(Hypertension. 1996;27:649-652.)
© 1996 American Heart Association, Inc.
Articles |
Influence of NaCl Concentration at the Macula Densa on Angiotensin II–Induced Constriction of the Afferent Arteriole
From the Hypertension and Vascular Research Division, Department of Internal Medicine and Heart and Vascular Institute, Henry Ford Hospital, Detroit, Mich.
Correspondence to YiLin Ren, MD, Hypertension and Vascular Research Division, Henry Ford Hospital, 2799 W Grand Blvd, Detroit, MI 48202.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Abstract The macula densa, a plaque of specialized tubular epithelial cells, monitors NaCl concentrations in tubular fluid and controls resistance of the glomerular afferent arteriole (AA). In vivo micropuncture studies suggest that there are significant interactions between angiotensin II (Ang II) and macula densa control of glomerular hemodynamics. We tested the hypothesis that Ang II causes stronger constriction of the AA when NaCl concentration at the macula densa is elevated. Rabbit AAs and the attached macula densa were simultaneously microperfused in vitro, and dose-response curves to Ang II were obtained when the macula densa was not perfused or was perfused with either low NaCl (Na+, 26 mEq/L; Cl-, 7 mEq/L) or high NaCl (Na+, 84 mEq/L; Cl-, 65 mEq/L). Ang II induced stronger constriction when the macula densa was perfused with high NaCl; the decrease in diameter at 100 pmol/L was 29±5.6% (n=7) compared with 2.1±1.2% (n=8) for the nonperfused macula densa or 6.1±4.2% (n=7) for low NaCl (P<.002). However, there was no such difference in the action of norepinephrine. Adding furosemide (10 µmol/L) to the macula densa perfusate abolished the difference in Ang II action between low and high NaCl at the macula densa. Since AA tone is higher when the NaCl concentration at the macula densa is elevated, we tested whether augmented Ang II action is due to higher AA tone. Preconstriction of the AA by 20% with norepinephrine had no effect on Ang II action. Thus, our results demonstrate that sensitivity of the AA to Ang II increases when NaCl concentration at the macula densa is elevated. Such modulation of Ang II action by macula densa NaCl concentration may be important in the control of glomerular hemodynamics.
Key Words: arterioles • tubuloglomerular feedback • furosemide • rabbit • hemodynamics
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Introduction |
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TopAbstractIntroductionMethods Results Discussion References |
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The macula densa, a plaque of specialized tubular epithelial cells, monitors NaCl concentrations in tubular fluid and controls resistance of the glomerular AA. In this mechanism, called TGF, increased NaCl concentration at the macula densa constricts the AA, thereby decreasing glomerular capillary pressure and hence single-nephron GFR. It has been reported that Ang II regulates renal hemodynamics not only by exerting a direct vasoconstrictor effect but also by modulating the sensitivity of the TGF response.1 2 3 4 5 In vivo micropuncture studies have demonstrated that blocking the action of Ang II with either ACE inhibitors or receptor antagonists attenuates but does not abolish TGF response.2 3 4 5 In addition, infusion of Ang II in rats already in a state of angiotensin blockade partially restored feedback response,2 3 4 5 6 while Ang II infused either intravenously or into peritubular capillaries enhanced TGF responses in normal rats.7 These findings suggest that there are significant interactions between Ang II and TGF.
To study the interactions between Ang II and macula densa control of glomerular hemodynamics directly, we performed in vitro microperfusion of both the AA and attached macula densa. We examined (1) whether NaCl concentration at the macula densa influences the vasoconstrictor action of Ang II on the AA and (2) whether adding furosemide (which blocks Na+, K+, Cl- cotransport) to the macula densa perfusate alters Ang II action in the AA.
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Methods |
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TopAbstractIntroductionMethods Results Discussion References |
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We used a method similar to that described previously to isolate and microperfuse AAs with macula densa attached.8 9 Briefly, young male New Zealand White rabbits fed standard rabbit chow (Ralston Purina) and given tap water ad libitum were anesthetized with ketamine (50 mg/kg IM) and given an injection of heparin (500 U IV). The kidneys were removed and sliced along the corticomedullary axis. Slices were placed in ice-cold MEM (Gibco) containing 5% BSA (Sigma Chemical Co) and dissected under a stereomicroscope (Olympus SZH) as described previously. From each rabbit, a single superficial AA and its intact glomerulus were microdissected together with adherent tubular segments consisting of portions of the thick ascending limb of the loop of Henle, the macula densa, and the early distal tubule. With a micropipette, the microdissected sample was transferred to a temperature-regulated chamber mounted on an inverted microscope (Olympus IMT-2) with Hoffman modulation. Both the AA and the end of either the distal tubule or thick ascending limb were cannulated with an array of glass pipettes as described previously8 9 (Fig 1
). Intraluminal pressure of the AA was measured by
Landis' technique, with a fine pipette introduced into the lumen
through the perfusion pipette. The AA was perfused with
oxygenated MEM (95% O2/5%
CO2) containing 5% BSA, and intraluminal pressure was
maintained at 60 mm Hg throughout the experiment. The macula densa was
perfused with a modified Krebs-Ringer bicarbonate buffer
(oxygenated to pH 7.4) at a rate of 10 nL/min.
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The bath consisted of 100 µL MEM containing 0.15% BSA and was exchanged continuously at a rate of 1 mL/min. Microdissection and cannulation were completed within 90 minutes at 8°C, after which the bath was gradually warmed to 37°C for the rest of the experiment. Once the temperature was stable, a 30-minute equilibration period was allowed before any measurements were taken. Images were displayed at magnifications up to x1980 and recorded with a Sony video system consisting of a camera (DXC-755), monitor (PVM1942), and video recorder (EDV-9500). The diameter of the distal AA was measured with an image-analysis system (Fryer).
Experimental Protocols
AA Responses to Ang II With
Various NaCl Concentrations at the
Macula Densa
While AAs were perfused at 60 mm Hg, the macula densa was
perfused with either low NaCl (Na+, 26 mEq/L;
Cl-, 7 mEq/L; n=7) or high NaCl
(Na+, 84 mEq/L; Cl-, 65
mEq/L; n=7) or was left unperfused with the tubular segments cannulated
(n=8). After a 30-minute equilibration period, increasing doses of Ang
II (1 pmol/L to 1 nmol/L) were added to the bath, and the AA was
observed for 5 minutes at each dose. Since we found that the action of
Ang II on the AA was enhanced by high NaCl at the macula densa, we also
examined the effect of another vasoconstrictor,
norepinephrine (1 nmol/L to 1 µmol/L added to the
bath).9 10
Effect of Furosemide at
the Macula Densa on AA Constriction Induced
by Ang II
We examined whether increased Ang II action with high NaCl
at
the macula densa is linked to active transport. Furosemide was added to
low- and high-NaCl macula densa perfusates at 10 µmol/L, and
Ang II action was examined as described above.
Effect of
Ang II Action on AAs Preconstricted With
Norepinephrine
Since increasing NaCl concentration at the macula densa
constricts AAs by 10% to 20%,9 10 we next examined
whether augmentation of Ang II action is due to higher basal AA tone.
While tubular segments containing the macula densa were cannulated but
left unperfused, AAs were perfused with norepinephrine (200
nmol/L) to decrease luminal diameter by approximately 20%, and the
effect of Ang II was examined 10 minutes later.
Statistics
Values are expressed as mean±SEM. A paired
t test
was used to examine whether the diameter at a given concentration was
different from the control value. ANCOVA was used to examine whether
dose-response curves differed between groups, and a two-sample
t test was used to examine whether the change in diameter at
a given concentration differed between groups. A value of
P<.013 was considered significant because of multiple
comparisons.
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Results |
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TopAbstractIntroductionMethods Results Discussion References |
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AA Responses to Ang II With Various NaCl Concentrations at the Macula Densa
Fig 2
shows an example of AA responses to
100
pmol/L Ang II when the macula densa was perfused with low- or high-NaCl
solution, and Fig 3 depicts AA diameter at each
concentration of Ang II. When the macula densa was not perfused, the
basal luminal diameter of the AAs was 22.6±1.6 µm; Ang II had no
effect on the AA until the concentration reached 1 nmol/L, which
decreased luminal diameter by 22.3±6.3% (P<.003). When
the macula densa was perfused with low NaCl, the basal luminal diameter
of the AAs was 18.7±1.7 µm, and AA responses to Ang II were similar
to the nonperfused group. However, when the macula densa was perfused
with high NaCl, the basal luminal diameter of the AAs was 21.9±0.8
µm, and Ang II caused much stronger constriction of AAs; the
decreases in diameter induced by Ang II were 15.2±3.2% and
29.2±5.6% at 10 and 100 pmol/L, respectively, compared with
0±1.6%
and 2.1±1.2% for unperfused macula densas and 0.3±3.2% and
6.1±4.2% for macula densas perfused with low NaCl. In contrast,
norepinephrine produced almost identical constriction
regardless of whether the macula densa was perfused with high NaCl or
was not perfused (Fig 4).
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) or low (L) NaCl (
) or was left unperfused (non-perf,
) throughout the experiment. After control measurements were
taken, Ang II (10-12 to
10-9 mol/L) was added to the bath and the
arteriole was observed for 5 minutes at each dose. *P<.006
for low vs high NaCl.
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Effect of Furosemide at the Macula Densa on AA Constriction Induced
by Ang II
When the macula densa was perfused with a low-NaCl solution
containing furosemide, the basal luminal diameter of AAs was 21.0±1.0
µm (n=7), whereas it was 19.1±1.2 µm (n=7) for
high NaCl plus
furosemide. In the presence of furosemide, Ang II–induced constriction
of AAs no longer differed whether the macula densa was perfused with
low or high NaCl (Fig 5). Dose-response curves were
similar to those obtained when the macula densa was not perfused or was
perfused with low NaCl.
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Effect of Ang II Action on AAs Preconstricted With
Norepinephrine
Pretreatment with norepinephrine reduced basal
diameter by 19%, from 22.9±0.8 to 18.7±1.1 µm (n=6).
However, it
did not affect the vasoconstrictor action of Ang II (Fig 6).
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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We directly examined the interaction between Ang II and macula densa control of glomerular hemodynamics. We found that AA sensitivity to Ang II was greatly augmented when NaCl concentration at the macula densa was elevated. Such interaction does not appear to be due to increased basal tone associated with high NaCl at the macula densa, since preconstriction with norepinephrine had no effect on Ang II sensitivity. In the presence of furosemide, however, no interaction was observed, suggesting that intact tubular transport at the macula densa is needed. Ang II may be unique in interacting with the macula densa at the level of the AA, since norepinephrine action was not altered by NaCl concentration at the macula densa.
It has been reported that there are significant interactions between Ang II and TGF in the control of glomerular hemodynamics. On the one hand, micropuncture studies have examined changes in single-nephron GFR or stop-flow pressure when NaCl concentration at the macula densa was varied while Ang II was maintained at a given level. These studies showed that in normal rats, Ang II infused either systemically or directly into the peritubular capillaries enhances TGF sensitivity, while blocking Ang II action with either ACE inhibitors or receptor antagonists attenuates TGF response.2 3 4 5 7 In addition, the reduced TGF sensitivity associated with ACE inhibition can be partially reversed by infusion of Ang II.2 6 Taken together, these results demonstrate that Ang II can enhance TGF response. On the other hand, there is little information as to whether the level of the TGF signal can modulate the vasoconstrictor action of Ang II in the renal microcirculation. In early studies, Hall et al11 and Hall and Granger12 reported that intravenous Ang II increased calculated resistance of both the AA and efferent arteriole in dogs treated with ACE inhibitors, while blocking TGF with ureteral occlusion abolished increased AA resistance without affecting the increase in efferent arteriole resistance. In contrast, norepinephrine action was well preserved even after ureteral occlusion. More recently, Ikenaga et al13 reported that in juxtamedullary nephrons perfused in vitro, AA constriction induced by Ang II was significantly attenuated either by blockade of TGF with furosemide or interruption of flow to the macula densa. Here, we provide direct evidence that vasoconstrictor action of Ang II in the AA is regulated by NaCl concentration at the macula densa and hence activity of TGF. Our results are fully in accord with previous studies, suggesting an important interaction between Ang II and macula densa control of glomerular hemodynamics.
Although the mechanism of interaction is not clear from the present study, it does not seem to be due to elevated AA tone induced by high NaCl at the macula densa but rather appears to involve tubular transport at the macula densa. It may be that Ang II increases tubular transport at the macula densa, thereby elevating levels of the vasoconstrictor signal (as yet undefined) sent from the macula densa to the AA. It is also possible that Ang II and the vasoconstrictor signal interact at the level of the AA in a synergistic manner. In this regard, it is interesting to note that Ang II and adenosine may enhance each other's action in the AA,14 and adenosine seems to play an important role in AA constriction induced by TGF.15 16 17
The interaction between Ang II and macula densa control of AA resistance may play an important role in various physiological and pathological conditions.12 18 Under physiological conditions, the activity of the renin-angiotensin system seems to be associated mainly with sodium balances. Despite the potent vasoconstrictor action of Ang II, changes in sodium intake usually cause little change in the GFR.19 Such stability may be due to a complex interplay of various renal paracrine hormones20 21 and well-integrated actions of Ang II on both tubules and the AA, which are controlled by NaCl concentration at the macula densa. For instance, during low NaCl intake, increased Ang II would stimulate proximal tubular reabsorption, leading to decreased NaCl delivery to the macula densa (and hence decreased NaCl concentration at the macula densa). Since TGF response is extremely sensitive to changes in NaCl concentration at the macula densa,17 22 it is conceivable that even a small change in NaCl would lessen Ang II–induced constriction of the AA, thereby maintaining the GFR. Thus, fine-tuning of Ang II action on the AA by NaCl concentration at the macula densa may serve as a basis for the stability of the GFR and hence homeostasis of body fluid and electrolytes despite daily variations in sodium intake.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by National Institutes of Health grants HL-28982 and HL-46518.
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References |
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