Biomedical subjects

Nikolay O Bukanov

Publications and source records attributed to Nikolay O Bukanov.

6 recordsLinked to original sources

PubMed2006

Long-lasting arrest of murine polycystic kidney disease with CDK inhibitor roscovitine.

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.

Animals↗

PubMed2006

Development of polycystic kidney disease in juvenile cystic kidney mice: insights into pathogenesis, ciliary abnormalities, and common features with human disease.

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.

Animals↗

PubMed2005

Impaired formation of desmosomal junctions in ADPKD epithelia.

Mutations in polycystin-1 (PC-1) are responsible for autosomal dominant polycystic kidney disease (ADPKD), characterized by formation of fluid-filled tubular cysts. The PC-1 is a multifunctional protein essential for tubular differentiation and maturation found in desmosomal junctions of epithelial cells where its primary function is to mediate cell-cell adhesion. To address the impact of mutated PC-1 on intercellular adhesion, we have analyzed the structure/function of desmosomal junctions in primary cells derived from ADPKD cysts. Primary epithelial cells from normal kidney showed co-localization of PC-1 and desmosomal proteins at cell-cell contacts. A striking difference was seen in ADPKD cells, where PC-1 and desmosomal proteins were lost from the intercellular junction membrane, despite unchanged protein expression levels. Instead, punctate intracellular expression for PC-1 and desmosomal proteins was detected. The N-cadherin, but not E-cadherin was expressed in adherens junctions of ADPKD cells. These data together with co-sedimentation analysis demonstrate that, in the absence of functional PC-1, desmosomal junctions cannot be properly assembled and remain sequestered in cytoplasmic compartments. Taken together, our results demonstrate that PC-1 is crucial for formation of intercellular contacts. We propose that abnormal expression of PC-1 causes disregulation of cellular adhesion complexes leading to increased proliferation, loss of polarity and, ultimately, cystogenesis.

Cadherins↗

PubMed2003

Functional analysis of PKD1 transgenic lines reveals a direct role for polycystin-1 in mediating cell-cell adhesion.

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 non-transgenic 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.

Animals↗

PubMed2002

Functional polycystin-1 expression is developmentally regulated during epithelial morphogenesis in vitro: downregulation and loss of membrane localization during cystogenesis.

Polycystin-1 is a protein mutated in the majority of cases of autosomal dominant polycystic kidney disease (ADPKD), but its role in the molecular pathway of tubulogenesis and cystogenesis is not understood. To define the role of polycystin-1 during dynamic changes in formation of intercellular contacts and cell polarity accompanying epithelial morphogenesis, we have utilized a 3D MDCK in vitro model of tubulogenesis and cystogenesis. Here we demonstrate that polycystin-1 is a novel component of desmosomal junctions of epithelial cells. A striking downregulation of polycystin-1 mRNA was detected in cysts as compared to tubules, leading to altered protein expression and localization. While polycystin-1 is localized to basolateral membranes of MDCK tubules, it is only detected in cytoplasmic pools in cystic cells. Furthermore, the expression of polycystin-1 is modulated during distinct stages of HGF-induced tubulogenesis from MDCK cysts. Thus, polycystin-1 is not detected in intercellular contacts at early steps of tubulogenesis, but assumes its basolateral localization at the time of cell polarization and lumen formation. An important role of polycystin-1 is further demonstrated using the pancreatic ductal epithelial cell line SU.86.86 which undergoes in vitro differentiation resulting in the formation of domes. Dome formation is thought to parallel tubular differentiation and morphogenesis in vivo. Our data reveal significant upregulation of polycystin-1 mRNA and protein levels in domes. Collectively, our results demonstrate a critical importance of controlled level of polycystin-1 expression for proper tubular differentiation and maturation. We suggest that the loss of polycystin-1 from its basolateral location in tubular epithelium may alter critical pathways controlling normal tubulogenesis leading to cystic transformation.

Adherens Junctions↗

PubMed2002

Canine PKD1 is a single-copy gene: genomic organization and comparative analysis.

Most cases of autosomal dominant polycystic kidney disease are caused by mutations in the gene PKD1, encoding polycystin-1. To gain insight into the role of polycystin-1 in tubulogenesis and cystogenesis using the well-characterized canine kidney epithelial cell line MDCK, we have now cloned and characterized the exon/intron structure of the canine gene PKD1. FISH analysis showed that the dog genome lacks the multiple PKD1 homologs present in human. Intron 21 of dog PKD1 lacked the polypyrimidine tract characteristic of the human gene, whereas pyrimidine-rich elements were identified in canine intron 30. Canine polycystin-1 showed a higher degree of homology with the human counterpart and lower homology with mouse and rat. A striking degree of conservation (97% identity) was determined for the leucine-rich repeat domain between dog and human. Also, the homology analysis indicated that 4 of 16 Ig-like repeats (IgIII, IgVII, IgX, and IgXV) are likely to be functionally significant. This is particularly important in light of our recent findings demonstrating that Iglike domains form strong homophilic interactions and can mediate cell-cell adhesion. These data enable detailed analysis of the role of polycystin-1 in cystogenesis and tubulogenesis using the canine MDCK cell line.

Amino Acid Sequence↗