The thick ascending limb of the loop of Henle (THAL) plays an important role in the regulation of NaCl and water reabsorption. In vivo studies have shown that the free radical superoxide (O ) stimulates Na and water reabsorption by the kidney. However, it is not known whether O regulates transport along the nephron in general or in the THAL specifically. We hypothesized that O stimulates THAL NaCl reabsorption. Cl absorption was measured in isolated, perfused THALs from Sprague-Dawley rats. First, we tested whether extracellular O stimulates Cl absorption. Addition of the O -generating system xanthine oxidase/hypoxanthine increased Cl absorption from 112.7 ± 12.0 to 146.2 ± 13.9 pmol · mm−1 · min−1, a 33% increase (P < 0.03). When superoxide dismutase (300 U/ml) was present in the bath, addition of xanthine oxidase/hypoxanthine did not significantly increase Cl absorption (116.9 ± 13.8 vs. 102.5 ± 8.5 pmol · mm−1 · min−1). Furthermore, adding 200 nM H2O2 to the bath did not significantly affect Cl absorption (from 130.3 ± 13.7 to 125.3 ± 19.6 pmol · mm−1 · min−1). Because extracellular O stimulated Cl absorption, we next tested whether endogenously produced O could stimulate transport. Under basal conditions, THALs produced detectable amounts of O , as measured by lucigenin-enhanced chemiluminescence. Adding the O scavenger tempol to the bath decreased Cl absorption from 198.1 ± 35.4 to 132.4 ± 23.5 pmol · mm−1 · min−1, a 31% decrease (P < 0.02). To make sure tempol was not exerting cytotoxic effects, we tested whether its effect was reversible. With tempol in the bath, Cl absorption was 117.2 ± 9.3 pmol · mm−1 · min−1. Sixty minutes after tempol was removed from the bath, Cl absorption had increased to 149.2 ± 9.1 pmol · mm−1 · min−1(P < 0.05). We concluded that both exogenous and endogenous O stimulate THAL NaCl absorption. To our knowledge, these are the first data showing a direct effect of O on nephron transport.
- superoxide dismutase
- sodium-potassium-2 chloride cotransport
- loop of Henle
- salt-sensitive hypertension
- urinary sodium excretion
the thick ascending limb of Henle's loop (THAL) plays an important role in the maintenance of salt and fluid homeostasis. This nephron segment reabsorbs ∼30% of NaCl filtered at the glomeruli and generates the corticomedullary osmotic gradient necessary for urine concentration (15, 18). NaCl absorption in the THAL occurs via a secondary active transport mechanism, which includes passive entry of NaCl through apical transporters (NKCC2, NHE3) and active extrusion of Na through basolateral Na-K-ATPase (26).
Superoxide (O ) is a free radical produced by one-electron reduction of oxygen (5). In mammals, O is produced by the mitochondria during aerobic respiration and by specific oxidases (1, 3, 7, 35), some of which are present in the THAL (4, 36). At low concentrations, O and other reactive oxygen species (ROS) have been shown to regulate ion transport (13, 22). However, it is not known whether O regulates transport along the nephron in general or in the THAL specifically.
In vivo animal experiments have recently shown that an increase in O production in the renal medulla decreases urinary Na and volume excretion, whereas infusion of a O scavenger into the renal medulla increases urinary Na and volume excretion (25, 44). Although these data suggest that O has a stimulatory effect on salt and water reabsorption by the nephron, we know of no studies that address this issue. We hypothesized that O can directly stimulate NaCl absorption by the rat THAL.
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% potassium (Purina, Richmond, IN) for at least 5 days before THAL perfusion and preparation of THAL suspensions. On the day of the experiment, rats were anesthetized with ketamine (100 mg/kg body wt ip) and xylazine (20 mg/kg body wt ip).
Suspensions of THALs.
Suspensions of medullary THALs (mTHAL) were prepared according to a modified protocol as described previously (33). Briefly, kidneys were perfused retrograde via the aorta with a solution containing 0.1% collagenase (Sigma, St. Louis, MO) and 100 U heparin. The inner stripe of the outer medulla was cut from coronal slices, minced, and incubated at 37°C for 30 min in 0.1% collagenase. The tissue was pelleted via centrifugation at 114 g, resuspended in cold solution, and stirred on ice for 30 min to release the tubules. The suspension was filtered through 250-μm nylon mesh and centrifuged at 114 g. The pellet was washed, centrifuged again, and finally resuspended in 0.1 ml cold perfusion solution.
Measurement of O production.
Suspensions of mTHALs were prepared as described above. Tubules were resuspended in 1 ml HEPES-buffered perfusion solution gassed with 100% O2. Aliquots (100 μl) placed in 1.6-ml polypropylene tubes were diluted in perfusion solution to a final volume of 900 μl and placed on ice. Lucigenin (100 μl; for a final concentration of 5 μM) was added to the diluted tubule suspensions, which were then incubated for 30 min at 37°C. Tubes were placed in a luminometer chamber (model 20e, Turner Designs, Mountain View, CA) maintained at 37°C. The average of 10 consecutive 30-s measurements was recorded for each sample. The metal-dependent O scavenger 4,5-dihydroxy-1,3-benzenedisulfonic acid disodium salt (tiron; 10 μl) was added to the sample for a final concentration of 10 mM, and 10 consecutive 30-s measurements were made; the average of the last 3 measurements was used. The difference in average luminescence between samples with and without tiron was used to calculate the amount of O . Measurements were normalized to protein content. The average luminescence of 10 consecutive measurements was calculated for a blank containing PBS and lucigenin. Arbitrary luminiscence units were converted to nanomoles per minute per milligram of protein by use of a calibration curve obtained as described previously (34).
THAL isolation and perfusion.
After the animals were anesthetized, the abdominal cavity was opened; the left kidney was bathed in ice-cold saline and removed. Coronal slices were placed in oxygenated physiological saline. Cortical THALs were dissected from the medullary rays under a stereomicroscope at 4–10°C. THALs ranging from 0.5 to 1.0 mm were transferred to a temperature-regulated chamber and perfused using concentric glass pipettes at 37 ± 1°C as described previously (9,33). The flow rate of the basolateral bath was 0.5 ml/min.
Measurement of Cl absorption.
THALs were mounted on concentric glass pipettes and perfused at 37°C as described previously. The luminal perfusion rate was set at 5–10 nl · min−1 · mm−1. Compounds that alter production of O or endogenous O levels were added as indicated. To avoid excess H2O2 formation in the bath during addition of xanthine oxidase/hypoxanthine, the enzyme and its substrate were mixed and warmed to 37°C 20 s before being introduced into the perfusion chamber through a continuous-flow system. HEPES-buffered perfusion solution gassed with 100% O2 (pH = 7.40) was used for the bath and perfusate. The composition of the solution was (in mM) 130 NaCl, 2.5 NaH2PO4, 4.0 KCl, 1.2 MgSO4, 6 l-alanine, 1.0 sodium citrate, 5.5 glucose, 2.0 calcium lactate, and 10 HEPES. The effect of xanthine oxidase/hypoxanthine, tempol, and H2O2 was studied in different sets of tubules. After initial perfusion, THALs were equilibrated for 20 min, and four measurements were made to calculate the Cl absorption rate. Then, compounds of interest were either added to or removed from the bath as indicated. After a 20-min reequilibration period, four additional collections were made. Cl concentration in the perfusate and collected fluid was measured by microfluorometry. Time control experiments showed no significant change in Cl absorption during the experimental period (153.7 ± 30.5 vs. 144.0 ± 12.5 pmol · mm−1 · min−1). All data were recorded and stored using data-acquisition software (DATAQ Instruments, Akron, OH). Data analysis was performed with software specifically designed for voltage-spike analysis. Because water is not reabsorbed by the THAL, chloride absorption (J Cl −) was calculated as follows where CR is the collection rate normalized per tubule length, Co Cl− is the chloride concentration in the perfusion solution and Cl Cl−is the chloride concentration in the collected fluid. All chemicals and enzymes were purchased from Sigma, with the exception of purified catalase (Oxis Research, Portland, OR).
Results are expressed as mean ± SE. Data were evaluated with Student's paired t-test. P < 0.05 was considered significant.
To investigate whether O has any effect on NaCl absorption by the THAL, we first tested whether an exogenous O -generating system could alter Cl absorption (Fig.1). Under basal conditions, Cl absorption was 112.7 ± 12.0 pmol · mm−1 · min−1. After xanthine oxidase (1 mU/ml) and hypoxanthine (0.5 mM) were added to the bath to increase O production, Cl absorption increased to 146.2 ± 13.9 pmol · mm−1 · min−1, a 33% increase (P < 0.03).
Because xanthine oxidase/hypoxanthine increases not only O but also H2O2, the effects of xanthine oxidase/hypoxanthine may be caused by H2O2, hypoxanthine, or xanthine oxidase itself. To make sure the observed effect was due to O , we repeated the above experiment in the presence of superoxide dismutase, which scavenges the O produced by xanthine oxidase/hypoxanthine (Fig. 2). In the presence of superoxide dismutase (300 U/ml), chloride absorption was 116.9 ± 13.8 pmol · mm−1 · min−1. After xanthine oxidase and hypoxanthine were added to the bath, Cl absorption was 102.5 ± 8.5 pmol · mm−1 · min−1, not significantly different from control.
To further test whether H2O2 could significantly affect transport, we directly tested its ability to stimulate Cl absorption by the THAL (Fig.3). During the control period, Cl absorption was 130.3 ± 13.7 pmol ·mm−1 · min−1. After 200 nM H2O2 was added to the bath, Cl absorption was 125.3 ± 19.6 pmol · mm−1 · min−1, not significantly different from control. Taken together, these data suggest that exogenously added O can stimulate NaCl absorption by the THAL.
Essentially all cells produce O during aerobic respiration (3). Many cells also possess specific oxidases that produce O , including THAL cells (4,36). To investigate the amount of O generated by THALs, we used lucigenin-enhanced chemiluminescence to directly measure production. We found that under basal conditions, THALs produced O at a rate of 35.8 ± 0.25 nmol · min−1 · mg protein −1.
To test whether endogenously produced O significantly affects Cl absorption by the THAL, we examined the effect of the O scavenger tempol on transport. Under basal conditions, Cl absorption was 198.1 ± 35.4 pmol · mm−1 · min−1. After tempol (50 μM) was added to the bath, chloride absorption decreased to 132.4 ± 23.5 pmol · mm−1 · min−1, a 31% decrease (P < 0.02) (Fig.4).
To show that the decrease caused by tempol was not due to a toxic effect, we next examined the change in transport caused by removing tempol from the bath. In the presence of tempol, Cl absorption was 117.2 ± 9.3 pmol · mm−1 · min−1. Sixty minutes after tempol was removed from the bath, Cl absorption increased to 149.2 ± 9.1 pmol · mm−1 · min−1, a 27% increase (P < 0.05) (Fig.5). Taken together, these data indicate that endogenously produced O stimulates NaCl reabsorption by the THAL.
We found that O generated by exogenously added xanthine oxidase and hypoxanthine stimulated NaCl absorption by rat THALs. Endogenously produced O also increased NaCl transport. In contrast, H2O2, another oxidizing agent, did not significantly alter NaCl absorption when added to the bath. These data indicate that O stimulates NaCl absorption by the THAL.
To examine the effects of O on NaCl absorption by THALs, we first investigated the ability of xanthine oxidase/hypoxanthine to stimulate transport. We found that when xanthine oxidase/hypoxanthine was added to the bath, it increased net Cl absorption by 33%. In the presence of oxygen and hypoxanthine, xanthine oxidase catalyzes the formation of both O and H2O2 (35). To study whether the effect of xanthine oxidase/hypoxanthine on Cl absorption was due to H2O2 or a direct effect of xanthine oxidase or hypoxanthine, we tested whether the response could be prevented by superoxide dismutase. We found that superoxide dismutase completely blocked the effect of xanthine oxidase/hypoxanthine on Cl absorption, suggesting that the increase in transport caused by xanthine oxidase/hypoxanthine cannot be due to either the enzyme or the substrate directly stimulating absorption. Because superoxide dismutase increases H2O2 levels by reducing O to H2O2, the increase in Cl absorption cannot be caused by H2O2 produced by xanthine oxidase/hypoxanthine. To ensure that the effect of xanthine oxidase/hypoxanthine was not mediated by H2O2, we tested it directly. We found that adding 200 nM H2O2 alone to the bath did not affect THAL Cl absorption. While this concentration is higher than reported H2O2 levels in the kidney (12), our data do not rule out the possibility that higher concentrations could affect THAL transport. Taken together, these data indicate that exogenously added O stimulates NaCl absorption by the THAL.
Because exogenously produced O enhanced THAL transport, we next tested whether endogenous O regulates NaCl absorption. Because O production by the THAL has not been measured previously to our knowledge, we first examined O production by measuring steady-state O levels in THAL suspensions using lucigenin-enhanced chemiluminescence. O production was detected in the absence of known stimulators or superoxide dismutase inhibitors, suggesting that THALs produce O under basal conditions.
To see whether altering endogenous O levels affects NaCl absorption, we used a superoxide dismutase mimetic, tempol. In isolated, perfused THALs, tempol decreased Cl absorption when added to the bath. Because this effect could be explained by either tempol scavenging O or toxic effects of tempol, we tested whether the effect of tempol was reversible. Sixty minutes after tempol was removed from the bath, THAL Cl absorption recovered significantly. Taken together, our data suggest that endogenously produced O exerts a tonic stimulatory effect on THAL NaCl transport. To our knowledge, this is the first evidence that endogenously produced O stimulates NaCl transport by the nephron.
While we found that THALs produce O under basal conditions, the sources and sites of O production are not known. Possible sources of O include the mitochondria (3), xanthine oxidase (35), and phagocyte-like NAD(P)H oxidase (16, 17). THAL cells contain numerous mitochondria. Approximately 1–5% of oxygen consumed during aerobic respiration is transformed to O in the inner mitochondrial membrane. However, the presence of superoxide dismutase in the matrix as well as in the intermembranous space (8, 28) makes it unlikely that O escapes from the mitochondria under physiological conditions. Thus mitochondria are unlikely sources for the O that stimulates transport.
Xanthine oxidase is also an important source of O in many cells (2, 14, 35), and it has also been detected in membrane fractions of isolated THALs (36). In addition, increased xanthine oxidase activity has been reported in the kidneys of Dahl salt-sensitive rats (23). Enhanced salt absorption by the THAL has been implicated in causing the hypertension in this model (30). These data suggest that xanthine oxidase-derived O may contribute to the development of salt-sensitive hypertension by increasing THAL NaCl absorption. Recently, the presence of NAD(P)H oxidase in renal cells and a kidney-specific homologous protein has been reported (10,40). NAD(P)H oxidase is a multimeric enzyme composed of two membrane subunits (p22 and gp91phox) and three cytosolic subunits (p47, p67, and p40phox) (1). The p22, p47, and p67phoxsubunits have been localized to the THAL by immunohistochemistry (4). NAD(P)H oxidase has been shown to generate both extracellular and intracellular O (1, 17, 21,27, 42). Consequently, O produced by NAD(P)H oxidase may stimulate THAL Cl absorption. Overall, the contribution of each of these pathways to endogenous O production and regulation of THAL NaCl transport is largely unknown, and additional research in this area is required.
The solutions used in the present experiments were gassed with 100% oxygen. Given the affinity constant of oxygen for O -producing enzymes, it is possible that high Po 2 could result in increased generation of O . However, it has recently been shown that O production in the THAL increases when cells are exposed to low rather than high Po 2(24). Thus it remains unclear whether high Po 2 may contribute to O2 - production.
We previously reported that O can decrease nitric oxide (NO) bioavailability in the THAL (32). Because NO inhibits THAL NaCl absorption (31, 33), it could be argued that the stimulation of THAL transport caused by O may be due to a decrease in NO levels. However, in the present experiments, the effects of xanthine oxidase/hypoxanthine and tempol were studied without including l-arginine, the substrate for NO synthase, in the bath. We have previously demonstrated that tempol does not increase NO levels in the THAL whenl-arginine is not present in the bath (32). In addition, blockade of THAL NO synthase in the absence ofl-arginine does not affect THAL NaCl absorption (29,37), suggesting that NO levels are very low under these conditions. Therefore, the stimulatory effect of O on THAL NaCl transport in the absence of l-arginine is most likely due to a direct effect of O and is independent of NO.
NaCl transport by the THAL is important for maintenance of high interstitial osmolality, which drives fluid reabsorption by the nephron. Therefore, stimulation of THAL NaCl transport by O should decrease both Na and fluid excretion by the kidney. In agreement with our data, Zou et al. (44) reported that increasing O levels in the renal medulla by infusion of a superoxide dismutase inhibitor decreased urinary Na and fluid excretion in anesthetized rats, suggesting that O stimulates nephron transport. Moreover, infusion of tempol into the renal medulla increased urinary flow and Na excretion, suggesting that O tonically stimulates NaCl and water reabsorption by the nephron. Recently, Majid and Nishiyama (25) reported that infusion of a superoxide dismutase inhibitor into the renal artery of anesthetized dogs decreased urinary flow and fractional Na excretion without significantly affecting glomerular filtration rate. These data suggest that O stimulates NaCl absorption by the nephron. Overall, our results are consistent with in vivo data that suggest a role for O as a physiological regulator of Na excretion by the kidney.
The transport mechanism by which O stimulates THAL Cl absorption is unknown. O could stimulate the apical Na-K-2Cl cotransporter and K channels or increase the activity of basolateral Na-K-ATPase or Cl channels. While little is known about the effects of O on nephron transport, in other cells O and other ROS have been shown to regulate ion transport by affecting different transporters (22). In myocytes, O was found to stimulate Na/Ca exchange (11, 38) and plasma membrane (41) or mitochondrial (43) ATP-sensitive K channels (KATP). In various cell types, including endothelial and smooth muscle cells, O increased intracellular Ca by increasing calcium entry or release from intracellular stores (13, 19, 20). Inhibitory effects of O on ion transport have also been reported. Sakai et al. (39) found a decrease in Cl channel activity in gastric parietal cells exposed to xanthine oxidase, while O decreased Na-K-ATPase activity in coronary arteries (6). Overall, these data suggest that the effects of O on ion transport may be cell specific and depend on which transport system is affected. The transport mechanism by which O stimulates net Cl absorption in the THAL remains to be studied.
We conclude that both exogenous and endogenous O stimulate THAL NaCl reabsorption. To our knowledge, these are the first data showing that O can directly stimulate nephron transport, and they support a role for O as a physiological modulator of Na and water excretion by the kidney.
This work was supported in part by National Heart, Lung, and Blood Institute Grant HL-28982 (to J. L. Garvin). P. A. Ortiz was supported in part by a fellowship (0020438Z) from the American Heart Association.
Address for reprint requests and other correspondence: J. L. Garvin, Div. of Hypertension and Vascular Research, Henry Ford Health Sciences Ctr., 2799 West Grand Blvd., Detroit, MI 48202 (E-mail:).
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.
July 16, 2002;10.1152/ajprenal.00102.2002
- Copyright © 2002 the American Physiological Society