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

Modulatory role of chelating agents in diet-induced hypercholesterolemia in rats

AbstractIntroductionHypercholesterolemia is a major risk factor for the development of atherosclerosis and endothelial dysfunction. Chelating agents may play a modulatory role in atherosclerosis by removal of calcium from atherosclerotic plaques.AimThe present study aimed to explore the effects of calcium disodium ethylenediaminetetraacetic acid (CaNa2EDTA) and meso-2,3-dimercaptosuccinic acid (DMSA) on diet-induced hypercholesterolemia in rats using simvastatin as a reference standard.MethodsHypercholesterolemia was induced by feeding rats with cholesterol-rich diet for six weeks. Rats were divided into five groups (n=8): normal control, hypercholesterolemic control, simvastatin (20mg/kg; p.o.), CaNa2EDTA (100mg/kg; i.p.) and DMSA (100mg/kg; i.p.). Treatments continued daily for the six weeks of diet feeding.ResultsDiet-induced hypercholesterolemia resulted in alterations in the lipid profile markers and a state of oxidative stress coupled by compensatory increase in serum nitric oxide (NO) level and decreased aortic endothelial nitric oxide synthase (eNOS) activity parallel to increased inducible nitric oxide synthase (iNOS) activity, aortic calcium content and aortic wall thickness. Treatment with simvastatin, CaNa2EDTA and DMSA improved lipid profile and oxidative stress markers. In addition, they attenuated hypercholesterolemia-induced changes in serum NO level, aortic eNOS and iNOS activities, calcium content and aortic wall thickness.ConclusionPretreatment of hypercholesterolemic rats with simvastatin, CaNa2EDTA or DMSA attenuated most of the changes induced by feeding rats with cholesterol-rich diet owing to their observed anti-hyperlipidemic and antioxidant properties

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

Relation of Leptin Gene Polymorphism to the Circulating Leptin, Insulin, Estradiol, and Progesterone Hormones in Mares with High Rump Fat

Equine metabolic syndrome is a growing concern usually associated with increased insulin and leptin concentrations. To investigate the relations of obesity determined by measuring the rump fat (RF) with ovarian hormones, leptin, and leptin gene polymorphism, cyclic mares (n=36) were categorized based on RF thickness estimated by the ultrasonography and body condition score (BCS) into obese (RF>5mm; BCS >5), moderate (RF>3≤5 mm;BCS >3≤5) and lean animals (RF<3mm; BCS<3). The circulating estradiol, progesterone, insulin, and leptin were analyzed. The polymorphism of the leptin gene was performed and compared for obese, moderate and lean mares. Depending on increased RF, the obesity was associated with increased BCS (P<0.0001) and leptin (P<0.0001), insulin (P<0.01), and P4 (P<0.0001). Obese mares of leptin genotype BB had the highest leptin (1146±420 pg/ml), insulin (7.42±0.89 μU/l) and progesterone (22.69±9.34 ng/ml), but minimum estradiol (101±54 pg/ml) concentrations. Mares of leptin genotype AA had the highest RF (P<0.0001). The interaction of leptin genotype and obesity had affected RF and all hormone concentrations with no significant effect on the BCS. In conclusion, obesity in cyclic mares altered ovarian hormones, insulin and leptin concentrations. The hyperleptinemia, and hyperinsulinemia were associated with the leptin genotype BB but neither to the adiposity (RF) nor BCS

Detection of A2142G, A2142C and A2143G clarithromycin mutations in Helicobacter pylori in Alexandria University Pediatric Hospital

Background: Helicobacter pylori (H. pylori)colonizes the stomach and affect almost 50% of the world’s population. Clarithromycin is considered a cornerstone for H. pylori treatment. Emergence of clarithromycin resistance (CLR-R) has played a major role in failure of H. pylori eradication both in adults and children.  Clarithromycin resistance is mostly due to mutations in 23S rRNA gene: A2142G, A2142C, and A2143G. The aim of the current study is to determine the prevalence of CLR-R among H. pylori infected children with prior clarithromycin treatment. Materials and Methods: Multiple endoscopic gastric biopsies were collected from 50 H. pylori infected children after cessation of clarithromycin-based treatment. Samples were subjected to histopathological examinations, rapid urease test (RUT) and simultaneous molecular detection of H. pylori infection as well as CLR-R by multiplex Real-Time polymerase chain reaction (PCR). Results: Histopathological examinations and RUT revealed H. pylori in 74% and 92% of samples respectively. Molecular detection of CLR-R showed that 62.5% positive H. pylori cases were not harboring any of the tested mutations, while 25% harbored 2143A-G single mutation. Double mutations (2142A-C and 2143A-G) were detected in only 4 cases. Statistical significant correlation existed between both RUT and PCR results as well as between histopathological findings and PCR test results. Conclusions: A combination of histopathogy, RUT and multiplex PCR procedures offers a real benefit in the simultaneous diagnosis of H. pylori infection along with clarithromycin resistance status. Other mechanisms of clarithromycin resistance need to be investigated to explain treatment failure in absence of the previously detected mutations

Characterization of a biosurfactant producing electroactive Bacillus sp. for enhanced Microbial Fuel Cell dye decolourisation

A biosurfactant producing Gram positive bacterium isolated from anodic biofilm of textile wastewater fed MFC was identified as Bacillus sp. MFC (Accession number: MT322244). Scanning Electron Microscopy of the bacterium showed appendages, the bacterium forms biofilm on Congo red agar medium. The obtained results showed that the addition of 5 mg/l endogenous biosurfactant to the bacterial cells resulted in 19-fold increase in bacterial surface-bound exopolysaccharides (EPS) and 1.94-fold increase in biofilm. However, when the biosurfactant concentration increased to 20 and 40 mg/l, EPS and biofilm decreased and the cells lost their colony forming ability. The dielectric properties of the bacterial cells showed increase in conductivity and relative permittivity with increasing biosurfactant concentrations. The shape of the voltammogram currents peak, their location and Electrochemical impedance spectroscopy (EIS) suggest the involvement of biofilm as direct electron transfer pathway. The average voltage obtained was 0.65 V as compared to 0.45 V for the control MFC. Decolourization was tested for Congo red in a double chamber Microbial Fuel Cell (MFC), the results showed 2-fold increase in decolourization when biosurfactant is added post biofilm formation. The results confirm that Bacillus sp. MFC possess electrogenic properties and that adding low concentrations of endogenous biosurfactant to 24 h biofilm accelerates electron transfer by inducing perforations in the cell wall and increasing EPS as an electron transfer transient medium. Therefore, MFC performance can be enhanced

Phytochemical Screening, Gas Chromatography-mass Spectrometry Analysis, and Antidiabetic Effects of Corchorus olitorius Leaves in Rats

BACKGROUND: Therapies for diabetes mellitus are still meeting failure in most cases, especially in the developed stages of the disease due to incredible associating complications. Hence, there is a need for continuous development of curative therapies for that stubborn disease. AIM: We aimed to investigate the antidiabetic effects of one of the most popular plants cultivated in Egypt, C. olitorius. METHODS: Phytochemical screening of total alcoholic extract of Corchorus olitorius leaves and its aqueous and chloroform fractions revealed the presence of flavonoids, saponins, carbohydrates, tannins, coumarins, and alkaloids. RESULTS: The gas chromatography-mass spectrometry analysis showed the presence of 12 and nine chemical compounds in aqueous and chloroform extracts, respectively. C. olitorius decreased serum glucose level and α-amylase activity. This effect was more pronounced in the total alcoholic extract and its chloroform fraction than the aqueous one. The extracts also adjusted the lipid profile, reduced liver injury parameters, and caused remarkable improvement and increase number, size, and density of functioning β-cells. CONCLUSION: The findings suggest the antihyperglycemic and antioxidant effects of C. olitorius besides its beneficial effect on diabetic complications such as hyperlipidemia and liver injury. The presence of some phytochemicals such as theophylline, trans-2, 3-dimethoxycinnamic acid, 7-hydroxy-4-methyl coumarin, apigenin 7-glucoside, and glycitein may contribute to such pharmacological effects

Blocking Type I Interferon Signaling Rescues Lymphocytes from Oxidative Stress, Exhaustion, and Apoptosis in a Streptozotocin-Induced Mouse Model of Type I Diabetes

Elevated levels of type I interferon (IFN) during type 1 diabetes mellitus (T1D) are associated with a defective immune response. In the present study, we investigated whether blocking type I IFN signaling during streptozotocin- (STZ-) induced T1D in mice improves lymphocyte proliferation and escape from continuous apoptosis. Three groups of mice were examined: diabetic mice, type I IFN signaling-incompetent diabetic mice, and control nondiabetic mice. We first found that diabetes induction was accompanied by an elevation in the plasma levels of reactive oxygen species (ROS), hydroperoxide, malondialdehyde (MDN), and the proinflammatory cytokines IL-1α, IL-1β, IL-6, and CXCL10. Blocking type 1 IFN signaling in diabetic mice significantly decreased the levels of oxidative stress and proinflammatory cytokines. In addition, lymphocytes from diabetic mice exhibited a marked reduction in their proliferative capacity, increased apoptosis, upregulation of the exhaustion marker PD-1, and aberrant phosphorylation of STAT1, STAT2, AKT and IκB-α. Interestingly, following the blocking of type I IFN signaling in diabetic mice, the lymphocytes exhibited restored proliferative capacity, decreased apoptosis, normal expression of PD-1, and normal phosphorylation of STAT1, STAT2, AKT and IκB-α. Our data suggest that elevated levels of type I IFN during T1D trigger lymphocyte exhaustion and a defective lymphocyte-medicated immune response

The mechanism of action of Spirulina as antidiabetic: a narrative review

Spirulina happens to be a special type of blue-green algae that originally emerged 3.5 billion years ago and was used as a source of nutrition. Spirulina gets its name from the filaments’ spiral or helical structure, but its true name is taxonomically Genus Arthrospira which encompasses several species. The most common species are S. fusiformis, S. maxima, and S. platensis. It is rich in various nutrients and chemical components including protein, carbohydrates, lipids, vitamins, minerals, pigments, chlorophyll, and enzymes. Spirulina’s active molecules and rich nutrients make it have several pharmacological activities and uses including antioxidant, anti-inflammatory, immunomodulatory, immune system booster, anticancer, antiviral activity, and neuroprotective properties. It is also utilized as a nutritional supplement and for weight loss. Moreover, several studies confirm that Spirulina improves insulin sensitivity and reduces blood glucose levels in rat models as well as diabetic patients. The reason behind this unique behavior could be credited to the presence of several active components in it, but the action’s fundamental mechanism is still a matter of debate. Several studies have suggested different mechanisms including anti-inflammatory activity, increased insulin sensitivity, inhibition of gluconeogenesis, antioxidant activity, modulating gut microbiota composition, improved glucose homeostasis, and insulin receptor activation. Therefore, it became clear that Spirulina is a mine of active substances used as a nutritional supplement and reduces blood glucose levels or used in conjunction with other treatments to tackle type 2 diabetes. Further exploration is required to fully explain its effects on human physiology and determine optimal dosages for treatment