Patent Application: US-201013508272-A

Abstract:
the invention relates to the field of biology and medicine . in particular , the invention relates to the field of pharmaceutical research and drug development , and especially to means for physiological , pharmacological and toxicological research . more in particular , the invention provides renal cell lines with proximal tubular characteristics including multiple influx and efflux transporters . these cell lines are termed ciptec . four representative examples have been deposited at the deutsche sammlung von mikroorganismen and zellkulturen gmbh at inhoffenstrasse 7 b , 38124 braun - schweig , germany under accession number dsm acc3019 , dsm ac - c3020 , dsm acc3021 and dsm acc3022 .

Description:
using a non - invasive technique of obtaining renal material from urine , we have developed conditionally immortalized human ptec ( ciptec ) cell lines from a healthy volunteer . these cell lines may be maintained for at least 45 passages and presents proximal tubular characteristics when cultured at a non - permissive temperature of 37 ° c . for 10 days . the activities of the apical ( brush border membrane ) atp - binding cassette ( abc ) transporter p - glycoprotein ( pgp / mdr1 / abcb1 ) and the basolateral membrane solute carrier ( slc ) organic cation transporter 2 ( oct2 , slc22a2 ) as well as the apical uptake of phosphate ( mediated by slc34a1 and slc34a3 ) and of albumin are demonstrated . in addition , the presence of the apical proximal tubular - specific atp - binding cassette ( abc ) transporter multidrug resistance protein 4 ( mrp4 / abcc4 ) was confirmed . approximately 10 % of the 38 collected mid stream urine sediments from 31 healthy volunteers contained viable cells with the ability to proliferate into single cell colonies . primary cultures showed heterogeneous morphology . cell cultures infected with sv40t and htert were found to be antibiotic - resistant to hygromycin b and geneticin ( g418 ), indicating successful immortalization . antibiotic resistance was maintained in ciptec cells for at least 40 passages , indicating that expression of sv40t and htert remained over time during proliferation at 33 ° c . proliferation was maintained at 33 ° c . and cells transferred to 37 ° c . at 70 % confluency grew into confluent monolayers within 10 days , while sv40t antigen expression gradually decreased ( fig1 ). subcloning of immortalized cells resulted in a cell culture with homogenous cobblestone morphology . electron microscopy ( em ) analysis showed moderate formation of microvilli and endocytic vesicles . proliferation of clones could be maintained for at least 45 passages . routinely , cell morphology was monitored by phase - contrast microscopy at each passage , which showed no marked difference up to passage 40 . to investigate the viability of the cells during maturation at 37 ° c . for 10 days , a resazurin assay was performed [ 31 , 16 ] showing intact viability during the maturation stage ( data not shown ). successfully immortalized cell lines obtained from two donors were subcloned , resulting in 15 and 9 clones respectively . these clones were characterized for expression of aminopeptidase n , pgp / mdr1 / abcb1 , aquaporin 1 ( aqp1 ) and dpp - iv expression . based on these results , one clone from one donor ( female , 12 years ) expressing the markers mentioned above , was designated ciptec - 14 . 4 ( dsm acc3019 ) and selected for further investigation . a characterization study was performed after subcloning the cell line to confirm its proximal tubular origin [ 7 ]. at 37 ° c ., monolayers of the subclones expressed zona occludens 1 ( zo - 1 ) protein , indicating the epithelial origin of cells with development of tight junctions . formation of tight monolayers was further supported by the inulin - fitc diffusion experiments . cells grown on permeable filter supports assembled to a monolayer , with 13 . 2 % (+/− 1 . 1 ) diffusion of inulin - fitc after 2 hr incubation at 37 ° c . proximal tubular specific brush border membrane enzyme aminopeptidase n ( cd13 ) was detected using flow cytometry ( fig2 ) and confirmed by immuno - fluorescence microscopy the activity of brush border membrane enzyme alkaline phosphatase was investigated and compared to activity in hk - 2 cell lines ( atcc ; manassas va ., usa ). in ciptec - 14 . 4 the activity of alkaline phosphatase was 0 . 96 +/− 0 . 21 and in hk - 2 0 . 59 +/− 0 . 03 munits sap / mg protein . endothelial marker cd31 did not bind to ciptec . ciptec - 14 . 4 ( dsm acc3019 ) could be clearly distinguished from human podocyte cell line [ 23 ] due to differences in morphology , the presence of cd13 antigen and alkaline phosphatase activity [ 30 ]. the presence of proximal tubular specific transporters and enzymes aqp1 , dpp - iv and mrp4 / abcc4 was demonstrated in cells cultured for 10 days at 37 ° c . ( fig3 ). cells originating from distal tubules or collecting ducts were excluded by positive expression of aqp1 in ciptec - 14 . 4 [ 15 ]. the variations in molecular size between ciptec - 14 . 4 and human kidney mrp4 was likely to be due to a difference in glycosylation of this abc - transporter [ 8 ]. the reabsorption of albumin was analyzed using fitc labelled bovine serum albumin ( bsa - fitc ) uptake in ciptec - 14 - 4 between passage number 20 - 25 . the results show that uptake was concentration - and temperature - dependent , indicating active and specific transport of bsa in ciptec - 14 . 4 ( fig4 a ). kinetic analysis of bsa - fitc uptake resulted in an apparent km of 126 μg / ml . to investigate the mechanism of bsa uptake , localization of bsa - fitc was analyzed and uptake was performed in presence of receptor associated protein ( rap ), a known inhibitor of albumin endocytosis by binding to multi - ligand receptor megalin [ 32 ], or excess unlabelled bsa . the vesicular pattern of bsa - fitc indicates uptake via endocytic vesicles ( fig4 b ). additionally , rap inhibited the uptake of bsa - fitc in a concentration - dependent manner ( fig4 c ). bsa - fitc uptake was significantly inhibited in the presence of 1 μm rap ( p & lt ; 0 . 05 ) or 200 - fold excess unlabelled fitc ( p & lt ; 0 . 01 ) by 41 % and 54 %, respectively ( fig4 d ). the uptake of phosphate in ptec is mediated by the sodium - dependent transporters napi - iia ( slc34a1 ) and napi - iic ( slc34a3 ) [ 9 ]. in the ciptec cell lines described herein , the uptake of 32 po 4 was concentration and sodium - dependent ( fig5 a ). maximum phosphate uptake rate ( vmax ) was 1717 μmol / 24 well / 5 min and an apparent km of 0 . 12 mm was calculated . in the absence of sodium , uptake was significantly decreased by approximately 86 % ( p & lt ; 0 . 001 ; fig5 b ). phosphate uptake was performed at passage numbers ranging from 30 to 39 with comparable results , suggesting sodium - dependent phosphate uptake remains functional at higher passage numbers . the ability of ciptec - 14 . 4 to transport xenobiotics was studied by the expression and activity of the basolateral transporter oct2 / slc22a2 and the apical efflux abc transporter pgp / mdr1 / abcb1 . western blotting using cell homogenates of ciptec - 14 . 4 cultured at 37 ° c . clearly showed presence of oct2 ( fig6 a ) and pgp ( fig7 a ). expression of both transporters was confirmed by western blotting in cell homogenates of passage number 40 , indicating tubular characteristics remained over time . following basolateral exposure , the fluorescent cation 4 -( 4 -( dimethyl - amino ) styryl )- n - methylpyridinium iodide ( asp ) was internalized by ciptec - 14 . 4 cultured on supporting membranes . this transport could be significantly inhibited using oct2 - inhibitor tetrapentylammonium ( tpa ) ( p & lt ; 0 . 05 ) or when uptake was performed at 4 ° c . ( p & lt ; 0 . 01 ) by respectively 36 and 30 % ( fig6 b ). significantly more intracellular fluorescent calcein accumulated when cells were incubated with psc - 833 ( ratio 1 . 6 ; p & lt ; 0 . 001 ; fig7 b ), indicating pgp - dependent transport activity in ciptec . to determine whether pgp activity remained over time during proliferation , the calcein assay was performed in cells of up to passage number 39 . this resulted in active export of calcein , which could be inhibited by psc - 833 ( ratio 2 . 3 ), indicating pgp was functionally expressed at a high passage number . three additional cell lines obtained by the process described hereinwere tested for the above parameters and yielded the same results , well within the experimental error margins . these cell lines were termed ciptec33 . 5 ( dsm acc3020 ), ciptec34 . 8 ( dsm acc3021 ) and ciptec10 . 3 ( dsm acc3022 ). in summary , the ciptec cell lines according to the invention have the following characteristics : cobblestone morphology expression of zona - occludens1 ( zo1 ), epitheliale tight junction marker resistance of monolayer on permeable filters transepithelial electrochemical resistance ( teer ) of monolayer expression of aminopeptidase n , enzyme on proximal brush border alkaline phosphatase activity , enzyme on proximal brush border expression of specific proximal tubular proteins : aquaporin1 ( aqp1 ) organic cation transporter 2 ( oct2 / slc22a2 ) dipeptidyl - peptidase iv ( dppiv ) multidrug resistance protein 4 ( mrp4 / abcc4 ) p - glycoprotein ( pgp / mdr1 / abcb1 ) albumine endocytosis , specifically inhibited by receptor associated protein ( rap ) sodium - dependent phosphate uptake functional basolateral transport of substrate asp via oct2 / slc22a2 functional efflux transport of calceïne via p - glycoprotein / mdr1 / abcb1 proliferation of more than 40 passage numbers , while maintaining morphology and expression of aqp1 , pgp / mdr1 / abcb1 and oct2 / slc22a2 and sodium - dependent phosphate uptake and pgp - activity . the above results show that we have developed a human conditionally immortalized proximal tubular cell line from urine of a healthy volunteer expressing transporters involved in renal reabsorption and excretion . the immortalization of non - invasively collected cells using sv40t and htert vectors enabled us to produce human cells maintaining proximal tubular characteristics , proliferating for at least 45 passages . expression of sv40t decreased gradually in ciptec - 14 . 4 cultured for 10 days at 37 ° c ., minimizing the influence of the transfection on cellular metabolism . subcloning improved homogeneity of the cell line as is shown by the decrease in morphological variations , probably caused by the exfoliation of various cell types originating from the renal - urinary tract into urine [ 7 ]. culturing ptec from control urine is usually hampered by low amounts of viable exfoliated cells in urine of healthy volunteers . interestingly , only some urine portions from a very few subjects contained viable ptec with variable morphology . in pharmacology and toxicology , the availability of a cell model of human origin expressing a broad range of functional transporters is of paramount importance . the provision of a cell line according to the invention may facilitate the study of the pathogenesis of inherited proximal tubular disorders , which is often hampered by the limited availability of renal tissue [ 6 , 19 , 5 ]. more specific , the ciptec cell lines as presented herein are the first human cell lines with expression of mrp4 / abcc4 , in concert with expression and activity of oct2 and pgp . we have confirmed mrp4 expression by quantitative pcr after rna isolation from ciptec . together with the formation of a tight monolayer , as was observed using the inulin diffusion experiments , these features make ciptec cells a valuable tool for identification of substrates and inhibitors of renal drug excretion and the prediction of potential drug - drug interactions in pharmacological research . next to functional organic cation excretion , the human ciptec cells maintain sodium - dependent phosphate uptake , as well as albumin endocytosis sensitive to inhibition by rap . the apparent km calculated for sodium - dependent phosphate transport in ciptec - 14 . 4 ( 0 . 12 mm ) was approximately one - third of the apical phosphate transport value demonstrated in opossum kidney ( ok ) cells ( 0 . 37 mm ) [ 20 ]. this suggests a higher affinity for phosphate in ciptec compared to currently available cell models . the reabsorption of albumin by ciptec in this study is most likely receptor - mediated endocytosis transport , since it is sensitive to rap inhibition and the intracellular vesicular pattern of bsa - fitc . inhibition of bsa - fitc in ciptec is similar to the inhibition found earlier in ok cells [ 32 ], while the apparent km calculated for ciptec - 14 . 4 ( 126 μg / ml ) was approximately 6 times higher than the value reported for ok cells ( 20 μg / ml ) [ 12 ]. this transport may be facilitated by the multi - ligand receptor megalin , however , we could not identify this receptor by western blot nor by immunofluorescence techniques ( data not shown ) [ 2 ]. this suggests involvement of alternative albumin reabsorption mechanisms [ 11 ]. in conclusion , the present study introduces the first human cell line featuring functional sodium - dependent and endocytosis mediated reabsorption together with functional secretion capacity by pgp / mdr1 / abcb1 and oct2 / slc22a2 and combined expression of mrp4 / abcc4 . the capacity of ciptec to proliferate for extended passages with maintained functional transport , allows for standardized and rapid investigation of renal drug handling and interactions in pharmacological and toxicological research . a cell line according to the invention may advantageously be used in the functional analysis of renal transporters . it may also be used in a bioassay for testing transport properties of substances through the kidney . in a preferred embodiment , such assays may comprise a solid or semi - solid phase comprising a cell line according the invention wherein said solid or semi - solid phase separates two fluid compartments . for example , said bioassay can be used for assessing epithelial barrier function using , for example a rems autosampler , an ussing chamber , or a chopstick electrode / evometer technique , in the presence or absence of one or more drugs and / or early drug discovery compounds . in a preferred embodiment , said bioassay can be performed in a medium - or high - throughput method for the identification of substrates of transporters and inhibitors of said transporters , and for predicting potential renal drug - drug interactions . for this , said compartments can be equipped with a fluidic - control system for automatic introduction of compounds , buffers , and gasses into the compartments and sampling from the compartments . the invention further provides a solid phase comprising a cell line according to the invention . said solid phase preferably comprises a suitable receptacle such as a tissue culture flask or a multi well plate . said solid phase can be coated , for example , with collagen or fibronectin a cell line according to the invention may also be used for specific reabsorption of electrolytes from ultrafiltrate when used in an ( bio ) artificial kidney . said cell line may be cultured on highly permeable filters covered with a monolayer of the cells to replace renal function in such a device . cell homogenates from ciptec - 14 . 4 cultured for various times at 37 ° c . were analyzed for sv40t antigen expression using western blotting . house - keeping protein gapdh was used as control . aminopeptidase n was detected using incubation of ciptec - 14 . 4 with anti - cd13 - fitc and analyzed by flow cytometry ( white histogram , negative control ; black histogram incubation with cd13 - fitc ). expression of proximal tubule specific proteins aquaporin - 1 ( aqp1 ), dipeptidyl peptidase iv ( dppiv ) and multi resistant protein 4 ( mrp4 / abcc4 ) in cell homogenates of ciptec - 14 . 4 were compared with expression in human kidney homogenate ( hukid ) by western blotting . albumin uptake in ciptec - 14 . 4 was analyzed using bsa - fitc . ( a ) uptake of bsa was concentration - and temperature - dependent ( black line , 37 ° c . ; dashed line , 4 ° c . ); data are expressed as means of duplo experiments . ( b ) bsa - fitc was located in intracellular vesicles . bar 10 μm ( c ) uptake of bsa - fitc ( 50 μg / ml ) was inhibited by rap in a concentration - dependent manner . data are expressed as means of duplo experiments . ( d ) uptake was significantly inhibited by 1 μm rap ( p & lt ; 0 . 05 ; grey bar ) or excess unlabelled bsa ( xs bsa , 200 - fold ; p & lt ; 0 . 01 ; white bar ). data are means of three independent experiments (+/− se ) and expressed as relative uptake compared to normal bsa - fitc uptake . uptake of 32 po 4 ( pi ) was analyzed in the presence and absence of sodium in four independent experiments . ( a ) uptake of pi was concentration - dependent and sodium - dependent ( in presence of sodium , black line ; with nmdg as sodium replacement , dashed line ). ( b ) uptake of 0 . 2 mm pi was significantly decreased ( p & lt ; 0 . 001 ) in the absence of sodium ( nmdg ). ( a ) presence of oct2 was shown using western blotting of ciptec - 14 . 4 homogenates . ( b ) activity of oct2 was analyzed by measuring the fluorescence of transported asp in absence ( black bar ) or presence ( white bar ) of oct2 inhibitor tpa . additionally , uptake was performed at 4 ° c . ( grey bar ). uptake was significantly decreased in ciptec - 14 . 4 in presence of tpa ( p & lt ; 0 . 05 ) or at 4 ° c . ( p & lt ; 0 . 01 ). data are expressed as mean values +/− se of three experiments . ( a ) presence of pgp was shown using western blotting of ciptec - 14 . 4 homogenates . ( b ) activity of pgp was analyzed by measuring the fluorescence of accumulated calcein in absence ( white bar ) or presence ( black bar ) of pgp inhibitor psc . accumulation was significantly increased in ciptec - 14 . 4 in presence of psc ( p & lt ; 0 . 001 ). data are expressed as mean values +/− se of three experiments . primary cells were cultured as described before by collecting mid stream urine after signing of informed consent by the parents of healthy volunteers with no clinical history of renal disease , nor with any other chronic disease . urine sediment was transferred to supplemented dmem - ham &# 39 ; s f12 medium ( lonza ; basel , switzerland ), and cultured at 37 ° c ., 5 % co2 [ 30 ]. primary cells were infected with sv40t and htert vectors containing respectively geneticin ( g418 ) or hygromycin resistance as described before [ 24 , 17 ]. subconfluent cell layers were transferred to 33 ° c . and selected using g418 ( 400 μg / ml ; sigma - aldrich ) and hygromycin b ( 25 μg / ml ( sigma - aldrich )) for 10 days . to obtain a homogenous cell culture , cells were subcloned using irradiated nih 3t3 fibroblast as non - dividing feeder cells [ 23 ]. after culturing for two weeks at 33 ° c ., single cell clones were visible and picked using cloning discs drained in trypsin / edta . for the following experiments , cells were cultured at 33 ° c . to 70 % confluency , followed by maturation for 10 days at 37 ° c . during which the cells formed a confluent monolayer . propagation of cells was maintained by reseeding the cells at a dilution of 1 : 3 to 1 : 6 at 33 ° c . experimental procedures were performed on the cloned cells between passages 15 and 40 . morphology of ciptec - 14 . 4 was investigated using phase contrast microscopy . additionally , cells cultured for 10 days at 37 ° c . were scraped off flask using a rubber policeman and embedded in paraffin for electron microscopy analysis . to investigate the epithelial origin of cells , confluent monolayers were fixed using 2 % paraformaldehyde , permeabilized in pbs - tween ( 0 . 1 %) and incubated with antibodies against the tight junction protein zo - 1 ( 1 : 25 dilution ; zymed laboratories , south san francisco , calif ., usa ). following secondary goat - anti - rabbit - alexa488 conjugate ( dako , glostrup , denmark ) and dapi ( molecular probes , invitrogen ) to stain nuclei , cells were analyzed using immuno - fluorescence microscopy . the presence of brush border membrane protein aminopeptidase n using mouse - anti - human cd13 - fitc antibody ( dako ) and endothelial marker cd31 - fitc ( dako ) was detected as described previously [ 30 ]. additionally , a sample of stained cells was transferred to a glass slide by cyto - spin ( 1000 × g , 10 min ) and analyzed using immuno - fluorescence microscopy . alkaline phosphatase activity was determined in at least three independent experiments using bm chemiluminescence elisa substrate ( ap ) kit ( roche diagnostics , mannheim , germany ) as described before [ 30 ]. values are compared to hk - 2 cell line using shrimp alkaline phosphate as positive control and expressed as mean +/− se . to investigate whether the monolayers assembled sufficiently tight for transport studies , ciptec - 14 . 4 was cultured on transwell ®- clear polyester membranes ( corning costar corporation , cambridge , mass ., usa ) for 10 days at 37 ° c . both apical and basal compartments were washed in hepes - tris buffer ( hepes - tris ( 10 mm ), nacl ( 132 mm ), kcl ( 4 . 2 mm ), cacl2 ( 1 mm ), mgcl2 ( 1 mm ), d - glucose ( 5 . 5 mm ), ph 7 . 4 ), prior to the addition of 0 . 1 mg / ml inulin - fitc ( sigma - aldrich ) to the apical compartment . inulin - fitc diffusion through the monolayer was monitored for 2 hours by sampling 100 μl of both apical and basal compartments and measuring fluorescence at 485 nm with emission at 535 nm . data are expressed as mean +/− se . cellular homogenates of cells cultured for various days at 37 ° c . were made by scraping cells off using a rubber policeman from 75 cm 2 tissue culture flask and lysed in 400 μl ripa buffer containing igepal ca630 ( 1 %), na - deoxycholate ( 0 . 5 %), sodium dodecyl sulfate ( sds ) ( 0 . 1 %), phenylmethanesulphonylfluoride ( pmsf ) ( 0 . 01 %), aprotinin ( 3 %) and na - orthovanadate ( 1 mm ). expression of sv40t antigen in cell homogenates was analyzed by western blotting using reduced 12 % sodium dodecyl sulphate polyacrylamide gel electrophoreses ( sds - page ) and blotted onto a pvdf membrane ( immobilon , millipore ; bedford , mass ., usa ). membranes were incubated with sv40t antibody ( 1 : 400 dilution ; santa cruz biotechnology , santa cruz calif ., usa ) and gapdh ( 1 : 5 . 000 dilution ; abcam , cambridge , uk ) as a house - keeping antigen , followed by incubation with goat - anti - mouse - hrp conjugate ( dako ) and visualization using pierce ecl western blotting substrate ( thermo fisher scientific , waltham mass ., usa ). cellular homogenates matured for 10 days at 37 ° c . were analyzed as described above using 6 or 12 % sds - page as indicated , using the following antibodies : rabbit anti - aquaporin 1 ( aqp1 ; 1 : 4000 ; 12 %; chemicon intl , millipore ), rabbit anti - oct2 ( 1 : 500 ; 12 %, alpha diagnostics , san antonio tex ., usa ), rabbit anti - cd26 ( dipeptidyl peptidase iv ( dpp - iv ); 1 : 200 , 12 %; santa cruz biotechnology ), rabbit anti - multidrug resistance protein 4 [ 27 ] ( mrp4 , abcc4 ; 1 : 5000 ; 6 %), mouse anti - pgp ( 1 : 200 ; 6 %; dako ) goat - anti - mouse - hrp conjugate ( dako ) and goat - anti - rabbit - hrp conjugate ( dako ). human kidney homogenate in ripa buffer was used as control . the ability of ciptec - 14 . 4 to reabsorb albumin was investigated by the incubation of confluent monolayers in 24 well plates with 50 μg / ml bsa - fitc ( sigma - aldrich ) for 30 min at 37 ° c . unless described otherwise . uptake was arrested using ice - cold pbs and cells were detached using trypsin , fixed by paraformaldehyde ( 0 . 5 %) in pbs and analyzed using flow cytometry or immuno - fluorescence microscopy . concentration - and temperature - dependent uptake , was investigated using a concentration range bsa - fitc ( 0 ; 3 . 7 ; 11 ; 33 ; 100 ; 300 μg / ml ) at 37 ° c . and on ice for 30 min . uptake inhibition was studied in three independent experiments by incubating the cells with bsa - fitc ( 50 μg / ml ) in addition of excess unlabelled bsa ( 10 mg / ml ) or recombinant rap ( 1 μm ), which was a kind gift of dr . m . nielsen ( university of aarhus , denmark ). uptake inhibition by rap was further examined using a dilution range of rap . bsa uptake in saturation experiments are plotted as mean fluorescence intensity and in inhibition experiments as mean (+/− se ) percentage uptake compared to control condition . phosphate uptake was performed in confluent monolayers using 32po4 ( perkin elmer , waltham mass ., usa ) as described earlier [ 14 ]. cells cultured for 10 days at 37 ° c . were incubated with 0 . 2 mm kh2po4 ( 10 μci / ml ) for 5 min in four independent experiments , in the presence of 137 mm sodium or 137 mm nmdg to study sodium - dependent transport . additionally , time - ( 0 . 5 ; 1 ; 2 ; 5 ; 10 ; 15 ; 30 or 60 min ) and concentration - dependent ( 0 . 02 ; 0 . 07 ; 0 . 22 ; 0 . 66 or 2 mm po4 ) uptake was studied . data are expressed as mean +/− se . transport of xenobiotics across the basolateral membrane was investigated in ciptec - 14 . 4 by measuring the activity and expression of the solute carrier tansporter ( slc ) oct2 / slc22a2 using a method adapted from brown et al [ 4 ]. cells were grown on transwell ®- clear polyester membranes as described before . activity of oct2 was measured by incubating 1 μm fluorescent oct2 substrate 4 -( 4 -( dimethylamino ) styryl )- n - methylpyridinium iodide ( asp , invitrogen ) in hepes - tris buffer for 1 min at 37 ° c . at the basal compartment . to inhibit oct2 mediated uptake , cells were exposed to 100 μm tetrapentylammonium ( tpa ) at both apical and basal compartments for 10 min prior to uptake of asp . additionally , one set of experiments was performed at 4 ° c . after incubation , transport was arrested using 1 mm ice - cold tpa . cells were homogenized using 250 μl hepes - tris - triton ( 0 . 1 %) buffer for 30 min , followed by analyzing fluorescence intensity ( excitation 450 nm , emission 642 nm ) using victor3 multiplate reader ( perkin elmer inc .). data are expressed as mean + i - se . the activity of the atp binding cassette ( abc ) efflux transporter pgp was assessed by measuring the accumulation of calcein as describe before [ 28 ]. briefly , matured cells were incubated in two independent experiments for 1 hr at 37 ° c . with lipophylic non - fluorescent pgp substrate calcein - am ( invitrogen ) in the presence or absence of inhibitor psc - 833 , which was a kind gift from novartis pharma ( basel , switzerland ). intracellularly , calcein - am is metabolized by esterase activity to the fluorescence calcein . fluorescence of cell lysates was measured at 488 nm with emission at 518 nm . fluorescence is expressed as mean +/− se . michaelis - menten curve fitting for calculation of km and vmax values was performed by non - linear regression analysis using graphpad prism 4 . 03 software . differences in substrate transport in presence or absence of inhibitors or unlabelled analogues were assessed by a paired t - test . the evaluation of the monolayer integrity was done by measuring the trans - epithelial electric resistance ( teer ) expressed as ohms . cm 2 . these were measured by using a millicell - ers teermeter at room temperature . the results are shown in table 1 . 3 . bodnar a g , ouellette m , frolkis m , holt s e , chiu c p , morin g b , harley c b , shay j w , lichtsteiner s , wright w e ( 1998 ) extension of life - span by introduction of telomerase into normal human cells . science 279 : 349 - 352 4 . brown c d , sayer r , windass a s , haslam i s , de broe m e , d &# 39 ; 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