Riboflavin transport by isolated perfused rabbit renal proximal tubules

Norimoto Yanagawa1,2, Remi N. G. Shih1,2, Oak D. Jo1,2, and Hamid M. Said3,4

1 Medical and Research Services, Sepulveda Veterans Affairs Medical Center, Sepulveda 91343; 2 Department of Medicine, School of Medicine, University of California at Los Angeles, Los Angeles 90024; 3 Medical Research Service, Long Beach Veterans Affairs Medical Center, Long Beach 90822; and 4 Departments of Medicine and Physiology/Biophysics, School of Medicine, University of California at Irvine, Irvine, California 92717


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Rabbit renal proximal tubular transport of riboflavin (RF) was examined by using the in vitro isolated tubule perfusion technique. We found that proximal tubules actively reabsorbed (Jlb) and secreted (Jbl) RF. At 0.1 µM RF concentration, Jbl was significantly higher than Jlb, resulting in a net secretion. This net secretion of RF was decreased at 0.01 µM RF concentration and increased at 1 µM RF concentration. Both Jlb and Jbl were inhibited by lowering temperature or by adding iodoacetate, a metabolic inhibitor, and lumichrome, an RF analog, suggesting the involvement of carrier-mediated transport mechanisms. Jbl was inhibited by probenecid, an anion transport inhibitor, and by para-aminohippuric acid, an organic anion, suggesting the relevance of RF secretion to renal organic anion transport. Jbl was also inhibited by alkaline pH (8.0) and by the calmodulin inhibitor trifluoperazine, indicating the influence of pH and Ca2+/calmodulin-dependent pathway on RF secretion. Finally, we found that addition of chlorpromazine, a phenothiazine derivative, inhibited both Jlb and Jbl, raising the concern about the nutritional status in patients receiving such a type of medication.

carrier-mediated transport; chlorpromazine


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

RIBOFLAVIN (RF) is an essential vitamin required for normal cellular functions. It serves in key metabolic reactions in the body, which include carbohydrate, amino acid, and lipid metabolism, and the conversion of folic acid and pyridoxine into their coenzyme forms (4, 14, 15). RF deficiency can thus cause a series of clinical abnormalities, including growth retardation, degenerative changes in the nervous system, anemia, skin lesions, and an increase in the susceptibility to carcinogens (4, 8, 16). Mammals cannot synthesize RF but obtain the vitamin from the diet through intestinal absorption. Elimination of RF from the body takes place mainly through the kidneys, so that kidneys play a key role in the regulation of normal body RF homeostasis. In attempts to elucidate the mechanisms whereby kidneys excrete RF, clearance studies have been performed in chickens (20), rats (3), dogs (10), and humans (9), which showed that RF is filtered through glomeruli and followed by tubular reabsorption. On the basis of the finding that RF excretion rate exceeded glomerular filtration rate, a component of tubular secretion was also implicated (3, 9, 10). Furthermore, stop-flow studies suggested proximal tubules to be the site where RF reabsorption and secretion take place (10). However, the limitations in these classical clearance studies made it difficult to disclose the individual transport process from this complex bidirectional RF transport. The aim of our present study is to examine proximal tubule RF transport by using the in vitro isolated tubule perfusion technique, an experimental system that allows unidirectional transport processes to be examined in a defined tubular segment. We found that both RF reabsorption and secretion occurred in rabbit proximal tubules with a net secretion of RF. These transport processes are likely to occur through carrier-mediated transport mechanisms, under the regulation of RF availability, the prevailing pH, and the intracellular Ca2+/calmodulin-mediated pathway. Our results also show that chlorpromazine, a widely used psychotropic agent, interfered with these RF transport processes.


    METHODS
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METHODS
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Animals. New Zealand White male rabbits, weighing 1.5-2.0 kg, were used in these studies. The animals were maintained on an ad libitum diet of standard rabbit chow with free access to water. This study protocol was approved by the local animal care committee.

In vitro isolated tubule perfusion. Superficial proximal straight and convoluted tubules were dissected from rabbit renal cortex free-hand under the dissecting microscope and perfused in vitro using the conventional method originally described by Burg et al. (2). The luminal perfusate contained (in mM) 125 NaCl, 25 NaHCO3, 5 KCl, 1.2 MgSO4, 2 NaH2PO4, and 1.5 CaCl2, pH 7.4. The peritubular bath solution was the identical solution plus 6% BSA. The bath pH was adjusted after being preequilibrated with 95% O2-5% CO2 at 37°C. The perfusate and bath were made isosmolar with NaCl. Twenty- to thirty-minute equilibration periods were allowed before the first sample collection and between each collection period. To minimize the potential photodecomposition of RF, the lights of the room and the microscope were dimmed, and the duration of these studies was limited to <2 h.

Measurements of RF fluxes. Extensively dialyzed [14C]inulin (15 µCi/ml) was added to the perfusate as the impermeant volume marker for the measurements of perfusion rate (Vi) and collection rate (Vo). The lumen-to-bath RF flux rate (Jlb) was measured by adding [3H]RF (15 µCi/ml) to the perfusate, whereas the bath-to-lumen RF flux rate (Jbl) was measured by adding [3H]RF (15 µCi/ml) to the bath solution. Jlb and Jbl were calculated as Jlb = [(ViCi - VoCo)/L][RF/Ci] and Jbl = VoCo/XbL, where Vi is the perfusion rate (nl/min), Vo is the collection rate (nl/min), L the tubule length (mm), Ci is the [3H]RF concentration in the perfusate (cpm/nl), Co is the [3H]RF concentration in the collected samples (cpm/nl), RF is the RF concentration (fmol/nl), and Xb is the specific activity of [3H]RF in the bath (cpm/fmol). It is not known to what extent RF is metabolized by the proximal tubules in these studies. Our computation of Jbl based on the rate of RF appearance from the peritubular bath into the luminal perfusate thus does not include the amount of RF metabolized by the proximal tubules.

Data analysis. Data presented are means ± SE of separate determinations on separate tubules. Statistic P values were analyzed by using Student's t-test for paired or unpaired data as appropriate.


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Jlb and Jbl across the isolated perfused proximal tubules. We first measured Jlb and Jbl across the isolated perfused proximal straight tubules (PST; S2-S3 segments) with both perfusate and bath solution containing 0.1 µM RF. As shown in Fig. 1, we found that Jbl was significantly higher than Jlb (0.58 ± 0.06 vs. 0.22 ± 0.02 fmol · mm-1 · min-1, n = 8, P < 0.01). The net RF transport across PST thus occurred in the direction of RF secretion from bath to lumen at a rate of ~0.36 fmol · mm-1 · min-1. Similar results were also obtained with superficial proximal convoluted tubules (PCT; S1-S2 segments), where Jbl was also significantly higher than Jlb (0.90 ± 0.15 vs. 0.45 ± 0.1 fmol · mm-1 · min-1, n = 9, P < 0.03), with net RF secretion at a rate (~0.45 fmol · mm-1 · min-1) not significantly different from that of PST.


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Fig. 1.   Riboflavin (RF) reabsorbtion from the lumen to bath (Jlb) and secretion from the bath to lumen (Jbl) across proximal straight tubules (PST) and proximal convoluted tubules (PCT). Jlb and Jbl across PST and PCT were measured with both perfusate and bath solution containing 0.1 µM RF. In these studies, Jlb and Jbl were measured in sequence with the same tubule. Jbl was significantly higher than Jlb in both PST (n = 8, *P < 0.01) and PCT (n = 9, *P < 0.03), with a net Jbl at comparable rates between PST and PCT.

Effects of RF concentrations. We next examined the effect of varying RF concentrations on Jlb and Jbl across PST. As shown in Fig. 2, lowering the perfusate and bath RF concentration from 0.1 to 0.01 µM significantly reduced both Jlb and Jbl. At this low RF concentration, Jlb was not significantly different from Jbl (0.03 ± 0.004 vs. 0.03 ± 0.008 fmol · mm-1 · min-1, n = 5, P = 0.6), so that no net RF transport across PST occurred at this low RF concentration. In contrast, raising the perfusate and bath RF concentration from 0.1 to 1 µM significantly increased both Jlb and Jbl. The net transport of RF across PST at this high RF concentration occurred in the direction of RF secretion at a rate (~5.13 fmol · mm-1 · min-1) significantly higher than that at 0.1 µM RF concentration.


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Fig. 2.   Effects of RF concentrations. The influence of RF levels on Jlb and Jbl was examined by changing RF concentrations in the perfusate and bath. Lowering RF concentration from 0.1 to 0.01 µM significantly reduced both Jlb and Jbl with minimal net RF secretion, whereas raising RF concentration from 0.1 to 1 µM significantly increased both Jlb and Jbl with increased net RF secretion (n = 6, *P < 0.01 vs. Jlb).

Effects of temperature and lumichrome. We found that both Jlb and Jbl across PST were temperature sensitive. Thus lowering bath temperature from 38 to 18°C significantly lowered Jlb (from 0.14 ± 0.02 to 0.04 ± 0.01 fmol · mm-1 · min-1, n = 4, P < 0.01) and Jbl (from 0.32 ± 0.08 to 0.09 ± 0.05 fmol · mm-1 · min-1, n = 4, P < 0.05). In separate studies, we also found that addition of the metabolic inhibitor iodoacetate (10 mM) to the bath medium completely abolished RF fluxes. In addition, we found that adding lumichrome (50 µM), an RF analog, to either the perfusate or the bath solution significantly lowered Jlb (from 0.16 ± 0.03 to 0.10 ± 0.01 fmol · mm-1 · min-1, n = 4, P < 0.05) or Jbl (from 0.44 ± 0.03 to 0.21 ± 0.06 fmol · mm-1 · min-1, n = 4, P < 0.05), respectively. Taken together, these results indicate the involvement of carrier-mediated active transport mechanisms in both Jlb and Jbl across PST.

Effects of probenecid and PAH. Because organic anion transport mechanism plays an important role in the secretion of organic metabolites by proximal tubules, we examined the involvement of this transport system in RF transport by testing the effects of probenecid, an anion transport inhibitor, and PAH, an organic anion. As shown in Fig. 3, we found that Jbl was significantly inhibited when 1 mM of probenecid (from 0.51 ± 0.12 to 0.31 ± 0.06 fmol · mm-1 · min-1, n = 5, P < 0.05) or PAH (from 0.52 ± 0.12 to 0.42 ± 0.12 fmol · mm-1 · min-1, n = 5, P < 0.05) was added to the bath solution. In contrast, no effect on Jlb was found with the addition of 1 mM of probenecid (from 0.19 ± 0.03 to 0.24 ± 0.04 fmol · mm-1 · min-1, n = 4) or PAH (from 0.19 ± 0.04 to 0.17 ± 0.03 fmol · mm-1 · min-1, n = 4) to the perfusate. These results thus indicate that the organic anion transport system is involved in RF secretion by PST.


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Fig. 3.   Effects of probenecid (A) and para-aminohippuric acid (PAH) (B). The involvement of organic anion transport system was examined by studying the effects of probenecid (1 mM) and PAH (1 mM). Addition of these agents to perfusate did not affect Jlb (n = 4), whereas addition of these agents to bath solution significantly inhibited Jbl (n = 5, *P < 0.05 vs. control).

Effect of pH. In another set of studies, we examined the effects of altering luminal and peritubular pH on RF transport across PST. Changes in perfusate pH from 7.4 to 7.0 or 8.0 did not affect Jlb significantly (from 0.16 ± 0.02 to 0.13 ± 0.02 or 0.14 ± 0.01 fmol · mm-1 · min-1, n = 6, respectively). In contrast, whereas lowering bath medium pH from 7.4 to 7.0 did not affect Jbl (from 0.39 ± 0.12 to 0.35 ± 0.07 fmol · mm-1 · min-1, n = 6), raising bath medium pH from 7.4 to 8.0 significantly lowered Jbl (from 0.39 ± 0.12 to 0.19 ± 0.03 fmol · mm-1 · min-1, n = 6, P < 0.05).

Effects of cAMP, phorbol ester, and trifluoperazine. Because protein kinase systems are known to regulate solute transport processes in proximal tubules, we examined the possible role played by these pathways in regulating the RF transport across PST. For PKA- or PKC-mediated pathways, we tested the effect of adding 8-bromo-cAMP (0.1 mM) or phorbol 12-myristate 13-acetate (PMA) (50 µM), respectively, to the bath solution. For the involvement of Ca2+/calmodulin-mediated pathways, we tested the effect of the calmodulin inhibitor trifluoperazine (TFP). We found that neither 8-bromo-cAMP nor PMA significantly affected Jlb (from 0.16 ± 0.03 to 0.17 ± 0.04 fmol · mm-1 · min-1 and from 0.22 ± 0.03 to 0.21 ± 0.01 fmol · mm-1 · min-1, respectively, n = 6) or Jbl (from 0.32 ± 0.08 to 0.33 ± 0.05 fmol · mm-1 · min-1 and from 0.75 ± 0.10 to 0.57 ± 0.11 fmol · mm-1 · min-1, respectively, n = 6). However, addition of TFP (250 µM) to the bath solution significantly lowered Jbl (from 0.45 ± 0.04 to 0.19 ± 0.05 fmol · mm-1 · min-1, n = 6, P < 0.05) but not Jlb (from 0.12 ± 0.02 to 0.13 ± 0.03 fmol · mm-1 · min-1, n = 6). These results therefore indicate that RF secretion, but not reabsorption, by PST is under the influence of Ca2+/calmodulin-mediated pathway.

Effect of chlorpromazine. Because a variety of psychotropic agents have been found to share structural similarity with RF and may thus affect RF transport/metabolism, we also tested the effect of chlorpromazine, a phenothiazine derivative, on RF transport across PST. As shown in Fig. 4, addition of chlorpromazine (1 mM) to the perfusate inhibited Jlb significantly (from 0.38 ± 0.03 to 0.17 ± 0.07 fmol · mm-1 · min-1, n = 5, P < 0.02). Similarly, addition of chlorpromazine (1 mM) to the bath solution also caused a significant inhibition of Jbl (from 0.65 ± 0.06 to 0.22 ± 0.05 fmol · mm-1 · min-1, n = 5, P < 0.01).


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Fig. 4.   Effect of chlorpromazine. The effects of adding chlorpromazine (1 mM) to the perfusate or bath on Jlb or Jbl, respectively, were examined. Addition of chlorpromazine caused significant inhibitions on both Jlb and Jbl (n = 5, *P < 0.02 vs. control).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of our present study is to examine RF transport in rabbit renal proximal tubules by using the in vitro isolated tubule perfusion technique. This experimental system is ideal for studying bidirectional renal tubular transport processes because it allows unidirectional fluxes to be measured separately in a defined tubule segment. We found that rabbit renal proximal tubules reabsorbed and secreted RF. In PST, the capacity of Jbl was significantly greater than that of Jlb, so that the net RF transport across PST occurred in the direction of RF secretion. Compared with PST, PCT exhibited higher Jlb and Jbl, probably due in part to their larger luminal and peritubular surface area per tubular length. However, the net RF transport across PCT also occurred in the direction of RF secretion at a rate not significantly different from that of PST. It thus appears that RF secretion occurs throughout different segments of proximal tubules at a comparable rate. These results are in agreement with in vivo clearance studies, where RF infusion induced a component of tubular secretion as implicated by the greater RF excretion rate than the glomerular filtration rate (3, 9, 10, 23).

In our present study, we found that both Jlb and Jbl were influenced by RF concentration. Thus, compared with the corresponding transport rates at 0.1 µM RF concentration, both Jlb and Jbl were significantly reduced when RF concentrations in the perfusate and bath solution were lowered to 0.01 µM. At this lower RF concentration, Jlb was not significantly different from Jbl, so that there was no net RF transport across PST. In contrast, both Jlb and Jbl were significantly increased when RF concentrations in the perfusate and bath solution were raised to 1 µM, leading to a significantly greater net RF secretion. Because the physiological plasma RF concentration has been found to be 0.01~0.03 µM in humans (23) and in rats (3), the secretion of RF by proximal tubules at higher RF concentrations may thus constitute an adaptive mechanism to maximize urinary RF excretion during RF-sufficient states. A similar adaptive change in urinary RF excretion according to blood RF concentration was also found in in vivo clearance studies in human (9) and rats (3). Our studies therefore lend further support to these previous observations and clarify the mechanism underlying this phenomenon.

To delineate the nature of RF transport mechanisms, we found that both Jlb and Jbl were energy dependent as indicated by the abolishment of these transport activities when energy supply was blocked by low temperature or iodoacetate. In separate studies, we found that changes in peritubular bath osmolality did not affect Jlb or Jbl. It is therefore likely that the RF transport across the proximal tubule occurs through transcellular, rather than paracellular, pathways. We also found that both Jlb and Jbl were inhibited by lumichrome (50 µM), an RF structural analog, suggesting the involvement of carrier-mediated transport mechanisms. In further support of this notion, we found that Jbl was inhibited by probenecid and PAH, indicating the relevance of RF secretion to renal organic anion transport. RF has been reported to behave as an anion when transported across biological membranes (9, 13), and probenecid was found to inhibit renal excretion of RF in rats (3), dogs (10), and humans (9). However, in vitro studies using kidney slices or isolated kidney cells have produced conflicting results with regard to the involvement of organic anion transport system. Thus, by using rabbit kidney slices, Spector (22) found that RF transport was inhibited by organic anions such as PAH and penicillin G. In contrast, by using isolated kidney cells in suspension, Bowers-Komro and McCormick (1) found no inhibition of RF uptake by these organic anions. Because we also found that both probenecid and PAH had no effect on Jlb, it is conceivable that the contradictory reports from these previous in vitro studies could have resulted from their inability to separate RF reabsorption from secretion. Nevertheless, the finding that organic anions may interfere with RF secretion in renal proximal tubules could be of clinical importance, because many commonly used drugs are organic anions and may thus have the potential of interacting with renal RF excretion.

In our further studies, we examined the regulatory mechanisms of RF transport in renal proximal tubules. We found that changes in luminal pH over the physiological range (7.0-8.0) did not affect Jlb, whereas changes in bath pH caused an inhibition of Jbl when it was increased to 8.0. It thus appears that changes in systemic acid-base balance may affect urinary excretion of RF by inhibiting proximal tubule RF secretion during systemic alkalemia. As for the intracellular regulatory pathways, we focused on the potential roles of protein kinase systems known to affect solute transports in renal proximal tubules (12). We found that activation of PKA by cAMP, or PKC by PMA, did not affect Jlb or Jbl. In contrast, inhibition of Ca2+/calmodulin-dependent pathway by TFP inhibited Jbl but not Jlb. These results therefore indicate that the intracellular Ca2+/calmodulin-dependent pathway may play an important role in mediating the regulation of proximal tubule RF transport through its effect on RF secretion. A similar result was also found in our recent studies using human proximal tubular cells (11) and colonic cells (21).

In a series of studies, Gabay and Harris (5-7) called attention to the structural similarities between RF and phenothiazine derivatives and showed the interference of these drugs with RF metabolism. Subsequent studies by Pinto and associates (17-19) also showed that treatment of rats with these drugs caused an increase in RF losses in the urine and accelerated the development of RF deficiency in animals fed RF-deficient diet. In support of these notions, we found in our present study that chlorpromazine, a phenothiazine derivative, caused a significant inhibition of both Jlb and Jbl. However, results of our study would indicate that the net effect of chlorpromazine was to reduce RF secretion by proximal tubules. This may appear to contradict the riboflavinuric effect of these drugs found in in vivo animal studies (17-19). Although the reason for this discrepancy is not immediately clear, it is conceivable that the inhibitory effect of these drugs on systemic RF metabolism may lead to increased levels of RF in the blood to be filtered through glomeruli. With the concomitant inhibition of proximal tubule RF reabsorption by these drugs, the increase in RF filtered may exceed the decrease in RF secretion and result in riboflavinuria. Regardless of the underlying mechanism, our studies did confirm the interference of proximal tubule RF transport by chlorpromazine and reiterate the concern about the nutritional status of psychiatric patients receiving these medications.

In summary, our current studies confirm the bidirectional RF transport activity in isolated perfused rabbit proximal tubules. Our results suggest the involvement of carrier-mediated transport systems in RF transport, which are under the influence of RF availability, pH, and intracellular Ca2+/calmodulin-dependent pathway. In view of the differing sensitivities between Jbl and Jlb to these transport modulators, it is likely that different transport systems are involved in the reabsorption and secretion of RF by renal proximal tubules. Further studies with eventual cloning and isolation of these RF transporters will help to further our understanding on their exact natures and provide new strategies to manage patients with compromised RF homeostasis.


    ACKNOWLEDGEMENTS

This study was supported by grants from the National Institute of Diabetes and Digestive and Kidney Diseases (DK-47203) and the Department of Veterans Affairs.


    FOOTNOTES

Address for reprint requests and other correspondence: N. Yanagawa, Nephrology Division (111R), Sepulveda VA Medical Center, 16111 Plummer St., Sepulveda, CA 91343 (E-mail: nori{at}ucla.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 22 February 2000; accepted in final form 31 July 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Cell Physiol 279(6):C1782-C1786