Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on October 11, 2007
Human Molecular Genetics 2007 16(24):3188-3196; doi:10.1093/hmg/ddm299

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Kidney-specific inactivation of the Pkd1 gene induces rapid cyst formation in developing kidneys and a slow onset of disease in adult mice

Irma S. Lantinga-van Leeuwen¹, Wouter N. Leonhard¹, Annemieke van der Wal², Martijn H. Breuning¹, Emile de Heer² and Dorien J.M. Peters^1,*

¹ Department of Human Genetics and ² Department of Pathology, Leiden University Medical Center, Leiden 2300RC, The Netherlands

^* To whom correspondence should be addressed. Tel: +31 715269490; Fax: +31 715268285; Email: d.j.m.peters{at}lumc.nl

Received August 14, 2007; Accepted October 8, 2007

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
Autosomal dominant polycystic kidney disease, caused by mutations in the PKD1 gene, is characterized by progressive deterioration of kidney function due to the formation of thousands of cysts leading to kidney failure in mid-life or later. How cysts develop and grow is currently unknown, although extensive research revealed a plethora of cellular changes in cyst lining cells. We have constructed a tamoxifen-inducible, kidney epithelium-specific Pkd1-deletion mouse model. Upon administration of tamoxifen to these mice, a genomic fragment containing exons 2–11 of the Pkd1-gene is specifically deleted in the kidneys and cysts are formed. Interestingly, the timing of Pkd1-deletion has strong effects on the phenotype. At 1 month upon gene disruption, adult mice develop only a very mild cystic phenotype showing some small cysts and dilated tubules. Young mice, however, show massive cyst formation. In these mice, at the moment of gene disruption, cell proliferation takes place to elongate the nephron. Our data indicate that Pkd1 gene deficiency does not initiate sufficient autonomous cell proliferation leading to cyst formation and that additional stimuli are required. Furthermore, we show that one germ-line mutation of Pkd1 is already associated with increased proliferation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disease characterized by progressive development of fluid-filled cysts in both kidneys. The formation of numerous cysts together with interstitial fibrosis usually causes chronic renal failure in 50% of patients by the age of 60 years (1). The kidney is the most severely affected organ, but the disease is systemic and extra-renal manifestations such as cyst formation in liver and pancreas, hypertension and cerebral aneurysms also can occur (2).

The majority of patients, 85%, carry a mutation in the PKD1 gene (3). The protein encoded by the PKD1 gene, polycystin-1, forms multiprotein complexes at the cell membrane and functions in cell–cell and cell–matrix interactions, signal transduction and in mechanosensation (4). The C-terminal region of polycystin-1 has been shown to interact with the PKD2 gene product, polycystin-2, which is mutated in 15% of the ADPKD patients, and functions as a Ca²⁺-permeable cation channel (5–7). Both polycystins have been found in primary cilia. Several proteins in this cell organelle have been reported to be associated with cystic diseases and are probably participating in sensing fluid flow or shear stress (8).

ADPKD patients carry one germ-line mutation and one normal PKD1 allele. Two genetic mechanisms for cyst formation have been postulated, e.g. somatic inactivation of PKD1 or PKD2 by a ‘two-hit’ mechanism has been shown for cysts in PKD1 and PKD2 kidneys (9,10) but reduced levels of polycystins may already be sufficient to cause polycystic kidney disease (11,12). In addition, cysts formation in mice overexpressing Pkd1 suggest that a balanced polycystin-1 and -2 dosage seem to be critical to maintain an intact renal epithelial architecture (13,14).

A plethora of cellular changes in cyst-lining cells have been observed, but the precise mechanisms of how cysts arise and grow are still not well understood. To study the pathogenesis of ADPKD several mouse models have now been generated. Homozygous Pkd1 and Pkd2 knock-out mice die at embryonic stages, around E15.5, or directly after birth due to severe cystic disease, vascular defects and/or abnormalities of the placental labyrinth (15–20). Heterozygous knock-out mice showed minimal renal cyst formation later on in life (21) and are therefore not very suitable to test therapeutic interventions. Hypomorphic Pkd1 models, on the other hand, are viable and have polycystic kidney disease just as Pkd2 mutant mice carrying one null allele and one unstable Pkd2 allele (10,11,22). These mice also show extra-renal manifestations like cysts in liver, pancreas and/or develop aortic aneurysms (10–12,23).

We now have generated a kidney-specific and tamoxifen-inducible Pkd1 mutant mouse line, using Cre/loxP recombination. Not hampered by early death due to malfunctioning of other organs these mice are highly suitable for long-term studies on the pathogenesis of polycystic kidney disease, for testing therapeutic interventions, and for studying the initial steps of the disease. We show that the proliferative status of the renal epithelium, at the time of Pkd1 inactivation, has a major effect on the development of PKD.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
Generation of inducible and kidney-specific Pkd1 mutant mice
To get more insight into cyst formation and cyst progression, we created a conditional and inducible Pkd1 deletion mouse model. For this model we have generated mouse lines in which exons 2-11 of Pkd1 are either deleted (Pkd1^del2–11) or flanked by loxP sites (Pkd1^lox2–11 hereafter Pkd1^lox) (Fig. 1). Homozygous Pkd1^lox,lox mice are viable and have no discernable phenotype analyzed up to 15–17 months of age (n  =  5). Homozygous Pkd1^{del2–11,del2–11} mice die embryonically as reported for conventional knock-out mice (15–17). In order to selectively knock-out genes in renal epithelium we also created tamoxifen-inducible tam-KspCad-CreER^T2 (Cre) mice with Cre-recombinase expression in renal epithelium and capable to delete the floxed fragment of Pkd1 (24). The transgenic mice, Cre;Pkd1^{del2–11,lox} or Cre;Pkd1^lox,lox, were viable and fertile and showed no histological abnormalities compared with littermates without the Cre-transgene and the same Pkd1-genotype.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 1. Generation of Pkd1 mutant mice. A Pkd1-targeted mouse line containing a PGK-neo-cassette flanked by two loxP sites in intron 1 of the Pkd1 gene and a third loxP in intron 11 was generated previously (11). Cre-mediated excision of the floxed sequences results in mouse lines in which exons 2–11 of Pkd1 are either deleted (Pkd1del2–11) or flanked by loxP sites (Pkd1lox2–11). PGK, phosphoglycerate kinase gene; Neo, neomycin phosphotransferase gene; Ex, exon.

 
As expected, tamoxifen administration resulted in formation of the deleted allele, as shown by allele-specific PCR on genomic DNA isolated from the kidneys (Fig. 2).

View larger version (40K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 2. Tam-KspCad-CreERT2-mediated recombination of the floxed Pkd1 allele. Agarose gel analysis of PCR products amplified from genomic DNA isolated from kidneys of adult Cre;Pkd1del2–11,lox, young Cre;Pkd1lox,lox and adult Cre;Pkd1lox,+ mice treated (+) or not treated (–) with tamoxifen. In DNA from the Cre;Pkd1del2–11,lox mouse a slight increase in the 366-bp deletion-specific PCR product and a slight decrease in the 296-bp floxed-allele-specific product was observed in tamoxifen-treated compared with untreated animals. In Cre;Pkd1lox,lox and Cre;Pkd1lox,+ mice the deletion-specific product was only present in tamoxifen-treated animals. In treated and untreated Cre;Pkd1lox,+ mice the 250 bp wild-type (wt)-specific product is present.

 
Inactivation of the Pkd1 gene in kidneys causes cyst formation
When we treated transgenic Cre;Pkd1^{del2–11,lox} mice at 3–6 month of age with tamoxifen, pathologic examination of these mice revealed a mild cystic phenotype in the kidneys. At 1 month upon tamoxifen administration dilated tubules and 1–5 microscopic cyst per cross-section could be detected (n  = 4) and after 3 months, 10 microscopic renal cysts per cross-section and occasionally a small macroscopic cyst (cyst area 0.8 mm²) could be seen (n  =  4) (Fig. 3A). Cysts were detected bilaterally, in the cortex and the medulla.

View larger version (142K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 3. Age-dependent renal cyst development in tamoxifen-induced triple transgenic mice. Pkd1 deletion induced in adult Cre;Pkd1del2–11,lox mice results in a minor cystic phenotype. (A) Grossly normal renal tissue with a cyst (cy) in a mouse treated with tamoxifen at age 3 months and sacrificed 3 months later (50x, f); (B) Pkd1 deletion induced in newborn Cre;Pkd1del2–11,lox mice (PN4) causes massive cyst formation after 1 month (50x, f); (C) already 13 days after the inactivation of the Pkd1 gene in newborn Cre;Pkd1del2–11,lox mice, cysts can be detected (100x, f); (D) the majority of cysts arise from distal parts of the nephron and are Uromodulin positive: PN4-treated Cre;Pkd1del2–11,lox mouse sacrificed after 1 month; (E) Pkd1 deletion induced in a newborn Cre;Pkd1lox,lox mouse sacrificed 1 month later, showing a somewhat milder phenotype (50x, f); (F) in time, 5 months after tamoxifen-treatment, adult-induced conditional Cre;Pkd1lox,lox mice develop polycystic kidneys (25x, m); (G–I) phenotypic differences in conditional Pkd1 mothers and pups: tamoxifen-treated pup, showing massive cyst formation, while her triple transgenic tamoxifen-treated mother shows only a few cysts; (G) PN4-treated Cre;Pkd1del2–11,lox pup sacrificed after 1 month (50x, f); (H) Cre;Pkd1del2–11,lox mother, treated at 6 months of age, and sacrificed 5 months later (50x); (I) female control littermate of the mother, also treated with tamoxifen and sacrificed at 11 months of age (wt; Pkd1del2–11,lox) (50x).

 
In contrast, when 4-days old mice (PN4) received tamoxifen via breast feeding by the weaning mother, massive cyst formation was observed when sacrificed 1 month later (n = 9, Fig. 3B). In these mice after 13 days small cysts can sometimes be observed (n = 15) (Fig. 3C). The majority of cysts arises from distal parts of the nephron and are Uromodulin-positive (Fig. 3D). Dilated collecting ducts were also frequently observed. Transgenic mice with two floxed Pkd1 alleles (n = 3), showed a slightly milder cystic phenotype compared with Cre;Pkd1^{del2–11,lox} littermates (n = 3)(Fig. 3E). No liver or pancreatic cysts were detected in the young conditional knock-out mice. Adult tamoxifen-treated Cre;Pkd1^{del2–11,lox} animals also showed no pancreatic cysts but in the liver 0–5 microscopic and occasionally a macroscopic cyst per cross-section could be observed, similar as in wt;Pkd1^del2–11,+ and wt;Pkd1^{del2–11,lox} mice of 4–11 months. These findings support previous data that deletion of the floxed Pkd1 gene mediated by our KspCad-CreER^T2 mouse line is restricted to the kidneys (24).

Prolonged follow-up of adult-induced mice revealed that in time these animals do develop more severe PKD. Mice sacrificed at 5 months after tamoxifen-administration showed kidneys that were paler and were more or less enlarged compared with controls. Four out of five mice had kidney/body weight ratios above 2%, up to 9.5% (Controls 1.3–1.8%, n = 5). Male mice tend to manifest a more severe cystic phenotype while the recombination efficiency was similar in female and male animals. Histological examination revealed many dilated tubules and microcysts, especially at the medullar-cortical boundaries (Fig. 3F). Staining with kidney-segment markers showed that dilated tubules originated from all nephron-segments and collecting ducts and that many cysts showed no staining.

Maternal administration of tamoxifen induces a progressive cystic phenotype in newborn mutant mice but a slow disease onset in the mutant mothers
In the experiments described above, adult mice had received tamoxifen via a feeding needle, while young mice received tamoxifen via the milk from the mother who was treated. In most of these experiments the mother herself did not have the triple mutant phenotype. To minimize, however, the possibility that a technical artifact was underlying the phenotypic differences observed between adult and young mice, we treated three Cre;Pkd1^{del2–11,lox} mothers with tamoxifen and carefully analyzed the pathology in mothers and progeny. In agreement with the phenotypes found in unrelated young and adult mice, the Cre;Pkd1^{del2–11,lox} progeny sacrificed after 1 month showed a much more severe polycystic phenotype than their mothers sacrificed after 1–5 months (Fig. 3G and H).

Efficient Cre-mediated deletion of the floxed Pkd1 allele
To ascertain that recombination of the floxed allele had occurred in tamoxifen-treated triple transgenic mice, we performed a quantitative PCR on genomic DNA, isolated from the kidneys. Essentially, we analyzed the increase in deletion allele using primers in intron 1 and intron 11, flanking the deletion. In addition kidneys of tamoxifen-treated Cre;Pkd1^lox,+ mice were analyzed as well. Since Cre;Pkd1^lox,+ mice do not show a phenotype, we assume that the percentage of deletion allele is not affected by pathologic alterations of the kidneys. Adult mice show 20–25% deletion allele upon tamoxifen-administration, indicating that recombination in renal tissues had occurred in up to 40–50% of the cells. In mice treated at postnatal day 4 (PN4), we measured a lower but distinct increase in deletion of 4–12% indicating that the Pkd1 gene was knocked out in 10–20% of the renal cells. In triple transgenic Cre;Pkd1^{del2–11,lox} mice, which have already one deleted Pkd1 allele, 54–59% deletion was measured in young mice and 61–85% deletion in adult mice (Fig. 4). From these experiments we conclude that in the triple transgenic mice the Pkd1-gene is deleted in a substantial number of cells. Even more, young mice that have a much more severe renal cystic phenotype compared with adult mice, show a slightly lower percentage of Pkd1-deletion. No deletion allele was measured in liver or in renal cells of non-treated Cre;Pkd1^lox,+ mice.

View larger version (7K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 4. Percentage deletion of the floxed Pkd1 allele in young weaning and in adult tamoxifen-induced Cre;Pkd1del2–11,lox mice and Cre;Pkd1lox,+ control mice. Quantitative real-time PCR performed on genomic DNA isolated from kidneys. Mean with standard error are shown for each animal. Controls, Pkd1del2–11,+ mice (n  =  6) with one deleted allele, show on an average 50% Pkd1-deletion, indicating that in 100% of cells one allele is deleted. Adult Cre;Pkd1lox,+ mice (n  =  3) show 19–22% Pkd1 deletion (mean 20%) indicating that in 40% of cells one allele is deleted. Adult Cre;Pkd1del2–11,lox mice (n  =  6) show 61–85% Pkd1 deletion (mean 76%), indicating that in 52% of cells both alleles are deleted. At young-age treated Cre;Pkd1lox,+ mice (n  =  3) and Cre;Pkd1del2–11,lox mice (n  =  3) show 4–12% (mean 7%) and 54–59% Pkd1 deletion (mean 57%), respectively, suggesting that in 10–20% of cells Cre-mediated recombination of the floxed Pkd1 allele has occurred. Significant higher percentage of deletion was measured in adult Cre;Pkd1del2–11,lox mice compared with young Cre;Pkd1del2–11,lox mice (Mann–Whitney test, P  =  0.024).

 
Since the lower percentage of recombination in the young mice might be explained by lower activity of the Ksp-Cadherin promoter at the time-point of tamoxifen administration, postnatal days 4–6, we measured Cre expression in 5-days-old mice and in adult mice by real time RT–PCR. Cre expression is 2-fold lower in young mice (PN5) compared with adult mice (mean 0.50 ± 0.06 versus mean 0.93 ± 0.18; median adult set at 1.0, n  =  6 for both groups).

Cell proliferation differs in newborn and adult mice
We hypothesized that the proliferation status of the tissue may underlie the observed pathogenic differences between the adult and newborn tamoxifen-treated triple mice. Therefore, we stained kidney sections with the proliferation marker Ki-67. A substantial number of proliferating cells can be observed in newborn wild-type mice, while in adult wild-type controls Ki-67 positive nuclei can hardly be found, indicating that at the moment of tamoxifen treatment the renal epithelium is proliferative in young mice but scarcely in adults (Fig. 5). The mean proliferation index (PI) in kidneys of young mice (postnatal week 1) was 8.2% (range 4.4–16%, n = 4) while in adult mice (>3 months) the mean PI was around 0.2% (range 0.1–0.4, n = 3) (Table 1).

View larger version (117K): [in this window] [in a new window] [Download PowerPoint slide]   Figure 5. Cell proliferation in normal and cystic kidneys. Only a few Ki-67-positive nuclei are detected in kidneys of a 3-month-old adult wild-type mouse (A). In contrast, multiple Ki-67-positive nuclei are seen in tubular cells of a 7-days-old wild-type mouse (B). In cystic Cre;Pkd1del2–11,lox kidneys occasionally Ki-67-positive nuclei can be observed in large cysts (C) whereas multiple Ki-67-positive cells were found in dilated tubules and small cysts (D) (A–D, 20x).

 

View this table: [in this window] [in a new window]   Table 1. Tubular cell proliferation index (PI) at the time-points of tamoxifen-induction

 
Pkd1 haploinsufficiency is associated with increased renal epithelial cell proliferation
In cystic kidneys, the epithelia of dilated tubules and small cysts show strong proliferation, as many nuclei are Ki-67 positive. Large cysts incidentally show a positive nucleus (Fig. 5). An association between haploinsuffiency of Pkd genes and an increased proliferating index has been postulated for renal tissues of ADPKD patients and adult heterozygous Pkd2 mice compared with controls (25). To address this issue in our mouse models we measured the PI of precystic tubular epithelial cells in different Pkd1 mutant mice and controls. Adult mice that carry only one Pkd1 allele show a higher PI compared with mice that carry no deletion allele (mean 1.2% versus mean 0.3%, P < 0.01), suggesting that a germ-line mutation of Pkd1 results in increased proliferation (Table 2). The adult conditional knock-out mice show roughly a 5–20-fold increase in Ki-67 index compared with mice that carry no deletion allele. These mice were sacrificed at 3 or 5 months after tamoxifen-treatment and displayed variable cystic phenotypes. In the group of mice sacrificed 3 months after treatment, which show grossly normal tubules, the PI was not significantly different from mice with one deletion allele (compare mean PI 1.9%, n = 5, to mean 1.2%, n = 8; P = 0.14). In the mice sacrificed 5 months after treatment, which show many dilated tubules, a significant increase in Ki-67 index was observed (mean PI 5.2%, n = 5; P = 0.02).

View this table: [in this window] [in a new window]   Table 2. Tubular cell proliferation index (PI) in adult and young mutant Pkd1 mice

 
In the young-age-treated mice the increase in PI upon Pkd1 disruption, was not significant (Table 2), either because there is not yet an increased PI or because base line proliferation of renal cells in young mice is too high to demonstrate any additional increase. All together these data support the idea that deletion of Pkd1 results in increased renal epithelial proliferation with conditional knock-outs displaying cystic changes showing the highest levels of proliferation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
In this paper we present the first study describing an inducible and kidney-specific Pkd1 knock-out mouse model to study ADPKD. Our findings show that timing of Pkd1 gene disruption has a major effect on the severity of cyst development. Adult mice showed a mild cystic phenotype 1 month after the tamoxifen-treatment that resulted in disruption of the Pkd1 gene in a substantial number of cells. Newborn mice, in contrast, and in spite of a lower percentage of Pkd1 deletion, showed rapid and massive cyst formation in the same timeframe, suggesting that in these mice renal tissue is highly susceptible to cystogenesis, while in adult mice, with a relatively mild phenotype, it is not. We hypothesized that the proliferation status of the tissue may underlie the observed pathogenic differences in adult and young mice. Indeed, staining with the proliferation marker Ki-67 showed a substantial number of proliferating cells in newborn wild-type mice, while in adult wild-type controls Ki-67 positive nuclei can hardly be found, indicating that at the moment of Pkd1 gene inactivation by tamoxifen treatment the renal epithelium is proliferative in young mice but not in adults.

Dilated tubules and cysts originate from all nephron-segments, which is in line with the Cre expression pattern of our conditional knock-out mouse model (24). In the adult mice, 5 months upon the induction of Pkd1-gene disruption, the majority of cysts did not show staining for any of the segment-markers, whereas in young mice at 1 month upon Pkd1-gene disruption, cysts frequently showed staining for Uromodulin. Since especially the loops of Henle are proliferating and enlarging in the developing kidneys of young mice, a large proportion of cysts seem to originate from this segment. In adult mice dilatations have been observed in different parts of the nephron, but cysts seem to have lost expression of their renal segment-specific markers.

The observation that in adult mice the number of renal epithelial cells, in which both alleles of Pkd1 are disrupted, does not rapidly expand and grows out to form cysts, suggests that in the context of the renal tissue Pkd1 gene deficiency does not initiate autonomous cell proliferation beyond control. Our finding seems to be in agreement with the observation by Nishio et al. (26), who showed that cystic epithelial cells of Pkd1 null mice do not grow in nude mice. Apparently, additional stimuli or events are required to trigger the process. In vitro, however, decreased levels of Pkd1 gene expression induced an increase in cell proliferation but cell culture conditions are usually permissive for growth factor-induced cell proliferation (26,27). In the absence of polycystin-1, cells continuously proliferate since a negative regulatory signal, regulated by polycystin-1, is missing. In the developing kidneys of neonatal mice, proliferation is induced by growth factors required for elongation of the nephron and renal development.

It is well documented that a number of signaling pathways, as Ras-Raf-ERK, JAK/STAT signaling and Akt/PI3 kinase activation can be induced by overexpression of full length or truncated Pkd1 constructs (28–30). In addition, in immortalized renal cystic epithelial cells, with reduced levels of polycystin-1, a hyperproliferative response to IGF-1 is associated with enhanced activation of the Ras-Raf-ERK signaling system compared with normal cells, suggesting that cells with reduced levels of polycystin-1 are more sensitive to growth factor stimulation (31). However, the exact signaling cascades critical for the initiation of cyst formation are not completely known.

Cell proliferation is not only critical for the initiation of cyst formation but proliferation, in addition to extra cellular matrix remodeling and fluid accumulation, is contributing to cyst growth as well. Indeed the epithelia of dilated tubules and small cysts show increased proliferation, as many nuclei are Ki-67 positive. Large cysts, however, scarcely show positive nuclei, as described for renal cystic tissues from humans and the Han:SPRD-rats. Our data support the notion that proliferation is important in the early steps of cyst formation and cyst growth while at later stages epithelial stretch induced by increased luminal pressure may contribute to cyst growth (32,33). Promising results obtained by treatment of two non-orthologues murine models for polycystic kidney disease with the cyclin-dependent kinase inhibitor Roscovitine, also suggest that proliferation may be an important target for therapeutic interventions in ADPKD-patients (34). Likely, the drug will be more effective in not too advanced stages of the disease. These non-orthologous mouse and rat models have proven to be very useful to study the pathogenesis of polycystic kidney disease and to test potential therapeutic interventions. Diets or drugs that have been tested, however, sometimes had opposing effects, emphasizing the importance of using models that mimic the human disease (35–38).

Aberrant functioning of primary cilia may also be a crucial step in cystogenesis, since most if not all proteins encoded by genes related to cyst formation appear to be involved in ciliary or basal body function. Cilia of renal epithelial cells act as mechanosensors responding to flow by creating a Ca²⁺ influx, a process hampered in Pkd1^–/– cells (8). Defective signaling may affect maintenance of the mature epithelial architecture and, indeed, in time the phenotype of adult mice becomes more severe, similar to the late-onset of PKD in human patients. Sensing the direction of urine flow by primary cilia may also be critical for proper orientation of tubular epithelial cells, e.g. planar cell polarity. Defects in these processes are correlated with tubular dilation and cyst formation (39,40).

In most experiments we used triple transgenic mice with one floxed and one deletion allele, thus mimicking the process of somatic inactivation by a second hit. We also analyzed triple transgenic mice with two floxed Pkd1 alleles, Cre;Pkd1^lox,lox. In general the phenotype of these mice was milder compared with Cre;Pkd1^{del2–11,lox} mice sacrificed after similar time periods. Possible explanations are that Cre is either not always deleting both floxed Pkd1 alleles, or the deletion of the second allele may occur with some delay. It may also take more time until the levels of polycystin-1, the protein encoded by Pkd1, fall below a critical threshold, or cells with one mutant Pkd1-allele may be more prone to Cre-mediated deletion of the other allele. In addition, haploinsufficiency of the surrounding cells may enhance cyst formation and progression.

Haploinsufficiency at Pkd2 can induce phenotypic changes in vascular smooth muscle cells and influence the proliferative activity of renal tubular epithelium in Pkd2 mutant mice of 9–12 months (12,25). It was hypothesized that the increase in cell proliferation may predispose the tubular cells to a somatic mutation, necessary for cystic changes (25). In support of this hypothesis, this study showed that heterozygous adult Pkd1 mice, like the adult heterozygous Pkd2 mice, had an 4-fold increased PI compared with wild-type animals (P < 0.05), strengthening the possibility that haploinsuffiency of Pkd1 may underlie the difference between Cre;Pkd1^{del2–11,lox} and Cre;Pkd1^lox,lox mice and that haploinsufficiency of Pkd genes may predispose tubular cells to a somatic mutation. Inactivation of the second allele eventually results in cystic changes and after that time-period of 5 months, a further increase in proportion of proliferating cells was observed in dilated tubules and starting cysts. Apparently, it takes time for additional stimuli to occur or to build up to sufficient levels for cystogenesis.

The difference in efficiency of Cre-mediated Pkd1 gene disruption in young compared with adult mice may be explained by the 2-fold higher expression of Cre in kidneys of adult mice compared with 5-days-old mice. Additionally, reduced intake of tamoxifen may underlie lower recombination efficiency. In the case of the young mice a whole litter received tamoxifen via breast-feeding from the mother who was treated, whereas adult animals received tamoxifen individually and directly via oral administration. Nevertheless, it is clear that the model presented here, can serve to disrupt the Pkd1 gene at different pre- and post-natal stages. Endogenous Ksp-Cadherin mRNA as well as a reporter gene under control of the Ksp-Cadherin promoter fragment have also been reported to be expressed in developing nephrons and in adult mice (41,42).

The cystic phenotype in adult conditional knock-out mice is in line with the observation that heterozygous Pkd1^+,- mice hardly develop cysts in contrast to ADPKD patients. In this respect, it may be good to realize that mice have a shorter lifespan, are kept in a highly controlled environment and are not exposed to putative harmful factors like infections, or toxic agents. The kidney has the capacity to regenerate upon renal ischemia, inflammation and drug or toxicant exposure. Renal injury followed by repair processes including proliferation and apoptosis, may be an important permissive condition for cystogenesis in adult mice and in patients.

In conclusion, we have generated the first inducible renal-specific knock-out mouse model for cystogenesis in ADPKD. We can selectively inactivate the Pkd1 gene in renal epithelium in an allele-specific fashion, bypassing the embryonic or postnatal lethality observed in Pkd1-deficient mice. Conditional knock-out mice do not show extra-renal manifestations like cysts in liver and pancreas and vascular defects as did Pkd1^–/– embryos and 4-week-old Pkd1 hypomorphic mice (11,15,16). Not hampered by early death due to malfunction of other organs our mice are highly suitable for long-term studies of the pathogenesis of ADPKD and for testing therapeutic interventions. Deletion of Pkd1 results in increased proliferation with conditional knock-outs displaying cystic changes showing the highest levels of proliferation. Kidney-specific inactivation of Pkd1 in newborn mice caused massive renal cyst formation, showing that Pkd1 is essential for nephron maturation in developing kidneys. In newborn mice kidney development is not yet completed and cell proliferation still occurs to elongate the nephron. Therefore, we hypothesize that proliferation in the absence of functional polycystins is a trigger for cyst formation.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
Generation of tam-KspCad-Cre;Pkd1^{del2–11,lox} mice and tamoxifen administration
Mice with the Pkd1^del2–11 and Pkd1^lox allele were generated as described for the hypomorphic Pkd1^nl allele (11). In short, a loxP-flanked PGK-neo-poly(A) cassette in antisense orientation was inserted into the Eco105I site in intron 1 and a synthetic double-stranded oligonucleotide containing a loxP site was cloned into the BglII site in intron 11. The linearized targeting construct was electroporated into 129/Ola embryonic stem (ES) cells, and homologous recombination was identified using 5'- and 3'-external probes. Chimeric mice were obtained by injection of Pkd1^nl,+ ES cells into C57BL/6 blastocysts and clone no. 4 and no. 18 were found to transmit the mutant Pkd1 allele through the germ line. Partial or complete excision of the loxP-flanked DNA sequences occurred by crossbreeding clone no. 18-derived mice with EIIaCre-mice (43), and the generated mice with the Pkd1^nl, the Pkd1^lox, and the Pkd1^del2–11 alleles were further bred (Fig. 1). Mice bred on a C57BL/6 background for more than eight generations were analyzed in this study. Genotypes were assessed by PCR analysis of tail genomic DNA using a reverse primer in intron 11 combined with a forward primer in intron 1 (del allele) or a forward primer in intron 11 (floxed and wild-type alleles).

Generation of tam-KspCad-CreER^T2 mice and tamoxifen treatment have been described previously (24). Adult mice, 3–4 months of age, have been treated with 5 mg tamoxifen for three consecutive days, using a feeding needle. Weaning mothers, 3–6-months-old, received a similar treatment starting at postnatal day 4 (PN4) of the progeny, and these newborn mice received tamoxifen via breast-feeding from the mother.

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

Quantitative PCR to detect deletions and Cre expression
To quantify the deletion a real-time PCR was performed on genomic DNA isolated from the kidneys of tamoxifen-treated and control mice. The increase in ‘deletion’ was analyzed as described previously (23). Essentially, primers have been selected in intron 1 and intron 11, flanking the deletion. As reference PCR, a fragment from exon 36 of the undeleted distal part of the Pkd1 gene was used. DNA isolated from heterozygous Pkd1^{del2–11,lox} mice (n = 6), with 50% of Pkd1 alleles containing a deletion, served as a 50% reference. All measurements were performed three times in triplicate.

RT–PCR measurements to quantify the Cre expression in PN5 (n = 6) and adult mice (n = 6) were expressed as mean ± SD, related to the median value of adult mice (set at 1.0). Measurements were done in triplicate and performed twice.

Histological analysis
Tissues were fixed in 10% buffered formalin, embedded in paraffin, sectioned at 4 or 5 µm and stained with hematoxylin-eosin (HE). Kidney-segment identity was characterized using lectin Dolichos biflorus, and antisera against Uromodulin, aquaporin-2 and megalin as described (11). Ki-67 (1 : 3000, Novocastra) staining was performed using standard protocol.

Proliferative index (PI)
The PI was assessed essentially as described by Chang et al. (25), by counting the percentage of Ki-67-positive renal cells in four to six randomly selected fields of a cross-section. Between 400 and 1300 tubular epithelial nuclei of normal and dilated tubules with a diameter of up to five times the normal tubule diameter were counted. Only clearly definable nuclei with heavy/or granular staining were identified as positive. Results are expressed as mean ± SD.

Statistical analysis
Statistical comparisons between groups were performed with the Mann–Whitney rank-sum test, at 95% confidence interval. A P-value of <0.05 was considered statistically significant.

	FUNDING

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 
This research was supported by grants from the Dutch Kidney Foundation (NSN C04-2086 and C05-2132) and the Netherlands Organization for Scientific Research (NWO 917.36.353).

	ACKNOWLEDGEMENTS

 
We thank the transgenic mouse facility for generating the transgenic mice, Ron Wolterbeek for statistical support and Klaas van der Ham for photographic assistance.

Conflict of Interest statement. None declared.

	REFERENCES

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS FUNDING REFERENCES

 

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