Circ_UBR4 Knockdown Alleviates Oxidized Low-Density Lipoprotein-Provoked Growth and Migration of Human Vascular Smooth Muscle Cells by Acting on the miR-637/FOXO4 Pathway.

ABSTRACT: Excessive proliferation and migration of human vascular smooth muscle cells (HVSMCs) induced by oxidized low-density lipoprotein (ox-LDL) are important pathological features of atherosclerosis. Emerging evidence indicates that circular RNAs deregulation is involved in this pathological process. The objective of this study was to explore the role of circular RNA ubiquitin protein ligase E3 component n-recognin 4 (circ_UBR4) in ox-LDL-treated HVSMCs. The expression of circ_UBR4, microRNA-637 (miR-637), and forkhead box O4 (FOXO4) mRNA was detected by quantitative real-time PCR. Cell cycle progression was examined by flow cytometry assay. Cell viability was examined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell migration was examined by transwell assay. The protein levels of proliferating cell nuclear antigen, matrix metalloproteinase 2, and FOXO4 were measured by western blot. The relationship between miR-637 and circ_UBR4 or FOXO4 was confirmed by dual-luciferase reporter assay. The results presented that the expression of circ_UBR4 was increased in atherosclerosis serum samples and ox-LDL-treated HVSMCs. Cell cycle progression, cell proliferation, and cell migration were promoted by ox-LDL, whereas circ_UBR4 knockdown inhibited HVSMCs proliferation and migration. MiR-637 was a target of circ_UBR4, and FOXO4 was a target of miR-637. Circ_UBR4 positively regulated FOXO4 expression by targeting miR-637. Circ_UBR4 knockdown-inhibited HVSMCs proliferation and migration were recovered by miR-637 inhibition, and miR-637 restoration-inhibited HVSMCs proliferation and migration were recovered by FOXO4 overexpression. In conclusion, circ_UBR4 knockdown inhibited ox-LDL-induced excessive proliferation and migration of HVSMCs by regulating FOXO4 via targeting miR-637.

Role of tea polyphenols in delaying hyperglycemia-induced senescence in human glomerular mesangial cells via miR-126/Akt-p53-p21 pathways.

PURPOSE: The aim of this study was to investigate the effects and possible mechanism of tea polyphenols (TPs) on the senescence of human glomerular mesangial cells (HGMCs) under high glucose conditions. METHODS: HGMCs were divided into the normal group (NG, 5.5 mmol/L glucose), mannitol group (MNT, 5.5 mmol/L glucose and 24.5 mmol/L mannitol), TP group (TP, 30 mmol/L glucose and 5 µg/mL TP) and high-dose D-glucose group (HG, 30 mmol/L glucose). The effects of TP on the cell morphology of HGMCs; the percentage of cells positive for senescence-associated ß-galactosidase (SA-ß-gal); the ratio of G1 phase of cell cycle; telomere length; and the expression of p-Akt, p53, p21 and Rb proteins of the Akt-p53-p21 signaling pathway and the expression miR-126 were examined. RESULTS: High glucose led to premature senescence of HGMCs, as evident from the increase in the percentage of SA-ß-gal-positive cells, decrease in telomere length, cell cycle arrest at G1 phase,decrease in the expression of miR-126 and p-Akt and increase in the expression of p53, p21 and Rb proteins in the HG group. In contrast, in the TP group, these effects of high glucose treatment were abrogated and this indicates that TP had a protective effect on HGMCs. CONCLUSIONS: High glucose induces the senescence of HGMCs in vitro via the miR-126 and Akt-p53-p21 signaling pathways. TP can delay the high glucose-induced senescence of HGMCs by regulating the activity of these signaling pathways. Thus, the polyphenols present in tea may have potential for the treatment of diabetic nephropathies associated with premature senescence.

Soluble Vascular Cell Adhesion Molecule-1 Is Associated With Disease Activity in Adult-Onset Minimal Change Disease.

BACKGROUND: Cell adhesion molecules have been documented to be elevated in numerous immune inflammatory diseases. Minimal change disease (MCD) is an immune disorder. This study aimed to evaluate whether levels of soluble vascular cell adhesion molecule-1 (sVCAM-1) and soluble E-selectin (sE-selectin) reflect disease activity in adult-onset MCD. METHODS: A sandwich enzyme-linked immunosorbent assay was used to measure the soluble adhesion molecules in 40 patients with nephrotic-range proteinuria and biopsy-proven MCD, obtained at the time of diagnosis and during remission. Thirty-five age- and sex-matched healthy volunteers served as controls. RESULTS: Patients with MCD during the active stage showed significantly higher levels of sVCAM-1 and sE-selectin when compared to controls. Moreover, sVCAM-1 had significantly positive correlations with both urine protein and serum cholesterol, and was negatively associated with serum albumin. Multiple analyses showed that serum albumin was an independent predictor of sVCAM-1. The correlations between sE-selectin and other clinical parameters were not statistically significant. At follow-up, these markers systematically decreased as the disease went into remission, but the increase in sVCAM-1 persisted even in patients obtaining complete remission for 6 months. CONCLUSIONS: Patients with active MCD had increased levels of sVCAM-1 and sE-selectin. The correlation between sVCAM-1 and proteinuria, serum albumin and cholesterol and its decline during remission indicate that sVCAM-1 is associated with disease activity.

Clinical Significance of Urinary Biomarkers in Patients With Primary Focal Segmental Glomerulosclerosis.

BACKGROUND: Focal segmental glomerulosclerosis (FSGS) is often accompanied with tubulointerstitial lesion. This study aimed to assess the role of urinary biomarkers in predicting tubulointerstitial lesion and treatment response in FSGS patients. METHODS: Urinary neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), N-acetyl-ß-d-glucosaminidase (NAG) and retinol-binding protein (RBP) were measured in 32 FSGS patients and 22 patients with minimal change nephrotic syndrome. Patients with FSGS were followed up to investigate the value of these markers in predicting treatment response. RESULTS: FSGS patients had higher urinary NGAL, NAG and RBP than patients with minimal change nephrotic syndrome with comparable proteinuria. A cutoff value of 15.87ng/mL NGAL demonstrated 87.1% sensitivity and 59.1% specificity for the diagnosis of FSGS, with an area under the receiver operator characteristic curve of 0.801. In FSGS, these markers correlated significantly with the degree of acute tubulointerstitial damage but not with chronic tubulointerstitial lesion. Response to immunosuppressive therapy was significantly different in patients with KIM-1, NAG and RBP levels below and above the cutoff values. CONCLUSIONS: Urinary NGAL, KIM-1, NAG and RBP are reliable biomarkers of tubulointerstitial lesion in FSGS patients. The measurements of these markers may be useful in diagnosing FSGS, detecting acute tubulointerstitial lesion and predicting treatment response.

Vitamin D supplementation improves endothelial dysfunction in patients with non-dialysis chronic kidney disease.

PURPOSE: Hypovitaminosis D is common in chronic kidney disease (CKD) and is associated with endothelial dysfunction and cardiovascular events. This study aimed to investigate the effects of vitamin D supplementation on endothelial dysfunction in non-dialysis CKD patients. MATERIALS AND METHODS: Seventy-one non-dialysis CKD patients with low vitamin D (serum 25(OH)D < 30 ng/mL) were recruited. Patients received oral cholecalciferol 50,000 units once a week for 12 weeks. Changes in endothelial function by brachial artery flow-mediated dilation (FMD), soluble vascular cell adhesion molecule-1 (sVCAM-1), and sE-selectin were studied. RESULTS: There was a significant increase in serum levels of 25(OH)D after cholecalciferol supplementation (33.7 ± 12.1 vs. 13.2 ± 5.4 ng/mL, P < 0.001). Multivariable regression analysis showed that higher proteinuria (ß = - 0.548, P < 0.001) and lower levels of 25(OH)D (ß = 0.360, P < 0.001) at baseline were related to lower 25(OH)D level after supplementation. FMD increased significantly from 4.4 ± 1.3 to 5.1 ± 1.5% (P < 0.001), and soluble endothelial biomarkers decreased: sVCAM-1 from 926.9 ± 158.0 to 867.0 ± 129.0 ng/mL (P < 0.001), and sE-selectin 69.7 ± 15.8 to 63.3 ± 14.7 ng/mL (P < 0.001). CONCLUSIONS: Vitamin D supplementation can improve endothelial dysfunction in pre-dialysis CKD patients.

Peritoneal catheter fixation combined with straight upward tunnel and low implant position to prevent catheter malfunction.

AIM: Catheter malfunction is the main reason for early peritoneal dialysis (PD) technique failure. This study aimed to evaluate the effect of a new surgery technique with catheter fixation to the lower abdominal wall combined with straight upward tunnel and low implant position in reducing catheter malfunction. METHODS: Patients with end stage renal disease who received PD in our centre from January 2013 to December 2015 were involved in this study. They were randomly divided into three groups according to surgical technique: traditional open surgery group, modified open surgery group and modified open surgery with catheter fixation group. All patients were followed up for six months after surgery. Catheter- related complications were analyzed. RESULTS: A total of 152 patients were involved. Among them, 49 received traditional open surgery (TOS group), 49 received modified open surgery (MOS group), and 54 received modified open surgery with catheter fixation (MOS-F group). During follow-up, no patients (0%) in MOS-F group developed catheter malfunction which was significantly lower than that of the TOS group (0 vs 16.33%, P = 0.002). Although not statistically significant, the incidence of catheter malfunction was lower in MOS-F group than that in MOS group (0 vs 4.08%, P = 0.134). No significant difference was observed in the episodes of infection, bleeding, leakage, inflow or outflow pain, hernia and delayed wound healing among the three groups (all P > 0.05). CONCLUSIONS: Catheter fixation combined with straight upward tunnel and low implant position can effectively prevent catheter malfunction in PD catheter placement.

Hypovitaminosis D is associated with endothelial dysfunction in patients with non-dialysis chronic kidney disease.

OBJECTIVE: Cardiovascular events are highly prevalent in chronic kidney disease (CKD). Hypovitaminosis D and vascular endothelial dysfunction are risk factors for cardiovascular morbidity and mortality and they both are common in CKD patients. This study aimed to investigate the association between hypovitaminosis D and endothelial dysfunction in non-dialysis CKD patients. METHODS: In 117 non-dialysis CKD patients, we assessed endothelial function by brachial artery flow-mediated dilation (FMD), soluble vascular cell adhesion molecule-1 (sVCAM-1) and sE-selectin. 25-hydroxyvitamin D [25(OH)D] was measured by electrochemiluminescence immunoassay. RESULTS: Brachial artery FMD was lower in vitamin D-deficient and -insufficient versus vitamin D-sufficient groups, with the lowest value observed in the vitamin D-deficient group. Conversely, sVCAM-1 and sE-selectin were higher in vitamin D-deficient and -insufficient groups versus vitamin D-sufficient, and the highest value was observed in the vitamin D-deficient group. There was a positive association between FMD and 25(OH)D (r = 0.556, p < 0.001) and negative correlations between both sVCAM-1 (r = -0.549, p < 0.001) and sE-selectin (r = -0.360, p < 0.001) and 25(OH)D. These associations remained significant after adjusting for confounders. CONCLUSIONS: Hypovitaminosis D is associated with endothelial dysfunction in non-dialysis CKD patients. Further studies are needed to confirm whether vitamin D supplementation can improve endothelial function and reduce cardiovascular events in these patients.

[Proapoptotic effect of angiotensin II on renal tubular epithelial cells and protective effect of Cordyceps sinensis].

OBJECTIVE: To investigate the mechanism of the protective effect of Cordyceps sinensis (C. sinensis) on the apoptosis of cultured NRK-52E induced by angiotension II (AngII). METHODS: NRK-52E cells were incubated with C. sinensis (0, 5, 10, 20, and 40 mg/L) and 10(-8) mol/ L AngII for 24, 48, 72 h. The optimal concentration of C. sinensis was selected. Either NRK-52E cells were incubated with different doses of AngII (0, 10(-12), 10(-10), 10(-8), and 10(-6) mol/L) for 24 h, or with 10(-8) mol/L AngII for 24, 48, and 72 h, to observe the effect of AngII on the apoptosis of NRK- 52E cells. The optimal concentration and time of AngII were selected. In another experiment cells were divided into 5 groups: a control, AngII (10(-8) mol/L), AngII (10(-8) mol/L)+ C. sinensis (40 mg/ L), Ang II (10(-8) mol/L)+ fosinopril (10(-5) mmol/L), and Ang II (10(-8) mol/L)+ fosinopril (10(-5) mol/ L)+C. sinensis (40 mg/L). MTT assay was used to test the changes in the proliferation of NRK-52E cultured with different concentration of C. sinensis for 24, 48, 72 h. The Annecxin V-FITC and PI stainings were applied to detect the apoptosis rate induced by AngII by flow cytometer (FCM) and to determine the eddects of C. sinensis. The activity of caspase-3 was assayed by spectrophotometry. RESULTS: Certain concentrations of C. sinensis (10-40 mg/L) promoted the proliferation of NRK- 52E cells inhibited by AngII(P<0.05). AngII induced the apoptosis of NRK-52E in a dose and timedependent manner, accompanied with increased activity of caspase-3 (P<0.05). C. sinensis partially suppressed the apoptosis of NRK-52E induced by AngII, and declined the activity of caspase-3 (P<0.05). No significant difference was shown as between the fosinopril group and the fosinopril+C. sinensis group (P>0.05). CONCLUSION: C. sinensis can suppress the apoptosis of NRK-52E by AngII, and the protective effect of C. sinensis may be inhibiting the activation of caspase-3 during the AngII-induced apoptosis of NRK-52E.

Fosinopril and losartan regulate klotho gene and nicotinamide adenine dinucleotide phosphate oxidase expression in kidneys of spontaneously hypertensive rats.

BACKGROUND/AIMS: Klotho, a newly identified antiaging gene, predominantly detected in the kidney, has pleiotropic protective effects on kidney diseases. Several studies have confirmed the association between Klotho and oxidative stress. The present studies were performed to explore effects of fosinopril (Fos) and losartan (Los) on Klotho and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase expression in kidneys of spontaneously hypertensive rats (SHR). METHODS: Twenty-four male 22-week-old SHR were randomly divided into three groups: model group, Fos group and Los group. Wistar-Kyoto rats were taken as control. After 8 weeks, urinary N-acetyl-ß-D-glucosaminidase (NAGase), 24 h urinary protein (Upro), serum creatinine (Scr), blood urea nitrogen (BUN) and renal pathological changes were detected. Renal mRNA and protein expression of Klotho and three subunits of NADPH oxidase protein expression were evaluated. RESULTS: As compared to the model group, NAGase, Upro, Scr and BUN were decreased; the typical renal pathological damage was relieved in the Fos or Los group. Fos or Los inhibited the reduction of Klotho expression, and reduced the elevation of NADPH oxidase expression. CONCLUSION: Abnormal expression of Klotho and NADPH oxidase plays important roles in progression of hypertensive renal damage. Fos and Los can increase Klotho expression, and inhibit NADPH oxidase expression, which may be one of the mechanisms of their protective effects in hypertensive renal damage.

[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.

[Effect of cordyceps sinensis extract on Klotho expression and apoptosis in renal tubular epithelial cells induced by angiotensin II].

OBJECTIVE: To investigate the effect of cordyceps sinensis (CS) extract and losartan (Los) on the expression of Klotho (Kl), P53, P21, and apoptosis in renal tubular epithelial cell NRK-52E induced by angiotensin II (Ang II), and to elucidate its therapeutical mechanism in Ang II induced renal tubular epithelial cell apoptosis. METHODS: NRK-52E cells were incubated with CS with or without Ang II for 24 hours. Experimental groups were divided according to the increasing concentrations of CS:0 (serving as controls), 5, 10, 20, 40, and 80 mg/L. The optimal concentration of CS was selected and cells were divided into 5 groups: controls, Ang II (1*10(-8) mol/L), Ang II (1*10(-8) mol/L)+CS (40 mg/L), Ang II (1*10(-8) mol/L)+Los (1*10(-5) mol/L), and Ang II (1*10(-8) mol/L)+CS (40 mg/L)+Los (1*10(-5) mol/L). After 24 hours, cell proliferation was evaluated by MTT assay. The mRNA and protein expression of Kl, P53 and P21 were measured by RT-PCR. Activity of caspase-3 was evaluated by caspase-3 activity assay Kit. Cell apoptosis was determined by Annexin V-FITC/PI double staining and flow cytometry. RESULTS: Certain concentrations of CS promoted the proliferation of NRK-52E cells and increased cells proliferation inhibited by Ang II (P<0.01 or P<0.05 ). Ang II significantly down-regulated the mRNA and protein expression of Kl, and up-regulated the levels of P53 and P21. Caspase-3 activity and apoptotic rates were decreased, too (all P values<0.01). CS or/and Los significantly increased the expression of Kl mRNA and protein down-regulated by Ang II, decreased P53 mRNA and protein expression, P21 mRNA and protein expression,and inhibited caspase-3 activity and apoptotic rates(all P values<0.05). No cooperative effects were observed in the two drugs (P>0.05). CONCLUSION: CS can increase the expression of Kl down-regulated by Ang II, decrease P53 and P21 expression and caspase-3 activity, and reduce Ang II induced NRK-52E cell apoptosis, which may be part of its mechanism of the protective effects on hypertensive renal damage.

[Effects of fosinopril and losartan on the expression of Toll- like receptor 4 in renal tubular epithelia cells].

OBJECTIVE: To determine the mechanism of Toll-like receptor 4(TLR4) in hypertensive renal injury and the protective effect of fosinopril(Fos) and losartan(Los). METHODS: NRK-52E was incubated into 5 groups: NRK-52E (normal control), NRK-52E+AngII, NRK-52E+AngII+Fos(10(-5) mmol/L),and NRK-52E+AngII+Los(10(-5) mmol/L), NRK-52E +AngII+Fos(10(-5) mmol/L)+Los(10(-5) mmol/L). TLR4-specific RNAi plasmids were stably transfected into NRK-52E. After 24 h, TLR4, IL-6, and TNF-alpha mRNAs were examined by reverse transcription-polymerase chain reaction(RT-PCR). TLR4 proteins were detected by Western blot, NF-kappaB nuclear translocations were tested by immunocytochemistry,and IL-6 and TNF-alpha supernatant levels were tested by enzyme linked immuno-sorbent assay(ELISA). RESULTS: TLR4, NF-kappaB, IL-6,and TNF-alpha were highly expressed in AngII induced NRK-52E(P<0.01). In NRK-52E that was stably transfected TLR4-special RNAi plamids, TLR4 protein and mRNA expression were obviously inhibited(P<0.05). After stimulation by AngII, the TLR4, IL-6, TNF-alpha levels in the stabe transfection group were increased compared with the normal group(P<0.05). Fos or/and Los down-regulated TLR4, IL-6, and TNF-alpha expressions(P<0.05), but no cooperation was observed. CONCLUSION: TLR4 may lead to inflammatory reaction in hypertensive renal injury. Fos or/and Los can decrease the expressions of TLR4 and correlate inflammatory factors, which may be part of the renal protective mechanism.

[Effect of bizhongxiao decotion on TNF-alpha and interleukin-1beta in plasma of rats with C II-induced rheumatoid arthritis].

OBJECTIVE: To observe the influence of bizhongxiao decotion (BZXD) on the plasma TNF-alpha and IL-1beta in rats with C II-induced arthritis (CIA) and to explore the mechanism of BZXD in the treatment of rheumatoid arthritis. METHODS: We divided 75 rats into 4 groups randomly. The rat experimented arthritis model was established by subcutaneous injection with collagen II. The plasma TNF-alpha and IL-1beta levels were detected with the radio-immunity assay at different time spots. RESULTS: The incidence of arthritis in the rats immunized with C II was approximately 88%. The plasma TNF-alpha and IL-1beta levels of the model group, BZXD group and methotrexate (MTX) group were notably higher than those of the normal group (P < 0.05). The plasma TNF-alpha and IL-1beta levels of the model group were higher than those of the MTX control group and BZXD treatment group at different time spots (P < 0. 01). The plasma TNF-alpha and IL-1beta levels rose step by step, but those of the BZXD group and MTX group decreased gradually. Moreover, the plasma TNF-alpha and IL-1beta levels of the rats of BZXD group were lower than those of the MTX group (P < 0. 05). CONCLUSION: TNF-alpha and IL-1beta play a very important role in the formation and development of rheumatoid arthritis. Both BZXD and MTX can notably decrease the plasma TNF-alpha and IL-1beta levels, but the effect of BZXD is better than that of MTX.