Objective To study the prognosis of IgA nephropathy(IgAN). Methods Forty-six out of 780 IgAN patients biopsied in a single unit of north China since 1997 showed segmental necrotizing lesions. Thirty-five of these patients were followed up for (26±26) months after biopsy. Their morphological features and natural history were compared with those of control group of 80 patients without segmental necrosis， who had comparable serum creatinine with a follow-up for (39±23) months. Progression was indicated as 50% elevation of serum creatinine or 33% decline of Ccr or development of ESRD. Their clinical outcomes were compared using Kaplan-Meier estimation. Cox regression was performed to identify predictive factors for clinical events. Results Only 2 necrotizing patients showed ARF. No difference was found in the clinical symptoms presented. The necrotizing group showed a more significant accumulation of monocytes and lymphocytes (P = 0.004) with lower percentage of glomerular sclerosis (P = 0.002). Among the groups of follow-up shorter than 24 months， 3 of 22 necrotizing IgAN patients and 1 of 28 non-necrotizing IgAN patients had futher progression (P = 0.08). In other groups， all necrotizing IgAN patients were event-free， 13 of 52 non-necrotizing IgAN patients progressed， in which necrotizing lesion was not an independent risk factor. Multivariate analysis showed that lesions with severe chronic background were the only prognostic factors (RR = 23.13， P < 0.01)， indicating prognosis was associated with the severity of universal lesions， not with necrotizing lesions alone. Conclusions Based on the present data， necrotizing lesion is not an independent risk factor and may not require aggressive therapy. Longer period of follow-up is needed for further confirmation.

Objective To investigate the role of urine albumin from nephrotic syndrome patients on expression of endothelin(ET)-1 in human proximal tubular epithelial cells (HK-2). Methods Proteins were precipitated with polyethylene glycol， and albumin were purified by Sephadex-75 column chromatography from urine taken from nephrotic syndrome patients. The HK-2 cells were divided into two groups: one group incubated with the urine albumin from nephrotic syndrome patients， another with HSA. The expression of ET-1 mRNA of HK-2 cells was assessed by RT-PCR， and the level of ET-1 protein was assessed by indirect immunofluorescence. Results The urine albumin and HSA up-regulated the expression of ET-1 mRNA and protein in HK-2 cells. The expression of ET-1 protein was associated with the stimulation time， and increased in dose-dependent manner. The expression of ET-1 mRNA in HK-2 cells increased significantly when cultured with urine albumin (0.5 mg/ml) from nephrotic syndrome (P < 0.05). However， only when the HK-2 cells were cultured with HSA at 2.5mg/ml， the ET-1 mRNA were markedly increased. Conclusion The urine albumin from nephrotic syndrome patients and normal serum albumin can up-regulate expression of ET-1 in HK-2 cells， and the urine albumin stimulates ET-1 synthesis by HK-2 cells to a higher extent as compare to that of normal serum albumin.

Objective To investigate a possible relationship between CD2AP mutation and focal segmental glomerulosclerosis (FSGS). Methods Genomic DNA from peripheral blood cells of FSGS patients were extracted， and CD2AP mutation was analyzed by polymerase chain reaction (PCR) and direct sequencing. Immunofluorescence staining， confocal laser scanning microscopy and LSM-510 graphic system were used to detect the expression and distribution of protein， including both CD2AP and podocin， in patients with CD2AP mutation. Eighty-two Chinese patients with idiopathic FSGS， including 43 males and 39 females whose age ranged from 12 to 76 years old were enrolled in this study. Of these， 55 had nephrotic syndrome (NS). Sixty genomic DNA from 60 healthy volunteers were selected as normal control group. Results (1) Two CD2AP heterozygous mutations were detected in exons. One was 160G > A in exon 2， which caused the 54th amino acid changed from valine to isoleucine， and occurred in one non-NS patient with renal failure. Another was 358A > G in exon 4， which caused the 120th amino acid changed from isoleucine to valine， and occurred in one NS patient with normal renal function and relapse twice. The same mutations were not found in the control group of 60 healthy people. (2) A decreased expression was observed in glomeruli stained with CD2AP antibody， accompanied with decreasing of podocin， in the patients with CD2AP mutation. (3) Moreover， 2 mutations in introns， 1 in promoter region and 1 SNP were first reported. Conclusions The mutations in CD2AP may cause FSGS in both NS and non-NS patient. The decreasing expression of CD2AP resulting from CD2AP gene mutation may affect the expression of podocin.

Objective To explore the relation between specific pathological features and clinical data in diabetic patients with renal damage. Methods Pathological features and clinical data were retrospectively analyzed in 64 diabetic patients with renal damage who underwent renal biopsy from 1992 to 2002. The pathological profiles were simultaneously compared with biopsies from non-diabetic patients during the same period. Results Patients were primarily divided into 3 groups according to their pathological features: the diabetic nephropathy (DN) group (42 cases， 65.6%)， the arterio-arteriolosclerosis and ischemic glomerular damage (BNS) group (6 cases， 9.4%)， and the primary glomerulonephritis (CGN) group (16 cases， 25.0%)， including 2 patients with glomerulonephritis superimposed on diabetic glomerulosclerosis. The specific pathological profiles of the CGN group were as following: IgA nephropathy 6 cases(37.5%)， focal segmental glomerulosclerosis (FSGS) 6 cases(37.5%)， minor change renal disease (MCND) 2 cases(12.5%)， and membranous nephropathy (MN) 2 cases (12.5%)， which were similar to those of non-diabetic patients， except that the ratio of FSGS was significantly increased (27.3% vs. 4.7%， P < 0.01). Linking with clinical data， DN and BNS groups had a longer diabetic duration than the CGN group (P < 0.05). The age of CGN groups was younger than that of other two groups (P < 0.05). No differences were showed in glucose and blood pressure levels among three groups. The amount of proteinuria in BNS group was significantly less than that of DN and CGN group[(0.45±0.33) g/d vs. (3.18±2.40) g/d and (2.68±1.27) g/d， P < 0.01]， and the latter two groups were not different. Similarly， the retinopathy incidence of the BNS group was markedly lower than that of DN and CGN group (0% vs. 38.1% and 37.5%， P < 0.01)， and no difference was found between DN and CGN group. Conclusions The CGN incidence in diabetic patients with renal damage is 25%， and its pathological profiles are similar to those of non-diabetic patients. Diabetic patients with renal damage should undergo early renal biopsy to differentiate DN and CGN， which would be beneficial for patients’ individual treatment.

Objective To study the effects of oxidants on the structure of albumin. Methods Using both AOPPs and protein carbonyl content as indices. The oxidative stress level in normal controls and uremia patients was evaluated. Albumin in plasma was purified by HPLC and then was subjected to amino acids composition assay. Results Both AOPPs level and protein carbonyl content in uremic patients were significantly higher than those in controls (P < 0.01). Most amino acids’ levels in albumin from uremic patients were down-regulated by 10% compared to the controls. A statistically significant difference of both cysteine and arginine in albumin was found between controls and uremic patients (P < 0.05). Conclusion The extensive loss of amino acids in uremic patients may play a pivotal role in affecting the structure and biological functions of albumin， provided that both cysteine and arginine are the major targets attacked by free radicals.

【Abstract】 Objective To dynamically observe the expression of slit diaphragm complex molecules, including nephrin, podocin, CD2AP, and cytoskeleton protein α-actinin-4, in adriamycin-induced nephrotic (ADN) rats, and to further explore the molecular behavior of podocyte proteins during the occurrence and development of proteinuria and their possible mechanisms. Methods Adriamycin nephropathy was induced by a single tail intravenous injection of adriamycin. Renal tissue samples were collected at day 3, 7, 14, and 28, respectively. The distribution, mRNA expression and protein expression of nephrin, podocin, CD2AP and α-actinin-4 were examined by indirect immunofluorescence, real-time PCR and Western blotting, respectively. Results (1) After the adriamycin injection, a significant increment of the 24-hour urinary protein was observed at day 14 and persisted up to day 28 (P < 0.01). (2) In ADN rats, the foot processes broadened to a different extent at day 14, and the diffuse fusion and effacement of foot processes were observed at day 28. (3) From the 7th day to the 28th day after adriamycin injection, nephrin and podocin staining gradually shifted from a linear-like pattern along the capillary loops of glomerulus to a discontinuous coarse granular pattern. Similarly, CD2AP shifted from an even GBM-like pattern to a coarse granular pattern, and α-actinin-4 changed from a dot linear-like pattern along capillary loops to a coarse granular pattern. (4) In ADN rats, nephrin mRNA expression was up-regulated at day 7 (P < 0.01) and returned to normal level at day 14 and 28. Podocin and CD2AP constantly increased from day 3 and reached a significant level at day 14 and day 28, respectively (P < 0.05). (5) The protein expression of nephrin started to increase at day 7 after adriamycin injection and was markedly elevated up to day 28 (P < 0.05). Likewise, compared with the control group, CD2AP protein expression of the ADN rats prominently increased at day 14 and persisted to day 28(P < 0.05). Interestingly, after the injection of adriamycin, podocin protein expression was dramatically upregulated at day 7 (P < 0.05), and thereafter recovered again, whereas it was significantly down-regulated at day 28 (P < 0.05). (6) Alpha-actinin-4 mRNA and protein expression showed no change at the time studied. Conclusion The increased expression of nephrin, podocin and CD2AP and their abnormal distributions are the molecular mechanism that leads to the occurrence and development of proteinuria in ADN rats. The enhanced expression may be a compensatory reaction of podocyte to injury.

Objective To design a method to characterize AOPPs. Methods Carbonyl groups were used as an oxidative index to link AOPPs and oxidized protein. Plasma-AOPPs were obtained by a series of preparation as follows. The native plasma was first determined for its protein contents followed by AOPPs level evaluation. Specimen was then washed with PBS and ultrafiltrated with an ultrafilter (10 000 cut-off membrane) to obtain clean plasma-AOPPs. A size-exclusion HPLC technique was used to verify which protein was oxidatively damaged. Fractions resulted from delipidation were also examined. Results The levels of AOPPs and total carbonyl groups in patient plasma were significantly higher than those in controls； both in native/delipidated plasma and CHCl3-resulted precipitate. HPLC revealed that serum albumin presented highest carbonyl levels. It was an exclusive protein with statistically significant difference between controls and patients (patients vs. controls in nmol carbonyl/mg protein: HSA:1.510±0.067 vs. 0.791±0.048， P < 0.01； Fg: 0.617±0.100 vs. 0.672±0.159， P = 0.773； IgG: 0.047±0.029 vs. 0.053±0.030， P = 0.791； Tf: 0.102±0.025 vs. 0.098±0.043， P = 0.385， respectively). Delipidation mainly removed fibrinogen and an unknown oxidized macromolecule. Conclusion Albumin is a more important contributor to AOPPs than other proteins， IgG in particular.

Objective To study the expression of angiopoietin-like protein 3(ANGPTL3)mRNA successively in kidney of adriamycin(ADR)-induced nephrotic rats during the development of proteinuria， and to disclose the possible assosiation of ANGPTL3 with proteinuria. Methods Adriamycin-induced nephrotic rat models were established by a single injection of adriamycin via the tail vein. Glomeruli and cortex tubuli of rats were dissected by laser microdissection. Real time quantitative RT-PCR was used to study the expression of ANGPTL3mRNA in kidney of ADR rats at 7， 14， 21 and 28 days successively following adriamycin injection. Results (1) In ADR rats， urinary protein and the level of triglyceride and cholesterol in serum increased significantly at day 14 (P < 0.01， 0.05 and 0.01， respectively)， at same time， serum albumin decreased significantly (P < 0.01). Changes of these indices reached the peak level at day 28. (2) Glomeruli and cortex tubuli of rats were dissected successfully by laser microdissection. (3) The mRNA of ANGPTL3 in total cortex of kidney in ADR rat increased significantly at day 21(P < 0.01) and at day 28 (P < 0.05) compared to that of control. (4) The tendency of ANGPTL3 mRNA expression in glomeruli of ADR rats was as the same as that in total cortex. ADR rats had a higher expression level of ANGPTL3mRNA in glomeruli at day 21 and day 28 compared to that of control (both P < 0.05). (5) There was no significantly change of ANGPTL3 mRNA expression in cortex tubuli following adriamycin injection. Conclusions ANGPTL3 mRNA can be expressed in the kidneys of normal and ARD rats. The expression of ANGPTL3 mRNA increases in glomeruli of ADR rats during the development of proteinuria.

Objective To explore whether LDL and oxLDL may induce kidney tubular epithelial-mesenchymal transition (EMT) and its mechanism. Methods The second generation human kidney tubular epithelial cells(TECs) were cultured for 24 hours in different conditions as (1)serum free as control， (2) treated with LDL(50 μg/ml)， (3) treated with oxLDL(50 μg/ml)， (4)treated with LDL(50 μg/ml) plus PD98059(5 μmlo/L)， (5) treated with oxLDL(50 μg/ml) plus PD98059(5 μmol/L). The expression of cytokeratin， E-cadherin， α-SMA and vimentin was assessed by immunofluorescence and Western-blot. Western-blot was also performed to test the expression of collagen I and phospho-ERK1/2MAPK and phospho-GSK-3β. Results oxLDL was more potently in inducing tubular EMT than LDL at 24 hours as demonstrated by de novo α-SMA expression， increased expression of vimentin， partial loss of cytokeratin and reduction of E-cadherin expression by TECs. The expression of collagen I and phospho-ERK1/2MAPK and phospho-GSK-3β was increased in TECs stimulated by LDL or oxLDL. MAPK inhibitor(PD98059) inhibited the phosphorylation of GSK-3β and almost completely blocked oxLDL-induced tubular EMT. However，PD98059 alone was able to inhibit LDL-induced tubular EMT partially. Conclusions oxLDL is more potently in inducing tubular EMT than LDL. The ERK1/2MAPK-GSK-3β signaling pathway mediates the LDL or oxLDL-induced tubular EMT.