

23. Wu, G., Markowitz, G.S., Li, L., D'Agati, V.D., Factor, S.M., Geng, L., Tibara, S., Tuchman, J., Cai, Y., Park, J.H. et al. (2000) Cardiac defects and renal failure in mice with targeted mutations in Pkd2. Nat. Genet., 24, 75–78.[CrossRef][Web of Science][Medline]

24. Hateboer, N., Dijk, M.A., Coto, E., Saggar-Malik, A.K., San Millan, J.L., Torra, R., Breuning, M. and Ravine, D. (1999) Comparison of phenotypes of polycystic kidney disease types 1 and 2. Lancet, 353, 103–107.[CrossRef][Web of Science][Medline]

25. Watnick, T., He, N., Wang, K., Liang, Y., Parfrey, P., Hefferton, D., St George-Hyslop, P., Germino, G. and Pei, Y. (2000) Mutations of PKD1 in ADPKD2 cysts suggest a pathogenic effect of trans-heterozygous mutations. Nat. Genet., 25, 143–144.[CrossRef][Web of Science][Medline]

26. Koptides, M., Mean, R., Demetriou, K., Pierides, A. and Deltas, C.C. (2000) Genetic evidence for a trans-heterozygous model for cystogenesis in autosomal dominant polycystic kidney disease. Hum. Mol. Genet., 9, 447–452.[Abstract/Free Full Text]

27. Brook-Carter, Ph.T., Peral, B., Ward, C.J., Thompson, P., Hughes, J., Maheshwar, M.M., Nellist, M., Gamble, V., Harris, P.C. and Sampson, J.R. (1994) Deletion of the TSC2 and PKD1 genes associated with severe infantile polycystic kidney disease—a contiguous gene syndrome. Nat. Genet., 8, 328–332.[CrossRef][Web of Science][Medline]

28. Cook, D.L., Gerber, A.N. and Tapscott, S.J. (1998) Modeling stochastic gene expression: implications for haploinsufficiency. Proc. Natl Acad. Sci. USA, 95, 15641–15646.[Abstract/Free Full Text]

29. Knudson, A.G. (1971) Mutation and cancer: statistical study of retinoblastoma. Proc. Natl Acad. Sci. USA, 68, 820–823.[Abstract/Free Full Text]

30. Song, W.J., Sullivan, M.G., Legare, R.D., Hutchings, S., Tan, X., Kufrin, D., Ratajczak, J., Resende, I.C., Haworth, C., Hock, R. et al. (1999) Haploinsufficiency of CBFA2 causes familial thrombocytopenia with propensity to develop acute myelogenous leukaemia. Nat. Genet., 23, 166–175.[CrossRef][Web of Science][Medline]

31. Fodde, R. and Smits, R. (2002) Cancer biology. A matter of dosage. Science, 298, 761–763.[Free Full Text]

32. Torra, R., Nicolau, C., Badenas, C., Bru, C., Perez, L., Estivill, X. and Darnell, A. (1996) Abdominal aortic aneurysms and autosomal dominant polycystic kidney disease. J. Am. Soc. Nephrol., 7, 2483–2486.[Abstract]

33. Chapman, A.B., Rubinstein, D., Hughes, R., Stears, J.C., Earnest, M.P., Johnson, A.M., Gabow, P.A. and Kaehny, W.D. (1992) Intracranial aneurysms in autosomal dominant polycystic kidney disease. N. Engl. J. Med., 327, 916–920.[Abstract]

34. Huston, J. III, Torres, V.E., Sulivan, P.P., Offord, K.P., Wiebers, D.O. (1993) Value of magnetic resonance angiography for the detection of intracranial aneurysms in autosomal dominant polycystic kidney disease. J. Am. Soc. Nephrol, 3, 1871–1877.[Abstract]

35. Wakabayashi, T., Fujita, S., Ohbora, Y., Suyama, T., Tamaki, N. and Matsumoto, S. (1983) Polycystic kidney disease and intracranial aneurysms. Early angiographic diagnosis and early operation for the unruptured aneurysm. J. Neurosurg., 58, 488–491.[Web of Science][Medline]

36. Qian, Q., Hunter, L.W., Li, M., Marin-Padilla, M., Prakash, Y.S., Somlo, S., Harris, P.C., Torres, V.E. and Sieck, G.C. (2003) Pkd2 haploinsufficiency alters intracellular calcium regulation in vascular smooth muscle cells. Hum. Mol. Genet., 12, 1875–1880.[Abstract/Free Full Text]

37. Guay-Woodford, L.M. (2003) Murine models of polycystic kidney disease: molecular and therapeutic insights. Am. J. Physiol. Renal Physiol., 285, F1034–F1049.[Abstract/Free Full Text]

38. Peters, D.J.M. and Breuning, M.H. (2001) Autosomal dominant polycystic kidney disease: modification of disease progression. Lancet, 358, 1439–1444.[CrossRef][Web of Science][Medline]

39. Torres, V.E., Sweeney, W.E. Jr., Wang, X., Qian, Q., Harris, P.C., Frost, P. and Avner, E.D. (2003) EGF receptor tyrosine kinase inhibition attenuates the development of PKD in Han:SPRD rats. Kidney Int., 64, 1573–1579.[CrossRef][Web of Science][Medline]

40. Torres, V.E., Sweeney, W.E., Wang, X., Qian, Q., Harris, P.C., Frost, P. and Avner, E.D. (2003) Inhibition of epidermal growth factor receptor tyrosin Kinase activity worsens the polycystic kidney disease in PCK rats. J. Am. Soc. Nephrol., 14, 109A–110A.

41. Gattone, V.H., Wang, X., Harris, P.C. and Torres, V.E. (2003) Inhibition of renal cystic disease development and progression by a vasopressin V2 receptor antagonist. Nat. Med., 9, 1323–1326.[CrossRef][Web of Science][Medline]

42. Torres, V.E., Wang, X., Qian, Q., Somlo, S., Harris, P.C. and Gattone, V.H. (2004) Effective treatment of an orthologous model of autosomal dominant polycystic kidney disease. Nat. Med., 10, 363–364.[CrossRef][Web of Science][Medline]

43. Verroust, P.J., Birn, H., Nielsen, R., Kozyraki, R. and Christensen, E.I. (2002) The tandem endocytic receptors megalin and cubilin are important proteins in renal pathology. Kidney Int., 62, 745–56.[CrossRef][Web of Science][Medline]

44. Koop, K., Eikmans, M., Baelde, H.J., Kawachi, H., De Heer, E., Paul, L.C. and Bruijn, J.A. (2003) Expression of podocyte-associated molecules in acquired human kidney diseases. J. Am. Soc. Nephrol., 14, 2063–2071.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				S. Prasad, J. P. McDaid, F. W. K. Tam, J. L. Haylor, and A. C. M. Ong Pkd2 Dosage Influences Cellular Repair Responses following Ischemia-Reperfusion Injury Am. J. Pathol., October 1, 2009; 175(4): 1493 - 1503. [Abstract] [Full Text] [PDF]

				H. Happe, W. N. Leonhard, A. van der Wal, B. van de Water, I. S. Lantinga-van Leeuwen, M. H. Breuning, E. de Heer, and D. J.M. Peters Toxic tubular injury in kidneys from Pkd1-deletion mice accelerates cystogenesis accompanied by dysregulated planar cell polarity and canonical Wnt signaling pathways Hum. Mol. Genet., July 15, 2009; 18(14): 2532 - 2542. [Abstract] [Full Text] [PDF]

				C. S. Bonnet, M. Aldred, C. von Ruhland, R. Harris, R. Sandford, and J. P. Cheadle Defects in cell polarity underlie TSC and ADPKD-associated cystogenesis Hum. Mol. Genet., June 15, 2009; 18(12): 2166 - 2176. [Abstract] [Full Text] [PDF]

				P. C. Harris 2008 Homer W. Smith Award: Insights into the Pathogenesis of Polycystic Kidney Disease from Gene Discovery J. Am. Soc. Nephrol., June 1, 2009; 20(6): 1188 - 1198. [Abstract] [Full Text] [PDF]

				Y. Pirson Does TNF-{alpha} enhance cystogenesis in ADPKD? Nephrol. Dial. Transplant., December 1, 2008; 23(12): 3773 - 3775. [Full Text] [PDF]

				A. Takakura, L. Contrino, A. W. Beck, and J. Zhou Pkd1 Inactivation Induced in Adulthood Produces Focal Cystic Disease J. Am. Soc. Nephrol., December 1, 2008; 19(12): 2351 - 2363. [Abstract] [Full Text] [PDF]

				M. R. Islam, S. Puri, M. Rodova, B. S. Magenheimer, R. L. Maser, and J. P. Calvet Retinoic acid-dependent activation of the polycystic kidney disease-1 (PKD1) promoter Am J Physiol Renal Physiol, December 1, 2008; 295(6): F1845 - F1854. [Abstract] [Full Text] [PDF]

				L. Battini, S. Macip, E. Fedorova, S. Dikman, S. Somlo, C. Montagna, and G. L. Gusella Loss of polycystin-1 causes centrosome amplification and genomic instability Hum. Mol. Genet., September 15, 2008; 17(18): 2819 - 2833. [Abstract] [Full Text] [PDF]

				J. P. Calvet Strategies to Inhibit Cyst Formation in ADPKD Clin. J. Am. Soc. Nephrol., July 1, 2008; 3(4): 1205 - 1211. [Abstract] [Full Text] [PDF]

				X. Fu, Y. Wang, N. Schetle, H. Gao, M. Putz, G. von Gersdorff, G. Walz, and A. G. Kramer-Zucker The Subcellular Localization of TRPP2 Modulates Its Function J. Am. Soc. Nephrol., July 1, 2008; 19(7): 1342 - 1351. [Abstract] [Full Text] [PDF]

				S. Shibazaki, Z. Yu, S. Nishio, X. Tian, R. B. Thomson, M. Mitobe, A. Louvi, H. Velazquez, S. Ishibe, L. G. Cantley, et al. Cyst formation and activation of the extracellular regulated kinase pathway after kidney specific inactivation of Pkd1 Hum. Mol. Genet., June 1, 2008; 17(11): 1505 - 1516. [Abstract] [Full Text] [PDF]

				C. Cadieux, R. Harada, M. Paquet, O. Cote, M. Trudel, A. Nepveu, and M. Bouchard Polycystic Kidneys Caused by Sustained Expression of Cux1 Isoform p75 J. Biol. Chem., May 16, 2008; 283(20): 13817 - 13824. [Abstract] [Full Text] [PDF]

				A.-R. Gallagher, E. L. Esquivel, T. S. Briere, X. Tian, M. Mitobe, L. F. Menezes, G. S. Markowitz, D. Jain, L. F. Onuchic, and S. Somlo Biliary and Pancreatic Dysgenesis in Mice Harboring a Mutation in Pkhd1 Am. J. Pathol., February 1, 2008; 172(2): 417 - 429. [Abstract] [Full Text] [PDF]

				I. S. Lantinga-van Leeuwen, W. N. Leonhard, A. van der Wal, M. H. Breuning, E. de Heer, and D. J.M. Peters Kidney-specific inactivation of the Pkd1 gene induces rapid cyst formation in developing kidneys and a slow onset of disease in adult mice Hum. Mol. Genet., December 15, 2007; 16(24): 3188 - 3196. [Abstract] [Full Text] [PDF]

				T. Weimbs Polycystic kidney disease and renal injury repair: common pathways, fluid flow, and the function of polycystin-1 Am J Physiol Renal Physiol, November 1, 2007; 293(5): F1423 - F1432. [Abstract] [Full Text] [PDF]

				S. Hassane, N. Claij, I. S. Lantinga-van Leeuwen, J. C. Van Munsteren, N. Van Lent, R. Hanemaaijer, M. H. Breuning, D. J.M. Peters, and M. C. DeRuiter Pathogenic Sequence for Dissecting Aneurysm Formation in a Hypomorphic Polycystic Kidney Disease 1 Mouse Model Arterioscler Thromb Vasc Biol, October 1, 2007; 27(10): 2177 - 2183. [Abstract] [Full Text] [PDF]

				Y. Tian, R. Kolb, J.-H. Hong, J. Carroll, D. Li, J. You, R. Bronson, M. B. Yaffe, J. Zhou, and T. Benjamin TAZ Promotes PC2 Degradation through a SCF{beta}-Trcp E3 Ligase Complex Mol. Cell. Biol., September 15, 2007; 27(18): 6383 - 6395. [Abstract] [Full Text] [PDF]

				Q. Qian, L. W. Hunter, H. Du, Q. Ren, Y. Han, and G. C. Sieck Pkd2+/- Vascular Smooth Muscles Develop Exaggerated Vasocontraction in Response to Phenylephrine Stimulation J. Am. Soc. Nephrol., February 1, 2007; 18(2): 485 - 493. [Abstract] [Full Text] [PDF]

				L. Battini, E. Fedorova, S. Macip, X. Li, P. D. Wilson, and G. L. Gusella Stable Knockdown of Polycystin-1 Confers Integrin-{alpha}2beta1-Mediated Anoikis Resistance J. Am. Soc. Nephrol., November 1, 2006; 17(11): 3049 - 3058. [Abstract] [Full Text] [PDF]

				P. C. Harris, K. T. Bae, S. Rossetti, V. E. Torres, J. J. Grantham, A. B. Chapman, L. M. Guay-Woodford, B. F. King, L. H. Wetzel, D. A. Baumgarten, et al. Cyst Number but Not the Rate of Cystic Growth Is Associated with the Mutated Gene in Autosomal Dominant Polycystic Kidney Disease J. Am. Soc. Nephrol., November 1, 2006; 17(11): 3013 - 3019. [Abstract] [Full Text] [PDF]

				A. R. Gallagher, S. Hoffmann, N. Brown, A. Cedzich, S. Meruvu, D. Podlich, Y. Feng, V. Konecke, U. de Vries, H.-P. Hammes, et al. A Truncated Polycystin-2 Protein Causes Polycystic Kidney Disease and Retinal Degeneration in Transgenic Rats J. Am. Soc. Nephrol., October 1, 2006; 17(10): 2719 - 2730. [Abstract] [Full Text] [PDF]

				M. Y. Chang, E. Parker, S. Ibrahim, J. R. Shortland, M. E. Nahas, J. L. Haylor, and A. C. M. Ong Haploinsufficiency of Pkd2 is associated with increased tubular cell proliferation and interstitial fibrosis in two murine Pkd2 models Nephrol. Dial. Transplant., August 1, 2006; 21(8): 2078 - 2084. [Abstract] [Full Text] [PDF]

				C. Thivierge, A. Kurbegovic, M. Couillard, R. Guillaume, O. Cote, and M. Trudel Overexpression of PKD1 Causes Polycystic Kidney Disease Mol. Cell. Biol., February 15, 2006; 26(4): 1538 - 1548. [Abstract] [Full Text] [PDF]

				T. Yamaguchi, S. J. Hempson, G. A. Reif, A.-M. Hedge, and D. P. Wallace Calcium Restores a Normal Proliferation Phenotype in Human Polycystic Kidney Disease Epithelial Cells J. Am. Soc. Nephrol., January 1, 2006; 17(1): 178 - 187. [Abstract] [Full Text] [PDF]

				N. Hang Le, A. van der Wal, P. van der Bent, I. S. Lantinga-van Leeuwen, M. H. Breuning, H. van Dam, E. de Heer, and D. J.M. Peters Increased Activity of Activator Protein-1 Transcription Factor Components ATF2, c-Jun, and c-Fos in Human and Mouse Autosomal Dominant Polycystic Kidney Disease J. Am. Soc. Nephrol., September 1, 2005; 16(9): 2724 - 2731. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 13/24/3069    most recent ddh336v1 Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Request Permissions Google Scholar Articles by Leeuwen, I. S. L.-v. Articles by Peters, D. J.M. Search for Related Content PubMed PubMed Citation Articles by Leeuwen, I. S. L.-v. Articles by Peters, D. J.M. Social Bookmarking          What's this?

Online ISSN 1460-2083 - Print ISSN 0964-6906

Copyright © 2009 Oxford University Press

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics