Autosomal Dominant Polycystic Kidney Disease Cells Research Articles

Acyl-CoA thioesterase 13 (ACOT13) attenuates the progression of autosomal dominant polycystic kidney disease in vitro via triggering mitochondrial-related cell apoptosis.

Autosomal dominant polycystic kidney disease (ADPKD) is the most common cause of end-stage kidney disease. It has been shown that Acyl-CoA thioesterase 13 (ACOT13) level was reduced in renal cystic tissues from ADPKD patients. However, the role of ACOT13 in ADPKD remains largely elusive. The data in the GSE7869 dataset were acquired from the GEO database to determine ACOT13 level between normal renal cortical tissues and renal cystic tissues. Next, the potential functions of ACOT13 were explored by gene set enrichment analysis (GSEA). Furthermore, ACOT13 level in ADPKD cells (WT9-12) was verified by RT-qPCR. The effects of ACOT13 on WT9-12 cell growth were evaluated using the EdU staining and flow cytometry assays. Compared to normal group, ACOT13 mRNA level was obviously reduced in renal cystic tissues and WT9-12 cells. Meanwhile, GSEA results showed that compared to the low ACOT13 expression group, PI3K-Akt and MAPK signaling pathways were inactivated, and PPAR signaling pathway and fatty acid metabolism were activated in high ACOT13 expression group. Furthermore, overexpression of ACOT13 notably reduced WT9-12 cell proliferation and triggered cell cycle arrest. Moreover, ACOT13 overexpression remarkably triggered apoptosis, increased cleaved caspase 3 protein level, reduced ATP production and induced loss of mitochondrial membrane potential in WT9-12 cells, suggesting that ACOT13 overexpression could trigger mitochondrial-related apoptosis in WT9-12 cells. Collectively, our results showed that overexpression of ACOT13 could suppress WT9-12 cell proliferation and trigger mitochondrial-mediated cell apoptosis, suggesting that ACOT13 may exert a protective role in ADPKD.

#2048 Exploring somatic mutation mechanisms in pre-cystic renal cells of autosomal dominant polycystic kidney disease patients

Abstract Background and Aims Genetic changes accumulate in our cells during a lifetime, at a rate that varies according to cell type and mutagen exposure [1]. Somatic mutations are the driving force of cancer and contribute to “second hit” disorders, such as Autosomal Dominant Polycystic Kidney Disease (ADPKD). ADPKD is caused by inherited mutations that disrupt one allele of PKD1 or PKD2 genes. Somatic loss of the second allele triggers tubule cell clonal expansion and the formation of renal cysts. Given the high number of cysts that form during a lifetime, somatic mutation rates may be abnormally high in the kidney of ADPKD patients. The identification of factors enhancing somatic mutation rates can lead to preventive strategies to limit cyst formation. Metabolic alterations, i.e. increased glutamine utilization and reduced urea cycle, have been shown to induce somatic mutations in cancer [2]. Loss of urea cycle increases pyrimidine synthesis, which unbalances the nucleotide pools and results in a distinct signature of single base substitutions (SBSs) [1]. Similar metabolic changes have been observed in ADPKD [3, 4]. Thus, we sought to test metabolism-driven somatic mutagenesis in pre-cystic kidneys and its contribution to second hit mutations in ADPKD. Method We clonally expanded normal kidney cells from human urine samples and performed a gene expression study by qPCR. We studied 31 clones from 4 ADPKD patients (age range: 25-45) with truncating mutations in PKD1 and normal kidney function, despite a clear cystic phenotype. ADPKD cells were compared to 55 clones from 5 healthy volunteers (age range: 24-53), and 32 clones from 6 patients with another cystic kidney disease, Von Hippel Lindau disease (VHL; age range: 29-56). A subset of clones (n = 19 controls, n = 12 ADPKD) was subjected to whole genome sequencing and somatic mutation analysis. Results Urines of ADPKD patients contained higher numbers of cells that expanded in vitro, compared to both control (p = 0.0008) and VHL individuals (p = 0.01). Gene expression analyses showed that irrespective of genetic background, all cultured clones originated from the kidney tubule epithelium (high PAX2 and PAX8), and some originated from damaged tubules (VCAM1, KIM1). We tested the hypothesized metabolic rewiring by analyzing expression levels of enzymes involved in glutamine utilization, urea cycle, and pyrimidine biosynthesis. Except for ASNS (Asparagine Synthetase), which was higher in ADPKD vs control clones (p = 0.0098), no gene-expression differences were observed. However, clones from ADPKD patients showed signs of the metabolic rewiring responsible for increased pyrimidine production, i.e. a positive correlation (r = 0.691; p < 0.0001) between urea cycle enzymes downregulation and upregulation of the pyrimidine synthesis enzyme CAD. Since excessive pyrimidines lead to mutation [2], we analyzed the number of somatic SBSs per genome, after filtering for germline variants. We did not find increased mutation rates in ADPKD compared to controls, but an analogous, linear increase of mutations with age and similar levels of the pyrimidine-rich mutational signature. Conclusion Urine-derived kidney tubule epithelial cells with heterozygous truncating mutations in PKD1 exhibit certain characteristics of metabolic reprogramming typical of kidneys from ADPKD patients with advanced pathology. Nevertheless, in the limited number of pre-cystic cells that we have analyzed, this metabolic reprogramming did not translate into an excess of somatic mutations.

The Link between Autosomal Dominant Polycystic Kidney Disease and Chromosomal Instability: Exploring the Relationship.

In autosomal dominant polycystic kidney disease (ADPKD) with germline mutations in a PKD1 or PKD2 gene, innumerable cysts develop from tubules, and renal function deteriorates. Second-hit somatic mutations and renal tubular epithelial (RTE) cell death are crucial features of cyst initiation and disease progression. Here, we use established RTE lines and primary ADPKD cells with disease-associated PKD1 mutations to investigate genomic instability and DNA damage responses. We found that ADPKD cells suffer severe chromosome breakage, aneuploidy, heightened susceptibility to DNA damage, and delayed checkpoint activation. Immunohistochemical analyses of human kidneys corroborated observations in cultured cells. DNA damage sensors (ATM/ATR) were activated but did not localize at nuclear sites of damaged DNA and did not properly activate downstream transducers (CHK1/CHK2). ADPKD cells also had the ability to transform, as they achieved high saturation density and formed colonies in soft agar. Our studies indicate that defective DNA damage repair pathways and the somatic mutagenesis they cause contribute fundamentally to the pathogenesis of ADPKD. Acquired mutations may alternatively confer proliferative advantages to the clonally expanded cell populations or lead to apoptosis. Further understanding of the molecular details of aberrant DNA damage responses in ADPKD is ongoing and holds promise for targeted therapies.

The tryptophan-metabolizing enzyme indoleamine 2,3-dioxygenase 1 regulates polycystic kidney disease progression.

Autosomal dominant polycystic kidney disease (ADPKD), the most common monogenic nephropathy, is characterized by phenotypic variability that exceeds genic effects. Dysregulated metabolism and immune cell function are key disease modifiers. The tryptophan metabolites, kynurenines, produced through indoleamine 2,3-dioxygenase 1 (IDO1), are known immunomodulators. Here, we study the role of tryptophan metabolism in PKD using an orthologous disease model (C57BL/6J Pkd1RC/RC). We found elevated kynurenine and IDO1 levels in Pkd1RC/RC kidneys versus wild type. Further, IDO1 levels were increased in ADPKD cell lines. Genetic Ido1 loss in Pkd1RC/RC animals resulted in reduced PKD severity, as measured by cystic index and percentage kidney weight normalized to body weight. Consistent with an immunomodulatory role of kynurenines, Pkd1RC/RC;Ido1-/- mice presented with significant changes in the cystic immune microenvironment (CME) versus controls. Kidney macrophage numbers decreased and CD8+ T cell numbers increased, both known PKD modulators. Also, pharmacological IDO1 inhibition in Pkd1RC/RC mice and kidney-specific Pkd2-knockout mice with rapidly progressive PKD resulted in less severe PKD versus controls, with changes in the CME similar to those in the genetic model. Our data suggest that tryptophan metabolism is dysregulated in ADPKD and that its inhibition results in changes to the CME and slows disease progression, making IDO1 a therapeutic target for ADPKD.

The inhibition of MDM2 slows cell proliferation and activates apoptosis in ADPKD cell lines.

Autosomal dominant polycystic kidney disease (ADPKD) is characterised by progressive cysts formation and renal enlargement that in most of cases leads to end stage of renal disease (ESRD). This pathology is caused by mutations of either PKD1 or PKD2 genes that encode for polycystin-1 (PC1) and polycystin-2 (PC2), respectively. These proteins function as receptor-channel complex able to regulate calcium homeostasis. PKD1/2 loss of function impairs different signalling pathways including cAMP and mTOR that are considered therapeutic targets for this disease. In fact, Tolvaptan, a vasopressin-2 antagonist that reduces cAMP levels, is the only drug approved for ADPKD treatment. Nevertheless, some ADPKD patients developed side effects in response to Tolvaptan including liver damage. Conversely, mTOR inhibitors that induced disease regression in ADPKD animal models failed the clinical trials. Here, we show that the inhibition of mTOR causes the activation of autophagy in ADPKD cells that could reduce therapy effectiveness by drug degradation through the autophagic vesicles. Consistently, the combined treatment with rapamycin and chloroquine, an autophagy inhibitor, potentiates the decrease of cell proliferation induced by rapamycin. To overcome the dangerous activation of autophagy by mTOR inhibition, we targeted MDM2 (a downstream effector of mTOR signalling) that is involved in TP53 degradation by using RG7112, a small-molecule MDM2 inhibitor used for the treatment of haematologic malignancies. The inhibition of MDM2 by RG7112 prevents TP53 degradation and increases p21 expression leading to the decrease of cell proliferation and the activation of apoptosis. The targeting of MDM2 by RG7112 might represent a new therapeutic option for the treatment of ADPKD.

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.

Secreted Neutrophil Gelatinase-Associated Lipocalin Shows Stronger Ability to Inhibit Cyst Enlargement of ADPKD Cells Compared with Nonsecreted Form.

Polycystic kidney disease (PKD) is one of the most common inherited diseases and is characterized by the development of fluid-filled cysts along multiple segments of the nephron. Autosomal dominant polycystic kidney disease (ADPKD) is the most common form of PKD, which is caused by mutations in either PKD1 or PKD2 genes that encode polycystin-1 (PC1) and polycystin-2 (PC2), respectively. As ADPKD progresses, cysts enlarge and disrupt normal kidney architecture, eventually leading to kidney failure. Our previous study showed that overexpression of exogenous kidney-specific neutrophil gelatinase-associated lipocalin (NGAL) reduced cyst progression and prolonged the lifespan of ADPKD mice (Pkd1L3/L3, 2L3 for short). In this study, we attempted to explore the underlying mechanism of reduced cyst progression in the presence of NGAL using immortalized 2L3 cells. The results of MTT and BrdU incorporation assays showed that recombinant mouse NGAL (mNGAL) protein significantly decreased the viability and proliferation of 2L3 cells. Flow cytometry and western blot analyses showed that mNGAL inhibited activation of the ERK and AKT pathways and induced apoptosis and autophagy in 2L3 cells. In addition, a 3D cell culture platform was established to identify cyst progression in 2L3 cells and showed that mNGAL significantly inhibited cyst enlargement in 2L3 cells. Overexpression of secreted mNGAL (pN + LS) and nonsecreted mNGAL (pN − LS) repressed cell proliferation and cyst enlargement in 2L3 cells and had effects on markers involved in proliferation, apoptosis, and autophagy. However, secreted mNGAL had a more pronounced and consistent effect than that of nonsecreted form. These results reveal that secreted mNGAL has stronger ability to inhibit cyst enlargement of ADPKD cells than that of nonsecreted form. These findings could help to identify strategies for the future clinical treatment of ADPKD.

Interdependent Regulation of Polycystin Expression Influences Starvation-Induced Autophagy and Cell Death.

Autosomal dominant polycystic kidney disease (ADPKD) is mainly caused by deficiency of polycystin-1 (PC1) or polycystin-2 (PC2). Altered autophagy has recently been implicated in ADPKD progression, but its exact regulation by PC1 and PC2 remains unclear. We therefore investigated cell death and survival during nutritional stress in mouse inner medullary collecting duct cells (mIMCDs), either wild-type (WT) or lacking PC1 (PC1KO) or PC2 (PC2KO), and human urine-derived proximal tubular epithelial cells (PTEC) from early-stage ADPKD patients with PC1 mutations versus healthy individuals. Basal autophagy was enhanced in PC1-deficient cells. Similarly, following starvation, autophagy was enhanced and cell death reduced when PC1 was reduced. Autophagy inhibition reduced cell death resistance in PC1KO mIMCDs to the WT level, implying that PC1 promotes autophagic cell survival. Although PC2 expression was increased in PC1KO mIMCDs, PC2 knockdown did not result in reduced autophagy. PC2KO mIMCDs displayed lower basal autophagy, but more autophagy and less cell death following chronic starvation. This could be reversed by overexpression of PC1 in PC2KO. Together, these findings indicate that PC1 levels are partially coupled to PC2 expression, and determine the transition from renal cell survival to death, leading to enhanced survival of ADPKD cells during nutritional stress.

Metabolic Reprogramming and Reconstruction: Integration of Experimental and Computational Studies to Set the Path Forward in ADPKD.

Metabolic reprogramming is a key feature of Autosomal Dominant Polycystic Kidney Disease (ADPKD) characterized by changes in cellular pathways occurring in response to the pathological cell conditions. In ADPKD, a broad range of dysregulated pathways have been found. The studies supporting alterations in cell metabolism have shown that the metabolic preference for abnormal cystic growth is to utilize aerobic glycolysis, increasing glutamine uptake and reducing oxidative phosphorylation, consequently resulting in ADPKD cells shifting their energy to alternative energetic pathways. The mechanism behind the role of the polycystin proteins and how it leads to disease remains unclear, despite the identification of numerous signaling pathways. The integration of computational data analysis that accompanies experimental findings was pivotal in the identification of metabolic reprogramming in ADPKD. Here, we summarize the important results and argue that their exploitation may give further insights into the regulative mechanisms driving metabolic reprogramming in ADPKD. The aim of this review is to provide a comprehensive overview on metabolic focused studies and potential targets for treatment, and to propose that computational approaches could be instrumental in advancing this field of research.

Metabolic derangement in polycystic kidney disease mouse models is ameliorated by mitochondrial-targeted antioxidants

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by progressively enlarging cysts. Here we elucidate the interplay between oxidative stress, mitochondrial dysfunction, and metabolic derangement using two mouse models of PKD1 mutation, PKD1RC/null and PKD1RC/RC. Mouse kidneys with PKD1 mutation have decreased mitochondrial complexes activity. Targeted proteomics analysis shows a significant decrease in proteins involved in the TCA cycle, fatty acid oxidation (FAO), respiratory complexes, and endogenous antioxidants. Overexpressing mitochondrial-targeted catalase (mCAT) using adeno-associated virus reduces mitochondrial ROS, oxidative damage, ameliorates the progression of PKD and partially restores expression of proteins involved in FAO and the TCA cycle. In human ADPKD cells, inducing mitochondrial ROS increased ERK1/2 phosphorylation and decreased AMPK phosphorylation, whereas the converse was observed with increased scavenging of ROS in the mitochondria. Treatment with the mitochondrial protective peptide, SS31, recapitulates the beneficial effects of mCAT, supporting its potential application as a novel therapeutic for ADPKD.

Role of DNA-Dependent Protein Kinase in Mediating Cyst Growth in Autosomal Dominant Polycystic Kidney Disease.

DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein involved in DNA damage response (DDR) signaling that may mediate kidney cyst growth in autosomal dominant polycystic kidney disease (ADPKD) due to its pleiotropic effects on proliferation and survival. To test this hypothesis, the expression of DNA-PK in human ADPKD and the in vitro effects of DNA-PK inhibition in a three-dimensional model of Madin-Darby Canine Kidney (MDCK) cyst growth and human ADPKD cells were assessed. In human ADPKD, the mRNA expression for all three subunits of the DNA-PK complex was increased, and using immunohistochemistry, the catalytic subunit (DNA-PKcs) was detected in the cyst lining epithelia of human ADPKD, in a focal manner. In vitro, NU7441 (a DNA-PK kinase inhibitor) reduced MDCK cyst growth by up to 52% after long-term treatment over 6–12 days. Although human ADPKD cell lines (WT9-7/WT9-12) did not exhibit synthetic lethality in response to DNA-PK kinase inhibition compared to normal human kidney cells (HK-2), the combination of low-dose NU7441 enhanced the anti-proliferative effects of sirolimus in WT9-7 and WT9-12 cells by 17 ± 10% and 11 ± 7%, respectively. In conclusion, these preliminary data suggest that DNA-PK mediates kidney cyst growth in vivo without a synthetically lethal interaction, conferring cell-specificity in human ADPKD cells. NU7441 enhanced the anti-proliferative effects of rapamycin complex 1 inhibitors, but the effect was modest.

Long-term dietary nitrate supplementation does not reduce renal cyst growth in experimental autosomal dominant polycystic kidney disease.

Augmentation of endogenous nitric oxide (NO) synthesis, either by the classical L-arginine-NO synthase pathway, or the recently discovered entero-salivary nitrate-nitrite-NO system, may slow the progression of autosomal dominant polycystic kidney disease (ADPKD). To test this hypothesis, the expression of NO in human ADPKD cell lines (WT 9-7, WT 9-12), and the effect of L-arginine on an in vitro model of three-dimensional cyst growth using MDCK cells, was examined. In addition, groups of homozygous Pkd1RC/RC mice (a hypomorphic genetic ortholog of ADPKD) received either low, moderate or high dose sodium nitrate (0.1, 1 or 10 mmol/kg/day), or sodium chloride (vehicle; 10 mmol/kg/day), supplemented drinking water from postnatal month 1 to 9 (n = 12 per group). In vitro, intracellular NO, as assessed by DAF-2/DA fluorescence, was reduced by >70% in human ADPKD cell lines, and L-arginine and the NO donor, sodium nitroprusside, both attenuated in vitro cyst growth by up to 18%. In contrast, in Pkd1RC/RC mice, sodium nitrate supplementation increased serum nitrate/nitrite levels by ~25-fold in the high dose group (P<0.001), but kidney enlargement and percentage cyst area was not altered, regardless of dose. In conclusion, L-arginine has mild direct efficacy on reducing renal cyst growth in vitro, whereas long-term sodium nitrate supplementation was ineffective in vivo. These data suggest that the bioconversion of dietary nitrate to NO by the entero-salivary pathway may not be sufficient to influence the progression of renal cyst growth in ADPKD.

Effect of Reducing Ataxia-Telangiectasia Mutated (ATM) in Experimental Autosomal Dominant Polycystic Kidney Disease.

The DNA damage response (DDR) pathway is upregulated in autosomal dominant polycystic kidney disease (ADPKD) but its functional role is not known. The ataxia-telangiectasia mutated (ATM) and AT and Rad3-related (ATR) protein kinases are key proximal transducers of the DDR. This study hypothesized that reducing either ATM or ATR attenuates kidney cyst formation and growth in experimental ADPKD. In vitro, pharmacological ATM inhibition by AZD0156 reduced three-dimensional cyst growth in MDCK and human ADPKD cells by up to 4.4- and 4.1-fold, respectively. In contrast, the ATR inhibitor, VE-821, reduced in vitro MDCK cyst growth but caused dysplastic changes. In vivo, treatment with AZD0156 by oral gavage for 10 days reduced renal cell proliferation and increased p53 expression in Pkd1RC/RC mice (a murine genetic ortholog of ADPKD). However, the progression of cystic kidney disease in Pkd1RC/RC mice was not altered by genetic ablation of ATM from birth, in either heterozygous (Pkd1RC/RC/Atm+/−) or homozygous (Pkd1RC/RC/Atm−/−) mutant mice at 3 months. In conclusion, despite short-term effects on reducing renal cell proliferation, chronic progression was not altered by reducing ATM in vivo, suggesting that this DDR kinase is dispensable for kidney cyst formation in ADPKD.

Up-Regulation of DNA Damage Response Signaling in Autosomal Dominant Polycystic Kidney Disease

DNA damage and alterations in DNA damage response (DDR) signaling could be one of the molecular mechanisms mediating focal kidney cyst formation in autosomal dominant polycystic kidney disease (ADPKD). The aim of this study was to test the hypothesis that markers of DNA damage and DDR signaling are increased in human and experimental ADPKD. In the human ADPKD transcriptome, the number of up-regulated DDR-related genes was increased by 16.6-fold compared with that in normal kidney, and by 2.5-fold in cystic compared with that in minimally cystic tissue (P<0.0001). In end-stage human ADPKD tissue, γ-H2A histone family member X (H2AX), phosphorylated ataxia telangiectasia and radiation-sensitive mutant 3 (Rad3)-related (pATR), and phosphorylated ataxia telangiectasia mutated (pATM) localized to cystic kidney epithelial cells. Invitro, pATR and pATM were also constitutively increased in human ADPKD tubular cells (WT 9-7 and 9-12) compared with control (HK-2). In addition, extrinsic oxidative DNA damage by hydrogen peroxide augmented γ-H2AX and cell survival in human ADPKD cells, and exacerbated cyst growth in the three-dimensional Madin-Darby canine kidney cyst model. In contrast, DDR-related gene expression was only transiently increased on postnatal day 0 in Pkd1RC/RC mice, and not altered at later time points up to 12 months of age. In conclusion, DDR signaling is dysregulated in human ADPKD and during the early phases of murine ADPKD. The constitutive expression of the DDR pathway in ADPKD may promote survival of PKD1-mutated cells and contribute to kidney cyst growth.

SNX9 Inhibits Cell Proliferation and Cyst Development in Autosomal Dominant Polycystic Kidney Disease via Activation of the Hippo-YAP Signaling Pathway.

Autosomal dominant polycystic kidney disease (ADPKD) is a complex process, involving the alteration of multiple genes and signaling pathways, and the pathogenesis of ADPKD remains largely unknown. Here, we demonstrated the suppressive role of sorting nexin 9 (SNX9) during ADPKD development. Sorting nexin 9 expression was detected in the kidney tissues of ADPKD patients, for the first time, and SNX9 expression was also detected in Pkd1 knockout (Pkd1–/–) and control mice. Subsequently, a series of gain- and loss-of-function studies were performed, to explore the biological roles and underlying molecular mechanisms of SNX9 in ADPKD progression. The expression of SNX9 was significantly downregulated in ADPKD patients and Pkd1–/– mice compared with control individuals and wild-type mice (Pkd1+/+), respectively. The ectopic expression of SNX9 significantly inhibited ADPKD cell proliferation, renal cyst formation and enlargement, whereas these effects were promoted by SNX9 silencing. Mechanistically, we found that SNX9 interacted directly with yes-associated protein (YAP) and increased the large tumor suppressor kinase 1-mediated phosphorylation of YAP, resulting in the cytoplasmic retention of YAP, the decreased transcriptional activity of the YAP/TEA domain transcription factor 4 complex, and, consequently, the inhibition of Hippo target gene expression and ADPKD development. Taken together, our findings provided novel insights into the role played by SNX9 during ADPKD pathogenesis and may reveal novel therapeutic approaches for ADPKD and related kidney diseases.

Cux1 regulates cilia length in polycystic kidney disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is one of the most common inherited disorders affecting the kidney. Renal cyst development in ADPKD results from mutations in the PKD1 or PKD2 genes, which encode the proteins polycstin1 (PC1) and polycystin2 (PC2), respectively. PC1 and PC2 proteins are localized to primary cilia where they are proposed to form a receptor channel complex that detects flow transmitting a calcium‐mediated signal. Primary cilia are critical to the pathogenesis of ADPKD, which is one of many ciliopathies that exhibit renal cystic disease. Cux1, a murine homolog of the Drosophila gene Cut, is a cell cycle dependent transcriptional repressor that regulates expression of the cyclin kinase inhibitor p27. Cux1 is highly and ectopically expressed in mice carrying a collecting duct specific mutation of Pkd1 (Pkd1CD) and in human ADPKD cells where it promotes cell proliferation. Mice carrying mutations in both Cux1 and Pkd1 have reduced cystic disease and an increased life span. A role for Cux1 in regulating genes involved in cilia assembly and function has recently been identified in the Galapagos cormorant, however, the role of Cux1 in cilia in the mammalian kidney is not known. To begin to determine whether Cux1 regulates ciliogenesis we evaluated cilia morphology and the expression of the ciliary protein, OFD1 (oral‐facial‐digital‐1), identified as a target of Cux1 in the Galapagos cormorant. Cilia analysis was performed on kidneys isolated from wild type, Cux1 transgenic, Pkd1CD, and Pkd1CD/Cux1 double mutant mice. Cilia morphology was assessed by immunofluorescence labeling of alpha‐tubulin and the collecting duct marker dolichos biflorus agglutinin (DBA) to identify cells in which Pkd1 was deleted. Cilia in Pkd1/Cux1 double knockout kidneys were significantly shorter than cilia in the Pkd1CD kidneys, consistent with previous studies showing that decreased cilia length corresponds to decreased cystic disease. OFD1 is an inhibitor of ciliogenesis and OFD1 expression in the various mouse models demonstrate that OFD1 expression corresponds to Cux1 expression. OFD1 protein levels were the lowest in the kidneys of mice constitutively expressing Cux1 and were highest in mice with deletions of Cux1. Taken together, our results reveal a novel role for Cux1 in regulating ciliogenesis in the kidney and suggest that the reduced cystic disease in the Pkd1/Cux1 double mutant mice results from reduced cilia length.Support or Funding InformationAmerican Association of Anatomy FGAP, Western Michigan University Homer Stryker MD School of Medicine

A high-throughput screening platform for Polycystic Kidney Disease (PKD) drug repurposing utilizing murine and human ADPKD cells

Autosomal dominant polycystic kidney disease (ADPKD) is one of the most common inherited monogenic disorders, characterized by a progressive decline in kidney function due in part to the formation of fluid-filled cysts. While there is one FDA-approved therapy, it is associated with potential adverse effects, and all other clinical interventions are largely supportive. Insights into the cellular pathways underlying ADPKD have revealed striking similarities to cancer. Moreover, several drugs originally developed for cancer have shown to ameliorate cyst formation and disease progression in animal models of ADPKD. These observations prompted us to develop a high-throughput screening platform of cancer drugs in a quest to repurpose them for ADPKD. We screened ~8,000 compounds, including compounds with oncological annotations, as well as FDA-approved drugs, and identified 155 that reduced the viability of Pkd1-null mouse kidney cells with minimal effects on wild-type cells. We found that 109 of these compounds also reduced in vitro cyst growth of Pkd1-null cells cultured in a 3D matrix. Moreover, the result of the cyst assay identified therapeutically relevant compounds, including agents that interfere with tubulin dynamics and reduced cyst growth without affecting cell viability. Because it is known that several ADPKD therapies with promising outcomes in animal models failed to be translated to human disease, our platform also incorporated the evaluation of compounds in a panel of primary ADPKD and normal human kidney (NHK) epithelial cells. Although we observed differences in compound response amongst ADPKD and NHK cell preparation, we identified 18 compounds that preferentially affected the viability of most ADPKD cells with minimal effects on NHK cells. Our study identifies attractive candidates for future efficacy studies in advanced pre-clinical models of ADPKD.

Mitochondrial TRPC3 promotes cell proliferation by regulating the mitochondrial calcium and metabolism in renal polycystin-2 knockdown cells.

Previous studies indicate that autosomal dominant polycystic kidney disease (ADPKD) cells exhibited dysregulated calcium homeostasis and enhanced cell proliferation. TRPC3 has been shown to function in the modulation of calcium and sodium entry, but whether TRPC3 plays a role in cellular abnormalities of ADPKD cells has not been defined. Human conditionally immortalized proximal tubular epithelial cells and mouse IMCD3 cells were used with polycystin-2 (PC2, TRPP2) knockdown. Cell proliferation assay was used to detect the cell proliferations upon different treatments. QRT-PCR and western blotting were used to measure the expression profiles of TRPP2 and other proteins. High-resolution respirometry, enzymic activities and ROS levels were detected to reflect the mitochondrial functions. Calcium and sodium uptakes were measured using Fura2-AM and SBFI dyes. We showed that PC2 knockdown promoted cell proliferation, ROS productions and ERK phosphorylation, compared with negative control. Meanwhile, we demonstrated that receptor-operated calcium entry (ROCE) exhibited less reductions compared with store-operated calcium entry (SOCE) upon PC2 knockdown. Inhibition of ROCE and SOCE by specific inhibitors partially reversed the enhanced cell proliferation, ROS productions and ERK phosphorylation induced by PC2 knockdown. Moreover, TRPC3 upregulation was observed upon PC2 knockdown, which acted as both SOC and ROC, promoting cation entry, cell proliferation and ERK phosphorylation. Furthermore, we showed that mitochondrial located TRPC3 was upregulated and modulating mitochondrial calcium uptake, thus promoting the ROS productions in the presence of PC2 knockdown. We demonstrated that TRPC3 upregulation upon PC2 knockdown aggravated the mitochondrial abnormalities and cell proliferation by modulating mitochondrial calcium uptake. Targeting TRPC3 might be a promising target for ADPKD treatment.

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.