Adsorptive Function Research Of Epithelial Cells From Distal Convoluted Renal Tubule

The transport function of renal tubular includes reabsorption and secretion, and the transport methods can be divided into passive transport and active transport.Madin-Darby canine kidney (MDCK) cell line originated from the kidney distal convoluted tubule epithelial (DCT) cells of an adult female cocker spaniel, and it can growth and differentiate into structurally and physiologically intact cell monolayer. The MDCK cell monolayer is very useful as in vitro model for drug reabsorption and excretion in kidney, absorption in intestine, and even transport across the blood brain barrier. It is easy to obtain, and economy, and with short culture period. It is clear in present days that small hydrophobic molecular can reabsorbed into bloodstream from renal proximal convoluted tubule and some drugs can actively secreted into the tubule lumen fluid. But, up to date, little is known about the role of the renal DCT in drug reabsorption with the exception of metal ions transport. Taking notice of the actuality, we have applied our energies to explore the absorptive function of DCT epithelial cell in recent years. In this thesis, probenecid (PBD), an organic acid (with a pKa 3.4) and a competitive substrate of organic anion transporter (OAT), was served as a model drug for the aim to achieve a positive knowledge of reabsorption function of DCT.Methods To reach this goal, the following methods were applied. (1) Parallel Artificial Membrane Permeability Assay (PAMPA) is used to examine the passive diffusion across plasma membrane of PBD for differentiating transporter mediated transport; (2) MDCK monolayer was established, and used to examine the absorption and secretion of PBD. And then, the data obtained from PAMPA and MDCK monolayer were analyzed to find the possible passive diffusion or transporter mediated transport. (3) The effects of some important factors on PBD absorption in MDCK monolayer were observed to characterized transporters which might be involved in PBD absorption as well as the possible absorption mechanism. The observed factors include the changes of pH, temperature, Na+ respectively, and the applications of P-glycoprotein (P-gp) inhibitor verapamil (Ver), organic anion transporter (OAT) competitive inhibitor taurine (TAU), energy metabolic poison sodium azide (SA), protein phosphatase inhibitor Microcystin-LR (MC-LR), respectively. Results (1) PBD can be transported in the form of passive diffusion cross the artificial membrane; (2) the absorption amount from MDCK model was as two folds higher as that from PAMPA model. It is indicated that the transport mechanisms in these two models are different, and there is a more effective, transporter-dependent absorption in MDCK monolayer. (3) Absorptions of PBD in buffers with different pH are significantly different in MDCK cell monolayer, so PBD absorption is pH-dependent. The passive diffusion is responsible for pH-dependent absorption change of PBD. (4) PBD absorption was temperature (ATP produce)-dependent. It is suggested that an active transport takes part in PBD absorption on MDCK cell monolayer. (5) The absorbed amount of PBD was decreased when MDCK cell monolayer was incubated in sodium-free buffer, and it indicated that PBD absorption is partly a secondary active transport. (6) It was found that taurine could effectively inhibit the absorption of PBD no matter given in the apical side or the opposite side. So far, we concluded that the transporter involved in the absorption of PBD is OAT, which mediates both reabsorption and secretion of PBD on the cellular model. (7) The absorption amount of PBD was increased when incubated with verapamil, and this indicates that P-glycoprotein (P-gp) is involved in the PBD absorption process on the MDCK cell model. (8) Lastly, we found that absorption amount of PBD was very markedly increased by MC-LR, a protein phosphatase inhibitor. The result suggestes strongly that protein phosphorylation is a key regulator of OAT which mediates the PBD absorption in the MDCK monolayer.Conclusion According to the experiments above, we concluded that:1 Renal DCT epithelial cell is of the reabsorption function, which includes active transport and passive diffusion.2 There is OAT located in membrane of DCT cells. OAT can mediate the reabsorption and secretion of organic acids in form of secondary active transport. And the activity of this certain OAT was regulated by phosphorylation.3 P-glycoprotein reduce the drug absorption of PBD by active secretion of drug out of cell.4 The approach that combining the MDCK cell model with PAMPA model is an ideal method in the research of drug transportation cross DCT cells as well as its mechanisms.