Objective To study the characteristics of glomerular filtration rate (GFR) and its influential factors in type 2 diabetes mellitus at different stages of albuminuria. Method GFR was measured in 630 cases of type 2 diabetes mellitus between 2002 and 2005 by plasma disappearance of 99m-techmetium-diethylene- triamine- penta-acetic acid (99mTc-DTPA). Body mass index (BMI), blood pressure, plasma glucose, HbA1c, Scr, BUN, uric acid (UA), profile of plasma lipid and 24 h-urinary albumin excretion (24 h-UAE) were also measured. All the patients were divided into 3 groups according to their 24 h-UAE: normoalbuminuric group (group A, 24 h-UAE<30 mg), microalbuminuric group (group B, 24 h-UAE from 30 mg to 300 mg) and macroalbuminuric group (group C, 24 h-UAE>300 mg). Results (1) The mean GFR was (99.8±26.3) ml/min, (96.1±31.2) ml/min and (69.7±29.8) ml/min in A, B and C groups respectively. The GFR in group C was significantly lower than that in group A and group B(P<0.01). (2) Negative correlations were found between GFR and age in all these groups (group A r= -0.533, group B r=-0.612 and group C r=-0.412，respectively, P<0.01）. (3) In each group, GFR of patients with hypertension was significantly lower than that of patients without hypertension（P<0.05）. (4) The Pearson correlation analysis adjusted by age showed that GFR was negatively correlated with 24 h-UAE in group B and group C (r=-0.283 and -0.24 respectively, all P<0.05). The multiple stepwise regression analysis showed that 24 h-UAE was the major influential factor of GFR in these 2 groups. Conclusions Measurement of both GFR performed by non-traumatic plasma disappearance of 99mTc-DTPA method and UAE provides a more precise evaluation on the the development and progression of diabetic nephropathy. Albuminuria should be controlled, especially in microalbuminuric stage.

Objective To report 2 cases of HIV infection complicated with kidney disease for the first time in China. Methods The clinical manifestation of 2 HIV-seropositive male patients and pathological findings by renal biopsy were collected. Results The HIV-1 seropositive male patient was 69 years old. He was diagnosed as nephrotic syndrome （NS） complicated with renal insufficiency. Renal biopsy revealed characteristic focal segmental glomerular sclerosis（FSGS）, non-collapsing, tubulointerstitial prominent lymphocytic infiltration. These characteristics were partially compatible with human immunodeficiency virus-associated nephropathy (HIVAN). According to NS and normal range of CD4, he was recommended with steroid and immunosuppressive therapy while absent of highly active anti-retroviral therapy (HAART). During one-year followed up, he had experienced several episodes of severe infection without remission of NS. After immunosuppressive therapy withdrawal and counterchecked low level of CD4, he received HAART for HIV treatment. The other patient was a hemophile-infected AIDS complicated with chronic hepatitis C， haemophilia A(lack of factor Ⅷ)， and diabetes mellitus（DM）. Proteinuria and chronic renal failure occurred after 1 year of HAART. During the therapy period, the renal function was impaired progressively and irreversibly entered into ESRD at the 3rd year of HAART. Conclusions The clinical and pathological manifestation of HIV infection complicated with kidney disease shonld be known in China. For HIV-infected kidney impaired patients suspected of HIVAN or other pathological pattern, it is important to establish the diagnosis and treatment by renal biopsy.

Objective To examine the effects of connective tissue growth factor (CTGF) on cell proliferation and production of collagen type Ⅰin cultured rat cortical myofibroblasts，and to investigate the role of ERK1/2 siganling pathway. Methods Myofibroblasts were obtained from normal rat renal cortex. 5’-bromodeoxyuridine (BrdU) incorporation assay and cell counting were used to detect cell proliferation. Western blot analysis was used to detect the levels of collagen typeⅠin the supernatant medium and the activation of ERK1/2 signaling pathway in cultured myofibroblasts. Results CTGF could induce the proliferation of myofibroblasts in a dose- and time-dependent manner. Cell number of 100 μg/L CTGF at day 4 was 1.6 folds of control group(P<0.05). Incubation with 100 μg/L CTGF also significantly increased secretion of collagen type I in the supernatant medium compared with control group (2.6±1.2 folds over control, P<0.01). ERK1/2 activation occurred as early as 5 minutes following 100 μg/L CTGF treatment, and persisted till 15 minutes later, and then declined back to the basal level after 30 minutes. Pretreatment with 50 μmol/L PD98059, a specific inhibitor of ERK1/2 pathway, abolished the effects of CTGF-induced cell proliferation (7%±5% vs 85%±7%, P<0.01) and CTGF-increased secretion of collagen type I (1.0±0.1 vs 1.6±0.3, P<0.05). Conclusion CTGF promotes cell proliferation and secretion of collagen type I through ERK1/2 pathway in primary cultured rat myofibroblasts.

Objective To investigate the changes of protein expression in rat kidney proximal tubular epithelial NRK52E cells during the epithelial to mesenchymal transitions (EMT) process. Methods Comparative proteomics was applied. Proteins extracted from NRK52E cells(three plates treated with TGF-β1 and three plates without treatment) were separated by two-dimensional gel electrophoresis(2-DE). Comparative analysis of 2-DE protein patterns between the two groups were carried out using computerized image analysis. Selected proteins exhibiting significant alternations were identified by mass spectrometry. Western blotting and RT-PCR were performed to examine the expression of the candidated proteins. Results The expression of 22 proteins including transgelin, ARP2/3 complex 21 000 subunit, destrin, ATP synthase α, ubiquitin-conjugating enzyme 9 (Ubc9), and malate dehydrogenase involved in the organization of the cytoskeleton, energy metabolism and post-translational modification were altered during the EMT process. Western blotting and RT-PCR confirmed the differences of several altered proteins, which was consistent with the findings of 2-DE. Conclusion TGF-β1 induces specific changes in the expression of structural, regulatory and metabolic proteins relevant to the complex process of EMT in NRK52E cells. This findings are helpful to understand the molecular mechanism of EMT and expound the chronic progress mechanism of the kidney disease.

Objective To investigate whether JAK/STAT signaling pathway is associated with the tissue inhibitor of metallproteinase-1(TIMP-1)-induced apoptosis in rat mesangial cells(RMC). Methods The human sense and antisense TIMP-1 recombinant plasmids were transfected into RMC through liposome. After RMC was stimulated under the condition of serum deprivation and AG490(JAK2 specific inhibitor) for 24 hours in vitro, the apoptosis rate was measured by flow cytometry. The expression of TIMP-1, bcl-xl, cyclin D1, p27kip1 and JAK2 mRNA was assayed by RT-PCR. The expression of JAK2, STAT3, STAT5, p-JAK2, p-STAT3 and p-STAT5 protein was analyzed by Western blot. Results The apoptosis rates of the non-transfected group, sense TIMP-1 group and antisense TIMP-1 group in the serum-deprived culture medium without serum and AG490 were （10.59±0.96）％, （7.08±0.43）％ and （21.91±0.25）％,respectively. There were statistical differences among those apoptosis rates. The expression of bcl-xl and cyclin D1 mRNA was the highest in sense group, while it was the lowest in antisense one. The expression of p27kip1 mRNA was the highest in antisense group. AG490 treatment enhanced the apoptosis rates of RMC significantly(P<0.01). AG490 decreased the expression of TIMP-1 mRNA and bcl-xl, as well as cyclin D1 mRNA, and increased the expression of p27kip1 mRNA. Before RMC was stimulated with AG490, the expression of p-JAK2, p-STAT3 and p-STAT5 was the highest in sense TIMP-1 group, and it was the lowest in antisense one. AG490 treatment significantly suppressed the expression of the phosphorylation proteins in all groups. Conclusions The expression of TIMP-1 can be regulated by JAK/STAT signaling pathway. The JAK/STAT can inhibit RMC apoptosis through up-regulating the expression of the TIMP-1. TIMP-1 inhibits the apoptosis of RMC induced by serum deprivation through JAK/STAT signal pathway. Bcl-xl, cyclin D1 and p27kip1 are involved in the above-mentioned courses

Objective To investigate the effect of TNF-α on the expression of bone alkaline phosphatase (BAP), osteopontin (OPN) and bone morphogenetic protein 2 (BMP-2), and the deposit of extracellular calcium in human umbilical artery smooth muscle cells (hUASMC). Method hUASMC were cultured primarily with tissue explants-attached method. The mRNA expression of BAP and OPN was determined by real time-PCR. The protein expression of BAP, OPN and BMP-2 was determined by Western blotting. The calcium deposit was detected by O-cresolphthalein complexone method. Results TNF-α promoted the proliferation of hUASMC and increased the calcium deposit in a time- and dose-dependent manner. The mRNA expression of BAP and OPN in hUASMC was up-regulated by TNF-α（50 μg/L） treatment at day 3(BAP mRNA 1.908±0.034 vs 1.000±0.033, OPN mRNA 3.600±0.073 vs 1.000±0.079, all P<0.05). The protein expression of BAP, OPN and BMP-2 in hUASMC was up-regulated by TNF-α（50 μg/L） treatment at day 5 (BAP protein 3.394±0.083 vs 1.000±0.030, OPN protein 1.967±0.134 vs 1.000±0.070, BMP-2 protein 2.745±0.289 vs 1.000±0.208, all P<0.05）. Conclusion TNF-α of certain concentrations can promote the proliferation of hUASMC, induce the ossific calcification of hUASMC, increase the calcium deposit, and participate in the development of vascular calcium

Objective To investigate the effects of TNF-α on the expression of matrix metalloproteinases (MMP) and their inhibitors (TIMP) in human peritoneal mesothelial cells,meanwhile, to investgate the effects of treatment of TNF-α+TGF-β1, TNF-α+TGF-β1+IL-1 or TNF-α alone on the MMP-9 activity of the HPMC. Methods After 24-hour stimulation by different concerntrations of cytokines（TNF-α，TGF-β， IL-1）, the serum-free conditioned HPMC culture supernatants and the HPMC were collected. The mRNA expression of MMP-2, MMP-9, TIMP-2 and TIMP-1 was detected by semi-quantitative RT-PCR. The activity of active and pro MMP-9 in the conditioned media was quantitatively measured by Biotrak MMP-9 activity assay system. Collagen Ⅰ protein level was examined by ELISA. Results In vitro by the treatment of TNF-α, the expression of MMP-9 mRNA of HPMC was significantly up-regulated in a dose- and time-dependent manner（2.3~4.9 folds of base value, P＜0.05）; the expression of TIMP-1 mRNA and TIMP-2 mRNA was slightly down-regulated（77.2%, 61.3% of base value, P＜0.05）, however the expression of MMP-2 mRNA did not change obviously. Activity and secretion of MMP-9 in the conditioned cell supernatant increased significantly by the treatment of TNF-α+TGF-β１, TNF-α+TGF-β１+IL-1 or TNF-α alone. Collagen Ⅰ protein expression of HPMC increased significantly as well by 24-hour stimulation of TNF-α. Conclusion The increase of MMP-9 activity and expression induced by TNF-α alone and TNF-α with other cytokines may play a role in the peritoneal fibrogenesis process.

Objective To investigate the effect of large dosage of spironolactone on renal fibrosis in spontaneously hypertensive rats. Methods Twenty-four 8-week aged spontaneously hypertensive rats were divided into low and large dosage of spironolactone group and control group. Eight normal Wistar-Kyoto rats were as normal group. Low and large dosage groups were given spironolactone 20 mg&#8226;kg-1&#8226;d-1 and 100 mg&#8226;kg-1&#8226;d-1 for 8 weeks. Then systolic blood pressure, proteiuria, albumin, K＋,Na＋, Scr, the aldosterone level of kidney tissue and plasma were measured. Renal tissue sections were stained by HE and Masson for evaluating glomerular injury and collagen deposition. The protein expression of TGF-β1 and aldosterone receptor in renal tissue were detected by immunohistochemical SABC method, and the mRNA levels were detected by RT-PCR. Results Compared to the controls, in low dosage of spironolactone group, urinary protein decreased (P<0.05), serum albumin increased (P<0.05), plasma and renal tissue aldosterone levels decreased, without significant diffeence. In large dosage of spironolactone group, blood pressure had no significant change, whereas the levels of urinary protein[(27.3±4.5) vs (24.5±3.2) mg/d] as well as plasma and renal tissue aldosterone [(28.3±1.5) vs (22.2±0.6) ng/g] increased significantly (P<0.05), and serum albumin [(20.2±4.2) vs (22.7±3.5) g/L] decreased significantly (P<0.05). Compared to the controls, in low dosage of spironolactone group, protein cast and inflammatory cells infiltration around tubules reduced (P<0.05); the renal tissue aldosterone receptor mRNA and protein expression did not change significantly, TGF-β1 mRNA and protein expression reduced significantly (P<0.05). In high-dose spironolactone group, protein cast and inflammatory cells infiltration around tubules increased significantly (P <0.05), and glomerular collagen formation also increased significantly (P<0.05); the renal tissue aldosterone receptor and TGF-β1 mRNA and protein expression were significantly increased (P<0.05). Conclusions Large dosage of spironolacton can aggravate renal fibrosis maybe through up-regulating level of renal aldosterone and its receptor.

Objective To promote the proliferation of seeding cells, obtain plenty of engineering cells for bioartificial kidney(BAK) in relatively short time and facilitate the construction of BAK system. Methods Recombinant adenovirus-associated virus II type harboring human Nanog gene (rAAV2- hNanog) was prepared, then rAAV2-hNanog was transfected into seeding cell HKC. The expression of human Nanog in seeding cells was detected by RT-PCR after transfection. The effects of human Nanog on HKC cell line were examined by tetrazolium salt colorimetry (MTT), immunofluorescence and confocal laser scanning microscope（CLSM）. Results The human Nanog could be expressed in engineering cells. The proliferation activity of transfected seeding cell was significantly improved（P<0.05）. Meanwhile, no obvious morphology changes could be found under inverted microscope in the seeding cells transfected by rAAV2-hNanog compared with control cells. Conclusions The proliferation of seeding cells modified by human Nanog gene is strikingly accelerated. This new approach will overcome the insufficiency of seeding cells for BAK system.

Objective To compare the efficacy between the two-cuff swan neck catheter and the Tenckhoff catheter in continuous ambulatory peritoneal dialysis (CAPD) patients prospectively. Methods One hundred and ten patients with end-stage renal disease (ESRD) were selected as candidates, who received catheter implantation and CAPD therapy for the first time. Patients were divided into group A (swan neck catheter group) and group B (Tenckhoff catheter group), 55 patients for each group. Catheters of both groups had a straight end and were implanted by routine surgical procedure. One-year follow-up was performed and information was recorded such as complications, survival time, quit of dialysis, death, etc. Survival analysis was carried out by Kaplan-Meier method and Log-Rank tests. Results At the end of follow-up, 17 patients died, 3 received renal transplantation, 8 were transferred to hemodialysis, 3 went to other hospitals, and 79 patients (71.8%) remained in our department for CAPD. Twenty-six patients of both groups had peritonitis with a total of 35 occurrences. The total incidence of peritonitis was 0.32 times/patient year, with the detailed figure of 0.35 times/patient year for group A and 0.29 times/patient year for group B respectively （P>0.05）. The time interval between the catheter implanting and the onset of peritonitis was (30±29) weeks and (29±24) weeks for group A and group B respectively （P>0.05）. The risk of developing peritonitis in both groups was 26.97% within 1 year. Tunnel infection occurred in 2 patients and exit-site infections in 9 patients of two groups. The incidence of tunnel plus exit-site infections was 0.1 times/patient year. Incidence of tunnel infection and the exit-site infection for group A was lower than that of group B (0 vs 0.036 times/patient year and 0.06 times/patient year vs 0.11 times/patient year respectively). However, the difference was not significant （P>0.05）. Mechanical complications of catheter (catheter migration, omentum wrapping, leakage of peritoneal dialysates, slip out of outer cuff), incidence of inguinal hernia and bellyache between two groups were not significantly different （P>0.05）. There were 4 cases of catheter drawing in each group. Both two groups had the same 12-month technical survival rate as 92.73%. Of 17 dead cases, 7 were in group A and 10 in group B （P>0.05）. The main death causes were cardiocerebral events (47.1%) and infections (23.5%). The 12-month survival rate was 86.34% for group A and 80.68% for group B（P>0.05）. Conclusions There are no significant differences of infection, mechanical complications, technical survival rate and patients’ survival rate between two groups. The efficacy of swan-neck catheter is similar to Tenckhoff catheter in CAPD patients.

Objective To study the effect of long-term salmon calcitonin on bone mineral density (BMD), bone metabolism biochemical indicators and subjective score of bone pain in maintenance hemodialysis (MHD) patients with osteopenia. Methods Thirty-four MHD patients diagnosed as osteopenia by dual-energy X-ray absorptiometry (DXEA) were enrolled in this study. All the patients were treated with hypodermic injection of salmon calcitonin (50 U, thrice a week) for 12 months. The detecting parameters were as follows: BMD with DEXA in lumbar spine (L1-L4), femoral neck, troch, inter, and Ward’s triangle before and after the study; serum bone metabolism biochemical indicators before and 6 and 12 months after the study; subjective scores of bone pain before and 1, 6, and 12 months after the study. Results Thirty-two patients were followed-up successfully. As compared to BMD parameters before study, the total T-score (-1.98±2.20 vs 1.26±1.88, P=0.009) and total Z-score (-0.90±2.15 vs 0.08±2.05, P=0.002) of lumbar spine, the total T-score (-1.72±1.53 vs 1.06±1.58, P=0.016) and totle Z-score (-0.66±0.80 vs 0.08±1.08, P=0.029) of hip, the T-score of L3 (-2.02±2.51 vs 1.24±2.02, P=0.033), the Z-score of L2 (-0.44±1.82 vs 0.06±1.63, P=0.016), the Z-score of femoral troch (－0.65±1.11 vs 0.48±1.12, P=0.034) and the Z-score of inter (-0.58±0.94 vs 0.02±1.12, P=0.006) were increased significantly after study. But there were no significant differences in other examined regions and serum biochemical parameters. The subjective scores of bone pain were decreased rapidly for 41.7% after 1 month (P<0.01) and 76.6% after 6 months (P<0.01). The subjective score of bone pain after 12 months was similar to 6 months. The side effects of salmon calcitonin included nausea and vomitting in 5 cases (14.71%, 5/34）, dizziness, blushing and flustered in 1 case respectively (3.13%,1/32）. Conclusions Long-term hypodermic injection of salmon calcitonin can improve BMD and bone pain for MHD patients with osteopenia but has no significant effect on serum bone metabolism biochemical indicators. Salmon calcitonin is safe for MHD patients with seldom side effects, such as nausea and vomitting.

Objective To investigate the effects of hemodialysis (HD) and peritoneal dialysis (PD) on the complications and outcomes after renal transplantation. Methods Clinical data of 402 renal transplant recipients maintained on dialysis for more than 3 months were retrospectively studied and divided into 2 groups: HD group（n＝303）and PD group（n＝99）. Among them, 345 recipients were followed up for an average of (30.2±15.2) months. The impact of HD and PD on the acute rejection, delayed graft function (DGF), infection, chronic rejection and the graft and patient survival rates were analyzed. Results The mean dialysis duration was significantly longer in PD group and the hepatitis B infection rate was significantly higher in HD group. There were no significant differences between the HD and PD groups in regarding to primary disease for end-stage renal disease, age, gender, blood pressure, hemoglobin, HLA match, hot and cold ischemia time, and hepatitis C virus infection. The incidence of DGF, acute and chronic rejection, and cytomegalovirus and other infections between HD and PD groups were not significantly different. However, the graft loss happened more frequently in hepatatis B patients than that in non hepatitis B patients （19.23% vs 8.86%, P=0.021）, and the post-transplant infection ocurred less in non hepatits B patients with PD. The acute rejection episodes were higher in HD patients who received pretransplant dialysis for more than 12 months (P<0.05). The overall recipients survival rates of HD and PD groups were similar( 1-year: HD 94.34%, PD 91.25%; 5-year: HD 92.83%, PD 90%), and the same as the graft survival rates in HD and PD groups (1-year: HD 93.21%, PD 96.25%; 5-year: HD 87.17%, PD 91.25%). Conclusions The influences of PD and HD on the complications after renal transplantaton, 1-year and 5-year recipients and graft survival rates are similar, so both HD and PD can be chosen as the pretransplant dialysis modality. As the incidence of acute rejection increases with time in HD, it is better to shorten the time of pretransplant dialysis to decrease the complication.

Objective To investigate the prevalence and risk factors of chronic kidney disease (CKD) in the adult urban population of Hezhou Guangxi. Methods One thousand and two hundred urban residents (older than 18 years) from Hezhou Guangxi were randomly selected using a random sampling. All the residents were interviewed. Their morning spot urine were tested to determine albumin to creatinine ratio (abnormal：≥30 mg/g), and renal function [abnomal：eMDRD <60 ml·min-1·(1．73 m2)-1] was assessed. Morning spot urine dipstick of hematuria (abnormal: ≥l+) was confirmed by microscopy (abnormal: > 3 red blood cells／HP)． The associations among demographic characteristics, health characteristics and indicators of kidney damage were examined． Results Eligible data of 1069 subjects were enrolled in the study． The prevalence of albuminuria was 7.5％, hematuria 4.8%, and reduced eGFR 3.6％. The prevalence of kidney disease was 14.4％ and the recognition was 1.4％. Age(OR 1.022, 95%CI 1.008-1.035）, gender (OR 2.249, 95%CI 1.502-3.367), diabetes mellitus (OR 7.422, 95%CI 3.985-13.825) and hypertension (OR 4.397, 95% CI 2.601-7.432) were independently associated with CKD. Conclusions The prevalence of chronic kidney disease is 14.4％ and the recognition is 1.4％ in adult urban population of Hezhou Guangxi. Independent risk factors associated with chronic kidney disease are age, gender, diabetes mellitus and hypertension which is similar to those in developed countries and domestic big cities.

Objective To investigate the current status and changing patterns of hemodialysis in Wenzhou areas of China from 1999 to 2006. Methods Data of blood purification centres of 18 hospitals in Wenzhou areas from 1999 to 2006 were collected. The incidence, prevalence, mortality, the etiology and the relevant factors such as age, gender, dialysis ages, outcomes and causes of death in end stage renal disease(ESRD) patients on hemodialysis(HD) were analyzed retrospectively. Results The incident number and the number of patients increased annually while the mortality remained steady. The male patients outnumbered the female every year, but the male/female ratio was decreasing. The percentage of both young and oldly patients was increasing. The first cause was chronic glomerulonephritis, although the constituent ratio of glomerulonephritis decreased year by year. The constituent ratio of diabetic nephropathy and hypertensive nephropathy increased. The constituent ratio of dialysis age 1-2 years group decreased, ≤1 year, 2-3 years, 3-4 years groups were relatively steady, and 4-5 years, 5-10 years, >10 years groups increased. The number of patients receiving renal transplantation and transferring to peritoneal dialysis increased annually. The leading cause of death was cardiovascular incidence (19.9%), followed by cerebrovascular disorder(10.8%), systemic failure(10.8%), hemorrhagic diseases(4.7%) and infectious diseases(4.3%). The constituent ratio of cardiovascular incidence and cerebrovascular disorder were relatively steady. The constituent ratio of hemorrhagic diseases and systemic failure showed great fluctuation. The constituent ratio of infectious diseases and malnutrition was decreasing. Conclusions In Wenzhou area from 1999 to 2006, the patients number has been increasing annually. The onset age of HD is 30-70 years old, and proves a younger tendency and older tendency. The leading cause is chronic glomerulonephritis. The constituent ratio of diabetic nephropathy and hypertensive nephropathy rises year by year. The long-term survival rate of HD patients is improved. The leading cause of death is cardiovascular accident.

Objective To investigate the effects of angiotensin Ⅱ(AngⅡ) on the expression of TLR4 and its role in lipopolysaccharide (LPS)-induced NF-κB activation and CD40 expression in rat peritoneal mesothelial cells (RPMCs). Methods RPMCs were harvested from Sprague-Dawley rat peritoneal cavity and maintained under defined in vitro condition. The cells were treated with AngⅡ at different concentrations (10-9, 10-8, 10-7, 10-6 mol/L) and exposed to AngⅡ (10-7 mol/L) for different times (1, 2, 4, 8, 12, 24, 48 h for mRNA and 6, 12, 24, 36, 48 h for protein, respectively). Meanwhile, the influence of AT1 receptor antagonist (AT1R, losartan, 10-5 mol/L) and AT2 receptor blocker (AT2R, PD123177, 10-5 mol/L) on the TLR4 induced by AngⅡ was observed. After synchronization for 24 hours, the cells were randomly assigned to four groups: the control group, the AngⅡ (10-7 mol/L) group, the LPS (1 mg/L) group, the AngⅡ (10-7 mol/L) plus LPS (1 mg/L) group, which were used to investigate the effects of AngⅡ on the NF-κB activation and CD40 expression induced by LPS. The mRNA expression of TLR4 and CD40 was measured by RT-PCR and the protein abundance of TLR4, NF-κB p65, phospho-p65, IκBα and phospho-IκBα were analyzed by Western blot. Immunofluorescence was performed to determine the subcellular localization of p65 subunit of NF-κB. Results (1) Treatment of RPMCs with AngⅡ resulted in a concentration-dependent increase in the expression of TLR4. AngⅡ at 10-9, 10-8, 10-7 and 10-6 mol/L increased TLR4 mRNA expression by 70.5%, 89.5%, 102.9%, and 121.9%, respectively and protein expression by 12.1%, 27.7%, 51.2%, and 41.6%, respectively (P<0.01). Treatment of RPMCs with 10-7 mol/L AngⅡ resulted in a time-dependent increase in the expression of TLR4, with the peak of mRNA expression at 8 and 12 h (P<0.01) and the protein expression at 12 and 24 h (P<0.01). (2) Losartan antagonized AngⅡ-stimulated expression of TLR4 by 33.5% (P<0.05), PD123177 had no such effect (P>0.05). (3) Treatment of RPMCs with LPS (1 mg/L) for 60 min significantly increased the ratio of phospho-IκBα to IκBα by 362.6% (P<0.01) , phospho-p65 to p65 by 67.4% (P<0.05), and LPS (1 mg/L) for 4 h significantly increased the expression of CD40 mRNA by 299.9% (P<0.01) compared to the control group. In comparison to the LPS (1 mg/L) group, preincubation of RPMCs with AngⅡ (10-7 mol/L) for 24 h then treated with LPS (1 mg/L) for 60 min significantly increased the ratio of phospho-IκBα to IκBα by 49.1% (P<0.01), phospho-p65 to p65 by 29.3%(P<0.05), and LPS (1 mg/L) for 4 h significantly increased the expression of CD40 mRNA by 56.8%(P<0.01). (4) The p65 subunit of NF-κB was dominantly distributed in the cytoplasm in the control and AngⅡ group. Following exposure to LPS for 60 min, p65 subunit labeling was upregulated and translocated into the nuclei. A significantly increased nuclear staining of p65 in cells treated with AngⅡ plus LPS were observed. Conclusions AngⅡ induces the expression of TLR4 in dose- and time-dependent manner in RPMCs, resulting in enhanced NF-κB signaling and induction of CD40 expression. Locally produced AngⅡ in the peritoneum may play an amplified role in LPS-induced peritoneal inflammation.

Objective To explore the glomerular change of renin-angiotensin system (RAS) expression in AT1aR gene knockout mice and its effects on extracellular matrix (ECM)remodeling under diabetic condition. Methods AT1aR knockout mice were generated previously. Hyperglycemia was induced by peritoneal injection of streptozotocin in AT1aR knockout mice and wild type mice. Normal AT1aR knockout mice and wild type mice were used as control group. Twelve weeks later, kidneys were harvested and frozen quickly in dry ice-acetone. Glomeruli were collected by laser capture microdissection and total RNA was extracted. mRNA expression of AT1aR, AT1bR, AT2R, angiotensinogen, ACE, renin, and CYP11B2 was assessed by real-time PCR. ECM accumulation was evaluated by PAS staining. Protein levels of transforming growth factor β1(TGF-β1), type 1 plasminogen activator inhibitor(PAI-1), monocyte chemotactic protein 1(MCP-1) and renin were semi-quantitated by immunostaining. Results Compared to the wild type, mRNA expression of AT1bR, angiotensinogen, renin, CYP11B2 within glomeruli was upregulated significantly in AT1aR knockout mice （P<0.05), but no change of ACE expression was found in these two groups. AT2R protein was poorly detected in AT1aR knockout glomeruli and downregulated in wild type glomeruli. ECM accumulation was significantly increased associated with the parallel increase in TGF-β1, PAI-1, MCP-1 and renin within glomeruli(P<0.05). Conclusions AT1aR gene knockout cannot improve ECM deposition in diabetic nephropathy. The compensate change of RAS components may be involved in this scenario: upregulation of AT1bR, downregulation of AT2R. CYP11B2 and renin may function in a novel pathway.

Objective To observe the impact of IL-1β on the expression of lectin-like oxidized LDL receptor 1(LOX-1) and ATP-binding cassette transporter A1(ABCA1) in human mesangial cell line (HMCL), and its association with cholesterol homeostasis of HMCL. Methods Levels of LOX-1 and ABCA1 of HMCL induced by IL-1β were examined by using real-time PCR and Western blot. Results IL-1β up-regulated LOX-1 mRNA and protein expression. Treated with 5 μg/L IL-1β, the levels of LOX-1 mRNA and protein reached the peak after 6 h and 24 h of stimulation and were 6.87 folds and 1.88 folds of control rspectively. The expression of ABCA1 mRNA and protein of lipid-loaded HMCL was down-regulated by IL-1β. Stimulated with 5 μg/L IL-1β, the expression of ABCA1 mRNA and protein decreased to the lowest level, 19.0％ and 50.62％ of the baseline respectively. Conclusions The expression of LOX-1 can be up-regulated while the expression of ABCA1 can be decreased by the stimulation of IL-1β. IL-1β can enhance dyslipidemia and influence the balance of cholesterol homeostasis of HMCL.

Objective To evaluate the nephrotoxicity induced by radiographic contrast media with different osmolality in rats with hypercholesterolemia, and to explore the protective effect of pentoxifylline. Methods Forty-eight healthy SD male rats were randomly divided into normal dietary group (NN, n=8) and high cholesterol supplemented dietary group (H, 4% cholesterol and 1% cholic acid, n=40). At the end of 8th week, the rats with high cholesterol diet were randomly divided into five subgroups (n=8, respectively): high cholesterol diet group(HN), high cholesterol plus iso-osmolar contrast media (iodixanol, IOCM) group (HI), high cholesterol plus low-osmolar contrast media (iohexle, LOCM) group(HL), high cholesterol diet plus high-osmolar contrast media (diatrizoate, HOCM) group (HH) and high cholesterol plus HOCM plus pentoxifylline group (HHP). Forty-eight hours after contrast media injection, the rats were executed and blood samples were prepared to determine total cholesterol, triglyceride, serum creatinine, creatinine clearance(Ccr), fractional excretion of sodium and potassium(FeNa, FeK), and angtensionⅡ(AngⅡ) levels. The renal injury was assessed by HE staining and TUNEL staining, respectively. The expression of NF-κB protein in the renal tissue was detected by using immunohistochemical method. Results An increase of cholesterol was observed in all the rats with high cholesterol diet. Scr, FeNa%, FeK% and AngⅡlevels of rats in HH group were obviously higher than those in HL and HI groups respectively. Ccr in HH group[(0.11±0.02) ml·min-1·(100 g)-1] was significantly lower than that in HHP group [(0.43±0.03) ml·min-1·(100 g)-1], HL group [(0.25±0.02) ml·min-1·(100 g)-1] or HI group [(0.27±0.03) ml·min-1·(100 g)-1] (P<0.05). TUNEL staining showed that the percentage of apoptotic cells in HH group [(89.60±6.40)%] was higher than that of the other groups [NN (2.40±0.77)％, HN (5.60±1.08)％, HHP (8.91±1.44)％, HL (63.34±11.97)％ and HI (61.50±9.40)％]. Immunohistochemistry staining showed that the average gray value of NF-κB positive cells in HH group decreased (P<0.05). There were no significant differences of all indices between HL and HI groups (P>0.05). Conclusions Contrast media can cause kidney injuries in the rats with hypercholesterolemia. PTX can protect the renal tissue from nephrotoxicity induced by HOCM in hypercholesterolemia.

Objective To investigate the role of LPL in enhancing VLDL uptake in mesangial cells and modulating VLDL-mesangial interaction. Methods Human wild type LPL (LPLwt), catalytically inactive LPL (LPL194) or control alkaline phosphatase (AP) were expressed in human mesangial cell line (HMCL) via adenoviral vectors. The expression of LPL mRNA and protein was detected by RT-PCR and immunochemistry staining, respectively. LPL activity was assayed by radioisotope labeled liposome substrate. Cellular lipid deposition was visualized by oil red O staining and analyzed quantitatively by standard enzymatic procedures. Effect of LPL on HMCL proliferation was evaluated by colorimetric assay using MTT. MCP-1 mRNA and protein levels in treated HMCLs were determined by real-time quantitative RT-PCR and enzyme-linked immunosorbent assay respectively. For adhesion study, HMCLs were treated with VLDL for six hours, followed by one-hour incubation with Tamm-Horsfall protein-1 (THP-1) cells. Results Compared with HMCLs transfected by Ad-AP, the lever of cellular triglyceride content was sharply increased in Ad-LPLwt transfected HMCLs [(109.11±5.01) mg/g protein vs (23.98±3.23) mg/g protein, P＜0.01] and was slightly increased in Ad-LPL194 transfected HMCLs[(36.33±2.64) mg/g protein vs (23.98±3.23) mg/g protein, P＜0.05]. LPLwt amplified VLDL-driven mesangial cells proliferation. Compared to the HMCL-Ad-AP, MCP-1 mRNA and protein expression increasd by 39% (P＜0.05） and 171% (P＜0.01） in HMCL-Ad-LPLwt, and the amount of THP-1 cells adhering to HMCL-Ad-LPLwt was increased by 1.69-fold (P＜0.05）, without significant difference between HMCL-Ad-LPL194 and HMCL-Ad-AP. Conclusions Overexpression of either active or inactive LPL in HMCLs accelerates VLDL-induced triglyceride accumulation, and enzymolysis action of LPL may be the major factor in this process. Active LPL significantly amplifies VLDL-induced proliferative effect on mesangial cells and enhances monocyte adhesion to mesangial cells through up-regulation of MCP-1. Hence, LPL may be an important contribution to initiation and progression of renal injury mediated by triglyceride-rich lipoproteins.

Objective To observe whether bone marrow mesenchymal stem cells (MSCs) can promote the repair of IgA nephropathy and to explore its possible mechanism. Methods Sprague-Dawley rats were randomly divided into three groups which were MSCs injection group, normal saline(NS) infusion group and healthy control group. IgA nephropathy model was established by the improving method with BSA+SEB+CCl4 in former two groups. MSCs of SD rats were continuously cultured in vitro and identified with specific surface antigens by flow cytometry and osteogenic and adipogenic differentiation. MSCs were labeled with bromodeoxyuridine (BrdU) in vitro before transplanted. At 1st and 4th week after MSCs injection, the changes of body weight, urine protein, renal function, histopathology and IgA immunofluorescence were observed. MCP-1, TGF-β1 in urine were detected by ELISA. The expression of MCP-1, TGF-β1 in kidney were examined by RT-PCR. The cytokines and BrdU labeled MSCs were detected by immunohistochemistry to observe the disposition in kidney. Results At the end of the first week of MSCs transplantation, MSCs group urine protein (36.86±4.78) mg/24 h, serum creatinine (53.50±6.28) μmol/L, and the NS group urine protein (66.98±5.86) mg/24 h, serum creatinine (82.50±8.36) μmol/L, the differences between two groups were significant(P<0.05). At the same time, the content of MCP-1, TGF-β1 in urine and expression in renal tissue of MSCs group were obviously less than those of NS group(P<0.05). At the end of the 4th week, the body weight, histopathology, IgA immunofluorescence of MSCs group were remarkably improved as compared with those of NS group. The content of MCP-1, TGF-β1 in urine and expression in renal tissue, and renal pathological change in MSCs group had no significant differences as compared with those of healthy control group. As the time passed, the disposition of BrdU-labeled MSCs in kidney was taper. Conclusions MSCs injection contributes to renal repair in rat IgA nephropathy. The mechanism may partly depend on adjusting the excretion of cytokines in renal microenvironment and/or other functions rather than completely depend on their differentiation to renal cells.

Objective To clarify the signaling mechanisms underlying angiotensin II biphasic regulation of renal proximal Na+-HCO3- transport. Methods Different concentration AngⅡ to the responses of Na+-HCO3- cotransporter(NBC) activity in isolated proximal tubules, with or without ATR, MAPK, cPLA2α, P450 blockade was compared in wild-type and AngⅡ type 1a receptor (AT1aR)-deficient mice. The phospholipase of ERK was examined by Western blotting. AT1aR mRNA was examined by RT-PCR from kidney proximal tubules. Results (1)In isolated wild-type mouse, renal proximal tubules showed biphasic effects of AngⅡ on NBC activity. Low concentration AngⅡ (10-10 mol/L) increased NBC activity, but high concentration AngⅡ (10-6 mol/L) decreased NBC activity. Olmesartan (AT1 antagonist) blocked both stimulatory and inhibitory effects of AngⅡ on NBC activity, but PD98059 (mitogen-activated protein kinase inhibitor) blocked only the stimulatory effect of low concentration AngⅡ（10-10 mol/L）. (2)In AT1aR-deficient mice, only the stimulatory effect by high concentration of AngⅡ (10-6 mol/L) was observed, which was blocked by olmesartan and PD98059. (3)In wild-type mice, pharmacological blockade of cPLA2 or P450 converted the inhibition effect by high concentration AngⅡ (10-6 mol/L) to the stimulation, which was blocked by olmesartan and PD98059. These results indicated that the extracellular signal-regulated kinase (ERK) activation via AT1 mediated only the stimulatory effect of AngⅡ, while the cPLA2α/P450 activation via AT1 mediated the inhibitory effect of AngⅡ independently of ERK. The analysis of ERK phosphorylation by AngⅡ also supported a view that the cPLA2α/P450 pathway worked to suppress the ERK activation. Conclusions AngⅡ activates ERK and cPLA2α with different concentration dependency via AT1. The balance between ERK and cPLA2α activities determines the final responses to AngⅡ in intact proximal tubules.