Hyperglycemia upregulates thioredoxin interacting protein (TXNIP) expression, which in turn induces ROS production, inflammatory and fibrotic responses in the diabetic kidney. Dysregulation of autophagy contributes to the development of diabetic nephropathy. However, the interaction of TXNIP with autophagy/mitophagy in diabetic nephropathy is unknown. In this study, streptozotocin-induced diabetic rats were given TXNIP DNAzyme or scrambled DNAzyme for 12 weeks respectively. Fibrotic markers, mitochondrial function and mitochondrial reactive oxygen species (mtROS) were assessed in kidneys. Tubular autophagy and mitophagy were determined in kidneys from both human and rats with diabetic nephropathy. TXNIP and autophagic signaling molecules were examined. TXNIP DNAzyme dramatically attenuated extracellular matrix deposition in the diabetic kidneys compared to the control DNAzyme. Accumulation of autophagosomes and reduced autophagic clearance were shown in tubular cells of human diabetic compared to non-diabetic kidneys, which was reversed by TXNIP DNAzyme. High glucose induced mitochondrial dysfunction and mtROS production, and inhibited mitophagy in proximal tubular cells, which was reversed by TXNIP siRNA. TXNIP inhibition suppressed diabetes-induced BNIP3 expression and activation of the mTOR signaling pathway. Collectively, hyperglycemia-induced TXNIP contributes to the dysregulation of tubular autophagy and mitophagy in diabetic nephropathy through activation of the mTOR signaling pathway.
Giant mitochondria are peculiarly shaped, extremely large mitochondria in hepatic parenchymal cells, the internal structure of which is characterised by atypically arranged cristae, enlarged matrix granules and crystalline inclusions. The presence of giant mitochondria in human tissue biopsies is often linked with cellular adversity, caused by toxins such as alcohol, xenobiotics, anti-cancer drugs, free-radicals, nutritional deficiencies or as a consequence of high fat Western diets. To date, non-alcoholic fatty liver disease is the most prevalent liver disease in lipid dysmetabolism, in which mitochondrial dysfunction plays a crucial role. It is not well understood whether the morphologic characteristics of giant mitochondria are an adaption or caused by such dysfunction. In the present study, we employ a complementary multimodal imaging approach involving array tomography and transmission electron tomography in order to comparatively analyse the structure and morphometric parameters of thousands of normal- and giant mitochondria in four patients diagnosed with non-alcoholic fatty liver disease. In so doing, we reveal functional alterations associated with mitochondrial gigantism and propose a mechanism for their formation based on our ultrastructural findings.
Cell-to-cell transmission of vaccinia virus can be mediated by enveloped virions that remain attached to the outer surface of the cell or those released into the medium. During egress, the outer membrane of the double-enveloped virus fuses with the plasma membrane leaving extracellular virus attached to the cell surface via viral envelope proteins. Here we report that F-actin nucleation by the viral protein A36 promotes the disengagement of virus attachment and release of enveloped virus. Cells infected with the A36YdF virus, which has mutations at two critical tyrosine residues abrogating localised actin nucleation, displayed a 10-fold reduction in virus release. We examined A36YdF infected cells by transmission electron microscopy and observed that during release, virus appeared trapped in small invaginations at the plasma membrane. To further characterise the mechanism by which actin nucleation drives the dissociation of enveloped virus from the cell surface, we examined recombinant viruses by super-resolution microscopy. Fluorescently-tagged A36 was visualised at sub-viral resolution to image cell-virus attachment in mutant and parental backgrounds. We confirmed that A36YdF extracellular virus remained closely associated to the plasma membrane in small membrane pits. Virus-induced actin nucleation reduced the extent of association, thereby promoting the untethering of virus from the cell surface. Virus release can be enhanced via a point mutation in the luminal region of B5 (P189S), another virus envelope protein. We found that the B5P189S mutation led to reduced contact between extracellular virus and the host membrane during release, even in the absence of virus-induced actin nucleation. Our results posit that during release virus is tightly tethered to the host cell through interactions mediated by viral envelope proteins. Untethering of virus into the surrounding extracellular space requires these interactions be relieved, either through the force of actin nucleation or by mutations in luminal proteins that weaken these interactions.
Autophagy is emerging as an important pathway in many diseases including diabetic nephropathy. It is acknowledged that oxidative stress plays a critical role in autophagy dysfunction and diabetic nephropathy, and KCa3.1 blockade ameliorates diabetic renal fibrosis through inhibiting TGF-β1 signaling pathway. To identify the role of KCa3.1 in dysfunctional tubular autophagy in diabetic nephropathy, human proximal tubular cells (HK2) transfected with scrambled or KCa3.1 siRNAs were exposed to TGF-β1 for 48 h, then autophagosome formation, the autophagy marker LC3, signaling molecules PI3K, Akt and mTOR, and oxidative stress marker nitrotyrosine were examined respectively. In vivo, LC3, nitrotyrosine and phosphorylated mTOR were examined in kidneys of diabetic KCa3.1+/+ and KCa3.1−/− mice. The results demonstrated that TGF-β1 increased the formation of autophagic vacuoles, LC3 expression, and phosphorylation of PI3K, Akt and mTOR in scrambled siRNA transfected HK2 cells compared to control cells, which was reversed in KCa3.1 siRNA transfected HK2 cells. In vivo, expression of LC3 and nitrotyrosine, and phosphorylation of mTOR were significantly increased in kidneys of diabetic KCa3.1+/+ mice compared to non-diabetic mice, which were attenuated in kidneys of diabetic KCa3.1−/− mice. These results suggest that KCa3.1 activation contributes to dysfunctional tubular autophagy in diabetic nephropathy through PI3K/Akt/mTOR signaling pathways.
Thioredoxin-interacting protein (TXNIP) expression is ubiquitous and is induced by a variety of cellular stresses, including high intracellular glucose. TXNIP is associated with activation of oxidative stress and tubulointerstitial fibrosis in diabetic nephropathy. Autophagy is a major pathway that delivers damaged proteins and organelles to lysosomes to maintain cellular homeostasis. This study aimed to investigate the dysregulation of autophagy and the regulation of TXNIP on autophagy in renal proximal tubular cells (PTCs) under diabetic conditions. The formation of autophagosomes was measured using transmission electron microscopy, and LC3-II, and the effectiveness of autophagic clearance was determined by p62 expression in diabetic kidney and in human PTCs exposed to high glucose (HG). The results collectively demonstrated increased expression of TXNIP, LC3/LC3-II and p62 in renal tubular cells of mice with diabetic nephropathy and in cultured human PTCs exposed to HG (30 mM/l) for 48 h compared with control. The formation of autophagic vacuoles was increased in HG-induced cells. Furthermore, silencing of TXNIP by siRNA transfection reduced autophagic vacuoles and the expression of LC3-II and p62 in human PTCs exposed to HG compared with control and partially reversed the accumulation of LC3-II and p62 induced by bafilomycin A 1 (50 nM/l), a pharmacological inhibitor of autophagy which blocks the fusion of autophagosomes with lysosomes and impairs the degradation of LC3-II and p62. Collectively, these results suggest that hyperglycemia leads to dysfunction of autophagy in renal tubular cells and decreases autophagic clearance. HG-induced overexpression of TXNIP may contribute to the dysfunction of tubular autophagy in diabetes.
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