Tonic and phasic influences of nitric oxide on renal blood flow autoregulation in conscious dogs

Armin Just, Heimo Ehmke, Uwe Wittmann, and Hartmut R. Kirchheim

I. Physiologisches Institut, Ruprecht-Karls-Universität Heidelberg, D-69120 Heidelberg, Germany


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The aim of this study was to investigate the influence of the mean level and phasic modulation of NO on the dynamic autoregulation of renal blood flow (RBF). Transfer functions were calculated from spontaneous fluctuations of RBF and arterial pressure (AP) in conscious resting dogs for 2 h under control conditions, after NO synthase (NOS) inhibition [NG-nitro-L-arginine methyl ester hydrochloride (L-NAME)] and after L-NAME followed by a continuous infusion of an NO donor [S-nitroso-N-acetyl-DL-penicillamine (SNAP)]. After L-NAME (n = 7) AP was elevated, heart rate (HR) and RBF were reduced. The gain of the transfer function above 0.08 Hz was increased, compatible with enhanced resonance of the myogenic response. A peak of high gain around 0.03 Hz, reflecting oscillations of the tubuloglomerular feedback (TGF), was not affected. The gain below 0.01 Hz, was elevated, but still less than 0 dB, indicating diminished but not abolished autoregulation. After L-NAME and SNAP (n = 5), mean AP and RBF were not changed, but HR was slightly elevated. The gain above 0.08 Hz and the peak of high gain at 0.03 Hz were not affected. The gain below 0.01 Hz was elevated, but smaller than 0 dB. It is concluded that NO may help to prevent resonance of the myogenic response depending on the mean level of NO. The feedback oscillations of the TGF are not affected by NO. NO contributes to the autoregulation below 0.01 Hz due to phasic modulation independent of its mean level.

renal hemodynamics; transfer function; tubuloglomerular feedback; myogenic response; nitric oxide donor


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

AS IN OTHER VASCULAR BEDS, nitric oxide (NO) exerts a strong vasodilator effect (24) and is tonically active in the renal circulation (3). It thereby is an important determinant for the mean level of renal blood flow (RBF). Irrespective of this tonic influence, there is consistent evidence that despite the shift of the mean level of RBF, the autoregulatory function does not seem to be affected by the presence of NO (2, 4, 22). However, more recent studies have shown that both the myogenic response (14, 19, 25) as well as the tubuloglomerular feedback (TGF) (6, 18, 33, 35-37) are attenuated, whereas neither of them seems to be mediated by NO. It is not clear why these modulating influences of NO were not found to affect the autoregulation of total RBF (2, 4, 22). However, in the latter studies, the autoregulatory function was assessed in response to stepwise artificial reductions of renal artery pressure, whereas under physiological conditions the kidney has to cope with dynamic fluctuations of arterial pressure (AP) distributed over a wide range of frequencies (20). Since one major stimulus for the release of NO is shear stress on the vascular wall (7), the influence of NO may be more pronounced during dynamic changes of AP. This might be of particular importance in those frequency ranges in which the autoregulatory efficiency is small (20), and thus fluctuations of pressure will be accompanied by relatively large variations of flow. Furthermore, due to the shear stress-induced release and the attenuating effect on the myogenic response, it is conceivable for NO to prevent resonance of the myogenic response. This effect may also be more important in response to dynamic changes.

Therefore, the aim of the present study was threefold: first, to find out whether NO might contribute to the prevention of resonance of the myogenic response; second, to test whether the attenuating effect of NO on the TGF can be observed in the conscious dog; and third, to investigate the contribution of NO to the autoregulatory efficiency in response to the physiological blood pressure variability. To this end, the transfer functions were calculated from the spontaneous fluctuations of AP and RBF recorded in conscious resting dogs with and without NO synthase (NOS) inhibition. To more specifically assess only the role of phasic modulation of NO release, the same experiments were also done after NO had been fixed at a physiological level by constant infusion of an NO donor after NOS inhibition.


    METHODS
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INTRODUCTION
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All data were derived from 24 experiments on 10 conscious, chronically instrumented foxhounds (23-33 kg body wt), held on a standard dog diet (SSniff, Soest, Germany; or Alma 5003, Botzenhardt, Kempten, Germany) and kept under artificial light-dark cycle (6:00 AM to 6:00 PM light, 6:00 PM to 6:00 AM dark). All experiments and procedures were done in accordance with the national law for the care and use of research animals (Regierungspräsidium Karlsruhe, license 37-9185.81/105/94).

Surgical procedures. The dogs were surgically prepared under sterile conditions. After premedication with atropine (0.5 mg sc; Braun, Melsungen, Germany) and propionylpromazine (0.64 mg/kg sc, Combelen; Bayer, Leverkusen, Germany), anesthesia was induced by pentobarbital sodium (20 mg/kg, Nembutal; Sanofi, Libourne Cedex, France) and maintained by halothane (0.8-1.0%, Fluothane; Zeneca, Planckstadt, Germany) and N2O (0.5 l/min). Through a left flank incision, the left renal artery and the abdominal aorta were exposed retroperitoneally. A polyurethane catheter was implanted into the abdominal aorta or into the renal artery. An ultrasonic transit time flow probe (Transonic, Ithaca, NY) was placed on the renal artery. In seven of the dogs, an inflatable cuff was implanted on the same artery distal to the flow probe to allow later assessment of the flow probe's zero flow offset. Catheter(s) and cable were subcutaneously led to the animal's neck, where they were exteriorized. At least 10 days were allowed before the experiments were done. During the first 9 days, the dogs received a combination of benzylpenicilline and sulfatolamide (3 ml sc every 3rd day, Tardomycel; Bayer). The catheter(s) was flushed every 2nd or 3rd day and filled with a solution of heparin (1,700 IU/ml) and cephtazidim (16 mg/ml, Fortum; Glaxo, Bad Oldesloh, Germany) in 0.9% saline.

Measurements. All experiments were done between 7:30 AM and 11:00 AM while the dogs were resting on their right side, as they had been trained to do previously. AP from the catheter was measured via a pressure transducer (Statham P23Db or P23XL; Gould, Valley View, OH) with a calibrated amplifier (Gould Pressure Processor). RBF was measured by the implanted flow probe connected to the flowmeter (Transonic T-106 or T-108), the output of which was low-pass filtered below 10 Hz by the built-in analog filter. The AP signal was not filtered, because its spectral content above 5 Hz is known to be negligible (see methods section in Ref. 20). AP and filtered RBF were continuously recorded on a computer (80286, or 80386 + Labtech Note Book V 7.11) at a sampling rate of 20 Hz, after analog-to-digital conversion (model DAS-16; Keithley-Metrabyte, Taunton, MA).

AP and RBF were continuously recorded for a duration of 2 h (6,750 s) while the dogs were quietly resting as described above. In each dog a control experiment was done and also one (in 8 dogs) or both (in 2 dogs) of the following two protocols.

For protocol 1 (n = 7), after a control period of 10 min, a bolus (50 mg/kg iv in 10 ml of saline, according to previous dose testing; see Ref. 2) of the NOS inhibitor NG-nitro-L-arginine-methyl ester hydrochloride (L-NAME; Sigma, Deisenhofen, Germany) was slowly injected intravenously. The recording was started 20 min after the bolus.

For protocol 2 (n = 5), after a control period of 20 min, L-NAME (50 mg/kg iv) was given. Then 20 min later, an intravenous infusion of the NO donor S-nitroso-N-acetyl-DL-penicillamine (SNAP; Alexis, Grünberg, Germany) was begun at an initial dose of 4 µg · kg-1 · min-1 in 12 ml/h of vehicle (11.06 ml/h saline + 40 µl/h DMSO). The dose was then adjusted so that mean RBF was restored to the level observed during the 20-min control period of the same experiment. The final dose was 4.6 ± 0.5 µg · kg-1 · min-1. The recording was started 10 min after the final dose adjustment.

In the control experiments, which were done on separate days, the recording was started after a control period of at least 30 min. To avoid interferences by possible long-lasting effects of L-NAME, the control experiments were always done first. An analysis of 20 control experiments done between day 12 and 90 after implantation surgery failed to show any time-dependent changes of either the gain below 0.01 Hz (linear regression: -0.0153 dB/day, r = 0.154) or of any other aspect of the transfer function between AP and RBF. In the two dogs in which both protocols with L-NAME were done, at least 7 days were allowed between the two experiments, and the order was changed between the dogs.

Data analysis. All calculations were done off-line by specifically designed programs as described in more detail previously (20). Heart rate (HR) was derived off line from the 20 Hz AP data. For the calculation of the transfer function, the 20 Hz data files were digitally low-pass filtered (cutoff 3.5 Hz, finite impulse response, order 50) and then decimated by 1:4 to a rate of 5 Hz. This will produce aliasing in the frequency range above 1.5 Hz, but it allows one to keep the ratio of sampling to cutoff frequency small and the cutoff frequency far outside of the frequency range of interest. These 5-Hz data were split into 4 blocks of 8,192 data points each (~27 min). Zero-offset of the flowmeter (RBF0) was determined in separate experiments. RBF was corrected by subtracting the individual RBF0. No correction was made in the three dogs without a renal artery occluder. AP was reduced by 16 mmHg in all dogs to account for renal artery occlusion pressure. The transfer function was calculated from the cross-spectral density of (AP - 16 mmHg) and (RBF - RBF0) (each normalized to the respective mean value) divided by the autospectral density of (AP - 16 mmHg) (Blackman-Tukey). After conversion of the gain values into decibels [20×log(gain)], a mean transfer spectrum was calculated from the consecutive spectra and averaged for all dogs of each group. Since the transfer function compares the fluctuations of RBF and AP, this method will only then give an ideal reflection of the underlying regulatory mechanisms if any fluctuation of RBF is predictably related to the fluctuations of AP. This ideal condition is impaired if RBF is not only determined by AP but is also influenced by extraneous noise or by some other input, or if the relation between AP and RBF is not always the same, i.e., is nonlinear. The coherence gives a measure of the extent to which fluctuations in RBF can be predicted from those of AP, similar to what the correlation coefficient does in the time domain (5), and is expressed in values between 0 and 1. Thus the coherence indicates to what extent the ideal condition for the transfer function is fulfilled. In general, a coherence of 0.5 is considered to indicate a sufficient degree of this condition (5). The coherence between AP and RBF and power density spectra (Blackman-Tukey) of AP were calculated from the same blocks of 8,192 values as the transfer function. The mean coherence for each experimental group of the present study was higher than 0.5 at all frequencies (Fig. 1). Integrated spectral density (iPSD) was derived by a modified rectangular rule (sum of spectral densities × frequency range of integration). Integration ranges were arbitrarily chosen from the characteristics of the transfer function. The phase was derived from the ratio between the imaginary and the real part of the transfer function. The direct current component and the lowest two frequencies of all spectra were discarded because of their inaccuracy resulting from the nonstationarity and nonrhythmicity of the data set (5). Calculation of less spectra of longer duration increased the scatter of the results so much that no valid conclusions could be drawn at the lower frequencies. It was also not possible to enhance the observation time, since RBF remained stabile for only 2 h and then gradually decreased during the continuous infusion of SNAP. Consequently, the lowest frequency for which valid information could be gained in this study was limited to 0.0018 Hz.


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Fig. 1.   Coherence between renal arterial pressure and flow. Group averages of the coherence for each experimental group: after NG-nitro-L-arginine methyl ester hydrochloride (L-NAME; solid line, n = 7), after L-NAME with substituted NO (dash-dot line, n = 5), and in respective control experiments [dashed (n = 7) and dotted (n = 5) lines].

Statistical methods. Differences in the hemodynamic mean values were tested by the Student's t-test for paired samples. Gain, phase, and iPSD values were compared with the paired control and to 0 dB by ANOVA in conjunction with the Student-Newman-Keuls test. P < 0.05 was considered significant. All values are expressed as means ± SE.


    RESULTS
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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After L-NAME mean AP was elevated from 107 ± 4 to 122 ± 5 mmHg (Fig. 2A). HR was markedly reduced from 83 ± 5 to 47 ± 1 beats/min after L-NAME. RBF greatly fell from 221 ± 32 to 117 ± 12 ml/min or by 42 ± 7%.


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Fig. 2.   Hemodynamic responses to L-NAME. Mean values of arterial pressure (MAP, A), heart rate (HR, B), and renal blood flow (RBF, C) averaged over the entire 2-h recording period in control experiments (open bars) and after L-NAME (solid bars). Values are means ± SE from 7 dogs. * P < 0.05 vs. control.

The transfer functions between AP and RBF are shown in Figs. 3 and 5. In the control experiments (broken lines in Figs. 3 and 5) the gain (Figs. 3A and 5A) was above 0 dB for frequencies higher than ~0.1 Hz. Below ~0.01 Hz, a plateau of small gain values was reached. Around 0.03 Hz, a peak of higher gain was observed. The phase (Figs. 3B and 5B) was positive at all frequencies, with maxima at around 0.1 Hz and 0.02 Hz, respectively, indicating that the changes in RBF occurred in advance to those in AP.


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Fig. 3.   Transfer function from spontaneous fluctuations of renal arterial pressure and flow: effect of L-NAME. A: mean gain in decibels (dB). B: phase in radians (rad). Values in control experiments (broken line) and after L-NAME (solid line) are means ± SE (dotted lines) from 7 dogs.

After L-NAME (solid line in Fig. 3) the gain between 0.1 Hz and 0.2 Hz was markedly elevated (6.7 ± 0.7 vs. 2.2 ± 0.4 dB, P < 0.05). The phase in this range was also significantly enhanced to values in excess of pi /4 (0.93 ± 0.10 vs. 0.41 ± 0.03 rad, Fig. 3). The iPSD of the fluctuations of AP, i.e., the input of the transfer function, was not significantly altered by L-NAME between 0.1 and 0.2 Hz (Table 1). However, above 0.2 Hz, iPSD was markedly elevated (35.0 ± 5.8 vs. 11.6 ± 1.7 × 10-3 mmHg2). The peak of high gain around 0.03 Hz was not substantially altered. iPSD between 0.02 and 0.04 Hz was not changed (Table 1). Below 0.01 Hz, the gain was slightly but significantly elevated from -7.6 ± 1.0 to -3.7 ± 1.3 dB; nevertheless, it was still significantly smaller than 0 dB (Fig. 3). The positive phase observed in the range up to 0.03 Hz was abolished (-0.20 ± 0.12 vs. 0.36 ± 0.12 rad, P < 0.05). iPSD in this range tended to be smaller, but this change was not statistically significant (Table 1). The coherence between AP and RBF was higher than 0.5 at all frequencies (Fig. 1).

                              
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Table 1.   Integrated spectral density of blood pressure

When L-NAME was combined with exogenous supplementation of NO (L-NAME + SNAP), mean AP (85 ± 1 mmHg) and RBF (209 ± 4 ml/min) were not significantly different from control (92 ± 4 mmHg and 201 ± 10 ml/min), whereas HR was slightly elevated from 68 ± 3 to 81 ± 5 beats/min (Fig. 4).


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Fig. 4.   Hemodynamic responses to L-NAME with substituted NO. Mean values of AP (MAP, A), HR (B), and RBF (C) averaged over the entire 2-h recording period in control experiments (open bars) and after L-NAME with continuous infusion of NO (solid bars). Values are means ± SE from 5 dogs. * P < 0.05 vs. control. SNAP, S-nitroso-N-acetyl-DL-penicillamine.

In the transfer function, the gain above 0.1 Hz was not affected (Fig. 5A, 1.9 ± 0.5 vs. 2.2 ± 0.1 dB). The phase (Fig. 5B; 0.58 ± 0.09 vs. 0.44 ± 0.05 rad) and iPSD (Table 1) were also not significantly altered in this range. Around 0.03 Hz, the peak of high gain was not appreciably affected in its magnitude. Although the center frequency of the peak may appear slightly shifted toward higher frequencies, this effect was almost exclusively observed in one dog. iPSD in this range was reduced (Table 1). Below 0.01 Hz, the gain was slightly but significantly elevated from -9.0 ± 1.0 dB to -6.0 ± 0.6 dB, while it remained significantly smaller than 0 dB (Fig. 5). iPSD in this range was slightly but not significantly reduced (Table 1). The phase below 0.03 Hz was not appreciably affected (Fig. 5; 0.46 ± 0.18 vs. 0.38 ± 0.10 rad). The mean coherence for each group was higher than 0.5 at all frequencies (Fig. 1).


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Fig. 5.   Transfer function from spontaneous fluctuations of renal arterial pressure and flow: effect of L-NAME with substituted NO. A: mean gain. B: phase. Values in control experiments (broken line) and after L-NAME with continuous infusion of NO (solid line) are means ± SE (dotted lines) from 5 dogs.

To find out whether the decrease in the transfer gain below 0.01 Hz might be explained by the slight depression of iPSD after L-NAME and after L-NAME combined with SNAP, the linear regression between iPSD and the transfer gain was evaluated and compared with the same relation from 20 control experiments done in our laboratory on dogs of the same strain. Although a very slight trend for higher gain values with smaller iPSD cannot be excluded (linear regression: gain = -0.21 dB/10-3 mmHg2 × iPSD - 5.4 dB, r = 0.37), the elevation of the gain observed after L-NAME or L-NAME with SNAP was higher than expected for the change in iPSD. Furthermore, in one dog the gain was elevated, although the iPSD was even higher after L-NAME. Calculation of the transfer function from data of linear trend corrected values of AP and RBF gave the same results.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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The dynamic response of the autoregulation under physiological conditions was assessed by the transfer function between the spontaneous fluctuations of AP and RBF. A transfer gain of less than 1 (0 dB) denotes efficient regulation, whereas values above 0 dB indicate that changes in RBF are enhanced compared with AP. At least two autoregulatory mechanisms can be discerned by the transfer function (10, 11, 16, 20): a fast mechanism at 0.1-0.2 Hz, recognized by the decrease of the gain below this frequency, and a slower one around 0.03 Hz, characterized by a peak of high gain (17). The faster mechanism probably corresponds to the myogenic response (17), whereas the slower one reflects the TGF, since it is accompanied by similar fluctuations of tubular flow and chloride concentration (15) and is abolished by ureter ligation (11) or furosemide (1, 20). Below 0.01 Hz a plateau of low gain is attained, the level of which reflects the resulting regulatory efficiency.

The tonic influence of the mean level of NO was investigated by the experiments after NOS inhibition. L-NAME caused a sustained hypertension, pronounced bradycardia, and a reduction of RBF by more than 40% (Fig. 2). The role of phasic modulation of NO release was assessed by clamping the ambient concentration of NO at a physiological level. The successful clamping was shown by the fact that the level of RBF remained the same throughout the 2-h recording period with constant infusion of SNAP. The slight elevation of mean HR at this dose (Fig. 4) suggests that the infused NO was more effective on the majority of the resistance vessels than on the renal vasculature. This is consistent with the view that the renal vessels receive a particularly abundant supply of endogenous NO compared with other vessels (29, 31).

Influence on the myogenic response. Above 0.1 Hz, there was a pronounced elevation of phase and gain after L-NAME. It should be noted that above 0.2 Hz there was a pronounced increase in the fluctuations of AP, so that no valid conclusions can be drawn in this range. However, between 0.1 and 0.2 Hz, there was no difference in the fluctuations of AP. The rise in phase and gain are both compatible with an enhanced activity of the myogenic response in the absence of NO. The fact that the gain was elevated to even more positive dB values than under control conditions means that the amplitude of the fluctuations of flow exceeded those of pressure even more than they do normally and therefore indicates the development or enhancement of resonance of the myogenic response. It should be noted in this respect that the occurrence of resonance requires a linear control system to be of at least second order. This means that the system would need a component that is not only responsive to the absolute level of the stimulus but also to the rate of its change. Because of its temporal characteristics, such a system would have a phase at the corner frequency of pi /2, whereas a first order system lacking such a rate-sensitive component would reach only pi /4. Therefore, the enhancement of the phase by L-NAME in excess of pi /4 is compatible with the view that a rate-sensitive component of the myogenic response, which has been described in isolated arterioles (12), may gain more influence in the absence of NO and may by this means render the system more susceptible to resonance.

It cannot be entirely excluded that the rise in gain and phase was also due to changes in capacitive effects of the renal vascular tree. However, since vascular compliance is expected to decrease in the absence of NO (13), this would rather depress the gain toward 0 dB. To explain a higher gain, one would have to assume more complexly that the increase in resistance much outranks the loss of compliance.

After clamping of NO, there was essentially no change in the gain nor in the phase, indicating that the effects of NO depend on the mean level and not on phasic modulation of NO. This is in contrast to findings in rats, in which the gain in this frequency range was elevated after clamping of NO (8). Because of the preliminary nature of that report, an explanation for the discrepancy is difficult to determine. However, it might be of importance that those experiments were done under anesthesia with halothane, which by itself has been shown to interfere with the characteristics of the transfer function especially in this frequency range (10).

Taken together, the previously described attenuating effect of NO on the myogenic response (14, 19, 25) is confirmed. However, at least from the present results, this influence seems to be due to the mean level of ambient NO and does not depend on phasic modulation of NO release. This attenuation contributes to the suppression of resonance of the myogenic response, mediated by the mean level of NO, which may predominantly affect the rate-sensitive component of the myogenic response.

Influence on the TGF. The peak of high gain around 0.03 Hz was essentially unchanged after elimination of NO (Fig. 3) as well as after clamping of NO at a physiological level (Fig. 5). The slight shift of the peak to higher frequencies in the latter case was not a consistent finding.

The findings are compatible with the view that the function of the TGF neither depends on the presence nor on the endogenous modulation of NO. This observation in the conscious dog is in line with the consistent finding in micropuncture studies that NO is not mediating the TGF (6, 18, 33, 36, 37). However, the latter studies had also shown that the gain of the TGF is enhanced in the absence of NO, and more recently this was also demonstrated after clamping endogenous modulations of NO (35). There are at least four possible explanations for this discrepancy. First, comparison of the peak of high gain in the two control groups shown in Figs. 3 and 5 demonstrates that there is considerable variation in the height of this peak and thus suggests that the accuracy of this method may be insufficient to detect small changes. However, it should be noted that after blockade of the TGF, the gain was markedly reduced to less than -5 dB (20). If the TGF should not be at its maximum strength under control conditions, then a considerable range of further variation may be expected, even more so on this logarithmic scale in the vicinity of 0 dB. Second, it might be that there is no change in the gain of the TGF in the conscious resting dog. In almost all of the mentioned micropuncture studies, the TGF had been tested only at zero and maximum tubular flow or chloride concentration, whereas it is well possible that in the conscious dog these extremes are not employed normally. More subtle determination of the TGF had shown that NO attenuated the TGF also at lower than maximum tubular flows, but the effect of NO on the sensitivity of the TGF to small changes along the function curve (slope) appeared to be much less pronounced (33). Nevertheless, if tested in response to small perturbations from the ambient state, the sensitivity of the TGF was still found to be augmented after NOS inhibition in rats (36). The sensitivity, however, depends critically on the location of the ambient state on the TGF function curve, and this might be different in the anesthetized rat and the conscious dog. A third explanation for the unchanged peak in the transfer function in our study would be that the TGF of each single nephron was indeed sensitized after L-NAME but that this effect was counteracted by a diminished synchronicity of the nephrons. Such a balanced effect might allow the kidney to modulate the TGF gain of the single nephrons while preserving the magnitude of the fluctuation of mean RBF and thus presumably of the average glomerular and postglomerular pressure in this frequency range. A fluctuation of this kind might be important for postglomerular functions such as medullary perfusion and pressure natriuresis (27). As a fourth possibility, it must be considered that there is evidence for important interaction between the TGF and the myogenic response at the frequency range around 0.03 Hz (9). Because of the serial arrangement of these two systems, an enhanced activity of the myogenic response will diminish the TGF, due to attenuation of the signal reaching the macula densa. On the other hand, any constriction induced by the TGF will activate the myogenic response in more upstream segments, because of the resulting increase in intravascular pressure. Furthermore, by L-NAME, NO production is inhibited in both the endothelium and the macula densa. It is thus conceivable that the loss of endothelial NO might have indirectly diminished the TGF via exaggeration of the myogenic response, whereas the elimination of NO from the macula densa might have directly strengthened the TGF, and that these two effects exactly canceled out each other.

Our study is in line with the notion that the TGF is not mediated by NO. Furthermore, the magnitude of the TGF-mediated fluctuations of total RBF in relation to pressure around 0.03 Hz is not modified by the mean level or by endogenous fluctuations of NO. Although our data do not allow a firm conclusion about possible changes of the TGF on the single-nephron level, the finding for total RBF demonstrates that such changes do not have a major impact on these fluctuations in the total RBF and thus on those in the average glomerular capillary pressure.

Influence on the regulatory efficiency below 0.01 Hz. In the frequency range below 0.01 Hz, the gain was slightly but significantly elevated, whereas it was still smaller than 0 dB both after elimination of NO and after clamping of NO at a physiological level. Thus the regulatory efficiency for fluctuations of AP occurring slower than with a cycle length of 100 s was attenuated, but not entirely abolished. This attenuation seems to hold true down to the lower limit of frequencies assessed, i.e., 0.0018 Hz or 9 min cycle length. Smaller frequencies could not be assessed because of limited accuracy of the transfer calculation as explained in the METHODS section.

Our data suggest that NO contributes to the autoregulation of RBF below 0.01 Hz. This effect is brought about by endogenous fluctuations of NO and is independent of the ambient level of NO. This is an unexpected result with regard to the well-supported attenuating effect of NO on both the myogenic response and the TGF as discussed above. It also contrasts to the unaltered autoregulation of total RBF after NOS inhibition (2, 4, 22). Methodological effects do not seem to account for this finding. The slight and nonsignificant depression of the iPSD of AP fluctuations, both after L-NAME and after L-NAME with substituted NO, was too small to account for the elevation of the gain. Furthermore, trend correction also did not change the results. With respect to this apparent discrepancy, it should be noted that the autoregulation of total RBF in the above-mentioned studies (2, 4, 22) had been assessed by consecutive stepwise artificial reductions of renal AP. Apart from the fact that step changes are different from dynamic fluctuations, it should be noted that the elevation of the gain was most pronounced below 0.003 or 0.004 Hz, i.e., corresponding to cycle lengths of 250-300 s or 4-5 min, whereas the step reductions had a duration of less than 5 min. In most of the micropuncture studies, the TGF had been investigated in response to zero and maximum stimulation, which most probably does not occur in the conscious resting dog. In one study, in which the TGF had been investigated in response to small perturbations from the ambient level (36), the perturbation steps had a duration of only 2 min.

The TGF and more slowly acting juxtaglomerular functions as mentioned in the following are unlikely to account for this regulatory effect of NO. Macula densa-mediated renin release may provide an adequate response time (<10 min) (30) but rather opposes the TGF and thus would allow only a very indirect influence of NO. Adaptation of the TGF to changes in body fluid volume (23) is unlikely to play a role in our experiments, since without drinking, the fluid volume can only have changed in one direction. Modulations of local gene expression (28) are most probably too slow to account for this effect. However, although the response time of resetting of the TGF during prolonged activation (32) may be slightly too slow (20-40 min, i.e., <0.0008 Hz), a partial contribution of this mechanism to the investigated frequency range above 0.0018 Hz is conceivable. Unfortunately, lower frequencies could not be assessed accurately by our method. As another possible explanation, providing a more adequate response time (more than 2 min, i.e., <0.008 Hz), a previously described chloride-sensitive release of NO from mesangial cells (34) should be considered.

Conclusions. It is concluded that NO may contribute to the suppression of resonance of the myogenic response. This does not depend on rapid counteracting changes of NO but rather on the mean level of ambient NO, and this may predominantly affect the rate-sensitive component of the myogenic response. The TGF-mediated oscillations of total RBF around 0.03 Hz are not affected by NO, neither tonically nor phasically, and thus are not mediated by NO. Whether the TGF gain of each single nephron is also not affected by NO under these conditions cannot be decided. NO contributes to the autoregulatory efficiency in the conscious dog in the frequency range below 0.01 Hz. This effect is brought about by phasic modulation of endogenous release independent of the mean level of NO.

Perspectives. The unexpected finding of a contribution of NO to RBF autoregulation below 0.01 Hz suggests the existence of an as yet unrecognized slowly acting regulatory mechanism that is mediated by NO. Although TGF resetting or completely unknown mechanisms also have to be considered, the hypothesis of a chloride-sensitive pathway that is mediated by NO release from mesangial cells (21, 26, 34) appears to be a likely explanation. Therefore, although the failure to detect any known constitutive isoform of NOS in these cells remains an objective (28), this mesangial pathway may well merit reconsideration in this newly defined frequency range.


    ACKNOWLEDGEMENTS

We thank PD Dr. H. Zanzinger, I. Physiolog. Institut, University of Heidelberg, for consultation about the choice of the NO donor. We gratefully acknowledge the excellent technical assistance by I. Keller, L. Mahl, A. Klein, and E. Röbel.


    FOOTNOTES

This study was supported by the Deutsche Forschungsgemeinschaft (project Ki 151/5-3 and Graduiertenkolleg für Experimentelle Nieren und Kreislaufforschung, Heidelberg).

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. §1734 solely to indicate this fact.

Address for reprint requests: A. Just, I. Physiologisches Institut, Universität Heidelberg, Im Neuenheimer Feld 326, D-69120 Heidelberg, Germany (E-mail: Justy{at}novsrv1.pio1.uni-heidelberg.de).

Received 17 August 1998; accepted in final form 30 November 1998.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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