RAPID COMMUNICATION
Real-time profiling of kidney tubular fluid nitric oxide concentrations in vivo

David Z. Levine1, Michelle Iacovitti1, Kevin D. Burns1
Xueji Zhang2
(With the Technical Assistance of Amy Slater)

1 Division of Nephrology, The Kidney Research Centre, Ottawa Health Research Institute, and University of Ottawa, Ottawa, Ontario, Canada K1H 8M5; and 2 World Precision Instruments, Sarasota, Florida 34240-9258


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

To directly determine intratubular nitric oxide concentrations ([NO]) in vivo, we modified amperometric integrated electrodes (WPI P/N ISO-NOP007), which are highly sensitive to NO and not affected by ascorbic acid, nitrite, L-arginine, or dopamine. Although reactive lengths were as short as 5 µm long, the electrode still responded rapidly. With the use of kidney surface fluid as the "zero point," the electrode tip was inserted into tubular segments along the track of a perforation made by a beveled glass pipette. The surface fluid zero point was usually stable as distal, late proximal, and early proximal tubule [NO] levels were measured sequentially in the same nephron. In eight normal rats, distal, late proximal, and early proximal [NO] concentrations were each ~110 nM. In contrast, in nine 5/6 nephrectomized rats 2 wk postsurgery, although [NO] also did not differ among distal, late proximal, and early proximal segments, levels were approximately fourfold higher than those in normal rats and were significantly reduced after NG-monomethyl-L-arginine administration. These are the first quantitative in vivo tubular fluid [NO] measurements and show a significant increase in tubular fluid [NO] after renal ablation.

kidney tubules; in vivo nitric oxide measurements; remnant kidney


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

HUNDREDS OF PUBLICATIONS HAVE appeared addressing the multifaceted intrarenal effects of nitric oxide (NO), such as in arteriolar tone, glomerular filtration rate, tubular transport, and renal damage. For example, studies using inhibitors of nitric oxide synthases (NOS) and NO donors suggest that the NO system can alter tubuloglomerular feedback (TGF) responses, likely via changes in arteriolar tone. However, direct intrarenal NO concentration ([NO]) measurements are needed to characterize and quantify effects of NOS inhibitors or NO donors to track short-term changes (seconds to minutes) in [NO] (11). In studies of ischemia-reperfusion injury, there is agreement that NO plays an important role; unfortunately, conflicting results have emerged that can only be resolved by direct intrarenal NO measurements, as has been already pointed out by Weight and Nicholson (14).

Of course, different approaches to NO measurement have been reported for more than a decade, with the electrochemical and chemiluminescence methods attracting most attention. The chemiluminescence approach requires stripping of NO from the aqueous sample to the gas phase, thereby precluding an ability to monitor [NO] in real-time biological systems (4, 5). The electrochemical approach has been used usually without integration of the reference system and with limitations in its specificity for NO detection (8, 13). Nonetheless, Noiri et al. (9) impaled 30-µm-diameter, NO-sensitive electrodes 1 mm into the kidney cortex of control and ischemic rats and showed clear differences in tissue NO release in their preparations, whereas Majid et al. (7), using coated platinum-iridium amperometric electrodes of 200 µm diameter, impaled their electrodes 5 mm into the cortex of dog kidneys and demonstrated clear current increases in response to intra-arterial injection of S-nitrosos-N-acetyl-penicillamine (SNAP), despite a varying baseline.

However, to better understand the short-term control of TGF and tubular transport, it would be desirable to quantitatively measure real-time [NO] in structures such as tubules and the glomerular capsule. Accordingly, we modified the WPI amperometric NO electrode (WPI P/N ISO-NOP007), with an integrated Ag/AgCl reference system, so that its reactive length was ~5-15 µm and the electrode could be completely inserted into a tubule. This electrode is not affected by ascorbic acid, nitrite, L-arginine, or dopamine, but it is very sensitive to NO and responds rapidly (15). Electrodes with sufficient stability and sensitivity for in vivo use in our laboratory permitted successful real-time in vivo measurements of tubular fluid [NO], and, assuming [NO] is in diffusion equilibrium, with that in the surrounding parenchyma.

The present study, therefore, describes a convenient system for measurement of real-time [NO] in tubular fluid of rats prepared for micropuncture. We show that [NO] in distal, late proximal, and early proximal tubular fluid in normal rats is ~110 nM, not different from each other, whereas 2 wk after 5/6 nephrectomy corresponding tubular fluid [NO] measurements are approximately fourfold higher. We believe this first demonstration of quantitative measurement of intratubular [NO] in vivo should be useful in validating previous conclusions derived from the use of nonspecific NOS inhibitors and NO donors, as well as tracking real-time, rapid changes in [NO] in discrete intrarenal structures.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Construction of NO electrodes. Carbon fibers were initially cleaned by sonication for 5 min first in acetone, then in nitric acid, and finally in distilled water. After being dried at room temperature, a single carbon fiber was mounted at the end of a copper wire, fixed with silver epoxy, and inserted into a prepulled glass capillary with a 10-µm tip. A small drop of epoxy secured the insertion point. An Ag-AgCl layer was coated near, but not at, the end of the capillary fiber and acted as a reference electrode. The exposed fiber was coated with a Nafion ion exchange membrane and other WPI membranes. The basic electrode design, supplied by World Precision Instruments (WPI P/N ISO-NOP007), is highly specific for NO (15). Electrodes, especially modified for our in vivo use, were constructed (See Fig. 1). In the first set of experiments, series A, the proximal electrode shaft was insulated with epoxy so that a length of ~7-15 µm was reactive, and the diameter was 7 µm. In a later design, used in series B experiments, glass was used to insulate the shaft to absolutely preclude NO reactivity, the reactive length was ~5-15 µm, and the tip diameter ranged from ~0.8 to 5 µm. Electrodes in both designs had variable life spans. These inherently fragile electrodes could be easily broken through operator error, kidney movement, or the trauma of tubular puncture through the track of the perforation made by a conventional glass micropuncture sampling pipette. Nevertheless, some electrodes were used for as many as 6 experiments involving ~60 tubular measurements.


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Fig. 1.   Modified WPI integrated amperometric nitric oxide (NO)-sensing electrode. The basic features of the electrode structure are shown. Arrow pointing to insulation layer refers to either epoxy or glass coatings. The NO-sensitive length, coated with Nafion and WPI membranes, was ~5-15 µm; the tip diameter varied from less than 1 to 7 µm. See METHODS for additional details.

Electrode in vitro calibration, tubular fluid, and intratubular "zero points." Electrodes, as delivered from WPI, were calibrated after modification usually two to four times in the course of an in vivo experiment. The SNAP-copper sulfate system was used as previously described (9). Invariably, each electrode showed a regression coefficient of better than 0.99. The picoampere and/or nanomolar sensitivity varied from electrode to electrode, and data were only accepted if sensitivity was stable, as determined by in vitro calibration before and after intratubular measurements. The electrode zero point shifted only moderately as it was moved from the copper sulfate solution to be immersed in the kidney surface fluid, which was made up of slowly perfused saline maintained within an agar enclave. Both the in vitro calibration and the surface fluid were maintained at ~37°C.

Tubular fluid measurements of [NO]. Our micropuncture and microperfusion techniques, as well as normal and 5/6 nephrectomy (Nx) rat preparations, have been recently described (6). In the present experiments, NO measurements were made by first perforating the tubule with a beveled glass pipette and inserting the electrode tip into the tubular lumen through the perforation. The picoampere difference between surface fluid and the stable intratubular reading was taken as the NO signal, and the corresponding nanomolar concentration was read off the mean of the bracketed SNAP-copper sulfate calibration curves. In many but not all tubules, immediately after the tip of the electrode was inserted, a spike was observed (see RESULTS), which immediately dropped to a more stable but much higher current, reflecting intratubular [NO]. This spike seemed to be associated with bending of the fiber tip, could be reproduced by bending the fiber on the kidney capsule, and was thereby assumed to be mechanical in origin.

Intratubular zero point. We presumed that if the picoampere signal was solely due to intratubular NO, the signal should be abolished by high-flow saline perfusion around the electrode. Indeed, 40 nl/min saline diminished the picoampere current in a repeated and reproducible fashion (Fig. 2) to values similar to that obtained from surface fluid. Finally, to confirm that the surface fluid was free of NO, 50-ul samples of surface fluid were pipetted into the copper sulfate solution without any significant response even after many tubular segments were punctured, presumably favoring an NO leak out of the tubules into the surface fluid.


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Fig. 2.   Intratubular recording (reading from right to left) of NO concentration ([NO]) with perfusion with saline and/or saline equilibrated with NO. First, a tubule was punctured, the electrode was inserted, and [NO] was measured in tubular fluid (TF). Next, an intratubular "zero point" was taken (A) after perfusion with NO-free saline at 40 nl/min. Then, stepwise increments of the saline/NO solution were perfused, with corresponding reduction of the saline perfusate to maintain an approximately constant 40 nl/min flow rate. B, C, D, E: 10, 20, 30, 40 nl/min perfusion with the saline/NO solution, respectively.

Response to intratubular perfusion of an NO gas-equilibrated saline solution. To confirm that the electrode readily senses NO in vivo, we equilibrated a saline solution with the gas and then perfused the solution upstream from the electrode. Figure 2 shows the in vivo response to delivering varying concentrations of NO at a flow of ~40 nl/min. To achieve constant flow, two perfusion pipettes were used: one contained saline equilibrated with NO, and the other contained NO-free saline. The rate of perfusion of the two pipettes was varied so as to maintain a constant 40 nl/min flow past the electrode while [NO] was varied. As shown in Fig. 2, after a stable ~40-pA (~50 nM) signal from the tubular fluid was obtained, 40 nl/min of saline reproducibly diminished the signal to intratubular zero values, similar to that of kidney surface fluid (not shown). Thereafter, when the NO perfusate was run at 10 nl/min and the saline perfusate was reduced to 30 nl/min, the picoampere response increased. When this was repeated using up to a 40 nl/min perfusion of only the NO perfusate, the picoampere response was threefold that of the endogenous tubular fluid signal. It is to be noted that these in vivo picoampere and/or nanomolar responses are not perfectly linear. At least three factors could account for this: the perfusion system was not closed (i.e., endogenous filtrate was not blocked); transtubular diffusion of NO may have occurred; and there may have been perfusion rate errors in this situation of rapid flow adjustment. Notwithstanding these considerations, Fig. 2 shows unequivocal, prompt responses to progressive increases and then decreases in perfused [NO]. These responses cover the range of [NO] seen in normal rat tubular fluid.

Lack of influence of glass vs. epoxy electrode insulation on measured [NO] in tubules of normal and Nx rats. To test the theoretical possibility that NO could permeate the epoxy insulation during calibration, which would result in falsely low tubular fluid readings, several rats from each group were studied using the same electrode. In series A experiments, carried out in experiments in 1999-2000, two epoxy-insulated electrodes were used for one or more rats in each of the two groups. In these individual experiments, tubular fluid [NO] remained higher in Nx vs. normal rats. In series B experiments, carried out in late 2000 and early 2001, two glass-insulated electrodes were also used for one or more rats in each of the two groups. Figure 3 shows actual recordings made by a glass-insulated electrode from three tubular sites on the same nephron of an Nx rat and those made the next day, using the same electrode, in a normal rat. As in the results from series A, tubular fluid [NO] almost invariably was higher in Nx rats (see also RESULTS).


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Fig. 3.   Intratubular recordings from normal and 5/6 nephrectomized (Nx) rats using the same glass-insulated electrode on successive days. A: recording showing NO measurements from a distal, late proximal (LP), and early proximal (EP) tubule from the same nephron of a normal rat kidney. The surface fluid (asterisk) is the zero point taken immediately before the electrode was inserted into the nephron segment. The picoampere difference is taken from the surface fluid to the shoulder of the response. The time scale is compressed so that stable and more level shoulders are apparent in real time. B: corresponding measurements from an Nx rat using the same electrode. See also RESULTS.

NG-monomethyl-L-arginine inhibitor experiments. These studies in 4 Nx rats (15 late proximal and 9 distal tubules) were designed to confirm that elevated NO measurements in Nx rats were truly NO based rather than caused by an unknown interfering substance in this renal ablation model. If NO readings fall after NG-monomethyl-L-arginine (NMMA; Sigma) inhibition, the significant influence of an unknown substance would be unlikely. With the use of the glass-insulated electrode and the protocol described by Deng and Baylis (3), a polyethylene catheter was inserted above the renal arteries by using a femoral approach. Control late proximal or distal tubular fluid was sampled, and immediately thereafter a bolus of 6 mg/kg body wt (BW) NMMA was injected just above the renal arteries, followed by an intravenous sustaining infusion of 0.4 mg · kg BW-1 · min-1 until the end of the experiment. The same tubules were repunctured at 40 and 60 min after the NMMA was administered (see Fig. 5, A and B).

Statistical methods. Tubular fluid [NO] differences were tested by one-way ANOVA and pairwise multiple comparisons using the Bonferroni method.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Most tubular fluid recordings were made in three segments from the same nephron, and distal tubules were first punctured so as not to change the loop of Henle flow, possibly eliciting secondary NO effects at proximal sites. The surface fluid zero point was taken to be the measurement made immediately before the nephron was punctured (see METHODS). The picoampere difference was taken from the surface fluid measurement to the shoulder of the response. (For the purposes of presentation, the time scale was compressed so that the stable and more level shoulder in real time is much more apparent in the late proximal tubule than is shown in Fig. 3.) Figure 3, A and B, shows [NO] from distal, late proximal, and early proximal segments in sequence from the same nephron from a normal and from an Nx rat.

As described in METHODS, in series A experiments 4 normal and 4 Nx rats were studied with epoxy-insulated electrodes, whereas in series B (4 normal and 5 Nx rats) glass-insulated electrodes were used. In each group of four rats, recordings from three segments were usually obtained from each of three to four nephrons in each rat. A few unpaired measurements were also included. When results from each early proximal, late proximal, and distal tubule site in series A were compared with those in series B, the data in only one of these six comparisons were significantly different. In normal rats, distal tubule [NO] in series A vs. series B was 141 ± 10 vs. 79 ± 19 nM, P < 0.01. No other significant differences were present in early proximal, late proximal, or distal samples from normal or Nx groups in either series A or series B. Figure 4 shows results from each nephron site from the combined groups in both series A and B, including the normal rat distal tubule groups whose series A vs. series B distal tubule data were significantly different (see above). Means of [NO] responses from normal and Nx rats (~30-40 tubules/site) are presented. There is no significant difference in [NO] among tubular sites within either the normal or Nx groups. However, there is a fourfold elevation in [NO] at all three sites in Nx rats vs. normal rats (P < 0.001).


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Fig. 4.   Tubular fluid [NO] at different nephron sites in normal and Nx rat tubules. In each group, recordings from 3 segments were usually obtained from each of 3-4 nephrons/rat. It is clear that normal rats show little difference in [NO] at the 3 sites. Corresponding [NO] measurements from Nx rats show an ~4-fold increase in mean values. Prox., proximal. *P < 0.001 by 1-way ANOVA and pairwise multiple comparisons using the Bonferroni method.

NMMA inhibitor studies. Figure 5, A and B, depicts results from 4 Nx rats (triplicate measurements in 15 late proximal and 9 distal tubules) showing the effects of an intrarenal bolus and a subsequent sustaining infusion of NMMA on serial tubular fluid [NO] determinations, as described in METHODS. Both late proximal and distal tubule [NO] fell significantly by 30-50% compared with measurements made before NMMA administration. In other experiments (not shown), tubular fluid [NO] was not altered by the duration of the micropuncture period up to ~60 min post-control period, in the absence of NMMA and with or without the aortic catheter in place.


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Fig. 5.   Effect of NMMA on tubular fluid [NO] in Nx rats. A: mean of 15 triplicate late proximal [NO] determinations in 4 rats before and after NMMA administration. i.v., Intravenous. B: mean of 9 triplicate distal [NO] determinations in 4 rats before and after NMMA administration. See METHODS. *P < 0.02 vs. control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study demonstrates for the first time that direct, in situ tubular fluid NO can be measured in vivo. By the modification of a World Precision Instrument combined reference and amperometric electrode with a 7-µm-diameter tip and perforation of the tubule first with a beveled glass pipette, rapid, sensitive, and stable recordings of tubular fluid [NO] could be made after in vitro calibration. We also show that, with one exception (see RESULTS), there was no significant difference in the ~110 nM [NO] in distal, late proximal, and early proximal tubular fluid of normal rats, whereas in Nx rats tubular fluid [NO] was greatly increased to ~400 nM with no differences among the three segments.

Significance of changes in intratubular [NO]. Our assumption is that the tubular fluid [NO], although possibly generated in part by tubular epithelium, mostly reflects the [NO] in the surrounding parenchyma. NO is generally agreed to be highly diffusable, as well as labile with a short half-life, so that tubular fluid concentrations most likely reflect that of a dynamic steady state, with high rates of dissipation and formation of the gas, as is likely the case with intrarenal PCO2. Indeed, our saline perfusion experience is consistent with the view that there is a constantly renewable peritubular gas tension that diffuses rapidly into the tubular lumen, with concentrations returning to the tubule's "basal" level after the cessation of rapid saline perfusion, stops dissipating the intratubular [NO].

Pathophysiological implications. With the assumption, therefore, that the entire cortex of normal rat kidneys has an [NO] of ~110 nM, local NO production must greatly exceed this level to be an effective signal. Thus it is likely that if macula densa cells do release NO to relax afferent arteriolar tone over a period of perhaps 10 s to 1 min, this local production of NO may be >400 nM. Of course, our results from intratubular measurements offer no insight into local interstitial [NO]. For this purpose, puncturing the interstitium in vivo would be necessary and, if successful, would facilitate defining the role of NO in TGF responses.

The striking finding of a fourfold increase in [NO] in the tubular fluid of Nx vs. normal rats is inconsistent with some reports of low [NO] in remnant kidneys. For example, Aiello et al. (1) reported that even after 7 days post-renal ablation, urinary nitrite/nitrate excretion rates fall and that they fall even further at 30 days. Aiello et al. concluded that intrarenal [NO] is reduced compared with that in control rats. In contrast, our 2 wk post-Nx rats showed increased [NO] by direct measurement of subcortical nephrons.

Indeed, it should be emphasized that our NO measurements come from single surviving cortical nephrons, which may not reflect medullary structures where countercurrent effects may reduce [NO], as is the case with PCO2. Nevertheless, this direct-measurement approach to [NO], instead of indirect inference, is required to firmly establish the significance and implications of inhibiting NOS and using NO donors or nitrate/nitrate ratios to influence and evaluate the NO system. Indeed, in our laboratories, using the same preparation as in the present study, Roczniak et al. (10) showed that 2 wk after 5/6 nephrectomy, both cortical neuronal NOS (nNOS) and the urinary nitrate/nitrite excretion are decreased. The present findings of an increase in [NO] in Nx rats not only underscores the importance of direct measurement but also stimulates analysis to reconcile the apparent contradiction. The following possibilities seem worthy of consideration. First, despite a decrease in nNOS, enhanced substrate delivery per surviving nephron may enhance [NO] in the remnant tissue. Second, there may be decreases in degradation and/or diffusion of NO in the remnant, so even with reduced rates of nNOS NO formation may achieve a higher steady state. Third, an atypical NOS in Nx rats, perhaps similar to that already described (12), may stimulate greater NO production. Fourth, perhaps the subcapsular tubules punctured in this study do differ from those in midcortex, from which the nNOS tissue sample was taken. Fifth, although unlikely, it is conceivable that, in the Nx rats, a non-NO product of renal ablation might elicit a signal from the electrode, notwithstanding the work already published by Xang et al. (15) excluding reactivity of several NO-like substances. For this reason, we undertook an additional series of experiments in which we injected a bolus of NMMA into the renal artery, followed by a nontoxic sustaining infusion. As shown in Fig. 5, A and B, [NO] fell significantly. With the foregoing in mind, we surmise that more examples of unexpected levels of [NO] will be revealed as direct NO measurements are more widely used. Indeed, Barker et al. (2) showed an increase in inducible NOS without an increase in NO formation in LPS/gamma -interferon-stimulated astrocytes.

In summary, we demonstrate for the first time that tubular fluid [NO] can be measured in real time, in vivo, with a modified commercial electrode. We have profiled these concentrations at three tubular sites in rats after 5/6 nephrectomy (2 wk postsurgery), showing approximately fourfold increases in [NO] compared with normal rats.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Medical Research Council of Canada (D. Z. Levine). Portions of this work were presented at The First Biennial International Conference of the Nitric Oxide Society, San Francisco, CA, June 2000.


    FOOTNOTES

Address for reprint requests and other correspondence: D. Z. Levine, Dept. of Medicine, Health Science Bldg., 451 Smyth Rd., Rm. 1333, Ottawa, Ontario, Canada K1H 8M5.

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.

Received 8 February 2000; accepted in final form 28 February 2001.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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2.   Barker, JE, Strangward HM, Brand MP, Hurst RD, Land JM, Clark JB, and Heales SJR Increased inducible nitric oxide synthase protein but limited nitric oxide formation occurs in astrocytes of the hph-1 (tetrahydrobiopterin deficient) mouse. Brain Res 804: 1-6, 1998[ISI][Medline].

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12.   Singh, R, Pervin S, Rogers NE, Ignarro LJ, and Chaudhuri G. Evidence for the presence of an unusual nitric oxide- and citrulline-producing enzyme in rat kidney. Biochem Biophys Res Commun, 232: 672-677, 1997[ISI][Medline].

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14.   Weight, SC, and Nicholson ML. Nitric oxide and renal reperfusion injury: a review. Eur J Vasc Endovasc Surg 16: 98-103, 1998[ISI][Medline].

15.   Zhang, X, Cardosa L, Broderick M, Fine H, and Lin J. An integrated nitric oxide sensor based on carbon fiber coated with selective membranes. Electroanalysis 12: 1113-1117, 2000[ISI].


Am J Physiol Renal Fluid Electrolyte Physiol 281(1):F189-F194
0363-6127/01 $5.00 Copyright © 2001 the American Physiological Society