High Glucose Promotes the CTGF Expression in Human Mesangial Cells via Serum and Glucocorticoid-induced Kinase 1 Pathway

The role of serum and glucocorticoid-induced kinase 1 （SGK1） pathway in the connective tissue growth factor （CTGF） expression was investigated in cultured human mesangial cells （HMCs） under high glucose. By using RT-PCR and Western blot, the effect of SGK1 on the CTGF expression in HMCs under high glucose was examined. Overexpression of active SGK1 in HMCs transfected with PIRES2-EGFP- S422D hSGK1 （SD） could increase the expression of phosphorylated SGK1 and CTGF as compared with HMCs groups transfected with PIRES2-EGFP （FP） under high glucose or normal glucose. Overexpression of inactive SGK1 in HMCs transfected with PIRES2-EGFP- K127N hSGK1 （KN） could decrease phosphorylated SGK1 and CTGF expression as compared with HMCs groups transfected with FP under high glucose. In conclusion, these results suggest that high glucose-induced CTGF expression is mediated through the active SGK1 in HMCs.

PKCα signaling pathway involves in TNF-α-induced IP_3R1 expression in human mesangial cells

BACKGROUND:This study aimed to explore the effects of TNF-a on the expression of IP_3R1mRNA and protein in human mesangial cells(HMCs),and to elucidate the mechanism of TNF-a relating to IP_3R1 expression in the occurrence of hepatorenal syndrome(HRS).METHODS:HMCs were stimulated by tumor(TNF-a) with 100 ng/mL for different hours(2,4,8,and 24 hours).The expression changes of IP_3R1 mRNA and protein were detected by quantitative real-time polymerase chain reaction and immunoblotting.Several inhibitors including D609,U73122,PP1,safingol,rottlerin and non-radioactive protein kinase C(PKC) were used to examine the mechanism of signal transduction ofTNF-a-regulated IP_3R1 in HMCs.RESULTS:The levels of IP_3R1 mRNA at 2 hours after TNF-a exposure were significantly enhanced and peaked at 8 hours in HMCs(P<0.01),then descended at 24 hours(P<0.01).The levels of IP_3R1 protein at 4 hours after TNF-a exposure were obviously increased and peaked at24 hours after TNF-a exposure(P<0.01).Compared to the control group,safingol(PKCa inhibitor)and D609(phosphatidylcholine-specific phospholipase C inhibitor) significantly blocked the TNF-ainduced expression of IP_3R1 mRNA(3.30±0.81 vs.1.95±0.13,P<0.05;2.10±0.49,P<0.01) and IP_3R1protein(3.09±0.13 vs.1.86+0.39,P<0.01;1.98±0.02,P<0.01).TNF-a promoted PKCa activation with maximal PKCa phosphorylation that occurred 8 hours after stimulation measured by non-radioactive PKC assay,and the effect was markedly attenuated by pretreatment with D609 or safingol.CONCLUSION:TNF-a increased the expression of IP_3R1 and this was mediated,at least in part,through the PC-PLC/PKCa signaling pathways in HMCs.

Differential effect of three mitogen-activated protein kinases on lipoprotein （a）-induced human mesangial cell proliferation

Background Mesangial hypercellularity is a critical early histopathological finding in human and experimental glomerular diseases. Hyperlipidemia and the glomerular deposition of lipoproteins are commonly associated with mesangial hypercellulafity and play an important pathobiological role in the development of glomerular diseases. The activated cytoplasmic mitogen-activated protein kinase （MAPK）, including mainly extracellular-signal regulated protein kinase （ERK）, c-Jun amino-terminal kinase （JNK）, and p38, has been thought to translocate into the nucleus and activate various transcription factors and protooncogenes associated with cell growth and proliferation. Lipoprotein （a） （Lp（a）） has been shown to stimulate proliferation of mesangial cells, but the events of Lp（a） signaling have not yet been characterized. The purpose of this study was to investigate the signal transduction pathways involved in Lp（a）-induced cell proliferation and provide an evidence for the participation of Lp（a） in intracellular signaling pathways for mesangial cell proliferation. Methods Lp（a） was isolated from a patient who was being treated with low density lipoprotein （LDL）-apheresis by density gradient ultracentrifugation and then chromatography. Human mesangial cells （HMCs） were isolated by the sequential sieving technique and stimulated with Lp（a） in different concentration and time course. The DNA synthesis of the cells was measured by [^3H] thymidine incorporation for detecting the proliferation. The expression of all the three members of MAPK family, including ERK1/ERK2, JNK, and p38, and their phosphorylation were detected by Western blotting. Results Lp（a） could induce a significant dose-dependent proliferation of HMCs. The 3H-TdR incorporation was 1.64±0.31, 1.69±0.48, 3.59±0.68 （P 〈0.01）, 4.14±0.78 （P 〈0.01）, and 4.05±0.55 （P 〈0.01） （103 cpm） at the Lp（a） concentration of 0, 5, 10, 25, and 50ug/ml, respectively. Lp（a） induced an increase in ERK1/ERK2 phosphorylation between 5 and 60 minutes, and in JNK phosphorylation between 15 and 30 minutes after incubating with HMCs, whereas the level of p38 and its phosphorylation was not changed. Conclusions Lp（a） could stimulate the proliferation of HMCs by activiating the phosphorylation of ERK1/ERK2 and JNK MAPK signaling pathway, whereas p38 pathway had no effect on the Lp（a）-induced HMC proliferation, which indicated that three MAPKs seem to be distinctly involved in the effect. In particular, it also provides the evidence that Lp（a） may act as one of the major endogenous modulators for mitogenic signaling response and cell proliferation within the glomerulus.

Expression of Nrf2/ARE pathway in diabetic nephropathy and renal protection of Qihuang Bushen Xiezhuo Formula

Objective:To investigate the effect of Qihuang Bushen Xiezhuo Formula on the expression of the nuclear factor E2 related factor 2(Nrf2)/antioxidant response element(ARE)signaling pathway of human glomerular mesangial cells(HMC)in high glucose medium and its protective effect on oxidative stress.Methods:The HMC were cultured in vitro to prepare normal rat serum.The rats were intragastrically administered with irbesartan and Qihuang Bushen Xiezhuo Formula.The serum containing the drugs was prepared after the blood concentration was reached.The rats were divided into the control group,the high glucose group,the irbesartan group and the Qihuang Bushen Xiezhuo Formula group.The HMC of the control group was cultured with normal rat serum(10%serum concentration)medium,while that of the high glucose group was cultured with high glucose medium of rat serum(10%serum concentration).The irbesartan group and the Qihuang Bushen Xiezhuo Formula group were respectively treated with 10%irbesartan and 10%Qihuang Bushen Xiezhuo Formula in serum high glucose medium.After 48 h of culture,the relevant indicators were collected and detected.The mRNA expression levels of Nrf2,gamma-glutamylcysteine synthetase(γ-GCS)and superoxide dismutase(SOD)in each group were detected by real-time PCR.The expressions ofγ-GCS and SOD were detected by immunohistochemistry.The protein expressions ofγ-GCS and SOD in HMC of each group were observed by Western Blot.Results:The expressions of Nrf2,γ-GCS,SOD mRNA and protein in experimental cells of each group were low in the control group,while those in the high glucose group were decreased as compared with the control group(P<0.05).After interference of irbesartan and Qihuang Bushen Xiezhuo Formula the expressions were increased,and the increase in Qihuang Bushen Xiezhuo Formula group was more significant(P<0.05).Conclusion:Qihuang Bushen Xiezhuo Formula can improve HMC oxidative stress injury in high glucose culture,and its mechanism may be achieved by activating Nrf2 and its related downstream proteinsγ-GCS and SOD.