Polycystic kidney diseases (PKDs) are primarily characterized by the growth of fluid-filled cysts in renal tubules leading to end-stage renal disease. Mutations in the PKD1 or PKD2 genes lead to autosomal dominant PKD (ADPKD), a slowly developing adult form. Autosomal recessive polycystic kidney disease results from mutations in the PKHD1 gene, affects newborn infants and progresses very rapidly. No effective treatment is currently available for PKD. A previously unrecognized site of subcellular localization was recently discovered for all proteins known to be disrupted in PKD: primary cilia. Because ciliary functions seem to be involved in cell cycle regulation, disruption of proteins associated with cilia or centrioles may directly affect the cell cycle and proliferation, resulting in cystic disease. We therefore reasoned that the dysregulated cell cycle may be the most proximal cause of cystogenesis, and that intervention targeted at this point could provide significant therapeutic benefit for PKD. Here we show that treatment with the cyclin-dependent kinase (CDK) inhibitor (R)-roscovitine does indeed yield effective arrest of cystic disease in jck and cpk mouse models of PKD. Continuous daily administration of the drug is not required to achieve efficacy; pulse treatment provides a robust, long-lasting effect, indicating potential clinical benefits for a lifelong therapy. Molecular studies of the mechanism of action reveal effective cell-cycle arrest, transcriptional inhibition and attenuation of apoptosis. We found that roscovitine is active against cysts originating from different parts of the nephron, a desirable feature for the treatment of ADPKD, in which cysts form in multiple nephron segments. Our results indicate that inhibition of CDK is a new and effective approach to the treatment of PKD.

Significant progress in understanding the molecular mechanisms of polycystic kidney disease (PKD) has been made in recent years. Translating this understanding into effective therapeutics will require testing in animal models that closely resemble human PKD by multiple parameters. Similar to autosomal dominant PKD, juvenile cystic kidney (jck) mice develop cysts in multiple nephron segments, including cortical collecting ducts, distal tubules, and loop of Henle. The jck mice display gender dimorphism in kidney disease progression with more aggressive disease in male mice. Gonadectomy experiments show that testosterone aggravates the severity of the disease in jck male mice, while female gonadal hormones have protective effects. EGF receptor is overexpressed and mislocalized in jck cystic epithelia, a hallmark of human disease. Increased cAMP levels in jck kidneys and activation of the B-Raf/extracellular signal-regulated kinase pathway are demonstrated. The effect of jck mutation on the expression of Nek8, a NIMA-related (never in mitosis A) kinase, and polycystins in jck cilia is shown for the first time. Nek8 overexpression and loss of ciliary localization in jck epithelia are accompanied by enhanced expression of polycystins along the cilia. The primary cilia in jck kidneys are significantly more lengthened than the cilia in wild-type mice, suggesting a role for Nek8 in controlling ciliary length. Collectively, these data demonstrate that the jck mice should be useful for testing potential therapies and for studying the molecular mechanisms that link ciliary structure/function and cystogenesis.

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A stable complex between duplex DNA and an oligonucleotide is assembled with the aid of a DNA synthetic mimic, peptide nucleic acid (PNA). Homopyrimidine PNAs are known to invade into short homopurine tracts in duplex DNA forming P-loops. We have found that P-loops, formed at two closely located purine tracts in the same DNA strand separated by a mixed purine-pyrimidine sequence, merge and open the double helix between them. The opposite DNA strand, which is not bound with PNA, exposes and becomes accessible for complexing with an oligonucleotide via Watson-Crick pairing. As a result, the PD-loop emerges, which consists of locally open duplex DNA, PNA ''openers,'' and an oligonucleotide. The PD-loop stability and sequence specificity are demonstrated by affinity capture of duplex DNAs by using biotinylated oligonucleotides and streptavidincovered magnetic beads. The type of complex formed by PNAs, an oligonucleotide and duplex DNA we describe, opens ways for development of various in vitro and in situ hybridization techniques with duplex DNA and may find applications in DNA nanotechnology and genomics.Linear, nonsupercoiled double-stranded DNA (dsDNA) is known to be able to accommodate an additional oligonucleotide strand with much less efficiency than single-stranded nucleic acids and supercoiled DNAs. Formation of intermolecular triplexes is mostly limited to long homopurinehomopyrimidine regions (1, 2). D-loops are formed in linear dsDNA only at the ends of the DNA duplex in case of binding of long single-stranded DNA molecules (3). R-loops may be formed inside linear dsDNA, but long RNAs and transient DNA denaturation are necessary (4). A complex between an oligodeoxyribonucleotide (ODN) and linear dsDNA can be formed with assistance of the RecA protein. However, the fidelity of recognition is lower than for protein-free DNA-DNA interactions and the complex is unstable upon deproteinization (5, 6). The possibility of binding to dsDNA of a pair of complementary modified ODNs due to their self-mediated invasion into DNA duplex has recently been demonstrated. However, these complexes were formed only at the ends of dsDNA (7). In addition, a few techniques exist for formation of specific complexes between ODNs and dsDNA based on either prior DNA denaturation or degradation of one DNA strand before ODN binding. These techniques, however, require subsequent reconstruction or reparation of targeted molecules into DNA duplex (8, 9).Here we describe a complex between linear dsDNA and an ODN with mixed sequence of purines and pyrimidines, which is formed via Watson-Crick pairing facilitated by the peptide nucleic acid (PNA) invasion. As a result, an unusual multicomponent structure emerges, which we call the PD-loop. It is characterized by exceptionally high sequence specificity. The PD-loop structure opens totally new ways for hybridizing oligonucleotides with dsDNA and for selective manipulation with DNA duplexes. We demonstrate that the PD-loop makes it possible to selectively isolate a dsDNA fragment from ...

Abstract. The PKD1 protein, polycystin-1, is a large transmembrane protein of uncertain function and topology. To study the putative functions of polycystin-1, conditionally immortalized kidney cells transgenic for PKD1 were generated and an interaction between transgenic polycystin-1 and endogenous polycystin-2 has been recently demonstrated in these cells. This study provides the first functional evidence that transgenic polycystin-1 directly mediates cell-cell adhesion. In non-permeabilized cells, polycystin-1 localized to the lateral cell borders with N-terminal antibodies but not with a C-terminal antibody; there was a clear difference in surface intensity between transgenic and non-transgenic cells. Compared with non-transgenic cells, transgenic cells showed a dramatic increase in resistance to the disruptive effect of a polycystin-1 antibody raised to the PKD domains of polycystin-1 (IgPKD) in both cell adhesion and cell aggregation assays. The differential effect on cell adhesion between transgenic and nontransgenic cells could be reproduced using recombinant fusion proteins encoding non-overlapping regions of the IgPKD domains. In contrast, antibodies raised to other extracellular domains of polycystin-1 had no effect on cell adhesion. Finally, the specificity of this finding was confirmed by the lack of effect of IgPKD antibody on cell adhesion in a PKD1 cystic cell line deficient in polycystin-1. These results demonstrate that one of the primary functions of polycystin-1 is to mediate cell-cell adhesion in renal epithelial cells, probably via homophilic or heterophilic interactions of the PKD domains. Disruption of cell-cell adhesion during tubular morphogenesis may be an early initiating event for cyst formation in ADPKD.Autosomal dominant polycystic kidney disease (ADPKD), the most common inherited human renal disease (incidence, 1 in 1000 live births) is caused by mutations in two genes, PKD1 (85%) and PKD2 (15%). It is a systemic disorder characterized by the formation of fluid-filled cysts mainly in the kidney but also commonly in the liver and pancreas. ADPKD is also associated with an increased incidence of non-cystic manifestations, including hypertension, cardiac valve abnormalities, diverticular disease, and intracranial aneurysms.Since the identification of PKD1 and PKD2, investigation into the putative functions of the two ADPKD proteins, polycystin-1 and polycystin-2, has been intense, but a consensus on their likely physiologic functions has not been reached. Several possible mutational mechanisms underlying cyst formation including haploinsufficiency and a two-hit model have been proposed, but it is not clear how mutations in either gene lead to cyst formation (1). Potentially more than one mechanism could be operative.Polycystin-1 is a large (Ͼ460 kD) heavily glycosylated integral membrane protein (2). It is predicted to have a large N-terminal extracellular domain (approximately 2500 aa), 11 transmembrane domains, and a short C-terminal cytoplasmic tail (3). The extracellular region a...

Abstract. Polycystic kidney diseases (PKDs) represent a large group of progressive renal disorders characterized by the development of renal cysts leading to end-stage renal disease. Enormous strides have been made in understanding the pathogenesis of PKDs and the development of new therapies. Studies of autosomal dominant and recessive polycystic kidney diseases converge on molecular mechanisms of cystogenesis, including ciliary abnormalities and intracellular calcium dysregulation, ultimately leading to increased proliferation, apoptosis and dedifferentiation. Here we review the pathobiology of PKD, highlighting recent progress in elucidating common molecular pathways of cystogenesis. We discuss available models and challenges for therapeutic discovery as well as summarize the results from preclinical experimental treatments targeting key disease-specific pathways.

Polycystic kidney diseases (PKDs) comprise a subgroup of ciliopathies characterized by the formation of fluid-filled kidney cysts and progression to end-stage renal disease. A mechanistic understanding of cystogenesis is crucial for the development of viable therapeutic options. Here, we identify CDK5, a kinase active in post mitotic cells, as a new and important mediator of PKD progression. We show that long-lasting attenuation of PKD in the juvenile cystic kidneys (jck) mouse model of nephronophthisis by pharmacological inhibition of CDK5 using either R-roscovitine or S-CR8 is accompanied by sustained shortening of cilia and a more normal epithelial phenotype, suggesting this treatment results in a reprogramming of cellular differentiation. Also, a knock down of Cdk5 in jck cells using small interfering RNA results in significant shortening of ciliary length, similar to what we observed with R-roscovitine. Finally, conditional inactivation of Cdk5 in the jck mice significantly attenuates cystic disease progression and is associated with shortening of ciliary length as well as restoration of cellular differentiation. Our results suggest that CDK5 may regulate ciliary length by affecting tubulin dynamics via its substrate collapsin response mediator protein 2. Taken together, our data support therapeutic approaches aimed at restoration of ciliogenesis and cellular differentiation as a promising strategy for the treatment of renal cystic diseases.

Autosomal dominant polycystic kidney disease (ADPKD) and other forms of PKD are associated with dysregulated cell cycle and proliferation. Although no effective therapy for the treatment of PKD is currently available, possible mechanism-based approaches are beginning to emerge. A therapeutic intervention targeting aberrant cilia-cell cycle connection using CDK-inhibitor R-roscovitine showed effective arrest of PKD in jck and cpk models that are not orthologous to human ADPKD. To evaluate whether CDK inhibition approach will translate into efficacy in an orthologous model of ADPKD, we tested R-roscovitine and its derivative S-CR8 in a model with a conditionally inactivated Pkd1 gene (Pkd1 cKO). Similar to ADPKD, Pkd1 cKO mice developed renal and hepatic cysts. Treatment of Pkd1 cKO mice with R-roscovitine and its more potent and selective analog S-CR8 significantly reduced renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD.