Objective To compare improved culture methods of pathogenic organism in peritoneal effluent with conventional methods, in order to decrease the culture-negative rate of pathogenic organism in patients with peritoneal dialysis(PD)-related infectious peritonitis. Methods Effluents of 45 peritonitis episodes from 27 patients were included. Effluents were cultured with different methods dividing into the following 6 culture groups: conventional plate group (P group), centrifugation plate group (CP group), BacT/Alert culture bottle groups (PF group, SA group and SN group) and frozen-lysis group (FL group). Results The culture-positive rate of CP group was significantly higher than that of P group (60.0% vs 44.4%, P<0.05). Compared with CP group, the culture-positive rates of PF group (84.4%), SA group (82.2%) and SN group (82.2%) were significantly increased (all P<0.01). Concerning the time to detect growth (TDG), compared with that of CP group [(24.31±9.80) h], those of PF group [(14.25±9.13) h], SA group [(12.75±7.47) h] and SN group [(9.79±4.14) h] were significantly shortened (all P<0.01). SN group had the shortest TDG compared with PF group and SA group (all P<0.01). Conclusions Centrifugation of effluent is proved to increase the culture-positive rate of pathogenic organism in patients with PD-related infectious peritonitis. Culturing the effluent with BacT/Alert system is superior to plate technique in the aspects of increased culture-positive rate and fastened speed to detect the microbiological growth. Culturing with SN bottle is the fastest method to detect microbiological growth in PD-related infectious peritonitis compared with PF bottle and SA bottle.

Objective To analyze and identify the mutations of SLC12A3 gene in Gitelman syndrome patients， and improve the cognition and apprehension to the disease. Methods Twelve patients [8 males and 4 females, age (37±13) years] hospitalized in Ruijin hospital from 7 unrelated families with the clinical and biochemical features of Gitelman syndrome were analyzed by direct sequencing of SLC12A3 gene. Fifty unrelated normal subjects were selected to evaluate all the mutations found by this study. Results Eight mutations were identified in SLC12A3 gene of 12 patients with Gitelman syndrome. Five were novel variants, including 2 missense mutations: Cys430Gly and Leu571Pro; two deletions: 1384delG and 346~353delACTGATGG; and one in-frame insertion: 997insCys. Three were recurrent ones including two missense mutations: Thr60Met and Asp486Asn, and one deletion: 2883~2884delAG. The homozygous or heterozygous mutation Thr60Met was found in 8 of 12 patients. The majority of the patients were compound heterozygotes. Conclusions Gene analysis is essential to diagnose Gitelman syndrome. Thr60Met may be a more common mutation in Chinese patients with Gitelman syndrome. Possible specific genotype-phenotype correlation is difficult to identify.

Objective To study the mutation and polymorphism of podocin coding NPHS2 gene in sporadic primary nephrotic syndrome (NS) children in order to predict the responsiveness to regular steroid therapy and prognosis. Methods Genome DNA was amplified by PCR. Mutation and /or polymorphism of NPHS2 gene in 38 steroid-sensitive NS children, 22 steroid-resistant NS children and 30 healthy controls were examined by direct sequencing. Results Three patients were found to carry novel heterozygous allelic variants leading to amino acid substitution (S260I,E188D) and one to carry a novel nonsense mutation leading to a truncated protein product (E237X). Two known polymorphisms were also found. Conclusions Mutation and polymorphism of NPHS2 gene are present in sporadic Chinese primary NS children. Genetic analysis may be beneficial to early predict the responsiveness to steroid and the prognosis of primary NS children.

Objective To explore the relationship between vascular smooth muscle cell (VSMC) apoptosis and uremic vascular calcification. Methods Pieces of radial artery were taken from 43 uremic patients at the time of internal arteriovenous fistula operation. Alizarin red staining was used to determine medial layer vascular calcification. Uremic patients were divided into two groups according to the results of calcification stain. Pieces of radial artery from age-matched normal renal function patients with periarteriole diseases were used as control. Apoptosis of VSMC was detected by terminal deoxynucleotidyl transferease-mediated biotinylated deoxyuridine triphosphate nick end labeling (TUNEL) and apoptosis index (AI) was calculated. Expressive levels of apoptosis-associated genes p53 and Bcl-2 were examined by immunohistochemistry. Related clinical and laboratory parameters were determined as well. Results Obvious VSMC apoptosis in medial layer of uremic patients’ radial artery was noticed both in calcification positive and negative groups. AI of the calcification positive group was significant higher than that of negative group (48.13±17.81 vs 19.50±8.25,P<0.01). There was little VSMC apoptosis in control group. The expression of Bcl-2 in calcification positive group was markedly decreased compared to control group (4.29±2.16 vs 14.87±5.62) while the expression of p53 was markedly increased in calcification positive group compared to the other two groups (31.22±14.25 vs 20.67±7.35， 4.17±2.61, P<0.01). AI was positively correlated to product of serum calcium and phosphorus, and the levels of serum phosphorus and LDL (P<0.05). Conclusions There is VSMC apoptosis in the medial layer of uremic patients’ radial artery. VSMC apoptosis occurs before overt artery calcification and it is more severe in calcification positive artery, which suggests that VSMC apoptosis takes part in the pathogenesis and development of vascular calcification in uremic patients. Managements lowering the level of serum phosphorus, calcium and phosphorus product and LDL may be beneficial to the prevention and treatment for uremic vascular calcification by means of decreasing VSMC apoptosis.

Objective To investigate the relationship between plasma asymmetric dimethylarginine (ADMA) and cardiovascular disease (CVD) in patients with non-dialytic chronic kidney disease (CKD). Methods Seventy-six patients with CKD (mean age 46 years, 39 female, 37 male) and 15 healthy controls were enrolled. Plasma ADMA was measured by HPLC-MS. The relationship between plasma ADMA level and CVD in non-dialytic CKD patients was investigated with Logistic regression model. Results Mean plasma ADMA level increased significantly in CKD group compared to control group [(41.56±12.76) vs (17.12±7.09) mg/L, P < 0.01], even in those patients in early stage of CKD. The plasma levels of ADMA were higher in renal patients with congestive heart failure or artherosclerosis cardiovascular diseases than that in those without (both P < 0.05). Plasma ADMA level was positively correlated with left ventricular mass index (r = 2.521, P < 0.01) and intima-media thickness of common carotid artery (r = 0.544, P = 0.002). Multiple regression analysis showed that plasma ADMA level was the independant risk factor of LVMI and intima-media thickness of common carotid artery in CKD patients. Logistic regression analysis further indicated that ADMA （β = 1.117，95%CI: 1.013~1.232, P = 0.027）was an independent risk factor of CVD in non-dialytic CKD patients. Conclusion Increased plasma ADMA concentration is found at even very early stage of CKD and may play a role in pathogenesis and progression of CVD in these patients.

Objective To observe hepatocyte growth factor receptor c-Met expression in high glucose-treated rat mesangial cells (MsC) and explore its significance and relevant mechanism. Methods RT-PCR and Western blot analysis were performed to detect c-Met expression in MsC cultured under high glucose conditions for different time (0, 12, 24, 48, 96 h). Sp1-binding inhibitor mithramycin A and c-Met inhibitor SU11274 were used to block Sp1 binding DNA and c-Met respectively. With electrophoretic mobility shift assay (EMSA), binding of Sp1 to c-Met promoter was evaluated. Intracellular reactive oxygen species was examined by loading MsC with dichlorodihydrofluorescein diacetate, a redox-sensitive fluorescent dye. Results Exposure MsC to high glucose resulted in an increase in c-Met mRNA and protein expression at first 48 h then a following decrease at 96 h. Mithramycin A suppressed high glucose-increased c-Met expression in a dose-dependent manner. Enhanced Sp1 binding to c-Met promoter was shown in high glucose-treated MsC. HGF/c-Met markedly reduced intracellular reactive oxygen species in MsC cultured in high glucose. Conclusions High glucose may activate c-Met expression in MsC through Sp1. HGF/c-Met can attenuate high glucose-mediated oxidative stress.

Objective To compare the efficacy of two hHGF gene electroporation methodologies(muscle and kidney) in order to reduce the deleterious effects induced by renal warm ischemia. Methods Thirty male SD rats were divided into three groups, 10 in each group. One group was warm ischemia without pretreatment as control. The other two groups received hHGF gene muscle injection and subsequent electroporation and hHGF gene kidney electroporation respectively 3 days before renal warm ischemia. Pharmacokinetic, function and histology were assessed in ischemic kidney. Scr levels were measured at day 1,3,5 after injury. Results Plasma hHGF levels and hHGF expression of renal tissue in kidney electroporation group were higher than those in muscle electroporation group（P<0.01）. Both treated groups showed lower Scr than control[(79.4±23.4), (109.7±18.6) vs (164.8±55.5) ?滋mol/L,P<0.05]. The kidney electroporation group showed faster recovery of renal function, with significantly lower Scr level compared to muscle electroporation group. The tubular necrosis score was lower in both HGF-treated groups(P<0.05). The tubular necrosis score of the hHGF gene kidney electroporation group(1.365±0.186) was lower than that of hHGF gene muscle electroporation group (1.864±0.389) (P<0.05). hHGF-treated groups had fewer macrophages and lymphocytes than control group, as well as lower values of MPO activity(P<0.01), but no significant difference was found between 2 hHGF-treated groups(P>0.05). Conclusion Kidney direct electrotransfer is shown to be more efficient not only in pharmacokinetic but also in therapy as compared to muscle electrotransfer, so it may become a clinically practical alternative in renal transplantation.

Objective 　To investigate the influence of irbesartan on the expression of integrin-linked kinase (ILK) and its relationship with epithelial-mesenchymal transition (EMT) in mice with unilateral ureteral obstruction (UUO). Methods Mice were randomly divided into three groups: sham operation (C, n=20), UUO (n=40) and UUO with irbesartan treatment (UUO＋Irb, n=40). Animals were sacrificed at day 1, 3, 7 and 14 respectively after the surgery. Tubulointerstitial fibrosis (TIF) was graded according to Masson staining. The expression of ILK was examined by immunohistochemistry, Western blot and real time PCR, respectively. mRNA expression of FN was measured by real-time PCR. Expression of α-SMA and E-cadherin was detected by immunohistochemistry and real-time PCR. Results The expression levels of ILK mRNA and protein were significantly increased in UUO group, which was significantly decreased by treatment with Irb（P < 0.01）. The expression of α-SMA and the mRNA level of FN were significantly increased, while E-cadherin was decreased in mice with UUO at day 3 after the surgery. Treatment with Irb significantly abrogated such effects（P < 0.01）. The protein expression of ILK was positively correlated with α-SMA, but negatively with E-cadherin. Conclusions Irbesartan attenuates renal tubulointerstitial fibrosis in UUO mice, which may be related to the inhibition of ILK expression and the subsequent prevention of tubular epithelial-mesenchymal transition.

Objective 　To investigate the influence of renal lymph circulation disorder on renal tubulointerstitial fibrosis and the expression of TGF-β1/Smad2/3 in rat kidney. Methods 　Forty-eight male Wistar rats were randomly divided into two groups, twenty-four rats in each group: (1)control group: sham operation; (2)model group: renal lymph ligation. Six rats in each group were sacrificed at week 1, 2, 4 and 8 respectively after operation. The renal tissue was examined by PAS and Masson stain. The sites and expressions levels of type I collagen(Col I),TGF-β1, Smad2/3, phosphorylated-Smad 2/3 (p-Smad2/3) protein were examined by immunohistochemical staining and/or Western blot. Real-time PCR was applied to determine the mRNA expression of Col I， TGF-β1, Smad2 and Smad3. Results 　Renal disfunction and tubulointerstitial fibrosis were found in model group, and the tubulointerstitial lesion was remarkably aggravated in week 8 compared with control group（P<0.05）. Expression levels of Col I， TGF-β1, Smad2/3, p-Smad2/3 protein and/or mRNA were significantly increased during 1～8 weeks after operation. Immunohistochemical staining indicated that these proteins were mainly expressed in renal tubulointerstitium. Conclusions Disorder of renal lymph circulation can damage renal structure, especially lead to tubulointerstitial fibrosis. The possible mechanism is the activation of TGF-β/Smad signaling pathway. The high expression levels of Smad2, Smad3, and TGF-β1 protein may be the major cause leading to tubulointerstitial fibrosis.

Objective To observe the effect of low protein diet supplemented with ketoacid on the protection of remnant kidney and the inhibition of inflammation infiltration in 5/6 nephrectomized rats. Methods Thirty male Sprague-Dawley rats were randomly assigned to following 3 groups and given different diets: low protein supplemented with ketoacid group(4% casein protein＋2%ketoacid), low protein group(6% casein protein) and high protein group (20% casein protein). Ten SD rats were uses as sham-operation group(20% casein protein). The former three groups were subjected to 5/6 nephrectomy. Urine and blood samples were collected before operation and 4, 8 and 12 weeks after operation. All rats were killed after 12 weeks and pathologic changes of kidney tissue were investigated by microscope. Immunohistochemistry was also employed to investigate the expression of MCP-1, RANTES, ED-1 within the residual kidney. Results Scr and BUN of operated rats increased gradually. Compared with high protein group and low protein group, Scr and BUN of low protein supplemented with ketoacid group were significantly lower[Scr (125.44±5.50) vs (172.00±9.54), (135.22±9.54) μmol/L, P<0.01]. The urinary albumin excretion (UAE) increased significantly at the 4th week and reached the peak at the 12th week. The UAE (mg/24 h) of low protein supplemented ketoacid group (28.36±3.69)was markedly lower than that of high protein group(92.32±34.06) and low protein group(46.62±6.19). Compared with the latter two groups, pathological lesion of low protein supplemented with ketoacid group was obviously improved (glomerular pathological score 0.38±0.13 vs 0.84±0.28, 0.49±0.11, P<0.01). The expression levels of MCP-1, RANTES, ED-1 in operation groups were significantly higher than those in sham-operation group, and above expression levels in low protein supplemented with ketoacid group were the lowest among three operation groups (positive ED-1 cell 3.59±0.78 vs 13.33±1.20, 6.50±0.99, P<0.01). Conclusion Low protein diet supplemented with ketoacid can attenuate pathological lesion of residual kidney through certain mechanisms such as inhibition of inflammatory infiltration.

Objective To construct hypoxia regulatory vectors which express selectively under hypoxia and to utilize the vectors to express regulatory transduced gene. Methods Hypoxia response element (HRE) oligonucleotides derived from hypoxia response genes were synthesized and linked with CMV promoter to drive luciferase reporter and therapeutic gene transcription. Then the hypoxic transcriptional activation of the constructs was measured by detecting relative luciferase activity and hEPO expression. Results Among five vectors, the combination of 3HRE/mPGK and CMV promoter exhibited excellent hypoxia responsiveness to the similar level to the intact CMV promoter under normoxia. Conclusion The 3HRE/mPGK/CMV vector may be especially useful to achieve regulatory therapeutic gene expression precisely.