1,25-Dihydroxyvitamin D3 Prevents Epithelial-Mesenchymal Transition of HMrSV5 Human Peritoneal Mesothelial Cells by Inhibiting Histone Deacetylase 3 (HDAC3) and Increasing Vitamin D Receptor (VDR) Expression Through the Wnt/ß-Catenin Signaling Pathway.

BACKGROUND Peritoneal dialysis is the most common treatment for end-stage renal disease. However, peritoneal fibrosis resulting from long-term peritoneal dialysis restricts peritoneal ultrafiltration. Previous studies have shown a role for 1,25-dihydroxyvitamin D3 (1,25[OH]2D3) in preventing fibrosis, but the potential mechanisms remain unknown. This study aimed to investigate the role of 1,25(OH)2D3 in epithelial-mesenchymal transition (EMT) and the downstream signaling pathway in HMrSV5 human peritoneal mesothelial cells in vitro. MATERIAL AND METHODS An in vitro cell model of peritoneal fibrosis was established using the HMrSV5 human peritoneal mesothelial cell line. High glucose and lipopolysaccharide (LPS) culture conditions, with or without 1,25(OH)2D3, were used. Wnt agonist 1, a Wnt signaling pathway activator, was applied. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to measure the vitamin D receptor (VDR) and histone deacetylase 3 (HDAC3) gene and protein expression levels, ß-catenin, and EMT-associated biomarkers. RESULTS High glucose plus LPS culture medium inhibited cell proliferation, induced cell apoptosis and promoted EMT in HMrSV5 cells, which was reversed by 1,25(OH)2D3 by down-regulation of HDAC3 and upregulation of VDR. HDAC3 inhibited VDR gene expression. The expression of EMT-associated biomarkers was increased by Wnt agonist 1 and inhibited by 1,25(OH)2D3. CONCLUSIONS In HMrSV5 human peritoneal mesothelial cells, 1,25(OH)2D3 reversed EMT by inhibiting the expression of HDAC3 and upregulating VDR gene expression via the Wnt/ß-catenin signaling pathway.

Spleen Tyrosine Kinase (SYK) in the Progression of Peritoneal Fibrosis Through Activation of the TGF-ß1/Smad3 Signaling Pathway.

BACKGROUND Long-term exposure to hypertonic and high glucose in peritoneal dialysis fluid can result in peritoneal fibrosis. Spleen tyrosine kinase (SYK) has a role in inflammation and fibrosis. This study aimed to investigate the role of SYK in an in vivo rat model of peritoneal fibrosis and in rat peritoneal mesothelial cells (PMCs) in vitro and to investigate the underlying mechanisms. MATERIAL AND METHODS Sprague-Dawley rats (N=24) were randomized into the sham control group (N=6); the peritoneal fibrosis group (N=6) treated with intraperitoneal chlorhexidine digluconate; the SYK inhibitor group (N=6), treated with chlorhexidine digluconate and fostamatinib; and the TGF-ß inhibitor group (N=6), treated with chlorhexidine digluconate and LY2109761. The rat model underwent daily intraperitoneal injection with 0.5 ml of 0.1% chlorhexidine digluconate. Rat peritoneal mesothelial cells (PMCs) were cultured in vitro in high glucose. SYK expression was measured by quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and Western blot. Enzyme-linked immunosorbent assay (ELISA) and qRT-PCR measured inflammatory mediators. Transforming growth factor-ß1 (TGF-ß1) and Smad3 were detected by Western blot. Short hairpin RNA (shRNA) was used to target the SYK gene. RESULTS SYK was upregulated in the rat model of peritoneal fibrosis and was induced rat PMCs cultured in high glucose. Knockdown of SYK and inhibition of TGF-ß1 significantly reduced fibrosis and inflammation. Findings in the in vivo rat model confirmed that SYK mediated peritoneal fibrosis by regulating TGF-ß1/Smad3 signaling. CONCLUSIONS In a rat model and in rat PMCs, expression of SYK increased peritoneal fibrosis through activation of the TGF-ß1/Smad3 signaling pathway.

Heparin-free continuous vena-venous hemofiltration as a veno-venous bypass in inferior vena cava reconstruction.

OBJECTIVE: This paper investigated the effects of continuous vena-venous hemofiltration on inferior vena cava reconstruction. METHOD: Totally, 11 patients were observed, vascular access in right internal jugular vein and femoral vein catheterization was established guided by ultrasound, and heparin-free continuous vena-venous hemofiltration was used to substitute for extracorporeal veno-venous bypass. Furthermore, blood pressure, central venous pressure, urine volume, blood platelet, serum albumin, renal function, serum cystatin C, CRP, TBil, AST, ALT, serum amylase, serum lipase, PLT, PT, APTT, Fig, D-mier, and adverse events were determined. RESULTS: All operations were completed successfully. Average time of continuous vena-venous hemofiltration was 2.96 ± 0.76 h. No hematoma and blood leakage was occurred when catheters were inserted, and no luminal stenosis and catheter-related infections were observed. Visceral congestion was observed when the inferior vena cava was clamped, but significantly improved immediately after the continuous vena-venous hemofiltration was begun. No hemofilter was changed due to clotting during continuous vena-venous hemofiltration therapy. Blood pressure, central venous pressure, and urine volume of the patients maintained stable. No significant change was observed in blood platelet, serum albumin, and serum creatinin. Serum cystatin and hsCRP increased after operation, but still in normal level. CONCLUSION: Heparin-free continuous vena-venous hemofiltration was an effective mode as veno-venous bypass in the treatment of inferior vena cava interruption and reconstruction.

Neferine Targeted the NLRC5/NLRP3 Pathway to Inhibit M1-type Polarization and Pyroptosis of Macrophages to Improve Hyperuricemic Nephropathy.

BACKGROUND: Neferine (Nef) has a renal protective effect. This research intended to explore the impact of Nef on hyperuricemic nephropathy (HN). METHODS: Adenine and potassium oxonate were administered to SD rats to induce the HN model. Bone marrow macrophages (BMDM) and NRK-52E were used to construct a transwell co-culture system. The polarization of BMDM and apoptosis levels were detected using immunofluorescence and flow cytometry. Renal pathological changes were detected using hematoxylin-eosin (HE) and Masson staining. Biochemical methods were adopted to detect serum in rats. CCK-8 and EDU staining were used to assess cell activity and proliferation. RT-qPCR and western blot were adopted to detect NLRC5, NLRP3, pyroptosis, proliferation, and apoptosis-related factor levels. RESULTS: After Nef treatment, renal injury and fibrosis in HN rats were inhibited, and UA concentration, urinary protein, BUN, and CRE levels were decreased. After Nef intervention, M1 markers, pyroptosis-related factors, and NLRC5 levels in BMDM stimulated with uric acid (UA) treatment were decreased. Meanwhile, the proliferation level of NRK-52E cells co-cultured with UA-treated BMDM was increased, but the apoptosis level was decreased. After NLRC5 overexpression, Nef-induced regulation was reversed, accompanied by increased NLRP3 levels. After NLRP3 was knocked down, the levels of M1-type markers and pyroptosis-related factors were reduced in BMDM. CONCLUSION: Nef improved HN by inhibiting macrophages polarized to M1-type and pyroptosis by targeting the NLRC5/NLRP3 pathway. This research provides a scientific theoretical basis for the treatment of HN.

Effects of Purine Metabolism-Related LINC01671 on Tumor Heterogeneity in Kidney Renal Clear Cell Carcinoma.

BACKGROUND: Renal cell carcinoma has several subtypes, with kidney renal clear cell carcinoma (KIRC) being the most common and heterogeneous. Purine metabolism is associated with cancer progression. However, the role of purine metabolism-related long non-coding RNAs (lncRNAs) in KIRC remains unknown. METHODS: KIRC were grouped into Cluster-1 and Cluster-2 based on purine genes. Limma package was used to identify differentially expressed lncRNAs between two classes of purine genes. Single-factor screening was used followed by random forest dimensionality reduction and Lasso method to screen lncRNAs. A risk score model (Purine Score) containing the 3 lncRNAs was developed using the Lasso method. RESULTS: A total of 22 differentially expressed lncRNAs were identified. These were reduced to a final set of three (LINC01671, ARAP1-AS1 and LINC02747). Age and metastasis (M) were identified as independent prognostic factors for KIRC using univariate and multivariate Cox analysis. An abnormal immune cell response was also associated with patient survival. The Purine Score correlated with abnormal expression of immune checkpoint genes. Genetic analysis of KIRC found somatic mutations in TP53, TRIOBP, PBRM1, PKHD1, VHL, NPHP3, TLN2, CABIN1, ABCC6, XIRP2, and CHD4. In vitro cell experiments showed that knockdown of LINC01671 promoted the proliferation and migration of 786-O cells, while inhibiting apoptosis. Overexpression of LINC01671 inhibited the proliferation and migration of CAKI-1 cells, while promoting apoptosis. Gene Set Enrichment Analysis (GSEA) analysis revealed that LINC01671 was significantly enriched in the MAPK, NF-kappa B, mTOR, PI3K-Akt, and Wnt signaling pathways. CONCLUSIONS: LINC01671 may be a novel prognostic marker with important therapeutic value for KIRC.

miR-128-3p inhibits high-glucose-induced peritoneal mesothelial cells fibrosis via PAK2/SyK/TGF-ß1 axis.

AIM: To elucidate the mechanism of miR-128-3p in peritoneal fibrosis (PF). METHODS: Peritoneal mesothelial cells (PMCs) were dealt with high glucose (HG) for 3 days. The expressions of miR-128-3p, p21-activated kinase 2 (PAK2), spleen tyrosine kinase (SyK), and transforming growth factor-ß1 (TGF-ß1) were detected with quantitative real-time reverse transcription polymerase chain reaction. The levels of IL-1ß, TNF-α, IL-6, and monocyte chemotactic protein-1 in supernatant were measured by ELISA. Proteins of TGF-ß1, SyK, PAK2, α-SMA, collagen I, vimentin, ERK/AP-1, and IκBα/NF-κB pathway related proteins were measured by Western blot. The correlation between miR-128-3p and PAK2 was found by bioinformatics analysis and luciferase reporter gene analysis. RESULTS: miR-128-3p was decreased while PAK2, SyK, and TGF-ß1 were increased in HG-induced PMCs. Moreover, miR-128-3p inhibited HG-induced fibrosis and inflammation in PMCs by targeting PAK2. PAK2 activated SyK, which induced TGF-ß1 expression through ERK/AP-1 and IκBα/NF-κB pathways to promote HG-induced fibrosis of PMCs. CONCLUSION: miR-128-3p inhibited HG-induced PMCs fibrosis via PAK2/SyK/TGF-ß1 axis.

[Effect of spironolactone on the expression of Toll-like receptor 4 in renal tubular epithelia cells exposed to high glucose].

OBJECTIVE: To study the expression of Toll-like receptor 4 (TLR4) in renal tubular epithelial cells exposed to high glucose and the effect of spironolactone on the TLR4 expression. METHODS: In vitro renal tubular epithelial cells (NRK-52E) were randomly exposed to DMEM culture solution with low glucose (5 mmol /L), high glucose (25 mmol/L) or 10(-7) mol/L spironolactone plus 25 mmol/L glucose. Immunohistochemistry, RT-PCR and Western blot were used to determine TLR4 protein and mRNA expression. The levels of IL-6 and TNF-alpha in the cell culture supernatant were determined using ELISA. RESULTS: The expression of TLR4 mRNA in the high glucose group began to increase 6 hrs and remained at a higher level up to 24 hrs after exposure as compared with the low glucose group. The TLR4 mRNA expression in the spironolactone treatment group was significantly lower than that in the high glucose group, although it was higher than that in the low glucose group between 6 and 24 hrs after exposure. TLR4 protein expression increased significantly in the high glucose group 24 and 48 hrs after exposure compared with that in the low glucose group. The TLR4 protein expression in the spironolactone treatment group was lower than that in the high glucose group, but higher than that in the low glucose group. IL-6 and TNF-alpha expression in the supernatant from the NRK-52E cells in the high glucose groups increased significantly as compared with the low glucose group. The spironolactone treatment group had significantly reduced IL-6 and TNF-alpha expression compared with the high glucose group. CONCLUSIONS: High glucose triggers an increase in the expression of TLR4 and inflammatory factors in NRK-52E cells. TLR4 may participate in the progress of diabetic nephropathy. Spironolactone can reduce expression of TLR4 and inflammatory factors, which might be attributed to one of the mechanisms of protection by spironolactone against diabetic nephropathy.