Objective To investigate the impact of peritoneal albumin leakage on malnutrition-inflammation-atherosclerosis （MIA） syndrome in continuous ambulatory peritoneal dialysis (CAPD) patients. Methods A cross-sectional study of a cohort of 130 CAPD patients without edema or active infection was performed. In order to identify peritoneal transport characteristics in CAPD patients, a standard peritoneal equilibration test (PET) was carried out. For malnutrition and inflammation, serum albumin and high-sensitivity C-reactive protein (hs-CRP) levels were measured. Mean-carotid artery intima media thickness (IMT) was used to determine atherosclerosis. Residual glomerular filtration rate (rGFR) was defined as the average of 24-hour urinary urea and creatinine clearances. Results Pearson and Spearman correlation analysis showed that peritoneal albumin leakage amount was positively correlated with age, body mass index, night dwell time, blood glucose, 4 h D/P creatinine levels and hs-CRP levels (r=0.204, P<0.05；r=0.314, P<0.01; r=0.265, P<0.01; r=0.212, P<0.05; r=0.401, P<0.01; r=0.216, P<0.05); whereas it was negatively correlated with diastolic perssure, serum albumin levels, glucose level of dialyzate and peritoneal Kt/V(r=-0.209, P<0.05; r=-0.123, P<0.05; r=-0.271, P<0.01; r=-0.212, P<0.01). Overall, there was no correlation between peritoneal albumin leakage and IMT. Patients were further stratified into tertiles according to rGFR. IMT of those with rGFR <1 ml·min^－1·（1.73 m²）^－1 was significantly greater (P<0.01), and there was a positive correlation between peritoneal albumin leakage amount and IMT (r=0.650, P<0.01). Conclusions Peritoneal albumin leakage is significantly associated with peritoneal transport characteristics, malnutrition and inflammatory state in CAPD patients. High peritoneal albumin leakage amount is a risk factor for atherosclerosis in patients with rGFR less than 1 ml·min^－1·（1.73 m²）^－1.

Objective To investigate the association between angiopoietin-2 (Angpt-2) and peritoneal angiogenesis in a uremic peritoneal dialysis (PD) rat model. Methods Uremic (subtotal nephrectomy) rats were established and divided into non-PD, 10 d-PD, 28 d-PD and 56 d-PD groups. Standard PD solution was applied in the study. Rats undergone sham operation without PD were used as control group. Vessel density of the peritoneum was detected and quantified with anti-CD31 immunohistochemical staining. Expressive levels of Angpt-2 and vascular endothelial growth factor (VEGF) were examined in the peritoneum by real-time PCR and Western blotting. Results The non-PD group was characterized by increased vessel density in the peritoneum compared with that of the control group [（5±3）/HP vs（1±1）/HP]. Progressive angiogenesis was found in 10 d-PD, 28 d-PD and 56 d-PD groups [（10±5）/HP, (17±5)/HP, (19±4)/HP]. Furthermore, expressive levels of Angpt-2 and VEGF increased significantly in the non-PD group compared with the control（P<0.01）, and such expressions were significantly higher in the PD groups as compared to non-PD group（P<0.01）, but no difference was found among the PD groups. Both VEGF and Angpt-2 levels were positively correlated with vessel density（r=0.7756, P<0.01; r=0.5223, P<0.05）. Conclusions Uremia and PD promote peritoneal angiogenesis in rats. Increased expression level of Angpt-2 in peritoneum is positively correlated with peritoneal angiogenesis. Angpt-2 may be a new therapeutic target of peritoneal angiogenesis.

Objective To investigate the pro-inflammatory effect of transforming growth factor β1(TGF-β1) in rat peritoneal mesothelial cells(RPMCs) and its machanism. Methods TGF-β1-induced RPMCs model in vitro was established, and the expression of MCP-1 in the TGF-β1-induced RPMCs was observed. The intervention of Smad7 on the expression of MCP-1 and p38 signal proteins induced by TGF-β1 in RPMCs was explored as well as the intervention of p38 inhibitor SB203580 on the expression of MCP-1 induced by TGF-β1 in RPMCs. Results TGF-β1 could stimulate MCP-1 expression in RPMCs. Compared with control group, MCP-1 mRNA levels were significantly increased after 3 h treatment with TGF- β1（P<0.05）, peak MCP-1 induction occurred at 6 h（P<0.01）, and the stimulatory effect of TGF- β1 persisted through 24 h（P<0.05）. MCP-1 protein levels were significantly increased after 6 h treatment with TGF- β1（P<0.05）, peak MCP-1 induction occurred at 48 h（P<0.01）. Over-expressed Smad7 and p38 inhibitor could reduce the expression of MCP-1 induced by TGF-β1（P<0.05）. TGF-β1 could activate p38 signaling pathway, but over-expressed Smad7 could inhibit this role of TGF-β1. Compared with control group, the expression level of p-p38 was increased in TGF-β1-stimulated group. Compared with TGF-β1-stimulated group, the expression level of p-p38 was reduced in Smad7 gene transfer group. Conclusions TGF-β1-induced MCP-1 expression in rat peritoneal mesothelial cells is p38MAPK dependent.

Objective To observe the influence of different dietary protein intake (DPI) on nitrogen balance and nutritional indices in peritoneal dialysis (PD) patients, and explore the minimal DPI to maintain nitrogen balance. Methods Thirty-four PD patients were randomly divided into group A, B and C with DPI as 1.2, 0.9 and 0.6 g&#8226;kg－1&#8226;d－1 respectively. All the patients admitted into our hospital and completed a 10-day assessment for nitrogen balance, as well as nutritional status including serum albumin(Alb), pre-albumin at baseline, the 7th and 10th day. Results The DPI of group A, B and C was (1.18±0.05), (0.87±0.02), (0.66±0.03) g&#8226;kg－1&#8226;d－1, whose differences were significant (P<0.01). The dietary energy intake (DEI) was 129.29 (117.57-133.89), 111.71 (100.42-133.47), 146.86 (128.03-163.18) kJ&#8226;kg－1&#8226;d－1 respectively. Nitrogen balance was positive in group A, B, C [2.99 (2.15－4.72） g, 1.20(0.59－1.89) g, 0.24 (－0.87－1.27) g]. The BUN decreased at the 7th and 10th day (P<0.01) in group C. The BUN and phosphorus in group A increased, but without significant difference as compared to baseline. No significant differences of nutritional status were found among three groups throughout the trial. Conclusion Minimal DPI 0.65 g&#8226;kg-1&#8226;d-1 plus the supplement of protein loss in dialysate can maintain the nitrogen balance in peritoneal dialysis patients.

Objective To investigate the effect and mechanism by which PPARγ ligand, rosiglitazone, regulates the expression of CD40 and intercellular adhesion molecule 1(ICAM-1) in the rat peritoneal mesothelial cells(RPMCs) induced by lipopolysaccharide (LPS). Methods RPMCs were harvested from Sprague-Dawley rat peritoneal cavity and maintained under defined in vitro conditions. The cells were randomly divided into groups as follows: medium, LPS(5 mg/L), LPS (5 mg/L)+BAY11-7085(5 μmol/L, NF-κB inhibitor), rosiglitazone (10 μmol/L or 20 μmol/L, peroxisome proliferator-activated receptor γ activator), LPS(5 mg/L)+rosiglitazone (10 μmol/L)+ GW9662(3 μmol/L, peroxisome proliferator-activated receptor γ antagonist), and LPS(5 mg/L)+vehicle (DMSO 0.2 ml/L). The expressions of CD40 and ICAM-1 RNA in RPMCs were examined by RT-PCR after 3 hour treatment, and the protein expressions of CD40, ICAM-1, p-NF-κB p65 and p-IκBα were examined by Western blot or immunofluorescence after 24 hour treatment. Results Following treatment with LPS, both the expressions of CD40 and ICAM-1 protein in RPMCs were up-regulated significantly (P<0.05), and the phosphoralation of p65 was increased greatly(1.10±0.17 vs 0.55±0.06, P<0.05). BAY11-7085 (5 μmol/L) significantly decreased the protein expression of p-p65 (0.22±0.11 vs 1.10±0.17, P<0.01), CD40 (0.34±0.02 vs 0.50±0.06, P< 0.05) and ICAM-1 (0.35±0.16 vs 0.74±0.03, P<0.05). Pretreated with rosiglitazone for 3 h then added with LPS for 1 h, the levels of p-p65, CD40 and ICAM-1 in RPMCs were significantly decreased compared with those of LPS group (0.77±0.08 vs 0.90±0.10, P<0.01; 0.79±0.16 vs 0.99±0.06, P<0.05; 0.83±0.20 vs 1.22±0.13, P<0.05）. However, for the group pretreated with rosiglitazone(10 μmol/L) and GW9662(3 μmol/L) for 3 h then added with LPS, the levels of p-p65 in RPMCs did not change significantly compared with those of rosiglitazone-pretreated group. The expressions of CD40 and ICAM-1 in RPMCs were significantly increased compared with those of rosiglitazone-pretreated group (0.95±0.19 vs 0.79±0.16, and 1.04±0.24 vs 0.83±0.20, P<0.05). Conclusion Rosiglitazone can decrease LPS-induced expression of CD40 and ICAM-1 in RPMCs by inhibition of NF-κB activation, which suggests that rosiglitazone may mediate its anti-inflammatory effect through a NF-κB dependent mechanism.

Objective To investigate the expression of endostatin (ES) in rat peritoneum and its association with peritoneal neoangiogensis. Methods Thirty-two male SD rats were randomly divided into 4 groups: normal control rats (C group), renal failure without PD rats (non-PD group), rats dialyzed with 1.5% PD solution (1.5% PD group) and 4.25% PD solution (4.25% PD group). After regular PD for 28 days, mRNA and protein expression of ES in peritoneal tissues of each group were detected by RT-PCR and immunohistochemistry respectively. Microvessel density (MVD) of peritoneal tissue was assessed using immunohistochemistry with CD34 monoclonal antibody. Results ES mRNA was expressed in each group, 0.47±0.05 in C group, 0.45±0.04 in non-PD group, 0.46±0.04 in 1.5%PD group, 0.47±0.03 in 4.25%PD group, and no significant differences were found among groups. Score of ES protein expression was 0 in C group, 2 in non-PD group, 4 in 1.5%PD group, and 9 in 4.25%PD group. MVD was 3.13±1.13 in C group, 5.13±1.14 in non-PD group, 9.00±1.51 in 1.5%PD group, 10.75±1.83 in 4.25%PD group, and significant differences were found among groups. Conclusion Uremia circumstance and non-physiological compatibility peritoneal dialysate can increase ES protein expression and MVD, which may participate in and have effects on the course of peritoneal neoangiogensis.

objective To compare the technical survival between Tenckhoff double-cuffed straight catheter（TC）and swan-neck curled tip catheter (SNC) in peritoneal dialysis (PD). Methods Clinical data of 208 patients received PD in the Peritoneal Dialysis Center of Peking Union Medical College Hospital from January 1999 to December 2007 were analyzed retrospectively. All the patients were divided into two groups according to indwelling catheter. Technical survival and complications associated with the catheter between two groups were compared. Results Demographics and basic information were similar in both groups. The exit-site infection (ESI) rates of TC and SNC were 22.1% and 19.8% (P=0.786）, and peritonitis rates of TC and SNC were 31.1% and 22.1% (P=0.159）, which were slightly lower in SNC group, but the difference was not significant. Removal of the catheter was found in 27（13.0%）patients, including 17 cases in TC group (13.9%) and 10 cases in SNC group (11.6%)(P=0.680）.The median survival times of catheter in TC group and SNC group were 25 months and 22 months respectively without significant difference (P=0.103）. Conclusions There are no significant differences of ESI rate, peritonitis rate and catheter survival between these two catheters in PD. The expensive swan-neck catheter offers no additional advantage. Doctors should choose the catheter according to the economic status of patients.

Objective To examine the relationship of the inhibitory effect of vascular endothelial growth factor(VEGF) on epithelial-mesenchymal transition (EMT) induced by TGF-β1 in HK2 cells with the expression of connective tissue growth factor (CTGF) and PI3K-Akt pathway. Methods The cultured HK2 cells were divided into the following groups: normal control group, TGF-β1(5 μg/L) group, VEGF (100 μg/L) group, TGF-β1 plus VEGF group. LY294002 (25 μmol/L), the blocker of PI3K-Akt pathway, was added to each of above-mentioned groups for the second part of the study. α-smooth muscle actin (α-SMA) and E-cadherin expressions of HK2 cells were assessed with double-stain immunocytochemistry method. The mRNA and protein expressions of α-SMA and CTGF of cells were assessed with RT-PCR and Western blot. The expressions of fibronectin(FN) and collagen I (ColⅠ) in medium were assessed with ELISA. Results The expressions of α-SMA and CTGF significantly increased in HK2 cells treated with TGF-β1 compared with those in normal control（P<0.05）, while significantly decreased in cells co-treated with TGF-β1 and VEGF compared with those treated with TGF-β1 alone (P<0.05, respectively). The expression of E-cadherin was exactly opposite to that of α-SMA. When LY294002 was added to TGF-β1 and VEGF co-treated cells, the expressions of α-SMA, CTGF, FN and ColⅠ were markedly up-regulated, when compared with those without LY294002 treatment(P<0.05). Conclusion Inhibitory effect of VEGF on TGF-β1-induced EMT of HK2 cells in vitro may be related to down-regulation of CTGF expression and reduction of FN and ColⅠ, which may be partly dependent on PI3K-Akt pathway.

Objective To investigate the effect of rosiglitazone on p38 mitogen-activated protein kinase (p38MAPK) pathway in polycystic kidney cyst-lining epithelial cells. Methods The cyst-lining epithelial cells(PKD cells) from human polycystic kidney were treated with rosiglitazone (10 μmol/L), peroxisome proliferator-activated receptor-γ (PPARγ) inhibitor GW9662 (10 μmol/L), rosiglitazone(10 μmol/L)+GW9662(10 μmol/L), p38MAPK specific inhibitor SB203580 (10 μmol/L), SB203580 (10 μmol/L)+ rosiglitazone（10 μmol/L) for 2 hours followed by epidermal growth factor (EGF) stimulation. Protein expressions of p38, phospho-p38(p-p38) and proliferating cell nuclear antigen (PCNA) were detected by Western blot. p38 mRNA was examined by RT-PCR. Expression of c-fos and c-jun was observed by immunocytochemistry. Results (1) EGF markedly up-regulated the expressions of p38, p-p38, PCNA, c-fos and c-jun compared with control group(P＜0.01). (2) Compared with EGF treated group, rosiglitazone significantly reduced p38 activation and mRNA expression (P＜0.01, respectively). Rosiglitazone, rosiglitazone+SB203580 could significantly down-regulated p-p38, PCNA, c-fos and c-jun expression (P＜0.01, respectively) with no significant difference between these two groups. (3) GW9662 partially reversed the reduction effect of rosiglitazone. Conclusions Rosiglitazone can inhibit proliferation of autosomal dominant polycystic kidney disease cyst-lining epithelial cells partially through down-regulating p38 activation and reducing c-fos, c-jun and PCNA expression. The above effect of rosiglitazone is in part PPARγ-independent.

Objective To investigate if rat enhancer of split- and hairy-related protein-2 (SHARP-2) short hairpin RNA interference (shRNAi) prolongs the survival time of rat kidney transplant recipients. Methods Gene recombinant procedures, transfection and co-transfection were carried out to introduce short hairpin RNA interference sequences target for SHARP-2 into 3rd generation self-inactivated lentiviral-ViraPower packaging mix. Limiting dilution method was used for viral titration. Real-time PCR was employed for quantification of gene expression. Rat kidney transplantation was utilized to investigate the effect of SHARP-2 gene silence on the recipient survival. Results A lentiviral-based shRNAi construct LV-SHARP-2iC showed 84% SHARP-2 gene silence efficiency in normal rat kidney cells. At multiplicity of infection 20, 57% T cells could be transfected by lentivirus with spinoculation method. In activated T cells, SHARP-2 gene silence resulted in 61.3% and 68.7% reduction of interleukin 2 (IL-2) and interferon γ (IFN-γ) gene expression. When donor kidney was perfused with 5×107 TU LV-SHARP-2iC, the median survival time prolonged for 4-5 days as compared to blank and scramble control groups. Conclusions A recombinant lentivirus LV-SHARP-2iC that effectively silence SHARP-2 gene expression is constructed successfully, leading to the inhibition of IL-2 and IFN-γ. LV-SHARP-2iC treatment can prolong the survival time of rat kidney transplant recipients.

Objective To investigate whether low-protein diet has protective effect on the progression of renal interstitial fibrosis in rats with cyclosporine A (CsA)-induced nephropathy. Methods Eighteen male Sprague-Dawley rats were randomly divided into three groups (6 rats in each group). The rats in control group (C group) received common diet; in model group (M group) low-salt diet; in intervention group (I group) low-salt and low-protein diet. After diet adaptation period of one week, the rats in C group received subcutaneous injection of olive oil 1 mg/kg daily for 5 weeks, while M group and I group subcutaneous injection of CsA (diluted into 25 g/L with olive oil) 1 ml/kg for 5 weeks. All the rats were sacrificed at the end of the 5th week. The food-intake and body weight were measured daily. The creatinine clearance (Ccr) was examined before rats were sacrificed. The semi-quantitative pathological analysis on kidney sections was performed. The mRNA and protein expression of transforming growth factor-β1 (TGF-β1) and typeⅠcollagen (ColⅠ) in kidney tissue was determined with real time PCR and immunohistochemical staining, respectively. Results The food-intake and body weight of rats in M and I groups were significantly lower than those in C group (P<0.05). Compared with C group, the Ccr levels in M and I groups were significantly reduced [(0.65±0.15) ml/min, (0.40±0.13) ml/min vs (1.55±0.29) ml/min, P<0.05], the relative fibrosis areas of kidney interstitium in M and I groups were significantly increased (3.60%±0.46%, 3.26%±0.75% vs 0.44%±0.24%, P<0.05), the mRNA and protein expression of TGF-β1 in M and I group was significantly up-regulated (by 2.6 and 3.1 times in mRNA and by 1.5 and 1.6 times in protein, respectively, P<0.05), and the mRNA and protein expression of ColⅠin M and I groups was also significantly up-regulated (by 3.0 and 3.5 times in mRNA and by 2.3 and 2.1 times in protein, respectively, P<0.05). There were no significant differences between M and I groups in every parameters above-mentioned except the rat body weight and Ccr. Both the body weight and Ccr in I group were significantly lower than those in M group (P<0.05). Compared with C group, the urine osmotic pressure in M group and in I group were deceased (for M group, P>0.05; for I group, P<0.05). Compared with C group, the serum cholesterol levels in M and I groups were significantly increased (P<0.05), and the serum phosphorus level in I group was significantly decreased (P<0.05). The levels of serum albumin and serum calcium of all three groups had no statistical differences (P>0.05). Conclusion Low-protein diet has no renoprotective effects on the rat model of cyclosporin A nephropathy, on the contrary, may induce body weight loss.

Objective To investigate the effect of tumor necrosis factor α (TNF-α) on monocyte-renal tubular epithelial cell adhesion and to explore its associated mechanism. Methods Human renal proximal tubular epithelial cell line （HK2 cells）and monocyte cell line （U937）were used to perform cell co-culture. TNF-α（10 μg/L）was used to stimulate HK2 cells and then U937 was put into the HK2 monolayers. Three locational parts of hyaluronan around HK2 cells were extracted by different ways and the HA expression was detected by enzyme-linked binding protein assay. Monocyte adhesion was detected by florescence recording assay. Flow cytometry was applied to assess intercellular adhesion molecule-1(ICAM-1) expression in HK2 cells. Results Stimulation of HK2 cells with TNF-α significantly increased U937 binding to HK2 cell monolayer [(2.25±0.05) folds vs control, P<0.01]. Incubation of TNF-α (10 μg/L) with HK2 cells for 24 hours did not induce the change of total HA expression surrounding the HK2 cells, whereas HA redistribution happened with conditioned medium fraction (CM-HA) increased [(1.17±0.16) fold vs control, P<0.05], the pericellular fraction (trypsin extract fraction, TE-HA) decreased (83%±11% vs control, P< 0.05) and the remaining cells-associated HA (cell associated fraction, CA) did not change significantly. In addition, pre-treatment of HK2 cells with TNF-α significantly increased the ICAM-1 expression [(1.85±0.22) folds vs control, P<0.01] and ICAM-1-dependent monocytes binding. Removal of HA from the surface of confluent monolayers of HK2 cells by hyaluronidase before addition of U937 cells increased monocyte binding[ (1.35±0.06) folds vs control, P<0.05]. In contrast, the presence of either sICAM-1 (400 μg/L) or anti-CD18 antibody (10 mg/L) led to a significant decrease in ICAM-1-dependent monocyte binding (78%±7%, P<0.05; 75%±8%, P<0.01 vs those before adding sICAM-1 or anti-CD18 antibody, respectively). Conclusion HA redistribution induced by TNF-α may facilitate ICAM-1-dependent monocyte-epithelium binding, and pericellular HA plays a protective role in monocyte-driven tissue inflammation.

Objective To investigate the differentiation of rat bone marrow mesenchymal stem cells (MSCs) to renal tubular epithelial-like cells under different conditions. Methods MSCs were obtained from rat marrow. MSCs were isolated by gradient density centrifugation and plastic adherence and then purified. Surface markers were identified with flow cytometry after amplification in vitro. The purified MSCs of the third passage were cultured respectively as follows: (1) control group: DMEM medium with fetal bovine serum(FBS). (2) all-trans retinoic acid (ATRA) group: DMEM medium with FBS, ATRA and ischemic reperfusion-injured kidney tissue homogenate. (3)combination group: DMEM medium with FBS, ATRA, ischemic reperfusion-injured kidney tissue homogenate, epidermal growth factor (EGF) and bone morphogenetic protein 7 (BMP-7). After 7 days, the MSCs were collected for alkaline phosphatase (AKP) staining, cytokeratin-18 and E-cadherin immunocytochemical analysis. Results The positive rates of the third passage MSCs in CD44, CD90 and CD29 were 97.8%±0.9%, 96.8%±1.4% and 97.6%±2.4%，respectively, but in CD11b/c and CD34 were only 13.2%±0.6% and 1.2%±0.5%. The MSCs in control group were spindle. The MSCs in ATRA group were round and elliptic. The MSCs in combination group became cobblestone-like cells after 7 days. AKP staining showed that tubular epithelial-like cells from MSCs in control group were negative, some above cells in ATRA group were positive and number of above cells increased in combination group. Compared with negative control group, the ratios of cytokeratin-18 positive cells in ATRA group and combination group were respectively increased by 29.47%±1.08% and 47.52%±2.13% (all P<0.05), the ratios of E-cadherin positive cells in ATRA group and combination group were respectively increased by 14.88%±2.46% and 36.15%±1.13% (all P<0.05). Conclusion MSCs may differentiate by renal tubular epithelial-like cells under the induction of ischemic reperfusion-injured kidney tissue homogenate and ATRA in vitro, which are further differentiated under the combined induction of EGF and BMP-7.