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.

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.

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.

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.

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.