Correction of cilia structure and function alleviates multi-organ pathology in Bardet-Biedl syndrome mice.

Bardet-Biedl syndrome (BBS) is a pleiotropic autosomal recessive ciliopathy affecting multiple organs. The development of potential disease-modifying therapy for BBS will require concurrent targeting of multi-systemic manifestations. Here, we show for the first time that monosialodihexosylganglioside accumulates in Bbs2-/- cilia, indicating impairment of glycosphingolipid (GSL) metabolism in BBS. Consequently, we tested whether BBS pathology in Bbs2-/- mice can be reversed by targeting the underlying ciliary defect via reduction of GSL metabolism. Inhibition of GSL synthesis with the glucosylceramide synthase inhibitor Genz-667161 decreases the obesity, liver disease, retinal degeneration and olfaction defect in Bbs2-/- mice. These effects are secondary to preservation of ciliary structure and signaling, and stimulation of cellular differentiation. In conclusion, reduction of GSL metabolism resolves the multi-organ pathology of Bbs2-/- mice by directly preserving ciliary structure and function towards a normal phenotype. Since this approach does not rely on the correction of the underlying genetic mutation, it might translate successfully as a treatment for other ciliopathies.

Glycosphingolipid metabolism and polycystic kidney disease.

Sphingolipids and glycosphingolipids are classes of structurally and functionally important lipids that regulate multiple cellular processes, including membrane organization, proliferation, cell cycle regulation, apoptosis, transport, migration, and inflammatory signalling pathways. Imbalances in sphingolipid levels or subcellular localization result in dysregulated cellular processes and lead to the development and progression of multiple disorders, including polycystic kidney disease. This review will describe metabolic pathways of glycosphingolipids with a focus on the evidence linking glycosphingolipid mediated regulation of cell signalling, lipid microdomains, cilia, and polycystic kidney disease. We will discuss molecular mechanisms of glycosphingolipid dysregulation and their impact on cystogenesis. We will further highlight how modulation of sphingolipid metabolism can be translated into new approaches for the treatment of polycystic kidney disease and describe current clinical studies with glucosylceramide synthase inhibitors in Autosomal Dominant Polycystic Kidney Disease.

Differences in the timing and magnitude of Pkd1 gene deletion determine the severity of polycystic kidney disease in an orthologous mouse model of ADPKD.

Development of a disease-modifying therapy to treat autosomal dominant polycystic kidney disease (ADPKD) requires well-characterized preclinical models that accurately reflect the pathology and biochemical changes associated with the disease. Using a Pkd1 conditional knockout mouse, we demonstrate that subtly altering the timing and extent of Pkd1 deletion can have a significant impact on the origin and severity of kidney cyst formation. Pkd1 deletion on postnatal day 1 or 2 results in cysts arising from both the cortical and medullary regions, whereas deletion on postnatal days 3-8 results in primarily medullary cyst formation. Altering the extent of Pkd1 deletion by modulating the tamoxifen dose produces dose-dependent changes in the severity, but not origin, of cystogenesis. Limited Pkd1 deletion produces progressive kidney cystogenesis, accompanied by interstitial fibrosis and loss of kidney function. Cyst growth occurs in two phases: an early, rapid growth phase, followed by a later, slow growth period. Analysis of biochemical pathway changes in cystic kidneys reveals dysregulation of the cell cycle, increased proliferation and apoptosis, activation of Mek-Erk, Akt-mTOR, and Wnt-ß-catenin signaling pathways, and altered glycosphingolipid metabolism that resemble the biochemical changes occurring in human ADPKD kidneys. These pathways are normally active in neonatal mouse kidneys until repressed around 3 weeks of age; however, they remain active following Pkd1 deletion. Together, this work describes the key parameters to accurately model the pathological and biochemical changes associated with ADPKD in a conditional mouse model.

CaMKII as a pathological mediator of ER stress, oxidative stress, and mitochondrial dysfunction in a murine model of nephronophthisis.

Polycystic kidney diseases (PKDs) are genetic diseases characterized by renal cyst formation with increased cell proliferation, apoptosis, and transition to a secretory phenotype at the expense of terminal differentiation. Despite recent progress in understanding PKD pathogenesis and the emergence of potential therapies, the key molecular mechanisms promoting cystogenesis are not well understood. Here, we demonstrate that mechanisms including endoplasmic reticulum stress, oxidative damage, and compromised mitochondrial function all contribute to nephronophthisis-associated PKD. Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) is emerging as a critical mediator of these cellular processes. Therefore, we reasoned that pharmacological targeting of CaMKII may translate into effective inhibition of PKD in jck mice. Our data demonstrate that CaMKII is activated within cystic kidney epithelia in jck mice. Blockade of CaMKII with a selective inhibitor results in effective inhibition of PKD in jck mice. Mechanistic experiments in vitro and in vivo demonstrated that CaMKII inhibition relieves endoplasmic reticulum stress and oxidative damage and improves mitochondrial integrity and membrane potential. Taken together, our data support CaMKII inhibition as a new and effective therapeutic avenue for the treatment of cystic diseases.

CDK inhibitors R-roscovitine and S-CR8 effectively block renal and hepatic cystogenesis in an orthologous model of ADPKD.

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.

Loss of GM3 synthase gene, but not sphingosine kinase 1, is protective against murine nephronophthisis-related polycystic kidney disease.

Genetic forms of polycystic kidney diseases (PKDs), including nephronophthisis, are characterized by formation of fluid-filled cysts in the kidneys and progression to end-stage renal disease. No therapies are currently available to treat cystic diseases, making it imperative to dissect molecular mechanisms in search of therapeutic targets. Accumulating evidence suggests a pathogenic role for glucosylceramide (GlcCer) in multiple forms of PKD. It is not known, however, whether other structural glycosphingolipids (GSLs) or bioactive signaling sphingolipids (SLs) modulate cystogenesis. Therefore, we set out to address the role of a specific GSL (ganglioside GM3) and signaling SL (sphingosine-1-phosphate, S1P) in PKD progression, using the jck mouse model of nephronopthisis. To define the role of GM3 accumulation in cystogenesis, we crossed jck mice with mice carrying a targeted mutation in the GM3 synthase (St3gal5) gene. GM3-deficient jck mice displayed milder PKD, revealing a pivotal role for ganglioside GM3. Mechanistic changes in regulation of the cell-cycle machinery and Akt-mTOR signaling were consistent with reduced cystogenesis. Dramatic overexpression of sphingosine kinase 1 (Sphk1) mRNA in jck kidneys suggested a pathogenic role for S1P. Surprisingly, genetic loss of Sphk1 exacerbated cystogenesis and was associated with increased levels of GlcCer and GM3. On the other hand, increasing S1P accumulation through pharmacologic inhibition of S1P lyase had no effect on the progression of cystogenesis or kidney GSL levels. Together, these data suggest that genes involved in the SL metabolism may be modifiers of cystogenesis, and suggest GM3 synthase as a new anti-cystic therapeutic target.

mTOR signaling in polycystic kidney disease.

Polycystic kidney diseases (PKDs) comprise a large group of genetic disorders characterized by formation of cysts in the kidneys and other organs, ultimately leading to end-stage renal disease. Although PKDs can be caused by mutations in different genes, they converge on a set of common molecular mechanisms involved in cystogenesis and ciliary dysfunction, and can be qualified as ciliopathies. Recent advances in understanding the mechanisms regulating disease progression have led to the development of new therapies that are being tested in both preclinical and clinical trials. In this article, we briefly review a network of molecular pathways of cystogenesis that are regulated by ciliary functions. We discuss the mTOR pathway in depth, highlighting recent progress in understanding its role in PKD and the current results of clinical trials.

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.

Hox/Pbx and Brn binding sites mediate Pax3 expression in vitro and in vivo.

Pax3 is a paired-homeodomain class transcription factor that serves a role in dorsal-ventral and medial-lateral patterning during vertebrate embryogenesis. Its expression is localized to dorsal domains within the developing neural tube and lateral domains within the developing somite. Additionally, modulation of its expression occurs along the rostral-caudal axis. Previous studies [Development 124 (1997) 617] have localized sequence elements required for expression of Pax3 in the neural tube and neural crest to a 1.6 kbp promoter fragment. In the present study, four discrete DNA elements within the 1.6 kbp promoter fragment are shown by electrophoretic mobility shift assays (EMSA) to exhibit sequence specific interactions with proteins present in nuclear extracts from P19 EC cells induced to express Pax3 by treatment with retinoic acid (RA). Proteins interacting at each of these elements are identified based on biochemical purification using DNA affinity chromatography or a candidate approach. These identifications were confirmed by the ability of specific antibodies to super-shift DNA-protein complexes in EMSA. Two of the four DNA sequence elements are shown to interact with the neural specific Pou-domain class III transcription factors Brn1 and Brn2. The remaining sites contain either consensus binding elements for heterodimers of Pbx and an anterior set of Hox family members, from paralogous groups 1-5, or monomeric Meis and are shown to interact with members of the Pbx and Meis families. Ectopic expression of Brn2 plus HoxA1 but not either factor alone, is sufficient to induce efficient expression from the endogenous Pax3 promoter in P19 EC stem cells under conditions where they would not otherwise express Pax3. Finally, in transgenic mice, mutation of either of the Pou-domain protein binding sites results in reduced expression throughout the neural tube while mutation of the Pbx/Hox binding site results in loss of expression in the anterior domain in which Hox family members from paralogous groups 1-5 are expressed. These observations demonstrate that binding elements for both neural and anterior-posterior position specific transcription factors mediate domains of Pax3 expression.

Wt1 functions in the development of germ cells in addition to somatic cell lineages of the testis.

The Wilms' tumor suppressor gene, Wt1, encodes a transcription factor critical for development of the urogenital system. To identify lineages within the developing urogenital system that have a cell-autonomous requirement for Wt1, chimeric mice were generated from Wt1-null ES cells. Males with large contributions of Wt1-/- cells showed hypoplastic and dysgenic testes, with seminiferous tubules lacking spermatogonia. Wt1-null cells contributed poorly to both somatic and germ cell lineages within the developing gonad, suggesting an unexpected role for Wt1 in germ cell development in addition to a role in the development of the somatic lineages of the gonad. Wt1 expression was detected in embryonic germ cells beginning at embryonic day 11.5 after migrating primordial germ cells (PGCs) have entered the gonad. Germ cells isolated from Wt1-null embryos showed impaired growth in culture, further demonstrating a role for Wt1 in germ cell proliferation or survival. Therefore, Wt1 plays important, and in some cases previously unrecognized, roles in multiple lineages during urogenital development.

Role of the WT1 tumor suppressor in murine hematopoiesis.

The WT1 tumor-suppressor gene is expressed by many forms of acute myeloid leukemia. Inhibition of this expression can lead to the differentiation and reduced growth of leukemia cells and cell lines, suggesting that WT1 participates in regulating the proliferation of leukemic cells. However, the role of WT1 in normal hematopoiesis is not well understood. To investigate this question, we have used murine cells in which the WT1 gene has been inactivated by homologous recombination. We have found that cells lacking WT1 show deficits in hematopoietic stem cell function. Embryonic stem cells lacking WT1, although contributing efficiently to other organ systems, make only a minimal contribution to the hematopoietic system in chimeras, indicating that hematopoietic stem cells lacking WT1 compete poorly with healthy stem cells. In addition, fetal liver cells lacking WT1 have an approximately 75% reduction in erythroid blast-forming unit (BFU-E), erythroid colony-forming unit (CFU-E), and colony-forming unit-granulocyte macrophage-erythroid-megakaryocyte (CFU-GEMM). However, transplantation of fetal liver hematopoietic cells lacking WT1 will repopulate the hematopoietic system of an irradiated adult recipient in the absence of competition. We conclude that the absence of WT1 in hematopoietic cells leads to functional defects in growth potential that may be of consequence to leukemic cells that have alterations in the expression of WT1.

A mutant form of the Wilms' tumor suppressor gene WT1 observed in Denys-Drash syndrome interferes with glomerular capillary development.

The Wilms' tumor suppressor gene WT1 encodes a zinc finger protein that is required for urogenital development. In the kidney, WT1 is most highly expressed in glomerular epithelial cells or podocytes, which are an essential component of the filtering system. Human subjects heterozygous for point mutations in the WT1 gene develop renal failure because of the formation of scar tissue within glomeruli. The relationship between WT1 expression in podocytes during development and glomerular scarring is not well understood. In this study, transgenic mice that expressed a mutant form of WT1 in podocytes were derived. The capillaries within transgenic glomeruli were dilated, indicating that WT1 might regulate the expression of growth factors that affect capillary development. Platelet endothelial cell adhesion molecule-1 expression was greatly reduced on glomerular endothelial cells of transgenic kidneys. These results suggest that WT1 controls the expression of growth factors that regulate glomerular capillary development and that abnormal capillary development might lead to glomerular disease.

A mammal-specific exon of WT1 is not required for development or fertility.

The WT1 tumor suppressor gene is a zinc finger-containing transcription factor which is required for development of the kidney and gonads. A mammal-specific alternative splicing event within this gene results in the presence or absence of a 17-amino-acid sequence within the WT1 protein. To determine the function of this sequence in vivo, gene targeting was utilized to specifically eliminate the exon encoding this sequence in mice. Mice lacking WT1 exon 5 develop normally. Adult mice lacking this exon are viable and fertile, and females are capable of lactation.