Published online before print October 30, 2003, doi: 10.1161/01.RES.0000104086.43830.6C
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© 2003 American Heart Association, Inc.
Integrative Physiology |
Essential Role for Vascular Gelatinase Activity in Relaxin-Induced Renal Vasodilation, Hyperfiltration, and Reduced Myogenic Reactivity of Small Arteries
From the Department of Obstetrics, Gynecology and Reproductive Sciences (A.J., J.N., L.J.K., S.L.O., K.P.C.), University of Pittsburgh School of Medicine and Magee-Women’s Research Institute, Pittsburgh, Pa; Department of Pathology (L.A.D.), University of New Mexico School of Medicine, Albuquerque, NM; Department of Cell Biology and Physiology (K.P.C.), University of Pittsburgh School of Medicine, Pittsburgh, Pa.
Correspondence to Kirk P. Conrad, Magee-Women’s Research Institute, 204 Craft Ave, Pittsburgh, PA 15213. E-mail rsikpc{at}mwri.magee.edu
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Abstract |
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During pregnancy, relaxin stimulates nitric oxide (NO)–dependent renal vasodilation, hyperfiltration and reduced myogenic reactivity of small renal arteries via the endothelial ETB receptor subtype. Our objective in this study was to elucidate the mechanisms by which relaxin stimulates the endothelial ETB receptor/NO vasodilatory pathway. Using chronically instrumented conscious rats, we demonstrated that a specific peptide inhibitor of the gelatinases MMP-2 and -9, cyclic CTTHWGFTLC (cyclic CTT), but not the control peptide, STTHWGFTLS (STT), completely reversed renal vasodilation and hyperfiltration in relaxin-treated rats. Comparable findings were observed with a structurally different and well-established, general antagonist of MMPs, GM6001. In contrast, phosphoramidon, an inhibitor of endothelin-converting enzyme, did not significantly change the renal vasodilatory response to relaxin administration. When small renal arteries were incubated with either of the general MMP inhibitors, GM6001 or TIMP-2 (tissue inhibitor of MMP), or with the specific gelatinase inhibitor, cyclic CTT, the reduced myogenic reactivity of these blood vessels from relaxin-treated nonpregnant and midterm pregnant rats was totally abolished. Moreover, a neutralizing antibody specific for MMP-2 completely abrogated the reduced myogenic reactivity of small renal arteries from relaxin-treated nonpregnant and midterm pregnant rats. In contrast, phosphoramidon did not significantly affect the reduction in myogenic reactivity. Using gelatin zymography, we showed increased pro and active MMP-2 activity in small renal arteries from relaxin-treated nonpregnant and midterm pregnant rats relative to the control animals. Thus, inhibitors of MMPs in general and of gelatinases in particular reverse the renal vascular changes induced by pregnancy or relaxin administration to nonpregnant rats. Finally, the typical reduction in myogenic reactivity of small renal arteries from relaxin-treated nonpregnant rats was absent in ETB receptor–deficient rats, despite an increase in vascular MMP-2 activity. These results indicate an essential role for vascular gelatinase, which is in series with, and upstream of, the endothelial ETB receptor/NO signaling pathway in the renal vasodilatory response to relaxin and pregnancy.
Key Words: nitric oxide • endothelin B receptor • pregnancy • renal circulation • matrix metalloproteinases
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Introduction |
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Vasodilation of nonreproductive organs is one of the earliest and most dramatic of maternal adaptations to human pregnancy. The gravid rat manifests comparable circulatory changes including renal vasodilation and hyperfiltration.1,2 Consequently, this animal model has been extensively investigated in order to elucidate the mechanisms underlying the renal vasodilatory response to pregnancy. Endothelin (ET) has been shown to mediate nitric oxide (NO)–dependent renal vasodilation and hyperfiltration during pregnancy, as well as reduced myogenic reactivity of small renal arteries via the ETB receptor subtype on endothelium.3–5 The pregnancy hormone, relaxin (RLX), also induces renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small renal arteries through endothelin, the endothelial ETB receptor, and NO when chronically administered to nonpregnant rats, thereby mimicking the pregnant condition.6–8 Relaxin is secreted by the corpus luteum of the ovary during rodent (and human) pregnancy, and circulating hormone becomes detectable by gestational day 9 in rats.9 In fact, by neutralizing endogenous circulating RLX with specific antibodies or eliminating circulating RLX by ovariectomy, the renal circulatory (and osmoregulatory) adaptations of pregnancy are completely abrogated.10
Relaxin confers matrix-degrading properties on various types of fibroblast cells by stimulating matrix metalloproteinases (MMPs), as well as inhibiting tissue inhibitors of matrix metalloproteinases (TIMPs) and collagen expression.11–13 Recently, vascular MMP-2 was shown to process big ET-1 to ET-11-32, which potently activates ET receptors.14,15 In this study, we considered that the confluence of these findings made by others11–15 and by us3–8,10 constitutes the cellular basis of renal vasodilation and hyperfiltration during pregnancy. That is, we hypothesized that relaxin upregulates vascular MMP-2 activity during pregnancy, thereby contributing to renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small renal arteries through activation of the endothelial ETB receptor-NO vasodilatory pathway.
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An expanded Materials and Methods section is available in the online data supplement at http://www.circresaha.org.
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Results |
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Influence of MMP and Endothelin-Converting Enzyme (ECE) Inhibitors on Renal Function in Conscious Nonpregnant Rats Chronically Treated With Recombinant Human RLX (rhRLX) or Vehicle
As previously reported,6,7 chronic relaxin treatment significantly increased GFR (3164±94 versus 2310±76 µL/min; P<0.0001), ERPF (10 334±361 versus 7154±189 µL/min; P<0.0001), and reduced ERVR (7.50±0.25 versus 10.23±0.47 mm Hg/mL · min-1; P<0.0001) relative to vehicle treatment. Administration of the gelatinase inhibitor, 1.0 µg/min cyclic CTT reduced both GFR, ERPF, and increased ERVR in the relaxin-treated (P<0.005 versus baseline), but not vehicle-treated rats (P=NS versus baseline; Figures 1A through 1C, top panels). In fact, in the first of the three 30-minute renal clearances during infusion of cyclic CTT, relaxin-induced increases in GFR and ERPF, as well as decreases in ERVR were already completely reversed and comparable to levels observed in vehicle-treated rats administered cyclic CTT (data not shown). Because there was no significant difference among the three 30-minute renal clearances obtained during cyclic CTT infusion (P=NS by one-factor repeated-measures ANOVA), they were combined for presentation in Figure 1. Administration of the control peptide, 1.0 µg/min STT, did not significantly affect either GFR, ERPF, or ERVR in relaxin- or vehicle-treated rats (Figures 1A through 1C, bottom panels). Nor was mean arterial pressure significantly altered by cyclic CTT or STT in the two groups of rats (Figure 1D). Elimination of renal vasodilation and hyperfiltration in rhRLX-treated rats was also obtained using a higher dose of cyclic CTT (3.0 µg/min, data not shown).
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In order to corroborate these results obtained with the gelatinase inhibitor, cyclic CTT, we also tested the general MMP inhibitor, GM6001—a compound that is structurally different from cyclic CTT.16 Once again, at baseline GFR and ERPF were significantly higher, and ERVR lower in the relaxin-treated rats in comparison to vehicle-treated animals (P<0.05). Administration of 30 ng/min GM6001 significantly reduced both GFR, ERPF, and increased ERVR in the relaxin-treated, but not vehicle-treated rats (Figures 2A through 2C, top panels). Similar to cyclic CTT, GM6001 completely reversed relaxin-induced increases in GFR and ERPF, as well as decreases in ERVR, although the time-course of reversal was not as rapid. Administration of the vehicle control, 0.025% DMSO, did not significantly affect either GFR, ERPF, or ERVR in relaxin- or vehicle-treated rats (Figures 2A through 2C, bottom panels). Mean arterial pressure was not significantly altered by GM6001 or its vehicle in the two groups of rats (Figure 2D). Abrogation of renal vasodilation and hyperfiltration in rhRLX-treated rats was also observed using a higher dose of GM6001 (100 ng/min, data not shown).
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P<0.06 vs vehicle.To further substantiate the pivotal role for gelatinase activity in the renal vasodilation and hyperfiltration induced by relaxin, we also investigated the more traditional mechanism of big ET-1 processing through endothelin-converting enzyme. That is, after measurement of baseline renal function, 30 µg/min phosphoramidon, an ECE inhibitor, was administered to the rats. Once again, baseline GFR and ERPF were significantly higher (P<0.005), and ERVR lower (P<0.05) in the relaxin-treated rats when compared with vehicle-infused animals. However, in contrast to cyclic CTT or GM6001, phosphoramidon failed to alter relaxin-induced renal vasodilation or hyperfiltration. Nor was mean arterial pressure affected (Figure 3).
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After the renal function studies on day 2 or 3 of rhRLX or vehicle administration, the efficacy of ECE blockade by phosphoramidon was tested in the same rats on day 5 or 6. Before administration of phosphoramidon, a bolus of big ET-1 resulted in a rise in mean arterial pressure of 16±2 and 18±3 mm Hg in the relaxin- and vehicle-treated rats, respectively. During phosphoramidon infusion, the rise in MAP by the same dose of big ET-1 was totally abolished in both groups of rats (0±0.8 and -0.8±0.5 mm Hg, respectively). See the online data supplement for additional Results.
Myogenic Reactivity of Small Renal Arteries Isolated From Virgin and Midterm Pregnant, as Well as From rhRLX- and Vehicle-Treated Nonpregnant Rats: Effect of Treating Arteries In Vitro With MMP and ECE Inhibitors
Figure 4 portrays the results for the gelatinase inhibitor, cyclic CTT, and the control peptide, STT. Incubation of small renal arteries isolated either from rhRLX- and vehicle-treated rats, or from midterm pregnant and virgin rats with STT did not have any affect on myogenic reactivity. That is, in the presence of STT, myogenic reactivity was significantly reduced in small renal arteries from midterm pregnant relative to virgin control animals, as we previously reported.5 Furthermore, this pregnancy-induced reduction in myogenic reactivity was mimicked by treating nonpregnant rats with rhRLX, again as previously documented.8 In contrast, incubation of small renal arteries with cyclic CTT abolished the reduced myogenic reactivity of small renal arteries from both the midterm pregnant and rhRLX-treated nonpregnant rats without affecting the robust myogenic reactivity of small renal arteries from the virgin and vehicle-treated nonpregnant control rats.
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Although cyclic CTT is relatively selective for MMP-2 over MMP-9 at the dosage used herein (30 µmol/L),17 the issue of selectivity was further explored by using a neutralizing antibody specific for MMP-2.18 The reduced myogenic reactivity of small renal arteries from midterm pregnant and rhRLX-treated nonpregnant rats was also abrogated by the MMP-2 neutralizing antibody (Figure 5). The general MMP inhibitors, GM6001 and TIMP-2, were also tested in small renal arteries from midterm pregnant and rhRLX-treated rats relative to their respective controls. Because the results were comparable, and relaxin mediates the reduced myogenic reactivity of small renal arteries from midterm pregnant rats,10 the two groups of rats and their respective controls were combined for analysis (Table). The results corroborate those obtained with the gelatinase inhibitor, cyclic CTT, and the MMP-2 neutralizing antibody. That is, the diminished myogenic reactivity elicited by pregnancy or rhRLX-treatment was virtually abolished by these general inhibitors of MMPs. Finally, in order to provide evidence against a role for the traditional pathway of ETB receptor activation by ET-11-21, we utilized the ECE inhibitor, phosphoramidon. Phosphoramidon did not alter the reduced myogenic reactivity of small renal arteries from midterm pregnant and rhRLX-treated nonpregnant rats (Table).
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, on myogenic reactivity of small renal arteries from midterm pregnant and virgin rats (A) or rats receiving rhRLX or vehicle (B). Two-factor randomized block design ANOVA demonstrated a significant effect of rat group of P<0.0001 and P<0.005 for A and B, respectively. **P<0.0005, *P<0.05 vs all other groups.
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Figure 6A depicts the data for myogenic reactivity of small renal arteries from the ETB receptor–deficient and wild-type rats. The typical reduction in myogenic reactivity induced by administration of rhRLX to rats was not observed in the small renal arteries from the ETB receptor–deficient animals.
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MMP-2 and -9 Activity in Small Renal Arteries Isolated From Virgin and Midterm Pregnant, as Well as From rhRLX- and Vehicle-Treated Nonpregnant Rats
Figure 7A depicts a representative gelatin zymogram demonstrating increased pro and active MMP-2 activity in small renal arteries from midterm pregnant rats. Comparable findings were observed for rhRLX-infused nonpregnant rats, and the results are summarized in Figures 7B and 7C. In order to combine the results from several zymograms, the densitometric ratios of rhRLX/vehicle and pregnant/virgin were calculated and averaged for presentation (Figure 7B and 7C, respectively). That is, a ratio of 1.0 would indicate that the MMP-2 activity is the same in rhRLX- and vehicle-treated or midterm pregnant and virgin rats. In fact, both rhRLX treatment and pregnancy increased the activity of pro and active MMP-2 in small renal arteries by approximately 50%. Relative to the vehicle, treatment with rhRLX increased pro MMP-2 activity in the small renal arteries from 10 of 14 pairs of rats, and active pro MMP-2 in the small renal arteries from 12 of 14 pairs of rats. In 8 of 11 pairs of rats, pregnancy increased both pro and active MMP-2 activity in small renal arteries relative to virgin controls. The ratio of pro to active MMP-2 was not significantly affected by either rhRLX administration or pregnancy. However, an interesting discrepancy between midterm pregnancy and rhRLX treatment of nonpregnant rats is that only in the former was pro MMP-9 activity consistently detected in the small renal arteries (see Figure 7A).
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Figure 6B portrays the gelatin zymogram of small renal artery homogenates from the same ETB receptor–deficient rats used for investigation of myogenic reactivity (Figure 6A). MMP-2 activity was increased in the small renal arteries from the rhRLX- relative to the vehicle-treated rats.
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Discussion |
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The objective of this study was to further elucidate the molecular mechanisms of pregnancy (relaxin)-mediated vasodilatory changes in the renal circulation. Our working hypothesis was based on the confluence of several observations: (1) our own, showing the crucial role for the endothelial ETB receptor and NO as described in the Introduction3–8,10; (2) the ability of relaxin to upregulate MMP activity (at least in fibroblasts11–13); and (3) the potential for vascular MMPs, in particular MMP-2, to process big ET-1 at the gly-leu bond to ET-11-32, the latter capable of activating the endothelial ETB receptor.14,15 Thus, we hypothesized that RLX upregulates MMP-2 activity in the renal vasculature during pregnancy, thereby leading to renal vasodilation, hyperfiltration and reduced myogenic reactivity of small renal arteries in an endothelin- and nitric oxide–dependent fashion.
The best (if not the only) approach to testing the physiological role of MMP-2 in mediating renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small renal arteries induced by relaxin is to block production or inhibit its action. We chose the latter approach, first utilizing a newly developed, specific inhibitor of gelatinases, MMP-2 and -9.17,19 This peptide inhibitor, cyclic CTT, isolated from phage display peptide libraries, preferentially inhibits the activity of MMP-2 relative to the activity of MMP-9. Cyclic CTT is 10-fold more potent than STT, and does not inhibit MT1-MMP, MMP-8, MMP-13, or serine proteases such as trypsin-2, neutrophil elastase, or cathepsin G. See the online data supplement for further description of cyclic CTT.
We initially conducted dose-finding studies in conscious rats. First, bolus administration of 3.5 mg cyclic CTT over 5 minutes to midterm pregnant rats significantly increased mean arterial pressure by approximately 50 mm Hg, and reduced GFR and ERPF by almost 50%, whereas the control peptide STT or linearized CTT did not affect these variables (data not shown). Next, in preparation for the renal studies, we infused the compound at 100, 30, 10, 3, or 1 µg/min. The largest infusion rate was based on pharmacokinetic estimations and a desired plasma concentration of 30 µmol/L. However, this dose, as well as the 30 and 10 µg/min infusion rates increased mean arterial pressure by approximately 20 mm Hg in both relaxin- and vehicle-treated rats, whereas the lower infusion rates of 1 and 3 µg/min did not significantly affect mean arterial pressure (see Results and Figure 1D). Because increases in systemic arterial pressure can confound the interpretation of changes in renal hemodynamics, our goal was to utilize a dose that would minimally perturb mean arterial pressure. Thus, we focused on the lower infusion rates of 1.0 and 3.0 µg/min and found that cyclic CTT, but not the control peptide, STT, completely reversed the renal vasodilation and hyperfiltration induced by relaxin. These results support the concept that gelatinase activity processes big ET-1 to ET-11-32, that, in turn, activate the endothelial ETB receptor, thereby mediating renal vasodilation and hyperfiltration via NO.
In order to corroborate the concept that gelatinase activity is critical to the renal vasodilatory response of relaxin, we used a general inhibitor of MMPs, GM6001, with an inhibitory constant in the low nmol/L range.16 This compound is well established and in the hydoxamate class of MMP inhibitors that bind the divalent zinc in the catalytic site necessary for enzyme activation. Again, we initially performed dose-finding studies by infusing the compound at 30 and 100 µg/min, the latter dose based on pharmacokinetic estimations and a desired plasma concentration of 100 nmol/L. Because the results were comparable, we performed most of the experiments using the lower dose and found that GM6001, but not the dilute DMSO vehicle, totally abrogated the renal vasodilation and hyperfiltration induced by relaxin.
The traditional pathway for endothelin formation is through the endothelin-converting enzyme, which processes big ET-1 to ET-11-21. In addition to providing evidence of a critical role for MMP in the formation of endothelin in relaxin-treated rats, we also wanted to marshal evidence against the traditional, ECE pathway. Thus, we tested phosphoramidon, an ECE inhibitor. We began with an infusion of 100 µg/min based on the regimen of McMahon and colleagues20 who verified pharmacological blockade using this dose. However, we found that a lower dose of 30 µg/min also completely blocked the pressor response to big ET-1, and therefore, conducted most of the experiments using this lower dose. Consistent with our hypothesis, phosphoramidon did not alter the renal circulation in relaxin-treated rats. Nor did the drug affect renal hemodynamics in vehicle-treated control animals. Thus, renal vasodilation and hyperfiltration induced by relaxin persisted in the presence of pharmacological blockade of ECE. Although phosphoramidon can clearly inhibit ECE on the plasma membrane, it is less certain whether the drug can penetrate the endothelial cell to block intracellular enzyme. Unfortunately, the literature is in conflict over this issue.21,22
One potential limitation to the present studies in conscious rats is that we only investigated relaxin-treated and not gravid animals. Instead, we elected to rely on our previous work demonstrating that relaxin is the pregnancy hormone responsible for the renal circulatory changes in pregnancy,10 rather than test the MMP and ECE inhibitors in conscious gravid rats. From this point of view, in fact, the study of conscious gravid rats might be considered redundant. However, we did directly test our hypothesis in relation to pregnancy per se by utilizing other technical approaches as discussed next.
So far, the phenomenon of reduced myogenic reactivity of small renal arteries has served as a faithful bioassay for the renal vasodilatory changes induced by pregnancy or relaxin-treatment of nonpregnant rats in vivo.5,8 Therefore, we tested whether incubation of small renal arteries from midterm pregnant or relaxin-treated nonpregnant rats with inhibitors of MMP or ECE would reverse the reduction of myogenic reactivity. One advantage of this technical approach over the whole animal studies is that additional inhibitors can be used that are either too expensive or unavailable in sufficient quantities to be infused in vivo.
Analogous to our findings in the conscious rats, both cyclic CTT and GM6001 reversed the reduced myogenic reactivity of small renal arteries isolated from relaxin-treated nonpregnant and midterm pregnant rats. Comparable results were obtained using the general MMP inhibitor, TIMP-2, and a specific MMP-2 neutralizing antibody. In contrast, and in keeping with the results in the conscious rats, phosphoramidon did not alter the reduction in myogenic reactivity. Thus, these results obtained from the study of myogenic reactivity of small renal arteries recapitulate those dealing with renal hemodynamics in the conscious rats, thereby further substantiating a pivotal role for MMPs, specifically MMP-2 in the renal circulatory changes of pregnancy or relaxin-treated nonpregnant rats. Moreover, they implicate vascular MMP-2, rather than circulating enzyme.
Although vascular MMP-2 activity may be essential for the renal circulatory changes of pregnancy or relaxin-treated nonpregnant rats, vascular MMP-2 itself may not be the actual locus of regulation. Based on our results so far, vascular MMP-2 is clearly in the vasodilatory pathway, but other elements in the pathway may be upregulated such as the endothelial ETB receptor or eNOS phosphorylation, thereby enhancing renal vasodilation. To address this issue, we measured gelatinase activity in small renal arteries. The data show an approximately 50% increase in both pro and active MMP-2 activity in small renal arteries from relaxin-treated and midterm pregnant rats relative to their respective controls. The ratio of pro to active MMP-2 was not significantly altered, suggesting that processing of the pro to the active form by MT1-MMP and other proteases is not facilitated by pregnancy or relaxin administration. Interestingly, we consistently detected MMP-9 in the small renal arteries from midterm pregnant rats, but not in small renal arteries from relaxin-treated rats. Thus, additional pregnancy factors besides relaxin might be at work here in the control of MMP-9 activity. This observation reinforces the notion that MMP-2 is likely to be the critical vascular gelatinase responsible for renal circulatory changes induced by pregnancy or relaxin-treatment of nonpregnant rats, because only MMP-2 is unequivocally upregulated in both conditions. We are currently investigating whether this upregulation occurs in the endothelium (as we hypothesize), the vascular smooth muscle, or both. Finally, it should be pointed out that alteration of vascular MMP-2 activity as a pivotal step in the circulatory adjustments of pregnancy by relaxin, and the potential for concurrent regulation of the ETB receptor or eNOS phosphorylation (as we have previously proposed4,5) are not mutually exclusive.
The precise mechanism for the increase in MMP-2 activity in small renal arteries from midterm pregnant and relaxin-treated rats as determined by gelatin zymography remains to be established. The increase may represent an upregulation of MMP-2 expression, a decrease in TIMPs or other MMP inhibitors such as 
2-macroglobulin or RECK,23 or a combination thereof. MMP-2/inhibitor complexes persist in gelatin zymography, but are difficult to detect because they are not very active.24 Thus, the increase in MMP-2 activity may primarily reflect a decrease in abundance of these inhibitor(s), thereby unmasking gelatinase activity. In addition to upregulating MMP expression,11–13 relaxin has been shown to downregulate TIMP expression in both human dermal11 and lower uterine segment13 fibroblasts. Investigation of MMP-2 by Western analysis and TIMP by reverse zymography and Western analysis will help resolve this issue.
We cannot exclude the possibility that other MMPs may be enhanced in small renal arteries by relaxin during pregnancy, because in this work we limited our investigation to the gelatinases. While this work was in progress, Kelly and colleagues25 reported increased mRNA expression of a several MMPs during pregnancy in rat uterine artery. Several MMPs other than the gelatinases may have the potential to hydrolyze the gly-leu bond in big ET-1.26 However, based on the results from (1) use of a selective MMP-2 and -9 inhibitor both in vivo and in vitro (cyclic CTT), (2) application of a specific MMP-2 antibody in vitro, and (3) gelatin zymography showing that only MMP-2 activity is increased in small renal arteries from both midterm pregnant and relaxin-treated nonpregnant rats (MMP-9 activity is consistently detected only in the former), we implicate MMP-2 rather than other MMPs in the renal circulatory changes induced by relaxin or pregnancy.
In the present work, we provide evidence for a pivotal role of vascular MMP-2 in the renal vasodilation, hyperfiltration, and reduced myogenic reactivity of small renal arteries during pregnancy or during relaxin administration to nonpregnant rats. In our previous work, we established a crucial role for the endothelial ETB receptor/NO vasodilatory pathway.3–8 We reasoned, therefore, that vascular MMP-2 is in series with, and upstream of, the endothelial ETB receptor and NO. The connection is inferred and based on the recent finding that vascular MMP-2 can process big ET-1 to ET-11-32 at Gly32-Leu33, and ET-11-32 in turn can elicit either vasoconstriction or vasorelaxation depending on the experimental circumstance through the vascular smooth muscle or endothelial ETB receptor, respectively, the latter via nitric oxide.14,15 It is highly unlikely that vascular MMP-2 and the endothelial ETB receptor/NO in this study are members of separate vasodilatory pathways, which operate in parallel. If that is the case, then on inhibition of vascular MMP-2 or the endothelial ETB receptor/NO pathway, one might expect compensation of one for the other. We observed no such compensation or even partial compensation. Each and every inhibitor of the ETB receptor,4,5,7,8 nitric oxide synthase,3,5,6,8 and MMP (present study) totally abolished the renal circulatory changes during pregnancy or relaxin treatment of nonpregnant rats.
In order to substantiate this linkage between vascular MMP-2 and the endothelial ETB receptor/NO vasodilatory pathway, we provide both functional and biochemical data. First, the reduction of myogenic reactivity in small renal arteries by relaxin was lacking in rats genetically deficient in the ETB receptor, consistent with our earlier work using ETB receptor antagonists.4,5,7,8 However, in other small renal arteries harvested from the same rats deficient in the ETB receptor, MMP-2 activity was increased by the relaxin treatment. This dissociation of increased vascular MMP-2 activity from the functional behavior of reduced myogenic reactivity when the ETB receptor is absent, strongly supports the concept that vascular MMP-2 is in series with, and upstream of, the endothelial ETB receptor/NO vasodilatory pathway. Second, we observed that the basal expression of cGMP in small renal arteries was 14.8 pmol/mg protein, and that after incubation with 0.1 mmol/L L-NAME, 30 µmol/L cyclic CTT, and 1.0 µmol/L GM6001, the levels were 2.2, 7.8, and 9.6 pmol/mg protein, respectively (n=2 rats). Thus, as expected, L-NAME dramatically reduced the cGMP content, because NO is the predominant stimulus for its production.4 Both MMP inhibitors, cyclic CTT and GM6001, also reduced cGMP further supporting a linkage between vascular MMP-2 and the ETB receptor/NO pathway, thereby corroborating an earlier report.15
Because the endothelial ETB receptor/NO pathway also contributes to the low vascular resistance of the renal circulation in nonpregnant female and male rats (4, and citations therein), we suggest that vascular gelatinase activity plays a role here, too. We mainly focused on lower doses of cyclic CTT in this investigation, in order to avoid significant elevations in blood pressure. Nevertheless, in our dose-finding studies, the higher doses of cyclic CTT (10, 30, and 100 µg/min) produced hypertension, reduced renal plasma flow and increased renal vascular resistance not only in the relaxin-infused rats, but also in the control animals administered vehicle. In addition, although the 3 µg/min infusion did not increase MAP, it did significantly decrease GFR, ERPF, and increase ERVR in both vehicle- and rhRLX-treated rats, only more so in the latter (data not shown). It is also noteworthy that phosphoramidon not only failed to alter renal hemodynamics in the relaxin-infused rats, but also in the control rats administered the vehicle for relaxin. Thus, vascular MMP-2 may be the major endothelin-converting enzyme responsible for provision of substrate to the endothelial ETB receptor/NO vasodilatory pathway even in the nonpregnant condition. Perhaps, the colocalization of MMP-2 and associated proteins in the caveolae of endothelial cells with eNOS, and possibly the ETB receptor, facilitates this interaction.27–29 To our knowledge, the concept that vascular gelatinase activity is a major regulator of renal hemodynamics is without precedent. Perhaps also unforeseen is that its physiological role is one of renal vasodilation rather than vasocontriction.14
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Acknowledgments |
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This project was supported by NIH R01 DK63321. We are grateful to Elaine Unemori, PhD, of Connetics Corporation, Palo Alto, Calif, for providing the rhRLX and antibodies for the rhRLX ELISA. We thank Cheryl Gariepy for providing the ETB receptor–deficient rats. The technical assistance of Michelle Fisher, Julianna Matthews, and Ketah Doty is also gratefully acknowledged.
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Footnotes |
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Portions of this work have appeared in abstract form (J Soc Gynecol Invest. 2003;10:198A, 244A, 245A).
*These authors contributed equally to this work. 
Original received March 21, 2003; resubmission received September 11, 2003; revised resubmission received October 17, 2003; accepted October 20, 2003.
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References |
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