p - glycoprotein ( abcb1/p - gp ) is a member of the atp binding cassette ( abc ) superfamily and is able to transport a broad range of uncharged and cationic compounds .
the apical localization of p - gp in renal proximal tubule epithelial cells is consistent with its importance in excretory transport into urine . in agreement with the detoxifying role of p - gp
, we recently found an upregulation of p - gp in rat kidney during endotoxemia   and in mouse kidney after ischemia reperfusion injury .
p - gp is likely to be regulated by nitric oxide ( no ) produced by renal inducible no - synthase ( inos ) , because co - administration of an inos - inhibitor attenuated the endotoxin - induced effects on its expression in rats .
an upregulation of important efflux pumps , like p - gp , may diminish the accumulation of toxic compounds and serves a protective function in acute kidney injury . using killifish renal proximal tubules
, we found previously that no has a regulatory role in the transport activity of multidrug resistance protein 2 ( abcc2/mrp2 ) via an intracellular signaling pathway in response to the in vitro action of several nephrotoxic chemicals .
this pathway involved at least endothelin ( et ) release , binding to the basolateral etb   receptor , and activation of nos , soluble guanylyl cyclase ( sgc ) and protein kinase c ( pkc ) .
a similar regulatory pathway was found for p - gp in rat brain capillaries , where it was recently shown to be correlated with an innate immune response [ 7 , 8 ] .
the inflammatory mediator , tumor necrosis factor  alpha ( tnf- ) signalled through the tnf - receptor 1 ( tnfr1 ) to increase p - gp expression and transport activity by triggering the et - nos - pkc pathway , finally activating the nuclear factor kappab , nf-b .  
as nf-b was previously shown to be involved in p - gp increase during renal toxicity [ 2 , 3 , 9 ] , we hypothesized that the same signaling pathway is responsible for the upregulation of p - gp in the kidney during endotoxemia and that no is the central player in the field . to test this hypothesis
, we used a spontaneously immortalized rat kidney proximal tubular cell line and determined p - gp expression and activity after treating cells with endotoxin ( lipopolysaccharide ; lps ) or the most important pro - inflammatory cytokine , tnf-. our findings indicate that exposure to tnf- in combination with lps increases p - gp activity in renal proximal tubule cells under influence of no produced by inos . upon exposure to tnf- alone
, p - gp upregulation seems to involve tlr4 activation and nf-b translocation , a pathway that is likely independent of no .
these findings indicate that at least two different pathways regulate p - gp expression during endotoxemia .
dulbecco 's modified eagle 's medium ( dmem ) , hanks ' balanced salt solution ( hbss ) , insulin - transferrin - selenium , and trizol reagent were from invitrogen life sciences ( breda , the netherlands ) , fetal calf serum from mp biomedicals ( asse - relegum , belgium ) , 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid ( hepes ) from roche diagnostics ( almere , the netherlands ) , calcein - acetoxymethylester ( calcein - am ) from molecular probes ( eugene , or ) , bisindolylmaleimide ( bim , bruschwig chemie , amsterdam , the netherlands ) , h398 from alexis ( lausen , switzerland ) , antihuman toll - like receptor ( tlr)4 cd284/md complex from biolegend ( san diego , usa ) , and 1h-[1,2 , 4]oxadiazolo[4,3 , -a]quinoxalin-1-one ( odq ) and sn50 from calbiochem ( nottingham , u.k . ) .
the p - gp inhibitor , psc-833 , was a gift from novartis pharma ( valspodar , arnhem , the netherlands ) .
all other chemicals were of analytical grade and purchased from sigma - aldrich ( zwijndrecht , the netherlands ) or merck ( darmstadt , germany ) .
the spontaneously immortalized epithelial cell line isolated from rat kidney proximal tubule ( gerp cells ) was a kind gift of the department of veterinary pharmacology , pharmacy and toxicology of the university of utrecht , the netherlands .
these cells ( passages 53201374 ) were cultured in collagen coated culture flasks in dmem supplemented with 5% fetal calf serum and 1% insulin - transferrin - selenium .
we previously demonstrated that p - gp activity in these cells was highest just after reaching confluency and decreased with culturing time .
cells were plated at a density of 40,00060,000  cells / well for 3 days in a 24-well plate .
culture medium was replaced for medium alone ( control group ) or medium supplemented with the tested compounds : lipopolysaccharide ( 10100  g / ml lps , escherichia coli 0127:b8 ) , or tnf- ( 1100  ng / ml ) , or the no - donor sodium nitroprusside ( 0.10.5  mm snp ) , or to a combination of lps and tnf- , with the no - scavenger 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide ( 0.010.5  mm ptio ) .
the concentration tnf- relates to plasma levels that can be reached in patients with septic conditions .
cells were also exposed to tnf- in combination with an inhibitor of sgc , odq , ( 0.01  mm ) , an inhibitor of pkc , bim , ( 100  nm ) , an inhibitor of tnfr1 , h398 , ( 10  g / ml ) , the anti - human tlr4 , cd284)/md complex , ( 5  g / ml ) , the selective i kappa b kinase ( ikk) inhibitor , imd-0354 , ( 1050  m ) , or the peptide inhibitor for nf-b nuclear translocation sn50 ( 11.8  m ) . after treatment the transport activity of p - gp was determined with the calcein accumulation assay , as previously described .
the activity was determined by calculating the ratio of cellular accumulation of fluorescent calcein in the presence and absence of the p - gp inhibitor , psc 833 , and control cells were set to 100% .
cells were exposed to medium ( control ) , lps , tnf- or to a combination of lps and tnf- for various time periods ( 2 , 6 , 24 hours ) and were , subsequently , harvested for rna isolation ( for 5 minutes at 1300  g at room temperature ) .
cell pellets were transferred in ice cold trizol reagent ( invitrogen , breda , the netherlands ) and rna was isolated as described previously .
quantitative real time - pcr ( rq - pcr ) on cdna was performed according to the taqman protocol in optical tubes using either the abi prism 7700 single reporter sequence detection system ( n = 6 , applied biosystems , zwijndrecht , the netherlands ) or the abi prism 7900ht gene expression micro fluidic card sequence detection system ( 3 pooled samples from 9 different rats , applied biosystems ) according to the manufacturer 's instructions .
all experiments were performed in triplicate . in rodents , two genes ( abcb1a and abcb1b ) encode for p - gp , however , during endotoxemia only abcb1b was found to be differentially expressed .
different rat genes ( gapdh : ( rn99999916_s1 ) , abcb1b : ( rn00561753_m1 ) , nosii : ( rn00561646_m1 ) , or ednrb / endothelin receptor b : rn00569139_m1 ) were amplified with a pre - developed gene expression assay , provided by applied biosystems .
the relative expression of genes in control cells were normalized for the average cycle threshold ( ct ) value for the housekeeping gene , gapdh ( ct = 15.9  1.0 ) and set to 1 . for the analysis of tnf- receptor 1 ( tnfr1 ) , tnfr2 , and toll - like receptor-4 ( tlr4 ) mrna expression in gerp cells exposed for 24 hours to tnf- , pcr amplification was performed as previously described .
gene - specific primers were purchased from biolegio ( nijmegen , the netherlands ) : rat tnfr1 ( m63122 ; nt.983 - 1382 ) , forward primer : gggattcagctcctgtcaaa , reversed primer ( atgaactccttccagcgtgt ; ( m63122 , nt.704 - 1902 ) forward primer : tcccctgtaaggagaaacagaa , reversed primer : gctttttctccacaatcacctc .
579 - 1034 ) , forward primer : gttctctgacaccacatcatcc , reversed primer : gtcaataggtgctgctgttcaa ; ( af498039 , nt.541 - 1242 ) , forward primer : aatggaaacgtgatatgcagtg , reversed primer : gctacagacgttcacgatgc .
rat tlr4 : ( nm_019178 , nt.2418 - 2546 ) , forward primer : gagccggaaagttattgtgg , reversed primer : agcaaggacttctccactttct .
the expected pcr products are : tnfr1-primer set 1 : 400  bp , tnfr1-primer set 2 : 1199  bp , tnfr2-primer set 1 : 456  bp , tnfr2-primer set 2 : 702  bp , tlr4 : 129  bp .
the expression of the housekeeping gene -actin was measured as a control for the performed pcr reaction .
cells were harvested after exposure to different inflammatory mediators and lysed with 0.1% triton x-100 supplemented with protease inhibitors ( 1  mm pmsf , 10  m e64 , 1  g / ml pepstatin , 5  g / ml leupeptin and 1  g / ml aprotinin ) during 30 minutes on ice .
the amount of protein in cell lysates was determined with the bio - rad protein assay ( bio - rad laboratories , hercules , ca ) using bovine serum albumin as standard and samples were subjected to western blotting as described previously .
samples were separated on a 6% sodium dodecylsulfate polyacrylamide gel and transferred to hybond - c pure nitrocellulose membrane ( amersham , buckinghamshire , uk ) .
the membrane was incubated overnight at 4c with the primary antibodies against inos ( 1 : 1000 , according to ) , abcb1/p - gp ( 1 : 200 , c219 , dakocytomation , denmark ) , or -actin ( 1 : 10000 , sigma - aldrich ) in tris - buffered saline supplemented with 0.1% tween-20 ( tbs - t ) and 1% non - fat dried milk . subsequently
, the membranes were washed three times in tbs - t , blocked in tbs - t containing 5% non - fat dried milk , washed three times in tbs - t again , and incubated with affinity - purified horseradish peroxidase - conjugated goat antirabbit igg ( sigma - aldrich ) for inos or goat antimouse igg ( sigma - aldrich ) for p - gp and -actin diluted 1 : 5000 in tbs - t for 1 hour at room temperature .
the washing steps were repeated , after which the membranes were visualized with enhanced chemiluminescence ( pierce chemical , rockford , il ) . for semiquantification , the pixel intensity of the bands was measured using scion image version beta 4.02 for windows ( scion corporation , frederick , md ) .
, san diego , ca , usa ) and spss ( version 12.0.1 for windows , chicago , il ) . differences between the experimental groups were tested using one - way anova with bonferroni 's correction or two - way repeated measurements anova .
treatment of gerp cells with 10  g / ml lps and/or 10100  ng / ml tnf- for 24 hours resulted in an increase in p - gp activity ( figures 1(a)1(c ) , p < .05 ) .
as 10  ng / ml is a clinically relevant concentration   and a 10-fold higher tnf- concentration or combination of tnf- and lps did not further increase p - gp activity , we used a concentration of 10  ng / ml tnf- for additional experiments . in accordance
, we also observed an upregulation in abcb1b expression after exposure to tnf- , lps , or a combination of tnf- and lps ( figure 1(d ) ) , which was accompanied by an increase in p - gp protein expression ( figure 1(e ) ) , suggesting de novo p - gp synthesis .
this finding is in agreement with previous studies in mice and rats in which the upregulation of renal p - gp was demonstrated after in vivo lps exposure [ 2 , 19 , 20 ] .  
we previously suggested that p - gp is likely to be regulated by no produced by inos , as coadministration of aminoguanidine reversed the induction in inos , reduced renal damage and attenuated the endotoxin - induced effects on its expression in rats . in agreement
, we show here that both inos mrna ( figure 2(a ) , p < .001 ) and protein ( figure 2(b ) ) are upregulated when cells are exposed to lps , either alone or in combination with tnf-. treatment with tnf- alone , however , only marginally induced nosii mrna , but had no effect on inos protein ( figures 2(a ) , and 2(b ) ) .   to determine the no - dependency of the upregulation of p - gp , gerp cells were co - treated with the no - scavenger ptio or exposed to the no donor snp . indeed , the p - gp efflux activity was not significantly different from control anymore after co - treatment with ptio ( figure 2(c ) ) .
remarkably , snp , did not affect abcb1b ( data not shown ) and p - gp protein expression ( figure 2(d ) ) and its activity was slightly , though not significantly , upregulated after 1 hour exposure and 5 hours recovery ( figure 2(e ) ) .
this is in contrast with previous findings demonstrating that no , released by the no donor s - nitroso - n - acetylpenicillamine , was able to upregulate p - gp expression in the human caco2 cell line .
however , snp produces besides no also iron and cyanide , so it is not clear whether only effects of no on p - gp expression and activity were measured .
in contrast to lps , our findings indicate that p - gp signaling seems largely no - independent after exposure to tnf-. in addition , this signaling pathway may be dependent on the type of cytokine as no mediated increased p - gp activity after stimulation with interferon- .
furthermore , it was recently demonstrated that in vivo inhibition of nos and subsequent no production with l - name did not affect blood - brain barrier p - gp function during endotoxemia , which contradicts with previous in vitro findings in isolated rat brain capillaries [ 7 , 8 ] .   to dissect the signaling pathway
through which exposure to tnf- increased p - gp expression and activity we used various pharmacological agents . since no is a well - known activator of sgc , which is in turn involved in the pkc signalling pathway , the effects of the sgc inhibitor odq ( figure 3(a ) ) and the pkc inhibitor bim ( figure 3(b ) ) on p - gp activity were examined .
importantly , none of the two substances could prevent the upregulation of p - gp caused by tnf- , suggesting that a novel pathway mediates p - gp activity in gerp cells and/or that the no - sgc - pkc pathway is not activated in our cell line .
this finding is in contrast with previous studies using killifish renal proximal tubules and brain capillary membranes [ 47 ] .
lps signals through toll - like receptor-4 ( tlr-4 ) and tnf- mediates their effect by binding to tnfr1 ( p55 ) and tnfr2 ( p75 ) , which are known to be expressed in the kidney and their expression patterns are modulated during immune - mediated and ischemic renal injury [ 24 , 25 ] .
figure 4(a ) shows that tnfr1 and tlr4 are expressed in the gerp cell line , whereas tnfr2 expression could not be detected . to confirm the results of the pcr analysis , we used inhibitors of and/or antibodies against tnfr1 ( h398 , figure 4(b ) ) and tlr4 ( anti - tlr4 , figure 4(c ) ) and determined p - gp activity .
exposure to h398 could not prevent the upregulation of p - gp by tnf- , suggesting that although tnfr1 is expressed in gerp cells it may not be involved in p - gp regulation ( figure 4(b ) ) .
many cell types require cooperation between tnfr1 and tnfr2 to generate tnf responses , and the absence of tnfr2 in our cells could explain why tnfr1 did not affect the regulation of p - gp activity by tnf-. on the other hand , inhibition of tlr4 resulted in an attenuation in p - gp activity ( figure 4(c ) ) , and an involvement of this toll - like receptor is in agreement with previous findings in brain capillaries .
since the transcription factor nf-b is the downstream effector of tnf- signalling in brain capillaries , we wondered whether the same holds true for the regulation of p - gp activity by tnf- in this proximal tubular cell line .
gerp cells were exposed for 24 hours to the selective i kappa b kinase ( ikk) inhibitor , imd-0354 ( figure 5(a ) ) , or sn50 ( figure 5(b ) ) .
remarkably , imd-0354 alone gave a significant upregulation of p - gp activity compared to control cells ( figure 5(a ) , p < .01 ) , indicating that the compound itself has an effect on p - gp .
an alternative explanation would be that imd-0354 may have potentiated the responses to tnf- and , therefore , nf-b inhibits p - gp activity and blockade of nuclear translocation of this transcription factor leads to enhancement of tnf- induction . exposing the cells to the combination of sn50 , a cell - permeable inhibitor peptide that interferes with nf-b nuclear translocation , and
tnf- slightly reversed the increase in p - gp activity upon tnf- treatment ( figure 5(b ) ns ) , suggesting a role for the transcription factor .
although higher sn50 concentrations have been used before   this was not feasible in our cell system due to solubility problems .
showed that an upregulation of p - gp by nf-b is potentially important for protecting renal proximal tubule cells from apoptosis induced by cadmium- and reactive oxygen species ( ros ) .
in addition , an increase in p - gp expression and/or activity by tnf- was reported for primary rat hepatocytes , a rat hepatoma cell line   and mouse liver , which are also nf-b dependent .
furthermore , an nf-b binding site was found in the promoter region of the abcb1 gene .
therefore , we suggest that nf-b might have a central role in the signal transduction mechanism and could be involved in two distinct signalling pathways of p - gp .
this nuclear factor is probably activated directly by proinflammatory cytokines and no , and indirectly via tlr4 . however , more research is needed to further unravel the regulation mechanism of p - gp in the gerp cell line and the inflammatory response on the activity of the efflux pump .
in accordance with previous rat endotoxemia studies , the upregulation of p - gp expression and activity after exposure to lps in combination with tnf- in the cultured renal proximal tubule cells are under influence of no produced by inos .
on the other hand , the signaling pathway of tnf- alone leading to p - gp upregulation seems to involve tlr4 activation and nf-b translocation , which results in de novo synthesis of p - gp .
our findings indicate that at least two different pathways regulate p - gp in renal proximal tubule cells during endotoxemia .
 during endotoxemia , the atp - dependent drug efflux pump p - glycoprotein ( abcb1/p - gp ) is upregulated in kidney proximal tubule epithelial cells . 
 the signaling pathway through which lipopolysaccharide ( lps ) or tumor necrosis factor- ( tnf- ) regulates p - gp expression and activity was investigated further in the present study . 
 exposure of rat kidney proximal tubule cells to tnf- alone or tnf- and lps increased p - gp gene and protein expression levels and efflux activity , suggesting de novo p - gp synthesis . upon exposure to tnf- in combination with lps , p - gp activity in renal proximal tubule cells 
 is increased under influence of nitric oxide ( no ) produced by inducible no synthase . upon exposure to tnf- alone 
 , p - gp upregulation seems to involve tlr4 activation and nuclear factor kappab ( nf-b ) translocation , a pathway that is likely independent of no . 
 these findings indicate that at least two pathways regulate p - gp expression in the kidney during endotoxemia . 
1. Introduction
2. Materials and Methods
3. Results and Discussion
4. Conclusions