Bingmei Yang

High glucose induction of DNA-binding activity of the transcription factor NFκB in patients with diabetic nephropathy

The aim of this study was to investigate whether high glucose induces aldose reductase (AKR1B1) expression through NFkappaB, which may contribute to the pathogenesis of diabetic nephropathy. 34 Caucasoid patients with type 1 diabetes were recruited; 20 nephropaths and 14 long-term uncomplicated subjects. Peripheral blood mononuclear cells (PBMCs) were cultured under normal or high glucose (25 mmol/l of d-glucose) with or without an aldose reductase inhibitor (ARI). High glucose increased NFkappaB binding activities in the PBMCs from nephropaths compared to the uncomplicated subjects (1.77+/-0.22 vs. 1.16+/-0.04, p=0.02). ARI induced a substantially greater decrease of NFkappaB binding activities in the nephropaths compared to the uncomplicated subjects (0.58+/-0.06 vs. 0.79+/-0.06, p=0.032). AKR1B1 protein levels in the nephropaths were increased under high glucose conditions and decreased in the presence of an ARI, whilst the silencing of the NFkappaB p65 gene in vitro reduced the transcriptional activities of AKR1B1 in luciferase assays. These results show that NFkappaB induces AKR1B1expression under high glucose conditions, and the pattern of expression differs between nephropaths and the uncomplicated subjects.

Chemokines MCP-1 and RANTES in Isolated Rat Pancreatic Acinar Cells Treated with CCK and Ethanol In Vitro

The role of cholecystokinin (CCK) and ethanol on the expression and secretion of monocyte chemoattractant protein-1 (MCP-1) and regulated on activation, normal T-cell expressed and secreted (RANTES) chemokines from isolated rat pancreatic acinar cells was investigated. CCK at concentrations of 1 n M and 100 n M and ethanol at concentrations of 75, 200, 400, and 600 m M were used to stimulate isolated acini. The levels of MCP-1 and RANTES in the incubation medium were determined by enzyme-linked immunoabsorbent assay (ELISA). In the control groups, MCP-1 and RANTES were secreted into the incubation medium, and both increased with time. MCP-1 increased from baseline 17.6 pg/ml to 74.1 pg/ml, whereas RANTES increased from 255.5 to 318.3 pg/ml at 390 min. CCK at 100 n M caused a sustained increase in MCP-1 levels to 89.6 pg/ml at 390 min in the incubation medium, whereas the levels of RANTES gradually decreased after 180 min and reached its lowest level at 390 min. Ethanol at a concentration of 600 m M increased the levels of RANTES in the incubation medium, but inhibited the levels of MCP-1 at all concentrations (75, 200, 400, and 600 m M). In summary, rat pancreatic acinar cells secrete MCP-1 and RANTES, and the stimulation of these chemokines by CCK and ethanol suggests that they may be involved in the pathogenesis of acute pancreatitis.

Study of polymorphisms in the CYP2E1 gene in patients with alcoholic pancreatitis

Cytochrome P450IIEI (CYP2E1) is an ethanol-inducible enzyme. Recently, several novel polymorphisms in the CYP2E1 gene have been identified. A polymorphism at position -35 [G(-35)T] appears to be of functional significance in transcription assays. The aim of this study was to investigate if this and other polymorphisms, at position -1019 [C(-1019)T], 4808 [G(4808)A], and 7668 [T(7668)A] of the CYP2E1 gene are associated with alcoholic pancreatitis. DNA was extracted from peripheral blood of 38 patients with alcoholic chronic pancreatitis (CP), 19 patients with alcoholic acute pancreatitis (AP), 46 alcoholic controls (AC), and 155 normal controls (NC). The polymorphisms were examined by digestion with the corresponding restriction endonucleases following PCR amplification. The results have shown that the frequencies of the rare alleles of these polymorphisms were not significantly different between the CP, AP, and AC groups and NC. Therefore, our study results suggest to us that the polymorphisms investigated in the CYP2E1 gene are unlikely to be involved in the susceptibility and pathogenesis of alcoholic pancreatitis.

Elevated activity of transcription factor nuclear factor of activated T-cells 5 (NFAT5) and diabetic nephropathy.

The expression of aldose reductase is tightly regulated by the transcription factor tonicity response element binding protein (TonEBP/NFAT5) binding to three osmotic response elements (OREs; OREA, OREB, and OREC) in the gene. The aim was to investigate the contribution of NFAT5 to the pathogenesis of diabetic nephropathy. Peripheral blood mononuclear cells (PBMCs) were isolated from the following subjects: 44 Caucasoid patients with type 1 diabetes, of whom 26 had nephropathy and 18 had no nephropathy after a diabetes duration of 20 years, and 13 normal healthy control subjects. In addition, human mesangial cells (HMCs) were isolated from the normal lobe of 10 kidneys following radical nephrectomy for renal cell carcinoma. Nuclear and cytoplasmic proteins were extracted from PBMCs and HMCs and cultured in either normal or high-glucose (31 mmol/l d-glucose) conditions for 5 days. NFAT5 binding activity was quantitated using electrophoretic mobility shift assays for each of the OREs. Western blotting was used to measure aldose reductase and sorbitol dehydrogenase protein levels. There were significant fold increases in DNA binding activities of NFAT5 to OREB (2.06 ± 0.03 vs. 1.33 ± 0.18, P = 0.033) and OREC (1.94 ± 0.21 vs. 1.39 ± 0.11, P = 0.024) in PBMCs from patients with diabetic nephropathy compared with diabetic control subjects cultured under high glucose. Aldose reductase and sorbitol dehydrogenase protein levels in the patients with diabetic nephropathy were significantly increased in PBMCs cultured in high-glucose conditions. In HMCs cultured under high glucose, there were significant increases in NFAT5 binding activities to OREA, OREB, and OREC by 1.38 ± 0.22-, 1.84 ± 0.44-, and 2.38 ± 1.15-fold, respectively. Similar results were found in HMCs exposed to high glucose (aldose reductase 1.30 ± 0.06-fold and sorbitol dehydrogenease 1.54 ± 0.24-fold increases). Finally, the silencing of the NFAT5 gene in vitro reduced the expression of the aldose reductase gene. In conclusion, these results show that aldose reductase is upregulated by the transcriptional factor NFAT5 under high-glucose conditions in both PBMCs and HMCs.

Analysis of immunoglobulin G subclass in the serum antibody responses of alveolar echinococcosis patients after surgical treatment and chemotherapy as an aid to assessing the outcome

Immunoglobulin G (IgG) subclass-specific antibody responses were evaluated for the follow-up of alveolar echinococcosis (AE) patients. Seventy-four sequentially collected sera from 25 Chinese and French AE cases who underwent surgery including hepatectomy, liver transplant and/or chemotherapy were analysed quantitatively and qualitatively during the clinical follow-up period. These AE patients were classified in 4 groups--cured, improved, stabilized, or aggravated. Serum antibody levels of the subclasses IgG1 and IgG4 were significantly higher in the AE patients than in healthy controls. IgG1 and IgG4 isotypes in AE patients were the most sensitive IgG antibody response in an enzyme-linked immunosorbent assay (ELISA) and in binding to antigens of 44kDa, 35kDa, 21kDa and 17.5kDa in an Echinococcus multilocularis protoscolex extract after Western blotting. In AE cases classed as cured or improved, IgG subclass antibody levels tended to decrease earlier than total IgG levels, especially IgG4 antibody levels which became negative within one year after successful treatment. IgG4 antibody levels also decreased in most of the improved cases. Increasing or unchanged levels of IgG4 and IgG1 antibodies were demonstrated in both stabilized and aggravated AE cases using both ELISA and immunoblot assays. Reappearance of specific IgG4 antibodies was a strong indication of recurrence, especially in liver transplant patients. Combined quantitative and qualitative assessment of IgG1 and IgG4 antibodies may be potentially useful for the serological follow-up of human AE.

Mutations of the Cationic Trypsinogen Gene in Hereditary and Non-Hereditary Pancreatitis

Background/Aims: Mutations in the cationic trypsinogen gene have been detected in patients with hereditary pancreatitis (HP). This study investigates the prevalence of the R122H, N29I, A16V and –28delTCC mutations in the common, non-hereditary forms of chronic pancreatitis and in a HP family. Methods: DNA was prepared from blood samples of 53 patients with chronic pancreatitis (36 alcoholic, 14 idiopathic and 3 hereditary), 20 alcoholic controls and 20 healthy, ethnically matched controls. The R122H and A16V mutations were identified by the polymerase chain reaction (PCR) and restriction enzyme digestion. A nested-PCR was used to identify the N29I mutation. The –28delTCC deletion and the C133807T polymorphism were sought by direct sequencing. Results: The R122H mutation was detected in 1 patient with alcoholic chronic pancreatitis and all 3 affected members of a HP family. The N29I, A16V and –28delTCC mutations were not detected in any of the study subjects. At the C133807T polymorphism, the C allele and C/C genotype were significantly increased in alcoholic chronic pancreatitis (p = 0.001 and p = 0.0004, respectively) while the T allele and CT genotype were significantly reduced (p = 0.001 and p = 0.004, respectively) compared to healthy controls. Conclusions: Mutations of the cationic trypsinogen gene are rarely found in chronic pancreatitis patients of typical aetiology. Screening for these mutations should be considered in those with a family history consistent with hereditary pancreatitis but may also be appropriate in a well-defined subgroup of patients with non-hereditary chronic pancreatitis, i.e. those who have developed the disease before the age of 30.

Endothelial nitric oxide synthase polymorphisms are associated with hypertension and cardiovascular disease in renal transplantation

Aim:  Nitric oxide (NO), produced by the polymorphic endothelial nitric oxide synthase (NOS3), plays an important role in endothelial function. The aim was to determine the effect of NOS3 polymorphisms on hypertension and cardiovascular disease (CVD) in renal allograft recipients.

Genomics of Diabetic Neuropathy

Diabetes is associated with considerable mortality and morbidity due to the long term complications of the disease. Diabetic neuropathy is a major complication of diabetes and there is increasing evidence to implicate genetic factors together with elevated blood glucose in the susceptibility to this condition. The majority of the studies on the genomics of diabetic neuropathy have focussed on the gene coding for aldose reductase (AKR1B1). Polymorphisms of this gene have been associated with neuropathy. However, the Human Genome Project is likely to provide many more novel candidate genes that play a key role in the pathogenesis of this condition.