Objective:To analyze the target signaling pathway of histone H3K27 methylation-induced podocyte injury, verify the regulatory effect of histone H3K27 methylation on podocyte injury in focal segmental glomerulosclerosis (FSGS) mice through target signaling pathway, and explore the mechanism of abnormal methylation of histone H3K27-induced podocyte injury in FSGS mice.Methods:(1) Cell experiments: primary cultured immortalized mouse podocytes MPC5 were cultured in vitro, and divided into control group, adriamycin (ADR) group, ADR+GSK-J4 (histone demethylase, KDM6B inhibitor) group, ADR+coumarin A1 (C-A1, JAK2 agonist) group and ADR+GSK-J4+C-A1 group. The transmission electron microscope was used to observe ultrastructure of podocytes. Immunofluorescence was used to detect the protein expression of H3K27me3 and nephrin in podocytes. The whole genome sequence of podocytes was obtained, the differentially expressed genes were screened, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for enrichment analysis. Real time-quantitative PCR and Western blotting were used to detect the gene and protein expression of JAK2-STAT3 signaling pathway in podocytes respectively. Enzyme-linked immunosorbent assay was used to detect interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), α-smooth muscle actin (α-SMA) and transforming growth factor β1 (TGF-β1). (2) Animal experiments: EZH2 gene knock out ( EZH2podKo)-FSGS (tail vein injection of ADR) mouse models were established, and divided into EZH2ctrl+control group ( n=20), EZH2ctrl+FSGS ( n=20), EZH2podKo+control group ( n=30) and EZH2podKo+FSGS group ( n=30). HE staining was used to observe the morphology of kidney tissues. Immunohistochemistry was used to detect the H3K27me3 protein expression in podocytes. Real time-quantitative PCR and Western blotting were used to verify the gene and protein expression of JAK2-STAT3 signaling pathway respectively. Enzyme-linked immunosorbent assay was used to detect IL-6, MCP-1, α-SMA and TGF-β1 of kidney tissues. Results:(1) Cell experiments: Compared with control group, the nucleus shrank and ruptured, the cytoplasm showed vacuole, and mitochondria and endoplasmic reticulum swelled in ADR group, which verified that the podocyte injury model of ADR nephropathy was successfully established. Compared with control group, the protein expression level of H3K27me3 in ADR group was significantly lower ( P<0.05). Compared with ADR group, the protein expression level of H3K27me3 in podocytes in ADR+GSK-J4 group was significantly higher ( P<0.05), and there were 502 increased genes and 443 decreased genes. GO enrichment analysis showed that the differentially enriched peaks were mainly in ribonucleoprotein complex biogenesis, ribosome biogenesis, establishment of protein localization to organelle, and involved in regulation of receptor signaling pathway via JAK-STAT and receptor signaling pathway via JAK-STAT. Differential expressed genes were Irf1, Tnfrsf1a, S ocs1, Notch1, Gadd45a, Hes1 and Socs3, involving in the regulation of JAK-STAT signaling pathway. KEGG enrichment analysis showed that the differentially enriched peaks were mainly in amyotrophic lateral sclerosis, and the target genes were Mcl1, Egfr, Socs1, Cdkn1a, Pdgfa and Socs3, involving in the regulation of JAK-STAT signaling pathway. Compared with ADR group, the mRNA and protein expression levels of JAK2 and STAT3 in the ADR+GSK-J4 group were significantly lower, and the downstream inflammatory factors of IL-6, MCP-1 and α-SMA were significantly higher (all P<0.05). Compared with ADR group, the protein expression level of nephrin in ADR+GSK-J4 group was higher ( P<0.05), and the protein expression level of nephrin in ADR+C-A1 group was lower ( P<0.05). Compared with ADR+GSK-J4 group, the protein expression level of nephrin in ADR+GSK-J4+C-A1 group was lower ( P<0.05). (2) Animal experiments: Compared with EZH2ctrl+FSGS group, EZH2podKo+FSGS group showed obvious renal tissue damage, matrix hyperplasia in mesangial area with massive homogeneous substance deposition, apoptosis and necrosis of renal tubular epithelial cells, obvious thickening and extensive fusion of glomerular epithelial cells, basement membrane collapse, and compression and narrowing of capillary structure. Compared with EZH2ctrl+FSGS group, the protein expression level of H3K27me3 in EZH2podKo+FSGS group was significantly lower, and the mRNA and protein expression levels of JAK2 and STAT3, and the levels of IL-6, MCP-1, α-SMA and TGF-β1 were higher (all P<0.05). Conclusions:Abnormal methylation modification of H3K27 leads to change of target gene expression, activation of JAK2-STAT3 signaling pathway, podocyte injury, glomerulosclerosis and renal tubular injury, participating in the development of FSGS.

Objective:To analyze the target signaling pathway of histone H3K27 methylation-induced podocyte injury, verify the regulatory effect of histone H3K27 methylation on podocyte injury in focal segmental glomerulosclerosis (FSGS) mice through target signaling pathway, and explore the mechanism of abnormal methylation of histone H3K27-induced podocyte injury in FSGS mice.Methods:(1) Cell experiments: primary cultured immortalized mouse podocytes MPC5 were cultured in vitro, and divided into control group, adriamycin (ADR) group, ADR+GSK-J4 (histone demethylase, KDM6B inhibitor) group, ADR+coumarin A1 (C-A1, JAK2 agonist) group and ADR+GSK-J4+C-A1 group. The transmission electron microscope was used to observe ultrastructure of podocytes. Immunofluorescence was used to detect the protein expression of H3K27me3 and nephrin in podocytes. The whole genome sequence of podocytes was obtained, the differentially expressed genes were screened, and Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were used for enrichment analysis. Real time-quantitative PCR and Western blotting were used to detect the gene and protein expression of JAK2-STAT3 signaling pathway in podocytes respectively. Enzyme-linked immunosorbent assay was used to detect interleukin 6 (IL-6), monocyte chemotactic protein 1 (MCP-1), α-smooth muscle actin (α-SMA) and transforming growth factor β1 (TGF-β1). (2) Animal experiments: EZH2 gene knock out ( EZH2podKo)-FSGS (tail vein injection of ADR) mouse models were established, and divided into EZH2ctrl+control group ( n=20), EZH2ctrl+FSGS ( n=20), EZH2podKo+control group ( n=30) and EZH2podKo+FSGS group ( n=30). HE staining was used to observe the morphology of kidney tissues. Immunohistochemistry was used to detect the H3K27me3 protein expression in podocytes. Real time-quantitative PCR and Western blotting were used to verify the gene and protein expression of JAK2-STAT3 signaling pathway respectively. Enzyme-linked immunosorbent assay was used to detect IL-6, MCP-1, α-SMA and TGF-β1 of kidney tissues. Results:(1) Cell experiments: Compared with control group, the nucleus shrank and ruptured, the cytoplasm showed vacuole, and mitochondria and endoplasmic reticulum swelled in ADR group, which verified that the podocyte injury model of ADR nephropathy was successfully established. Compared with control group, the protein expression level of H3K27me3 in ADR group was significantly lower ( P<0.05). Compared with ADR group, the protein expression level of H3K27me3 in podocytes in ADR+GSK-J4 group was significantly higher ( P<0.05), and there were 502 increased genes and 443 decreased genes. GO enrichment analysis showed that the differentially enriched peaks were mainly in ribonucleoprotein complex biogenesis, ribosome biogenesis, establishment of protein localization to organelle, and involved in regulation of receptor signaling pathway via JAK-STAT and receptor signaling pathway via JAK-STAT. Differential expressed genes were Irf1, Tnfrsf1a, S ocs1, Notch1, Gadd45a, Hes1 and Socs3, involving in the regulation of JAK-STAT signaling pathway. KEGG enrichment analysis showed that the differentially enriched peaks were mainly in amyotrophic lateral sclerosis, and the target genes were Mcl1, Egfr, Socs1, Cdkn1a, Pdgfa and Socs3, involving in the regulation of JAK-STAT signaling pathway. Compared with ADR group, the mRNA and protein expression levels of JAK2 and STAT3 in the ADR+GSK-J4 group were significantly lower, and the downstream inflammatory factors of IL-6, MCP-1 and α-SMA were significantly higher (all P<0.05). Compared with ADR group, the protein expression level of nephrin in ADR+GSK-J4 group was higher ( P<0.05), and the protein expression level of nephrin in ADR+C-A1 group was lower ( P<0.05). Compared with ADR+GSK-J4 group, the protein expression level of nephrin in ADR+GSK-J4+C-A1 group was lower ( P<0.05). (2) Animal experiments: Compared with EZH2ctrl+FSGS group, EZH2podKo+FSGS group showed obvious renal tissue damage, matrix hyperplasia in mesangial area with massive homogeneous substance deposition, apoptosis and necrosis of renal tubular epithelial cells, obvious thickening and extensive fusion of glomerular epithelial cells, basement membrane collapse, and compression and narrowing of capillary structure. Compared with EZH2ctrl+FSGS group, the protein expression level of H3K27me3 in EZH2podKo+FSGS group was significantly lower, and the mRNA and protein expression levels of JAK2 and STAT3, and the levels of IL-6, MCP-1, α-SMA and TGF-β1 were higher (all P<0.05). Conclusions:Abnormal methylation modification of H3K27 leads to change of target gene expression, activation of JAK2-STAT3 signaling pathway, podocyte injury, glomerulosclerosis and renal tubular injury, participating in the development of FSGS.

This study aims to establish a rat model of renal ischemia reperfusion injury (RIRI) to observe the alterations in the expression of phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway following various exosome treatments. Additionally, differential miRNA expression analysis will be conducted to elucidate the molecular mechanisms underlying the effects of exosomes derived from ischemic preconditioned (IPC) renal tubular cells in mitigating RIRI in rats. Initially, ten SD rats were subjected to bilateral nephrectomy under general anesthesia to prepare primary renal tubular cells. The second-generation renal tubular cells were then subjected to the following treatments for 12 hours: normoxia (38% O 2, 5% CO 2), hypoxia (1% O 2, 5% CO 2), and hypoxia plus inactivation (heated at 65 ℃ for 30 minutes). Following these treatments, exosomes were extracted, yielding normoxic exosomes, IPC exosomes, and inactivated exosomes, respectively. A subsequent cohort of 50 SD rats was randomly divided into five groups: Sham group, RIRI group, RIRI + normoxic exosome group (NC group), RIRI + IPC exosome group (IPC group), and RIRI + inactivated exosome group (INA group). RIRI model was established in the latter four groups. Twenty-four hours after RIRI modeling, the NC, IPC, and INA groups received intravenous injections of 200 μg of normoxic exosomes, IPC exosomes, and inactivated exosomes via the tail vein, respectively. Six days later, venous blood samples were collected, and both kidneys were excised to observe renal function, histopathological changes in kidney tissue, and alterations in the PI3K/AKT/mTOR signaling pathway among the five groups. Furthermore, differential miRNA expression analysis [ P<0.05, |log 2(Fold Change)|≥1] was conducted between the NC and IPC groups to investigate the changes in the miRNA expression profile. Subsequently, GO analysis and KEGG pathway enrichment analysis were performed. The results revealed that: (1) Compared with the Sham group, the RIRI and INA groups exhibited elevated levels of serum creatinine and urea nitrogen (all P<0.01). Histopathological examination of kidney tissues showed substantial inflammatory cell infiltration in the interstitium accompanied by varying degrees of edema, degenerative swelling of tubular structures, necrosis, and detachment of tubular epithelial cells. Notably, the number of TUNEL-positive cells was significantly increased, while the number of Ki67-stained positive cells was markedly decreased. Additionally, the mRNA and protein expression of PI3K/AKT/mTOR signaling pathway in RIRI group and INA group were down-regulated. (2) Compared to the NC group, the IPC group demonstrated lower levels of serum creatinine and urea nitrogen (both P<0.01). Notably, there was a significant decrease in the accumulation of inflammatory cells in the renal interstitium, and tissue edema was markedly improved. Moreover, the number of TUNEL-positive cells was reduced, while the number of Ki67-stained positive cells was significantly increased. Additionally, the mRNA and protein expressions of PI3K, PDK1, AKT, and mTOR were all up-regulated (all P<0.05). (3) Compared to the NC group, 56 miRNAs were up-regulated and 42 miRNAs were down-regulated in the IPC group. The target genes of GO enrichment analysis were PIK3C2A, PIK3CA, PIK3CB, PIK3CD, PIK3C2G, AKT1, mTOR, Rheb, and KEGG enrichment analysis revealed significant enrichment in PI3K/AKT signal pathway and mTOR signal pathway. In conclusion, this study reveals that during the course of RIRI, exosomes derived from IPC renal tubular cells induce differential miRNA expression in kidney tissues, resulting in enhanced expression of the PI3K/AKT/mTOR signaling pathway, which plays a pivotal role in mitigating RIRI in rats.

The stenosis and embolization of internal fistula vessels directly affect the clinical treatment effect of maintenance hemodialysis patients, and the study of the mechanism of internal fistula stenosis has become a research hotspot in recent years. Previous studies mainly focused on the hemodynamics and pathophysiology of blood vessel wall, and there were few studies on molecular biology and its related signaling pathways. This paper reviews the hemodynamics of the vascular pathway of internal arteriovenous fistula (AVF), the pathophysiological mechanism, molecular biology, and changes in various signaling pathways of AVF dysfunction at home and abroad, in order to provide references for the study of AVF dysfunction.

Objective:Clamping bilateral renal arteries with refined surgical methods to establish the rat renal ischemia-reperfusion injury (RIRI) model, and study the protective mechanism of ischemic preconditioning renal (IPC) tubular cell-derived exosomes in RIRI.Methods:25 female Sprague Dawley (SD) rats were divided into sham group, model group, inactivated group, normoxic group, IPC group. In the sham operation group, after bilateral renal arteries were dissociated, the back incision was disinfected and closed. The model group established RIRI model; RIRI models were established in inactivated group, normoxia group and IPC group, and then 200 μg of inactivated exosomes, normal exosomes and IPC exosomes were injected into the caudal vein 24 hours after operation. Serum creatinine (Scr) and urea nitrogen (BUN) levels were detected. The pathological changes of renal tissue were observed under light microscope. Transmission electron microscopy (TEM) was used to observe the shape and size of renal tubular exosomes. Nanoparticle tracking analysis (NTA)was used to detect the concentration and size of renal tubular exosomes.Results:Compared with the sham group, the Scr and BUN levels in the model group were significantly elevated ( P<0.01). Renal pathological changes in the model group showed damaged of the tubular structure, necrosis and shedding of tubular epithelial cells, and a large number of inflammatory cells accumulated in the renal interstitial tissue with varying degrees of edema. Compared with the inactivated group, the Scr and BUN levels significantly decreased in the normoxic group and IPC group ( P<0.01). Renal pathological changes in the normoxic group and IPC group showed that the renal tubular cell necrosis alleviated, inflammatory was reduced, the improved edema. Compared with the normoxic group, the Scr and BUN levels in the IPC group were further reduced ( P<0.01). Renal pathological changes in the IPC group showed that the inflammatory cells were significantly reduced, the cell edema was significantly improved, and the cell apoptosis was significantly reduced. Conclusions:Clamping bilateral renal arteries with refined surgical methods is the main and optimal way to build a rat model of RIRI. IPC tubular cell-derived exosomes have protective and repair effects on RIRI.

Objective:To establish a mouse model of intra-jugular arteriovenous fistula (AVF) to screen differentially expressed genes in the process of intimal stenosis of AVF for investigating the abnormal expression signaling pathways and the mechanisms.Methods:Forty-six male C57BL/6 mice were randomly divided into AVF group ( n=23) and sham-operated group ( n=23). The AVF group underwent internal jugular arteriovenous fistuloplasty, and the sham-operated group separated the right external jugular vein and common carotid artery and then sutured the incision. The whole-genome sequences of mice with AVF stenosis were determined by transcriptomic reversible chain terminator and synthetic sequencing. The microarray data set was established, and the Benjamini & Hochberg method of gene microarray data analysis was applied to screen the differentially expressed genes. The differentially expressed genes were screened by R-language enrichment analysis. Then, gene ontology (GO) and Kyoto encyclopedia of genes and genomes (KEGG) were performed. The subcellular localization of the differentially expressed genes was performed by BUSCA software. The protein network interaction of differentially expressed genes was analyzed by using STRING database and Cytoscape software. Results:In the AVF group, 21 mice were successfully modeled and 2 mice failed. Therefore, there were 21 mice in the AVF group and only 21 mice in the sham-operated group. This mouse internal jugular AVF model was innovated using the continuous-interrupted suture method, which improved the success rate of modeling this model. The differential gene sequencing analysis showed that there were 2 514 differentially expressed genes in the AVF process, including 1 323 up-regulated genes and 1 191 down-regulated genes. GO functional enrichment analysis showed that the differential genes were mainly enriched in metabolic process, activation, redox, mitochondria and so on. KEGG pathway enrichment analysis showed that the differential genes were enriched in metabolism, energy substance synthesis, diabetes, oxidative stress and so on. Statistical analysis of subcellular localization showed that the differences were mainly in mitochondrial proteins (24.24%), cytoplasmic proteins (17.51%), nuclear proteins (13.13%), cell membrane proteins (11.45%), and extracellular proteins (10.77%).Conclusions:Mitochondrial oxidative stress injury may be involved in the pathological damage process of endothelial proliferation stenosis in the AVF.

Objective:To investigate the effects and underlying mechanisms of phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT)/NF-κB signaling pathway in human kidney-2(HK-2) cells of hyperuricemic nephropathy.Methods:HK-2 cells were cultured in vitro and randomly divided into control group and experimental group. The experimental group was induced by high uric acid (720 μmol/L) immersion for 48 h to establish a cell model of hyperuricemic nephropathy in vitro and subsequently divided into hyperuricemic group, overexpressed protease activated receptor 2 (PAR2) and knockdown PAR2 group. The expressions of PAR2, PI3K, AKT, NF-κB mRNA were measured by real-time PCR. The expressions of PAR2, PI3K, AKT and NF-κB protein were measured by Western blotting. The expressions of tumor necrosis factor-α (TNF-α), monocyte chemotactic protein-1 (MCP-1), interleukin-6 (IL-6), pro-interleukin-1β (pro-IL-1β), interleukin-1β (IL-1β) and transforming growth factor-β1 (TGF-β1) were detected by enzyme linked immunosorbent assay (ELISA). Results:(1) Compared with the control group, the expressions of PAR2, PI3K, AKT and NF-κB mRNA and protein in hyperuricemic group were significantly increased (all P<0.05), the expressions of TNF-α, MCP-1, IL-6, pro-IL-1β, IL-1β and TGF-β1 in the supernatant in hyperuricemic group were significantly increased (all P<0.01). (2) Compared with the hyperuricemic group, the expressions of PAR2, PI3K, AKT and NF-κB mRNA and protein in overexpressed PAR2 group were significantly increased (all P<0.05), the expressions of TNF-α, MCP-1, IL-6, IL-1β and TGF-β1 in the supernatant were significantly increased (all P<0.05). (3) Compared with the hyperuricemic group, the expression of PAR2, PI3K, AKT and NF-κB mRNA and protein in knockdown PAR2 group were significantly decreased (all P<0.05), the expressions of IL-6, pro-IL-1β, IL-1β and TGF-β1 in the supernatant were significantly decreased (all P<0.05). Conclusions:In the process of uric acid-induced HK-2 cell damage, uric acid significantly up-regulates the expression of PI3K/AKT/NF-κB signaling pathway by activating PAR2, leading to a marked increase in inflammatory damage. Knocking down PAR2 inhibits the expression of PI3K/AKT/NF-κB signaling pathway, which can effectively reduce the inflammatory damage of HK-2 cells.

Objective To observe the level of CD4+CD25+ regulatory T cells (CD4+CD25+ Treg cells) with positive fork head transcription factor 3 (Foxp3) and changes of T-box transcription factor TBX1 (TBX1) and myocyte specific enhancer 2D (MEF2D) expression in peripheral blood of rats with acute rejection after renal transplantation,and to investigate its regulatory mechanisms by combined with renal function,plasma interleukin-10 (IL-10),interferon-γ (IFN-γ) and renal histopathological changes.Methods Rat renal transplantation model was established and divided into two groups:acute rejection group (AR group) and non-acute rejection group (non-AR group).Their renal function including serum creatinine (Scr) and blood urea nitrogen (BUN) in plasma was measured.The renal histopathology was observed by HE staining.Levels of IL-10 and IFN-γ in plasma were detected by ELISA.The proportion of CD4+CD25+ Treg cells was measured by flow cytometry.The mRNA expressions of Foxp3,TBX1 and MEF2D in CD4+CD4+Treg cells were detected by real-time PCR,and their protein expressions were tested by Western blotting.Results Compared with these in the non-AR group,the levels of BUN,Scr and IFN-γ significantly increased in AR group (all P ＜ 0.05),while IL-10 decreased (P ＜ 0.05).Renal histopathology in the acute rejection group showed glomerular hypertrophy and mesangial cell proliferation,capillary proliferation and neutrophil infiltration;renal interstitial edema and tubular necrosis,accompanied by lymphocytes,plasma cells and neutrophils infiltration.Compared with that in the non-AR group,the percentage of CD4+CD25+ Treg cells in peripheral blood was notably lowered in AR group (4.50％±0.50％ vs 5.74％±1.96％,P ＜ 0.05).The mRNA and protein expressions of Foxp3 and MEF2D were lower in AR group than those in non-AR group,while the expressions of TBX1 was elevated (all P ＜ 0.05).Conclusions In rats with acute renal allograft rejection,the percentage of CD4+CD25+ Treg cells and expressions of Foxp3,MEF2D and IL-10 decrease,while the expressions of TBX1 and IFN-γ enhance.These participate in the development of acute rejection after renal transplantation,and aggravate the renal damage.