ABSTRACT
BACKGROUND: Nitroalkene derivatives of oleic acid (OA-NO2) serve as high-affinity ligand for PPAR-γ, which regulates apoptosis, oxidation and inflammation and plays a central role in ischemia-reperfusion injury. In the present study, we elucidated the protective mechanisms of OA-NO2 against renal ischemia-reperfusion injury. METHODS: HK-2 cells were subjected to oxygen and glucose deprivation followed by re-oxygenation (OGD/R) to mimic renal ischemia-reperfusion injury. Cell apoptosis was analyzed by flow cytometry. Bax mitochondrial translocation, cytochrome c and apoptosis-inducing factor (AIF) cytosolic leakage and Akt/Gsk 3ß phosphorylation were evaluated by Western blotting. Bax activation was visualized by immunocytochemistry. GW9662 and siRNA transfection were employed to examine the involvement of PPAR-γ. RESULTS: OGD/R injury promoted mitochondrial translocation and activation of Bax, leakage of cytochrome c and AIF, subsequent caspase-3 activation, and eventually cell apoptosis. Pre-incubation with OA-NO2 (1.25 µM, 45min) inhibited Bax activation and blocked apoptotic cascade, while the protective effects were negated by GW9662 or PPAR-γ siRNA. Moreover, OA-NO2 restored Akt and Gsk 3ß phosphorylation in a PPAR-γ-dependent way. CONCLUSION: These findings suggest that OA-NO2 attenuates OGD/R-induced apoptosis by inhibiting Bax translocation and activation and the subsequent mitochondria-dependent apoptotic cascade in a PPAR-γ dependent manner.
Subject(s)
Kidney/drug effects , Oleic Acid/administration & dosage , PPAR gamma/biosynthesis , Reperfusion Injury/genetics , bcl-2-Associated X Protein/biosynthesis , Anilides/administration & dosage , Apoptosis/drug effects , Apoptosis Inducing Factor/biosynthesis , Caspase 3/biosynthesis , Cell Hypoxia/drug effects , Cell Hypoxia/genetics , Cytochromes c/biosynthesis , Glycogen Synthase Kinase 3/biosynthesis , Glycogen Synthase Kinase 3 beta , Humans , Kidney/injuries , Kidney/pathology , Mitochondria/drug effects , PPAR gamma/genetics , Proto-Oncogene Proteins c-akt/biosynthesis , Proto-Oncogene Proteins c-bcl-2/biosynthesis , Reperfusion Injury/drug therapy , Reperfusion Injury/pathology , Signal Transduction/drug effects , bcl-2-Associated X Protein/metabolismABSTRACT
BACKGROUND: Overwhelming evidences suggest epithelial to mesenchymal transition (EMT) of tubular epithelial cells contributes to renal fibrosis of chronic kidney disease (CKD). Connective tissue growth factor (CTGF) plays an important role in the pathogenesis of EMT. However, the molecular mechanisms that regulate cell behaviors are not clear. OBJECTIVE: The purpose of this study was to investigate whether CTGF induces EMT via activation of canonical Wnt signaling in renal tubular epithelial cells. METHODS: Human renal proximal tubular epithelial cells (HK-2) were divided into control group, CTGF group and dickkopf (Dkk)-1 plus CTGF group. We assessed the biological changes of canonical Wnt signaling, including phosphorylation of low-density lipoprotein receptor-related protein (LRP6) and glycogen synthase kinase-3ß (GSK-3ß) and accumulation and nuclear localization of ß-catenin. Meanwhile, morphological changes of the three groups were observed and tubular EMT was further confirmed by detecting the expression of α-SMA and E-cadherin. RESULTS: The phosphorylation levels of LRP6 and GSK-3ß and the expression of ß-catenin in CTGF group were higher than control group (p < 0.05). The accumulation and nuclear localization of ß-catenin was induced in CTGF group. Meanwhile, CTGF group cells showed a mesenchymal morphological phenotype and exhibited increased expressions of E-cadherin and decreased expressions of α-SMA compared to control group (p < 0.05), suggesting tubular EMT. Furthermore, we also found that Dkk-1 blocked the above CTGF's effects by binding with LRP6. CONCLUSION: CTGF induces EMT via activation of canonical Wnt signaling in HK-2 cells in vitro, which may play an important role in the renal fibrosis of CKD.
Subject(s)
Connective Tissue Growth Factor/metabolism , Epithelial Cells , Kidney Tubules, Proximal , Renal Insufficiency, Chronic , Wnt Signaling Pathway/physiology , Cell Line , Epithelial Cells/metabolism , Epithelial Cells/pathology , Epithelial-Mesenchymal Transition , Fibrosis , Glycogen Synthase Kinase 3/metabolism , Glycogen Synthase Kinase 3 beta , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Kidney Tubules, Proximal/metabolism , Kidney Tubules, Proximal/pathology , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Phosphorylation , Renal Insufficiency, Chronic/metabolism , Renal Insufficiency, Chronic/pathology , Renal Insufficiency, Chronic/physiopathology , beta Catenin/metabolismABSTRACT
Neutrophil-to-lymphocyte ratio (NLR), a novel inflammatory marker, is strongly associated with the risk of sarcopenia. Notably, being overweight has been found to accelerate the loss of skeletal muscle mass and function in chronic kidney disease (CKD) patients. However, the effect of overweight status on the relationship between NLR and sarcopenia risk has been poorly studied. We conducted a cross-sectional study at a hemodialysis center in Chengdu, China, from September to December 2022. The prevalence of sarcopenia was determined according to the Asian Working Group for Sarcopenia (AWGS). Participants were stratified based on body mass index (BMI) categories for the Asian population (non-overweight < 23 kg/m2 and overweight ≥ 23 kg/m2). 272 participants aged 18-85 years were included, with 144 being male. The overall prevalence of sarcopenia was 32.72% (89/272). After adjusting for covariates, NLR was significantly associated with sarcopenia risk in overweight participants (OR 1.60, 95% CI 1.15-2.24, p = 0.006), whereas it was not significant in the non-overweight group (OR 0.88, 95% CI 0.70-1.10, p = 0.254). Moreover, subgroup analysis showed a significant interactive association between NLR and overweight status with respect to sarcopenia. These findings emphasize the potential significance of regular screening of NLR for the early detection of sarcopenia in overweight patients undergoing maintenance hemodialysis.
Subject(s)
Sarcopenia , Humans , Male , Female , Sarcopenia/epidemiology , Sarcopenia/etiology , Sarcopenia/diagnosis , Overweight/complications , Neutrophils , Cross-Sectional Studies , Renal Dialysis/adverse effects , Lymphocytes , Prevalence , Hand Strength/physiologyABSTRACT
Unenhanced CT scans exhibit high specificity in detecting moderate-to-severe hepatic steatosis. Even though many CTs are scanned from health screening and various diagnostic contexts, their potential for hepatic steatosis detection has largely remained unexplored. The accuracy of previous methodologies has been limited by the inclusion of non-parenchymal liver regions. To overcome this limitation, we present a novel deep-learning (DL) based method tailored for the automatic selection of parenchymal portions in CT images. This innovative method automatically delineates circular regions for effectively detecting hepatic steatosis. We use 1,014 multinational CT images to develop a DL model for segmenting liver and selecting the parenchymal regions. The results demonstrate outstanding performance in both tasks. By excluding non-parenchymal portions, our DL-based method surpasses previous limitations, achieving radiologist-level accuracy in liver attenuation measurements and hepatic steatosis detection. To ensure the reproducibility, we have openly shared 1014 annotated CT images and the DL system codes. Our novel research contributes to the refinement the automated detection methodologies of hepatic steatosis on CT images, enhancing the accuracy and efficiency of healthcare screening processes.
Subject(s)
Deep Learning , Fatty Liver , Liver , Tomography, X-Ray Computed , Humans , Tomography, X-Ray Computed/methods , Fatty Liver/diagnostic imaging , Fatty Liver/pathology , Liver/diagnostic imaging , Liver/pathology , Male , Reproducibility of Results , FemaleABSTRACT
PURPOSE: To investigate the expression and function of serum response factor in podocyte epithelial-mesenchymal transition of diabetic nephropathy. METHODS: The expression of serum response factor, epithelial markers and mesenchymal markers was examined in podocytes or renal cortex tissues following high glucose. Serum response factor was upregulated by its plasmids and downregulated by CCG-1423 to investigate how it influenced podocyte epithelial-mesenchymal transition in diabetic nephropathy. Streptozotocin was used to generate diabetes mellitus in rats. RESULTS: In podocytes after high glucose treatment, serum response factor and mesenchymal markers increased, while epithelial markers declined. Similar changes were observed in vivo. Serum response factor overexpression in podocytes induced expression of Snail, an important transcription factor mediating epithelial-mesenchymal transition. Blockade of serum response factor reduced Snail induction, protected podocytes from epithelial-mesenchymal transition and ameliorated proteinuria. CONCLUSION: Together, increased serum response factor activity provokes podocytes' epithelial-mesenchymal transition and dysfunction in diabetic nephropathy. Targeting serum response factor by small-molecule inhibitor may be an attractive therapeutic strategy for diabetic nephropathy.
Subject(s)
Arabidopsis Proteins/drug effects , Diabetes Mellitus, Experimental/metabolism , Diabetic Nephropathies/metabolism , Epithelial-Mesenchymal Transition/drug effects , Glucose/pharmacology , Intramolecular Transferases/drug effects , Kidney/pathology , Podocytes/drug effects , Serum Response Factor/drug effects , Sweetening Agents/pharmacology , Actins/drug effects , Actins/genetics , Anilides/pharmacology , Animals , Arabidopsis Proteins/metabolism , Benzamides/pharmacology , Cell Line , Diabetic Nephropathies/pathology , Fibronectins , Immunohistochemistry , Intramolecular Transferases/metabolism , Male , Mice , Podocytes/metabolism , Podocytes/pathology , Rats , Rats, Wistar , Reverse Transcriptase Polymerase Chain Reaction , S100 Calcium-Binding Protein A4 , S100 Proteins/drug effects , S100 Proteins/genetics , Serum Response Factor/genetics , Serum Response Factor/metabolism , Snail Family Transcription Factors , Trans-Activators/drug effects , Trans-Activators/genetics , Transcription Factors/metabolism , Zonula Occludens-1 Protein/drug effects , Zonula Occludens-1 Protein/geneticsABSTRACT
BACKGROUND: Label-retaining cells (LRCs) have been recognized as rare stem and progenitor-like cells, but their complex biological features in renal repair at the cellular level have never been reported. This study was conducted to evaluate whether LRCs in kidney are indeed renal stem/progenitor cells and to delineate their potential role in kidney regeneration. METHODS: We utilized a long-term pulse chase of 5-bromo-2'-deoxyuridine (BrdU)-labeled cells in C57BL/6J mice to identify renal LRCs. We tracked the precise morphological characteristics and locations of BrdU(+)LRCs by both immunohistochemistry and immunofluorescence. To examine whether these BrdU(+)LRCs contribute to the repair of acute kidney injury, we analyzed biological characteristics of BrdU(+)LRCs in mice after ischemia/reperfusion (I/R) injury. RESULTS: The findings revealed that the nuclei of BrdU(+) LRCs exhibited different morphological characteristics in normal adult kidneys, including nuclei in pairs or scattered, fragmented or intact, strongly or weakly positive. Only 24.3 ± 1.5 % of BrdU(+) LRCs co-expressed with Ki67 and 9.1 ± 1.4 % of BrdU(+) LRCs were positive for TUNEL following renal I/R injury. Interestingly, we found that newly regenerated cells formed a niche-like structure and LRCs in pairs tended to locate in this structure, but the number of those LRCs was very low. We found a few scattered LRCs co-expressed Lotus tetragonolobus agglutinin (LTA) in the early phase of injury, suggesting differentiation of those LRCs in mouse kidney. CONCLUSIONS: Our findings suggest that LRCs are not a simple type of slow-cycling cells in adult kidneys, indicating a limited role of these cells in the regeneration of I/R injured kidney. Thus, LRCs cannot reliably be considered stem/progenitor cells in the regeneration of adult mouse kidney. When researchers use this technique to study the cellular basis of renal repair, these complex features of renal LRCs and the purity of real stem cells among renal LRCs should be considered.
Subject(s)
Cell Nucleus/ultrastructure , Cell Tracking , Epithelial Cells/cytology , Regeneration/physiology , Reperfusion Injury/pathology , Animals , Apoptosis , Bromodeoxyuridine , Cell Differentiation , Cell Nucleus/metabolism , Cell Proliferation , Cell Survival , Epithelial Cells/metabolism , Gene Expression , In Situ Nick-End Labeling , Ki-67 Antigen/genetics , Ki-67 Antigen/metabolism , Kidney , Lectins/genetics , Lectins/metabolism , Male , Mice , Mice, Inbred C57BL , Reperfusion Injury/genetics , Reperfusion Injury/metabolismABSTRACT
Nitroalkene derivative of oleic acid (OA-NO2), due to its ability to mediate revisable Michael addition, has been demonstrated to have various biological properties and become a therapeutic agent in various diseases. Though its antioxidant properties have been reported in different models of acute kidney injury (AKI), the mechanism by which OA-NO2 attenuates intracellular oxidative stress is not well investigated. Here, we elucidated the anti-oxidative mechanism of OA-NO2 in an in vitro model of renal ischemia/reperfusion (I/R) injury. Human tubular epithelial cells were subjected to oxygen and glucose deprivation/re-oxygenation (OGD/R) injury. Pretreatment with OA-NO2 (1.25 µM, 45 min) attenuated OGD/R triggered reactive oxygen species (ROS) generation and subsequent mitochondrial membrane potential disruption. This action was mediated via up-regulating endogenous antioxidant defense components including superoxide dismutase (SOD1), heme oxygenase 1 (HO-1), and γ-glutamyl cysteine ligase modulatory subunits (GCLM). Moreover, subcellular fractionation analyses demonstrated that OA-NO2 promoted nuclear translocation of nuclear factor-E2- related factor-2 (Nrf2) and Nrf2 siRNA partially abrogated these protective effects. In addition, OA-NO2 inhibited NADPH oxidase activation and NADPH oxidase 4 (NOX4), NADPH oxidase 2 (NOX2) and p22phox up-regulation after OGD/R injury, which was not relevant to Nrf2. These results contribute to clarify that the mechanism of OA-NO2 reno-protection involves both inhibition of NADPH oxidase activity and induction of SOD1, Nrf2-dependent HO-1, and GCLM.
Subject(s)
Glucose/metabolism , NADPH Oxidases/metabolism , NF-E2-Related Factor 2/metabolism , Oxygen/metabolism , Humans , Kidney/drug effects , Oleic Acid , Oxidative Stress , Reactive Oxygen Species , Signal Transduction , TransfectionABSTRACT
Diabetic nephropathy is a major cause of morbidity and mortality in diabetic patients. Effective therapies to prevent the development of this disease are required. Berberine (BBR) has several preventive effects on diabetes and its complications. However, the molecular mechanism of BBR on kidney function in diabetes is not well defined. Here, we reported that activation of AMP-activated protein kinase (AMPK) is required for BBR-induced improvement of kidney function in vivo. AMPK phosphorylation and activity, productions of reactive oxygen species (ROS), kidney function including serum blood urea nitrogen (BUN), creatinine clearance (Ccr), and urinary protein excretion, morphology of glomerulus were determined in vitro or in vivo. Exposure of cultured human glomerulus mesangial cells (HGMCs) to BBR time- or dose-dependently activates AMPK by increasing the thr172 phosphorylation and its activities. Inhibition of LKB1 by siRNA or mutant abolished BBR-induced AMPK activation. Incubation of cells with high glucose (HG, 30 mM) markedly induced the oxidative stress of HGMCs, which were abolished by 5-aminoimidazole-4-carboxamide ribonucleoside, AMPK gene overexpression or BBR. Importantly, the effects induced by BBR were bypassed by AMPK siRNA transfection in HG-treated HGMCs. In animal studies, streptozotocin-induced hyperglycemia dramatically promoted glomerulosclerosis and impaired kidney function by increasing serum BUN, urinary protein excretion, and decreasing Ccr, as well as increased oxidative stress. Administration of BBR remarkably improved kidney function in wildtype mice but not in AMPKα2-deficient mice. We conclude that AMPK activation is required for BBR to improve kidney function in diabetic mice.
Subject(s)
AMP-Activated Protein Kinases/metabolism , Berberine/administration & dosage , Diabetes Mellitus, Experimental/metabolism , Diabetic Nephropathies/metabolism , Animals , Berberine/pharmacology , Cell Line , Diabetes Mellitus, Experimental/drug therapy , Diabetic Nephropathies/drug therapy , Gene Expression Regulation/drug effects , Humans , Kidney Function Tests , Mesangial Cells/metabolism , Mice , Oxidative Stress/drug effects , Phosphorylation , StreptozocinABSTRACT
PURPOSE: To investigate whether mesenchymal stem cells (MSCs) could inhibit transforming growth factor beta (TGF-ß) signalling pathway by paracrine action. METHODS: Bone marrow-derived MSCs were transplanted to streptozotocin-induced diabetic rats via tail vein. MSC-conditioned media were used with a model of mesangial cell fibrosis induced by high glucose in vitro. RESULTS: At 8 weeks after MSC treatment, the renal function and the glomerulosclerosis as revealed by periodic acid Schiff stain was dramatically attenuated. The expression of collagen I, collagen IV and α-smooth muscle actin (SMA) in diabetic kidney was decreased, and E-cadherin increased after MSC treatment. The TGF-ß signalling pathway was suppressed both in vivo and in vitro. MSCs secreted a significant amount of bone morphogenetic protein 7 (BMP7), in vitro, MSC-conditioned media inhibited TGF-ß signalling stimulated by high glucose, and BMP7 neutralizing antibody blocked the inhibitory effect of MSC-conditioned media. CONCLUSION: MSCs ameliorated glomerular fibrosis in vivo and in vitro by inhibiting TGF-ß/Smad signalling pathway via secretion of BMP7.
ABSTRACT
Mesenchymal stem cells (MSCs) treatment has been shown to be effective in diabetic nephropathy (DN). However, the mechanisms involved in the renoprotective effects of MSCs have not been clearly demonstrated. Especially, there was no study on the relationship of MSCs and macrophages in diabetic kidney. To explore the effect of MSCs on macrophages in DN, streptozotocin-induced diabetes animals received no treatment or treatment with MSCs (2×10(6), via tail vein) for two continuous weeks. Eight weeks after treatment, physical, biochemical and morphological parameters were measured. Immunohistochemistry for fibronectin (FN), CollagenI, ED-1, monocyte chemoattractant protein-1 (MCP-1) was performed. Expressions of pro-inflammatory cytokines and hepatocyte growth factor (HGF) at gene level and protein level were determined by real-time reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. Blood glucose, urinary albumin excretion, creatinine clearance were significantly reduced after MSCs treatment. The glomerulosclerosis as revealed by periodic acid Schiff stain and expression of FN and CollagenI was also dramatically attenuated. Most importantly, the expression of MCP-1 and the number of infiltrated macrophages in kidney were effectively suppressed by MSCs treatment. The expression of HGF in MSCs group was up-regulated. Meanwhile, the expressions of IL-1ß, IL-6 and TNFα were significantly down-regulated by MSCs treatment. Our study suggest that MSCs treatment ameliorates DN via inhibition of MCP-1 expression by secreting HGF, thus reducing macrophages infiltration, down-regulating IL-1ß, IL-6, TNFα expression in renal tissue in diabetic rats.