Di (2-Ethylhexyl) Phthalate (DEHP) Mediates Oxidative Stress And Activates P38MAPK/NF-κB To Exacerbate Diabetic Nephropathy

The plasticizer di(2-ethylhexyl)phthalate(DEHP)is used in the manufacture of polyethylene polymers.Several studies in epidemiology and toxicology suggest that long-term exposure to DEHP may be harmful to the body,including nephrotoxicity,and exacerbate kidney damage in cases of underlying disease.There has been a dramatic increase in the diabetic population worldwide,with diabetic nephropathy(DN)as one of the most serious complications,and exposure to DEHP may exacerbate kidney damage.However,studies on the toxicity of DEHP in DN have been rarely reported.In this study,we observed the toxic effects of DEHP on a high-fat diet(HFD)combined with streptozotocin(STZ)-induced diabetic nephropathy and explored the possible mechanisms of toxicity in depth.The results showed that DEHP exposure significantly promoted renal inflammation and oxidative stress damage in mice,increased P-p38 and P-p65 protein levels,and exacerbated the loss of podocin,a characteristic protein of podocytes.Also,DEHP exposure increased levels of oxidative stress and secretion of inflammatory factors.The same findings were observed in vitro after stimulation of podocytes with high glucose(30 mmol/L)and exposure to DEHP.Our results suggest that DEHP may exacerbate DN by mediating oxidative stress and activating p38MAPK/NF-κB.OBJECTIVE:The effect of DEHP on the pathological process of diabetic nephropathy in mice was explored using HFD combined with STZ to induce a mouse model of diabetic nephropathy.The role of the signaling pathway p38MAPK/NF-κB in diabetic nephropathy was explored in an in vitro experiment using podocytes.METHODS:In an in vivo experiment,mice were given HFD daily and fed for four weeks,and a model of diabetic nephropathy was induced by intraperitoneal injection of STZ at week five.Different doses of DEHP(0.1,10,1000 mg/kg)were given daily by gavage to the model group,while the control group was given 1000 mg/kg DEHP by gavage.Feeding was continued for five weeks.After five weeks,24 h urine was collected and the mice were executed according to animal ethics.The kidney tissues were stained with HE and PAS to observe the pathological damage of DEHP on the kidneys of mice under diabetic nephropathy;ELISA was used to measure the levels of tumor necrosis factor(TNF-α)and interleukin 6(IL-6)as well as malondialdehyde(MDA),superoxide dismutase(SOD)and reduced glutathione(GSH)in the kidney tissues;specialized kits were used to measure the levels of creatinine and urea in the mice The kits are used to detect creatinine and urea nitrogen(BUN)levels in serum and creatinine and urine protein in 24 h urine;laser confocal method to detect the expression of podocin protein in mouse kidney tissues;immunohistochemistry to detect the expression of NF-κB in mouse kidney tissues;Western blot method to detect the expression of P-p38,P-p65 and podocin protein.In vitro experiments were also divided into six groups: normal group,high glucose group(30 mmol/L),high glucose + DEHP(12.5,50 and 200 μmol/L),and DEHP 200 μmol/L control group.After 24 h of culture,the effects of DEHP on the proliferation and migration ability of podocytes were detected by CCK-8 and Transwell assays;the DCFH-DA probe labeled podocyte ROS,and the effects of DEHP exposure on ROS were detected by flow cytometry;the podocin protein expression in podocytes was detected by immunofluorescence and q RT-PCR;the Western blot assay detected P p38,P-p65 and podocin protein expression in podocytes by Western blot.RESULTS:1.Effects of DEHP on renal index and functional parameters in miceThe kidney index of DN mice increased significantly after DEHP exposure;ELISA kits were used to test the serum and 24 h urine of mice.The results found that medium to high doses of DEHP(10,1000 mg/kg)exposure significantly increased the creatinine and urea nitrogen levels in serum as well as creatinine and urine albumin in the urine.This suggests that DEHP exposure exacerbates renal damage in DN.2.Effect of DEHP on histopathological damage in DN miceThe results of HE staining and PAS staining showed that the normal group had normal tubular structure with neat arrangement and intact glomerular structure.Compared with the normal group,the kidney tissues of the model group showed proliferation of thylakoid cells,proliferation of thylakoid stroma and thickening of glomerular basement membrane.The renal injury was further aggravated by DEHP exposure compared to the model group.Inflammatory cell infiltration occurred in the 10 mg/kg DEHP group and further increased in the 1000 mg/kg DEHP group with periglomerular fibrosis.It is suggested that DEHP accelerates the process of kidney tissue damage.3.Effect of DEHP on the oxidative stress indicators and the levels of TNF-α and IL-6 in the renal tissues of DN miceCompared with normal group,MDA content in model group was significantly increased,while SOD and GSH levels were significantly decreased.After DEHP exposure,medium and high doses of DEHP(10,1000 mg/kg)significantly increased the MDA content and decreased the SOD and GSH levels compared to the model group.The levels of IL-6 and TNF-α were significantly increased in the kidney tissues of the model group compared to the normal group,and the levels of IL-6 and TNF-α were significantly increased in the model group after DEHP exposure.Notably,levels were also increased to some extent in the low-dose DEHP(0.1 mg/kg)group.It is suggested that DEHP exposure may exacerbate DN by exacerbating oxidative stress,causing an imbalance between oxidative and antioxidant systems,and promoting the release of proinflammatory factors.4.Effects of DEHP on P-p38,NF-κB and podocin protein expression in kidney tissues of DN miceImmunohistochemical results showed that DEHP exposure significantly increased the expression of NF-κB protein in kidney tissue.According to immunofluorescence analysis,DEHP exposure could significantly reduce the expression of podocin protein,especially at high dose.Based on the results of WB,compared to the normal group,the levels of P-p38 and P-p65 were significantly increased and the levels of podocin were significantly decreased in the kidneys of DN mice.After DEHP exposure,the levels of P-p38 and Pp65 protein expression were significantly increased and the levels of podocin protein expression were significantly decreased in the medium and high doses of DEHP(10 and 1000 mg/kg)groups compared to the model group.Meanwhile,the level of podocin protein expression was also decreased in the 0.1 mg/kg DEHP group.It is suggested that DEHP may activate the p38MAPK/NF-k B pathway to exacerbate the loss of podocin protein and aggravate DN.5.Effect of DEHP on proliferative migration and ROS levels of podocytesCompared to the normal group,cell proliferation viability was significantly reduced after exposure to high glucose,and even more significantly inhibited after exposure to medium to high doses of DEHP(25,50,100,and 200 μmol/L).Analysis of the Transwell results showed that the migration capacity of the high-sugar group was significantly greater than that of the normal group.The number of podocytes in the lower chamber also increased significantly with increasing doses of DEHP exposure compared to the high glucose group.Flow cytometry data showed that high glucosestimulated podocytes produced significantly more ROS than the normal group,and ROS production also increased significantly with DEHP(50,200 μmol/L)exposure compared to the high glucose group.6.Effect of DEHP on P-p38,P-p65 and podocin protein expression in podocytesAnalysis of immunofluorescence and qRT-PCR results revealed that podocin protein levels were significantly decreased in the high glucose-stimulated group compared to the normal group.The podocin protein levels were significantly decreased in the 50 and 200 μmol/L DEHP groups compared to the high glucose group,and no significant changes were observed in the 12.5 μmol/L group.The WB results showed that the overall trend of the in vitro experiments was generally consistent with the in vivo results,with DEHP significantly increasing P-p38 and P-p65 protein levels and significantly decreasing podocin protein levels.CONCLUSIONS1.DEHP exacerbates HFD combined with STZ-induced DN in mice.DEHP elevates renal index,exacerbates renal injury and promotes ROS production in podocytes,which may be related to mechanisms such as promoting inflammatory response and exacerbating oxidative stress.2.DEHP aggravation of DN may be related to mediating oxidative stress and activating P38MAPK/NF-κB signaling pathway.In a series of animal and cell experiments,it was demonstrated that DEHP exposure increased the level of oxidative stress indicators,and up-regulated the protein expressions of pathway related proteins P-p38 and P-p65.It is suggested that DEHP may aggravate DN through P38MAPK/NF-κB.