Adenosine A2A receptors modulate BDNF both in normal conditions and in experimental models of Huntington 's disease Brain-derived neurotrophic factor -LRB- BDNF -RRB- , a member of the neurotrophin family , enhances synaptic transmission and regulates neuronal proliferation and survival . Functional interactions between adenosine A2A receptors -LRB- A2ARs -RRB- and BDNF have been recently reported . In this article , we report some recent findings from our group showing that A2ARs regulate both BDNF functions and levels in the brain . Whereas BDNF -LRB- 10 ng/ml -RRB- increased the slope of excitatory postsynaptic field potentials -LRB- fEPSPs -RRB- in hippocampal slices from wild-type -LRB- WT -RRB- mice , it was completely ineffective in slices taken from A2AR knock-out -LRB- KO -RRB- mice . Furthermore , enzyme immunoassay studies showed a significant reduction in hippocampal BDNF levels in A2AR KO vs. WT mice . Having found an even marked reduction in the striatum of A2AR KO mice , and as both BDNF and A2ARs have been implicated in the pathogenesis of Huntington 's disease -LRB- HD -RRB- , an inherited striatal neurodegenerative disease , we then evaluated whether the pharmacological blockade of A2ARs could influence striatal levels of BDNF in an experimental model of HD-like striatal degeneration -LRB- quinolinic acid-lesioned rats -RRB- and in a transgenic mice model of HD -LRB- R6/2 mice -RRB- . In both QA-lesioned rats and early symptomatic R6/2 mice -LRB- 8 weeks -RRB- , the systemic administration of the A2AR antagonist SCH58261 significantly reduced striatal BDNF levels . These results indicate that the presence and the tonic activation of A2ARs are necessary to allow BDNF-induced potentiation of synaptic transmission and to sustain a normal BDNF tone . The possible functional consequences of reducing striatal BDNF levels in HD models need further investigation . Introduction Brain-derived neurotrophic factor -LRB- BDNF -RRB- is an endogenous glycoprotein belonging to neurotrophins , a family of signalling molecules that play a key role in regulating neuronal proliferation , differentiation and survival -LSB- 1 -- 3 -RSB- . Among the neurotrophins , BDNF has the widest distribution in the central nervous system -LSB- 4 , 5 -RSB- . In the adult hippocampus , BDNF is critically involved in the regulation of synaptic plasticity -LSB- 6 -RSB- and facilitates long-term potentiation -LRB- LTP -RRB- -LSB- 7 -- 10 ; see 11 for review -RSB- . Most biological effects of BDNF are mediated by the tyrosine kinase receptor TrkB . Both BDNF and its receptors are highly expressed in the hippocampus , and the activation state of the complex BDNF/TrkB appears critical for modulating synaptic efficacy -LSB- 12 -RSB- and the response to excitotoxic injury -LSB- 13 , 14 -RSB- . In hippocampal neurones , adenosine has been reported to transactivate TrkB receptors , an effect involving the A2A receptor subtype -LRB- A2ARs -RRB- -LSB- 15 -RSB- . Furthermore , Diogenes et al. -LSB- 16 -RSB- reported that A2ARs facilitate the excitatory action of BDNF on hippocampal synaptic transmission . Besides its involvement in hippocampal functions , BDNF is also very important for the survival of striatal neurons and the activity of corticostriatal synapses -LSB- 17 -RSB- . Impairment in BDNF function is though to play a major role in Huntington 's disease -LRB- HD -RRB- , an inherited neurodegenerative disease caused by a mutation in the protein huntingtin and characterised by marked striatal degeneration -LRB- see -LSB- 18 -RSB- for review -RRB- . Specifically , it has been shown that the activity of BDNF depends on the presence of normal huntingtin -LSB- 19 -- 21 -RSB- . In vitro and in vivo data showed that wild-type huntingtin , but not the mutant protein , stimulates cortical BDNF production by acting at the level of Bdnf gene transcription -LSB- 20 , 21 -RSB- . At the corticostriatal synapse , BDNF controls glutamate release and its exogenous administration allows striatal neurons to survive excitotoxin-induced neurodegeneration -LSB- 22 -RSB- . Mounting evidence also indicates an involvement of striatal A2A receptors in HD -LRB- see -LSB- 23 -RSB- and -LSB- 24 -RSB- for reviews -RRB- . First , A2ARs are mainly localised on the neurons that degenerate early in HD -LSB- 25 -RSB- . Second , A2ARs and underlying signalling systems undergo profound changes in cellular and animal models of HD -LRB- see -LSB- 24 -RSB- for review -RRB- . Thus , not only A2ARs seem to regulate BDNF functions in the brain , but both BDNF and A2ARs seem to be implicated in HD . In this article we report and critically discuss some recent findings on the following issues obtained by our group : -LRB- 1 -RRB- the regulatory role exerted by hippocampal A2ARs on BDNF functions and levels as revealed by studies performed in A2AR KO mice , and -LRB- 2 -RRB- the effects of A2AR blockade on striatal BDNF levels in experimental models of HD or HD-like striatal degeneration . In hippocampal slices from wild-type mice , the facilitatory effects exerted by BDNF on synaptic transmission require the endogenous activation of A2AR In hippocampal slices from wild-type -LRB- WT -RRB- mice -LRB- 400-μm thick , see -LSB- 26 -RSB- for a detailed methodological description -RRB- , BDNF -LRB- 10 ng/ml over 30 min -RRB- induced a long-lasting increase in the slope of field excitatory postsynaptic potential -LRB- fEPSP -RRB- recorded in the CA1 area -LRB- Fig. 1 -RRB- . This effect was abolished not only by the inhibitor of Trk phosphorylation K-252a , but also by the selective adenosine A2A antagonists ZM241385 and SCH58261 -LRB- Tebano et al. , manuscript in preparation -RRB- . The effects of A2ARs seem to involve the cyclic adenosine monophosphate/protein kinase A -LRB- cAMP-PKA -RRB- pathway , the main transduction mechanism operated by A2ARs , as the selective PKA inhibitor KT 5720 abolished the excitatory action of BDNF -LRB- Tebano et al. , manuscript in preparation -RRB- . Fig. 1Brain-derived neurotrophic factor -LRB- BDNF -RRB- facilitates synaptic transmission in hippocampal slices from wild-type -LRB- WT -RRB- but not A2A receptor -LRB- R -RRB- knock-out -LRB- KO -RRB- mice . In hippocampal slices from WT mice , BDNF -LRB- 10 ng/ml -RRB- induces an increase of the excitatory postsynaptic field potentials -LRB- fEPSP -RRB- slope , whereas a lower concentration -LRB- 5 ng/ml -RRB- is ineffective . BDNF -LRB- 5 , 10 and 20 ng/ml -RRB- was totally ineffective in hippocampal slices from A2AR KO mice . * P < 0.05 vs. baseline -LRB- paired t test -RRB- The finding that BDNF enhances synaptic transmission in mice hippocampal slices confirms and strengthens previous data on the acute synaptic effects of BDNF at adult central synapses -LSB- 17 , 27 -RSB- . Although the mechanisms responsible for such synaptic effects are not completely understood , they do not seem to be related to the neuroprotective ability of BDNF . That the above functions of BDNF recognise different molecular mechanisms is indicated by the fact that ligand-induced TrB translocation into lipid rafts is required for short-term synaptic modulation , but not neuronal survival , by BDNF -LSB- 28 -RSB- . Whatever the mechanisms , the blocking effect of ZM241385 and SCH58261 suggests that A2ARs have to be tonically activated by endogenous adenosine to manifest BDNF effects . To further explore the apparently `` permissive '' role of adenosine A2ARs on the synaptic effects of BDNF , A2AR KO mice were used . BDNF is no longer able to facilitate synaptic transmission in hippocampal slices from A2A KO mice A2AR KO mice -LRB- A2AR - / - -RRB- were generated , as previously described -LSB- 29 -RSB- . In hippocampal slices from A2AR KO mice , BDNF was no longer able to increase the fEPSP slope -LRB- Tebano et al. , manuscript in preparation -RRB- . As shown in Fig. 1 , none of the tested concentrations of BDNF -LRB- 5 , 10 and 20 ng/ml -RRB- potentiated the synaptic response in slices from A2AR KO mice . Since the effectiveness of BDNF may depend on the proper expression of its receptors , we compared the levels of TrkB protein in WT and A2A KO mice by Western blotting . By using primary anti-TrkB antibody -LRB- Bioscience -RRB- , we found that the levels of full-length TrkB isoforms were very similar in hippocampal extracts from WT and A2AR KO mice -LRB- Tebano et al. , manuscript in preparation -RRB- . Thus , the loss of BDNF activity observed in A2AR KO mice is not associated with a reduced density of TrkB receptors . BDNF protein levels are significantly reduced in the brain of A2AR KO vs. WT mice The BDNF protein levels were then measured on hippocampal extracts from A2AR KO and WT mice using the BDNF immunoassay system -LSB- enzyme-linked immunosorbent assay -LRB- ELISA -RRB- kit ; Promega -RSB- . Interestingly , hippocampal BDNF levels were significantly lower in the A2AR KO mice compared with the WT littermates . As shown in Fig. 2 , such an effect was not confined to the hippocampus , as a significant loss of BDNF levels was observed also in the striatum of A2A KO vs. WT mice . Fig. 2Brain-derived neurotrophic factor -LRB- BDNF -RRB- levels are significantly reduced in the hippocampus and striatum of A2A receptor -LRB- R -RRB- knock-out -LRB- KO -RRB- mice . BDNF concentration was significantly reduced in extracts of hippocampal and striatal tissues prepared from A2AR mice compared with wild-type WT mice . The amount of BDNF -LRB- total -RRB- is expressed as pg/mg of wet tissue -LSB- means ± standard error of the mean -LRB- SEM -RRB- from five mice/group -RSB- . * P < 0.05 vs. WT This result indicates that the presence of A2ARs is required to maintain normal BDNF levels in the brain . Although the mechanism by which A2ARs regulate BDNF is yet undetermined , it is worth mentioning that the cAMP-PKA pathway -LRB- the main transduction system operated by A2ARs -RRB- has been implicated not only in `` gating '' BDNF functions -LSB- 30 -RSB- , but also in modulating BDNF gene transcription -LSB- 31 -RSB- and release -LSB- 32 -RSB- . Furthermore , Kolarow et al. -LSB- 33 -RSB- showed that the PKA inhibitor Rp-cAMP significantly inhibited and delayed BDNF secretion in hippocampal neurons . Interestingly , the endogenous state of activation of A2ARs seems to be adequate to sustain a normal BDNF tone in WT mice , as no increase in the hippocampal levels of BDNF were observed in WT mice treated with a single -LRB- 0.5 mg/kg i.p sacrifice 24 h later -RRB- or with repeated -LRB- 0.5 mg/kg per day over 5 days -RRB- doses of the A2A agonist CGS 21680 . On their whole , the experiments performed in A2A KO mice indicate that A2ARs are essential for the tone and synaptic activity of BDNF . It should be noted , however , that the stimulation or the blockade of A2ARs can induce effects very different from those achieved after stimulation or blockade of the BDNF/TRkB system . For instance , whereas BDNF is thought to play an important role in memory processes -LSB- 34 , 35 -RSB- , adenosine A2ARs seem to negatively influence them . Indeed , an improvement in spatial memory has been reported in A2AR KO mice -LSB- 36 -RSB- , whereas working memory was impaired in rats overexpressing A2ARs -LSB- 37 -RSB- . Another consideration that apparently weakens the importance of the A2AR/BDNF interaction is that A2ARs and TrkB Rs undergo opposite age-related changes in the hippocampus -LSB- 38 -RSB- . According to the authors ' interpretation , however , the relationship between age-related changes in the density of TrkB and A2AR receptors does play a role in the modulation of BDNF effects , thus even reinforcing the hypothesis of a close functional interplay between A2ARs and BDNF . A2ARs regulate striatal BDNF levels in experimental models of HD or HD-like striatal degeneration Two different models of HD were used : the quinolinic-acid -LRB- QA -RRB- - lesioned rats -LRB- pathogenetic model of HD-like striatal degeneration , -LSB- 39 -RSB- -RRB- and transgenic R6/2 mice -LRB- genetic HD model , -LSB- 40 -RSB- -RRB- . For the method of QA-induced striatal lesions , see -LSB- 41 -RSB- . Briefly , QA -LRB- 180 nmol/1 μl -RRB- was unilaterally injected in the striatum of anaesthetised rats , whereas vehicle -LSB- 1 μl phosphate-buffered saline -LRB- PBS -RRB- -RSB- was injected in the other side . SCH58261 was administered at the doses of 0.01 and 1 mg/kg i.p. 20 min before QA lesion . The levels of BDNF were measured 24 h after surgery . As shown in Fig. 3 , BDNF levels were significantly increased in the QA-lesioned vs. the control side . In animals pretreated with both doses of SCH58261 the rise in striatal BDNF was completely prevented . Worthy of note , SCH58261 markedly reduced BDNF levels in the control -LRB- vehicle-injected -RRB- side also -LRB- Fig. 3 -RRB- . As the early rise in BDNF levels most probably reflects a compensatory mechanism following QA injection , one can consider that SCH58261 may worsen QA effects by preventing BDNF increase . Previous studies have , however , demonstrated that this is not the case . We found , indeed , that whereas SCH58261 exerted very clear neuroprotective effects towards QA at 0.01 mg/kg i.p. , it was no longer neuroprotective when administered at 1 mg/kg -LSB- 39 -RSB- . The finding that SCH58261 reduces BDNF levels irrespective of the dose -LRB- neuroprotective or non-neuroprotective -RRB- indicates that early changes in the striatal BDNF levels do not seem to correlate with the clinical and neuropathological outcome in the QA model . Fig. 3The A2A receptor -LRB- R -RRB- - antagonist SCH58261 reduces striatal brain-derived neurotrophic factor -LRB- BDNF -RRB- levels in control and quinolinic-acid -LRB- QA -RRB- - lesioned rats . QA -LRB- 180 nmol/1 μl -RRB- was unilaterally injected in the striatum of anaesthetised rats , whereas vehicle -LSB- 1 μl phosphate-buffered saline -LRB- PBS -RRB- -RSB- was injected in the other side . SCH58261 was administered at the doses of 0.01 and 1 mg/kg i.p. , 20 min before QA lesion . The levels of free mature BDNF were measured 24 h after surgery . BDNF levels were significantly increased in the QA-lesioned vs. the control side . In animals pretreated with both doses of SCH58261 , the rise in striatal BDNF was completely prevented . N 5/group . ° P < 0.05 vs. PBS . * P < 0.05 vs. corresponding vehicle R6/2 HD mice may represent a more suitable model to evaluate whether A2AR antagonists have a worsening influence through the reduction of striatal BDNF levels . Indeed , the administration of BDNF has been reported to have beneficial effects in transgenic models of HD -LSB- 42 , 43 -RSB- . Considering that in the transgenic mice the neurodegenerative process progressively takes place over several weeks , to ascertain whether A2AR blockade could influence striatal BDNF levels , we decided to treat R6/2 mice chronically with SCH58261 . The drug was administered at the dose of 0.01 mg/kg per day i.p. over 1 or 3 weeks starting from the fifth week of age . Another group of R6/2 mice was treated with vehicle over 3 weeks . BDNF levels were then assayed at the beginning of the symptomatic phase -LRB- 8 weeks of age -RRB- . Three groups of age-matched WT littermates were used as controls and treated in the same way described as above . As shown in Fig. 4 , BDNF protein levels were not reduced , and showed instead a nonsignificant tendency to increase , in the striatum of early symptomatic R6/2 vs. WT mice . This finding was unexpected , as a significant decrease in BDNF mRNA has been recently reported in the striatum of R6/2 mice of the same age -LSB- 43 -RSB- . Although we have not explored the mechanisms responsible for such a discrepancy , a dissociation between the levels of BDNF protein and mRNA has already been observed in the brains of transgenic R6/1 HD mice -LSB- 44 -RSB- . Another reason for which one would expect a reduction in BDNF levels in the striatum of R6/2 mice is that a marked reduction in the expression of A2ARs -LRB- which , as mentioned above , are very important to maintain normal BDNF levels -RRB- was reported in these mice -LSB- 45 -RSB- . More recently , however , the A2AR-stimulated adenylyl cyclase , the A2AR density , and their affinity for the agonist CGS21680 were found unchanged in the striatum of clearly symptomatic R6/2 mice -LSB- 46 -RSB- . Fig. 4The A2A receptor -LRB- R -RRB- - antagonist SCH58261 reduces striatal brain-derived neurotrophic factor -LRB- BDNF -RRB- levels in R6/2 HD mice SCH58261 was administered at the dose of 0.01 mg/kg per day i.p. over 1 or 3 weeks starting from the fifth week of age . Free mature BDNF levels were then assayed at the beginning of the symptomatic phase -LRB- 8 weeks of age -RRB- . Three groups of age-matched wild-type -LRB- WT -RRB- littermates were used as controls and treated in the same way as described above . Both schedules -LRB- 1 and 3 weeks -RRB- of SCH treatment significantly reduced BDNF levels in the striatum of either R6/2 and WT mice . N 4 -- 7/group . * P < 0.05 vs. corresponding control group -LRB- two-tailed unpaired t test -RRB- As for the influence of A2AR blockade , both schedules -LRB- 1 and 3 weeks -RRB- of SCH treatment significantly reduced BDNF levels in the striatum of either R6/2 and WT mice -LRB- Fig. 4 -RRB- . Again , however , such an effect does not necessarily imply a negative influence of SCH58261 on HD mice . Indeed , in recent experiments , we found that SCH treatment -LRB- 0.01 mg/kg per day i.p. between the 5th and the 6th weeks of age -RRB- exerted some beneficial effects , namely , normalisation of emotional behaviour and restoration of a normal sensitivity to NMDA in corticostriatal slices -LRB- Domenici et al. , manuscript in preparation -RRB- . Thus , although the pharmacological blockade of A2ARs significantly reduces striatal BDNF levels in experimental models of HD or HD-like neurodegeneration , this does not seem to have a negative impact on the course of the disease . Of course , it is still possible that reducing BDNF levels at other time points of the pathological process has a much marked influence on the disease . This issue , as well as the evaluation of BDNF protein changes at different stages of the disease , will be the object of future studies . Conclusions Over the last several years , BDNF has emerged not only as a potent neuromodulator but also as a substance exerting fast excitatory actions in neurons , controlling resting membrane potential , neuronal excitability and synaptic transmission and participating in the induction of long-term changes in synaptic transmission -LSB- 47 -RSB- . In agreement with the recent evidence that A2ARs play a major role in regulating BDNF functions -LSB- 16 , 48 -RSB- , data reported in this article indicate that the presence and tonic activation of A2ARs are necessary to allow BDNF-induced potentiation of synaptic transmission . As for the mechanisms responsible for the permissive effects of A2ARs , data indicate an involvement of the cAMP-PKA pathway , the main transduction mechanism operated by A2ARs . The finding of reduced BDNF levels in the brain of A2AR KO mice indicates that A2ARs are critical not only to allow the synaptic effects of BDNF but also to maintain normal BDNF levels . Findings also point out that A2ARs tonically regulate BDNF levels in different areas of the brain , irrespective of their levels of expression . The tonic effect exerted by A2ARs in the regulation of BDNF levels is also evident in experimental models of HD -LRB- R6/2 mice -RRB- and of HD-like striatal degeneration -LRB- QA lesion in rats -RRB- . Given that BDNF delivery has beneficial effects in models of HD -LSB- 42 , 43 -RSB- , the possible functional consequences of reducing striatal BDNF levels in those models need to be clarified . In particular , further investigations on trophic factors and associated signal pathways are warranted before A2AR antagonists can be considered as a suitable therapeutic approach to HD -LSB- 24 -RSB- .

B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O B I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O 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O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

adenosine a2a receptors
bdnf levels
hippocampus
striatum
synaptic transmission


Nitric oxide differentially regulates renal ATP-binding cassette transporters during endotoxemia Nitric oxide -LRB- NO -RRB- is an important regulator of renal transport processes . In the present study , we investigated the role of NO , produced by inducible NO synthase -LRB- iNOS -RRB- , in the regulation of renal ATP-binding cassette -LRB- ABC -RRB- transporters in vivo during endotoxemia . Wistar -- Hannover rats were injected with lipopolysaccharide -LRB- LPS + -RRB- alone or in combination with the iNOS inhibitor , aminoguanidine . Controls received detoxified LPS -LRB- LPS − -RRB- . After LPS + , proximal tubular damage and a reduction in renal function were observed . Furthermore , iNOS mRNA and protein , and the amount of NO metabolites in plasma and urine , increased compared to the LPS − group . Coadministration with aminoguanidine resulted in an attenuation of iNOS induction and reduction of renal damage . Gene expression of 20 ABC transporters was determined . After LPS + , a clear up-regulation in Abca1 , Abcb1/P-glycoprotein -LRB- P-gp -RRB- , Abcb11/bile salt export pump -LRB- Bsep -RRB- , and Abcc2/multidrug resistance protein -LRB- Mrp2 -RRB- was found , whereas Abcc8 was down-regulated . Up-regulation of Abcc2/Mrp2 was accompanied by enhanced calcein excretion . Aminoguanidine attenuated the effects on transporter expression . Our data indicate that NO , produced locally by renal iNOS , regulates the expression of ABC transporters in vivo . Furthermore , we showed , for the first time , expression and subcellular localization of Abcb11/Bsep in rat kidney . Introduction The free radical nitric oxide -LRB- NO -RRB- has been shown to play an important role in various physiological processes in the kidney , including salt and fluid reabsorption , renal hemodynamics , renin secretion , and tubuloglomerular feedback -LSB- 1 -RSB- . Endogenous NO is enzymatically produced from conversion of the amino acid l-arginine to l-citrulline , a reaction that is catalyzed by the enzyme NO synthase -LRB- NOS -RRB- . This enzyme exists in three isoforms ; the neuronal -LRB- nNOS -RRB- and endothelial , both of which are Ca2 + - and calmodulin-dependent constitutive isoforms , and an inducible -LRB- iNOS -RRB- isoform -LSB- 2 -RSB- . Mediators associated with sepsis , such as endotoxin and the proinflammatory cytokines , interleukin-1β -LRB- IL-1β -RRB- , IL-2 , and tumor necrosis factor-α -LRB- TNF-α -RRB- , have been shown to induce iNOS , thereby increasing production of NO . Sustained , high-output generation of NO by iNOS can cause cell death and tissue damage through lipid peroxidation , DNA damage , and proapoptotic effects -LSB- 3 -RSB- . This is the result of NO itself and the oxidant peroxynitrite , which is generated after reaction of NO with superoxide anions -LSB- 4 -RSB- . In contrast to many other organs , iNOS is constitutively expressed in the kidney , in particular in the medulla and in proximal tubules -LSB- 5 -RSB- . Many in vivo animal studies showed the importance of iNOS as a contributor to the pathophysiology of acute kidney injury -LRB- AKI -RRB- . For example , it has been shown that selective chemical inhibitors of iNOS -LSB- 6 , 7 -RSB- , NO scavengers -LSB- 8 -RSB- , iNOS gene knockout mice -LSB- 9 -RSB- , or antisense oligodeoxynucleotides to iNOS -LSB- 10 -RSB- resulted in less renal tubular injury and improved structural and functional outcome . This renoprotective effect was partly due to the rescue of tubular epithelial cells from injury by NO -LSB- 3 -RSB- . Using killifish renal proximal tubules , we found previously that NO has a regulatory role in the transport activity of the ATP-binding cassette -LRB- ABC -RRB- transporter multidrug resistance protein 2 -LRB- Abcc2/Mrp2 -RRB- via an intracellular signaling pathway in response to the action of several nephrotoxic chemicals in vitro -LSB- 11 -RSB- . This pathway involved at least endothelin -LRB- ET -RRB- release , binding to the basolateral ETB receptor , and activation of NOS , soluble guanylyl cyclase , and protein kinase C -LSB- 12 -RSB- . A similar regulatory pathway was found for the ABC transporter P-glycoprotein -LRB- Abcb1/P-gp -RRB- in rat brain capillaries -LSB- 13 -RSB- . Both studies indicate that the activation of NOS is most likely Ca2 + - independent and we concluded that the production of NO is a result of the activation of iNOS . The proximal tubule is responsible for the excretory transport of xenobiotics , xenobiotic metabolites , and waste products of metabolism from blood into urine . Consequently , the proximal tubule is also an important target for toxic effects and is often the first site of damage in AKI -LSB- 14 -RSB- . Luminal transport proteins , like the ABC transporters ABCB1/P-gp , ABCC2/MRP2 , and ABCC4/MRP4 , are important efflux transporters in the kidney -LSB- 15 -RSB- and , therefore , play a role in the detoxification of tubule cells . The observed effects of NO on Abcc2/Mrp2 in killifish proximal tubule may be part of the normal progression of cellular events that occur during AKI . In the present study , we hypothesized that iNOS has a role in the regulation of renal ABC transporters during nephrotoxicity in vivo . An endotoxemia model was used in which rats were exposed to lipopolysaccharide -LRB- LPS -RRB- , and the gene expression of 20 ABC transporters was evaluated . In addition , we examined the effect of the iNOS inhibitor , aminoguanidine , to determine whether the change in expression of the renal efflux transporters is caused directly by NO generated by iNOS . Materials and methods Animals and experimental design Specified pathogen-free male Wistar -- Hannover rats -LRB- 240 -- 295 g ; purchased from Harlan , Zeist , The Netherlands -RRB- were kept under routine laboratory conditions at the Central Animal Laboratory of the Radboud University Nijmegen Medical Centre . This strain was used because they do not develop hydronephrosis spontaneously like normal Wistar rats , which in turn could have a down-regulating effect on iNOS expression -LSB- 16 -RSB- . The local Animal Care Committee approved all experimental procedures . Different pilot studies have been conducted to determine the route of administration , intravenously or intraperitoneally -LRB- IP -RRB- , concentration of the endotoxin LPS -LRB- LPS + , Escherichia coli 0127 : B8 , Sigma-Aldrich , Zwijndrecht , The Netherlands -RRB- and aminoguanidine -LRB- Sigma-Aldrich -RRB- , and the time point of coadministration in this strain . LPS and aminoguanidine were dissolved in sterile Hank 's balanced salt solution -LRB- HBSS , Gibco , Paisly , UK -RRB- supplemented with 10 mM 4 - -LRB- 2-hydroxyethyl -RRB- -1-piperazine ethanesulfonic acid -LRB- HEPES , Gibco -RRB- , and set to pH 7.4 with NaOH . Thirty rats were , stratified to body weight , injected IP with 5 mg/kg LPS + and were killed after 3 , 6 , 12 , 24 , or 48 h -LRB- n = 6 for each experimental group -RRB- . Fifteen rats served as controls and received 10 mg/kg detoxified LPS -LRB- LPS − , n = 3 per group -RRB- , in which the biologically active component , lipid A , essential for the activation of NOS , is lacking -LSB- 17 -RSB- . Although the control group is small , standard errors appeared to be satisfactory to allow a reduction and refinement of animals in this group . Twelve rats were injected IP with both 5 mg/kg LPS + and , after 1 h , 100 mg/kg of the iNOS inhibitor aminoguanidine -LRB- Sigma-Aldrich -RRB- , and were killed 6 and 12 h after LPS + -LRB- n = 6 per group -RRB- . For the isolated perfused kidney experiments , six rats received LPS + , four received LPS − , and four were untreated and served as controls , and were killed after 12 h as described under the `` Isolated kidney perfusion '' section . To collect urine before and after the injection , rats were housed in metabolic cages , washed with 2 % chlorohexidine -LRB- Genfarma , Zaandam , The Netherlands -RRB- to prevent bacterial growth . Urine volumes were recorded and samples were snap-frozen in liquid nitrogen and stored at − 80 °C until assayed . During the experiment , body weight and body temperature were determined . At indicated time points after LPS injection , the rats were anesthetized with 5 % isoflurane and heparin -LRB- 125 IU/100 g -RRB- was injected via the penile vein to minimize blood clotting . The left kidney was removed , divided in four pieces , and snap-frozen in liquid nitrogen for immunohistology or fixed in Bouin 's solution for routine histology . Arterial blood was obtained by aortic puncture and the rats were killed by exsanguination . The right renal vein was punctured and , subsequently , the kidney was perfused with HBSS -- HEPES for 2 min at 7.5 ml/min -LRB- Minipuls 2 , Gilson , Villiers-le-Bel , France -RRB- . After perfusion , the kidney was removed , divided , and snap-frozen in liquid nitrogen for quantitative real-time polymerase chain reaction -LRB- RQ-PCR -RRB- and Western blotting . Thrombocyte and leukocyte counts were determined in blood as inflammatory parameters , and the concentrations of creatinine , urea , protein , glucose , and sodium were analyzed in blood and urine by routine clinical chemistry . The activity of alkaline phosphatase , a proximal tubule injury marker , was determined in 26-fold diluted urine samples , as described before -LSB- 18 -RSB- . Determination of mRNA expression RNA was isolated using a Mikro-dismembrator U -LRB- Sartorius B. Braun Biotech , Melsungen , Germany -RRB- . To prevent RNA degradation , we washed the metal cylinders with 0.5 M NaOH . Perfused kidneys were pulverized -LRB- 2,000 rpm , 30 s -RRB- and transferred in ice-cold TRIzol reagent -LRB- Invitrogen , Breda , The Netherlands -RRB- , and then RNA was isolated as described previously -LSB- 19 -RSB- . 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 -LRB- n = 6 , Applied Biosystems , Zwijndrecht , The Netherlands -RRB- or the ABI/PRISM 7900HT Gene Expression Micro Fluidic Card Sequence Detection System -LRB- three pooled samples from nine different rats , Applied Biosystems -RRB- according to the manufacturer 's instructions . All experiments were performed in triplicate . Different rat genes were amplified with a predeveloped Gene Expression Assay , provided by Applied Biosystems . Western blotting Total membrane and cytosolic fractions from perfused kidneys were isolated using a Mikro-dismembrator U -LRB- Sartorius BBI Systems GmbH , Melsungen , Germany ; 2,000 rpm for 30 s -RRB- and subjected to Western blotting as described previously -LSB- 20 -RSB- . Protein concentration was determined with the Bio-Rad protein assay -LRB- Bio-Rad , Hercules , CA , USA -RRB- using bovine serum albumin -LRB- BSA -RRB- as standard . Samples were separated on a 6 or 12 % sodium dodecylsulfate polyacrylamide gel and transferred to Hybond-C pure nitrocellulose membrane -LRB- Amersham , Buckinghamshire , UK -RRB- . The membrane was incubated overnight at 4 °C with antibodies against NOSII/iNOS -LRB- 1:1,000 , according to Vos et al. -LSB- 21 -RSB- -RRB- , ABCB1/P-gp -LRB- 1:200 , C219 , DakoCytomation , Dako , Denmark -RRB- , Abcc2/Mrp2 -LRB- 1:250 , M2III-6 , Alexis Biochemicals , San Diego , CA , USA -RRB- , ABCC4/MRP4 -LRB- 1:1,000 , as described by van Aubel et al. -LSB- 22 -RSB- -RRB- , Abcb11/bile salt export pump -LRB- Bsep -RRB- -LRB- 1:1,000 , kindly provided by Dr. Bruno Stieger , University Hospital Zürich , Switzerland -RRB- , or β-actin -LRB- 1:10,000 , Sigma -RRB- in Tris-buffered saline supplemented with 0.1 % Tween-20 and 1 % nonfat dried milk . Nitrate and nitrite determination The total amount of the stable NO metabolites -LRB- NOx -RRB- , nitrate and nitrite , were determined as a measure of NO radicals production , using the Griess reaction , according to Moshage et al. -LSB- 23 -RSB- . Heparinized plasma was diluted 1:4 and urine samples were diluted 1:40 or 1:80 with distilled water . Each sample was determined in triplicate . Calibration curves were made with sodium nitrite and sodium nitrate in distilled water . The nitrate recovery was 90 -- 100 % over the entire range tested -LRB- 0 -- 50 μmol/l -RRB- . The coefficient of variation between the different experiments was less than 5 % . Histology and immunohistology Kidneys fixed in Bouin 's solution were dehydrated , embedded in paraplast -LRB- Amsterstad , Amsterdam , The Netherlands -RRB- , and cut into 3-μm sections , which were stained with periodic acid-Schiff for routine histology . For immunohistologic studies , serial tissue sections of 1.5 μm were cut from frozen kidneys using a cryostat as described previously -LSB- 24 -RSB- . The slices were incubated for 60 min at room temperature with antibodies against either NOSII/iNOS -LRB- 1:200 , according to Vos et al. -LSB- 21 -RSB- -RRB- or ABCB1/P-gp -LRB- 1:50 , C219 , DakoCytomation -RRB- in PBS supplemented with 1 % BSA . For the antibodies peroxynitrite -LRB- 1:500 , Sigma -RRB- and Abcb11/Bsep -LRB- 1:1,000 , as described by Huls et al. -LSB- 24 -RSB- -RRB- , cryostat slices of 4 μm were used , fixed in acetone for 5 min . Endogenous peroxidase was blocked by incubation with 5 % H2O2 for 5 min , and nonspecific protein binding was blocked with 10 % normal goat serum -LRB- Vector , Burlingame , CA , USA -RRB- in PBS for 10 min at room temperature . Slices were incubated with the primary antibody in PBS 1 % BSA overnight at 4 °C . Primary antibody was detected using the Vectastain Elite peroxidase ABC kit -LRB- Vector -RRB- with 3,3 ′ - diaminobenzidine as substrate . Finally , slices were counterstained with hematoxylin , dehydrated , and mounted in mounting medium . Sections were examined with a confocal laser-scanning microscope -LRB- Leica lasertechnik GmbH , Heidelberg , Germany -RRB- . Isolated kidney perfusion Twelve hours after the administration of LPS + -LRB- n = 6 -RRB- or LPS − -LRB- n = 4 -RRB- , the transport activity of Abcc2/Mrp2 was determined by monitoring the urinary excretion of calcein in an isolated perfused rat kidney , as described in detail previously -LSB- 25 -RSB- . Briefly , rats were anesthetized IP with pentobarbital -LRB- 6 mg/100 g -RRB- , and furosemide was injected IP -LRB- 1 mg/100 g -RRB- to prevent deterioration of the distal nephron . Heparin -LRB- 125 IU/100 g -RRB- was injected in the spleen . The kidney was excised and placed in a fluid bath with a constant temperature of 37.5 °C and rats were killed by exsanguination . Vitamin B12 was used for the determination of the glomerular filtration rate -LRB- GFR -RRB- and calcein-acetoxymethylester was used as a source for the fluorescent substrate calcein -LSB- 25 -RSB- . Statistical analysis Values are given as mean ± SE . Differences between the experimental groups were tested using one-way ANOVA with Bonferroni 's correction or two-way ANOVA corrected for repeated measurements . A two-sided P value < 0.05 was considered significant . Results Signs of inflammation A few hours after LPS + administration , inflammatory symptoms appeared , like diarrhea and piloerection . Body temperature was elevated after 24 h and the loss in body weight increased from − 3 % -LRB- t = 3 h -RRB- , due to normal circadian rhythm , to 12 % -LRB- t = 48 h -RRB- , compared to the controls -LRB- Table a in the supplementary material 1 -RRB- . Furthermore , administration of LPS + resulted in the expected leucopenia -LRB- t = 3 h -RRB- followed by leukocytosis -LRB- t = 48 h -RRB- and thrombocytopenia , with a maximum at 48 h. Coadministration of LPS + with aminoguanidine did not affect the inflammatory parameters , except for a greater loss in body weight 12 h after LPS administration . iNOS induction after LPS treatment RQ-PCR was used to determine the iNOS mRNA levels in kidney samples . The relative expression of iNOS in LPS − rats was normalized for the average cycle threshold -LRB- CT -RRB- value for the housekeeping gene , GAPDH -LRB- CT = 16.06 ± 0.04 , n = 15 -RRB- , and set to 1 . GAPDH expression remained stable during endotoxemia -LRB- CT = 16.22 ± 0.09 , n = 42 -RRB- . Furthermore , no circadian rhythm for the different genes was detected in control animals . Analysis of three separate experiments revealed a more than 40,000 times induction in iNOS mRNA 3 h after LPS + compared to controls -LRB- Fig. 1a -RRB- , which decreased with time , but was still 100 times higher than control values 48 h after LPS + . Treatment with both LPS + and aminoguanidine slightly , but not significantly , reversed iNOS mRNA induction compared to LPS + alone -LRB- Fig. 1a -RRB- . Maximal iNOS protein expression was observed in cytosolic fractions 6 h after LPS + , and returned to LPS − level at 48 h -LRB- Fig. 1b -RRB- . In contrast to its effect on iNOS mRNA expression , aminoguanidine reversed the induction in protein expression -LRB- Fig. 1c -RRB- . Fig. 1iNOS mRNA and protein . a iNOS mRNA expression in rat kidney at different time points after LPS + was determined with RQ-PCR without -LRB- closed bars , n = 6 -RRB- or with coadministration of aminoguanidine -LRB- gray bars , n = 6 -RRB- . The iNOS mRNA expression was normalized for the GAPDH CT value -LRB- 16.06 ± 0.04 , n = 15 -RRB- , and ΔCT values for the LPS − were set to 1 . Data are expressed as mean ± SE . Significantly different compared to the LPS − -LRB- double asterisksP < 0.01 , triple asterisksP < 0.001 , quadruple asterisksP < 0.0001 -RRB- . Cytosolic fractions of rat kidney were isolated and expression of iNOS was determined by Western blotting . Representative images -LRB- n = 4 -RRB- show maximal iNOS protein expression 6 h after LPS + administration -LRB- b -RRB- , and coadministration with aminoguanidine reversed this induction -LRB- c -RRB- NOx levels in plasma and urine were measured using the Griess assay . An increase in NOx concentration in plasma was already visible 3 h after LPS + , with a peak concentration at 12 h. Coadministration with aminoguanidine reversed the increase in NOx significantly at both 6 and 12 h -LRB- Fig. 2a -RRB- . The urinary excretion of NOx increased 12 h after LPS + , and was still elevated 48 h later . Aminoguanidine returned this increase to baseline levels at both time points -LRB- Fig. 2b -RRB- . Fig. 2NOx in plasma and urine . NOx levels in plasma and urine were measured at various times after LPS − -LRB- open bars , n = 3 -RRB- or LPS + -LRB- closed bars , n = 6 -RRB- treatment or LPS + together with aminoguanidine -LRB- gray bars , n = 6 -RRB- . a Blood samples show a peak concentration at 12 h and coadministration with aminoguanidine reversed this increase at both 6 and 12 h. b The urinary excretion of NOx increased 12 h after LPS + treatment , and aminoguanidine returned this increase to baseline levels . Data are expressed as mean ± SE . Significantly different compared to the LPS − -LRB- single asterisksP < 0.05 , double asterisksP < 0.01 , triple asterisksP < 0.001 -RRB- or LPS + -LRB- double sharp signsP < 0.01 , triple sharp signsP < 0.001 -RRB- Renal localization of iNOS and formation of 3-nitrotyrosine-protein adducts , a reliable biomarker of peroxynitrite generation -LSB- 26 -RSB- , were determined by immunohistology . There was no iNOS staining visible in kidney sections of rats treated with LPS − . Positive iNOS staining was seen in both experimental groups after LPS + , in inflammatory cells , and in proximal tubule cells . Forty-eight hours after LPS + , iNOS staining was no longer detectable . Figure 3 shows representative immunohistochemical images with iNOS expression in all segments of the proximal tubule -LRB- 3A + C -RRB- . After colocalization with Abcb1/P-gp -LRB- 3B -RRB- , iNOS expression in the vicinity of the apical membrane was visible -LRB- 3A + C -RRB- . 3-Nitrotyrosine-protein adducts were predominantly present in the cortex in peritubulary capillaries and proximal tubules after LPS + -LRB- Fig. 3d , e -RRB- . Coadministration with aminoguanidine attenuated the formation of adducts in the cortex -LRB- Fig. 3f , g -RRB- . Fig. 3Immunofluorescence microscopic analysis of iNOS and peroxynitrite in the kidney . a -- c Representative immunohistochemical images show that iNOS -LRB- green staining -RRB- is expressed in the vicinity of the apical membrane -LRB- P-gp counterstaining , red -RRB- in all segments of the proximal tubule 12 h after LPS + treatment . d , e Formation of 3-nitrotyrosine-protein adducts , a reliable biomarker of peroxynitrite generation , were visible in the cortex in peritubulary capillaries and proximal tubules 12 h after LPS + treatment . f , g Coadministration with aminoguanidine resulted in attenuated formation of 3-nitrotyrosine-protein adducts at 12 h. G glomerulus , asterisks peritubular staining , P proximal tubule , D distal tubule . Original magnification d , f × 200 and a -- c , e , g × 400 Aminoguanidine restores the slightly impaired kidney function and loss of brush border membrane Kidney function was slightly impaired after LPS + as indicated by increased blood urea nitrogen -LRB- BUN -RRB- , an increase in plasma creatinine , and an increased urinary glucose excretion -LRB- Table 1 -RRB- . Furthermore , the urine/plasma creatinine ratio was decreased -LRB- data not shown -RRB- as was the creatinine clearance , although not significantly . Table 1Renal functional parametersKidney functionTime -LRB- h -RRB- Controls -LRB- LPS − -RRB- LPS+LPS + and aminoguanidineTotal urine volume -LRB- ml -RRB- 31.6 ± 0.241.7 ± 0.2762.4 ± 0.654.45 ± 0.987.3 ± 0.76123.2 ± 0.5710.0 ± 0.70 a9 .4 ± 0.65 a2411 .5 ± 0.2513.7 ± 2.114823.5 ± 3.4718.0 ± 1.57 BUN -LRB- mmol/l -RRB- 36.6 ± 0.209.9 ± 0.7464.6 ± 0.7214.0 ± 1.48 b12 .1 ± 0.82 b126 .3 ± 0.2412.3 ± 1.38 a15 .4 ± 1.27 b247 .0 ± 0.5810.6 ± 0.58486.5 ± 0.109.1 ± 0.38 Blood creatinine -LRB- mmol/l -RRB- 30.035 ± 0.0020.049 ± 0.002 b60 .039 ± 0.0020.053 ± 0.002 b0 .043 ± 0.019 d120 .037 ± 0.0020.048 ± 0.002 c0 .036 ± 0.032 e240 .038 ± 0.0010.051 ± 0.001 a480 .042 ± 0.0010.052 ± 0.001 Glucose excretion -LRB- μmol/h -RRB- 30.290 ± 0.0450.481 ± 0.34160.333 ± 0.0860.420 ± 0.1000.478 ± 0.051120.425 ± 0.1060.537 ± 0.0540.332 ± 0.080240.254 ± 0.0060.984 ± 0.039 c480 .325 ± 0.0370.606 ± 0.128 Creatinine clearance -LRB- ml/min -RRB- 31.33 ± 0.120.78 ± 0.1961.06 ± 0.150.87 ± 0.090.93 ± 0.35121.01 ± 0.050.90 ± 0.031.01 ± 0.13241.19 ± 0.020.91 ± 0.20481.00 ± 0.071.08 ± 0.37 FENa -LRB- % -RRB- 30.816 ± 0.0040.365 ± 0.07361.046 ± 0.1450.358 ± 0.094 b1 .423 ± 0.310 e121 .094 ± 0.0970.458 ± 0.075 a1 .610 ± 0.282 e241 .070 ± 0.0240.429 ± 0.065 c481 .033 ± 0.0770.121 ± 0.024 aData are expressed as mean ± SE , n = 6 for each time point for both LPS + and LPS + + aminoguanidine and n = 3 for LPS − FENa fractional sodium excretionaSignificantly different compared to the control group ; P < 0.01 bSignificantly different compared to the control group ; P < 0.001 cSignificantly different compared to the control group ; P < 0.05 dSignificantly different compared to the LPS + group ; P < 0.01 eSignificantly different compared to the LPS + group ; P < 0.001 Histological examination after LPS + revealed damage to the proximal tubules , compared to LPS − -LRB- Fig. 4a , b -RRB- . Loss of brush border membranes and formation of vacuoles were observed . Aminoguanidine reduced LPS + - induced damage to the proximal tubule -LRB- Fig. 4c -RRB- . The activity of alkaline phosphatase in urine samples was assayed to get an indication of proximal tubule injury . Twelve hours after LPS + , the activity of alkaline phosphatase was increased in urine samples and was still significantly increased after 48 h -LRB- Fig. 4d -RRB- . Furthermore , the expression of kidney injury molecule 1 -LRB- Kim-1 -LSB- 27 -RSB- -RRB- was strongly up-regulated , 400 times more than control values , 6 and 12 h after LPS + -LRB- Table b in the supplementary material 1 -RRB- . Except for BUN , aminoguanidine improved all parameters of renal function , as shown in Table 1 and Fig. 4 . No differences in urinary protein excretion were found and , remarkably , the fractional excretion of filtered sodium was decreased -LRB- Table 1 -RRB- . Fig. 4Proximal tubular damage during endotoxemia . Histological examination after LPS + revealed damage to the proximal tubules compared to LPS − . a LPS − - treated rat with intact brush border membrane in proximal tubules . b Twelve hours after LPS + , damage to the proximal tubules is observed with loss of brush border membranes and formation of vacuoles . c Treatment with both LPS + and aminoguanidine resulted in less damage to the proximal tubule . Kidneys of LPS + - treated rats killed after 24 or 48 h also showed signs of proximal tubule damage -LRB- data not shown -RRB- . Proximal tubules are indicated by arrows . Original magnifications -LRB- a -- c -RRB- × 400 . d Histological data are supported by an increase in the activity of alkaline phosphatase , marker for proximal tubule damage , in urine samples 12 h after LPS + administration -LRB- closed bars , n = 6 -RRB- , compared to controls -LRB- open bars , n = 3 -RRB- . Coadministration with aminoguanidine -LRB- gray bars , n = 6 -RRB- reduced this proximal tubule damage . Data are expressed as mean ± SE . Significantly different compared to the LPS − -LRB- double asterisksP < 0.01 -RRB- or LPS + -LRB- sharp signP < 0.05 -RRB- Furthermore , the mRNA expression of different biotransformation enzymes -LSB- 28 -RSB- , enzymes involved in the NO pathway -LSB- 2 -RSB- or known to be activated during oxidative stress -LSB- 29 , 30 -RSB- was determined . Twelve and 24 h after LPS + treatment , expression of the metabolizing enzymes glutathione S-transferases -LRB- GST-α2 and GST-α5 -RRB- and uridine diphosphate-glucuronosyltransferases -LRB- UDPGT1a6 -RRB- was down-regulated -LRB- Table b in the supplementary material 1 , at least a 2 times lower expression -RRB- . The arginine metabolic enzymes , argininosuccinate synthetase and arginase 2 , were not changed after LPS + . In contrast , arginase 1 mRNA expression was reduced . The gene expression of the ETB receptor , which gets activated as part of the NO-dependent signaling pathway regulating Abcb1/P-gp and Abcc2/Mrp2 -LSB- 13 , 31 -RSB- , did not change during endotoxemia . Three and 6 h after LPS + the mRNA expressions of super oxide dismutase 2 , heam-oxygenase 1 , and nuclear factor κB -LRB- NF-κB -RRB- were up-regulated -LRB- i.e. , at least a 2 times higher expression -RRB- . Coadministration of aminoguanidine had no effect on the gene expression of all these proteins . In contrast , aminoguanidine reversed the down-regulation of nNOS gene expression during endotoxemia -LRB- Table b in the supplementary material 1 -RRB- . LPS + treatment affects ABC drug transporter expression in the kidney The mRNA expression of 20 renal ABC transporters was profiled . Abcb1a/P-gp , Abcb1b/P-gp , Abcc2/Mrp2 and Abcc4/Mrp4 expression was determined with the ABI PRISM 7700 single reporter sequence detection system . In rodents , Abcb1/P-gp exists as two functional isoforms , which are encoded by two different genes : Abcb1a and Abcb1b -LSB- 32 -RSB- . The other ABC transporters were assessed with the ABI/PRISM 7900HT Gene Expression Micro Fluidic Card -LRB- Table 2 -RRB- . Their expression was normalized for the average CT value for GAPDH , as described for iNOS . Other housekeeping genes , like glucuronidase and hypoxanthine guanine phosphoribosyl transferase 1 , gave comparable values to GAPDH -LRB- data not shown -RRB- . Abcb1b/Pg-p and Abcc2/Mrp2 were clearly up-regulated 3 h after LPS + and were still elevated 12 and 24 h later -LRB- Fig. 5b , c -RRB- . Furthermore , Abcb1a/P-gp and Abcc4/Mrp4 were slightly , but not significantly , increased 12 h after LPS + treatment -LRB- Fig. 5a , d -RRB- . Table 2 presents a list of the expression of other transporter genes during endotoxemia . Abca1 showed a slight up-regulation 3 h after LPS + , but returned to basal levels within 12 h. Furthermore , Abcb11 was up-regulated , but only significantly after 48 h . A down-regulation was observed for Abcb4 and Abcc8 -LRB- at least a 2 times lower expression -RRB- . The expression of the Abcg genes was not affected by endotoxemia . Table 2Gene expression of efflux transporters during endotoxemiaGene symbolProtein synonymAssay IDsDelta CT valuesaLPS − -LRB- controls -RRB- LPS + t = 3 hLPS + t = 6 hLPS + t = 12 hLPS + t = 24 hLPS + t = 48 hLPS + + amino t = 6 hLPS + + amino t = 12 hAbca1Abca 1Rn00710172_m17 .1 ± 0.315.1 ± 0.165.8 ± 0.227.3 ± 0.557.3 ± 0.356.8 ± 0.236.1 ± 0.238.0 ± 0.12 Abca2Abca 2Rn00577821_m13 .5 ± 0.403.5 ± 0.354.1 ± 0.154.5 ± 0.384.3 ± 0.353.6 ± 0.064.4 ± 0.384.4 ± 0.12 Abca5Abca 5Rn00597824_m19 .8 ± 0.069.8 ± 0.359.8 ± 0.109.5 ± 0.1210.0 ± 0.2310.2 ± 0.219.5 ± 0.259.7 ± 0.35 Abcb1aP_gpRn00591394_m18 .4 ± 0.01 Fig. 5aAbcb1bP_gpRn00561753_m110 .8 ± 0.04 Fig. 5bAbcb4Abcb 4Rn00562185_m110 .7 ± 0.6212.3 ± 0.1612.5 ± 0.2112.8 ± 0.1510.9 ± 0.157.3 ± 0.4213.2 ± 0.4612.4 ± 0.24 Abcb6Abcb 6Rn00589801_m15 .5 ± 0.355.9 ± 0.266.4 ± 0.256.5 ± 0.525.9 ± 0.385.4 ± 0.146.4 ± 0.256.2 ± 0.32 Abcb9Abcb 9Rn00573757_m18 .1 ± 0.208.5 ± 0.389.3 ± 0.298.8 ± 0.178.6 ± 0.108.7 ± 0.179.2 ± 0.428.9 ± 0.15 Abcb11BsepRn00582179_m110 .5 ± 0.408.6 ± 0.3710.3 ± 0.2411.9 ± 0.218.7 ± 0.175.4 ± 0.499.9 ± 0.219.7 ± 0.21 Abcc1Mrp1Rn00574093_m15 .9 ± 0.256.2 ± 0.266.1 ± 0.256.1 ± 0.256.3 ± 0.215.9 ± 0.236.4 ± 0.406.2 ± 0.15 Abcc2Mrp2Rn00563231_m16 .7 ± 0.31 Fig. 5cAbcc3Mrp3Rn00589786_m18 .4 ± 0.158.7 ± 0.128.8 ± 0.5088.6 ± 0.068.0 ± 0.358.2 ± 0.128.8 ± 0.328.7 ± 0.23 Abcc4Mrp4Rn01465699_q16 .6 ± 0.06 Fig. 5dAbcc5Mrp5Rn00588341_m17 .1 ± 0.017.9 ± 0.037.6 ± 0.156.4 ± 0.066.5 ± 0.506.5 ± 0.387.6 ± 0.456.4 ± 0.36 Abcc6Mrp6Rn00578778_m17 .0 ± 0.147.0 ± 0.018.4 ± 0.218.4 ± 0.127.8 ± 0.056.8 ± 0.358.6 ± 0.078.3 ± 0.14 Abcc8Sur1Rn00564778_m111 .5 ± 0.4011.4 ± 0.2111.4 ± 0.2613.4 ± 0.1514.8 ± 0.1513.2 ± 0.2511.7 ± 0.2313.7 ± 0.31 Abcc9Sur2Rn00564842_m16 .8 ± 0.017.7 ± 0.268.4 ± 0.318.1 ± 0.157.2 ± 0.326.8 ± 0.408.0 ± 0.358.2 ± 0.32 Abcg1Abcg1Rn00585262_m16 .8 ± 0.157.8 ± 0.256.8 ± 0.257.1 ± 0.406.8 ± 0.316.7 ± 0.216.4 ± 0.317.3 ± 0.36 Abcg2Abcg2Rn00710585_m14 .1 ± 0.244.5 ± 0.074.6 ± 0.215.0 ± 0.064.5 ± 0.144.5 ± 0.074.9 ± 0.425.2 ± 0.07 Abcg5Abcg5Rn00587092_m118 .8 ± 0.29 baData of nine pooled samples , measured in triplicate , are expressed as mean ± SDbLow expressed transport protein : CT value is not considered as validFig . 5mRNA expression of different renal ABC transporters . Gene expression of Abcb1a/P-gp -LRB- a -RRB- and Abcb1b/P-gp -LRB- b -RRB- , Abcc2/Mrp2 -LRB- c -RRB- , and Abcc4/Mrp4 -LRB- d -RRB- were determined in rat kidney at different time points after LPS − -LRB- open bars , n = 3 -RRB- or LPS + -LRB- closed bars , n = 6 -RRB- treatment or LPS + together with aminoguanidine -LRB- gray bars , n = 6 -RRB- . ΔCT values for the LPS − , given in Table 2 , were set to 1 . Data are expressed as mean ± SE . Significantly different compared to the LPS − -LRB- single asterisks P < 0.05 , double asterisks P < 0.01 , triple asterisks P < 0.001 -RRB- or LPS + -LRB- sharp signs P < 0.05 -RRB- Coadministration with aminoguanidine blocked the induction in mRNA expression of both Abcb1b/P-gp and Abcc2/Mrp2 at 6 and 12 h , respectively -LRB- Fig. 5b , c -RRB- . Furthermore , aminoguanidine reduced the increased protein expression of both transporters 12 h after LPS + treatment -LRB- Fig. 6b , d -RRB- , indicating a regulatory role for NO in the expression of these efflux transporters . Abcc4/Mrp4 protein levels , on the other hand , decreased even more after treatment with both LPS + and aminoguanidine . Gene expression levels of other transporters were not affected by aminoguanidine -LRB- Table 2 -RRB- . Fig. 6Protein expression of different renal ABC transporters . Total membrane fractions of rat kidney were isolated and expression of Abcb1/P-gp -LRB- a , b -RRB- , Abcb11/Bsep -LRB- c -RRB- , Abcc2/Mrp2 -LRB- d , e -RRB- , and Abcc4/Mrp4 -LRB- f , g -RRB- was determined by Western blotting . Representative images -LRB- n = 4 -RRB- show maximal Abcb1/P-gp protein expression 12 and 24 h after LPS + -LRB- a -RRB- , and coadministration with aminoguanidine reversed this induction -LRB- b -RRB- . The protein expression of Abcb11/Bsep was up-regulated 12 h after LPS + treatment , which was reversed by aminoguanidine -LRB- c -RRB- . Maximal Abcc2/Mrp2 protein expression was seen 12 h after LPS + -LRB- d -RRB- and aminoguanidine reversed this induction -LRB- e -RRB- . Abcc4/Mrp4 protein expression was down-regulated during endotoxemia -LRB- f -RRB- , which was intensified by aminoguanidine -LRB- g -RRB- The expression patterns of Abcb1/P-gp , Abcb11 , Abcc2/Mrp2 , and Abcc4/Mrp4 were investigated further . Using total membrane fractions , we found an up-regulation of Abcb1/P-gp 6 h after LPS + , with maximal protein expression 12 and 24 h after LPS + -LRB- Fig. 6a -RRB- . Abcb11 is the major liver canalicular bile salt export pump -LRB- Bsep -RRB- , but surprisingly , this transporter is also expressed at the apical membrane of mice renal proximal tubule , as we discovered recently -LSB- 24 -RSB- . In agreement with an increased mRNA expression , Abcb11/Bsep protein was up-regulated after LPS + -LRB- Fig. 6c -RRB- . Although we are not completely certain whether the C219 antibody against Abcb1/P-gp also detects Abcb11/Bsep -LSB- 33 -RSB- , our Western blot results showed expression of Abcb1/P-gp , with a molecular weight of 130 kDa , and it does not seem to cross-react with Bsep at 160 kDa . By using immunohistochemistry , the expression and localization of Abcb11/Bsep was investigated in endotoxemic rat kidneys . In agreement with mouse kidney , positive staining was observed in the apical membranes of the proximal tubule 12 h after LPS + treatment -LRB- Fig. 7 -RRB- . In addition , we observed staining in rat glomerular endothelium and peritubular capillaries . Fig. 7Distribution of Abcb11/Bsep in rat kidney cortex . a , b Representative immunohistochemical images show apical staining in proximal tubules -LRB- indicated by arrows -RRB- . In addition , glomerular and peritubular staining of Bsep in the cortex was found . G glomerulus , asterisks peritubular staining , P proximal tubule , D distal tubule . Original magnification in a × 200 , and in b × 400 Furthermore , a clear increase in Abcc2/Mrp2 protein was observed after LPS + -LRB- Fig. 6d -RRB- , but a down-regulation in Abcc4/Mrp4 was found -LRB- Fig. 6f -RRB- . We measured the function of Abcc2/Mrp2 by monitoring the urinary excretion of calcein in an isolated perfused rat kidney 12 h after LPS + or LPS − . A 16 ± 3 % increase in calcein excretion was observed after LPS + , as shown in Fig. 8 . We do not expect calcein to be a Bsep substrate because Mrp2-deficient -LRB- TR − -RRB- rats clearly showed that calcein excretion was highly reduced -LSB- 25 -RSB- , despite the presence of Bsep in these strains -LRB- unpublished data -RRB- . Twenty-four hours after LPS + , Abcc2/Mrp2 activity was still elevated , although not significantly . In accordance , endotoxemia affected renal function as observed by diminished GFR and fractional reabsorption of water in isolated perfused rat kidneys -LRB- Table 3 -RRB- . Fig. 8Renal calcein excretion in isolated perfused rat kidneys . Secretion of the fluorescent calcein into the urine was measured after 12 h in untreated -LRB- closed squares control ; n = 4 -RRB- , LPS − -LRB- open squaresn = 4 -RRB- , or LPS + -LRB- trianglesn = 6 -RRB- rats . Urinary excretion was corrected for GFR . Significantly different compared to the LPS − -LRB- asterisk P < 0.05 -RRB- Table 3Functional parameters of isolated perfused rat kidneys in control rats -LRB- no additional treatment -RRB- and after exposure to LPS − or LPS + for 12 hParameterControls -LRB- n = 4 -RRB- LPS − -LRB- n = 4 -RRB- LPS + -LRB- n = 6 -RRB- GFR -LRB- μl/min -RRB- 350 ± 25380 ± 17290 ± 20aDiuresis -LRB- μl/min -RRB- 18.6 ± 1.821.1 ± 1.317.4 ± 1.0 FRwater -LRB- % -RRB- 94.7 ± 0.294.5 ± 0.392.7 ± 0.6 aRPP -LRB- mmHg -RRB- b88 .4 ± 1.584.5 ± 1.788.7 ± 1.0 Data are expressed as mean ± SE of four to six perfused kidney experiments over the period 25-115 min . Untreated rats served as controls for this model . Kidneys were perfused for 150 min as described in the `` Materials and methods '' sectionaSignificantly different from perfused control kidney -LRB- P < 0.05 -RRB- bConstant flow during the experiments was 18 ± 0.5 ml/minFR fractional reabsorption , RPP renal perfusion pressure Discussion In the present study , we investigated the role of NO , produced by iNOS , in the differential expression of renal ABC transporters in rats in vivo . A clear up-regulation of Abca1 , Abcb1/P-gp , Abcb11/Bsep , and Abcc2/Mrp2 expression was found after LPS + . In addition , we observed a functional up-regulation of Abcc2/Mrp2 in perfused kidneys . This supports a detoxifying role of the efflux pump , which is in accordance with the up-regulation of Abcc2/Mrp2 protein in killifish renal tubules -LSB- 34 , 35 -RSB- , in rat kidney , and in MDCKII cells overexpressing human ABCC2/MRP2 after long-term exposure to the nephrotoxicant gentamicin -LSB- 20 -RSB- , and of abcb1/P-gp in mouse kidneys after ischemic reperfusion injury -LSB- 24 -RSB- . Indeed , blocking Abcb1/P-gp during endotoxemia leads to further renal damage -LRB- Heemskerk , Wouterse , Russel , and Masereeuw , preliminary findings -RRB- . The renal drug transporters are likely to be under direct influence of NO produced by iNOS , as coadministration of the iNOS inhibitor , aminoguanidine , attenuated the LPS-induced effects on transporter expression . Aminoguanidine treatment results in decreased NO production and , subsequently , reduced oxidative stress by decelerating the formation of peroxynitrite . Under these conditions , less toxic metabolites may be produced , explaining the restoration in transporter expression and reversal of proximal tubular damage . In contrast to our findings , Cherrington et al. -LSB- 36 -RSB- reported that coadministration of aminoguanidine failed to change the expression of different hepatic drug transporters during endotoxemia . This discrepancy might be due to organ differences and the fact that Cherrington et al. pretreated the animals with the NOS inhibitor , whereas we gave aminoguanidine 1 h after LPS + . The lack of an effect of aminoguanidine on the expression of different biotransformation enzymes , enzymes involved in the iNOS pathway and during oxidative stress , supports a direct function of NO itself in ABC transporter regulation . The role of two up-regulated efflux pumps , Abca1 and Abcb11/Bsep , in the kidney is unknown . In humans , ABCA1 is widely expressed and found to be the causative gene in Tangier disease , a disorder of cholesterol transport between tissues and the liver , mediated by binding of the cholesterol onto high-density lipoprotein particles -LSB- 37 -RSB- . ABCA1 controls the extrusion of membrane phospholipid and cholesterol toward specific extracellular acceptors ; however , the exact role of the protein in this process is not known . Moreover , its expression and function in the kidney requires further investigation . The presence of Abcb11/Bsep , the major canalicular bile salt export pump , was recently demonstrated by us in mouse kidney -LSB- 24 -RSB- , and here we show a clear apical expression in rat kidney proximal tubules for the first time as well . Hence , the function of Abcb11/Bsep in the kidney still needs to be determined . In addition to their up-regulation in the kidney , we found a down-regulation of Abcb1/P-gp , Abcc2/Mrp2 , and Abcb11/Bsep in the liver -LRB- data not shown -RRB- . This is in agreement with previous findings for the hepatic expression of the transporters during inflammation -LSB- 36 , 38 -- 40 -RSB- , obstructive jaundice -LSB- 41 -RSB- , and cholestasis -LSB- 42 -RSB- . An induction of the transporters in the kidney during liver failure may function as a compensatory elimination mechanism to reduce enhanced levels of circulating bile salts caused by endotoxin-induced cholestasis -LSB- 39 -RSB- and/or to protect proximal tubular cells against the higher exposure via primary urine . A differential regulation was found for Abcc4 . Although this transporter is expressed in the same nephron segments as Abcb1/P-gp and Abcc2/Mrp2 , the apically expressed efflux pump clearly has a distinct regulation . In killifish , we previously found renal proximal tubules exposed to nephrotoxicants or ET-1 that Abcc2/Mrp2-mediated transport and Mrp2 expression in the luminal membrane were increased after 24 h -LSB- 34 -RSB- , whereas the expression of Abcc4/Mrp4 remained unchanged -LSB- 35 -RSB- . Furthermore , Abcc4/Mrp4 protein levels in rat kidney were decreased during cholestasis in contrast to the Abcc2/Mrp2 levels -LSB- 43 -RSB- . No changes in Abcc4/Mrp4 mRNA were found , suggesting a posttranscriptional regulation of transporter expression , including phosphorylation and protein routing . A down-regulation of ABC transporters , as observed for Abcc8/Sur1 and initially also for Abcb4 , may be explained by the different functions of these ABC members in the kidney . LPS itself may affect transporter expression after binding to the Toll-like receptor 4 and a second , not yet identified , factor through activation of NOS-dependent pathways , as was found for Abcb1/P-gp in rat brain capillaries -LSB- 44 -RSB- . Furthermore , systemic TNF-α , released during endotoxemia , may regulate efflux pumps , including ABC transporters -LSB- 45 , 46 -RSB- . This cytokine might act through NOS activation -LSB- 44 -RSB- , but also through a pathway that does not involve iNOS-derived NO during endotoxin-associated renal failure -LSB- 47 -RSB- . Furthermore , regulation of iNOS after exposure to LPS occurs at the level of gene transcription and may require activation of the NF-κB pathway -LSB- 48 -RSB- . It has been shown previously that an up-regulation of Abcb1/P-gp in renal failure , i.e. , after ischemic -- reperfusion damage -LSB- 24 -RSB- or exposure to cadmium -LSB- 49 -RSB- , might be signaled through an NF-κB-dependent pathway . Consistent with these findings , we observed a clear induction -LRB- sevenfold -RRB- of NF-κB expression 3 h after LPS + as well . Selective inhibition of renal iNOS has important implications for the treatment of septic acute kidney disease . Changes in the expression levels of renal drug transporters alters the pharmacokinetics of many clinically important drugs -LSB- 50 -RSB- and has implications for the prediction of overall drug disposition during sepsis . For example , glucocorticoids are substrates for Abcb1/Pg-p and are often prescribed for their anti-inflammatory properties in septic patients . An up-regulation of Abcb1/Pg-p , as observed in our study , might have consequences for therapeutic levels of these substrates -LSB- 51 -RSB- . In conclusion , after LPS + , a clear up-regulation in Abca1 , Abcb1/P-gp , Abcb11/Bsep , and Abcc2/Mrp2 was found , whereas Abcc8/Sur1 was down-regulated . Abcb4 and Abcc4/Mrp4 show a differential pattern during the course of endotoxemia . The renal drug transporters are likely to be under direct influence of NO produced by iNOS , as coadministration of the iNOS inhibitor , aminoguanidine , attenuated the LPS-induced effects on their expression . Apparently , an up-regulation of important efflux pumps diminishes the renal accumulation of toxic compounds and attenuates further proximal tubular damage during endotoxemia . Electronic supplementary material Below is the link to the electronic supplementary material . Supplementary data -LRB- DOC 47.6 kb -RRB-

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