Preliminary study on 24p3 / neutrophil gelatinase-associated lipocalin (NGAL) ferroptosis inhibition in renal tubular epithelial cells.

The 24p3/neutrophil gelatinase-associated lipocalin (NGAL) protein plays an important protective role in acute kidney injury (AKI), but the exact mechanism remains unclear. Therefore, we have made a preliminary exploration of its mechanism. The experimental group was formed by constructing and transfecting 24P3 overexpressed plasmid into renal tubular epithelial cells. Western Bolt was used to detect NGAL expression. Cell proliferation was detected by CCK8 kit, cell death was detected by Hoechst 33342 and PI kit, mitochondrial morphology was observed under light microscope, reactive oxygen species (ROS) content was detected by fluorescence probe, and iron level and glutathione peroxidase 4 (GPX4) activity were detected by kit. Furthermore, the mechanism of NGAL action was further demonstrated by adding ferrostein-1 (Fer-1), an ferroptosis inhibitor, and erastin (containing DMSO),an ferroptosis inductor. We found that ferroptosis-related indicators were lower in the NGAL overexpression group than in the control group. At the same time, we found that NGAL alleviated ferroptosis induced by erastin and coordinated with Fer-1 to alleviate ferroptosis. In conclusion, NGAL inhibits ferroptosis in renal tubular epithelial cells, which may be associated with the progression of AKI and may provide a new therapeutic target for the transition from acute kidney injury to chronic kidney injury.

CKD autophagy activation and skeletal muscle atrophy-a preliminary study of mitophagy and inflammation.

BACKGROUND/OBJECTIVES: Long-lived proteins and organelles, such as mitochondria and the sarcoplasmic reticulum, are degraded by autophagy. However, the specific role of autophagy in chronic kidney disease (CKD) muscle atrophy is still undefined. SUBJECTS/METHODS: This was a cross-sectional study with 20 subjects and 11 controls. Autophagy induction was studied in human skeletal muscle biopsies from CKD patients and controls by comparing the cross-sectional areas of muscle fibers, protein, and mRNA expression of autophagy-related genes and the appearance of autophagosomes. RESULTS: The cross-sectional area of muscle fibers was decreased in CKD patients as compared with the control group. CKD was associated with activated autophagy and mitophagy, as measured by the elevated mRNA and protein expression of BNIP3, (microtubule-associated proteins 1 A/1B light chain 3, also MAP1LC3) LC3, p62, PINK1, and PARKIN in the skeletal muscle and isolated mitochondria of the CKD group. Electron microscopy and immunohistofluorescence analysis showed mitochondrial engulfment by autophagosomes. Mitophagy was further demonstrated by the colocalization of LC3 and p62 puncta with the mitochondrial outer membrane protein TOM20. In addition, degradative FOXO3 (Forkhead box O3) was activated and synthetic mTOR (mammalian target of rapamycin) was inhibited, whereas the upstream mediators VPS34 (class III PI3-kinase) and AKT (protein kinase B, PKB) were activated in CKD patients. CONCLUSIONS: Hyperactive autophagy and mitophagy may play important roles in CKD muscle atrophy. Autophagy was activated by FOXO3 translational factors in the skeletal muscle tissues of CKD patients, which maybe a new way of intervention for CKD muscle atrophy.

Phosphate stimulates myotube atrophy through autophagy activation: evidence of hyperphosphatemia contributing to skeletal muscle wasting in chronic kidney disease.

BACKGROUND: Accelerated muscle atrophy is associated with a three-fold increase in mortality in chronic kidney disease (CKD) patients. It is suggested that hyperphosphatemia might contribute to muscle wasting, but the underlying mechanisms remain unclear. Although evidence indicates that autophagy is involved in the maintenance of muscle homeostasis, it is not known if high phosphate levels can result in activation of autophagy, leading to muscle protein loss. METHODS: Immortalized rat L6 myotubes were exposed to a high concentration of phosphate, with or without autophagy inhibition. Myotube atrophy was examined by phase contrast microscopy. Autophagic activity was assessed by measuring the expression of microtubule-associated protein 1 light chain 3 (LC3) and p62 using quantitative real-time polymerase chain reaction and western blot. RESULTS: Phosphate induced cell atrophy in L6 myotubes in a dose- and time-dependent manner, and these responses were not associated with calcification or osteogenesis. Phosphate also dose- and time-dependently increased the LC3-II/LC3-I ratio. Inhibition of autophagy with wortmannin or knockdown of Atg5 significantly suppressed myotube atrophy caused by high phosphate concentration. CONCLUSIONS: High phosphate concentration induces muscle cell atrophy through the activation of autophagy. Targeting autophagy could be a therapeutic strategy for preventing muscle wasting caused by hyperphosphatemia.

Methylglyoxal and advanced glycation end-products promote cytokines expression in peritoneal mesothelial cells via MAPK signaling.

BACKGROUND: Peritoneal dialysis fluid degrades glucose into glucose degradation products that impair peritoneal mesothelial cell functions. These compounds are known to interfere with many cellular functions and to promote the formation of advanced glycation end products (AGEs). This study aimed to investigate the biological effects and the underlying mechanism of glucose degradation products and AGEs on mesothelial cells. METHODS: Cell proliferation was determined using [3H]-thymidine incorporation assay. Real-time polymerase chain reaction and enzyme linked immunosorbent assay (ELISA) were used to determine the mRNA and protein expression of cytokines. Reactive oxygen species production in mesothelial cells was determined by flow cytometry. Western blot was used to measure the protein expression of p38 MAPK. RESULTS: Methylglyoxal (MGO) and AGE-human serum albumin (AGE-HSA) inhibited human peritoneal mesothelial cells proliferation in a dose- and time-dependent manner. The mRNA and protein expression of cytokines including vascular endothelial growth factor (VEGF) and monocyte chemotactic protein-1 (MCP-1) was significantly increased after treatment with MGO and AGE-HSA. Also, the antioxidant N-acetylcysteine (NAC) inhibited MGO- or AGE-HSA-induced reactive oxygen species generation. Western blot showed that MGO and AGE increased the phosphorylation levels of p38 MAPK, which was significantly attenuated after treatment of NAC or p38 MAPK inhibitor SB203580. Furthermore, AGE- or MGO-induced increased expression of VEGF and MCP-1 was significantly reduced in the presence of NAC or SB203580. CONCLUSIONS: Together, this study suggested that AGE or MGO promoted VEGF and MCP-1 expression through activation of p38 MAPK signaling.

[Effect of Notch1 signal on hepatitis B virus X-mediated abnormal immune activity in renal tubular epithelial cells].

OBJECTIVE: To explore the expression of Notch1 receptor in renal tubular epithelial cells transfected with hepatitis B virus X(HBx) gene and its role in immunologic activity. METHODS: The eukaryotic vector pcDNA3.1/myc-HBx containing HBx gene or vector pcDNA3.1/myc-Notch1 containing Notch1 gene was transiently transfected into HK-2 cells. And the shRNA technique was used to silence Notch1. HK-2 cells were divided into 7 groups, including normal culture, pcDNA3.1/myc, HBx, HBx +pcDNA3.1/myc, HBx+Notch1, HBx+ shRNA and HBx+ Notch1 shRNA. Real-time PCR and Western blotting were used to detect the expression of Notch1. The expressions of MHC-IIand CD40 were examined by flow cytometry. And the supernatant contents of IL-4 and IFN-γ were measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: The results of real-time PCR and Western blotting verified that HBx and Notch1 were successfully expressed in HK-2 cells after transfection. The transfection efficiency of shRNA was 70%. Compared with normal culture and pcDNA3.1/myc groups, the expression of Notch1 increased. The expressions of MHC-II and CD40 also significantly increased in the HBx+Notch1 group (9.69% ± 0.52% vs 4.90% ± 0.32%, 21.56% ± 0.71% vs 15.74% ± 0.20%, both P < 0.05) . The supernatant level of IFN-γ was lower in HBx+Notch1 group (11.9 ± 1.7 vs 18.8 ± 0.8, P < 0.05) while the level of IL-4 was higher than control groups (50.2 ± 0.6 vs 28.1 ± 1.2, P < 0.05). And the HBx+Notch1 shRNA group had the opposite results. CONCLUSIONS: An over-expression of HBx gene may up-regulate the expression of Notch1. And Notch1 promotes the expression of immune molecules of renal tubular epithelial cell and regulates the secretion of cytokines so as to cause injury of cells and dysfunction of immune microenvironment.

[Autophagy-lysosome pathway in skeletal muscle of diabetic nephropathy rats and the effect of low-protein diet plus α-keto acids on it].

OBJECTIVE: To explore the regulation of autophagy-lysosome pathway (ALP) in skeletal muscle of diabetic nephropathy and examine the effect of low protein diet plus α-keto acid on ALP. METHODS: A total of 45 24-week-old Goto-Kakizaki rats were randomized to receive normal protein (22%) diet (NPD), low-protein (6%) diet (LPD) or low-protein (5%) plus α-keto acids (1%) diet (Keto) (n = 15 each). Wistar control rats had a normal protein diet. The mRNA and protein levels of ALP markers LC3B, Bnip3, Cathepsin L in soleus muscle were evaluated at 48 weeks. Electron microscopy was used to confirm the changes of autophagy. RESULTS: Compared with CTL group, the mRNA levels of LC3B, Bnip3, Cathepsin L in soleus muscle of rats on NPD were higher, and protein levels of LC3B-I, LC3B-II, Bnip3, Cathepsin L in soleus muscle of rats on NPD also higher than CTL group (0.82 ± 0.33 vs 0.25 ± 0.07, 0.76 ± 0.38 vs 0.20 ± 0.12, 1.25 ± 0.30 vs 0.56 ± 0.19, 1.29 ± 0.40 vs 0.69 ± 0.20). The mRNA levels of LC3B, Bnip3 and Cathepsin L in LPD group were slightly lower, compared with NPD group. However there was no statistical significance. Similarly the protein levels of LC3B-I, LC3B-II, Bnip3 and Cathepsin L in LPD group were slightly lower with no statistical significance. In contrast, the mRNA levels of LC3B, Bnip3 and Cathepsin L were greatly lower in Keto group in comparison with NPD and LPD. And protein levels of LC3B-I, LC3B-II, Bnip3 and Cathepsin L were also greatly lower in Keto group in comparison with NPD and LPD. Additionally, autophagosome or auto-lysosome was found in NPD and LPD groups by electron microscopy. CONCLUSIONS: ALP is activated in skeletal muscle of diabetic nephropathy rats. And low protein plus α-keto acid decrease the activation of ALP and improve muscle wasting.