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【摘要】 Superoxide (O 2 - ) regulates renal function and is implicated in hypertension. O 2 - production increases in response to increased ion delivery in thick ascending limbs (TALs) and macula densa and mechanical strain in other cell types. Tubular flow in the kidney acutely varies causing changes in ion delivery and mechanical stress. We hypothesized that increasing luminal flow stimulates O 2 - production by NADPH oxidase in TALs via activation of Na-K-2Cl cotransport. We measured intracellular O 2 - in isolated rat TALs using dihydroethidium in the presence and absence of luminal flow and inhibitors of NADPH oxidase, Na-K-2Cl cotransport, and Na/H exchange. In the absence of flow, the rate of O 2 - production was 5.8 ± 1.4 AU/s. After flow was initiated, it increased to 29.7 ± 4.3 AU/s ( P < 0.001). O 2 - production was linearly related to flow. Tempol alone and apocynin alone blocked the flow-induced increase in O 2 - production (3.5 ± 1.7 vs. 4.5 ± 2.8 AU/s and 8.2 ± 2.1 vs. 10.6 ± 2.8 AU/s, respectively). Furosemide decreased flow-induced O 2 - production by 55% (37.3 ± 5.2 to 16.8 ± 2.8 AU/s; P < 0.002); however, dimethylamiloride had no effect. Finally, we examined whether changes in mechanical forces are involved in flow-induced O 2 - production by using a Na-free solution to perfuse TALs. In the absence of NaCl, luminal flow enhanced O 2 - production (1.5 ± 0.5 to 13.5 ± 1.1 AU/s; P < 0.001), 50% less stimulation than when flow was increased in the presence of luminal NaCl. We conclude that flow stimulates O 2 - production in TALs via activation of NADPH oxidase and that NaCl absorption due to Na-K-2Cl cotransport and flow-associated mechanical factors contribute equally to this process.
【关键词】 reactive oxygen species ion delivery Na transport mechanical strain
SUPEROXIDE (O 2 - ) is an important regulator of kidney function ( 45, 47 ). This reactive oxygen species favors salt and water retention and has been implicated in salt-sensitive hypertension ( 20, 29, 42 ). The thick ascending limb of the loop of Henle (TAL) is involved in inappropriate NaCl retention in salt-sensitive hypertension ( 16, 19, 36 ), and we found that O 2 - increases TAL transport by enhancing Na-K-2Cl cotransport and Na/H exchange ( 17, 18 ). However, few studies have addressed how O 2 - production is regulated along the nephron.
Luminal flow through the TAL normally varies over a wide range ( 7, 37, 38 ). Recently, it was reported that increasing luminal flow in the TAL stimulates O 2 - production due to enhanced NaCl delivery ( 1 ). However, the source of O 2 - activated by increasing luminal NaCl delivery was not studied. O 2 - can be produced by NADPH oxidase, xanthine oxidase, uncoupled nitric oxide synthase (NOS), and the mitochondria ( 45 ). O 2 - production in the renal outer medulla has been attributed to NADPH oxidase ( 22, 23, 47 ). TALs express NADPH oxidase and comprise most of the outer medulla. Therefore, NADPH oxidase is the mostly likely source of flow-induced O 2 - production in the TAL. However, other potential sources of O 2 - exist in this nephron segment. The TAL expresses xanthine oxidase ( 33 ) and NOS ( 43 ) and possesses numerous mitochondria ( 26 ) in addition to NADPH oxidase.
In addition to the uncertainty of the source of O 2 - activated by enhanced luminal NaCl delivery, the specific transporter involved is unclear ( 1 ). The TAL expresses both a luminal amiloride-sensitive Na/H exchanger (NHE) and a furosemide-sensitive Na-K-2Cl cotransporter. Enhanced NHE activity has been proposed as the cause of O 2 - generation in nonperfused TALs when NaCl in the bath is elevated from 150 to 250 mM ( 31 ). However, Na-K-2Cl cotransport may also be involved. In the TAL activation of Na-K-2Cl cotransport induces depolarization ( 11 ), and depolarization has been shown to stimulate NADPH oxidase activity ( 28, 41 ). In the macula densa increased luminal NaCl causes depolarization via Na-K-2Cl cotransport ( 21 ) and enhances O 2 - production by depolarization ( 24 ).
Increasing luminal flow in the TAL not only enhances NaCl delivery but also causes mechanical strain. Mechanical stress has been shown to enhance O 2 - production in several cell types ( 4, 12, 39 ). Thus it is possible that increasing luminal flow in the TAL stimulates O 2 - production by altering one or more physical factors in addition to increasing NaCl delivery.
We hypothesized that increasing NaCl delivery by augmenting luminal flow stimulates O 2 - production in the TAL via activation of Na-K-2Cl cotransport and consequently NADPH oxidase. To test this hypothesis, we examined the effect of acutely increasing luminal flow on O 2 - production in isolated TALs. Our findings indicate that 1 ) luminal flow activates NADPH oxidase, resulting in an increase in O 2 - production; 2 ) 50% of flow-induced O 2 - production can be attributed to an increase in NaCl delivery and enhanced Na-K-2Cl cotransporter activity; and 3 ) the other 50% is due to a physical effect of flow unrelated to transport.
MATERIALS AND METHODS
Chemicals and solutions. Dihydroethidium was purchased from Molecular Probes (Eugene, OR). Apocynin (acetovanillone), tempol, and dimethylamiloride were obtained from Sigma (St. Louis, MO) and furosemide from Hospira (Lake Forest, IL). The composition of the physiological saline used to perfuse and bathe the TALs was (in mM) 130 NaCl, 2.5 NaH 2 PO 4, 4 KCl, 1.2 MgSO 4, 6 L -alanine, 1 trisodium citrate, 5.5 glucose, 2 calcium dilactate, and 10 HEPES, pH 7.4, at 37°C. Where noted, a Na-free solution composed of 270 mM mannitol and 10 mM HEPES, pH 7.4 (titrated with KOH), at 37°C was used instead of physiological saline. All solutions were adjusted to 290 ± 3 mosmol/kgH 2 O.
Isolation and perfusion of rat TAL. All protocols involving animals were approved by the Institutional Animal Care and Use Committee (IACUC). Male Sprague-Dawley rats (Charles River Breeding Laboratories, Wilmington, MA) were maintained on a diet containing 0.22% sodium and 1.1% potassium (Purina, Richmond, IN) for at least 5 days. On the day of the experiment, rats weighing 120-150 g were anesthetized with ketamine and xylazine (100 and 20 mg/kg body wt ip, respectively). The abdominal cavity was opened and the left kidney was bathed in ice-cold saline and removed. Coronal slices were placed in oxygenated physiological saline. Medullary thick ascending limbs (mTALs) were dissected from the outer medulla under a stereomicroscope at 4-10°C. Tubules measuring 0.5-1.0 mm were transferred to a temperature-regulated chamber and perfused using concentric glass pipets at 37 ± 1°C as described previously ( 10 ). For the first few experiments, we used nonphysiological perfusion flow rates greater than 20 nl/min to determine whether flow had any effect on the rate of O 2 - production. For all subsequent experiments, we perfused tubules at a physiological flow rate of 20 nl/min using a nanoliter syringe pump (Harvard Apparatus, Holliston, MA). Because we observed no difference in the degree of O 2 - stimulation, data were pooled in Fig. 1 A.
Fig. 1. Flow stimulates O 2 - production in the thick ascending limb. A : initiation of flow ( 20 nl/min) induced an increase in O 2 - generation as measured by an increase in the rate of change in oxyethidium/dihydroethidium fluorescence intensity ratios. The rate changed from 5.8 ± 1.4 to 29.7 ± 4.3 AU/s (* P < 0.001; n = 6). B : addition of the O 2 - scavenger tempol (100 µM) blunted the flow-induced increase in O 2 - generation (3.5 ± 1.7 vs. 4.5 ± 2.8 AU/s; n = 4).
Measurement of O 2 - using dihydroethidium. O 2 - converts dihydroethidium to oxyethidium ( 9, 46 ). An increase in the rate of change in the ratio of the emitted fluorescence intensities (oyxethidium/dihydroethidium) indicates an increase in the rate of O 2 - production. Isolated, perfused mTALs were loaded with 10 µM dihydroethidium for 20 min and then washed for 30 min. Oxyethidium was measured by exciting 515 nm was recorded every 5 s for 60-75 s. Dihydroethidium was excited at 435 nm was recorded every 5 s for 60-75 s. Fluorescence was imaged digitally with an image intensifier in conjunction with a CCD camera and recorded utilizing the Metafluor system (Universal Imaging, West Chester, PA). For each measurement period, regression analysis of the fluorescence ratios was performed and differences in slopes were statistically evaluated.
Statistical analysis. Results are expressed as means ± SE. Data were evaluated with Student?s t -test or linear regression analysis (for Fig. 2 ) and P < 0.05 was considered significant.
Fig. 2. O 2 - production in the thick ascending limb varies in response to flow rate. O 2 - generation increases linearly ( y = 0.0004 x + 0.0031; P < 0.01) at flow rates of 0 ( n = 4), 10 ( n =6), and 20 nl/min ( n = 6).
RESULTS
To study whether increasing flow in the TAL enhances O 2 - production, we subjected isolated TALs to enhanced luminal flow and measured the rate of O 2 - production using the dye dihydroethidium. O 2 - converts dihydroethidium to oxyethidium. Increases in the rate of change in the fluorescence ratio (oyxethidium/dihydroethidium) indicate an increase in the rate of O 2 - production. Figure 1 A shows that in tubules in which there was no luminal flow, the rate of change in the fluorescence ratio was 5.8 ± 1.4 AU/s. After flow was initiated, it increased significantly to 29.7 ± 4.3 AU/s ( P < 0.001; n = 6).
To confirm that the increase was due to an increase in O 2 - production, the O 2 - scavenger tempol (100 µM) was added to the bath at the beginning of the experiment. Figure 1 B shows that in the presence of tempol, the rate of change in the fluorescence ratio did not increase significantly after initiating flow (3.5 ± 1.7 to 4.5 ± 2.8 AU/s; n = 4). Therefore, the flow-induced stimulation of the rate of change in the oyxethidium/dihydroethidium fluorescence ratio was caused by an increase in the rate of O 2 - production.
Since physiological luminal flow rates in the TAL can vary from 0 to 20 nl/min, we determined whether O 2 - production varies in response to different flow rates. We measured O 2 - production by TALs at flow rates of 0, 10, and 20 nl/min and found that there is a significant correlation between flow rate and O 2 - generation ( Fig. 2 ). The fitted regression line is y = 0.0004 x + 0.0031 ( P < 0.01).
Although there are other possible sources of O 2 -, the most likely one in the TAL is NADPH oxidase. Therefore, to determine whether the source of O 2 - was NADPH oxidase, apocynin (10 µM), an inhibitor of NADPH oxidase assembly, was added to the bath before initiating flow. As shown in Fig. 3, in the absence of flow and apocynin, the rate of O 2 - production was 8.2 ± 2.1 AU/s. After incubating the tubules with apocynin, the rate of change in the fluorescence ratio did not increase significantly after flow was initiated (10.6 ± 2.8 AU/s; n = 5). Therefore, flow-induced O 2 - production was blocked in the presence of apocynin. These data indicate that luminal flow stimulates O 2 - production by NADPH oxidase.
Fig. 3. Flow-induced stimulation of O 2 - production is blocked by apocynin, an NADPH oxidase inhibitor. In the presence of 10 µM apocynin (apo) flow had no effect on O 2 - (8.2 ± 2.1 vs. 10.6 ± 2.8 AU/s; n = 5). A perfusion flow rate of 20 nl/min was used.
Increased flow enhances ion delivery and transport. Recently, it has been reported that increases in NaCl delivery augment O 2 - production in the TAL ( 1 ). However, the transporters involved were not studied. Therefore, we examined the luminal Na + transporter(s) that may play a role in flow-stimulated O 2 - generation by adding the Na-K-2Cl cotransporter inhibitor furosemide (100 µM) or the NHE inhibitor dimethyl amiloride (DMA; 100 µM) to the lumen. Figure 4 shows that in the absence of furosemide, tubules perfused with physiological saline generated O 2 - at a rate of 37.3 ± 5.2 AU/s. When furosemide was added to the lumen, the rate decreased by 55% to 16.8 ± 2.8 AU/s ( P < 0.002; n = 6). Figure 5 shows that in the absence of DMA, TALs perfused with physiological saline generated O 2 - at a rate of 35.0 ± 5.4 AU/s. After DMA was added to the lumen, the rate did not change (32.2 ± 5.5 AU/s; n = 5). Thus it appears that luminal Na-K-2Cl cotransport accounts for a significant portion of flow-induced O 2 - production, whereas Na/H exchange plays little or no role.
Fig. 4. Effect of inhibiting Na-K-2Cl cotransport on flow-induced O 2 - production. Addition of 100 µM furosemide (furos) decreased the rate of O 2 - generation by 55%, from 37.3 ± 5.2 to 16.8 ± 2.8 AU/s (* P < 0.002; n = 6). The flow rate was 20 nl/min.
Fig. 5. Effect of inhibiting Na/H exchange on flow-induced O 2 - production. In the presence of 100 µM dimethylamiloride (DMA), there was no change in flow-induced O 2 - production (35.0 ± 5.4 to 32.2 ± 5.5 AU/s; n = 5). Tubules were perfused at a rate of 20 nl/min.
In addition to increasing ion delivery and transport, initiation of flow alters several mechanical factors that could trigger signals that affect O 2 - production. To test whether the inability of furosemide to completely block flow-induced O 2 - production was due to activation of other transporters or mechanical factors, we replaced the physiological saline used to perfuse the tubules with a Na-free solution ( Fig. 6 ). In control experiments using physiological saline containing Na to perfuse the TALs, the rate of O 2 - generation was 1.4 ± 0.6 AU/s in the absence of luminal flow. It increased to 25.8 ± 3.0 AU/s after luminal flow was established. In separate experiments using the Na-free solution, the rate increased from 1.5 ± 0.5 to 13.5 ± 1.1 AU/s, 52% of the increase observed with physiological saline [ P < 0.001, flow with physiological saline containing Na ( n = 5) vs. flow with Na-free solution ( n = 8)]. These data indicate that flow-associated changes in physical factors can stimulate O 2 - production independent of increases in ion delivery and Na-K-2Cl cotransport.
Fig. 6. Effect of Na + removal on flow-induced O 2 - production. Na + removal decreased flow-activated O 2 - generation by 48%, from 25.8 ± 3.0 to 13.5 ± 1.1 AU/s [* P < 0.001, flow with physiological saline containing Na ( n = 5) vs. flow with Na-free solution ( n = 8)]. A flow rate of 20 nl/min was used.
DISCUSSION
We demonstrated that flow stimulates O 2 - production in the TAL and that there is a significant correlation between flow rate and O 2 - generation. This effect is blocked by the O 2 - scavenger tempol and by the NADPH oxidase inhibitor apocynin. Two main types of parameters are altered when luminal flow is changed in TALs: 1 ) mechanical factors such as pressure, stretch, and shear stress, and 2 ) ion delivery. Since enhanced ion delivery affects ion transport, we examined what transporters might be involved. We found that in the presence of the Na-K-2Cl cotransporter inhibitor furosemide, flow-induced O 2 - production was blocked by only 50%, whereas the NHE inhibitor dimethylamiloride had no effect. Thus at least 50% of O 2 - production could be attributed to increased transport via the Na-K-2Cl cotransporter. To clarify how flow might augment O 2 - generation in TALs, Na + in the luminal perfusate was removed. TALs perfused with Na-free solution generated O 2 -, but about half as much compared with perfusion with physiological saline. Therefore, 50% of flow-stimulated O 2 - generation appears to be due to increased Na-K-2Cl cotransport and the remaining 50% appears to be a result of mechanical factors. Taken together, these data indicate that luminal flow stimulates O 2 - production in the TAL via activation of NADPH oxidase and that an increase in NaCl transport via the Na-K-2Cl cotransporter and changes in physical factors contribute equally to this process.
O 2 - can be generated by the mitochondria, xanthine oxidase, NADPH oxidase, and uncoupled NOS ( 45 ). We found that the increase in O 2 - generated in response to flow could be blocked by the NADPH oxidase inhibitor apocynin. This is consistent with reports attributing O 2 - production in the renal medulla and the TAL to NADPH oxidase ( 22, 23, 47 ). Although the kidney expresses the NADPH oxidase isoforms, Nox1, Nox2 (gp91 phox ), and Nox4 ( 3 ), we do not know which isoform is involved in flow-induced O 2 - production.
Our results indicate that 50% of the flow-induced increase in O 2 - in the TAL is due to changes in ion delivery. Because altering ion delivery influences NaCl transport, we investigated the possible luminal transporters involved. The main luminal transporters involved in NaCl absorption in the TAL are the Na-K-2Cl cotransporter and NHE. Absorption of NaCl by the TAL can be described as a two-step process. In the first step, Na + crosses the luminal membrane of the cell via Na-K-2Cl cotransport and Na/H exchange. In the second step, Na + exits across the basolateral membrane via Na-K-ATPase while Cl - exits via either K/Cl cotransport or Cl - channels ( 30 ).
We used furosemide to block the Na-K-2Cl cotransporter and DMA to inhibit NHE. We did not use ouabain to block transport because it will inhibit Na-K-ATPase directly and both luminal transporters indirectly by eliminating the Na + gradient that is necessary to drive them.
Na-K-2Cl cotransport accounts for 70-80% of absorbed Na +, while Na/H exchange comprises 20-30%. We found that only the Na-K-2Cl cotransporter, and not NHE, is involved in flow-induced increase of O 2 -. While this could be due to the differences in the amount of Na + absorbed by each transporter, we believe that it is far more likely due to the nature of the transport. In the TAL activation of Na-K-2Cl cotransport induces depolarization by increasing intracellular Cl - which stimulates basolateral electrogenic Cl - exit ( 11 ). In other cells, depolarization has been shown to stimulate NADPH oxidase activity ( 28, 41 ). For example, we found that in the macula densa increased luminal NaCl stimulates Na-K-2Cl cotransport ( 21 ) and also increases O 2 - production by depolarization ( 24 ). Such findings may explain why Na + transport by NHE does not stimulate O 2 - production.
Our data appear to conflict with a report that amiloride-sensitive O 2 - production increased with increasing concentrations of NaCl in the bath of nonperfused TALs ( 31 ). However, bath NaCl was increased from 150 to 250 mM in this study. Essentially, all NHEs, including those in the TAL, have a K 1/2 for Na + of 10 mM. Thus increasing NaCl above 150 mM should not enhance Na/H exchange activity because it is already saturated.
The results showing that amiloride inhibits O 2 - production stimulated by increased bath NaCl may be due to cell volume changes. Basolateral NHE1 plays a key role in cell volume regulation. NHE1 activity increases in response to cell shrinkage initiating a volume-regulatory increase ( 34 ). Increasing NaCl from 150 to 250 mM would cause TAL cells to shrink and activate basolateral NHE1. Adding DMA to the bath of nonperfused TALs would inhibit NHE1 and consequently block the volume-regulatory response. Therefore, the cells would remain shrunken.
Recently, Abe et al. ( 1 ) reported that O 2 - is affected by changes in Na + delivery. Increasing luminal Na + concentration from 60 to 149 mM at a fixed flow rate of 15 nl/min and raising luminal flow from 5 to 20 nl/min at 60 mM Na + increased O 2 - generation. In contrast, changing flow had a negligible effect at 149 mM Na +. The authors concluded that these results were solely due to increases in Na + transport. Our data partially corroborate their findings. We found that 50% of flow-induced O 2 - production was due to NaCl transport by the Na-K-2Cl cotransporter. However, we also found that about half of flow-induced O 2 - was due to mechanical strain. Stimulation of Na + transport is unlikely to be the sole explanation for the data of Abe, given the literature concerning this transporter. Transport rates of NaCl in the TAL are 100-200 pmol·mm -1 ·min -1 ( 13 ). Given an initial Na + concentration of 60 mM and a flow rate of 15 nl/min, the calculated Na + at the end of the tubule would be 47-53 mM, well above the affinities of the Na-K-2Cl cotransporter and NHE. Therefore, increasing luminal Na + from 60 to 149 mM should not significantly alter ion transporter activity. The possible explanation of Abe?s data is unclear but may involve pressure transients during switching of the solutions.
The effect of flow and its associated mechanical stress on O 2 - has been widely studied in endothelial cells ( 5, 27, 39 ). O 2 - generation in response to mechanical stress has also been described in vascular smooth muscle cells ( 12 ) and pulmonary epithelial cells ( 4 ). Because the TAL is one of the most compliant nephron segments ( 37 ), the pressure that accompanies an increase in luminal flow can stretch TAL cells and thereby trigger O 2 - production. Flow can also exert changes that trigger a number of signaling pathways, such as PKC ( 15, 44 ), Ca 2+ ( 25 ), and Akt ( 14, 32 ), which in turn can stimulate O 2 - production. Abe et al. ( 1 ) saw enhanced O 2 - production at 149 mM Na + in the presence of ouabain in response to increased flow, indicating that Na + transport alone cannot account for a flow-induced increase in O 2 -. This confirms our finding that some mechanical component in addition to Na + delivery and transport plays a role in flow-induced O 2 - stimulation in the TAL. To our knowledge, the present study is the first to address the effect of the mechanical component of flow on O 2 - production in the TAL. Although several physical factors can be affected by changes in flow, including shear stress, stretch, and intramural pressure, determination of which factors are involved in flow-stimulated O 2 - generation is beyond the scope of this study.
Regulation of O 2 - production in the TAL may play a key role in maintaining NaCl homeostasis. Previously, we showed that O 2 - stimulates Na + absorption in the TAL by enhancing the activity of the luminal Na-K-2Cl cotransporter ( 17 ) and Na/H exchanger ( 18 ) and that this effect is PKC dependent ( 40 ). Therefore, flow-induced changes in O 2 - production can lead to inappropriate NaCl retention. Increased flow in the TAL may occur chronically due to high salt intake ( 8 ), extensive renal ablation ( 2 ), hypertension ( 6 ), or diabetes ( 35 ). Although other factors that increase O 2 - may be involved, increased flow in the TAL may play a role in the O 2 - -associated nephropathy observed in diseases such as hypertension and diabetes.
We previously showed that flow induces endothelial NOS activation in the TAL ( 32 ). On the surface it would appear that flow-induced O 2 - would simply scavenge flow-induced NO and the net result would be no effect on transport. However, with normal variations in flow that occur in the TAL, the rates at which O 2 - and NO production are stimulated and the amounts produced may differ. Without precisely knowing all parameters it is difficult, if not impossible, to state the final effect of a given increase in luminal flow vis-à-vis NO and O 2 -. However, when the balance is upset, Na retention and hypertension may result as when animals are infused with subpressor doses of ANG II. ANG II stimulates NADPH oxidase expression ( 22 ). While animals are on low salt, flow through the TAL would be expected to be relatively low. Thus O 2 - production would also be expected to be low. In contrast, if animals were placed on high salt, flow would increase. Now instead of NO and O 2 - balancing each other, the ANG II infusion has caused an increase in NADPH oxidase expression. Thus the increase in flow causes a greater stimulation of O 2 - production. This results in less NO and more O 2 - compared with the low-salt state. Both of these effects contribute to increased NaCl absorption and lead to salt retention and hypertension.
In summary, we found that luminal flow stimulates NADPH oxidase-dependent O 2 - production by the TAL. The increase in Na + retention caused by enhanced O 2 - in the TAL may be important in the pathogenesis of hypertension and other diseases associated with Na + retention.
GRANTS
This work was supported by National Institutes of Health Grants HL-28982 and HL-70985 to J. L. Garvin.
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作者单位：1 Division of Hypertension and Vascular Research, Henry Ford Hospital, Detroit; and 2 Department of Physiology, Wayne State University, Detroit, Michigan