| Background:Diabetic nephropathy(DN)is an important complication of diabetes and the leading cause of end-stage renal disease(ESRD)in the world.Research on the mechanisms and treatment strategies of DN is important to improve the quality of life of patients.Tubular injury is a key link in the occurrence and development of DN,and it is also the pathological basis for the progression of DN tubulointerstitial fibrosis.Therefore,exploring the mechanism of renal tubular injury plays an important role in the research of DN.It has important clinical significance for determining early intervention targets and improving the long-term prognosis of DN.Lipoxygenase(LO)is a family of enzymes that insert oxygen molecules into polyunsaturated fatty acids such as arachidonic acid and linoleic acid.It is classified into 5-,8-,12-and 15-LO according to the position of oxygen inserted in arachidonic acid.12-LO is closely related to the pathogenesis of DN.Studies have shown that the levels of 12-LO m RNA and protein increase in glucose-stimulated mesangial cells(MCs)and DN animal models.12-LO and its downstream product12(S)-HETE can directly activate p38 MAPK,induce cell hypertrophy and the expression of Fibronectin(FN)in the extracellular matrix(ECM).LO activation can directly cause oxidative stress through the production of superoxide and mediate DN cell damage.The current research on 12-LO and DN is mainly focused on the mechanism of 12-LO's damage to the glomerulus,while the effect of 12-LO on the renal tubules of DN and its mechanism are still unclear.Renalase(RNLS)is an amine oxidase mainly produced and secreted by renal tubular epithelial cells.It can degrade catecholamines in the circulation and regulate heart function and blood pressure.In acute and chronic renal tubular injury,the expression of RNLS in the renal tissue decreases significantly.The latest research shows that RNLS has cytokine-like effects in addition to enzyme activity.Exogenous RNLS reduces the level of activated ERK phosphorylation to improve renal tubulointerstitial fibrosis in mice with chronic kidney disease(CKD)induced by unilateral ureteral obstruction(UUO).Another study found that RNLS and its de-enzyme active subtype RP200 quickly inhibited the phosphorylation of p38 MAPK and at the same time inhibited JNK to slow down the toxic injury of human proximal tubular epithelial cells induced by cisplatin or hydrogen peroxide.However,the expression changes of RNLS in DN renal tubules and the mechanism of their effect on DN renal tubule damage still need to be further explored.Many studies have shown that the increase of renal p38 MAPK activity is related to the progression of DN.In animal models of diabetes,the activity of p38 MAPK in the glomeruli and tubules increases rapidly under the stimulation of hyperglycemia.The latest study found that p38 MAPK activation is related to diabetic renal tubular hypertrophy and interstitial fibrosis,and the expression of p38 MAPK is mainly increased in renal tubular epithelial cells.P38 MAPK activation induces the production of angiotensinogen in rat renal tubular cells,stimulates TGF-? to induce the accumulation of fibronectin in renal interstitial fibroblasts,and increases the expression of TGF-? in renal tubular cells.At the same time,phosphorylation of p38 MAPK induces an increase of the apoptosis marker caspase-3in renal tubules.These findings suggest that the activation of p38 MAPK and its downstream signaling pathway is one of the important mechanisms of DN renal tubular injury.In summary,this study puts forward the hypothesis: 12-LO expression increases in DN renal tubules,while down-regulating the expression of RNLS in DN renal tubules,and then activates the p38 MAPK signaling pathway to aggravate renal tubular epithelial cell damage and apoptosis.Methods:(1)Rat renal tubular epithelial cells were divided into 2 groups: control group and high glucose stimulation group(HG).Cells were collected after 24 h and 48 h respectively.Western blot was used to detect the levels of RNLS,BCL-2,BAX,and caspase-3.(2)Rat renal tubular epithelial cells were divided into 2 groups: control group and high glucose stimulation group(HG).Cells were collected after 24 h and 48 h respectively.Western blot was used to detect the levels of RNLS,p38 MAPK,p-p38 MAPK,ERK,p-ERK.(3)Rat renal tubular epithelial cells were divided into 3 groups: control group,high glucose stimulation group(HG),intracellular overexpression of renalase plus high glucose stimulation group(RNLS+HG group).Cells were collected after 48 h.Western blot was used to detect the levels of RNLS,BCL-2,BAX,caspase-3,p38 MAPK,and p-p38 MAPK.(4)High-dose streptozotocin(STZ)(65 mg/kg,intraperitoneal injection)was used to induce type 1 diabetes rat model,with fasting blood glucose> 16.7 mmol/L as the success criterion for type 1 DM modeling.The increase of 24 h-urinary excretions of albumin indicates that the experimental model rats meet the characteristics of diabetic nephropathy.After successful induction of type 1 diabetes model,it was randomly divided into 2 groups: type 1 diabetic nephropathy group(T1DN)and CDC(Cinnamyl-3,4-dihydroxy-?-cynanocinnamate,12-LO inhibitor,subcutaneous injection in the leg,8 mg/kg/d,3 times/week)treatment group(DN+CDC).Rats with regular normal diet served as control group.The rats were sacrificed 8 weeks later,and the rat kidneys were extracted.During the experiment,the rats' fasting blood glucose was monitored,and urine was collected for 24 hours.To assess the damage of glomeruli and podocytes,quantitative reverse transcription polymerase chain reaction(RT-q PCR)was used to detect the expression levels of Podocin and Nephrin,the marker proteins of podocytes.(5)In order to prove that 12-LO is involved in DN renal tubular injury and the apoptosis of renal tubular epithelial cells,the rat kidney tissues of the above 3groups were extracted,and periodic acid-Schiff(PAS)staining was used to observe the glomerular and renal tubular damage.Td T-mediated d UTP Nick-End Labeling(TUNEL)staining was used to observe the apoptosis of renal tubular epithelial cells.Masson staining was used to observe the renal tubular interstitial fibrosis.Immunohistochemistry(IHC)was used to observe the expression level of 12-LO in renal tubular tissues,while RT-q PCR detected the m RNA expression of 12-LO,BAX,and BCL-2,and Western blot detected 12-LO,p-p38 MAPK,BAX,BCL-2,caspase-3protein levels.(6)Based on the above experiments,in order to prove that 12-LO changes cause RNLS changes,and then aggravate renal tubular epithelial cell damage,the kidney tissues of the above three groups of rats were extracted.IHC was used to observe the expression level of RNLS in renal tubular tissues.RT-q PCR and western blot were used to detect the expression of RNLS m RNA and protein in each group.(7)Based on the above experiments,in order to further prove that RNLS reduces renal tubular epithelial cell damage through the p38 MAPK pathway,the kidney tissues of the above three groups of rats were extracted,and the expression level of p-p38 MAPK in renal tubular tissues was observed by IHC.Western blot was used to detect p-p38 MAPK protein level.Enzyme-linked immunosorbent assay(ELISA)was used to detect urine albumin level,western blot,RT-q PCR to detect related protein and m RNA expression,IHC to observe renal tubular tissue 12-LO,RNLS,p38 MAPK expression,PAS staining was used to observe glomerular and tubular damage,Masson staining to observe tubular interstitial fibrosis,TUNEL staining to observe renal tubular epithelial cell apoptosis.Results:(1)Compared with the control group,the blood glucose,kidney weight/body weight and 24 h urine albumin of rats in the DN group increased significantly(P<0.05),and the expression levels of podocyte marker proteins Podocin and Nephrin increased significantly(P<0.05).The glomerulus volume increased significantly.These changes were in line with typical changes in DN.(2)The 12-LO transcription and protein expression levels in the kidney tissue of rats in the DN group were significantly higher than those in the control group(P<0.05).IHC staining also showed that compared with the control group,the expression of 12-LO in the renal tubules of the DN group was significantly increased.PAS staining showed that the structure of the glomeruli and tubules in the control group were normal,and the capillaries were clear.In the DN group,the lumen of the renal tubules was narrow,the tubules were swollen and partially atrophied,and the number of epithelial cells decreased;Masson staining showed that compared with the control group,in the DN group the renal tubule lumen was narrowed,renal tubular epithelial cells atrophied,and interstitial collagen fibers increased significantly.(3)CDC mainly inhibited the enzyme activity of 12-LO,and the protein level of12-LO in the kidney tissue of the DN+CDC group was not significantly different from that of the DN group.IHC staining also showed that the expression level of renal tubular 12-LO in the DN+CDC group was not significantly different from that of the DN group.PAS staining and Masson staining showed that compared with the DN group,the renal tubule stenosis,swelling,and atrophy of the DN+CDC group were significantly reduced,and the interstitial exudation and collagen fibers were significantly reduced.(4)The expression of RNLS in the kidney tissue of the DN group was significantly lower than that of the control group(P<0.05).IHC staining showed that the RNLS protein level in the renal tubules of the DN group was significantly lower than that of the control group.The expression of RNLS protein showed a downward trend 24 hours after high glucose stimulated in rat renal tubular epithelial cells.After48 hours of maintenance,the level of RNLS protein decreased significantly compared with the control group(P<0.05).(5)The expression of RNLS which reduced in the kidney tissue of the DN group was significantly higher in the DN+CDC group(P<0.05).The RNLS transcription level which decreased in kidney tissue of DN group was significantly up-regulated in DN+CDC group(P<0.05).IHC staining showed that the 12-LO level in the renal tubules of the DN group was significantly increased,and the RNLS expression was low,while the RNLS protein level in the renal tubules of the DN+CDC group was significantly increased after 8 weeks of CDC administration.(6)The protein level of p38 phosphorylation product p-p38 MAPK in the DN group was significantly higher than that of the control group(P<0.05).The p-p38 MAPK in the renal tissue of the DN+CDC group was significantly lower than that of the DN after the 12-LO inhibitor CDC was given for 8 weeks(P<0.05).The phosphorylation level of ERK signaling pathway did not change significantly in the DN group and DN+CDC group.IHC staining showed that the p-p38 MAPK protein level in the renal tubules of the DN group was significantly increased.The p-p38 MAPK protein level which was significantly up-regulated in the DN group wassignificantly reduced in the DN+CDC group,while the p-ERK protein level showed no significant difference between the 3 groups.(7)The expression of apoptosis-related proteins BAX and Cleaved Caspase-3 in the kidney tissue of rats in the DN group was significantly higher than that of the control group(P<0.05),and the expression of anti-apoptotic protein BCL-2 was decreased(P<0.05).The increase of BAX and Cleaved Caspase-3 in the DN+CDC group was significantly reduced,and the expression of BCL-2 showed a rebound compared with DN group(P<0.05).The BAX/BCL-2 transcription ratio in the kidney tissue of the DN group was significantly higher than that of the control group(P<0.05),while the DN+CDC group was significantly lower than the DN group(P<0.05).TUNEL staining showed that the number of brown-yellow nuclei in the renal tubules of the DN group was significantly higher than that of the control group,while the number of brown-yellow nuclei staining in the renal tubules of the DN+CDC group was significantly less than that of the DN group after 8 weeks of CDC administration in DN rats.(8)The level of RNLS protein under HG stimulation was significantly lower than that of the control group(P<0.05),while the level of p-p38 MAPK was significantly increased(P<0.05),and the level of p-ERK protein was not significantly different from that of the control group.The expression of apoptosis-related proteins BAX and Cleaved Caspase-3 in renal tubular epithelial cells of rats in the DN group was significantly higher than that of the control group,and the expression of anti-apoptotic protein BCL-2 was reduced,both of which were significantly different than those of the control group(P<0.05).(9)The increase of p-p38 MAPK,apoptosis-related proteins BAX and Cleaved Caspase-3 levels accompanying the low expression of RNLS induced by DN state was significantly reduced in the RNLS high expression HG stimulation group(P<0.05).The down-regulated expression of anti-apoptotic protein BCL-2 in the HGgroup was significantly increased in the RNLS high expression HG stimulation group(P<0.05).Conclusions:(1)12-LO is involved in DN renal tubular injury,and the up-regulated expression of 12-LO in DN renal tubules is closely related to renal tubular injury and cell apoptosis.(2)12-LO reduces the level of RNLS protein in DN renal tubules and inhibiting the activity of 12-LO can reverse the low expression of RNLS.(3)RNLS/p38 MAPK signaling pathway is involved in 12-LO regulation of DN renal tubular damage and cell apoptosis. |
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