Roles Of Organic Anion Transporters (Oats) And Chymase/Ang? In Chronic Kidney Injury Induced By Adefovir Or Acute Kidney Injury Induced By Methotrexate

AimsDrug-induced kidney injury has become the most common disease induced by drugs in the clinic.The pathological changes of drug-induced kidney injury are renal tubular injury,renal interstitial injury or tubulointerstitial injury.It is of great significance to investigate the mechanism of drug-induced kidney injury for its clinical diagnosis and treatment.Transporters and renal micro-blood flow have important effects on the clearance of drugs in the kidney and are closely related to renal injury.Chymase/Ang? is important factor affecting renal micro-blood flow.Therefore,roles of Oats and chymase/Ang? in chronic kidney injury induced by adefovir or acute kidney injury induced by methotrexate were investigated in vivo or vitro.Methods(1)Rats were randomly divided into control group,adefovir(ADV)group,adefovir + probenecid(ADV+PR)group,adefovir + probenecid + sodium cromoglycate(ADV+PR+SCG)group and adefovir + probenecid + valsartan(ADV+PR+VAL)group.Rats in control group were treated with 0.5% CMC-Na(1 mL/100 g,i.g.)+ saline(0.25 mL/100 g,i.p.).Rats of other groups were treated with ADV(10 mg/kg,i.g.)+ saline(0.25 mL/100 g,i.p.),ADV(10 mg/kg,i.g.)+ PR(30 mg/kg,i.g.)+ saline(0.25 mL/100 g,i.p.),ADV(10 mg/kg,i.g.)+ PR(30 mg/kg,i.g.)+ SCG(25 mg/kg,i.p.),ADV(10 mg/kg,i.g.)+ PR(30 mg/kg,i.g.)+ VAL(50 mg/kg,i.g.)+ saline(0.25 mL/100 g,i.p.),respectively.Drugs were administrated once a day for 2 weeks or 4 weeks.The renal interstitial dialysis fluids were collected for 1 h by microdialysis technology at 2 h after the last administration.Blood was collected via the abdominal aorta and then the kidneys were removed.The concentrations of ADV in blood,kidney tissues and renal interstitium were determined by LC-MS/MS.The concentrations of Ang? in plasma and renal interstitium were measured by ELISA.Creatinine,urea nitrogen and cystatin C in serum were determined by fully automatic biochemical analyser.Renal interstitial mast cells,kidney pathology and renal microvascular were observed by toluidine blue staining,hematoxylin-eosin staining(HE)and CD31-immunofluorescence,respectively.Western blotting was used to evaluate the expression of Oat1,Oat3,Mrp2 and Mrp4 in kidney.(2)The chymase release was measured by ELISA in RBL-2H3 cells after intervention by ADV.(3)Rats were randomly divided into control group,methotrexate(MTX)group,methotrexate + indometacin(MTX+INDO)group and indometacin(INDO)group.Rats in control group were treated with saline(0.25 mL/100 g,i.p.)+ purified water(1 mL/100 g,i.g.).Rats of other groups were administrated with MTX(360 mg/kg,i.p.)+ purified water(1 mL/100 g,i.g.),MTX(360 mg/kg,i.p.)+ INDO(10 mg/kg,i.g.),INDO(10 mg/kg,i.g.)+ saline(0.25 mL/100 g,i.p.),respectively.Urine was collected for 4 h at 48 h after single administration.Blood was collected via the abdominal aorta at 72 h after the administration and then the kidneys were removed.The concentration of MTX in kidney was measured by HPLC.The concentration of Ang? in plasma was measured by ELISA.Creatinine and urea nitrogen in serum,cystatin C and albumin in urine were determined by fully automatic biochemical analyser.Renal interstitial mast cells and kidney pathology were observed by toluidine blue staining and HE,respectively.Western blotting was used to evaluate the expression of Oat1,Oat3,Mrp2 and Mrp4 in kidney.(4)Rats were grouped and administrated according to the same method in item(3).Renal interstitial dialysis fluid was collected for 1 h at 72 h after single administration.The concentration of Ang? in renal interstitium was measured by ELISA.(5)The chymase release was measured by ELISA in RBL-2H3 cells after incubation with MTX or INDO.(6)Rats were randomly divided into control group,MTX group,MTX+SCG group,MTX+INDO group,MTX+INDO+SCG group,INDO group and INDO+SCG group.Rats in control group were treated with saline(0.25 mL/100 g,i.p.)+ purified water(1 mL/100 g,i.g.).Rats of other groups were administrated with MTX(360 mg/kg,i.p.)+ purified water(1 mL/100 g,i.g.),MTX(360 mg/kg,i.p.)+ purified water(1 mL/100 g,i.g.)+ SCG(50 mg/kg,i.p.),MTX(360 mg/kg,i.p.)+ INDO(10 mg/kg,i.g.),MTX(360 mg/kg,i.p.)+ INDO(10 mg/kg,i.g.)+ SCG(50 mg/kg,i.p.),INDO(10 mg/kg,i.g.)+ saline(0.25 mL/100 g,i.p.),INDO(10 mg/kg,i.g.)+ SCG(50 mg/kg,i.p.),respectively.Urine was collected for 4 h at 48 h after single administration.Blood was collected via the abdominal aorta at 72 h after administration.After the samples were processed,creatinine and urea nitrogen in serum,cystatin C and albumin in urine were determined by fully automatic biochemical analyser.Results(1)After 2 weeks of administration in rats,the amount of mast cells in renal interstitium of the ADV+PR group was significantly increased compared with the control group(P<0.05).Compared with the control group,renal microvessels were severely lost and the concentration of Ang? in plasma was significantly increased in the ADV+PR group(P<0.05).The concentration of Ang? in renal interstitium of the ADV+PR group was increased but no significant difference compared with the control group.Compared with the ADV+PR group,mast cells in renal interstitium of the ADV+PR+SCG group and the ADV+PR+VAL group were significantly reduced(P<0.05).The concentrations of Ang? in renal interstitium and plasma of the ADV+PR+SCG group were decreased but no significant difference compared with the ADV+PR group.There was no significant change in the concentrations of Ang? in plasma and renal interstitium in the ADV+PR+VAL group compared with the ADV+PR group.Compared with the ADV+PR group,the loss of renal microvessels were relieved and the fibrosis were reduced in the ADV+PR+SCG group and the ADV+PR+VAL group.Creatinine,cystatin C and urea nitrogen in serum showed no significant difference in the ADV+PR+SCG group and the ADV+PR+VAL group compared with the ADV+PR group.The concentrations of ADV in the blood and kidneys were significantly reduced in the ADV+PR+SCG group and the ADV+PR+VAL group(P<0.05/0.01),and which in the renal interstitial dialysate fluids did not change significantly compared with the ADV+PR group.In addition,compared with the ADV+PR group,the expression of Oat1,Mrp2 and Mrp4 in the kidney tissues of the ADV+PR+SCG group did not significantly change,and the expression of Oat3 was significantly increased(P<0.05).In the ADV+PR+VAL group,the expression of Oat1 and Oat3 were significantly increased(P<0.05/0.01)compared with the ADV+PR group.Compared with the ADV+PR group,Mrp4 expression increased but no significant difference and there was no significant change in Mrp2 protein expression in the ADV+PR+VAL group.After 4 weeks of administration in rats,the amount of mast cells in renal interstitium in the ADV+PR group was significantly increased compared with the control group(P<0.05).Compared with the control group,renal microvessels were severely lost and the concentrations of Ang? in blood and renal interstitium were greatly significantly increased in the ADV+PR group(P<0.01).Compared with the ADV+PR group,the amount of mast cells in renal interstitium in the ADV+PR+SCG group was significantly reduced(P<0.05).In the ADV+PR+SCG group,the concentrations of Ang ? in renal interstitium and plasma were extremely significantly reduced compared with the ADV+PR group(P<0.01).Besides,renal microvascular loss was relieved and renal interstitial fibrosis was reduced.Compared with the ADV+PR group,creatinine and cystatin C in serum were significantly decreased in the ADV+PR+SCG group(P<0.05)and there was no significant difference in serum urea nitrogen.The concentrations of ADV in kidney,renal interstitial dialysis fluids and blood were significantly reduced in the ADV+PR+SCG group compared with the ADV+PR group(P<0.05/0.01).In addition,the expression of Oat1 and Oat3 were greatly significantly increased in the ADV+PR+SCG group compared with the ADV+PR group(P<0.01).Compared with the ADV+PR group,the amount of renal interstitial mast cells in the ADV+PR+VAL group was significantly reduced(P<0.05).There was no significant difference in the concentrations of Ang? in plasma and renal interstitium between the ADV+PR+VAL group and the ADV+PR group.In the ADV+PR+VAL group,renal microvascular loss was relieved and renal interstitial fibrosis was reduced compared with the the ADV+PR group.No significant difference observed in serum creatinine,urea nitrogen and cystatin C between the ADV+PR+VAL group and the ADV+PR group.The concentrations of ADV in blood,kidney and renal interstitial dialysis fluids were significantly reduced in the ADV+PR+SCG group compared with the ADV+PR group(P<0.05/0.01).Besides,the expression of Oat1,Oat3 and Mrp4 were significantly increased in the ADV+PR+SCG group compared with the ADV+PR group(P<0.05/0.01).(2)Chymase release of RBL-2H3 was time and concentration dependent on ADV.(3)After a single administration,the volume of urine was significantly reduced and the urea nitrogen in the serum was significantly increased in the MTX+INDO group compared with the control group and the MTX group(P<0.05/0.01).Renal tubular atrophy was obvious and renal interstitial mast cells were significantly increased in the MTX+INDO group(P<0.05).Compared with the MTX group,the concentration of MTX in kidney was significantly increased in the MTX+INDO group(P<0.05).Compared with the control group,the concentrations of Ang? in the renal interstitium of the MTX+INDO group and the INDO group were greatly significantly increased(P<0.01),while which of the MTX group did not significantly change.Compared with the MTX+INDO group,the concentration of Ang? in the renal interstitium of the INDO group was extremely significantly increased(P<0.01).Compared with the control group and the INDO group,the expression of Oat1,Oat3 and Mrp4 increased significantly in the MTX+INDO group,while the expression of Mrp2 decreased significantly(P<0.05/0.01).(4)Chymase release of RBL-2H3 was time and concentration dependent on MTX or INDO.(5)Compared with the MTX+INDO group,the volume of urine,creatinine and urea nitrogen in serum,albumin and cystatin C in urine showed no significant difference in the MTX+INDO + SCG group.Conclusions(1)The long-term accumulation of ADV in renal interstitium caused by inhibition of Oats could promote the recruitment of renal interstitial mast cells,accelerate chymase release,and then cause increased production of renal Ang?,lead to renal microvascular loss and might cause tissue ischemia and hypoxia and finally induced renal injury.Mast cells membrane stabilizer SCG and Ang? receptor antagonist VAL significantly improved renal microvascular loss by inhibiting chymase release of mast cells and antagonizing the physiological effects of Ang?,respectively.They also increased the expression of Oat1,Oat3 as well as Mrp4,accelerated the clearance of ADV in the kidney,effectively prevented the accumulation of ADV in renal interstitiuml and alleviated renal damage caused by ADV.(2)After MTX combined with the non-steroidal anti-inflammatory drug INDO,the expression of uptake transporters Oat1 and Oat3 in kidney increased and that of the efflux transporter Mrp2 decreased,which indicated that the MTX might accumulate in renal tubular epithelial cells instead of renal interstitium and caused renal tubular injury.In addition,coadministration of SCG did not influence the biochemical parameters in each group compared with no SCG group,which indicated that chymase/Ang?was not the key factor in acute renal injury induced by MTX.