ABSTRACT
BACKGROUND: Endoplasmic reticulum stress is associated with podocyte apoptosis in the pathogenesis of diabetic nephropathy (DN). A previous study has demonstrated that emodin has a protective effect in the kidney by suppressing proliferation of mesangial cells and inhibiting the renal tubular epithelial-to-mesenchymal transition. However, the effects of emodin on the podocyte apoptosis in DN and its mechanisms are unknown. AIM: This study aimed to explore the effect of emodin on DN model KK-Ay mice and high glucose induced podocytes apoptosis via the PERK-eIF2α pathway. METHODS: KK-Ay mice model of DN were treated with emodin at dose of 40 and 80 mg/kg/day for 8 weeks. Urine albumin, serum creatinine, blood urea nitrogen levels and the renal histopathology in mice were performed. In vitro, conditionally immortalized mouse podocytes exposed to HG (30mM) were incubated with emodin. Cell viability was measured by CCK-8 assay. Additionally, we performed RNA interference and measured the apoptosis in cultured podocytes treated with emodin. Immunohistochemistry, immunofluorescence, western blot, and real-time PCR were used to detect gene and protein expression both in vivo and in vitro. RESULTS: The results showed that emodin treatment ameliorated urine albumin, serum creatinine, and blood urea nitrogen of DN mice. The pathological damage of kidney tissue was also improved after treatment with emodin. Moreover, emodin increased nephrin expression. Podocytes apoptosis and endoplasmic reticulum stress markers (GRP78) were significantly reduced upon emodin treatment. Furthermore, emodin treatment decreased the expression of phosphorylated protein kinase RNA-like endoplasmic reticulum kinase (P-PERK), phosphorylated P-eIF2α, ATF4, and CHOP. In vitro, emodin treatment was further found to decrease the GRP78 level induced by high glucose or tunicamycin (TM). Besides, emodin and PERK knockdown inhibited the apoptosis of podocytes cultured in high glucose by counteracting the upregulation of phosphorylated PERK, phosphorylated eIF2α, ATF4, and CHOP. CONCLUSION: Overall, the findings indicate that emodin mitigates podocytes apoptosis by inhibiting the PERK-eIF2α signaling pathway in vivo and in vitro, and, therefore, exerts a protective action on podocytes in DN.
Subject(s)
Apoptosis/drug effects , Diabetic Nephropathies/drug therapy , Emodin/pharmacology , Endoplasmic Reticulum Stress/drug effects , Protein Kinases/pharmacology , eIF-2 Kinase/antagonists & inhibitors , Animals , Cell Proliferation/drug effects , Cells, Cultured , Diabetic Nephropathies/metabolism , Diabetic Nephropathies/pathology , Dose-Response Relationship, Drug , Emodin/administration & dosage , Emodin/chemistry , Endoplasmic Reticulum Chaperone BiP , Epithelial-Mesenchymal Transition/drug effects , Male , Mice , Mice, Inbred Strains , Molecular Structure , Podocytes/drug effects , Podocytes/metabolism , Podocytes/pathology , Protein Kinases/administration & dosage , Protein Kinases/chemistry , Structure-Activity Relationship , eIF-2 Kinase/metabolismABSTRACT
New data indicate that abnormal glomerular endothelial cell (GEC)-podocyte crosstalk plays a critical role in diabetic nephropathy (DN). The aim of our study is to investigate the role of exosomes from high glucose (HG)-treated GECs in the epithelial-mesenchymal transition (EMT) and dysfunction of podocytes. In this study, exosomes were extracted from GEC culture supernatants and podocytes were incubated with the GEC-derived exosomes. Here, we demonstrate that HG induces the endothelial-mesenchymal transition (EndoMT) of GECs and HG-treated cells undergoing the EndoMT secrete more exosomes than normal glucose (NG)-treated GECs. We show that GEC-derived exosomes can be internalized by podocytes and exosomes from HG-treated cells undergoing an EndoMT-like process can trigger the podocyte EMT and barrier dysfunction. Our study reveals that TGF-ß1 mRNA is enriched in exosomes from HG-treated GECs and probably mediates the EMT and dysfunction of podocytes. In addition, our experimental results illustrate that canonical Wnt/ß-catenin signaling is involved in the exosome-induced podocyte EMT. Our findings suggest the importance of paracrine communication via exosomes between cells undergoing the EndoMT and podocytes for renal fibrosis in DN. Thus, protecting GECs from the EndoMT and inhibiting TGF-ß1-containing exosomes release from GECs is necessary to manage renal fibrosis in DN.
Subject(s)
Endothelial Cells/metabolism , Epithelial-Mesenchymal Transition , Exosomes/metabolism , Glucose/metabolism , Kidney Glomerulus/metabolism , Podocytes/metabolism , Animals , Biomarkers , Cells, Cultured , Diabetic Nephropathies/etiology , Diabetic Nephropathies/metabolism , Endocytosis , Endothelial Cells/drug effects , Exosomes/ultrastructure , Gene Expression , Glucose/pharmacology , Kidney Glomerulus/pathology , Mice , Permeability , Phenotype , Podocytes/drug effects , Transforming Growth Factor beta1/genetics , Transforming Growth Factor beta1/metabolism , Wnt Signaling PathwayABSTRACT
Glomerular mesangial cells (GMCs) activation is implicated in the pathogenesis of diabetic nephropathy (DN). Our previous study revealed that high glucose (HG)-treated glomerular endothelial cells (GECs) produce an increased number of TGF-[Formula: see text]1-containing exosomes to activate GMCs through the TGF-[Formula: see text]1/Smad3 signaling pathway. We also identified that Tongxinluo (TXL), a traditional Chinese medicine, has beneficial effects on the treatment of DN in DN patients and type 2 diabetic mice. However, it remained elusive whether TXL could ameliorate renal structure and function through suppression of intercellular transfer of TGF-[Formula: see text]1-containing exosomes from GECs to GMCs. In this study, we demonstrate that TXL can inhibit the secretion of TGF-[Formula: see text]1-containing exosomes from HG-treated GECs. Furthermore, exosomes produced by HG induced-GECs treated with TXL cannot trigger GMC activation, proliferation and extracellular matrix (ECM) overproduction both in vitro and in vivo. These results suggest that TXL can prevent the transfer of TGF-[Formula: see text]1 from GECs to GMCs via exosomes, which may be one of the mechanisms of TXL in the treatment of DN.
