The Role And Mechanisms Of TAB1-regulated Glycolysis In The Activation Of Macrophages In Diabetic Nephropathy

Background and objectiveDiabetic nephropathy(DN)is considered to be the trickiest complication in diabetes.The infiltration and activation of macrophages(especially M1 macrophages)and the inflammation mediated by macrophages play a critical role in the pathogenesis of diabetic nephropathy.The metabolic reprogramming of macrophages draws more attention.In different pathological environments,macrophages exhibit different phenotypes,play different biological functions,and also exhibit different energy metabolism mode.M1 type macrophages prefer to obtain energy through glycolysis.Transforming growth factor beta activated kinase 1(TAB1)can bind to transforming growth factor beta activated kinase 1(TAK1),exert an inflammatory effect.TAB1/TAK1 can activate nuclear factor ?B(NF-?B)and regulate the activation of macrophages;activation of the NF-?B signaling pathway can control over the HIF signaling pathway.HIF-1?(hypoxia-inducible factor-1)is involved in regulating the glycolysis,and increase the activity of glycolytic enzymes,such as Hexokinase-1(HK1),6-phosphofructose-2-kinase/fructose-2,6-bisphosphatase 3(6-Phosphofructo-2)-Kinase/Fructose-2,6-Biphosphatase 3(PFKFB3)and lactate dehydrogenase A(LDHA).At present,the regulation of TAB1/NF-?B/HIF-1? in macrophage glycolysis in diabetic nephropathy has not been reported.We had investigated the glycolysis of macrophages in diabetic nephropathy,and the regulatory role of TAB1/NF-?B/HIF-1? in glycolysis and activation of macrophages in DN.Methods Part1:We collected patients and renal biopsy tissue with diabetic nephropathy in the first affiliated hospital of Anhui Medical University.ELISA was used for assessment of inflammation;PAS staining was used for assessment of pathological manifestations.Immunohistochemical staining was used for assessment of CD68,TNF-?,HK1,PFKFB3,LDHA.Double immunofluorescence staining was used for assessment of the co-express of CD68 with HK1,PFKFB3,LDHA.Part2:Lentiviral vector-mediated TAB1 knockdown was used in streptozotocin(STZ)-induced diabetic mice.After 12 weeks,the blood glucose,relative kidney weight(kidney weight/body weight)and 24 h urine albumin excretion rate of mice in each group were measured;the pathological manifestations of renal tissue were observed by light microscope;Immunohistochemical staining was used for assessment of TAB1,CD68,IL-1?,TNF-1?,HK1,PFKFB3 and LDHA.Western blot was used for assessment of TAB1/NF-?B/HIF-1?,Inducible nitric oxide synthase(i NOS),IL-1?,TNF-1?,HK1,PFKFB3 and LDHA.ELISA was used for assessment of IL-1?,TNF-1?;Immunohistochemical double staining was used to detecte the co-expression of CD68 and HK1,PFKFB3,LDHA,i NOS.Part3:Bone marrow-derived macrophages(BMMs)were identified by flow cytometry;the experiment was divided into the following groups: mannitol control group(D-mannitol),negative si RNA control group(control si RNA),blank control group(control),high glucose stimulation group(HG),TAB1 si RNA group(TAB1 si RNA),TAB1 si RNA high glucose stimulation group(TAB1 si RNA+HG).Western blot,flow cytometry,laser confocal were used to observe the expression of M1 macrophage marker i NOS.Laser confocal,Western blot,q RT-PCR were used to detect the expression of HK1,PFKFB3 and LDHA;ELISA was used for assessment of MCP-1 and IL-1?.Lactic acid production kit and glucose absorption kit was used for assessment of lactic acid production and glucose absorption function;Western blot was used for assessment of TAB1,NF-?B p65,HIF-1? protein expression changes in each group of cells.Laser confocal was used to observe the nuclear transfer of NF-?B p65;Ch IP was used to detect the combination of NF-?B p65 and HIF-1? promoter sequence.Result Part1:We found that the inflammatory factor TNF-? and IL-1? were up-regulated in DN;the positive rate of the macrophage marker CD68 and TNF-? in diabetic nephropathy was significantly higher than normal;Renal tissue in DN showed higher glycolytic enzymes HK1,PFKFB3,LDHA.And the level of glycolytic enzymes in the kidney of DN patients has a strong correlation with clinical indicators.The results of immunofluorescence double staining suggested the co-localization of CD68 with glycolytic enzymes HK1,PFKFB3,and LDHA in DN.Part2:Compared with the control group,the blood glucose and 24 h urine albumin excretion rate of diabetic mice was significantly increased.PAS staining suggested that diabetic mice had typical pathological changes of diabetic nephropathy.Diabetic mice exhibit activation of macrophages,increased levels of glycolytic enzymes HK1,PFKFB3,LDHA,and increased levels of inflammation in kidney.Downregulation of TAB1 could inhibit glycolysis/polarization of macrophages and inflammation.The albuminuria,the tubulointerstitial damage index and glomerular mesangial expansion index of STZ-induced diabetic nephropathy mice were decreased by TAB1 knockdown.Part3:High glucose stimulation increased the expression of M1-type marker i NOS in macrophages,increased secretion of inflammatory factors MCP-1 and IL-1?,and increased expression levels of glycolytic enzymes HK1,PFKFB3,LDHA.TAB1 si RNA inhibited the increase in the levels of glycolytic enzymes caused by HG and suppressed macrophage activation and inflammation.The Ch IP results suggest specific binding of NF-?B to the promoter of HIF-1? in response to high glucose stimulation.TAB1 si RNA inhibits the nuclear translocation of NF-?B p65 and suppresses the expression of NF-?B/HIF-1?.Conclusion1.In patients with diabetic nephropathy,the inflammatory factors were elevated,glycolytic enzymes HK1,PFKFB3,and LDHA were elevated,and the correlation analysis results indicate that the expression of glycolytic enzymes PFKFB3 and LDHA in the DN group is positively correlated with 24-hour urine albuminand.Renal macrophages are infiltrated,accompanied by the activation of macrophages and the enhancement of glycolysis.2.Diabetic mice have typical kidney pathological changes,with an increase in the 24-hour urine albumin excretion rate and an increase in renal tissue inflammation.Knockdown of TAB1 can improve the clinical condition and pathological changes of diabetic mice;TAB1/NF-?B/HIF-1?signaling pathway is activated in the kidney tissue of diabetic mice,accompanied by the glycolysis level of kidney tissue and macrophages.With the inhibition of TAB1,the expression level of NF-?B/HIF-1? decreases,and the glycolysis level is also inhibited.3.High glucose stimulates the polarization of macrophages to M1 type,with the release of inflammatory factors and the enhancement of glycolysis level,TAB1 can regulate macrophage activation,inflammatory release and glucose glycolysis through the NF-?B / HIF-1? signaling pathway.