Objective To evaluate the effects of alendronate sodium on bone mineral density (BMD) in maintenance haemodialysis (MHD) patients with osteoporosis. Methods Twenty-eight MHD patients with osteoporosis diagnosed by dual energy X-ray absorptiometry were randomly divided into control group (n＝15) and treatment group (n=13). Patients in treatment group were treated with oral 70 mg alendronate sodium once a week for 18 months. BMD of hip and lumbar spine was measured by dual energy X-ray absorptiometry at baseline and the end of the 6th, 12th and 18th month. Parathyroid hormone, calcium, phosphorous, alkaline phosphatase levels, blood routine, hepatic and renal function were assayed at baseline and the end of the 18th month, Kt/V was calculated, new bone fracture was recorded. Results The BMD, T-scores and Z-scores in the lumbar spine and specific regions of the hip were stable in the treatment group and obviously decreased in the control group (P<0.01). New bone fracture was found in 1 patient of the treatment group and 5 patients of the control group. The side-effect of alendronate sodium was epigastric discomfort in 1 cases. Conclusions Oral alendronate sodium appears to be well tolerated in MHD patients and keep the BMD stable in the lumbar spine and specific regions of the hip.

Objective To investigate the removal effect of immunoadsorption (IA) on associated antibodies and the efficacy in late-onset myasthenia gravis (MG). Methods A total of 25 late-onset MG patients were randomly selected to enroll in this study. IA therapy was given to 10 patients(IA group), while immunoglobin (0.4 g·kg-1·d-1) was administrated to the other 15 patients for 5 days(Ig group). The titers of Titin antibody (Titin-ab), acetylcholine receptor antibody (AchR-ab) and presynaptic membrane antibody (PrsmR-ab) were detected before and after the treatment. Quantitive MG (QMG) score was assessed before and immediately after the entire course of treatment. The clinical efficacy, the duration of respiratory support and in-hospital were compared between two groups. The correlation between three antibodies and QMG score was also analyzed. Results Compared with that before treatment, the Titin-ab P/N values, the AchR-ab P/N values, and the PrsmR-ab P/N values of IA group were all decreased significantly after treatment(P<0.05, respectively). The P/N value of Titin-ab in IA group was decreased by 54.7%±3.5%, which was significantly higher than that in Ig group(19.9%±3.1%) (P<0.01). QMG score reduced by 42.4%±4.2% and 23.8%±3.7% in IA group and Ig group respectively (P<0.01, respectively). Symptoms were effectively ameliorated by both treatments, but the effective power of IA group was higher than that of Ig group(70% vs 40%, P<0.05). Remission time of IA group was significantly shorter than that of Ig group [(5.38±0.42) d vs (8.4±1.54) d, P=0.008), so was the duration of in-hospital [(13.50±0.50) d vs (16.50±0.50) d, P<0.05). The number of respiratory support in IA group was less than that in Ig group (1/10 vs 6/15, P<0.05). By the Pearson correlation analysis, the decrease of Titin-ab showed a better longitudinal correlation with the decrease of QMG score than the other two antibodies (r=0.6315, P<0.01). Conclusion IA can rapidly and effectively clear the pathogenic antibodies of late-onset MG patients and its short-term clinical efficacy is better than immunoglobin.

Objective To observe the efficacy of the treatment of mycophenolate mofetil (MMF) combined with prednisone on steroid-resistant idiopathic membranoproliferative glomerulonephritis (IMPGN) patients with moderate to severe proteinuria. Methods Thirteen cases were diagnosed as IMPGN by renal biopsy after excluding secondary factors. Among 13 patients, 9 had severe proteinuria and another 4 had moderate proteinuria, 9 with hypertension and 11 with decreased renal function. Before MMF therapy, all of the cases were resistant to the treatment of glucocorticoid (prednisone 1 mg·kg-1·d-1) for 8 weeks or more. The dose of MMF was 1.5 g/d. Patients were followed up every month for blood pressure, urinary protein excretion, liver and kidney function, complete blood count, and adverse effects. Results At the initiation, the 24 h urinary protein excretion was (4.1±1.4 ) g, Scr (131.0±44.9) μmol/L, and estimated glomerular filtration rate (eGFR) (63.3±26.8) ml·min-1·（1.73 m2）-1. After prednisone therapy for at least 2 months, the 24 h urinary protein excretion （4.2±1.5） g, Scr（133.2±52.8）μmol/L and eGFR（63.3±27.1） ml·min-1·（1.73 m2）-1 did not change significantly. After 3 months of the addition of MMF, 24 h urinary protein excretion declined slightly [(3.8±1.2) g, P>0.05]. After 6 months, 24 h urinary protein excretion declined significantly [（2.5±0.9） g, P<0.05], with decrease in Scr and eGFR[(97.2±27.3) μmol/L and (81.3±24.2) ml·min-1·（1.73 m2）-1, P<0.05)]. At the end of 1 year, 24 h urinary protein excretion was only (1.5±0.6) g（P<0.01）, Scr and eGFR were (95.9±22.5) μmol/L and (81.2±23.8) ml·min-1·（1.73 m2）-1 (P<0.01). All the patients experienced a partial remission of proteinuria (urinary protein excretion decreased by 50% or more). Adverse event including stomach upset was found in 1 patient. Conclusion MMF combined with glucosteroids can effectively decrease proteinuria and improve renal function without obvious side effect in steroid-resistant IMPGN.

Objective To investigate the survival rate and the influencing factors in lupus nephritis (LN) patients with neuropsychiatric systemic lupus erythematosus(NPSLE). Methods Clinical characteristics and biochemical markers of 78 patients including 59 variances were analyzed. Patients were followed up from the onset of NPSLE to death. Patient survival rate was estimated by Kaplan-Meier method. Cox regression model was used to analyze influencing factors. Results Sixteen (20.5%) of 78 patients died of SLE or its complications. Infection was the main cause of death (31.3%). One-, 3-, 5- and 10-year survival rates were 83.2%, 81.7%, 76.7% and 76.7%, respectively. Hypertension（RR＝6.965，95%CI:1.578-30.746, P＝0.010）, pulmonary infection（RR＝8.171，95%CI:1.954-34.177, P＝0.004）and acute renal failure （RR＝6.978，95%CI: 2.063-23.609, P＝0.002） were risk factors of mortality, while cyclophosphamide(CTX) impulse therapy（RR＝0.130，95%CI:0.031-0.541, P＝0.005） and resolution of NPSLE（RR＝0.169， 95%CI: 0.042-0.679, P＝0.012）were protective factors. Conclusions Infection is the main cause of death in patients of LN complicated with NPSLE. Survival rate of LN patients with NPSLE in this study is lower than those of LN and NPSLE alone reported by other authors. Hypertension, pulmonary infection and acute renal failure are risk factors of mortality, while CTX impulse therapy and resolution of NPSLE reduce the mortality and improve the prognosis.

Objective To study the effect of overexpression of TRPC6 on AngⅡ-induced apoptosis of mouse podocytes in vitro and to explore the possible mechanisms. Methods Mouse TRPC6 cDNA eukaryotic expression vector pEGFP-N1-mTRPC6 was transfected to conditionally immortalized murine podocyte cell line by liposome. The fluorescent microscopy was used to examine the expression of EGFP after 24 hours. The change of TRPC6 protein expression was observed by Western-blot. Podocytes were treated by different concentrations of AngⅡ. The podocyte intracellular calcium concentration was measured with laser-scanning confocal microscope. The expression of Bax and Bcl-2 mRNA was assessed by RT-PCR and the expression of Bax and Bcl-2 protein was measured by Western-blot. The apoptotic ratio of podocytes was monitored by flow cytometry and Hoechst staining. Results About 35% of the cells expressed EGFP. An up-regulation of protein expression of TRPC6 was detected in podocytes when transfected with pEGFP-N1-mTRPC6（P<0.01）. The overexpression of TRPC6 promoted the AngⅡ-induced influx of extracellular calcium and elevated the expression of Bax but decreased the expression of Bcl-2（P<0.01, P<0.05）. The apoptotic ratio of podocyte was (2.50±0.72)% when treated by low-dose AngⅡ（10-10 mol/L）, and it was increased to (4.33±0.45)% when transfected with pEGFP-N1-mTRPC6（P <0.05）. Transfection with pEGFP-N1-mTRPC6 increased apoptosis rate from (15.46±1.40)% to (18.33±0.87)%（P<0.01） by high-dose AngⅡ（10-6 mol/L）. Conclusion TRPC6 plays an important role in the AngⅡ-induced apoptosis of podocytes by promoting the influx of extracellular calcium, which leads to the apoptosis cascade initiation.

Objective To study the effects of the interaction of advanced glycation end products(AGEs) and the receptor of AGEs(RAGE) on apoptosis of mice podocytes. Methods Podocytes were exposed to soluble AGEs such as bovine serum albumin (BSA), carboxymethyl-lysin(CML)-BSA, AGE-BSA and matrix-bound AGEs (AGE-modified collagenⅣ), and to different concentrations of AGE, such as 10 mg/L, 50 mg/L, 100 mg/L. Apoptosis was assessed by TUNEL staining. Fluorescence-activated cell sorting(FACS) was used for the quantification of apoptotic and necrotic podocytes after Annexin Ⅴ-fluorescein isothiocyanate(FITC) and propidium iodide(PI) labeling. Apoptosis was described as the ratio of apoptotic cells to the total number cells under the high-power field. siRNA was transfected into podocytes through combining Dharmacon on Targetplus SMART pool siRNA reagents and Amaxa RNAi nucleofection kit. Results The apoptosis rate was higher in podocytes exposed to either CML-BSA or AGE-BSA than that exposed to BSA. There was a two- to three-fold increase in apoptosis when podocytes were cultured in AGE-modified collagen Ⅳ as compared with native collagen Ⅳ. The apoptotic response of podocytes to AGE-BSA exposure occurred in a dose-dependent manner. Podocyte necrosis occurred only at the highest concentration of AGE-BSA(100 mg/L). AGE-BSA failed to induce apoptosis in podocytes transfected with RAGE siRNA. RAGE-specific gene knockdown did not significantly reduce the apoptosis of podocytes cultured in AGE-modified collagen Ⅳ. Conclusions The AGE-RAGE interaction plays a major role in the apoptosis of podocytes triggered by soluble AGEs, but not by matrix-bound AGEs. Reduction of AGE burden and RAGE expression may be important therapeutic approaches to prevent the progression of kidney disease.

Objective To study the combination effects of telmisartan and pioglitazone on the expression of heparanases (HPA) and podocin in the kidney of diabetic nephropathy (DN) rats and its possible mechanism. Methods DN model rats were established by intraperitoneal injection with STZ for 12 weeks. All the DN rats were randomly divided into telmisartan group （T group）, pioglitazone group (P group), combination of telmisartan and pioglitazone group （L group）, and DN group (D group). Healthy rats were chosen as healthy control group(N group). After gavage with drugs for 12 weeks, 24-h urinary protein and serum biochemical indicators were examined. RT-PCR and immunohistochemistry methods were applied to detect the mRNA and protein expression of HPA and podocin. Results Compared with T group and P group, 24-h urinary protein of L group was markedly decreased(P<0.05). Compared with the N group, the level of fasting blood glucose, relative renal weight, BUN and Scr in other 4 groups were markedly increased (P<0.05). Compared with T group and P group, the Scr level and the expression of HPA mRNA and protein in L group was markedly decreased(P<0.05), and the protein and mRNA expression of podocin in L group was markedly increased (P＜0.05). Conclusion Combination of telmisartan and pioglitazone can down-regulate the mRNA and protein expression of HPA of glomerular basement membrane and up-regulate the protein expression of podocin of podocyte in DN rats, which may ameliorate the proteinuria.

Objective To investigate the effect of losartan on tubulointerstitial fibrosis of rat unilateral ureteral obstruction(UUO) model, and to study whether the use of large dose could be better exert a superior renoprotective effect than conventional dose. Methods Three days after UUO, rats were randomly assigned to conventional dose and large dose of losartan group, sham-operated group and operation group. Two treatment groups were administered orally with a daily dose of losartan 50 mg/kg or 500 mg/kg by gastric gavage. Sham-operated group and operation group received the same volume of physiological saline. Rats were sacrificed 7, 14, 21 days after obstruction. Daily urinary albumin excretion(UalbV), tail-cuff pressure(TCP), the percentage of renal tubular lesions, fractional interstitial area(INT), macrophage infiltration and the expression of transforming growth factor(TGF)-β1 mRNA were assessed and compared. Results The TCP, UalbV, percentage of tubular lesions and interstitial fibrosis, the number of interstitial macrophages and the expression of TGF-β1 mRNA were significantly increased in operation group as compared with other groups(P<0.05). Furthermore, in comparison with conventional dose, the large dose treatment significantly reduced TCP and UalbV, attenuated interstitial fibrosis and tubular lesions, suppressed macrophages infiltration and the expression of TGF-β1 mRNA. Conclusion The large dose of losartan provides superior renoprotection compared to the treatment with conventional dose in UUO model, which interrupts the positive feedback involved in the vicious cycle between inflammatory cell and AngⅡ.

Objective To investigate the mechanism of renal damage due to rupture of atherosclerotic plaque of renal artery in apolipoprotein E (ApoE) knock-out mice. Methods The model for atherosclerotic renal artery stenosis (ARAS) was established by using ApoE knock-out mice. The model mice with renal artery stenosis <50% were divided into the plaque rupture group and the non-plaque rupture group. Wild-type C57BL/6J mice were used as the control group. All the mice were raised under the same conditions. The renal arteries and kidneys were collected for the following analysis. Nuclear factor-kappa-Bp65 (NF-κBp65), intercellular adhesion molecule 1 (ICAM-1) and P-selectin (P-sel) were determined by Western blotting. The expression of interleukin 6 (IL-6) mRNA was detected by RT-PCR. Immunohistochemistry was performed by using serial sections to detect F4/80-related macrophages. Urine n-acetyl-β-d-glucosaminidase (NAG) activity was determined by direct enzyme-substrate coloration. Results In comparison with the non-plaque rupture group and the control group, the expression of NF-κBp65 protein in the blood, renal artery and kidney increased significantly in the plaque rupture group (P<0.05). The expression of F4/80, ICAM-1, P-sel, and IL-6 mRNA were increased significantly in the plaque rupture group (P<0.05), as compared with the non-plaque rupture group and the control group. The Scr and the activity of urine NAG in the plaque rupture group were higher than those in the non-plaque rupture group. The expression of NF-κBp65 protein differed insignificantly between the control group and the non-plaque rupture group (P>0.05). The group differences in the expression of F4/80, ICAM-1, P-sel, and IL-6 mRNA were similar to those in the expression of NF-κBp65 protein. The group differences in the activity of urine NAG and the Scr were similar to those in the expression of NF-κBp65 protein. Conclusion Rupture of atherosclerotic plaque of renal artery causes renal pathology change and renal function damage, which is mediated by inflammation.

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 scrambing 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 podocytes and different PAN concentrations incubated podocytes were detected by flow cytometer. The distribution of F-actin was observed under laser scanning confocal microscope. Nephrin protein expression and its phosphorylation level were examined by immunofluorescence and Western blot. Results The relative cell 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 be partly responsible for the decline of cell adhesion and spreading.

Objective To investigate the effects of resistin on mesangial cells proliferation induced by high glucose and subsequent change of p38MAPK signal pathway. Methods Human macrophrages were cultured and treated with adenovirus encoding for resistin (Ad-resistin) for 48 h and were then co-cultured with human mesangial cells stimulated by high glucose for another 48 h. Mesangial cells were harvested and their proliferation was measured by 3H-TdR. Activator protein 1 (AP-1) was examined by immunocytochemistry and laminin of excellular matrix was observed with immuofluorescence. Protein levels of p38MAPK and TGF-β1 were measured by Western blot. Smad2 phosphatase activity was aslo detected by Western blot. Results The mRNA and protein levels of resistin were significantly higher in Ad-resistin treated macrophages than those in Ad treated cells (P<0.01). Over-expression of resistin up-regulated p38MAPK protein levels of human mesangial cells(P<0.05). Resistin also promoted the proliferation of mesangial cells (P<0.01） and the synthesis of laminin stimulated by high glucose. The expression of TGF-β1 and phosphorylation of Smad2 were up-regulated in the mesangial cells(P<0.05). Conclusion Macrophage cytokine resistin may promote mesangial cells proliferation and abnormal accumulation of excellular matrix stimulated by high glucose via activating p38MAPK signal passway.

Objective To investigate the influence of albumin on the expression of angiotensin-converting enzyme (ACE) in cultured human proximal tubular cells (HK-2). Methods HK-2 cells were exposed to 2.5, 5, 10 g/L bovine serum albumin (BSA) for 6 h and 12 h respectively. The expression of ACE in HK-2 cells was detected by real-time RT-PCR and Western blot. Results Compared to the control group, the expression of HK-2 cells ACE mRNA treated for 12 h with different concentrations of BSA (2.5, 5, 10 g/L) significantly increased (P<0.05). Furthermore, Western blot analysis showed that the expression of ACE protein induced by BSA significantly increased (P< 0.05). Treated with BSA (10 g/L) for 6 h and 12 h, the expression of ACE mRNA significantly increased in a time-dependent manner (P<0.05, respectively), and the ACE protein expression significantly increased (P<0.05, respectively). Conclusion BSA can induce ACE up-regulation in proximal tubular cells, which may lead to the increased production of local AngⅡ and finally contributes to the intrarenal activation of renin angiotensin system (RAS).