Myeloid-derived Growth Factor(MYDGF) Protects Against Podocyte Injury In Diabetic Nephropathy

BackgroundDiabetic nephropathy(DN)is one of the most common complications of diabetes and is a major cause of end stage renal disease(ESRD).At present,the pathogenesis of DN is not fully understood,In addition,there is no effective drug in the clinic to completely reverse the occurrence and development of diabetic nephropathy.Therefore,the search for new potential therapeutic targets is of great significance for the treatment of diabetic nephropathy.Podocytes are major components of glomerular basement membrane(GBM),and podocyte injury plays an important role in the development and progression of DN,mainly manifesting the reduction of podocyte density and number,podocyte hypertrophy and foot process fusion and disappearance.Therefore,improving podocyte injury and finding effective therapeutic targets have great significance for the treatment of diabetic nephropathy.A very recent study has identified a new secreted protein in bone marrow cells with therapeutic potential.This substance was first named Myeloid-derived growth factor(MYDGF).Bone marrow-derived growth factor is a novel secreted growth factor that plays an important role in maintaining the normal function of cells.Related studies of myeloid growth factor and its important protective effects on acute myocardial infarction(AMI)have been reported in recent literature.This secreted protein promotes cardiomyocyte survival and angiogenesis by activating PI3K-Akt signaling pathway to improve cardiomyocyte apoptosis in myocardial ischemia and promoting endothelial cell proliferation by regulating MAPK1/3,cyclinD1 and STAT3 related pathways.Treatment with recombinant protein MYDGF can significantly reduce the infarct size of myocardial infarction mice and improve cardiac contractile function.However,the expression and function of MYDGF in the kidney,especially in diabetic nephropathy,has not been reported so far.Research purposes1.To examine the expression of MYDGF in DN,and to determine whether MYDGF participates in the occurrence and development of DN by affecting podocytes.2.To explore the role and mechanism of MYDGF in podocyte injury in mice with DN.Research methodsPart I:The Expression changes of MYDGF in DN1.Detection of MYDGF expression in various organs and renal parenchyma cells of WT mice by RT-PCR and real-time quantitative PCRFirst,the mRNA levels of MYDGF in various organs of mice was detected by RT-PCR and real-time quantitative RT-PCR.Human podocyte(HPC),mouse podocyte(MPC),human renal tubular epithelial cell(HK-2),rat mesangial in vitro Rat messangial cell(RMC)and rat glomerular endothelial cells(GENC)were also detected by RT-PCR and real-time quantitative PCR in the expression of MYDGF in renal parenchymal cells.2.To verify the establishment of a mouse model of DN induced by streptozotocin(STZ)Ten weeks old of wild-type C57BL/6J(WT)mice were selected.After one kidney excision and recovered for one week.STZ was intraperitoneally injected at a dose of 100 mg/kg,and DN was induced by continuous injection for three days.Glucose concentration(GLU)in whole blood of mouse peripheral vein was measured using a blood glucose meter,renal weight,renal kidney weight(RKW)was measured,and urinary albumin and creatinine(urinary)in diabetic mice were detected by ELISA kit.Albumin:creatinine ratio(UACR),simultaneous detection of glycogen deposition and expression of nephrin and Podocin by periodic acid-schiff stain(PAS)and immunofluorescence(IF).The above indicators and methods were used to verify the successful modeling of the diabetic nephropathy model.3.Characterization of physiological indicators related to db/db miceWe purchased a batch of db/db 16-week mice.Mouse GLU was detected using a blood glucose meter,kidney weight was measured,RKW was calculated,and UACR of diabetic mice was detected by ELISA kit.The expression of glycogen deposition and Nephrin and Podocin were detected by PAS staining and immunofluorescence.The above indicators and methods were used to verify the successful modeling of the diabetic nephropathy model.4.Detection of MYDGF expression in renal cortex of diabetic nephropathy miceWhen the mouse model was successfully constructed,Western blot(WB)and Real-time RT-PCR were used to detect the expression of MYDGF in the kidney of diabetic nephropathy mice.5.Changes in expression levels of MYDGF after stimulation of different renal parenchymal cells by advanced glycation end products(AGE)Human podocyte(HPC),rat glomerular endothelial cells(GEnC),human renal tubular epithelial cells(HK-2),rat system Rat messangial cell(RMC)and mouse glomerular podocyte(MPC)were stimulated with advanced glycation end products(AGE)for 48 hours to extract intracellular proteins for protein.Western blot analysis was performed to detect changes in protein expression of MYDGF in various renal parenchymal cells under AGE stimulation.6.Expression of MYDGF in the supernatant of each renal parenchyma cells by AGE stimulation in vitro.Human podocyte(HPC),rat glomerular endothelial cells(GEnC),human renal tubular epithelial cells(HK-2),rat system Rat messangial cell(RMC)and mouse podocyte(MPC)were stimulated with advanced glycation end products(AGE)for 48 hours,and then the cell supernatant was taken through Elisa.The experiment verified the expression of MYDGF in the supernatant of each renal parenchyma.7.Detection and distribution of MYDGF in the kidney of diabetic nephropathy mice by immunofluorescenceMale wild type(WT)C57BL/6J mice of about 10 weeks old were selected and recovered for one week after single kidney excision.Streptozotocin(STZ)was intraperitoneally injected at a dose of 100 mg/kg.Day-induced diabetic nephropathy.After the mouse model was successfully constructed,the expression of MYDGF in glomeruli of diabetic nephropathy mice was detected by immunofluorescence double staining.Part Ⅱ:The role and mechanism of MYDGF in diabetic nephropathy1.Flow cytometry detection of overexpression of MYDGF on AGE-induced podocyte apoptosis.Human podocyte(HPC)was cultured in vitro and transfected into MYDGF overexpression plasmid.After stimulation with advanced glycation end products(AGE)for 48 hours,the podocytes were detected by flow cytometry..2.Western blotting was used to detect the effect of overexpression of MYDGF on AGE-induced podocyte injury.Human podocyte(HPC)was cultured in vitro,transfected with MYDGF overexpression plasmid,and stimulated by advanced glycation end products(AGE)for 48 hours.Western blot was used to detect podocyte function protein Podocin,WT1 and Nephrin.3.Western blotting was used to detect the effect of overexpression of MYDGF on the level of autophagy in podocytes under high glucose conditions.Human podocyte(HPC)was cultured in vitro and transfected into MYDGF overexpression plasmid.The cells were stimulated with advanced glycation end products(AGE)for 48 hours.Western blot was used to detect autophagy-associated protein ATG12,LC3Ⅱ,P62,Beclin-1,ATG5 and VPS34 expression levels.4.Using gene chip technology to detect differential genesThe differentially expressed genes between the AGE-induced podocyte injury group and the MYDGF overexpression group were analyzed by gene chip technology,and the Wnt family molecular heat map was drawn.Research resultsPart I:Expression changes of MYDGF in diabetic nephropathy1.In the main organs of WT mice,the expression of MYDGF in liver and kidney was relatively high;the expression of MYDGF in RMC and GENC was relatively high in different renal parenchyma cells.Firstly,the expression of MYDGF in mouse heart,liver,spleen,lung,kidney and brain was characterized.The results of common PCR and real-time quantitative PCR showed that the content of MYDGF mRNA in kidney and liver was relatively high.The content in the heart,spleen,lungs and brain is relatively low.The expression level of MYDGF in rat mesangial cells and rat glomerular endothelial cells is relatively high.2.STZ-induced type 1 diabetic nephropathy model successfully establishedThe results of glucose,kidney weight,urinary albumin(creatinine ratio,UACR)showed that STZ-induced diabetic nephropathy mice had blood glucose compared with control mice.The ratio of kidney weight to urine albumin to creatinine was significantly increased.The results of periodic acid-schiff stain(PAS)also showed a significant increase in glycogen deposition and mesangial proliferation in STZ-induced diabetic nephropathy mice compared with control mice.The results of immunofluorescence experiments also showed that the expression levels of nephrin and Podocin in STZ-induced diabetic nephropathy mice were significantly lower than those in the control group.3.The db/db mouse model is consistent with the type 2 diabetic nephropathy model.The results of glucose,kidney weight,urinary albumin(creatinine ratio,UACR)showed that db/db mice had blood glucose and kidney compared with control mice.The ratio of weight ratio,urinary albumin to creatinine was significantly increased.The results of periodic acid-schiff stain(PAS)also showed a significant increase in glycogen deposition and mesangial proliferation in db/db mice compared with control mice.The results of immunofluorescence experiments also showed that the expression levels of nephrin and Podocin in db/db mice were significantly lower than those in the control group.4.The expression of MYDGF is significantly decreased in the kidney of diabetic nephropathy mice.Western blot(WB)and Real-time RT-PCR showed that MYDGF was induced by Streptozocin(STZ)in diabetic nephropathy model group and db/db mouse kidney tissue.The expression was significantly decreased compared with the control group.5.AGE stimulation can significantly down-regulate MYDGF protein levels in podocytesWestern blotting results showed that advanced glycation end products(AGE)can significantly induce MYDGF level of expression in human podocyte(HPC)and mouse podocyte(MPC)is significantly reduced.The expression of MYDGF protein in human renal tubular epithelial cells(HK-2)also decreased.There was no significant change of MYDGF protein expression in rat glomerular endothelial cells(GEnC)and rat mesangial cells(RMC).It indicates that MYDGF has relative cell-specific changes under high glucose conditions.6.AGE stimulation significantly down-regulate MYDGF protein levels in human glomerular podocytes and mouse glomerular podocyte supernatantsElisa results showed that the expression of MYDGF was decreased in the cell supernatants of human podocyte(HPC)and mouse podocyte(MPC).7.The expression level of MYDGF was significantly decreased in glomeruli Podocytes of diabetic nephropathy mice.Immunofluorescence double-labeled experiments showed that MYDGF was expressed in both glomerular podocytes and renal tubules.At the same time,it was found that the diabetic nephropathy mice induced by STZ decreased more significantly in the podocytes than the control group.Part Ⅱ:The role and mechanism of MYDGF in diabetic nephropathy1.Overexpression of MYDGF significantly attenuated AGE-induced podocyte apoptosis.Flow cytometry results showed that AGE can significantly aggravate apoptosis of podocytes,while overexpression of MYDGF can significantly improve podocyte apoptosis.2.Overexpression of MYDGF can improve AGE-induced podocyte injury.Western blot results showed that AGE can significantly down-regulate the expression of Podocin and WT1,while over-expression of MYDGF can significantly reverse the down-regulation of Podocin and WT1.3.Overexpression of MYDGF upregulated the level of autophagy in AGE-induced diabetic nephropathy.Western blot results showed that AGE can significantly down-regulate the expression of ATG12 and LC3Ⅱ,while over-expression of MYDGF can significantly restore the expression of ATG12 and LC3Ⅱ.AGE can significantly up-regulate the expression of P62,while over-expression of MYDGF can significantly reduce the expression of P62.4.Using gene chip technology to detect differential genesThrough gene chip technology analysis,it was found that some members of Wnt family were significantly decreased in AGE-induced podocyte injury group,and Wntl was the most obvious.Overexpression of MYDGF significantly restored the expression changes of these genes induced by injury.Conclusion and innovation:1.MYDGF was significantly decreased in the mouse model of diabetic nephropathy,and the level of MYDGF protein in the podocytes and the level of MYDGF in the supernatant were significantly down-regulated under AGE stimulation,suggesting that MYDGF is closely related to the pathogenesis of diabetic nephropathy and may be involved in podocyte injury.112.Overexpression of MYDGF may further protect the podocytes and kidneys by regulating autophagy levels and podocyte apoptosis.