OBJECTIVE: To observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC). METHODS: A monoclonal antibody against AM developed by ...OBJECTIVE: To observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC). METHODS: A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry. The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC) and MsC were investigated by Northern blot assay, and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H]thymidine incorporation as an index. RESULTS: A specific monoclonal antibody against AM was succesfully developed. AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells), some cortical proximal tubules, medullary collecting duct cells, interstitial cells, vascular smooth muscle cells and endothelial cells. Northern blot assay showed that AM mRNA was expressed only on cultured GEC, but not on MsC, however, AM receptor CRLR mRNA was only expressed on MsC. GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect. CONCLUSION: AM produced by GEC inhibits the proliferation of MsC, which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.展开更多
Diabetes is a growing global health issue,and effective diagnostic tools are needed to support early detection.This study proposes an enhanced deep learning framework,SE-ResNet50,which integrates a squeeze-and-excitat...Diabetes is a growing global health issue,and effective diagnostic tools are needed to support early detection.This study proposes an enhanced deep learning framework,SE-ResNet50,which integrates a squeeze-and-excitation(SE)block into the conventional ResNet50 architecture to improve the classification of diabetic kid-ney pathology from glomerular images.The SE block adaptively recalibrates feature responses,enabling the model to emphasize diagnostically relevant structures better.The proposed framework was trained and validated on a kidney tissue dataset from Kanazawa Medical University,achieving superior performance with an accuracy of 97.02%,precision of 0.96,and an AUC of 0.9856.SE-ResNet50 exhibited superior robustness and general-izability compared to established CNN architectures such as EfficientNet B0,Inception V3,and ConvNeXt.Further visualization via Grad-CAM revealed that the model effectively localizes critical regions within glomerular images.These results highlight the potential of SE-ResNet50 as a reliable and interpretable tool for advancing diabetes-related CKD diagnosis in clinical settings.展开更多
Objective To investigate the role of hepatitis B virus X protein(HBx)in glomerular podocyte immune disorder and its regulatory mechanism.Methods Fourteen 6-week-old male hepatitis B virus(HBV)transgenic(HBVTg)mice wer...Objective To investigate the role of hepatitis B virus X protein(HBx)in glomerular podocyte immune disorder and its regulatory mechanism.Methods Fourteen 6-week-old male hepatitis B virus(HBV)transgenic(HBVTg)mice were selected,and age-matched wild type(WT)mice were as controls.They were fed to different weeks,and 24 h urinary protein,blood biochemistry,renal pathology and podocyte changes under electron microscope were detected.The expression of HBx and the infiltration of immune cells in kidney tissue of HBVTg mice were observed by immunohistochemistry.Human podocyte cell line was transfected with pcDNA3.1/myc-HBx plasmid,and the localization of HBx and Nephrin in podocytes was detected by immunofluorescence.The expression of major histocompatibility complex I(MHCII)and co-stimulatory molecule CD40 on the cell surface was detected by flow cytometry.The contents of multiple cytokines in cell culture supernatants were determined by enzyme-linked immunosorbent assay.Transcriptome sequencing(RNA-seq)was used to screen the downstream related genes regulated by HBx,and real-time quantitative PCR was used to verify their expressions.After overexpression or silencing of Notch1 gene with overexpressed plasmids or short hairpin RNA(shRNA)in podocytes,the effect on the expression of immune molecules and cytokines secretion was observed.The Notch receptor inhibitor N-[N-(3,5-difluorophenyll-alanyl)]-(s)-phenylglycine tert-butyl ester(DAPT)was used to block Notch1 signaling pathway in HBV-Tg mice,and then blood biochemistry,renal pathological changes and infiltration of immune cells in kidney tissue were observed.Results Twenty-four-hour urine protein,serum creatinine and urea nitrogen levels were markedly increased(all P<0.05)and renal pathological injury was significantly aggravated in HBV-Tg mice than those in WT mice.Also,HBx was up-regulated and immune cells infiltrated in the glomerulus of HBV-Tg mice.After transfection with HBx in podocytes,the expression of MHC-I and CD40 on the cellular surface was upregulated(all P<0.05),the contents of monocyte chemotactic protein-1(MCP-1),tumor necrosis factor-α(TNF-α)and interleukin(IL)-1βin the supernatants were increased(all P<0.05),and the secretion of IL-4 and interferonγ(IFN-γ)was unbalanced.RNA-seq screened downstream genes of HBx,such as Notch1,PLA2R,TLR4,etc.,and further confirmed that HBx could promote the up-regulation of Notch1 mRNA and protein(all P<0.05).After over-expression of Notch1 gene,HBx-induced expression of MHC-II and CD40 on the cellular surface was significantly up-regulated(all P<0.05),and the contents of MCP-1,TNF-αand IL-1βin the supernatants were obviously increased(all P<0.05),and the imbalance of IL-4/IFN-γwas further aggravated.After Notch1 gene silencing,the above results showed the opposite changes.In vivo,the results indicated that serum creatinine levels were obviously decreased(all P<0.05),renal pathological injury and immune cell infiltration were significantly alleviated in HBV-Tg+DAPT group than those in HBV-Tg+DMSO group.Conclusion HBx protein can promote the upregulation of Notch1 signaling pathway in podocytes.And Notch1 signaling pathway promotes the expression of immune molecules on the surface of podocytessand regulates the imbalance of cytokines,then causes glomerular injury and dysfunction of immune microenvironment.展开更多
文摘OBJECTIVE: To observe the localization of adrenomedullin (AM) in rat kidney tissue and its inhibitory effect on the growth of cultured rat mesangial cells (MsC). METHODS: A monoclonal antibody against AM developed by our laboratory was used to detect the localization of AM protein in rat kidney tissue by avidin-biotin complex immunohistochemistry. The expressions of AM and its receptor CRLR mRNA on cultured glomerular epithelial cells (GEC) and MsC were investigated by Northern blot assay, and the possible effect of AM secreted by GEC on MsC proliferation was observed using [3H]thymidine incorporation as an index. RESULTS: A specific monoclonal antibody against AM was succesfully developed. AM was immunohistochemically localized mainly in glomeruli (GEC and endothelial cells), some cortical proximal tubules, medullary collecting duct cells, interstitial cells, vascular smooth muscle cells and endothelial cells. Northern blot assay showed that AM mRNA was expressed only on cultured GEC, but not on MsC, however, AM receptor CRLR mRNA was only expressed on MsC. GEC conditioned medium containing AM can inhibit MsC growth and AM receptor blocker CGRP8-37 may partially decreased this inhibitory effect. CONCLUSION: AM produced by GEC inhibits the proliferation of MsC, which suggests that AM as an important regulator is involved in glomerular normal physiological functions and pathologic processes.
基金approved by the ethical review board of Kanazawa Medical University(Kanazawa Medical University Hospital Medical Research Ethics Committee,approval No.I493).
文摘Diabetes is a growing global health issue,and effective diagnostic tools are needed to support early detection.This study proposes an enhanced deep learning framework,SE-ResNet50,which integrates a squeeze-and-excitation(SE)block into the conventional ResNet50 architecture to improve the classification of diabetic kid-ney pathology from glomerular images.The SE block adaptively recalibrates feature responses,enabling the model to emphasize diagnostically relevant structures better.The proposed framework was trained and validated on a kidney tissue dataset from Kanazawa Medical University,achieving superior performance with an accuracy of 97.02%,precision of 0.96,and an AUC of 0.9856.SE-ResNet50 exhibited superior robustness and general-izability compared to established CNN architectures such as EfficientNet B0,Inception V3,and ConvNeXt.Further visualization via Grad-CAM revealed that the model effectively localizes critical regions within glomerular images.These results highlight the potential of SE-ResNet50 as a reliable and interpretable tool for advancing diabetes-related CKD diagnosis in clinical settings.
文摘Objective To investigate the role of hepatitis B virus X protein(HBx)in glomerular podocyte immune disorder and its regulatory mechanism.Methods Fourteen 6-week-old male hepatitis B virus(HBV)transgenic(HBVTg)mice were selected,and age-matched wild type(WT)mice were as controls.They were fed to different weeks,and 24 h urinary protein,blood biochemistry,renal pathology and podocyte changes under electron microscope were detected.The expression of HBx and the infiltration of immune cells in kidney tissue of HBVTg mice were observed by immunohistochemistry.Human podocyte cell line was transfected with pcDNA3.1/myc-HBx plasmid,and the localization of HBx and Nephrin in podocytes was detected by immunofluorescence.The expression of major histocompatibility complex I(MHCII)and co-stimulatory molecule CD40 on the cell surface was detected by flow cytometry.The contents of multiple cytokines in cell culture supernatants were determined by enzyme-linked immunosorbent assay.Transcriptome sequencing(RNA-seq)was used to screen the downstream related genes regulated by HBx,and real-time quantitative PCR was used to verify their expressions.After overexpression or silencing of Notch1 gene with overexpressed plasmids or short hairpin RNA(shRNA)in podocytes,the effect on the expression of immune molecules and cytokines secretion was observed.The Notch receptor inhibitor N-[N-(3,5-difluorophenyll-alanyl)]-(s)-phenylglycine tert-butyl ester(DAPT)was used to block Notch1 signaling pathway in HBV-Tg mice,and then blood biochemistry,renal pathological changes and infiltration of immune cells in kidney tissue were observed.Results Twenty-four-hour urine protein,serum creatinine and urea nitrogen levels were markedly increased(all P<0.05)and renal pathological injury was significantly aggravated in HBV-Tg mice than those in WT mice.Also,HBx was up-regulated and immune cells infiltrated in the glomerulus of HBV-Tg mice.After transfection with HBx in podocytes,the expression of MHC-I and CD40 on the cellular surface was upregulated(all P<0.05),the contents of monocyte chemotactic protein-1(MCP-1),tumor necrosis factor-α(TNF-α)and interleukin(IL)-1βin the supernatants were increased(all P<0.05),and the secretion of IL-4 and interferonγ(IFN-γ)was unbalanced.RNA-seq screened downstream genes of HBx,such as Notch1,PLA2R,TLR4,etc.,and further confirmed that HBx could promote the up-regulation of Notch1 mRNA and protein(all P<0.05).After over-expression of Notch1 gene,HBx-induced expression of MHC-II and CD40 on the cellular surface was significantly up-regulated(all P<0.05),and the contents of MCP-1,TNF-αand IL-1βin the supernatants were obviously increased(all P<0.05),and the imbalance of IL-4/IFN-γwas further aggravated.After Notch1 gene silencing,the above results showed the opposite changes.In vivo,the results indicated that serum creatinine levels were obviously decreased(all P<0.05),renal pathological injury and immune cell infiltration were significantly alleviated in HBV-Tg+DAPT group than those in HBV-Tg+DMSO group.Conclusion HBx protein can promote the upregulation of Notch1 signaling pathway in podocytes.And Notch1 signaling pathway promotes the expression of immune molecules on the surface of podocytessand regulates the imbalance of cytokines,then causes glomerular injury and dysfunction of immune microenvironment.
