Microparticles as Potential Mediators of High Glucose-Induced Renal Cell Injury.

Diabetic nephropathy (DN) is the most common cause of chronic kidney disease worldwide. Activation of signaling pathways such as the mammalian target of rapamycin (mTOR), extracellular signal-regulated kinases (ERK), endoplasmic reticulum (ER) stress, transforming growth factor-beta (TGF-β), and epithelial-mesenchymal transition (EMT), are thought to play a significant role in the etiology of DN. Microparticles (MPs), the small membrane vesicles containing bioactive signals shed by cells upon activation or during apoptosis, are elevated in diabetes and were identified as biomarkers in DN. However, their exact role in the pathophysiology of DN remains unclear. Here, we examined the effect of MPs shed from renal proximal tubular cells (RPTCs) exposed to high glucose conditions on naïve RPTCs in vitro Our results showed significant increases in the levels of phosphorylated forms of 4E-binding protein 1 and ERK1/2 (the downstream targets of mTOR and ERK pathways), phosphorylated-eIF2α (an ER stress marker), alpha smooth muscle actin (an EMT marker), and phosphorylated-SMAD2 and nuclear translocation of SMAD4 (markers of TGF-β signaling). Together, our findings indicate that MPs activate key signaling pathways in RPTCs under high glucose conditions. Pharmacological interventions to inhibit shedding of MPs from RPTCs might serve as an effective strategy to prevent the progression of DN

Comparison Of In Vitro Models Of Diabetic Nephropathy Using Renal Tubular Cells

Background: Diabetic nephropathy (DN) is a chronic and serious complication associated with diabetes. The standardization of an in vitro model to best represent DN is very challenging due to the chronic nature of the condition. Therefore, two different renal tubule cell lines - Madin-Darby canine kidney cells (MDCK) and Normal rat kidney cells (NRK-52E) - were used to investigate the effects of high glucose on kidney cells. Objective: To determine the effects of high glucose concentrations on cell viability (using MTT assay), oxidative stress (using dichlorofluorescein (DCF) staining), and expression of proteins activated in DN such as aldose reductase and glucose-regulated protein-78 (GRP78), an endoplasmic reticulum chaperone (using western blotting). Results: MDCK cells showed a subtle decrease in viability when exposed to high glucose concentrations (30 mM and 1% FBS) for 48 h. Furthermore, there was a slight increase in aldose reductase expression after 48 h of high glucose exposure, however; the GRP78 levels remained unchanged. NRK-52E cells showed more consistent decrease in viability after 48 and 72 h of high glucose exposure (30 mM and 1% FBS). In addition, the DCF staining also demonstrated an increase in oxidative stress after 24 h of high glucose exposure. Furthermore, a 30% increase in aldose reductase expression has been observed after 48 h of high glucose exposure. Conclusion: Although the 48 h high glucose exposure in MDCK cells can be used as a model for in vitro DN, the results are less reproducible, whereas NRK-52E cells seem to be a better and more reliable cell line to mimic the features of DN in vitro. Key words: Diabetic nephropathy; In vitro; Kidney; Oxidative Stress; ER Stress.qscienc

Metformin Induces Different Responses in Clear Cell Renal Cell Carcinoma Caki Cell Lines.

Clear cell renal cell carcinoma (ccRCC) is the most common and lethal form of urological cancer diagnosed globally. Mutations of the von Hippel-Lindau tumor-suppressor gene and the resultant overexpression of hypoxia-inducible factor (HIF)-1α protein are considered hallmarks of ccRCC. Persistently activated HIF-1α is associated with increased cell proliferation, angiogenesis, and epithelial⁻mesenchymal transition (EMT), consequently leading to ccRCC progression and metastasis to other organs. However, the status alone cannot predict the differential sensitivity of ccRCC to cancer treatments, which suggests that other molecular differences may contribute to the differential response of ccRCC cells to drug therapies. In this study, we investigated the response to metformin (an antidiabetic drug) of two human ccRCC cell lines Caki-1 and Caki-2, which express wild-type . Our findings demonstrate a differential response between the two ccRCC cell lines studied, with Caki-2 cells being more sensitive to metformin compared to Caki-1 cells, which could be linked to the differential expression of HIF-1 despite both cell lines carrying a wild-type . Our study unveils the therapeutic potential of metformin to inhibit the progression of ccRCC in vitro. Additional preclinical and clinical studies are required to ascertain the therapeutic efficacy of metformin against ccRCC.Qatar University grants QUUG-CPH\2017-2 and QUCG-CPH-2018\2019-

Sestrin2 as a Novel Biomarker and Therapeutic Target for Various Diseases

Sestrin2 (SESN2), a highly conserved stress-inducible metabolic protein, is known to repress reactive oxygen species (ROS) and provide cytoprotection against various noxious stimuli including genotoxic and oxidative stress, endoplasmic reticulum (ER) stress, and hypoxia. Studies demonstrate that the upregulation of Sestrin2 under conditions of oxidative stress augments autophagy-directed degradation of Kelch-like ECH-associated protein 1 (Keap1), which targets and breaks down nuclear erythroid-related factor 2 (Nrf2), a key regulator of various antioxidant genes. Moreover, ER stress and hypoxia are shown to induce Sestrins, which ultimately reduce cellular ROS levels. Sestrin2 also plays a pivotal role in metabolic regulation through activation of the key energy sensor AMP-dependent protein kinase (AMPK) and inhibition of mammalian target of rapamycin complex 1 (mTORC1). Other downstream effects of Sestrins include autophagy activation, antiapoptotic effects in normal cells, and proapoptotic effects in cancer cells. As perturbations in the aforementioned pathways are well documented in multiple diseases, Sestrin2 might serve as a potential therapeutic target for various diseases. Thus, the aim of this review is to discuss the upstream regulators and the downstream effectors of Sestrins and to highlight the significance of Sestrin2 as a biomarker and a therapeutic target in diseases such as metabolic disorders, cardiovascular and neurodegenerative diseases, and cancer.This publication was made possible by a NPRP award [NPRP8-1750-3-360] from the Qatar National Research Fund (a member of The Qatar Foundation). The statements made herein are solely the responsibility of the authors

Synthesis And Pharmacological Screening Of Novel Piperine Analogs For Potential In Vitro Protection From Endoplasmic Reticulum Stress

Background: The endoplasmic reticulum (ER) is the chief organelle involved in protein homeostasis. Perturbations to the ER protein folding machinery caused by hyperlipidemia, hyperglycemia or hypoglycemia has been shown to trigger ER stress and activate the unfolded protein response (UPR) as a defense mechanism. Accumulating evidences implicate the role of ER stress in the development of chronic kidney disease. Thus there is an urgent need for novel compounds, which have the ability to ameliorate ER stress to treat or prevent any organ damage. Among the natural compounds, piperine and its analogs have been reported to exhibit multiple pharmacological activities, however, the efficacy of piperine and its analogs against ER stress in kidney cells is still unknown. Thus, the goal of the current study is to synthesize a range of piperine analogs and screen them for pharmacological activity to relieve ER stress using an in vitro model of tunicamycin-induced ER stress in rat renal proximal tubular (NRK-52E) cells. Methods: To perform a structure-activity relationship study, several piperine analogs were prepared using piperic acid as a starting material. The structures of the obtained compounds were confirmed by liquid chromatography-mass spectrometry (LC/MS), differential scanning calorimetry (DSC), fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR). The in vitro ER stress model was developed using tunicamycin. Results: Several piperine analogs were synthesized and their structures were elucidated. The preliminary findings indicate that exposure to tunicamycin induces the expression of ER chaperone GRP 78 in NRK-52E cells. The MTT assay confirms the reduction in cell viability even with a low concentration of 1 ug/mL of tunicamycin for 15 minutes. The developed in vitro model will be used to evaluate the effect of piperine analogs on ER stress markers. Conclusion: The synthesis, structural elucidation and the results of the preliminary screening of selected piperine analogs will be presented.qscienc

Molecular Mechanisms Underpinning Microparticle-Mediated Cellular Injury in Cardiovascular Complications Associated with Diabetes.

Microparticles (MPs) are small vesicles shed from the cytoplasmic membrane of healthy, activated, or apoptotic cells. MPs are very heterogeneous in size (100-1,000 nm), and they harbor proteins and surface antigens specific to cells they originate from. Virtually, all cells can shed MPs, and therefore, they can be found in all body fluids, but also entrapped in tissues. Of interest and because of their easy detection using a variety of techniques, circulating MPs were recognized as biomarkers for cell activation. MPs were also found to mediate critical actions in intercellular communication and transmitting biological messages by acting as paracrine vehicles. High plasma numbers of MPs were reported in many cardiovascular and metabolic disturbances that are closely associated with insulin resistance and low-grade inflammation and have been linked to adverse actions on cardiovascular function. This review highlights the involvement of MPs in cardiovascular complications associated with diabetes and discusses the molecular mechanisms that underpin the pathophysiological role of MPs in the onset and progression of cellular injury in diabetes.NPRP award (NPRP8-1750-3-360) from Qatar National Research Fund (a member of Qatar Foundation) and a Qatar University high collaborative grant (QUCG-CPH-2018\2019-2

Renoprotective Effects of Aldose Reductase Inhibitor Epalrestat against High Glucose-Induced Cellular Injury

Diabetic nephropathy (DN) is the leading cause of end stage renal disease worldwide. Increased glucose flux into the aldose reductase (AR) pathway during diabetes was reported to exert deleterious effects on the kidney. The objective of this study was to investigate the renoprotective effects of AR inhibition in high glucose milieu in vitro. Rat renal tubular (NRK-52E) cells were exposed to high glucose (30 mM) or normal glucose (5 mM) media for 24 to 48 hours with or without the AR inhibitor epalrestat (1 M) and assessed for changes in Akt and ERK1/2 signaling, AR expression (using western blotting), and alterations in mitochondrial membrane potential (using JC-1 staining), cell viability (using MTT assay), and cell cycle. Exposure of NRK-52E cells to high glucose media caused acute activation of Akt and ERK pathways and depolarization of mitochondrial membrane at 24 hours. Prolonged high glucose exposure (for 48 hours) induced AR expression and G1 cell cycle arrest and decreased cell viability (84% compared to control) in NRK-52E cells. Coincubation of cells with epalrestat prevented the signaling changes and renal cell injury induced by high glucose. Thus, AR inhibition represents a potential therapeutic strategy to prevent DN

Crosstalk Between Oxidative Stress and Endoplasmic Reticulum (ER) Stress in Endothelial Dysfunction and Aberrant Angiogenesis Associated With Diabetes: A Focus on the Protective Roles of Heme Oxygenase (HO)-1

Type-2 diabetes prevalence is continuing to rise worldwide due to physical inactivity and obesity epidemic. Diabetes and fluctuations of blood sugar are related to multiple micro- and macrovascular complications, that are attributed to oxidative stress, endoplasmic reticulum (ER) activation and inflammatory processes, which lead to endothelial dysfunction characterized, among other features, by reduced availability of nitric oxide (NO) and aberrant angiogenic capacity. Several enzymatic anti-oxidant and anti-inflammatory agents have been found to play protective roles against oxidative stress and its downstream signaling pathways. Of particular interest, heme oxygenase (HO) isoforms, specifically HO-1, have attracted much attention as major cytoprotective players in conditions associated with inflammation and oxidative stress. HO operates as a key rate-limiting enzyme in the process of degradation of the iron-containing molecule, heme, yielding the following byproducts: carbon monoxide (CO), iron, and biliverdin. Because HO-1 induction was linked to pro-oxidant states, it has been regarded as a marker of oxidative stress; however, accumulating evidence has established multiple cytoprotective roles of the enzyme in metabolic and cardiovascular disorders. The cytoprotective effects of HO-1 depend on several cellular mechanisms including the generation of bilirubin, an anti-oxidant molecule, from the degradation of heme; the induction of ferritin, a strong chelator of free iron; and the release of CO, that displays multiple anti-inflammatory and anti-apoptotic actions. The current review article describes the major molecular mechanisms contributing to endothelial dysfunction and altered angiogenesis in diabetes with a special focus on the interplay between oxidative stress and ER stress response. The review summarizes the key cytoprotective roles of HO-1 against hyperglycemia-induced endothelial dysfunction and aberrant angiogenesis and discusses the major underlying cellular mechanisms associated with its protective effects

Adoption of Retail Internet Banking: A Study of Demographic Factors

This study focuses on the adoption of retail internet banking among consumers in the Klang Valley, Malaysia and the impact of demographics factors on such adoption behavior. This study adopts a quantitative approach using surveys conducted at banks. The results indicate that while the hypotheses pertaining to gender, race, income, educational level and occupation were not supported but age is supported. Therefore, the current research indicates that the age of retail banking consumers affects the adoption of internet banking among Malaysian consumers. The result also suggests that consumers in the age group below 25 years old are the major contributor to the differences. Based on these findings, we conclude that younger consumers are more likely to adopt internet banking. Therefore, the result suggests implications for retail bankers in Malaysia to adopt appropriate strategies to encourage retail internet banking for other age categories of consumers in Klang Valley. By doing so, it enables the banks in Malaysia to save costs of maintaining physical distribution systems through providing bigger scale of Internet retail banking services

Anti-Neoplastic Effects of Annonacin against Renal Cell Carcinoma

Background and Objectives: Renal cell carcinoma is the most common and lethal form of all renal cancers, and accounts for 4.1% of all cancer cases in Qatar. Mutations to Von-Hippel Lindeau (VHL) gene in renal cells activates hypoxia inducible factor-1 alpha (HIF-1?) response pathway, and contributes to increased proliferation and progression to renal cell carcinoma. Hence, chemotherapeutic modalities available to treat renal cell carcinoma are targeted toward modulation of the VHL-HIF response pathway. Annonacin, a potent cytotoxic mono-tetrahydrofuran acetogenin found in Annonaceae plants, has been demonstrated to exert anticancer activity against breast cancer; however, its therapeutic potential against renal cell carcinoma is yet to be determined. Hence the objective of this study is to investigate anti-neoplastic potential of annonacin in renal carcinoma cells. Methods: We investigated the effect of annonacin - at concentrations ranging from 0.5 to 2 ?M - on cell viability (using MTT assay and Alamar blue assay), and the protein expression of markers of HIF signaling pathway (HIF-1?), mTOR pathway (Thr-389 phosphorylation of p70S6 kinase), cell cycle progression (p21 levels), and apoptosis (caspase-3 expression) in CaKi-2 cells, a human renal carcinoma cell line. The cells were treated with annonacin for 24 or 48 hours and assessed for the aforementioned parameters. Results: hour annonacin treatment caused a significant and dose-dependent decrease in the viability of CaKi-2 cells, i.e., 42% in 0.5 ?M, 36% in 1 ?M and 29% in 2 ?M annonacin treatment groups as compared to control set at 100%. This was further confirmed by Alamar blue assay, which revealed a significant decrease in the viability of CaKi-2 cells upon treatment with annonacin for 48 h. The expression of HIF-1? was reduced by 68% at 24 h in CaKi-2 cells treated with 2 ?M annonacin. In addition, the expression of p21 (a key molecule that inhibits transition of cells from G1 to S phase in cell cycle) was induced by 1.34-fold in 0.5 ?M annonacin-treated cells indicating an arrest in G1 phase of cell cycle. This was further confirmed through cell cycle analysis using Tali cytometer, in which annonacin treated groups (0.5 ?M and 1 ?M) showed cell cycle arrest at G1 phase, i.e., 57% of cells in G1 phase with 0.5 ?M annonacin treated vs. 7% of cells in G1 phase in control group. In addition, a dose-dependent decrease in the phosphorylation of p70S6 kinase (a downstream target of mTOR) was observed with annonacin treatment at both 24 and 48 h end-points. This suggests that treatment of annonacin has possibly led to the inhibition of mTOR, in addition to suppression of HIF-1? activation, and underscores the cross-talk between HIF pathway and mTOR signaling pathway in renal cell carcinoma. Conclusions: Our findings demonstrate that annonacin treatment (at concentrations ranging from 0.5 to 2 ?M) inhibits HIF-1? and mTOR activation and causes cell cycle arrest at G1 phase and induces apoptosis in renal cell carcinoma. These findings indicate that annonacin exerts anti-cancer effects via modulation of HIF and mTOR signaling pathways, resulting in alterations in the cell cycle and activation of apoptosis in renal cell carcinoma. In conclusion, our study for the first time unveils the therapeutic potential of annonacin to inhibit the progression of renal cell carcinoma. Further studies in vivo are required to establish its efficacy to treat patients with renal cell carcinoma.qscienc