Effects Of Uremic Serum On The Transport Of Atorvastatin In CACO-2 Cell Monolayers

OBJECTIVEChronic kidney Disease (CKD) can alter the metabolic kinetic parameters of the drugs that are excreted by kidney, it needs to adgust the dosage during the application of theses drugs in the patients with CKD, but the dosage is seldom adgusted for the drugs that are extensively metabolized by the liver or excreted in the bile. More and more evidences show the uremic toxins accumulated in the ESRD patient accelerate the deterioration of renal function、damage cells and matrix proteins, meanwhile, they can also change the diposition process of drugs in the intestine and liver. Studies have shown uremic serum can alter the mRNA level and protein expression of the intestinal transporters, but there are few studies about the effect of uremic serum on the drugs transpoted by the intestinal transporters. Atorvastatin(ATV) is one of the drugs that are used commonly in patients with chronic kidney disease, it is metabolized in the liver and excreted in the bile,only 2% is excreted in the kidney. Studies have shown ATV is uptaked by Monocarboxylate transporter 1(MCT1) and effluxed by P-glycorprotein (P-gp) in the Caco-2 cell model.The overall objective of this study is to evaluate the effect of uremic serum on the transport of ATV in the intestinal epithelial cells,including the effect on MCT1 and P-gp,and to carry out a preliminary discussion of the absorption of ATV in the intestine in patients with chronic kidney disease,to provide more theoretical basis for its clinical application.METHODS1.Establishment of the Caco-2 cell modelCaco-2 cells in logarithmic growth phase were seeded on Transwell filters for 21 days, and then determined the apparent permeability (Papp) of digoxin, the positive substrate of P-gp, and observed the cell morphology with Scanning electron micrograph, to evaluate the density and the integrity of Caco-2 cell monolayer model.2.Validation of the transport of ATV across the Caco-2 cell monolayerTo determine the apparent permeability of ATV in the basolateral(BL)-to-apical(AP) direction and in the AP-to-BL direction,calculate the ratio of the Papp(BL→AP)/Papp(AP→BL), to evaluate the transport of ATV across the Caco-2 cell monolayer.3.The effect of uremic serum on the transport of ATV across the Caco-2 cell monolayerIn the Caco-2 cell model, evaluating the efflux process of ATV mediated by P-gp in the normal serum, compared with in the uremic serum and inhibitor GG918.Comparing the apparent permeability of ATV at 40、80、120min in above groups, to evaluate the effect of uremic serum on the efflux of ATV mediated by P-gp.4.The effect of uremic serum on the uptake of ATV mediated by MCT1In the Caco-2 cell model, evaluating the uptake process of ATV mediated by MCT1 in the normal serum, compared with in the uremic serum and inhibitor GG918.Comparing the apparent permeability of ATV at 40、80、120min in above groups, to evaluate the effect of uremic serum on the uptake of ATV mediated by MCT1.RESULTS1.Establishment of the Caco-2 cell modelAfter 21 days of cell culture cycle, Caco-2 cells differentiated completely and formed cellular tight junction.Choosing filters whose transepithilure electrical resistance(TEER)>400Ω·cm2 for transporting. With the increase of reaction time,the apparent permeability of digoxin in the BL-to-AP direction was 11.3±0.7×10 cm·s-1 and that in the AP-to-BL direction was 3.41±1.4×106cm·s-1,the ratio of the Papp(BL→AP)/Papp(AP→BL) was 3.31,indicating the absoption direction played a primary role,which was in accordance with other reported values,the results showed the Caco-2 cell model was can be used in the following experiments.2. Validation of the transport of ATV across the Caco-2 cell modelIn the eligible Caco-2 cell monolayers, with the increase of reaction time,both the apparent permeability of ATV in the BL-to-AP direction and in the AP-to-BL direction were increasing,and which existed differences significantly at 40 and 80 min. At 120 min,the apparent permeability of ATV in the BL-to-AP direction was 39.5±9.4×106c·-1 and that in the AP-to-BL direction was 2.55±0.8×106cm·s-1, the ratio of the Papp(BL→AP)/Papp(AP→BL) was 15.4, indicating the absoption direction played a primary role, which was closed to results of other papers.3.The effect of uremic serum on the efflux of ATV mediated by P-gpComparing with the group of normal serum, both the Papp(BL→AP) of ATV in the uremic serum group and in the inhibitor GG918 group were reduced, and uremic serum can reduced the Papp(BL→AP) of ATV at 80 and 120min significantly. Comparing with the group of inhibitor GG918, the Papp(BL→AP) of ATV in the uremic serum group were all lower than the group of inhibitor,and exist significant differences at 120 min (p<0.05)4.The effect of uremic serum on the uptake of ATV mediated by MCT1Comparing with the group of normal serum, at 40、80、120min, the Papp(AP→BL) of ATV in the inhibitor benzoic acid group was reduced, and the Papp(AP→BL) of ATV in uremic serum was reduced more significantly.(p<0.05、 P<0.05、p<0.01)); The Papp(AP→BL) of ATV in the uremic serum group were all lower than the group of inhibitor, and existed differences at 120min significantly (p<0.05)CONCLUSION1. Caco-2 cells in logarithmic growth phase were seeded on Transwell filters for 21 days, then Caco-2 cells differentiated completely, and formed cellular tight junction. whose TEER>400Ω·cm2, the Caco-2 cell model was can be used to in the follow-up experiment.The apparent permeability of ATV in the BL-to-AP direction and in the AP-to-BL direction were closed to that of other papers, indicating the Caco-2 cell model is reliable.3. In the Caco-2 cell model, uremic serum can inhibit the efflux and uptake process mediated by P-gp and MCT1, respectively, suggesting that there are some factors existing in the serum of patients with end-stage renal disease,which have effects on the transport of ATV in the intestine.Further investigations are needed to study the effect of uremic serum on the transport and metabolism of ATV throughout the body, to provide more theoretical basis for its clinical application in patients with ESRD and improving the safety and effectiveness for clinical use.