Chloride dependency of renal brush-border membrane phosphate transport

Norimoto Yanagawa, Chi Pham, Remi N. J. Shih, Stephen Miao, and Oak Don Jo

Division of Nephrology, Medical and Research Services, Sepulveda Veterans Affairs Medical Center, University of California at Los Angeles School of Medicine, Los Angeles, California 91343


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In our present study, we examined the effect of Cl- on rabbit renal brush-border membrane (BBM) phosphate (Pi) uptake. It was found that the Na+-dependent BBM 32P uptake was significantly inhibited by Cl- replacement in the uptake solution with other anions, or by Cl- transport inhibitors, including DIDS, SITS, diphenylamine-2-carboxylate (DPC), niflumic acid (NF), and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB). Intravesicular formate or Cl- increased BBM 36Cl- uptake but did not affect BBM 32P uptake. BBM 22Na+ uptake was lowered by Cl- replacement in the uptake solution but not by Cl- transport inhibitors. Changes in transmembrane electrical potential altered BBM 36Cl- and 32P uptake in directions consistent with a net inward movement of negative and positive charges, respectively. However, the Cl--dependent BBM Pi uptake was not affected by changes in transmembrane electrical potential. Finally, a similar Cl- dependency of Pi uptake was also found with BBM derived from rat and mouse kidneys. In summary, our study showed that a component of Na+-dependent Pi uptake was also Cl- dependent in rabbit, rat, and mouse renal BBM. The mechanism underlying this Cl- dependency remains to be identified.

proximal tubule; chloride channel; chloride channel inhibitor; sodium cotransport; hereditary nephrolithiasis


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE KIDNEY plays a pivotal role in maintaining body phosphate (Pi) homeostasis through regulation of urinary Pi excretion (2). Pi reabsorption in the kidney occurs mainly at the proximal tubule, where the uptake of Pi across the apical brush-border membrane (BBM) represents the rate-limiting step and the main target of regulation (10). BBM Pi uptake is an active process that occurs together with sodium (Na+) along the inwardly directed Na+ gradient via the Na-Pi cotransport (23). Recently, however, several lines of evidence have been reported to suggest that chloride (Cl-) may in some way interact with this cotransport process.

First, recent cloning studies have identified two distinctive types of mammalian renal Na-Pi cotransporters, i.e., type I Na-Pi cotransporters in the kidneys of rabbit (NaPi-1) (29), rat (rNaPi-1) (19), mouse (Npt-1) (8), and human (NPT-1) (7), and type II Na-Pi cotransporters in the kidneys of rat (NaPi-2) (21), human (NaPi-3) (21), opossum (NaPi-4) (26), flounder (NaPi-5) (30), rabbit (NaPi-6) (27), and mouse (NaPi-7) (9, 14). Although the type II Na-Pi cotransporters are generally considered to be the BBM Na-Pi cotransporter system responsible for dietary adaptation and hormonal regulation (18), the functional role of type I Na-Pi cotransporters remains less well defined. In addition to its role as a Na-Pi cotransporter, the rabbit type I Na-Pi cotransporter, NaPi-1, was also found to function as an anion channel permeable to Cl- and other organic anions (6). It is not known whether this unique function of NaPi-1 as an anion channel affects BBM Pi transport.

Another line of evidence supporting a potential role of Cl- in renal Pi reabsorption derives from studies on a group of hereditary kidney stone diseases, including Dent's disease (31), X-linked recessive nephrolithiasis (XRN) (12), and X-linked recessive hypophosphatemic rickets (XLRH) (3). These disorders are associated with Fanconi-like proximal tubular dysfunction such as low-molecular-weight proteinuria, hypercalciuria, and hyperphosphaturia (24). These disorders have recently been found to be associated with a common defect in a gene, CLCN5, which encodes a Cl- channel, CLC-5, expressed predominantly in the kidney (20). The function of CLC-5 and the mechanism whereby CLC-5 defect leads to the characteristic proximal tubular transport dysfunction remain unknown (15).

Our present study was designed to examine the possible effect of Cl- on Pi transport by renal BBM. We found that up to 40% of the Na+-dependent Pi uptake by rabbit renal BBM was Cl- dependent and was sensitive to inhibition by Cl- transport inhibitors. A similar Cl- dependency of Pi uptake was also found with BBM prepared from rat and mouse kidneys.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 chow with free access to tap water for drinking.

BBM vesicle preparation and uptake measurements. Purified BBM vesicles were prepared from renal cortex by the conventional magnesium-precipitation method. Purification of BBM preparation, as assessed by the enrichment of BBM enzyme markers, was monitored routinely as reported previously (22). Final BBM vesicles were suspended in a medium that comprised (in mM) 300 mannitol, 10 MgSO4, 10 Tris, and 16 HEPES, pH 7.4. Uptake was measured by a Millipore rapid-filtration procedure at 24°C and was initiated by mixing 10 µl BBM vesicle suspension with 90 µl of uptake medium that comprised (in mM) 100 NaCl, 80 mannitol, and 20 HEPES-Tris, pH 7.4. For Pi or glucose uptake, the uptake solution also contained 0.1 mM of 32Pi or [3H]glucose, respectively. For Na+ or Cl- uptake, the uptake solution also contained 100 mM 22Na+ or 36Cl-. To examine the effect of Cl- replacement, BBM uptake was measured with Cl--free uptake solutions where NaCl was replaced by sodium gluconate. Incubation was terminated at indicated times by adding 2 ml of ice-cold stop medium that comprised (in mM) 100 NaCl, 100 mannitol, and 20 K2HPO4, pH 7.4. The suspension was filtered and washed twice with 2 ml of stop solution. The filter membrane was then dissolved in 5 ml of scintillation fluid (UltimaGold, Packard) and counted for radioactivity in a liquid scintillation counter (model 1600 TR, Packard). All measurements were carried out in triplicate and expressed as nanomoles per milligram protein per unit time. The protein concentration was assayed using Coomassie brilliant blue G250 with bovine serum albumin as the reference protein (25). For those studies where outward formate or Cl- gradients were imposed, BBM vesicles were preequilibrated for 3 h before uptake measurements with a solution that comprised (in mM) 100 mannitol, 100 K, 100 gluconate, 10 Tris, and 16 HEPES, pH 7.4, as control, or with 40 gluconate replaced by formate or Cl-.

Materials. Radioisotopes including [32P]phosphoric acid, 22Na, 36Cl, and [3H]glucose were purchased from Amersham (Arlington Heights, IL). All other chemicals were purchased from Sigma (St. Louis, MO).

Data analysis. Measurements made in triplicate were averaged. These values were pooled, and means ± SE were determined for the number of experiments indicated. Significance of difference was determined by two-tailed Student's t-test for paired and unpaired data as appropriate.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Effect of Cl- replacement. To examine the effect of Cl- replacement, BBM 32P uptake was measured using Cl--free uptake solution where NaCl was replaced by sodium gluconate. As shown in Fig. 1, Cl- replacement significantly lowered BBM 32P uptake. With Na+-free uptake solution where Na+ was replaced by tetramethylammonium, the BBM 32P uptake was significantly lowered, and the replacement of Cl- with gluconate did not further lower the 32P uptake. Thus Cl- replacement affected only the Na+-dependent BBM 32P uptake. The effect of Cl- on BBM 32P uptake was concentration dependent, so that a stepwise decrease in Cl- concentration from 150 mM caused a gradual decrease in BBM 32P uptake, reaching a maximal inhibition of up to 40% at 0 mM Cl- (Fig. 2). The effect of Cl- replacement on BBM 32P uptake kinetic parameters was analyzed by varying Pi concentrations in the uptake solution (0.01-3 mM). These studies revealed an effect of Cl- replacement to lower the Vmax values (from 2.18 ± 0.22 to 1.11 ± 0.10 nmol · min-1 · mg protein-1; n = 5, P < 0.01) without altering the apparent Km for Pi (from 0.24 ± 0.03 to 0.22 ± 0.02 mM; n = 5, P > 0.1). To test whether there is a reciprocal effect of Pi on Cl- uptake, we examined the effect of Pi on BBM 36Cl- uptake. As shown in Fig. 3, varying Pi concentrations in the uptake solution from 0 to 10 mM had no significant effect on BBM 36Cl- uptake. Similar to gluconate, replacement of Cl- in the uptake solution with other anions, such as NO-3, SO-4, isethionate, or SCN-, also caused a significant decrease in BBM 32P uptake (Table 1). In contrast, Cl- replacement with these anions had varying effects on BBM [3H]glucose uptake. As shown in Table 1, BBM [3H]glucose uptake was significantly lowered with gluconate, SO-4 or isethionate, but was significantly increased with SCN- and not altered with NO-3.


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Fig. 1.   Effect of Cl- replacement on brush-border membrane (BBM) 32P uptake. BBM 32P uptake measured at indicated time periods was significantly lower with Cl--free uptake solution where NaCl was replaced with sodium gluconate (open circle ), compared with the corresponding control uptake (). BBM 32P uptake was significantly lowered when Na+ in uptake solution was replaced by tetramethylammonium (+) and was not further lowered when Cl- was also replaced with gluconate (down-triangle). Results are means ± SE (n = 5).



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Fig. 2.   Inhibition of BBM 32P uptake by lowering Cl- concentrations. BBM 32P uptake at 1 min was measured with uptake solutions where a stepwise decrease in Cl- concentrations was induced by replacing NaCl with increasing concentrations of sodium gluconate. BBM 32P uptake was expressed as the percentage of uptake at 150 mM Cl-. Lowering Cl- concentration below 150 mM caused a gradual decrease in BBM 32P uptake, reaching a maximal inhibition up to 40% at 0 mM Cl-. Results are means ± SE (n = 6).



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Fig. 3.   Effect of Pi on BBM 36Cl uptake. BBM 36Cl uptake at 1 min was measured in uptake solutions containing varying concentrations of Pi. Presence of Pi at concentrations up to 10 mM had no significant effect on BBM 36Cl- uptake. Results are means ± SE (n = 4).


                              
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Table 1.   Effects of different anions on BBM Pi and glucose uptake

Effect of Cl- transport inhibitors. To examine whether the Cl- dependency of BBM 32P uptake involves Cl- uptake, we tested the effect of Cl- transport inhibitors. As shown in Table 2, BBM 36Cl- uptake was significantly lowered by anion transport inhibitors including DIDS (1 mM), SITS (1 mM), and by Cl--channel inhibitors including diphenylamine-2-carboxylate (DPC, 1 mM), niflumic acid (NF, 1 mM), and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, 0.1 mM). As shown in Fig. 4, these Cl- transport inhibitors also significantly lowered BBM 32P uptake, which occurred in a Cl--dependent manner so that no further inhibition on BBM 32P uptake occurred when Cl- was replaced with gluconate in the uptake solution. These results thus indicate that the Cl- dependency of BBM 32P uptake involves not only the presence, but also the uptake of Cl-.


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Fig. 4.   Effect of Cl- transport inhibitors on BBM 32P uptake. BBM 32P uptake at 1 min was measured in control uptake solution (open bars) or in Cl--free uptake solution where NaCl was replaced with sodium gluconate (hatched bars). Addition of Cl- transport inhibitors, including DIDS (1 mM), SITS (1 mM), diphenylamine-2-carboxylate (DPC, 1 mM), niflumic acid (NF, 1 mM), and 5-nitro-2-(3-phenylpropylamino)benzoate (NPPB, 0.1 mM), significantly lowered BBM 32P uptake in control, but not in Cl--free uptake solution. Results are means ± SE (n = 5). * P < 0.05 vs. control.

Effects of intravesicular formate and Cl-. Since BBM Cl- uptake has been shown to involve anion-exchange mechanism, such as Cl-/formate exchange (16), we studied the effect of intravesicular formate on BBM Pi uptake. In addition, to test whether the Cl- dependency of BBM 32P uptake involves a Pi/Cl- exchange transport process that may occur subsequent to Cl- uptake, we studied the effect of intravesicular Cl-. As shown in Fig. 5, preloading BBM vesicles with 40 mM of either formate or Cl- increased BBM 36Cl- uptake but did not affect BBM 32P uptake. These results therefore provide no evidence for the involvement of anion-exchange mechanisms in the effect of Cl- on BBM Pi uptake.


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Fig. 5.   Effects of intravesicular formate and Cl-. BBM vesicles were preequilibrated for 3 h before uptake measurements with a solution that comprised (in mM) 100 mannitol, 100 K, 100 gluconate, 10 Tris, and 16 HEPES, pH 7.4, as control, or with 40 gluconate replaced by formate or Cl-. Outward formate and Cl- gradients increased BBM uptake of 36Cl- (top) but not 32P (bottom). Results are means ± SE (n = 4). * P < 0.05 vs. control.

Effect of transmembrane electrical potential. The effect of transmembrane electrical potential on BBM Cl- and Pi uptake was examined by imposing a negative or positive intravesicular potential with the use of nigericin (10 µg/mg protein) and an outwardly or inwardly directed K+ gradient, respectively (intracellular and extracellular K+ concentrations are indicated by [K+]in and [K+]out, respectively). The uptake of 36Cl- at voltage-clamp control condition ([K+]in/[K+]out, 50/50 mM: 79.6 ± 5.8 nmol · min-1 · mg protein-1, n = 5) was decreased with a negative intravesicular potential ([K+]in/[K+]out, 50/0 mM: 63.4 ± 9.4 nmol · min-1 · mg protein-1; n = 5, P < 0.02) and increased with a positive intravesicular potential ([K+]in/[K+]out, 0/50 mM: 113.5 ± 11.9 nmol · min-1 · mg protein-1; n = 5, P < 0.005), consistent with a net inward movement of negative charges with Cl- uptake. In contrast, as shown in Fig. 6, BBM 32P uptake was higher with a negative intravesicular potential ([K+]in/[K+]out, 50/0 mM) and lower with a positive intravesicular potential ([K+]in/[K+]out, 0/50 mM), consistent with a net inward movement of positive charges with Pi uptake. Nevertheless, Cl- replacement continued to lower BBM 32P uptake irrespective of changes in transmembrane electrical potential (Fig. 6). The net inward movement of negative charges induced by Cl- uptake therefore cannot be accountable for the Cl- dependency of BBM Pi uptake. Also evident from Fig. 6, the Cl--dependent component of BBM Pi uptake, i.e., the difference between BBM 32P uptake in the presence and absence of Cl-, remained constant at different transmembrane electrical potentials (1.32 ± 0.29 nmol · min-1 · mg protein-1 at [K+]in/[K+]out of 50/0 mM; 1.44 ± 0.32 nmol · min-1 · mg protein-1 at [K+]in/[K+]out of 50/50 mM; and 1.40 ± 0.32 nmol · min-1 · mg protein-1 at [K+]in/[K+]out of 0/50 mM; n = 5, P > 0.6). This observation suggests that the Cl--dependent BBM Pi uptake is an electroneutral transport process. In contrast, Cl--independent component of BBM Pi uptake, i.e., BBM 32P uptake in the absence of Cl-, is electrogenic with a net inward movement of positive charges (Fig. 6).


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Fig. 6.   Effect of transmembrane electrical potential. BBM vesicles were preequilibrated for 3 h before uptake measurements with a solution that comprised (in mM) 200 mannitol, 50 K+, 50 gluconate, 20 HEPES, pH 7.4 (for intracellular K+ concentration, [K+]in, of 50 mM); or 300 mannitol, 20 HEPES, pH 7.4 (for [K+]in = 0). Nigericin (10 µg/mg protein) was added during preincubation. BBM 32P uptake at 1 min was measured in control uptake solution (open bars) that comprised (in mM) 50 Na+, 50 K+, 100 Cl-, 100 mannitol, 20 HEPES, pH 7.4 (for extracellular K+ concentration, [K+]out, of 50 mM); or 50 Na+, 50 Cl-, 200 mannitol, 20 HEPES, pH 7.4 (for [K+]out = 0); or in Cl--free uptake solutions with similar compositions except for the replacement of Cl- with gluconate (hatched bars). Compared with voltage-clamp control condition ([K+]in/[K+]out, 50/50 mM), BBM 32P uptake was higher with a negative intravesicular potential ([K+]in/[K+]out, 50/0 mM) and lower with a positive intravesicular potential ([K+]in/[K+]out, 0/50 mM). Cl- replacement significantly lowered BBM 32P uptake (P < 0.05) by similar extent regardless of changes in transmembrane electrical potential. Results are means ± SE (n = 6). * P < 0.05 vs. respective group at [K+]in/[K+]out, 50/50 mM.

Effect of Cl- replacement on BBM 22Na+ uptake. To examine whether the effect of Cl- on BBM Pi uptake was mediated through its effect on BBM Na+ uptake, we studied the effects of Cl- replacement and Cl- transport inhibitors on BBM 22Na+ uptake. We found that replacement of Cl- in the uptake solution with gluconate lowered BBM 22Na+ uptake from 208 ± 4.5 to 154 ± 7.5 nmol · min-1 · mg protein-1 (n = 5, P < 0.005). However, as shown in Table 2, Cl- transport inhibitors, including DIDS, SITS, DPC, NF, and NPPB, did not affect BBM 22Na+ uptake. Thus the Cl- dependency of BBM Na+ uptake may involve the presence of Cl- and/or the uptake of Cl- through mechanisms not inhibited by these Cl- transport inhibitors. Combined with the fact that these Cl- transport inhibitors significantly lowered BBM 32P uptake, it seems unlikely that Cl- interacts with BBM Pi uptake through its effect on Na+ uptake.

                              
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Table 2.   Effects of Cl- transport inhibitors on BBM 36Cl- and 22N+ uptake

Effect of Cl- replacement on rat and mouse BBM Pi uptake. The effect of Cl- replacement on 32Pi uptake was also examined with BBM vesicles isolated from rat and mouse kidneys. In these studies, kidneys from at least two rats (Sprague-Dawley, 250-300 g) or four mice (C57BL/6J, 25-30 g) were pooled, and BBM vesicles were prepared from kidney cortex with the enrichment of BBM marker enzymes monitored in a fashion similar to that with rabbit kidneys. As shown in Fig. 7, replacement of Cl- in the uptake solution by gluconate inhibited 32Pi uptake to a similar extent in both rat and mouse renal BBM compared with rabbit renal BBM.


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Fig. 7.   Effect of Cl- replacement on rat and mouse BBM Pi uptake. 32Pi uptake at 1 min was measured with BBM vesicles isolated from rat and mouse kidneys, either in control uptake solution or in Cl--free uptake solution where NaCl was replaced with sodium gluconate. Results are means ± SE (n = 3) and are expressed as the percentage inhibition by Cl- replacement.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In our present study, we found that Cl- replacement in the uptake solution with other anions, including gluconate, NO-3, SO-4, isethionate, and SCN-, lowered BBM Pi uptake up to 40% (Table 1; Figs. 1 and 2). Cl- replacement affected only the Na+-dependent BBM Pi uptake, so that a component of BBM Na+-dependent Pi uptake appears to be also Cl- dependent. The Cl- dependency of BBM Pi uptake involves the uptake of Cl-, because inhibition of BBM Cl- uptake by Cl- transport inhibitors similarly lowered BBM Pi uptake in a Cl--dependent manner (Fig. 4). In contrast, as shown in Fig. 3, Pi did not affect BBM 36Cl- uptake. The lack of effect of Pi on Cl- uptake suggests that the interaction between BBM Pi and Cl- uptake consists mainly of a Cl- dependency of BBM Pi uptake. However, we cannot completely exclude the possibility that a component of Pi-dependent BBM Cl- uptake may exist that is sufficiently small compared with the total Cl- uptake to remain undetected by our uptake measurements.

It has been well demonstrated that the anion-exchange mechanism such as Cl-/formate exchange constitutes an important transport process responsible for Cl- uptake by renal BBM (1, 16). Our current study supports this notion, as BBM Cl- uptake was stimulated when an outwardly directed formate gradient was imposed (Fig. 5). However, we found that this maneuver did not affect BBM Pi uptake (Fig. 5), suggesting that the interaction between BBM Cl- uptake and Pi uptake does not involve the Cl-/formate exchange. We also found that an outwardly directed Cl- gradient did not affect BBM Pi uptake (Fig. 5), suggesting that the Cl- dependency of BBM Pi uptake is not mediated by a transport process such as Cl-/Pi exchange following an initial uptake of Cl- into the intravesicular space. Our data therefore do not provide evidence for the involvement of anion exchangers in the interaction between BBM Cl- uptake and Pi uptake. Since anion conductances have been shown to be present on the apical membrane of the proximal tubule (17, 28), our data may be more in favor of the involvement of Cl- channels.

Our current study shows that changes in transmembrane electrical potential affected both BBM Cl- and Pi uptake. BBM Cl- uptake was enhanced by a positive, and suppressed by a negative, intravesicular potential, consistent with a net inward movement of negative charges with BBM Cl- uptake. In contrast, BBM Pi uptake was enhanced by a negative, and suppressed by a positive, intravesicular potential, consistent with a net inward movement of positive charges with BBM Pi uptake (Fig. 6). These results raised the possibility that the effect of Cl- on BBM Pi uptake may be due to the inward movement of negative charges with Cl- uptake. However, this possibility seems unlikely given the continuing lowering effect of Cl- replacement on BBM Pi uptake at varying intravesicular potentials (Fig. 6).

Although Cl- replacement lowered BBM 22Na+ uptake up to 25%, Cl- transport inhibitors, including DIDS, SITS, DPC, NF, and NPPB, did not affect BBM 22Na+ uptake (Table 2). Since these Cl- transport inhibitors were effective in lowering BBM Pi uptake, the Cl- dependency of BBM Pi uptake thus seems unlikely to be mediated through changes in Na+ uptake. The diverse effects of different anions on BBM glucose uptake may also lend support to this notion. However, it is possible that the portion of BBM Na+ uptake coupled to Pi uptake may constitute only a small fraction of the total Na+ uptake and cannot be easily detected with our uptake measurements using Cl- transport inhibitors. We therefore cannot completely exclude the possibility that Cl- affects BBM Pi uptake through its effect on BBM Na+ uptake that is coupled to Pi uptake.

It is not clear from our current study whether the Cl- dependency of BBM Pi uptake is related to the anion channel function of the rabbit renal type I Na-Pi cotransporter, NaPi-1. As is evident from Fig. 6, although the Cl--independent component of BBM Pi uptake varied with changes in intravesicular potential, the Cl--dependent component of BBM Pi uptake remained constant with varying intravesicular potentials. It thus appears that, although Cl--independent BBM Pi uptake is electrogenic and carries a net inward movement of positive charges, Cl--dependent BBM Pi uptake is electroneutral. These results may be relevant to the recently described electrophysiological characteristics of Na-Pi cotransporters expressed in Xenopus oocytes (5, 6). These studies showed that type II Na-Pi cotransporters exhibited an electrogenic transport with inward movement of positive charges (5), whereas type I Na-Pi cotransporter, NaPi-1, exhibited electrogenic transport only at high extracellular Pi concentrations (3 mM) (6). In our current studies with rabbit renal BBM at low Pi concentration (0.1 mM), it is possible that the type I Na-Pi cotransporter, NaPi-1, mediated the Cl--dependent and electroneutral component of Pi uptake while the type II Na-Pi cotransporter, NaPi-6, mediated the Cl--independent and electrogenic component of Pi uptake. However, it is also possible that a component of BBM Pi uptake mediated by type II Na-Pi cotransporter may be Cl- dependent, which occurs in an electroneutral fashion when Cl- is cotransported through Cl- channels either related or unrelated to type I Na-Pi cotransporter and nullifies the positive charges. Indeed, it has been reported recently that both Pi uptake and Cl- conductance induced by NaPi-1 in Xenopus oocytes appeared to be separate functions, and the NaPi-1-induced Pi uptake was not affected by Cl- channel inhibitor, NPPB (4). Furthermore, preliminary observations with Xenopus oocytes expressing type II Na-Pi cotransporters showed a decrease in Pi-induced inward current under voltage-clamp condition when extracellular Cl- was replaced with gluconate (I. Forster, personal communication). Resolution of these issues clearly requires further studies. In separate studies, we also found that BBM derived from rat and mouse kidneys exhibited a similar component of Pi uptake which is Cl- dependent (Fig. 7). Thus the Cl- dependency is a feature of BBM Pi uptake not only limited to rabbit kidneys.

Relevant to our current finding of a Cl--dependent component of BBM Pi uptake is the recent report that a group of hereditary kidney stone diseases associated with proximal tubular dysfunctions, including decreased Pi reabsorption (24), shares a common defect in the renal Cl- channel, CLC-5 (20). CLC-5 belongs to a growing family of Cl- channels without similarity in molecular structure with NaPi-1 (11). The function of CLC-5 and the mechanism whereby its defect leads to proximal tubular dysfunction are currently not known. However, CLC-5 has recently been found to be expressed in the proximal tubular cells, where it is localized closely with H+-ATPase in a region below BBM densely packed with endocytic vesicles (13). Although it is not clear whether CLC-5 is directly involved in BBM Cl- transport, it seems likely that CLC-5 may serve as the Cl- channel of proximal tubular endosomes. Defect in CLC-5 can thus affect BBM transport indirectly through the disrupted endocytotic process. Although these possibilities still need to be examined, it does appear that Cl- is emerging as an important anion capable of regulating BBM Pi transport, either directly or indirectly.


    ACKNOWLEDGEMENTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant RO1-DK-47203 and by funds from the Department of Veterans Affairs.


    FOOTNOTES

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. §1734 solely to indicate this fact.

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

Received 13 November 1998; accepted in final form 25 May 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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Am J Physiol Renal Physiol 277(4):F506-F512
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society




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