Chinese Journal of Nephrology

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Expression of IgA class switching gene in tonsillar mononuclear cells in patients with IgA nephropathy

LIU Hong;PENG You-ming;LIU Fu-you;XIAO Wei-wei;LI Wei-wei;ZHANG Yu;LIU Yang.

2012, 28(2): 83-88.

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Objective To investigate the molecular mechanism of the mal-production of IgA and IgA1 by tonsillar mononuclear cells(TMCs) in IgA nephropathy (IgAN) patients by measuring the mRNA expression of Iα-Cα germline transcript and the mRNA and protein expression of activated induced cytidine deaminase (AID) in cultured TMCs stimulated with lipopolysaccharide (LPS) or hemolytic streptococcus (HS) in IgAN patients as well as the chronic tonsilitis patients. Methods Twent-seven IgAN patients admitted into our hospital from Jan. 2009 to Feb. 2010 were enrolled. Twent-seven patients with chronic tonsillitis without renal disease were selected as control. Tonsillar mononuclear cells were isolated by density gradient centrifugation in lymphocyte separation medium. The amount of IgA or IgA1 secreted in the culture supernatants was determined by specific enzyme-linked immunosorbent assay (ELISA). Expressions of Iα-Cα germline transcript and AID mRNA were examined by real-time PCR. The AID protein was determined by Western blot. Results The production of IgA and IgA1 protein, especially the ratio of IgA1/IgA and the expression of AID protein in TMCs were significantly increased in IgAN group compared with chronic tonsillitis group(all P<0.05). The IgA and IgA1 level of stimulated TMCs were obviously increased in patients with IgAN compared with control group(P<0.05). And the expressions of Iα-Cα mRNA, AID mRNA and AID protein were up-regulated significantly in stimulated TMCs (all P<0.05). Conclusions Both LPS and HS can induce the production of IgA and IgA1 and up-regulate the expressions of AID and Iα-Cα in TMCs of IgAN patients. Our results indicate that the TMCs are capable of producing high level of IgA and IgA1 stimulated by LPS or HS, which may be due to the increased expression of AID and Iα-Cα.

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Serum nutritional markers are predictors of early mortality in hospital-acquired acute kidney injury

MA Shuai;XIE Qiong-hong;YOU Huai-zhou;ZHOU Ying;QIAN Jing;KUANG Ding-wei;LIU Jun-feng;HE Qi-liu;HAO Chuan-ming;GU Yong;LIN Shan-yan;DING Feng.

2012, 28(2): 89-94.

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Objective To evaluate the role of nutritional parameters in prognosis, especially in the early and late mortality of hospital-acquired acute kidney injury (AKI) patients. Methods This study was a prospective cohort study conducted in a hospital comprising 1500 beds in Shanghai, China. One hundred ninety-four patients with hospital-acquired AKI, as determined using the RIFLE staging criteria, were enrolled as subjects after obtaining informed consent. Patients with AKI caused by postrenal obstruction, glomerulonephritis, interstitial nephritis or vasculitis were excluded. Nutritional evaluation, including subjective global assessment (SGA), anthropometric and laboratory examination, was conducted. Other laboratory measurements and clinical data were recorded. The primary outcome was early mortality (≤7 days) and late mortality (8-28 days) after enrolling into the study. Results AKI patients at enrollment were characterized by a high prevalence of malnutrition as determined by SGA, anthropometric and laboratory examination. Univariate analysis showed that the SGA, the serum levels of prealbumin, cholesterol and total lymphatic cells, and the Maastricht index were significantly different among early mortality, late mortality and survival groups. The serum prealbumin and cholesterol levels in the early death group were significantly lower than those in the survival and late death groups (P<0.05). Multivariate analysis revealed that SGA, albumin, prealbumin and cholesterol remained independently and significantly associated with early mortality after adjusting for age, sex, dialysis, ventilation, hemoglobin, platelets, bilirubin, and Glasgow coma score. The areas under the receiver operating characteristic curve to predict early mortality for albumin, prealbumin and cholesterol were 0.591, 0.736 and 0.603, respectively, with that of prealbumin significantly higher than others (P<0.05). Conclusion Low levels of serum prealbumin, albumin and cholesterol at enrollment are independently associated with increased early mortality in hospital-acquired AKI patients.

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Mutational analysis of NPHS1 gene in children with sporadic steroid-resistant nephrotic syndrome in Southern Chinese Han ethnic group

WANG Dao-jing;YU Zi-hua;MENG Da-chuan;FU Rong;WANG Jing-jing;FENG Dong-ning;YANG Yong-hui.

2012, 28(2): 95-100.

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Objective To elucidate the mutations of NPHS1 gene in children with sporadic steroid-resistant nephrotic syndrome (SRNS) in Southern Chinese Han ethnic group. Methods Peripheral blood samples were collected for genetic analysis from 40 patients with sporadic SRNS and 50 healthy volunteers as control. Genomic DNA was isolated from peripheral blood leucocytes. Twenty-nine exons and exon-intron boundaries of the NPHS1 gene were amplified by polymerase chain reaction. Mutational analysis was performed by DNA sequencing directly. Results Seven variants, 928G＞A(D310N), 2677A＞G (T893A), 2869G＞C (V957L), IVS8+30C＞T, IVS21+14G＞A, IVS25-23C＞T and *142T＞C, of NPHS1 gene were found in 6 of 40 children with sporadic SRNS, whereas they were not found in 50 healthy controls. 2677A＞G, IVS8+30C＞T, IVS21+14G＞A, IVS25-23C＞T and *142T＞C were novel. Moreover, thirteen already reported NPHS1 polymorphisms, 294C＞T, 349G＞A, IVS3+15C＞T, IVS3+61A＞G, 803G＞A, IVS8+68A＞G, 1339G＞A, 1802G＞C, 2223C＞T, 2289C＞T, IVS24+36C＞T, 3315G＞A and IVS27+45C＞T, were detected in some patients and controls. Conclusions NPHS1 mutations in 6 of 40 children with sporadic SRNS in Southern Chinese Han ethnic group (15%) are detected. NPHS1 mutations are existed in Southern Chinese children, so it is necessary to perform the mutation analysis of NPHS1 gene in those children patients.

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Epidemiologic study of chronic kidney disease in adult population of Anhui province

WANG De-guang;HAO Li;DAI Hong;CHEN Wei-dong;ZHANG Dao-you;LI Long-hai;LIU Zhi;HAN Jiu-huai;YE Li-ping;BAI You-wei.

2012, 28(2): 101-105.

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Objective To explore the prevalence, awareness and risk factors of chronic kidney disease (CKD) in general adult population of Anhui province, China. Methods A total of 3800 residents (older than 18 years) from Anhui province were randomly selected using a stratified, multi-stage sampling. All the residents were interviewed and tested for urinary albumin to creatinine ratio (abnormal: ≥30 mg/g) and reduced estimated GFR [abnormal: ＜60 ml?min-1?(1.73 m2)-1]. The associations of CKD with demographic characteristics, healthy characteristics (hypertension, diabetes and hyperuricemia) were examined. Results Eligible data of 3374 subjects were enrolled in the study. After the adjustment of age and gender component, the prevalence of albuminuria was 9.8% (95%CI 8.8%-10.9%), and reduced eGFR was 2.1% (95% CI 1.7%-2.7%). Approximately 10.4%(95%CI 9.4%-11.5%）subjects had at least one indicator of kidney damage. The awareness rate of CKD was 6.5%. Female, age, hypertension, diabetes mellitus, and hyperuricemia were independently associated with CKD. While obesity, hyperlipdemia. smoking and alcohol drinking were not associated with the prevalence of CKD. Conclusions The prevalace of chronic kidney disease is 10.4% and the awareness rate is 6.5% in general adult population of Anhui province. Independent risk factors associated with CKD are female, age, hypertension, diabetes mellitus and hyperuricemia.

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Clinical analysis of arrhythmia in chronic kidney disease patients without renal replacement therapy

ZHANG Hai-ying;XUE Qin;WANG Nian-song;HU Wei-guo;LI Qing;JIAN Gui-hua;GAO Xu-ping;CHEN Hai-bing;JIA Wei-ping.

2012, 28(2): 106-110.

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Objective To observe the occurrence and risk factors of arrhythmia in chronic kidney disease (CKD) patients in different stages of renal function. Methods A total of 405 CKD patients were enrolled in this study and none of them received renal replacement therapy. The 24 h dynamic electrocardiogram (DCG) was performed, and baseline characteristics were compared. Multivariable Logistic regression analysis was used to examine the relationship between the severe arrhythmia and the potential risk factors, such as age, gender, CKD stage, diabetes, hypertension, hyperpotassaemia, left ventricular hypertrophy (LVH), etc. Results There were 69 patients (17.04%), 79 patients (19.51%), 82 patients (20.25%), 88 patients (21.73 %) and 87 patients (21.48%) in CKD stage 1, 2, 3, 4 and 5, respectively. As high as 45.68% of all the patients had severe arrhythmia, represented by 27.54%, 29.11%, 42.68%, 57.95% and 65.52% in CKD stages 1-5 respectively. The occurrence of severe arrhythmia increased as the eGFR decreased in CKD stages 2, 3, 4 (P<0.05). On multivariable Logistic regression analysis, the occurrence of severe arrhythmia was related to LVH, CKD stage, diabetes, hypertension and hyperpotassaemia. Conclusions The occurrence of severe arrhythmia in CKD patients without renal replacement therapy is high. LVH, CKD stage, hypertension, diabetes and hyperpotassaemia are significantly associated with severe arrhythmia.

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Correlation of anti-M-type phospholipase A2 receptor antibody with disease severity in adult patients with idiopathic membranous nephropathy

ZHOU Guang-yu;JIN Ling;YU Jing;ZHANG Zhi-ping.

2012, 28(2): 111-114.

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Objective To investigate the correlation of serum anti-M-type phospholipase A2 receptor (PLA2R) antibody with laboratory parameters of idiopathic membranous nephropathy (IMN) in adult patients with membranous nephropathy (MN), and to explore the role of anti-PLA2R antibody in the pathogenesis of IMN. Methods Forty-six adult patients with biopsy-proved glomerular diseases were involved in this study, including 20 cases with IMN, 7 cases with IgA nephropathy (IgAN), 6 cases with hepatitis B-associated membranous nephropathy (HBV-MN), 6 cases with minimal change nephropathy (MCN), 4 cases with focal segmental glomerulosclerosis (FSGS) and 3 cases with class Ⅴ lupus nephritis. Total RNA was extracted from human glomeruli and was reversely transcribed to the first-strand cDNA. The full-length human M-type PLA2R was amplified by PCR and the 605 bp product was subcloned into eukaryotic expression vector containing CMV promoter. The recombinant human M-type PLA2R plasmid vector was transiently transfected into human embryonic kidney (HEK) 239T cell line using the FuGene6 transfection reagent. Western blotting was used to detect serum anti-PLA2R antibodies. Correlations of anti-PLA2R antibody level with laboratory parameters, including serum albumin, total cholesterol, Scr and 24-hour urine protein, of IMN patients were evaluated. Results Among 20 cases with IMN, 15 cases showed positive anti-PLA2R antibodies (positive rate 75%). Of 7 cases with IgAN and 6 cases with HBV-MN, only 1 case showed positive anti-PLA2R antibody respectively (positive rate 14.29% and 16.67% respectively). Anti-PLA2R antibody was negative in other patients. The positive rate of anti-PLA2R antibody in IMN patients was significantly higher than that in patients with secondary MN and other types of glomerlonephritis (all P<0.01). Furthermore, anti-PLA2R antibody level was positively correlated with 24-hour urine protein(r=0.803, P<0.01) and negatively correlated with serum albumin in IMN patients (r=-0.816, P<0.01). Conclusions The high positive ratio of anti-PLA2R antibody may indicate that it is the specific autoantibody in IMN. Anti-PLA2R antibody is correlated with IMN disease severity, which indicates that it may be the pathogenic autoantibody in IMN.

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Erythropoietin inhibits complement 3a-induced renal tubular epithelial to mesenchymal transition

ZHANG Feng-xia;WAN Jian-xin;FU Bin-bin;CUI Jiong;XU Yan-fang;YOU Dan-yu.

2012, 28(2): 115-120.

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Objective To investigate the effects of erythropoietin (EPO) on complement 3a (C3a)-induced renal tubular epithelial to mesenchymal transition. Methods The HK-2 cells were divided into 6 groups namely control group, EPO group, TGF-β group, TGF-β+EPO group, C3a group and EPO+C3a group. The mRNA and protein expressions of α-SMA, E-cadherin and C3 were investigated by RT-PCR, Western blot and immunofluorescence respectively. Results Compared with control group and EPO group, the mRNA and protein expressions of α-SMA in HK-2 cells were up-regulated after the intervention of C3a or TGF-β(all P<0.05). On the contrast, the mRNA and protein expressions of E-cadherin were down-regulated(P<0.05), the mRNA and protein expressions of C3 were enhanced (all P<0.05). However, all those above effects of C3a or TGF-β were inhibited after the intervention of EPO (all P<0.05). Conclusion EPO is capable of suppressing the epithelial to mesenchymal transition induced by C3a.

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Effects of recombinant human augmenter of liver regeneration on renal inflammation after renal ischemia reperfusion injury in rats

LIAO Xiao-hui;SUN Hang;LIU Qi;GUO Hui;ZHANG Ling.

2012, 28(2): 121-126.

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Objective To investigate the effects of recombinant human augmenter of liver regeneration (rhALR) on renal inflammation in acute kidney injury (AKI) induced by renal ischemia reperfusion(IR). Methods SD rats were randomly divided into sham-operated group, IR group, rhALR1 group (100 μg/kg) and rhALR2 group (200 μg/kg). Both renal pedicles of rats were identified and occluded with microvascular clamps for 60 min to induce acute kidney injury (AKI). Blood urea nitrogen and serum creatinine levels were evaluated using a Hitachi 747 automatic analyzer. For histological examination, sections were stained with HE. The activity of myeloperoxidase (MPO) was detected by spectrophotometer. Expression of TNF-α, ICAM-1, MCP-1 was determined by Western blotting. Results Blood urea nitrogen, serum creatinine levels and the injury of kidney were improved significantly in rhALR group as compared with IR group (all P< 0.05). They were improved more significantly in rhALR2 group as compared to in rhALR1 group(all P<0.05). The protein levels of TNF-α, ICAM-1, MCP-1 and the activity of MPO in kidneys from the sham-operated rats were low, and increased significantly after renal ischemia reperfusion injury(all P<0.05). After treated with rhALR, the expression of TNF-α, ICAM-1, MCP-1 and the activity of MPO were decreased significantly in kidneys as compared to those in IR group (all P<0.05), which decreased more significantly in rhALR2 group than those in rhALR1 group (all P<0.05). Conclusions rhALR can protect kidneys from ischemia reperfusion injury in rats. The mechanism may be associated with the inhibition of renal inflammatory cells infiltration and down-regulated expressions of TNF-α, ICAM-1 and MCP-1 in the kidney.

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Effects of selective cyclooxygenase 2 inhibitor on the hyperplasia of parathyroid glands from uremic rats

QIU Jun-si;ZHANG Qian;LU Yan-wen;LI Shen-sen;LI Hai-ming;YOU Li;GU Yong;HAO Chuan-ming;CHEN Jing.

2012, 28(2): 127-132.

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Objective To investigate the effects of selective cyclooxygenase 2 inhibitor on the hyperplasia of parathyroid glands from uremic rats. Methods Sixty-five 5/6-nephrectomized (Nx) and fifteen sham operated rats were assigned to 4 groups: (1)Sham group(n=14): sham-operated +normal phosphate diet (P 0.8%, Ca 1.2%); (2) Nx-HP group (n=17): Nx+high phosphate(HP) diet (P 1.2%, Ca 1.2%); (3)Prophylactic COX2 inhibition group (Prev group, n=18): Nx+HP+celecoxib 100 mg?kg-1?d-1 for 3 months; (4)Therapeutic group (Ther group, n=18): Nx+HP+celecoxib 100 mg?kg-1?d-1 starting at the second month of the 5/6 nephrectomy. At the end of 3 month, blood, urine and parathyroid samples were collected. The expressions of COX2 and PCNA were determined by immunohistochemistry, Western blotting and real-time PCR. Results All of the Nx rats fed with high phosphate diet for 3 months manifested progressively increasing serum creatinine, serum iPTH as well as augmentation of parathyroid gland volume, suggesting that secondary parathyroid hyperplasia animal model was established successfully. Celecoxib significantly decreased serum iPTH levels [Sham (34.77±0.83), Nx-HP(100.73±4.35), Prev (87.36±2.18), Ther(87.47±1.76) ng/L, P<0.05], the size of the parathyroid glands in Nx rats[Sham(0.461±0.089), Nx-HP(2.436±0.372), Prev (0.987±0.254), Ther(1.27±0.305) mm2/kg, P<0.05] and PCNA expression in PG determined by Western blotting (decreased to 52.91% in Prev group and 34.68% in Ther group respectively, P<0.05). No significant difference was observed between the two COX2 inhibition groups. The levels of COX2 expression in parathyroid gland were greatly increased in three Nx groups compared with that in sham group (2.47-fold in Nx-HP, 2.34-fold in Prev group, 3.04-fold in Ther group, P<0.05). COX2 inhibitor had no effects on COX2 expression in PGs. Real-time PCR analysis demonstrated the same trends of mRNA expression of COX2 and PCNA in PGs of rats. Conclusion Selective inhibition of COX2 may help to suppress the hyperplasia of parathyroid glands in uremic rats.

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Expression of activin A in high glucose-induced human kidney tubular epithelial cells

LIU Gang;REN Xiao-jun;GUAN Guang-ju.

2012, 28(2): 133-137.

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Objective To investigate the expression of activin A and the intervention effect of follistatin on high glucose-cultured human proximal tubular epithelial cells (HK-2) in vitro. Methods HK-2 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM). By 80% confluent, the cells were incubated in a serum-free medium for 24 h and then exposed to the following experimental conditions for different time periods (12, 24 and 48 h): 5 mmol/L glucose (normal glucose group, NG), 5 mmol/L glucose plus 25 mmol/L mannitol (osmotic control, mannitol group, MG), 25 mmol/L glucose (high glucose group, HG), 25 mmol/L glucose plus 100 μg/L rh-follistatin (100 μg/L rh-follistatin intervention group, HG+FS100) or 25 mmol/L glucose plus 500 μg/L rh-follistatin (500 μg/L rh-follistatin intervention group, HG+FS500). Then, the cells and cellular supernatant were collected for further detection. Activin A and p-Smad2/3 expression were determined by Western blotting. TGF-β and fibronectin released into media were quantitatively analyzed by enzyme-linked immunosorbent assay(ELISA). Results Activin A had very slight activation in the NG group, but its production was enhanced in the HG group after 12 h culture (P<0.05) and increased gradually until study completion. Follistatin blocked its production in a dose-dependent manner. p-Smad2/3 was significantly increased after 24 h culture in the HG group compared with the NG group and follistatin treatment decreased its overexpression in the HG group (P<0.05）. TGF-β was markedly higher in the HG group than that in the NG group after 12 h culture(P<0.05） and increased in a time-dependent manner, but both doses of follistatin failed to modify the overexpression of TGF-β in HG. Compared with the NG group, fibronectin in the HG group was obviously upregulated after 24 h culture(P<0.01). Follistatin reduced its overexpression in a dose-dependent manner. Conclusions High glucose can induce activin A activation and then promote FN production in HK-2 cells. Decreased fibronectin production inhibited by follistatin in HK-2 cells in high glucose condition is associated with blocking of activin A activation.

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Sulodexide prevents diabetic nephropathy through inhibiting renal NF-κB activation and MCP-1 expression

JIANG Zhao-shun;ZHAO Jian-qin;LIU Yuan-tao;HAN Chen-dong;QU Wei.

2012, 28(2): 138-142.

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Objective To study the effects of sulodexide on renal NF-κB activation and monocyte chemotactic protein 1 (MCP-1) expression in diabetic rats and elucidate the possible mechanism of sulodexide in preventing diabetic nephropathy(DN). Methods Wistar rats were fed with high-sucrose-high-fat diet and injected with a low dose of STZ(streptozotocin,35 mg/kg) into abdominal cavity to induce diabetes. DM rats were randomly divided into non-treated group (DM) and sulodexide treated group(DMS). Normal rats were served as control (NC). After 12 weeks of treatment, blood glucose (BG), triglyceride (TG), cholesterol, serum creatinine (Scr), urea nitrogen (BUN), 24 h urinary albumin excretion (UAE) were measured. HE staining was performed in renal tissues for light microscopy examination of mean glomerular volume (MGV). MCP-1 expression was detected by immunohistochemical method. NF-κB activation was determined by Western blot. Results Compared with NC group, DM group and DMS group had significant elevated BG, TG and TC levels (all P<0.01). There were no significant differences of BG, TG or TC levels between DM group and DMS group. Compared with NC group, DM group and DMS group had significant increased Scr, BUN, UAE levels (all P<0.01). Scr, BUN, UAE levels were significantly lower in DMS group than those in DM group [(39.1±0.88) μmol/L vs (41.0±2.16) μmol/L, (9.12±1.06) mmol/L vs (9.87±0.19) mmol/L; (19.92±0.96) mg/24 h vs (25.99±0.52) mg/24 h, all P<0.05]. Compared with NC group, the MGV of DM group was significantly increased [(7.47±1.11)×105 μm3 vs (4.22±1.09)×105 μm3, P<0.01]. Compared with DM group, the MGA of DMS group was significantly reduced [(6.64±0.71)×105 μm3 vs (7.47±1.11)×105 μm3, P<0.05], but was still increased compared with that of NC group(P<0.01). Compared with NC group, the MCP-1 expression of DM group was significantly higher [(12.17±1.94)/HPF vs (1.19±0.70)/HPF，P<0.01]. MCP-1 expression in DSM group was significantly lower than that of DM group[(9.22±1.61)/HPF vs (12.17±1.94)/HPF, P<0.01], but still higher than that of control group (P<0.01). Compared with NC group, the NF-κB activity was significantly higher in DM group [(0.89±0.07) vs (0.24±0.03), P<0.01]. Compared with DM group, NF-κB activity of DMS group was significantly lower [(0.27±0.01) vs (0.89±0.07), P<0.01]. There was no significant difference of NF-κB activity between DMS group and NC group. Conclusion Sulodexide has protective effects on diabetic nephropathy, and one of the mechanisms may involve the inhibition of NF-κB activation as well as the suppression of MCP-1 expression

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临床研究

基础研究

临床研究

基础研究