Study On The Correlation Of Renal Excretion Between Several Drugs And Creatinine In Different Degrees Of Acute Kidney Injury

ObjectivesThe kidney is critically involved in the excretion of various endogenous and exogenous substances.The renal injury usually alters the function of glomerular filtration and tubular transport.For drugs metabolized and eliminated by renal,evaluating the changes of excretory pathway in varying functional states of the kidney and assessing their influence on pharmacokinetics are important to guide rational drug use for clinical patients.Creatinine levels are commonly used as the permanent standard for the evaluation of renal function.However,the changes in the renal excretion pathway and the reference value of creatinine for drug dose adjustment in different degrees of acute kidney injury（AKI）have not been fully clarified.Therefore,this study used animal experiments to explore the alteration of renal excretion pathway and the correlation of renal excretion between several drugs and creatinine in different degrees of AKI.Methods（1）The establishment of the AKI modelRats were randomly divided into 5 groups（n=6）:the Control group,the gentamicin（GEN）（25）group,the GEN（50）group,the GEN（75）group,and the GEN（100）group.Gentamicin sulfate injection at 25 mg/kg,50 mg/kg,75 mg/kg,and 100mg/kg were injected intraperitoneally for 7 consecutive days in the GEN（25）,GEN（50）,GEN（75）,and GEN（100）groups,respectively,while the Control group was administered with an equal volume of physiological saline.The rats were fasted for 12h,and arterial serum samples were collected after anhydrous ether anesthesia on the 8th day.Serum creatinine（Scr）,blood urea nitrogen（BUN）,uric acid（UA）,and serum cystatin C（Cys C）levels were measured by the automatic biochemical analyzer.The kidney tissues of rats in each group were collected.Part of them was fixed in 4%paraformaldehyde buffer for histopathological analysis.The other parts of the kidney tissue were stored at-80°C until analysis to determine the protein expression levels of uptake transporters,including r OAT1,r OAT2,r OAT3,and r OCT2 by using a western blotting（WB）assay.（2）Effects of different degrees of AKI on pharmacokinetics in ratsRats were divided randomly into 5 groups（n=5）:the Control group,the GEN（25）group,the GEN（50）group,the GEN（75）group,and the GEN（100）group.The administration method is the same as shown in（1）.The mixed exogenous substrate solution,including furosemide（5 mg/kg）,metformin（20 mg/kg）,p‐aminohippurate（20mg/kg）,5-fluorouracil（20 mg/kg）,and inulin（100 mg/kg）was injected into the tail vein of model animals with different degrees of AKI after fasting for 12 hours on the8th day.Urine samples were collected during 0-2,2-4,4-6,6-8,8-10,and 10-12 h,respectively.The net volume of each urine sample was calculated.The concentration of inulin in urine samples was determined by the enzyme labeling method after processing accordingly.The concentrations of metformin in urine were determined by high-performance liquid chromatography（HPLC）.The concentrations of furosemide,p‐aminohippurate,5-fluorouracil and creatinine in urine were determined by liquid chromatography-mass spectrometry（LC-MS）.The cumulative urinary excretion（Ae）of each drug at different degrees of AKI was calculated.The group setting and administration were as same as shown in（1）（n=5）,the cocktail renal markers,except for inulin,were administered through intravenous injection.Each rat’s arterial blood was collected via femoral artery intubation at 5,10,15,30,60,90,120,240,360,480,and 600 min,respectively.After processing accordingly,the concentrations of metformin in serum were determined by HPLC.The concentrations of furosemide,p-aminohippurate,5-fluorouracil,and creatinine in the serum were determined by LC-MS.Drug and Statistics 2.0（DAS 2.0）was used for pharmacokinetic data processing.（3）The correlation of AUC,CL,and Ae between serum creatinine and substrates for renal transporters in different degrees of AKIThe Pearson correlation coefficient was used to analyze the correlation between the area under the concentration-time curve（AUC）,clearance rate（CL）,and Ae of creatinine and substrates.（4）The correlation of AUC,Ae between creatinine and metformin in different degrees of AKIRats were randomly divided into 4 groups（n=6）:the Control group,the GEN（25）group,the GEN（50）group,and the GEN（75）group.The administration method is the same as shown in（1）.After establishing the AKI model,metformin（10 mg/kg）was injected through the tail vein.Urine and serum samples of rats were collected to evaluate the correlation between creatinine and metformin in AUC and Ae.Results（1）The evaluation of rat models with different degrees of AKIAs compared to the Control group,levels of serum biochemical indexes and ATN scores in the GEN（25）group did not show any significant differences.The level of Cys C,Scr,BUN,UA and ATN scores increased significantly in the GEN（50）group,the GEN（75）group,and the GEN（100）group,which indicated the amplitude increased with the risen in GEN dose.These results suggest that gentamicin in diverse doses can cause different degrees of AKI in rats,and the severity of the injury is proportional to the dose in the range of 25-100 mg/kg.（2）The effects of different degrees of AKI on the expression of transportersCompared with the Control group,the level of r OAT2 in the GEN（25）group was significantly reduced.The expressions of r OAT1,r OAT2,and r OAT3 in the GEN（50）,GEN（75）and GEN（100）groups were significantly lower than those in the Control group.The level of r OCT2 had no significant change in mild to moderate renal injury,but decreased in severe renal injury.（3）The effects of different degrees of AKI on pharmacokinetics in ratsAs compared with the Control group,the Ae of metformin in GEN（25）group increased significantly at 4,8,and 10 h,the Ae of inulin increased significantly at 2,4,6,8,and 10 h,and the Ae of creatinine tended to increase.In the GEN（50）group,the Ae of metformin showed an increasing trend,and the Ae of inulin increased significantly at 2,4,6,8,and 10 h when compared to the Control group.Additionally,in the GEN（75）group,the Ae of 5-fluorouracil and furosemide at 2,4,6,8,and 10 h and the Ae of metformin at 2 h were significantly lower than those of the Control group.In the GEN（100）group,the Ae of 5-fluorouracil,furosemide and metformin at 2,4,6,8,10 h,the Ae of inulin and p‐aminohippurate at 2 h,the Ae of creatinine at 2,8,10 h were significantly lower than those of Control group.Compared with the Control group,the serum creatinine concentration in the GEN（75）group increased significantly at 5,15,30,60,90,120,240,360,480,and 600 min,and that in the GEN（100）group at 5,10,15,30,60,90,120,240,360,480,and 600min was observably higher.Compared with the Control group,there were no remarkable changes in the pharmacokinetic parameters of serum creatinine in the GEN（25）group.the area under the concentration-time curve（AUC）and terminal elimination half-life(t_1/2)of serum creatinine in the GEN（50）group were observably higher than those in the Control group.The AUC,mean residence time（MRT）,and t_1/2 of serum creatinine in the GEN（75）and GEN（100）group were markedly increased compared with the Control group,while the CL was dramatically reduced.Furthermore,the terminal elimination rate（Ke）and volume of distribution（V1）of creatinine in the GEN（100）group were significantly lower than those in the Control group.As compared with the Control group,there was no significant difference in serum concentration and pharmacokinetic parameters of substrates in the GEN（25）group.In the GEN（50）group,the serum concentration of 5-fluorouracil,furosemide,and p‐aminohippurate increased significantly,the MRT,t_1/2,and V1 of furosemide were dramatically lower than the Control group,and the Ke was significantly increased.However,the pharmacokinetic parameters of 5-fluorouracil,metformin and p‐aminohippurate were not changed in the GEN（50）group.The serum concentrations of5-fluorouracil,furosemide,metformin and p‐aminohippurate in the GEN（75）and GEN（100）groups increased in a dose-dependent manner,while the AUC and MRT of 5-fluorouracil,metformin and p‐aminohippurate increased notably and the CL decreased remarkably.AUC of furosemide was observably increased,and CL had no significant change.（4）The correlation of AUC,CL,Ae between serum creatinine and renal transporter substrates in different degrees of AKIThere was no significant linear correlativity in AUC between serum creatinine and5-fluorouracil,furosemide in rats with different degrees of AKI（P>0.05）,and there was a weakly linear correlativity in AUC between serum creatinine and metformin,p-aminohippurate（P<0.05,0.00.05）and there was a weak correlation in CL between serum creatinine and 5-fluorouracil,p-aminohippurate（P<0.05,0.0