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【摘要】 Ischemic acute renal failure (iARF) was described to reduce renal extraction of the organic anion para -aminohippurate (PAH) in humans. The rate-limiting step of renal organic anion secretion is its basolateral uptake into proximal tubular cells. This process is mediated by the organic anion transporters OAT1 and OAT3, which both have a broad spectrum of substrates including a variety of pharmaceutics and toxins. Using a rat model of iARF, we investigated whether impairing the secretion of the organic anion PAH might be associated with downregulation of OAT1 or OAT3. Inulin and PAH clearance was determined starting from 6 up to 336 h after ischemia-reperfusion (I/R) injury. Net secretion of PAH was calculated and OAT1 as well as OAT3 expression was analyzed by RT-PCR and Western blotting. Inulin and PAH clearance along with PAH net secretion were initially diminished after I/R injury with a gradual recovery during follow-up. This initial impairment after iARF was accompanied by decreased mRNA and protein levels of OAT1 and OAT3 in clamped animals compared with sham-operated controls. In correlation to the improvement of kidney function, both mRNA and protein levels of OAT1 and OAT3 were upregulated during the follow-up. Thus decreased expression of OAT1 and OAT3 is sufficient to explain the decline of PAH secretion after iARF. As a result, this may have substantial impact on excretion kinetics and half-life of organic anions. As a consequence, the biological effects of a variety of organic anions may be affected after iARF.
【关键词】 organic anion transporter organic anion transporter proximal tubule basolateral uptake renal plasma flow glomerular filtration inulin clearance PAH net secretion
THE ORGANIC ANION TRANSPORT system of the renal proximal tubule plays a crucial role in the excretion of a variety of potentially toxic compounds ( 22, 35 ). This system consists of organic anion exchangers located at the basolateral membrane and a less well-characterized transport step at the apical membrane ( 8 ). The classical basolateral organic anion exchanger is the terminal step in a tertiary active transport system, dependent on an inward-directed Na + gradient to drive the uptake of -ketoglutarate ( KG), which is then exchanged for organic anions ( 7, 11, 23 ). It has been shown that OAT1 represents characteristics of the basolateral, polyspecific transporter for organic anions ( 30 ), which had been functionally described since substantial time ( 23 ). Recently, new evidence has indicated that OAT3 ( 16 ), which is also located in the basolateral renal proximal tubular membrane, works as an exchanger for organic anions and dicarboxylates, too ( 32 ). Moreover, additional homologs have been cloned and were called OAT2 ( 28 ), OAT4 ( 4 ), OAT5 ( 40 ), and OAT6 ( 20 ). These clones show 40 to 60% homology in amino acid sequence compared with OAT1 or OAT3, and they differ in substrate specificity and expression pattern. Furthermore, these latter proteins are not anion exchangers like OAT1 or OAT3 but seem to work as facilitators of anion diffusion. In summary, the classical renal basolateral polyspecific uptake transporter for organic anions is represented by OAT1 and OAT3 ( 9, 29 ).
Meanwhile, there is increasing evidence that renal basolateral organic anion uptake is regulated. An increasing number of studies published concentrate on acute regulatory phenomena and deal with regulation by PKC and PKA (for a review, see Refs. 9 and 33 ). With respect to long-term regulation of renal organic anion transport, much less data are available. There is evidence that renal organic anion transport is regulated by sex steroids since substantial time ( 25 ), which is now shown to be most probably due to regulation of OAT1 and OAT3 ( 3, 17 ) and possibly OAT2 ( 3 ). Moreover, hyperuricemia was shown to reversibly downregulate OAT1 and OAT3 ( 12, 13 ). In the rat kidney, OAT1 and OAT3 are both downregulated after ureteral obstruction ( 36 ). Only recently, it was shown that prostaglandin E 2 leads to downregulation of the expression of both OAT1 and OAT3 in rat proximal tubular cell line NRK-52E after long-term exposure (up to 72 h) ( 26 ).
In human renal allograft, the clearance of the prototypical organic anion PAH was reduced for at least 7 days after transplantation ( 5 ). In this model of postischemic renal injury, the renal extraction of para -aminohippurate (PAH) was found to be profoundly reduced; leading to the assumption that proximal tubular transport of organic anions itself is impaired. The rate-limiting step of organic anion secretion, its basolateral uptake, is mediated by OAT1 and OAT3, which in principle is now known to be regulated on the level of protein expression ( 3, 12, 13, 17, 26, 36 ). Thus we hypothesized that the impairment of organic anion extraction mentioned above may be due to a decreased expression of OAT1 or OAT3. We investigated this in a well-established rat model of ischemic acute renal failure (iARF) ( 24, 27 ). In fact, the determination of inulin clearance, PAH clearance, and net secretion of PAH and the amount of mRNA and protein of OAT1 and OAT3 indicate that expression of both OAT1 and OAT3 is transiently downregulated after ischemia-reperfusion (I/R) injury.
MATERIALS AND METHODS
Experimental Procedure
Experiments were performed as published recently ( 24, 27 ), where I/R injury was induced by bilateral clamping of renal arteries for 45 min. Female Sprague-Dawley rats (200 to 250 g body wt) were obtained from Charles River (Kissleg, Germany). After a period of at least 24 h in cages within a temperature-controlled room with 12:12-h light-dark cycle and standard food with free access to tap water, anesthesia was performed by intraperitoneal application of xylacin hydrochloride (10 mg/kg body wt) and ketamine (100 mg/kg body wt). All operative procedures were performed on thermoregulated heating boards to maintain body temperature at 37°C. Animals were divided into the following subgroups.
Clamping group (bilateral clamping and supplementation with saline). Both kidneys were prepared carefully by a bilateral flank incision. Renal arteries were prepared and temporarily ligated on both sides to start clamping with microclips simultaneously. Dispensable fluid loss was prevented by closing wounds with sterile strips during the clamping phase. Intraperitoneal application of 0.5 ml 0.9% NaCl was started 15 min after clamping. Following the clamping period, both microclips were removed and sutures of muscular layers and skin were made to close the wound. For postoperative pain relieving therapy tramadol (0.05 mg/kg body wt) was subcutaneously applied and postoperative dehydration was prevented by subcutaneously administration of additional 0.3 ml 0.9% NaCl. Animals were kept in a warmed environment as long as animals became awake.
Sham group (sham operation and supplementation with saline). Identical procedure was performed in analogy as described for clamping group, except that in these animals no clamping of renal arteries was performed.
Control group (untreated animals). Animals with no previous treatment were investigated. These animals reflect day 0.
The care of animals and experimental procedures performed in this study were in accordance with the German law for animal protection and the guidelines of the American Journal of Physiology Guiding Principles in the Care and Use of Animals.
Measurement of Clearances and PAH Net Secretion
Inulin and PAH clearances were determined as described recently ( 27 ). Thereafter, net secretion of PAH (PNS) was calculated as described below.
In the anesthetized animal, the left femoral vein was cannulated with a PE-10 (polyethylene) catheter. Fluorescein-isothiocyanate-inulin (inulin) and PAH (1 mg of each substance solved in 0.25 ml 0.9% NaCl) were applied as a bolus injection, followed by constant infusion of both substances (5 mg/h inulin, 5 mg/h PAH) using a Secura FT perfusor (B. Braun, Melsungen, Germany). After suprapubic incision, the urine bladder was cannulated with a PE-50 (polyethylene) catheter to measure urine flow and obtain urine samples. When reaching a steady state after 30 min of infusion, urine was collected for 20 min and blood samples were drawn subsequently. Samples were centrifuged and stored at -20°C. Inulin concentrations in urine and plasma were determined by fluorescence spectrometry (1420 Victor 2 Multilabel Counter), whereas PAH concentrations were measured by photospectrometry (Dynatech Lab, Guernsey, UK) using the anthrone method. Calculations of inulin clearance, PAH clearance, and PNS were performed according to the equations: inulin clearance = ( I U x V U )/( I P x t ); PAH clearance = (PAH U x V U )/(PAH P x t ); and PNS = [(PAH U x V U )/ t ] - ; where I U is inulin concentration in urine; PAH U is PAH concentration in urine; I P is inulin concentration in plasma; PAH P is PAH concentration in plasma; V U is urine volume; and t is time of measurement.
Organ Preparation and Tissue Harvesting
After blood samples were drawn, both kidneys were perfused under pressure-controlled conditions (100 mmHg) with ice-cold 0.9% NaCl for 20 s. Subsequently, samples of renal cortex were snap-frozen in liquid nitrogen and stored at -80°C.
Protein Immunoblot
For Western blot analysis, frozen kidney cortex was homogenized using a stainless steel mortar cooled by liquid nitrogen, dissolved in lysis buffer containing 25 mmol/l Tris·HCl, 7 mmol/l reduced glutathione, 0.5 mmol/l EDTA, 0.2 mol/l PMSF, 1 µmol/l leupeptin, 1 µmol/l pepstatin, 1 µmol/l trans -epoxysuccinyl- L -leucylamido butane, and 1 mg/ml trypsin inhibitor, and further minced with an ultrasonic disperser UW 70 (Bandelin Electronic, Berlin, Germany). Total protein was measured in samples using the Bradford method ( 1 ). Samples of protein (5 to 40 µg) were analyzed by Western blot with the respective antibodies. Rabbit OAT1 polyclonal antibody (diluted 1:500) and rabbit OAT3 polyclonal antibody (diluted 1:500) were from alpha diagnostic (San Antonio, TX). Blots were subsequently incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:2,000, Dako, Hamburg, Germany) and were developed using a chemiluminescence kit (ECL Plus) following the manufacturer's instruction (Amersham Pharmacia Biotech, Buckinghamshire, UK). Blots were analyzed densitometrically using the Quantity One software (Bio-Rad Laboratories, Philadelphia, PA).
RT-PCR
RNA from kidney cortex was extracted using AquaPure RNA Isolation Kit (Bio-Rad). In brief, RT-PCR was performed according to Superscript One-Step RT-PCR system protocol (Invitrogen, Carlsbad, CA). cDNA was generated at 50°C for 30 min and then the samples were denatured at 94°C for 2 min. PCR amplification was performed in 40 cycles of 94°C for 15 s, then 55°C for 30 s, and 72°C for 60 s. The final elongation step was 72°C for 10 min. For OAT1, the primers were 5'-aga gtc aca gag ccc tgc at-3' (sense) and 5'-gcc cag gct gta gac ata gc-3' (antisense), resulting in a 402-bp RT-PCR product. For OAT3, the primers were 5'-tcc tgg tgg gta cca gag tc-3' (sense) and 5'-ctg cat ttc tga agg cac aa-3' (antisense), resulting in an 468-bp RT-PCR product. For GAPDH, the primers were 5'-cgg caa ctt caa cgg cac agt ca-3' (sense) and 5'-ggt ttc tcc agg cgg cat gtc a-3' (antisense), resulting in a 560-bp RT-PCR product. The RT-PCR products generated with primers for OAT1 and OAT3 were tested by sequencing (MWG Biotech, München, Germany) and were found to represent the predicted parts of the respective mRNAs.
Data Analysis
Data are presented as means ± SE. The n value is given in the text or in the figures. For all experiments, n equals the number of rats or the number of experiments (RT-PCR, Western blot) with tissue or tissue extractions from distinctive rats. Statistical significance was determined by unpaired Student's t -test. Data from sham-operated animals were tested against untreated controls and data from clamped animals were tested against sham-operated animals. Differences were considered statistically significant when P < 0.05.
Materials
Fluorescein-isothiocyanate-inulin and PAH were from ICN Pharmaceuticals (Costa Mesa, CA), tramadol (Tramal) was from Grünenthal (Aachen, Germany), xylacin hydrochloride (Rompun) was from Bayer (Leverkusen, Germany), and ketamine (Ketanest) was from Pharmacia and Upjohn (Erlangen, Germany). If not indicated otherwise, all substances were further diluted in 0.9% NaCl (wt/vol). If not stated otherwise, chemicals were from Sigma (St. Louis, MO).
RESULTS
Bilateral clamping of renal arteries led to a transient decrease of GFR as measured by renal clearance of inulin, which was almost total 6 h after start of reperfusion ( Fig. 1 ). Renal clearance of inulin gradually recovered until day 7 (168 h) after I/R injury. Subsequently, no significant difference between animals with clamped renal arteries and sham-operated animals was measured in the follow up beginning with day 7. Thus 7 days (168 h) after ischemic insult renal filtration appears to have already completely recovered compared with sham-operated animals.
Fig. 1. Effect of renal ischemia and reperfusion on the renal clearance of inulin in Sprague-Dawley rats. Renal clearance of inulin was determined as a measure of glomerular filtration rate (GFR) as described in MATERIALS AND METHODS [GFR = ( I U x V U )/( I P x t )]. After either a sham operation or a bilateral clamping of the renal arteries for 45 min, the renal clearance of inulin was determined after 6, 24, 72, 168, or 336 h. Renal clearance of inulin was additionally determined in untreated control rats. n Is given in or beside the respective bars. *Statistical significant difference between sham-operated and clamped animals.
As there are numerous studies indicating a decrease in RPF after iARF, we investigated the effect of I/R injury on PAH clearance in our model of bilateral clamping of renal arteries in rats. In analogy to what was measured for inulin clearance, PAH clearance almost completely ceased 6 h after reperfusion followed by a steady increase with time ( Fig. 2 ). Similar to what was observed for inulin clearance, PAH clearance recovery was completed 7 days (168 h) after I/R injury compared with sham-treated animals.
Fig. 2. Effect of renal ischemia and reperfusion on the renal clearance of para -aminohippuric acid (PAH) in Sprague-Dawley rats. Renal clearance of PAH was determined as described in MATERIALS AND METHODS [PAH clearance = (PAH U x V U )/(PAH P x t )]. After either a sham operation or a bilateral clamping of the renal arteries for 45 min, the renal clearance of PAH was determined after 6, 24, 72, 168, or 336 h. Renal clearance of PAH was additionally determined in untreated control rats. n Is given in or beside the respective bars. *Statistical significant difference between sham-operated and clamped animals.
PAH clearance was diminished for up to 24 h after I/R injury, which can in principle be due to reduced renal delivery of PAH or reduced proximal tubular secretion of PAH. In particular, the study from Corrigan et al. ( 5 ) indicated a reduced proximal tubular secretion of the organic anion PAH after I/R injury. Therefore, we calculated the PAH net secretion (PNS) as a measure of organic anion secretion by the renal proximal tubule. As indicated in Fig. 3, PNS strongly declines 6 h after I/R injury and gradually increases with time. Surprisingly, 24 h after I/R injury PNS was slightly reduced in sham-operated animals compared with untreated control. At present, we have no ensured explanation for this phenomenon except the putative peri- and postoperative stress. More important however, the PNS in clamped animals is substantially different from sham-operated animals. In accordance to inulin clearance and PAH clearance as well, PNS reaches values of sham-treated animals 7 days (168 h) after I/R injury.
Fig. 3. Effect of renal ischemia and reperfusion on the renal net secretion of of PAH in Sprague-Dawley rats. Renal net secretion of PAH (PNS) was determined as described in MATERIALS AND METHODS {PNS = [(PAH U x V U )/ t ] - }. After either a sham operation or a bilateral clamping of the renal arteries for 45 min, the renal net secretion of PAH was determined after 6, 24, 72, 168, or 336 h. Renal net secretion of PAH was additionally determined in untreated control rats. n Is given in or beside the respective bars. #Statistical significant difference between sham-operated and control animals. *Statistical significant difference between sham-operated and clamped animals.
A reasonable explanation for a reduced PNS in general is an impairment of the proximal tubules capacity to secrete organic anions after renal I/R injury. As already mentioned in the introduction, the rate-limiting step of proximal tubular organic anion secretion is basolateral uptake into the proximal tubular epithelial cells, which is mediated by OAT1 and OAT3. Since the expression of both proteins is known to be regulated in principle, we hypothesized that the observed decline in PNS after I/R injury may be due to a diminished amount of OAT1 and/or OAT3 present in the proximal tubular cells.
To further test this hypothesis, we first investigated the effect of renal ischemia and subsequent reperfusion on the amount of mRNA of both OAT1 and OAT3 present in kidney cortex extracts. Figure 4 displays a typical RT-PCR result showing the relative amount of mRNA of OAT1 and OAT3 compared with glycerinaldehyd dehydrogenase (GAPDH). As indicated in Fig. 4 B, the relative amount of OAT1 mRNA is dramatically decreased 6 h after I/R injury, which is also the case after 24 h. The amount of mRNA is again indifferent 72 h after I/R injury in clamped and sham-operated animals. The relative amount of OAT1 mRNA in sham-operated animals never differs from the amount in untreated controls. As indicated in Fig. 4 C, a similar regulation was determined for the relative amount of OAT3 mRNA after I/R injury. Thus the relative amount of mRNA for both OAT1 and OAT3 is strongly decreased 6 h and is totally restored after a 72-h postischemic reperfusion period.
Fig. 4. Effect of renal ischemia and reperfusion on the mRNA levels of OAT1 and OAT3 in renal cortex from Sprague-Dawley rats. Total RNA was generated from kidney cortex. RT-PCR against OAT1, OAT3, and GAPDH was performed as described in MATERIALS AND METHODS. A : typical pattern of RT-PCR products. RT-PCR leads to products of the predicted lengths for OAT1 (402 bp), OAT3 (468 bp), and GAPDH (560 bp). After a reperfusion interval of 72 h, no difference was detected between the untreated controls, the sham-operated animals, and the clamped group. B : effect of renal ischemia and reperfusion on the mRNA levels of OAT1. Total RNA was generated from kidney cortex. Amount of OAT1 mRNA signal normalized to the respective signal from GAPDH. *Statistical significant difference between sham-operated and clamped animals. n Is given in the respective bars. C : effect of renal ischemia and reperfusion on the mRNA levels of OAT3. Total RNA was generated from kidney cortex. Amount of OAT3 mRNA signal normalized to the respective signal from GAPDH. *Statistical significant difference between sham-operated and clamped animals. n Is given in the respective bars.
The latter data strongly point to a reduced expression level of OAT1 and OAT3 after I/R injury. As this would be a potential explanation for the decline in PNS after renal ischemia, we investigated the amount of OAT1 and OAT3 protein in kidney cortex extracts in our rat model of iARF. Anti-OAT1 antibody recognized a band in the range just below 60 kDa, which is well within the range of data published before for OAT1 ( 14, 18, 26 ). As indicated in Fig. 5 B, the relative expression of OAT1 is strongly reduced 6 h after reperfusion and then gradually recovers with time. The expression of OAT1 is no more different in clamped animals compared with sham-operated animals 7 days after I/R injury. Expression of OAT1 in sham-operated animals never differs from untreated controls. As indicated in Figs. 5 A and 6 A, the expression of -actin in renal cortex was not affected by I/R injury.
Fig. 5. Effect of renal ischemia and reperfusion on the protein levels of OAT1 in renal cortex from Sprague-Dawley rats. Total protein was generated from kidney cortex. Western blot against OAT1 and -actin was performed as described in MATERIALS AND METHODS. A : Western blot against OAT1 and -actin. Antibody against OAT1 recognized a band in the range of 57 kDa, and the anti- -actin antibody recognized a band at 42 kDa. Western blotting was performed using protein extracts from kidney cortex either from sham-operated or from clamping animals after 6, 24, 72, 168, or 336 h. Kidney cortex extracts from untreated animals were used as controls. B : effect of renal ischemia and reperfusion on the relative expression of OAT1. Amount of OAT1 Western blot signal was normalized to the respective signal from -actin. Western blotting was performed using protein extracts from kidney cortex either from sham-operated or from clamping animals after 6, 24, 72, 168, or 336 h. Kidney cortex extracts from untreated animals were used as controls. *Statistical significant difference between sham-operated and clamped animals. n Is given in the respective bars.
Fig. 6. Effect of renal ischemia and reperfusion on the protein levels of OAT3 in renal cortex from Sprague-Dawley rats. Total protein was generated from kidney cortex. Western blot against OAT3 and -actin was performed as described in MATERIALS AND METHODS. A : Western blot against OAT3 and -actin. Antibody against OAT3 recognized a band in the range of 110 kDa, and the anti- -actin antibody recognized a band at 42 kDa. Western blotting was performed using protein extracts from kidney cortex either from sham-operated or from clamping animals after 6, 24, 72, 168, or 336 h. Kidney cortex extracts from untreated animals were used as controls. B : effect of renal ischemia and reperfusion on the relative expression of OAT1. Amount of OAT1 Western blot signal was normalized to the respective signal from -actin. Western blotting was performed using protein extracts from kidney cortex either from sham-operated or from clamping animals after 6, 24, 72, 168, or 336 h. Kidney cortex extracts from untreated animals were used as controls. *Statistical significant difference between sham-operated and clamped animals. n Is given in the respective bars.
Anti-OAT3 antibody recognized two prominent bands in the range of 130 and 110 kDa, the latter being the more pronounced one. Preabsorption of the antibody to the epitope peptide led to strongly diminished signal of both bands (data not shown), indicating that the positive bands were specific for the antigen peptide. In accordance to what was described by Ljubojevic et al. ( 17 ), we used the lower band for comparison of the effect of renal ischemia in our study. Relative protein expression of OAT3 is similar compared with OAT1: relative expression of OAT3 is strongly reduced 24 h after renal ischemia and then gradually recovers with time ( Fig. 6 B ). Starting with day 7 (168 h) after I/R injury, expression of OAT3 is no more different in clamped and sham-operated animals. Expression of OAT3 in sham-operated animals did not differ from untreated controls.
DISCUSSION
In the present study, we addressed the question of whether the decline of PAH clearance measured after renal ischemia may be accompanied by a decreased expression of the basolaterally located transport proteins OAT1 and/or OAT3, which mediate the rate-limiting step of renal organic anion secretion. In a first approach, we therefore characterized the behavior of the renal clearance of inulin and PAH at different time points in the reperfusion period. The effect of ischemia observed in the reperfusion period is in good agreement with data published before ( 27 ), indicating a good reliability and reproducibility of this particular model system of ischemic acute renal failure. As expected, renal ischemia leads to a similar pattern of inhibition of PAH clearance and inulin clearance, due to the fact that reduced plasma flow should lead to reduced filtration. Thus, considering PAH clearance and inulin clearance alone, it is impossible to decide to what extent a reduced renal clearance of PAH is really due to impaired renal perfusion or to diminished proximal tubular secretion. Therefore, we calculated the PNS (as indicated in MATERIALS AND METHODS ) to extinguish the effect of changed glomerular filtration within PAH clearance data to describe the secretion of organic anions by the proximal tubular epithelium.
PNS was diminished in clamped animals and recovered to values of untreated controls or sham-operated animals at day 7 (168 h). This is in parallel to the pattern of the clearances of both inulin and PAH. Thus the reduction of proximal tubular PAH secretion is, at least in part, responsible for the decrease in PAH clearance observed in the reperfusion period. This is in agreement with Corrigan et al. ( 5 ) who concluded that the reduced extraction of PAH after ischemia has a major impact on PAH clearance.
As I/R injury decreases PNS, we speculated whether this may be due to a reduced expression of the basolaterally located, rate-limiting ( 9 ) exchange proteins OAT1 and/or OAT3. The amount of OAT1 and OAT3 steeply decreases on the level of mRNA and protein after intervention. The relative amount of mRNA from both OAT1 and OAT3 in kidney cortex of clamped animals is restabilized to the range of sham-operated animals 3 days (72 h) after I/R injury, whereas equalization of protein amount is achieved 7 days (168 h) after intervention. This is strong evidence that transcriptional recovery of OAT1 and OAT3 leads to a reconstitution of mRNA amount followed by a delayed increase of the respective proteins.
The amount of protein from OAT1 and OAT3 recovered 7 days after reperfusion. This is sufficient to explain the parallel recovery of PNS at the same time, although it is certainly no proof that restored expression of OAT1 and OAT3 alone is responsible for PNS recovery. We are aware of the fact that other effects may be involved in the recovery of PNS. Beside a recovery of renal perfusion, the regain of proximal tubular surface area ( 31 ) or the restoration of epithelial polarity, which both were demonstrated to be lost after I/R injury ( 37 ), may be involved. Moreover, the apical export steps for organic anions ( 35, 39 ) may also be affected by I/R injury and will thus influence the PNS and its recovery. Unfortunately, up to now there are no data available on expression of luminal organic anion transport proteins in the proximal tubule after renal I/R injury. Therefore, we cannot exclude that changes in expression or activity of luminal organic anion transport proteins may be involved in the behavior of PNS. Whether this is the case in our model will be investigated in future studies.
As mentioned earlier, it has already been shown that expression of OAT1 and/or OAT3 may be regulated in principle. Only recently, in a cell line from rat proximal tubule we could show that prostaglandin E 2 downregulates the expression of OAT1 and OAT3 in a time- and dose-dependent manner ( 26 ). Prostaglandin E 2 is a metabolite of arachidonic acid playing an important role in inflammation processes and intracellular signal transduction. Prostaglandin E 2 is known to be an important regulator of renal perfusion ( 10 ) and its generation is significantly increased in the kidney after I/R injury ( 19 ). In the latter, it is thought to restore and stabilize renal perfusion [together with other vasoactive regulators, e.g., nitric oxide (NO) ( 6, 34 )]. COX-2, one source of prostaglandin E 2 generation, is induced and activated in a NO-dependent manner ( 38 ). As NO is generated in iARF ( 27 ), this may represent a signaling pathway leading to COX-2 activation and subsequent generation of prostaglandin E 2. A recent study from Myers et al. ( 21 ) indicates that upregulation of COX-2 takes also place in the kidney cortex, which is also the case in our model (preliminary, unpublished results). Interestingly, upregulation of COX-2 is reported to be maximal in the period up to 5 h after ischemia and then gradually declines ( 19 ). This is in good agreement with our data showing the maximum detrimental effects 6 h after I/R injury. Supportive preliminary evidence is obtained using a simple in vitro model of proximal tubular ischemia. Therein, we applied hypoxia and hypoglycemia (2 h) to a cell line from rat proximal tubule (NRK-52E), leading to a decrease of basolateral organic anion uptake (and expression of OAT1 and OAT3) 48 h after ischemia. Application of the COX-Inhibitor indomethacin after hypoxia/hypoglycemia completely restored organic anion uptake (unpublished results), which is supportive to the above mentioned mechanistic considerations. In summary, we therefore hypothesize that prostaglandin E 2 is generated in the kidney cortex after I/R injury, which then downregulates the expression of OAT1 and OAT3. Whether this speculation holds true will be tested in future studies.
As OAT1 and OAT3 play a crucial role in the renal excretion of a variety of anionic drugs or other potentially toxic compounds, the data presented here are in agreement with dose reduction necessities in case of ARF. Thus, renal ischemia will affect the pharmacokinetics of the mentioned substances, leading to increased plasma levels and half-lives. Prostaglandin E 2 is an organic anion itself and thus a substrate for both OAT1 and OAT3 ( 15 ). According to the data presented here, the proximal tubular elimination of prostaglandin E 2 will be inhibited after renal I/R injury. As a consequence, this would prevent a washout of prostaglandin E 2 and could thus represent a rescue mechanism for maintenance of renal circulation after renal ischemia.
GRANTS
This work was in part supported by the Deutsche Forschungsgemeinschaft Grant DFG Ge 905/4-4 and the Interdiziplinaeres Klinisches Forschungszentrum (IZKF) Wuerzburg Grant E-34( 1 ).
【参考文献】
Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254, 1976.
Brandoni A, Anzai N, Kanai Y, Endou H, Torres AM. Renal elimination of p-aminohippurate (PAH) in response to three days of biliary obstruction in the rat. The role of OAT1 and OAT3. Biochim Biophys Acta In press.
Buist SC, Cherrington NJ, Klaassen CD. Endocrine regulation of rat organic anion transporters. Drug Metab Dispos 31: 559-564, 2003.
Cha SH, Sekine T, Kusuhara H, Yu E, Kim JY, Kim DK, Sugiyama Y, Kanai Y, Endou H. Molecular cloning and characterization of multispecific organic anion transporter 4 expressed in the placenta. J Biol Chem 275: 4507-4512, 2000.
Corrigan G, Ramaswamy D, Kwon O, Sommer FG, Alfrey EJ, Dafoe DC, Olshen RA, Scandling JD, Myers BD. PAH extraction and estimation of plasma flow in human postischemic acute renal failure. Am J Physiol Renal Physiol 277: F312-F318, 1999.
Cristol JP, Thiemermann C, Mitchell JA, Walder C, Vane JR. Support of renal blood flow after ischaemic-reperfusion injury by endogenous formation of nitric oxide and of cyclo-oxygenase vasodilator metabolites. Br J Pharmacol 109: 188-194, 1993.
Dantzler WH. Comparative aspects of renal organic anion transport. Cell Physiol Biochem 6: 28-38, 1996.
Dantzler WH. Renal organic anion transport: a comparative and cellular perspective. Biochim Biophys Acta 1566: 169-181, 2002.
Dantzler WH, Wright SH. The molecular and cellular physiology of basolateral organic anion transport in mammalian renal tubules. Biochim Biophys Acta 1618: 185-193, 2003.
Epstein M. Nonsteroidal anti-inflammatory drugs and the continuum of renal dysfunction. J Hypertens Suppl 20: S17-S23, 2002.
Friis C. Renal transport and metabolism of xenobiotics. Acta Vet Scand 32: 33-43, 1991.
Habu Y, Yano I, Okuda M, Fukatsu A, Inui Ki. Restored expression and activity of organic ion transporters rOAT1, rOAT3 and rOCT2 after hyperuricemia in the rat kidney. Biochem Pharmacol 69: 993-999, 2005.
Habu Y, Yano I, Takeuchi A, Saito H, Okuda M, Fukatsu A, Inui Ki. Decreased activity of basolateral organic ion transports in hyperuricemic rat kidney: roles of organic ion transporters, rOAT1, rOAT3 and rOCT2. Biochem Pharmacol 66: 1107-1114, 2003.
Hosoyamada M, Sekine T, Kanai Y, Endou H. Molecular cloning and functional expression of a multispecific organic anion transporter from human kidney. Am J Physiol Renal Physiol 276: F122-F128, 1999.
Kimura H, Takeda M, Narikawa S, Enomoto A, Ichida K, Endou H. Human organic anion transporters and human organic cation transporters mediate renal transport of prostaglandins. J Pharmacol Exp Ther 301: 293-298, 2002.
Kusuhara H, Sekine T, Utsunomiya-Tate N, Tsuda M, Kojima R, Cha SH, Sugiyama Y, Kanai Y, Endou H. Molecular cloning and characterization of a new multispecific organic anion transporter from rat brain. J Biol Chem 274: 13675-13680, 1999.
Ljubojevic M, Herak-Kramberger CM, Hagos Y, Bahn A, Endou H, Burckhardt G, Sabolic I. Rat renal cortical OAT1 and OAT3 exhibit gender differences determined by both androgen stimulation and estrogen inhibition. Am J Physiol Renal Physiol 287: F124-F138, 2004.
Lu R, Chan BS, Schuster VL. Cloning of the human kidney PAH transporter: narrow substrate specificity and regulation by protein kinase C. Am J Physiol Renal Physiol 276: F295-F303, 1999.
Matsuyama M, Yoshimura R, Hase T, Kawahito Y, Sano H, Nakatani T. Study of cyclooxygenase-2 in renal ischemia-reperfusion injury. Transplant Proc 37: 370-372, 2005.
Monte JC, Nagle MA, Eraly SA, Nigam SK. Identification of a novel murine organic anion transporter family member, OAT6, expressed in olfactory mucosa. Biochem Biophys Res Commun 323: 429-436, 2004.
Myers SI, Wang L, Liu F, Bartula LL. Suprarenal aortic clamping and reperfusion decreases medullary and cortical blood flow by decreased endogenous renal nitric oxide and PGE 2 synthesis. J Vasc Surg 42: 524-531, 2005.
Perri D, Ito S, Rowsell V, Shear NH. The kidney-the body's playground for drugs: an overview of renal drug handling with selected clinical correlates. Can J Clin Pharmacol 10: 17-23, 2003.
Pritchard JB, Miller DS. Proximal tubular transport of organic anions and cations. In: The Kidney: Physiology and Pathophysiology (2nd ed.), edited by Seldin DW and Giebisch G. New York: Raven, 1992, p. 2921-2945.
Raff U, Schneider R, Gambaryan S, Seibold S, Reber M, Vornberger N, Freund R, Schramm L, Wanner C, Galle J. L -arginine does not affect renal morphology and cell survival in ischemic acute renal failure in rats. Nephron Physiol 101: p39-p50, 2005.
Reyes JL, Melendez E, Alegria A, Jaramillo-Juarez F. Influence of sex differences on the renal secretion of organic anions. Endocrinology 139: 1581-1587, 1998.
Sauvant C, Holzinger H, Gekle M. Prostaglandin E 2 inhibits its own renal transport by downregulation of organic anion transporters rOAT1 and rOAT3. J Am Soc Nephrol 17: 46-53, 2006.
Schneider R, Raff U, Vornberger N, Schmidt M, Freund R, Reber M, Schramm L, Gambaryan S, Wanner C, Schmidt HHHW, Galle J. L -Arginine counteracts nitric oxide deficiency and improves the recovery phase of ischemic acute renal failure in rats. Kidney Int 64: 216-225, 2003.
Sekine T, Cha SH, Tsuda M, Apiwattanakul N, Nakajima N, Kanai Y, Endou H. Identification of multispecific organic anion transporter 2 expressed predominantly in the liver. FEBS Lett 429: 179-182, 1998.
Sekine T, Miyazaki H, Endou H. Molecular physiology of renal organic anion transporters. Am J Physiol Renal Physiol 290: F251-F261, 2006.
Sekine T, Watanabe K, Hosoyamada M, Kanai N, Endou H. A novel multispecific organic anion transporter (PAH transporter): its structure and functional characteristics. Nova Acta Leopoldina 306: 119-126, 1998.
Sheridan AM, Bonventre JV. Cell biology and molecular mechanisms of injury in ischemic acute renal failure. Curr Opin Nephrol Hypertens 9: 427-434, 2000.
Sweet DH, Chan LM, Walden R, Yang XP, Miller DS, Pritchard JB. Organic anion transporter 3 (Slc22a8) is a dicarboxylate exchanger indirectly coupled to the Na + gradient. Am J Physiol Renal Physiol 284: F763-F769, 2003.
Terlouw SA, Masereeuw R, Russel FG. Modulatory effects of hormones, drugs, and toxic events on renal organic anion transport. Biochem Pharmacol 65: 1393-1405, 2003.
Tokuyama H, Hayashi K, Matsuda H, Kubota E, Honda M, Okubo K, Takamatsu I, Ozawa Y, Saruta T. Role of nitric oxide and prostaglandin E 2 in acute renal hypoperfusion. Nephrology (Carlton) 8: 65-71, 2003.
Van Montfoort JE, Hagenbuch B, Groothuis GM, Koepsell H, Meier PJ, Meijer DK. Drug uptake systems in liver and kidney. Curr Drug Metab 4: 185-211, 2003.
Villar SR, Brandoni A, Anzai N, Endou H, Torres AM. Altered expression of rat renal cortical OAT1 and OAT3 in response to bilateral ureteral obstruction. Kidney Int 68: 2704-2713, 2005.
Woroniecki R, Ferdinand JR, Morrow JS, Devarajan P. Dissociation of spectrin-ankyrin complex as a basis for loss of Na-K-ATPase polarity after ischemia. Am J Physiol Renal Physiol 284: F358-F364, 2003.
Yang T, Zhang A, Pasumarthy A, Zhang L, Warnock Z, Schnermann JB. Nitric oxide stimulates COX-2 expression in cultured collecting duct cells through MAP kinases and superoxide but not cGMP. Am J Physiol Renal Physiol In press.
You G. Structure, function, and regulation of renal organic anion transporters. Med Res Rev 22: 602-616, 2002.
Youngblood GL, Sweet DH. Identification and functional assessment of the novel murine organic anion transporter Oat5 (Slc22a19) expressed in kidney. Am J Physiol Renal Physiol 287: F236-F244, 2004.
作者单位：1 Institute of Physiology, 2 Clinic of Internal Medicine I, Division of Nephrology, University of Wuerzburg, Wuerzburg, Germany