Digoxin Transport by Renal Proximal Tubule Cells Is Enhanced
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Digoxin transport by renal proximal tubule cells is enhanced on adhesive synthetic RGD peptide On-line Number 0679 Hiroyuki Ijima, Shohei Kuroda, Koei Kawakami Department of Chemical Engineering, Faculty of Engineering, Graduate School, Kyushu-University 6-10-1 Higashi-ku, Fukuoka 812-8581, Japan E-mail: [email protected] ABSTRACT INTRODUCTION: Dialyzer apparatus have widely been used as an artificial kidney in medical treatment. However, some side effects as amyloidosis and so on have been occurred during the long term treatment. Therefore, we forcused on development of a hybrid artificial kidney with not only filtration but also reabsorption apparatus, but extensive spreading is easily occurred and difficult to maintain uniform monolayer state on collagen coated substrate. The purpose of this study is improvement of the cell adhesion, uniform stable monolayer formation and active transport function by immobilization of RGD on the culture substratum. MATERIALS AND METHODS: Polycarbonate semipermeable membrane separating culture well was coated by collagen, fibronectin, laminin and synthetic polypeptide including RGD (PnF). Cell adhesive and digoxin transport activities were estimated using renal proximal tubule cell line overexpressed p-glycoprotein gene. RESULTS AND DISCUSSION: Immobilized cell densities at initial and confluent conditions on PnF-coated membrane were higher than those on the other conditions in culture well. Transepithelial electrical resistance and digoxin transport activity on PnF coated membrane were the highest of all conditions. This might be caused by uniform cell morphology and high cell density. KEYWORDS RGD, Renal proximal tubule cells, Active transport, Digoxin INTRODUCTION Number of renal failure patients was 200,000 in Japan on 2000, but the patients who received kidney transplantation was only 4000 (2%). Hemodialysis therapy is applied to the most of patients, but the complete functional substitute is very difficult. Hemodialysis can substitute only glomerular filtration on water and waste excretion, but impossible to substitute reabsorption at renal proximal tubule cells and metabolism, etc. Therefore, novel therapeutic method is required. On the other side, hybrid type artificial kidney for resolve the problems of hemodialysis by maintaining renal tubular cell functions is studying. This hybrid type artificial kidney is expected that inhibiting protein adsorption to hollow fiber membrane, and realizing selective transport by reabsorption, metabolism and secretion, by covering hollow fiber membrane with glomerular cells and renal tubule 1 cells. Recently, there is a report that hybrid type artificial kidney was applied for medical treatment for severe renal failure patients (Humes, et al., 2003). However, cell function was decreased because of the instability of monolayer cell morphology, and the performance of the devise was decreased with time. It should be improved in hybrid type artificial kidney by improvement of renal tubular cell immobilization on hollow fiber membrane (Fujita, et al., 2002). On the other side, primary hepatocytes rapidly formed stable monolayer structure with uniform cell morphology, and it was maintained for a long period on Pronectin FTM (PnF) coated culture dish, and expressed well liver specific function of ammonia matabolism (Minguell, et al., 1993; Ijima, et al., 2001). PnF is a genetic synthesized culture substratum and has 13 RGD sequences, which is an active site for cell adhesion of fibronectin in the molecule. Primary hepatocytes on PnF coated surface was suppressed extensive spreading, improved cell adhesion and cell functions, etc. than that on ECM (collagen, fibronectin, etc.) coated surfaces. The purpose of this study is renal proximal tubule cell adhesion supported by PnF coating on culture surface for the stability of monolayer state and improvement of an active transport of digoxin. A selective active transport of digoxin shows a drug elimination ability of the cell, and a non-specific diffusion via cell layer is estimated using inulin and multidrug resistance protein inhibitor, verapamil (Tsuruoka, et al., 1999). MATERIALS AND METHODS Cells and cell culture Proximal tubule cell line induced human multidrug resistance protein-1 gene (PCTL-MDR) (Tsuruoka, et al., 1999) was used in this study. Culture medium was consisted of DMEM with low glucose/Ham’s F12 (1:1) as a basal medium supplemented with 25 mM NaHCO3, 40 mg/ml neomaycin (Sigma Chemical Co., St. Louis, MO, U.S.A). 12 well MULTIWELL™ PLATE (Nippon Becton Dickinson Co., Ltd. Tokyo, Japan) and microporous polycarbonate membrane filter (0.4 µm pore size, Transwell®3404; Corning Corstar, Acton, MA, U.S.A) were used as the culture substratum. Cells were cultured in 5% CO2/95% air incubator at 37 ºC under the stationary condition. Treatment of culture surface Culture surface was coated by Pronectin F (PnF: Sanyo Chemical industries, Ltd. Kyoto, Japan), Fibronectin (FN: Sigma), Laminin (LN: Sigma) or Type I collagen (Cellmatirix I (Col): Nitta Gelatin Inc. Osaka, Japan). Coating solution concentrations of PnF were 500, 50, 5 µg/ml, of FN were 50, 25, 10 µg/ml, of LN were 40, 20, 10 µg/ml and of Col was 100 µg/ml. Culture surface was covered by each solutions by adding 0.1 ml/cm2. Then each substratum was dried at room temperature (PnF) or at 4 ºC (FN, LN, Col) during over night. Finally, remained solution was removed and culture substrata were washed by CMF-PBS for 3 times. Culture plate and Transwell without these treatments were used as control experiments. 2 Number of cells Number of PCTL-MDR cells was estimated by using Cell Counting Kit-8 (DOJINDO LABORATORIES, Kumamoto, Japan). Briefly, after waste medium was removed from each well, 0.5 ml fresh medium and 50 µl WST-8 solution (2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium) were added into a well of 12 well multiwell plate. In the case of membrane filter, cell-adhered membrane was taken off from the holder by knife and the cell-adhered polycarbonate membrane put into 24 well multiwell plate, and added the same volume of solutions. Then, the plate was incubated at 37 ºC on a rotary shaker at 60 rpm for 1.5 h (initial of culture) or 1.0 h (confluent). WST-8 formazan concentration was detected using spectrometer (450 nm). Immunofluorescent stain of multidrug resistance protein (MDR) PCTL-MDR adhered on porous polycarbonate membrane was stained by FITC-labeled antibody as follows. 100 % cold methanol (-20 ºC) was added into the membrane-placed well and left for 10 minutes to fixation. Then, the membrane was washed using PBS with 0.1 % bovine serum albumin. This procedure was repeated for 3 times. The membrane was soaked in mouse anti-multidrug resistance 1 antibody (Zymed® Laboratories, Inc. South San Francisco, U.S.A) solution and left for overnight at 4 ºC. Then, the membrane soaked in FITC-labeled gout ant-mouse IgG2a (Santa Cruz Biotechnology, Inc, California, U.S.A) solution and left for overnight at 4 ºC. Mouse anti-multidrug resistance 1 antibody and FITC-labeled gout ant-mouse IgG2a were diluted 30 and 100 times by PBS, respectively. The membrane was observed by confocul microscopy (R2100 AG-2QST, Bio-Rad Laboratories, Hercules, CA, U.S.A.). Active transport of 3H-digoxin via membrane covered by PCTL-MDR cell layer Confluent condition on polycarbonate porous membrane was checked by transepithelial electrical resistance (TER) with Millicell®-ERS (Millipore Corporation, MA, U.S.A). Active transport experiment was started when the TER indicated peak value. 1.5 ml and 0.5 ml of fresh medium containing 5.5 nM 3H-digoxin (PerkinElmer Life and Analtical Sciences, Boston, MA, U.S.A) were pored into basolateral and apical side, respectively. After 3 h incubation in 5%CO2/95%air incubator at 37 ºC on a rotary shaker at 20 rpm, 3H-digoxin transport from basolateral side to apical side was assayed using 100 µl medium sample from apical side. Furthermore, 3H-digoxin diffusion ratio was estimated by adding MDR inhibitor, verapamil at the final concentration was 20 µM (Sigma), to apical side using the same well. On the other hand, 14C-inulin(4.5µM; PerkinElmer) was added to basolateral side and cultured for 3 hours, then 14C-inulin diffusion amount was measured using 100 µl sample from apical side to check paracellular leak. Radioactivity of 3H and 14C was measured by liquid scintillation counter (LS6500 Scintillation System, Beckman Instruments, Ins. Fullerton, CA, U.S.A.) 3 RESULTS Cell adhesion and morphology on culture plate Initial adhesion efficiency of PCTL-MDR cells on 12 multiwell plate was estimated at 24 h after cell inoculation (Fig.1). There is no effect of FN and LN concentrations on the initial adhesion efficiency (data not shown). Therefore, 25 µg/ml FN and 20 µg/ml LN were used for coating the culture surface in the following studies. Those are the sufficient coating amount for cell immobilization (Mooney, et al., 1992). On the other side, the efficiency was increased with PnF concentration (Fig.1a). PnF (500 µg/ml)-coated well (PnF-well) indicates the highest initial cell adhesion efficiency of all culture substrata (Fig.1b). Therefore, 500 µg/ml-PnF was used for the following studies. Number of cells at confluent condition was measured at 2 days after the cell covered the whole culture surface (Fig.2). PnF-coated well (PnF-well) indicates the highest cell density of all. FN and LN, which are well known as extracellular matrix (ECM) component, coated wells (FN-well, LN-well) indicate slightly lower cell densities than tissue culture well (TC-well) surface. 4 Kidney epithelial cells usually form blister in in vitro cell culture on tissue culture dish. Though many blisters were formed on Col-well and LN-well (Figs. 3b, 3d), blister formation was not so much and cell aggregates were formed on TC-well (Fig.3e). On the other side, blister formation on PnF-well was few than that on the others, and cell aggregate was not formed on PnF-well (Fig.3a).