Part Ⅱ：Overexpression Of PKD1 Causes Polycystic Kidney Disease

Contact: Audrey Hu Whatsapp/hp: 0086 13880143964 Email: audrey.hu@wecistanche.com

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

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 (polycystic kidney disease 1) bacterial artificial chromosome PKD1 (polycystic kidney disease 1)-BAC) was modified by homologous recombination to solely target a sustained PKD1 (polycystic kidney disease 1) expression preferentially to the adult kidney. Several transgenic lines were generated that specifically overexpressed the PKD1 (polycystic kidney disease 1) transgene in the kidneys 2- to 15-fold over PKD1 (polycystic kidney disease 1) 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 (polycystic kidney disease 1) 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 a critical in vivo downstream effector of the PKD1 (polycystic kidney disease 1) molecular pathway. 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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RESULTS

Production of PKD1 (polycystic kidney disease 1)-Ac-BAC by homologous recombination. To determine whether PKD1 (polycystic kidney disease 1) 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 PKD1 (polycystic kidney disease 1) gene in a BAC vector 129/Sv library. This library was screened by PCR with two sets of primers for the PKD1 (polycystic kidney disease 1) 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 (polycystic kidney disease 1) gene was identified that included the entire adjacent Tsc2 gene body. The PKD1 (polycystic kidney disease 1) 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 (polycystic kidney disease 1) 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 (polycystic kidney disease 1)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.

FIG. 2. Production of SBPkdlrAc constructs and transgenic mice. (a)Successive homologous recombination events were carried out on the murine Pkdl-BAC to introduce two modifications: the SB regulatory elements inserted immediately upstream of the Pkdl initiation codon and a silent point mutation of EcoRI (RI*)introduced in exon(ex)10. The BAC recombination vector contains an R6Ky origin of replication, an ampicillin-resistant gene, a SacB gene, a RecA gene, and a unique Smal 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 Pkdl-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 Pkdl regions of the BAC cointegrates were eliminated in a second selection step. Resolved Pkdl-BACs can either revert to wild type or include the intended modification. Subsequent homologous recombination into the newly modified BAC can then be introduced.(b)(ienomc DNA ot SBPkd l-etransgenic mxce was anakZed bv Sou thern blot.AVicToiD1ecLon 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 Pkdl probe (exon 7-15)is shown (middle panel).

This SBPKD1 (polycystic kidney disease 1)-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 (polycystic kidney disease 1)-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-trans-gene to restrict expression to the kidneys(36, 38)(Fig. 2a).

This new SBPkdlrAG-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 T'sc2 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).

CISTANCHE BENEFIT: TREATING KIDNEY DISEASES

Production and analysis of SBPkdl-ac transgenic mice. Four transgenic founders carrying several copies of the SBPkdlrAc transgene consistently developed PKD. From the four SBPKD1 (polycystic kidney disease 1)RAC founder mice determined by Southern analysis, three SBPKD1 (polycystic kidney disease 1)TAG 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 SBPkdlrAc trans-gene 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 (polycystic kidney disease 1) 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 SBPkdl-Ac transgenic mice. Expression of the SBPkdl-AG. transgene and PKD1 (polycystic kidney disease 1) 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 SBPkd 1 A mouse lines had consistently increased transcript expression compared to normal PKD1 (polycystic kidney disease 1) 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 (polycystic kidney disease 1) levels (Fig.3a).Particularly,transgenic line39(n = 4)showed higher PKD1 (polycystic kidney disease 1) levels than lines 3 (n = 3)and 41(n = 4).Furthermore, PKD1 (polycystic kidney disease 1)expression levels measured by Northern blot analysis correlated with those obtained by real-time PCR using primers in exons 1 and 2(Fig. 3b).

FIG.3. Renal expression analysis of SBPkdlrA mice. (a) Expression analysis of Pkd1 endogenous(endo) and SBPkdlrA 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 Pkdl transcript of normal control age-matched mice from the same genetic background (C5BI 6I ×CBA/DE, Kidney RNA samples were obtained from transgenic mice prior to end-stage renal disease. Transcripts from endo and Tg are both ～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 Pkd1endogenous levels from control mice arbitrarily set at 1. (b) Schematic representation of SBPkdl-。 transgene and primers were used to amplify total Pkdl 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 SBPkdlAc transgene expression was quantified in renal and extrarenal tissues. A representative semiquantitative evaluation of SBPkdl.Ac 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 SBPkd1rAc transgene produced a specific 307-bp amplicon, whereas the S16 internal control generated a 102-bp amplicon. M,100-bp marker; H, O, negative control for PCR amplification. (c)Real-time PCR expression analysis of the SBPkdlr. The transgene was determined from several independent mice. The SBPkdl.Ac 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 SBPkdlrAc 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 the brain, heart, liver, pancreas, spleen, and lung. (d)Expression of the endogenous c-mc gene in the SBPkdl.Ac 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-Mye is detected in all adult SBPkdlrAc kidneys of the three lines, as observed in the adult transgenic SBM kidneys used as a 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, β-globin promoter) and in exon 2 of PKD1 (polycystic kidney disease 1)(Fig.3b). The SBPKD1 (polycystic kidney disease 1) RAC 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 (polycystic kidney disease 1) transgene expression and for renal PKD1 (polycystic kidney disease 1)total(transgene and endogenous)expression by real-time PCR. Interestingly. lines 39 and 41 relative to line 3 showed that the increased PKD1 (polycystic kidney disease 1)transgene renal expression was similar to or above that of PKD1 (polycystic kidney disease 1)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 SBPKD1 (polycystic kidney disease 1) RAC; 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 1,000-fold lower level of the transgene expression in extrarenal tissues relative to kidney expression (Fig. 3c). The"SB" regulatory elements of the SBPkdlrAc 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-Mye, a downstream effector of PKD1 (polycystic kidney disease 1) signaling pathways in SBPkdlrAc: mice. To gain insight into the intracellular pathogenetic mechanism of SBPkdlrAc 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-Mye induced in SBPkdlrAa mice relative to control mice of similar age. Interestingly, the level of c-Myc expression in some SBPkdlrAG 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 SBPKD1 (polycystic kidney disease 1) ARC 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). SBPkdlrAc. 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)

Fig. 4

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 1), 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 SBPkdlrsc 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 (autosomal dominant polycystic kidney disease). and consequently showed decreased urinary BUN. creatinine, protein, and iron concentrations. Transgenic SBPKD1 (polycystic kidney disease 1)rAc.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 SBPkdlAc transgenic mice of3 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 trans-genic 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.

EFFECTS OF CISTANCHE: TREATING KIDNEY DISEASES

DISCUSSION

Herein we report the isolation and characterization of a murine PKD1 (polycystic kidney disease 1)-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 SBPkdlrAG gene showed a 2-to 15-fold increase in PKD1 (polycystic kidney disease 1)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 (polycystic kidney disease 1) overexpression mechanism responsible for this phenotype is mediated by signaling activation of c-Myc in vivo. This study demonstrates that the murine PKD1 (polycystic kidney disease 1) gain of function in the kidneys is sufficient to produce a PKD renal phenotype.

Since the murine PKD1 (polycystic kidney disease 1) gene is not duplicated as it is in humans (27), we have directly identified and isolated a BAC clone that contained the entire PKD1 (polycystic kidney disease 1) gene. Complete characterization of the 129/Sv murine PKD1 (polycystic kidney disease 1)-BAC. indirect comparison with two other inbred mouse strains confirmed the integrity of the PKD1 (polycystic kidney disease 1)locus. The PKD1 (polycystic kidney disease 1)-BACinsert contained～37 to 39 kb of upstream and downstream sequences from the PKD1 (polycystic kidney disease 1) gene. Our analysis demonstrated that the PKD1 (polycystic kidney disease 1) 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 (polycystic kidney disease 1) per se is a sufficient proximate cause of cystogenesis. In transgenic mice bearing the human PKD1 (polycystic kidney disease 1), TSC2. RAB26. 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 (polycystic kidney disease 1) overexpression in cystogenesis. Our model differs, as two to nine wild-type copies of PKD1 (polycystic kidney disease 1)alone, without contiguous genes, were integrated in transgenic mice. Since the PKD1 (polycystic kidney disease 1)gene has essential functions in various organs or tissues, as described for numerous mice with ablation of the PKD1 (polycystic kidney disease 1)gene, a systemic overexpression of PKD1 (polycystic kidney disease 1) could lead to additional confounding effects. Consequently, we have addressed the role of PKD1 (polycystic kidney disease 1)gain of function using an approach that targets PKD1 (polycystic kidney disease 1) specifically to the kidneys. By homologous recombination, we have first substituted the PKD1 (polycystic kidney disease 1) upstream regulatory region with the"SB" renal restricted regulatory elements, thereby preventing the decreased gene expression normally seen for PKD1 (polycystic kidney disease 1)in adulthood as well as potential secondary feedback loop regulation(36,38). Second, we have marked the murine PKD1 (polycystic kidney disease 1) transgene(Pkdlr)with a silent point mutation in exon 10but 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 SBPkdlrAG 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 SBPkdlrActransgenic 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 SBPKD1 (polycystic kidney disease 1) rAc 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 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 (polycystic kidney disease 1)expression. Indeed, expression of the PKD1 (polycystic kidney disease 1) 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 (polycystic kidney disease 1) expression was caused by the transgene and not by an indirect endogenous PKD1 (polycystic kidney disease 1) activation. Hence, our results provide clear evidence that the gain of function of a wild-type functional PKD1 (polycystic kidney disease 1) can produce multiple renal cysts. Importantly, these SBPKD1 (polycystic kidney disease 1) practices constitute the first mouse model generated by the sole overexpression of the mouse orthologue of the human PKD1 (polycystic kidney disease 1) gene.

The SBPkdl-Ac 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 (polycystic kidney disease 1) overexpression in the development of SBPKD1 (polycystic kidney disease 1)-Ac 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 under-going 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 (polycystic kidney disease 1) 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-Mye is a prime mediator of PKD1 (polycystic kidney disease 1)cystogenesis.

Our results from the PKD1 (polycystic kidney disease 1)gain-of-function model, together with murine PKD1 (polycystic kidney disease 1) haploinsufficiency and loss of function, indicate that any PKD1 (polycystic kidney disease 1) 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 (polycystic kidney disease 1) 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 SBPkdlrAc; mice constitute a powerful model of renal cystogenesis that can provide major insights into the pathophysiology of PKD, 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 (polycystic kidney disease 1) multimeric complex.

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