A porcine kidney-derived clonal cell line with clear genetic annotation is highly susceptible to African swine fever virus.

African Swine Fever virus (ASFV), the causative agent of African swine fever, is a highly lethal hemorrhagic virus affecting domestic pigs and wild boars. The primary target cells for ASFV infection are porcine alveolar macrophages (PAMs), which are difficult to obtain and maintain in vitro, and less subjective to genetic editing. To overcome these issues and facilitate ASFV research, we obtained a subclonal cell line PK1-C5 by subcloning LLC-PK1 cells that support stable ASFV proliferation. This consequential cell line exhibited high ASFV infection levels and similar viral growth characteristics to PAMs, while also allowing high-efficiency genomic editing through transfection or lentivirus transduction of Cas9. Taken together, our study provided a valuable tool for research aspects including ASFV-host interactions, pathogenicity, and vaccine development.

Protective effect of Peucedanum praeruptorum Dunn extract on oxidative damage of LLC­PK1 cells induced by H2O2.

Peucedanum praeruptorum Dunn extract (PPDE) is a well-known treatment used in traditional Chinese medicines, where it is most commonly used to treat coughs and symptoms such as headaches and fever. In the present study, the antioxidant capacity of PPDE in vitro was determined by scavenging experiments using DPPH, ABTS+·, ·OH, and ·O2-. The cell survival rate was determined by MTT assay. The MDA, SOD, CAT, GSH, and GSH-Px content were determined by colorimetry assays. The expression levels of antioxidant genes SOD, CAT, GSH, and GSH-Px were assessed by reverse transcription-quantitative PCR. HPLC was used to identify the PPDE components. The results suggested that PPDE had scavenging effects on DPPH, ABTS, hydroxyl, and superoxide anion radicals in a concentration-dependent manner; H2O2 treatment resulted in oxidative stress in LLC-PK1 cells, and the degree of injury of LLC-PK1 cells following PPDE treatment was improved, which was positively correlated with its concentration. Peucedanum praeruptorum Dunn extract treatment reduced the content of MDA and increased the content of CAT, SOD1, GSH, and GSH-Px. The mRNA expression levels of antioxidant genes detected by quantitative PCR were consistent with changes in CAT, SOD, GSS, and GSH-Px. Additionally, the trend in CAT, SOD1, GSH, and GSS protein expression levels was also consistent at the mRNA level. PPDE was found to consist of isochlorogenic acid C, myricetin, baicalin, luteolin, and kaempferol. Therefore, PPDE, which was formed of products derived from natural substances, functioned in the inhibition of oxidative damage. The present study aimed to obtain a better understanding of the traditional Chinese medicine Peucedanum praeruptorum Dunn and preliminarily elucidate its antioxidant mechanism at the cellular level. Further animal or human experiments are required to verify the antioxidant effects of PPDE for further development and utilization.

Spectroscopic Characterization and Biological Activity of Hesperetin Schiff Bases and Their Cu(II) Complexes.

The three Schiff base ligands, derivatives of hesperetin, HHSB (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]isonicotinohydrazide), HIN (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]benzhydrazide) and HTSC (N-[2,3-dihydro-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)chromen-4-ylidene]thiosemicarbazide) and their copper complexes, CuHHSB, CuHIN, and CuHTSC were designed, synthesized and analyzed in terms of their spectral characterization and the genotoxic activity. Their structures were established using several methods: elemental analysis, FT-IR, UV-Vis, EPR, and ESI-MS. Spectral data showed that in the acetate complexes the tested Schiff bases act as neutral tridentate ligand coordinating to the copper ion through two oxygen (or oxygen and sulphur) donor atoms and a nitrogen donor atom. EPR measurements indicate that in solution the complexes keep their structures with the ligands remaining bound to copper(II) in a tridentate fashion with (O-, N, Oket) or (O-, N, S) donor set. The genotoxic activity of the compounds was tested against model tumour (HeLa and Caco-2) and normal (LLC-PK1) cell lines. In HeLa cells the genotoxicity for all tested compounds was noticed, for HHSB and CuHHSB was the highest, for HTSC and CuHTSC-the lowest. Generally, Cu complexes displayed lower genotoxicity to HeLa cells than ligands. In the case of Caco-2 cell line HHSB and HTSC induced the strongest breaks to DNA. On the other side, CuHHSB and CuHTSC induced the highest DNA damage against LLC-PK1.

Elucidation of protective effects of oxime derivatives against cisplatin-induced cytotoxicity in LLC-PK1 kidney cells.

This study aimed to explore the renoprotective effects of oxime derivatives against cisplatin-mediated cell death in LLC-PK1 porcine kidney epithelial cells. Treatment with compounds 161-A and 161-F improved cisplatin-mediated LLC-PK1 cell damage and increased cell viability by more than 80% of the control value when compared with that of cisplatin-treated cells. In addition, 161-A and 161-F reduced cisplatin-induced apoptosis. Analysis of the molecular mechanisms underlying the effects exerted by these compounds revealed that treatment with 161-A and 161-B inhibited the protein expression of extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK) and cleaved caspase-3 in cisplatin-treated LLC-PK1 cells. Thus, these findings provide in vitro scientific evidence that oxime derivatives may be useful as pharmacological candidates for the prevention of cisplatin-mediated nephrotoxicity.

Molecular Mechanisms Linking Empagliflozin to Renal Protection in the LLC-PK1 Model of Diabetic Nephropathy.

Aims: Chronic diabetes complications, including diabetic nephropathy (DN), frequently result in end-stage renal failure. This study investigated empagliflozin (SGLT2i) effects on collagen synthesis, oxidative stress, cell survival, and protein expression in an LLC-PK1 model of DN. Methods: Combinations of high glucose (HG) and increasing empagliflozin concentrations (100 nM and 500 nM), as well as combinations of HG, H2O2, and empagliflozin, were used for cell culture treatment. The cell viability, glutathione (tGSH), ECM expression, and TGF-ß1 concentration were measured. In addition, the protein expression of Akt, pAkt, GSK3, pGSK3, pSTAT3, and SMAD7 was determined. Results: The addition of both concentrations of empagliflozin to cells previously exposed to glucose and oxidative stress generally improved cell viability and increased GSH levels (p < 0.001, p < 0.05). In HG30/H2O2/Empa500-treated cells, significant increase in pSTAT3, pGSK3ß, GSK3ß, SMAD7, and pAKT levels (p < 0.001, p < 0.001, p < 0.05) was observed except for AKT. Lower drug concentrations did not affect the protein expression levels. Furthermore, empagliflozin treatment (100 nM and 500 nM) of HG30/H2O2-injured cells led to a decrease in TGF-ß1 levels (p < 0.001). In cells exposed to oxidative stress and hyperglycemia, collagen production remained unchanged. Conclusion: Renoprotective effects of empagliflozin, in this LLC-PK1 cell model of DN, are mediated via activation of the Akt/GSK-3 signalling pathway, thus reducing oxidative stress-induced damage, as well as enhanced SMAD7 expression leading to downregulation of TGF-ß1, one of the key mediators of inflammation and fibrosis.

Signaling Cascade Mediating the Effect of FTY720P on the Na+/K+ ATPase in LLC-PK1.

BACKGROUND/AIMS: In renal ischemia, the Na+/K+ ATPase of the kidney epithelial cells translocates to intracellular compartments, resulting in altered kidney functions. Sphingosine-1-phosphate (S1P) was shown to play a protective role against this ischemic injury. Whether the sphingolipid targets the Na+/K+ ATPase is a possibility that has not been explored before. This work aims at investigating the effect of S1P on renal Na+/K+ ATPase using its analogue FTY720P and LLC-PK1 cells. METHODS: The activity of the Na+/K+ ATPase was assayed by measuring the amount of inorganic phosphate liberated in presence and absence of ouabain, a specific inhibitor of the enzyme while its protein expression was studied by western blot analysis. RESULTS: FTY720P increased the activity of the ATPase in a dose and time dependent manner, with a highest effect observed at 15 minutes and a dose of 80 nM. The protein expression was also increased. The stimulation of the Na+/K+ ATPase disappeared completely in presence of JTE-013, a specific blocker of S1PR2, as well as in presence of Y-27632, a Rho kinase inhibitor, BAPTA-AM, a Ca2+ chelator, wortmannin, a PI3K inhibitor, carboxy-PTIO, a scavenger for nitric oxide (NO), and KT 5823, a PKG inhibitor. CYM 5520, a S1PR2 agonist mimicked the effect of FTY720P. FTY720P increased the expression of p-Akt, a direct effector of PI3K, however, this increase disappeared when Rho kinase was inhibited, revealing that Rho kinase acts upstream PI3K. Glyco-SNAP-1, a NO donor, activated the pump in both presence and absence of wortmannin, indicating that PI3K is upstream NO. Interestingly, glyco-SNAP-1 and 8-bromo-cGMP, a PKG activator, exerted no effect on the Na+/K+ ATPase in absence of free Ca2+ revealing that the NO mediated effect is calcium-dependent. The involvement of calcium was further confirmed by the translocation of NFAT to the nucleus. The presence of verapamil or extracellular EGTA abolished the stimulatory effect of FTY720P, indicating that the source of calcium is extracellular. CONCLUSION: The results suggest that FTY720P activates sequentially S1PR2, Rho kinase, PI3K, leading to NO release and PKG stimulation. The latter phosphorylates calcium channels in the cell membrane, leading to calcium influx, and translocation of the ATPase units to the membrane.

Renoprotective Effect of Liraglutide Is Mediated via the Inhibition of TGF-Beta 1 in an LLC-PK1 Cell Model of Diabetic Nephropathy.

Background: Recently published research demonstrated direct renoprotective effects of the glucagon-like peptide-1 receptor agonist GLP 1 RA, but the relevant molecular mechanisms are still not clear. The aim of this research was to assess the effects of Liraglutide in a cell culture model of diabetic nephropathy on cell viability, antioxidant (GSH) and transforming growth factor beta 1 (TGF- ß1) levels and extracellular matrix (ECM) expression. The metabolic activity in hyperglycemic conditions and the effect of Liraglutide treatment were assessed by measuring Akt, pAkt, GSK3ß, pGSK3ß, pSTAT3, SOCS3, iNOS and NOX4 protein expression with Western blot. F actin distribution was used to assess the structural changes of the cells upon treatment. Materials and methods: The cells were exposed to high glucose (HG30 mM) followed by 0.5 mM H2O2 and a combination of glucose and H2O2 during 24 h. Subsequently, the cells were treated with different combinations of HG30, H2O2 and Liraglutide. Cell viability was determined by an MTT colorimetric test, and the GSH, TGF-ß1 concentration and ECM expression were measured using a spectrophotometric/microplate reader assay and an ELISA kit, respectively. Western blotting was used to detect the protein level of Akt, pAkt, GSK3ß, pGSK3ß, pSTAT3, SOCS3, iNOS and NOX4. The F-actin cytoskeleton was visualized with Phalloidin stain and subsequently quantified. Results: Cell viability was decreased as well as GSH levels in cells treated with a combination of HG30/H2O2, and HG30 alone (p < 0.001). The addition of Liraglutide improved the viability in cells treated with HG30, but it did not affect the cell viability in the cell treated with the addition of H2O2. GSH increased with the addition of Liraglutide in HG30/H2O2 (p < 0.001) treated cells, with no effect in cells treated only with HG30. TGF-ß1 levels (p < 0.001) were significantly increased in HG30 and HG30/H2O2. The addition of Liraglutide significantly decreased the TGF-ß1 levels (p < 0.01; p < 0.05) in all treated cells. The synthesis of collagen was significantly increased in HG30/H2O2 (p < 0.001), while the addition of Liraglutide in HG30/H2O2 significantly decreased collagen (p < 0.001). Akt signaling was not significantly affected by treatment. The GSK3b and NOX4 levels were significantly reduced (p < 0.01) after the peroxide and glucose treatment, with the observable restoration upon the addition of Liraglutide suggesting an important role of Liraglutide in oxidative status regulation and mitochondrial activity. The treatment with Liraglutide significantly upregulated STAT3 (p < 0.01) activity, with no change in SOCS3 indicating a selective regulation of the STAT 3 signaling pathway in glucose and the oxidative overloaded environment. A significant reduction in the distribution of F-actin was observed in cells treated with HG30/H2O2 (p < 0.01). The addition of Liraglutide to HG30-treated cells led to a significant decrease of distribution of F-actin (p < 0.001). Conclusion: The protective effect of Liraglutide is mediated through the inhibition of TGF beta, but this effect is dependent on the extent of cellular damage and the type of toxic environment. Based on the WB analysis we have revealed the signaling pathways involved in cytoprotective and cytotoxic effects of the drug itself, and further molecular studies in vitro and vivo are required to elucidate the complexity of the pathophysiological mechanisms of Liraglutide under conditions of hyperglycemia and oxidative stress.

Panax ginseng and its ginsenosides: potential candidates for the prevention and treatment of chemotherapy-induced side effects.

Chemotherapy-induced side effects affect the quality of life and efficacy of treatment of cancer patients. Current approaches for treating the side effects of chemotherapy are poorly effective and may cause numerous harmful side effects. Therefore, developing new and effective drugs derived from natural non-toxic compounds for the treatment of chemotherapy-induced side effects is necessary. Experiments in vivo and in vitro indicate that Panax ginseng (PG) and its ginsenosides are undoubtedly non-toxic and effective options for the treatment of chemotherapy-induced side effects, such as nephrotoxicity, hepatotoxicity, cardiotoxicity, immunotoxicity, and hematopoietic inhibition. The mechanism focus on anti-oxidation, anti-inflammation, and anti-apoptosis, as well as the modulation of signaling pathways, such as nuclear factor erythroid-2 related factor 2 (Nrf2)/heme oxygenase-1 (HO-1), P62/keap1/Nrf2, c-jun N-terminal kinase (JNK)/P53/caspase 3, mitogen-activated protein kinase (MEK)/extracellular signal-regulated kinases (ERK), AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR), mitogen-activated protein kinase kinase 4 (MKK4)/JNK, and phosphatidylinositol 3-kinase (PI3K)/AKT. Since a systemic review of the effect and mechanism of PG and its ginsenosides on chemotherapy-induced side effects has not yet been published, we provide a comprehensive summarization with this aim and shed light on the future research of PG.

Identification of Renoprotective Phytosterols from Mulberry (Morus alba) Fruit against Cisplatin-Induced Cytotoxicity in LLC-PK1 Kidney Cells.

The aim of this study was to explore the protective effects of bioactive compounds from the fruit of the mulberry tree (Morus alba L.) against cisplatin-induced apoptosis in LLC-PK1 pig kidney epithelial cells. Morus alba fruit is a well-known edible fruit commonly used in traditional folk medicine. Chemical investigation of M. alba fruit resulted in the isolation and identification of six phytosterols (1-6). Their structures were determined as 7-ketositosterol (1), stigmast-4-en-3ß-ol-6-one (2), (3ß,6α)-stigmast-4-ene-3,6-diol (3), stigmast-4-ene-3ß,6ß-diol (4), 7ß-hydroxysitosterol 3-O-ß-d-glucoside (5), and 7α-hydroxysitosterol 3-O-ß-d-glucoside (6) by analyzing their physical and spectroscopic data as well as liquid chromatography/mass spectrometry data. All compounds displayed protective effects against cisplatin-induced LLC-PK1 cell damage, improving cisplatin-induced cytotoxicity to more than 80% of the control value. Compound 1 displayed the best effect at a relatively low concentration by inhibiting the percentage of apoptotic cells following cisplatin treatment. Its molecular mechanisms were identified using Western blot assays. Treatment of LLC-PK1 cells with compound 1 decreased the upregulated phosphorylation of p38 and c-Jun N-terminal kinase (JNK) following cisplatin treatment. In addition, compound 1 significantly suppressed cleaved caspase-3 in cisplatin-induced LLC-PK1 cells. Taken together, these findings indicated that cisplatin-induced apoptosis was significantly inhibited by compound 1 in LLC-PK1 cells, thereby supporting the potential of 7-ketositosterol (1) as an adjuvant candidate for treating cisplatin-induced nephrotoxicity.

Antioxidant and Anti-Inflammatory Effects of 3-Dehydroxyceanothetric Acid 2-Methyl Ester Isolated from Ziziphus jujuba Mill. against Cisplatin-Induced Kidney Epithelial Cell Death.

Cisplatin is a platinum-based chemotherapeutic agent for treating solid tumors; however, it presents a risk factor for nephropathy. In the present study, we investigated the antioxidant and anti-inflammatory effects of 3-dehydroxyceanothetric acid 2-methyl ester (3DC2ME) isolated from Ziziphus jujuba Mill. in LLC-PK1 cells following cisplatin-induced cytotoxicity. These cells were exposed to 3DC2ME for 2 h, followed by treatment with cisplatin for 24 h. The treated cells were subjected to cell viability analysis using the Ez-Cytox assay. Reactive oxygen species (ROS) were detected via 2', 7'- dichlorodihydrofluorescein diacetate (DCFH-DA) staining. In addition, western blotting and fluorescent immunostaining were performed to evaluate protein expressions related to oxidative stress and inflammation pathways. Pretreatment with 3DC2ME protected LLC-PK1 cells from cisplatin-induced cytotoxicity and oxidative stress. In addition, pretreatment with 3DC2ME upregulated heme oxygenase 1 (HO-1) via the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway in the cisplatin-treated LLC-PK1 cells. Furthermore, the increase in the expressions of IκB kinase α/ß (IKKα/ß), inhibitor of kappa B alpha (IκBα), nuclear factor kappa B (NF-κB), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) in these cells was inhibited. These results provide basic scientific evidence for understanding the antioxidant and anti-inflammatory effects of 3DC2ME isolated from Z. jujuba against cisplatin-induced kidney epithelial cell death.

Human Proximal Tubule Epithelial Cells (HK-2) as a Sensitive In Vitro System for Ochratoxin A Induced Oxidative Stress.

Ochratoxin A (OTA) is a mycotoxin that is potentially carcinogenic to humans. Although its mechanism remains unclear, oxidative stress has been recognized as a plausible cause for the potent renal carcinogenicity observed in experimental animals. The effect of OTA on oxidative stress parameters in two cell lines of LLC-PK1 and HK-2 derived from the kidneys of pig and human, respectively, were investigated and compared. We found that the cytotoxicity of OTA on LLC-PK1 and HK-2 cells was dose- and time-dependent in both cell lines. Furthermore, increased intracellular reactive oxygen species (ROS) induced by OTA in both cell lines were observed in a time-dependent manner. Glutathione (GSH) was depleted by OTA at >48 h in HK-2 but not in LLC-PK1 cells. While the mRNA levels of glucose-6-phosphate dehydrogenase (G6PD) and glutathione peroxidase 1 (GPX1) in LLC-PK1 were down-regulated by 0.67- and 0.66-fold, respectively, those of catalase (CAT), glutathione reductase (GSR), and superoxide dismutase 1 (SOD) in HK-2 were up-regulated by 2.20-, 2.24-, and 2.75-fold, respectively, after 72 h exposure to OTA. Based on these results, we conclude that HK-2 cells are more sensitive to OTA-mediated toxicity than LLC-PK1, and OTA can cause a significant oxidative stress in HK-2 as indicated by changes in the parameter evaluated.

Preventive Effect of Muscone against Cisplatin Nephrotoxicity in LLC-PK1 Cells.

Cisplatin, one of the most common antitumor agents, is widely applied to treat various cancerous diseases and is included in the World Health Organization Model List of Essential Medicines. Cisplatin therapy is used to treat 10-20% of all cancerous cases, and its cure rate is especially high in testicular cancer (over 90%). However, a major side effect of this anticancer drug is nephrotoxicity, limiting treatment effect and reducing the quality of life in cancer patients. Muscone, an odoriferous constituent of musk, was confirmed to inhibit cisplatin-induced LLC-PK1 kidney proximal tubule cell death in a dose-dependent manner. In term of renal protective mechanism, muscone inhibited cisplatin oxidative toxicity by decreasing reactive oxygen species (ROS) level and stimulating HO-1 expression. Muscone also exerted anti-inflammation effect through inhibition of p38 phosphorylation. Furthermore, muscone mitigated cisplatin-induced apoptosis in LLC-PK1 cells via both intrinsic and extrinsic pathways by inhibiting pro-apoptotic protein Bax expression, and cleaved caspase-3, 7, and 8; and increase of anti-apoptotic protein Bcl-2 level. In addition, the anti-apoptotic effect of muscone also was enhanced by preventing p53 expression and its phosphorylation. Our study showed that muscone may be a potential protective agent against cisplatin-induced nephrotoxicity.

Impact of In Vitro Passive Permeability in a P-gp-transfected LLC-PK1 Model on the Prediction of the Rat and Human Unbound Brain-to-Plasma Concentration Ratio.

PURPOSE: More accurate prediction of the extent of drug brain exposure in early drug discovery and understanding potential species differences could help to guide medicinal chemistry and avoid unnecessary animal studies. Hence, the aim of the current study was to validate the use of a P-gp transfected LLC-PK1 model to predict the unbound brain-to-plasma concentration ratio (Kpuu,brain) in rats and humans. METHODS: MOCK-, Mdr1a- and MDR1-transfected LLC-PK1 monolayers were applied in a transwell setup to quantify the bidirectional transport for 12 specific P-gp substrates, 48 UCB drug discovery compounds, 11 compounds with reported rat in situ brain perfusion data and 6 compounds with reported human Kpuu,brain values. The in vitro transport data were introduced in a minimal PBPK model (SIVA®) to determine the transport parameters. These parameters were combined with the differences between in vitro and in vivo passive permeability as well as P-gp expression levels (as determined by LC-MS/MS), to predict the Kpuu,brain. RESULTS: A 10-fold difference between in vitro and in vivo passive permeability was observed. Incorporation of the differences between in vitro and in vivo passive permeability and P-gp expression levels resulted in an improved prediction of rat (AAFE 2.17) and human Kpuu,brain (AAFE 2.10). CONCLUSIONS: We have succesfully validated a methodology to use a P-gp overexpressing LLC-PK1 cell line to predict both rat and human Kpuu,brain by correcting for both passive permeability and P-gp expression levels.

Chlorpromazine Induces Basolateral Aquaporin-2 Accumulation via F-Actin Depolymerization and Blockade of Endocytosis in Renal Epithelial Cells.

We previously showed that in polarized Madin-Darby canine kidney (MDCK) cells, aquaporin-2 (AQP2) is continuously targeted to the basolateral plasma membrane from which it is rapidly retrieved by clathrin-mediated endocytosis. It then undertakes microtubule-dependent transcytosis toward the apical plasma membrane. In this study, we found that treatment with chlorpromazine (CPZ, an inhibitor of clathrin-mediated endocytosis) results in AQP2 accumulation in the basolateral, but not the apical plasma membrane of epithelial cells. In MDCK cells, both AQP2 and clathrin were concentrated in the basolateral plasma membrane after CPZ treatment (100 µM for 15 min), and endocytosis was reduced. Then, using rhodamine phalloidin staining, we found that basolateral, but not apical, F-actin was selectively reduced by CPZ treatment. After incubation of rat kidney slices in situ with CPZ (200 µM for 15 min), basolateral AQP2 and clathrin were increased in principal cells, which simultaneously showed a significant decrease of basolateral compared to apical F-actin staining. These results indicate that clathrin-dependent transcytosis of AQP2 is an essential part of its trafficking pathway in renal epithelial cells and that this process can be inhibited by selectively depolymerizing the basolateral actin pool using CPZ.

Investigation of MDR1-overexpressing cell lines to derive a quantitative prediction approach for brain disposition using in vitro efflux activities.

MDR1-overexpressing Lilly Laboratories cell porcine kidney 1 cells (LLC-PK1-MDR1) and Madin-Darby canine kidney cells (MDCK-MDR1) are widely used in drug discovery to evaluate the in vivo relevance of MDR1-mediated efflux. However, as the in vitro efflux ratio (ER) of these cell lines are variable among research facilities, the in vitro ER of these cell lines that would affect quantitative predictivity of brain disposition has not been fully clarified. The aim of this study was to examine the effect of ER on the quantitative predictivity of brain disposition toward compounds with MDR1 and/or breast cancer resistant protein (BCRP) liabilities. Test compounds including internal molecules and five typical substrates of MDR1 and/or BCRP were assessed via an in vitro transporter assay to determine the corrected flux ratio (CFR) and an in vivo animal study using wild-type (WT) and Mdr1a (-/-)/Bcrp(-/-) (dual KO) rats. To assess the in vivo ER for MDR1, the two cell lines LLC-PK1-MDR1 and MDCK-MDR1 were used. After intravenously administering 29 test compounds to rats, the Kp,brain ratio (ratio of Kp,brain,WT to Kp,brain,dual KO), which is considered to be the unbound plasma-to-brain ratio (Kp,uu,brain) that does not require correction for protein binding in both plasma and brain, was determined by measuring their concentrations in the plasma and brain. The Kp,brain ratio of these compounds was predicted by fitting scaling factor that was extrapolated from the in vitro to in vivo ER for MDR1 and BCRP, defined as α and ß, respectively. Kp,brain ratio values of 83% and 68% of compounds were predicted by using MDCK-MDR1 and LLC-PK1-MDR1, respectively, within a 2-fold range of the actual corresponding values. The α predicted from CFRs of MDCK-MDR1 was 47-fold smaller than that of LLC-PK1-MDR1; however, a dramatic change in ß was not observed. This result appears to be consistent with the data of in vitro transport activity of MDR1, which was estimated to be ~28-fold higher in MDCK-MDR1 than in LLC-PK1-MDR1 by correlation analysis with CFR. Through this study, we revealed that 1) brain disposition in rats was well-predicted by considering the in vitro efflux activities for both MDR1 and BCRP, and 2) MDCK-MDR1 was the superior cell line for the quantitative prediction of brain disposition.

Structural Insights into Escherichia coli Shiga Toxin (Stx) Glycosphingolipid Receptors of Porcine Renal Epithelial Cells and Inhibition of Stx-Mediated Cellular Injury Using Neoglycolipid-Spiked Glycovesicles.

Shiga toxin (Stx) producing Escherichia coli (STEC) cause the edema disease in pigs by releasing the swine-pathogenic Stx2e subtype as the key virulence factor. Stx2e targets endothelial cells of animal organs including the kidney harboring the Stx receptor glycosphingolipids (GSLs) globotriaosylceramide (Gb3Cer, Galα1-4Galß1-4Glcß1-1Cer) and globotetraosylceramide (Gb4Cer, GalNAcß1-3Galα1-4Galß1-4Glcß1-1Cer). Since the involvement of renal epithelial cells in the edema disease is unknown, in this study, we analyzed the porcine kidney epithelial cell lines, LLC-PK1 and PK-15, regarding the presence of Stx-binding GSLs, their sensitivity towards Stx2e, and the inhibitory potential of Gb3- and Gb4-neoglycolipids, carrying phosphatidylethanolamine (PE) as the lipid anchor, towards Stx2e. Immunochemical and mass spectrometric analysis revealed various Gb3Cer and Gb4Cer lipoforms as the dominant Stx-binding GSLs in both LLC-PK1 and PK-15 cells. A dihexosylceramide with proposed Galα1-4Gal-sequence (Gal2Cer) was detected in PK-15 cells, whereas LLC-PK1 cells lacked this compound. Both cell lines were susceptible towards Stx2e with LLC-PK1 representing an extremely Stx2e-sensitive cell line. Gb3-PE and Gb4-PE applied as glycovesicles significantly reduced the cytotoxic activity of Stx2e towards LLC-PK1 cells, whereas only Gb4-PE exhibited some protection against Stx2e for PK-15 cells. This is the first report identifying Stx2e receptors of porcine kidney epithelial cells and providing first data on their Stx2e-mediated damage suggesting possible involvement in the edema disease.

Hybrid Polyketides from a Hydractinia-Associated Cladosporium sphaerospermum SW67 and Their Putative Biosynthetic Origin.

Five hybrid polyketides (1a, 1b, and 2-4) containing tetramic acid core including a new hybrid polyketide, cladosin L (1), were isolated from the marine fungus Cladosporium sphaerospermum SW67, which was isolated from the marine hydroid polyp of Hydractinia echinata. The hybrid polyketides were isolated as a pair of interconverting geometric isomers. The structure of 1 was determined based on 1D and 2D NMR spectroscopic and HR-ESIMS analyses. Its absolute configuration was established by quantum chemical electronic circular dichroism (ECD) calculations and modified Mosher's method. Tetramic acid-containing compounds are reported to be derived from a hybrid PKS-NRPS, which was also proved by analyzing our 13C-labeling data. We investigated whether compounds 1-4 could prevent cell damage induced by cisplatin, a platinum-based anticancer drug, in LLC-PK1 cells. Co-treatment with 2 and 3 ameliorated the damage of LLC-PK1 cells induced by 25 µM of cisplatin. In particular, the effect of compound 2 at 100 µM (cell viability, 90.68 ± 0.81%) was similar to the recovered cell viability of 88.23 ± 0.25% with 500 µM N-acetylcysteine (NAC), a positive control.

[Protective effects of flavonoids from Gynostemma pentaphyllum on oxidative damage in LLC-PK1 cells].

Four flavonoids were isolated from Gynostemma pentaphyllum by chromatography methods and their structures were identified by MS and NMR spectra data as quercetin-3-O-( 2″,6″-di-α-L-rhamnosyl)-ß-D-galactopyranoside( 1),quercetin-3-O-( 2″,6″-di-α-L-rhamnosyl)-ß-D-glucopyranoside( 2),quercetin-3-O-( 2″-α-L-rhamnosyl)-ß-D-galactopyranoside( 3),and quercetin-3-O-( 2″-α-L-rhamnosyl)-ß-D-glucopyranoside( 4). Among them,compounds 1-3 were obtained from the Cucurbitaceae family for the first time.The four flavonoids showed potent antioxidant effects against the DPPH,·OH and ■radicals in vitro,especially for DPPH radical scavenging activity with the IC50 values of 71. 4,29. 5,48. 3 and 79. 2 µmol·L~(-1),respectively. Moreover,the four flavonoids displayed strong cytoprotection against AAPH-induced oxidative damage in LLC-PK1 cells by suppressing the increase of malondialdehyde( MDA) and the decrease of the superoxide dismutase( SOD) and glutathione( GSH). Since further research is needed to prove its efficacy in vivo and clinical trial,the study may provide four potential antioxidants from G. pentaphyllum.

Protective effects of flavonoids from Gynostemma pentaphyllum on oxidative damage in LLC-PK1 cells / 中国中药杂志

Four flavonoids were isolated from Gynostemma pentaphyllum by chromatography methods and their structures were identified by MS and NMR spectra data as quercetin-3-O-( 2″,6″-di-α-L-rhamnosyl)-β-D-galactopyranoside( 1),quercetin-3-O-( 2″,6″-di-α-L-rhamnosyl)-β-D-glucopyranoside( 2),quercetin-3-O-( 2″-α-L-rhamnosyl)-β-D-galactopyranoside( 3),and quercetin-3-O-( 2″-α-L-rhamnosyl)-β-D-glucopyranoside( 4). Among them,compounds 1-3 were obtained from the Cucurbitaceae family for the first time.The four flavonoids showed potent antioxidant effects against the DPPH,·OH and ■radicals in vitro,especially for DPPH radical scavenging activity with the IC50 values of 71. 4,29. 5,48. 3 and 79. 2 μmol·L~(-1),respectively. Moreover,the four flavonoids displayed strong cytoprotection against AAPH-induced oxidative damage in LLC-PK1 cells by suppressing the increase of malondialdehyde( MDA) and the decrease of the superoxide dismutase( SOD) and glutathione( GSH). Since further research is needed to prove its efficacy in vivo and clinical trial,the study may provide four potential antioxidants from G. pentaphyllum.

Paliperidone, a relatively novel atypical antipsychotic drug, is a substrate for breast cancer resistance protein.

Paliperidone (PAL) is a relatively novel atypical antipsychotic drug for schizophrenia that induces markedly varying responses. Breast cancer resistance protein (BCRP) is a recently discovered member of the ATP-binding cassette superfamily that has been used to control drug absorption, distribution and elimination, and especially to impede drug entry into the brain. To the best of our knowledge, the present study is the first to investigate the possibility of using PAL as a BCRP substrate. The intracellular accumulation and bidirectional transport were investigated using transfected 293 cell/BCRP and porcine renal endothelial cell (LLC-PK1)/BCRP cell monolayers and BCRP overexpression was confirmed by reverse transcription-quantitative polymerase chain reaction and western blot analysis. The in vitro affinity to BCRP was assessed in human BCRP (Arg482) membranes. The intracellular accumulation and bidirectional transport investigations demonstrated that BCRP can efflux PAL from cells and significantly decrease its cellular concentration over a concentration range of 0.1-50 µM. The in vitro affinity experiments indicated that PAL has a moderate affinity to BCRP at 0.1-100 µM. These results together suggest that PAL is a substrate for BCRP and that it can affect the blood-brain barrier penetration of PAL at therapeutic dosages.