Toxic tubular injury in kidneys from Pkd1-deletion mice accelerates cystogenesis accompanied by dysregulated planar cell polarity and canonical Wnt signaling pathways.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by large fluid-filled cysts and progressive deterioration of renal function necessitating renal replacement therapy. Previously, we generated a tamoxifen-inducible, kidney epithelium-specific Pkd1-deletion mouse model and showed that inactivation of the Pkd1 gene induces rapid cyst formation in developing kidneys and a slow onset of disease in adult mice. Therefore, we hypothesized that injury-induced tubular epithelial cell proliferation may accelerate cyst formation in the kidneys of adult Pkd1-deletion mice. Mice were treated with the nephrotoxicant 1,2-dichlorovinyl-cysteine (DCVC) after Pkd1-gene inactivation, which indeed accelerated cyst formation significantly. After the increased proliferation during tissue regeneration, proliferation decreased to basal levels in Pkd1-deletion mice just as in DCVC-treated controls. However, in severe cystic kidneys, 10-14 weeks after injury, proliferation increased again. This biphasic response suggests that unrestricted cell proliferation after injury is not the underlying mechanism for cyst formation. Aberrant planar cell polarity (PCP) signaling and increased canonical Wnt signaling are suggested to be involved in cyst formation. Indeed, we show here that in Pkd1 conditional deletion mice expression of the PCP component Four-jointed (Fjx1) is decreased while its expression is required during tissue regeneration. In addition, we show that altered centrosome position and the activation of canonical Wnt signaling are early effects of Pkd1-gene disruption. This suggests that additional stimuli or events are required to trigger the process of cyst formation. We propose that during tissue repair, the integrity of the newly formed Pkd1-deficient cells is modified rendering them susceptible to subsequent cyst formation.

Elevated TGFbeta-Smad signalling in experimental Pkd1 models and human patients with polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited renal disease characterized by many fluid-filled cysts and interstitial fibrosis in the kidneys, leading to chronic renal failure. During cystogenesis the renal tubules undergo extensive structural alterations that are accompanied by altered cellular signalling, directly and/or indirectly regulated by the PKD1 and PKD2 proteins. Since transforming growth factor (TGF)-beta signalling modulates cell proliferation, differentiation, apoptosis, adhesion and migration of various cell types, we studied the activation of this signalling pathway in Pkd1-mutant mouse models at different stages of the disease. Therefore, we analysed expression of the TGFbeta-Smad signalling pathway and its target genes in different Pkd1 mutant mouse models in various stages of polycystic disease. Nuclear accumulation of P-Smad2 in cyst lining epithelial cells was not observed in the initiation phase but was observed at mild and more advanced stages of PKD. This coincides with mild fibrosis and increased mRNA levels of TGFbeta target genes, such as fibronectin, collagen type I, plasminogen activator inhibitor 1 and matrix metalloproteinase-2. At this stage many interstitial fibroblasts were found around cysts, which also showed nuclear localization for P-Smad2. However, bone morphogenetic protein (BMP) signalling, which can antagonize TGFbeta signalling, is not affected, since nuclear expression of P-Smad1/5/8 and expression of the BMP target gene, inhibitor of DNA binding/differential-1 (ID-1) is not altered compared to wild-type controls. Also, human kidneys with progressive ADPKD showed increased nuclear localization of P-Smad2, while in general expression of P-Smad1/5/8 was weak. These results exclude TGFbeta signalling at the initiation of cystogenesis, but indicate an important role during cyst progression and in fibrogenesis of progressive ADPKD.

Quantification of Cre-mediated recombination by a novel strategy reveals a stable extra-chromosomal deletion-circle in mice.

BACKGROUND: Inducible conditional knockout animals are widely used to get insight in the function of genes and the pathogenesis of human diseases. These models frequently rely on Cre-mediated recombination of sequences flanked by Lox-P sites. To understand the consequences of gene disruption, it is essential to know the efficiency of the recombination process. RESULTS: Here, we describe a modification of the multiplex ligation-dependent probe amplification (MLPA), called extension-MLPA (eMLPA), which enables quantification of relatively small differences in DNA that are a consequence of Cre-mediated recombination. eMLPA, here applied on an inducible Pkd1 conditional deletion mouse model, simultaneously measures both the reduction of the floxed allele and the increase of the deletion allele in a single reaction thereby minimizing any type of experimental variation. Interestingly, with this method we were also able to observe the presence of the excised DNA fragment. This extra-chromosomal deletion-circle was detectable up to 5 months after activation of Cre. CONCLUSION: eMLPA is a novel strategy which easily can be applied to measure the Cre-mediated recombination efficiency in each experimental case with high accuracy. In addition the fate of the deletion-circle can be followed simultaneously.

Pathogenic sequence for dissecting aneurysm formation in a hypomorphic polycystic kidney disease 1 mouse model.

OBJECTIVE: Autosomal Dominant Polycystic Kidney Disease (ADPKD) is a multi-system disorder characterized by progressive cyst formation in the kidneys. Serious complications of ADPKD are intracranial and aortic aneurysms. The condition is mainly caused by mutations in the PKD1 or PKD2 gene. We have carefully analyzed vascular remodeling in hypomorphic Pkd1(nl/nL) mouse model with dissecting aneurysms in the aorta. METHODS AND RESULTS: Quantitative real-time polymerase chain reaction revealed that in the aorta the expression of normal Pkd1 is reduced to approximately 26%. Using (immuno)histochemistry we have characterized the pathogenetic sequence for dissecting aneurysm formation. The aorta shows regions with accumulation of matrix components between the elastin lamellae. This is followed by increased numbers of smooth muscle cells and locally weakening of the media. In the intima, accumulation of matrix components and detachment of endothelial cells from the elastin lamellae results in a tear. The combination of weak media and a tear in the intima leads to rupture of the vessel wall resulting in intramural bleeding. CONCLUSIONS: The Pkd1(nl/nl) mouse reveals that polycystin1 is implicated in maintenance of the vessel wall structural integrity, and it is a useful model for dissecting aneurysm formation studies.

Common regulatory elements in the polycystic kidney disease 1 and 2 promoter regions.

The PKD1 and PKD2 genes are mutated in patients with autosomal dominant polycystic kidney disease (ADPKD), a systemic disease, with the formation of renal cysts as main clinical feature. The genes are developmentally regulated and aberrant expression of PKD1 or PKD2 leads to cystogenesis. To date, however, the transcription factors regulating expression of these genes have hardly been studied. To identify conserved putative transcription factor-binding sites, we cloned and characterized the 5'-flanking regions of the murine and canine Pkd1 genes and performed a multispecies comparison by including sequences from the human and Fugu rubripes orthologues as well as the Pkd2 promoters from mouse and human. Sequence analysis revealed a variety of conserved putative binding sites for transcription factors and no TATA-box element. Nine elements were conserved in the mammalian Pkd1 promoters: AP2, E2F, E-Box, EGRF, ETS, MINI, MZF1, SP1, and ZBP-89. Interestingly, six of these elements were also found in the mammalian Pkd2 promoters. Deletion studies with the mouse Pkd1 promoter showed that a approximately 280 bp fragment is capable of driving luciferase reporter gene expression, whereas reporter constructs containing larger fragments of the Pkd1 promoter showed a lower activity. Furthermore, mutating a potential E2F-binding site within this 280 bp fragment diminished the reporter construct activity, suggesting a role for E2F in regulating cell cycle-dependent expression of the Pkd1 gene. Our data define a functional promoter region for Pkd1 and imply that E2F, EGRF, Ets, MZF1, Sp1, and ZBP-89 are potential key regulators of PKD1 and PKD2 in mammals.

Cloning and characterization of the 5'-flanking region of the canine growth hormone gene.

The growth hormone (GH) gene is expressed in a variety of tissues outside the pituitary, including the mammary gland. GH expression in the mammary gland is stimulated by progestins. The local synthesis of mammary GH may provide a highly proliferative environment within the mammary gland that may contribute to the development or progression of mammary tumours. To elucidate the mechanism regulating mammary GH expression, we cloned the 5'-flanking region of the canine GH gene using inverse polymerase chain reaction. Gel-shift experiments showed that several sequences in the 5'-flanking region of the GH gene bind mammary nuclear proteins and may be involved in basal and progesterone-induced mammary GH expression. Sequence analysis and comparison with the GH promoters of human, rat, and mouse genes revealed a number of shared binding sites for transcription factors such as Pit-1, which is involved in pituitary GH expression, and for factors involved in the differentiation of lymphoid cells. Moreover, a putative binding site for the progesterone receptor (PR) was identified in all promoters, indicating that the progestin-induced expression of GH in mammary tissue is most probably a direct effect of activated PRs on the GH gene promoter and that this may occur in various species.

Growth hormone gene expression in canine normal growth plates and spontaneous osteosarcoma.

The indirect growth-promoting action of pituitary-derived growth hormone (GH) on skeletal growth is thought to be mediated by systemically released insulin-like growth factor-I (IGF-I) and by locally produced IGF-I. The aim of the present study was to document whether GH is expressed locally in canine bone and spontaneous osteosarcoma. Using RT-PCR the expression of GH mRNA was demonstrated in the metaphyseal, but not in the majority of epiphyseal ends of the canine growth plate. GH mRNA was also present in 25% of the canine osteosarcoma specimens. The expression of GH mRNA in predominantly active osteoid forming areas was associated with the presence of immunoreactive GH in osteoblasts, as shown by immunohistochemistry. It is concluded that locally produced GH is involved in osteoid formation and may play a role in the growth of neoplastic bone lesions in the dog.

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

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.

Transgenic mice expressing tamoxifen-inducible Cre for somatic gene modification in renal epithelial cells.

Gene inactivation often leads to an embryonic-lethal phenotype. In focal diseases like renal cell carcinomas and polycystic kidney disease, somatic gene inactivation in subsets of cells is likely to occur at later stages. We generated a transgenic mouse line with an inducible form of Cre recombinase for conditional gene modifications in kidney epithelial cells. To this end a 1.4-kb promoter fragment of the kidney-specific cadherin gene (KspCad) was cloned upstream of a tamoxifen-inducible Cre recombinase (CreER(T2)) encoding sequence. Expression and activity of Cre was evaluated using reverse transcriptase polymerase chain reaction (RT-PCR) analysis and by crossbreeding to Z/EG reporter mice. One KspCad-CreER(T2) line showed kidney-specific Cre expression and mediated recombination upon tamoxifen treatment in Z/EG reporter mice. No reporter gene expression was detected in untreated animals or in extrarenal tissues upon treatment. Within the kidneys, enhanced green fluorescent protein (EGFP) fluorescence was observed in epithelial cells in several nephronic segments. In addition, the system successfully recombined a floxed Pkd1 gene.

Increased activity of activator protein-1 transcription factor components ATF2, c-Jun, and c-Fos in human and mouse autosomal dominant polycystic kidney disease.

Autosomal dominant polycystic kidney disease is a common inherited disorder that predominantly manifests with the formation of fluid-filled cysts in both kidneys. The disease can be accounted for by a mutation in either the PKD1 or the PKD2 gene. It was demonstrated previously that aberrant expression of the PKD1 gene product, polycystin-1, results in modification of activator protein-1 (AP-1) transcription factor activity in cultured renal epithelial cells. Here, it is reported that activity of the AP-1 components c-Jun, ATF2, and c-Fos is altered in renal cystic tissue of patients with autosomal dominant polycystic kidney disease and of hypomorphic Pkd1 mice with polycystic kidney disease. Data were obtained using immunohistochemical and Western blot analysis. Significant upregulation of Thr71- and Thr69/71-phosphorylated ATF2 and Ser73-phosphorylated c-Jun and increased c-Fos were detected in small cysts and (dilated) ducts and tubules surrounded by fibrotic interstitium. The data indicate that various AP-1 components are constitutively activated in polycystic kidney disease and suggest that aberrant AP-1 activity is relevant for cyst formation.

Lowering of Pkd1 expression is sufficient to cause polycystic kidney disease.

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