Superoxide (O2−) increases Na+ reabsorption in the thick ascending limb (THAL) by enhancing Na/K/2Cl cotransport. However, the effects of O2− on other THAL transporters, such as Na+/H+ exchangers, are unknown. We hypothesized that O2− stimulates Na+/H+ exchange in the THAL. We assessed total Na+/H+ exchange activity by measuring recovery of intracellular pH (pHi) after acid loading in isolated perfused THALs before and after adding xanthine oxidase (XO) and hypoxanthine (HX). We found that XO and HX decreased total pHi recovery rate from 0.26 ± 0.05 to 0.21 ± 0.04 pH units/min (P < 0.05), and this net inhibition decreased steady-state pHi from 7.52 to 7.37. Because THALs have different Na+/H+ exchanger isoforms on the luminal and basolateral membrane, we tested the effects of xanthine oxidase and hypoxanthine on luminal and basolateral Na+/H+ exchange by adding dimethylamiloride to either the bath or lumen. Xanthine oxidase and hypoxanthine increased luminal Na+/H+ exchange from 3.5 ± 0.8 to 6.7 ± 1.4 pmol·min−1·mm−1 (P < 0.01) but decreased basolateral Na+/H+ exchange from 10.8 ± 1.8 to 6.8 ± 1.1 pmol·min−1·mm−1 (P < 0.007). To ascertain whether these effects were caused by O2− or H2O2, we examined the ability of tempol, a superoxide dismutase mimetic, to block these effects. In the presence of tempol, xanthine oxidase and hypoxanthine had no effect on luminal or basolateral Na+/H+ exchange. We conclude that O2− inhibits basolateral and stimulates luminal Na+/H+ exchangers, perhaps because different isoforms are expressed on each membrane. Inhibition of basolateral Na+/H+ exchange may enhance stimulation of luminal Na+/H+ exchange by providing additional protons to be extruded across the luminal membrane. Together, the effects of O2− on Na+/H+ exchange may increase net HCO3− reabsorption by the THAL.
- reactive oxygen species
- intracellular pH
- superoxide dismutase
the thick ascending limb (THAL) reabsorbs 25–30% of filtered NaCl and generates a corticomedullary osmotic gradient that drives the countercurrent system of urine concentration (15). The Na+/K+/2Cl− cotransporter (NKCC2) accounts for 70–80% of Na+ reabsorption in the THAL, while Na+/H+ exchange accounts for the remaining 20–30% (24). Sodium reabsorption brought about by luminal Na+/H+ exchange causes reabsorption of HCO3−, which is important for acid/base balance (10, 12). The THAL also expresses basolateral Na+/H+ exchangers (13), which play a major role in regulating cytosolic pH and cell volume (5).
Reactive oxygen species (ROS) have recently been shown to regulate excretion of salt and water. Increases in ROS have been implicated in salt-sensitive hypertension (34). Recent data from our laboratory demonstrated that O2− enhances net NaCl absorption by the THAL. Endogenous O2− was shown to indirectly augment transport by decreasing the bioavailability of nitric oxide (NO), a molecule that inhibits NaCl absorption (26). O2− also increases NaCl absorption in THALs in the absence of NO, suggesting a direct effect of O2− (9, 27). This stimulatory effect is due to enhanced Na+/K+/2Cl– cotransport activity rather than K+ channel or ATPase activity (19). However, the impact of reactive oxygen species in general and O2− in particular on Na+/H+ exchanger activity in this segment has not been evaluated to our knowledge. We hypothesized that the stimulatory effect of O2− on net NaCl absorption in the THAL is due, in part, to increased Na+/H+ exchange. To test this hypothesis, we examined the effect of exogenous O2− on total, apical, and basolateral Na+/H+ exchange in isolated perfused THALs. Our findings indicate that exogenous O2− stimulates apical Na+/H+ exchange and inhibits basolateral Na+/H+ exchange in isolated perfused THALs.
Isolation and perfusion of rat THALs.
All protocols were approved by the Institutional Animal Care and Use Committee. Male Sprague-Dawley rats weighing 120–150 g (Charles River Breeding Laboratories, Wilmington, MA) were fed a diet containing 0.22% Na and 1.1% K (Purina, Richmond, IN) for at least 7 days. On the day of the experiment, they were anesthetized with ketamine (100 mg/kg) and xylazine (20 mg/kg) by intraperitoneal injection. The left kidney was bathed in ice-cold saline, removed, and sliced along the coronal axis. The slices were placed in physiological saline containing (in mM) 140 NaCl, 4 KCl, 1.2 MgSO4, 5.5 glucose, 2 CaCl2 and 10 HEPES (pH 7.4) at 10°C. THALs were dissected from the outer medulla, transferred to a temperature-regulated chamber, and perfused using concentric glass pipettes at 37 ± 1°C (7, 28) with the above saline solution.
Measurement of intracellular pH of perfused THALs.
We used a ratiometric optical technique that uses a pH-sensitive dye, 2′,7′-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein, acetoxymethyl ester (BCECF-AM; Molecular Probes, Eugene, OR) to measure the intracellular pH (pHi) of the THALs, as described previously (8). Briefly, isolated perfused THALs were loaded by adding 1 μM BCECF-AM to the bath for 15 min at 37 ± 1°C, followed by a 20-min wash. We then excited the dye alternately at two wavelengths, 446 and 500 nm. Light was directed to the tubule using a 510-nm long-pass dichroic mirror. Emitted fluorescence was collected after passing through a 515-nm long-pass filter. The emitted light was digitally imaged with an image intensifier and charge-coupled device camera, and recorded using Image One Metafluor software (Universal Imaging, West Chester, PA). At the end of the experiment, the fluorescence ratio of each tubule was measured and converted to pHi using a three-point nigericin technique for in situ pHi calibration (8).
After the initial washout period, fluorescence measurements were taken once every 15 s until a steady state was reached and maintained for 2 min (control). THAL cells were then acid-loaded by replacing 30 mM NaCl in the bath with Na acetate. The sampling rate was increased to once every 3 s during this procedure and recorded for 1.5 min to monitor pHi recovery rate. The bath was then switched back to normal perfusate for a washout period of 3 min. After washout, we added 1 mU/ml xanthine oxidase (XO) and 0.5 mM hypoxanthine (HX) to the bath for 15 min. The cells were then acidified again, and fluorescence was measured as before. The initial rate of recovery was calculated from the first 8–10 time points after pHi reached a minimum caused by Na acetate.
To determine whether XO/HX can affect luminal and basolateral Na+/H+ exchangers differently, we repeated the above experiments with 5-(N,N-dimethyl)amiloride (DMA; 100 μM; Sigma Chemical, St. Louis, MO) in either the bath or luminal solution. In addition, we tested whether the effects of XO/HX are due to O2− or H2O2 by repeating the experiments with 1 mM tempol (Sigma), a superoxide dismutase mimetic, in the bath.
Determination of intrinsic buffering capacity.
We determined the intrinsic buffering capacity (βi) of the THAL, as described previously (8). THALs were exposed to solutions containing 0, 20, and 40 mM Na acetate, and changes in pHi were recorded. A pKa of 4.76 for acetic acid was used to calculate the intracellular acetate concentration for each extracellular acetate concentration. For the midpoint of each range of changes in pHi, βi was calculated as the change in intracellular acetate concentration divided by the change in pHi. In situ calibrations for pHi were performed with nigericin, as described above.
Calculation of H+ flux rates.
H+ flux rates were calculated as a measure of Na+/H+ exchange using the equation: JH = (dpHi/dt)·βi·V, where dpHi/dt is the initial recovery rate (in pH units/min), βi is the intrinsic buffering capacity (mM/pH unit), and V is the cell volume per millimeter tubule length (in nl/mm). V was calculated by measuring external and internal tubule diameters. Fluxes were expressed per unit tubule length.
Results are expressed as means ± SE. Data were evaluated with Student's paired t-test. P < 0.05 was considered significant.
Previously, we showed that ROS, particularly O2−, enhance Na+/K+/2Cl− cotransport activity in the THAL. However, to our knowledge the effects of O2− on Na+/H+ exchange activity have not been studied in this segment. To address this issue, we first examined pHi recovery rate in THALs before and after treatment with the ROS-generating system XO/HX. Figure 1 represents pHi recovery in an isolated perfused THAL before and after treatment. In this experiment, the pHi recovery rate during the control period was 0.19 pH units/min. After treatment with XO/HX, recovery decreased to 0.15 pH units/min. Mean H+ flux rates for all experiments were calculated as 11.0 ± 2.1 and 8.6 ± 1.7 pmol·min−1·mm−1 (P < 0.05) (Fig. 2), representing a 22% decrease in total JH. XO alone and HX alone had no significant effect on pHi recovery. Time controls demonstrated stable Na+/H+ exchange activity within the time constraints of our experiments. Thus, contrary to our hypothesis, ROS decreased total Na+/H+ exchange in the THAL.
THALs express Na+/H+ exchangers on both luminal and basolateral membranes, raising the possibility that the luminal and basolateral Na+/H+ exchangers may be regulated differently. Consequently, we next tested whether XO/HX can exert different effects on luminal and basolateral Na+/H+ exchange. For this, we first investigated the effects of XO/HX on luminal Na+/H+ exchange by adding 1 mM DMA to the bath. In a representative experiment, XO/HX increased pHi recovery rate from 0.08 to 0.18 pH units/min (Fig. 3). During the control period, mean H+ flux was 3.5 ± 0.8 pmol·min−1·mm−1. After XO/HX treatment, it increased to 6.7 ± 1.4 pmol·min−1·mm−1 (P < 0.01) (Fig. 4). Thus XO/HX increased luminal Na+/H+ exchange by 92%.
Next, we examined the effect of XO/HX on basolateral Na+/H+ exchange by adding DMA to the luminal perfusate to inhibit luminal Na+/H+ exchange. In a representative experiment, pHi recovery was 0.39 pH units/min during the control period and decreased to 0.29 pH units/min after treatment (Fig. 5). Calculated mean H+ flux decreased from 10.8 ± 1.8 to 6.8 ± 1.1 pmol·min·−1mm−1 (P < 0.007) (Fig. 6). Thus, in contrast to their effect on luminal Na+/H+ exchange, XO/HX decreased basolateral Na+/H+ exchange by 37%.
XO/HX can produce H2O2 in addition to O2−. Consequently, we next investigated whether O2− or H2O2 induces increases in luminal and decreases in basolateral Na+/H+ exchange by adding XO/HX in the presence of 1 mM tempol, a superoxide dismutase mimetic, to decrease O2− and enhance H2O2 levels. We found that in the presence of tempol, XO/HX had no effect on either luminal or basolateral H+ flux after an acid load (Δ 0.04 ± 0.03 and 0.8 ± 0.8 pmol·min−1·mm−1, respectively). These results suggest that H2O2 does not exert a significant effect on luminal or basolateral Na+/H+ exchange.
The THAL reabsorbs 25–30% of the filtered Na+ load and generates a corticomedullary NaCl gradient. Na+ absorption occurs via a two-step process. Na+ first enters the cell from the tubular lumen via Na+/K+/2Cl− cotransport and Na+/H+ exchange. In the second step, it is extruded across the basolateral membrane by Na+-K+-ATPase. Recycling of K+ across the luminal membrane is necessary for full activity of Na+/K+/2 Cl− cotransport (17). Na/H exchangers on the basolateral membrane play no direct role in net Na+ absorption but may affect the activity of other transporters through changes in intracellular pH. We have previously shown that O2− generated by the ROS-generating system XO/HX stimulates NaCl absorption in the THAL (26) and augments Na+/K+/2Cl− cotransport without altering K+ channel or Na+-K+-ATPase activity (19). In the present study, we found that XO/HX had a net inhibitory effect on total Na+/H+ exchange activity, as measured by the rate of recovery of intracellular pH after an acid load.
THALs express Na+/H+ exchangers on both luminal and basolateral membranes (3, 6). This raises the possibility that the luminal and basolateral Na+/H+ exchangers may be regulated differently. Consequently, we next tested whether XO/HX can exert different effects on luminal and basolateral Na+/H+ exchange by adding the Na+/H+ exchanger inhibitor DMA to either the bath or lumen, respectively. We found that XO/HX stimulated luminal Na+/H+ exchange by 92%, while at the same time, they inhibited basolateral Na+/H+ exchange by 37%. This differential response makes it unlikely that DMA crosses the cell to affect the Na+/H+ exchanger on the opposite membrane. Furthermore, we have previously found that adding DMA to both the bath and lumen blocks virtually all hydrogen efflux (8). Opposite responses on luminal and basolateral Na+/H+ exchange raise the question of what effect XO/HX might have on Na+ or HCO3− absorption; certainly, given the above data, stimulation of luminal Na+/H+ exchange would be expected to facilitate transepithelial transport of Na+ and HCO3−, and simultaneous inhibition of basolateral Na+/H+ exchange could promote this process by providing additional protons for luminal Na+/H+ exchangers to transport out of the cell.
Because the effects of XO/HX on Na+/H+ exchange could be due to either O2− or H2O2, we tested the ability of tempol, a superoxide dismutase mimetic (4), to block these effects. Tempol catalyzes dismutation of O2− to H2O2 (31), thereby decreasing O2− while simultaneously increasing H2O2. We found that in the presence of tempol, XO/HX had no effect on either basolateral or luminal Na+/H+ exchange activity. Thus we conclude that O2− rather than H2O2 stimulates luminal and inhibits basolateral Na+/H+ exchange.
We have previously found that O2− can affect the bioavailability of NO, which itself is an important regulator of NaCl absorption (26). Thus the effects of XO/HX on Na+/H+ exchange could be due to the interaction of O2− and NO. However, in the isolated, perfused tubule, l-arginine must be added for NO production by the THAL. Because of the flowing bath and the few number of cells in a tubule, isolated perfused tubules quickly lose all of their arginine. In the absence of l-arginine, the addition of the NO synthase inhibitor l-NAME has no effect on transport (29). Therefore, by not including l-arginine in the solutions in this study, we can eliminate the role of NO.
On the face of it, the fact that O2− stimulates luminal Na+/H+ exchange while inhibiting basolateral Na+/H+ exchange appears contradictory. However, the family of Na+/H+ exchangers comprises at least eight different proteins (6, 16). In the THAL, type 3 is primarily expressed in the luminal membrane (3), along with lesser amounts of type 2 (25). In contrast, type 1 is the predominant family member on the basolateral membrane (2), followed by type 4 (14, 25). On the basis of this distribution, we conclude that O2− has different effects depending on which Na+/H+ exchanger is involved. The differential regulation of Na+/H+ exchanger (NHE) isoforms is not unique. Enteropathogenic E. coli stimulate apical NHE2 and basolateral NHE1 activity but inhibit apical NHE3 activity (18). cAMP and hyperosmolality also stimulate NHE1 (1) but inhibit NHE3 (11, 33).
ROS have been shown to regulate ion transport in various cell types (20). However, we believe our findings are the first to show that O2− stimulates NHE3 while inhibiting NHE1. Our data concerning the inhibition of NHE1 by O2− appear to conflict with data from the heart. H2O2 rather than O2− has been shown to induce rapid activation of NHE1 through MAP kinase in cardiac myocytes (30). Although we did not directly test the effects of H2O2 on Na+/H+ exchange activity in the THAL, our data suggest that it has little or no effect on any of the NHE family members expressed in this segment. These data may indicate that the effects of ROS depend more on the tissue being studied and the signaling cascades activated than the specific transporter.
Our finding that O2− rather than H2O2 regulates Na+/H+ exchange activity in the THAL is consistent with our previous work showing that O2−, not H2O2, stimulates net NaCl absorption (27). The fact that simultaneously stimulating luminal Na+/H+ exchange and inhibiting basolateral Na+/H+ exchange would accelerate net NaHCO3 absorption also fits with the observed stimulatory effect on net NaCl absorption.
ROS such as O2−, H2O2 and hydroxyl radicals have been implicated in the pathogenesis of hypertension (23), especially salt-sensitive hypertension (21, 32). Increasing medullary O2− levels by inhibiting superoxide dismutase reduces renal Na+ and volume excretion (22). In contrast, reducing O2− levels by infusing a O2− scavenger increases renal Na+ and volume excretion (34). Our data suggest that the effects of O2− on THAL NaHCO3 (this study) and NaCl absorption (19, 27) may account for at least part of the Na+-retaining actions of O2−.
In summary, we found that O2− stimulates luminal Na+/H+ and inhibits basolateral Na+/H+ in the THAL. This disparate effect may be due to the different types of Na/H exchangers being expressed on each membrane. Our results predict that O2− would enhance NaHCO3 absorption. Regulation of THAL Na/HCO3 absorption by O2− may play an important role in the pathogenesis of several forms of hypertension and acid/base disturbances.
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