Study On The Mechanism Of Creatinine Transport And Screening/Validating The Markers Of Organic Cation/Anion Transport Pathway In Renal Tubule

ObjectivesKidney is one of the important organs for drug excretion,and thus accurate evaluation of its excretion ability is vital for precision medication.As a marker of glomerular filtration,creatinine is also the only marker used to evaluate drug excretion ability and determine/adjust drug dosage.Some studies have shown that there may be a renal tubular secretion process of creatinine,but the renal tubular secretion mechanism of creatinine is not clear.Therefore,whether creatinine can be used as an ideal marker for the evaluation of drug renal excretion depends on clarifying the renal tubular secretion mechanism of creatinine.Renal tubular secretion is one of the important ways of drug renal excretion.More than 92%of renal excretory drugs exist in renal tubular secretion.The secretion process of renal tubule is mediated by vector transport pathways composing of uptake and efflux transporters.Organic cation transporter 2（OCT2）and Multidrug and toxin efflux protein 1（MATE1）constitute the transport pathway of cationic drugs,and organic anion transporters（OATs）and Multidrug resistance associated protein 4（MRP4）constitute the transport pathway of anionic drugs.However,there are still no markers to evaluate the function of OCT2-MATE1 and OATs-MRP4 pathway.Therefore,screening the markers of OCT2-MATE1 and OATs-MRP4 pathway provides support for predicting the drug excretion ability of their substrates and determining/adjusting drug dosage.Methods（1）Study on the renal tubular transport mechanism of creatinineCreatinine probe was synthesized chemically and its biological activity was verified.The proteome interacting with creatinine was screened by activity-based protein profiling technology.HEK293T cells overexpressing of human renal tubular OCT2,MATE1,OAT1,OAT2,OAT3,MRP4,OATP4C1,P-gp,PEPT2,URAT1 and OAT4 were constructed,and the uptakes of d3-creatinine were performed in the control and overexpressed cell lines to identify the transporters which could mediate the uptake/efflux of d3-creatinine.Time-and concentration-dependent uptake of d3-creatinine was performed in cells overexpressing OCT2,and K_m and V_max were calculated according to Michaelis equation.In OCT2-overexpressing cells,different concentrations of creatinine and cimetidine（OCT2 inhibitor）were used to competitively inhibit the uptake of d3-creatinine,and the IC50 was calculated respectively.Different dosages of creatinine and cimetidine were used to compete for the renal excretion of d3-creatinine in mice,respectively,and the effect of cimetidine on glomerular filtration rate was measured by FITC sinistrin.Alpha Flod2 was used to predict the interaction between OCT2 and creatinine.E448A,Y362A and G242A stably expressed cell lines were obtained by site mutation,and the uptake of d3-creatinine was verified.（2）Screening and validating OCT2-MATE1 pathway markersThe serum was collected from patients with chronic kidney disease to prepare serum powder as endogenous substances,uptake of which in control and OCT2-overexpressing cells was performed.Metabolomics technology was used to screen the differential metabolism with significantly increased uptake in OCT2-overexpressing cells.Differential metabolites and endogenous substrates of OCT2 or MATE1 reported were used as candidate markers.The candidate markers were verified by uptake test in OCT2-overexpressing cell lines,and uptake of the verified OCT2 substrates in MATE1-overexpressing cell lines was carried out to obtain the common substrates of OCT2 and MATE1 as the potential markers.Potential markers were validated in vitro（4℃/37℃,inhibitors）and in vivo;the specificity was evaluated in cell lines overexpressing OAT1,OAT2,OAT3,MRP4,OATP4C1,P-gp,PEPT2,URAT1 and OAT4,respectively;K_m was calculated through time and concentration dependent uptake in OCT2-and MATE1-overexpressing cell lines,and the endogenous marker of OCT2-MATE1 was obtained.Finally,after administration of cimetidine in rats,the correlation of renal excretion between OCT2-MATE1 pathway marker and its classical substrate of metformin was investigated.（3）Screening and validating OATs-MRP4 pathway markers26 Uremic toxins（UTs）were selected as candidate compounds from the European Uremic Toxin Work Group（EUTox）database and related literatures,and the human serum albumin（HSA）binding rates were measured in vivo/in vitro respectively.UTs uptakes were carried out in cell lines overexpressing OAT1,OAT3 and MRP4,separately.The UTs with HSA binding rate>90%and substrates of OAT1,OAT3 and MRP4 were obtained as candidate markers.Candidate markers were verified by in vitro（4℃/37℃,inhibitors）,in vivo（inhibitors and gene knockout）and Transwell vector transport to obtain potential markers of OATs-MRP4 pathway.The specificity of potential markers was verified in cell lines overexpressing OCT2,MATE1,MATE2-K,OAT2,OATP4C1,P-gp,PEPT2,URAT1 and OAT4,respectively;K_m was calculated by time-and concentration-dependent uptake in OAT1-and OAT3-overexpressing cell lines;t_1/2 of renal clearance was obtained by administering of deuterated potential markers in rats;the effects of different factors on markers were analyzed in patients with different nephropathy,and the markers of OATs-MRP4 pathway were obtained.Finally,the AUC_0-t correlation between OATs-MRP4 pathway markers and substrate of cefmetazole was evaluated after administration of probenecid in rats.Results（1）Study on the renal tubular transport mechanism of creatinine（1）No interaction between creatinine and renal tubular transporters was found in ABPP experiment.（2）The uptake of d3-creatinine was significantly increased in OCT2-and OAT4-overexpressing cell lines（p<0.01）.The K_m and V_max of d3-creatinine uptake mediated by OCT2 were 3.1 m M and 408 pmol/mg protein/min,respectively.In the OCT2-overexpressing cell lines,the IC50 of creatinine on d3-creatinine uptake was 10.3 m M,and that of cimetidine on d3-creatinine uptake was 99.04μM.（3）Different dosages of creatinine did not affect the renal excretion of d3-creatinine in mice（p>0.05）,while cimetidine could significantly reduce the renal excretion of d3-creatinine（p<0.01）and did not affect the glomerular filtration rate（p>0.05）.（4）Computer molecular docking showed that OCT2 residue GLN242 formed a hydrogen bond interaction of 2.5（?）with creatinine,and there may beπ-πforce between TYR362and creatinine.Site mutation experiment demonstrated that TYR362 and GLN242 were the key sites of OCT2 and creatinine interaction.（2）Screening and validating OCT2-MATE1 pathway markers（1）Screening candidate markers:blood samples from chronic kidney disease patients with creatinine level of 443-706μM were collected,and the serum was prepared into powder.Metabolomics analysis was performed after ingestion in control and OCT2-overexpressing cell lines to obtain differential metabolites with significantly increased intake in OCT2-overexpressing cell lines.（2）Screening potential markers:After differential metabolism combined with the reported OCT2/MATE1 substrates,the potential markers thiamine,histamine and 5-hydroxytryptamine mediated by OCT2and MATE1 were obtained.（3）Evaluation of HSA binding rate:The HSA binding rate of thiamine,histamine and 5-hydroxytryptamine was less than 15%.（4）In vitro/in vivo validation:compared with 4℃,the uptake of thiamine,histamine and 5-hydroxytryptamine mediated by OCT2 and MATE1 was increased significantly at 37℃（p<0.01）;Cimetidine significantly inhibited uptake of thiamine,histamine and 5-hydroxytryptamine mediated by OCT2,while pyrimidine significantly inhibited uptake of thiamine and histamine mediated by MATE1.The cumulative urinary excretion of thiamine decreased significantly at 2 h and 4 h after administration of cimetidine via tail vein（p<0.05）;after cimetidine was administered for 7 consecutive days,the level of serum thiamine increased significantly（p<0.05）;after continuous administration of pyrimidine for 7 days,thiamine levels in serum and renal tissue were increased significantly（p<0.01）.（5）Specificity evaluation:there were no significant changes in the uptake or efflux of thiamine and histamine in cell lines overexpressing OAT1,OAT2,OAT3,MRP4,OATP4C1,P-gp,PEPT2,URAT1 and OAT4;the uptake of histamine was increased markedly in OATP4C1-overexpressing cell lines;the uptake of 5-hydroxytryptamine was increased significantly in OAT1-and OAT4-overexpressing cell lines,respectively（p<0.01）.（6）Suitability evaluation:K_m of OCT2for thiamine,histamine and 5-hydroxytryptamine was 67.88μM,784.5μM and 622.8μM,respectively;K_m of MATE1 for thiamine and histamine were 6.83μM and 1176μM,respectively.（7）Marker application:Cimetidine could significantly reduce the urinary excretion of thiamine,and the cumulative excretion of thiamine at 2 h and 4 h was strongly correlated between with metformin（R²>0.6）.（3）Screening and validating OATs-MRP4 pathway markers（1）Screening of candidate markers:26 UTs were selected as candidate compounds from EUTox database and literatures.Among them,the HSA binding rate of indole-3-acetic acid,3-indoxyl sulfate,3-indolyl-β-D-glucopyranoside,kynurenic acid,CMPF,p-cresol sulfate,4-ethylphenyl sulfonate and N-（cinnamoyl）glycine was 86%-100%,and the uptake of free and bound kynurenic acid,CMPF and N-（cinnamoyl）glycine in OAT1-and OAT3-overexpression cell lines was significantly increased,respectively（p<0.01）,but the accumulation of free kynurenic acid,CMPF and N-（cinnamoyl）glycine in MRP4 overexpression cell lines was decreased dramatically（p<0.05）.（2）In vitro/in vivo validation:compared with 4℃,the uptake of kynurenic acid,CMPF and N-（cinnamoyl）glycine in OAT1-and OAT3-overexpressing cell lines was increased significantly at 37℃,respectively,while the accumulation of kynurenic acid,CMPF and N-（cinnamoyl）glycine in MRP4 overexpressing cell lines was decreased significantly（p<0.05）;Probenecid significantly inhibited the uptake of kynurenic acid,CMPF and N-（cinnamoyl）glycine mediated by OAT1/3,and MK-571significantly inhibited the efflux mediated by MRP4;the transport of kynurenic acid,CMPF and N-（cinnamoyl）glycine from basolateral membrane to apical membrane increased significantly in the double transfected OATs-MRP4 cell line,but the transport from apical membrane to basolateral membrane did not change.Probenecid increased serum kynurenic acid and CMPF levels and decreased renal uptake in rats with dose dependence;compared with the wild type,the levels of serum kynurenic acid and CMPF in OAT1^-/-and OAT3^-/-rats increased significantly（p<0.01）,and the renal uptake rate and renal clearance rate decreased significantly（p<0.01）.MK-571remarkably increased the levels of kynurenic acid and CMPF in renal tissue,but did not affect the levels of serum and urine;compared with the wild type,the level of CMPF in kidney tissue of MRP4^-/-rats,but not kynurenic acid in plasma and renal tissue,was significantly increased.（3）Specificity evaluation:Efflux/uptake of kynurenic acid and N-（cinnamoyl）glycine were not mediated by OCT2,MATE1,MATE2-K,OAT2,OATP4C1,P-gp,PEPT2,URAT1 and OAT4.The uptake of CMPF was increased significantly in OAT4-overexpressing cell lines.（4）Suitability evaluation:K_m of OAT1 and OAT3 for kynurenic acid was 496.7μM and 382.2μM,respectively.The renal clearance t_1/2 of d5-kynurenic acid was 3.7±0.7 h.d5-kynurenic acid was metabolized into d4-xanthurenic acid in vivo,and its metabolic rate was 5.2-11.8%.The K_m of OAT1 and OAT3 for CMPF was 1483μM and 447.1μM,respectively.The renal clearance t_1/2 of CMPF was 1.9±0.3 h.K_m of OAT1 and OAT3for N-（cinnamoyl）glycine was 4265μM and 1984μM,respectively.Kynurenic acid and CMPF were hardly affected by serum protein level,liver function,cardiac function,glomerular filtration and other factors in vivo.（5）Marker application:After administration of probenecid in rats,cefmetazole had significant correlation with kynurenic acid AUC_0-t,but had no significant correlation with creatinine.Conclusions（1）OCT2 mediates the renal tubular excretion of creatinine with low affinity and the TYR362 and GLN242 of OCT2 are the two key function sites during this process.In addition,OAT4 can also mediate the reabsorption of creatinine.These findings indicate that the renal excretion process of creatinine includes glomerular filtration,OCT2-mediated secretion and OAT4-mediated a small amount of reabsorption in vivo,and thus it has poor specificity as an endogenous marker to evaluate renal drug excretion.（2）Thiamine can be filtered by glomerulus because of its low binding rate to HSA.However,thiamine is specifically secreted by OCT2-MATE1 pathway in renal tubules with appropriate affinity.Besides,there is a significant correlation between the cumulative excretion of metformin and thiamine in urine.These results imply that thiamine has certain reference value in evaluating the function of OCT2-MATE1pathway.（3）The binding rate of kynurenic acid to HSA is 99%.Kynurenic acid is not filtered by glomerulus in vivo,but it is specifically secreted through OATs-MRP4pathway in renal tubule with appropriate affinity and renal clearance half-life.Moreover,AUC_0-tof kynurenic acid has a significant correlation with that of cefmetazole.The above results suggest that kynurenic acid is expected to be an endogenous marker for the functional evaluation of OATs-MRP4 pathway and the determination/adjustment of the dosage of its substrate drugs.