Division of Hypertension and Vascular Research, Henry Ford Health Sciences Center, Detroit, Michigan 48202
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ABSTRACT |
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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 (O1 · 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
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
superoxide dismutase; sodium-potassium-2 chloride cotransport; loop of Henle; salt-sensitive hypertension; urinary sodium excretion
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INTRODUCTION |
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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
In vivo animal experiments have recently shown that an increase in
O
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METHODS |
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Animals. 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
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 · min1 · mm
1.
Compounds that alter production of O
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
(JCl
) was calculated
as follows
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Statistics. Results are expressed as mean ± SE. Data were evaluated with Student's paired t-test. P < 0.05 was considered significant.
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RESULTS |
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To investigate whether O1 · min
1.
After xanthine oxidase (1 mU/ml) and hypoxanthine (0.5 mM) were added
to the bath to increase O
1 · min
1,
a 33% increase (P < 0.03).
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Because xanthine oxidase/hypoxanthine increases not only
O1 · 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.
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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 ·mm1 · 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
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Essentially all cells produce O1 · mg
protein
1.
To test whether endogenously produced O1 · 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).
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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 · mm1 · 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
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DISCUSSION |
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We found that O
To examine the effects of O
Because exogenously produced O
To see whether altering endogenous O
While we found that THALs produce O
Xanthine oxidase is also an important source of O
The solutions used in the present experiments were gassed with 100%
oxygen. Given the affinity constant of oxygen for
O
We previously reported that O
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
The transport mechanism by which O
We conclude that both exogenous and endogenous O
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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: jgarvin1{at}hfhs.org).
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
Received 15 March 2002; accepted in final form 13 July 2002.
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