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Molecular and Cellular Biology, February 2006, p. 1538-1548, Vol. 26, No. 4
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.4.1538-1548.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Overexpression of PKD1 Causes Polycystic Kidney Disease

Caroline Thivierge, Almira Kurbegovic, Martin Couillard, Richard Guillaume, Olivier Coté, and Marie Trudel*

Institut de Recherches Cliniques de Montreal, Molecular Genetics and Development, Faculte de Medecine de l'Universite de Montreal, Montreal, Quebec, Canada

Received 7 October 2005/ Returned for modification 3 November 2005/ Accepted 4 December 2005


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ABSTRACT
 
The pathogenetic mechanisms underlying autosomal dominant polycystic kidney disease (ADPKD) remain to be elucidated. While there is evidence that Pkd1 gene haploinsufficiency and loss of heterozygosity can cause cyst formation in mice, paradoxically high levels of Pkd1 expression have been detected in the kidneys of ADPKD patients. To determine whether Pkd1 gain of function can be a pathogenetic process, a Pkd1 bacterial artificial chromosome (Pkd1-BAC) was modified by homologous recombination to solely target a sustained Pkd1 expression preferentially to the adult kidney. Several transgenic lines were generated that specifically overexpressed the Pkd1 transgene in the kidneys 2- to 15-fold over Pkd1 endogenous levels. All transgenic mice reproducibly developed tubular and glomerular cysts and renal insufficiency and died of renal failure. This model demonstrates that overexpression of wild-type Pkd1 alone is sufficient to trigger cystogenesis resembling human ADPKD. Our results also uncovered a striking increased renal c-myc expression in mice from all transgenic lines, indicating that c-myc is a critical in vivo downstream effector of Pkd1 molecular pathways. This study not only produced an invaluable and first PKD model to evaluate molecular pathogenesis and therapies but also provides evidence that gain of function could be a pathogenetic mechanism in ADPKD.


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INTRODUCTION
 
Autosomal dominant polycystic kidney disease (ADPKD) is one of the most frequent genetic diseases in humans. It is characterized by progressive development of multiple renal cysts affecting all segments of the nephron. Other manifestations include formation of cysts in the liver and pancreas as well as intracranial aneurysms and cardiovascular defects. ADPKD typically leads to renal insufficiency with progression to end-stage renal disease by late middle age.

Approximately 85% of ADPKD cases are associated with mutations in the PKD1 gene. This PKD1 gene is large, spans 54 kb, and consists of 46 exons. It generates a 14.2-kb transcript and encodes a 4,302-amino-acid protein called polycystin-1 (4, 9-11). Human PKD1 and polycystin-1 expression have been analyzed in normal and ADPKD kidneys. During renal development, polycystin-1 is readily detected in glomerular and tubular epithelial cells (reviewed in reference 37 and references therein). In normal adults, we and others have shown that PKD1 RNA and protein are expressed at moderate to low levels in the collecting and distal tubules, whereas levels increased (~2-fold) in ADPKD kidneys (22, 39). Interestingly, persistent or enhanced polycystin-1 expression is detected in the majority of renal epithelial cysts, although staining was absent in a significant minority of cysts (29). In addition to the kidneys, PKD1 expression is normally widespread in other adult tissues, including epithelial and nonepithelial cell types (6, 14, 18, 29, 30, 39).

More than 200 different PKD1 mutations have been described, most of which are deletion-insertion, frameshift, or nonsense mutations. These are predicted to result in truncated forms of the protein, consistent with inactivation of one allele. However, a significant proportion are missense or in-frame mutations that are found throughout the gene and are often unique to a particular family (33, 34). As the name implies, ADPKD is dominant and the transmitted mutated PKD1 allele is sufficient to produce the disease. However, the focal nature of the renal cysts in ADPKD suggests that the mutational mechanism for PKD1 could be a two hit or a loss of heterozygosity. Support for this mechanism was obtained by detection of PKD1 clonal somatic mutations in cells from a significant proportion of cysts (3, 21, 32). A mechanism of loss of heterozygosity could account for the widely varying phenotype commonly observed in individual families.

Studies on the mouse Pkd1 gene may provide valuable insights into PKD1 function(s) because of the close similarity between the human and murine gene and gene product. In normal development, murine Pkd1 is expressed at high levels from the morula stage and is detected in all neural crest cell derivatives, including adult brain, aortic arch, cartilage, and mesenchymal condensation (16, 17). Homozygous mutant mice targeted for Pkd1 deletion have been reported to die in utero and to develop renal and pancreatic cysts (2, 19, 24-26, 40). These previous attempts to generate mouse models unfortunately did not provide viable animals postnatally. Nevertheless, the occurrence of renal cysts in these homozygous Pkd1 mutant mice would be consistent with the hypothesis of a two-hit mutational mechanism in humans that involves a germ line mutation and somatic inactivation of the normal allele. However, mice heterozygous for the Pkd1 targeted deletion also displayed PKD with occasional liver and pancreatic cysts despite a late adult onset, supporting a mechanism of haploinsufficiency or gene dosage reduction. Moreover, loss of heterozygosity or haploinsufficiency may not be the sole mechanisms for ADPKD pathogenesis. Indeed, these mechanisms are at variance with the persistent or enhanced expression of PKD1 seen in the majority of human renal cysts unless nonfunctional proteins are produced. This finding of sustained or increased PKD1 expression raises the question of whether a gain of function or overexpression may be operant.

To investigate the Pkd1 gain-of-function pathogenetic mechanism, we have isolated and characterized a murine Pkd1 bacterial artificial chromosome (Pkd1-BAC) that was subsequently modified by homologous recombination in Escherichia coli to target expression of Pkd1 specifically to the kidneys. We report the production of three transgenic lines that expressed the Pkd1 transgene at different levels. All mice reproducibly displayed a number of similarities to human ADPKD and consistently developed early onset with rapid progression of renal morphological and functional alterations and died of renal failure by middle age. In addition, the current study describes an in vivo mechanism by which Pkd1 can mediate this PKD phenotype. These mice represent the first model of PKD produced by the sole renal overexpression of the orthologous PKD1 gene.


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MATERIALS AND METHODS
 
Isolation of BAC clones containing the Pkd1 locus. Pkd1-BAC clones from the bacterial host strain E. coli DH10B (RecA– RecBC+) were isolated from a 129Sv mouse pBelo11BAC library (Research Genetics). Screening of BAC superpools was performed by PCR with the following primers: 5'region, 5'-CTGATGAGTTCTGGCCATGGATG-3'(forward Pkd1 exon 1) and 5'-CTGCCAGCCAATGCCATAGTCAC-3'(reverse Pkd1 exon 1); and 3' region, 5'-TCGGCCCTAGCGTCTGCAGCC-3'(forward Pkd1 exon 39) and 5'-TCCAGTCCCACCTACAGCCAAC-3'(reverse Pkd1 exon 40). One positive clone for both amplifications was identified and analyzed on standard gel and pulsed-field gel electrophoresis (PFGE) followed by Southern blotting. For Southern blot analysis, seven mouse Pkd1 probes have been designed: genomic exon 1 (516 bp; nucleotides [nt] 1 to 516; NCBI accession number U70209), genomic exon 2-3 (220 bp), genomic exon 7-15 (8,479 bp), cDNA exon 15-20 (1,724 bp; nt 6455 to 8179), cDNA exon 25-34 (1,315 bp; nt 9415 to 10730), cDNA exon 36-45 (1,655 bp; nt 10963 to 12618), and genomic exon 45-46 (1,640 bp) (16). Several regions including the BAC insert extremities of this murine Pkd1-BAC have also been sequenced and were confirmed to be orthologous to the human PKD1 gene and contiguous regions. This Pkd1-BAC clone of ~129 kb contains the entire sequence of Pkd1, with ~37 kb and ~39.1 kb of upstream and downstream flanking sequences, including the Tsc2 gene body.

Targeting vector constructs for homologous recombination. To modify the original wild-type Pkd1-BAC by homologous recombination, two constructs were produced in the pLD53.SC-AB BAC recombination vector (15). The first construct was carried out in order to modify the Pkd1 regulatory elements by insertion of the renal epithelial specific regulatory elements (SB) directly upstream of the Pkd1 start codon. SB regulatory elements consist of two 72-bp repeats of the simian virus 40 enhancer linked to the ß-globin promoter contained within a 687-bp fragment (36). A recombination cassette containing the SB regulatory elements flanked by two homology arms was introduced in the BAC recombination vector. The first homology arm was obtained from a 997-bp KpnI-SstII fragment (Pkd1 promoter-exon1) of a restriction enzyme digest, and the second arm was from a PCR product of 1,374 bp amplified with the following primers: 5'-ATGCCGCCCGGCGCGCCTGCT-3'(forward Pkd1 exon1) and 5'-AGCCGGGCAGTGGTGGTGCACACC-3'(reverse Pkd1 intron1). The second construct consisted of introducing a silent point mutation by substitution of a G-to-A nucleotide. This substitution created a new EcoRI restriction site in Pkd1 exon 10 that distinguished the transgene from the endogenous gene and transcript (nt 2355; accession number U70209). In this case, the recombination cassette included two Pkd1 homology arms that extended on both sides of the EcoRI silent point mutation site. The two homology arms were obtained by PCR amplification, the first arm with primers 5'-CCTCTGCATCGATTGGCACAG-3'(forward Pkd1 exon 8) and 5'-GGCACAGAAAAAAAGAATTCCTTCC-3'(reverse Pkd1 exon 10) and the second arm with 5'-GGAAGGAATTCTTTTTTTCTGTGCC-3'(forward Pkd1 exon 10) and 5'-GTTTTGCCTGGATCCGCTGTTG-3'(reverse Pkd1 exon 11), 1,180 bp and 869 bp in length, respectively.

Modification of BAC clones by homologous recombination in E. coli. Each of the two BAC recombination vectors was used in a two-step RecA strategy for BAC modifications as previously described (15). Cointegrates were selected by progressive ampicillin concentrations to 100 µg/ml. Approximately 20 cointegrates were analyzed for each recombination by Southern blot to monitor for appropriate integration events. Five proper cointegrates were chosen for the second recombination event, and resolved BACs were selected by adding 6% sucrose and UV exposure. Positive clones from the resolved BACs were further analyzed with seven probes spanning the entire sequence of the modified BAC by Southern blotting following standard gel electrophoresis and PFGE. Subsequently, modified Pkd1 gene regions were sequenced to confirm that the intended recombined BAC clones were achieved. Following these two modifications the BAC clone was referred to as SBPkd1TAG-BAC.

Production and analysis of BAC transgenic mice. SBPkd1TAG-BAC (40 to 50 µg of DNA) was digested with the restriction enzymes ClaI and NotI. The 70-kb transgene fragment was isolated on 1% low-melt agarose (SeaPlaque GTG agarose; Mandel) by PFGE with the following conditions: 20 h at 14°C in 0.5x Tris-borate-EDTA with a switch time of 5 s. The SBPkd1TAG-linearized DNA fragment was purified by a 10-min incubation at 65°C, followed by a digestion with 2 to 3 U of ß-agarase per 100 µl for 2 to 3 h at 42°C. Once the digestion was completed, the DNA was concentrated to a volume of 50 to 100 µl by centrifugation at 1,200 x g on Millipore filters (Ultrafree-MC 100000 NMWL Filter Unit). Prior to oocyte microinjection, the fragment preparation was verified for integrity by PFGE. The SBPkd1TAG fragment obtained was diluted (1 ng/µl) in buffer (10 mM Tris, pH 7.5, 0.1 mM EDTA, pH 7.5, 100 mM NaCl) and microinjected into the pronuclei of (C57BL/6J x CBA/J)F2-fertilized eggs (36). Transgenic founder mice and progenies were identified by Southern analysis of DNA (10 µg) obtained from tail biopsies (36). Genomic DNA was digested with HindIII, EcoRI, and/or KpnI, and blots were respectively hybridized with the seven mouse Pkd1 probes and the SB (831 bp) transgene probe to verify integrity of the 5', internal, and 3' regions of the transgene.

RNA expression analysis. Total RNA was extracted from various tissues, including kidneys, lungs, spleen, brain, heart, pancreas, and liver, of 1- to 12-month-old animals using the guanidium thiocyanate or the Trizol-chloroform method (12). The integrity of all RNA preparations was monitored by electrophoresis on formaldehyde-agarose gels (38).

SBPkd1TAG transgene expression in all tissues was analyzed by semiquantitative reverse transcription-PCR (RT-PCR) and quantitative real-time PCR. All RNA samples were reverse transcribed as previously described (12). The primers used for both analyses were as follows: SBPkd1TAG transgene, 5'-CATTTGCTTCTGACACAACTGTGTTC-3'(forward ß-globin promoter) and 5'-CCAGCGTCTGAAGTAGGTTGTGGG-3'(reverse Pkd1 exon 2); an additional primer was used for real-time PCR analysis for endogenous and transgene Pkd1, 5'-TCAATTGCTCCGGCCGCTG-3'(forward Pkd1 exon 1). The S16 ribosomal gene product served as an internal control with the following primers: 5'-AGGAGCGATTTGCTGGTGTGGA-3'(forward S16 exon 3) and 5'-GCTACCAGGCCTTTGAGATGGA-3'(reverse S16 exon 4). Each pair of primers was designed such that only spliced mRNA would produce the predicted amplification products of 307 bp for SBPkd1TAG transgene, 100 bp for total Pkd1 (endogenous gene and transgene), and 102 bp for S16. For semiquantitative PCR, control reactions were performed a priori using varying quantities of RT aliquots to ensure that the conditions used were within the linear range. Reverse-transcribed aliquots were subsequently amplified semiquantitatively in PCR buffer (10 mM Tris, pH 8.5, 50 mM KCl, 0.75 mM MgCl2) containing 0.2 mM of each deoxynucleoside triphosphate, 0.5 µM of each primer, and 2.5 U of Taq polymerase. Conditions for amplification were 5 min at 94°C followed by 26 cycles of 95°C, 30 s; 55°C, 30 s; and 72°C, 30 s, with a final elongation of 7 min at 72°C. Samples were analyzed on 8% polyacrylamide gels and visualized by ethidium bromide staining. For quantitative real-time PCR analysis, all reactions were performed in triplicate in a master mix (QIAGEN, Mississauga, Canada) containing 0.3 µM of each primer. Conditions for amplification were 15 min at 95°C followed by 40 cycles of 94°C, 30 s; 58°C, 30 s; and 72°C, 30 s in an MX4000 Mutiplex quantitative PCR analyzer.

Analysis of c-myc expression was performed by semiquantitative RT-PCR with the same reverse-transcribed conditions used for the SBPkd1TAG transgene. Primers for c-myc were (forward) 5'-CTCTCAACGACAGCAGCTCG-3' (exon 2) and (reverse) 5'-AGATGAGCCCGACTCCGACC-3' (exon 3), with the same amplification conditions described previously (35). Samples were analyzed on 6% polyacrylamide gels, and the predicted c-myc amplification product is 250 bp. For quantitative evaluation, gels were scanned and analyzed by Image Quant 5.0 software.

Expression analysis of Pkd1 (endogenous gene and transgene) was also performed by Northern blotting. Total RNA from each sample (30 µg) was electrophoresed on a 0.8% agarose-0.6 M formaldehyde gel and transferred to nylon membranes. cDNA probes for Pkd1 (exon 36-45) and for the internal control glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1.2 kb) were synthesized using a random primer DNA-labeling system with [{alpha}-32P]dCTP (16). Hybridization was performed at 65°C overnight, and membranes were exposed to X-ray film (Biomax MS) for 24 to 48 h. For quantitative evaluation, membranes were scanned by a phosphorimager and quantified with the Image Quant 5.0 software.

In situ hybridization techniques were performed on paraformaldehyde-fixed, paraffin-embedded renal tissues. Sections were collected on 3-aminopropyl triethoxy silane-coated slides. Each section was hybridized with a uniformly digoxigenin-labeled antisense RNA produced from the exon 36-45 probe (16), washed, and incubated 2 h with antidigoxigenin-alkaline phosphatase-conjugated antibody (Roche) and revealed overnight in nitroblue tetrazolium-5-bromo-4-chloro-3-indolylphosphate according to the manufacturer's recommendation. Controls consisted of adjacent tissue sections incubated with phosphate-buffered saline containing 10 µg per microliter RNase A1 at room temperature for 30 min, washed, and hybridized as described above.

Renal physiological function analysis. The renal function was evaluated by analysis of mouse serum, obtained from centrifuged capillaries (2-3) of tail blood at 5,000 rpm for 3 min. Blood urea nitrogen (BUN) and creatinine in the serum were measured with a CX9 Beckmann apparatus. For urine analysis, 500 µl to 1 ml of urine was collected per mouse. Urine BUN, creatinine, and ion concentrations were measured with a CX9 Beckmann apparatus, whereas urine osmolality was determined with a radiometer. Aliquots of urine containing 25 µg of total protein were qualitatively analyzed on sodium dodecyl sulfate-10% polyacrylamide gel electrophoresis (SDS-10% PAGE) and stained with Coomassie blue as described previously (13).

The hematological parameters were determined on aliquots of 250 µl of blood collected from the tail vein in EDTA-treated tubes (Capijek T-MQK tubes). Blood was analyzed using an ADVIA 120 apparatus as we had done previously (1).

Histological analysis. Renal tissues were analyzed from newborn to adult transgenic mice. Five-micrometer-thick paraffin sections of formalin-fixed tissues were deparaffinized, hydrated in graded alcohols, and stained with hematoxylin and eosin for morphological analysis.

Statistical analysis. Values were expressed as means ± standard deviations. Unpaired two-sample Student's t test was used for statistical analysis; P < 0.05 was considered significant.


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RESULTS
 
Production of SBPkd1TAG-BAC by homologous recombination. To determine whether Pkd1 gain of function alone is sufficient to produce the ADPKD phenotype, we first isolated a genomic clone containing the entire Pkd1 gene in a BAC vector 129/Sv library. This library was screened by PCR with two sets of primers for the Pkd1 gene that spanned exon 1 at the 5' end and exons 39 to 40 toward the 3' end (Fig. 1). A positive BAC clone for the Pkd1 gene was identified that included the entire adjacent Tsc2 gene body. The Pkd1 insert was characterized in detail to ensure that the genomic structure matched that of the endogenous Pkd1 gene of the 129/Sv mouse strain from which the insert was derived and from the C57BL/6J inbred strain. Genomic maps of the Pkd1 locus in the BAC and in these inbred strains by Southern blot analysis, with four restriction enzyme digestions and seven probes covering the entire Pkd1 gene, appeared identical with no evidence of rearrangements (Fig. 1). This BAC contained a ~121-kb insert including ~37 kb of upstream and ~39 kb of downstream sequence of the Pkd1 gene as determined by electrophoresis and sequencing.


Figure 1
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  		FIG. 1. Schematic representation and detailed restriction map analysis of a murine Pkd1-BAC. Genomic DNA digestion patterns of the Pkd1-BAC were compared to that of the Pkd1 locus in the 129Sv and C57Bl/6J inbred mouse strains. Seven probes encompassing most of the murine Pkd1 gene were produced: (a) exon 1, (b) exon 2-3, (c) exon 7-15, (d) exon 15-20, (e) exon 25-34, (f) exon 36-45, and (g) exon 45-46, labeled a to g on the genomic Pkd1 representation and over individual blots. Southern blot analysis following restriction digests (BamHI, EcoRI, HindIII, and KpnI) of genomic DNA from the Pkd1-BAC and murine Pkd1 loci showed identical patterns with all seven probes. M, {lambda} HindIII marker; 129, 129/Sv; C57, C57Bl/6J.

This Pkd1-BAC clone was modified by two successive homologous recombination events in E. coli. The Pkd1 gene was tagged in exon 10 by substituting a nucleotide (G to A) to create a novel EcoRI site at position 2355 on the cDNA map. This silent point mutation was produced to distinguish the Pkd1 gene and transcript of the BAC from that of endogenous origin. In addition, we have replaced the 5' regulatory elements of the Pkd1-BAC gene by taking advantage of previously identified "SB" renal epithelial-specific elements from the SBM (linked to c-Myc) or SBF linked to c-fos) construct-transgene to restrict expression to the kidneys (36, 38) (Fig. 2a). This new SBPkd1TAG-BAC was digested with NotI, a unique site located immediately upstream of the SB elements, and ClaI within the Tsc2 gene body, truncating the Tsc2 regulatory elements and the 5' half of the gene body to ensure lack of Tsc2 exogenous expression in all tissues and to remove the prokaryotic BAC vector sequences (Fig. 1 and 2). This 70-kb NotI-ClaI linearized fragment was isolated, purified, and quantified for oocyte microinjection (36).


Figure 2
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  		FIG. 2. Production of SBPkd1TAG construct and transgenic mice. (a) Successive homologous recombination events were carried out on the murine Pkd1-BAC to introduce two modifications: the SB regulatory elements inserted immediately upstream of the Pkd1 initiation codon and a silent point mutation of EcoRI (RI*) introduced in exon (ex) 10. The BAC recombination vector contains an R6K{gamma} origin of replication, an ampicillin-resistant gene, a SacB gene, a RecA gene, and a unique SmaI cloning site in which the "SB" regulatory elements (or the exon 10 silent point mutation) were cloned with flanking Pkd1 gene arms. The BAC recombination vector was electroporated into E. coli (DH10B) cells containing the Pkd1-BAC wild type, and following selection a first homologous recombination event occurred via one of the two Pkd1 arms to produce BAC cointegrates. The recombination vector and duplicated Pkd1 regions of the BAC cointegrates were eliminated in a second selection step. Resolved Pkd1-BACs can either revert to wild type or include the intended modification. A subsequent homologous recombination into the newly modified BAC can then be introduced. (b) Genomic DNA of SBPkd1TAG transgenic mice was analyzed by Southern blot. Microinjection of linearized fragments caused insertion of the transgene in a head-to-head, head-to-tail, and/or tail-to-tail orientation. The 5' end was analyzed by digestion of genomic DNA with HindIII and hybridized with the specific transgene SB probe (left panel). All three transgenic mouse lines generated the expected 10.9-kb band for head-to-tail insertion; the additional band observed in line 39 most likely represents a junction fragment between SB and the mouse genome. Internal integrity of the transgene was monitored by several restriction enzyme digestions, and one representative blot of genomic DNA digested with EcoRI and hybridized with the Pkd1 probe (exon 7-15) is shown (middle panel). Two bands are expected at 6.9 kb and 2.5 kb for the transgene due to the silent EcoRI site and a single band at 9.4 kb for the endogenous gene. The 3' end (right panel) was analyzed by a KpnI digestion of genomic DNA and hybridized with the exon 45-46 Pkd1 probe. The three transgenic lines produced the expected 7.1-kb genomic fragment for both the Pkd1 endogenous gene and SBPkd1TAG transgene. M, {lambda} HindIII marker; 1C and 10C, 1 and 10 copies of the transgene as positive controls for hybridization; SB, simian virus 40 enhancer and ß-globin promoter; RI*, novel restriction site introduced by homologous recombination; H, HindIII; RI, EcoRI; K, KpnI.

Production and analysis of SBPkd1TAG transgenic mice. Fourtransgenic founders carrying several copies of the SBPkd1TAGtransgene consistently developed PKD. From the four SBPkd1TAG founder mice determined by Southern analysis, three SBPkd1TAG transgenic lines were established with two to nine copies of the transgene (Fig. 2b). Characterization of the transgene integrity in these lines was monitored with 5', internal, and 3' probes as shown by representative examples in Fig. 2b. Transgenic lines revealed with the 5' "SB" probe a band at 10.9 kb consistent with the SBPkd1TAG transgene being integrated in a head-to-tail orientation and revealed with the 3' probe a 7.1-kb band (Fig. 2b). In addition, the internal probe detected the 9.4-kb endogenous Pkd1 band as well as the 6.9-kb and 2.5-kb bands of the transgene due to the EcoRI insertion site in exon 10 (Fig. 2b). These mice contained complete copies of the transgene based on the genomic overlapping structure analysis.

Pkd1 gain of function in adult SBPkd1TAG transgenic mice. Expression of the SBPkd1TAG transgene and Pkd1 gene was investigated in various organs. Quantification of transcript levels from the transgene and/or endogenous gene was carried out by Northern blot analysis (Fig. 3a). As expected, the transgene and endogenous gene transcripts were of similar length (14.2 kb). Based on control GAPDH expression, kidneys from all SBPkd1TAG mouse lines had consistently increased transcript expression compared to normal Pkd1 levels in adult kidneys (n = 3) of similar age. Renal transgene and endogenous expression for the different transgenic lines displayed a range of 2- to 15-fold above the control renal endogenous Pkd1 levels (Fig. 3a). Particularly, transgenic line 39 (n = 4) showed higher Pkd1 levels than lines 3 (n = 3) and 41 (n = 4). Furthermore, Pkd1 expression levels measured by Northern blot analysis correlated with those obtained by real-time PCR using primers in exons 1 and 2 (Fig. 3b).


Figure 3
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  		FIG. 3. Renal expression analysis of SBPkd1TAG mice. (a) Expression analysis of Pkd1 endogenous (endo) and SBPkd1TAG transgene (Tg) transcripts in kidneys from three transgenic lines by Northern blotting. Two samples from each transgenic line, 3, 39, and 41, were compared to endogenous renal Pkd1 transcript of normal control age-matched mice from the same genetic background (C57BL/6J x CBA/J)F1. Kidney RNA samples were obtained from transgenic mice prior to end-stage renal disease. Transcripts from endo and Tg are both of ~14.2 kb in length. A systematic overexpression of the transcripts was observed in kidneys of all transgenic mice relative to nontransgenic controls. GAPDH was used as an internal control for loading. Quantification of renal expression in these transgenic mice ranged from 2- to 15-fold relative to Pkd1 endogenous levels from control mice arbitrarily set at 1. (b) Schematic representation of SBPkd1TAG transgene and primers used to amplify total Pkd1 including endogenous and transgene (exon 1 and exon 2) and only Pkd1 transgene (B, exon 2) by real-time PCR and semiquantitative RT-PCR. RT-PCR analysis of the SBPkd1TAG transgene expression was quantified in renal and extrarenal tissues. A representative semiquantitative evaluation of SBPkd1TAG transgene (Tg) is shown that includes a renal tissue sample (K) from one mouse of all three transgenic lines (3, 39, and 41) and of extrarenal tissues. H, heart; Lu, lung; B, brain; Li, liver; and S, spleen from a mouse of line 39. Expression of the transgene is readily detectable in the kidneys of all transgenic mice, whereas it is low to undetectable in extrarenal tissues. Expression from the SBPkd1TAG transgene produced a specific 307-bp amplicon, whereas the S16 internal control generated a 102-bp amplicon. M, 100-bp marker; H2O, negative control for PCR amplification. (c) Real-time PCR expression analysis of the SBPkd1TAG transgene was determined from several independent mice. The SBPkd1TAG transgene from the three transgenic mouse lines 3 (n = 5), 39 (n = 7), and 41 (n = 5) showed that lines 39 and 41 had the highest renal expression levels. Expression of SBPkd1TAG transgene in extrarenal tissues from mice (n = 3) of the three transgenic lines was evaluated by real-time PCR. In comparison to the kidneys of each transgenic line (100%), analysis of extrarenal tissues showed that transgene expression levels were consistently lower by 10- to 1,000-fold in brain, heart, liver, pancreas, spleen, and lung. (d) Expression of endogenous c-myc gene in the SBPkd1TAG kidneys by semiquantitative RT-PCR. A schematic illustration shows the primers used to amplify c-myc. As expected, expression of c-myc is minimal in adult nontransgenic kidneys (controls). In contrast, increased expression of c-myc is detected in all adult SBPkd1TAG kidneys of the three lines, as observed in the adult transgenic SBM kidneys used as positive control. The amplicon of c-myc was 250 bp; the amplicon of S16, an internal control, was 102 bp. M, 100-bp marker.

Quantification of the transgene expression levels specifically was carried out by real-time PCR and semiquantitative RT-PCR in the three transgenic lines at adult age by using primers in the 5' untranslated region (B, ß-globin promoter) and in exon 2 of Pkd1 (Fig. 3b). The SBPkd1TAG expression in transgenic mice was compared to the S16 ribosomal protein gene product as an internal standard. Conditions used for semiquantitative RT-PCR amplification were within the linear range. Transgene expression by real-time PCR and semiquantitative RT-PCR consistently and specifically showed the highest expression in the kidney of all transgenic lines relative to other organs (Fig. 3b and c). Renal expression levels for an individual sample were reproducible with any of the detection techniques used. Highest levels of Pkd1 transgene renal expression were measured for lines 39 and 41. To monitor whether the increased Pkd1 expression resulted from the transgene or the endogenous gene, the same group of mice from the three transgenic lines was compared for renal Pkd1 transgene expression and for renal Pkd1 total (transgene and endogenous) expression by real-time PCR. Interestingly, lines 39 and 41 relative to line 3 showed that the increased Pkd1 transgene renal expression was similar to or above that of Pkd1 total renal expression, pointing to the transgene as specifically responsible for this induced expression. In various organs (including heart, lung, brain, liver, pancreas, and spleen), the SBPkd1TAG transgene showed very weak expression occasionally detected in spleen and lung, with little to undetectable expression in other organs (Fig. 3b). Quantification by real-time PCR demonstrated a 10- to 1,000-fold lower level of the transgene expression in extrarenal tissues relative to kidney expression (Fig. 3c). The "SB" regulatory elements of the SBPkd1TAG transgene conferred preferential renal expression; this particular organ distribution was also determined when used in transgenes linked to c-myc (SBM) and c-fos (SBF) (36, 38).

c-myc, a downstream effector of Pkd1 signaling pathways in SBPkd1TAG mice. To gain insight into the intracellular pathogenetic mechanism of SBPkd1TAG transgenic mice, we next sought to monitor c-myc renal expression level based on our previous observation of c-myc deregulation in human ADPKD kidneys (22). Analysis of kidneys was carried out from all three transgenic lines 3 (n = 4), 39 (n = 7), and 41 (n = 4) as well as controls (n = 4). As shown in Fig. 3d, there is a substantial expression of endogenous c-myc induced in SBPkd1TAG mice relative to control mice of similar age. Interestingly, the level of c-myc expression in some SBPkd1TAG kidneys, in particular line 39, reached levels comparable to that observed in the PKD SBM transgenic mouse model produced by renal c-myc expression.

Renal anomalies in SBPkd1TAG mice similar to PKD. To characterize the phenotype caused by the transgene expression, gross and histologic examinations were undertaken on transgenic kidneys. Adult kidneys from all transgenic lines were affected bilaterally. Kidneys contained numerous cortical cysts that varied from microscopic to macroscopic in size (Fig. 4a and b). SBPkd1TAG kidneys were pale, a typical finding in PKD. On histologic examination, all transgenic founder mice and progenies (n = 25; n > 6 for each line) developed multiple tubular (T) and glomerular cysts (G) (Fig. 4d, f, and g). Cysts were observed in tubules from the cortical and medullary regions as well as collecting tubules from the papilla (Fig. 4d and e). Transgenic mice displayed tubular epithelial hyperplasia (arrowhead) involving both cystic and noncystic tubules and frequent hypertrophy (Fig. 4g and h), but the severity varied between individual mice. Interstitial fibrosis (F), perivascular lymphoid infiltrates, and proteinaceous casts (P) were frequently observed (Fig. 4d and e).


Figure 4
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  		FIG. 4. Renal PKD phenotype in SBPkd1TAG mice. (a) Laparotomy of a control mouse (4 months) that exhibited healthy kidneys with normal vascularization. (b) Laparotomy of a 4.5-month-old SBPkd1TAG mouse showed that both kidneys are pale and studded with numerous cysts on the cortical surfaces. (c) Overview of renal cortical sections from adult control that displayed normal tubules (T) and glomeruli (G). High-power views of glomeruli and tubule are depicted in the inset (hematoxylin and eosin, x100; inset, x200). (d) Overview of renal cortical sections from an SBPkd1TAG mouse showed several scattered tubular cysts (T) and glomerular cysts (G). Noticeably, the presence of interstitial fibrosis (F) is frequently observed (hematoxylin and eosin, x100). (e) Renal section of an SBPkd1TAG mouse displaying a papilla with the presence of elongated dilated collecting tubules (T) associated with proteinaceous casts (P) (hematoxylin and eosin, x50). (f) Renal section of an SBPkd1TAG mouse showing the presence of several tubular cysts (T) (hematoxylin and eosin, x200). (g) Renal section of an SBPkd1TAG mouse that developed multiple glomerular cysts (G) associated with epithelial hypertrophy (arrowhead) (hematoxylin and eosin, x200). (h) Cysts from SBPkd1TAG mice are frequently lined with epithelial hyperplasia and hypertrophy (arrowhead) (hematoxylin and eosin, x200). (i) In situ hybridization control of transgenic SBPkd1TAG mouse kidney treated with RNase. No signal is detected over normal or cystic tubules and glomeruli (x200). (j) Detection of Pkd1 expression by in situ hybridization in transgenic SBPkd1TAG kidney tissue. Intense signal is present over the epithelium of cystic tubules and glomeruli as well as in slightly dilated tubules (x200). (k) Newborn SBPkd1TAG mouse displayed scattered tubular dilatations in all sections of the kidneys and also glomerular dilatations due to expansion of Bowman's space (hematoxylin and eosin, x50). (l) Higher-power view of newborn renal cortex of an SBPkd1TAG mouse with highlighted glomerular and tubular dilatations (hematoxylin and eosin, x200).

To more precisely define the localization site of increased Pkd1 expression in the kidneys, we carried out in situ hybridization using the exon 36-45 probe previously used (16). The hybridization signal was localized specifically to the epithelial cells lining cyst and hyperplastic tubules as well as glomerular cysts. In addition, some signal was seen over the epithelium of noncystic or slightly dilated tubules, likely identifying tubules predestined to undergo future cystic changes (Fig. 4i and j).

Renal histologic analysis was also carried out on transgenic mice at birth (n = 8), postnatal day 10 (P10) (n = 3), P20 (n = 5), P35 (n = 3), and P45 (n = 3) in comparison to negative littermates of the same age group (n = 2 to 4). Interestingly, all newborn transgenic mice displayed tubular and glomerular dilatation relative to control negative littermates (Fig. 4k and l), indicating that renal anomalies initiated in utero as observed in SBM mice and in ADPKD patients. The tubular and glomerular dilatation increased in size and number with progressive age. By P35, transgenic mice displayed more severe hyperplasia and evidence of glomerulosclerosis.

Altered renal physiological functions in SBPkd1TAG mice. Renal physiologic functions of all transgenic mice displayed features similar to PKD, while the nontransgenic littermates never developed the disease. Within a few months after birth, the affected animals developed chronic renal insufficiency. These animals were monitored for renal functional parameters by measurement of serum and urinary levels, blood urea nitrogen (BUN) and creatinine, urine osmolality, urine protein, and ion excretion (Table 1). All mice from the three lines compared to controls displayed concentrating defects, a common finding in ADPKD, and consequently showed decreased urinary BUN, creatinine, protein, and ion concentrations. Transgenic SBPkd1TAG founders and progenies (n = 6) from each line were monitored qualitatively for proteinuria on urine samples by SDS-PAGE (Fig. 5). Mice older than 2 months displayed nonselective proteinuria that progressed with age. In addition, levels of the serum BUN and serum creatinine were increased, revealing renal insufficiency (Table 2). Because chronic renal insufficiency commonly leads to alterations in hematologic parameters, these were examined in SBPkd1TAG transgenic mice of 3 to 14 months of age (Table 2). These transgenic mice were anemic as evidenced by the significantly decreased red blood cell count, with hemoglobin and hematocrit reaching half the normal levels. Other red blood cell parameters, like the percentage of reticulocytes, were unaffected, as expected when induced by a renal defect. These animals consistently died of renal failure at ~5.9 ± 2.8 months of age (n = 42) for transgenic line 39 and at later ages, ~14.6 ± 3.1 months (n = 20) and ~11.7 ± 6.5 months (n = 7), for lines 3 and 41, respectively.


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  		TABLE 1. Evidence of altered renal function in SBPkd1TAG mice by urine analysisb


Figure 5
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  		FIG. 5. Qualitative analysis of urinary proteins by SDS-PAGE. Urinary protein samples from the three transgenic mouse lines 3 (n = 1), 39 (n = 2), and 41 (n = 2) of different ages (mouse age) were analyzed in comparison to aliquots from negative control mice (F1) of the same genetic background (C57BL/6J x CBA/J)F1 used to produce the transgenic mice and from an SBM transgenic mice, a PKD mouse model, as a positive control for proteinuria. Proteins from normal control mouse serum (S) served to compare the protein distribution obtained with urine samples. Urine from all mice showed low-molecular-weight bands (bottom arrow) that represent the normally excreted major urinary proteins (MUPs). The SBPkd1TAG transgenic mice from all lines displayed nonselective protein spillage, mainly albumin (top arrow). The albumin levels in some mice appeared comparable to those of SBM transgenic mice that exhibit renal insufficiency. M, molecular mass markers of 14.1 to 200 kDa.


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  		TABLE 2. Evidence of chronic renal insufficiency in SBPkd1TAG mice by hematologic analysish


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DISCUSSION
 
Herein, we report the isolation and characterization of a murine Pkd1-BAC. This Pkd1 gene was tagged and regulatory elements were replaced to target expression specifically to the kidneys by two successive homologous recombination events. Transgenic mice produced with this novel SBPkd1TAG gene showed a 2- to 15-fold increase in Pkd1 expression and reproducibly developed early renal morphological alterations typical of PKD. Renal insufficiency is apparent in middle age, and mice die prematurely of renal failure. Our results also indicate that the Pkd1 overexpression mechanism responsible for this phenotype is mediated by signaling activation of c-myc in vivo. This study demonstrates that the murine Pkd1 gain of function in the kidneys is sufficient to produce a PKD renal phenotype.

Since the murine Pkd1 gene is not duplicated as it is in humans (27), we have directly identified and isolated a BAC clone that contained the entire Pkd1 gene. Complete characterization of the 129/Sv murine Pkd1-BAC, in direct comparison with two other inbred mouse strains, confirmed the integrity of the Pkd1 locus. The Pkd1-BAC insert contained ~37 to 39 kb of upstream and downstream sequences from the Pkd1 gene. Our analysis demonstrated that the Pkd1 gene in this BAC was a bona fide murine wild-type locus that could serve for further studies.

Although there is strong evidence that cyst formation in ADPKD can result from loss of heterozygosity following somatic inactivation of the normal PKD1 allele (3, 21, 32), there is also suggestive evidence for sustained or even increased polycystin-1 expression in cystic tubular epithelium (22, 29). The latter observation raises the question of whether overexpression of Pkd1 per se is a sufficient proximate cause of cystogenesis. In transgenic mice bearing the human PKD1, TSC2, RAB 26, NTHL1, and SLC9A3R2 genes, only a minority of mice developed cysts and none had detectable transgene expression in adulthood despite 30 copies of the transgene (31). In those transgenic mice, it was difficult to establish a clear role for Pkd1 overexpression in cystogenesis. Our model differs, as two to nine wild-type copies of Pkd1 alone, without contiguous genes, were integrated in transgenic mice. Since the Pkd1 gene has essential functions in various organs or tissues, as described for numerous mice with ablation of the Pkd1 gene, a systemic overexpression of Pkd1 could lead to additional confounding effects. Consequently, we have addressed the role of Pkd1 gain of function using an approach that targets Pkd1 specifically to the kidneys. By homologous recombination, we have first substituted the Pkd1 upstream regulatory region with the "SB" renal restricted regulatory elements, thereby preventing the decreased gene expression normally seen for Pkd1 in adulthood as well as potential secondary feedback loop regulation (36, 38). Second, we have marked the murine Pkd1 transgene (Pkd1TAG) with a silent point mutation in exon 10 but did not insert an epitope tag to ensure that a fully functional "wild-type" protein with conserved structure and integrity would be produced. From this modified BAC, an SBPkd1TAG fragment was purified away from the Tsc2 gene and BAC vector to prevent interference by the Tsc2 gene, which can also induce a cystic phenotype (8, 20, 28), as well as to avoid the inhibitory effect of prokaryotic sequences (5).

Four different SBPkd1TAG transgenic founder mice and three independent lines were produced with specific renal Pkd1-enhanced expression. Particularly striking is the complete penetrance of the phenotype in these transgenic mice. The SBPkd1TAG founder and mouse lines shared several physiopathologic features in common with ADPKD. These include the development of cysts in cortex, medulla, and glomeruli together with epithelial hyperplasia, interstitial fibrosis, and focal interstitial inflammation.

Because the PKD phenotype was consistently observed in all different transgenic founder mice and the transgene integration into the mouse genome is a random phenomenon, the phenotype cannot result from chromosomal position effect but only from increased Pkd1 expression. Indeed, expression of the Pkd1 transgene in all lines was demonstrated to be renal restricted, as previously observed for other transgenes regulated by the "SB" elements (36, 38). Moreover, this increased Pkd1 expression was caused by the transgene and not by an indirect endogenous Pkd1 activation. Hence, our results provide clear evidence that gain of function of a wild-type functional Pkd1 can produce multiple renal cysts. Importantly, these SBPkd1TAG mice constitute the first mouse model generated by the sole overexpression of the mouse orthologue of the human PKD1 gene.

The SBPkd1TAG mice demonstrate that Pkd1 overexpression is a primary pathogenetic mechanism of renal cystogenesis. Importantly, the highest transgene expression levels in kidneys appeared to correlate with the progression and severity of the phenotype. We also found that Pkd1 overexpression in the development of SBPkd1TAG phenotype is likely to signal activation of c-myc in vivo. Conceivably, this activation could even be direct through the polycystin-1 C-terminal tail undergoing proteolytic cleavage and nuclear translocation (7). Since enhanced renal expression of c-myc in adult mice was shown to induce PKD, it would be highly consistent to support c-myc as a major downstream effector of Pkd1 signaling pathways. This result also correlated with our previous findings of increased c-myc expression in kidneys of all human ADPKD analyzed (22). Altogether, these results indicate that c-myc is a prime mediator of Pkd1 cystogenesis.

Our results from the Pkd1 gain-of-function model, together with murine Pkd1 haploinsufficiency and loss of function, indicate that any Pkd1 dysregulation could lead to cystogenesis (2, 19, 23-26, 31, 40). Severe Pkd1 imbalance in mice induced by Pkd1 ablation or transgenic overexpression caused early onset and rapid progression of renal cysts and affected a high proportion of tubules. By contrast, a milder Pkd1 imbalance such as a haploinsufficiency led to a slower progression of PKD with more focal cysts. The apparent paradoxical development of a similar phenotype by means of opposite polycystin-1 dysregulation could be explained by the common result, namely a relative protein concentration imbalance that could alter the formation orthe function of an active polycystin multiprotein complex. Taken together, our results and those of other investigators argue that the mechanism of cyst formation in ADPKD is likely to arise from three pathogenetic mechanisms: gain of function, loss of function, and gene dosage effects.

The novel SBPkd1TAG mice constitute a powerful model of renal cystogenesis that can provide major insights into the pathophysiology of PKD, Pkd1 signal transduction pathways, and interacting partners. Study of this model may also lead to the development of new therapeutic strategies to restore normal protein balance within the Pkd1 multimeric complex.


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ACKNOWLEDGMENTS
 
We thank V. D'Agati and M. Aubry for critical reading of the manuscript and N. Heintz for providing the pLD53 vector.

This work was supported by a Canadian Institute of Health Research (CIHR) grant to M.T. and a studentship from the CIHR and FRSQ to M.C.


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FOOTNOTES
 
* Corresponding author. Mailing address: Molecular Genetics and Development, IRCM, 110 ave. des Pins ouest, Montreal, Quebec, Canada H2W 1R7. Phone: (514) 987-5712. Fax: (514) 987-5585. E-mail: trudelm{at}ircm.qc.ca. Back


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Molecular and Cellular Biology, February 2006, p. 1538-1548, Vol. 26, No. 4
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