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The fetal kidney receives 2-3% of cardiac output(1, 2). The mechanisms regulating fetal renal hemodynamics must be developed by the end of gestation for the kidney to function properly after birth. After birth, the fraction of cardiac output increases to 15-18% in the newborn and 20-25% in the adult(3, 4). The lower fetal kidney perfusion relative to postnatal perfusion is associated with a relatively higher renal vascular resistance(5). The mechanisms previously explored to explain the elevated fetal renal vascular resistance include responses to renal sympathetic nerve activation(5), the renin angiotensin system(5), and thromboxane(6). Fetal renal blood flow increases gradually with gestation as pressure increases and vascular resistance decreases(5, 7–9). The vasodilator PG also contribute to regulation of fetal renal hemodynamics(10).

Recently, NO has been implicated as a modulator of fetal renal blood flow(11, 12). The basal release of NO contributes to the developmental decrease in renal vascular resistance during the late third trimester (0.8 gestation) of fetal lambs(11). Renal blood flow increases in response to an ACh infusion and is inhibited by NOS inhibition(11). There is additional evidence supporting the hypothesis that endothelium-derived NO regulates renal hemodynamics in the developing piglet(12) and in the adult renal circulation of animals and humans(13–16). Based on differences between the fetal, newborn, and adult renal responses to NO synthesis inhibition, we hypothesize that NO release increases with progressive maturation of the kidney. There are no studies which examine the direct effect of NO-mediated relaxation of fetal renal arteries or the ontogeny of renal NOS. The purpose of this study is to determine whether relaxation to ACh, the prototypic endothelium-dependent relaxing agent, and SNP, an NO donor, is age-dependent before and after birth. To test this hypothesis we measured the relaxation of isolated renal arteries from fetal, neonatal, and adult guinea pigs to ACh and SNP, and the effect of inhibition of NOS and cyclooxygenase on ACh relaxation. These findings were then compared with Ca2+-dependent NOS (constitutive) activity of the fetal, neonatal and adult renal cortex.

METHODS

The protocols used for this investigation were approved by the University of Iowa Animal Care Committee.

Animals. Newborn (1-50 d old; n = 8) and reproductively mature adult nonpregnant female guinea pigs (n = 11) were anesthetized with xylazine (1 mg/kg) and ketamine (80 mg/kg; intraperitoneally). Fetuses were obtained from anesthetized time-mated pregnant guinea pigs via hysterotomy while the sow was under general anesthesia. A single fetus was obtained from each pregnant sow and selected from a similar location in the uterus. Fetuses (n = 23) of varying gestation were obtained and grouped into three gestational ages: fetal age 1(F1; n = 8) = 35-50 d (0.5-0.8); fetal age 2 (F2; n = 8) = 51-58 d (0.8-0.9); and fetal age 3 (F3; n = 7) = 60-63 d (0.9-0.97 gestation) (term = 65 ± 3 d).

Tissue preparation. An abdominal incision was made in all animals, and the left kidney was exposed for excision. The section of the abdominal aorta where the renal artery attaches was cleared of adherent tissue. The aorta was cut proximal and distal to the renal artery insertion, and the kidney along with its main renal artery was removed en masse. It was quickly placed in a dish containing ice-cold physiologic buffer solution for dissection. The kidney was cut sagittally to fully expose the proximal end of the main renal artery entering the kidney.

The main renal artery was isolated and excised from the kidney and cut into 3-4-mm long segments. Arteries were cleaned of adherent fat and connective tissue and cut into two rings under a dissecting microscope, mounted onto two tungsten wires (23-μm diameter each), and placed in a horizontal myograph apparatus for measurement of isometric force. Tissues were maintained in temperature-regulated baths containing Krebs bicarbonate buffer [(in mM): 130 NaCl, 4.7 KCl, 1.6 CaCl2, 1.18 KH2PO4, 14.9 NaHCO3, 1.17 MgSO4, 0.03 EDTA, and 11 dextrose] at 37°C, gassed with 95% oxygen and 5% carbon dioxide. Rings were allowed to equilibrate for 1 h before the start of each experiment. Optimal passive force was determined for each ring using submaximal KCl (60 mM) after incremental stretch.

Relaxation responses. Relaxation to cumulative addition of the endothelium-dependent relaxing agent, ACh (10-9 M to 10-5 M), and the endothelium-independent relaxing agent, SNP (10-9 M to 10-5 M), was measured in renal arteries contracted submaximally with PGF2α (5 × 10-6 M). We have previously shown that ACh relaxation of guinea pig renal arteries is abolished after endothelium-removal(17). Responses to ACh were compared between renal arteries obtained from the three fetal age groups (F1, F2, and F3). Responses to both ACh and SNP were measured in rings from fetal (a combined group of F1-F3; age range 35-65 d; n = 13), neonatal (1-50 d after birth), and adult (mature) guinea pigs. The concentration-response curves to ACh and SNP were each measured in rings obtained from separate animals.

To determine the role of NO and prostanoids, respectively, on ACh relaxation, rings from renal arteries obtained from fetal, neonatal, and adult kidneys were treated with either the NOS inhibitor, LNA (10-4 M; 30 min incubation) or the cyclooxygenase inhibitor, indomethacin (10-5 M; 30 min incubation). The concentration of PGF2α was adjusted so that contractile responses measured in the presence of either LNA or indomethacin treatment were not different from control and the duration of treatment with the inhibitor and the contractile agent before adding the relaxing agent was identical. The effect of each inhibitor was measured on a separate ring segment in parallel to an untreated control. For experiments using the fetal arteries, where the number of rings obtained was limited by the short length of the renal artery, the effect of each treatment was measured on the same artery segment as control. Preliminary control experiments were conducted demonstrating that there was no effect of time on the repeated ACh dose-response curve (not shown).

NOS activity. Because NOS activity could not be measured directly in the fetal renal microvasculature, we measured constitutive enzyme activity in the vasculature of the renal cortex by the conversion of L-arginine to L-citrulline plus NO. In a separate series of animals, kidneys were obtained from fetal, neonatal, and adult guinea pigs ranging in age from 0.5 gestation to reproductively mature females (5-10 animals per time point). Briefly, renal cortex was excised from the left kidney and immediately frozen in liquid nitrogen and stored at -80°C until assayed. The frozen tissue was homogenized (with an Ystral homogenizer) at 0°C in 3 volumes of buffer containing 320 mM sucrose, 50 mM Tris, 1 mM EDTA, 1 mM DL-diothreitol, phenylmethylsulfonyl fluoride (100 μg/mL), leupeptin (10 μg/mL), soybean trypsin inhibitor (100 μg/mL), and aprotinin (2 μg/mL) brought to pH 7.0 at 20°C with KCl. NOS activity was determined within 1 h of homogenization by measuring in duplicate the conversion of L-[U-14C]arginine to[U-14C]citrulline, as described in detail previously(18). L-Valine (50 mM) was added to the reaction buffer to minimize arginase activity. The total NOS activity was determined as the difference between the [14C]citrulline produced from control samples and samples containing both 1 mM EGTA to bind calcium and 2 mMNω-monomethyl-L-arginine, an inhibitor of NOS. Ca2+-independent activity was determined as the difference between samples containing 1 mM EGTA and samples containing both 1 mM EGTA and 2 mMNω-monomethyl-L-arginine. Ca2+-dependent activity was then calculated by subtracting calcium-independent activity from total activity. Intra-and interassay variations were each <8%(19).

Statistics. Relaxation responses are measured and illustrated in the figures as a percent of the contractile level to PGF2α from the baseline. Maximal relaxation to baseline is equivalent to 100%. Comparisons were made using a two-way ANOVA with repeated measures with relaxation response as the dependent variable and concentration, age, and treatment as independent variables. If the ANOVA demonstrated a significant difference at p < 0.05 among dose-response curves, then either a Duncan's or Newman-Keuls multiple comparison test was performed on individual concentrations. Agonist potency was measured as the -log ED50 value and determined as the -log concentration which produces 50% of the agonist's maximal response. It is obtained using regression analysis over the linear portion of the dose-response curve.

RESULTS

Relaxation to ACh of fetal renal arteries was compared between the three fetal age groups shown in Figure 1. Maximal relaxation to ACh of renal arteries from the earliest age group (F1: 66 ± 8%,n = 8) was significantly (p < 0.05) less than relaxation of arteries from the other gestational ages (F2, 96 ± 5%,n = 8; F3, 82 ± 8%, n = 7). Sensitivity to ACh(measured by the -log ED50 values) increased with advancing gestation in a similar pattern. The -log ED50 value of arteries from F1 was 5.70± 0.17 and significantly (p < 0.05) increased to 6.20± 0.06 and 6.23 ± 0.20 at F2 and F3, respectively. There was a trend for an increase in contractile responses to PGF2α with advancing gestation (F1, 20.3 ± 2.8 mg; F2, 27.1 ± 3.0 mg; F3, 33.6 ± 7.1 mg; ANOVA, p = 0.14).

Figure 1
figure 1

Fetal renal artery. Effect of fetal age (term = 65± 3 d gestation) on relaxation of renal arteries to cumulative addition of ACh. Responses are mean ± SEM (* = F1 vs F3; + = F1vs F2; p < 0.05).

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Relaxation to ACh and SNP was compared between renal arteries from fetal, neonatal, and adult guinea pigs (Fig. 2). Maximal relaxation to ACh was greater in renal arteries of both the neonatal and adult guinea pigs compared with the fetal guinea pigs. The -log ED50 values(Table 1) for ACh were similar between arteries from fetal(6.25 ± 0.20, n = 13) and neonatal (6.30 ± 0.08,n = 11) guinea pigs but both values differed significantly from those arteries obtained from the adult (6.71 ± 0.08, n = 5). Maximal relaxation to SNP did not differ among the three age groups(Fig. 2). However, the -log ED50 values for SNP of arteries from fetal guinea pigs (6.15 ± 0.13, n = 7) were significantly reduced (p < 0.01) from those arteries of neonatal(6.87 ± 0.17, n = 6) and adult (7.15 ± 0.22,n = 6) guinea pigs. There was a trend for an increase in contraction to a single concentration of PGF2α with advancing age (fetal, 20.6 ± 3.1 mg; neonatal, 55.2 ± 19.4 mg; adult, 73 ± 17.8 mg; p = 0.054).

Figure 2
figure 2

Relaxation of renal arteries of near term fetal(n = 13), neonatal (n = 11), and adult (n = 5) guinea pig kidneys to ACh (left) and SN (right). Responses are mean ± SEM (* = fetal vs adult; + = fetal vs neonatal; # = neonatal vs adult; p < 0.05).

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Table 1 Effect of age on sensitivity (-log ED50 values) to ACh and SN of guinea pig renal arteries
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There were also age-dependent differences in the effect of LNA and indomethacin on ACh relaxation. In fetal renal arteries (Fig. 3), LNA but not indomethacin reduced the maximal relaxation to ACh. However, the sensitivity to ACh (-log ED50 values;Table 1) was significantly reduced after inhibition of either NOS or cyclooxygenase by LNA and indomethacin, respectively. In contrast, only LNA (6.20 ± 0.12 versus 5.76 ± 0.08;n = 8; control versus LNA; Table 1 and Fig. 4) and not indomethacin (6.36 ± 0.16 versus 6.27± 0.12; n = 5; control versus indomethacin) reduced-log ED50 values for ACh of neonatal renal arteries. Maximal relaxation to ACh of rings from neonatal animals was not affected by either LNA or indomethacin. In a manner similar to neonatal renal arteries, LNA caused a rightward shift of the dose-response curve to ACh (Fig. 5) and significantly reduced -log ED50 values of arteries obtained from adult guinea pigs (Table 1). Indomethacin had no effect on either the maximal relaxation or the -log ED50 values of ACh of adult renal arteries.

Figure 3
figure 3

Fetal renal artery. Effect of and LNA (left, LNA: 10-4 M; n = 13) and indomethacin (right, 10-5 M; n = 7) on ACh relaxation of renal arteries of near term fetal guinea pigs. Responses are mean ± SEM (* = p < 0.05 vs control).

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Figure 4
figure 4

Neonatal renal artery. Effect of LNA (left, LNA, 10-4 M; n = 8) and indomethacin (right, 10-5 M; n = 5) on ACh relaxation of renal arteries of neonatal guinea pigs. Responses are mean ± SEM (* = p < 0.05 vs control).

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Figure 5
figure 5

Adult renal artery. Effect of LNA (left, LNA; 10-4 M; n = 5) and indomethacin (right, 10-5 M; n = 6) on ACh relaxation of renal arteries of adult guinea pigs. Responses are mean ± SEM (* = p < 0.05 vs control).

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Ca2+-dependent NOS activity was measured in separate groups of animals each representing different ages. NOS activity of fetal guinea pig renal cortex progressively increased from 0.5 to 0.87 at which time activity was significantly greater than the adult (p < 0.05)(Fig. 6). After birth, NOS activity of neonatal kidneys at 1 d was not significantly different from 0.87 gestation. Although the values remained elevated through the 10th d, there was considerable inter-animal variation.

Figure 6
figure 6

Effect of age on Ca2+-dependent NOS activity(pmol of [14C]citrulline/min/g of tissue) of the guinea pig renal cortex. Responses are mean ± SEM (5-10 animals were used per time point).

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CONCLUSIONS

This study is the first to demonstrate that the relaxation of isolated renal arteries to ACh and Ca2+-dependent NOS activity of the renal cortex increases during fetal maturation. Both maximal relaxation and sensitivity to ACh of fetal renal arteries increases by late and near term gestation compared with early mid term gestation. ACh relaxation further increases with maturation after birth. Maximal ACh relaxation increases in arteries from neonatal guinea pigs compared with fetal guinea pigs, whereas sensitivity to ACh is not different. Maximal responses to ACh are similar between arteries from neonatal and adult guinea pigs. With further maturation, both the maximal relaxation and the sensitivity to ACh increases in the arteries from adult guinea pigs compared with arteries from the fetal guinea pigs. The significant differences in the maximal ACh relaxation within the fetal groups and between fetal and postnatal groups do not correlate with the contractile levels and thus do not provide a likely explanation for the age-dependent increase in the responses. These results suggest that agonist-stimulated NO release of guinea pig renal arteries increases with maturation. Further, the clear ontogenetic increase in Ca2+-dependent NOS activity of the renal cortex supports the conclusion that the capacity of the microvasculature to produce NO also increases with maturation. Although we cannot exclude the contribution of neuronal NOS to the activity measurement, we eliminated much of it by restricting our study to the renal cortex.

ACh relaxation of renal arteries of adult nonpregnant female guinea pigs is endothelium-dependent and LNA-sensitive(17). The present study extends this finding to the renal arteries of fetal and neonatal guinea pigs. LNA inhibits ACh relaxation of renal arteries of all animal groups and suggests that agonist-stimulated NO release mediates the relaxation. Indomethacin has no effect on ACh relaxation of renal arteries of adult and neonatal guinea pigs but inhibits ACh sensitivity of arteries from fetal guinea pigs. This finding suggests that a vasodilator prostanoid may contribute to ACh relaxation of renal arteries before birth, but its role diminishes after birth. This conclusion is supported by others who have reported that renal PG are important in regulating blood flow of the fetal kidney(20, 21), but do not appear to impact on renal hemodynamics in the newborn(22, 23). Thus, the contribution of NO in modulating renal artery responsiveness varies during fetal development and continues after birth, whereas the contribution of vasodilator prostanoids decreases with maturation. Kon et al.(24) observed that the main renal artery of the rat is a major source of endothelium-derived NO and that stimulated NO release can act downstream to modulate renal blood flow. Thus, differences in stimulated NO release of fetal, neonatal, and adult guinea pig renal arteries may be physiologically significant and contribute to the increase in renal perfusion with maturation. Although further study is needed to confirm the finding by Kon et al.(24), the ontogenetic increase in NOS activity of the renal cortex supports the conclusion that a similar maturation in the microvasculature occurs.

Sensitivity to the NO donor, SNP, also increases with maturation, although maximal relaxation is unaffected. Fetal arteries were less sensitive to SNP than arteries from both neonatal and adult guinea pigs. This suggests that changes in NO sensitivity as well as NO release mediate renal vascular responses with advancing maturation. However, the differences in vascular sensitivity to SNP do not completely exclude changes in endothelium-derived mechanisms between the three age groups as an explanation for differences in ACh sensitivity. For example, the maximal relaxation to ACh is inhibited in arteries from fetal kidneys compared with arteries from neonatal and adult kidneys but does not differ among the three groups for SNP. Therefore, the reduced maximal response to ACh in the young fetuses is unlikely due to a reduced ability of NO to relax the fetal vascular smooth muscle, but rather an age-dependent difference in ACh-stimulated NO synthesis or release. Secondly, although there are differences in ACh sensitivity between arteries from neonatal and adult kidneys, there are no differences in SNP sensitivity between these same groups. This, too, argues that the difference in ACh sensitivity between these age groups is not due solely to differences in NO sensitivity of the vascular smooth muscle, but rather to differences in NO synthesis-either due to differences in muscarinic receptors or NOS activity. It is likely that both the endothelium and the vascular smooth muscle undergo maturational changes which contribute to the changes in renal hemodynamics associated with fetal and postnatal maturation(5).

The contribution of NO to decreasing vascular tone during fetal maturation has been demonstrated in other vascular beds. Dilator responses to ACh of the fetal lamb pulmonary circulation increases with progressive maturation in the latter third of gestation(25–27). Both basal NO and stimulated NO of the fetal lamb pulmonary circulation increase with advancing gestation and contribute to the decrease in pulmonary vascular resistance(27–29). The role of NO in mediating the developmental decrease in pulmonary vascular resistance has been further supported by an increase in endothelial NOS-specific mRNA(30). In contrast to the results of the present study, no difference in NO sensitivity of the vascular smooth muscle was observed in ovine pulmonary arteries(15). It is unclear whether this reflects the differences among species or vascular beds. In cerebral arteries, cGMP-mediated relaxation is fully functional in the term fetus and the newborn lamb but then decreases in the adult lamb(31, 32). In the present study, we demonstrate a maturational increase in NO sensitivity to SNP, presumably mediated by an increase in either cGMP content or sensitivity. Thus, maturational changes in the NO:cGMP pathway are not a generalized phenomenon but differ among vascular beds.

This study does not distinguish between an age-dependent increase in NO synthesis/release from an increase in muscarinic receptors. Clearly, a maturational increase in receptor population on endothelial cells has been demonstrated for several agonists(32) and accounts for some of the age-dependent changes in endothelial function. Although not tested in the current study, it is a possible explanation for some or all of the increase in ACh sensitivity in the renal vasculature. However, the increase in NOS activity with age also indicates that an age-related increase in potential NO synthesis occurs in the microvasculature of the kidney independent of muscarinic receptor maturation.

Another explanation for age-dependent changes in ACh relaxation is that the endothelium is damaged in the arteries from the younger fetuses because the lumen diameters are smaller. We believe this is unlikely. First, the maximal SNP relaxation does not differ among the age groups, suggesting that the arteries are capable of being mounted without age-related damage to the vascular smooth muscle. Second, arteries of the younger fetuses compared with older fetuses were not significantly different in size (inner diameter-195± 37 μm, 198 ± 20 μm, and 208 ± 16 μm for F1, F2, and F3, respectively). Further, some arteries from F1 actually had larger diameters than arteries from F3 and still had lower maximal ACh relaxation compared with F3. Finally, the fetal artery diameters are sufficiently large for easy mounting, and each mounting wire is only 23 μm in diameter-well within our technical expertise for setting up this preparation without damaging the endothelium. Thus, an age-dependent increase in technical difficulties is an unlikely explanation for our findings.

Overall, this study demonstrates an age-dependent increase in endothelium-dependent relaxation to ACh of the guinea pig renal artery and of Ca2+-dependent NOS activity in the renal cortex. The former suggests that the increase in ACh-stimulated relaxation could be related to differences in either the muscarinic receptor population and/or NO synthesis and release. The increase in NOS activity suggests the increase in NO is not confined to the main renal artery, but also occurs in the renal microvasculature. As a result, it may contribute to the changes in renal vascular resistance before and after birth(5). Further, we demonstrate that cyclooxygenase inhibition by indomethacin decreases, albeit small, the ACh sensitivity of fetal arteries but has no effect on neonatal and adult arteries. Thus, the relative contribution of NO and vasodilator PG in modulating vascular tone changes with maturation. Finally, we demonstrate that NO sensitivity of renal arteries based on SNP relaxation increases after birth. In conclusion, both the endothelium and vascular smooth muscle undergo changes during fetal guinea pig maturation and after birth which contribute to the altered renal artery responsiveness.