The effect of trehalose on autophagy-related proteins and cyst growth in a hypomorphic Pkd1 mouse model of autosomal dominant polycystic kidney disease

Autosomal dominant polycystic kidney disease (ADPKD) is a common inherited disorder characterized by kidney cyst growth often resulting in end-stage renal disease. There is growing attention on understanding the role of impaired autophagy in ADPKD. Trehalose (TRE) has been shown to increase both protein stability and aggregate clearance and induce autophagy in neurodegenerative diseases. TRE treatment in wild type mice compared to vehicle resulted in increased expression in the kidney of Atg12–5 complex and increased Rab9a, autophagy-related proteins that play a role in the formation of autophagosomes. Thus, the aim of the study was to determine the effect of TRE on cyst growth and autophagy-related proteins, in the hypomorphic Pkd1^RC/RC mouse model of ADPKD. Pkd1^RC/RC mice were treated 2% TRE in water from days 50 to 120 of age. TRE did not slow cyst growth or improve kidney function or affect proliferation and apoptosis in Pkd1^RC/RC kidneys. In Pkd1^RC/RC vs. wild type kidneys, expression of the Atg12–5 complex was inhibited by TRE resulting in increased free Atg12 and TRE was unable to rescue the deficiency of the Atg12–5 complex. Rab9a was decreased in Pkd1^RC/RC vs. wild type kidneys and unaffected by TRE. The TRE-induced increase in p62, a marker of autophagic cargo, that was seen in normal kidneys was blocked in Pkd1^RC/RC kidneys. In summary, the autophagy phenotype in Pkd1^RC/RC kidneys was characterized by decreases in crucial autophagy-related proteins (Atg12–5 complex, Atg5, Atg16L1), decreased Rab9a and increased mTORC1 (pS6^S240/244, pmTOR^S2448) proteins. TRE increased Atg12–5 complex, Rab9a and p62 in normal kidneys, but was unable to rescue the deficiency in autophagy proteins or suppress mTORC1 in Pkd1^RC/RC kidneys and did not protect against cyst growth.     

Increased mTOR and suppressed autophagic flux in the heart of a hypomorphic Pkd1 mouse model of autosomal dominant polycystic kidney disease

Cardiac hypertrophy is common in autosomal dominant polycystic kidney disease (ADPKD) patients. We found increased heart weight in Pkd1^RC/RC and Pkd2^WS25/+ mouse models of ADPKD. As there is a link between increased heart weight and mammalian target of rapamycin (mTOR), the aim of the study was to determine mTOR complex 1 and 2 signaling proteins in the heart in the Pkd1^RC/RC mouse model of PKD. In 70 day old Pkd1^RC/RC hearts, on immunoblot analysis, there was a large increase in p-AMPK^Thr172, a known autophagy inducer, and an increase in p-Akt^Ser473 and p-Akt^Thr308, but no increase in other mTORC1/2 proteins (p-S6^Ser240/244, p-mTOR^Ser2448). In 150 day old Pkd1^RC/RC hearts, there was an increase in mTORC1 (p-S6^Ser240/244) and mTOR-related proteins (p-Akt^Thr308, p-GSK3β^Ser9, p-AMPK^Thr172). As the mTOR pathway is the master regulator of autophagy, autophagy proteins were measured. There was an increase in p-Beclin-1 (BECN1), an autophagy regulator and activating molecule in Beclin-1-regulated autophagy (AMBRA1), a regulator of Beclin that play a role in autophagosome formation, an early stage of autophagy. There was a defect in the later stage of autophagy, the fusion of the autophagosome with the lysosome, known as autophagic flux, as evidenced by the lack of an increase in LC3-II, a marker of autophagosomes, with the lysosomal inhibitor bafilomycin, in both 70 day old and 150 day old hearts. To determine the role of autophagy in causing increased heart weight, Pkd1^RC/RC were treated with 2-deoxyglucose (2-DG) or Tat-Beclin1 peptide, agents known to induce autophagy. 2-DG treatment from 150 to 350 days of age, a time period when increased heart weight developed, did not reduce the increased heart weight. Unexpectedly, Tat-Beclin 1 peptide treatment from 70 to 120 days of age resulted in increased heart weight. In summary, there is suppressed autophagic flux in the heart at an early age in Pkd1^RC/RC mice. Increased mTOR signaling in older mice is associated suppressed autophagic flux. There was a large increase in p-AMPK^Thr172, a known autophagy inducer, in both young and old mice. 2-DG treatment did not impact increased heart weight and Tat-Beclin1 peptide increased heart weight.     

Overexpression of PKD1 causes polycystic kidney disease

The pathogenetic mechanisms underlying autosomal dominant polycystic kidney disease (ADPKD) remain to be elucidated. While there is evidence that Pkd1 gene haploinsufficiency and loss of heterozygosity can cause cyst formation in mice, paradoxically high levels of Pkd1 expression have been detected in the kidneys of ADPKD patients. To determine whether Pkd1 gain of function can be a pathogenetic process, a Pkd1 bacterial artificial chromosome (Pkd1-BAC) was modified by homologous recombination to solely target a sustained Pkd1 expression preferentially to the adult kidney. Several transgenic lines were generated that specifically overexpressed the Pkd1 transgene in the kidneys 2- to 15-fold over Pkd1 endogenous levels. All transgenic mice reproducibly developed tubular and glomerular cysts and renal insufficiency and died of renal failure. This model demonstrates that overexpression of wild-type Pkd1 alone is sufficient to trigger cystogenesis resembling human ADPKD. Our results also uncovered a striking increased renal c-myc expression in mice from all transgenic lines, indicating that c-myc is a critical in vivo downstream effector of Pkd1 molecular pathways. This study not only produced an invaluable and first PKD model to evaluate molecular pathogenesis and therapies but also provides evidence that gain of function could be a pathogenetic mechanism in ADPKD.     

Disruption of polycystin-1 cleavage leads to cardiac metabolic rewiring in mice

Cardiovascular manifestations account for marked morbi-mortality in autosomal dominant polycystic kidney disease (ADPKD). Pkd1- and Pkd2-deficient mice develop cardiac dysfunction, however the underlying mechanisms remain largely unclear. It is unknown whether impairment of polycystin-1 cleavage at the G-protein-coupled receptor proteolysis site, a significant ADPKD mutational mechanism, is involved in this process. We analyzed the impact of polycystin-1 cleavage on heart metabolism using Pkd1^V/V mice, a model unable to cleave this protein and with early cardiac dysfunction. Pkd1^V/V hearts showed lower levels of glucose and amino acids and higher lipid levels than wild-types, as well as downregulation of p-AMPK, p-ACCβ, CPT1B-Cpt1b, Ppara, Nppa and Acta1. These findings suggested decreased fatty acid β-oxidation, which was confirmed by lower oxygen consumption by Pkd1^V/V isolated mitochondria using palmitoyl-CoA. Pkd1^V/V hearts also presented increased oxygen consumption in response to glucose, suggesting that alternative substrates may be used to generate energy. Pkd1^V/V hearts displayed a higher density of decreased-size mitochondria, a finding associated with lower MFN1, Parkin and BNIP3 expression. These derangements were correlated with increased apoptosis and inflammation but not hypertrophy. Notably, Pkd1^V/V neonate cardiomyocytes also displayed shifts in oxygen consumption and p-AMPK downregulation, suggesting that, at least partially, the metabolic alterations are not induced by kidney dysfunction. Our findings reveal that disruption of polycystin-1 cleavage leads to cardiac metabolic rewiring in mice, expanding the understanding of heart dysfunction associated with Pkd1 deficiency and likely with human ADPKD.     

NS398 as a potential drug for autosomal-dominant polycystic kidney disease: Analysis using bioinformatics,and zebrafish and mouse models

Autosomal-dominant polycystic kidney disease (ADPKD) is characterized by uncontrolled renal cyst formation, and few treatment options are available. There are many parallels between ADPKD and clear-cell renal cell carcinoma (ccRCC); however, few studies have addressed the mechanisms linking them. In this study, we aimed to investigate their convergences and divergences based on bioinformatics and explore the potential of compounds commonly used in cancer research to be repurposed for ADPKD. We analysed gene expression datasets of ADPKD and ccRCC to identify the common and disease-specific differentially expressed genes (DEGs). We then mapped them to the Connectivity Map database to identify small molecular compounds with therapeutic potential. A total of 117 significant DEGs were identified, and enrichment analyses results revealed that they are mainly enriched in arachidonic acid metabolism, p53 signalling pathway and metabolic pathways. In addition, 127 ccRCC-specific up-regulated genes were identified as related to the survival of patients with cancer. We focused on the compound NS398 as it targeted DEGs and found that it inhibited the proliferation of Pkd1^−/− and 786-0 cells. Furthermore, its administration curbed cystogenesis in Pkd2 zebrafish and early-onset Pkd1-deficient mouse models. In conclusion, NS398 is a potential therapeutic agent for ADPKD.     

Epigenetics and autosomal dominant polycystic kidney disease

The roles of epigenetic modulation of gene expression and protein functions in autosomal dominant polycystic kidney disease (ADPKD) have recently become the focus of scientific investigation. Evidence generated to date indicates that one of the epigenetic modifiers, histone deacetylases (HDACs), are important regulators of ADPKD. HDACs are involved in regulating the expression of the Pkd1 gene and are the target of fluid flow-induced calcium signal in kidney epithelial cells. Pharmacological inhibition of HDAC activity has been found to reduce the progression of cyst formation and slow the decline of kidney function in Pkd1 conditional knockout mice and Pkd2 knockout mice, respectively, implicating the potential clinical application of HDAC inhibitors on ADPKD. Since the expression of HDAC6 is upregulated in cystic epithelial cells, the potential roles of HDAC6 in regulating cilia resorption and epidermal growth factor receptor (EGFR) trafficking through deacetylating α-tubulin and regulating Wnt signaling through deacetylating β-catenin are also discussed. This article is part of a Special Issue entitled: Polycystic Kidney Disease.     

Pkd1 is required for male reproductive tract development

Reproductive tract abnormalities and male infertility have higher incidence in ADPKD patients than in general populations. In this work, we reveal that Pkd1, whose mutations account for 85% of ADPKD cases, is essential for male reproductive tract development. Disruption of Pkd1 caused multiple organ defects in the murine male reproductive tract. The earliest visible defect in the Pkd1^?/? reproductive tract was cystic dilation of the efferent ducts, which are derivatives of the mesonephric tubules. Epididymis development was delayed or arrested in the Pkd1^?/? mice. No sign of epithelial coiling was seen in the null mutants. Disruption of Pkd1 in epithelium alone using the Pax2-cre mice was sufficient to cause efferent duct dilation and coiling defect in the epididymis, suggesting that Pkd1 is critical for epithelium development and maintenance in male reproductive tract. In-depth analysis showed that Pkd1 is required to maintain tubulin cytoskeleton and important for Tgf-β/Bmp signal transduction in epithelium of male reproductive tract. Altogether, our results for the first time provide direct evidence for developmental roles of Pkd1 in the male reproductive tract and provide new insights in reproductive tract abnormalities and infertility in ADPKD patients.     

CDK inhibitors R-roscovitine and S-CR8 effectively block renal and hepatic cystogenesis in an orthologous model of ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) and other forms of PKD are associated with dysregulated cell cycle and proliferation. Although no effective therapy for the treatment of PKD is currently available, possible mechanism-based approaches are beginning to emerge. A therapeutic intervention targeting aberrant cilia-cell cycle connection using CDK-inhibitor R-roscovitine showed effective arrest of PKD in jck and cpk models that are not orthologous to human ADPKD. To evaluate whether CDK inhibition approach will translate into efficacy in an orthologous model of ADPKD, we tested R-roscovitine and its derivative S-CR8 in a model with a conditionally inactivated Pkd1 gene (Pkd1 cKO). Similar to ADPKD, Pkd1 cKO mice developed renal and hepatic cysts. Treatment of Pkd1 cKO mice with R-roscovitine and its more potent and selective analog S-CR8 significantly reduced renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD.     

Rapamycin treatment dose‐dependently improves the cystic kidney in a new ADPKD mouse model via the mTORC1 and cell‐cycle‐associated CDK1/cyclin axis

Although translational research into autosomal dominant polycystic kidney disease (ADPKD) and its pathogenesis has made considerable progress, there is presently lack of standardized animal model for preclinical trials. In this study, we developed an orthologous mouse model of human ADPKD by cross‐mating Pkd2 conditional‐knockout mice (Pkd2^f3) to Cre transgenic mice in which Cre is driven by a spectrum of kidney‐related promoters. By systematically characterizing the mouse model, we found that Pkd2^f3/f3 mice with a Cre transgene driven by the mouse villin‐1 promoter (Vil‐Cre;Pkd2^f3/f3) develop overt cysts in the kidney, liver and pancreas and die of end‐stage renal disease (ESRD) at 4–6 months of age. To determine whether these Vil‐Cre;Pkd2^f3/f3 mice were suitable for preclinical trials, we treated the mice with the high‐dose mammalian target of rapamycin (mTOR) inhibitor rapamycin. High‐dose rapamycin significantly increased the lifespan, lowered the cystic index and kidney/body weight ratio and improved renal function in Vil‐Cre;Pkd2^f3/f3 mice in a time‐ and dose‐dependent manner. In addition, we further found that rapamycin arrested aberrant epithelial‐cell proliferation in the ADPKD kidney by down‐regulating the cell‐cycle‐associated cyclin‐dependent kinase 1 (CDK1) and cyclins, namely cyclin A, cyclin B, cyclin D1 and cyclin E, demonstrating a direct link between mTOR signalling changes and the polycystin‐2 dysfunction in cystogenesis. Our newly developed ADPKD model provides a practical platform for translating in vivo preclinical results into ADPKD therapies. The newly defined molecular mechanism by which rapamycin suppresses proliferation via inhibiting abnormally elevated CDK1 and cyclins offers clues to new molecular targets for ADPKD treatment.     

Conditional Mesenchymal Disruption of Pkd1 Results in Osteopenia and Polycystic Kidney Disease

Conditional deletion of Pkd1 in osteoblasts using either Osteocalcin(Oc)-Cre or Dmp1-Cre results in defective osteoblast-mediated postnatal bone formation and osteopenia. Pkd1 is also expressed in undifferentiated mesenchyme that gives rise to the osteoblast lineage. To examine the effects of Pkd1 on prenatal osteoblast development, we crossed Pkd1 ^flox/flox and Col1a1(3.6)-Cre mice, which has been used to achieve selective inactivation of Pkd1 earlier in the osteoblast lineage. Control Pkd1 ^flox/flox and Pkd1 ^flox/+, heterozygous Col1a1(3.6)-Cre;Pkd1 ^flox/+ and Pkd1 ^flox/null, and homozygous Col1a1(3.6)-Cre;Pkd1 ^flox/flox and Col1a1(3.6)-Cre;Pkd1 ^flox/null mice were analyzed at ages ranging from E14.5 to 8-weeks-old. Newborn Col1a1(3.6)-Cre;Pkd1 ^flox/null mice exhibited defective skeletogenesis in association with a greater reduction in Pkd1 expression in bone. Conditional Col1a1(3.6)-Cre;Pkd1 ^flox/+ and Col1a1(3.6)-Cre;Pkd1 ^flox/flox mice displayed a gene dose-dependent decrease in bone formation and increase in marrow fat at 6 weeks of age. Bone marrow stromal cell and primary osteoblast cultures from homozygous Col1a1(3.6)-Cre;Pkd1 ^flox/flox mice showed increased proliferation, impaired osteoblast development and enhanced adipogenesis ex vivo. Unexpectedly, we found evidence for Col1a1(3.6)-Cre mediated deletion of Pkd1 in extraskeletal tissues in Col1a1(3.6)-Cre;Pkd1 ^flox/flox mice. Deletion of Pkd1 in mesenchymal precursors resulted in pancreatic and renal, but not hepatic, cyst formation. The non-lethality of Col1a1(3.6)-Cre;Pkd1 ^flox/flox mice establishes a new model to study abnormalities in bone development and cyst formation in pancreas and kidney caused by Pkd1 gene inactivation.     

Polycystin-2 expression is regulated by a PC2-binding domain in the intracellular portion of fibrocystin

Autosomal dominant (ADPKD) and autosomal recessive (ARPKD) polycystic kidney disease are caused by mutations in Pkd1/Pkd2 and Pkhd1, which encode polycystins (PCs) and fibrocystin/polyductin (FPC). Our recent study reported that a deficiency in FPC increases the severity of cystic disease in Pkd2 mutants and down-regulates PC2 in vivo, but the precise molecular mechanism of these effects is unknown (Kim, I., Fu, Y., Hui, K., Moeckel, G., Mai, W., Li, C., Liang, D., Zhao, P., Ma, J., Chen, X.-Z., George, A. L., Jr., Coffey, R. J., Feng, Z. P., and Wu, G. (2008) J. Am. Soc. Nephrol. 19, 455-468). In this study, through the use of deletion and mutagenesis strategies, we identified a PC2-binding domain in the intracellular C terminus of FPC and an FPC-binding domain in the intracellular N terminus of PC2. These binding domains provide a molecular basis for the physical interaction between PC2 and FPC. In addition, we also found that physical interaction between the binding domains of PC2 and FPC is able to prevent down-regulation of PC2 induced by loss of FPC. In vivo, we generated a mouse model of ADPKD with hypomorphic Pkd2 alleles (Pkd2nf3/nf3) and show that PC2 down-regulation is accompanied by a phenotype similar to that of Pkhd1(-/-) mice. These findings demonstrate a common mechanism underlying cystogenesis in ADPKD and ARPKD and provide insight into the molecular relationship between PC2 and FPC.     

CDK inhibitors R-roscovitine and S-CR8 effectively block renal and hepatic cystogenesis in an orthologous model of ADPKD

Autosomal dominant polycystic kidney disease (ADPKD) and other forms of PKD are associated with dysregulated cell cycle and proliferation. Although no effective therapy for the treatment of PKD is currently available, possible mechanism-based approaches are beginning to emerge. A therapeutic intervention targeting aberrant cilia-cell cycle connection using CDK-inhibitor R-roscovitine showed effective arrest of PKD in jck and cpk models that are not orthologous to human ADPKD. To evaluate whether CDK inhibition approach will translate into efficacy in an orthologous model of ADPKD, we tested R-roscovitine and its derivative S-CR8 in a model with a conditionally inactivated Pkd1 gene (Pkd1 cKO). Similar to ADPKD, Pkd1 cKO mice developed renal and hepatic cysts. Treatment of Pkd1 cKO mice with R-roscovitine and its more potent and selective analog S-CR8 significantly reduced renal and hepatic cystogenesis and attenuated kidney function decline. Mechanism of action studies demonstrated effective blockade of cell cycle and proliferation and reduction of apoptosis. Together, these data validate CDK inhibition as a novel and effective approach for the treatment of ADPKD.     

Impaired glomerulogenesis and endothelial cell migration in Pkd1-deficient renal organ cultures

The PKD1 gene is essential for a number of biological functions, and its loss-of-function causes autosomal dominant polycystic kidney disease (ADPKD). The gene is developmentally regulated and believed to play an essential role in renal development. Previous studies have shown that manipulating murine renal organ cultures with dominant-negative forms of the Pkd1 gene impaired ureteric bud (UB) branching. In the current study, we analyzed different stages of renal development in two distinct mouse models carrying either a null mutation or inactivation of the last two exons of Pkd1. Surprisingly, metanephric explants from Pkd1-deleted kidneys harvested at day E11.5 did not show defects of UB branching and elongation, estimated by cytokeratin staining on fixed tissues or by Hoxb7-GFP time-lapse imaging. However, renal explants from Pkd1-mutants isolated at day E14.5 showed impaired nephrogenesis. Notably, we observed cell migratory defects in the developing endothelial compartment. Previous studies had implicated the Pkd1 gene in controlling cell migration and collagen deposition through PI3 kinases. In line with these studies, our results show that wild-type explants treated with PI3-kinase inhibitors recapitulate the endothelial defects observed in Pkd1 mutants, whereas treatment with VEGF only partially rescued the defects. Our data are consistent with a role for the Pkd1 gene in the endothelium that may be required for proper nephrogenesis.     

Genome-wide methylation profiling of ADPKD identified epigenetically regulated genes associated with renal cyst development

Autosomal dominant polycystic kidney disease (ADPKD) is a common human genetic disease characterized by the formation of multiple fluid-filled cysts in bilateral kidneys. Although mutations in polycystic kidney disease 1 (PKD1) are predominantly responsible for ADPKD, the focal and sporadic property of individual cystogenesis suggests another molecular mechanism such as epigenetic alterations. To determine the epigenomic alterations in ADPKD and their functional relevance, ADPKD and non-ADPKD individuals were analyzed by unbiased methylation profiling genome-wide and compared with their expression data. Intriguingly, PKD1 and other genes related to ion transport and cell adhesion were hypermethylated in gene-body regions, and their expressions were downregulated in ADPKD, implicating epigenetic silencing as the key mechanism underlying cystogenesis. Especially, in patients with ADPKD, PKD1 was hypermethylated in gene-body region and it was associated with recruitment of methyl-CpG-binding domain 2 proteins. Moreover, treatment with DNA methylation inhibitors retarded cyst formation of Madin-Darby Canine Kidney cells, accompanied with the upregulation of Pkd1 expression. These results are consistent with previous studies that knock-down of PKD1 was sufficient for cystogenesis. Therefore, our results reveal a critical role for hypermethylation of PKD1 and cystogenesis-related regulatory genes in cyst development, suggesting epigenetic therapy as a potential treatment for ADPKD.     

Anti-fibrotic effects of nintedanib in lung fibroblasts derived from patients with idiopathic pulmonary fibrosis

Background

Idiopathic pulmonary fibrosis (IPF) is a progressive lung disease with poor prognosis. The kinase inhibitor nintedanib specific for vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR) significantly reduced the rate of decline of forced vital capacity versus placebo.

Aim

To determine the in vitro effect of nintedanib on primary human lung fibroblasts. Methods: Fibroblasts were isolated from lungs of IPF patients and from non-fibrotic controls. We assessed the effect of VEGF, PDGF-BB and basic FGF (bFGF) ± nintedanib on: (i) expression/activation of VEGFR, PDGFR, and FGFR, (ii) cell proliferation, secretion of (iii) matrix metalloproteinases (MMP), (iv) tissue inhibitor of metalloproteinase (TIMP), and (v) collagen.

Results

IPF fibroblasts expressed higher levels of PDGFR and FGFR than controls. PDGF-BB, bFGF, and VEGF caused a pro-proliferative effect which was prevented by nintedanib. Nintedanib enhanced the expression of pro-MMP-2, and inhibited the expression of TIMP-2. Transforming growth factor-beta-induced secretion of collagens was inhibited by nintedanib.

Conclusion

Our data demonstrate a significant anti-fibrotic effect of nintedanib in IPF fibroblasts. This effect consists of the drug’s anti-proliferative capacity, and on its effect on the extracellular matrix, the degradation of which seems to be enhanced.