Abstract After secretion by the heart, atrial natriuretic factor (ANF) circulates in plasma, whereas C-type natriuretic peptide (CNP), which is found in abundance in the endothelium, may regulate vascular tone in a paracrine manner. However, there is little information on the effect of CNP on renal microvessels. We hypothesized that CNP dilates the afferent arteriole via the nitric oxide pathway, whereas ANF acts directly on vascular smooth muscle cells. When we perfused rat kidneys with minimal essential medium and bovine serum albumin at 100 mm Hg and examined the juxtamedullary afferent arterioles, neither CNP nor ANF was found to have any effect. When the peptides were added to arterioles preconstricted with norepinephrine, CNP and ANF dilated them in a similar fashion; diameters increased by 25±4% (n=7) and 29±6% (n=6) at 10−7 mol/L, respectively (P<.008). Pretreatment with 10−4 mol/L N-nitro-l-arginine methyl ester (L-NAME) or 5×10−6 mol/L indomethacin blocked CNP-induced dilation; dilation by ANF was unaffected by indomethacin (52±25%, n=5) and potentiated by L-NAME (73±14%, n=5). Thus, CNP dilates the afferent arterioles via the prostaglandin/nitric oxide pathway, whereas ANF dilates them directly. This difference may be important in controlling glomerular hemodynamics.
Natriuretic peptides are important regulators of cardiovascular homeostasis through their actions on the vasculature, adrenal glands, kidneys, and brain.1 2 Although structurally similar, these peptides are encoded by separate genes and differ in their tissue distribution, receptor affinities, and biological actions.1 2 ANF and BNP are secreted largely by the heart and originally were considered circulating hormones. CNP was initially isolated from the porcine brain3 and was described as a neuromodulator because of its abundance in the central nervous system.4 5 In contrast to ANF or BNP, CNP is either undetectable5 or found at low levels6 7 in plasma; therefore, its peripheral actions are likely to be autocrine or paracrine in nature.
In the kidney, mRNA coding for CNP has been detected in the glomerulus, vasa recta bundle, and arcuate artery,8 whereas mRNA for its receptor is widely distributed, with relatively large amounts localized to the above structures as well as to distal nephron segments. Although CNP may have a role in the regulation of renal function, there is little information regarding its direct action on the renal microcirculation. In the present study, we compared the actions of CNP and ANF in the juxtamedullary Af-Art and determined whether NO or PGs might be involved.
We used the in vitro perfused juxtamedullary nephron preparation originally developed by Casellas and Navar.9 10 Male Sprague-Dawley rats weighing 350 to 400 g were anesthetized with pentobarbital sodium (40 mg/kg IP) and given an intravenous injection of heparin (300 U). The right renal artery was cannulated via the superior mesenteric artery and immediately perfused with ice-cold MEM (pH 7.4) containing 50 g/L BSA (Intergen). While still being perfused via the cannula, the kidney was removed and sectioned longitudinally, leaving the papilla intact in the dorsal two thirds. With the use of a stereomicroscope (model SZH; Olympus), small incisions were made in the lateral fornices, and the papilla was reflected back to expose the underlying pelvic cavity. The pelvic mucosa and the adipose and connective tissues overlying the inner cortical surface were removed, exposing the main branches of the renal artery, tubules, glomeruli, and related microvasculature of the juxtamedullary nephrons. Tight ligatures were placed around the distal ends of the large arteries so that the perfusate flowed only into the juxtamedullary nephrons arising from the proximal segments of the arterioles. The perfusate was administered with the use of 60-mL syringes, with driving force provided by a tank containing 95% O2/5% CO2. A small cannula positioned in the tip of a double-barreled perfusion cannula was connected to a DPM-II Universal pressure meter (Bio-Tek), allowing continuous measurement of renal perfusion pressure. Pressure was maintained at 100 mm Hg by adjusting an air regulator positioned between the tank and the syringes. The inner cortical surface of the kidney was continuously superfused with warmed MEM (37°C) containing 10 g/L BSA.
The perfusion chamber containing the prepared kidney was mounted on the movable stage of a Nikon microscope (Optiphot-2) equipped with water-immersion objectives (×4, ×10, ×20, and ×40). The tissue was transilluminated, and the focused image was transferred via a high-resolution Newvicon camera (model NC-70m; Dage-MTI) and displayed on a video monitor (model PVM-1343MD; Sony) while simultaneously recording the video signal (model SLV-R5UC; Sony). An image-analysis system (Fryer) was used to measure luminal diameter.
After the 20-minute equilibration period, the Af-Art image was recorded and the luminal perfusate was replaced with MEM containing either CNP or ANF (Peninsula Laboratories) in concentrations increasing from 10−12 to 10−7 mol/L. The Af-Art was observed for 10 minutes at each concentration.
Vessels perfused in vitro with a synthetic solution have little intrinsic tone, making it difficult to observe vasodilator responses. Therefore, to examine the vasodilator action of CNP or ANF, norepinephrine was added to the luminal perfusate to decrease the diameter by ≈30% to ≈40%. Then either CNP or ANF (10−12 to 10−7 mol/L) was added, and dose-response curves were obtained in the presence of norepinephrine. Luminal diameter is reportedly reduced by ≈20% when juxtamedullary Af-Arts are perfused with blood.11
After the equilibration period, L-NAME (Sigma Chemical Co), a compound that inhibits synthesis of NO, was added to the arterial perfusate at 10−4 mol/L and continued until the end of the experiment. Ten minutes later, we examined the effect of either CNP or ANF as described above.
Indomethacin (Sigma), a cyclooxygenase inhibitor, was dissolved in 0.02 mol/L Trizma base solution at a concentration of 5×10−3 mol/L. Indomethacin was added to the dissection solution at a final concentration of 5×10−6 mol/L from the beginning. In addition, indomethacin was added to the bath and arteriolar perfusate from the equilibration period to the end of the experiment and the effect of either CNP or ANF examined as described above.
Values are expressed as mean±SEM, and all statistical analyses were carried out with absolute values. Student’s paired t test was used to examine whether the diameter at a given concentration was different from the controls. 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<.008 was considered significant using Bonferroni’s adjustment for multiple comparisons.
Response to CNP or ANF in Nonpreconstricted Af-Arts
Luminal diameter was 17.8±2 μm (n=4) and 18.4±1.3 μm (n=4) before the addition of CNP and ANF, respectively. It remained unchanged during the application of CNP or ANF (17.1±2.3 μm at 10−7 mol/L CNP and 18.4±1.3 μm at 10−7 mol/L ANF).
Response to CNP or ANF in Norepinephrine-Preconstricted Af-Arts
Fig 1⇓ depicts the effects of CNP and ANF on Af-Art luminal diameter. Basal diameter in the CNP group was 20.1±1.3 μm (n=7), which was reduced to 13.8±1.3 μm by norepinephrine. Subsequent infusion of CNP at 10−12, 10−11, 10−10, 10−9, 10−8, and 10−7 mol/L dilated Af-Arts in a dose-dependent manner; diameter increased from 13.8±1.3 μm to 15.1±1.2, 15.3±1.4, 15.3±1.2, 16.0±0.9, 16.7±0.9, and 17.0±1.1 μm, respectively.
In the ANF group, basal Af-Art diameter was 18.5±1.5 μm (n=6), which was reduced to 11.2±1.4 μm by norepinephrine. Subsequent infusion of ANF at 10−12, 10−11, 10−10, 10−9, 10−8, and 10−7 mol/L dilated Af-Arts in a similar dose-dependent fashion, increasing from 11.2±1.4 μm to 12.5±1.7, 12.6±1.3, 13.3±1.5, 14.6±1.5, 15.0±1.9, and 14.5±1.7 μm, respectively. There was no significant difference in vasodilator response between CNP and ANF (P>.05).
Pretreatment With L-NAME
L-NAME decreased basal diameter significantly (from 18.9±0.8 to 16.6±0.9 μm), and it was reduced further to 11.7±1.9 μm with norepinephrine (n=5). As shown in Fig 2A⇓, the addition of CNP failed to induce dilation in the presence of L-NAME. Fig 3⇓ shows a comparison of CNP-induced changes in diameter from the preconstricted level in the presence and absence of L-NAME. Although CNP increased diameter by 3.2±0.3 μm at 10−7 mol/L in the absence of L-NAME, this vasodilator response was completely blocked by L-NAME.
In the ANF group, L-NAME decreased basal diameter from 21.0±2.4 to 16.7±1.1 μm (n=5), and it was reduced further to 11.9±1.2 μm by norepinephrine. Fig 2B⇑ shows ANF action in the absence and presence of L-NAME. In marked contrast to the action of CNP, the vasodilator action of ANF was not inhibited by L-NAME but rather was augmented at high concentrations. As shown in Fig 4⇓, the increase in diameter induced by ANF at 10−7 mol/L was 3.2±0.6 μm without L-NAME and 8.4±1.7 μm with L-NAME.
Pretreatment With Indomethacin
The diameter of the indomethacin-treated Af-Arts in the CNP group was 18.4±1.0 μm, which was reduced to 13.6±1.4 μm by norepinephrine (n=5). As with L-NAME, indomethacin completely blocked the vasodilator action of CNP (Fig 3⇑).
In the ANF group, the diameter of the indomethacin-treated Af-Arts was 19.8±1.8 μm, which was reduced further to 13.5±2.1 μm by norepinephrine (n=5). In contrast to CNP, ANF still dilated Af-Arts even in the presence of indomethacin, increasing by 5.2±1.5 μm in diameter at 10−7 mol/L (Fig 4⇑). The change in diameter with and without indomethacin did not differ for any dose of ANF.
There are at least three receptors for natriuretic peptides.1 Two of them—GC-A and GC-B—contain intracellular guanylyl cyclase moieties. These receptors have different affinities for natriuretic peptides; GC-A specifically binds ANF and BNP, whereas GC-B binds CNP. The best understood peripheral CNP system is that found in the vasculature, where endothelial cells synthesize CNP7 12 and GC-B receptors are believed to be localized predominantly to vascular smooth muscle cells.
In the kidney, immunoreactivity and mRNA for CNP as well as GC-B receptor mRNA5 8 have been detected in the vasculature, including the arcuate artery, glomeruli, and vasa recta. However, little information is available regarding the action of CNP on the renal microcirculation. Our results clearly demonstrate that CNP and ANF have similar vasodilator actions on the juxtamedullary Af-Art; however, the mechanism involved appears to differ. Although CNP-induced dilation appears to require intact synthesis of NO and PG, ANF-induced dilation may be due to direct action on the vascular smooth muscle cells of the Af-Art. Such differences are inconsistent with the notion that the mechanisms of action of ANF and CNP are both mediated by elevation of intracellular cGMP. However, recent studies indicate that at least in some tissues, CNP can exert its action without elevating cGMP and is therefore independent of GC-B receptors.13
Despite the potent hypotensive action of CNP, intravenous or intrarenal infusion has been shown to cause little change in renal hemodynamics in anesthetized dogs14 and conscious sheep.15 The reason for the discrepancy between our findings and those of other investigators may be related to species differences, the presence or absence of factors such as changes in hemodynamics and neurohormones, or both. In addition, it is possible that unlike juxtamedullary nephrons, which represent only a small proportion of the total, Af-Arts of superficial nephrons may not dilate in response to CNP. Because the juxtamedullary nephron has an important role in the mechanism of urine concentration and therefore sodium homeostasis, selective dilation of the juxtamedullary Af-Art may help explain the natriuretic and diuretic actions of CNP in the absence of changes in total renal blood flow and glomerular filtration rate.
Consistent with its systemic hypotensive effect, CNP has been shown to relax strips obtained from the aorta and saphenous arteries and veins.16 Although renal veins dilated in response to CNP, renal arteries were unresponsive. Furthermore, removal of the endothelium either had no effect (in arteries) or potentiated the vasodilator response (in veins). In contrast, we observed that CNP dilated juxtamedullary Af-Arts and that this dilation was completely blocked by either L-NAME or indomethacin. Such blockade is not due to the inability of Af-Arts to dilate because ANF-induced dilation was not impaired. Although the reason for the discrepancy between our study and others is not clear, it may be due to differences in the species and vascular segments that were studied. It may also be related to differences in the preparation used; in our experiments, the Af-Art endothelium was exposed to luminal flow, which is a potent stimulus of NO and PG synthesis, and thus there might be some interaction between CNP and flow, as seen with angiotensin II.17
We observed that the vasodilator action of CNP on the juxtamedullary Af-Art can be blocked by either L-NAME or indomethacin. If CNP acts through both NO and PGs, then blockade of one system would at best blunt (not abolish) the response because the other system would compensate. Lahera et al18 19 reported that in anesthetized dogs, the renal vasodilator action of acetylcholine or bradykinin was not affected by blocking the synthesis of either NO or PGs alone. Thus, our results indicate that there are significant interactions between NO and PGs in mediating CNP action. Although the mechanism remains speculative, there are several previous studies (including ours) that indicate an interaction between NO and PGs (particularly PGI2) at various intracellular levels.20 21 22 23 24
The vasodilator action of ANF in the present study is consistent with that reported in a number of previous studies that examined the effects of ANF on the renal microvasculature.25 In marked contrast to the complete blockade of CNP-induced dilation, neither L-NAME nor indomethacin blocked ANF-induced dilation. Although the lack of effect of indomethacin is consistent with previous studies that involved isolated rat Af-Arts,26 the potentiation of ANF-induced dilation with L-NAME (although significant only at high ANF concentrations) is surprising. It may be that both ANF and NO exert dilator action via a common mechanism; thus, inhibition of endogenous NO (and therefore the mechanism involved) would enhance the response to activation of the same mechanism by ANF. For example, Moncada et al27 reported that supersensitivity to nitrovasodilators develops after inhibition of endogenous NO synthesis.
In conclusion, we have provided evidence that CNP and ANF dilate juxtamedullary Af-Arts via discrete mechanisms. Although CNP appears to act via NO and PGs, ANF-induced dilation may be due to its direct action on vascular smooth muscle cells. Such differences may be important under certain physiological and pathological conditions.
Selected Abbreviations and Acronyms
|ANF||=||atrial natriuretic factor|
|BNP||=||brain-type natriuretic peptide|
|BSA||=||bovine serum albumin|
|CNP||=||C-type natriuretic peptide|
|GC-A||=||guanylyl cyclase type A|
|GC-B||=||guanylyl cyclase type B|
|L-NAME||=||N-nitro-l-arginine methyl ester|
|MEM||=||minimal essential medium|
This study was supported by National Institutes of Health grants HL-46518 and HL-28982.
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