Notch3 as a novel therapeutic target for the treatment of ADPKD by regulating cell proliferation and renal cyst development.

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenic kidney disease. Emerging research indicates that the Notch signaling pathway plays an indispensable role in the pathogenesis of numerous kidney diseases, including ADPKD. Herein, we identified that Notch3 but not other Notch receptors was overexpressed in renal tissues from mice with ADPKD and ADPKD patients. Inhibiting Notch3 with γ-secretase inhibitors, which block a proteolytic cleavage required for Notch3 activation, or shRNA knockdown of Notch3 significantly delayed renal cyst growth in vitro and in vivo. Subsequent mechanistic study elucidated that the cleaved intracellular domain of Notch3 (N3ICD) and Hes1 could bind to the PTEN promoter, leading to transcriptional inhibition of PTEN. This further activated the downstream PI3K-AKT-mTOR pathway and promoted renal epithelial cell proliferation. Overall, Notch3 was identified as a novel contributor to renal epithelial cell proliferation and cystogenesis in ADPKD. We envision that Notch3 represents a promising target for ADPKD treatment.

Photochemical behavior and photo-induced toxicity of chiral pesticides and their chiral monomers in aqueous environment.

The photochemical behaviors of chiral pollutants in aqueous solutions are rarely studied using chiral monomers, which may hamper their precise risk assessment and lead to suspicious conclusions. In this study, we systematically investigated the phototransformation behavior and toxicity evolution of two widely used chiral pesticides (triadimefon (TF) and triadimenol (TN)) at enantiomer and diastereomer levels, and proposed a calculation method of total photolysis rate constants of chiral mixture. Results show that TF and TN could be photodegraded faster in pure water than in natural waters, and the observed photolysis rate constants (kobs) of TN with two chiral centers exhibit enantioselectivity, i.e., kobs(TN-RS) = kobs(TN-SR) > kobs(TN-RR) = kobs(TN-SS). The photolysis of TF and TN mainly occurs through their excited singlet and triplet states, respectively. Their photodegradation pathways mainly include dechlorination and elimination of triazole ring. TF could also undergo ether bond cleavage. It is also found that, both TF and TN exhibit photo-induced toxicity to V. fischeri, due to the generation of more toxic products than parent compounds. Furthermore, TN exhibits enantioselective photo-induced toxicity after 240-min irradiation, which could be ascribed to the formation of chiral products. These results could benefit the understanding of enantioselective environmental behavior of chiral pollutants.

PF-06409577 inhibits renal cyst progression by concurrently inhibiting the mTOR pathway and CFTR channel activity.

Renal cyst development and expansion in autosomal dominant polycystic kidney disease (ADPKD) involves over-proliferation of cyst-lining epithelial cells and excessive cystic fluid secretion. While metformin effectively inhibits renal cyst growth in mouse models of ADPKD it exhibits low potency, and thus an adenosine monophosphate-activated protein kinase (AMPK) activator with higher potency is required. Herein, we adopted a drug repurposing strategy to explore the potential of PF-06409577, an AMPK activator for diabetic nephropathy, in cellular, ex vivo and in vivo models of ADPKD. Our results demonstrated that PF-06409577 effectively down-regulated mammalian target of rapamycin pathway-mediated proliferation of cyst-lining epithelial cells and reduced cystic fibrosis transmembrane conductance regulator-regulated cystic fluid secretion. Overall, our data suggest that PF-06409577 holds therapeutic potential for ADPKD treatment.

Identification of active or inactive agonists of tumor suppressor protein based on Tox21 library.

Exposure of cells to xenobiotic human-made products can lead to genotoxicity and cause DNA damage. It is an urgent need to quickly identify the chemicals that cause DNA damage, and their toxicity should be predicted. In this study, recursive partitioning (RP), binary logistic regression, and one machine learning approach, namely, random forest (RF) classifier, were used to predict the active and inactive compounds of a total 5036 data based on the assay conducted by a ß-lactamase reporter gene under control of the p53 response element (p53RE) from Tox21 library. Results show that the binary logistic regression model with a threshold of 0.5 has a high accuracy rate (83%) to distinguish active and inactive compounds. The RF classifier method has satisfactory results, with an accuracy rate (84.38%) approximately higher than that of binary logistic regression. The models established can identify compounds that induce DNA damage and activate p53, and provide a scientific basis for the risk assessment of organic chemicals in the environment.

Benzodiazepine Derivatives as Potent Vasopressin V2 Receptor Antagonists for the Treatment of Autosomal Dominant Kidney Disease.

Cyst formation and enlargement in autosomal dominant kidney disease (ADPKD) is mainly driven by aberrantly increased cytosolic cAMP in renal tubule epithelial cells. Because the vasopressin V2 receptor (V2R) regulates intracellular cAMP levels in kidneys, a series of benzodiazepine derivatives were developed targeting the V2R. Among these derivatives, compound 25 exhibited potent binding affinity to the V2R (Ki = 9.0 ± 1.5 nM) and efficacious cAMP inhibition (IC50 = 9.2 ± 3.0 nM). This led to the suppression of cyst formation and growth in both an MDCK cell model and an embryonic kidney cyst model. Further advancing compound 25 in a murine model of ADPKD demonstrated a significantly improved in vivo efficacy compared with the reference compound tolvaptan. Overall, compound 25 holds therapeutic potential for the treatment of ADPKD.

Application of machine learning to predict the inhibitory activity of organic chemicals on thyroid stimulating hormone receptor.

With the promotion of carbon neutrality, it is also important to synchronously promote the assessment and sustainable management of chemicals so as to protect public health. Humans and animals are possibly exposed to endocrine disruptors that have inhibitory effects on thyroid stimulating hormone receptor (TSHR). As such, it is important to identify chemicals that inhibit TSHR and to develop models to predict their inhibitory activity. In this study, 5952 compounds derived from a cyclic adenosine monophosphate (cAMP) analysis, a key signaling pathway in thyrocytes, were used to establish a binary classification model comparing methods that included random forest (RF), extreme gradient boosting (XGB), and logistic regression (LR). The prediction model based on RF showed the highest identification accuracy for revealing chemicals that may inhibit TSHR. For the RF model, recall was calculated at 0.89, balance accuracy was 0.85, and its receiver operating characteristic (ROC) curve-area under (AUC) was 0.92, indicating that the model had very high predictive capacity. The lowest CDocker energy (CE) and CDocker interaction energy (CIE) for chemicals and TSHR were determined and were subsequently introduced into the predictive model as descriptors. A regression model, extreme gradient boosting-Regression (XGBR), was successfully established yielding an R2 = 0.65 to predict inhibitory activity for active compounds. Parameters that included dissociation characteristics, molecular structure, and binding energy were all key factors in the predictive model. We demonstrate that QSAR models are useful approaches, not only for identifying chemicals that inhibit TSHR, but for predicting inhibitory activity of active compounds.

Comparison of modes of action between fish, cell and mitochondrial toxicity based on toxicity correlation, excess toxicity and QSAR for class-based compounds.

Mitochondria are significant targets in cells for many environmental chemicals. Mitochondrial damage and dysfunction can lead to apoptosis and death of fish. The objectives of this study were to compare the modes of action (MOAs) between fish, cell and mitochondrial toxicity. To achieve the goal, toxicity correlation, excess toxicity and quantitative structure-activity relationship (QSAR) were investigated between these three toxicity endpoints for a wide range of compounds. Results showed that fish toxicity is well correlated to cytotoxicity, but overall fish toxicity is relatively greater than the cytotoxicity. On the other hand, fish or cell toxicity is poorly related to mitochondrial toxicity, suggesting some compounds share same toxic mechanism but some not. The excess toxicity calculated from toxicity ratio (TR) shows that specifically-acting compounds in cytotoxicity, such as insecticides, fungicides, herbicides, dyes and medications used to treat cancer, depression, heart failure and blood pressure, are active compounds in mitochondrial toxicity. However, the less inert compounds identified in fish and cell toxicity exhibit greatly mitochondrial toxicity. QSAR models reveal that fish or cell toxicity is closely related to the chemical hydrophobicity, ionization, energy of lowest unoccupied molecular orbital, hydrogen bonding potential and stability. These descriptors reflect chemical bio-uptake, reactivity and interaction with target receptors. On the other hand, binomial model reveals that mitochondrial toxicity is closely related to the chemical hydrophobicity and polarizability/dipolarity, indicating bio-uptake and Van der Waals interaction play key roles in mitochondrial toxicity. Theoretical equations have been used to explain the toxicity correlation, excess toxicity and QSAR for fish, cell and mitochondrial toxicity. Above results suggest that cytotoxicity can serve as a surrogate for fish toxicity and be used in the safety evaluation of organic pollutants in aqueous environment, but not mitochondrial toxicity, although some compounds share same modes of action between fish or cell toxicity and mitochondrial toxicity.

Comparative analysis on the photolysis kinetics of four neonicotinoid pesticides and their photo-induced toxicity to Vibrio Fischeri: Pathway and toxic mechanism.

Neonicotinoids are widely used pesticides all over the world and pose severe water pollution. Although they can be degraded via absorbing sunlight, few attentions have been paid to the environmental risks of their photolysis products. In this paper, the photo-toxicity was investigated for four neonicotinoids (dinotefuran, nitenpyram, thiamethoxam and clothianidin) based on a series of experiments (i.e., photolysis kinetics, radical scavenging, bioluminescent inhibition test to Vibrio Fischeri and intermediate identification) and in-silico calculation of photolysis pathway. The results show that direct photolysis dominates the photolysis of the four neonicotinoids under simulated sunlight radiation. The bioluminescent inhibition kinetics shows that all four neonicotinoids have photo-induced toxicity to V. fischeri, but with different light-induced responses. Scavenging radicals (·OH and 1O2) will decrease the photo-induced toxicity of all the four neonicotinoids, indicating radicals play important roles to the photo-chemical reactions of intermediates. Dissolved organic matters exhibit slightly shading effect to the photolysis rates of four parent compounds. However, the ROSs generated by DOM can accelerate the photo-chemical reactions of intermediates, leading to different photo-induced toxicity in present of DOM. According to the detected intermediates and Gaussian calculations, there are different photolysis pathways and mechanisms for the four neonicotinoids. The calculation for photo-sensitization reactions with 3O2 indicates that both energy transfer reactions and electron transfer reactions can be produced under simulated sunlight radiation, which further consolidate that reactive oxygen species are involved in the photolysis process. A theoretical model has been developed to explain the toxicity variations of four neonicotinoids in different aqueous conditions.

Preventive and therapeutic effect of Ganoderma lucidum on kidney injuries and diseases.

Ganoderma lucidum (G. lucidum, Lingzhi) is a well-known Chinese traditional medicine to improve health and to treat numerous diseases for over 2000 years in Asian countries. G. lucidum has the abundant chemical components such as triterpenes and polysaccharides, which have various biological activities including anti-oxidation, anti-inflammation, anti-liver disorders, anti-tumor growth and metastasis, etc. Recently, many lines of studies have elucidated the therapeutic effects of G. lucidum and its extractions on various acute kidney injury (AKI) and chronic kidney disease (CKD) pathogenesis, including autosomal dominant polycystic kidney disease, diabetic nephropathy, renal proximal tubular cell oxidative damage and fibrotic process, renal ischemia reperfusion injury, cisplatin-induced renal injury, adriamycin-induced nephropathy, chronic proteinuric renal diseases, etc. Clinical researches also showed potent anti-renal disease bioactivities of G. lucidum. In this chapter, we review experimental and clinical researches and provide comprehensive insights into the renoprotective effects of G. lucidum. In recent years, renal diseases have gradually aroused attention on account of their booming prevalence worldwide and lack of effective therapies. Although the complicated pathogenesis of kidney diseases, such as acute kidney injury (AKI) and chronic kidney diseases (CKD) have been intensively studied. The morbidity and mortality of AKI and CKD still rise continuously. Thanks to the conventional experience and the multi-target characteristics, natural products have been increasingly recognized as an alternative source for treating renal diseases.

Preventive and Therapeutic Effect of Ganoderma (Lingzhi) on Renal Diseases and Clinical Applications.

The mechanisms of kidney diseases, such as acute kidney injury (AKI) and chronic kidney disease (CKD), have been intensively studied. Nonetheless, the morbidity and mortality of AKI and CKD increased in recent years. Recently, natural products have been increasingly recognized as an alternative source for treating renal diseases on account of the conventional experience and the multi-target characteristics. Ganoderma lucidum (G. lucidum, Lingzhi) has been used for centuries as nutraceuticals and alternative medicine to improve health and to treat numerous diseases. Benefiting from various biological activities, such as anti-oxidation, anti-inflammation, anti-tumor growth and metastasis, etc., G. lucidum has been proved to exhibit significant role in preventing and treating various kidney diseases. In this chapter, we review certain researches and provide comprehensive insights into the renoprotective effects of G. lucidum.

Silencing long non-coding RNA SNHG6 restrains proliferation, migration and invasion of Wilms' tumour cell lines by regulating miR-15a.

Wilms' tumour (WT) is a frequent primary malignant tumour of urinary system in children. LncRNAs small nucleolar RNA host gene 6 (SNHG6) modulates kinds of biological procedures of cancer cells. Our research is to explore the effect and associated regulatory mechanism of SNHG6 in WT. CCK8 assays and bromodeoxyuridine were used to determine cell viability and cell proliferation, respectively. Flow cytometric analysis was performed to measure cell apoptosis rate. Cell mobility was tested through transwell and migration assays. Western blotting was employed to test the expression of proteins related to cell proliferation, cell apoptosis and signal pathways. In the results, overexpression of SNHG6 was found in WT tissues. The knockdown of SNHG6 suppressed cell proliferation, migration and incursion, but promoted apoptosis. Further study found that the knockdown of SNHG6 elevated the expression of miR-15a. Then, the combination of miR-15a inhibitor abolished the inhibiting effect of si-SNHG6 on WT progression. We also found that the TAK1/JNK and Wnt/ß-catenin signal pathways were inactivated by the knockdown of SNHG6 through elevating the expression of miR-15a. In summary, SNHG6 is an oncogene of WT development by targeting miR-15a.

Ezrin directly interacts with AQP2 and promotes its endocytosis.

The water channel aquaporin-2 (AQP2) is a major regulator of water homeostasis in response to vasopressin (VP). Dynamic trafficking of AQP2 relies on its close interaction with trafficking machinery proteins and the actin cytoskeleton. Here, we report the identification of ezrin, an actin-binding protein from the ezrin/radixin/moesin (ERM) family as an AQP2-interacting protein. Ezrin was first detected in a co-immunoprecipitation (co-IP) complex using an anti-AQP2 antibody in a proteomic analysis. Immunofluorescence staining revealed the co-expression of ezrin and AQP2 in collecting duct principal cells, and VP treatment caused redistribution of both proteins to the apical membrane. The ezrin-AQP2 interaction was confirmed by co-IP experiments with an anti-ezrin antibody, and by pulldown assays using purified full-length and FERM domain-containing recombinant ezrin. By using purified recombinant proteins, we showed that ezrin directly interacts with AQP2 C-terminus through its N-terminal FERM domain. Knocking down ezrin expression with shRNA resulted in increased membrane accumulation of AQP2 and reduced AQP2 endocytosis. Therefore, through direct interaction with AQP2, ezrin facilitates AQP2 endocytosis, thus linking the dynamic actin cytoskeleton network with AQP2 trafficking.

Ganoderma triterpenes retard renal cyst development by downregulating Ras/MAPK signaling and promoting cell differentiation.

Autosomal dominant polycystic kidney disease (ADPKD) is a common monogenetic disease characterized by the progressive development of renal cysts with further need for effective therapy. Here our aim was to investigate the effect of Ganoderma triterpenes (GT) on the development of kidney cysts. Importantly, GT attenuated cyst development in two mouse models of ADPKD with phenotypes of severe cystic kidney disease. Assays for tubulogenesis showed that GT promoted epithelial tubule formation in MDCK cells, suggesting a possible effect on epithelial cell differentiation. The role of GT in regulating key signaling pathways involved in the pathogenesis of PKD was further investigated by immune blotting. This showed that GT specifically downregulated the activation of the Ras/MAPK signaling pathway both in vitro and in vivo without detectable effect on the mTOR pathway. This mechanism may be involved in GT downregulating intracellular cAMP levels. Screening of 15 monomers purified from GT for their effects on cyst development indicated that CBLZ-7 (ethyl ganoderate C2) had a potent inhibitory effect on cyst development in vitro. Additionally, like GT, CBLZ-7 was able to downregulate forskolin-induced activation of the Ras/MAPK pathway. Thus, GT and its purified monomer CBLZ-7 may be potential therapeutic regents for treating ADPKD.

Repulsive guidance molecule b inhibits renal cyst development through the bone morphogenetic protein signaling pathway.

Autosomal dominant polycystic kidney disease (ADPKD) is a monogenetic disease that still lacks effective therapy. Repulsive guidance molecule b (RGMb), a co-receptor for bone morphogenetic proteins (BMPs) and a ligand for neogenin, is expressed in renal tubular epithelial cells. Previous studies showed that RGMb plays negative roles in several types of tumors and prevents the immune system from over activation. The present study was designed to explore the effects of RGMb in ADPKD development. We found that expression of RGMb in kidney was less in PKD mice than wild-type mice. With stimulation of 8-bromo-cAMP, RGMb-null embryonic kidneys had greater cyst index, though their ureteric bud branched less than wild-type mice at E13.5. Postnatal RGMb-null kidneys showed interstitial hyperplasia and decreased tubular structures, especially in the boundary area of renal cortex and medulla. RGMb overexpression dramatically inhibited cyst development and promoted tubulogenesis in MDCK cells grown in 3D collagen gels. Biochemical analysis showed increased p-Smad1/5/8 and decreased p-ERK in RGMb-overexpressing MDCK cells, suggesting modulated BMP signaling. Specific inhibition of p-Smad1/5/8 by LDN193189 reversed the suppression of RGMb on MDCK cyst model. These results reveal RGMb as a novel regulator for ADPKD by promoting renal tubule branching and regulating BMP signaling pathway. Elevating RGMb and enhancing p-Smad1/5/8 are promising new strategies to treat ADPKD.

The Effect of cAMP-PKA Activation on TGF-ß1-Induced Profibrotic Signaling.

BACKGROUND: The cAMP-PKA signaling pathway and TGF-ß1-dependent fibrosis pathways are of particular importance in ADPKD progression, but the cross-talk between these pathways remains unclear. Therefore, we used an MDCK-cell model and embryonic kidney-cyst model to study the regulatory role of cAMP-PKA signaling in the TGF-ß1 induced fibrotic process. METHOD AND RESULTS: Pkd1(flox/flox); Ksp-Cre and Pkd1(+/+); Ksp-Cre mice were used as an in vivo model. Increased kidney volume, renal cysts formation and up-regulation of the fibrosis-related proteins TGF-ß1, connective tissue growth factor (CTGF), and fibronectin (FN) can be observed in Pkd1(flox/flox); Ksp-Cre mice. In an embryonic kidneys-cyst model, TGF-ß1, FN and collagen type I were highly expressed. Western blotting revealed the obviously up-regulation of TGF-ß1, CTGF, FN and collagen type I expression following forskolin treatment in MDCK cells. Selective PKA inhibition with H89 may partially reversed the above effects. Pretreatment with the TGF-ß RI kinase inhibitor VI SB431542 suppressed the increased expression of CTGF, FN and collagen type I caused by forskolin. Our data also indicate that forskolin inhibited TGF-ß-induced ERK1/2 phosphorylation and FN up-regulation. ERK inhibition useing PD98059 significantly inhibited the expression of CTGF, FN and collagen type I caused by TGF-ß1. CONCLUSIONS: The cAMP-PKA signaling pathway can directly promote the production of TGF-ß1 and/or TGF-ß1-dependent fibrogenetic molecules in MDCK cells and embryonic kidney cysts, but when TGF-ß1 and its downstream pathways were highly expressed in MDCK cells, cAMP-PKA had a significantly negative effect on TGF-ß1 induced p-ERK1/2 and FN expression.

Synthesis and pharmacological evaluation of novel bisindolylalkanes analogues.

In an effort to develop potent anti-cancer chemopreventive agents that act on topoisomerase II, a novel series of bisindolylalkanes analogues such as 3,3'-(thiochroman-4,4-diyl)bis(1H-indole) are synthesized. Structures of all compounds are elucidated by (1)H NMR, (13)C NMR and HRMS. Anti-proliferative activities for all of these compounds are investigated by the method of MTT assay on 7 human cancer lines. Most of them showed antitumor activities in vitro, the half maximal inhibitory concentration (IC50) value is 7.798 µg/mL of 3a against MCF7. Compound 3a showed comparable topoisomerase II inhibitory activity to etoposide (VP-16) at 100 µM concentration. The rest of the compounds also showed varying degree topoisomerase II inhibitory activity.