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Am J Physiol Regul Integr Comp Physiol 289: R620-R626, 2005. First published March 24, 2005; doi:10.1152/ajpregu.00610.2004
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INNOVATIVE METHODOLOGY

WATER AND ELECTROLYTE HOMEOSTASIS

Flash photolysis of caged nitric oxide inhibits proximal tubular fluid reabsorption in free-flow nephron

Kay-Pong Yip1

1Department of Physiology and Biophysics, University of South Florida, Tampa, Florida

Submitted 8 September 2004 ; accepted in final form 20 March 2005


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A nonobstructing optical method was developed to measure proximal tubular fluid reabsorption in rat nephron at 0.25 Hz. The effects of uncaging luminal nitric oxide (NO) on proximal tubular reabsorption were investigated with this method. Proximal fluid reabsorption rate was calculated as the difference of tubular flow measured simultaneously at two locations (0.8–1.8 mm apart) along a convoluted proximal tubule. Tubular flow was estimated on the basis of the propagating velocity of fluorescent dextran pulses in the lumen. Changes in local tubular flow induced by intratubular perfusion were detected simultaneously along the proximal tubule, indicating that local tubular flow can be monitored in multiple sites along a tubule. The estimated tubular reabsorption rate was 5.52 ± 0.38 nl·min–1·mm–1 (n = 20). Flash photolysis of luminal caged NO (potassium nitrosylpentachlororuthenate) was induced with a 30-Hz UV nitrogen-pulsed laser. Release of NO from caged NO into the proximal tubule was confirmed by monitoring intracellular NO concentration using a cell-permeant NO-sensitive fluorescent dye (DAF-FM). Emission of DAF-FM was proportional to the number of laser pulses used for uncaging. Photolysis of luminal caged NO induced a dose-dependent inhibition of proximal tubular reabsorption without activating tubuloglomerular feedback, whereas uncaging of intracellular cGMP in the proximal tubule decreased tubular flow. Coupling of this novel method to measure reabsorption with photolysis of caged signaling molecules provides a new paradigm to study tubular reabsorption with ambient tubular flow.

tubular flow; tubuloglomerular feedback; ultraviolet pulse laser; caged cGMP

PROXIMAL TUBULAR FLUID REABSORPTION is conventionally measured by using either shrinking split-droplet (6) or intraluminal perfusion and recollection techniques (14, 21). The shrinking split-droplet method offers higher temporal resolution in measuring reabsorption than the fluid recollection method, but the latter is more popular because it directly measures the reabsorbed fluid volume per unit time per unit tubular length. However, these techniques suffer drawback because of the lack of continuous ambient tubular flow and the exclusion of a portion of proximal tubule by the wax/oil block. To measure tubular reabsorption in physiological conditions, ambient tubular flow is required to maintain intranephron feedbacks such as glomerular-tubular balance and tubuloglomerular feedback (TGF). One approach to alleviate this limitation is to measure tubular flow at two locations along a proximal tubule by using nonobstructing optical techniques. The difference of tubular flow is then equivalent to the fluid reabsorption rate at that segment of tubule. To date, there are two nonobstructing optical techniques available to measure tubular flow, namely, the fluorescent photobleaching recovery technique and the dual-slit method. Flamion et al. (5) perfused isolated inner medullary collecting duct with fluorescein sulfonate (an impermeant fluorophore) and induced localized photobleaching with an argon laser. They demonstrated that the recovery rate of fluorescent photobleaching is a direct function of perfusate velocity. However, this technique has not been evaluated in vivo. The only optical method that has been applied successfully in vivo to measure proximal tubular flow was developed by Chou and Marsh (3). This technique measures the propagating velocity of fluorescent dextran, which is injected periodically into the ambient tubular flow with a micropipette, by monitoring the time delay of fluorescence transients between two adjacent slits.

In the present study, the dual-slit technique was modified to measure local tubular flow at two locations simultaneously along a convoluted proximal tubule by employing two micropipettes to independently inject fluorescent dextran pulses. This novel method was applied in conjunction with flash photolysis of caged nitric oxide (NO) in luminal fluid to examine the important role of NO in the regulation of proximal tubular reabsorption (4, 13). Our results demonstrate that it is feasible to monitor proximal tubular fluid reabsorption continuously in a nephron with ambient tubular flow at 0.25 Hz and that controlled release of caged signaling molecules with the use of a UV nitrogen-pulse laser can be applied to intact renal epithelium without interrupting data acquisition.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. Experiments were carried out in accordance with guidelines for the care and use of research animals. All experiments were performed under protocols approved by the Institutional Animal Care and Use Committee at the University of South Florida, in accordance with Public Health Service Policy on Human Care and Use of Laboratory Animals. Experiments were performed in male Sprague-Dawley rats (220–280 g body weight). All rats were purchased from Harlan. The rats had free access to food and tap water before the experiments. Anesthesia was induced by placing each rat in a chamber containing 5% halothane administered in 25% oxygen and 75% nitrogen through a Fluotec Mark-3 vaporizer. A tracheostomy was performed, and the rats were placed on a servo-controlled heated operating table, which maintained body temperature at 37°C. The tracheostomy tube was connected to a small animal respirator (Harvard model 683) adjusted to maintain arterial blood pH between 7.35 and 7.45 with a mixture of 25% oxygen-75% nitrogen. Tidal volume ranged from 1.9 to 2.5 ml, depending on body weight, with a frequency of 57–60 breaths/min. The final concentration of halothane needed to maintain sufficient anesthesia was ~1%. A polyethylene catheter (PE-50) was placed in the right jugular vein for infusions. After a priming dose of smooth muscle vasorelaxant (pancuronium, 1 mg/kg body wt) in 1 ml 0.9% saline, a continuous infusion of pancuronium (1 mg·kg–1·h–1) in 0.9% saline was given at 20 µl/min. The left kidney was exposed through a flank incision, immobilized with a Lucite ring, and superfused with saline preheated at 37°C. The renal capsule was left intact. Arterial pressure was measured in the left carotid artery with a Statham-Gould P23dB pressure transducer connected to a transducer amplifier (TBM4; WPI). Rats were anesthetized with Inactin [5-ethyl-5-(L-methylpropyl)-2-thiobarbituric acid, 100 mg/kg ip] instead of halothane in experiments designed for examining the effects of NO on tubular fluid reabsorption. No pancuronium was administered when Inactin was used as anesthetic.

Measurement of tubular flow and reabsorption rate. A bolus of lissamine green dye was injected intravenously to identify early segments of proximal tubules. Proximal tubules were selected for observation only if they had a long segment (>1 mm) that ran on the surface, which permitted measurement of tubular flow on two different locations simultaneously (Fig. 1). In each selected location, proximal tubular flow was measured using a method developed by Chou and Marsh (3) as described previously. In brief, a micropipette (2- to 3-µm outer diameter) was filled with synthetic proximal tubular fluid containing a 1% solution of rhodamine-isothiocyanate 20S-labeled dextran (MW 17,200; Sigma) and was inserted into the proximal convoluted tubule. Injection was driven by a triggered pneumatic picopump (PV830; WPI). Injection frequency was set at 15 pulses/min (0.25 Hz) with an injection pressure of 20 lb./in.2 and an injection duration of 5–10 ms. The volume of each injection pulse was ~15 pl under these conditions (7). Fluorescent dextran was excited with a green helium-neon laser (1 mW, 534 nm; Melles Griot) aimed at the renal surface and was detected with an intensified charge-coupled device (CCD) video camera (IC-300; PTI) through a long-pass filter at 560 nm. Injections of dextran pulses at upstream and downstream sites were synchronized with the use of a signal generator.



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  		Fig. 1. Schematic drawing of tubular flow measurement at 2 locations on a convoluted proximal tubule. Arrows indicate direction of tubular flow. Fluid propagating velocity is estimated with a pair of sampling windows distal to the injection pipette at each site.

 
To determine the velocity of the fluid stream, the composite video signal was digitized into a 512 x 480-pixel array at 8-bit resolution with a Matrox IP-8 image processing board housed on a desktop computer and displayed on a 15-in. monitor. The imaging board allowed four sampling windows of variable size to be placed independently on the digitized image. A pair of sampling windows was positioned downstream in each dextran injection site (Fig. 1). The board returned four digital signals at 60 Hz. Each signal is proportional to the light intensity in the area defined by the sampling windows. The upstream signal in each injection site served as a template for the downstream signal of the same site. The transit time delay for the passage of the fluorescent dextran bolus between the two sampling windows was calculated for each pulse with a cross-correlation routine. A digital low-pass filter (5-Hz cutoff) was applied before the computation of cross-correlation. The distance separating the two sampling windows and the tubule diameter was measured on the digitized image. Fluid velocity was calculated by dividing the distance by the time delay. Tubular fluid flow was calculated as the product of the velocity and the cross-sectional area. The difference of tubular flow measured simultaneously at two injection sites was then normalized by the tubular length between the two injection sites and was used as the fluid reabsorption rate along that specific segment of proximal tubule. For measurements of mean fluid reabsorption rate, the reabsorption rate was averaged over a period of 5 min.

To test whether this method has sufficient spatial and temporal resolution to detect local variations of tubular flow, we induced local variations in tubular flow by performing intraluminal perfusion with and without furosemide. The intraluminal perfusion pipette was inserted either distal to both dextran-injecting pipettes or in between the two dextran-injecting pipettes.

Flash photolysis of caged NO in cultured renal vascular smooth muscle cells. The efficiency of the UV nitrogen-pulsed laser in uncaging NO was first tested in vitro. A primary culture of renal vascular smooth muscle cells was prepared using the iron oxide method as described previously (2). Vascular smooth muscle cells of second passage in a collagen-coated coverslip were loaded with 10 µM 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM diacetate), a cell-permeant NO-sensitive fluorescent dye (Molecular Probes) for 30 min, washed, and incubated with Hanks' balanced salt solution (HBSS) for 15 min for deacetylation. The cells were then bathed with fresh HBSS containing 2-µM caged NO (potassium nitrosylpentachlororuthenate; Molecular Probes). Emission of DAF-FM was measured at 0.5 Hz by exciting DAF-FM at 488 nm with an argon laser and collecting emission at 522 nm via a band-pass filter (bandwidth 22 nm) with a Bio-Rad confocal scanning unit (MRC-1000) at x20 (N.A. 0.75; Zeiss). Flash photolysis of caged NO was then induced with a UV nitrogen-pulse laser (337 nm, 4 ns/pulse; Laser Science 337ND) through a quartz optical fiber of 1 mm in diameter. The firing frequency and number of laser pulses in each burst were controlled by an analog-to-digital board (Data Translation DT-2801A) housing on a Pentium computer.

Flash photolysis of caged NO in proximal tubule. To test whether photolysis of luminal caged NO increases NO concentration ([NO]) in proximal tubules, we first loaded proximal tubular cells with DAF-FM via intraluminal perfusion (10 µM in synthetic tubular fluid, 10 nl/min for 30 min). Luminal perfusate was then switched to synthetic tubular fluid containing 2 µM caged NO. DAF-FM in proximal tubule was excited at 490 nm via the objective lens (x10, N.A. 0.25) with a UV nitrogen-pulse laser (Laser Science 337) coupled to a laser dye module (22). Emission from DAF-FM was collected by a photomultiplier through a band-pass filter of 522 nm (bandwidth 32 nm) at 2 Hz. Flash photolysis of caged NO was initiated by shining a burst of UV laser pulses on the surface of kidney via a quartz optical fiber (1 mm in diameter). The firing frequency and number of laser pulses in each burst were controlled by an analog-to-digital board (Data Translation DT-2801A) housing on a Pentium computer. The trigger of excitation and collection of emission were controlled by an analog-to-digital board (Data Translation DT-2813) housing on another Pentium computer as described previously (22).

To test the effects of uncaging NO in proximal tubular reabsorption, we used two dextran-injecting micropipettes to measure tubular flow at 0.25 Hz in the same convoluted proximal tubule as described above. Caged NO (2 µM) was included in the dextran solution of the upstream injection pipette. The holding pressure of the upstream injection pipette was then set at <2 lb./in.2, which was just sufficient to overcome the intratubular hydrostatic pressure and provide a continuous leaking of fluorescent dextran into the lumen. Photolysis of caged NO was induced by delivering a burst of UV laser pulses on the kidney surface while tubular flow was simultaneously monitored on two locations along the tubule. Caged NO was introduced into the lumen solely by the upstream injection pipette.

Flash photolysis of caged cGMP in the proximal tubule. To test the effects of uncaging intracellular cGMP in proximal tubular flow, we loaded caged cGMP [guanosine 3',5'-cyclic monophosphate, P-1-(2-nitrophenyl)ethyl ester, a cell-permeant form of caged cGMP; Calbiochem] into proximal tubular cells by luminal perfusion. Caged cGMP (0.4 mM) was perfused intraluminally at 10 nl/min for 30 min. Photolysis of caged cGMP was then induced by a UV nitrogen-pulse laser while proximal tubular flow was monitored at a site proximal to the cGMP loading site. It has been suggested that NO inhibits proximal tubular reabsorption through a cGMP-linked pathway (4).

Solutions. The synthetic proximal tubular fluid used contained (in mM) 127 NaCl, 25 NaHCO3, 3 KCl, 1 MgSO4·7H2O, 1 K2HPO4, 5 urea, and 1.8 CaCl2.

Statistics. Paired t-tests were used wherever applicable. Results are reported as means ± SE. Only one proximal tubule was studied in each rat.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Proximal tubular flow in halothane-anesthetized rats is known to oscillate at about 2 cycles/min because of the operation of TGF (7). When tubular flow was monitored simultaneously at two sites along a convoluted proximal tubule, synchronized oscillations were found, as expected (Fig. 2). Activation of TGF by intraluminal perfusion of tubular fluid distal to both dextran injection sites triggered synchronous reduction in tubular flow (Fig. 2). The decrease in tubular flow was proportional to the intraluminal perfusion rate (5–20 nl/min at 5 nl/min step). The reduction in tubular flow was due to TGF-mediated vasoconstriction in afferent arterioles. Conversely, inhibition of TGF by intraluminal perfusion of furosemide-containing perfusate (0.4 mM, 10 nl/min) increased tubular flow (Fig. 3). Intraluminal perfusion of furosemide distal to both dextran injection sites elicited a synchronous increase in tubular flow (Fig. 3A). When intraluminal perfusion of furosemide was applied between two dextran injection sites, there was an immediate increase of flow at the downstream site because of luminal perfusion, followed by a synchronous increase of flow on both sites because of TGF inhibition (Fig. 3B). Termination of intraluminal perfusion resulted in an immediate decrease of tubular flow at the downstream site, followed by a synchronous decrease of flow on both sites. These observations established the fact that local tubular flow could be monitored simultaneously at two different locations along a proximal tubule every 4 s. Therefore, proximal tubular fluid reabsorption rate could be monitored continuously at 0.25 Hz without interrupting ambient tubular flow. However, the resolution in measuring tubular flow is not constant but is a nonlinear function of fluid stream velocity, or the transit time delay between two sampling windows. The resolution of time delay is limited to 1/60 s (1 video frame). The resolution of flow measurement at a specific time delay was defined as the increase in tubular flow when the time delay is reduced by 1/60 s. Figure 4 is a plot of resolution of flow measurement as a function of transit time delay in a typical tubule with a diameter of 30 µm and a distance of 150 µm between sampling windows. The resolution was 3.5 nl/min when the time delay was 10/60 s, whereas the resolution was 0.6 nl/min when the time delay was 25/60 s. The resolution deteriorated very rapidly when the time delay was <10/60 s. Typical transit time delay was ~10/60 s at the rising phase of tubular flow oscillations in halothane-anesthetized rats (Figs. 2 and 3). In Inactin-anesthetized rats, typical transit time delay varied between 15/60 and 25/60 s in each oscillatory cycle. Therefore, Inactin was the choice of anesthetic in measuring tubular flow and tubular reabsorption rate. The mean proximal tubular reabsorption rate in Inactin-anesthetized rats was 5.52 ± 0.38 nl·min–1·mm–1 (n = 20) in the present study. The calculated reabsorption rate ranged from 4 to 8 nl·min–1·mm–1. The distance between dextran injection sites ranged from 0.8 to 1.8 mm. Proximal fluid reabsorption measured using microperfusion and fluid recollection in rats with similar body weights was in the range of 2.5–5.2 nl·min–1·mm–1 (14, 21).



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  		Fig. 2. Time course of changes in proximal tubular flow at 2 locations measured simultaneously when tubuloglomerular feedback (TGF) is activated by intratubular perfusion of synthetic tubular fluid. Dotted line represents perfusion rate.

 


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  		Fig. 3. Time course of changes in proximal tubular fluid flow at 2 locations measured simultaneously when TGF was inhibited by intratubular furosemide perfusion (0.4 mM, 10 nl/min). Furosemide was perfused distal to both flow measuring sites (A) and in between 2 measuring sites (B). Horizontal bars indicate duration of perfusion.

 


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  		Fig. 4. Resolution in tubular flow measurement as a function of transit time delay in a typical proximal tubule. The resolution of flow measurement is defined as the difference in tubular flow when the time delay is reduced by 1/60 s. Distance between sampling windows was 150 µm. Tubular diameter was 30 µm.

 
To examine whether the laser pulses delivered through a 1-mm-diameter optical fiber (~150 µJ/pulse measured by laser meter) are capable of triggering photolysis of caged NO, we first tested the uncaging capability of the laser system in cultured renal vascular smooth muscle cells using confocal fluorescent microscopy. The [NO] in cultured smooth muscle cells was monitored with a cell-permeant NO-sensitive fluorescent dye, DAF-AM. In the presence of 2-µM caged NO in the bathing solution, 30 pulses of UV laser at 30 Hz were sufficient to induce a detectable increase of DAF-FM fluorescence (Fig. 5). A graded accumulative increase of DAF-FM fluorescence was detected when the number of laser pulses was increased in each successive burst. These observations indicate that 30 pulses of UV laser delivered within 1 s are sufficient to initiate photolysis of caged NO and that the amount of NO released can be controlled by the number of pulses in each laser burst.



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  		Fig. 5. Mean normalized time course of changes in 4-amino-5-methylamino-2',7'-difluorofluorescein diacetate (DAF-FM) fluorescence emission intensity from cultured renal vascular smooth muscle cells. Flash photolysis of caged nitric oxide (NO) was induced by bursts of 30-Hz UV laser pulses. Each arrow indicates a burst of laser pulses; 30, 60, 90, 120, 150, 180, 210, and 240 pulses of UV laser were applied sequentially. Dotted lines represent SE (n = 30 cells/3 coverslips).

 
To further examine whether photolysis of caged NO in luminal fluid could raise the intracellular [NO] of proximal tubule in situ, we microperfused the tubule with luminal perfusate containing 2 µM caged NO and monitored intracellular [NO] in proximal tubular cells with DAF-FM. The time course of changes in DAF-FM emission intensity from a proximal tubule during photolysis is shown in Fig. 6A. Sixty pulses of laser triggered a detectable spike of DAF-FM emission, and increasing the number of laser pulses in each burst increased the magnitude of DAF-FM emission. The rise in DAF-FM emission was more transient in intact tubules than in cultured smooth muscle cells. It probably is the consequence of luminal dilution of caged NO, removal of uncaged NO by diffusion, and ambient tubular flow in intact tubule. Figure 6B depicts the mean normalized time course of changes in DAF-FM emission when the tubule was exposed to 30-Hz UV laser pulses for 120 s. There was a sustained elevation of DAF-FM emission after the initiation of photolysis.



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  		Fig. 6. Time course of changes in DAF-FM emission in proximal tubules induced by photolysis of luminal caged NO. A: changes in DAF-FM emission in a proximal tubule. Each arrow indicates a burst of laser pulses; 60, 90, 120, and 240 pulses of UV laser were applied sequentially. B: mean normalized time course of changes in DAF-FM. Horizontal bar indicates duration of uncaging laser burst. Dotted lines represent SE (n = 6 tubules/ 6 rats).

 
Figure 7A shows the time course of changes in tubular flow induced by photolysis of luminal caged NO. Consecutive application of 1,200, 2,400, and 3,600 pulses of UV laser preferentially increased the tubular flow in the downstream site, indicating that tubular fluid reabsorption was inhibited in the proximal tubule between the two dextran injection sites. Inhibition of proximal tubular reabsorption occurred without reduction in mean tubular flow (Table 1), suggesting that TGF was attenuated (16, 18). Laser illumination did not induce changes in tubular flow in the absence of luminal caged NO (Table 1). The dose-dependent effects of uncaging NO on reabsorption rate are summarized in Fig. 8. Inhibition of tubular reabsorption was enhanced when the number of laser pulses for uncaging was increased. cGMP is known as the mediator of NO to inhibit proximal tubular reabsorption (4). Uncaging of intracellular cGMP in proximal tubule to inhibit reabsorption triggered TGF and reduction of tubular flow (Fig. 9). cGMP released by photolysis was membrane impermeant, whereas uncaged NO was free to diffuse from the releasing site (9). The different responses in tubular flow induced by uncaging luminal NO and uncaging intracellular cGMP were probably due to their differences in diffusion capacity.



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  		Fig. 7. Effects of photolysis on variations of tubular flow rates measured simultaneously at 2 sites on a convoluted proximal tubule with 2 µM caged NO in the lumen (A) and a timed control (B). Horizontal bars indicate duration of 30-Hz laser burst; 1,200, 2,400, and 3,600 pulses of UV laser were applied sequentially.

 

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  		Table 1. Mean normalized proximal tubular flow at upstream and downstream sites after exposure to uncaging laser pulses in the absence and presence of caged NO in the lumen

 


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  		Fig. 8. Changes in mean normalized proximal tubular fluid reabsorption rate when an increasing number of laser pulses was used to induce photolysis of caged NO in luminal fluid. The mean baseline reabsorption rates are 5.23 ± 0.46 (n = 6) and 5.53 ± 0.69 nl·min–1·mm–1 (n = 8) for the timed control and NO uncaging, respectively. *P < 0.05, significant inhibition.

 


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  		Fig. 9. Mean normalized time course of changes in proximal tubular flow induced by photolysis of intracellular caged cGMP. Photolysis was induced by a burst of 30-Hz UV laser pulses for 120 s. Horizontal bar indicates duration of uncaging. Dotted lines represent SE (n = 5 tubules/5 rats).

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The present study successfully implemented a novel approach to measure proximal tubular fluid reabsorption in vivo at 0.25 Hz without interrupting tubular flow. This was achieved by measuring tubular flow at two locations along a proximal convoluted tubule based on the method originally developed by Chou and Marsh (3). A major challenge of applying this new method was to locate a segment of convoluted proximal tubule that permitted insertion of two micropipettes to inject fluorescent dextran without interfering with each other. A separation of 0.8 mm between two injection sites was in general far enough to avoid cross-detection at the downstream site due to injection from the upstream site. The validity of the method was indicated by the detection of differential increases and decreases in local tubular flow along a proximal convoluted tubule (Fig. 3). These observations indicate that this method has sufficient temporal and spatial resolution to detect local changes in tubular flow.

The transit time delay detected between two sampling windows was not continuous but discrete because of the CCD camera framing rate. The resolution in measuring tubular flow deteriorated rapidly when the transit time delay was <10/60 s (Fig. 4). Typical transit time delay was ~10/60 s at the rising phase of tubular flow oscillations in halothane-anesthetized rats. The amplitude of TGF-mediated oscillations is smaller in Inactin-anesthetized rats than in halothane-anesthetized rats because of the difference in proximal tubular compliance (12). The transit time delays in Inactin-anesthetized rats were in the range of 15/60–25/60 s in each oscillatory cycle, values that were in the more linear range of Fig. 4. The estimated mean proximal tubular fluid reabsorption rate was 5.52 ± 0.38 nl·min–1·mm–1 in the present study, consistent with values reported in the literature (14, 21). The proximal tubular fluid reabsorption rates measured with microperfusion and fluid recollection (using radioactive inulin as volume marker) in rats with similar body weight are in the range of 2.5–5.2 nl·min–1·mm–1 (14, 21). Proximal tubular fluid reabsorption rate measured using the perfusion/recollection method depends on the tubular perfusion rate used. In the optical method, the tubular perfusion rate is not an experimental variable because ambient tubular flow is not disturbed. The temporal resolution of the perfusion/recollection method is on the order of minutes, whereas that of the optical method is on the order of seconds. A silicon rubber cast of the proximal tubule is required to determine the tubular length for the normalization of reabsorption rate in the perfusion/recollection method, whereas the tubular length is determined from the digitized image in the optical method. Therefore, the optical method not only provides a means to measure native tubular fluid reabsorption but also improves the temporal resolution in monitoring tubular reabsorption.

Flash photolysis of caged signaling molecules allows rapid manipulation of intra- and extracellular environment with minimal disturbance in data acquisition. Conventional flash photolysis is induced by a flash lamp through the objective lens, which limits the field of illumination, interrupts image acquisition, and imposes a finite time delay between consecutive flashes. These issues were alleviated in the present study by using a UV nitrogen-pulse laser coupled to an optical fiber to induce photolysis (23). One advantage of using the UV pulse laser is the ability to apply consecutive flashes over a long uncaging period with relatively short laser dwelling time. Each laser pulse lasts only 4 ns. The laser dwelling time for 1 min of uncaging at 30 Hz is 7.2 µs, and thus the potential tissue damage due to UV radiation is minimized. With the use of confocal fluorescence microscopy, it was found that 30 pulses of UV laser were sufficient to initiate photolysis of caged NO and that the extent of uncaging could be controlled by the number of laser pulses in each burst (Fig. 5). The imaging system used for detecting DAF-FM emission from proximal tubule is similar to that used for detecting BCECF emission as described previously (20, 22). Uncaging of NO in luminal perfusate was achieved by delivering the laser pulses to the kidney surface via an optical fiber. The laser spot on the kidney surface was positioned with a visible light source through the optic fiber before uncaging. Similar to the observations in cultured vascular smooth muscle cells, DAF-FM emission intensity from proximal tubule was proportional to the number of laser pulses used for uncaging.

Uncaging of NO in luminal perfusate inhibited tubular reabsorption. The degree of inhibition was increased as the number of laser pulses used for uncaging was increased (Fig. 8). Luminal application of sodium nitroprusside (0.1–1 mM) has been shown to inhibit proximal tubular reabsorption with the use of the shrinking split-droplet technique (4), which is consistent with the present study. Inhibition of proximal tubular reabsorption in free-flow nephron increases distal NaCl delivery, which activates TGF-mediated vasoconstriction and decreases tubular flow. Therefore, pharmacological inhibition of proximal tubular reabsorption will not manifest as an increase of tubular flow unless TGF is deactivated. This prediction has been verified using EIPA to inhibit apical Na+/H+ exchanger in the proximal tubule of free-flow nephron. Intratubular perfusion of EIPA into the proximal tubule decreases proximal tubular flow when TGF is intact, and the same maneuver increases proximal tubular flow when TGF is inhibited by furosemide (20). cGMP is the mediator of NO to inhibit proximal tubular reabsorption (4). Uncaging of intracellular cGMP within the proximal tubule triggered reduction of tubular flow, which indicated inhibition of proximal tubular reabsorption and activation of TGF. However, the NO-induced inhibition of fluid reabsorption did not trigger TGF-mediated vasoconstriction. One plausible explanation is that intracellular cGMP released by photolysis is membrane impermeant, whereas diffusion of NO from the uncaging site is very rapid and far reaching (9).

NO production is an integral part of the TGF signaling event at the macula densa to buffer excessive TGF-mediated vasoconstriction (8, 18). There are three potential mechanisms by which uncaged NO can reach the macula densa and afferent arterioles to inhibit TGF. Uncaged NO from convoluted proximal tubules might diffuse directly to the macula densa and renal arteriole. NO has a high diffusion constant (3,300 µm2/s) and is very widely diffusible (15, 19). The range of NO diffusion is ~150–300 µm for 4–15 s, and the half-life of NO is in the range of 5–15 s (10). The time delay between initiation of NO uncaging and increase of tubular flow was in the range of 16–28 s (Fig. 7). Therefore, it is spatially and temporally possible that the effect of increased delivery of NaCl to the macula densa due to reabsorption inhibition is nullified by NO diffused from the proximal tubule. Uncaged NO might also be carried into the loop of Henle with tubular flow. There is indirect evidence that NO produced in the loop of Henle was detected well beyond the macula densa (11). However, it is very unlikely that uncaged NO can reach the macula densa through advective transport because of the high diffusion constant of NO (17). All uncaged NO should have diffused across the tubular wall in a few seconds after entering the loop of Henle, whereas the fluid transit time in the loop of Henle is at least 30 s (1). Finally, uncaging of NO might occur in close proximity to the macula densa if the UV laser penetrates into the cortex and there is a sufficient amount of caged NO in the lumen of early distal tubule.

In summary, an optical technique was successfully developed and implemented to measure proximal fluid reabsorption at 0.25 Hz without interrupting ambient tubular flow. Manipulation of intracellular [NO] and [cGMP] of proximal tubular cells by flash photolysis demonstrates the potential of using caged signaling molecules to rapidly manipulate the intracellular environment of intact renal epithelium. The unique approach to measure tubular reabsorption with ambient tubular flow and to deliver luminal NO via photolysis reveals that NO inhibits proximal tubular reabsorption without activating TGF-mediated vasoconstriction.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by National Institutes of Health Grants HL-59156 and DK-60501.


   ACKNOWLEDGMENTS
 
I acknowledge Drs. N.-H. Holstein-Rathlou, D. J. Marsh, and A. J. Wagner for helpful suggestions and C. Landon for technical assistance.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K.-P. Yip, Dept. of Physiology and Biophysics, College of Medicine, Univ. of South Florida, MDC 8, 12901 Bruce B. Downs Blvd., Tampa, FL 33612 (E-mail: dyip{at}hsc.usf.edu)

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.


   REFERENCES
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 ABSTRACT
 METHODS
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