RESUMO
INTRODUCTION: Diabetic nephropathy (DN) is an important microvascular complication of uncontrolled diabetes. The features of DN include albuminuria, extracellular matrix alterations, and progressive renal insufficiency. Rice bran protein hydrolysates (RBPs) have been reported to have antihyperglycemic, lipid-lowering, and anti-inflammatory effects in diabetic rats. Our study was to investigate the renoprotective effects of RBP in diabetic animals and mesangial cultured cells. METHODS: Eight-week-old male db/m and db/db mice were orally treated with tap water or RBP (100 or 500 mg/kg/day) for 8 weeks. At the end of the experiment, diabetic nephropathy in kidney tissues was investigated for histological, ultrastructural, and clinical chemistry changes, and biomarkers of angiogenesis, fibrosis, inflammation, and antioxidant in kidney were analyzed by Western blotting. Protection against proangiogenic proteins and induction of cytoprotection by RBP in cultured mesangial cells was evaluated. RESULTS: RBP treatment improved insulin sensitivity, decreased elevated fasting serum glucose levels, and improved serum lipid levels and urinary albumin/creatinine ratios in diabetic mice. RBP ameliorated the decreases in podocyte slit pore numbers, thickening of glomerular basement membranes, and mesangial matrix expansion and suppressed elevation of MCP-1, ICAM-1, HIF-1α, VEGF, TGF-ß, p-Smad2/3, and type IV collagen expression. Moreover, RBP restored suppressed antioxidant Nrf2 and HO-1 expression. In cultured mesangial cells, RBP inhibited high glucose-induced angiogenic protein expression and induced the expression of Nrf2 and HO-1. CONCLUSION: RBP attenuates the progression of diabetic nephropathy and restored renal function by suppressing the expression of proangiogenic and profibrotic proteins, inhibiting proinflammatory mediators, and restoring the antioxidant and cytoprotective system.
Assuntos
Diabetes Mellitus Tipo 2/dietoterapia , Nefropatias Diabéticas/prevenção & controle , Hipoglicemiantes/uso terapêutico , Resistência à Insulina , Oryza/química , Proteínas de Vegetais Comestíveis/uso terapêutico , Hidrolisados de Proteína/uso terapêutico , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Linhagem Celular , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patologia , Nefropatias Diabéticas/imunologia , Indústria de Processamento de Alimentos/economia , Hiperglicemia/prevenção & controle , Hipoglicemiantes/economia , Hipoglicemiantes/metabolismo , Resíduos Industriais/análise , Resíduos Industriais/economia , Rim/imunologia , Rim/metabolismo , Rim/patologia , Rim/ultraestrutura , Masculino , Células Mesangiais/imunologia , Células Mesangiais/metabolismo , Células Mesangiais/patologia , Células Mesangiais/ultraestrutura , Camundongos Mutantes , Microscopia Eletrônica de Transmissão , Epiderme Vegetal/química , Proteínas de Vegetais Comestíveis/economia , Proteínas de Vegetais Comestíveis/metabolismo , Hidrolisados de Proteína/economia , Hidrolisados de Proteína/metabolismo , Insuficiência Renal/complicações , Insuficiência Renal/imunologia , Insuficiência Renal/prevenção & controle , Sementes/química , TailândiaRESUMO
Transcription factors of the peroxisome proliferator-activated receptor (PPAR) family are ligand-activated receptors that play key roles in lipid metabolism and inflammation. The γ isoform (PPARγ) is involved in adipocyte differentiation, insulin sensitization, and vascular pathophysiology, including inflammation and atherosclerosis, for which it is considered an important drug target. PPARγ ligands display varied structures and include fatty acids, electrophilic lipids, and certain drugs. These agonists promote conformational changes allowing interaction of PPARγ with coactivators and hence transcriptional regulation. Here we present a panoply of methods to study PPARγ interactions with ligands and activation in vitro and in cells. The first method is based on the competition of the fluorescent dye 1-anilinonaphthalene-8-sulfonic acid (ANS) with PPARγ ligands for the ligand binding pocket, allowing detection and quantification of ligand binding to PPARγ. This method is specific for PPARγ while ANS displays negligible interaction with other nuclear receptors such as PPARα and retinoid X receptor α (RXRα). The ANS competition assay has been validated through comparison of the affinities determined for well-known PPARγ ligands by this method with those reported in the literature. We also describe here gel-based competition assays that show limited performance with non-covalently bound ligands. In addition, we present a fluorescence anisotropy assay to analyze PPARγ activation by ligands in vitro through their capacity of eliciting PPARγ interaction with a fluorescently labeled peptide derived from one of its coactivators (SRC-1). Finally, we show cell-based assays to investigate PPARγ activation by interaction with its ligands. We believe that combined approaches using ANS, fluorescent coactivator peptides, and in-cell assays to monitor PPARγ binding and interactions may provide valuable strategies for the identification and characterization of PPARγ ligands.
Assuntos
Biofísica/métodos , Células Mesangiais/metabolismo , PPAR gama/metabolismo , Naftalenossulfonato de Anilina/metabolismo , Ligação Competitiva , Polarização de Fluorescência , Genes Reporter/genética , Humanos , Ligantes , PPAR gama/química , Ligação Proteica , EspectrofotometriaRESUMO
Measurement of cell membrane integrity has been widely used to assess chemical cytotoxity. Several assays are available for determining cell membrane integrity including differential labeling techniques using neutral red and trypan blue dyes or fluorescent compounds such as propidium iodide. Other common methods for assessing cytotoxicity are enzymatic "release" assays which measure the extra-cellular activities of lactate dehydrogenase (LDH), adenylate kinase (AK), or glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in culture medium. However, all these assays suffer from several practical limitations, including multiple reagent additions, scalability, low sensitivity, poor linearity, or requisite washes and medium exchanges. We have developed a new cytotoxicity assay which measures the activity of released intracellular proteases as a result of cell membrane impairment. It allows for a homogenous, one-step addition assay with a luminescent readout. We have optimized and miniaturized this assay into a 1536-well format, and validated it by screening a library of known compounds from the National Toxicology Program (NTP) using HEK 293 and human renal mesangial cells by quantitative high-throughput screening (qHTS). Several known and novel membrane disrupters were identified from the library, which indicates that the assay is robust and suitable for large scale library screening. This cytotoxicity assay, combined with the qHTS platform, allowed us to quickly and efficiently evaluate compound toxicities related to cell membrane integrity.