Integrative analysis and risk model construction for super­enhancer­related immune genes in clear cell renal cell carcinoma.

Clear cell renal cell carcinoma (ccRCC) is the most common type of kidney cancer associated with poor prognosis, and accounts for the majority of RCC-related deaths. The lack of comprehensive diagnostic and prognostic biomarkers has limited further understanding of the pathophysiology of ccRCC. Super-enhancers (SEs) are congregated enhancer clusters that have a key role in tumor processes such as epithelial-mesenchymal transition, metabolic reprogramming, immune escape and resistance to apoptosis. RCC may also be immunogenic and sensitive to immunotherapy. In the present study, an Arraystar human SE-long non-coding RNA (lncRNA) microarray was first employed to profile the differentially expressed SE-lncRNAs and mRNAs in 5 paired ccRCC and peritumoral tissues and to identify SE-related genes. The overlap of these genes with immune genes was then determined to identify SE-related immune genes. A model for predicting clinical prognosis and response to immunotherapy was built following the comprehensive analysis of a ccRCC gene expression dataset from The Cancer Genome Atlas (TCGA) database. The patients from TCGA were divided into high- and low-risk groups based on the median score derived from the risk model, and the Kaplan-Meier survival analysis showed that the low-risk group had a higher survival probability. In addition, according to the receiver operating characteristic curve analysis, the risk model had more advantages than other clinical factors in predicting the overall survival (OS) rate of patients with ccRCC. Using this model, it was demonstrated that the high-risk group had a more robust immune response. Furthermore, 61 potential drugs with half-maximal inhibitory concentration values that differed significantly between the two patient groups were screened to investigate potential drug treatment of ccRCC. In summary, the present study provided a novel index for predicting the survival probability of patients with ccRCC and may provide some insights into the mechanisms through which SE-related immune genes influence the diagnosis, prognosis and potential treatment drugs of ccRCC.

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.

Mechanism of miRNA-141-3p in Calcium Oxalate-Induced Renal Tubular Epithelial Cell Injury via NLRP3-Mediated Pyroptosis.

BACKGROUND/AIMS: Renal calculi represent a prevalent disorder associated with mineral deposition in renal calyces and the pelvis. Aberrant microRNA (miRNA) expression is implicated in renal injury. This study investigated the mechanism of miR-141-3p in calcium oxalate (CaOx) crystal-induced renal tubular epithelial cell (RTEC) injury. METHODS: Human RTECs HK-2 cells were treated with CaOx crystals to induce RTEC injury. Cell viability was evaluated using Cell Counting Kit-8 assay, and apoptosis was measured using flow cytometry. The contents of lactate dehydrogenase (LDH), malondialdehyde (MDA), superoxide dismutase (SOD), interleukin (IL)-1ß, and IL-18 were measured using enzyme-linked immunosorbent assay kits. The expressions of NLRP3, cleaved caspase-1, and GSDMD-N were detected using Western blot. miR-141-3p and NLRP3 expressions were determined using reverse transcription quantitative polymerase chain reaction. The binding of miR-141-3p and NLRP3 was validated using a dual-luciferase assay. The role of NLRP3 in the protection of miR-141-3p on RTEC injury was verified using functional rescue experiments. RESULTS: CaOx crystals induced RTEC injury, manifested as attenuated cell viability, enhanced apoptosis, elevated intracellular LDH and MDA levels, and decreased SOD level. Pyroptosis of RTECs was enhanced by CaOx crystal induction, evidenced by elevated expressions of cleaved caspase-1, GSDMD-N, IL-1ß, and IL-18. miR-141-3p expression was reduced in CaOx crystal-induced RTECs. miR-141-3p overexpression alleviated CaOx crystal-induced RTEC injury and suppressed pyroptosis of RTECs. miR-141-3p bound to NLRP3 and thereby repressed NLRP3 expression. NLRP3 overexpression reversed the protective effect of miR-141-3p overexpression on RTECs. CONCLUSION: miR-141-3p repressed NLRP3-mediated pyroptosis by suppressing NLRP3 expression, thus protecting CaOx crystal-induced RTEC injury.

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.

MRP-1 and BCRP Promote the Externalization of Phosphatidylserine in Oxalate-treated Renal Epithelial Cells: Implications for Calcium Oxalate Urolithiasis.

OBJECTIVE: To investigate the possible involvement of multidrug resistance-associated protein 1 (MRP-1) and breast cancer resistance protein (BCRP) in the oxalate-induced redistribution of phosphatidylserine (PS) in renal epithelial cell membranes. METHODS: A western blot analysis was used to examine the MRP-1 and BCRP expression levels. Surface-expressed PS was detected by the annexin V-binding assay. The cell-permeable fluorogenic probe 2,7-dichlorofluorescein diacetate was used to measure the intracellular reactive oxygen species (ROS) level. A rat model of hyperoxaluria was obtained using 0.5% ethylene glycol and 1.0% ammonium chloride. In addition, certain animals received verapamil (50 mg/kg body weight), which is a common inhibitor of MRP-1 and BCRP. The degree of nephrolithiasis was assessed histomorphometrically using sections stained by Pizzolato method and by measuring the calcium oxalate crystal content in the renal tissue. RESULTS: Oxalate produced a concentration-dependent increase in the synthesis of MRP-1 and BCRP. Treatment with MK571 and Ko143 (MRP-1- and BCRP-specific inhibitors, respectively) significantly attenuated the oxalate-induced PS externalization. Adding the antioxidant N-acetyl-l-cysteine significantly reduced MRP-1 and BCRP expression. In vivo, markedly decreased nephrocalcinosis was observed compared with that in the rat model of hyperoxaluria without verapamil treatment. CONCLUSION: Oxalate induces the upregulation of MRP-1 and BCRP, which act as phospholipid floppases causing PS externalization in the renal epithelial cell membrane. The process is mediated by intracellular ROS production. The ROS-mediated increase in the synthesis of MRP-1 and BCRP can play an important role in hyperoxaluria-promoted calcium oxalate urolithiasis by facilitating phosphatidylserine redistribution in renal epithelial cells.

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.

Externalization of phosphatidylserine via multidrug resistance 1 (MDR1)/P-glycoprotein in oxalate-treated renal epithelial cells: implications for calcium oxalate urolithiasis.

OBJECTIVES: We investigated the possible involvement of multidrug resistance protein 1 P-glycoprotein (MDR1 P-gp) in the oxalate-induced redistribution of phosphatidylserine in renal epithelial cell membranes. METHODS: Real-time PCR and western blotting were used to examine MDR1 expression in Madin-Darby canine kidney cells at the mRNA and protein levels, respectively, whereas surface-expressed phosphatidylserine was detected by the annexin V-binding assay. RESULTS: Oxalate treatment resulted in increased synthesis of MDR1, which resulted in phosphatidylserine (PS) externalization in the renal epithelial cell membrane. Treatment with the MDR1 inhibitor PSC833 significantly attenuated phosphatidylserine externalization. Transfection of the human MDR1 gene into renal epithelial cells significantly increased PS externalization. CONCLUSIONS: To our knowledge, this study is the first to show that oxalate increases the synthesis of MDR1 P-gp, which plays a key role in hyperoxaluria-promoted calcium oxalate urolithiasis by facilitating phosphatidylserine redistribution in renal epithelial cells.

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.

Expressions of voltage-gated K+ channel 2.1 and 2.2 in rat bladder with detrusor hyperreflexia.

BACKGROUND: Voltage-gated K+ channel (Kv) plays a critical role in the modulation of detrusor contraction. This study was conducted to investigate the expressions of Kv2.1 and Kv2.2 in rat bladder with detrusor hyperreflexia (DH). METHODS: Thirty adult female Sprague-Dawley rats (200-220 g) were randomly divided into the control group and the experimental group. The experimental group was subjected to spinal cord injury (SCI). In the controls, the surgical procedure was identical with the exception that dura and spinal cord were transected. Four weeks after SCI, in vivo cystometry and mechanical pulling tests of isolated detrusor strips were performed. mRNA was extracted from the detrusors of normal and DH rats for the detection of expression of Kv2.1 and Kv2.2 by RT-PCR. Differences in expression between normal and overactive detrusors were identified by gel imaging. RESULTS: Fourteen rats in the experimental group exhibited uninhibited bladder contraction (>8 cmH2O) before voiding after SCI. One rat died from infection. The frequency of DH in the experimental group was significantly different from that in the control group with or without treatment with 4-aminopyridine (4-AP) (P < 0.05), while the amplitude of DH did not change markedly. The rates of variation of the automatic contractile frequency and amplitude were (66.8 +/- 12.4)% and (42.6 +/- 12.6)% respectively in the control group, and (38.4 +/- 9.8)% and (28.0 +/- 4.6)% respectively in the DH group. 4-AP increased the automatic contractile frequency apart from the automatic contractile amplitude in both the control and DH groups (P < 0.05). 4-AP increased the rate of variation of the automatic contractile frequency more markedly in the control group than in the DH group (P < 0.05). Significant expression of Kv2.2 was not detected in bladders in the control group. Compared to the mRNA levels of beta-actin, the mRNA level of Kv2.1 was 1.26 +/- 0.12 in the control group and 0.66 +/- 0.08 in the DH group. SCI significantly reduced the mRNA level of Kv2.1 in rat bladders with DH (P < 0.05). CONCLUSIONS: Our study showed that the mRNA level of Kv2.1 decreased significantly in rat bladder with DH, which was one of the important pathogenetic mechanisms for DH, and suggested that Kv2.1 might be one of the therapeutic targets for bladder overactivity.

Effects of alcohol intake on penile structure and function in rats.

OBJECTIVE: To investigate the effects of alcohol intake on penile structure and function in rats. METHODS: Thirty adult male Wistar rats were randomly divided into two groups: control group and alcohol intake group. They were administered with 2 mL of normal saline and 40% alcohol solution respectively through gastric tubes every day. Three months later, the animal model of alcohol intake was evaluated by modified Nayagida's method, and the effects of alcohol on the rats were studied by sexual behavior, the number of apomorphine-induced penile erection, level of testosterone in the sera, and the content of penile smooth muscle. RESULTS: The scores of animal model of alcohol intake evaluated by Nayagida's method were 0.66 +/- 2.05 in the control group and 9.26 +/- 5.50 in the alcohol intake group (P < 0.05), which indicated that an animal model of alcohol intake was successfully established. Sexual behavior, the number of apomorphine-induced penile erection, testosterone level in the sera, and the content of penile smooth muscle of the alcohol intake group were all statistically different as compared with the control group (P < 0.05). CONCLUSION: Alcohol intake induces sexual dysfunction in rats, which may be due to the decline of testosterone level in the sera and decline of penile smooth muscle.

[The effect of ethanol on sexual function of males and its mechanism].

Though an adequate volume of ethanol relieves nervousness and enhances sexual desire,long term and excessive intake of ethanol can induce sexual dysfunction. The reasons that ethanol results in sexual dysfunction are as follows: ethanol inhibits the hypothalamo-pituitary-testes axis and decreases serum testosterone level. The decline of smooth muscle, choline acetyltransferase and nitric oxide synthase in the penis may be responsible for it.

[Relationship between ethanol intake and sexual function in rats].

OBJECTIVE: To investigate the relationship between the intake of ethanol and sexual function of rats. METHODS: Sixty adult male Wistar rats were randomly divided into five groups: the control, 10% , 20% , 30% and 40 ethanol groups, which received. . 9% sodium chloride, 10% , 20% , 30% and 40% ethanol solutions respectively at a dose of 2 ml through gastric tubes once a day. Three months later, we observed the effects of ethanol on the sexual function of the rats by their sexual behaviors, the number of apomorphine-induced penile erections, and the content of testosterone in the serum and nitric oxide synthase ( NOS) in the penis. RESULTS: Compared with the control group, the number of apomorphine-induced penile erections in the 10% and 20% ethanol groups was not inhibited significantly (P > 0.05), but the latent period of mounting and intromission in the 10% ethanol group was prolonged and the sexual behaviors in the 20% ethanol group were inhibited except the latent period of ejaculation. The sexual behaviors and the number of apomorphine-induced penile erections of the 30% and 40% ethanol groups were inhibited significantly (P < 0.05). Testosterone in the serum and NOS activity in the penis of the experimental groups were reduced (Pat < 0.05). CONCLUSION: An adequate volume of ethanol does not induce sexual dysfunction in rats, but long term and excessive intake of ethanol may cause penile erectile dysfunction.

[The dynamic effects of allogenic transplantations with the bladder acellular matrix grafts of rabbits].

OBJECTIVE: To study the dynamic effects of allogenic transplantations with the bladder acellular matrix grafts (BAMG) of rabbits. METHODS: Hemi-cystectomies were performed in 25 rabbits, and the defects were repaired with BAMG about half bladder size. The rabbits underwent postoperative assessment of bladder function at 8 weeks, including cystometry, vesical volume, vesical compliance and cystography. The allografts were observed by light microscope and electron microscope at 1, 2, 4, 8, 12, 16 weeks after surgery. RESULTS: Macroscopic observation revealed that BAMG regenerated gradually. All urodynamic results of 8 weeks after surgery were not different statistically as compared with these of preoperation (P > 0.05). Cystography revealed that the morphous of bladder was recovered. Epithelialization and neovascularity occurred accompanied by infiltration of inflammatory cell at 1 week. Smooth muscle cell and stratified epithelium regenerated 2 weeks after grafting. Neural elements formed around smooth muscle bundles as early as 4 weeks. Each component regenerated on the frame of BAMG sequentially. After 16 weeks, it was difficult to delineate the junction between the host bladder and BAMG by histology. CONCLUSION: After allogenic transplantation with rabbits' BAMG, the constitution and function of the allografts regenerate completely and gradually on the frame of BAMG.