Polycystin-1 Interacting Protein-1 (CU062) Interacts with the Ectodomain of Polycystin-1 (PC1).

The PKD1 gene, encoding protein polycystin-1 (PC1), is responsible for 85% of cases of autosomal dominant polycystic kidney disease (ADPKD). PC1 has been shown to be present in urinary exosome-like vesicles (PKD-ELVs) and lowered in individuals with germline PKD1 mutations. A label-free mass spectrometry comparison of urinary PKD-ELVs from normal individuals and those with PKD1 mutations showed that several proteins were reduced to a degree that matched the decrease observed in PC1 levels. Some of these proteins, such as polycystin-2 (PC2), may be present in a higher-order multi-protein assembly with PC1-the polycystin complex (PCC). CU062 (Q9NYP8) is decreased in ADPKD PKD-ELVs and, thus, is a candidate PCC component. CU062 is a small glycoprotein with a signal peptide but no transmembrane domain and can oligomerize with itself and interact with PC1. We investigated the localization of CU062 together with PC1 and PC2 using immunofluorescence (IF). In nonconfluent cells, all three proteins were localized in close proximity to focal adhesions (FAs), retraction fibers (RFs), and RF-associated extracellular vesicles (migrasomes). In confluent cells, primary cilia had PC1/PC2/CU062 + extracellular vesicles adherent to their plasma membrane. In cells exposed to mitochondrion-decoupling agents, we detected the development of novel PC1/CU062 + ring-like structures that entrained swollen mitochondria. In contact-inhibited cells under mitochondrial stress, PC1, PC2, and CU062 were observed on large, apically budding extracellular vesicles, where the proteins formed a reticular network on the membrane. CU062 interacts with PC1 and may have a role in the identification of senescent mitochondria and their extrusion in extracellular vesicles.

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.

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.

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.

Overexpression of TGF-ß1 induces renal fibrosis and accelerates the decline in kidney function in polycystic kidney disease.

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the presence of numerous fluid-filled cysts, extensive fibrosis, and the progressive decline in kidney function. Transforming growth factor-ß1 (TGF-ß1), an important mediator for renal fibrosis and chronic kidney disease, is overexpressed by cystic cells compared with normal kidney cells; however, its role in PKD pathogenesis remains undefined. To investigate the effect of TGF-ß1 on cyst growth, fibrosis, and disease progression, we overexpressed active TGF-ß1 specifically in collecting ducts (CDs) of phenotypic normal (Pkd1RC/+) and Pkd1RC/RC mice. In normal mice, CD-specific TGF-ß1 overexpression caused tubule dilations by 5 wk of age that were accompanied by increased levels of phosphorylated SMAD3, α-smooth muscle actin, vimentin, and periostin; however, it did not induce overt cyst formation by 20 wk. In Pkd1RC/RC mice, CD overexpression of TGF-ß1 increased cyst epithelial cell proliferation. However, extensive fibrosis limited cyst enlargement and caused contraction of the kidneys, leading to a loss of renal function and a shortened lifespan of the mice. These data demonstrate that TGF-ß1-induced fibrosis constrains cyst growth and kidney enlargement and accelerates the decline of renal function, supporting the hypothesis that a combined therapy that inhibits renal cyst growth and fibrosis will be required to effectively treat ADPKD.

Extracellular matrix, integrins, and focal adhesion signaling in polycystic kidney disease.

In autosomal dominant polycystic kidney disease (ADPKD), the inexorable growth of numerous fluid-filled cysts leads to massively enlarged kidneys, renal interstitial damage, inflammation, and fibrosis, and progressive decline in kidney function. It has long been recognized that interstitial fibrosis is the most important manifestation associated with end-stage renal disease; however, the role of abnormal extracellular matrix (ECM) production on ADPKD pathogenesis is not fully understood. Early evidence showed that cysts in end-stage human ADPKD kidneys had thickened and extensively laminated cellular basement membranes, and abnormal regulation of gene expression of several basement membrane components, including collagens, laminins, and proteoglycans by cyst epithelial cells. These basement membrane changes were also observed in dilated tubules and small cysts of early ADPKD kidneys, indicating that ECM alterations were early features of cyst development. Renal cystic cells were also found to overexpress several integrins and their ligands, including ECM structural components and soluble matricellular proteins. ECM ligands binding to integrins stimulate focal adhesion formation and can promote cell attachment and migration. Abnormal expression of laminin-332 (laminin-5) and its receptor α6ß4 stimulated cyst epithelial cell proliferation; and mice that lacked laminin α5, a component of laminin-511 normally expressed by renal tubules, had an overexpression of laminin-332 that was associated with renal cyst formation. Periostin, a matricellular protein that binds αVß3- and αVß5-integrins, was found to be highly overexpressed in the kidneys of ADPKD and autosomal recessive PKD patients, and several rodent models of PKD. αVß3-integrin is also overexpressed by cystic epithelial cells, and the binding of periostin to αVß3-integrin activates the integrin-linked kinase and downstream signal transduction pathways involved in tissue repair promoting cyst growth, ECM synthesis, and tissue fibrosis. This chapter reviews the roles of the ECM, integrins, and focal adhesion signaling in cyst growth and fibrosis in PKD.

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.

Phosphodiesterase Isoform Regulation of Cell Proliferation and Fluid Secretion in Autosomal Dominant Polycystic Kidney Disease.

cAMP stimulates cell proliferation and Cl(-)-dependent fluid secretion, promoting the progressive enlargement of renal cysts in autosomal dominant polycystic kidney disease (ADPKD). Intracellular cAMP levels are determined by the balance of cAMP synthesis by adenylyl cyclases and degradation by phosphodiesterases (PDEs). Therefore, PDE isoform expression and activity strongly influence global and compartmentalized cAMP levels. We report here that PDE3 and PDE4 expression levels are lower in human ADPKD tissue and cells compared with those of normal human kidneys (NHKs), whereas PDE1 levels are not significantly different. Inhibition of PDE4 caused a greater increase in basal and vasopressin (AVP)-stimulated cAMP levels and Cl(-) secretion by ADPKD cells than inhibition of PDE1, and inhibition of PDE4 induced cyst-like dilations in cultured mouse Pkd1(-/-) embryonic kidneys. In contrast, inhibition of PDE1 caused greater stimulation of extracellular signal-regulated kinase (ERK) and proliferation of ADPKD cells than inhibition of PDE4, and inhibition of PDE1 enhanced AVP-induced ERK activation. Notably, inhibition of PDE1, the only family of Ca(2+)-regulated PDEs, also induced a mitogenic response to AVP in NHK cells, similar to the effect of restricting intracellular Ca(2+). PDE1 coimmunoprecipitated with B-Raf and A-kinase anchoring protein 79, and AVP increased this interaction in ADPKD but not NHK cells. These data suggest that whereas PDE4 is the major PDE isoform involved in the regulation of global intracellular cAMP and Cl(-) secretion, PDE1 specifically affects the cAMP signal to the B-Raf/MEK/ERK pathway and regulates AVP-induced proliferation of ADPKD cells.

Ouabain Regulates CFTR-Mediated Anion Secretion and Na,K-ATPase Transport in ADPKD Cells.

Cyst enlargement in autosomal dominant polycystic kidney disease (ADPKD) requires the transepithelial secretion of fluid into the cyst lumen. We previously showed that physiological amounts of ouabain enhance cAMP-dependent fluid secretion and cyst growth of human ADPKD cyst epithelial cells in culture and formation of cyst-like dilations in metanephric kidneys from Pkd1 mutant mice. Here, we investigated the mechanisms by which ouabain promotes cAMP-dependent fluid secretion and cystogenesis. Ouabain (3 nM) enhanced cAMP-induced cyst-like dilations in embryonic kidneys from Pkd1 (m1Bei) mice, but had no effect on metanephroi from Pkd1 (m1Bei) mice that lack expression of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, ouabain stimulation of cAMP-induced fluid secretion and in vitro cyst growth of ADPKD cells were abrogated by CFTR inhibition, showing that CFTR is required for ouabain effects on ADPKD fluid secretion. Moreover, ouabain directly enhanced the cAMP-dependent Cl(-) efflux mediated by CFTR in ADPKD monolayers. Ouabain increased the trafficking of CFTR to the plasma membrane and up-regulated the expression of the CFTR activator PDZK1. Finally, ouabain decreased plasma membrane expression and activity of the Na,K-ATPase in ADPKD cells. Altogether, these results show that ouabain enhances net fluid secretion and cyst formation by activating apical anion secretion via CFTR and decreasing basolateral Na(+) transport via Na,K-ATPase. These results provide new information on the mechanisms by which ouabain affects ADPKD cells and further highlight the importance of ouabain as a non-genomic stimulator of cystogenesis in ADPKD.

Periostin promotes renal cyst growth and interstitial fibrosis in polycystic kidney disease.

In renal cystic diseases, sustained enlargement of fluid-filled cysts is associated with severe interstitial fibrosis and progressive loss of functioning nephrons. Periostin, a matricellular protein, is highly overexpressed in cyst-lining epithelial cells of autosomal-dominant polycystic disease kidneys (ADPKD) compared with normal tubule cells. Periostin accumulates in situ within the matrix subjacent to ADPKD cysts, binds to αVß3 and αVß5 integrins, and stimulates the integrin-linked kinase to promote cell proliferation. We knocked out periostin (Postn) in pcy/pcy mice, an orthologous model of nephronophthisis type 3, to determine whether periostin loss reduces PKD progression in a slowly progressive model of renal cystic disease. At 20 weeks of age, pcy/pcy:Postn(-/-) mice had a 34% reduction in kidney weight/body weight, a reduction in cyst number and total cystic area, a 69% reduction in phosphorylated S6, a downstream component of the mTOR pathway, and fewer proliferating cells in the kidneys compared with pcy/pcy:Postn(+/+) mice. The pcy/pcy Postin knockout mice also had less interstitial fibrosis with improved renal function at 20 weeks and significantly longer survival (51.4 compared with 38.0 weeks). Thus, periostin adversely modifies the progression of renal cystic disease by promoting cyst epithelial cell proliferation, cyst enlargement, and interstitial fibrosis, all contributing to the decline in renal function and premature death.

Calmodulin-sensitive adenylyl cyclases mediate AVP-dependent cAMP production and Cl- secretion by human autosomal dominant polycystic kidney cells.

In autosomal dominant polycystic kidney disease (ADPKD), binding of AVP to the V2 receptor (V2R) increases cAMP and accelerates cyst growth by stimulating cell proliferation and Cl(-)-dependent fluid secretion. Basal cAMP is elevated in human ADPKD cells compared with normal human kidney (NHK) cells. V2R mRNA levels are elevated in ADPKD cells; however, AVP caused a greater increase in global cAMP in NHK cells, suggesting an intrinsic difference in cAMP regulation. Expression, regulatory properties, and receptor coupling of specific adenylyl cyclases (ACs) provide temporal and spatial regulation of the cAMP signal. ADPKD and NHK cells express mRNAs for all nine ACs. Ca(2+)-inhibited ACs 5 and 6 are increased in ADPKD cells, while Ca(2+)/CaM-stimulated ACs 1 and 3 are downregulated. ACs 1, 3, 5, and 6 were detected in cyst cells in situ, and codistribution with aquaporin-2 suggests that these cysts were derived from collecting ducts. To determine the contribution of CaM-sensitive ACs to AVP signaling, cells were treated with W-7, a CaM inhibitor. W-7 decreased AVP-induced cAMP production and Cl(-) secretion by ADPKD cells. CaMKII inhibition increased AVP-induced cAMP, suggesting that cAMP synthesis is mediated by AC3. In contrast, CaM and CaMKII inhibition in NHK cells did not affect AVP-induced cAMP production. Restriction of intracellular Ca(2+) switched the response in NHK cells, such that CaM inhibition decreased AVP-induced cAMP production. We suggest that a compensatory response to decreased Ca(2+) in ADPKD cells switches V2R coupling from Ca(2+)-inhibited ACs 5/6 to Ca(2+)/CaM-stimulated AC3, to mitigate high cAMP levels in response to continuous AVP stimulation.

Endogenous concentrations of ouabain act as a cofactor to stimulate fluid secretion and cyst growth of in vitro ADPKD models via cAMP and EGFR-Src-MEK pathways.

In autosomal-dominant polycystic kidney disease (ADPKD), renal cysts develop by aberrant epithelial cell proliferation and transepithelial fluid secretion. We previously showed that ouabain increases proliferation of cultured human ADPKD cells via stimulation of the EGF receptor (EGFR)-Src-MEK/ERK signaling pathway. We examined whether ouabain affects fluid secretion and in vitro cyst growth of human ADPKD cell monolayers, ADPKD cell microcysts cultured in a three-dimensional collagen matrix, and metanephric organ cultures from Pkd1(m1Bei) mice. Physiological concentrations of ouabain alone did not affect net transepithelial basal-to-apical fluid transport in ADPKD monolayers or growth of cultured ADPKD microcysts. In contrast, in the presence of forskolin or 8-bromo-cAMP, ouabain significantly enhanced ADPKD fluid secretion and microcyst expansion. Ouabain exerted this effect by enhancing cAMP-dependent Cl(-) secretion via the CFTR. Similarly, ouabain accelerated cAMP-dependent cyst enlargement in Pkd1(m1Bei) mice metanephroi, with a more prominent response in homozygous than heterozygous mice. Ouabain had no effect on fluid secretion and cystogenesis of normal human kidney cells and caused only slight cystic dilations in wild-type mouse kidneys. The effects of ouabain in ADPKD cells and Pkd1(m1Bei) metanephroi were prevented by inhibitors of EGFR (AG1478), Src (PP2), and MEK (U0126). Together, our results show that ouabain, used in physiological concentrations, has synergistic effects on cAMP-mediated fluid secretion and cyst growth, via activation of the EGFR-Src-MEK pathway. These data provide important evidence for the role of ouabain as an endogenous hormone that exacerbates ADPKD cyst progression.

Tolvaptan inhibits ERK-dependent cell proliferation, Cl⁻ secretion, and in vitro cyst growth of human ADPKD cells stimulated by vasopressin.

In autosomal dominant polycystic kidney disease (ADPKD), arginine vasopressin (AVP) accelerates cyst growth by stimulating cAMP-dependent ERK activity and epithelial cell proliferation and by promoting Cl(-)-dependent fluid secretion. Tolvaptan, a V2 receptor antagonist, inhibits the renal effects of AVP and slows cyst growth in PKD animals. Here, we determined the effect of graded concentrations of tolvaptan on intracellular cAMP, ERK activity, cell proliferation, and transcellular Cl(-) secretion using human ADPKD cyst epithelial cells. Incubation of ADPKD cells with 10(-9) M AVP increased intracellular cAMP and stimulated ERK and cell proliferation. Tolvaptan caused a concentration-dependent inhibition of AVP-induced cAMP production with an apparent IC(50) of ∼10(-10) M. Correspondingly, tolvaptan inhibited AVP-induced ERK signaling and cell proliferation. Basolateral application of AVP to ADPKD cell monolayers grown on permeable supports caused a sustained increase in short-circuit current that was completely blocked by the Cl(-) channel blocker CFTR(inh-172), consistent with AVP-induced transepithelial Cl(-) secretion. Tolvaptan inhibited AVP-induced Cl(-) secretion and decreased in vitro cyst growth of ADPKD cells cultured within a three-dimensional collagen matrix. These data demonstrate that relatively low concentrations of tolvaptan inhibit AVP-stimulated cell proliferation and Cl(-)-dependent fluid secretion by human ADPKD cystic cells.

Ouabain activates the Na-K-ATPase signalosome to induce autosomal dominant polycystic kidney disease cell proliferation.

The Na-K-ATPase is part of a cell signaling complex, the Na-K-ATPase signalosome, which upon activation by the hormone ouabain regulates the function of different cell types. We previously showed that ouabain induces proliferation of epithelial cells derived from renal cysts of patients with autosomal dominant polycystic kidney disease (ADPKD cells). Here, we investigated the signaling pathways responsible for mediating the effects of ouabain in these cells. Incubation of ADPKD cells with ouabain, in concentrations similar to those found in blood, stimulated phosphorylation of the epidermal growth factor receptor (EGFR) and promoted its association to the Na-K-ATPase. In addition, ouabain activated the kinase Src, but not the related kinase Fyn. Tyrphostin AG1478 and PP2, inhibitors of EGFR and Src, respectively, blocked ouabain-dependent ADPKD cell proliferation. Treatment of ADPKD cells with ouabain also caused phosphorylation of the caveolar protein caveolin-1, and disruption of cell caveolae with methyl-ß-cyclodextrin prevented Na-K-ATPase-EGFR interaction and ouabain-induced proliferation of the cells. Downstream effects of ouabain in ADPKD cells included activation of B-Raf and MEK and phosphorylation of the extracellular regulated kinase ERK, which translocated into the ADPKD cell nuclei. Finally, ouabain reduced expression of the cyclin-dependent kinase inhibitors p21 and p27, which are suppressors of cell proliferation. Different from ADPKD cells, ouabain showed no significant effect on B-Raf, p21, and p27 in normal human kidney epithelial cells. Altogether, these results identify intracellular pathways of ouabain-dependent Na-K-ATPase-mediated signaling in ADPKD cells, including EGFR-Src-B-Raf-MEK/ERK, and establish novel mechanisms involved in ADPKD cell proliferation.