OBJECTIVE:To prepare and characterize Fluorescent dye 1,1′-octacosyl-3,3,3′,3′-tetramethylindocarbocyanine iodide(DiR)-loading polyethylene glycol-poly lactic-co-glycolic acid(DiR-PEG-PLGA)nanocapsules,and to evaluate its biocompatibility in vitro. METHODS:Using PLGA and PEG-PLGA as carrier,DiR-PEG-PLGA nanocapsules were prepared by modified ultrasonic emulsification method. The particle size,Zeta potential,morphology,stability and fluorescence in vitro of nanocapsules were detected respectively. MTT assay was used to evaluate cytotoxicity in vitro of nanocapsules to human-derived HL7702 hepatocytes,and hemolysis test was carried out to investigate its hemolysis effects. RESULTS:Prepared DiR-PEG-PLGA nanocapsules were spherical with a clear core-shell structure. The average particle size was(507.53 ± 7.87)nm,polydispersity coetficient of particle size was 0.306 1±0.001 5 and Zeta potential was(-35.20±0.92)mV with good stability within 6 months under 4℃. Fluorescence signal intensity(y)of nanocapsules was increased linearly with DiR mass concentration(x)in vitro. The linear eguation was y=0.345 2x+0.433 4(R2=0.997 3).The toxicity of nanocapsules to HL7702 cells was between 0-1 degree,and no hemolytic effect was observed. CONCLUSIONS:The study successfully prepare fluorescent DiR-PEG-PLGA nanocapsules with high biocompatibility in vitro,which is further expected to become a safe optical drug carrier.

Objective To evaluate the changes of left ventricular(LV) 3-dimension peak displacement (3D-D) during different cardiac pacing patterns. To provide a reliable mechanical data base for the optimization cardiac pacing. Methods Cardiac pacings in open-chest Beagle canine models( n = 10) were performed using three patterns[I, e. , right ventricular apical pacing (RVA-P), LV lateral pacing (LVL-P)and LV apical pacing(LVA-P)],3D full volumetric real-time imaging were acquired in a completed cardiac cycle. The 3D-D,3D-D peak time (3D-DTc) and the standard deviation of TC(3D-DTSD) were calculated and analyzed in different pacing patterns for difference and spatial correlationship. Results ① The 3D-D of LVL-P and LVA-P state decreased compared with BASE and RVA-P state, there were significant 3D-D difference of mid anterior,mid anteriorspetal, mid interior,mid posterior, mid lateral between LVL-P and BASE, RVA-P patterns( P ＜0.05). There were significant 3D-D difference of mid anterior,mid lateral,mid posterior between LVA-P and RVA-P patterns groups( P ＜0.05). There were significant 3D-D difference of all segments in apical level between LVL-P,LVA-P and BASE, RVA-P states( P ＜0.05). ② Corrected by the heart rate,the 3D-DTC of different cardiac pacing patterns were shorter than BASE state. ③ There were no significant 3D-DTSD difference between different cardiac pacings and BASE patterns. There were significant 3D-DTSD difference between RVA-P and LVA-P patterns (P ＜ 0.05). Conclusions LV mechanical activation and synchronization could be maintained during RVA-P rather than LVA-P and LVL-P. Echocardiographic study of left ventricular 3D-D can actually reveal myocardial mechanical state during different cardiac pacings and BASE patterns.

Objective To access the effects of aldosterone (ALD) and its receptor antagonist- spironolactone (SPI) on the production of reactive oxygen species (ROS) and apoptosis in podocytes and to explore the possible mechanism involved. Methods Conditionally immortalized mouse podocytes were divided into control group, ALD group, SPI group and SPI +ALD group. The level of ROS production and the expression of nephrin protein were assayed by fluorescent spectrophotometry and indirect immunofluorescence technology. The apoptosis rate of podocytes was monitored by flow cytometry. The expression of bax and bcl-2 mRNA and protein was detected by RT-PCR and Western blotting methods. The anti-oxidant N-acetylcysteine (NAC)was applied to determine whether the effects of ALD on podocytes were mediated by ROS pathway.Results Compared with the control group, ALD significantly increased ROS production in podocytes (P＜0.05). SPI completely abolished the above-mentioned effect of ALD (P＜0.05). ALD induced the down-regulation of the expression of nephrin and the up-regulation of podocytes apoptosis (P＜0.05), which was accompanied with the elevated expression of bax mRNA and protein and the reduced expression of bcl-2 mRNA and protein (P＜0.05). SPI or NAC prevented the above-mentioned changes induced by ALD (P＜0.05). Conclusion ALD increases theexpression of pro-apoptotic factor (bax) but decreases the expression of anti-apoptotic factor (bcl-2)to induce podocytes apoptosis through the mineralocorticoid receptor (MR) possibly via the mechanisms involving ROS pathway.

This study examined the effect of sulodexide on podocyte injury in rats with adriamycin nephropathy (AN). A total of 36 healthy male SD rats were randomly assigned to three groups:control group,AN group and sulodexide treatment group. Rat models of AN were established by a single tail intravenous injection of adriamycin (6.5 mg/kg) in both AN group and sulodexide treatment group. Sulodexide (10 mg/kg) was administered the rats in the treatment group once daily by garage from the fast day of model establishment until the 14th day or the 28th day. Samples of 24-h urine and renal cortex tissues were harvested at day 14,28 after the model establishment. Excretion of 24-h urinary protein was measured by Coomassie brilliant blue method. The pathological changes in renal tissues were observed by light microscopy and electron microscopy respectively. Heparanase mRNA was detected by RT-PCR. Expressions of desmin,CD2AP and heparanase were determined by immunohistological staining. The results showed that the expressions of heparanase mRNA and proteinwere increased in the glomeruli of AN rats at day 14 and 28 after the model establishment,which was accompanied by the increased expression of desmin and CD2AP. The mRNA and protein expression of heparanase was decreased in the sulodexide-treated rats as compared with AN rats at day 14 and 28. And,the protein expression of desmin and CD2AP was reduced as with heparanase in the sulodexide-treated rats. Proteinuria and podocyte foot process effacement were alleviated in the AN rats after sulodexide treatment. There was a positive correlation between the expression of heparanase and the expression of desmin and CD2AP (as well as 24-h urinary protein excretion). It was concluded that increased heparanase is involved in podocyte injury. Sulodexide can maintain and restore podocyte morphology by inhibiting the expression of heparanase in AN.

Purpose To observe the pathologic types of glomerular diseases which have high renal interstitial foam cells infiltration and to evaluate the relationship between infiltration of foam cells (FC) in renal interstitial tissue and pathologic parameters.Methods A total of 2 862 patients who had received renal biopsy were enrolled in this study. Patients with Alport syndrome (AS,n=5), membranous proliferative glomerular nephritis (MPGN,n=28), focal segmental glomerulosclerosis (FSGS,n=144), idiopathic membranous nephropathy (IMN,n=132), IgA nephropathy (IgAN,n=893) were divided into two groups:FC+ group with foam cells and FC- group without foam cells.Results Infiltration of foam cells in renal interstitial tissue was commonly seen in AS.The frequency of interstitial foam cells was 46.43% in MPGN, 20.14% in FSGS, 13.64% in IMN, and 6.27% in IgAN. It was found that the segmental glomerular sclerosis and interstitial fibrosis were more severe in FC+ group than that in FC- group.Conclusions The renal interstitial foam cells are most common in patients with AS, but also seen in patients with MPGN, FSGS, IMN and IgAN. There might be a relationship between glomerular sclerosis, interstitial fibrosis and infiltration of foam cells. The present of foam cells in the renal interstitial tissue may contribute to the progression of renal diseases.

This study examined the effect of sulodexide on podocyte injury in rats with adriamycin nephropathy (AN). A total of 36 healthy male SD rats were randomly assigned to three groups: control group, AN group and sulodexide treatment group. Rat models of AN were established by a single tail intravenous injection of adriamycin (6.5 mg/kg) in both AN group and sulodexide treatment group. Sulodexide (10 mg/kg) was administered the rats in the treatment group once daily by garage from the first day of model establishment until the 14th day or the 28th day. Samples of 24-h urine and renal cortex tissues were harvested at day 14, 28 after the model establishment. Excretion of 24-h urinary protein was measured by Coomassie brilliant blue method. The pathological changes in renal tissues were observed by light microscopy and electron microscopy respectively. Heparanase mRNA was detected by RT-PCR. Expressions of desmin, CD2AP and heparanase were determined by immunohistological staining. The results showed that the expressions of heparanase mRNA and protein were increased in the glomeruli of AN rats at day 14 and 28 after the model establishment, which was accompanied by the increased expression of desmin and CD2AP. The mRNA and protein expression of heparanase was decreased in the sulodexide-treated rats as compared with AN rats at day 14 and 28. And, the protein expression of desmin and CD2AP was reduced as with heparanase in the sulodexide- treated rats. Proteinuria and podocyte foot process effacement were alleviated in the AN rats after sulodexide treatment. There was a positive correlation between the expression of heparanase and the expression of desmin and CD2AP (as well as 24-h urinary protein excretion). It was concluded that increased heparanase is involved in podocyte injury. Sulodexide can maintain and restore podocyte morphology by inhibiting the expression of heparanase in AN.

Objective To study the effects of CD2-associated protein (CD2AP) on podocyte adhesion and extension ability and to explore its possible mechanism. Methods Conditionally immortalized murine podocyte cell line was cultured in RPMI 1640 medium at 33℃permissive conditions. The podocytes were transfected with CD2AP small interfering RNA (siRNA) and serambing sequences labeled with fluorescein were taken as control. The transfected podocytes were trypsinized and seed into collagen IV coated plates. The relative cell adhesion and cell area were examined 90 min later. Apoptotic rates of CD2AP siRNA transfected podoeytes and different PAN concentrations incubated podoeytes were detected by flow cytometer. The distribution of F-actin was observed under laser scanning confoeal microscope. Nephrin protein expression and its phosphorylation level were examined by immunofluorescence and Western blot. Results The relative ceil adhesion of CD2AP siRNA transfected podocytes was apparently lower than that of control group[(41.72±6.07)% vs (64.46±8.53)%, P<0.05]. The cell area analysis had the similar result. The apoptotic rate of CD2AP siRNA transfected podocytes was significantly higher than that of the controls [(5.73±0.61)% vs (3.26±0.45)%, P<0.05]. 100 mg/L PAN could markedly induce podocytes to apoptosis and impair cell adhesion ability (P<0.05). Nevertheless, no significant difference was found in cell body spreading (P>0.05). The distribution of F-actin in CD2AP depletion podocytes was apparently altered. The expression of nephrin protein and its phosphorylation level was conspicuously descended to some degree (P<0.05). Conclusions CD2AP depletion facilitates podocyte apoptosis and impairs cell adhesion function. Cytoskeleton confusion and nephrin signaling weakness caused by CD2AP depletion may he partly responsible for the decline of cell adhesion and spreading.

Objective To evaluate the mechanical homogeneity pattern of isolated carotid atherosclerotic plaque intima on longitudinal axis view using ultrasonic velocity vector imaging.Methods Fourty six patients with 48 isolated plaques were undergone the high frequency ultrasound scanning,the real-time two-dimensional longitudinal-axis gray scale view at the maximal thickness of the plaque was obtained in three complete cardiac cycles.A dedicated velocity vector imaging(ⅤⅤⅠ)workstation was used for the off-line dynamic two-dimensional gray-scale image analysis and mechanics parameter(i.e.,the peak strain and the absolute difference of the peak strain)assessment at the sites of the upper,middle and down stream separately.The length and direction distribution of the velocity vectors at the carotid atherosclerotic plaque intima were observed.Wilcoxon test was utilized for the comparison of the peak strain and the absolute difference of the peak strain between two sites.Results The chaos phenomenon of velocity vector distribution at the sites of plaque intima occurred significantly differed from those uniform velocity vectors at the reference sites.According to the two groups of the higher strain value side and the lower strain value side on the upper stream and the down stream of the plaque intima,the peak strain at the higher strain value side was significantly higher than the lower strain value side(P=0.000).There was a significantly difference between the absolute difference of peak strain from the higher strain value side to the middle stream point of the plaque intima and the absolute difference of peak strain from the lower strain value side to the higher strain value side of the plaque intima(P＜0.017).Conclusions The length and direction distribution of velocity vectors of the isolated carotid atherosclerotic plaque intima on longitudinal axis view are uneven,the mechanical heterogeneity pattern exists at the plaque intima and could be visualized and quantitatively evaluated using ultrasonic velocity vector imaging.

In current study, the effect of angiotensin receptor blocker Micardis on the localization and expression of aquaporin-2 (AQP2) was investigated in the renal medullary collecting duct of mice with diabetic nephropathy (DN). Mice were divided into three groups: normal group, DN group and Micardis-treated group. Six weeks after establishment of STZ-induced DN model in mice, the expression of AQP2 in renal medulla was detected measured by semiquantitative immunofluorescence histochemistry and Western blot techniques, and the localization of AQP2 by confocal immunofluorescence laser scanning microscopy. The results showed that the urinary osmolality was decreased in DN group as compared with normal group (2.39±0.11 vs 3.16±0.16, P<0.05). Although the localization of AQP2 on the renal medulla was unchanged, the expression of AQP2 was increased significantly in DN group as compared with normal group. Micardis could partly attenuate above changes. It was concluded that treatment with Micardis could partly rectify the abnormal expression of AQP2 in renal medulla of DN mice, which suggested that rennin-angiotensin system (RAS) is implicated in the pathogenesis of DN by regulating the expression of AQP2.

Summary: The role of serum and glucocorticoid-induced kinase 1 (SGK1) pathway in the connective tissue growth factor (CTGF) expression was investigated in cultured human mesangial cells (HMCs) under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with pIRES2-EGFP-S422D hSGK1 (SD) could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with pIRES2-EGFP (FP) under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with pIRES2-EGFP-K127N hSGK1 (KN) could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.