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Department of Physiology, Medical College of Wisconsin, Milwaukee, Wisconsin
Submitted 13 March 2008 ; accepted in final form 24 June 2008
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ABSTRACT |
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 TOP ABSTRACT METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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blood pressure; free radicals; hypertension; kidney; reduced nicotinamide adenine dinucleotide phosphatase oxidase
Our laboratory has previously demonstrated that both the Na+-K+-ATPase inhibitor ouabain and the Na+/H+ exchanger (NHE) inhibitor amiloride reduce O2– production in mTAL in response to increased extracellular NaCl (20). Furthermore, Li et al. (18) have recently provided evidence that amiloride-sensitive H+ efflux stimulates O2– production in mTAL and that amiloride-sensitive pathways account for more than 50% of outer medullary oxidative stress in anesthetized Sprague-Dawley rats. Given these data, the first aim of the present study was to determine whether O2– production is enhanced in mTAL isolated from SS rats, providing evidence either for or against a role of mTAL epithelial cells in the development of oxidative stress in the outer medulla of these animals. Our second aim was to determine whether N-methyl-amiloride (NMA)-sensitive O2– production was elevated in SS rats and whether these pathways may be enhanced during high-sodium feeding and the development of hypertension in these animals.
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METHODS |
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Solutions. Hanks’ balanced salt solution (HBSS) was purchased from Invitrogen (Grand Island, NY) and prepared by adding HEPES (Sigma) to a final concentration of 20 mM. The pH of the resulting solution was adjusted to 7.40 by addition of NaOH. Apocynin, ouabain, NMA, choline chloride (ChCl), and NaCl were purchased from Sigma (St. Louis, MO). Apocynin was dissolved in methanol before being added to the experimental buffer. Dihydroethidium (DHE) and sodium-binding benzofuran isophthalate were purchased from Molecular Probes (Eugene, OR). DHE was prepared in DMSO, as instructed, before being diluted to appropriate concentration in HBSS.
Determination of NaCl concentration in renal medullary interstitial fluid. Interstitial fluid samples from the outer medulla of SS and SS.13BN rats fed a 0.4% NaCl diet were obtained using the method described by Lee et al. (14, 15).
The Na+ concentration of interstitial fluid samples was determined using the Na+-sensitive fluorescent dye sodium-binding benzofuran isophthalate (Molecular Probes), using a fluorescent plate reader (Spectrafluor Plus; TECAN U.S.). The final sodium concentration of interstitial fluid samples was then determined by comparing the excitation ratio x340/x405 to samples of known sodium concentration on a standard curve.
Since unstimulated mTAL in our in vitro preparation produce only low levels of O2–, it was necessary to stimulate mTAL O2– production to identify drug treatment or strain effects on mTAL O2– production. As in vivo mTAL are exposed to relatively large fluctuations in both osmolality and extracellular NaCl levels in response to variations in Na+ and water intake (24), we chose to stimulate mTAL O2– production by incrementing superfusate NaCl concentration from 154 (physiologically low) to 254 (physiologically normal) to 500 mM (physiological high). The mean interstitial Na+ concentration determined from fluid obtained from SS and SS.13BN rats was used to estimate physiologically low, normal (x1), and high (x2), the average NaCl levels to which mTAL are exposed in vivo.
Preparation of mTAL for in vitro studies. Isolation of mTAL was performed as described previously (9). Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg ip), and the left kidney was perfused with 6 ml of chilled HBSS at 3 ml/min via the abdominal aorta. The left kidney was then excised and sliced sagitally using a razor blade, and a segment of the renal outer medulla was dissected using fine scissors. The segment of outer medulla was then placed in chilled HBSS and microdissected under a Lecia M3Z stereomicroscope to remove a thin strip of tissue from the inner stripe of the outer medulla containing mTAL. The thin tissue strip containing mTAL was placed on a glass coverslip coated with the tissue adhesive Cell-Tak (BD Biosciences, Bedford, MA) for fluorescence imaging.
Fluorescence detection of dyes. Fluorescence measurements were made using a Nikon TE2000 inverted microscope with a x60 magnification water immersion (numerical aperture 1.2) objective lens. The signal was detected using a high-resolution digital camera (Photometrics Cascade 512B, Roper Scientific, Tucson, AZ). Excitation was provided by a Sutter DG-4 175 W xenon arc lamp (Sutter Instruments, Novato, CA) that allowed high-speed excitation wavelength switching. DHE and ethidium (Eth) fluorescent signals were stimulated by dual-wavelength excitation at 380 and 480 nm. A 445/40-nm and a 605/55-nm band-pass emission filter were used to collect DHE (excitation 380 nm/emission 445 nm) and Eth (excitation 480 nm/emission 605 nm) signals, respectively. The excitation intensity was adjusted on the DG-4 to minimize bleaching and to balance excitation intensities.
Determination of mTAL O2– production. Tissue strips containing mTAL were loaded with DHE (50 mmol/l) in HBSS for 1 h at room temperature. Loading buffer was then replaced with HBSS, and tissues rested for a further 15 min before being imaged. Coverslips were placed on a heated imaging chamber maintained at 37°C (Warner Instruments, Hamden, CT) that allowed the rapid exchange of superfusion buffer and were mounted on the stage of an inverted microscope. Five to 10 mTAL epithelial cells were selected within each tissue strip to quantify changes in fluorescent intensity of the DHE dye using Metafluor imaging software (Universal Imaging, Downingtown, PA). The rate of O2– production was calculated as the change in the ratio of Eth fluorescent signal to DHE fluorescent signal. We have found that using the ratio Eth/DHE to determine O2– production in mTAL reduces measurement artifacts associated with cell volume changes and dye bleaching (19).
O2– production in mTAL was stimulated by increasing bath NaCl concentration (20) through 154, 254, and 500 mM, for 200 s at each increment, over a 600-s period, representing approximately physiological isotonic and one and two times normal in vivo NaCl levels in the renal outer medulla. mTAL O2– production was determined in tissue strips obtained from the inner stripe of the outer medulla of SS and SS.13BN rats fed a 0.4% NaCl diet in response to incrementing bath NaCl concentration, and SS and SS.13BN rats fed a 4.0% NaCl diet for 3 days before tissue harvest. Responses to incrementing bath NaCl concentration in mTAL of SS and SS.13BN rats were also determined in the presence of ouabain (4 mM; to inhibit Na+-K+-ATPase) and NMA (100 µM; to inhibit NHE activity). Responses to incrementing bath NaCl concentration in mTAL of 0.4% NaCl-fed SS and SS.13BN rats were also determined in the presence of ouabain (4 mM) + NMA (100 µM; to inhibit both Na+-K+-ATPase and NHE) and apocynin [100 µM; to inhibit NAD(P)H oxidase]. In all cases, tissue strips were incubated with inhibitors for 15 min before recording. In some tissue strips, osmolality was increased in increments of 300, 500, and 1,000 mosmol/kgH2O using ChCl in the presence of ouabain (4 mM; to determine the active Na+ transport independent effects of osmolality) and ChCl in the presence of NMA (100 µM) or urea (to determine the effects of changes in cell volume on mTAL O2– production independent of other osmotic effects).
Eth and DHE fluorescent signals were recorded for 200 s at each increment at 3-s intervals. To conserve time and maximize the number of observations that could be obtained from each animal, positive control stimuli were not tested in all tissue strips. In some tissue strips, diethyldithiocarbamic acid (1 mmol/l; Sigma) to inhibit superoxide dismutase and menadione sodium bisulfite (500 mol/l; Sigma) to stimulate mitochondrial O2– production were added to serve as positive control stimuli to test for dye loading and cell viability. Strong positive control responses were observed in all coverslips tested, indicating that none of the protocols utilized exhausted the DHE dye or adversely affected cell viability.
Western blots.
Rats were prepared as described above. The left kidney was flushed with chilled HBSS, and the outer medulla was snap frozen in liquid nitrogen. Tissue was homogenized, and 
30 mg of protein were extracted from the outer medulla for Western blot. The membrane was immunoblotted with primary antibody (p22phox-polyclonal antibody; Santa Cruz, Biotechnology, sc-20781) in 1:500 dilution and secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit antibody, Abcam, ab6721) in 1:5,000 dilution. The densitometry values were normalized to the band of β-actin reprobed in the same sample.
Data and statistical analysis. Background fluorescent signals were subtracted, and the DHE and Eth signals were normalized so that the ratio Eth/DHE at time = 0 was equal to 1. Data are expressed as means ± SE. The responses of intracellular O2– production to incrementing bath NaCl concentration were evaluated with a two-way ANOVA using a Bonferroni post hoc test to compare responses between SS and SS.13BN rat strains (PStrain*Sodium). To determine the effects of treatments, where appropriate, responses were also compared with control responses of mTAL to incrementing sodium from low-sodium (0.4% NaCl)-fed SS.13BN and SS rat in the absence of inhibitors (PTreatment). Where indicated, slopes represent rate of change in ratio of Eth/DHE over the final 100 s of each stimulus period. For all other data, significance was evaluated using an unpaired t-test. The level required to reach significance was P < 0.05.
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RESULTS |
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O2– responses in mTAL to incrementing NaCl concentration.
Incrementing bath NaCl concentrations (154 to 254 to 500 mM) stimulated O2– production in mTAL of both SS and SS.13BN rats (Fig. 1A). The rate of O2– production, however, was significantly greater in mTAL of SS rats (n = 13 mTAL, 8 rats) compared with mTAL of SS.13BN rats (n = 16 mTAL, 10 rats) (Fig. 1A: PStrain*Sodium < 0.001), with SS rats producing 20% more O2– during the 600-s protocol. Differences in the rate of O2– production between mTAL obtained from SS and SS.13BN rats became significant only during the final 200 s of the protocol in which bath NaCl concentration was raised to 500 mM. During the final 100 s of this period, the rate of O2– production was nearly twice as great in mTAL from SS rats (slope = 0.009 ± 0.002 U/s) compared with mTAL from SS.13BN rats (slope = 0.004 ± 0.001 U/s).
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Effect of Na+ transport inhibitors on O2– responses to incrementing NaCl concentration. As shown in Fig. 2A, addition of NMA (100 µM) to the bath attenuated O2– production in response to incrementing bath NaCl levels in mTAL obtained from 0.4% NaCl-fed SS rats (n = 8 mTAL, 5 rats; PTreatment*Sodium < 0.001) but not 0.4% NaCl-fed SS.13BN rats (n = 7 mTAL, 5 rats; note: SS and SS.13BN traces superimposed in Fig. 2A). NMA also inhibited O2– production in both 4.0% NaCl-fed SS rats (n = 7 mTAL, 7 rats; PTreatment*Sodium < 0.001) and 4.0% NaCl-fed SS.13BN rats (n = 7 mTAL, 7 rats; PTreatment*Sodium < 0.001; Fig. 2B). Importantly, in both 0.4% and 4.0% NaCl-fed rats, NMA abolished differences in the rate of O2– production between strains (note: SS and SS.13BN traces superimposed in Fig. 2B).
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mTAL O2– responses to incrementing bath osmolality and osmotic cell shrinkage. As shown in Fig. 3B, in the presence of ouabain (4 mM) to inhibit active Na+ transport, incrementing bath osmolality using ChCl, in equimolar amounts to that using NaCl in other protocols, stimulated O2– production in mTAL from both SS (n = 6 mTAL, 5 rats) and SS.13BN rats (n = 6 mTAL, 4 rats). Increases in O2– production induced by ChCl were not significantly different from those observed in response to stimulation with NaCl. Consistent with our results using NaCl, O2– production was significantly greater in mTAL obtained from SS rats compared with that from SS.13BN rats in response to increasing bath osmolality in increments of 300, 500, and 1,000 mosmol/kgH2O using ChCl (PStrain+ChCl < 0.001). Importantly, differences in O2– production in response to incrementing ChCl between SS and SS.13BN rats were abolished by addition of NMA, suggesting ChCl was stimulating O2– production through a similar pathway to that stimulated by NaCl (Fig. 3A; note: SS and SS.13BN traces superimposed). Increasing bath osmolality to the same degree using the cell-permeable urea as that achieved using NaCl or ChCl did not stimulate significant O2– production in mTAL (studies performed in mTAL obtained from SS rats only; n = 6 mTAL, 4 rats), indicating that cell shrinkage was required to stimulate O2– production.
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DISCUSSION |
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Since in vivo high-sodium feeding results in increased outer medullary oxidative stress and the development of hypertension in SS rats (27), in addition to prehypertensive 0.4% NaCl-fed rats, we also examined responses in mTAL from SS and SS.13BN rats fed a high (4.0%) NaCl diet for 3 days before tissue harvest. A relatively early time point (3 days) after initiation of high-sodium feeding was chosen, since hypertension is only beginning to develop at this time, limiting the confounding effects of severe chronic hypertension and associated renal tubular damage (21). Our results demonstrate that this 4.0% NaCl diet increased the rate of O2– production in mTAL obtained from both rat strains in response to acute increments of bath NaCl. Importantly, however, O2– production in mTAL of SS rats remained elevated above that of SS.13BN rats.
Both amiloride- and ouabain-sensitive pathways of O2– production were enhanced following 3 days of high (4.0%) NaCl feeding in mTAL of SS and SS.13BN rats compared with mTAL of 0.4% NaCl-fed rats. Importantly, while amiloride-sensitive O2– production was greater in both mTAL of SS or SS.13BN rats by high NaCl, strain differences in O2– production between mTAL of SS and SS.13BN rats remained. The factors that result in upregulation of mTAL O2– production in the early stages of a high-sodium diet remain incompletely understood. Enhanced O2– production could, however, be mediated by a variety of factors, including increased active transport of Na+, enhanced mitochondrial-derived O2– production, upregulation of NAD(P)H oxidase, or reduced levels of free radical scavengers, such as superoxide dismutase, as our laboratory has previously demonstrated that these processes are altered in the outer medulla of high-NaCl-fed SS rats (27, 28). Our data are consistent with the published data of Taylor et al., who, using in vivo microdialysis techniques, demonstrated differences in outer medullary O2– levels in both 0.4% NaCl-fed SS and SS.13BN rats (27), and SS and SS.13BN rats fed 4.0% NaCl for 6 wk (28).
Evidence that the activity of NHE linked to O2– production in rat mTAL. The amiloride-sensitive O2– production observed in our studies appears to be mediated primarily by cell shrinkage. A direct effect of increased extracellular Na+ concentration or changes in active Na+ transport can be excluded for two reasons. First, when ChCl was used to increase bath osmolality, extracellular NaCl concentration remained unchanged, yet O2– responses were the same as during stimulation with NaCl, and these responses were also sensitive to amiloride. Second, amiloride-sensitive O2– responses to increases in bath NaCl were present when Na+ transport was inhibited by ouabain. Unlike NaCl and ChCl, mTAL are highly permeable to urea, so incrementing bath urea concentration does not largely affect cell volume (13). Since increasing bath osmolality using urea to the magnitude of that reached with NaCl and ChCl did not stimulate significant mTAL O2– production, we conclude that the amiloride-sensitive component of the O2– response was mediated by cell shrinkage rather than other factors associated with increasing bath osmolality.
Our data are consistent with the results of Li et al. (18), who demonstrated H+ efflux through amiloride-sensitive, NHE-stimulated, NAD(P)H oxidase-dependent O2– production in mTAL obtained from Sprague-Dawley rats. The hypothesis that cellular extrusion of H+ through NHE mediates O2– responses to incrementing bath NaCl levels is attractive, since NHE-1 is known to be present on the basolateral membrane of mTAL (2) and the activity of NHE-1 is known to be increased in response to cell shrinkage (8, 10). It should also be noted here that, in protocols in which we incremented bath osmolality using ChCl, NaCl (154 mM) was present, and thus NHE would not have been inhibited (23). While the analog of amiloride used in our study, NMA, is a relatively specific inhibitor of the basolateral NHE, this amiloride analog does not allow us to confirm which isoform of NHE may be involved in the observed O2– responses, or if nonspecific inhibition of other transporters may have occurred. Further studies utilizing novel analogs of amiloride capable of specific inhibition of NHE isoforms will be required to resolve these issues.
Addition of apocynin (100 µM) ameliorated the O2– response to incrementing bath NaCl in mTAL from SS but not SS.13BN rats, and no differences in the rate of O2– production between rat strains were detected in the presence of apocynin. We conclude from these data that the enzyme NAD(P)H oxidase is linked to the enhanced rate of O2– production observed in mTAL from SS rats. These data are consistent with our laboratory's previous data, indicating that, in vivo, O2– production in the intact renal outer medulla of SS rats can be reduced by interstitial infusion of apocynin (27). Furthermore, we have previously demonstrated that NAD(P)H oxidase activity is enhanced in outer medullary tissue homogenates of SS rats (27). Many of the subunits of NAD(P)H oxidase have been found to be more highly expressed in the outer medulla of SS rats compared with SS.13BN rats, including gp47phox and p22phox (27), as confirmed by Western blot analysis of protein expression of p22phox in the present study. While our data indicate that NAD(P)H oxidase is an important mediator of the enhanced O2– production observed in mTAL from SS rats, it has recently been demonstrated that apocynin acts as a nonspecific antioxidant in vascular smooth muscle cells and other nonphagocytic cells lacking the enzyme myeloperoxidase, which converts apocyinin to its active form, diapocynin (11). Whether other peroxidases capable of converting apocynin to its active form are present in mTAL is currently unknown. The renal medulla does, however, exhibit endogenous peroxidase activity (3–5, 29). Our data are also consistent with previous studies demonstrating that NAD(P)H oxidase is a major producer of O2– in mTAL using various pharmacological approaches (6, 18). At present, the cellular pathways by which NHE may stimulate NAD(P)H oxidase remain unclear. Further studies will be required to elucidate such pathways and to determine whether, in addition to NAD(P)H oxidase, NHE expression or other constituents of such a pathway may be upregulated in mTAL of SS rats.
Physiological significance. In vivo mTAL are exposed to large fluctuations in extracellular osmolality and NaCl concentration in response to variations in Na+ and water intake (24). These osmotic fluctuations lead changes in cell volume, which, to maintain cellular function, must be offset by activating regulatory transporters, such as NHE-1, which return cell volume to normal (8, 10). While in vivo relatively small changes in cell volume and the production of reactive oxygen species in the renal medulla may have powerful consequences on renal function and tubular Na+ reabsorption, experimentally these alterations may be short lived and are difficult to quantify. To identify differences between strains of animals in pathways of O2– production within the tubular segment responsible for the greatest amount of O2– production (mTAL), in vitro studies are required. Given that we were more likely to identify differences between strains under conditions of high O2– production, large concentrations of NaCl were used, recognizing that such concentrations occur in vivo only in conditions of severe dehydration (15, 16). While recognizing this caveat, we have found good consistency between in vitro data, using similar approaches in freshly isolated mTAL and results from whole animal in vivo studies in which physiologically relevant stimuli are utilized (9, 22, 25, 31).
There is evidence indicating that the pathways identified under maximal NaCl stimulation in the present study are of consequence under normal physiological conditions. Consistent with the data of Zhou et al. (30), indicating that TAL produce the majority of O2– in the renal medulla, our present data indicate that mTAL are likely to be responsible in large part for differences in outer medullary oxidative stress observed in SS and SS.13BN rats (27, 28). Our data are also consistent with the data of Li et al. (18), who provided evidence that amiloride-sensitive pathways account for >50% of outer medullary oxidative stress in Sprague-Dawley rats in vivo under normal physiological conditions. We confirm that much of the O2– generated by mTAL is amiloride sensitive and have found that these amiloride-sensitive pathways are upregulated in SS rats. The observation that amiloride-sensitive pathways of O2– production may be upregulated in SS rats has immediate physiological relevance, given that our laboratory has previously demonstrated that enhanced outer medullary oxidative stress in SS rats leads to the development of hypertension and renal injury in these animals (21, 27). We believe that the present findings provide a strong impetus for future experimental studies aimed at ameliorating renal damage and hypertension in SS animals with amiloride analogs directed at inhibition of NHEs such as NMA.
Our finding that amiloride-sensitive pathways account for much of the excess O2– produced by mTAL of SS rats does not exclude the possibility that other pathways of reactive oxygen species production contribute to the outer medullary oxidative stress observed in SS hypertension. Our laboratory has previously demonstrated that both increased luminal Na+ concentration and increased luminal flow increased the rate of O2– production in isolated, perfused mTAL of Sprague-Dawley rats (1). Furthermore, Hong and Garvin (12) have demonstrated that, in response to increased luminal NaCl delivery, O2– production is increased in mTAL via a furosemide-inhibitable pathway, indicating that active transport of Na+ stimulates mTAL O2– production. This group has also recently demonstrated that tubular stretch increases mTAL O2– production. Importantly, Hong and Garvin (12) were unable to inhibit O2– production in NaCl-perfused mTAL with amiloride, indicating that the pathways of O2– production that they observed are different from the amiloride-sensitive pathways observed in the present study. As both mTAL luminal NaCl delivery and flow are likely to be increased during high-NaCl feeding, it is probable that many of these alternative mechanisms of mTAL O2– production are activated and contribute to elevated outer medullary oxidative stress in high-sodium-fed SS rats. While it is clear from the present study that amiloride-sensitive pathways of O2– production are differentially regulated in SS and SS.13BN and likely contribute to the elevated hypertension and renal damage observed in SS rats compared with salt-resistant SS.13BN rats, whether alternative pathways are also elevated in SS mTAL remain unclear (27, 28).
Summary and conclusions. The primary findings of our study are that O2– responses in mTAL obtained from SS rats in response to increments in bath NaCl are greater than that of mTAL obtained from salt-resistant SS.13BN rats. Addition of both amiloride and apocynin normalized the rate of O2– production in mTAL from SS to that of SS.13BN rats, indicating that the amiloride-sensitive pathways may mediate enhanced O2– production in mTAL from SS rats via an interaction with NAD(P)H oxidase. Amiloride-sensitive O2– production in SS rats is largely independent of active Na+ transport and is mediated by hyperosmotic cell shrinkage, consistent with activation of NHE-1. Whereas both amilioride-sensitive and amiloride-insensitive pathways of O2– production are elevated in mTAL of SS and SS.13BN rats fed a high-NaCl diet, differences in O2– production between the strains are abolished by amiloride. We conclude that amiloride-sensitive pathways active in mTAL epithelial cells are likely to be responsible for the enhanced levels of O2– observed in the outer medulla of the SS kidney. Future studies aimed at the further characterization of such pathways, as well as studies utilizing amiloride analogs directed toward inhibition of NHE, may be important in understanding and preventing oxidative stress and salt-sensitive hypertension.
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FOOTNOTES |
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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.
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, data obtained from SS rats; 
, data obtained from SS.13BN rats. *Area identified under time point = P < 0.05 for differences between strains, as determined by Bonferroni post hoc test.
significant difference between treatment groups.
