CaMK4 overexpression in polycystic kidney disease promotes mTOR-mediated cell proliferation.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressive enlargement of fluid-filled cysts, causing nephron loss and a decline in renal function. Mammalian target of rapamycin (mTOR) is overactive in cyst-lining cells and contributes to abnormal cell proliferation and cyst enlargement; however, the mechanism for mTOR stimulation remains unclear. We discovered that calcium/calmodulin (CaM) dependent kinase IV (CaMK4), a multifunctional kinase, is overexpressed in the kidneys of ADPKD patients and PKD mouse models. In human ADPKD cells, CaMK4 knockdown reduced mTOR abundance and the phosphorylation of ribosomal protein S6 kinase (S6K), a downstream target of mTOR. Pharmacologic inhibition of CaMK4 with KN-93 reduced phosphorylated S6K and S6 levels and inhibited cell proliferation and in vitro cyst formation of ADPKD cells. Moreover, inhibition of calcium/CaM-dependent protein kinase kinase-ß and CaM, two key upstream regulators of CaMK4, also decreased mTOR signaling. The effects of KN-93 were independent of the liver kinase B1-adenosine monophosphate-activated protein kinase (AMPK) pathway, and the combination of KN-93 and metformin, an AMPK activator, had additive inhibitory effects on mTOR signaling and in vitro cyst growth. Our data suggest that increased CaMK4 expression and activity contribute to mTOR signaling and the proliferation of cystic cells of ADPKD kidneys.

The lonidamine derivative H2-gamendazole reduces cyst formation in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is a debilitating renal neoplastic disorder with limited treatment options. It is characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl- secretion. We tested the effectiveness of the indazole carboxylic acid H2-gamendazole (H2-GMZ), a derivative of lonidamine, to inhibit these processes using in vitro and in vivo models of ADPKD. H2-GMZ was effective in rapidly blocking forskolin-induced, Cl--mediated short-circuit currents in human ADPKD cells, and it significantly inhibited both cAMP- and epidermal growth factor-induced proliferation of ADPKD cells. Western blot analysis of H2-GMZ-treated ADPKD cells showed decreased phosphorylated ERK and decreased hyperphosphorylated retinoblastoma levels. H2-GMZ treatment also decreased ErbB2, Akt, and cyclin-dependent kinase 4, consistent with inhibition of heat shock protein 90, and it decreased levels of the cystic fibrosis transmembrane conductance regulator Cl- channel protein. H2-GMZ-treated ADPKD cultures contained a higher proportion of smaller cells with fewer and smaller lamellipodia and decreased cytoplasmic actin staining, and they were unable to accomplish wound closure even at low H2-GMZ concentrations, consistent with an alteration in the actin cytoskeleton and decreased cell motility. Experiments using mouse metanephric organ cultures showed that H2-GMZ inhibited cAMP-stimulated cyst growth and enlargement. In vivo, H2-GMZ was effective in slowing postnatal cyst formation and kidney enlargement in the Pkd1flox/flox: Pkhd1-Cre mouse model. Thus, H2-GMZ treatment decreases Cl- secretion, cell proliferation, cell motility, and cyst growth. These properties, along with its reported low toxicity, suggest that H2-GMZ might be an attractive candidate for treatment of ADPKD.NEW & NOTEWORTHY Autosomal dominant polycystic kidney disease (ADPKD) is a renal neoplastic disorder characterized by the formation of large fluid-filled cysts that develop from kidney tubules through abnormal cell proliferation and cyst-filling fluid secretion driven by cAMP-dependent Cl- secretion. This study shows that the lonidamine derivative H2-GMZ inhibits Cl- secretion, cell proliferation, and cyst growth, suggesting that it might have therapeutic value for the treatment of ADPKD.

Expression of active B-Raf proto-oncogene in kidney collecting ducts induces cyst formation in normal mice and accelerates cyst growth in mice with polycystic kidney disease.

Polycystic kidney disease (PKD) is characterized by the formation and progressive enlargement of fluid-filled cysts due to abnormal cell proliferation. Cyclic AMP agonists, including arginine vasopressin, stimulate ERK-dependent proliferation of cystic cells, but not normal kidney cells. Previously, B-Raf proto-oncogene (BRAF), a MAPK kinase kinase that activates MEK-ERK signaling, was shown to be a central intermediate in the cAMP mitogenic response. However, the role of BRAF on cyst formation and enlargement in vivo had not been demonstrated. To determine if active BRAF induces kidney cyst formation, we generated transgenic mice that conditionally express BRAFV600E, a common activating mutation, and bred them with Pkhd1-Cre mice to express active BRAF in the collecting ducts, a predominant site for cyst formation. Collecting duct expression of BRAFV600E (BRafCD) caused kidney cyst formation as early as three weeks of age. There were increased levels of phosphorylated ERK (p-ERK) and proliferating cell nuclear antigen, a marker for cell proliferation. BRafCD mice developed extensive kidney fibrosis and elevated blood urea nitrogen, indicating a decline in kidney function, by ten weeks of age. BRAFV600E transgenic mice were also bred to Pkd1RC/RC and pcy/pcy mice, well-characterized slowly progressive PKD models. Collecting duct expression of active BRAF markedly increased kidney weight/body weight, cyst number and size, and total cystic area. There were increased p-ERK levels and proliferating cells, immune cell infiltration, interstitial fibrosis, and a decline in kidney function in both these models. Thus, our findings demonstrate that active BRAF is sufficient to induce kidney cyst formation in normal mice and accelerate cystic disease in PKD mice.

Generation of primary cells from ADPKD and normal human kidneys.

Autosomal dominant polycystic kidney (ADPKD) is a common genetic disorder characterized by the presence of numerous fluid-filled cysts that lead to a progressive decline in renal function. Cystic tissues and primary cyst epithelial cells obtained from discarded human ADPKD kidneys provide unique biomaterials for the investigation of cellular mechanisms involved in cyst growth and changes in the microenvironment adjacent to the cysts. ADPKD cells have been used to develop straightforward in vitro cell model assays to study events down-stream of the mutant proteins in carefully controlled experimental conditions, test specific hypotheses, and evaluate the cellular response to potential therapeutic drugs. Normal cadaver kidneys deemed unsuitable for transplantation and "non-involved" portions of nephrectomy specimens removed for the treatment of kidney cancer provide important control tissues and the source of primary normal human kidney (NHK) cells for comparison to ADPKD specimens. This chapter describes the methods used in the collection of cystic and non-cystic tissues from ADPKD and normal kidneys and the generation of primary cell cultures. We also highlight strengths and weaknesses of using immortalized isogenic normal and PKD mutant cell lines.

In vitro cyst formation of ADPKD cells.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by the relentless growth of numerous fluid-filled cysts in the kidneys. Mutations in PKD1 and PKD2, genes that encode polycystin 1 and 2, respectively, are responsible for most cases of ADPKD. Currently, the cellular mechanisms responsible for cyst formation remain poorly understood. In vitro models have been used by researchers to investigate cellular processes for cyst formation in carefully controlled experimental conditions. Madin-Darby canine kidney (MDCK) cells, a distal tubule epithelial cell line, were first used to form 3-dimensional (3-D) cysts within a hydrated collagen gel. This method was applied to epithelial cells cultured from cysts of human ADPKD kidneys, allowing investigators to study cellular mechanisms for cyst growth using cells that harbor the genetic mutations responsible for ADPKD in humans. Studies using ADPKD in vitro cysts have provided insight into cellular processes regulating cell proliferation, fluid secretion, and cell polarity. These assays were used to demonstrate the central role of cAMP agonists, such as arginine vasopressin, on cyst growth; and to test the effectiveness of potential therapeutic agents, including tolvaptan. Results obtained from in vitro cyst experiments demonstrate the translational value of cell model systems for investigating the mechanisms for cyst formation in human ADPKD. In this chapter, we describe protocols for growing ADPKD cells in a 3-D in vitro cyst assay and measuring total cyst volume by microscopy and image analysis.

ADPKD cell proliferation and Cl--dependent fluid secretion.

Autosomal dominant polycystic kidney disease (ADPKD) is a common genetic disorder characterized by bilateral fluid-filled cysts, renal inflammation and extensive fibrosis, leading to the progressive decline in kidney function. Renal cyst formation begins in utero from aberrant proliferation of tubule epithelial cells; however, the mechanisms for cystogenesis remain unclear. Cell proliferation and Cl--dependent fluid secretion, which drives the accumulation of cyst fluid, are responsible for inexorable growth of cysts and the remarkable appearance of massively enlarged ADPKD kidneys. Investigators have used in vitro assays to explore cellular and molecular mechanisms involved in ADPKD cyst epithelial cell proliferation and Cl--dependent fluid secretion in experimentally controlled environments. These assays have been used to evaluate potential therapeutic approaches to inhibit cellular pathways involved in cyst growth. This chapter discusses methods for measuring ADPKD cell proliferation, transepithelial Cl- secretion, and net fluid transport across cyst epithelial cell monolayers.

Periostin overexpression in collecting ducts accelerates renal cyst growth and fibrosis in polycystic kidney disease.

In polycystic kidney disease (PKD), persistent activation of cell proliferation and matrix production contributes to cyst growth and fibrosis, leading to progressive deterioration of renal function. Previously, we showed that periostin, a matricellular protein involved in tissue repair, is overexpressed by cystic epithelial cells of PKD kidneys. Periostin binds αVß3-integrins and activates integrin-linked kinase (ILK), leading to Akt/mammalian target of rapamycin (mTOR)-mediated proliferation of human PKD cells. By contrast, periostin does not stimulate the proliferation of normal human kidney cells. This difference in the response to periostin is due to elevated expression of αVß3-integrins by cystic cells. To determine whether periostin accelerates cyst growth and fibrosis, we generated mice with conditional overexpression of periostin in the collecting ducts (CDs). Ectopic CD expression of periostin was not sufficient to induce cyst formation or fibrosis in wild-type mice. However, periostin overexpression in pcy/pcy ( pcy) kidneys significantly increased mTOR activity, cell proliferation, cyst growth, and interstitial fibrosis; and accelerated the decline in renal function. Moreover, CD-specific overexpression of periostin caused a decrease in the survival of pcy mice. These pathological changes were accompanied by increased renal expression of vimentin, α-smooth muscle actin, and type I collagen. We also found that periostin increased gene expression of pathways involved in repair, including integrin and growth factor signaling and ECM production, and it stimulated focal adhesion kinase, Rho GTPase, cytoskeletal reorganization, and migration of PKD cells. These results suggest that periostin stimulates signaling pathways involved in an abnormal tissue repair process that contributes to cyst growth and fibrosis in PKD.

Deficient transient receptor potential vanilloid type 4 function contributes to compromised [Ca2+]i homeostasis in human autosomal-dominant polycystic kidney disease cells.

Autosomal-dominant polycystic kidney disease (ADPKD) is a devastating disorder that is characterized by a progressive decline in renal function as a result of the development of fluid-filled cysts. Defective flow-mediated [Ca2+]i responses and disrupted [Ca2+]i homeostasis have been repeatedly associated with cyst progression in ADPKD. We have previously demonstrated that the transient receptor potential vanilloid type 4 (TRPV4) channel is imperative for flow-mediated [Ca2+]i responses in murine distal renal tubule cells. To determine whether compromised TRPV4 function contributes to aberrant Ca2+ regulation in ADPKD, we assessed TRPV4 function in primary cells that were cultured from ADPKD and normal human kidneys (NHKs). Single-channel TRPV4 activity and TRPV4-dependent Ca2+ influxes were drastically reduced in ADPKD cells, which correlated with distorted [Ca2+]i signaling. Whereas total TRPV4 protein levels were comparable in NHK and ADPKD cells, we detected a marked decrease in TRPV4 glycosylation in ADPKD cells. Tunicamycin-induced deglycosylation inhibited TRPV4 activity and compromised [Ca2+]i signaling in NHK cells. Overall, we demonstrate that TRPV4 glycosylation and channel activity are diminished in human ADPKD cells compared with NHK cells, and that this contributes significantly to the distorted [Ca2+]i dynamics. We propose that TRPV4 stimulation may be beneficial for restoring [Ca2+]i homeostasis in cyst cells, thereby interfering with ADPKD progression.-Tomilin, V., Reif, G. A., Zaika, O., Wallace, D. P., Pochynyuk, O. Deficient transient receptor potential vanilloid type 4 function contributes to compromised [Ca2+]i homeostasis in human autosomal-dominant polycystic kidney disease cells.

Integrin-Linked Kinase Signaling Promotes Cyst Growth and Fibrosis in Polycystic Kidney Disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by innumerous fluid-filled cysts and progressive deterioration of renal function. Previously, we showed that periostin, a matricellular protein involved in tissue repair, is markedly overexpressed by cyst epithelial cells. Periostin promotes cell proliferation, cyst growth, interstitial fibrosis, and the decline in renal function in PKD mice. Here, we investigated the regulation of these processes by the integrin-linked kinase (ILK), a scaffold protein that links the extracellular matrix to the actin cytoskeleton and is stimulated by periostin. Pharmacologic inhibition or shRNA knockdown of ILK prevented periostin-induced Akt/mammalian target of rapamycin (mTOR) signaling and ADPKD cell proliferation in vitro Homozygous deletion of ILK in renal collecting ducts (CD) of Ilkfl/fl ;Pkhd1-Cre mice caused tubule dilations, apoptosis, fibrosis, and organ failure by 10 weeks of age. By contrast, Ilkfl/+ ;Pkhd1-Cre mice had normal renal morphology and function and survived >1 year. Reduced expression of ILK in Pkd1fl/fl ;Pkhd1-Cre mice, a rapidly progressive model of ADPKD, decreased renal Akt/mTOR activity, cell proliferation, cyst growth, and interstitial fibrosis, and significantly improved renal function and animal survival. Additionally, CD-specific knockdown of ILK strikingly reduced renal cystic disease and fibrosis and extended the life of pcy/pcy mice, a slowly progressive PKD model. We conclude that ILK is critical for maintaining the CD epithelium and renal function and is a key intermediate for periostin activation of signaling pathways involved in cyst growth and fibrosis in PKD.

Ouabain promotes partial epithelial to mesenchymal transition (EMT) changes in human autosomal dominant polycystic kidney disease (ADPKD) cells.

The hormone ouabain has been shown to enhance the cystic phenotype of autosomal dominant polycystic kidney disease (ADPKD). Among other characteristics, the ADPKD phenotype includes cell de-differentiation and epithelial to mesenchymal transition (EMT). Here, we determined whether physiological concentrations of ouabain induces EMT in human renal epithelial cells from patients with ADPKD. We found that ADPKD cells respond to ouabain with a decrease in expression of the epithelial marker E-cadherin and increase in the expression of the mesenchymal markers N-cadherin, α smooth muscle actin (αSMA) and collagen-I; and the tight junction protein occludin and claudin-1. Other adhesion molecules, such as ZO-1, ß-catenin and vinculin were not significantly modified by ouabain. At the cellular level, ouabain stimulated ADPKD cell migration, reduced cell-cell interaction, and the ability of ADPKD cells to form aggregates. Moreover, ouabain increased the transepithelial electrical resistance of ADPKD cell monolayers, suggesting that the paracellular transport pathway was preserved in the cells. These effects of ouabain were not observed in normal human kidney (NHK) cells. Altogether these results show a novel role for ouabain in ADPKD, inducing changes that lead to a partial EMT phenotype in the cells. These effects further support the key role that ouabain has as a factor that promotes the cystic characteristics of ADPKD cells.