Carol Yi-Ki Szeto

Differential effects of transforming growth factor-beta on the synthesis of connective tissue growth factor and vascular endothelial growth factor by peritoneal mesothelial cell.

Background: Previous studies found that transforming growth factor-β (TGF-β) plays a conflicting role in peritoneal fibrosis. We hypothesise that TGF-β acts on peritoneal mesothelial cells (PMC) via VEGF and CTGF as downstream mediators. Methods: The effect of TGF-β in primary culture of rat PMC was studied. VEGF and CTGF mRNA expression was examined by real time quantitative polymerase chain reaction (RT-QPCR), and VEGF antigen level in cell supernatant by ELISA. Results: Incubation of rat PMC with TGF-β resulted in a time- (3–72 h) and concentration- (0–50 pg/ml) dependent increase in VEGF mRNA expression, and VEGF protein level in the cell supernatant. When stimulated with TGF-β 100 pg/ml, there was a 20-fold up-regulation of VEGF mRNA expression (p < 0.001). The CTGF mRNA expression and protein level of PMC was slightly increased at low concentration of TGF-β (50 pg/ml) but decreased at a higher concentration (100 pg/ml or above). The effect of TGF-β on PMC CTGF, but not VEGF, gene expression was inhibited by Smad decoy oligodeoxynucleotide. The effect of TGF-β on PMC VEGF gene expression and protein synthesis was inhibited by PD98059 (a specific MAP kinase inhibitor) and chelerythrine (a specific protein kinase C inhibitor), but not cholera toxin (activator of cyclic AMP) or herbimycin A (inhibitor of protein tyrosine kinase). The up-regulation of CTGF mRNA expression was inhibited by PD98059, but not chelerythrine, cholera toxin or herbimycin A. Furthermore, CTGF gene expression in TGF-β-stimulated PMC was inhibited by co-administration of recombinant VEGF. Conclusions: Our data demonstrate that TGF-β induces PMC production of VEGF and CTGF via different signalling pathways. At high concentration of TGF-β, VEGF production predominates and CTGF production was inhibited. Since CTGF and VEGF have different biologic effects, our results may explain the complex activity of TGF-β in peritoneal physiology.

Association of ENOS polymorphism with basal peritoneal membrane function in uremic patients

BACKGROUND Basal peritoneal permeability has a major impact on the outcome of peritoneal dialysis (PD) patients, but the determinant of this is unknown. Early evidence suggests that peritoneal permeability is affected by nitric oxide (NO) activity. Recently, a gene polymorphism of the endothelial NO synthase (ENOS) gene was identified that is associated with circulating nitrate levels. METHODS We performed a cross-sectional study to examine the relationship between ENOS4(a/b) gene polymorphism and basal peritoneal function in 86 Chinese incident PD patients. ENOS genotypes for variable number tandem repeats in intron 4 (a/b) were identified by polymerase chain reaction. Patients were classified into 2 groups according to results of a basal peritoneal equilibration test (PET) performed within 2 months of dialysis therapy: group A consisted of patients with low (L)/L average (LA) PET results, and group B consisted of those with H and HA PET results. RESULTS Group A (L/LA) had a significantly greater prevalence of ENOS aa/ab genotype than group B (H/HA; 30% versus 12%; P < 0.05). Frequencies of the ENOS a allele also were greater in group A (L/LA) than group B (H/HA) (16% versus 6%; P = 0.03). ENOS genotype remained an independent predictor for peritoneal transport after adjustment for sex, body weight, and prevalence of diabetes by multivariate analysis (adjusted odds ratio, 3.3; confidence interval, 1.1 to 3.7; P = 0.03). Subjects with the aa/ab genotype had significantly lower mass transfer area coefficients (7.35 +/- 3.4 versus 9.48 +/- 5.21 mL/min; P = 0.023) and dialysate-plasma creatinine ratios at 4 hours (0.55 +/- 0.13 versus 0.62 +/- 0.14; P = 0.048) than those with the bb genotype. CONCLUSION ENOS4(a/b) gene polymorphism is associated with basal peritoneal permeability in uremic Chinese patients.

Connective tissue growth factor is responsible for transforming growth factor-beta-induced peritoneal mesothelial cell apoptosis.

Background: Previous studies found that transforming growth factor-β (TGF-β) induces mesothelial production of connective tissue growth factor (CTGF), which may be downstream mediators of TGF-β. Since high dose TGF-β induces apoptosis of peritoneal mesothelial cells (PMC), we study the effect of CTGF blockade in the system of TGF-β-induced PMC apoptosis. Method: We examined the effect of TGF-W in primary culture of rat peritoneal mesothelial cells (PMC). PMC apoptosis was studied by flow cytometry. The effect of CTGF was blocked by antibody and short-interfering RNA (siRNA). Expression of apoptotic gene was studied by real-time polymerase chain reaction. Result: In cultured unstimulated rat PMC, there is a low but definite incidence of spontaneous apoptosis. Stimulation with TGF-β 50 pg/ml induces an upregulation of apoptotic gene BAX expression and a downregulation of anti-apoptotic gene BCL-2L expression, and a 4-fold increase in PMC apoptosis. The effect of TGF-β-induced PMC apoptosis was partly prevented by antibody against CTGF, and completely abolished by CTGF-specific siRNA, while CTGF-blockade by siRNA had no effect on PMC necrosis. CTGF silencing by siRNA prevented the down-regulation of BCL-2L expression induced by TGF-β, had no effect on the BAX expression. Conclusion: Our results indicate that CTGF is an important downstream mediator of TGF-β-induced PMC apoptosis.

Isolation and transcript analysis of two-component histidine kinase gene Le.nik1 in Shiitake mushroom, Lentinula edodes

Le.nik1, a two-component histidine kinase gene of Lentinula edodes, the Shiitake mushroom, was identified. The relationship between this two-component signal transduction system and mushroom development was studied. We used a modified RNA arbitrarily-primed PCR (RAP-PCR) method to isolate Le.nik1 as a differentially expressed gene during L. edodes development. We determined the 6.29kb full-length cDNA sequence of Le.nik1. It had high sequence homology to Neurospora crassa nik1, which encoded a histidine kinase essential for development and osmotic response. In L. edodes, the expression level of Le.nik1 was highest during primordium formation and fruiting body maturation. The transcripts were localized predominantly in the developing hymenophores, or mushroom gills, which may indicate the role of a two-component signal transduction system in cell differentiation during mushroom development. Mannitol stress influenced transcript expression of Le.nik1, suggesting that it may be involved in osmo-sensing and regulation. To our knowledge, this is the first report on the two-component system in mushrooms and the first analysis on the distribution of Le.nik1 transcript in the course of fruiting body formation and in parts of fruiting bodies.

Relationship Between β1-Adrenergic Receptor Polymorphisms and Cardiovascular Disease in Peritoneal Dialysis Patients

Background Recent studies show that a common gain-of-function polymorphism of β1-adrenergic receptor (389 Gly→Arg) plays an important role in the pathogenesis of hypertension and heart failure in patients with normal renal function. We studied the relationship between β1-adrenergic receptor polymorphism and cardiovascular disease in peritoneal dialysis (PD) patients. Methods We studied 189 new PD patients. The β 1-adrenergic receptor genotype was determined by polymerase chain reaction-restriction fragment length polymorphism assay. They were then prospectively followed for the development of cardiovascular events. All-cause mortality and duration of hospitalization were also recorded. Results There were 95 male cases. The mean age was 56.2 ± 14.8 years. Eighty-six (45.5%) patients were diabetic; 81 (42.9%) received beta-blocker therapy. Only one case was homozygous for the mutant CC genotype. The prevalence of GG, GC, and CC genotypes were 34.9%, 64.6%, and 0.5%, respectively. The genotype distribution was significantly different from that predicted by the assumption of Hardy-Weinberg equilibrium ( p p = 0.53). Event-free survival was 63.6% and 71.5% at 24 months, respectively ( p = 0.26), and the duration of hospitalization was 15.9 ± 3.0 and 16.6 ± 2.3 days per year, respectively ( p = 0.8). The results remained similar when patients with and without beta-blocker treatment were separately analyzed. Conclusion Our study demonstrates that the β 1-adrenergic receptor polymorphism is not related to cardiovascular disease in PD patients. Nevertheless, the low prevalence of mutant CC genotype in new PD patients suggests that PD patients represent a highly biased population.