The Role Of BMP4/Smad1 Signaling Pathway In Diabetic Nephropathy Mesangial Matrix Proliferation

Objective: Diabetic nephropathy(DN)is one of the common complications of diabetes mellitus(DM),which can lead to end-stage renal disease and a series of cardiovascular complications.The pathogenesis of diabetic nephropathy is complex,the basic pathological changes were mesangial matrix proliferation and basement membrane thickening.Bone morphogenetic protein 4(BMP4)belongs to the transforming growth factor-?(TGF-?)superfamily and participates in regulating morphological differentiation,proliferation,migration,and apoptosis of cells.It play a important role in the nervous system,cardiovascular system,urinary tract physiological and pathological processes.BMP4 regulates the transcription of target genes through Smad and non-Smad signaling pathways.Recent studies have found that BMP4/Smad1 signaling pathway may be closely related to the onset of diabetic nephropathy,especially in the involvement of mesangial stromal hyperplasia.Therefore,this study will researchthe relationship between BMP4/Smad1 signaling pathway and diabetic nephropathy mesangial matrix hyperplasia.Methods:1.Animal experiments: Forty male Sprague-Dawley Sprague-Dawley(SDF)healthy male Sprague-Dawleyrats were randomly divided into control group(18 animals in CON group)and diabetic nephropathy group(22 cases in DN group).DN group was given high-fat diet for 4 weeks.A single intraperitoneal injection of 1%streptozotocin(STZ)solution(35 mg/kg)induces diabetes,and CON is injected with an equal volume of 0.1 mmol/L citrate buffer.72 hours later,blood glucose monitoring was performed on the DN group.If the blood glucose was greater than 16.7 mmol/L for three consecutive days,diabetes was defined as a model.The DN group model was successfully defined as 2 weeks later,the urine volume of the model group was greater than 50% of the original urine volume,and the urine protein amount was greater than 30 mg at 24 hours.The serum and urine of each group were collected at each time point for detection of relevant indicators.Rat kidney tissues were collected for HE staining and PASM staining to observe the basic pathological changes of the glomerulus;Immunohistochemical staining was used to detect BMP4,ALK3,Smad1,Col4,Col1,and MMP9.2.Cell experiments: Rat mesangial cells were divided into low glucose group(LG group: medium containing 5.6mmol/L glucose),high glucose group(HG group: medium containing 30mmol/L glucose),and osmotic pressure control group.(OP group:medium containing 5.6mmol/L glucose + 24.4mmol/L mannitol),cultured for 24 h,48h,and 72 h respectively,immunofluorescence was used to detect the expression of BMP4,Smad1,and Col4,and the best time pointwas selected based on the above results.Use the CCK-8 method to detect mesangial cell proliferation at the optimal time.The rat mesangial cells were divided into low glucose group(LG group),high glucose group(HG group),high glucose + BMP4-si RNA group(BMP4-si RNA group),high glucose + scramble-siRNA group(scramble-siRNA group),and cultured to the optimal time point.BMP4,ALK3,Smad1,Col4,Col1,MMP9 expression were detected by western bolt.NOGGIN recombinant protein is BMP4 antagonists,to further verify the role of BMP4/Smad1 in mesangial stromal hyperplasia in diabetic nephropathy,the rat mesangial cells were divided into low glucose group(LG group),high glucose group(HG group),high glucose + NOGGIN Group(NOGGIN group),high glucose + BSA group(BSA group),cultured to the optimal time point,the use of western bolt detection of ALK3,Smad1,Col4,Col1,MMP9 expression.Rusults: Animal experiments: 1)General data of rats in each group: The general condition of SD male rats: After the injection of STZ,the male rats of DN group gradually increased in drinking water,increased diet,and increased urine output.In the 8th week,the activity decreased,the response was slow,the body weight decreased,and the skin gloss decreased.In the 12 th week,there was further weight loss.Some of the rats developed symptoms of abdominal bulging,tail ulceration,and submandibular mass.At 16 weeks,they showed various symptoms,the symptoms are all worsening.The rats in the CON grouphad no significant increase in drinking water,increased diet,and increased urine output.The response was rapid,the activity was more,and the body mass gradually increased.Body weight: The weight of rats in DN group gradually decreased with time,and the body weight of CON rats gradually increased.The body weight of rats in DN group was significantly lower than that of CON group at the same time point(P<0.05).Blood glucose: Compared with the CON group,the blood glucose levels of the DN group were significantly higher at each time point(P<0.05).24-hour Urine protein(24-h Upro): 24 h Upro in DN rats at each time point was higher than that in CON group(P<0.05),and gradually increased with time.Renal function:Blood urea nitrogen(BUN),serum creatinine(Scr)and cystatin C(CysC): BUN levels and cystatin C levels in the DN group at 8 weeks,12 weeks,and 16 weeks were significantly higher than those in the CON group at the same time(P<0.05).At 8th week,Scr values in DN group rats were not significantly different from those in CON group.Scr levels in DN group were significantly higher than those in CON group at 12 and 16 weeks(P<0.05).2)Renal tissue: HE staining: The glomerular size of the CON group was normal at each time point,and the morphology was normal.There was no thickening of the basement membrane and no proliferation of the mesangial stroma.In the DN group,the glomerular volume increased slightly,the basement membrane was slightly thickened,andthe mesangial area was widened at the end of the 8th week.At the end of the 12 th week,the thickening of basement membrane and the proliferation of mesangial matrix were more obvious than those of the 8th week.Obviously,at the end of the 16 th week,the renal tissue was aggravated,the basement membrane was thickened,and segmental sclerosis was visible.PASM staining: There was no obvious abnormality at each time point in the CON group.At each time point,a little black collagen deposition was observed in the mesangial area,and the basement membrane was not thickened.The deposition of black collagen in the mesangial area was significantly increased in the DN group at the 8th weekend compared with the CON group at the same time point,with thickening of the basement membrane(P<0.05).Mesangial matrix proliferation and basement membrane were more obvious at the end of the 12 th week.(P<0.05);At the end of the 16 th week,the mesangial matrix of rats in DN group showed marked hyperplasia,had diffuse or nodular distribution,and the basement membrane was further thickened(P<0.05).3)Immunohistochemical staining: in CON group,the expression of BMP4,ALK3,Smad1,Col4,Col1 protein was scattered in various time points,and there was no significant change with time.The expression of BMP4,ALK3,Smad1,Col4,Col1 protein in kidney tissue of DN group was significantly higher than that in CON group at the same time,and gradually increased with time(P<0.05);There was nosignificant change in MMP9 at each time point in the CON group.The expression of MMP9 in the DN group at the 8th week was not significantly different from that in the CON group at the 8th week,while the MMP9 expression at the 12 th and 16 th week was lower than that in the DN group(P<0.05).2.Cell experiments: 1)Immunofluorescence staining of rat mesangial cells: The green fluorescence of mesangial cells in LG group and OP group was weak,and there was no significant change with time.The expression of BMP4,Smad1 and Col4 in the HG group was higher than that in the LG group and the OP group at each time point.Compared with the LG group and the OP group,the expression of BMP4,Smad1,Col4 was significantly enhanced at the 48 th hour.According to the above results,48 hours were used as the optimal time point for follow-up experiments.The cell proliferation was detected by CCK8: the proliferation of HG group at 48 hours was higher than that of LG group and OP group,but there was no significant difference(P>0.05).2)The optimal transfection conditions have been screened out through pre-experiments,and the related protein expression in each group was detected by westerm blot.Compared with LG group,the expression of BMP4,ALK3,Smad1,Col4,Col1 in HG group increased significantly(P<0.05),while the expression of MMP9 decreased(P<0.05).Compared with HG group,the expression of BMP4,ALK3,Smad1,Col4,Col1 in BMP4-siRNA group was significantly decreased(P<0.05),and theexpression of MMP9 was increased(P<0.05).3)The optimal concentration of NOGGIN protein was selected by pre-experiment.Westerm blot was used to detect the expression of related proteins in each group.Compared with LG group,the expression of ALK3,Smad1,Col4,Col1 in HG group was significantly increased(P<0.05),the expression of MMP9 was decreased(P<0.05).The expression of ALK3,Smad1,Col4,Col1 in NOGGIN group was lower than that in HG group(P<0.05),and the expression of MMP9 was increased(P<0.05).Conclusions:1.The activation of BMP4/Smad1 signaling pathway occurs in the mesangial matrix hyperplasia of diabetic nephropathy;2.The blocking of BMP4/Smad1 signaling pathway can significantly reduce the mesangial matrix hyperplasia of diabetic nephropathy.