Endocrinology, doi:10.1210/en.2003-0351
Endocrinology Vol. 144, No. 11 4931-4938
Copyright © 2003 by The Endocrine Society
Altered Renal Hemodynamics in Mice Overexpressing the Parathyroid Hormone (PTH)/PTH-Related Peptide Type 1 Receptor in Smooth Muscle
William T. Noonan,
Jin Qian,
William D. Stuart,
Thomas L. Clemens and
John N. Lorenz
Departments of Molecular and Cellular Physiology (W.T.N., J.N.L.) and Medicine (J.Q., W.D.S., T.L.C.), University of Cincinnati College of Medicine, Cincinnati, Ohio 45267
Address all correspondence and requests for reprints to: John N. Lorenz, University of Cincinnati College of Medicine, Department of Molecular and Cellular Physiology, 231 Albert Sabin Way, Cincinnati, Ohio 45267-0576. E-mail: john.lorenz{at}uc.edu.
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Abstract
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PTH-related protein (PTHrP) is an autocrine/paracrine peptide expressed in renal tubules and vasculature and may play an important role in regulating overall renal function. To evaluate the potential role of endogenous PTHrP in the control of renal hemodynamics, we performed clearance measurements in transgenic (TG) mice in which the SMP8 
-actin promoter was used to drive overexpression of the PTH/PTHrP type 1 receptor in smooth muscle. In protocol I, responses to acute saline volume expansion (SVE, 0.75 µl/min·g body weight) were measured in TG and nontransgenic (NTG) mice. Mean arterial pressure was significantly lower in TG mice throughout the experiment, and it decreased comparably in both groups in response to SVE. SVE significantly increased effective renal plasma flow in both groups of mice, but the increase was greater in TG than in NTG. Glomerular filtration rate decreased in response to SVE in NTG but did not change in TG animals. In protocol II, renal responses to angiotensin II (ANG II) infusion were determined (0.5 ng/min·g body weight). Baseline arterial pressure was again significantly lower in TG, compared with NTG mice, and TG mice had a blunted pressor response to ANG II. Also, ANG II decreased effective renal plasma flow and glomerular filtration rate in both groups of animals, but the reductions were less in TG than in NTG mice. Our findings indicate that smooth-muscle-specific overexpression of the PTH/PTHrP type 1 receptor resulted in augmentation of the vasodilatory response to SVE and attenuation of the vasoconstrictor response to ANG II. We conclude that endogenous PTHrP can act as an endogenous vasorelaxant factor to modulate renal responses to vasoactive stimuli.
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Introduction
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P THrP WAS FIRST identified as a tumor-derived protein responsible for the syndrome humoral hypercalcemia of malignancy (1, 2, 3). PTHrP is homologous to PTH in the N-terminus region, perhaps indicating why this peptide’s actions are similar to those of PTH when it is secreted into the systemic circulation by malignant tumors (4). PTHrP mRNA transcripts are found not only in malignant tumors, but they are also widely expressed in endocrine and nonendocrine tissues. In the adult animal, immunohistochemical staining revealed that PTHrP expression in smooth muscle was particularly widespread, including the uterus (5), bladder (6), stomach (7), and blood vessels (8). PTHrP is expressed in a variety of vascular tissues, including rat aorta, vena cava, and the renal vascular tree (9). In addition, PTHrP has been localized in glomerular epithelial cells and in both proximal and distal tubular cells in the rat (10). Although the precise physiological function of PTHrP in smooth muscle is still unknown, marked induction of protein expression by mechanical stretch (11) and vasoconstrictor agents (12), together with its ability to relax smooth muscle (13), indicate that perhaps this peptide functions in an autocrine or paracrine role to control blood flow or response to contractile stimuli in these organs. To date, very few studies have examined the role of PTHrP and its receptor, the PTH/PTHrP type 1 receptor (PTH/PTHrP-R), in renal function. In one study using the split hydronephrotic rat kidney model, Endlich et al. (14) found that the renal vasculature was highly sensitive to vasodilation by PTHrP. Work by Massfelder et al. (15) further showed that renal blood flow (RBF) and glomerular filtration rate (GFR) increased in response to intrarenal infusion of PTHrP.
Previous work from our laboratory has shown that overexpression of PTH/PTHrP-R, using the smooth muscle-specific SMP8 -actin promoter, significantly reduces blood pressure in awake mice (16). This observation suggested a possible renal perturbation, because it is well established that the kidneys play a dominant role in long-term blood pressure regulation. Therefore, the experiments presented here were designed to evaluate renal function in these mice under baseline conditions and under conditions that alter fluid volume homeostasis and renal hemodynamics. Parameters of renal function were determined in SMP8-PTH/PTHrP-R transgenic (TG) mice and wild-type cohorts before and after: 1) acute isotonic saline volume expansion (SVE); and 2) systemic infusion of angiotensin II (ANG II). We hypothesized that enhanced local effects of endogenous PTHrP in the TG animals would cause the vasodilatory effects of volume expansion to be potentiated and the vasoconstrictor effects of ANG II to be blunted.
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Materials and Methods
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The studies described herein were performed in accordance with the guidelines established by the Institutional Animal Care and Use Committee at the University of Cincinnati College of Medicine.
Animals
These studies used mice in which the TG expression of the PTH/PTHrP type 1 receptor was targeted to smooth muscle using the SMP8 -actin promoter as described previously (16). It has been confirmed that this promoter directs protein expression exclusively to smooth muscle as demonstrated by in situ hybridization studies with several different transgenes and in a variety of tissues, including renal vascular smooth muscle (17, 18). To confirm renal expression of the PTH/PTHrP-R and PTHrP in these mice, Northern blot analysis was performed. Briefly, 10 µg kidney total RNA was separated with a 1.2% agarose gel, transferred to a nylon membrane, and then hybridized with a random primer labeled mouse PTH/PTHrP-R cDNA or PTHrP cDNA. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA hybridization was used to evaluate equivalency of RNA loading. Animals were obtained from a colony that was bred to homozygosity on an FVB-N background. Age- and weight-matched NTG controls were obtained from a separate colony of FVB/N mice. The genotype of the mice was confirmed by Southern blotting of EcoRI-restricted genomic DNA as described (16).
Renal hemodynamic studies
SMP8-PTH/PTHrP-R-overexpressing male and NTG male mice from 8–12 wk of age were anesthetized with separate ip injections of Ketalar [50 µg/g body weight (BW)] followed by Inactin (100 µg/g BW; Research Biochemicals International, Natick, MA). Mice were placed on a heated surgical table to maintain body temperature at 37 C; and after tracheostomy, using PE-90 tubing, mice were provided with a steady stream of 100% O2 to breathe. The right femoral artery and vein were cannulated with heat-stretched polyethylene tubing hand drawn to a fine tip (0.3–0.5 mm outside diameter). The arterial catheter was connected to an Argon model CDXIII transducer (Maxxim Medical, Athens, TX) for measurement of arterial blood pressure, and the venous catheter was connected to a syringe pump for infusion. The bladder was cannulated with flared PE-10 for the collection of urine. Blood pressure and heart rate (HR) were monitored using a MacLab data acquisition system (AD Instruments, Colorado Springs, CO). Immediately after surgery, a 3-µl/g BW bolus of 1% fluorescein isothiocyanate (FITC)-inulin (Sigma, St. Louis, MO) and 4% para-aminohippuric acid sodium salt (PAH; ICN Biomedicals Inc., Aurora, OH) in isotonic saline was administered. This was followed by a maintenance infusion of the same solution at 0.15 µl/min·g BW.
Protocol I.
After a 30-min equilibration period, baseline renal function was determined in TG (n = 8) and NTG mice (n = 8) through two 30-min urine collections. At the midpoint of each baseline collection, an arterial blood sample (60 µl) was obtained for determination of plasma FITC-inulin and PAH concentrations. Donor blood was immediately administered after all blood samples, to replace lost volume. At the end of the second baseline collection, another blood sample was obtained for electrolyte determinations. Next, mice were volume expanded by initiating an infusion of isotonic saline at 0.75 µl/min·g BW, which was continued for the remainder of the experiment. After a 30-min equilibration period, two additional 30-min urine collections were obtained, with arterial blood samples taken at the midpoint of each collection. At the end of the last collection period, a final arterial blood sample was obtained for electrolyte determinations.
Protocol II.
In the second set of experiments, two baseline clearance periods were performed as described above. After these collections, an infusion of ANG II (Sigma) was initiated at a rate of 0.5 ng/min·g BW via a second catheter placed in the femoral vein during surgery. After another 30-min equilibration period, two 30-min experimental clearance periods were performed, with arterial blood samples taken at the midpoint of each collection. At the end of the last collection period, a final arterial blood sample was obtained for electrolyte determinations.
PTH and total calcium measurements
Because circulating PTH is also a ligand for the PTH/PTHrP receptor, we sought to confirm that the observed differences between TG and NTG animal results were due to the autocrine nature of PTHrP, and not influenced by changes in circulating PTH as a result of the treatments. In a separate set of experiments, therefore, we measured plasma PTH and serum calcium concentration under baseline and experimental conditions. NTG and TG mice were surgically prepared as detailed above. After a 30-min recovery period, a baseline blood sample (200 µl) was taken while being simultaneously replaced with mouse donor blood (200 µl). The animals were then volume expanded or ANG II treated as described above, and a second blood sample was obtained. Serum samples were stored frozen for later analysis, and samples were assayed together. PTH was measured in plasma samples by ELISA (ALPCO Diagnostics, Windham, NH), and total plasma calcium was determined by colorimetric assay (Sigma).
Analytical procedures and statistical analysis
Midpoint clearance blood samples, obtained in heparinized tubes, were centrifuged, and plasma aliquots were transferred into microcentrifuge tubes for FITC-inulin (19) and PAH determinations. The PAH assay was a modification of the method used by Waugh and Beall (20) specifically modified for use on small volumes (21). Electrolyte determinations of the blood samples obtained at the end of an experimental period were performed using a Chiron Diagnostics 348 pH/Blood Gas Analyzer (Chiron Diagnostics Corp., Medfield, MA). Urine volumes and kidney weights were determined gravimetrically. Sodium and potassium concentrations in the urine were determined with a Corning 480 Flame Photometer (Ciba Corning Diagnostics Corp., Medfield, MA). Urine osmolality was determined by freezing point depression (Model One-Ten; Fiske Associates, Norwood, MA). GFR was calculated from the clearance of inulin, and PAH clearance was used as an index of effective renal plasma flow (ERPF). Renal vascular resistance (RVR) was calculated by the formula: mean arterial pressure (MAP)/(PAH clearance/1 - hematocrit). Statistical analysis was performed using a single-factor ANOVA or a two-factor ANOVA (2 x 2) with repeated measures on the second factor. Where necessary, individual comparisons of group means were accomplished using individual contrasts. P < 0.05 defined statistical difference. Results are expressed as mean ± SEM.
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Results
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SMP8-PTHrP-R TG mice
As shown in Fig. 1
, Northern analysis of total kidney RNA demonstrated that PTH/PTHrP-R was expressed at markedly higher levels in TG line 127, compared with NTG controls. Interestingly, we found that the expression of PTHrP ligand mRNA was also increased in the kidneys of TG animals. Although our previous studies had reported a reduced arterial blood pressure in two lines of SMP8-PTH/PTHrP TG mice (lines 122 and 127), the difference in the 127 line did not reach statistical significance in that study (16). However, subsequent measurements from a large number of experiments in anesthetized line 127 mice revealed a modest, but consistent, decrease in blood pressure (unpublished observations). Furthermore, experiments exploring the relaxant properties of PTHrP(1–34) in precontracted aortic rings from line 127 mice demonstrated an enhanced relaxation response, compared with wild-type mice (16). Based on these findings, we chose to perform the present series of experiments on line 127 mice.
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FIG. 1. Northern blot analysis of SMP8-PTHrP receptor transgene expression (top panel) and of PTHrP ligand expression (middle panel) in kidney from TG (line 127) and wild-type (WT) mouse. Ten micrograms of total RNA were gel separated, transferred to a nylon membrane, and then hybridized with mouse PTH/PTHrP receptor cDNA or PTHrP cDNA. GAPDH (bottom panel) was used as an index of RNA loading.
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There were no differences in total BW and kidney weights between protocols I and II, so the data were pooled. The average BW of nontransgenic (NTG) mice were 26.6 ± 0.7 g and kidney weights were 0.46 ± 0.01 g. The average body and kidney weights of TG mice were 25.6 ± 0.6 and 0.44 ± 0.01 g, respectively, and there were no significant differences.
Protocol I: cardiovascular and renal effects of SVE
Mean arterial blood pressure (MAP) and HR effects of SVE in anesthetized NTG and TG mice are shown in Fig. 2. Blood pressure was lower in the TG animals throughout the experiment (genotype effect, P = 0.03), and MAP decreased comparably in response to SVE in both groups (treatment effect, P = 0.0004; interaction, P = 0.2). Heart rate was not different between the genotypes during the control period; however, it increased significantly in response to SVE in NTG (P = 0.0001) and TG (P = 0.004) mice. The increase in HR was greater in the NTG than the TG mice (interaction, P = 0.05). SVE decreased the hematocrit from 48 ± 1 to 45 ± 1 in NTG mice and from 46 ± 1 to 43 ± 1 in TG mice (treatment effect, P = 0.0001). The degree of plasma volume expansion estimated from these values was not different (11% and 12%, respectively).
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FIG. 2. Effect of overexpression of the SMP8-PTHrP-R transgene on MAP (A) and HR (B) in anesthetized NTG (n = 8) and TG (n = 8) mice before and during SVE (0.75 µl/min·g BW). *, Significant difference, compared with baseline value;  , significant difference, compared with corresponding value in the NTG group. Values are mean ± SEM.
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As shown in Table 1, there were no differences in baseline urine flow, sodium excretion, potassium excretion, or solute excretion between the NTG and TG mice. In response to SVE, urine flow rate, sodium excretion, and total solute excretion increased significantly (compared with baseline) in both NTG and TG mice, whereas potassium excretion decreased significantly. The magnitude of the changes was the same between the groups. As shown in Fig. 3A, there were no differences in baseline GFR between the NTG and TG mice; but in response to volume expansion, GFR decreased significantly in NTG (but not in TG) animals (interaction, P = 0.001). PAH clearance was comparable between the two groups during the baseline period (Fig. 3B), and it increased in response to SVE in both NTG (P = 0.03) and TG (P = 0.0001) mice. Importantly, however, the increase in PAH clearance was markedly greater in the TG (compared with NTG) group (interaction, P = 0.04). In a separate series of time-control experiments (n = 7), we found that GFR remained stable over the 180-min time course of these experiments in NTG mice (438 ± 27 vs. 430 ± 25 µl/min, P = 0.68).
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TABLE 1. Renal measurements in nontransgenic and transgenic anesthetized mice before and during saline volume expansion
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FIG. 3. GFR (A) and PAH clearance (B) in anesthetized NTG (open circles, n = 8) and TG (closed circles, n = 8) mice before and during SVE (0.75 µl/min·g BW). *, Significant difference, compared with baseline value; , significant difference, compared with corresponding value in the NTG group. Values are mean ± SEM.
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Protocol II: cardiovascular and renal effects of ANG II infusion
Consistent with results from the previous set of experiments, blood pressure was again lower in TG (compared with NTG) mice (P = 0.0001) throughout the experiment (Fig. 4). Although ANG II infusion increased blood pressure in both NTG (P = 0.0001) and TG animals (P = 0.0001), the magnitude of the blood pressure change was significantly less in the TG animals when compared with NTG (interaction, P = 0.04). There were no differences in baseline HR between the groups, and ANG II increased HR comparably in both the NTG and TG animals (P = 0.0001).
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FIG. 4. Effect of overexpression of the SMP8-PTHrP-R transgene on MAP (A) and HR (B) in anesthetized NTG (n = 7) and TG (n = 7) mice before and during systemic ANG II infusion (0.5 ng/min·g BW). *, Significant difference, compared with baseline value; , significant difference, compared with corresponding value in the NTG group. Values are mean ± SEM.
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During the baseline period, there were again no differences in urine flow, sodium excretion, potassium excretion, and solute excretion between NTG and TG animals (Table 2). In response to ANG II infusion, there was a significant diuresis and natriuresis in both groups, but the increase in urine flow rate was less in the TG (compared with NTG) animals (43% vs. 162%, respectively; interaction, P = 0.008). Sodium excretion increased similarly in both groups during ANG II infusion, probably reflecting a pressure-induced natriuresis. There were no differences in potassium or solute excretion responses between the groups. As shown in Fig. 5A, baseline GFR was not different between the NTG and TG animals, and ANG II infusion decreased GFR comparably in both groups studied (P = 0.0001). PAH clearance was not different between NTG and TG animals during the baseline period (Fig. 5B), and ANG II infusion significantly reduced PAH clearance in both groups (P = 0.0001). Importantly, however, the decrease was significantly less in the TG mice, compared with NTG (interaction, P = 0.05).
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TABLE 2. Renal measurements in nontransgenic and transgenic anesthetized mice before and during angiotensin II infusion
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FIG. 5. GFR (A) and PAH clearance (B) in NTG (open circles, n = 7) and TG (closed circles, n = 7) mice before and during ANG II infusion (0.5 ng/min·g BW). *, Significant difference, compared with baseline value; , significant difference, compared with corresponding value in the NTG group. Values are mean ± SEM.
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RVR during SVE and ANG II infusion
RVR was calculated for both experimental protocols. In both experiments (Fig. 6), baseline RVR was not different between the two groups of mice. SVE (Fig. 6A) caused a significant decrease in RVR in NTG (P = 0.05) and TG mice (P = 0.001), but the vasodilatory response to volume expansion was greater in TG mice, compared with NTG (interaction, P = 0.01). In protocol II, ANG II infusion (Fig. 6B) increased RVR in both NTG (P = 0.0004) and TG mice (P = 0.05); and in this case, the vasoconstrictor response to ANG II was blunted in the TG animals, compared with NTG animals (interaction, P = 0.05).
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FIG. 6. Calculated RVR (MAP/(PAH clearance/1 - hematocrit) in NTG (open circles) and TG (closed circles) mice before and during SVE (A, n = 8) or ANG II (B, n = 7). *, Significant difference, compared with baseline value; , significant difference, compared with corresponding value in the NTG group. Values are mean ± SEM.
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Plasma PTH and total calcium during baseline and experimental conditions
Plasma PTH was measured during baseline and experimental conditions to determine whether circulating PTH concentrations were affected by the experimental conditions and ultimately influenced the observed results (Table 3). No differences in plasma PTH were observed between TG and NTG animals before treatment with either SVE or ANG II. In addition, no changes in PTH were observed in either group of animals in response to SVE (protocol I). In response to ANG II administration in protocol II, plasma PTH concentrations were found to increase (P = 0.0003) modestly and comparably in both TG and NTG mice. No differences in total serum calcium concentration were observed within or between the groups.
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TABLE 3. Plasma PTH and total calcium measurements in nontransgenic and transgenic mice during baseline or experimental conditions
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Discussion
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In these studies, we sought to explore the effects of endogenously produced PTHrP on renal hemodynamics using mice with smooth muscle-specific overexpression of the PTH/PTHrP type 1 receptor. Consistent with earlier reports, we observed that mice overexpressing the SMP8-PTHrP-R transgene had lower blood pressure than their NTG cohorts. These findings support a previously postulated role for PTHrP as a local vasorelaxant factor, in that the observed alteration in baseline blood pressure may represent an amplification of tonic influence of local PTHrP on basal smooth muscle tone (16). Here, we further demonstrate that renal hemodynamic responses to known vasoactive maneuvers are also altered in this animal model and may therefore contribute to the long-term decrease in arterial blood pressure. The primary finding in this study was that animals overexpressing the PTH/PTHrP-R in smooth muscle have exaggerated renal vasodilatory responses (see Fig. 6
). Specifically, saline-induced decreases in RVR were larger in TG animals than in NTG. Likewise, ANG II-induced increases in RVR were blunted in TG animals, suggesting an elevated level of underlying vasodilatory activity during the ANG II infusion. These findings are therefore consistent with the hypothesis that an increased population of PTH/PTHrP type 1 receptors on the renal vasculature in TG animals results in an exaggerated vasodilatory influence of locally produced PTHrP during the vasodilatory or vasoconstrictor maneuvers. This conclusion is supported by our observation that renal PTHrP ligand expression is also increased in the TG animals, although we are uncertain of the mechanisms that may account for this up-regulation. Although we cannot discount the possibility that this up-regulation of PTHrP ligand is due to some tubular compensatory mechanism, it is useful to point out that baseline renal excretory function was not altered in these animals. These studies provide evidence that locally produced PTHrP can act as an important modulator of renal vascular function.
In protocol I, SVE caused a significant increase in ERPF in both groups studied, but the increase observed in the TG animals was markedly enhanced, compared with the increase observed in NTGs. When considered on a relative basis, the difference was even more dramatic: 48% increase in ERPF in the TG and only 17% increase in the NTG mice. From this data, it is readily apparent that the vasodilatory response to SVE is markedly enhanced in animals overexpressing the PTH/PTHrP-R in smooth muscle. Furthermore, although ERPF increased modestly in the NTG animals in response to SVE, GFR significantly declined, a result which may suggest a predominantly postglomerular vasodilatory effect. By contrast, in the TG animals, RBF increased markedly in response to SVE, whereas GFR did not change, suggesting a more balanced effect of SVE on pre- and postglomerular resistance. This finding leads us to speculate that PTH/PTHrP-R overexpression in the TG animals results in augmented vasodilation at a primarily preglomerular site. Evidence supporting this notion comes from the work of Endlich et al. (14) in the hydronephrotic rat kidney, in which they showed that PTHrP dilates the preglomerular vascular segments, including the afferent arteriole, without influencing the efferent arteriole. Furthermore, when PTHrP was infused directly into the left renal artery of anesthetized rats, RBF and GFR increased by 10 and 20%, respectively (15).
These changes in hemodynamics during SVE in the TG mice might be explained by mechanical induction of PTHrP release by vascular smooth muscle cells, as suggested by Pirola et al. (22). They demonstrated that aortic expression of PTHrP mRNA was increased within 2 h of balloon distension. Additionally, rocking vascular smooth muscle cells in primary culture, to create a flow of overlying medium, increased PTHrP mRNA expression 4-fold within 4 h. Thus, shear stress, rather than wall tension, may be the important factor modulating the local production of PTHrP by smooth muscle cells.
Although much of the current literature suggests that SVE normally increases GFR to some degree, we found that volume expansion produced a slight decrease in GFR in NTG mice and no change in GFR in TG mice. In this study, isotonic saline was infused at a rate of 0.75 µl/min·g BW (approximately 5% of BW per hour), and this resulted in a modest expansion of plasma volume in both groups (11–12%). This is in contrast to other species, such as the rat, in which saline infusion at this rate normally increases plasma volume by 25–40% (23). Although we cannot fully explain the differences observed here, time control experiments demonstrated that renal hemodynamic variables largely remain stable in mice over the time course used here. In addition, we have made similar measurements, on numerous occasions, in FVB/N and other strains of mice using this volume expansion protocol, with similar results.
In protocol II, systemic ANG II infusion resulted in increased blood pressure in both groups of animals, but the increase was less in the TG mice, compared with NTG littermates. These data support the hypothesis that PTHrP may act to buffer the effects of vasoconstrictor agents and attenuate their overall effect. In the kidney, ANG II infusion decreased GFR in both groups, but the decrease tended to be slightly less in the TG animals (although not significantly). Moreover, the decrease in ERPF induced by ANG II infusion was markedly blunted in the TG animals, compared with NTG. Because the relatively higher RBF in TG animals during ANG II infusion was associated with a relatively higher GFR, the data are again consistent with a predominately preglomerular site of action for locally produced PTHrP.
That the changes in renal hemodynamics in the TG animals in this portion of the study could be related to a direct induction of local PTHrP by ANG II is supported by several reported studies. ANG II treatment of cultured vascular smooth muscle cells resulted in a marked induction of PTHrP mRNA by 2 h, with a peak (6- to 10-fold) at 4–6 h (12). Studies have also shown that stimulation of rat aortic vascular smooth muscle cells, by cyclic stretch and ANG II, induces a synergistic, marked increase in PTHrP mRNA levels (24). Interestingly, it has also been reported that PTHrP gene expression in response to ANG II is impaired in smooth muscle cells from spontaneously hypertensive rats (25). Thus, the current observation, that RVR increased less in animals overexpressing the PTH/PTHrP-R in smooth muscle, supports the hypothesis that ANG II may induce local production of PTHrP in the vascular tree and thereby serve as a negative regulatory factor for the actions of ANG II. In addition, the data provide footing for the notion that derangements in PTHrP expression could hypothetically contribute to the pathogenesis of hypertension.
Because it has been well demonstrated that circulating PTH can have vasoactive effects in a variety of vascular beds (26), the possibility arises that the effects of PTH/PTHrP receptor overexpression may be due to hypersensitivity to circulating PTH, rather than to changes in local PTHrP production. For example, Crass et al. (27) investigated the effects of PTH infusion on different regional circulations in the dog, and they observed large increases in coronary and celiac blood flow but smaller effects on the renal and pulmonary vasculatures. We therefore performed separate experiments to evaluate circulating levels of PTH before and after SVE and ANG II infusion, and we found that PTH did not change in response to SVE, and increased modestly, but equally (
30–35 pg/ml), in response to ANG II. Relevant to this latter finding, Massfelder et al. (15) found that administration of exogenous PTH(1–34) in rats resulted in only a slight (3.5%) increase in RBF when infused at a rate that was calculated to raise renal plasma PTH levels by approximately 80 pg/ml. Because circulating levels of PTH are similar in mice and rats, it seems unlikely that the modest increase of 30–35 pg/ml after ANG II infusion, in the present experiments, could account for a substantial component of the vasodilation observed in Fig. 6B. Furthermore, in the case of SVE, a slight decrease in PTH levels was actually observed in the TG animals, whereas a small increase was observed in wild-types (although both changes were not significant). It is therefore difficult to reconcile the observed differences in the vascular responses between TG and wild-type animals with the lack of significant change in circulating PTH levels. Specifically, if the vascular effects of PTH/PTHrP receptor overexpression were mediated solely by an increased responsiveness to circulating PTH (which remains constant), then RVR would be expected to be lower in TG animals throughout the experiment, but the response to SVE should be parallel to that seen in NTG. To the contrary, the responses were not parallel, but rather substantially augmented in the TG mice, compared with NTG. Thus, although our present data cannot directly exclude the possibility of a role for PTH in mediating the observed responses, they appear to be more consistent with a role for local production of PTHrP in the regulation of renal hemodynamics.
No differences were observed in baseline urine flow, sodium excretion, potassium excretion, and solute excretion between the NTG and TG animals in either protocol. In response to SVE, the urine flow, sodium excretion, and solute excretion increased within each group, and these changes were not different between the groups. Potassium excretion decreased within each group from SVE, and this decrease was not different between the groups after SVE. ANG II induced increases in both urine flow and sodium excretion in both groups. However, the increase in urine flow was significantly less in the TG (compared with NTG) mice, whereas sodium excretion increased similarly in both groups in response to ANG II infusion. PTHrP has been shown to act in the same manner as PTH on renal tubular cells: it increases calcium reabsorption in the distal tubule, while inhibiting phosphate and bicarbonate reabsorption, and it stimulates 1-hydroxylase activity in the proximal tubule (28, 29). In the current study, the observed effects are limited to vascular alterations, without obvious tubular effects, because there were no remarkable differences in electrolyte excretion.
In summary, the data from this study demonstrate that animals with smooth muscle-specific overexpression of the PTH/PTHrP type 1 receptor show exaggerated responses to the vasodilator influences associated with SVE, and blunted responses to the vasoconstrictor influence of ANG II. These data support the postulate that locally produced PTHrP functions as a vasorelaxant factor in the kidney that may serve as a homeostatic mechanism to modulate renal hemodynamics.
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Footnotes
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This work was supported by NIH Grants DK57552, HL022619, and HL47811.
Abbreviations: ANG II, Angiotensin II; BW, body weight; ERPF, effective renal plasma flow; FITC, fluorescein isothiocyanate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GFR, glomerular filtration rate; MAP, mean arterial pressure; NTG, nontransgenic; PAH, para-aminohippuric acid sodium salt; PTHrP, PTH-related protein; -R, receptor; RBF, renal blood flow; RVR, renal vascular resistance; SVE, saline volume expansion; TG, transgenic.
Received March 20, 2003.
Accepted for publication July 11, 2003.
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Abstract
Introduction
