Construction And Evaluation Of An Organic Anion Transporter 1/3 Double-knockout Rat Model Based On CRISPR/Cas9 Technology

ObjectiveRenal organic anion transporter 1（OAT1/SLC22A6）and organic anion transporter 3（OAT3/SLC22A8）are primarily in charge of the renal excretion of organic anionic and a few organic cationic drugs,poisons and endogenous substances,which are the main rate-limiting steps in the renal elimination of these compounds.Meanwhile,OAT1 and OAT3 have overlapping substrates such that when one of the transporters is dysfunctional the other can exert a compensatory effect.Therefore,the knockout of r/m OAT1 or r/m OAT3 does not fully reflect the changes in the excretion of organic anionic substrates.However,no r/m OAT1 and r/m OAT3 double-knockout models are currently available.Based on this,this study used CRISPR/Cas9technology to construct the first rOAT1 and rOAT3 double-knockout（KO）rat model and evaluated its biological properties,with a view to the model rat becoming a model animal for studying the role of rOAT1 and rOAT3 in metabolic diseases,drug interactions and pharmacokinetics.Method（1）Construction of the KO rat model and genotype identification（1）Model construction:The target sites were selected at the second exon of Slc22a8 gene and the tenth exon of Slc22a6 gene.Firstly,guide RNA（gRNA）was synthesized artificially,and then the gRNA-Cas9 complex was co-injected into the fertilized rat eggs by a microinjection technique.After incubation,the surviving fertilized eggs were transplanted into pseudo-pregnant rats.When delivery,gene sequencing analysis and PCR technology were used to identify the KO rats.（2）Genotype identification:Total DNA was extracted and purified with kit using newborn rat tails when they reached 10 days or so of age.And then,using PCR technology for amplification and the amplified products were separated by electrophoresis on a 1.5%agarose gel.The genotype was judged according to the size of the bands.（2）Evaluation of the basic characteristics of KO rat model（1）Physiological phenotyping:After fasting for 12 h,the wild-type（WT）and KO rats at 4-week-old and 7-week-old were weighed and their 12 h urine samples were collected and weighed.Then,the rats were anesthetized and blood samples were collected via abdominal aorta.Subsequently,the kidneys of the rats were removed and weighed,and their liver tissues were collected.The blood samples were measured by automatic biochemical analyzer for serum renal function,liver function,blood lipid and blood glucose indexes.The urine samples were used to determine the early renal injury markers.HE,masson,toluidine blue and CD31 immunofluorescence staining were performed to investigate the renal histopathology,fibrosis,inflammation and microvascular loss respectively.HE staining was performed to observe the liver histopathology.（2）Endogenous uremic toxins（UTs）:We collected blood,12 h urine and tissue samples from WT and KO rats at the age of 4 weeks and 7 weeks and determined the levels of 26 endogenous UTs by HPLC-MS/MS.（3）Compensatory effects:The mRNA expression of Slc22a1,Slc22a2,Slc22a7,Slc22a11,Slc22a12,Slco4c1,Slc47a1,Abcc2,Abcc4,Abcb1a,Abcb1b and Abcg2 in the kidney of WT and KO rats at 7 weeks old was determined by q PCR technology.The Western blot was used to investigate the protein expression of OAT2,organic cation transporter 2（OCT2）,multidrug and toxin efflux transporter 1（MATE1）,multidrug resistance associated protein 2（MRP2）and MRP4.（3）Functional evaluation of the KO rat model and pharmacokinetic investigation of furosemide and metforminAfter the rats were fasted for 12 h and catheterized in the left femoral artery,a drug cocktail of p-aminohippuric acid,metformin and inulin was administered to3-month-old WT and KO rats via the tail vein and then blood and urine samples were collected at the time point to investigate the pharmacokinetics characteristics of these three drugs.In addition,in order to examine the pharmacokinetic properties of furosemide,3-month-old WT and KO rats were fasted for 12 h and administered furosemide by gavage,and blood and urine samples were collected at time points.Results（1）Construction of the KO rat model and genotype identificationCompared with WT rats,there were almost no Slc22a6 and Slc22a8 mRNA expressions,and rOAT1 and rOAT3 proteins were partially expressed but not functional in the kidneys of KO rats（p<0.001）.Agarose gel electrophoresis of the amplified genomic DNA from the tail of WT rats showed only a band of 613 bp size and KO rats showed only a band of 370 bp size.（2）Evaluation of the basic characteristics of KO rat model（1）Physiological phenotyping:The body weight,kidney weight and kidney/body ratio in KO rats at 4 weeks old were significantly lower than WT rats（p<0.01）,while they showed no visible differences at 7 weeks old.The serum urea nitrogen,creatinine,aspartate transaminase,alanine transaminase,total protein,globulin,albumin,alkaline phosphatase,total bile acid levels and urine N-acetyl-β-D-glucosidase level were much higher in KO rats than in WT rats at 4 weeks old（p<0.05）,but the levels of triglyceride and glucose were significantly lower（p<0.001）.The levels of serum biochemical indicators and urinary early kidney injury markers in KO rats were not significantly different at 7 weeks old.There were no obvious histopathological changes in the kidneys and livers of KO rats and fibrosis and inflammation in the kidneys of KO rats at 4 and 7 weeks of age.The number of renal microvessels was slightly higher in WT rats than in KO rats at 4-week-old,while it was comparable between WT and KO rats at 7-week-old.All of these results demonstrated that deletion of rOAT1 and rOAT3 did not lead to inflammation or pathological changes in the kidneys of 4-and 7-week-old rats.（2）Endogenous UTs:Compared with the WT rats,the plasma concentrations of3-indoxyl sulfate potassium salt,hippuric acid,p-cresol sulfate,N-acetyl-L-arginine,4-ethylphenyl sulfate and N-acetylcytidine in KO rats were significantly increased（p<0.05）and the renal uptake ratios obviously decreased（p<0.05）in both 4-and7-week-old KO rats.The plasma concentrations,cumulative urine excretions and renal uptake ratios of D-neopterin,3-（3,4-dihydroxyphenyl）-L-alanine and1-methyl-5-carboxylamide-2-pyridone were not affected by Slc22a6 and Slc22a8double-knockout at both the age of 4 and 7 weeks.There were 9 UTs with higher plasma concentrations at 4-week-old than at 7-week-old（p<0.05）.（3）Compensatory effects:Compared with the WT rats,Slc22a11（p<0.01）and Abcc4（p<0.05）mRNA levels were significantly downregulated in the kidneys of KO rats,Slco4c1 mRNA level was upregulated（p<0.05）,while mRNA levels of other genes were not different.The protein expressions of rOAT2,rOCT2,r MATE1,r MRP2 and r MRP4 were not affected by the knockout of rOAT1 and rOAT3.（3）Functional evaluation of the KO rat model and pharmacokinetic investigation of furosemide and metforminCompared with the WT rats,the plasma concentrations of p-aminohippuric acid and inulin were significantly higher（p<0.01）and the cumulative urinary excretions were much lower（p<0.05）in KO rats.Serum pharmacokinetic parameters AUC_0-12were significantly increased（p<0.001）and V_z was significantly decreased（p<0.05）for para-aminomaluric acid;the AUC_0-12 and AUC_0-∞of inulin were significantly increased（p<0.001）.The concentration of the anionic drug furosemide was significantly higher in the serum of KO rats（p<0.001）and the cumulative urinary excretion was significantly lower（p<0.001）.Meanwhile,the AUC₀–₁₂,AUC₀–∞,C_max and T_max of furosemide in KO rats increased by 300.38%,322.79%,183.24%,and 110.00%respectively（p<0.01）,while the V_z reduced by 72.73%（p<0.01）.The concentration of the cationic drug metformin was significantly higher in the serum of KO rats（p<0.05）and there was no difference in cumulative urinary excretion.The pharmacokinetic parameters AUC₀–₁₂ and AUC₀–∞significantly increased（p<0.01）.ConclusionIn this study,we successfully constructed and preliminarily evaluated a Slc22a6and Slc22a8 double-knockout rat model firstly based on a CRISPR/Cas9 technology,which was deficient in the physiological function of rOAT1 and rOAT3 and no renal pathological phenotype was observed.Knockdown of rOAT1 and rOAT3 resulted in a near-absence of renal excretion of the anionic drug furosemide,which was not found to be compensated by other types of renal tubular secretion,and unaffected urinary excretion of the cationic drug metformin,but with a significant decrease in glomerular filtration function.This model rat is expected to be one of the important model animals for studying the role of rOAT1 and rOAT3 in metabolic diseases,drug interactions mediated by rOATs and pharmacokinetics.