The Role And Mechanisms Of TGF-β Activated Kinase 1 In The Macrophage Activation Of Diabetic Nephropathy

Objective: Diabetic nephropathy(DN) is the most serious chronic microvascular complications of diabetes mellitus(DM) and gradually becomes the major cause of end-stage renal failure. Recently, many researches have focused on how to prevent and treat early DN effectively. Although previous studies revealed that macrophages play an important role in the development and progress of DN, little information is available about the signal pathway activating macrophages in DN.TGF-β-activated kinase 1(TAK1) is a member of MAP3 K family, which can activate the MAPK and IKK pathways. After activation by certain factors, TAK1 could further activate MAPK pathway, and phosphorate NF-κB induced kinase(NIK) which is the activator of NF-κB. Thus, TAK1 is crucial in cell growth, differentiation, apoptosis and transcription of pro-inflammatory mediators, cell adhesion and growth factor expression. To investigate the role of TAK1 in the development of DN, we studied the profile of TAK1 pathway, inflammatory factors, including monocyte chemoattractant protein-1(MCP-1), intercellular cell adhesion molecule-1(ICAM-1), from the whole, cellular level to molecular level. Additionally, TAK1 pathway was blocked in gene and protein level, which provide a novel direction to study the molecular mechanism of DN.Methods: Part 1:36 male mice were divided radomly into three groups: wild type(WT, n=12),db/db mice(db/db, n=12) and db/db mice with 5Z-7-oxozeaenol treatment(db/db+OZ, n=12). The concentrations of blood glucose(BG) were evaluated in db/db mice and the db/db+OZ mice with BG higher than 16.7mmol/L were used as disease model. In week 8 and week 12 after 5Z-7-oxozeaenol treatment, mice blood, urine and kidney tissue were sampled. Blood glucose(BG), body weight(BW), kidney weight(KW) and urinary albumin excretion rate(UAER) were evaluated. Kidney pathololical lesions were detected by light and electron microscopy. ED-1, NF-κB p65, MCP-1 and TNF-α were detected by immunohistochemistry. Western blot were used to detected p-TAK1, TAB1, p-p38 MAPK and IL-1β expression, while ICAM-1, MCP-1 m RNA level was evaluated by q RT-PCR.Part 2:Mice macrophages were seeded on 96-well cell culture plate, cell viability was tested after treatment with different concentration of TAK1 inhibitors. Cells were divided into groups(MN; BSA; NC; HG; AGEs; HG+OZ30, 100, 300; AGEs+OZ30, 100, 300) and treated for given time periods. Flow cytometry analysis the macrophage activated by HG and AGEs. Expression levels of p-TAK1, TAB1, p-JNK, p-p38 MAPK, NF-κB p65, IL-1β were detected by western blot. TNF-α and MCP-1 m RNA levels were evaluated by q RT-PCR.Results: Part 1:① Compared with control group, the levels of BG, BW, KW and UAER were much higher(P<0.01) in db/db mice group, while BW, KW and UAER level decreased significantly in db/db+OZ group(P<0.05, P<0.01). ②In week 8 and 12, glomeruli of kidney enlarged, total number of mesangial cell increased along with basal membrane thickening and extracelluar matrix increasing. Through electronic microscopic observasion, irregular thickening of basal membrane of glomeruli was detected and cell foot processes were effaced. In db/db+OZ group, pathological lesions in kidney tissue were positively improved. ③Immunohistochemistry experiment results showed thatED-1, NF-κB p65, MCP-1 and TNF-α expression level increased apparently in db/db mice group(P<0.05), while in db/db+OZ group expression of such proteins decreased(P<0.05). ④ Similar with the results of immunohistochemistry experiment, p-TAK1, TAB1, p-p38 MAPK and IL-1β expression level was higher in db/db mice group(P<0.05) and lower in db/db+OZ group(P<0.05). There is no statistical difference in the level of TAK1, p38MAPK(P>0.05). ⑤Also, transcription level of ICAM-1 and MCP-1 was higher in db/db mice group and lower in db/db+OZ group(P<0.05, P<0.01).Part 2:①Compared with control group, treatment with less than 300 nmol/L inhibitor has no negative effect on viability of macrophages cultured with medium containing HG or AGEs(P>0.05), while 1000nmol/L inhibitor was harmful to macrophages(P<0.05). ② Through q RT-PCR, we found higher transcription level of MCP-1 and TNF-α in HG and AGEs group(P<0.01). After treatment with inhibitor, transcription level of MCP-1 and TNF-α decreased significantly(P<0.05, P<0.01). ③ Through flow cytometry analysis, we found that both HG and AGEs could induce macrophages to M1 polarization. However, treatment with inhibitor could decrease the percentage of markers on the membrane of M1 macrophages(P<0.05, P<0.01). ④Western blot results showed that inhibitors could down regulate the expression of p-TAK1, TAB1, p-JNK, p-p38 MAPK, NF-κB p65 and IL-1β(P<0.05), which were highly expressed in HG and AGEs groups(P<0.05). The level of TAK1, JNK, p38 MAPK showed similar expression pattern in different groups(P>0.05).Conclusions: 1. In the condition of diabetes, p-TAK1 increasingly expressed in kidney tissue in vivo. And, mesangial cells proliferiated, accompanied by extracellular matrix clustering and macrophages activation, indicating that p-TAK1 may involved in the development of DN.2. Expression level of p-TAK1, TAB1, p-p38 MAPK and NF-κB p65 were significantly higher in kidney tissue of db/db mice. Similar phenomena occurred in the level of ICAM-1, MCP-1, TNF-α and IL-1β. Inhibitor of TAK1 could down regulate MAPK and NF-κB pathway and the expression of inflammatory factors, which needs further investigation. 3. With HG or AGEs treatment, macrophages activated and expression levels of p-TAK1, TAB1, MAPK and NF-κB p65 were significantly higher. These activated macrophages expressed more IL-1β, MCP-1, TNF-α. Inhibitor of TAK1 could down regulate the biological processes mentioned above.