Quinomycin A reduces cyst progression in polycystic kidney disease.
ABSTRACT: Polycystic kidney disease (PKD) is a genetic disorder characterized by aberrant renal epithelial cell proliferation and formation and progressive growth of numerous fluid-filled cysts within the kidneys. Previously, we showed that there is elevated Notch signaling compared to normal renal epithelial cells and that Notch signaling contributes to the proliferation of cystic cells. Quinomycin A, a bis-intercalator peptide, has previously been shown to target the Notch signaling pathway and inhibit tumor growth in cancer. Here, we show that Quinomycin A decreased cell proliferation and cyst growth of human ADPKD cyst epithelial cells cultured within a 3D collagen gel. Treatment with Quinomycin A reduced kidney weight to body weight ratio and decreased renal cystic area and fibrosis in Pkd1RC/RC ; Pkd2+/- mice, an orthologous PKD mouse model. This was accompanied by reduced expression of Notch pathway proteins, RBPjk and HeyL and cell proliferation in kidneys of PKD mice. Quinomycin A treatments also normalized cilia length of cyst epithelial cells derived from the collecting ducts. This is the first study to demonstrate that Quinomycin A effectively inhibits PKD progression and suggests that Quinomycin A has potential therapeutic value for PKD patients.
Project description:Polycystic kidney disease (PKD) is a genetic disorder characterized by fluid-filled cysts in the kidney and liver that ultimately leads to end-stage renal disease. Currently there is no globally approved therapy for PKD. The Notch signaling pathway regulates cellular processes such as proliferation and de-differentiation, which are cellular hallmarks of PKD. Thus we hypothesized that the Notch pathway plays a critical role in PKD. Evaluation of protein expression of Notch signaling components in kidneys of Autosomal Recessive PKD (ARPKD) and Autosomal Dominant PKD (ADPKD) mouse models and of ADPKD patients revealed that Notch pathway members, particularly Notch3, were consistently upregulated or activated in cyst-lining epithelial cells. Notch3 expression correlated with rapidly growing cysts and co-localized with the proliferation marker, PCNA. Importantly, Notch inhibition significantly decreased forskolin-induced Notch3 activation and proliferation of primary human ADPKD cells, and significantly reduced cyst formation and growth of human ADPKD cells cultured in collagen gels. Thus our data indicate that Notch3 is aberrantly activated and facilitates epithelial cell proliferation in PKD, and that inhibition of Notch signaling may prevent cyst formation and growth.
Project description:Autosomal-dominant polycystic kidney disease (ADPKD) is the most common inherited kidney disease and is characterized by progressive growth of fluid-filled cysts. Growth factors binding to receptor tyrosine kinases (RTKs) stimulate cell proliferation and cyst growth in PKD. Nintedanib, a triple RTK inhibitor, targets the vascular endothelial growth-factor receptor (VEGFR), platelet-derived growth-factor receptor (PDGFR), and fibroblast growth-factor receptor (FGFR), and is an approved drug for the treatment of non-small-cell lung carcinoma and idiopathic lung fibrosis. To determine if RTK inhibition using nintedanib can slow ADPKD progression, we tested its effect on human ADPKD renal cyst epithelial cells and myofibroblasts in vitro, and on Pkd1<sup>f/f</sup>Pkhd1<sup>Cre</sup> and Pkd1<sup>RC/RC</sup>, orthologous mouse models of ADPKD. Nintedanib significantly inhibited cell proliferation and in vitro cyst growth of human ADPKD renal cyst epithelial cells, and cell viability and migration of human ADPKD renal myofibroblasts. Consistently, nintedanib treatment significantly reduced kidney-to-body-weight ratio, renal cystic index, cystic epithelial cell proliferation, and blood-urea nitrogen levels in both the Pkd1<sup>f/f</sup>Pkhd1<sup>Cre</sup> and Pkd1<sup>RC/RC</sup> mice. There was a corresponding reduction in ERK, AKT, STAT3, and mTOR activity and expression of proproliferative factors, including Yes-associated protein (YAP), c-Myc, and Cyclin D1. Nintedanib treatment significantly reduced fibrosis in Pkd1<sup>RC/RC</sup> mice, but did not affect renal fibrosis in Pkd1<sup>f/f</sup>Pkhd1<sup>Cre</sup> mice. Overall, these results suggest that nintedanib may be repurposed to effectively slow cyst growth in ADPKD.
Project description:Renal M2-like macrophages have critical roles in tissue repair, stimulating tubule cell proliferation and, if they remain, fibrosis. M2-like macrophages have also been implicated in promoting cyst expansion in mouse models of autosomal dominant polycystic kidney disease (ADPKD). While renal macrophages have been documented in human ADPKD, there are no studies in autosomal recessive polycystic kidney disease (ARPKD). Here we evaluated the specific phenotype of renal macrophages and their disease-impacting effects on cystic epithelial cells. We found an abundance of M2-like macrophages in the kidneys of patients with either ADPKD or ARPKD and in the cystic kidneys of cpk mice, a model of ARPKD. Renal epithelial cells from either human ADPKD cysts or noncystic human kidneys promote differentiation of naive macrophages to a distinct M2-like phenotype in culture. Reciprocally, these immune cells stimulate the proliferation of renal tubule cells and microcyst formation in vitro. Further, depletion of macrophages from cpk mice indicated that macrophages contribute to PKD progression regardless of the genetic etiology. Thus, M2-like macrophages are two-pronged progression factors in PKD, promoting cyst cell proliferation, cyst growth, and fibrosis. Agents that block the emergence of these cells or their effects in the cystic kidney may be effective therapies for slowing PKD progression.
Project description:Polycystic kidney diseases (PKDs) are inherited disorders characterized by the formation of fluid filled renal cysts. Elevated cAMP levels in PKDs stimulate progressive cyst enlargement involving cell proliferation and transepithelial fluid secretion often leading to end-stage renal disease. The glycogen synthase kinase-3 (GSK3) family of protein kinases consists of GSK3? and GSK3? isoforms and has a crucial role in multiple cellular signaling pathways. We previously found that GSK3?, a regulator of cell proliferation, is also crucial for cAMP generation and vasopressin-mediated urine concentration by the kidneys. However, the role of GSK3? in the pathogenesis of PKDs is not known. Here we found that GSK3? expression and activity were markedly upregulated and associated with cyst-lining epithelia in the kidneys of mice and humans with PKD. Renal collecting duct-specific gene knockout of GSK3? or pharmacological inhibition of GSK3 effectively slowed down the progression of PKD in mouse models of autosomal recessive or autosomal dominant PKD. GSK3 inactivation inhibited cAMP generation and cell proliferation resulting in reduced cyst expansion, improved renal function, and extended life span. GSK3? inhibition also reduced pERK, c-Myc, and cyclin-D1, known mitogens in proliferation of cystic epithelial cells. Thus, GSK3? has a novel functional role in PKD pathophysiology, and its inhibition may be therapeutically useful to slow down cyst expansion and progression of PKD.
Project description:Cyclic AMP promotes cyst growth in polycystic kidney disease (PKD) by stimulating cell proliferation and fluid secretion. Previously, we showed that the primary cilium of renal epithelial cells contains a cAMP regulatory complex comprising adenylyl cyclases 5 and 6 (AC5/6), polycystin-2, A-kinase anchoring protein 150, protein kinase A, and phosphodiesterase 4C. In Kif3a mutant cells that lack primary cilia, the formation of this regulatory complex is disrupted and cAMP levels are increased. Inhibition of AC5 reduces cAMP levels in Kif3a mutant cells, suggesting that AC5 may mediate the increase in cAMP in PKD. Here, we examined the role of AC5 in an orthologous mouse model of PKD caused by kidney-specific ablation of Pkd2. Knockdown of AC5 with siRNA attenuated the increase in cAMP levels in Pkd2-deficient renal epithelial cells. Levels of cAMP and AC5 mRNA transcripts were elevated in the kidneys of mice with collecting duct-specific ablation of Pkd2. Compared with Pkd2 single mutant mice, AC5/Pkd2 double mutant mice had less kidney enlargement, lower cyst index, reduced kidney injury, and improved kidney function. Importantly, cAMP levels and cAMP-dependent signaling were reduced in the kidneys of AC5/Pkd2 double mutant compared to the kidneys of Pkd2 single mutant mice. Additionally, we localized endogenous AC5 in the primary cilium of renal epithelial cells and showed that ablation of AC5 reduced ciliary elongation in the kidneys of Pkd2 mutant mice. Thus, AC5 contributes importantly to increased renal cAMP levels and cyst growth in Pkd2 mutant mice, and inhibition of AC5 may be beneficial in the treatment of PKD.
Project description: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 α<sub>V</sub>β<sub>3</sub>-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 α<sub>V</sub>β<sub>3</sub>-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.
Project description:<h4>Background</h4>Transepithelial chloride<sup>-</sup> secretion, through the chloride channels cystic fibrosis transmembrane conductance regulator (CFTR) and TMEM16A (anoctamin 1), drives cyst enlargement in polycystic kidney disease (PKD). Polycystic kidneys are hypoxic, and oxidative stress activates TMEM16A. However, mechanisms for channel activation in PKD remain obscure.<h4>Methods</h4>Using tissue samples from patients with autosomal dominant PKD, embryonic kidney cultures, and an MDCK <i>in vitro</i> cyst model, we assessed peroxidation of plasma membrane phospholipids in human and mouse polycystic kidneys. We also used electrophysiologic Ussing chamber and patch clamp experiments to analyze activation of TMEM16A and growth of renal cysts.<h4>Results</h4>Peroxidation of phospholipids in human and mouse kidneys as well as MDCK cysts <i>in vitro</i> is probably due to enhanced levels of reactive oxygen species. Lipid peroxidation correlated with increased cyst volume as shown in renal cultures and MDCK cysts in three-dimensional cultures. Reactive oxygen species and lipid peroxidation strongly activated TMEM16A, leading to depletion of calcium ion stores and store-operated calcium influx. Activation of TMEM16A- and CFTR-dependent chloride secretion strongly augmented cyst growth. Exposure to scavengers of reactive oxygen species, such as glutathione, coenzyme Q10, or idebenone (a synthetic coenzyme Q10 homolog), as well as inhibition of oxidative lipid damage by ferrostatin-1 largely reduced activation of TMEM16A. Inhibition of TMEM16A reduced proliferation and fluid secretion <i>in vitro</i>.<h4>Conclusions</h4>These findings indicate that activation of TMEM16A by lipid peroxidation drives growth of renal cysts. We propose direct inhibition of TMEM16A or inhibition of lipid peroxidation as potentially powerful therapeutic approaches to delay cyst development in PKD.
Project description:<h4>Background</h4>Autosomal-dominant polycystic kidney disease (ADPKD) is the leading inherited renal disease worldwide. The proproliferative function of macrophages is associated with late-stage cyst enlargement in mice with PKD; however, the way in which macrophages act on cyst-lining epithelial cells (CLECs) has not been well elucidated.<h4>Methods</h4>We generated a rapid-onset PKD mouse model by inactivating <i>Pkd1</i> on postnatal day 10 (P10) and compared cell proliferation and differential gene expression in kidney tissues of the PKD mice and wild-type (WT) littermates.<h4>Results</h4>The cystic phenotype was dominant from P18. A distinct peak in cell proliferation in polycystic kidneys during P22-P30 was closely related to late-stage cyst growth. Comparisons of gene expression profiles in kidney tissues at P22 and P30 in PKD and WT mice revealed that arginine metabolism was significantly activated; 204 differentially expressed genes (DEGs), including <i>Arg1</i>, an arginine metabolism-associated gene, were identified in late-stage polycystic kidneys. The <i>Arg1</i>-encoded protein, arginase-1 (ARG1), was predominantly expressed in macrophages in a time-dependent manner. Multiple-stage macrophage depletion verified that macrophages expressing high ARG1 levels accounted for late-stage cyst enlargement, and inhibiting ARG1 activity significantly retarded cyst growth and effectively lowered the proliferative indices in polycystic kidneys. <i>In vitro</i> experiments revealed that macrophages stimulated CLEC proliferation, and that L-lactic acid, primarily generated by CLECs, significantly upregulated ARG1 expression and increased polyamine synthesis in macrophages.<h4>Conclusions</h4>Interactions between macrophages and CLECs promote cyst growth. ARG1 is a key molecule involved in this process and is a potential therapeutic target to help delay ADPKD progression.
Project description:Autosomal Dominant Polycystic Kidney Disease (ADPKD) is caused by mutation of PKD1 or PKD2, which encode polycystin 1 and 2, respectively. The polycystins localize to primary cilia and the functional loss of the polycystin complex leads to the formation and progressive growth of fluid-filled cysts in the kidney. The pathogenesis of ADPKD is complex and molecular mechanisms connecting ciliary dysfunction to renal cystogenesis are unclear. Primary cilia mediate Hedgehog signaling, which modulates cell proliferation and differentiation in a tissue-dependent manner. Previously, we showed that Hedgehog signaling was increased in cystic kidneys of several PKD mouse models and that Hedgehog inhibition prevented cyst formation in embryonic PKD mouse kidneys treated with cAMP. Here, we show that in human ADPKD tissue, Hedgehog target and activator, Glioma 1, was elevated and localized to cyst-lining epithelial cells and to interstitial cells, suggesting increased autocrine and paracrine Hedgehog signaling in ADPKD, respectively. Further, Hedgehog inhibitors reduced basal and cAMP-induced proliferation of ADPKD cells and cyst formation in vitro. These data suggest that Hedgehog signaling is increased in human ADPKD and that suppression of Hedgehog signaling can counter cellular processes that promote cyst growth in vitro.
Project description: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 ?V?3 and ?V?5 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.