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【摘要】 Superoxide (O 2 - ) enhances tubuloglomerular feedback by scavenging nitric oxide at the macula densa. However, the singling pathway of O 2 - production in the macula densa is not known. We hypothesized that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces O 2 - production by the macula densa via NAD(P)H oxidase, which is activated by macula densa depolarization. We isolated and microperfused the thick ascending limb of the loop of Henle and attached macula densa in rabbits. A fluorescent dye, dihydroethidium, was used to detect O 2 - production at the macula densa. When luminal NaCl was switched from 10 to 80 mM, a situation of initiating maximum tubuloglomerular feedback response, O 2 - production significantly increased. To make sure that the shifts in the oxyethidium/dihydroethidium ratio were due to changes in O 2 -, we used tempol (10 -4 M), a stable membrane-permeant superoxide dismutase mimetic. With tempol present, when we switched from 10 to 80 mM NaCl, the increase in oxyethidium/dihydroethidium ratio was blocked. To determine the source of O 2 -, we used the NAD(P)H oxidase inhibitor apocynin. When luminal NaCl was switched from 10 to 80 mM in the presence of apocynin, O 2 - production was inhibited by 80%. To see whether the effect of increasing luminal NaCl involves Na-K-2Cl cotransporters, we inhibited them with furosemide. When luminal NaCl was switched from 10 to 80 mM in the presence of furosemide, O 2 - production was blocked. To test whether depolarization of the macula densa induces O 2 - production, we artificially induced depolarization by adding valinomycin (10 -6 M) and 25 mM KCl to the luminal perfusate. Depolarization alone significantly increases O 2 - production. We conclude that increasing luminal NaCl induces O 2 - production during tubuloglomerular feedback. O 2 - generated by the macula densa is primarily derived from NAD(P)H oxidase and is induced by depolarization.
【关键词】 Depolarization superoxide production
tubuloglomerular feedback
TUBULOGLOMERULAR FEEDBACK refers to a negative feedback loop between the epithelial cells of the macula densa and the vascular smooth muscle cells of the afferent arteriole. Tubuloglomerular feedback regulates distal tubular sodium load by adjusting glomerular filtration rate in response to signals received from the macula densa. When the macula densa senses a decline in the delivery of NaCl to the distal tubule, it signals the afferent arteriole to dilate, which raises glomerular capillary hydraulic pressure and glomerular filtration rate and thus increases renal tubular flow and sodium delivery to the distal tubule. Increased distal NaCl delivery constricts the afferent arteriole, lowers capillary hydraulic pressure, slows glomerular filtration rate, and diminishes tubular flow ( 35, 56 ). Tubuloglomerular feedback can be regulated by a number of factors, including ANG II ( 40 ), adenosine ( 47 ), arachidonic acid metabolites ( 24 ), ATP ( 20 ), atrial natriuretic factor ( 19 ), nitric oxide (NO) ( 28, 30 ), and superoxide (O 2 - ) ( 31 ). Enhanced tubuloglomerular feedback may contribute to high blood pressure. In animal models of genetic hypertension, tubuloglomerular feedback is enhanced ( 53, 54 ), and resetting of tubuloglomerular feedback may also contribute to salt-sensitive hypertension in humans ( 1, 23, 38 ).
O 2 - plays an important pathophysiological role in the development of hypertension. Oxidative stress has been demonstrated in patients with essential hypertension, renovascular hypertension, malignant hypertension, and preeclampsia ( 17, 26, 27, 52 ), as well as in experimental models such as ANG II-mediated, Dahl salt-sensitive, lead-induced, obesity-associated, mineralocorticoid- and aldosterone-induced hypertension ( 8, 13, 22, 25, 36, 50, 51 ). Oxidative stress per se can lead to arterial hypertension in otherwise intact genetically normotensive animals ( 49 ). The macula densa expresses genes for the main components of NAD(P)H oxidase, and subunits of the NAD(P)H oxidase complex are overexpressed in the macula densa in spontaneously hypertensive rats (SHR) ( 6 ), indicating that they play an important role in oxidative stress.
Tubuloglomerular feedback is initiated by increasing luminal NaCl and activating the luminal Na-K-2Cl cotransporter (NKCC2), which increases intracellular NaCl in the macula densa. The increased intracellular Cl - stimulates basolateral Cl - efflux and results in cell depolarization ( 43 ). We found that depolarization of the macula densa is essential to initiate tubuloglomerular feedback ( 42 ). We previously reported that O 2 - enhances tubuloglomerular feedback both directly and by scavenging NO at the macula densa ( 39 ). Thus both the interaction and the resulting balance between O 2 - and NO in the macula densa will modify tubuloglomerular feedback in physiological and pathophysiological situations. However, the singling pathway of O 2 - production in the macula densa is not known. We hypothesize that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces O 2 - production by the macula densa via NAD(P)H oxidase. We also postulate that this increase in O 2 - is activated by macula densa depolarization.
MATERIALS AND METHODS
Isolation and microperfusion of the rabbit afferent arteriole and attached macula densa. We used methods similar to those described previously to isolate and microperfuse the thick ascending limb (TAL) of the loop of Henle and attached macula densa ( 28 ). Briefly, young male New Zealand White rabbits (1.5 to 2.0 kg) were anesthetized with ketamine (50 mg/kg im) and given an injection of heparin (500 U iv). The kidneys were sliced along the corticomedullary axis, and the slices were placed in ice-cold minimum essential medium (MEM; GIBCO, Grand Island, NY) containing 5% BSA (Sigma, St. Louis, MO). With the use of a stereomicroscope (SZH; Olympus, Tokyo, Japan), a single superficial intact glomerulus was microdissected together with adherent tubular segments consisting of portions of the TAL, macula densa, and early distal tubule. With the use of a micropipette, the sample was transferred to a temperature-regulated chamber mounted on an inverted microscope (TE2000-S, Nikon). The TAL was cannulated with an array of glass pipettes ( 30 ) while using another pipette to hold and stabilize the glomerulus. The sample was arranged so that each macula densa cell could be clearly visualized on the edge of the glomerulus. The macula densa was perfused with physiological saline consisting of (in mM) 10 HEPES, 1.0 CaCO 3, 0.5 K 2 HPO 4, 4.0 KHCO 3, 1.2 MgSO 4, 5.5 glucose, 0.5 Na acetate, 0.5 Na lactate, and either 80 or 10 mM NaCl, pH 7.4. We used mannitol to adjust the 10 mM NaCl solution to the same osmolality as 80 mM NaCl (180 mosmol/kgH 2 O) to avoid changes in cell volume when switching luminal NaCl ( 28 ). The bath consisted of MEM containing 0.15% BSA and was exchanged continuously at a rate of 1 ml/min. Microdissection and cannulation were completed within 60 min at 8°C, after which the bath was gradually warmed to 37°C for the rest of the experiment. Once the temperature was stable, a 30-min equilibration period was allowed before taking any measurements. The imaging system consisted of a microscope (TE2000-S, Nikon, Yuko, Japan), digital CCD camera (IEEE 1394, Hamatsu Photonics K.K., Hamatsu, Japan), and optical filter changer (Lambda 10-2, Sutter Instruments, Novato, CA). Images were analyzed with SimplePCI imaging software (Compix, Tualatin, OR).
Measurement of superoxide. A O 2 - -sensitive fluorescent dye, dihydroethidium, was used to detect O 2 - production as we described recently ( 18 ). Once the TAL was perfused as described above, macula densa cells were loaded with 10 µM dihydroethidium in 0.1% DMSO plus 0.1% pluronic acid from the lumen for 30 min and then washed for 20 min. Dihydroethidium is irreversibly converted to oxyethidium in the presence of O 2 - ( 12, 58 ). Therefore, the rate of oxyethidium accumulation reflects O 2 - production, and an increase or decrease in this rate indicates enhanced or blunted O 2 - production. Dihydroethidium and oxyethidium both fluoresce but at different wavelengths. The excitation and emission wavelength (Ex/Em) for dihydroethidium is Ex:380/Em:440 nm, whereas for oxyethidium it is Ex:490/Em:620 nm. O 2 - production is expressed as the rate of increase in the ratio of oxyethidium to dihydroethidium fluorescence intensity (U/min). The loaded macula densa cells were exposed to 380- and 490-nm light to excite dihydroethidium and oxyethidium, respectively. Emitted fluorescence from dihydroethidium was recorded using a 420-nm dichroic mirror with a 460/50-nm band-pass filter; for oxyethidium, we used a 565-nm dichroic mirror with a 605/55-nm band-pass filter (Chroma). Square-shaped regions of interest were set inside the cytoplasm of macula densa cells and mean intensity within them was recorded every 5 s for 5 min. We recorded oxyethidium and dihydroxyethidium emission signals subsequently and calculated the rate of the changes for both wavelengths. Since we found that the rate of changes for both oxyethidium and dihydroxyethidium was constant after equilibrium period, the intensities of oxyethidium and dihydroxyethidium as well as the ratio of oxyethidium to dihydroxyethidium at different points of time can be calculated by the rate. Changes in O 2 - concentration were expressed as the increase in oxyethidium/dihydroethidium ratio (U/s). Since we previously found that increased luminal NaCl activates neuronal NO synthase and induces NO release by the macula densa ( 28, 30 ), we added the NOS inhibitor inhibitor N -nitro- L -arginine methyl ester (10 -4 M) to the bath and lumen while measuring O 2 - to eliminate its reaction with NO.
Chemicals. Dihydroethidium, BCECF-AM, DMSO, and pluronic acid were obtained from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma.
Statistics. Data were collected as repeated measures over time under different conditions. We tested only the effects of interest, using a paired t -test or ANOVA for repeated measures. Significance was judged as P < 0.05 or an adjusted value using Hochberg's method for multiple testing.
RESULTS
First, we tested whether increasing luminal NaCl induces O 2 - production. Macula densa cells were initially perfused with 10 mM NaCl. The increase in the oxyethidium/dihydroethidium ratio reached a constant value in 30 to 40 min. Figure 1 shows a representative O 2 - measurement in macula densa cells loaded with dihydroethidium. When luminal NaCl was switched from 10 to 80 mM to mimic initiation of tubuloglomerular feedback, the intensity of dihydroethdium decreases, the intensity of oxyethidium increases, and the ratio increases. Figure 2 is the average data. In the presence of 10 mM NaCl, the rate of increase was 0.35 ± 0.07 U/min. After the luminal NaCl was switched to 80 mM, a situation to initiate maximum tubuloglomerular feedback response ( 31 ), it increased to 2.78 ± 0.05 U/min ( P < 0.01). To show the increase was repeatable, we performed the switch again and the rate of increase went from 0.31 ± 0.03 to 2.58 ± 0.04 U/min ( P < 0.01; n = 6; Fig. 2 ). Thus the increase in the oxyethidium/dihydroethidium ratio caused by increasing NaCl is stable over time.
Fig. 1. A : emission signals from dihydroethdium. The intensity of dihydroethdium decreases when luminal NaCl increases from 10 mM ( A1 ) to 80 mM ( A2 ). B : emission signals from oxyethidium. The intensity of oxyethidium increases when luminal NaCl increases from 10 mM ( B1 ) to 80 mM ( B2 ). C : ratio images of oxyethidium/dihydroethidium. The ratio increases when luminal NaCl increases from 10 mM ( C1 ) to 80 mM ( C2 ). D : light microscopy image.
Fig. 2. O 2 - production measured by oxyethidium/dihydroethidium ratio. When luminal NaCl was switched from 10 to 80 mM, the rate of increase in the oxyethidium/dihydroethidium ratio increased from 0.35 ± 0.07 to 2.78 ± 0.05 U/min. When the luminal perfusate was switched back to 10 mM NaCl, then 20 min later switched to 80 mM NaCl, the oxyethidium/dihydroethidium ratio increased from 0.31 ± 0.03 to 2.58 ± 0.04 U/min ( n = 6). There was no significant difference in oxyethidium/dihydroethidium ratio between the first and second switch of NaCl.
To determine whether the changes in oxyethidium/dihydroethidium ratio were due to changes in O 2 -, we examined changes in the presence of tempol, a stable membrane-permeant superoxide dismutase (SOD) mimetic, and measured the oxyethidium/dihydroethidium ratio while switching luminal NaCl. After the perfused macula densa was loaded with dihydroethidium, tempol (10 -4 M) was added to the lumen and bath for 30 min. When the lumen was perfused with 10 mM NaCl, the increase in oxyethidium/dihydroethidium ratio was 0.48 ± 0.37 U/min. When NaCl was switched to 80 mM, it was 0.66 ± 0.33 U/min ( n = 6; Fig. 3 ). These data indicate that changes in the ratio of oxyethidium/dihydroethidium ratio are due to changes in O 2 - production and that increasing luminal NaCl induces O 2 - production by the macula densa.
Fig. 3. Effect of tempol on superoxide production by the macula densa. When luminal NaCl was switched from 10 to 80 mM in the presence of the SOD mimetic tempol (10 -4 M), superoxide generation expressed by oxyethidium/dihydroethidium ratio showed no significant change (from 0.48 ± 0.37 to 0.66 ± 0.33 U/min; n = 6).
To study the source of O 2 -, we used the NAD(P)H oxidase inhibitor apocynin and measured O 2 - production. During the control period in the absence of apocynin, when luminal NaCl was switched from 10 to 80 mM, intracellular O 2 - production increased from 0.47 ± 0.21 to 2.72 ± 0.21 U/min. Then, we switched luminal NaCl back to 10 mM and added apocynin (10 -5 M) to the lumen and bath for 60 min. When NaCl was again switched from 10 to 80 mM in the presence of apocynin, O 2 - production increased from 0.53 ± 0.25 to 0.92 ± 0.40 U/min ( P < 0.01, without vs. with apocynin; n = 5; Fig. 4 ). These data indicate that NAD(P)H oxidase is the primary source of O 2 - produced by the macula densa during an increase in luminal NaCl.
Fig. 4. Effect of apocynin on superoxide production by the macula densa. When luminal NaCl was switched from 10 to 80 mM, O 2 - increased from 0.47 ± 0.21 to 2.72 ± 0.21 U/min. When NaCl was again switched from 10 to 80 mM in the presence of apocynin, O 2 - increased from 0.53 ± 0.25 to 0.92 ± 0.40 U/min ( P < 0.01, without vs. with apocynin; n = 5).
To study whether the effect of increasing luminal NaCl involves the NKCC2s, we used furosemide to inhibit the cotransporters and measured O 2 - production. During the control period, when luminal NaCl was switched from 10 to 80 mM, O 2 - production increased from 0.35 ± 0.09 to 2.68 ± 0.43 U/min ( P < 0.01; n = 4). Then, we switched NaCl back to 10 mM and added furosemide (10 -4 M) to the lumen for 10 min. When NaCl was switched from 10 to 80 mM in the presence of furosemide, O 2 - did not increase significantly (from 0.34 ± 0.09 to 0.39 ± 0.17 U/min; P < 0.01, with vs. without furosemide; n = 5; Fig. 5 ). These data indicate that cotransporter activity plays a crucial role in the macula densa O 2 - production induced by increasing luminal NaCl.
Fig. 5. Effect of furosemide on superoxide production by the macula densa. When luminal NaCl was switched from 10 to 80 mM, O 2 - production increased from 0.35 ± 0.09 to 2.68 ± 0.43 U/min ( P < 0.01; n = 4). When NaCl was switched from 10 to 80 mM in the presence of furosemide, O 2 - increased from 0.34 ± 0.09 to 0.39 ± 0.17 U/min ( P < 0.01, with vs. without furosemide; n = 5).
The macula densa is depolarized upon increasing luminal NaCl ( 43 ). To test whether depolarization induces O 2 - production, we artificially depolarized the macula densa and measured O 2 -. The macula densa was perfused with 10 mM NaCl, and fluorescent signals were recorded for 5 min. Then, valinomycin (10 -6 M) and 25 mM KCl were added to the luminal perfusate to depolarize the macula densa ( 42 ). When the macula densa was perfused with 10 mM NaCl, O 2 - production was 0.37 ± 0.08 U/min. When the macula densa was depolarized, O 2 - production increased to 2.17 ± 0.49 U/min ( P < 0.01; n = 6; Fig. 6 ). These data indicate that depolarization triggers O 2 - production by the macula densa.
Fig. 6. Effect of depolarization on superoxide production by the macula densa. When the macula densa was perfused with 10 mM NaCl, O 2 - production was 0.37 ± 0.08 U/min. When the macula densa was depolarized by valinomycin (10 -6 M) and 25 mM KCl, O 2 - increased to 2.17 ± 0.49 U/min ( P < 0.01; n = 6).
DISCUSSION
The new findings of present study: 1 ) increasing luminal NaCl enhances O 2 - production by the macula densa, 2 ) NAD(P)H oxidase is the primary source of O 2 - production, and 3 ) depolarization of the macula densa induces O 2 - production.
We found that when we switched luminal NaCl from 10 to 80 mM, a situation mimicing to initiate tubuloglomerular feedback, there was a significant increase in oxyethidium/dihydroethidium ratio ( Figs. 1 and 2 ). To see whether this is due to changes in O 2 - generation, we used a stable membrane-permeant SOD mimetic, tempol. We found that tempol significantly blocked the increase in oxyethidium/dihydroethidium ratio induced by increasing luminal NaCl ( Fig. 3 ). These data indicate that O 2 - generation by the macula densa increases during tubuloglomerular feedback. High salt has been reported to enhance O 2 - generation in the cultured renal medullary cells ( 57 ) and human embryonic kidney cells ( 59 ), which are in agreement with our findings. We found recently that NO production enhances during tubuloglomerular feedback ( 28, 30 ). In addition, O 2 - generated by the macula densa enhances tubuloglomerular feedback by scavenging NO ( 31 ). Both NO and O 2 - generated by the macula densa enhance during tubuloglomerular feedback. Their interaction and resulting balance may play an important role in tubuloglomerular feedback regulation both in physiological and pathophysiological situations. We recognize that generation of O 2 - induced by increasing NaCl is not unique to the macula densa. Several types of cells, including cells from cultured and perfused TALs, show enhanced O 2 - production with increasing NaCl ( 16, 34 ). However, the effect of O 2 - produced in the macula densa is unique. O 2 - is not cell membrane permeable ( 48 ); hence, O 2 - produced in the macula densa acts within the macula densa and O 2 - from other cells do not. We found that O 2 - generated only by the macula densa uniquely enhances tubuloglomerular feedback via actions in the macula densa ( 31 ). Therefore, O 2 - produced by other surrounding cells is not likely to affect tubuloglomerular feedback via macula densa.
The macula densa expresses genes for the main components of NAD(P)H oxidase ( 6 ), and subunits of the NAD(P)H oxidase complex are overexpressed in the macula densa in SHR ( 6 ), indicating that they play an important role in oxidative stress. To study the source of O 2 - production during tubuloglomerular feedback, we used the NAD(P)H oxidase inhibitor apocynin. We found that apocynin significantly inhibited O 2 - production, indicating that NAD(P)H oxidase is the primary source of O 2 - produced by the macula densa during tubuloglomerular feedback. Apocynin, a methoxy-substituted catechol, is a well-characterized inhibitor of NADPH oxidase. It acts by preventing serine phosphorylation of the cytosolic p47 phox subunit and blocking its assembly with gp91 phox in the cell membrane ( 11, 14, 33 ). At the present time, the exact isoform(s) of NAD(P)H oxidase (NOX) at the macula densa is unknown. Five isoforms of NOX proteins with distinct tissue distributions have been found: NOX1 is expressed mainly in the colonic epithelium ( 3, 46 ), NOX2 (gp91 phox ) in phagocytes ( 2 ), NOX3 in the embryonic kidney ( 21 ), NOX4 in the renal cortex ( 15, 44 ), and NOX5 in T- and B-lymphocytes of the spleen and lymph nodes as well as sperm precursors in the testis ( 4 ). Recently, two new members of the gp91 phox -homolog family have been reported ( 7 ): dual oxidase (DUOX) 1/thyroid oxidase 1, found primarily in the thyroid and lung; and DUOX2/thyroid oxidase 2, primarily located in the thyroid and colon ( 9, 10 ). The potential NAD(P)H oxidase isoforms expressed in the adult kidney are NOX1, NOX2, and NOX4, since NOX3, NOX5, and DUOX are not likely to be present ( 4, 7, 9, 21 ). Of these three main NOX isoforms, NOX1 and NOX2 require p47phox for optimal activation, whereas NOX4 activity is believed to be independent of p47phox ( 32 ). Therefore, NOX2 and NOX1 would most likely be affected by apocynin and these data suggest that either a NOX2- or NOX1-based oxidase is involved. This is currently the focus of intense study in our laboratory.
Next, we studied the mechanism of O 2 - generation. During tubuloglomerular feedback, increasing luminal NaCl activates the luminal NKCC2, which increases intracellular NaCl in the macula densa. The increased intracellular Cl - stimulates basolateral Cl - efflux and results in cell depolarization ( 43 ). Increasing luminal NaCl activates the luminal Na/H exchanger and alkalinizes the macula densa ( 28 ). Increasing luminal NaCl also alters intracellular calcium ( 29, 37 ). Depolarization, elevated intracellular pH or calcium induces release of ATP from the basolateral membrane of the macula densa ( 5 ). ATP released from the macula densa is broken down to form adenosine, which constricts the afferent arteriole ( 41 ). To find out whether NKCC2 is involved in O 2 - generation, we used furosemide to inhibit NKCC2 and measured the O 2 - generation induced by increasing luminal NaCl. We found that O 2 - generation was blocked by furosemide ( Fig. 4 ), indicating that O 2 - production during tubuloglomerular feedback is NKCC2 dependent.
The results of the present study demonstrate that depolarization of the macula densa induces O 2 - production during tubuloglomerular feedback. The macula densa is reportedly depolarized by up to 31 mV upon increasing luminal NaCl, measured with a microelectrode ( 43 ). Inhibiting NKCC2 with furosemide induces hyperpolarization and blocks depolarization in response to increased luminal NaCl ( 43 ). Whitin et al. ( 55 ) first described the correlation between changes in membrane potential and O 2 - production in human granulocytes. Sohn et al. ( 45 ) directly demonstrated that depolarization induced O 2 - production in human endothelial cells. The cells were depolarized by 90 mM potassium buffer, the nonselective potassium channel blocker tetrabutylammonium chloride, or the nonselective cation ionophore gramicidin. O 2 - formation was significantly elevated to a similar degree (60%) by all three treatments. These reports are in agreement with our findings. The mechanism of how depolarization activates NAD(P)H oxidase is not clear and needs further investigation.
In conclusion, we found that increasing luminal NaCl induces O 2 - production during tubuloglomerular feedback. O 2 - generated by the macula densa is derived primarily from NAD(P)H oxidase and is induced by depolarization. Thus the interaction and resulting balance between O 2 - and NO in the macula densa will modify the tone of tubuloglomerular feedback. Inappropriately enhanced O 2 - generation and/or impaired NO production by the macula densa might contribute to pathophysiological changes in hypertension. We will further study the source of O 2 - and downstream signaling of O 2 - production after depolarization.
GRANTS
This work was supported by American Heart Association Grant SDG-0630288N (to R. Liu) and National Institutes of Health Grant HL-28982 (to O. A. Carretero).
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作者单位：Hypertension and Vascular Research Division, Henry Ford Hospital, Detroit, Michigan