Machine Learning Reveals Ets2 as a Novel Target for Membranous Nephropathy Treatment and Its Role in Immune Infiltration.

Background: Membranous nephropathy (MN) is a common pathological phenotype for adult nephrotic syndrome (NS). The occurrence of MN is increasing across China, but diagnostic methods for MN still rely on kidney biopsy and PLA2R and THSD7A detection in plasma and kidney tissue, and there has been no new biomarker for MN discovered since 2014. Immune infiltration status in MN patients suffers from the dearth of associated studies. In the present study, we aimed to find new bio-markers for MN and evaluate the role of immune cells infiltration in MN pathology. Methods: We downloaded MN expression profile from the Gene Expression Omnibus database and used R-project to screen differentially expressed genes (DEGs) and performed functional correlation analysis. Least absolute shrinkage and selection operator (LASSO) logistic regression and Radom Forest algorithms were used to screen and verify the bio-markers of MN. Finally, CIBERSORT was used to evaluate the infiltration of immune cells in MN tissues. Results: A total of 463 DEGs were screened from the MN tissue in this study. ETS2 was identified as bio-marker for MN. The CIBERSORT results showed that there were statistical differences in monocytes, plasma cells, regulatory T cells, and memory B cells. In addition, ETS2 was positively related to monocytes, M1 phase macrophages, and neutrophils and negatively correlated to plasma cells, CD4+ T memory cells, M2 macrophages, CD8+ T cells, memory B cells, and resting mast cells. Conclusion: (1) Machine learning algorithms reveals Ets2 as a novel target for membranous nephropathy patients. (2) Immune infiltration plays an important part in membranous nephropathy. (3) Ets2 expression is related to immune cells infiltration.

Involvement of YTHDF1 in renal fibrosis progression via up-regulating YAP.

Renal fibrosis is a progressive, fatal renal disease characterized by the aberrant accumulation of myofibroblasts that produce excess extracellular matrix (ECM) in the renal interstitium and glomeruli. Yes-associated protein (YAP) has been regarded as a crucial modulator in myofibroblast transformation, but its upstream regulator remains a mystery. In the present study investigating the participation of m6A methylation during renal fibrosis through bioinformatics analysis, we identified YTHDF1, a modulator of m6A methylation, as a key contributor for renal fibrosis because it was highly expressed in human fibrotic kidneys and had a significant correction with YAP. Their co-localization in human fibrotic kidneys was additionally shown by immunofluorescence. We then found that YTHDF1 was also up-regulated in fibrotic mouse kidneys induced by unilateral ureteral obstruction (UUO), high-dose folic acid administration, or the unilateral ischemia-reperfusion injury, further supporting a causal role of YTHDF1 during renal fibrosis. Consistent with this notion, YTHDF1 knockdown alleviated the progression of renal fibrosis both in cultured cells induced by transforming growth factor-beta administration and in the UUO mouse model. Meanwhile, YAP was accordingly down-regulated when YTHDF1 was inhibited. Furthermore, the specific binding of YTHDF1 to YAP mRNA was detected using RNA Binding Protein Immunoprecipitation, and the up-regulation of fibrotic related molecules in cultured cells induced by YTHDF1 over-expression plasmid was attenuated by YAP siRNA. Taken together, our data highlight the potential utility of YTHDF1 as an indicator for renal fibrosis and suggest that YTHDF1 inhibition might be a promising therapeutic strategy to alleviate renal fibrosis via downregulating YAP.

Targeting Mechanosensitive Piezo1 Alleviated Renal Fibrosis Through p38MAPK-YAP Pathway.

Renal fibrosis is the most common pathological manifestation of a wide variety of chronic kidney disease. Increased extracellular matrix (ECM) secretion and enhanced microenvironment stiffening aggravate the progression of renal fibrosis. However, the related mechanisms remain unclear. Here, we evaluated the mechanism by which ECM stiffness aggravates renal fibrosis. In the present study, renal mesangial cells (MCs) were cultured on polyacrylamide hydrogels with different stiffness accurately detected by atomic force microscope (AFM), simulating the in vivo growth microenvironment of MCs in normal kidney and renal fibrosis. A series of in vitro knockdown and activation experiments were performed to establish the signaling pathway responsible for mechanics-induced MCs activation. In addition, an animal model of renal fibrosis was established in mice induced by unilateral ureteral obstruction (UUO). Lentiviral particles containing short hairpin RNA (sh RNA) targeting Piezo1 were used to explore the effect of Piezo1 knockdown on matrix stiffness-induced MCs activation and UUO-induced renal fibrosis. An in vitro experiment demonstrated that elevated ECM stiffness triggered the activation of Piezo1, which increased YAP nuclear translocation through the p38MAPK, and consequently led to increased ECM secretion. Furthermore, these consequences have been verified in the animal model of renal fibrosis induced by UUO and Piezo1 knockdown could alleviate UUO-induced fibrosis and improve renal function in vivo. Collectively, our results for the first time demonstrate enhanced matrix stiffness aggravates the progression of renal fibrosis through the Piezo1-p38MAPK-YAP pathway. Targeting mechanosensitive Piezo1 might be a potential therapeutic strategy for delaying the progression of renal fibrosis.

A-Lipoic Acid Alleviates Folic Acid-Induced Renal Damage Through Inhibition of Ferroptosis.

Folic acid (FA)-induced acute kidney injury (AKI) is characterized by the disturbance of redox homeostasis, resulting in massive tubular necrosis and inflammation. Α-lipoic acid (LA), as an antioxidant, has been reported to play an important role in renal protection, but the underlying mechanism remains poorly explored. The aim of this study is to investigate the protective effect of LA on FA-induced renal damage. Our findings showed that LA could ameliorate renal dysfunction and histopathologic damage induced by FA overdose injection. Moreover, FA injection induced severe inflammation, indicated by increased release of pro-inflammatory cytokines tumor necrosis factor (TNF)-α and IL-1ß, as well as infiltration of macrophage, which can be alleviated by LA supplementation. In addition, LA not only reduced the cellular iron overload by upregulating the expressions of Ferritin and ferroportin (FPN), but also mitigated reactive oxygen species (ROS) accumulation and lipid peroxidation by increasing the levels of antioxidant glutathione (GSH) and glutathione peroxidase-4 (GPX4). More importantly, we found that LA supplementation could reduce the number of Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive tubular cells caused by FA, indicating that the tubular cell death mediated by ferroptosis may be inhibited. Further study demonstrated that LA supplementation could reverse the decreased expression of cystine/glutamate antiporter xCT (SLC7A11), which mediated GSH synthesis. What is more, mechanistic study indicated that p53 activation was involved in the inhibitory effect of SLC7A11 induced by FA administration, which could be suppressed by LA supplementation. Taken together, our findings indicated that LA played the protective effect on FA-induced renal damage mainly by inhibiting ferroptosis.

Ibudilast Attenuates Folic Acid-Induced Acute Kidney Injury by Blocking Pyroptosis Through TLR4-Mediated NF-κB and MAPK Signaling Pathways.

Folic acid (FA)-induced renal tubule damage, which is characterized by extensive inflammation, is a common model of acute kidney injury (AKI). Pyroptosis, a pro-inflammatory form of cell death due to the activation of inflammatory caspases, is involved in AKI progression. Ibudilast, a TLR4 antagonist, has been used in the clinic to exert an anti-inflammatory effect on asthma. However, researchers have not explored whether ibudilast exerts a protective effect on AKI by inhibiting inflammation. In the present study, ibudilast reversed FA-induced AKI in mice, as indicated by the reduced serum creatinine and urea nitrogen levels, and improved renal pathology, as well as the downregulation of kidney injury marker-1. In addition, ibudilast significantly increased the production of the anti-inflammatory factor IL-10 while suppressing the secretion of the pro-inflammatory cytokine TNF-α and macrophage infiltration. Moreover, in the injured kidney, ibudilast reduced the levels of both inflammasome markers (NLRP3) and pyroptosis-related proteins (caspase-1, IL1-ß, IL-18, and GSDMD cleavage), and decreased the number of TUNEL-positive cells. Further mechanistic studies showed that ibudilast administration inhibited the FA-induced upregulation of TLR4, blocked NF-κB nuclear translocation, and reduced the phosphorylation of NF-κB and IκBα, p38, ERK, and JNK. Thus, this study substantiates the protective effect of ibudilast on FA-induced AKI in mice and suggests that protection might be achieved by reducing pyroptosis and inflammation, likely through the inhibition of TLR4-mediated NF-κB and MAPK signaling pathways.

Metformin alleviates ß-glycerophosphate-induced calcification of vascular smooth muscle cells via AMPK/mTOR-activated autophagy.

The aim of the present study was to investigate the effect of metformin on ß-glycerophosphate-induced calcification of vascular smooth muscle cells (VSMCs) and the possible mechanisms underlying this. Using an established VSMC calcification model, VSMCs were first treated with ß-glycerophosphate, before metformin, 3-methyladenine and compound C were added to the cell cultures in different combinations. Calcium deposition in the cells was examined by Alizarin Red S staining and using the O-cresolphthalein complexone method. To assess the occurrence of autophagy, autophagosomes inside the cells were studied using a transmission electron microscope and green fluorescent microtubule-associated protein 1 light chain 3 (LC3) puncta were examined using a fluorescent microscope. Additionally, protein expression levels of α-smooth muscle actin (α-SMA), runt-related transcription factor 2 (RUNX2), LC3II/I, beclin 1 and 5' adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway-associated proteins were determined by western blot analysis. Metformin increased the number of autophagosomes, green fluorescent LC3 puncta and the levels of LC3II/I, beclin 1, α-SMA and phosphorylated (p)-AMPK in the VSMCs that were treated with ß-glycerophosphate when compared to controls; whereas, calcium deposition and the expression levels of RUNX2 and p-mTOR were found to be decreased. Treating the VSMCs with 3-methyladenine or compound C reversed the effects of metformin. The results of the present study suggested that metformin may alleviate ß-glycerophosphate-induced calcification of VSMCs, which may be attributed to the activation of AMPK/mTOR signaling pathway-dependent autophagy.

Effect of local anti-vascular endothelial growth factor therapy to prevent the formation of stenosis in outflow vein in arteriovenous fistula.

BACKGROUND AND OBJECTIVES: Vascular stenosis and angiogenesis are the major causes of short expectancy of arteriovenous fistula (AVF). Increased expression of vascular endothelial growth factor-A (VEGF-A) has been suggested to play an important role in the pathophysiologic process. Anti-VEGF has been proved to be effective on anti-angiogenesis and applied in clinical practice, but its effect on anti-stenosis remains to be verified before it could be applied to prevent stenosis of AVF. This study was aimed to evaluate the effect of local anti-VEGF therapy to prevent the formation of stenosis in the outflow vein in AVF and its mechanism. METHODS: Bioinformatics of VEGF-A and its downstream-regulated molecules from the STRING PPI database were analyzed in this study. The biopsy samples from outflow veins of AVF in patients and C57BL/6 mouse models were analyzed to examine the mechanisms of pathologic vascular stenosis associated with VEGF pathways and their potential therapeutic targets. RESULTS: We found that the reduction of VEGF-A could downregulate downstream molecules and subsequently reduce the intimal hyperplasia and abnormal vascular remodeling by analyzing the STRING PPI database. Venous wall thickening, intimal neointima formation, and apoptosis of vascular endothelial cells in the proliferative outflow vein of the AVF were significantly more obvious, and upregulation of expression of VEGF was observed in dysfunctional AVF in patients. In mouse models, the expression of VEGF, Ephrin receptor B4 (EphB4), matrix metalloproteinase (MMP)2, MMP9, tissue inhibitor of metalloproteinase (TIMP)1, TIMP2, and caspase 3 in the control-shRNA surgical group was significantly higher than in the sham group (P < 0.05), and all of these indicators were significantly lower in lentiviral transfection group and Avastin group than in control-shRNA surgical group (P < 0.05) on the 14th day after AVF operation. CONCLUSION: VEGF expression is significantly increased in vascular endothelial cells in stenosed or occluded outflow veins of dysfunctional AVF. Local injection of Avastin into the adventitia of the proximal outflow vein in autologous AVF procedure has an excellent potential to prevent the subsequent local stenosis of the proximal outflow vein.

Efficacy and safety of cinacalcet compared with other treatments for secondary hyperparathyroidism in patients with chronic kidney disease or end-stage renal disease: a meta-analysis.

BACKGROUND: It is controversial for the effect and safety between cinacalcet and other treatments in treating secondary hyperparathyroidism for patients with chronic kidney disease (CKD) or end-stage renal disease (ESRD). METHODS: Embase, PubMed, and Cochrane Library were searched through Feb 2017. 21 randomized controlled trials were included. We calculated the pooled mean difference (MD), relative risk (RR) and corresponding 95% confidence interval (CI). RESULT: Patients received calcimimetic agents had significantly decreased serum parathyroid hormone (MD = - 259.24 pg/mL, 95% CI: - 336.23 to - 182.25), calcium (MD = - 0.92 mg/dL, 95% CI: - 0.98 to - 0.85) and calcium phosphorus product (MD = - 5.97 mg2/dL2, 95% CI: - 9.77 to - 2.16) concentration compared with control treatment. However, the differences in cardiovascular mortality and all-cause mortality between calcimimetics agents and control group were not statistically significant. The incidence of nausea (RR = 2.13, 95% CI: 1.62 to 2.79), vomiting (RR = 1.99, 95% CI: 1.78 to 2.23) and hypocalcemia (RR = 10.10, 95% CI: 7.60 to 13.43) in CKD patients with calcimimetics agents was significantly higher than that with control treatment. CONCLUSION: Cinacalcet improved the biochemical parameters in CKD patients, but did not improve all-cause mortality and cardiovascular mortality. Moreover, cinacalcet can cause some adverse events.

Pretreatment with Roxadustat (FG-4592) Attenuates Folic Acid-Induced Kidney Injury through Antiferroptosis via Akt/GSK-3ß/Nrf2 Pathway.

Folic acid- (FA-) induced kidney injury is characterized by the tubule damage due to the disturbance of the antioxidant system and subsequent interstitial fibrosis. FG-4592 is an inhibitor of prolyl hydroxylase of hypoxia-inducible factor (HIF), an antioxidant factor. The present study investigated the protective role of FG-4592 pretreatment at the early stage of the kidney injury and long-term impact on the progression of renal fibrosis. FG-4592 was administrated two days before FA injection in mice. On the second day after FA injection, the mice with FG-4592 pretreatment showed an improved renal function, compared with those without FG-4592 pretreatment, indicated by biochemical and histological parameters; meanwhile, the cellular content of iron, malondialdehyde, and 4-hydroxynonenal histologically decreased, implying the suppression of iron accumulation and lipid peroxidation. Simultaneously, upregulation of HIF-1α was found, along with Nrf2 activation, which was reflected by increased nuclear translocation and high-expression of downstream proteins, including heme-oxygenase1, glutathione peroxidase4, and cystine/glutamate transporter, as well as ferroportin. Correspondingly, the elevated levels of antioxidative enzymes and glutathione, as well as reduced iron accumulation, were observed, suggesting a lower risk of occurrence of ferroptosis with FG-4592 pretreatment. This was confirmed by reversed pathological parameters and improved renal function in FA-treated mice with the administration of ferrostatin-1, a specific ferroptosis inhibitor. Furthermore, a signal pathway study indicated that Nrf2 activation was associated with increased phosphorylation of Akt and GSK-3ß, verified by the use of an inhibitor of the PI3K that phosphorylates Akt. Moreover, FG-4592 pretreatment also decreased macrophage infiltration and expression of inflammatory factors TNF-α and IL-1ß. On the 14th day after FA injection, FG-4592 pretreatment decreased collagen deposition and expression of fibrosis biomarkers. These findings suggest that the protective role of FG-4592 pretreatment is achieved mainly by decreasing ferroptosis at the early stage of FA-induced kidney injury via Akt/GSK-3ß-mediated Nrf2 activation, which retards the fibrosis progression.

Specific knockdown of WNT8b expression protects against phosphate-induced calcification in vascular smooth muscle cells by inhibiting the Wnt-ß-catenin signaling pathway.

In the last 10 years, the prevalence, significance, and regulatory mechanisms of vascular calcification (VC) have gained increasing recognition. The aim of this study is to explore the action of WNT8b in the development of phosphate-induced VC through its effect on vascular smooth muscle cells (VSMCs) in vitro by inactivating the Wnt-ß-catenin signaling pathway. To explore the effect of WNT8b on the Wnt-ß-catenin signaling pathway and VC in vitro, ß-glycerophosphate (GP)-induced T/G HA-VSMCs were treated with small interfering RNA against WNT8b (Si-WNT8b), Wnt-ß-catenin signaling pathway activator (LiCl) and both, respectively. Reverse transcription quantitative polymerase chain reaction and western blot analysis were used to determine the messenger RNA and protein levels of WNT8b, α-smooth muscle actin (α-SMA), calcification-associated molecules, and molecules related to the Wnt signaling pathway. The TOP/FOP-Flash reporter assay was performed to detect the transcription activity mediated by ß-catenin. Si-WNT8b reduced calcium deposition and the activity of alkaline phosphatase (ALP), increased the α-SMA level, and decreased bone morphogenetic protein 2, Pit1, MSX2, and Runt-related transcription factor 2 levels, whereas stimulation of LiCl worsened ß-GP-induced calcium deposition, increased the activity of ALP, and reduced the α-SMA expression level. Si-WNT8b reduced the levels of WNT8b, frizzled-4, ß-catenin, phospho-GSK-3ß (p-GSK-3ß), and cyclin-D, whereas it increased the levels of p-ß-catenin and GSK-3ß, indicating that si-WNT8b could alter the Wnt-ß-catenin signaling pathway and thus hamper the VC in T/G HA-VSMC, which was further demonstrated by the TOP/FOP-Flash assay and detection of the ß-catenin expression level in the nucleus. Altogether, we conclude that WNT8b knockdown terminates phosphate-induced VC in VSMCs by inhibiting the Wnt-ß-catenin signaling pathway.

Glomus tumor of uncertain malignant potential of the lung: a case report and review of literature.

Glomus tumor is an uncommon tumor usually presenting in the dermis. Rarely, it occurred in visceral organs including stomach, liver and long. The majority of glomus tumors were benign. Herein, we present a case of glomus tumor located in the left lobe of the lung in a 49 year-old Chinese male. An irregular mass measuring 3 cm was detected by imaging examination because of his suffering from cough, dyspnea and chest pain. Histologically, the tumor is composed predominantly of sheets of ovoid to round cells with clear border, pale cytoplasm and fine granular chromatin. The mitotic count was less than 5 per 50 HPF. The tumor focally invaded the surrounding normal bronchial and alveolar tissue. Immunohistochemical staining showed that the cells were diffusely positive for SMA, caldesmon, and vimentin. The Ki-67 proliferation index was approximately 20%. Based on morphologic features and the immunohistochemical profile, the tumor was consistent with glomus tumor of uncertain malignant potential.