Objective To establish a relatively concise semi-quantitative pathological scoring and approach to predict the prognosis of IgA nephropathy (IgAN). Methods One hundred and fifty-five cases diagnosed as primary IgA nephropathy with 2 years follow-up were enrolled into our study. In order to examine intra- and inter-observer reproducibility of classification according to the scores, pathological data of 91 cases were reviewed twice by one pathologist, and data of 56 cases were reviewed again by another pathologist. Eight histological indices were analyzed at the beginning: (1) endothelial proliferative index (endoI). (2) active crescents and segmental necrosis of glomerular capillary wall (dGAI). (3) mesangial hypercellularity index (MsHI). (4) increment of mesangial matrix area (MsMI). (5) glomerular chronicity index (GCI). (6) interstitial inflammatory cells infiltration (infI). (7) tubular atrophy and interstitial fibrosis (TCI). (8) vascular chronicity index (VCI). Results Twenty-five (16.13%) patients progressed into irreversible end stage renal disease (ESRD) during follow-up period [(69.07±28.66) months, ranged from 10 to 170 months]. Serum creatinine at biopsy was（112.18±83.13） μmol/L. The initial histological variables were analyzed using Cox proportional hazard model and three variables, including dGAI，GCI and TCI, were finally chosen. GCI and TCI were added up to indicate the chronicity index (CI). dGAI and CI were both found as independent factors in predicting renal outcome (RR=1.255 and 1.691, P<0.05, respectively). Patients in group with both higher dGAI (≥4) and CI (≥6) had the worst renal prognosis (P<0.01). For patients in CKD grade 1 and grade 2, multivariate analysis including clinical and histological variables showed CI was the only independent risk factor of bad prognosis. The reproducibility of the scoring system was proved acceptable (inter- and intra-observer’s kappa values >0.4). Conclusions This relatively concise pathological scoring method containing dGAI and CI has good reproducibility and can predict renal outcome of IgAN.

Objective To investigate the clinicopathological characteristics of IgA nephropathy (IgAN) with urinary podocyte excretion（UPE）. Methods (1) Among the 36 enrolled patients with IgAN, 20 were male and 16 were female, with an average age of (34.1±12.2) years. All these patients were diagnosed by renal biopsy. Ten healthy volunteers were served as controls. (2)The UPE level was calculated by urinary sediment smear counting. (3) The correlation between UPE level and renal pathological changes was examined. Results (1)The patients with IgA nephropathy had a higher frequency of podocyturia (61%). No podocyturia was found in healthy controls. (2)The UPE level, as well as UPE/urinary creatinine or UPE/urinary renal epithelial excretion ratio, were significantly elevated in IgAN patients with massive proteinuria (≥3.0 g/24 h). Moreover，the UPE level was positively correlated with 24 h urinary protein excretion（r＝0.446，P＝0.007）. (3)Compared with negative staining patients, patients with positive podocalyxin stain showed significantly higher 24 h urinary protein excretion and urinary renal epithelial cells excretion, while their plasma albumin levels were significantly lower. These two groups had similar distribution in age, gender and frequency of hypertension, and no significant variations were observed concerning the levels of Scr, Hb, cholesterol and triglyceride（P > 0.05）. (4) UPE was related to the injuries of cellular crescent, glomerular disruption of capillaries and extensive epithelial foot process effacement, but not related to the mesangial proliferation, or focal capillary wall thickening. Patients with glomerular crescents showed more severe glomerular sclerosis and interstitial fibrosis (P<0.05). Conclusions Podocyturia is not only a clinical evidence of renal damage, but also a hallmark of the activity of IgAN. Urinary podocyte excretion level in IgAN patients is correlated with 24 h urinary protein excretion and specific pathological damages.

Objective To investigate somatic mutation of C1GALT1C1, a coding gene for molecular chaperone cosmc of β1,3 galactosyltransferase, in patients with IgA nephropathy(IgAN). Methods Twenty-seven IgAN patients and 19 normal healthy controls were enrolled in the study. Firstly, the coding region of C1GALT1C1 was amplified by PCR from genomic DNA in peripheral blood and PCR products were directly sequenced. Meantime, DNA from peripheral blood B lymphocyte was extracted from 15 IgAN patients and 7 normal controls. The coding region of C1GALT1C1 was amplified from DNA in peripheral blood B lymphocyte, and then, PCR products were subcloned into PGEM-T vector. Total 202 clones, including average 8 to 10 clones per individual, were randomly selected for directly sequencing. Results T393A polymorphism in the coding region of C1GALT1C1 was found in 2 patients and 1 control from their peripheral blood genomic DNA. The minor allele frequency was 6.9%, which was a bit lower than that reported in dbSNP (9.5%). No mutations or polymorphisms were found in total 202 clones from B lymphocyte DNA in 22 persons (15 patients and 7 controls). Conclusions A polymorphism, T393A, in the coding region of C1GALT1C1 gene is identified in present study. Minor allele frequency is lower than that in previous report. Somatic mutation can not be found in B lymphocyte of patients with IgAN.

Objective To investigate the effects of transforming growth factor β1(TGF-β1) and bone morphogenetic protein 7 (BMP-7) in IgA nephropathy(IgAN). Methods The study included 47 mild mesangial proliferative IgAN (group A) cases, twenty nine moderate and severe mesangial proliferative IgAN (group B) cases and thirteen proliferative-sclerosing or sclerosing IgAN (group C) cases. Blood pressure, 24 h urinary protein, serum creatinine and Ccr were monitored. Levels of TGF-β1 and BMP-7 in serum and urine were measured by ELISA. The expression of TGF-β1 and BMP-7 in renal frozen setions was examined by immunohistochemistry. The ratios of sclerosing glomeruli, crescents and area of interstitial fibrosis were calculated. Results Glomerular demage was positively correlated to atrophy tubule and interstitial fibrosis, the levels of blood pressure, 24 h urinary protein and serum creatinine. As for the above parameters, each group had significant difference（P < 0.05）except 24 h urinary protein between group B and C. Compared to Group C, levels of TGF-β1 in serum and urine were much higher in group A and B.（P < 0.01）. The level of BMP-7 in serum and urine was gradually decreased with the progression of renal damage (P < 0.01), and it was positively correlated with Ccr, and negatively correlated with blood pressure, proteinuria excretion rate, glomerular sclerosis, crescent and tubulointerstitial fibrosis. Conclusions TGF-β1 is up-regulated in early stage of mesangial proliferation while down-regulated in renal fibrosis, suggesting TGF-β1 may take part in renal fibrosis. The level of BMP-7 is gradually reduced with the progression of renal damage, leading to the inhibition of its protection against renal fibrosis.

Objective To investigate the alpha-galactosidase A activity and the mutations of GLA gene in Chinese pedigrees with Fabry disease. Methods All 7 exons and their neighboring intronic sequences of the GLA gene of the probands were analyzed by PCR and direct sequencing. The α-gal A enzymatic activity in peripheral leukocytes was measured by fluorogenic substrate, 4-methylumbelliferyl-a-D-galactopyranoside. Mutation screening in the pedigrees of the probands was also performed. Results Nine mutations including 4 novel mutations was identified in 11 probands. The mutations comprised of missense mutations (R301Q, I91T, G132R, F273L, D165Y), nonsense mutations (R342X, W236X), as well as a single nucleotide deletion (1082delG) and a gross deletion (44 bp nt. cd 1077). Forteen carriers of the relevant mutations including 3 hemizygotes and 11 heterozygotes in the pedigrees and less than 50% probands offered a positive family history at the beginning of the study. All the hemizygotes showed low enzymatic activity (<1 nmol&#8226;hr^－1&#8226;mg^－1) but 1/4 of heterozygotes had the enzymatic activity within normal range. Conclusions Nine mutations including 4 novel mutations in 11 Chinese pedigrees with Fabry disease are identified by PCR-DNA sequencing. Gene analysis and enzymatic activity detection should be performed to improve the identification of the disease. Identification of new mutations in this disease and mutation screening in the pedigrees will be helpful to the genetic counselling and proper treatment.

Objective To report a rare case of renal amyloidosis secondary to Castleman’s disease, and to review literatures concerned. Methods and Results A 43-year-old man, who was followed up for three years, presented proteinuria, anemia and poly-site enlarged lymph nodes. Renal amyloidosis of AA type was diagnosed by pathological examination of renal biopsy samples, and Castleman’s disease of multicentric and plasma cells type was diagnosed by immunology and pathological examination of lymph node biopsy samples. Conclusion Castleman’s disease can result in renal amyloidosis, which pathogenic mechanism may be associated with the abnormal immune states caused by Castleman’s disease.

Objective To observe the change and possible role of connexin 43 as well as gap junction intercellular communication function in TGF-β1 triggered renal tubular epithelial-myofibroblast transdifferentiation (TEMT) of cultured human renal tubular epithelial cells (HKC). Methods The normal HKC was stimulated with rhTGF-β1 of 10 μg/L. Simultaneously，pLNCX2-connexin 43 vector was transferred in TGF-β1-stimulated HKC. The morphological alterations were observed under an inverted microscope. The cells were collected at 48th hours after rhTGF-β1 stimulation. The expression levels of connexin 43，E-cadherin, α-smooth muscle actin (α-SMA), vimentin mRNA and protein were detected by RT-PCR and Western blotting. The gap junction intercellular communication function was measured by fluorescence recovery after photobleaching assay (FRAP). Results rhTGF-β1 stimulation altered the HKC’s shape from oval to fusiform, down-regulated the connexin 43 and E-cadherin expression and up-regulated α-SMA and vimentin expression (P<0.05), which resulted in down-regulation of intercellular communication function. The fluorescence recovery rate and recovery period of TGF-β1-stimulation group were significantly decreased compared to control (P<0.05). In the pLNCX2-connexin 43 vector transferred group, the fluorescence recovery rate and recovery period were significantly increased compared to TGF-β1 stimulated group (P<0.05). Conclusion Up-regulating connexin 43 expression can delay TEMT induced by exogenous TGF-β1.

Objective To investigate the mechanism of nephron deficit in the rat model of intrauterine growth retardation (IUGR). Methods A rat model of IUGR was established by maternal low-protein (6%) diet throughout pregnancy. Newborn male mice were chosen as objects. The cell proliferation and apoptosis in kidney were detected by Ki-67 and TUNEL method. Expression levels of WT1, Bcl-2, Bax and p53 mRNA were examined by real-time PCR. Immunohistochemistry and Western blot were used to examine the expression of WT1 and Bcl-2 gene products in renal tissue. The number of glomeruli was determined at age of 2 weeks when nephrogenesis finished. Results At two weeks postnatally, IUGR offspring had fewer glomeruli per kidney than those in controls (P<0.01). Compared to the controls, more TUNEL positive cells were located in the nephrogenic zone of IUGR newborns. In IUGR newborns, renal WT1 and Bcl-2 mRNA levels were significantly reduced, the Bcl-2 mRNA/Bax mRNA ratio was decreased, whereas the expression of p53 mRNA remained unchanged. In IUGR newborns, the expression levels of WT1 and Bcl-2 protein were significantly decreased, and their immunostaining were also suppressed in the nephrogenic zone. Conclusions Reduction of nephron number in IUGR rat may be associated with enhanced apoptosis in kidney development. Decreased WT1 and Bcl-2 expression as well as reduction of the Bcl-2/Bax ratio may contribute to the molecular mechanism.

Objective To determine the role of oxidative stress and mitogen-activated protein kinase (MAPK) in the pathogenesis of aldosterone plus salt-induced renal injury. Methods Male Sprague-Dawley rats were randomly treated with one of the following combinations for 6 weeks: Group 1, tap water+vehicle (0.5% ethanol); Group 2, 1%NaCl+vehicle; Group 3, 1%NaCl+aldosterone+vehicle; Group 4, 1%NaCl+aldosterone+eplerenone+vehicle; and Group 5, 1%NaCl+aldosterone+tempol+vehicle. Renal cortical mRNA expression of NADPH oxidase components p22phox, Nox-1 and gp91phox was quantitatively analyzed by real-time PCR. The activities of MAPKs, including extracellular signal-regulated kinases (ERK)1/2, c-Jun-NH2-terminal kinases (JNK) and ERK5 in renal cortical tissues were measured by Western blot. Results Compared with 1%NaCl-infused rats, aldosterone plus salt-infused rats showed higher blood pressure[(165±5) vs (118±3) mm Hg], urinary excretion of protein [(101.0±24.0) vs (9.1±3.0) mg/24 h] and renal cortical thiobarbituric acid-reactive substances (TBARS) level [(0.23±0.02) vs (0.09±0.01) nmol/mg protein] (all P<0.05). Renal cortical NADPH oxidase components p22phox,Nox-4 and gp91phox mRNA expression increased significantly by aldosterone plus salt-infusion [(2.3±0.2)， (4.3±0.8) and (3.0±0.3) folds，respectively, P<0.05]. Aldosterone plus salt-infused rats showed increased renal cortical ERK1/2 ，JNK and ERK5 activities [(3.3±0.3), (2.3±0.3) and (3.0±0.2) fold, respectively, P<0.05]. Eplerenone and tempol prevented aldosterone plus salt-induced hypertension [(127±2), (125±5) mm Hg, respectively] and elevation of urinary excretion of protein [(10±2)， (9.3±2) mg/24 h, respectively]. Furthermore, eplerenone and tempol normalized renal cortical TBARS level and ERK1/2，JNK as well as ERK5 activities in aldosterone plus salt-infused rats. Conclusion Oxidative stress and MAPK signaling pathway play an essential role in the progression of aldosterone plus salt chronic infusion-induced renal injury.

Objective To investigate the role of NADPH oxidase-dependent reactive oxygen species(ROS) in angiotensin (Ang)Ⅱ-induced epithelial-mesenchymal transition (EMT) and accumulation of extracellular matrix(ECM) in rat peritoneal mesothelial cells (RPMC). Methods Primary rat peritoneal mesothelial cells were cultured in vitro. After synchronization for 24 hours, the cells were randomly assigned to four groups: the control group, the AngⅡ(10^-7 mol/L) group,the AngII type Ⅰ receptor antagonist losartan (10 μmol/L) treatment group(AngⅡ+losartan), and the NADPH oxidase inhibitor diphehylendieodonium (DPI) (10 μmol/L) treatment group (AngⅡ+DPI). The DCF-sensitive cellular ROS was measured by fluorometric assay and confocal microscopy. RT-PCR was employed to detect the mRNA expression for NADPH oxidase subunit p47phox and PAI-1, E-cadherin. α-smooth muscle actin and p47phox protein expression were examined by Western blot. Results (1)AngⅡ significantly induced the production of intracellular ROS by (3.64±0.53) folds compared with control (P < 0.05). DPI and losartan inhibited AngⅡ-induced ROS generation (P < 0.05). (2)AngⅡ stimulated NADPH oxidase subunit p47phox mRNA and protein overexpression. Both losartan and DPI inhibited the up-regulation of p47phox mRNA overexpression. (3)AngⅡ stimulated α-SMA mRNA and protein synthesis in RPMC and down-regulated E-cadherin mRNA expression, which were ameliorated by losartan and DPI. (4)AngⅡ significantly up-regulated PAI-1 mRNA expression after 8 h stimulation by (3.06±0.77) folds compared with control (P < 0.05), which was significantly down-regulated by losartan and DPI (P < 0.05). Conclusions NADPH oxidase-dependent ROS mediates EMT and the accumulation of ECM induced by AngⅡ in peritoneal mesothelial cells. AngⅡ and NADPH oxidase may be the potential therapeutic targets in the progress of peritoneal fibrosis.