Objective To evaluate the clinical value of detecting serum underglycosylated IgA1 in diagnosis and differentiation of IgA nephropathy(IgAN). Methods 　Serum underglycosylated IgA1 was isolated by microspincolumn coupled with vicia villosa lectin(VVL) from 48 cases with IgAN and 43 cases with other primary glomerulonephritis. All the patients were diagnosed by renal biopsy. Sera from 20 healthy persons were used as control group. After isolation, the eluant with rich underglycosylated IgA1 was detected by incubation with biotin-labeled horseradish peroxidase(HRP) and Helix aspersa（HAA, recognizing N-acetylgalactosamine specifically）in enzyme-linked immunosorbent assay (ELISA). The sensitivity and specificity of diagnosis and differentiation of IgAN with elevated serum underglycosylated IgA1 were analyzed. Results The level of serum underglycosylated IgA1 in IgAN patients [(83.7±41.0) U] was significantly higher than that in healthy control group [(52.6±22.9) U] and the patients with other primary glomerular diseases[(49.2±27.3) U] (all P<0.01). Twenty-two cases of non-IgA mesangial proliferative glomerulonephritis accounted for 51% of other primary glomerular disease, whose underglycosylated IgA1 level [（47.6±21.5） U] （all P<0.01） was significantly lower as compared to IgAN patients. Taking the renal biopsy diagnosis as golden diagnostic criteria, the ROC curve was performed. The area under the curve was 0.797 with a standard error 0.047(P<0.01). The sensitivity as a diagnostic test was 72.9%, with specificity 72.1% and accuracy 72.5%. Conclusion Detection of serum underglycosylated IgA1 level by microspincolumn method and ELISA assay has certain clinical value in diagnosis and differentiation of IgAN.

Objective To clarify whether the Cosmc gene down-regulation in IgA nephropathy (IgAN) patients is resulted from genetic disorders or external suppressions. Methods Forty IgAN patients, 16 non-IgAN glomerulonephritis patients and 21 healthy controls were enrolled in the study. Genomic DNA was extracted and then Cosmc gene was amplified and sequenced. Peripheral B lymphocytes were isolated and cultured with RPMI-1640 alone or plus lipopolysaccharide (LPS). The Cosmc mRNA expression levels at baseline, after RPMI-1640 culture or RPMI-1640+LPS treatment were measured respectively by real-time RT-PCR. Results (1) Only 2 missense mutations and 2 silent mutations were detected in coding frame region of Cosmc gene in 2 IgAN patients. (2) The baseline Cosmc gene expression level was significantly lower in IgAN patients(31% of that in healthy controls) than that in healthy controls. (3) Relative quantification PCR indicated that Cosmc mRNA expression level was significantly increased (219% of baseline) after RPMI-1640 culture, and treatment of LPS could strongly inhibit this effect. (4) The Cosmc gene expression of healthy control was not affected by RPMI-1640 or LPS. Conclusion It is not genetic disorders but external suppression to cause the down-regulation of Cosmc gene mRNA expression in IgAN.

Objective To investigate the injury of podocyte and its association with proteinuria in IgA nephropathy (IgAN). Method Thirty-five patients of IgA nephropathy with proteinuria more than 1.0 g/24 h were enrolled in the study, and eight cases of renal harmatomaectomy or renal cancinomaectomy were as controls. Cell cycle regulatory proteins (p21, p27), podocyte-associated molecules (integrin-β1, nephrin, α-actinin 4, nestin), foot process width (FPW) and the amount of podocyte were examined by immunohistochemistry and real-time PCR, respectively. Patients were divided into two groups according to podocyte number per volume (Nv): podocytopenia group (n=15, Nv<52.49×106/μm3) and normal number group (n=20, Nv≥52.49×106/μm3). Proteinuria was followed up for eighteen months. Results Compared with the controls, podocyte p21 was re-expressed, while the expression of p27 was decreased (0.71±0.12 vs 0.91±0.07, P<0.05) in IgAN. The nestin protein level was markedly decreased in IgAN(13.4%±0.04% vs 17.6%±0.04%, P<0.05). The mRNA expression of integrin-β1 was significantly increased (12.54±5.20 vs 1.02±0.30, P<0.05), while the amount of nephrin, α-actinin4 remained unchanged. Effacement of foot processes and podocyte detachment from the glomerular basement membrane were observed in some cases. Nv was significantly less than that of controls (161.27±225.92 vs 323.22±138.12, P<0.05）， which was associated with the Lee’s grade of IgA nephropathy. The integrin-β1 mRNA expression and Nv were negatively correlated with baseline proteinuria by univariate analysis (r=－0.840, P=0.034; r=－0.4483, P=0.014, respectively). Proteinuria in podocytopenia group was decreased more slowly than that in normal number group. Conclusions Podocyte injury exsists in IgAN with proteinuria, which manifests alterations in cell cycle regulatory protein and some podocyte-associated molecules, as well as foot process effacement and loss of podocyte. Podocyte injury may be involved in proteinuria by affecting the progression of proteinuria in IgAN.

Objective To investigate the effects of supernatant of cultured mesangial cells with serum IgA1 from IgA nephropathy patients on apoptosis of podocyte. Methods Jacalin affinity chromatography and Sephacryl S-200 molecular sieve chromatography were used to isolate IgA1. Apoptosis rate of podocyte was assessed by flow cytometer. Monomeric IgA1(mIgA1) was transformed to aggregated IgA1(aIgA1) by heating. IgA-mesangial cell supernatant was prepared by collecting spent medium in which growth-arrested mesangial cells were incubated with different aIgA1, then the medium with RPMI 1640 containing 0.5%FBS was cultured with growth-arrested podocyte. Real time PCR was used to detect the mRNA expression of Bcl-2, Bax, Fas and Fas-L. Results Apoptosis rate of podocyte by supernatant of cultured mesangial cell with aIgA1 from IgAN patients was higher than that from healthy and control groups [(28.5±5.9 ) % vs (22.5±5.8)%, (20.5±4.5)%, all P<0.05]. Fas mRNA expression of podocyte exposed to supernatant of cultured mesangial cells with aIgA1 from IgAN patients increased significantly and was 1.89 folds of control (P<0.05), while Bcl-2 mRNA expression significantly decreased and was 72% of control (P<0.05). The concentrations of AngⅡand TGF-β1 in supernatant of cultured mesangial cells with IgA1 from IgA nephropathy were significantly higher than those from healthy control [(13.2±3.4) ng/L vs (8.2±2.3) ng/L, P<0.05; (15.4±3.4 ) ng/L vs (10.8±3.2) ng/L, P<0.05]. Conclusion Supernatant of cultured mesangial cells with IgA1 from IgA nephroapthy patients can induce apoptosis of podocyte, which may play a role in the progression of IgAN.

Objective To explore the clinicopathological features of IgA nephrolpathy associated with malignant hypertension (IgAN-MHT) and to analyze their correlation with renal vascular lesions. Methods Twenty-nine patients of IgAN-MHT were screened from 2000 biopsy-proven cases with primary IgA nephropathy (IgAN) in our department from April 1997 to May 2007. Data of clinicopathology and follow-up of these 29 patients were collected. Semi-quantitative analysis was performed to evaluate the pathological changes. Inner lumen, outer lumen, intimal thickness, tunica media-to-internal lumen ratio of 436 arterioles, 124 interlobular arteries and 5 arcuate arteries were measured. The primary endpoint was the composite of a doubling of serum creatinine level and ESRD. Correlations of renal vascular lesions with clinical manifestation, pathological change and prognosis were examined by Spearman and Cox methods. Results 1.5% of all the IgAN patients presented malignant hypertension. The common clinical features were renal failure (100%), hyperuricacidemia (62.7%) and hypertriglyceridemia (51.7%). The average amount of urine protein excretion was 2.8 g/d. The common pathological changes were moderate mesangial proliferation, severe global sclerosis, severe interstitial inflammation and severe interstitial-tubular fibrosis. The small arteries (arcuate arteries and interlobular arteries) and arterioles (afferent arterioles) were both involved in IgAN-MHT. The characteristic lesions of intrarenal arteries included vascular occlusion, media thickening, proliferative endarteritis (onionskin lesion, musculomucoid intimal hyperplasia), hyaline arteriosclerosis, but mainly vascular occlusion (86.2%). The arteriole lesion was negatively correlated with age and total protein level; vascular occlusion was positively correlated with uric acid level. The average follow-up period was 21.1 months. Forteen patients reached the endpoint. The arteriole lesion was the main independent risk factor for the progression of IgAN-MHT (RR=10.21, 95%CI=1.16~89.67). Conclusions The main clinical feature of IgAN-MHT is renal failure. The main histological feature of intrarenal vascular lesions is occludes arterioles. Arteriole lesion is the main independent risk factor for the progression of IgAN-MHT.

Objectives To study the relationship of atypical membranous nephropathy (AMN) with idiopathic membranous nephropathy (IMN) and lupus membranous nephropathy (LMN), and to explore the predictive clinical and pathological features for LMN diagnosis. Methods The patients undergone renal biopsy in PUMCH between 2003 and 2006 were selected, and were divided into group AMN (n=28), IMN(n=100) and LMN(n=45). Clinical manifestations and pathological features were compared among three groups retrospectively. The intensity of glomerular IgG subclasses was analyzed by immunohistochemical staining among three groups semi-quantitatively. The spatial arrangement of IgG and C3 deposits was investigated by immunofluorescence double staining among three groups by confocal laser scanning microscopy. Results (1) The onset age of AMN was (38±17) years and female/male ratio(F/M) was 2.5:1 in group LMN and IMN. The onset age was significantly different among three groups(P<0.01), and F/M ratio was significantly different between AMN and IMN (P=0.017). (2) The incidence of most extra-renal manifestations was less than 20% in AMN except for hematological disorder (21.4%) and serum anti-SSA antibody positivity(40.7%). (3) The incidence of subendothelial electron dense deposits in either LMN or AMN was significantly higher than that in IMN （P<0.01）. (4) The percentage of IgG3 predominance in AMN and LMN glomeruli was 78.9% and 73.9%, respectively, while the percentage of IgG4 predominance in IMN was 61.1%. The difference was significant(IMN vs AMN and LMN, P<0.01). (5) IMN had an overlapping distribution of IgG and C3 in subepithelial deposition, which was rarely found in AMN or LMN. (6) Among the indexes differentiating LMN and IMN, the high sensitive one was non-IgG4 predominance in glomeruli (91.3%), while the high specific ones included subendothelial electron dense deposits (100.0%), serum anti-SSA antibody (95.5%), glomerular IgG3 predominance (94.4%). Conclusions AMN with serum ANA positivity is similar to LMN in respect to pathological features and glomerular IgG subclasses, although it has few extra-renal clinical manifestations. It may represent a latent subgroup of lupus nephritis.

Objective To investigate the expression of nestin, a type Ⅵ intermediate filament protein in the glomeruli with foot process effacement and the potential relationship between nestin expression in the kidney and the degree of proteinuria. Method Immunohistochemistry was used to determine the localization of nestin in the kidney samples obtained from needle biopsies of normal human and patients with minimal change disease (MCD). Puromycin aminonucleoside(PAN) nephrosis rat models were established by a single intraperitoneal injection of PAN. Both real time quatitative reverse PCR and Western blot methods were applied to evaluate the levels of nestin expression at day 1, 4, 10 and 20 after PAN injection. Results Immunohistochemistry showed that the expression of nestin in glomeruli of MCD patients was significantly reduced compared with normal samples (0.93±0.08 vs 1.65±0.12， P<0.05) . The mRNA and protein expressions of nestin in the rat kidney were transitorily increased by 1.23 folds and 1.48 folds of control group (NC) after 1 day of PAN injection (P<0.05), then decreased quickly in the following days. The mRNA levels of nestin in the kidney were 35.8% and 12.1% of NC after 4 days and 10 days of PAN injection, respectively, (P<0.01) as determined by real time PCR. After 20 days of PAN injury, nestin mRNA expression partly recovered to 65.8% of NC（P<0.05）. The protein levels of nestin detected by Western blot presented the similar trend, which were 77.0%, 58.0% and 83.4% of NC after 4 days, 10 days and 20 days of PAN injection, respectively (P<0.05). The degree of proteinuria in puromycin aminonucleoside nephrosis rats was negatively correlated with both mRNA and protein levels of nestin in the kidney（r=－0.667，P<0.05 and r=－0.621，P<0.05, respectively）. Conclusions The expression of intermediate filament protein nestin is down-regulated in the kidney characterized with foot process effacement and negatively correlated with the degree of proteinuria in puromycin aminonucleoside nephrosis rats. Nestin may play a potential role in modulating the structure and function of podocyte.

Objective To investigate the role of Notch signaling in the progression of peritoneal fibrosis in a rat model induced by high glucose dialysate. Methods Male Sprague Dawley rats were subjected to daily peritoneal dialysis (PD) with a lactate-buffered solution containing 4.25% glucose. They were sacrificed at 2 and 4 weeks after PD. The parietal thickness was measured with Masson staining. The expression of TGF-β1, E-cadherin, α-SMA and collagen I was examined by immunoblotting. The expression of Notch ligand Jagged-1 and the negative Notch signaling regulator——Numb was analyzed by both immunoblotting and RT-PCR. The expression of a Notch nuclear target gene Hes-1 was examined by RT-PCR. Results Both HE and Masson trichrome staining revealed an increase in peritoneal thickness with a loss of mesothelial cells and a rich of collagen matrix deposition in the submesothelial zone was evident at 4 weeks after PD. Meanwhile, compared to healthy rats, the expression of TGF-β1, α-SMA and collagen I was significantly increased, but the expression of E-cadherin was decreased in peritoneum after PD treatment. It was difficult to detect the Jagged-1 and Hes-1 expression in normal peritoneum, but their expression was gradually increased after PD. In contrast, the expression level of Numb, a negative regulator of Notch signaling, was dramatically decreased after PD. Conclusions Notch signaling is activated during the process of PD-induced peritoneal fibrosis and the activation of Notch signaling is associated with the loss of negative regulation of Notch signaling via decreased expression of Numb. Inhibition of Notch signaling via overexpression of its negative regulators such as Numb may be a novel therapeutic approach for peritoneal fibrosis in PD patients.

Objective To investigate the protective effects of recombination rat augmenter of liver regeneration (rrALR) on tubular cell injury and renal dysfunction in rats with acute renal failure（ARF）induced by gentamicin. Methods One hundred fifty female Wistar rats were randomly divided into 5 groups: group A (control), group B (gentamincin 140 mg/kg+saline 100 μg/kg), group C (gentamincin 140 mg/kg+blank plasmid 100 μg/kg), group D1 (gentamincin 140 mg/kg+rrALR 80 μg/kg), group D2 (gentamincin 140 mg/kg +rrALR 160 μg/kg). Rats were sacrificed at day 4, 8, 12, 16 and 21 after gentamicin first injection. Blood urea nitrogen (BUN), serum creatinine (Scr) and urine N-acetyl-β-D-glucosaminidase (UNAG) were measured. The histopathologic changes were observed by periodic acid-Schiff (PAS) staining. Protein expression of ALR and PCNA in renal tissue was detected using immunohistochemistry and Western blot. Results Compared with control rats, the levels of BUN, Scr and UNAG were significantly increased in ARF rats at day 4, 8, 12 and 16（P<0.05）, however, the renal dysfunction and the renal histopathological injury were significantly improved in ARF rats simultaneously administered with rrALR (group D) compared with ARF rats (group B and C). The protein level of ALR, the number of PCNA positive tubular cells and the proliferation index (PI) in group D were significantly higher than those of group B and C (P<0.05). Conclusion rrALR can promote the regeneration of tubular cells in nephrotoxic ARF rats and ameliorate renal dysfunction.

Objective To evaluate the effect of parathyroid hormone (PTH) on the expression of connective tissue growth factor (CTGF) in human renal tubular epithelial cells, and to explore the role of MAPK signaling pathway. Methods Real time RT-PCR, Western blot, and reporter gene assay were employed to detect PTH-induced CTGF expression in HK-2 cells. Inhibitors (PD98059 and U0126) of MAPK signaling pathway were used to confirm involved signal pathway. Results HK-2 cells had basic expression level of CTGF mRNA and protein, which were increased significantly after treatment with PTH. The luciferase activity was up-regulated to a higher level as compared with control group after treatment with 10-10 mol/L PTH for 12 h [(1.8884±0.0780) vs (0.9891±0.0300) A, P＜0.01]. Moreover, a small amount of p-ERK1/2 was detected in normal HK-2 cells, but it was increased significantly in response to PTH activation, most remarkably when treated with 10-10 mol/L PTH for 30 min. Inhibitors of MAPK signaling pathway, PD98059 and U0126, noticeably inhibited the expression of CTGF mRNA and protein as well as gene promoters in HK-2 cells. Conclusion PTH can induce higher expression of CTGF in HK-2 cells probably via MAPK signaling pathway.

Objective To investigate the respective effects of highly concentrated amino acids, highly concentrated glucose and mixture of the above two on the proliferation and extracellular matrix synthesis of glomerular mesangial cells. Methods Mesangial cells were cultured with mannitol(13.3 mmol/L ), highly concentrated amino acids(13.3 mmol/L), highly concentrated glucose(30.5 mmol/L) and the mixture of highly concentrated amino acids and highly concentrated glucose repectively. Mesangial cell proliferation was examined by MTT and flow cytometry. The mRNA expression of TGF-β1，ColⅣ and fibronectin (FN) was detected by semi-quantitative RT-PCR, and the protein expression was detected by ELISA, Western blot and radioimmunoassay. Results (1) Cellular proliferation was increased in response to amino acids and was further enhanced by increased levels of both amino acids and glucose in a time-depentent manner. And the most prominent action occured at 48 hours. (2) Among the three group, mesangial cell cycle was affected, and the number of G0/G1 mesangial cells was reduced, but the number of cycle S cells was improved. (3) In the three group, the mRNA and protein expression of TGF-β1 was improved [protein unit: ng&#8226;ml－1&#8226;(105 cells)－1, 3023±85, 3163±80, 3321±85 vs 1506±63, P<0.05]. （4）In the three group, the mRNA and protein expression of ColⅣ and FN was enhanced[protein unit: ng&#8226;ml－1&#8226;(105 cells)－1, Col Ⅳ:16.78±2.56, 18.65±2.56, 22.83±2.45 vs 10.22±2.54, P<0.01; FN:18.47±2.34, 13.58±3.13, 17.98±2.56 vs 8.22±2.54, P<0.05]. The expression of ColⅣ mRNA and protein was stronger when the cells were cultured with the mixture of highly concentrated amino acids and highly concentrated glucose. Conclusions Highly concentrated amino acid can promote cell proliferation similarly to highly concentrated glucose, which may be through increasing the activity of mesangial cells and the synthesis of extracellular matrix via TGF-β1 signaling way. Highly concentrated amino acid has the synergism with highly concentrated glucose.

Objective To study whether the uremic toxins accumulated long-term in uremia patients may be involved in oxidation of protein by forming advanced oxidative protein products (AOPPs). Methods Malonylaldehyde (MDA), hippuric acid (HA) and p-cresol were used as the representatives of uremic toxins. Human albumin serum (HSA), plasma specimens from normal or uremia patients were incubated respectively with MDA (10 mmol/L), HA (20 mmol/L) and p-cresol (10 mmol/L) or PBS (20 mmol/L, pH 7.4, as control groups) at 37℃ for 30 minutes or 24 hours, respectively. Those indices such as AOPPs, protein thiol groups (Pt-SH) and dityrosine were used as biomarkers of protein injury. High performance liquid chromatography (HPLC) was employed to identify the aggregation and cross-links of modified proteins. Results AOPPs levels in all groups containing poison compounds were significantly increased by 121.5％(P<0.05) compared to that in control groups. Uremic toxins also resulted in over 14.7％ loss in Pt-SH (P<0.05) and 119.2％ increment in dityrosine, respectively (P<0.05). Meanwhile, the formation of HMW-AOPPs in a time-dependent manner was observed by HPLC and cross-linked protein levels were significantly increased by 148.45％～333.3％ in comparison with control groups. Conclusion Uremic toxins can directly mediate the damage of proteins by inducing the formation of HMW-AOPPs in a time-dependent manner, which is also one of the mechanism of AOPPs production in vivo besides the activation of the myeloperoxidase-H2O2-Cl- pathway.