Transcription factor 7 like 2 gene polymorphism and advanced glycation end products as risk factors for diabetic nephropathy.

Hepatocellular carcinoma (HCC) is a multifactorial disease with both genetic and environmental factors contributing to its pathogenesis. ACYP2 is a gene that is related to cell differentiation, apoptosis and prevention of malignant tumors. The ACYP2 gene also affects telomere length. The aim of this study was to evaluate the association between ACYP2 single nucleotide polymorphisms (SNPs) (rs843711), and (rs843706) and incidence of HCC in Egyptian HCC patients. The study included 30 patients with HCC and 30 normal controls. Detection of ACYP2 gene SNPs rs843711, and rs843706 in all study participants was done using real time polymerase chain reaction (RT-PCR). The results showed that all participants including HCC patients and controls carried the heterozygous CA (100%) of the rs843706 SNP (p> 0.05). As for the rs843711, 3.3% of HCC patients had the homozygous TT genotype, 46.7% had the heterozygous CT genotype and 50% had the wild CC genotype, while in the control group, 60% had the heterozygous CT genotype and 40% had the wild CC genotype with no significant difference between both groups (p>0.05). We concluded that there was no association between SNPs ACYP2 rs843706 and rs843711 and occurrence of HCC.

Thiourea derivatives inhibit key diabetes-associated enzymes and advanced glycation end-product formation as a treatment for diabetes mellitus.

This study was designed to screen novel thiourea derivatives against different enzymes, such as α-amylase, α-glucosidase, protein tyrosine phosphatase 1 B, and advanced glycated end product (AGEs). A cytotoxicity analysis was performed using rat L6 myotubes and molecular docking analysis was performed to map the binding interactions between the active compounds and α-amylase and α-glucosidase. The data revealed the potency of five compounds, including E (1-(2,4-difluorophenyl)-3-(3,4-dimethyl phenyl) thiourea), AG (1-(2-methoxy-5-(trifluoromethyl) phenyl)-3-(3-methoxy phenyl) thiourea), AF (1-(2,4-dichlorophenyl)-3-(4-ethylphenyl) thiourea), AD (1-(2,4-dichlorophenyl)-3-(4-ethylphenyl) thiourea), and AH (1-(2,4-difluorophenyl)-3-(2-iodophenyl) thiourea), showed activity against α-amylase. The corresponding percentage inhibitions were found to be 85 ± 1.9, 82 ± 0.7, 75 ± 1.2, 72 ± 0.4, and 65 ± 1.1%, respectively. These compounds were then screened using in vitro assays. Among them, AH showed the highest activity against α-glucosidase, AGEs, and PTP1B, with percentage inhibitions of 86 ± 0.4% (IC50  = 47.9 µM), 85 ± 0.7% (IC50  = 49.51 µM), and 85 ± 0.5% (IC50  = 79.74 µM), respectively. Compound AH showed an increased glucose uptake at a concentration of 100 µM. Finally, an in vivo study was conducted using a streptozotocin-induced diabetic mouse model and PTP1B expression was assessed using real-time PCR. Additionally, we examined the hypoglycemic effect of compound AH in diabetic rats compared to the standard drug glibenclamide.

The receptor for advanced glycation end products (RAGE) is involved in mitochondrial function and cigarette smoke-induced oxidative stress.

The mechanisms underlying muscle dysfunction with Chronic Obstructive Pulmonary Disease (COPD) are poorly understood. Indirect evidence has recently suggested a role of Advanced Glycation End Products (AGEs) and their receptor (RAGE) in the pathophysiology of COPD. Accordingly, this study aimed to examine the redox balance and mitochondrial alterations in the skeletal muscle of a mouse model deficient in the receptor for AGE (RAGE-KO) and wild-type C57BL/6 exposed to cigarette smoke for 8-months using immunoblotting, spectrophotometry, and high-resolution respirometry. Cigarette smoke exposure increased by two-fold 4-HNE levels (P < 0.001), a marker of oxidative stress, and markedly downregulated contractile proteins, mitochondrial respiratory complexes, and uncoupling proteins levels (P < 0.001). Functional alterations with cigarette smoke exposure included a greater reliance on complex-I supported respiration (P < 0.01) and lower relative respiratory capacity for fatty acid (P < 0.05). RAGE knockout resulted in 47% lower 4-HNE protein levels than the corresponding WT control mice exposed to cigarette smoke (P < 0.05), which was partly attributed to increased Complex III protein levels. Independent of cigarette smoke exposure, RAGE KO decreased mitochondrial specific maximal respiration (P < 0.05), resulting in a compensatory increase in mitochondrial content measured by citrate synthase activity (P < 0.001) such that muscle respiratory capacity remained unaltered. Together, these findings suggest that knockout of RAGE protected the skeletal muscle against oxidative damage induced by 8 months of cigarette smoke exposure. In addition, this study supports a role for RAGE in regulating mitochondrial content and function and can thus serve as a potential therapeutic target.

Ezetimibe attenuates experimental diabetes and renal pathologies via targeting the advanced glycation, oxidative stress and AGE-RAGE signalling in rats.

The current in-vivo study was premeditated to uncover the protective role of ezetimibe (EZ) against advanced glycation endproducts (AGEs)-related pathologies in experimental diabetes. Our results showed that EZ markedly improved the altered biochemical markers of diabetes mellitus (DM) (FBG, HbA1c, insulin, microalbumin, and creatinine) and cardiovascular disease (in-vivo lipid/lipoprotein level and hepatic HMG-CoA reductase activity) along with diminished plasma carboxymethyl-lysine (CML) and renal fluorescent AGEs level. Gene expression study revealed that EZ significantly down-regulated the renal AGEs-receptor (RAGE), nuclear factor-κB (NFκB-2), transforming growth factor-ß (TGF-ß1), and matrix metalloproteinase-2 (MMP-2) mRNA expression, however, the neuropilin-1 (NRP-1) mRNA expression was up-regulated. In addition, EZ also maintained the redox status via decreasing the lipid peroxidation and protein-bound carbonyl content (CC) and increasing the activity of high-density lipoprotein (HDL)-associated-paraoxonase-1 (PON-1) and renal antioxidant enzymes as well as also protected renal histopathological features. We conclude that EZ exhibits antidiabetic and reno-protective properties in diabetic rats.

Effects of RAGE Deletion on the Cardiac Transcriptome during Aging.

Cardiac aging is characterized by increased cardiomyocyte hypertrophy, myocardial stiffness, and fibrosis, which enhance cardiovascular risk. The receptor for advanced glycation end-products (RAGE) is involved in several age-related diseases. RAGE knockout (Rage-/-) mice show an acceleration of cardiac dimension changes and interstitial fibrosis with aging. This study identifies the age-associated cardiac gene expression signature induced by RAGE deletion. We analyzed the left ventricle transcriptome of 2.5-(Young), 12-(Middle age, MA), and 21-(Old) months-old female Rage-/- and C57BL/6N (WT) mice. By comparing Young, MA, and Old Rage-/- versus age-matched WT mice, we identified 122, 192, and 12 differently expressed genes, respectively. Functional inference analysis showed that RAGE deletion is associated with: (i) down-regulation of genes involved in antigen processing and presentation of exogenous antigen, adaptive immune response, and cellular responses to interferon beta and gamma in Young animals; (ii) up-regulation of genes related to fatty acid oxidation, cardiac structure remodeling and cellular response to hypoxia in MA mice; (iii) up-regulation of few genes belonging to complement activation and triglyceride biosynthetic process in Old animals. Our findings show that the age-dependent cardiac phenotype of Rage-/- mice is associated with alterations of genes related to adaptive immunity and cardiac stress pathways.

Integrated Analysis of Crucial Genes and miRNAs Associated with Osteoporotic Fracture of Type 2 Diabetes.

Purpose: The aim of this study is to explore pathological mechanisms of bone fragility in type 2 diabetes mellitus (T2DM) patients. Methods: Identifying common genes for T2DM and osteoporosis by taking the intersection is shared by the Comparative Toxicogenomics Database (CTD), DISEASES, and GeneCards databases. The differentially expressed genes (DEGs) and the differentially expressed miRNAs (DEMs) were identified by analyzing the Gene Expression Omnibus (GEO) datasets (GSE35958, GSE43950, and GSE70318). FunRich and miRNet were applied to predict potential upstream transcription factors and downstream target genes of candidate DEMs, respectively. The Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed to explore potential mechanisms using Metascape. Eventually, a miRNA-gene network was constructed by Cytoscape software. Results: 271 common targets and 35 common DEGs between T2DM and osteoporosis were screened out in the above databases, and a total of ten DEMs were obtained in the GSE70318. SP1 was predicted to potentially regulate most of the DEMs. Enrichment analysis showed the PI3K-Akt signaling pathway and AGE-RAGE signaling pathway in diabetic complications may play an important role in diabetic skeletal fragility. Two genes (NAMPT and IGFBP5) were considered as key genes involving in the development of diabetic osteoporosis. Through the construction of the miRNA-gene network, most of the hub genes were found to be potentially modulated by miR-96-5p and miR-7-5p. Conclusion: The study uncovered several important genes, miRNAs, and pathological mechanisms involved in diabetic skeletal fragility, among which the PI3K-Akt signaling pathway and AGE-RAGE signaling pathway in diabetic complications may play important roles.

RAGE management: ETS1- EGR1 mediated transcriptional networks regulate angiogenic factors in wood frogs.

Freeze-tolerant species, such as wood frogs (Rana sylvatica), are susceptible to multiple co-occurring stresses that they must overcome to survive. Freezing is accompanied by mechanical stress and dehydration due to ice crystal formation in the extracellular space, ischemia/anoxia due to interruption in blood flood, and hyperglycemia due to cryoprotective measures. Wood frogs can survive dehydration, anoxia, and high glucose stress independently of freezing, thereby creating a multifactorial model for studying freeze-tolerance. Oxidative stress and high glucose levels favors the production of pro-oxidant molecules and advanced glycation end product (AGE) adducts that could cause substantial cellular damage. In this study, the involvement of the high mobility group box 1 (HMGB1)-AGE/RAGE (receptor for AGE) axis and the regulation of ETS1 and EGR1-mediated angiogenic responses were investigated in liver of wood frogs expose to freeze/thaw, anoxia/reoxygenation and dehydration/rehydration treatments. HMGB1 and not AGE-adducts are likely to induce the activation of ETS1 and EGR1 via the RAGE pathway. The increase in nuclear localization of both ETS1 and EGR1, but not DNA binding activity in response to stress hints to a potential spatial and temporal regulation in inducing angiogenic factors. Freeze/thaw and dehydration/rehydration treatments increase the levels of both pro- and anti-angiogenic factors, perhaps to prepare for the distribution of cryoprotectants or enable the repair of damaged capillaries and wounds when needed. Overall, wood frogs appear to anticipate the need for angiogenesis in response to freezing and dehydration but not anoxic treatments, probably due to mechanical stress associated with the two former conditions.

Intracellular Toxic Advanced Glycation End-Products in 1.4E7 Cell Line Induce Death with Reduction of Microtubule-Associated Protein 1 Light Chain 3 and p62.

BACKGROUND: The death of pancreatic islet ß-cells (ß-cells), which are the insulin-producing cells, promote the pathology in both Type 1 and Type 2 diabetes mellitus (DM) (T1DM and T2DM), and they are protected by autophagy which is one of the mechanisms of cell survival. Recently, that some advanced glycation end-products (AGEs), such as methylglyoxial-derived AGEs and Nε-carboxymethyllysine, induced the death of ß-cells were revealed. In contrast, we had reported AGEs derived from glyceraldehyde (GA, the metabolism intermediate of glucose and fructose) are considered to be toxic AGEs (TAGE) due to their cytotoxicity and role in the pathogenesis of T2DM. More, serum levels of TAGE are elevated in patients with T1 and T2DM, where they exert cytotoxicity. AIM: We researched the cytotoxicity of intracellular and extracellular TAGE in ß-cells and the possibility that intracellular TAGE were associated with autophagy. METHODS: 1.4E7 cells (a human ß-cell line) were treated with GA, and analyzed viability, quantity of TAGE, microtubule-associated protein 1 light chain 3 (LC3)-I, LC3-II, and p62. We also examined the viability of 1.4E7 cells treated with TAGE-modified bovine serum albumin, a model of TAGE in the blood. RESULTS: Intracellular TAGE induced death of 1.4E7 cells, decrease of LC3-I, LC3-II, and p62. Extracellular TAGE didn't show cytotoxicity in the physiological concentration. CONCLUSION: Intracellular TAGE induced death of ß-cells more strongly than extracellular TAGE, and may suppress autophagy via reduction of LC3-I, LC3-II, and p62 to inhibit the degradation of them.

Protein degradation-associated mechanisms that are affected in Alzheimer´s disease.

Alzheimer's disease (AD) is the most common type of dementia associated with age-related neurodegeneration. Alteration of several molecular mechanisms has been correlated with the progression of AD. In recent years, dysregulation of proteostasis-associated pathways has emerged as a potential risk factor for neurodegenerative diseases. This review investigated the ubiquitin-proteasome system, lysosome-associated degradation, endoplasmic-reticulum-associated degradation, and the formation of advanced glycation end products. These pathways involved in proteostasis have been reported to be altered in AD, suggesting that their study may be critical for identifying new biomarkers and target molecules for AD.

Comparison of bovine serum albumin glycation by ribose and fructose in vitro and in vivo.

Advanced glycation end products (AGEs) play a critical pathogenic role in the development of diabetic complications. Recent studies have shown that diabetes is associated with not only abnormal glucose metabolism but also abnormal ribose and fructose metabolism, although glucose is present at the highest concentration in humans. The glycation ability and contribution of ribose and fructose to diabetic complications remain unclear. Here, the glycation ability of ribose, fructose and glucose under a mimic physiological condition, in which the concentration of ribose or fructose was one-fiftieth that of glucose, was compared. Bovine serum albumin (BSA) was used as the working protein in our experiments. Ribose generated more AGEs and was markedly more cytotoxic to SH-SY5Y cells than fructose. The first-order rate constant of ribose glycation was found to be significantly greater than that of fructose glycation. LC-MS/MS analysis revealed 41 ribose-glycated Lys residues and 12 fructose-glycated residues. Except for the shared Lys residues, ribose reacted selectively with 17 Lys, while no selective Lys was found in fructose-glycated BSA. Protein conformational changes suggested that ribose glycation may induce BSA into amyloid-like monomers compared with fructose glycation. The levels of serum ribose were correlated positively with glycated serum protein (GSP) and diabetic duration in type 2 diabetes mellitus (T2DM), respectively. These results indicate that ribose has a greater glycation ability than fructose, while ribose largely contributes to the production of AGEs and provides a new insight to understand in the occurrence and development of diabetes complications.

Effects of advanced glycation end products on dental pulp calcification.

OBJECTIVE: The main aim of this study was to elucidate the effects of advanced glycation end products (AGEs) on the calcification of cultured rat dental pulp cells (RDPCs) and to investigate the crystallisation ability of glycated collagen. MATERIALS AND METHODS: AGEs were prepared via non-enzymatic glycation of a dish coated with type I collagen using dl-glyceraldehyde. To investigate the effects of AGEs on RDPCs, we performed WST-1 and lactate dehydrogenase assays; alkaline phosphatase, Alizarin Red S and immunohistochemical staining; and real-time quantitative reverse transcription PCR. In addition, we performed crystallisation experiments on glycated collagen. All microstructures were analysed using scanning electron microscopy/energy-dispersive X-ray spectroscopy and transmission electron microscopy/diffraction pattern analysis. RESULTS: AGEs did not affect the proliferation or differentiation of RDPCs, but enhanced the calcification rate and cytotoxicity. No major calcification-related genes or proteins were involved in these calcifications, and glycated collagen was found to exhibit a negative polarity and form calcium phosphate crystals. Cytotoxicity due to drastic changes in the concentration of pericellular ions led to dystrophic calcification, assumed to represent an aspect of diabetic pulp calcifications. CONCLUSION: Glycated collagen-containing AGEs provide a nurturing environment for crystallisation and have a significant effect on the early calcification of RDPCs.

DJ-1 Acts as a Scavenger of α-Synuclein Oligomers and Restores Monomeric Glycated α-Synuclein.

Glycation of α-synuclein (αSyn), as occurs with aging, has been linked to the progression of Parkinson's disease (PD) through the promotion of advanced glycation end-products and the formation of toxic oligomers that cannot be properly cleared from neurons. DJ-1, an antioxidative protein that plays a critical role in PD pathology, has been proposed to repair glycation in proteins, yet a mechanism has not been elucidated. In this study, we integrate solution nuclear magnetic resonance (NMR) spectroscopy and liquid atomic force microscopy (AFM) techniques to characterize glycated N-terminally acetylated-αSyn (glyc-ac-αSyn) and its interaction with DJ-1. Glycation of ac-αSyn by methylglyoxal increases oligomer formation, as visualized by AFM in solution, resulting in decreased dynamics of the monomer amide backbone around the Lys residues, as measured using NMR. Upon addition of DJ-1, this NMR signature of glyc-ac-αSyn monomers reverts to a native ac-αSyn-like character. This phenomenon is reversible upon removal of DJ-1 from the solution. Using relaxation-based NMR, we have identified the binding site on DJ-1 for glycated and native ac-αSyn as the catalytic pocket and established that the oxidation state of the catalytic cysteine is imperative for binding. Based on our results, we propose a novel mechanism by which DJ-1 scavenges glyc-ac-αSyn oligomers without chemical deglycation, suppresses glyc-ac-αSyn monomer-oligomer interactions, and releases free glyc-ac-αSyn monomers in solution. The interference of DJ-1 with ac-αSyn oligomers may promote free ac-αSyn monomer in solution and suppress the propagation of toxic oligomer and fibril species. These results expand the understanding of the role of DJ-1 in PD pathology by acting as a scavenger for aggregated αSyn.

Personalized diet study of dietary advanced glycation end products (AGEs) and fatty acid desaturase 2 (FADS2) genotypes in obesity.

Obesity prevalence have tripled in the past decades. It is logical to consider new approaches to halt its prevalence. In this concept, considering the effect of interaction between fatty acid desaturase 2 (FADS2) gene variants and dietary advanced glycation end products (AGEs) on obesity-related characteristics seems to be challenging. The present cross-sectional study conducted among 347 obese individuals. A validated semi-quantitative 147-item food frequency questionnaire (FFQ) was used to estimate dietary intakes and American multiethnic database was used to calculate AGEs content of food items which were not available in Iranian Food Composition Table (FCT). FADS2 gene variants were determined according to Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Analysis of covariance (ANCOVA) was used to evaluate the modifier effect of FADS2 gene-dietary AGEs on biochemical values. Based on our findings, no significant differences was reported in term of biochemical variables between AGEs tertiles. In contrast, percent of macronutrients (carbohydrate, protein and fat) of total calorie intake, amount of daily intake of fiber and meat groups showed a significant differences among AGEs tertiles. Furthermore, statistical assays clarified the modifier effects of FADS2 gene-AGEs on weight (Pinteraction = 0.04), fat mass (Pinteraction = 0.03), waist circumference (Pinteraction = 0.008) and cholesterol (Pinteraction = 0.04) level. Accordingly, higher consumption of protein or fat based foods constitute high amount of AGEs and heterozygote genotype for FADS2 tended to show lower level of AGEs content. These findings address further investigation to develop new approaches for nutritional interventions.

Generation, biochemical characterizations and validation of potent nanobodies derived from alpaca specific for human receptor of advanced glycation end product.

A detrimental role of the receptor for the advanced glycation end product (RAGE) has been identified in the immune response, and various pathological conditions and its V and C1 domains in the extracellular region of RAGE are believed to be the main ligand-binding domains. Consequently, specific inhibitors targeting those domains could be of clinical value in fighting against the pathological condition associated with RAGE over-activation. Single-domain antibodies, also called nanobodies (Nbs), are antibody fragments engineered from the heavy-chain only antibodies found in camelids, which offer a range of advantages in therapy. In this study, we report the development and characterization of the V-C1 domain-specific Nbs. Three Nbs (3CNB, 4BNB, and 5ENB) targeting V-C1 domain of human RAGE were isolated from an immunized alpaca using a phage display. All of these Nbs revealed high thermostability. 3CNB, 4BNB, and 5ENB bind to V-C1 domain with a dissociation constant (KD) of 27.25, 39.37, and 47.85 nM, respectively, using Isothermal Titration Calorimetry (ITC). After homodimerization using human IgG1-Fc fusion, their binding affinity improved to 0.55, 0.62, and 0.41 nM, respectively, using Surface Plasmon Resonance (SPR). Flow cytometry showed all the Fc fusions Nbs can bind to human RAGE expressed on the cell surface. Competitive ELISA further confirmed their V-C1-hS100B blocking ability in solution, providing insights into the applicability of Nbs in treating RAGE-associated diseases.

MicroRNA-210 repression facilitates advanced glycation end-product (AGE)-induced cardiac mitochondrial dysfunction and apoptosis via JNK activation.

Hyperglycemia results in the formation of reactive oxygen species which in turn causes advanced glycation end products (AGEs) formation, leading to diabetic cardiomyopathy. Our previous study showed that AGE-induced reactive oxygen species-dependent apoptosis is mediated via protein kinase C delta (PKCδ)-enhanced mitochondrial damage in cardiomyocytes. By using microRNA (miRNA) database, miRNA-210 was predicted to target c-Jun N-terminal kinase (JNK), which were previously identified as downstream of PKCδ in regulating mitochondrial function. Therefore, we hypothesized that miR-210 mediates PKCδ-dependent upregulation of JNK to cause cardiac mitochondrial damage and apoptosis following AGE exposure. AGE-exposed cells showed activated cardiac JNK, PKCδ, and apoptosis, which were reversed by treatment with a JNK inhibitor and PKCδ-KD (deficient kinase). Cardiac miR-210 and mitochondrial function were downregulated following AGE exposure. Furthermore, JNK was upregulated and involved in AGE-induced mitochondrial damage. Interestingly, luciferase activity of the miR-210 mimic plus JNK WT-3'-untranslated region overexpressed group was significantly lower than that of miR-210 mimic plus JNK MT-3'UTR group, indicating that JNK is a target of miR-210. Moreover, JNK activation induced by AGEs was reduced by treatment with the miR-210 mimic and reversed by treatment with the miR-210 inhibitor, indicating the regulatory function of miR-210 in JNK activation following AGE exposure. Additionally, JNK-dependent mitochondrial dysfunction and apoptosis were reversed following treatment with the miR-210 mimic, while the miR-210 inhibitor showed no effect on JNK-induced mitochondrial dysfunction and apoptosis in AGE-exposed cardiac cells. Taken together, our study showed that PKCδ-enhanced JNK-dependent mitochondrial damage is mediated through the reduction of miR-210 in cardiomyocytes following AGE exposure.

DNA-Aptamer Raised against Receptor for Advanced Glycation End Products Improves Survival Rate in Septic Mice.

Despite remarkable scientific advances in the understanding of molecular mechanisms for sepsis, therapeutic options are far from satisfactory. High mobility group box 1 (HMGB1), one of the ligands of receptor for advanced glycation end products (RAGE), is a late mediator of lethality in septic mice. We have recently found that the DNA-aptamer raised against RAGE (RAGE-aptamer) significantly blocks experimental diabetic nephropathy and melanoma growth and metastasis. We examined the effects of RAGE-aptamer on sepsis score, survival rate, and inflammatory and oxidative stress responses in serum, peripheral monocytes, kidneys and livers of lipopolysaccharide- (LPS-) injected mice, and on LPS-exposed THP-1 cells. RAGE-aptamer inhibited the binding of HMGB1 to RAGE in vitro. RAGE-aptamer significantly (P = 0.002) improved sepsis score at 8 hours after LPS injection and survival rate at 24 hours (P < 0.01, 70%) in septic mice compared with LPS+vehicle- or LPS+control-aptamer-treated mice. RAGE-aptamer treatment significantly decreased expression of p-NF-κB p65, an active form of redox-sensitive transcriptional factor, NF-κB and gene or protein expression of TNF-α, IL-1ß, IL-6, and HMGB1 in serum, peripheral monocytes, and kidneys of septic mice in association with the reduction of oxidative stress and improvement of metabolic acidosis, renal and liver damage. LPS-induced oxidative stress, inflammatory reactions, and growth suppression in THP-1 cells were significantly blocked by RAGE-aptamer. Our present study suggests that RAGE-aptamer could attenuate multiple organ damage in LPS-injected septic mice partly by inhibiting the inflammatory reactions via suppression of HMGB1-RAGE interaction.

Phenethyl isothiocyanate attenuates diabetic nephropathy via modulation of glycative/oxidative/inflammatory signaling in diabetic rats.

Diabetic nephropathy (DN) is a diabetic complication characterized by disruption of renal microvasculature, reactive oxygen species accumulation and increased inflammation, all of which contribute to renal injury. Phenethyl isothiocyanate (PEITC) is a naturally occurring isothiocyanate well known for its antioxidant and anti-inflammatory effects, yet its reno-preventive effects against DN has not been investigated. The current study looked into the in vivo reno-protective effects of PEITC in STZ-induced DN in rats. PEITC (3, 10 and 30 mg/kg) was administered orally for 8 weeks post DM establishment. PEITC treatment significantly improved kidney and liver functions, renal histopathological features, tissue fibrosis, macrophage infiltration and blood glucose levels compared to DN control. Mechanistically, PEITC treatment alleviated DN-induced renal damage via modulating glycation and oxidative stresses and inflammatory response. As such, PEITC activated glyoxalase 1 (GLO1) that induced a retraction in renal tissue expression of advanced glycation end products (AGEs) and its receptor (RAGE). PEITC activated nuclear erythroid 2-related factor 2 (Nrf2) and increased expression of its downstream targets, hemeoxygenase-1 (HO-1) and gamma glutamate-cysteine (γ-GCS). Additionally, PEITC treatment decreased the expression of Nrf2 repressor protein, keap1. The anti-inflammatory effect of PEITC was driven, at least in part, via reducing the NLRP3 inflammasome activation as indicated by down regulation of NLRP3, TXNIP, capsase-1 and IL-1ß, TNF-alpha and IL-6. In conclusion; PEITC attenuated DN progression in a dose dependent manner mainly via interruption of AGE/RAGE and NLPR3/TXNIP/NrF2 crosstalk.

Inhibition of p53/miR-34a/SIRT1 axis ameliorates podocyte injury in diabetic nephropathy.

Podocyte injury is associated with albuminuria and the progression of diabetic nephropathy (DN). MiR-34a, a p53-regulated miRNA, directly targets SIRT1 and contributed to DN progression. MiR-34a represses SIRT1 to activate p53 and establish a positive feedback loop. However, whether p53/miR-34a/SIRT1 signaling is activated in podocytes and contributes to DN pathogenesis remains elusive. In this study, we observed that serum miR-34a level was positively correlated with podocyte injury in DN patients. The expression of acetylated p53 and miR-34a was upregulated, SIRT1was downregulated in glomeruli from patients with DN and STZ induced diabetic mice, as well as in human podocytes treated with advanced glycation end (AGE). MiR-34a antagonism in vitro and vivo in STZ induced diabetic mice developed alleviated glomerulus injury as reflected by attenuated albuminuria, reduced podocyte loss and restored autophagic flux. In human podocyte, inhibition of AGE formation by pyridoxamine prevented miR-34a dependent repression of SIRT1, p53 acetylation and activate podocyte autophagy in a dose-dependent manner. MiR-34a overexpression increases acetylation of p53 by translational repression of SIRT1. SIRT1 overexpression also impacts AGE induced apoptosis through deacetylating p53, whereas silencing of SIRT1 by EX527 attenuated the cytoprotective functions of miR-34a knockdown. Moreover, blockade of p53 acetylation significantly rescued miR-34a-induced apoptosis through SIRT1 restoration. Collectively, we demonstrate that by activation of p53, AGE induced the transcription of miR-34a, miR-34a in turn repressed SIRT1 to activate p53, resulting in a positive-feedback loop and contributing to podocyte injury. Targeting modulation of p53/miR-34a/SIRT1 feedback by miR-34a knockdown or overexpression of SIRT1 could rescue podocyte injury during DN.

Plasma concentrations of advanced glycation end-products and colorectal cancer risk in the EPIC study.

Advanced glycation end-products (AGEs) are a heterogeneous group of compounds formed by the non-enzymatic reaction between amino acids and reducing sugars, or dicarbonyls as intermediate compounds. Experimental studies suggest that AGEs may promote colorectal cancer, but prospective epidemiologic studies are inconclusive. We conducted a case-control study nested within a large European cohort. Plasma concentrations of three protein-bound AGEs-Nε-(carboxy-methyl)lysine (CML), Nε-(carboxy-ethyl)lysine (CEL) and Nδ-(5-hydro-5-methyl-4-imidazolon-2-yl)-ornithine (MG-H1)-were measured by ultra-performance liquid chromatography-tandem mass spectrometry in baseline samples collected from 1378 incident primary colorectal cancer cases and 1378 matched controls. Multivariable-adjusted odds ratios (ORs) and 95% confidence intervals (CIs) were computed using conditional logistic regression for colorectal cancer risk associated with CML, CEL, MG-H1, total AGEs, and [CEL+MG-H1: CML] and [CEL:MG-H1] ratios. Inverse colorectal cancer risk associations were observed for CML (OR comparing highest to lowest quintile, ORQ5 versus Q1 = 0.40, 95% CI: 0.27-0.59), MG-H1 (ORQ5 versus Q1 = 0.73, 95% CI: 0.53-1.00) and total AGEs (OR Q5 versus Q1 = 0.52, 95% CI: 0.37-0.73), whereas no association was observed for CEL. A higher [CEL+MG-H1: CML] ratio was associated with colorectal cancer risk (ORQ5 versus Q1 = 1.91, 95% CI: 1.31-2.79). The associations observed did not differ by sex, or by tumour anatomical sub-site. Although individual AGEs concentrations appear to be inversely associated with colorectal cancer risk, a higher ratio of methylglyoxal-derived AGEs versus those derived from glyoxal (calculated by [CEL+MG-H1: CML] ratio) showed a strong positive risk association. Further insight on the metabolism of AGEs and their dicarbonyls precursors, and their roles in colorectal cancer development is needed.

Acute glucose fluctuation promotes RAGE expression via reactive oxygen species­mediated NF­κB activation in rat podocytes.

Diabetic nephropathy (DN) is a common chronic complication of diabetes, for which acute glucose fluctuation (AGF) is a potential risk factor. Fluctuating hyperglycemia has been confirmed to induce more serious kidney damage than hyperglycemia in diabetic rats; however, the mechanism remains unknown. The purpose of this study was to explore the potential role of AGF in the progression of DN. Viability of rat podocytes following 72­h AGF treatment was detected using Cell Counting­Kit­8. The rates of apoptosis and the level of reactive oxygen species (ROS) in rat podocytes were assessed by flow cytometry. Western blotting and reverse transcription­quantitative PCR were performed to measure relative protein and mRNA expression levels, respectively. Transfection with an mRFP­GFP­LC3 adenoviral vector was used to track autophagic flux under confocal microscopy. The results indicated that AGF could inhibit cell proliferation, promote TNF­α, interleukin­1ß (IL­1ß), and reactive oxygen species (ROS) generation, and increase autophagy in rat podocytes. Moreover, AGF upregulated receptor for advanced glycation end products (RAGE) expression via activation of NF­κB/p65 and IκBα. Pretreatment with 5 mM N­Acetyl­L­cysteine or 10 µM pyrrolidine dithiocarbamate effectively reduced cellular damage and inhibited activation of the NF­κB/RAGE signaling pathway. Thus, AGF induces rat podocyte injury by aggravating oxidative stress, promoting the inflammatory response, and regulating ROS­mediated NF­κB/RAGE activation.