Human Kidney Epithelial Cells Research Articles

Cytotoxic and Mutagenic Properties of C1' and C3'-Epimeric Lesions of 2'-Deoxyribonucleosides in Human Cells.

Genomic integrity is constantly challenged by exposure to environmental and endogenous genotoxic agents. Reactive oxygen species (ROS) represent one of the most common types of DNA damaging agents. While ROS mainly induce single-nucleobase lesions, epimeric 2-deoxyribose lesions can also be induced upon hydrogen atom abstraction from the C1', C3', or C4' carbon and the subsequent incorrect chemical repair of the resulting carbon-centered radicals. Herein, we investigated the replicative bypass of the C1'- and C3'-epimeric lesions of the four 2'-deoxynucleosides in HEK293T human embryonic kidney epithelial cells. Our results revealed distinct bypass efficiencies and mutagenic properties of these two types of epimeric lesions. Replicative bypasses of all C1'-epimeric lesions except α-dA are mutagenic in HEK293T cells, and their mutagenic properties are further modulated by translesion synthesis (TLS) DNA polymerases. By contrast, none of the four C3'-epimeric lesions are mutagenic, and the replicative bypass of these lesions is not compromised upon depletion of polymerase η, ι, κ, or ζ. Together, our results provide important new knowledge about the cytotoxic and mutagenic properties of C1' and C3' epimeric lesions, and reveal the roles of TLS DNA polymerases in bypassing these lesions in human cells.

Candesartan inhibits inflammation through an angiotensin II type 1 receptor independent way in human embryonic kidney epithelial cells.

Besides stimulating vasoconstriction, Angiotensin II is also well known in inducing reactive oxygen species and promoting inflammatory phenotype switch via its type 1 receptor. In clinic, Angiotensin II type 1 (AT1) receptor blocker like candesartan has been widely applied as an antihypertensive medication. We previous have demonstrated that a higher dose of candesartan plays a protective role after kidney injury. However, whether candesartan could exhibit anti-inflammatory effects remains unclear. Here, by stimulating isolated human embryonic kidney epithelial cells with tumor necrosis factor-α (TNF-α), we observed the anti-inflammation capacity of candesartan ex vivo. It was found that pre-treat with candesartan significantly suppressed transforming growth factor-β (TGF-β) and interleukin-6 (IL-6) expression after incubation with TNF-α. Surprisingly, silence of angiotensin II type 1 receptor has little effects on reducing TGF-β or IL-6 products. Furthermore, candesartan inhibited TNF-α-induced oxidative stress in the primary cultured tubular epithelial cells. Overall, our data indicates that candesartan suppresses TNF-α-induced inflammatory cytokine production by inhibiting oxidative stress, rather than block AT1 receptor activity.

Evaluation of nonlinear dynamics of the cell structure damage as a promising method for personalized cancer diagnostics

Background:Modulation interference microscopy is one of the promising technologies for early personalized cancer diagnostics, while it gives the possibility to obtain real-time images of living functioning cells at a nanometer resolution, as well as to assess spatio-temporal changes of their intracellular structures.Aim:To identify the markers of malignant transformation of cells based on a multiple scale spatio-temporal of the data obtained by lase interferometry duringin situregistration of changes in intracellular structures.Materials and methods:The study was performed with the breast adenocarcinoma MCF-7 cell line and human kidney epithelial cells HEK 293 provided by the N.N. Blokhin National Medical Research Centre of Oncology (Moscow, Russia). Changes in fluctuation of the living cell phase thickness were measured with a laser modulation interference microscope MIM-340 (PA UOMZ, Russia). The multifractal detrended fluctuation analysis (MF-DFA) was used to assess the results obtained.Results:By comparison of the scaling exponential spectra and corresponding spectra of the singular fluctuations in the minimal and maximal diameters of the phase pictures of HEK 293 cells and MCF-7 cells, it was shown that the cancer cell dynamics has definite monofractal properties: a linear dependency of the scaling exponentsτfrom the actualqparameter and “dotted” singularity spectrum (the spectral width does not exceed 0.1). The dynamics of healthy cell demonstrated multifractal properties, such as a non-linear dependencyτ(q)and a bell shaped singularity spectrum (the spectral width over 0.1).Conclusion:We have proposed a new approach to assess the intracellular structural damage during oncogenesis. The signs of monofractality are objective criteria of malignization. The results obtained seems promising as early diagnostic markers of cancer transformation (damage) of cells and determination of tumor cell sensitivity / resistance to anti-tumor agents in an individual patient.

Generation of 3D Soluble Signal Gradients in Cell-Laden Hydrogels Using Passive Diffusion.

Soluble signal gradients play an important role in organ patterning, cell migration, and differentiation. Currently, signal gradients in 2D cell culture are realized using microfluidics and here cells are exposed to high and nonphysiological shear stress. Tissue morphogenesis (organogenesis) however occurs in 3D and therefore there is a need for simple and practical systems to impose gradients to cells dispersed in 3D matrix. Herein, a 3D gradient generator based on passive diffusion elements that recapitulates interstitial flow and is capable of imposing predictable gradients over long length scales (6 mm) lasting up to 48 h to cells dispersed in a hydrogel environment is reported. Using recombinant human WNT3A (rhWNT3A), the spatiotemporal activation of the canonical WNT pathway in human epithelial kidney cells and human mesenchymal stems cells expressing a green fluorescence protein reporter on a transcription factor/lymphoid enhancer-binding factor (TCF/LEF) promoter is demonstrated. By refining computation models based on experimental findings, the diffusion coefficient of rhWNT3A in presence of human cells in 3D is determined. Furthermore, the formation of rhBMP4 gradients is visualized using immunohistochemistry by staining for phospho-SMAD1/5, the downstream targets of the bone morphogenetic protein (BMP) pathway. The simplicity of the gradient generator is expected to spur its adoption in studying developmental biology paradigms in vitro.

Evaluation of the reproductive potential of Encephalitozoon intestinalis in four different cell line

Microsporidia are parasites that can cause infections in many vertebrate and invertebrate organisms and produce small spores resistant to environmental conditions. As they are obligate intracellular parasites, axenic cultures cannot be performed. The aim of this study was to investigate the reproductive potential of the parasite in human colon epidermal adenocarcinoma (Caco-2), human monocytic (U937), African green monkey renal epithelial (VERO) and human kidney epithelial (HEK-293) cell lines of tissue and organs where the parasite is located by following the culture of the parasites and the amount of spores for six weeks. RPMI-1640 medium was used for the cultivation of U937 cells, while DMEM was used for other cell lines and the immature U937 cells were stimulated with Phorbol-12-Myristate-13-Acetate before infection. All of the host cell groups were infected with freshly collected Encephalitozoon intestinalis spores in ratio 1:30 and free spores in the culture media were removed after overnight incubation at 37°C under 5% CO2 condition for parasite invasion. The first release of the spores from the infected cells was observed and recorded by following for six weeks. Furthermore, the spore density released from each cell groups was evaluated by measuring the parasite load by Thoma cell counting chamber and quantified by real-time PCR. As a result of the study, it was observed that four cell lines could be infected by E.intestinalis and the spore production can be maintained for six weeks. It was observed that the monolayer macrophages and CaCo-2 cells, started to be detached from the culture flasks in few days following the parasite invasion, thus decreasing the number of host cells. After 1-2 weeks, HEK-293 cells were also detached from the surface, thus negatively affected the pure spore production by contaminating the media with dead host cell suspension. Spores started to appear in VERO cell media at the end of the second week after initial infection, while it took longer time for other cells to start releasing spores. Over the course of six weeks, the VERO cell line had the highest spore-producing potential among the other cell lines. In conclusion, this study compared the potential for reproduction of E.intestinalis in three human cell lines and monkey originated VERO cell line. This study demonstrated that cells derived from the tissues or organs where Microsporidia species causes disseminated infections could be infected by the parasitic spores in vitro. Additionally, the parasite can survive and propagate longer than six weeks. The authors believe that the results of this study will contribute to the further studies related to the parasite in the area of genetics, pharmacology, biochemistry, immunology and eradication studies.

Structural Characterization, Antioxidant Activity, and Biomedical Application of Astragalus Polysaccharide Degradation Products

To study the antioxidant capacity of Astragalus polysaccharides (APS) with different molecular weights, we used hydrogen peroxide to degrade original Astragalus polysaccharide (APS0) with an initial molecular weight of 11.03 kDa and obtained three degraded polysaccharides with molecular weights of 8.38 (APS1), 4.72 (APS2), and 2.60 kDa (APS3). The structures of these polysaccharides were characterized by 1H NMR, 13C NMR, FT-IR, and GC/MS. The degradation process did not cause significant changes in the main chain structure of APS. The monosaccharide component of APS before and after degradation was slightly changed. The antioxidant ability in vitro (removing hydroxyl and ABTS radicals and reducing ability) and in cells (superoxide dismutase and malondialdehyde generation) of these polysaccharides is closely related to their molecular weight. If the molecular weight of APS is very high or low, it is not conducive to their activity. Only APS2 with moderate molecular weight showed the greatest antioxidant activity and ability to repair human kidney epithelial (HK-2) cells. Therefore, APS2 can be used as a potential antistone polysaccharide drug.

Arsenic induces fibrogenic changes in human kidney epithelial cells potentially through epigenetic alterations in DNA methylation.

Arsenic contamination is a significant public health issue, and kidney is one of the target organ for arsenic-induced adverse effects. Renal fibrosis is a well-known pathological stage frequently observed in progressive chronic kidney disease (CKD). Epidemiological studies implicate arsenic exposure to CKD, but the role of arsenic in kidney fibrosis and the underlying mechanism is still unclear. It is in this context that the current study evaluated the effects of long-term arsenic exposure on the cellular response in morphology, and marker genes expression with respect to fibrosis using human kidney 2 (HK-2) epithelial cells. Results of this study revealed that in addition to increased growth, HK-2 cells underwent phenotypic, biochemical and molecular changes indicative of epithelial-mesenchymal transition (EMT) in response to the exposure to arsenic. Most importantly, the arsenic-exposed cells acquired the pathogenic features of fibrosis as supported by increased expression of markers for fibrosis, such as Collagen I, Fibronectin, transforming growth factor β, and α-smooth muscle actin. Upregulation of fibrosis associated signaling molecules such as tissue inhibitor of metalloproteinases-3 and matrix metalloproteinase-2 as well as activation of AKT was also observed. Additionally, the expression of epigenetic genes (DNA methyltransferases 3a and 3b; methyl-CpG binding domain 4) was increased in arsenic-exposed cells. Treatment with DNA methylation inhibitor 5-Aza-2'-dC reversed the EMT properties and restored the level of phospho-AKT. Together, these data for the first time suggest that long-term exposure to arsenic can increase the risk of kidney fibrosis. Additionally, our data suggest that the arsenic-induced fibrotic changes are, at least in part, mediated by DNA methylation and therefore potentially can be reversed by epigenetic therapeutics.

Preparation and evaluation of anti-renal fibrosis activity of novel truncated TGF-β receptor type II.

Production of excessive transforming growth factor-beta 1 (TGF-β1) with elevated TGF-β1 activity has been implicated in renal fibrosis via renal epithelial cells activation and collagen deposition. As such, attenuating the binding of TGF-β1 to its receptor TGF-beta receptor type II (TGF-βRII) in TGF-β1-dependent signaling is an attractive target for the control of renal fibrosis. Here, we verified the interaction between novel truncated human TGF-βRII (thTβRII, Thr23-Gln166) and TGF-β1, prepared thTβRII in Escherichia coli, and assessed the effects of thTβRII on TGF-β1-induced human kidney epithelial cells (HK-2) and unilateral ureteral obstruction (UUO) model of renal fibrosis. Our data showed that thTβRII accounted for up to 20% of the total protein and 40% of the inclusion bodies of whole cell lysates under the optimal conditions (0.8 mM IPTG and 25°C for 6 H). Most of the expressed protein in inclusion body was refolded by dialysis refolding procedures and purified by Ni2+ -IDA affinity chromatography. Furthermore, thTβRII decreased type I collagen and α-smooth muscle actin protein expression in TGF-β1-induced HK-2 cells, and ameliorated kidney morphology and fibrotic responses in fibrosis animal. These findings indicate that thTβRII holds great promise for developing new treatments for renal fibrosis.

Identification and Characterization of a Phase-Variable Element That Regulates the Autotransporter UpaE in Uropathogenic Escherichia coli.

ABSTRACTUropathogenic Escherichia coli (UPEC) is the most common etiologic agent of uncomplicated urinary tract infection (UTI). An important mechanism of gene regulation in UPEC is phase variation that involves inversion of a promoter-containing DNA element via enzymatic activity of tyrosine recombinases, resulting in biphasic, ON or OFF expression of target genes. The UPEC reference strain CFT073 has five tyrosine site-specific recombinases that function at two previously characterized promoter inversion systems, fimS and hyxS. Three of the five recombinases are located proximally to their cognate target elements, which is typical of promoter inversion systems. The genes for the other two recombinases, IpuA and IpuB, are located distal from these sites. Here, we identified and characterized a third phase-variable invertible element in CFT073, ipuS, located proximal to ipuA and ipuB. The inversion of ipuS is catalyzed by four of the five CFT073 recombinases. Orientation of the element drives transcription of a two-gene operon containing ipuR, a predicted LuxR-type regulator, and upaE, a predicted autotransporter. We show that the predicted autotransporter UpaE is surface located and facilitates biofilm formation as well as adhesion to extracellular matrix proteins in a K-12 recombinant background. Consistent with this phenotype, the ipuS ON condition in CFT073 results in defective swimming motility, increased adherence to human kidney epithelial cells, and a positive competitive kidney colonization advantage in experimental mouse UTIs. Overall, the identification of a third phase switch in UPEC that is regulated by a shared set of recombinases describes a complex phase-variable virulence network in UPEC.

Potential bactericidal activity of S. nux-vomica–ZnO nanocomposite against multidrug-resistant bacterial pathogens and wound-healing properties

Multidrug resistance in bacterial strains has become the greatest challenge for healthcare professionals for treating non-healing ulcers such as diabetic foot infections (DFI). Plant-mediated synthesis of S. nux-vomica–ZnO nanocomposite appears as a potential new alternative therapeutic agent that might be capable of tackling antibiotic-resistant bacterial pathogens and for treating a non-healing ulcer. The aim of the study was to investigate the antibacterial potential of S. nux-vomica–ZnO nanocomposite biosynthesised from Strychnos nux-vomica against multidrug-resistant organisms (MDROs) from DFU, wound-healing properties, and cytotoxic effects. The antibacterial potential was assessed by minimum inhibitory concentration (MIC)/ minimum bactericidal concentration (MBC) assays, time-kill kinetics, protein-leakage, and flow cytometric analysis. The wound-healing properties were assessed by scratch assay on mouse L929 fibroblastic cell line to quantify cell migration towards the injured area. Cytotoxicity was assessed using 3-[4,5-dimethyl-2-thiazol-yl]-2,5-diphenyl- 2H-tetrazolium bromide (MTT) cellular viability assay on the L929 cell line and human embryonic kidney epithelial (HEK-293) cell line. Strychnos nux-vomica–ZnO nanocomposite at a size range of 10–12 nm exhibited significant bactericidal potency at a concentration of 100–200 μg/ml against MDR–Methicillin-resistant Staphylococcus aureus, MDR–Escherichia coli, MDR–Pseudomonas aeruginosa, MDR–Acinetobacter baumannii, and also against standard bacterial strains S. aureus ATCC 29213, E. coli ATCC 25922, P. aeruginosa ATCC 27853, E. faecalis ATCC 29212. S. nux-vomica–ZnO nanocomposite also exhibited wound-healing and reduced cytotoxic properties at the antimicrobially active concentrations. Our findings thus suggested remarkable bactericidal properties of S. nux-vomica–ZnO nanocomposite and can be further exploited towards for the development of an antibacterial agent against the threatening superbugs

Abstract 3080: Chronic exposure to arsenic induces malignant transformation in human kidney epithelial cells

Abstract Arsenic contamination is a significant environmental and public health issue worldwide including the United States. Accumulating evidence suggests that kidney is one of the target organ for arsenic-induced carcinogenesis and toxicological effects. However, the mechanism of arsenic-induced renal carcinogenesis is not well understood. Therefore, the objective of this study was to evaluate the carcinogenicity of chronic exposure to low dose arsenic on kidney epithelial cells, and identify the molecular mechanism underlying this process. HK-2 kidney epithelial cells derived from normal human kidney were treated with arsenic for acute, long-term and chronic durations and cellular response to arsenic exposure at these time points was determined by evaluating the changes in growth, morphology, and expression of genes. The results revealed a significant dose-dependent growth inhibition after acute exposure, whereas increased growth after long-term and chronic exposure to arsenic of HK-2 cells as compared to untreated control cells. The anchorage-independent growth assay for colony formation further confirmed malignant transformation of arsenic exposed cells. Additionally, the morphological changes of EMT and stemness were also observed in long-term arsenic exposed cells. These changes observed above were further confirmed by analysis of gene and protein expression. Our data suggest that cellular response of EMT and stemness act a driving factor and mechanistic basis for increased survival and malignant transformation in arsenic exposed cells. In summary, results of this study suggest that acute exposure to arsenic can cause loss of kidney epithelial cells with potential significance to acute kidney injury, whereas the chronic exposure to arsenic can increase the risk of kidney cancer through enhancing cell proliferation. Citation Format: Yu-Wei Chang, Kamaleshwar Singh. Chronic exposure to arsenic induces malignant transformation in human kidney epithelial cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 3080.

Applications of Nanomaterials Based on Magnetite and Mesoporous Silica on the Selective Detection of Zinc Ion in Live Cell Imaging.

Functionalized magnetite nanoparticles (FMNPs) and functionalized mesoporous silica nanoparticles (FMSNs) were synthesized by the conjugation of magnetite and mesoporous silica with the small and fluorogenic benzothiazole ligand, that is, 2(2-hydroxyphenyl)benzothiazole (hpbtz). The synthesized fluorescent nanoparticles were characterized by FTIR, XRD, XRF, 13C CP MAS NMR, BET, and TEM. The photophysical behavior of FMNPs and FMSNs in ethanol was studied using fluorescence spectroscopy. The modification of magnetite and silica scaffolds with the highly fluorescent benzothiazole ligand enabled the nanoparticles to be used as selective and sensitive optical probes for zinc ion detection. Moreover, the presence of hpbtz in FMNPs and FMSNs induced efficient cell viability and zinc ion uptake, with desirable signaling in the normal human kidney epithelial (Hek293) cell line. The significant viability of FMNPs and FMSNs (80% and 92%, respectively) indicates a potential applicability of these nanoparticles as in vitro imaging agents. The calculated limit of detections (LODs) were found to be 2.53 × 10−6 and 2.55 × 10−6 M for Fe3O4-H@hpbtz and MSN-Et3N-IPTMS-hpbtz-f1, respectively. FMSNs showed more pronounced zinc signaling relative to FMNPs, as a result of the more efficient penetration into the cells.

Glycogen-nucleic acid constructs for gene silencing in multicellular tumor spheroids

The poor penetration of nanocarrier-siRNA constructs into tumor tissue is a major hurdle for the in vivo efficacy of siRNA therapeutics, where the ability of the constructs to permeate the 3D multicellular matrix is determined by their physicochemical properties. Herein, we optimized the use of soft glycogen nanoparticles for the engineering of glycogen-siRNA constructs that can efficiently penetrate multicellular tumor spheroids and exert a significant gene silencing effect. Glycogen nanoparticles from different bio-sources and with different structural features were investigated. We show that larger glycogen nanoparticles ranging from 50 to 80 nm are suboptimal systems for complexation of nucleic acids if fine control of the size of constructs is required. Our studies suggest that 20 nm glycogen nanoparticles are optimal for complexation and efficient delivery of siRNA. The chemical composition, surface charge, and size of glycogen-siRNA constructs were finely controlled to minimize interactions with serum proteins and allow penetration into 3D multicellular spheroids of human kidney epithelial cells and human prostate cancer cells. We introduced pH sensitive moieties within the construct to enhance early endosome escape and efficiently improve the silencing effect in vitro. Glycogen-siRNA constructs were found to mediate gene silencing in 3D multicellular spheroids causing ∼60% specific gene silencing. The optimized construct exhibited an in vivo circulation lifetime of 8 h in mice, with preferential accumulation in the liver. No accumulation in the kidney, lung, spleen, heart or brain, or signs of toxicity in mice were observed. Our results highlight the potential for screening siRNA nanocarriers in 3D cultured prostate tumor models, thereby improving the predictive therapeutic efficacy of glycogen-based platforms in human physiological conditions.

A novel mechanism of NO synthase uncoupling involving isolevuglandin adduction

We have previously shown that hypertension leads to modification of self‐proteins in antigen presenting cells by isolevuglandin (isoLG) lipid oxidation products. These modified proteins seem to be immunogenic. IsoLG‐modified peptides are presented in major histocompatibility complex (MHC) class 1 and drive CD8+ T cell proliferation. To understand alterations in the peptidome presented by MHC1 in hypertension, we created transgenic mice expressing a truncated form of MHC1 Kb lacking the transmembrane domain, driven by the CD11c promoter. The lack of a transmembrane domain causes MHC1 to be shed into the media when DCs are cultured. We treated these mice with either 2 weeks sham or angiotensin (ang) II infusion, isolated splenic DCs and placed these in culture for 72 hours. The shed soluble MHC1 Kb was harvested and associated peptides were analyzed by mass spectroscopy. One peptide identified in DCs from hypertensive mice was an identical match for a portion of the neuronal nitric oxide synthase (nNOS) containing an isoLG adduct at lysine 225. Because this lysine is positioned between the heme and arginine binding sites, we hypothesized that its modification would disrupt normal NO production and instead lead to superoxide production, i.e. NOS uncoupling. In subsequent studies, we found that nNOS expression is increased 5‐fold in DCs of ang II‐treated compared to sham‐treated mice (n = 6). Importantly, ang II infusion increased superoxide production of DCs from 37±3 to 111±5 pmol/mg protein (mean ± SE, p = 0.0001), and this was completely blocked by treatment of the cells with the selective nNOS inhibitor L‐vinyl NIO, suggesting that nNOS is a source of superoxide in DCs in hypertension. Next we expressed wild type nNOS and a mutant nNOS in which lysine 225 was mutated to an alanine (nNOS K225A) in human epithelial kidney (HEK) cells. Untransfected HEK cells failed to produce NO, as measured by electron spin resonance and the spin trap Fe[DETC]2. HEK cells transfected with either the WT or K225A nNOS produced 152 ± 5 vs 156 ± 6 arbitrary units of NO/mg protein (p = NS). However, exposure of the transfected HEK cells to tertbutyl hydroperoxide, which promotes isoLG formation, reduced NO production by cells expressing the wild‐type enzyme to 86 ± 6 units/mg protein, while having no effect on cells transfected with the mutant K225A (p = 0.003, n = 5 – 7), further implicating lysine 225 as a site of oxidative modification in nNOS. In summary, hypertension increases nNOS expression in DCs and this enzyme is altered by isoLG adduction at lysine 225. This leads to an increase in superoxide and reduced NO production in these immune cells. We conclude that isoLG adduction represents a novel mechanism of NOS uncoupling and a source of reactive oxygen species in dendritic cells.Support or Funding InformationAmerican Heart Association grants POST290900 and SFRN204200, and National Institutes of Health grants K01HL130497, R01HL125865, R01HL039006 and P01HL129941This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Glutamine metabolism via glutaminase 1 in autosomal-dominant polycystic kidney disease.

Metabolism of glutamine by glutaminase 1 (GLS1) plays a key role in tumor cell proliferation via the generation of ATP and intermediates required for macromolecular synthesis. We hypothesized that glutamine metabolism also plays a role in proliferation of autosomal-dominant polycystic kidney disease (ADPKD) cells and that inhibiting GLS1 could slow cyst growth in animal models of ADPKD. Primary normal human kidney and ADPKD human cyst-lining epithelial cells were cultured in the presence or absence of two pharmacologic inhibitors of GLS1, bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide 3 (BPTES) and CB-839, and the effect on proliferation, cyst growth in collagen and activation of downstream signaling pathways were assessed. We then determined if inhibiting GLS1 in vivo with CB-839 in the Aqp2-Cre; Pkd1fl/fl and Pkhd1-Cre; Pkd1fl/fl mouse models of ADPKD slowed cyst growth. We found that an isoform of GLS1 (GLS1-GAC) is upregulated in cyst-lining epithelia in human ADPKD kidneys and in mouse models of ADPKD. Both BPTES and CB-839 blocked forskolin-induced cyst formation in vitro. Inhibiting GLS1 in vivo with CB-839 led to variable outcomes in two mouse models of ADPKD. CB-839 slowed cyst growth in Aqp2-Cre; Pkd1fl/fl mice, but not in Pkhd1-Cre; Pkd1fl/fl mice. While CB-839 inhibited mammalian target of rapamycin (mTOR) and MEK activation in Aqp2-Cre; Pkd1fl/fl, it did not in Pkhd1-Cre; Pkd1fl/fl mice. These findings provide support that alteration in glutamine metabolism may play a role in cyst growth. However, testing in other models of PKD and identification of the compensatory metabolic changes that bypass GLS1 inhibition will be critical to validate GLS1 as a drug target either alone or when combined with inhibitors of other metabolic pathways.

Duration-dependent effects of nicotine exposure on growth and AKT activation in human kidney epithelial cells.

Exposure to nicotine is known to cause adverse effects in many target organs including kidney. Epidemiological studies suggest that nicotine-induced kidney diseases are prevalent worldwide. However, the impact of duration of exposure on the nicotine-induced adverse effects in normal kidney cells and the underlying molecular mechanism is still unclear. Hence, the objective of this study was to evaluate both acute and long-term effects of nicotine in normal human kidney epithelial cells (HK-2). Cells were treated with 1 and 10µM nicotine for acute and long-term duration. The result of cell viability showed that the acute exposure to 1µM nicotine has no significant effect on growth. However, the 10µM nicotine caused significant decrease in the growth of HK-2 cells. The long-term exposure resulted in significantly increased cell growth in both 1 and 10µM nicotine-treated groups. Analysis of cell cycle and expression of marker genes related to proliferation and apoptosis further confirmed the effects of nicotine. Additionally, the analysis of growth signaling pathway revealed the decreased level of pAKT in cells with acute exposure whereas the increased level of pAKT in long-term nicotine-exposed cells. This suggests that nicotine, through modulating the AKT pathway, controls the duration-dependent effects on the growth of HK-2 cells. In summary, this is the first report showing long-duration exposure to nicotine causes increased proliferation of human kidney epithelial cells through activation of AKT pathway.

Protective Effect of Nicotinamide Adenine Dinucleotide Phosphate on Renal Ischemia-Reperfusion Injury

Background/Aims: Renal ischemia-reperfusion injury (IRI) is a common consequence of acute kidney injury. Nicotinamide adenine dinucleotide phosphate (NADPH), which is derived from the pentose phosphate pathway, is essential for the proper functioning of essential redox and antioxidant defense systems. Previous studies have indicated that NADPH is responsible for protecting the brain from ischemic injury. The goal of this study was to analyze the protective function of NADPH in renal IRI. Methods: The IRI animal model was generated through a midline laparotomy surgery that clamped both sides of the renal pedicles for 40 min to induce renal ischemia. The in vitro model was generated by removing oxygen and glucose from human kidney epithelial cells (HK-2 cells), followed by reoxygenation to imitate IRI. Renal function and histopathological changes were observed and evaluated. Additionally, malondialdehyde and glutathione levels were determined in renal tissue homogenate as indicators of oxidative stress. ROS production in cells was determined by DHE staining. Protein biomarker expression was evaluated by western blot, apoptosis was analyzed by TUNEL staining, and p65 nuclear translocation was visualized by immunofluorescence. Results: Our data indicated that NADPH safeguarded the kidneys from histological and functional damage, and significantly reduce cell injury along with preventing potential increases in blood urea nitrogen and creatinine levels. Furthermore, we observed that NADPH increased glutathione levels, while reducing levels of malondialdehyde and reactive oxygen species. Additionally, our results suggested that NADPH treatment may alleviate IRI-induced apoptosis and inflammation. Conclusion: NADPH treatment may protect against renal IRI and should be further developed as a new treatment for acute kidney injury.

An in vitro method for nephrotoxicity evaluation using HK-2 human kidney epithelial cells combined with biomarkers of nephrotoxicity.

The kidney is one of the major target organs for drug-induced toxicity. During drug development, the traditional markers of nephrotoxicity indicate only severe and late damage, which leads to high costs. The new biomarkers are needed for a more sensitive and reliable evaluation of nephrotoxicity, especially for the regulatory accepted and validated in vitro model. We developed an in vitro model based on the HK-2 cell using the biomarkers of nephrotoxicity as endpoints for the evaluation of nephrotoxicity. The predictive performance of the biomarkers including LDH, GGT, KIM-1, clusterin, CysC, NGAL, TIMP-1, GSTπ and osteopontin was evaluated with 22 well characterized compounds. The area under the curve (AUC) values of KIM-1, clusterin, CysC and osteopontin ranged between 0.79 and 0.84. The combination of clusterin, KIM-1 and/or osteopontin improved the AUC value (ranging between 0.88 and 0.95) compared to one biomarker. Taken together, these results suggest that the model based on the HK-2 cell using clusterin, osteopontin, CysC and KIM-1 as endpoints would allow the prediction of nephrotoxicity at early preclinical stages.

Gaining Access to Bacteria through (Reversible) Control of Lipophilicity.

The development of antimicrobial photodynamic therapy (aPDT) is highly dependent on the development of suitable photosensitizers (PSs); ideally, affinity of a PS towards bacterial cells should be much higher than that towards mammalian cells. A cationic charge on a PS may lead to its selective binding to bacteria mediated through electrostatic interaction; however, the photodynamic outcome is highly dependent on the lipophilicity of the PS. Herein, we report the aPDT effect of silicon(IV) phthalocyanine derivatives bearing four positive charges and methyl, phenyl, or naphthyl substituents at the periphery of the macrocycle. We show that through modulation of lipophilicity, it is possible to find a therapeutic window in which bacteria, but not mammalian cells, are effectively killed. The photobiological activity of these PSs was significantly lower when they were deployed as host-guest complexes with cucurbit[7]uril (CB[7]). CB[7] blocks the hydrophobic part of the PS and reduces its lipophilicity, indicating that a hydrophobic interaction with the outer membrane of bacterial cells is essential for aPDT activity. The efficacies of the obtained PSs have been evaluated by using different uropathogenic E. coli isolates and human kidney epithelial carcinoma cells.

Effect and mechanism of dioscin from Dioscorea spongiosa on uric acid excretion in animal model of hyperuricemia

Ethnopharmacology relevanceDioscin, a spirostane glycoside, the rhizoma of Dioscorea septemloba (Diocoreacea) is used for diuresis, rheumatism, and joints pain. Given the poor solubility and stability of Dioscin, we proposed a hypothesis that Dioscin's metabolite(s) are the active substance(s) in vivo to contribute to the reducing effects on serum uric acid levels. Aim of the studyThe aim of this study is to identify the active metabolite(s) of Dioscin in vivo and to explore the mechanism of its antihyperuricemic activity. Materials and methodsAfter oral administration of Dioscin in potassium oxonate (PO) induced hyperuricemia rats and adenine-PO induced hyperuricemia mice models, serum uric acid and creatinine levels, clearance of uric acid and creatinine, fractional excretion of uric acid, and renal pathological lesions were determined were used to evaluate the antihyperuricemic effects. Renal glucose transporter-9 (GLUT-9) and organic anion transporter-1 (OAT-1) expressions were analyzed by western blotting method. Renal uric acid excretion was evaluated using stably urate transporter-1 (URAT-1) transfected human epithelial kidney cell line. Intestinal uric acid excretion was evaluated by measuring the transcellular transport of uric acid in HCT116 cells. ResultsIn hyperuricemia rats, both 25 and 50mg/kg of oral Dioscin decreased serum uric acid levels over 4h. In the hyperuricemia mice, two weeks treatment of Dioscin significantly decreased serum uric acid and creatinine levels, increased clearance of uric acid and creatinine, increased fractional excretion of uric acid, and reduced renal pathological lesions caused by hyperuricemia. In addition, renal GLUT -9 was significantly down-regulated and OAT-1 was up-regulated in Dioscin treated hyperuricemia mice. Dioscin's metabolite Tigogenin significantly inhibited uric acid re-absorption via URAT1 from 10 to 100μM. Diosgenin and Tigogenin increased uric acid excretion via ATP binding cassette subfamily G member 2 (ABCG2). ConclusionDecreasing effect of Dioscin on serum uric acid level and enhancing effect on urate excretion were confirmed in hyperuricemia animal models. Tigogenin, a metabolite of Dioscin, was identified as an active substance with antihyperuricemic activity in vivo, through inhibition of URAT1 and promotion of ABCG2.