Dynamic interaction between myogenic and TGF mechanisms in afferent arteriolar blood flow autoregulation

Matthew Walker III, Lisa M. Harrison-Bernard, Anthony K. Cook, and L. Gabriel Navar

Department of Physiology, Tulane University School of Medicine, New Orleans, Louisiana 70112


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The dynamic activity of afferent arteriolar diameter (AAD) and blood flow (AABF) responses to a rapid step increase in renal arterial pressure (100-148 mmHg) was examined in the kidneys of normal Sprague-Dawley rats (n = 11) before [tubuloglomerular feedback (TGF)-intact] and after interruption of distal tubular flow (TGF-independent). Utilizing the in vitro blood-perfused juxtamedullary nephron preparation, fluctuations in AAD and erythrocyte velocity were sampled by using analog-to-digital computerized conversion, video microscopy, image shearing, and fast-frame, slow-frame techniques. These assessments enabled dynamic characterization of the autonomous actions and collective interactions between the myogenic and TGF mechanisms at the level of the afferent arteriole. The TGF-intact and TGF-independent systems exhibited common initial (0-24 vs. 0-13 s, respectively) response slope kinetics (-0.53 vs. -0.47% Delta AAD/s; respectively) yet different maximum vasoconstrictive magnitude (-11.28 ± 0.1 vs. -7.02 ± 0.9% Delta AAD; P < 0.05, respectively). The initial AABF responses similarly exhibited similar kinetics but differing magnitudes. In contrast, during the sustained pressure input (13-97 s), the maximum vasoconstrictor magnitude (-7.02 ± 0.9% Delta AAD) and kinetics (-0.01% Delta AAD/s) of the TGF-independent system were markedly blunted whereas the TGF-intact system exhibited continued vasoconstriction with slower kinetics (-0.20% Delta AAD/s) until a steady-state plateau was reached (-25.9 ± 0.4% Delta AAD). Thus the TGF mechanism plays a role in both direct mediation of vasoconstriction and in modulation of the myogenic response.

renal hemodynamics; frequency analysis; vascular resistance; myogenic response; dynamic analysis; tubuloglomerular feedback


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IT IS WELL ESTABLISHED THAT in response to changes in renal arterial perfusion pressure over a wide range from as low as 70 to 180 mmHg, the intrarenal indices of renal microvascular function exhibit highly efficient autoregulatory behavior such that steady-state renal blood flow, glomerular filtration rate, glomerular pressure, proximal tubule pressure, and peritubular capillary pressure remain relatively unchanged (22-24). This autoregulatory behavior is considered to be mediated by the myogenic and tubuloglomerular feedback (TGF) mechanisms; however, the dynamic influence of these two mechanisms on a single afferent arteriole has not been examined under conditions where the responses, with both mechanisms operant, can be compared with those after one has been neutralized. Furthermore, it has been difficult to separate experimentally these autoregulatory mechanisms without the confounding influence of the other. Although steady-state evaluations have been used to characterize the magnitudes of the autoregulatory range, our purpose was to characterize the time-dependent nature of the controllers and investigate possible interactions between the two systems. Under in vivo conditions, fluctuations in arterial pressure are partially transmitted to the renal microvasculature and are dampened by renal autoregulatory mechanisms. Arterial pressure fluctuations that oscillate faster than the response speed of inherent renovascular control mechanisms may be transmitted to the glomerulus undampened. Hence it is important to characterize the dynamic and steady-state efficiency of renal autoregulation by directly examining the vascular segments involved in resistance alterations.

Investigations using admittance gain and fast Fourier transform frequency domain analysis have demonstrated that the two mechanisms that contribute to renal autoregulation have distinct kinetics that enable attenuation of spontaneous or induced fluctuations of arterial pressure at different frequencies. The faster of the two mechanisms, the myogenic mechanism, operates at 0.1-0.2 Hz (i.e., 5-10 s/cycle), whereas the slower, the TGF mechanism, operates at 0.03-0.05 Hz (i.e., 20-30 s/cycle) (9, 18, 28). Although mathematical assessments have been performed, it has proven difficult to separate the individual contributions of these two mechanisms due to the potential interactions where activation of one system reinforces the responsiveness of the second (12). Hence there has been little direct experimental characterization of their dynamic efficiency or of their interactions. Accordingly, we designed experiments to define these control systems as they act on single afferent arterioles. We used the in vitro blood-perfused juxtamedullary nephron technique (7) in conjunction with video microscopy (4), analog-to-digital acquisition and postprocessing, and photodiode-based erythrocyte velocimetry (13). A unique advantage of this preparation is that the TGF mechanism can be interrupted by transection of the papilla, which contains the loops of Henle of the nephrons being studied (27). The dynamic efficiency, interaction, and contributions of the two systems were determined by characterizing the autoregulatory response before and after interruption of distal tubular flow by papillectomy. The afferent arteriolar diameters (AADs), erythrocyte velocities (RBC-Vs), and calculated afferent arteriolar blood flows (AABFs) were assessed in response to a rapid step increase in pressure in the presence (TGF-intact) and absence (TGF-independent) of TGF influence on the afferent arteriole. The resulting response patterns were analyzed by exponential stripping, linear regression, and curve fitting. These studies reveal a distinct modulation of the myogenic mechanism by the TGF mechanism, suggesting a significant interaction between the two systems.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The in vitro blood-perfused juxtamedullary nephron preparation was slightly modified to allow measurements of transient afferent arteriolar responses to changes in perfusion pressure. Two male Sprague-Dawley rats (blood donor and kidney donor), weighing between 350 and 420 g (Charles River, MA), were utilized for each experiment. In compliance with the guidelines for the care and use of research animals, the rats were anesthetized with pentobarbital sodium (50 mg/kg ip). Bilateral nephrectomy was performed on the blood donor rat to minimize renin release, and blood was then collected via the carotid artery into a heparinized syringe. The other rat provided the kidney that was prepared as previously described (7, 27).

AAD, RBC-V, renal arterial perfusion pressure, and calculated AABF were determined and converted to a digital form by using a Pentium 200-MHz computer tethered to an analog-to-digital data-acquisition system (model MP1000, Biopac Systems, Santa Barbara, CA). The sampling rate was determined in consideration of the Nyquist criterion, whereby the sampling rate must be at least twice the frequency of the highest frequency of desired detection. Because previous studies in the rat have reported that the myogenic mechanism naturally oscillates at frequencies between 90 and 110 mHz, whereas the slower TGF mechanism oscillates in the range of 12-30 mHz, the sampling rate of 1 Hz satisfies the Nyquist.

Afferent arteriolar inside diameters were measured from videotaped images by using a digital image-shearing monitor (Instrumentation for Physiology and Medicine, San Diego, CA) at a distance from the glomerulus sensitive to the TGF mechanism (<100 µm) (27). Microvessel diameters were measured at 1 Hz by using a fast-frame, slow-frame technique to allow measurement of vessel diameter in the freeze-frame mode at 1-s intervals.

Analog centerline RBC-V was monitored by an RBC-V-tracking correlator (model 102, Instruments for Physiology and Medicine) with the use of the photodiode-based, dual-slit, cross-correlation technique (14, 27). Although centerline RBC-V exceeds the mean velocity within the vessel, there is a linear relationship between centerline RBC-V and mean velocity with the constant of 0.625, which is independent of both hematocrit and diameter observed in this study (16). Thus the calculated mean velocity and measured inside-vessel diameter (D) were used to estimate the volumetric flow (F), according to the formula
Flow<IT>=0.625V&pgr;</IT><FENCE><FR><NU><IT>D</IT></NU><DE><IT>2</IT></DE></FR></FENCE><SUP><IT>2</IT></SUP> (1)
where V is centerline RBC-V.

This calculation was conducted on the digitized data within the software program so that each data point would be included in the calculation and the subsequent waveform would be phase matched with waveforms of AAD and RBC-V.

Protocol. The experimental protocol involved establishment of control diameter and RBC-V measurement during constant renal arteriolar perfusion pressure of 100 mmHg for 15 min. Responses were obtained in control conditions with both myogenic and TGF mechanisms operant and after interruption of distal nephron volume delivery by transaction of the loops of Henle (papillectomy). After 5 min of steady-state measurements, a rapid unit pressure increase was imposed to determine the afferent arteriolar response in the presence of both operant myogenic and TGF mechanisms. Rapid increases in renal arterial pressure were elicited by adjusting the sensitivity of the tank regulator to insure optimized and consistent responsiveness. This elevated pressure was sustained for 3 min to allow the AAD and RBC-V to reach their respective steady states. The pressure was then returned to 100 mmHg and maintained at that pressure for 5 min. Afferent arteriolar responses to an increase in pressure were reassessed in paired fashion after interruption of distal nephron volume delivery by transection of the loops of Henle as performed by Takenaka et al. (27).

Data analysis. The noncompartmental method of residuals (also called curve stripping or feathering) was used to resolve the response curve into a series of exponential terms corresponding to the percentage change in the AAD per unit time. By identifying the number of different linear components within each primary response curve, each residual was fitted to an exponential function. The curve-stripping approach assumes that the response curves of the TGF-intact and -independent systems follow first-order rate processes as evidenced by linearity in the terminal portion of a semilog plot of each respective primary curve. The curve-stripping software program creates three dynamic modules that enable the analysis of a set of curves that identify and define the terminal linear portion of a semilogarithmic plot. The modules work from left to right, stripping the curve term by term. This arrangement defines up to three exponential terms
D=<LIM><OP>∑</OP></LIM> D<SUB>n</SUB>e<SUP>−&lgr;<SUB><IT>n</IT></SUB><IT>t</IT></SUP> (2)
where Dn and gamma n are the zero-time intercept and rate constant, respectively, for each exponential term.

The rate constants associated with the composite primary curve were extracted by an automated curve-stripping software algorithm (PK Solutions Software, Ashland, OH). A primary curve with two significantly linear portions will result in a biexponential with a first residual and no second residual. A primary curve with three significantly linear portions will result in a triexponential with a terminal phase, a first residual, and a second residual.

The time constants were calculated as exponential decay functions characterized as the time (tau ) for the primary curve to reach 37% of its initial value. The exponential frequency is then calculated by Eq. 3
&ohgr;=<FR><NU>1</NU><DE>&tgr;</DE></FR> (3)
Values are reported as means ± SE. Statistical comparisons of differences in the responses were conducted with the use of ANOVA, followed by the Newman-Keuls test. Differences in the mean values were deemed significant at P < 0.05.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The rapid (step completed in 2.66 ± 0.23 s) renal arterial pressure step input from 100 to 148 mmHg, as shown in Fig. 1, enabled assessment of the autoregulatory responses in both the TGF-intact and TGF-independent cases. Figure 2 illustrates a representative digitized (1 sample/s) tracing of the coupled responses of AAD, RBC-V, and AABF to a rapid step elevation in renal arterial pressure before (TGF-intact) and after interruption (TGF-independent) of the TGF mechanism. In the TGF-intact system (A), the AAD changed from 18.0 to 13.0 µm as the RBC-V increased from 18 to 52 mm/s transiently and remained elevated throughout the pressure step-input period. Initially, AABF transiently increased from 105 to 350 nl/min but began to return to baseline within 5-6 s after the pressure step. Within 100 s, AABF returned to control. Figure 2B illustrates the responses of AAD, RBC-V, and AABF in the same vessel to the same pressure perturbation after interruption of distal tubular flow. The AAD in this representation shows an increased control diameter (19.8 µm) and a diminished vasoconstriction (Delta D = 1.8. µm) relative to the TGF-intact response to increased renal arterial pressure, where Delta  denotes change. Similarly, there is a reduction in the AABF autoregulatory efficiency relative to the TGF-intact response.


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Fig. 1.   Mean rapid arterial pressure step (100 to 148 ± 1.1 mmHg). The 2.66 ± 0.23-s rapid step from control to the final pressure was consistently faster than the afferent arteriolar autoregulatory response (n = 22, 2 steps/kidney).



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Fig. 2.   A representative digital tracing (1 sample/s) of the afferent arteriolar diameter (AAD), erythrocyte velocity (RBC-V), and afferent arteriolar blood flow (AABF) responses to a step increase in renal arterial perfusion pressure (RAP) in a single vessel, before [tubuloglomerular feedback (TGF)-intact; A] and after [TGF-independent (Indep.); B] interruption of distal tubular flow.

The steady-state assessment of AAD and AABF are shown in Fig. 3. The figure indicates that at 100 mmHg, the vessels without TGF feedback have larger mean control diameters than those with intact TGF mechanisms at 100 mmHg (P < 0.05, n = 11). After pressure was raised and maintained at 145 mmHg, there was a greater maximum adjustment in diameter in the TGF-intact system. This indicates that, in the absence of the TGF mechanism, there remains a control system with less vasoconstrictive capacity. At 100 mmHg, the difference in baseline AABF (P = 0.08) is not statistically significant; however, after the pressure increase, there is a significant difference between the steady-state AABF (P < 0.05) values of the TGF-intact vs. TGF-independent responses.


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Fig. 3.   A summary plot of mean steady-state AAD (µm; n = 11; A) and AABF (nl/min; n = 8; B) responses to a unit pressure step in TGF-intact () and TGF-independent (open circle ) systems. *P < 0.05 vs. baseline value at 100 mmHg. #P < 0.05 vs. TGF-intact (n = 11).

The AABF, as calculated from AAD and RBC-V data, revealed (Fig. 4) that, despite the common passive increase in the AABF from baseline to peak of the two systems, the durations of the initial attenuations from peak AABF to trough (initiation of second slope) were different. The TGF-intact system returned peak flow (260.4 ± 31.1 nl/min) to trough (181.3 ± 22.2 nl/min) in 10 ± 1.1 s. The TGF-independent system returned peak flow (305.5 ± 79.2 nl/min) to trough (216.4 ± 32.1 nl/min) in 5 ± 0.8 s. The trough indicated the end of the initial autoregulatory control system and the onset of the secondary control system in the TGF-intact case. However, after congruent 14 s, the initial trough of the TGF-independent system is sustained with no significant slope, indicating the end of the initial autoregulatory response and no active secondary control system. The TGF-intact secondary response returns the AABF to control flow with a slight steady-state error of congruent 7 ± 2.7%. TGF-independent system, with a steady-state error of congruent 29 ± 3.1% is a much less efficient control system. The sustained oscillations in the TGF-independent steady-state system may suggest that the TGF inputs could provide a stabilizing effect.


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Fig. 4.   The transient autoregulatory blood flow response to step increase in RAP. The initial TGF-intact () and TGF-independent (open circle ) systems exhibit different response durations (Delta t = congruent 14 s vs. Delta t = congruent 6 s, respectively) during the return of AABF toward the first change in slope. The TGF intact initial decrease in AABF continued for 6 s longer than that of the TGF-independent initial decrease in AABF. After 16 s, the TGF-intact response supplies more efficient autoregulation (steady-state error of congruent 7%) than the TGF-independent response (steady-state error of congruent 30%). *P < 0.05 vs. TGF-intact response ( n = 7).

The AAD (n = 11) responses to a step change in renal arterial perfusion pressure before and after removal of the TGF mechanism are illustrated in Fig. 5A. The criterion for onset of vasoconstriction was defined as a change in AAD of at least 0.5 µm. In the TGF-intact kidneys, the criterion was met when the AADs changed from the control diameter of 16.9 ± 0.7 to 16.3 ± 03 µm. Despite a significant (P < 0.05) increase in the baseline AAD after papillectomy, neither the initial vasoconstrictive response time (0 to 6.3 ± 0.9 s) nor the magnitude (Delta D = -0.4 ± 0.l µm) of the TGF-independent response differed from the initial responses of the TGF-intact system. This initial change in diameter was used to signify the initial point of measurement. The mean control AAD of the TGF-independent system of 17.7 ± 0.7 µm (n = 11) was significantly greater than the baseline diameters of the TGF-intact system. In addition, the magnitude of the response to the pressure perturbation after interruption of the TGF influence (Delta D = -1.2 ± 0.02 µm) was significantly less than the magnitude of the afferent arteriolar vasoconstrictive response (Delta D = -4.4 ± 0.2 µm) to the same pressure perturbation when the TGF mechanism was intact.


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Fig. 5.   AAD (µm) responses to step increases in RAP before and after interruption of distal tubular flow (A). After interruption of distal flow, the vessels exhibited both an increased baseline diameter and a diminished autoregulatory response. *P < 0.05 vs. TGF-intact baseline. #P < 0.05 vs. control diameter at 100 mmHg. B: the superimposed mean afferent arteriolar autoregulatory percent changes in TGF-intact and TGF-independent vessels. *P < 0.05 vs. TGF-intact (n = 11).

The superimposed percent changes in AADs in the TGF-intact and -independent systems are illustrated in Fig. 5B. Overlaying the response patterns enabled a comparison assessment of the relative vasoconstrictive responses of the TGF-intact (Delta AADmax = -25.9 ± 0.4%) and TGF-independent (Delta AADmax = -7.02 ± 0.9%) systems, where AADmax is maximal AAD. Figure 6 represents the analysis of initial (A) and secondary (B) TGF-intact and -independent responses. The exponential stripping algorithm revealed that both the TGF-intact and -independent responses exhibited two significantly different slopes that could be fitted to two exponentials. The secondary response slope of the TGF-intact curve was found to be most linear in the time region bracketed by 24 and 97 s (Fig. 6B) whereas the TGF-independent curve was found to have the terminal linear portion bracketed by 13 and 97 s. The secondary responses of the TGF-intact and -independent slopes were significantly different (-0.20 vs. -0.01%/s, respectively), with the magnitude of the TGF-intact response being much greater than that of the TGF-independent response (Delta D = -14.6 vs. -0.83%). The plateau (-25.9 ± 0.4%) of the TGF-intact secondary curve was reached 58 ± 4.5 s after the completion of the initial response. In the TGF-independent system, the maximum vasoconstrictive response (-7.02 ± 0.9%) was reached on completion of the initial response (13 s), and there was no further significant vasoconstriction. The dynamic subtraction of these terminal linear portions from their respective composite primary curves resulted in the first-residual curves seen in Fig. 6A. The TGF-intact first-residual curve was bracketed by 6 and 24 s (Fig. 6A). The first residual of the TGF-independent curve was bracketed by 6 and 13 s. There was not a significant difference between the TGF-intact (-0.53%/s, r2 = 0.95) and the TGF-independent (-0.47%/s, r2 = 0.94) rates. However, the magnitude of the TGF-intact initial response (Delta D = -12.72%) was greater than that of the TGF-independent response (Delta D = -6.11%) and lasted an additional 11 s. The comparison of observed diameters and the fitted biexponential functions for each primary curve is shown in Fig. 7 and suggests that there is a dual control system operating in the TGF-intact system. Although the TGF-independent response is characterized by a biexponential function, the absence of a significant slope in the terminal portion of the primary curve suggests that the initial exponential slope represents only one control system involved in the response to the rapid pressure step input.


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Fig. 6.   TGF-intact () and independent (open circle ) initial (A) and final (B) dynamic response to pressure step. Exponential stripping analysis revealed that the TGF-independent (striped bar) initial response starts at congruent 6 and ends at congruent 13 s. The same analysis revealed a modulation of the initial response (A) such that the TGF intact (checkered bar) system starts at congruent 6 and ends at congruent 24 s. The secondary (B) response of the TGF-intact system starts at congruent 24 and plateaus after congruent 68 s. The secondary TGF-independent system exhibits no slope (-0.01%/s) after 13 s (n = 11). *P < 0.05 vs. TGF intact.



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Fig. 7.   The exponential fit to the mean TGF-intact primary curve for AAD (% control) after step increase in RAP (100-148 mmHg) (r2 = 0.94; A). B: exponential fit to the mean TGF-independent primary curve (r2 = 0.93).

Figure 8 shows the plot of the exponential fit to the TGF-intact initial curve (A), identified as operating in the range of 6 to 24 s by the exponential stripping program. The fitted exponential to the curve indicates that, although there is a significant difference between the TGF-intact and -independent initial response magnitudes, there is no significant difference between their relative slopes (B). The plot for the exponential fit to the TGF-intact secondary diameter response curve was identified to operate in the range of 25-97 s by the stripping program (C). The fitted exponential to this curve demonstrates a significant difference between the secondary responses of the TGF-intact and -independent (D) exponential curves.


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Fig. 8.   The initial (A and B) and final (C and D) exponential equations fitted to model the TGF-intact (A and C) and TGF-independent (B and D) responses. A: TGF-intact initial exponential model of the response (r2 = 0.94). B: TGF-independent initial exponential model of the response (r2 = 0.95). C: TGF-intact secondary exponential model of the response (r2 = 0.83). D: TGF-intact final (r2 = 0.62) exponential model of the response. pap, Papillectomy; myo, myogenic.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In previous studies of dynamic renal autoregulation, the kinetic activity of renal blood flow (28) and tubular fluid oscillations (29) has been used as an indirect index of myogenic and TGF activity as they control the afferent arteriolar vasculature in response to pressure perturbations. In the present study, however, linear assessments of AAD fluctuations in the time domain were used to directly characterize the two controlling mechanisms as they act on the vascular wall. Measuring vascular wall instead of blood flow dynamics complies with the control systems concept that the controlling element (afferent arteriole) is separate and distinct from its controlled variable, which is, in this case, the AABF (25). This approach has also revealed the control character of the largely unstudied, dynamic autoregulatory role of the renovascular myogenic mechanism in kidneys with both mechanisms operant.

Steady-state autoregulation of blood flow by normal kidneys in vivo is highly efficient and involves active constriction and dilation of the preglomerular microvasculature, particularly, the afferent arteriole (3, 5, 6). Several studies have shown that afferent arterioles of preparations that allow direct visualization of the vasculature, such as the perfused juxtamedullary nephron and the hydronephrotic kidney preparations, exhibit vasoconstriction in response to increases in perfusion pressure (3, 5, 6, 8, 15, 27). A previous study performed by Takenaka et al. (27) of steady-state AAD and AABF responses to a gradual pressure increase from 100 to 150 mmHg revealed a decrease of congruent 20% in the AADs of TGF-intact vessels and an autoregulation of AABF to within 8% of its control. The present steady-state pressure-induced AAD response (-22%) and the autoregulation of AABF to within 10% of its control confirm autoregulation and are consistent with this previous study. A comparison of the present TGF-independent impairment of afferent arteriolar steady-state autoregulation is consistent with previous evaluations (20, 27).

The present dynamic evaluation of the TGF-intact and -independent afferent arteriolar response curves revealed that both the initial and final slopes exhibit distinct kinetics and is consistent with the operation of two different control mechanisms. The TGF-intact initial response exhibited a time constant of 11.01 s (0.091 mHz) that was consistent with the kinetics of the myogenic mechanism (90-120 mHz) as obtained from renal blood flow studies (18, 28). Similarly, the TGF-independent initial response exhibited a time constant of 6.1 s (0.16 Hz) that was within the operating range of the myogenic mechanism. The secondary response of the TGF-intact system exhibited a time constant of 37.1 s (0.027 Hz), indicating a substantially different operating range of the TGF mechanism. However, the secondary response of the TGF-independent system exhibited no significant slope and an effective time constant of tau  = infinity , suggesting no operating control system. Cupples and Loutzenhiser (8) examined the dynamic autoregulation of the perfusate flow in the hydronephrotic kidney in which chronic ligation and tubular atrophy eliminate the possibility of vasoconstriction mediated by the TGF mechanism. The myogenic mechanism exhibited a 31% contribution to the autoregulation of flow (8), which is similar to our finding that the -7.0 ± 0.9% decrease in AAD of the TGF-independent system after 13 s exhibits a 30% contribution to the complete autoregulatory response. Although this myogenic contribution was significant, the inability of this mechanism to return the system to baseline suggests that the myogenic mechanism is an inefficient controller of blood flow when acting alone. Despite similarities in the magnitudes of the myogenic responses between the two studies, the kinetics differ in that the myogenic mechanism in the hypdronephrotic kidney exhibited a faster response rate (0.3-0.35 Hz). One reason for the difference may be attributable to the differences in viscosity and constituents of perfusion media. The colloid-free modified Dulbecco's medium used in their evaluation vs. whole blood (hematocrit = 33%) used in the present study may have also contributed to the difference. A second difference may be the result of the difference in objects measured, as the perfusate flow (controlled variable) kinetics may be faster than the kinetics of the vascular wall due to influences on flow that are upstream of the vascular wall site measured. Holstein-Rathlou and Marsh (17) developed a mathematical model that specifically predicts the transfer function of the myogenic mechanism as derived from renal blood flow. In that study, the kinetic response of the myogenic component of autonomous oscillations predicted at 90-110 mHz was more consistent with that of the present study and with other studies (11, 18).

The present study suggests that there is a delay in the action of both myogenic and TGF inputs at the level of the afferent arteriole. The delay in the onset of the myogenic mechanism after the pressure step is evident in the AAD and the AABF responses of both the TGF-intact and TGF-independent systems. In both systems, there is a passive increase in AABF from 0 until congruent 6 s. After this delay, there is an onset of rapid and continuous attenuation of AABF until the onset of a different slope. This myogenic delay is also seen in the latency of vasoconstriction onset in both the TGF-intact and -independent response to the pressure. However, in a study by Just et al. (19), the myogenic delay was found to be only 2 s. The difference in these findings may be due to both our strict criterion for vasoconstriction onset of 0.5 µm and our 2.6-s pressure step-input time. The TGF delay, as reflected in the AAD, has received less direct investigation. The TGF delay is defined as the time delay between the change in pressure and the onset of the slower secondary response curve. This delay in the present study was found to be congruent 13 s. The TGF delay is most evident in the TGF-independent system, where the plateau at 13 s marks the beginning of what would have been the onset of the TGF mechanism. This is further apparent when one looks at the absence of the plateau at 13 s in the TGF-intact system. The time course of the onset of the TGF-mediated vasoconstriction of the afferent arteriole we observed was similar to that of a study by Daniels et al. (10), which revealed that the delay time of the TGF mechanism in stop-flow pressure measurements was 15.7 s in response to changes in loop perfusion rates. Bell et al. (2) found that there was a delay of congruent 11 s before the orthograde perfusion of the loop of Henle resulted in a decrease in stop-flow pressure in dogs. This same input resulted in a further decrease in stop-flow pressure of congruent 8 mmHg over a variable range of 77 s (2). The primary conclusions we draw from our data on this issue is that the 13-s TGF delay is due to the cumulative durations of 1) pressure-mediated changes in distal tubular flow, 2) signal transmission from the macula densa to the afferent arteriole, and 3) activation of smooth muscle-mediated vasoconstriction to produce a change in AAD of at least 0.5 µm.

The single-nephron afferent arteriolar dynamics examined in the present study reveal both the autonomous temporal components of the TGF and myogenic mechanisms and the modulatory interactions between the two. The prolongation of the myogenic mechanism during the period from 6 through 24 s in the TGF-intact system relative to the TGF-independent myogenic duration (6-13 s) suggests that there is a modulation of the myogenic mechanism by the TGF control system. Similarly, the prolonged attenuation of the AABF before the slope change in the TGF-intact vessels supports such a modulatory interaction. This experimental evidence for interaction supports the model of myogenic and TGF interaction developed by Moore et al. (21). In that mathematical model, the interaction between the two systems is provided by the strong TGF response localized near the end of the afferent arteriole, which augments the myogenic response in upstream vascular segments. Although the results of the present study indicate that the TGF inputs start at congruent 13 s, the sustained myogenic kinetics from 13 to 24 s in the TGF-intact system suggests that TGF inputs during this period prolong the response. However, the TGF inputs independently provide the additional decrease (after 24 s) in AAD that renders the complete autoregulatory response. There is further support for this dominant threshold of TGF autoregulation in a study by Schnermann and Briggs (26), which revealed that, at high-loop flows (maximum TGF stimulation), the difference in pressure-dependent elevations in stop-flow pressure seen at lower loop flow rates was eliminated. Therefore, despite previous indirect, transfer function, evaluations (1, 10, 19) suggesting that the dynamics of the myogenic component are not largely altered after blockade of the TGF mechanism, our data suggest a strong modulation of the myogenic magnitude by the TGF mechanisms.

We have identified the temporal components of the myogenic and TGF mechanisms as they act and interact on the afferent arteriole to autoregulate AABF and have identified the kinetics, magnitude, and delay of the myogenic mechanism in the afferent arteriole in response to a rapid pressure step. These results help define the role and the limitations of the myogenic mechanism in renal blood flow autoregulation. Similarly, these results confirm the kinetics, magnitude, and delay of the TGF system in the afferent arteriole. In conclusion, the communication between the TGF mechanism and the myogenic mechanism determines both the baseline diameter and the autoregulatory response of afferent arterioles. The faster kinetics of the myogenic mechanism enables an initial decrease in AAD followed by superimposed TGF vasoconstriction. Therefore, the TGF mechanism makes a substantial contribution to the autoregulatory efficiency of AABF through both its indirect modulatory interaction with the myogenic mechanism and its direct vasoconstrictive actions on the afferent arteriole.


    ACKNOWLEDGEMENTS

The authors acknowledge valuable discussions with Drs. W. A. Cupples and K. D. Mitchell during the preparation of this manuscript.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-18426. During portions of this research, M. Walker III was a William T. Porter Fellow of the American Physiological Society and is presently a UNCF Dissertation Fellow of the Merck Foundation.

Portions of this work were presented at the Experimental Biology meeting in Washington, DC, in April 1999 and at the Annual Meeting of the American Society of Nephrology in Miami Beach, FL, in November 1999, and has been published in abstract form (FASEB J 13: 797.6, 1999 and J Am Soc Nephrol 10: A1967, 1999).

Address for reprint requests and other correspondence: M. Walker, III, Dept. of Physiology, Tulane Univ. School of Medicine, New Orleans, LA 70112 (E-mail: mwalker3{at}tulane.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.

Received 16 December 1999; accepted in final form 19 July 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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