| Renal disease is one of the leading causes of morbidity and mortality in patients with diabetic mellitus. High glucose-induced growth of mesangial cells is a characteristic feather of diabetes-induced renal complications. The results of the Diabetics Control and Complication Trial have shown that strict glycemic control can prevent the onset and progression of diabetic complications. Excessive cellular growth is a major contributor to pathological changes associated with diabetic nephropathy. One of the basic underlying mechanisms of diabetic nephropathy seems to involve high glucose. High glucose (HG) causes mesangial cell (GMC) growth, production of transforming growth factor (TGF)-β, and increased synthesis of matrix proteins such as fibronectin, collagenⅣ, contributing to diabetic nephropathy. GMCs cultured under HG conditions produce more TGF-β,CTGF and extracellular matrix molecular (ECM) than those under normal glucose (NG) conditions.It has been shown that both the DAG/PKC, MAP kinase, as well as the JAK/STAT pathway are altered under HG conditions and these alteration could be responsible for some of the vascular dysfunction observed in the diabetic state, thus suggesting that glucose and its metabolites may mediate their adverse effects by altering the various signal transduction pathways employed by certain hormones. These signaling pathways have been implicated in a variety of cells, such as GMC that seem to play a prominent role in diabetic glomerulosclerosis. However, to date the effect of HG in relation to the activation of the JAK/STAT pathway in GMC has not been elucidated. The JAK enzymes, involves JAK1, JAK2, JAK3 and TYK2, are responsible for the activation of the STATs (STAT1, STAT2, STAT3, STAT4, STAT5A/B, STAT6), which are latent cytoplasmic transcription factors. STATs, when activated by tyrosine and/or serine phosphorylation, form homo- and hetero-dimers and translocate to the nucleus where they regulate the expression of various genes involved in cellular proliferation.Traditionally, three intracellular signaling pathways have been implicates in the activation of protooncogenes: the JAK/STAT, p21ras/raf-1/MAPK, and the plc-γ1 cascades.Extensive studies have clarified that both the MAPK and JAK/STAT pathways are activated by multistep phosphorylation cascades after ligand-cell surface receptor binding and that they transmit signals to cytosolic and nuclear targets, leading to alterations in cell growth, proliferation and other cellular functions. It is reported recently that exposure of cells to HG can actives the growth-promoting enzyme janus kinase 2 (JAK2) and its latent signal transducers and transcription factors (STATs). Our purpose was to determine the effect that if HG can actives JAK2/STAT3 pathway of GMC and the effect that inhibition of JAK2/STAT3 has on the HG-induced increase in TGF-β1 and ECM. So we have designed and finished three experiments as followed:1,To investigate the alteration of p-STAT3,STAT3 cultured under either NG or HG or mannitol with or without AG-490.2,To explain the effect of STAT3 in the over expression of TGF-β1,CTGF and FN.3,To investigate the role of JAK2/STAT3 in the expression of TGF-β1, CTGF, FN in GMC under high glucose and angiotensinⅡ.1,The activation of JAK/STAT pathway under high glucose in rat GMC.Objectives: To investigate the alteration of p-STAT3,STAT3 cultured under either NG or HG or mannitol with or without AG-490.Methods: First, GMC of rats were cultured under DMEM with normal glucose (NG) to 80% confluence, washed once with serum-free DMEM, and growth-arrested in serum-free DMEM in NG for 24 hours to synchronize the cell growth. After this time the medium was changed to fresh serum-free medium containing either NG (5.5mmol/l) or HG (25mmol/l) or mannitol (19.5mmol/l ) for 24~48 hours. At the end of desired periods , cells were lysed for further detection by Western blotting for p-STAT3,STAT3. Second, GMCs were incubated for 24 hours in serium-free medium containing either NG (5.5mmol/l) and HG (25mmol/l) with or without 10 ummol/1 AG-490 (specific inhibition of JAK2). At the end of cultured cell lysate was then immunoblotted with p-STAT3,STAT3 antibodies .Results: We found that HG can actives phosphorylation of STAT3 which were significantly inhibited by preincubation with AG-490.Conclusions: The JAK/STAT signaling pathway can be activated by HG in GMC.2,The alteration of TGF-β1,CTGF,FN under high glucose and the affection of AG-490 in over expression of ECM and TGF-β1.Objectives: To investigate the role of JAK/STAT in the expression of TGF-β1,CTGF,FN in GMC under high glucose.Methods: (1)GMC cultured under either NG (5.5mmol/l) or HG (25mmol/l) or mannitol (19.5mmol/l) with or without AG-490 (specific blocker of JAK2, 1.0μmol/l) for 24～48 hours, then detect the expression of TGF-β1,CTGF,FN mRNA by RT-PCR and FN protein in supernatant by ELISA.(2)GMC cultured under either NG (5.5mmol/l) or HG (25mmol/l) or mannitol (19.5mmol/l ) with or without SB-431542 (specific blocker of TGF-β1 receptor, 10μmol/l ) for 24~48 hours, then detect the expression of TGF-β1,CTGF,FN mRNA by RT-PCR and FN protein in supernatant by ELISA.Results: (1) HG can actives significantly increase in synthesis of TGF-β1,CTGF,FN mRNA and proteins, and the over expression of these factors were significantly inhibited by preincubation with AG-490 .(2) The expression of TGF-β1 wasn't been affected, the increased expression of CTGF and FN were only partly blocked by SB-431542.Conclusions: (1) Activation of the JAK/STAT pathway by HG may be of importance in the over expression of TGF-β1 and ECM in GMC that is seen in diabetic nephropathy.(2) Except for TGF-β1/CTGF/FN pathway , the activation of JAK/STAT could increase the expression ECM through CTGF derectly.3,To investigate the role of JAK2/STAT3 in the expression of TGF-β1,CTGF,FN in GMC under high glucose and angiotensinⅡ.Objectives: To investigate the role of JAK2/STAT3 in the expression of TGF-β1,CTGF,FN in GMC under high glucose and angiotensinⅡ.Methods: GMC cultured under high glucose for 48h, then stimulate GMC with angiotensin II of different concentrations with different time. At the end of cultured cell lysate was then immunoblotted with p-STAT3,STAT3 antibodies and detect the expression of TGF-β1, CTGF,FN mRNA by RT-PCR and FN protein in supernatant by ELISA.Results: angiotensin II can actives phosphorylation of STAT3 and significantly increase in synthesis of TGF-β1,CTGF,FN mRNA and proteins under high glucose, and the over expression of these factors were significantly inhibited by preincubation with AG-490.Conclusions: AngiotensinⅡand HG maybe of cooperation in activation of STAT3 and increase in synthesis of TGF-β1,CTGF,FN in GMC.In summaryThese results provide evidence that activation of the JAK/STAT pathway by HG may be of importance in the overproduction of TGF-β1 , CTGF and ECM in GMC that is seen in diabetic nephropathy . Expect for TGF-β1, the activation of JAK/STAT may increase the synthesis of ECM by other cell factors such as CTGF. AngiotensinⅡ and HG maybe of cooperation in activation of STAT3 and increase in synthesis of TGF-β1,CTGF,FN in GMC.
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