Objective To investigate the change of plasma marinobufagenin (MBG) level and the expression of its receptor Na+-K+-ATPase (NKA) in renal biopsy specimens of chronic glomerulonephritis (CGN) patients. Methods Twenty-eight CGN patients and 14 healthy people were enrolled in the study. The plasma MBG concentration was measured by competitive inhibition ELISA system. Immunofluorescence and immunohistochemistry staining were applied to detect the expression of NKA in renal biopsy specimens of 28 CGN patients and analyzed by semi-quantitively. Results Compared with healthy controls, CGN patients had significant lower plasma MBG concentration [(0.579±0.214) nmol/L vs (0.715±0.154) nmol/L, P<0.05], without further significant difference between CGN patients with hypertension and with normal blood pressure [(0.595±0.231) nmol/L vs (0.557±0.197) nmol/L, P>0.05]. Meanwhile, proximal tubular staining of NKA was decreased compared with normal controls. The NKA positive staining area of the CGN group was lower than that of normal controls [2.1% (0.5%-6.2%) vs 5.6% (3.5%-10.8%), P<0.01] and correlated with 24-hour urinary sodium excretion (r=0.551, P<0.01). Conclusion Decreased plasma MBG level and its receptor expression on proximal tubules may play a role in the regulation of sodium in CGN.

Objective To observe the efficacy and safety of in-center nocturnal hemodialysis (INHD) in uremic patients. Methods Thirty-two maintenence hemodialysis (MHD) patients received INHD (3 times per week and 7.5 hours each session) for 6 months. Before and 1, 3 and 6 months after entering INHD, blood routine, hepatic and renal function, serum electrolyte, lipids, parathyroid hormone and β2-microglobulin(β2-MG) were assayed, Kt/V and URR were calculated. Blood pressure of each dialysis session 2 months before and 6 months after INHD was recorded. Cardiac ultrasound and SF-36 questionnaire before and after INHD were performed. Use of drugs was recorded. Results Compared with 2 months before INHD, pre-dialysis BP decreased [(130.3/86.0) vs (139.3/88.6) mm Hg, P<0.01], while post-dialysis BP raised significantly [(121.1/80.5) vs (115.0/77.8) mm Hg, P<0.01] 6 months after INHD. Intradialysis hypertension (9.8%vs 24.0%) and hypotension (7.3% vs 14.9%) both reduced (all P<0.01). Serum phosphorus [(1.37±0.27) vs (2.08±0.49) mmol/L, P<0.01] and iPTH [(355.4±139.6) vs (632.3±750.0) ng/L, P<0.01] decreased, while calcium increased [(2.64±0.25) vs (2.28±0.37) mmol/L, P<0.01], HDL[(1.27±0.29) vs (0.75±0.08) mmol/L] increased, LDL [(2.04±0.52) vs (2.75±0.75) mmol/L] decreased (all P<0.05). URR [(79.7±0.1)% vs (64.7±4.7)%] and Kt/V (1.40±0.44 vs 0.89±0.25, P<0.01) increased. Serum β2-MG decreased [(17.3±3.9) vs (24.6±5.9) mg/L, P<0.01]. LVMI decreased [(99.8±29.0) vs (114.8±72.7), P<0.05]. Physical functioning, role-physical and role-emotional of SF-36 increased (all P<0.01). The types of antihypertension drug, dosage of EPO, Vitamin D3 and phosphorus binder decreased (all P<0.01). Patients of drug withdrawal increased (P<0.05). Conclusion The hypertension, anemia, calcium-phosphorus metabolism, lipid disorder, cardiac malfunction and the quality of life are improved in INHD patients.

Objective To investigate the cell types of renal allograft glomerulitis in post-transplantation patients and its relationship with peritubular capillary (PTC) C4d deposition. Methods Fifty-one allograft kidney transplant recipients diagnosed as acute rejection from June 2006 to June 2009 in our center were enrolled in this retrospective study. Fifty-one biopsy specimens of these patients accompanied with glomerulitis were examined by immunostain for macrophages, T cells, granzyme B, Foxp3 and C4d. Glomeular macrophages, T cells, cytotoxic T cells, regulatory T cells were counted. The recipients were divided into C4d+ and C4d- groups according to the C4d deposition in peritubular capillaries. Results The total number of glomerular infiltrating cells in C4d+ group was significantly higher than that in C4d- group (17.79±7.70 vs 8.17±3.80, P<0.01). The infiltrating cell was predominantly macrophage in C4d+ group and significantly higher as compared to C4d- group (13.73±7.03 vs 2.57±1.22, P<0.01). However, the infiltrating cell was predominantly T cell in C4d- group and significantly higher as compared to C4d+ group (5.60±2.81 vs 4.05±2.60, P=0.023). The infiltrating T cells in both groups were predominantly cytotoxic T cells. Conclusions The total number of infiltrating cells in glomerulitis is related to peritubular capillary C4d deposition. The infiltrating cell is predominantly macrophage in C4d+ group and predominantly T cell in C4d- group. Meanwhile the infiltrating T cell in both groups is predominantly cytotoxic T cell.

Objective To study the association of bone metabolism with the degree of proteinuria in patients of chronic kidney diseases (CKD). Methods A total of 71 CKD patients diagnosed as primary glomerulopathy were randomly selected from 2008.1-2009.5 in the First People’s Hospital of Shanghai. They were classified into three groups according to proteinuria: group A of 25 patients, proteinuria <1.0 g/24 h; group B of 16 patients, proteinuria 1.0-<3.5 g/24 h; group C of 30 patients, proteinuria ≥ 3.5 g/24 h. Fifty-eight healthy persons were selected from our medical examination center at the same time as control. Serum albumin, calcium, phosphorus, PTH, 25 hydroxy vitamin D3, bone gla protein (BGP), degradation products of C-terminal telopeptides of type I collagen (CTx), 24-h urinary protein excretion, and the ratio of urinary calcium to creatinine (UCa/Cr) were measured. Bone mineral density (BMD) was detected by dual-energy X-ray absorptiometry. Results Compared with control group, serum levels of calcium [(2.23±0.08), (2.13±0.09), (2.04±0.06) vs (2.37±0.12) mmol/L], 25-(OH)D3 [(50.19±6.58), (47.78±6.69), (42.42±10.85) vs (56.34±8.34) nmol/L] were significantly lower and UCa/Cr was significantly higher in A, B, C groups respectively (all P<0.05). In group B and C, BGP was lower [(18.69±7.35), (16.13±5.76) vs (22.88±6.21) μg/L] and CTx was higher [(413.59±114.93), (516.21±314.25) vs (304.53±234.15) ng/L] (all P<0.05). BMD was lower only in group C[(1.028±0.090) vs (1.090±0.062) g/cm2, P<0.05]. Pearson analysis showed that 24-h urinary protein excretion was negatively correlated with serum calcium and 25 hydroxy vitamin D3, and positively correlated with UCa/Cr. UCa/Cr was positively correlated with serum CTx and negatively correlated with BGP. 25-(OH) D3 was positively correlated with BGP and negatively correlated with CTx. Conclusion Bone metabolism disorder exists in CKD patients, presenting the decrease of bone formation and the increase of bone resorption, which is associated with as the degree of proteinuria, especially in patients with nephrotic syndrome.

Objective To analyze the protein expression profile of human glomerular mesangial cells (HMCs) under high glucose and to characterize molecular functions and biological processes. Methods HMCs were divided into high glucose cultured group (30 mmol/L) and normal glucose cultured group (5 mmol/L). The total proteins were extracted after culture for 48 hours. The total proteins of the two groups were separated using two-dimensional fluorescence difference in gel electrophoresis (2-D DIGE) and analyzed using DeCyder 2-D difference analysis software. The differentially expressed proteins were further identified using in-gel digestion with trypsin, of which peptide extracts were prepared for MALDI-TOF-MS analysis. Protein identifications were searched in the NCBI protein database using the Mascot search engine. Results One hundred and forty-seven protein spots whose expression levels were significantly increased or decreased more than 1.5 folds under high glucose were identified. Ninety-six differentially expression protein spots were analyzed by peptide mass fingerprinting and 37 kinds of proteins were identified. The protein spots of phosphatidylethanolamine binding protein 1(PEBP-1), granulysin, ATP synthase H+ transporting mitochondrial F0 complex subunit F2 were observed only in high glucose group. The expression of 24 proteins was up-regulated by high glucose, including eosinophil cationic protein, RGS membrane-interacting proteins 16 (MIR16), peptidyl-prolyl cis-trans isomerase, disks large homolog DLG2, breast cancer 2, early onset (BRCA2), Catechol-O-methyltransferase etc. The expression of 5 proteins was down-regulated by high glucose, including O-GlcNAc transferase-interacting protein 106 000 isoform 1, proteasome beta 6 subunit precursor, NEFA-interacting nuclear protein NIP30 etc. Conclusions Expression of 147 proteins in HMCs alters under high glucose. These proteins are involved in the regulation of cytoskeleton, glucose metabolism, cell division, gene transcription, signal transduction, phosphorylation, cell proliferation, apoptosis etc. In-depth analysis of these differentially expressed proteins’ function and crosstalk is expected to provide an important experimental basis for clarifying the pathogenesis of diabetic nephropathy.

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

Objective To elucidate the mechanism of inflammation in vascular endothelial cells induced by hypochlorite-modified albumin (HOCl-Alb). Methods HOCl-Alb-induced NADPH oxidase activity in human umbilical vein endothelial cells (HUVEC) was measured by lucigenin-enhanced chemiluminescence. Phosphorylation of p47phox and binding of p47phox and p22phox were measured with immunoprecipitation and Western blotting. Membrane translocation of p47phox was measured with immunofluorescence. RT-PCR and Western blotting were used to determine the intercellular adhesion molecule-1 (ICAM-1) mRNA and protein expression in the presence or absence of apocynin, respectively. Results Co-incubation of HUVEC with HOCl-Alb resulted in the enhancement of NADPH oxidase activity in time- and dose-dependent manner. Compared with bovine serum albumin group, exposure of the cells with 200 mg/L HOCl-Alb for 15 min resulted in a 6.16-fold increase in NADPH oxidase activity. Phosphorylation and membrane translocation of p47phox and binding of it with p22phox were also induced by HOCl-Alb. ICAM-1 expression was up-regulated after exposure to HOCl-Alb and this effect was significantly abolished by apocynin, a specific inhibiter of NADPH oxidase, in dose-dependent manner. Preincubation of the cells with 500 μmol/L apocynin inhibited the expression of ICAM-1 protein induced by HOCl-Alb by 68.97% (P＜0.01). Conclusion NADPH oxidase plays a central role in HOCl-Alb-mediated ICAM-1 expression and provides a mechanism for HOCl-Alb-related vascular endothelial inflammation.

Objective To investigate the location and expression of hypoxia inducible factor(HIF) subunits in the remnant kidney of 5/6 nephrectomy rats. Methods Remnant kidneys were produced in adult male SD rats by 5/6 nephrectomy. The renal function and histopathological changes were evaluated at week 1, 2, 4, 6, 8 and 12 after operation. Tissues of remnant kidneys were collected to detect the location and expression of HIF-1α and HIF-2α by immunohistochemistry staining and Western blotting. The mRNA levels of HIF targeted genes vascular endothelial growth factor (VEGF) and heme oxygenase-1(HO-1) were determined by RT-PCR. Results (1) 5/6 nephrectomy rats underwent one week of acute renal failure at first[Scr (122.8±22.1) μmol/L] and then developed compensative chronic renal failure [(66.0±3.7)－(66.4±8.4) μmol/L], but the level of Scr increased quickly after week 6[(66.4±8.4)－(127.8±22.7) μmol/L], concomitantly with progressive tubulointerstitial fibrosis in remnant kidney cortex. (2) In cortex, HIF-1α was expressed only in the atrophic and dilated tubular cells while HIF-2α was located in endothelial, interstitial fibroblasts, and vascular smooth muscle cells. The semiquantitative results of imunohistochemistry and Western blotting revealed that HIF-1α and HIF-2α were both gradually up-regulated during the early stage of remnant kidney, peaked at week 4 and 6, and then gradually down-regulated. (3) The mRNA levels of HIF targeted genes VEGF and HO-1 transiently peeked at week 4 and 6, and then decreased gradually. Conclusions The increased stabilization of HIF-α protein and transcription of HIF targeted genes at the early stage of this model is a compensation reaction towards hypoxia. The mechanism of decreased expression of HIF-α at the end stage of chronic kidney disease deserves further investigation.

Objective To investigate the effects of chronic renal failure rabbit serum on proliferation and nuclear factor kappa B(NF-κB) activation of rabbit arterial smooth muscle cells(ASMCs) and to explore the possible mechanism. Methods Rabbit model of chronic renal failure was established by the ligation of renal arterial branches. ASMCs were incubated in the media with various concentrations of chronic renal failure serum cultured in vitro. Cell proliferation was assessed by MTT. Cell apoptosis was detected by Hoechst33342 staining. NF-κB p65 nuclear translocation was analyzed by immunofluorescence. Expression of proliferating cell nuclear antigen (PCNA) and NF-κB p65 proteins in response to chronic renal failure serum in ASMCs was determined by Western blotting. Results Lower concentrations of chronic renal failure serum (≤10%) could significantly promot the proliferation of ASMCs in a dose- and time-dependent manner. Higher concentrations of chronic renal failure serum (>10%) could significantly inhibit the proliferation and induce apoptosis of ASMCs compared to the normal control (P<0.05). Under the stimulation of lower concentrations of chronic renal failure serum, the expression of PCNA and NF-κB p65 increased significantly compared to the normal control (P<0.01), while decreased markedly under the stimulation of higher concentrations of chronic renal failure serum compared to the normal control (P<0.01). Under the stimulation of 10% chronic renal failure serum, nuclear translocation of NF-κB p65 in ASMCs was found. Conclusions Different concentrations of chronic renal failure rabbit serum can effectively induce ASMCs proliferation or apoptosis. The mechanism of promoting proliferation may be mediated by activating NF-κB, which will be useful for the treatment of accelerated atherosclerosis in chronic renal failure.

Objective To investigate the protective effect and possible mechanism of adiponectin (ADPN) and its receptors in diabetic nephropathy (DN). Methods A total of 64 Sprague-Dawley female rats were equally divided into diabetes mellitus(DM) group with streptozotocin(STZ) 60 mg/kg injection, and normal control (NC) group with the same dose of citric acid buffer randomly. At the end of 2, 6, 10 and 12 weeks, weight, fast blood glucose, 24-hour urinary albumin excretion, serum creatinine and serum fast blood insulin were measured. The level of adiponectin in urine and serum was examined by ELISA. The pathological change of kidney tissue was studied by periodic acid-Schiff’s staining (PAS) and the expression of adiponectin receptor 1 (adipoR1), adiponectin receptor 2(adipoR2) was determined by immunohistochemistry. The NRK-52E cells were cultured with 5 mmol/L glucose(control group), 30 mmol/L glucose (high glucose group), 30 mmol/L glucose+different concentrations of adiponectin (1 mg/L, 5 mg/L, 10 mg/L, ADPN groups), respectively. The mRNA expression of monocyte chemoattractant protein 1 (MCP-1) was measured by RT-PCR after 12 h. Results (1)Both in urine and serum, the levels of ADPN in DM group were higher than that in NC group after 6 weeks (P<0.01) and increased gradually during the whole experiment. (2)Compared with NC group, the expressions of AdipoR1 and AdipoR2 in kidney tissues were elevated gradually in DM group. There was a significant positive correlation of ADPN with AdipoR1 and AdipoR2 in DM group(r=0.666, P<0.01; r= 0.684, P<0.01). (3)The expression of MCP-1 increased in high glucose group, but decreased in 1 mg/L, 5 mg/L, 10 mg/L adiponectin group. Conclusions The level of ADPN in urine and serum is positively correlated with its receptors and elevates with the process of diabetic kidney disease. High level of serum adiponectin plays a protective role through the direct action of AdipoR and the alleviation of MCP-1 expression in renal tubules.

Objective To observe the effect of neutralizing monoclonal antibodies to anti-glomerular basement membrane (GBM) antibody on anti-GBM nephritis rats. Methods Wistar rats were randomly divided into five groups: control group Ⅰ was a negative control and was injected with healthy human lgG via the caudal vein. Control group Ⅱ was injected with neutralizing monoclonal antibodies to anti-GBM antibody only. Anti- GBM nephritis group was injected with human anti-GBM antibody via the caudal vein only. Intervention group Ⅰ was injected with human anti-GBM antibody via the caudal vein and then with neutralizing monoclonal antibodies to anti-GBM antibody at day 7. Intervention group Ⅱ was injected with human anti-GBM antibody via the caudal vein and then with neutralizing monoclonal antibodies to anti-GBM antibody at day 14. The blood, urine and kidney tissue were collected at day 7, 14, 21 for analysis of 24-hour urinary protein, BUN, Scr and histological study. Results At day 21, there were significant decreases in intervention group I compared with anti-GBM nephritis group in 24-hour proteinuria [(16.62±5.53) g], BUN[(11.53±2.26) mmol/L] and Scr [(102.46±16.86) μmol/L] (P<0.05), and also in intervention group Ⅱ as compared to anti-GBM nephritis group, but no significant difference was found (P>0.05) . There was obvious decrease of renal cell proliferation, crescent formation and deposition of immune complexes in intervention group Ⅰand intervention group Ⅱcompared with anti-GBM nephritis group, while such improvement in intervention group Ⅰ was more significant. There was no significant change in control group Ⅰ and control group Ⅱ. Conclusion The early application of neutralizing monoclonal antibodies to anti-GBM antibodies can effectively improve the kidney lesions of anti-GBM nephritis rats.