Inhibition of glucosylceramide accumulation results in effective blockade of polycystic kidney disease in mouse models.

Polycystic kidney disease (PKD) represents a family of genetic disorders characterized by renal cystic growth and progression to kidney failure. No treatment is currently available for people with PKD, although possible therapeutic interventions are emerging. Despite genetic and clinical heterogeneity, PKDs have in common defects of cystic epithelia, including increased proliferation, apoptosis and activation of growth regulatory pathways. Sphingolipids and glycosphingolipids are emerging as major regulators of these cellular processes. We sought to evaluate the therapeutic potential for glycosphingolipid modulation as a new approach to treat PKD. Here we demonstrate that kidney glucosylceramide (GlcCer) and ganglioside GM3 levels are higher in human and mouse PKD tissue as compared to normal tissue, regardless of the causative mutation. Blockade of GlcCer accumulation with the GlcCer synthase inhibitor Genz-123346 effectively inhibits cystogenesis in mouse models orthologous to human autosomal dominant PKD (Pkd1 conditional knockout mice) and nephronophthisis (jck and pcy mice). Molecular analysis in vitro and in vivo indicates that Genz-123346 acts through inhibition of the two key pathways dysregulated in PKD: Akt protein kinase-mammalian target of rapamycin signaling and cell cycle machinery. Taken together, our data suggest that inhibition of GlcCer synthesis represents a new and effective treatment option for PKD.

Pkd1 and Nek8 mutations affect cell-cell adhesion and cilia in cysts formed in kidney organ cultures.

Development of novel therapies for polycystic kidney disease (PKD) requires assays that adequately reflect disease biology and are adaptable to high-throughput screening. Here we describe an embryonic cystic kidney organ culture model and demonstrate that a new mutant allele of the Pkd1 gene (Pkd1(tm1Bdgz)) modulates cystogenesis in this model. Cyst formation induced by cAMP is influenced by the dosage of the mutant allele: Pkd1(tm1Bdgz) -/- cultures develop a larger cystic area compared with +/+ counterparts, while Pkd1(tm1Bdgz) +/- cultures show an intermediate phenotype. A similar relationship between the degree of cystogenesis and mutant gene dosage is seen in cystic kidney organ cultures derived from mice with a mutated Nek8 gene (Nek8(jck)). Both Pkd1- and Nek8- cultures display altered cell-cell junctions, with reduced E-cadherin expression and altered desmosomal protein expression, similar to ADPKD epithelia. Additionally, characteristic ciliary abnormalities are identified in cystic kidney cultures, with elevated ciliary polycystin 1 expression in Nek8 homozygous cultures and elevated ciliary Nek8 protein expression in Pkd1 homozygotes. These data suggest that the Nek8 and Pkd1 genes function in a common pathway to regulate cystogenesis. Moreover, compound Pkd1 and Nek8 heterozygous adult mice develop a more aggressive cystic disease than animals with a mutation in either gene alone. Finally, we validate the kidney organ culture cystogenesis assay as a therapeutic testing platform using the CDK inhibitor roscovitine. Therefore, embryonic kidney organ culture represents a relevant model for studying molecular cystogenesis and a rapid tool for the screening for therapies that block cystic growth.

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.

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.