Response of descending vasa recta to luminal pressure

【摘要】  We tested whether luminal perfusion and pressurization induce an endothelial cytoplasmic Ca 2+ ([Ca 2+ ] CYT ) response in descending vasa recta (DVR). DVR isolated from the rat outer medulla were cannulated and subjected to free-flow microperfusion (5 nl/min); the onset of which increased [Ca 2+ ] CYT from a baseline of 76 ± 13 to 221 ± 65 nM. A graded increase in luminal pressure from 0 to 45 mmHg in stopped-flow experiments induced a parallel increase in [Ca 2+ ] CYT from a baseline of 74 ± 24 to 194 ± 33 nM at 45 mmHg, with a tendency for [Ca 2+ ] CYT 25 mmHg. The removal of extracellular Ca 2+ and blockade by either La 3+ (10 µM) or SKF-96365 (100 µM) eliminated the response. Luminal pressurization to 25 mmHg increased nitric oxide (NO) generation, a response blocked by NO synthase inhibition or removal of extracellular Ca 2+. The NO generation was not affected by the superoxide dismutase mimetic tempol. We conclude that DVR endothelia are mechanosensitive and respond to luminal pressure by elevating [Ca 2+ ] CYT and generating NO. That response might augment medullary perfusion and saliuresis.

medulla; kidney; microcirculation; shear; fura 2; 4,5-diaminofluoroscein

【关键词】  Response descending pressure

DESCENDING VASA RECTA (DVR) are small generation resistance vessels that carry blood flow from the cortex to the medulla of the kidney. They originate as branches of the juxtamedullary efferent arteriole and traverse the outer medulla in vascular bundles to supply the inner and outer medulla. Their radial distribution within vascular bundles implies that they distribute and regulate regional blood flow within the medulla ( 34 ). DVR are lined with a continuous endothelium and enveloped by smooth muscle/pericytes that impart contractility ( 30, 33 ). The importance of endothelial secretion of paracrine mediators to modulate adjacent smooth muscle is well established. Motivated by the importance of nitric oxide (NO) secretion in the maintenance of renal medullary perfusion and blood pressure ( 7, 22, 23, 28 ) we employed microperfusion and fluorescence microscopy to test whether DVR endothelial cytoplasmic Ca 2+ ([Ca 2+ ] CYT ) is affected by the transmural pressure gradient imposed across the DVR wall. We exploited the property of the Ca 2+ -sensitive probe, fura 2, to preferentially load into DVR endothelia (while sparing pericytes) to quantify the effect of raising intraluminal pressure on [Ca 2+ ] CYT. In addition, the probe 4,5-diaminofluorescein (DAF-2), which increases fluorescence when covalently modified by NO, was used to monitor NO generation. The results verify that an increase in luminal pressure is accompanied by a parallel rise in DVR endothelial [Ca 2+ ] CYT and a robust increase in NO synthesis. NO functions as a vasodilator and "endogenous diuretic" ( 23, 28 ). We hypothesize that the vasa recta endothelium might serve as a transducer that senses microvascular pressure to secondarily regulate medullary perfusion and salt and water handling by adjacent nephrons.

METHODS

Isolation and microperfusion of DVR. Kidneys were harvested from Sprague-Dawley rats (70-150 g; Harlan), sliced, placed in buffer, and maintained at 0 to 4°C on ice. DVR were dissected from outer medullary vascular bundles and transferred to the stage of an inverted microscope as previously described ( 30, 33 ). Vessels were mounted on concentric pipettes, cannulated at one end, and held at the other end in a manner that either permitted free flow into the collection pipette or crimped the end to enable pressurization of the lumen without flow ( Fig. 1 ). The pipettes and vessel were positioned with micromanipulators (Instruments Technology and Machinery, San Antonio, TX) near a thermocouple in the narrow entrance region of a custombuilt chamber. Temperature was maintained with a feedback system (CN9000A, Omega Engineering, Bridgeport, NJ). The buffer used for dissection, perfusion, and the bath was (in mM) 140 NaCl, 10 Na acetate, 5 KCl, 1.2 MgCl 2, 2 NaHPO 4 /NaH 2 PO 4, 1 CaCl 2, 5 alanine, 0.1 L -arginine, 5 glucose, 5 HEPES, and 0.5 g/dl albumin, pH 7.4. Zero Ca 2+ bath was obtained by omitting CaCl 2 and including 100 µM EGTA.

Fig. 1. Schematic diagram showing free-flow ( A ) and stopped-flow ( B ) microperfusion methods used to modulate flow and intraluminal pressure in these experiments, respectively.

All investigations involving animal use were performed according to protocols approved by the Institutional Animal Care and Use Committee of the University of Maryland.

Videomicroscopy and measurement of vessel diameters. To quantify changes in vessel diameter, microperfusion experiments were monitored with a video camera (Dage, MTI) and recorded on tape (Panasonic AG 1960 VCR). As previously described ( 30, 31 ), tapes were played back to measure DVR outer diameter with calipers.

Measurement of endothelial intracellular calcium. DVR were loaded with the Ca 2+ -sensitive fluorescent indicator fura 2 by exposure to bath containing 2 µM fura 2 AM ester (Molecular Probes, Eugene, OR) for 20 min ( 25, 27 ). We previously showed that fura 2 preferentially loads into endothelial cells rather than pericytes ( 34 ). For measurement of [Ca 2+ ] CYT, fura 2-loaded DVR were excited using a 350/380-nm dual-wavelength combination. The background-subtracted ratio of fluorescent emission (R 350/380 ) was calculated for conversion to the equivalent intracellular calcium concentration assuming a dissociation constant for fura 2 at 37°C of 224 nM. R max and R min were measured as previously described by exposing vessels to buffer containing 5 mM CaCl 2 or 0 mM CaCl 2, 0.5 mM EGTA, respectively, along with 10 µM calcium ionophore ( 31 ).

Fluorescent detection of NO with DAF-2 probe. 4,5-Diaminofluorescein diacetate (DAF-2DA) is a molecule that is loaded into cells by deesterification to form DAF-2. When covalently modified by NO, DAF-2 forms a fluorescent triazofluorescein. Thus DAF-2 produces an integrated measure of local NO concentration within the loaded cells. DAF-2 was excited at 485 nm from a xenon arc lamp (Photon Technology International, Lawrenceville, NJ), and emission was isolated with a band-pass filter at 530 nm (Omega Optical, Brattleboro, VT) and measured with a photon-counting detection assembly (D104B, PTI). Each DVR was transferred to the chamber and cannulated for microperfusion. The vessel was warmed to 37°C and loaded for 20 min with 10 µM DAF-2DA ester. We previously showed that DAF-2 loads into both endothelia and pericytes and increases its emission at 530 nm during excitation between 450 and 500 nm without a spectral shift ( 34, 36 ).

Reagents. DAF-2DA was purchased from Calbiochem (La Jolla, CA) and stored at -20°C (5 mM in DMSO). DAF-2DA was diluted to 10 µM to load DVR. N -nitro- L -arginine methyl ester ( L -NAME), L -arginine, 1-{ -[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride (SKF-96365), and 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (tempol) were purchased from Sigma. SKF-96365, tempol, L -NAME, and L -arginine were dissolved in water and stored at -20°C in aliquots that were thawed on the day of the experiment. The excess was discarded daily.

Statistics. Data are shown as means ± SE. Where data collection rate was high, the majority of error bars have been suppressed for clarity of presentation in the figures. Statistical testing employed Student's t -test (paired or unpaired, as appropriate) and ANOVA. For ANOVA, the Student-Newman-Keuls test was used to test significance.

RESULTS

DVR endothelial Ca 2+ responses to free-flow microperfusion. To test whether perfusion of DVR is accompanied by an elevation of [Ca 2+ ] CYT, fura 2 responses were measured before, during, and after pressurization of the perfusion pipette to induce nominal collection rates of 2.5 and 5 nl/min. Baseline was recorded for 2 min in the absence of flow following which the perfusion was abruptly initiated (10 min) and stopped (5 min) by returning the perfusion pipette pressure to zero. In control vessels, perfusion pressure was held at 0 mmHg throughout. As shown in Fig. 2, a rapid and reversible rise in endothelial [Ca 2+ ] CYT was observed.

Fig. 2. Descending vasa recta (DVR) endothelial cytoplasmic Ca 2+ ([Ca 2+ ] CYT ) during free-flow microperfusion. Endothelial [Ca 2+ ] CYT measured in perfused ( n = 5) and nonperfused (control, n = 5) DVR. After obtaining a 2-min baseline, pressure was abruptly increased to levels that yielded a 2.5-nl/min perfusion rate. After briefly returning pressure to zero, it was again increased to achieve 5 nl/min flow. Perfused vessels showed a brisk and reversible [Ca 2+ ] CYT response. Values are means ± SE; most error bars have been suppressed for clarity.

DVR endothelial Ca 2+ responses to luminal pressurization. In this preparation, it is technically impossible to vary luminal flow rate and shear stress on the endothelium without also altering transmural pressure. Accepting this limitation, we chose instead to isolate the effects of transmural pressure using "stop flow" microperfusion. This was accomplished by crimping the end of the vessel in the collection pipette ( Fig. 1 B ) and then incrementally increasing the perfusion pressure to the desired level. Where necessary, due to the need to turn on room lights and see the pressure manometer, the monitoring of fura 2 fluorescence was interrupted during perfusion pressure adjustments. Endothelial [Ca 2+ ] CYT progressively increased from a baseline of 74 ± 24 to 194 ± 33 nM, as pressure was incrementally raised from 0 to 45 mmHg. There was a tendency for [Ca 2+ ] CYT to plateau at pressures greater than 30 mmHg ( Fig. 3 ).

Fig. 3. Graded [Ca 2+ ] CYT response during stopped-flow microperfusion. Endothelial [Ca 2+ ] CYT was measured in DVR in which the inlet was cannulated to enable pressurization of the lumen and the outlet was occluded to prevent flow (see Fig. 1 B ). A : example of an experiment in which fura 2 emission was monitored by fluorescent microscopy. Measurements were taken for 2-min periods, between which the room lights were turned on (arrows) to adjust the pressure in the perfusion pipette. [Ca 2+ ] CYT was measured at pressures of 0, 10, 15, 20, 25, 35, and 45 mmHg. B : means ± SE ( n = 8) of [Ca 2+ ] CYT averaged for 10 s at the end of each period from experiments similar to A (* P < 0.01 vs. 0 mmHg).

Dependence of [Ca 2+ ] CYT response on extracellular Ca 2+. To verify specificity of fura 2 to detect [Ca 2+ ] CYT during these maneuvers, we tested whether removal of extracellular Ca 2+ (0 CaCl 2, 100 µM EGTA) could block the pressure-induced response. Two protocols were executed ( Fig. 4 ). In the first, endothelial Ca 2+ was recorded for 2 min after which the cells were exposed to 0 Ca 2+ bath for 10 min. Subsequently, luminal pressure was readjusted to 25 mmHg and [Ca 2+ ] CYT was measured before and after readdition of Ca 2+ to the bath (1 mM CaCl 2 ). Two features are apparent. Exposure of the endothelia to 0 Ca 2+ bath tended to reduce basal [Ca 2+ ] CYT (52 ± 13 nM, time 0 ) to low levels (30 ± 4 nM, time = 10 min, P < 0.05) and, removal of external Ca 2+ completely eliminated the pressure-induced response ( Fig. 4 A ). In the second protocol, cells were exposed to 0 Ca 2+ bath after pressurization/[Ca 2+ ] CYT elevation ( Fig. 4 B ). This reversed [Ca 2+ ] CYT from an elevated level (146 ± 25 nM, time = 8 min) to (39 ± 12 nM, time = 15 min, P < 0.01) a value that is lower than baseline (66 ± 16 nM, P < 0.05, time 0 vs. 15 min). In both protocols, refilling of the cytoplasm on readdition of Ca 2+ to the bath led to an overshoot of [Ca 2+ ] CYT, suggesting enhancement of conductance of Ca 2+ entry pathways into the DVR endothelium. The latter may imply depletion of intracellular Ca 2+ stores during the 0 CaCl 2 /100 µM EGTA incubation. An example of the temporal variations of the individual fura 2 F 350 and F 380 signals caused by pressurization and external Ca 2+ removal is shown in Fig. 4 C.

Fig. 4. Effect of omission of extracellular Ca 2+ on the DVR [Ca 2+ ] CYT response to luminal pressure. A : [Ca 2+ ] CYT was recorded from fura 2-loaded DVR ( n = 6) for 2 min before and 10 min after removal of Ca 2+ from the bath (0 CaCl 2, 100 µM EGTA). Recording was interrupted (arrow) as room lights were turned on to readjust pressure to 25 mmHg. Recording was resumed for 10 min after which CaCl 2 (1 mM) was replaced into the extracellular buffer. Omission of extracellular Ca 2+ eliminated the pressure-induced response and reintroduction of CaCl 2 led to rapid refilling and a sustained, elevated plateau [Ca 2+ ] CYT above the original baseline. B : [Ca 2+ ] CYT was recorded ( n = 6) for 2 min after which luminal pressure was readjusted to 25 mmHg (arrow). After a subsequent 5-min recording period, the extracellular buffer was exchanged to 0 CaCl 2 /100 µM EGTA for 10 min and then exchanged back to 1 mM CaCl 2. Elimination of extracellular Ca 2+ reversed the pressure-induced [Ca 2+ ] CYT response. Values are means ± SE; most error bars have been suppressed for clarity. C : individual record of the F 350 and F 380 fluorescence changes during the protocol shown in B. Values on the ordinate have been normalized by dividing by the F 350 value at time 0.

Blockade of Ca 2+ entry pathways. It is generally recognized that Ca 2+ entry into endothelia is mediated by nonselective cation channels rather than voltage-gated channels ( 27 ). The former are likely to be transient receptor potential (TRP) channels, many isoforms of which can be expressed in any given cell ( 5, 42 ). Specific blockers for TRP channels do not exist; however, nonselective blockade can be achieved with various agents. We examined the ability of La 3+ (10 µM) and SKF-96365 (100 µM) to affect the pressure-induced increase in [Ca 2+ ] CYT. Pretreatment with La 3+ reduced DVR endothelial Ca 2+ from a resting value of 71 ± 22 nM, time 0 to 45 ± 13 nM, time = 7 min, and prevented pressurization from raising [Ca 2+ ] CYT ( Fig. 5 A ). As demonstrated in the hour-long record shown in Fig. 5 B, reversal of the effects of La 3+ after washout was very slow. The nonselective blocker SKF-96365 also prevented the [Ca 2+ ] CYT rise associated with pressurization ( Fig. 6 ). Reversal of SKF-96365 after washout was also slow. Time controls in Figs. 5 and 6 are the same; water is the vehicle for both La 3+ and SKF-96365.

Fig. 5. Blockade of the [Ca 2+ ] CYT response by La 3+. A : [Ca 2+ ] CYT was recorded from fura 2-loaded DVR ( n = 6) for 2 min before and after addition of La 3+ (10 µM, n = 7) or vehicle ( n = 8) to bath. At time = 7 min, luminal pressure was raised from 0 to 25 mmHg. La 3+ eliminated the pressure-induced [Ca 2+ ] CYT response. B : individual record of a 1-h recording during which La 3+ 17 min). La 3+ prevented the [Ca 2+ ] CYT response to pressure but its effects were very slow to reverse after washout.

Fig. 6. Blockade of the [Ca 2+ ] CYT response by SKF-96365. [Ca 2+ ] CYT was recorded from fura 2-loaded DVR ( n = 6) for 2 min before and after addition of SKF-96365 (100 µM, n = 9) or vehicle ( n = 8) to bath. At time = 5 min, luminal pressure was raised from 0 to 25 mmHg. SKF-96365 eliminated the pressure-induced [Ca 2+ ] CYT response ( P 5 min). Washout of SKF-96365 at time = 15 min led to a slow rise of [Ca 2+ ] CYT.

Effect of luminal pressure on DVR diameter. In a separate series of experiments, we documented the effect of pressurizing the DVR lumen on outer diameter ( Fig. 7 ). Raising pressure successively from 0 to 25 mmHg increased diameter steeply. No constriction occurred during progressive luminal pressure elevation. This reinforces prior observations that were obtained under free-flow conditions ( 30, 33 ).

Fig. 7. Effect of luminal pressure elevation on DVR outer diameter. A : images of cannulated DVR pressurized to 0, 5, 10, 25, and 35 mmHg. B : means ± SE ( n = 5) of outer diameters of DVR pressurized to values on the abscissa. Elevation of luminal pressure progressively dilated the vessels (* P < 0.01 vs. 0 mmHg).

NO generation by pressurized DVR. To determine whether enhanced NO generation accompanies the [Ca 2+ ] CYT response to luminal pressure, we measured cellular NO activity by monitoring DAF-2 fluorescence. Protocols were executed under free-flow ( Fig. 8 ) and stopped-flow ( Fig. 9 ) conditions. With both approaches, pressurization markedly increased the rate of NO generation as documented by the conversion of DAF-2 to its fluorescent form. The ability of nonspecific NO synthase inhibition ( L -NAME) to block the DAF-2 response was verified under stopped-flow conditions ( Fig. 9 A ). Bioavailability of NO is modulated through its reaction with superoxide to form peroxynitrite. We previously observed that bradykinin-stimulated DVR endothelial NO can be enhanced by the cell-permeant superoxide dismutase mimetic tempol ( 34, 36 ). We chose to test the effect of tempol on pressure-stimulated NO generation because reactive oxygen species have been found to play a role in mechanosensitive signaling in other endothelia ( 1 ). In these experiments, tempol had no effect to enhance the rise in DAF-2 fluorescence that accompanies luminal pressurization, implying little role for superoxide to modulate the response ( Fig. 9 A ). The removal of extracellular Ca 2+, a maneuver that prevents [Ca 2+ ] CYT elevation ( Fig. 4 ), also reduces NO generation ( Fig. 9 B ).

Fig. 8. Nitric oxide generation by microperfused DVR. 4,5-Diaminofluorescein (DAF-2) fluorescence was measured in control DVR and DVR perfused at a nominal rate of 5 nl/min in free-flow configuration ( Fig. 1 A, n = 5, each group). DAF-2 fluorescence increased more rapidly in perfused than nonperfused vessels ( P 3 min).

Fig. 9. A : DAF-2 fluorescence was measured in 3 groups of DVR pressurized to 25 mmHg in the stopped-flow configuration ( Fig. 1 B ). The effects of tempol (1 mM, n = 8) and L -NAME (100 µM, n = 7) were compared with controls ( n = 7). L -NAME markedly reduced the rate of DAF-2 conversion to its fluorescent form ( P 4 min). The superoxide dismutase activity of tempol failed to augment the response. B : DAF-2 fluorescence was measured in DVR pressurized to 25 mmHg in the stopped-flow configuration under control conditions ( n = 7) and in zero Ca 2+ buffer ( n = 8). Elimination of extracellular Ca 2+ attenuated the DAF-2 response ( P 5.5 min). C : calcein fluorescence ( n = 6) was monitored in DVR pressurized to 25 mmHg from time = 3 to 6 min. A - C : data are expressed as means ± SE. Most error bars have been suppressed for clarity.

Calcein is a fluorescent probe that is loaded into cells by deesterification and monitored at excitation and emission wavelengths similar to DAF-2. Calcein is insensitive to intracellular pH, Ca 2+, and ionic strength. Pressurization of DVR to 25 mmHg under stopped-flow conditions had no obvious effect on calcein fluorescence ( Fig. 9 C ), although a slow decline of the signal was observed. The decline is similar to that seen with DAF-2 when NO generation is blocked with L -NAME ( Fig. 9 A ). We showed that the decline is due to a leak of DAF-2 from the cytoplasm and not photobleaching ( 36 ). The failure of calcein fluorescence to be affected by pressurization supports the interpretation that the rise in DAF-2 fluorescence is attributable to pressure-associated NO generation and not to a nonspecific effect of optics or cellular geometry on fluorescent emission.

DISCUSSION

The principal finding of this study is that DVR endothelia respond to pressurization of the lumen by increasing [Ca 2+ ] CYT and generating NO. This is a fundamental observation that could have important physiological implications. Most obviously, DVR luminal pressure elevation is expected to favor NO production that counteracts the effects of constrictors on adjacent smooth muscle/pericytes. NO production by DVR endothelia might also favor saliuresis because NO inhibits NaCl reabsorption at multiple sites along the nephron ( 22, 28 ). The possibility of such NO-mediated signaling between tubular and vascular structures in the kidney has been frequently postulated ( 7, 9, 22, 28, 31, 34 ).

These studies cannot readily distinguish between effects of shear stress tangent to the endothelial surface and stretch of the endothelial plasma membrane induced by a transmural pressure gradient. Shear forces are generated by the velocity gradient in the vicinity of the cell membrane ( 1, 8, 40 ), whereas an increase in intraluminal pressure might stretch the vessel wall in both radial and longitudinal directions, deforming the endothelium. For technical reasons, we are unable to separately examine shear and stretch. DVR are 13-15 µm in outer diameter requiring pipette tips typically 5-µm internal diameter x 500 µm long to achieve cannulation. Collecting pipettes with small openings are needed to anchor DVR during microperfusion and seal the collectate from the bath so that accurate timed collections of effluent flow rate can be obtained ( 30, 33 ). In addition to constriction of the vessel outlet by the collection pipette, adherence of the extracellular matrix at the cut end across the opening often partially blocks outflow. As such, under free-flow conditions, the pressure drop from the perfusion pipette to the lumen and the pressure drop from the lumen to the collection pipette cannot be readily controlled or measured ( 30 ). For these reasons, the endothelial [Ca 2+ ] CYT ( Fig. 2 ) and NO responses ( Figs. 8 ) to perfusion cannot be clearly attributed to shear stress; increasing perfusion rate also invariably pressurizes and dilates microperfused DVR. As an alternative, we determined whether stretch of the vessel wall in the absence of flow ( Fig. 3 ) elicits a graded [Ca 2+ ] CYT response. Occlusion of the vessel to eliminate flow is expected to yield a luminal pressure equal to perfusion pipette pressure. Those experiments verified that raising pressure between 0 and 45 mmHg yields a steep rise of [Ca 2+ ] CYT that tends to plateau at 30 mmHg ( Fig. 3 ). This is associated with brisk NO production ( Fig. 9 ).

Our observation that elimination of extracellular Ca 2+ prevents a [Ca 2+ ] CYT response from occurring when the DVR lumen is pressurized ( Fig. 4 ) verifies the specificity of fura 2 but cannot be taken as clear evidence that the source of [Ca 2+ ] CYT is entirely from the extracellular fluid. Incubation of endothelial cells in 0 Ca 2+ medium can rapidly deplete both cytoplasmic and endoplasmic/sarcoplasmic reticulum store Ca 2+ ( 1, 8 ). The lack of a classical peak and plateau [Ca 2+ ] CYT response ( Fig. 2 ) typical of endothelium-dependent vasodilators ( 31, 34 ) mitigates against internal store release but this is unproven. The overshoot that occurred on reintroduction of CaCl 2 into the bath ( Fig. 4 ) probably implies enhanced conductance of Ca 2+ entry pathways resulting from Ca 2+ store depletion.

It is generally accepted that voltage-gated Ca 2+ channels do not play a major role in Ca 2+ entry into endothelial cells. Rather, Ca 2+ entry occurs through nonselective cation channels ( 27 ). It is increasingly recognized that such pathways are provided by the family of TRP channels originally identified in Drosophila. TRP channels are ubiquitous, have many splice variants, and most cells express several TRP channels ( 5, 42 ). The precise role of TRP channels in receptor-operated and -capacitative Ca 2+ entry continues to be a topic of much debate ( 5, 26 ). Selective inhibitors of TRP isoforms do not exist; however, La 3+ and SKF-96365 can be used to inhibit currents through TRP/nonselective cation channels ( 42 ). Based on these considerations, we investigated the ability of these agents to block the [Ca 2+ ] CYT rise associated with pressurization. Both were effective to prevent the response and both were slow to reverse after washout from the bath ( Figs. 5 and 6 ). These data do little to identify the precise channel(s) involved; however, they reinforce a probable role for activation of Ca 2+ entry pathway(s).

Graded pressurization of DVR failed to elicit compensatory vasoconstriction ( Fig. 5 ). This points to a possible lack of a myogenic response and reinforces observations previously obtained with free-flow microperfusion ( 30 ). Myogenic constriction is prominent in the renal afferent arteriole where it appears to be mediated by purinergic receptors ( 17, 21 ). These experiments were not designed to rule out the existence of a myogenic mechanism; however, if one exists, it is not prominent under these stop-flow conditions.

Luminal pressures in DVR in vivo have been measured by servo-nulling methods. The inner one-third of the inner medulla can be accessed for micropuncture in young anesthetized Munich-Wistar rats by isolating the kidney and excising the ureter to expose the papilla. Under hydropenic conditions, in that preparation, pressures between 6 and 16 mmHg have been found near the papillary tip (reviewed in Ref. 29 ). Those puncture sites are several millimeters downstream of outer medullary DVR and are therefore likely to have yielded pressures lower than those that exist in outer medullary DVR in vivo. We previously measured luminal pressure in papillary DVR during furosemide diuresis at a point just upstream of a paraffin wax block placed in the DVR lumen. Pressures of 20 to 22 mmHg were observed. Those pressures are likely to be similar to luminal pressures in outer medullary DVR because they were obtained above the wax blocks under stop-flow conditions ( 32 ). Outer medullary vasa recta pressures in the range of 15 to 20 mmHg were obtained by micropuncture of the blood-perfused juxtamedullary nephron preparation where access to vasa recta that arise from juxtamedullary efferent arterioles is possible ( 4 ). Taken together, we conclude that the luminal pressures that generate [Ca 2+ ] CYT elevation and NO production in current experiments ( Figs. 3, 4 - 6, 9 ) can occur in vivo.

The importance of NO synthesis to the maintenance of medullary perfusion and sodium handling by the kidney has been the topic of many studies ( 7, 23, 28 ). If NO has a sufficiently long half-life for it to diffuse from vasa recta to the adjacent thick ascending limb or collecting duct in the outer medullary interbundle region, an effect of pressure-induced NO release on salt reabsorption might exist. Low oxygen tensions and low hematocrit in the medulla favor reduced rate of elimination of NO and an enhanced range of effect. The ability of a denervated kidney to undergo saliuresis when perfusion pressure is raised has been dubbed "pressure natriuresis" and hypothesized to provide a fundamental feedback mechanism to control extracellular fluid volume ( 6, 14, 20, 37 ). The ability of DVR endothelia to release NO in response to luminal pressure elevation ( Figs. 8 and 9 ) might be a mechanism through which microvascular pressure within the medulla is transduced to affect adjacent nephrons and yield saliuresis.

The current studies demonstrate the ability of DVR endothelia to respond to mechanical stimuli but do not provide evidence for a specific signaling mechanism through which this occurs. The ability of endothelia from conduit vessels and microvessels to increase [Ca 2+ ] CYT ( 11, 39, 41 ), remodel ( 2, 8, 40 ), modulate permeability ( 24 ), and release NO ( 10, 12, 13, 15 ) is firmly established. A single predominant mechanism through which endothelia sense and then respond to mechanical forces has not clearly emerged ( 1, 8 ). Cellular responses may be localized to the cell membrane or transmitted to the cell interior via the cytoskeleton. Integrins have been implicated in such mechanosensation. Of interest, 1 -integrins are associated with caveolae within which caveolin and signaling complexes regulate the activity of endothelial NO synthase ( 15, 25, 35, 38 ). Direct or indirect activation of ion channel activity has also been implicated as an endothelial mechanosensor ( 1, 3, 8, 16, 18, 19, 43 ). Taken together, myriad signaling pathways could account for the observed DVR endothelial response to luminal pressure. Additional effort will be needed to delineate mechanisms.

In summary, we measured [Ca 2+ ] CYT responses with fura 2 to demonstrate that the DVR endothelium responds to luminal pressure elevation between 0 and 30 mmHg with a steep rise in [Ca 2+ ] CYT. This fura 2 response is specific because it is eliminated when extracellular Ca 2+ is omitted from the bath or Ca 2+ entry is blocked. We employed the NO-sensitive probe DAF-2 to show that the [Ca 2+ ] CYT response is associated with brisk production of NO. The latter response is also specific because it is eliminated by NO synthase inhibition ( L -NAME) and elimination of extracellular Ca 2+. It is possible that this DVR endothelial response modulates perfusion and salt handling in the renal medulla.

GRANTS

The studies were supported by National Institutes of Health Grants DK-42495, HL-62220, and HL-68686.

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作者单位：Department of Medicine, Division of Nephrology, and Department of Physiology, University of Maryland School of Medicine, Baltimore, Maryland 21201-1595