The Cause Of Polycystic Kidney Disease

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Overexpression of PKD1 Causes Polycystic Kidney Disease

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

The pathogenetic mechanisms underlying autosomal dominant polycystic kidney disease (ADPKD) remain to be elucidated. While there is evidence that Pkd1(polycystic kidney disease 1) gene haploinsufficiency and loss of heterozygosity can cause cyst formation in mice, paradoxically high levels of Pkd1(polycystic kidney disease 1) expression have been detected in the kidneys of ADPKD (autosomal dominant polycystic kidney disease) patients. To determine whether Pkd1(polycystic kidney disease 1) 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 Pkdl transgene in the kidneys 2- to 15-fold over Pkd1 endogenous levels. All transgenic mice reproducibly developed tubular and glomerular costs 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 (autosomal dominant polycystic kidney disease). Our results also uncovered a striking increased renal c-myc expression in mice from all transgenic lines, indicating that c-myc is critical in vivo downstream effector of Pkd1(polycystic kidney disease 1) molecular Dathwavs, This study not only produced an invaluable and first PKD(polycystic kidney disease) model to evaluate molecular pathogenesis and therapies but also provides evidence that gain of function could be a pathogenetic mechanism in ADPKD (autosomal dominant polycystic kidney disease).

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

Approximately 85% of ADPKD (autosomal dominant polycystic kidney disease) cases are associated with mutations in the PKD1(polycystic kidney disease 1) gene. This PKD1(polycystic kidney disease 1) gene is large, spans 54 kb, and consists of 46 exons. It generates a 14.2-kb transcript and encodes a 4., a 302-amino-acid protein called polycystin-1(4, 9-11). Human PKD1 and polycystin-1 expression have been analyzed in normal and ADPKD (autosomal dominant polycystic kidney disease) 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(polycystic kidney disease 1) RNA and protein are expressed at moderate to low levels in the collecting and distal tubules, whereas levels increased (~2-fold) in ADPKD (autosomal dominant polycystic kidney disease) 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(polycystic kidney disease 1) 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(polycystic kidney disease 1) 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 the inactivation of one allele.

However, a significant proportion is a 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 (autosomal dominant polycystic kidney disease) is dominant and the transmitted mutated PKD1(polycystic kidney disease 1) allele is sufficient to produce the disease. However, the focal nature of the renal cysts in ADPKD (autosomal dominant polycystic kidney disease) suggests that the mutational mechanism for PKD1(polycystic kidney disease 1) could be a two-hit or a loss of heterozygosity. Support for this mechanism was obtained by the detection of PKD1(polycystic kidney disease 1) 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(polycystic kidney disease 1) 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 the 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 germline 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 mechanism for ADPKD (autosomal dominant polycystic kidney disease) pathogenesis. Indeed, these mechanisms are at variance with the persistent or enhanced expression of PKD1(polycystic kidney disease 1) 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(polycystic kidney disease 1) 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(polycystic kidney disease 1) gene.

MATERIALS AND METHODS

Isolation of BAC clones containing the Pkd1(polycystic kidney disease 1) 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 super pools was performed by PCR with the following primers: 5 regions, 5-CTG ATGAGTTCTGGCCATGGATG-3 (forward Pkd1 exon 1) and 5-CTGCCA GCCAATGCCATAGTCAC-3 (reverse Pkd1 exon 1); and 3 regions, 5-TCG GCCCTAGCGTCTGCAGCC-3 (forward Pkd1 exon 39) and 5-TCCAGTCC CACCTACAGCCAAC-3 (reverse Pkd1 exon 40). One positive clone for both amplification 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.

RESULTS

Production of SBPkdl(polycystic kidney disease 1) rAc-BAC by homologous recombination. To determine whether Pkd1 gain of function alone is sufficient to produce the ADPKD (autosomal dominant polycystic kidney disease) phenotype, we first isolated a genomic clone containing the entire Pkdl 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(polycystic kidney disease 1) 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 BACand in these inbred strains by Southern blot analysis, with four restriction enzyme digestions and seven probes covering the entire Pkdl 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 a downstream sequence of the Pkd1(polycystic kidney disease 1) gene as determined by electrophoresis and sequencing.

This Pkd1-BAC clone was modified by two successive homologous recombination events in E.coli. The Pkd1(polycystic kidney disease 1) 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(polycystic kidney disease 1) gene and transcript of the BAC from that of endogenous origin. In addition, we have replaced the 5'regulatory elements of the Pkd1-BACgene 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-trans-gene to restrict expression to the kidneys (36,38)(Fig. 2a).

This new SBPkd1TAc-BAC was digested with Notl, 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 BACvector sequences(Fig.1 and 2). This 70-kb Notl-Clal linearized fragment was isolated, purified, and quantified for oocyte microinjection (36).

Production and analysis of SBPkd1rAc transgenic mice. Four transgenic founders carrying several copies of the SBPkd1rAc transgene consistently developed PKD(polycystic kidney disease). From the four SBPkdlrAc founder mice determined by Southern analysis, three SBPkd1rAG 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 SBPkd1rAc trans-gene being integrated into 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(polycystic kidney disease 1) gain of function in adult SBPkdlrAc transgenic mice. Expression of the SBPkdlrAG 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 Pkdl 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(polycystic kidney disease 1) 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).

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,β-globin promoter) and in exon 2 of Pkd1(polycystic kidney disease 1) (Fig.3b). The SBPkdlrAc 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. The highest levels of Pkd1(polycystic kidney disease 1) transgene renal expression was measured for lines 39 and 41. To monitor whether the increased Pkd1(polycystic kidney disease 1) 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 Pkdl 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 SBPkd1rAg transgene showed very weak expression occasion-ally detected in spleen and lung, with little to undetectable expression in other organs (Fig. 3b). Quantification by real-time PCR demonstrated a 10- to a 1,000-fold lower level of the transgene expression in extrarenal tissues relative to kidney expression(Fig. 3c). The"SB" regulatory elements of the SBPkdlrA 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(polycystic kidney disease 1) signaling pathways in SBPkdlrAc mice. To gain insight into the intracellular pathogenetic mechanism of SBPkdlrAg transgenic mice, we next sought to monitor c-myc renal expression level based on our previous observation of c-myc deregulation in human ADPKD (autosomal dominant polycystic kidney disease) 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 SBPkd1-c 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 SBPkd1raG mice similar to PKD(polycystic kidney disease). 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). SBPkdl-AG 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 non-cystic 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).

To more precisely define the localization site of increased Pkd1(polycystic kidney disease 1) 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 (autosomal dominant polycystic kidney disease) 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(polycystic kidney disease), 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 iron 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.

FIG. 1. Schematic representation and detailed restriction map analysis of a murine Pkd1-BAC.

Genomic DNA digestion patterns of the Pkd1(polycystic kidney disease 1)-BAC were compared to that of the Pkd1(polycystic kidney disease 1) 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(polycystic kidney disease 1) 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(polycystic kidney disease 1) loci showed identical patterns with all seven probes. M, HindIII marker; 129, 129/Sv; C57, C57Bl/6J.

DISCUSSION

Herein, we report the isolation and characterization of a murine Pkd1-BAC. This Pkd1(polycystic kidney disease 1) 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, indirect 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 (autosomal dominant polycystic kidney disease) can result from loss of heterozygosity following somatic inactivation of the normal PKD1(polycystic kidney disease 1) allele (3, 21, 32), there is also suggestive evidence for sustained or even increased polycystin-1 expression in the 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(polycystic kidney disease 1), 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, 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 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(polycystic kidney disease 1)-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 (autosomal dominant polycystic kidney disease). These include the development of cysts in the cortex, medulla, and glomeruli together with epithelial hyperplasia, interstitial fibrosis, and focal interstitial inflammation.

Because the PKD(polycystic kidney disease) 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 the 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(polycystic kidney disease 1) 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 the 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 (autosomal dominant polycystic kidney disease) analyzed (22). Altogether, these results indicate that c-myc is a prime mediator of Pkd1(polycystic kidney disease 1) 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(polycystic kidney disease 1) imbalance in mice induced by Pkd1(polycystic kidney disease 1) 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 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 or the 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 (autosomal dominant polycystic kidney disease) 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(polycystic kidney disease), Pkd1(polycystic kidney disease 1) signal transduction pathways, and interacting partners. The study of this model may also lead to the development of new therapeutic strategies to restore normal protein balance within the Pkd1 multimeric complex.

REFERENCES

1. Blouin, M.J., H.Beauchemin, A.Wright, M.E.De Paepe, M.Sorette, A.-M. Bleau.B. Nakamoto. C.-N, Ou, G. Stamatovannopoulos,and M. Trudel 2000. Genetic correction of sickle cell disease: insights using transgenic mouse models. Nat. Med. 6:177-182.

2. Boulter, C., S.Mulroy, S.Webb, S. Fleming, K. Brindle, and R. Sandford. 2001.Cardiovascular, skeletal, and renal defects in mice with a targeted disruption of the Pkd1(polycystic kidney disease 1) gene.Proc. Natl. Acad. Sci. USA 98:12174-12179.

3. Brasier, J.L, and E.P.Henske.1997.Loss of the polycystic kidney disease (PKD1)region of chromosome 16p13 in renal cyst cells supports a loss-of-function model for cyst pathogenesis. J. Clin. Investig.99:194-199.

4. Burn, T. C., T.D. Connors, W. R.Dackowski, L.R.Petry, T.J.Van Raay, J.M. Millholland, M. Venet, G.Miller, R. M. Hakim, G. M.Landes, K.W. Klinger, F. Qian, L.F. Onuchic, T.Watnick, G. G. Germino, and N.A. Doggett.1995.Analysis of the genomic sequence for the autosomal dominant polycystic kidney disease(PKD1) gene predicts the presence of a leucine-rich repeat. Hum. Mol. Genet.4:575-582.

5.Chada,K, J.Magram, K. Raphael,G.Radice, E.Lacy, and F.Costantini.1985. Specific expression of a foreign β-globin gene in erythroid cells of transgenic mice. Nature 314:377-380.

6. Chauvet, V, F. Qian, N. Boute, Y. Cai, B.Phakdeekitacharoen, L. F. Onuchic, T.Attie-Bitach, L.Guicharnaud, O.Devuyst,G.G. Germino,and M.-C. Gubler.2002. Expression of PKD1(polycystic kidney disease 1) and PKD2(polycystic kidney disease 2) transcripts and proteins in the human embryo and during normal kidney development. Am. J. Pathol 160:973-983.

7.Chauvet, V., X. Tian, H.Husson, D.H.Grimm,T.Wang, T.Hiesberger, P. Igarashi, A. M. Bennett, O. Ibraghimov-Beskrovnava, S. Somlo,and M.J. Caplan.2004. Mechanical stimuli induce cleavage and nuclear translocation of the polycystin-1 C terminus. J. Clin. Investig. 114:1433-1443.

8. Cheadle,J.P. M.P. Reeve,J.R. Sampson, and D.J. Kwiatkowski.2000. Molecular genetic advances in tuberous sclerosis. Hum. Genet.107:97-114. 9. Consortium, E.P. K. D.1993.Identification and characterization of the tuberous sclerosis gene on chromosome 16. Cell 75:1305-1315.

10. Consortium, E. P. K. D.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.

11. Consortium, I.P. K. D.1995. Polycystic kidney disease: the complete structure of the PKD1(polycystic kidney disease 1) gene and its protein. Cell 81:289-298.

12.Couillard, M., R. Guillaume,N. Tanji, V. DAgati, and M. Trudel.2002. c-myc-Induced apoptosis in polycystic kidney disease is independent of FasL Fas interaction. Cancer Res. 62:2210-2214.

13. De Paepe, M. E., and M. Trudel.1994. The transgenic SAD mouse: a model of human sickle cell glomerulopathy. Kidney Int. 46:1337-1345.

14. Geng, L., Y. Segal, B.Peissel, N.Deng, Y. Pei,F. Carone,H.G.Rennke, A.M. Glücksmann-Kuis, M. C. Schneider, M. Ericsson, S.T.Reeders, and J.Zhou. 1996.Identification and localization of polycystin, the PKD1(polycystic kidney disease 1) gene product. J. Clin. Investig. 98:2674-2682.

15. Gong, S., X. W.Yang, C. Li, and N.Heintz.2002.Highly efficient modification of bacterial artificial chromosomes (BACs)using novel shuttle vectors containing the R6Ky origin of replication. Genome Res. 12;1992-1998.