Role of Natural Killer Cells in Antibody-mediated Rejection of Kidney Transplantation.

Objective: This study explicitly demonstrates the roles of natural killer (NK) cells in different types of kidney transplantation. Methods: We'd done the whole study from October 2022 to October 2023. To further explore the significance of NK cells during renal transplantation, we provide a theoretical basis for clinically overcoming immune rejection after renal transplantation by developing new anti-rejection drugs. We selected twelve male mice and divided them into three groups (Syngeneic transplant group allograft transplant group allograft transplant (priming) group) by random. Initially, the morphological and histopathological changes in the kidney transplantation graft model of mice in different groups are observed. Further, the DSA-IgG levels in peripheral blood and C3d and IgG deposition in mice are detected by ELISA and immunohistochemical staining. Then, the Banff 2015 score is recorded to screen a suitable AMR mouse model. Finally, the expression of NK cells in different rejection modes is detected by flow cytometry, and the expressions of various cytokines (INF-γ, perforin, granzyme B, TNF-α) in peripheral blood are detected by enzyme-linked immunosorbent assay (ELISA). Results: In the allogeneic transplantation (priming) group, peritubular capillary inflammatory cell infiltration, moderate endarteritis, and small arterial fibrinoid necrosis are evident. The Banff score showed that the allogeneic transplantation (pre-sensitized) group is significantly higher than the syngeneic and allogeneic transplantation groups. The C3H→C57BL/6 mice are pre-sensitized by skin transplantation, and then kidney transplantation is performed to establish the antibody-mediated rejection (AMR) model. After kidney transplantation, the expression levels of NK cells in the peripheral blood, spleen, and transplanted kidney tissue of mice in the pre-sensitized group are significantly higher than in the allogeneic transplantation and control groups. In the C3H→C57BL/6 mouse model of AMR, NK cells and the related cytokines in the peripheral blood are highly expressed after kidney transplantation, proving that NK cells play an essential role in the occurrence of AMR. Conclusion: Our study proved the significance of NK cells in the occurrence of AMR by systematically monitoring the expression of NK cell-related cytokines in different types, which provided some ideas for the clinical treatment of AMR.

RAB3D, upregulated by aryl hydrocarbon receptor (AhR), promotes the progression of prostate cancer by activating the PI3K/AKT signaling pathway.

Many patients with prostate cancer (PCa) cannot be diagnosed until an advanced stage, which make PCa become a large threat to human health. It is an urgent need to explore novel biomarkers for accurate diagnosis and targets for the effective treatment of PCa. This study aimed to investigate the effects of RAB3D (which belongs to the secretory Rab GTPases) on the progression of PCa. The results showed that RAB3D was highly expressed in PCa tissues compared to normal tissues according to the gene expression omnibus dataset. Consistent with the bioinformatics results, RAB3D exhibited a higher expression in PCa cells. Overexpression of RAB3D promoted the proliferation, migration, and invasion of PCa cells, whereas the knockdown of RAB3D led to the opposite results. The procancer effects of RAB3D were further confirmed by the in vivo growth of xenograft model. Subsequently, RAB3D upregulated the PI3K/AKT signaling pathway both in vivo and in vitro. LY294002 (a PI3K inhibitor) rescued the RAB3D upregulation-induced promotion of malignant phenotypes of PCa cells. Furthermore, the transcription activity of RAB3D was found to be enhanced by aryl hydrocarbon receptor (AhR; a transcription factor). The AhR silencing-induced inhibition of the proliferation and migration of PCa cells was reversed by the overexpression of RAB3D. Taken together, RAB3D, upregulated by AhR, promotes the PCa progression by activating the PI3K/AKT signaling pathway.

Construction and Analysis of lncRNA-miRNA-mRNA ceRNA Network Identify an Eight-Gene Signature as a Potential Prognostic Factor in Kidney Renal Papillary Cell Carcinoma (KIRP).

Aims: This study was conducted to establish the potential competing endogeneous RNA (ceRNA) network for predicting prognoses in kidney papillary renal cell carcinoma (KIRP) and explore novel therapeutic targets. Methods: The edgeR package in R was used to determine differentially expressed messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs) and microRNAs (miRNAs), based on data from The Cancer Gene Atlas Program (TCGA) and the Genotype Expression (GTEx) databases. Weighted gene co-expression network analysis (WGCNA) was performed to filter out the mRNAs or lncRNAs that were strongly related to KIRP. The miRNAs that possibly sponged by differentially expressed RNAs lncRNAs (DElncRNAs) were screened using miRcode. Starbase, miRDB, and TargetScan sets were utilized to predict target mRNAs to corresponding miRNAs. LASSO and multivariate Cox regression analyses were applied for the determination of potential prognostic significance. Finally, the lncRNA-miRNA-mRNA ceRNA network was constructed. Results: A total of 1739 DEmRNAs and 1599 DElncRNAs were identified in KIRP. WGCNA analysis suggested that DEmRNAs in the blue module and DElncRNAs in the turquoise module were closely correlated with KIRP. An 8-gene signature was constructed, which had prognostic significance and predictive value in KIRP. Of note, a lncRNA-miRNA-mRNA ceRNA network (including 18 lncRNAs, 5 miRNAs, and 7 mRNAs) was established. Conclusion: This investigation constructed a new lncRNA-miRNA-mRNA ceRNA network, and proposed some genes that may be novel targets, as well as a theoretical basis for the treatment of patients with KIRP.

CREB1 protects against the renal injury in a rat model of kidney stone disease and calcium oxalate monohydrate crystals-induced injury in NRK-52E cells.

Kidney stone disease (KSD) is a common urinary disease with increasing prevalence worldwide. In this study, we investigated the effect of cyclic AMP responsive element binding protein (CREB) 1 in a KSD model of rat and calcium oxalate monohydrate (COM) crystals-treated NRK-52E cells. Rats were pretreated with lentivirus (LV)-CREB1 vector or LV-control vector and administrated with ethylene glycol + ammonium chloride to induce KSD. It was found that CREB1 was activated in the renal tissue of non-treated KSD rats. Pretreating with LV-CREB1 vector significantly enhanced CREB1 expression in KSD rats. Biochemical analysis for serum and urine showed that upregulation of CREB1 could improve the renal function of KSD rats. Histological analysis confirmed that upregulation of CREB1 alleviated the renal injury in KSD rats. Moreover, the upregulation of CREB1 suppressed the apoptosis in renal tissue of KSD rats through regulating apoptosis-associated proteins. Further study showed that the upregulation of CREB1 could attenuate the oxidative stress in KSD rats as well. More interestingly, the upregulation of CREB1 enhanced the activity of complex I and complex III and the expression of mitochondrial cytochrome c, implicating the effect of CREB1 on improving mitochondrial function in KSD rats. In vitro study confirmed that upregulation of CREB1 inhibited the apoptosis and oxidative stress, while improved the mitochondrial function of NRK-52E cells treated with COM crystals, demonstrating the protective effect of CREB1 on COM crystals-induced renal epithelial cell injury. Therefore, CREB1 might be served as a promising target in the prophylaxis and treatment of KSD.

Identification of potential crucial genes associated with carcinogenesis of clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is a common genitourinary malignancy with high mortality. However, the molecular pathogenesis of ccRCC remains unclear and effective biomarkers for daily practice are still limited. Thus, we aimed to identify the potential crucial genes and pathways associated with carcinogenesis of ccRCC and further analyze the molecular mechanisms implicated in tumorigenesis. In the present study, expression profiles GSE 66270, GSE 53757, GSE 36895, and GSE 76351 were downloaded from GEO database, including 244 matched primary and adjacent normal tissues, furthermore, the level 3 RNAseq dataset (RNAseqV2 RSEM) of KIRC was also downloaded from The Cancer Genome Atlas (TCGA), which consist of 529 ccRCC tumors and 72 normal tissues. Then, differentially expressed genes (DEGs) and pathway enrichment were analyzed by using R software. A total of 129 up- and 123 down-regulated genes were identified, which were aberrantly expressed both in GEO and TCGA data. Second, Gene ontology (GO) analyses revealed that most of the DEGs were significantly enriched in integral component of membrane, extracellular exosome, plasma membrane, cell adhesion, and receptor binding. Signaling pathway analyses indicated that DEGs had common pathways in signal transduction, metabolism, and immune system. Third, hub genes were identified with protein-protein interaction (PPI) network, including PTPRC, TGFB1, EGF, MYC, ITGB2, CTSS, FN1, CCL5, KNG1, and CD86. Additionally, sub-networks analyse was also performed by using MCODE plugin. In conclusion, the novel DEGs and pathways in ccRCC identified in this study may provide new insight into the underlying molecular mechanisms that facilitates RCC carcinogenesis.

Aryl hydrocarbon receptor enhances the expression of miR-150-5p to suppress in prostate cancer progression by regulating MAP3K12.

It has been reported that mircoRNAs (miRNAs) can act as tumor inhibitors in multiple malignant tumors. As a tumor suppressor, miR-150-5p has been reported in some cancers. However, the biological impacts of miR-150-5p in prostate cancer is not fully elaborated. This study aims to explore the biological role and mechanism of miR-150-5p in prostate cancer. The expression level of miR-150-5p was examined with Quantitative real time polymerase chain reaction (qRT-PCR). Moreover, Kaplan Meier analysis revealed that downregulation of miR-150-5p predicted unfavorable prognosis for patients with prostate cancer. To identify the inhibitory effects of miR-150-5p on the cellular processes of prostate cancer, gain-of function assay was conducted. Next, the inhibitory effects of Tetrachlorodibenzo-p-dioxin (TCDD) and 3,3'-Diindolylmethane (DIM) on the proliferation and invasion of prostate cancer cells were demonstrated. Knockdown of Ahr reversed the TCDD/DIM-mediated proliferation and invasion. The expression level of CYP1A1 also was measured to confirm that Ahr was activated by TCDD or DIM in prostate cancer cells. Mechanism experiments revealed that MAP3K12 is a target mRNA of miR-150-5p in prostate cancer cells. In conclusion, Aryl hydrocarbon receptor enhances the expression of miR-150-5p to suppress cell proliferation and invasion in prostate cancer by regulating MAP3K12.

Calcium oxalate crystals induces tight junction disruption in distal renal tubular epithelial cells by activating ROS/Akt/p38 MAPK signaling pathway.

Tight junction plays important roles in regulating paracellular transports and maintaining cell polarity. Calcium oxalate monohydrate (COM) crystals, the major crystalline composition of kidney stones, have been demonstrated to be able to cause tight junction disruption to accelerate renal cell injury. However, the cellular signaling involved in COM crystal-induced tight junction disruption remains largely to be investigated. In the present study, we proved that COM crystals induced tight junction disruption by activating ROS/Akt/p38 MAPK pathway. Treating Madin-Darby canine kidney (MDCK) cells with COM crystals induced a substantial increasing of ROS generation and activation of Akt that triggered subsequential activation of ASK1 and p38 mitogen-activated protein kinase (MAPK). Western blot revealed a significantly decreased expression of ZO-1 and occludin, two important structural proteins of tight junction. Besides, redistribution and dissociation of ZO-1 were observed by COM crystals treatment. Inhibition of ROS by N-acetyl-l-cysteine (NAC) attenuated the activation of Akt, ASK1, p38 MAPK, and down-regulation of ZO-1 and occludin. The redistribution and dissociation of ZO-1 were also alleviated by NAC treatment. These results indicated that ROS were involved in the regulation of tight junction disruption induced by COM crystals. In addition, the down-regulation of ZO-1 and occludin, the phosphorylation of ASK1 and p38 MAPK were also attenuated by MK-2206, an inhibitor of Akt kinase, implying Akt was involved in the disruption of tight junction upstream of p38 MAPK. Thus, these results suggested that ROS-Akt-p38 MAPK signaling pathway was activated in COM crystal-induced disruption of tight junction in MDCK cells.

Identification of biological targets of therapeutic intervention for clear cell renal cell carcinoma based on bioinformatics approach.

BACKGROUND: We aimed to discover the potential microRNA (miRNA) targets and to explore the underlying molecular mechanisms of clear cell renal cell carcinoma (ccRCC). METHODS: Microarray data of GSE16441 was downloaded from Gene Expression Omnibus database. Differentially expressed genes (DEGs) and differentially expressed miRNAs between ccRCC tumors and matched non-tumor samples were analyzed. Target genes of differentially expressed miRNAs were screened. Besides, functional enrichment analysis of DEGs was performed, followed by protein-protein interaction (PPI) network construction and sub-module analysis. Finally, the integrated miRNA-DEGs network was constructed. RESULTS: A total of 1758 up- and 2465 down-regulated DEGs were identified. Moreover, 15 up- and 12 down-regulated differentially expressed miRNAs were screened. The up-regulated DEGs were significantly enriched in pathways such as cell adhesion molecules and focal adhesion. Besides, the down-regulated DEGs were enriched in oxidative phosphorylation, and citrate cycle (TCA cycle). Moreover, eight sub-modules of PPI network were obtained. Totally, eight down-regulated miRNAs were identified to significantly regulate the DEGs and miRNA-200c that could regulate collagen, type V, alpha 2 (COL5A2) as well as COL5A3 was found to be the most significant. Additionally, 10 up-regulated miRNAs were identified to be significantly associated with the DEGs. Thereinto, miRNA-15a that could regulate ATPase, H(+) transporting, lysosomal 21 kDa, V0 subunit b (ATP6V0B) and miRNA-155 were found to be the most significant. CONCLUSIONS: miRNA-200c that could regulate COL5A2 and COL5A3, miRNA-15a that could regulate ATP6V0B and miRNA-155 may play key roles in ccRCC progression. These miRNAs may be potential targets for ccRCC treatment.

Reduced ING4 Expression Is Associated with the Malignancy of Human Bladder.

INTRODUCTION: Inhibitor of growth 4 (ING4) is a tumor suppressor. However the role of ING4 in human bladder malignancy is unknown. In this study, ING4 expression in human bladder cancer and its potential effects were studied. MATERIALS AND METHODS: ING4 expression in 47 human bladder cancer tissues and paired adjacent normal tissues was detected by Western blotting, quantitative reverse transcription-polymerase chain reaction, and immunohistochemistry. The migration and cell cycle progression of SV-HUC-1 and T24 cells with aberrant ING4 expression were examined. RESULTS: ING4 protein and mRNA were significantly decreased in bladder cancer tissues. ING4 protein level was significantly lower in the group of patients over 50 years of age. ING4 knockdown caused more rapid cell migration and increased the population of SV-HUC-1 and T24 cells in the G2-M phase. CONCLUSION: Our data suggest a close connection between aberrant ING4 expression and the carcinogenesis of human bladder cells. ING4 may be a potential target for bladder cancer chemotherapy.

USP28 is a potential prognostic marker for bladder cancer.

This study was conducted to analyze the expression of the ubiquitin-specific protease Usp28 and assess its clinical significance in human bladder cancer. mRNA and protein expression levels of Usp28 were determined by real-time polymerase chain reaction (PCR) and Western blot in 24 paired bladder cancers and the adjacent non-cancerous tissues. In addition, the expression of Usp28 protein in 186 bladder cancers was also determined by immunohistochemistry. The relationship between expression of Usp28 and clinico-pathologic features and prognosis was finally evaluated. Usp28 was expressed at a higher level in bladder cancers compared to adjacent non-cancerous tissues at both the mRNA and protein levels in 24 paired samples (all P < 0.01). In immunohistochemical examination, 78 (41.9%) of 186 cases displayed low Usp28 expression in cancerous tissues, whereas 108 (58.1%) cases displayed high Usp28 expression. In the universal analysis, Usp28 correlated strongly with histopathological grade, clinical stage, tumor number and recurrence rate (P = 0.0001, 0.0001, 0.0001 and 0.0051, respectively), but did not correlate with gender or age (P = 0.5588 and 0.6574). After multiple analysis of the above factors and consideration of confounding factors, tumor number, histological grade, clinical stage, and recurrence were related to Usp28 expression (P = 0.001, 0.001, 0.001 and 0.001, respectively). Finally, Usp28 expression was indentified as a independent predictors of survival (P = 0.001). Usp28 protein expression is potentially valuable in prognostic evaluation of bladder cancer.

Anti-tumor effect of ribavirin in combination with interferon-α on renal cell carcinoma cell lines in vitro.

BACKGROUND: Ribavirin is an anti-viral drug; however, recent data suggest that it may also be effective in cancer therapy. This study investigated the effect of ribavirin alone or in combination with IFN-α on biological processes: proliferation, apoptosis, and migration of murine (Renca) and human renal carcinoma (RCC) cells (786-0) in vitro. METHODS: Renca and 786-0 cells were treated with IFN-α, ribavirin, or a combination of IFN-α and ribavirin at varying concentrations. Cell proliferation was evaluated using CCK-8 assay. Induction of apoptosis and distribution of cell cycle were determined by flow cytometry. The migratory capacity of cells was quantified using a transwell migration assay. The toxic effect of these drugs was examined using MTT assay in HEK-293 cells. ELISA was used to measure IL-10 and TGF-ß content in the culture supernatants. RESULTS: Our results showed that both ribavirin alone and in combination with IFN-α could significantly inhibit the cell proliferation and arrest the cell cycle progress at the G2/M phase. These treatments also inhibited cell migration and IL-10 production, in a concentration-dependent manner, in 786-0 and Renca cells. Moreover, they significantly induced apoptosis of RCC cells and increased TGF-ß production in concentration-dependent manner. No significant toxic effect was observed in HEK-293 cells. We also found that the effect of combined treatment was more pronounced than that of ribavirin or IFN-α alone. However, the combined effect of the two drugs was not synergistic. CONCLUSION: Our findings suggest that ribavirin can negatively affect biological processes of RCC cells. This agent might become a new candidate for the treatment of RCC in the clinical setting.

N-acetylcysteine regulates oxalate induced injury of renal tubular epithelial cells through CDKN2B/TGF-ß/SMAD axis.

This study was aimed to investigate the preventive effects of N-acetyl-L-cysteine (NAC) against renal tubular cell injury induced by oxalate and stone formation and further explore the related mechanism. Transcriptome sequencing combined with bioinformatics analysis were performed to identify differentially expressed gene (DEG) and related pathways. HK-2 cells were pretreated with or without antioxidant NAC/with or silencing DEG before exposed to sodium oxalate. Then, the cell viability, oxidative biomarkers of superoxidase dismutase (SOD) and malondialdehyde (MDA), apoptosis and cell cycle were measured through CCK8, ELISA and flow cytometry assay, respectively. Male SD rats were separated into control group, hyperoxaluria (HOx) group, NAC intervention group, and TGF-ß/SMAD pathway inhibitor group. After treatment, the structure changes and oxidative stress and CaOx crystals deposition were evaluated in renal tissues by H&E staining, immunohistochemical and Pizzolato method. The expression of TGF-ß/SMAD pathway related proteins (TGF-ß1, SMAD3 and SMAD7) were determined by Western blot in vivo and in vitro. CDKN2B is a DEG screened by transcriptome sequencing combined with bioinformatics analysis, and verified by qRT-PCR. Sodium oxalate induced declined HK-2 cell viability, in parallel with inhibited cellular oxidative stress and apoptosis. The changes induced by oxalate in HK-2 cells were significantly reversed by NAC treatment or the silencing of CDKN2B. The cell structure damage and CaOx crystals deposition were observed in kidney tissues of HOx group. Meanwhile, the expression levels of SOD and 8-OHdG were detected in kidney tissues of HOx group. The changes induced by oxalate in kidney tissues were significantly reversed by NAC treatment. Besides, expression of SMAD7 was significantly down-regulated, while TGF-ß1 and SMAD3 were accumulated induced by oxalate in vitro and in vivo. The expression levels of TGF-ß/SMAD pathway related proteins induced by oxalate were reversed by NAC. In conclusion, we found that NAC could play an anti-calculus role by mediating CDKN2B/TGF-ß/SMAD axis.

Arsenic trioxide increases the sensitivity of 786-0 renal carcinoma cells to radiotherapy.

We evaluated the combined effect of As(2)O(3) and ionizing radiation on cultured renal carcinoma cells. The cells receiving both As(2)O(3) and radiotherapy had more DNA damage, more apoptosis, more reactive oxygen species produced, more cells in G(2)/M phase, and a lower mitochondrial membrane potential than cells treated with As(2)O(3) only or with radiotherapy only (for all comparisons, p < .05). Renal carcinoma cells can be sensitized to ionizing radiation with As(2)O(3), and combining As(2)O(3) and radiation had larger effects than As(2)O(3) only or radiation only.

A Randomized Study on the Effect of Metformin Combined with Intensive-Exercise Diet Therapy on Glucose and Lipid Metabolism and Islet Function in Patients with Renal Cell Carcinoma and Diabetes.

Objective: To evaluate the effect of metformin combined with intensive-exercise diet therapy on glucose and lipid metabolism and islet function in diabetes patients with localized renal cell carcinoma after laparoscopic resection. Methods: A total of 120 renal cancer patients with diabetes mellitus treated in the oncology department of our hospital from January 2018 to December 2020 were recruited and assigned via random number table method at a ratio of 1 : 1 to receive either metformin (control group) or metformin plus intensive exercise diet therapy (study group) after laparoscopic nephrectomy. Outcome measures included glucose and lipid metabolism, pancreatic islet function, lifestyle, clinical efficacy, and adverse reactions. Results: After the intervention, the fasting blood glucose (FBG), 2 h postprandial blood glucose (PBG), glycosylated hemoglobin (HbA1c), triglyceride (TG), total cholesterol (TC), and low-density lipoprotein cholesterol (LDL-C) of the two groups of patients decreased significantly, and the study group had significantly lower results. After treatment, the two groups had elevated levels of high-density lipoprotein cholesterol (HDL-C), fasting serum insulin (FINS), and homeostasis model assessment of ß-cell function (HOMA-ß), and higher results were obtained in the study group (P < 0.05). After the intervention, the study group showed higher results of health promoting lifestyle profile-II (HPLP-II) and a 12-month progression-free survival rate than the control group. There were no significant differences in the incidence of adverse reactions between the two groups. Conclusion: Metformin combined with intensive-exercise diet therapy significantly improves the glucose and lipid metabolism and islet function of renal cancer patients with diabetes and effectively enhances the 12-month progression-free survival. Further trials are, however, required prior to clinical application.

MiRNA-148a inhibits cell growth and drug resistance by regulating WNT10a expression in renal cell carcinoma.

Background: We aimed to explore miR-148a exerts a tumor suppressor effect and arsenic trioxide (As2O3) sensitivity on renal cell carcinoma (RCC). Methods: We performed polymerase chain reaction (PCR) on 42 pairs of tumor and paracancerous samples collected from RCC patients to investigate the miR-148a expression; meanwhile, we analyzed the interplay between clinical indicators and miR-148a expression of RCC. Then, the influence of miR-148a overexpression on the functions of RCC cells were analyze using transwell migration assay, Cell Counting Kit-8 (CCK-8), and cell wound healing assay. Furthermore, the ability of miR-148a to sensitize Caki-1 cells treated with As2O3 were detected using flow cytometry. Finally, the relevant mechanism of miR-148a on the downstream gene Wnt family member 10A (WNT10a) was explored by cell reverse method. Results: The results from RCC patients indicated a significantly lower miR-148a level than adjacent tissues. The low miR-148a expression increased prevalence of distant metastasis and decreased survival rate compared to those with high expression in patients. In the RCC cell lines, the proliferation and metastasis ability of the miR-148a mimic group was remarkably lower than the miR-NC group. At the same time, it was verified that WNT10a was remarkably higher cell lines and RCC tissues; and negatively related to miR-148a expression. In addition, miR-148a mimics were found to remarkably reduce the protein expression of WNT10a. In the cell reverse experiment, overexpression of WNT10a was confirmed to offset the miR-148a mimics effect on metastasis and proliferation of RCC cells. In addition, an increase in relative apoptosis was detected in As2O3 treated with/without miR-148a mimics for 48 hours, and apoptosis was significantly reduced after transfection with WNT10a in the Caki-1 cell line and significantly reduced after combined treatment. Conclusions: The study revealed that miR-148a is associated with distant metastases and leads to poor prognosis in RCC patients. Moreover, miR-148a inhibit the malignant progression and increase the sensitivity of RCC cells to As2O3 by regulating WNT10a.

Puerarin prevents calcium oxalate crystal-induced renal epithelial cell autophagy by activating the SIRT1-mediated signaling pathway.

Calcium oxalate (CaOx) crystals can activate autophagy, causing damage to renal tubular epithelial cells (TECs). Puerarin has been shown to have protective and therapeutic effects against a variety of diseases by inhibiting autophagy activation. However, the protective effect of puerarin against CaOx crystals and the underlying molecular mechanisms are unclear. Cell Counting Kit-8 (CCK-8) assays were used to evaluate the effects of puerarin on cell viability. Intracellular reactive oxygen species (ROS) levels were measured by the cell-permeable fluorogenic probe 2,7-dichlorofluorescein diacetate (DCFH-DA). Immunofluorescence, immunohistochemistry, and western blotting were used to examine the expression of SIRT1, Beclin1, p62, and LC3, and explore the underlying molecular mechanisms in vivo and in vitro. Puerarin treatment significantly attenuated CaOx crystal-induced autophagy of TECs and CaOx cytotoxicity to TECs by altering SIRT1 expression in vitro and in vivo, whereas the SIRT1-specific inhibitor EX527 exerted contrasting effects. In addition, we found that the protective effect of puerarin was related to the SIRT1/AKT/p38 signaling pathway. The findings suggest that puerarin regulates CaOx crystal-induced autophagy by activating the SIRT1-mediated signaling pathway, and they suggest a series of potential therapeutic targets and strategies for treating nephrolithiasis.

Erratum: Inhibition of SMYD2 suppresses tumor progression by down-regulating microRNA-125b and attenuates multi-drug resistance in renal cell carcinoma: Erratum.

[This corrects the article DOI: 10.7150/thno.37628.].

Oxalate impairs aminophospholipid translocase activity in renal epithelial cells via oxidative stress: implications for calcium oxalate urolithiasis.

PURPOSE: We evaluated the possible involvement of phospholipid transporters and reactive oxygen species in the oxalate induced redistribution of renal epithelial cell phosphatidylserine. MATERIALS AND METHODS: Madin-Darby canine kidney cells were labeled with the fluorescent phospholipid NBD-PS in the inner or outer leaflet of the plasma membrane and then exposed to oxalate in the presence or absence of antioxidant. This probe was tracked using a fluorescent quenching assay to assess the bidirectional transmembrane movement of phosphatidylserine. Surface expressed phosphatidylserine was detected by annexin V binding assay. The cell permeable fluorogenic probe DCFH-DA was used to measure the intracellular reactive oxygen species level. RESULTS: Oxalate produced a time and concentration dependent increase in phosphatidylserine, which may have resulted from impaired aminophospholipid translocase mediated, inward directed phosphatidylserine transport and from enhanced phosphatidylserine outward transport. Adding the antioxidant N-acetyl-L-cysteine significantly attenuated phosphatidylserine externalization by effectively rescuing aminophospholipid translocase activity. CONCLUSIONS: To our knowledge our findings are the first to show that oxalate induced increased reactive oxygen species generation impairs aminophospholipid translocase activity and decreased aminophospholipid translocase activity has a role in hyperoxaluria promoted calcium oxalate urolithiasis by facilitating phosphatidylserine redistribution in renal epithelial cells.

MicroRNA-dependent regulation of PTEN after arsenic trioxide treatment in bladder cancer cell line T24.

Arsenic trioxide has shown remarkable biological activity against bladder cancer in some clinical studies. However, the mechanism of its action is unknown. Our aim was to find the relationship between miRNAs and arsenic trioxide treatment by using T24 human bladder carcinoma cells. By performing microRNA microarray and quantitative real-time PCR after ATO treatment, we found that expression levels of several miRNAs, in particular, miRNA-19a, were significantly decreased in T24 cell line. Furthermore, cell proliferation assay, flow cytometry analysis, prediction of miRNA targets, Western blot analysis, and luciferase reporter assay were performed to determine the role of mir-19a in affecting the biological behaviors of T24 cells. Several miRNAs were up-regulated or down-regulated in T24 cells treated with arsenic trioxide compared to their controls. If only changes above two folds were considered, two miRNAs were identified, miRNA-19a was down-regulated, while miRNA-222* was up-regulated. Among them, knockdown of miRNA-19a by anti-miRNA-19a transfection showed a positive therapeutic effect in bladder cancer cells by inhibiting cell growth and inducing cell apoptosis targeting PTEN through the PTEN/Akt pathway. Besides this, a synergy effect was detected between knockdown of miRNA-19a and arsenic trioxide. Arsenic trioxide altered miRNA expression profile in T24 cells. It seems miRNA-19a plays a critical role in the mechanism of arsenic trioxide treatment in bladder cancer. The synergy effect between miRNA-19a and arsenic trioxide that advocates targeting the mir-19a may represent a potential approach to enhance the efficacy and safety of ATO to treat bladder cancer by a decrease in dose.

FK506 binding protein 10: a key actor of collagen crosslinking in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of malignant tumor in the kidney. With numbers of patients whose physical condition or tumor stage not suitable for radical surgery, they only have a narrow choice of using VEGF/mTOR targeted drugs to control their tumors, but ccRCC often shows resistance to these drugs. Therefore, identifying a new therapeutic target is of urgent necessity. In this study, for the first time, we concluded from bioinformatics analyses and in vitro research that FK506 binding protein 10 (FKBP10), together with its molecular partner Lysyl hydroxylase 2 (LH2/PLOD2), participate in the process of type I collagen synthesis in ccRCC via regulating crosslinking of pro-collagen chains. Our findings may provide a potential therapeutic target to treat ccRCC in the future.