Inhibition of the K+ channel K(Ca)3.1 reduces TGF-ß1-induced premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells.

OBJECTIVE: K(Ca)3.1 channel participates in many important cellular functions. This study planned to investigate the potential involvement of K(Ca)3.1 channel in premature senescence, myofibroblast phenotype transition and proliferation of mesangial cells. METHODS & MATERIALS: Rat mesangial cells were cultured together with TGF-ß1 (2 ng/ml) and TGF-ß1 (2 ng/ml) + TRAM-34 (16 nM) separately for specified times from 0 min to 60 min. The cells without treatment served as controls. The location of K(Ca)3.1 channels in mesangial cells was determined with Confocal laser microscope, the cell cycle of mesangial cells was assessed with flow cytometry, the protein and mRNA expression of K(Ca)3.1, α-smooth muscle actin (α-SMA) and fibroblast-specific protein-1 (FSP-1) were detected with Western blot and RT-PCR. One-way analysis of variance (ANOVA) and Student-Newman-Keuls-q test (SNK-q) were used to do statistical analysis. Statistical significance was considered at P<0.05. RESULTS: Kca3.1 channels were located in the cell membranes and/or in the cytoplasm of mesangial cells. The percentage of cells in G0-G1 phase and the expression of K(ca)3.1, α-SMA and FSP-1 were elevated under the induction of TGF-ß1 when compared to the control and decreased under the induction of TGF-ß1+TRAM-34 when compared to the TGF-ß1 induced (P<0.05 or P<0.01). CONCLUSION: Targeted disruption of K(Ca)3.1 inhibits TGF-ß1-induced premature aging, myofibroblast-like phenotype transdifferentiation and proliferation of mesangial cells.

Premature senescence and cellular phenotype transformation of mesangial cells induced by TGF-B1.

BACKGROUND: Transforming growth factor-ß1 (TGF-ß1) is a polypeptide member of the transforming growth factor ß superfamily of cytokines and performs many cellular functions. Its overexpression may lead to renal fibrosis. AIM: This study planed to investigate the effects of TGF-ß1 on the cell cycle and phenotype of mesangial cells. METHODS: Rat mesangial cells were cultured together with different concentrations (0, 1, 2, 5, and 10 ng/mL) of TGF-ß1 for specified times from 0 min to 72 h. 0 ng/mL TGF-ß1 and 0 min served as controls. Cell cycles were assessed by flow cytometry and α-smooth muscle actin expression (α-SMA) protein expression by western blot analysis. All data were presented as Mean ± SD. Statistical analysis was performed by using one-way analysis of variance and correlation analysis. Results were considered significant at p < 0.05. RESULTS: After 15 min of co-culture with different concentrations of TGF-ß1, the percentage of mesangial cells in G0/G1 phase was significantly elevated compared to the control (p < 0.05). 12 h co-culture induced cell hyperplasia, 24 h co-culture obvious up-regulation of α-SMA (p < 0.01) and one or two cells' myofibroblast phenotype transition, and 36 h co-culture several cells' phenotype transition. Correlation analysis prompted that the TGF-ß1-induced premature aging was time-dependent (p < 0.01). CONCLUSION: TGF-ß1 may induce mesangial cells' premature senescence and myofibroblast-like phenotype transformation time-dependently, which may contribute to the development of early stage of glomerulosclerosis.

[Evaluation of immune status of kidney transplant recipients by combined HLA-G5 and sCD30].

OBJECTIVE: to study the relationship between the expression of serum human leucocyte antigen-G5 (HLA-G5)/soluble CD30 (sCD30) and the function of renal graft in kidney transplant recipients and investigate the immune status of recipients with combined HLA-G5 and sCD30. METHODS: from January 2002 to November 2008, a total of 66 kidney transplant recipients in our centre were selected as subjects and divided into three groups: stable function of renal graft (n = 38), acute rejection (n = 15) and chronic rejection (n = 13). The expressions of serum HLA-G5 and sCD30 were detected. There were two different immune conditions with acute/chronic allograft rejection and normal renal graft in kidney transplant recipients as evaluated by combined HLA-G5 and sCD30. The sensitivity, specificity and critical value of the method were analyzed by the curve of receiver operating characteristic. RESULTS: the levels of HLA-G5 and sCD30 were significantly correlated with serum creatinine (r = -0.493, 0.691, both P < 0.01). Within the first year post-transplantation, the sensitivity was 78.6% and the specificity 85.7% when HLA-G5 critical value 82 microg/L and sCD30 critical value 12.2 microg/L. After one year post-transplantation: the sensitivity was 92.3% and the specificity 84.6% when HLA-G5 critical value 141 microg/L and sCD30 critical value 10.3 microg/L. CONCLUSION: the immune state of recipients are evaluated by combine HLA-G5 and sCD30 which may be a simple and valid method.

Magnetic cell sorting and flow cytometry sorting methods for the isolation and function analysis of mouse CD4+ CD25+ Treg cells.

OBJECTIVE: In this paper we compared the two methods of cell sorting (magnetic cell sorting and flow cytometry sorting) for the isolation and function analysis of mouse CD4(+) CD25(+) regulatory T (Treg) cells, in order to inform further studies in Treg cell function. METHODS: We separately used magnetic cell sorting and flow cytometry sorting to identify CD4(+) CD25(+) Treg cells. After magnetic cell separation, we further used flow cytometry to analyze the purity of CD4(+) CD25(+) Treg cells, trypan blue staining to detect cell viability, and propidium iodide (PI) staining to assess the cell viability. We detected the immune inhibition of CD4(+) CD25(+) Treg cells in the in vitro proliferation experiments. RESULTS: The results showed that compared to flow cytometry sorting, magnetic cell sorting took more time and effort, but fewer live cells were obtained than with flow cytometry sorting. The CD4(+) CD25(+) Treg cells, however, obtained with both methods have similar immunosuppressive capacities. CONCLUSION: The result suggests that both methods can be used in isolating CD4(+) CD25(+) Treg cells, and one can select the best method according to specific needs and availability of the methodologies.