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Am J Physiol Renal Physiol 285: F95-F104, 2003. First published March 18, 2003; doi:10.1152/ajprenal.00396.2002
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Bidirectional regulation of renal hemodynamics by activation of PAR1 and PAR2 in isolated perfused rat kidney

Yu Gui,1 Rodger Loutzenhiser,1 and Morley D. Hollenberg1,2

Canadian Institutes of Health Group on the Regulation of Vascular Contractility, Smooth Muscle Research Group, Departments of 1Pharmacology and Therapeutics and 2Medicine, University of Calgary, Calgary, Alberta, Canada T2N 4N1

Submitted 6 November 2002 ; accepted in final form 11 March 2003


   ABSTRACT
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 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Proteinase-activated receptors (PARs) are activated by either serine proteinases or synthetic peptides corresponding to the NH2-terminal tethered ligand sequences that are unmasked by proteolytic cleavage. Although PARs are highly expressed in the kidney, their roles in regulating renal function are not known. In the present study, we evaluated the impact of PAR activation on renal hemodynamics using PAR1- and PAR2-activating peptides (TFLLR-NH2 and SLIGRL-NH2) and proteinases (thrombin and trypsin) as PAR agonists in the isolated perfused rat kidney preparation. PAR1 activation resulted in renal vasoconstriction and a marked reduction in the glomerular filtration rate (GFR). In contrast, PAR2 activation caused vasodilation, partially reversing the vasoconstriction induced by TFLLR-NH2 and ANG II and increasing GFR that had been prereduced by ANG II. The vasoconstrictor actions of PAR1 activation were abolished by protein kinase C inhibition. The PAR2-induced vasodilation was only partially blocked by NG-nitro-L-arginine methyl ester, suggesting both nitric oxide-dependent and -independent mechanisms. Although PAR4 mRNA was detected in renal parenchyma, the PAR4-activating peptide AYPGKF-NH2 had no effect on renal perfusion flow rate. We conclude that PAR1 and PAR2 play bidirectional roles in the regulation of renal hemodynamics.

nitric oxide; trypsin; thrombin; endothelium-derived relaxing factor; glomerular filtration rate

PROTEINASE-ACTIVATED RECEPTORS (PARs), a unique class of G protein-coupled receptors, are activated by the proteolytic unmasking of NH2-terminal sequences that then act as tethered ligands, binding to and activating the receptors (6, 8, 20). To date, four members of this receptor family have been cloned (PARs 1 to 4). PARs 1, 3, and 4 are targets for thrombin, whereas PAR2 is sensitive to trypsin and in certain circumstances mast cell tryptase but not to thrombin. Although PARs 1, 2, and 4 are able to generate intracellular signals after proteolytic activation, PAR3 on its own appears not to signal in response to thrombin but rather acts as a cofactor for the activation of PAR4 (reviewed in Ref. 8). Interestingly, except for PAR3, PARs can also be activated in the absence of proteolytic cleavage by synthetic peptide agonists (so-called PAR-activating peptides, or PAR-APs) corresponding to their proteolytically revealed tethered ligand sequences (9, 29). Structure-activity studies with the PAR-APs have led to the synthesis of receptor-selective agonists: TFLLR-NH2 for PAR1, SLIGRL-NH2 for PAR2, and AYPGKF-NH2 for PAR4 (reviewed in Ref. 8). Importantly, reverse-sequence peptides having the same overall amino acid composition as the PAR-APs (e.g., RLLFT-NH2 or LSIGRL-NH2) are unable to activate the receptors and can therefore act as "control" peptides for studies done in vitro or in vivo.

Considerable evidence indicates that activation of PARs can regulate vascular function both in vivo and in vitro (reviewed in Ref. 20). However, in a highly vascularized organ, such as the kidney, the role of PAR activation has not yet been characterized. Several studies have shown that the kidney expresses an abundance of PAR1 and PAR2 mRNA and immunohisto-chemistry has demonstrated the presence of PAR1 and PAR2 in human and murine kidney, with localization detected on renal vascular and tubular cells (3, 5). A functional role for PAR1 and PAR2 in renal pathophysiology has been suggested by recent work indicating that PAR2 can regulate chloride secretion in murine tubular cells (3) and that PAR1 may play a role in renal inflammation (4). Given the impact of PAR1 and PAR2 activation on peripheral vascular function (1, 9, 10, 26, 27), we hypothesized that PAR activation may regulate renal hemodynamics. The present studies were thus aimed at determining the effects of PAR1 and PAR2 activation on renal perfusion flow rate (RPF) and glomerular filtration rate (GFR) in an isolated perfused rat kidney preparation. The signaling pathways whereby PAR activation affected renal vascular function were also assessed. In addition, a potential role for PAR4 activation in regulating renal flow was evaluated.


   METHODS
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 METHODS
RESULTS
 DISCUSSION
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In vitro normal rat isolated kidney perfusion. Male albino Sprague-Dawley rats (250–300 g) were used in accordance with the Canadian Council on Animal Care. Animals were anesthetized with halothane and methoxyflurane. The perfused kidney preparation (19) was employed to examine the effects of PAR activation on renal hemodynamics. The left ureter was cannulated with PE-10 tubing to collect the urine, followed by cannulation in situ of the left renal artery. Then, the left kidney was excised with continuous perfusion using oxygenated (95% O2-5% CO2), modified Krebs-Ringer buffer, containing 30 mM bicarbonate, 5 mM D-glucose, and 5 mM HEPES. To avoid the use of albumin, which would preclude testing the system with trypsin or thrombin and that might sequester the synthetic peptides, 4.5% dextran (mass molecular 64–76 kDa; Sigma, Oakville, Ontario, Canada) was used as an oncotic agent. In our studies, renal arterial pressure was maintained constant at 100 mmHg to ensure adequate glomerular filtration. The perfusion medium was recirculated. The initial total volume of the perfusion system was 80 ml. The RPF was monitored by a transit-time flowmeter (model T106, Transonic Systems, Ithaca, NY). Kidneys were allowed to recover during an initial 20-min equilibration period before any measurements were obtained. In the ANG II-preconstricted preparations, ANG II was added as a single bolus, plus constant infusion, at 0.1 nM for 10 min, followed by a single bolus and constant infusion of either the PAR2-activating peptides or trypsin for 10 or 20 min at the concentrations calculated according to the total volume (80 ml) of the perfusion system. In the experiments using pharmacological inhibitors, the compounds were added 5 min before the application of agonists.

Determination of GFR. GFR was estimated from the clearance of FITC-labeled inulin (Sigma) (18), which was added to the perfusion medium at the start of the equilibration period. The urine was collected at 10-min intervals, and urine volume was determined gravimetrically. The perfusate samples were collected at the time points midway through urine collection period. The concentration of FITC-inulin in the urine samples and the concentration in each corresponding perfusate sample, obtained at the midpoint of each urine collection, were determined fluorometrically (480-nm excitation/530-nm emission). These measurements were used to determine the urine-to-perfusate ratio of FITC-inulin. Inulin clearance was then calculated as the product of the urine flow rate and the urine-to-perfusate ratio of FITC-inulin (measured fluorometrically). The filtration fractions (FF; %) were calculated from the measured GFR and RPF according to the equation FF = (GFR/RPF) x 100.

RT-PCR detection of PAR1, PAR2, and PAR4 mRNA. Cortical and medullary tissues were harvested from fresh kidneys (following a 3-min perfusion to flush out resident blood cells) and from kidneys that had been perfused for 1 h in vitro. Total RNA was extracted with the TRI-reagent protocol (Molecular Research Center, Cincinnati, OH). One microgram of total RNA was reverse-transcribed (RT) with a first-strand cDNA synthesis kit using (N)6 primer (Pharmacia LKB Biotechnology, Uppsala, Sweden) at 37°C for 1 h. Two microliters of RT product were used to amplify the fragments of PAR1, PAR2, PAR4, and actin. The primer pairs for PAR1 (expected product 394 bp) were 5'-AAAAGCTTCCCGCTCATTTTTT CTCAGGAA-3' (forward) and 5'-GGGAATTCAATCGGTGCCGGAGAAAGT-3' (reverse). The primer pairs of PAR2 (expected product 190 bp) were 5'-CAACAGCTGCAT(T/A)GACCCCTT-3' (forward) and 5'-CCCGGGCTCAGTAGGAGGTTTTAA CAC-3' (reverse). The primer pairs for PAR4 (expected product 498 bp), derived from published PAR4 sequences (11), were 5'-ACAACAGTGACACGCTGGAG-3' (forward) and 5'-GCAGACCTTCCTATTGGCTG-3' (reverse). Actin message was used as an internal control for the RT-PCR reaction. The actin primers were 5'-CGTGGGCCGCCCTAG GCACCA-3' (forward) and 5'-TTGGCCTTAGGGTTCAGGGGG-3' (reverse). The detection of a 243-bp PCR product using the actin primers, which span an intron, can establish the absence of intron sequences in the RT product obtained from tissue RNA. Ten microliters of the PCR products were separated using 1.5% agarose gel electrophoresis and visualized by ethidium bromide staining. The PCR products amplified by PAR1 and PAR2 primers were purified with the Magic DNA purification Kit (Pharmacia LKB Biotechnology) for DNA sequencing analysis (DNA sequencing facility, University of Calgary, Calgary, Alberta).

Peptides and other reagents. The synthetic peptides, SLIGRL-NH2, LSIGRL-NH2, TFLLR-NH2, RLLFT-NH2, and AYPGFK-NH2, were prepared using solid-phase synthesis by either BioChem Therapeutic (Laval, Quebec, Canada) or by the peptide synthesis facility at the University of Calgary. Peptide purity, assessed by HPLC and mass spectral analysis, was >95%. The concentration of stock peptide solutions (dissolved in 25 mM HEPES, pH 7.4) was verified by quantitative amino acid analysis. Dextran, ANG II, NG-nitro-L-arginine methyl ester (L-NAME), and chelerythrine were purchased from Sigma.

Data analysis. Data are presented as either representative figures or as means ± SE. The number of replicates (n) represents the number of isolated kidney preparations used in each study. Differences between means were evaluated by Student's t-test (paired or independent) for two-group comparisons and by ANOVA followed by Bonferroni's correction for comparisons involving three or more groups. For comparing the same groups under different conditions (such as different time points), repeated-measures ANOVA was used. P < 0.05 was considered significant. When Bonferroni's correction was used, P < 0.05/n (where n = number of comparisons) was considered significant. All statistical analyses were performed using the Statistical Package for the Social Sciences software (SPSS, version 9.0, Chicago, IL).


   RESULTS
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 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Detection of PAR1 and PAR2 mRNA in the kidney. With the use of RT-PCR, we were able to detect both PAR1 and PAR2 mRNA expression in both the cortical and medullary tissues obtained from either the fresh kidney or kidneys that had been perfused in vitro for 1 h during three experiments (Fig. 1). A representative RT-PCR result shows that both the cortex and medulla of fresh and perfused kidney yielded strong RT-PCR signals for PAR1 mRNA, detected as an expected 394-bp product (Fig. 1B), and PAR2 mRNA, detected as an expected 190-bp product (Fig. 1A). The oligonucleotide sequences obtained from the PCR products using the PAR1 and PAR2 primers corresponded precisely with published rat PAR1 and PAR2 sequences (24, 32). The detection of an expected 243-bp product for actin confirmed the absence of genomic DNA in the RT-PCR procedure.



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  		Fig. 1. Expression of proteinase-activated receptor 1 (PAR1) and PAR2 mRNA in rat kidney. A: PAR2 expression in fresh kidney and perfused kidney (1 h). RT-PCR analysis using primer pairs for PAR2 (lanes 1-4) and actin primers as internal control (lanes 5-8). Lanes 1 and 5: cortex of fresh kidney. Lanes 2 and 6: medulla of fresh kidney. Lanes 3 and 7: cortex of perfused kidney. Lanes 4 and 8: medulla of perfused kidney. B: PAR1 expression in fresh and perfused kidney. Lane 1: cortex of fresh kidney. Lane 2: medulla of fresh kidney. Lane 3: cortex of perfused kidney. Lane 4: medulla of perfused kidney. Internal controls (actin) for lanes 1-4 are shown in lanes 5-8, respectively. The positions of the oligonucleotide size markers in base pairs (bp) are shown to the left of each gel.

 

Effects of the PAR2-activating peptide SLIGRL-NH2 on RPF. The PAR2-activating peptide SLIGRL-NH2 (1 to 25 µM) did not alter basal RPF when administered in the absence of a vasoconstrictor (22 ± 1.2 vs. the basal 21 ± 1.6 ml · min-1 · g-1, P > 0.05, n = 5). However, following preconstriction with ANG II (0.1 nM bolus followed by an infusion of 0.1 nm ANG II), SLIGRL-NH2 elicited a vasodilation, as reflected by an increase in RPF. A representative tracing is depicted in Fig. 2A. ANG II evoked a rapid and sustained decrease in RPF (from 22 to 10 ml · min-1 · g-1). In this setting, 10 µM SLIGRL-NH2 caused a biphasic vasodilator response, consisting of an initial transient peak (20 ml · min-1 · g-1) followed by a sustained increase in flow (18 ml · min-1 · g-1). In contrast, the partial reverse sequence peptide LSIGRL-NH2, which is an inactive control for SLIGRL-NH2, did not affect RPF (Fig. 2B). The mean data summarizing the effects of SLIGRL-NH2 are presented in Fig. 2D (open bars). ANG II reduced RPF from 23 ± 2.5 to 14 ± 2.7 ml · min-1 · g-1 (P < 0.01, n = 6). The subsequent addition of 10 µM SLIGRL-NH2 increased RPF to a peak, 21 ± 2.5 ml · min-1 · g-1 (P < 0.01, n = 6), followed by a sustained level of 20 ± 2.4 ml · min-1 · g-1 (P < 0.01, n = 6, vs. ANG II alone).



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  		Fig. 2. Vasodilator effect of SLIGRL-NH2 on the isolated perfused rat kidney. A: representative tracings illustrating reversal of ANG II (A II; 0.1 nM)-induced vasoconstriction by infusion of PAR2-AP SLIGRL-NH2 (10 µM). B: lack of effect of the reverse peptide LSIGRL-NH2 on flow. C: representative tracing showing the inhibitory effect of NG-nitro-L-arginine methyl ester (L-NAME) on the SLIGRL-NH2-mediated vasodilatation. D: mean data (n = 6) summarizing the vasodilator effects of SLIGRL-NH2 in the presence of ANG II-induced renal vasoconstriction (open bars) and the inhibitory effects of L-NAME pretreatment on this response (gray bars). *P < 0.05 vs. control perfusate flow.

 

In our previous studies, PAR2 activation has been observed to cause an endothelium-dependent vasodilation in a number of vascular preparations (1, 24). We therefore determined the effects of blocking cyclooxygenase (COX) and nitric oxide synthase (NOS) on the actions of SLIGRL-NH2 in the isolated kidney preparations. In these studies, 10 µM ibuprofen alone did not alter the activity of SLIGRL-NH2 to increase RPF in preparations pretreated with ANG II (data not shown, n = 5). In contrast, L-NAME (100 µM) significantly inhibited the SLIGRL-NH2-induced vasodilatation. As shown by the representative tracing in Fig. 2C, in the presence of L-NAME, 10 µM SLIGRL-NH2 elicited only a transient vasodilation. The effects of L-NAME on the SLIGRL-NH2-induced vasodilator responses in the ANG II-constricted preparations are summarized in Fig. 2D (gray bars). The magnitudes of both the peak and sustained phases of the SLIGRL-NH2-induced vasodilation were reduced by L-NAME treatment. To illustrate further the effects of L-NAME on the response to SLIGRL-NH2, the data were expressed as a percent vasodilation during the peak and sustained phase, relative to the magnitude of the ANG II-mediated reduction in RPF (calculated as shown in Fig. 3). In the absence of L-NAME, the peak and sustained dilation were 81 ± 6 and 67 ± 3% (open bars), whereas following L-NAME treatment, these values were 39 ± 7 and 17 ± 5% (filled bars, P < 0.05, n = 5; Fig. 3). Notwithstanding, SLIGRL-NH2 did cause a vasodilation, even in the presence of L-NAME with or without the concurrent presence of the guanylyl cyclase inhibitor 1 H-[1,2,4]oxiadiazolo[4,3-a]quinoxalin-1-one (ODQ) (results not shown). This L-NAME-resistant response to SLIGRL-NH2 was not prevented by 10 µM ibuprofen, or a P-450 enzyme inhibitor, 17-octadecynoic acid (ODYA; 10 µM) (data not shown). In addition, we observed that in preparations (n = 3) pretreated with 30 mM KCl, which reduced RPF from the basal 20 ± 0.5 to 10 ± 2.5 ml · min-1 · g-1, subsequent infusion of SLIGRL-NH2 (10 mM) in the presence of L-NAME still increased RPF to a peak value of 15 ± 1.2 ml · min-1 · g-1 followed by a sustained level of 12 ± 2.1 ml · min-1 · g-1.



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  		Fig. 3. Inhibitory effect of L-NAME on the vasodilatory effect of SLIGRL-NH2, expressed as % dilatation. Mean data are expressed as a percentage dilatation relative to ambient flow {% dilatation = [the increase in renal perfusion flow rate (RPF) caused by SLIGRL-NH2 divided by the decrease in RPF caused by ANG II] x 100}. * And ** indicate that the inhibition by L-NAME of both the peak and sustained vasodilatation caused by SLIGRL-NH2 (filled bars) was significant compared with the action of SLIGRL-NH2 in the absence of L-NAME (open bars) (P < 0.05, n = 5).

 

Effects of the PAR2-AP SLIGRL-NH2 on GFR. In a separate series of experiments, we determined the effects of SLIGRL-NH2 on GFR during ANG II-induced vasoconstriction (Fig. 4). After the equilibration period, the GFRs and RPFs during the first 10-min interval in the control group (subsequently treated with ANG II alone) were 0.6 ± 0.07 and 20 ± 1.2 ml · min-1 · g-1 and in the treated group (subsequently treated with ANG II and SLIGRL-NH2) were 0.6 ± 0.12 and 22 ± 1.9 ml · min-1 · g-1, respectively. These control values were used to calculate the percent changes in GRF and RPF shown in Fig. 4. The administration of 0.1 nM ANG II resulted in a sustained reduction in GFRs in both control group (25 ± 3% of basal) and the treated group (25 ± 5% of basal) (Fig. 4A). In the treated group (ANG II + SLIGRL-NH2), SLIGRL-NH2 increased GFR from 25 ± 5 to 68 ± 10 and 65 ± 15% over the ensuing 20 min. However, in the control kidneys (ANG II alone), GFR fell from 25 ± 5 to 18 ± 4 and 16 ± 4% during the same time period (P < 0.01, n = 5). The corresponding effects of SLIGRL-NH2 on RPF are shown in Fig. 4B. The infusion of 0.1 nM ANG II reduced RPF to 62 ± 8% of the control values in the control groups and 62 ± 3% of the control values in the SLIGRL-NH2-treated groups. RPF remained stable during the ensuing 20 min of ANG II administration in the control groups. However, treatment with 10 µM SLIGRL-NH2 increased RPF to 89 ± 2 and 85 ± 4% of the control values (P < 0.05, n = 5) at the same time periods of ANG II administration in the treated group. FF averaged 3.0 ± 0.4% in the control period and was reduced to 1.6 ± 0.2 and 1.6 ± 0.3% in the two periods following the administration of ANG II. In the studies examining the effects of SLIGRL-NH2, ANG II reduced FF from 2.8 ± 0.3 to 1.3 ± 0.3%. The subsequent administration of SLIGRL-NH2 increased FF to 2.1 ± 0.3.



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  		Fig. 4. Reversal of angiotensin-mediated reduction in glomerular filtration rate (GFR) by SLIGRL-NH2. A: ANG II (0.3 nM) induced a marked reduction in GFR. SLIGRL-NH2 (10 µM: dotted line) reversed the ANG II-mediated reduction in GFR. [P < 0.05 among the SLIGRL-treated groups and ANG II-treated groups (repeated-measures ANOVA)]. B: RPF corresponding to the GFR results shown above. The data summarize results from 2 separate series of experiments. ANG II was infused from 20 to 50 min; when present, SLIGRL-NH2 was infused from 30 to 50 min. The GFR and RPF are expressed as a percentage (%) of the basal values of GFR and RPF observed in each preparation under basal conditions of flow (0 to 10 min).

 

Effect of trypsin on RPF. The effects of the PAR2-activating proteinase trypsin on RPF were also evaluated. A representative tracing (Fig. 5A) illustrates that the administration of 0.1 nM ANG II reduced RPF. The reduction in RPF was reversed by the addition of 2 U/ml trypsin. On average, in this series of experiments, RPF in response to the administration of 0.1 nM ANG II was reduced from 22 ± 0.7 to 11 ± 1.9 ml · min-1 · g-1 (Fig. 5C, solid histograms), and the subsequent infusion of trypsin increased RPF from 11 ± 1.9 to 17 ± 1.8 ml · min-1 · g-1 (P < 0.05, n = 5). In a separate series of experiments, the responses to trypsin were assessed following L-NAME treatment (Fig. 5, B and C). A representative tracing (Fig. 5B) shows that in the presence of L-NAME, trypsin elicited a biphasic response, which was characterized by an initial vasodilation followed by a transient vasoconstriction. These responses lasted for 4–5 min, whereafter RPF returned to the initial level of ANG II-induced vasoconstriction. In the series of experiments done in the presence of L-NAME, on average, 0.1 nM ANG II reduced RPF from 22 ± 1.2 to 8 ± 2.6 ml · min-1 · g-1 (n = 5), and trypsin initially increased RPF to 11 ± 1.7 ml · min-1 · g-1 (Fig. 5C, first open histogram on the far right, **P < 0.05), followed by a decrease in RPF to 6 ± 2.3 ml · min-1 · g-1 (Fig. 5C, second open histogram on the far right, P < 0.05). In Fig. 6, effects on RPF are expressed as the percent vasodilation from ambient flow, relative to the magnitude of the ANG II-induced reduction in flow, to illustrate further the effects of L-NAME on the response to trypsin. In the absence of L-NAME, trypsin evoked a 65 ± 10% dilatation (open histogram), whereas in the presence of L-NAME, trypsin caused a much smaller initial vasodilation (18 ± 3% left shaded histogram, *P < 0.01, n = 5, vs. L-NAME-untreated group), followed by a transient vasoconstriction (-10 ± 5%) (Fig. 6, right shaded histogram, P < 0.01), with a subsequent return to basal flow (Fig. 5B).



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  		Fig. 5. Trypsin-induced vasodilatation of ANG II-preconstricted preparations. A: representative tracing illustrates the reversal by trypsin (2 U/ml) of the vasoconstriction caused by ANG II (0.1 nM). B: representative tracing demonstrates that L-NAME treatment inhibits the trypsin-induced vasodilatation and also reveals a transient, biphasic response; an initial vasodilatation followed by a small and transient constriction. C: mean data summarizing the vasodilator effect of trypsin and inhibition of trypsin-induced vasodilatation by L-NAME. *Reversal of the ANG II effect by trypsin was significant compared with the ANG II-treated group (P < 0.01, n = 5); **ANG II + trypsin + L-NAME group vs. ANG II + trypsin - L-NAME group (P < 0.05).

 


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  		Fig. 6. Vasodilator effect of trypsin and the inhibitory effect of L-NAME expressed as %dilatation. Mean data are expressed as percent dilatation relative to ambient flow [%dilatation = (the trypsin-induced increase in RPF divided by the ANG II-mediated reduction in RPF) x 100]. The average constrictor response to trypsin in the presence of L-NAME is also shown. *Inhibition of trypsin-mediated vasodilatation by L-NAME was significant (P < 0.05, n = 5), relative to dilatation caused in the absence of L-NAME.

 

Effects of PAR1 activation on RPF. Administration of the PAR1-activating peptide TFLLR-NH2 resulted in a renal vasoconstriction under basal perfusion conditions, as shown by the representative tracing (Fig. 7A). In contrast, the reverse sequence peptide RLLFT-NH2 that cannot activate PAR1 not only had no effect on RPF but also failed to act as an antagonist for lower concentrations of the agonist peptide TFLLR-NH2 (Fig. 7B). The summarized data show that infusion of 2 µM TFLLR-NH2 reduced RPF from 24 ± 1.5 ml · min-1 · g-1 to a nadir level of 9 ± 1.4 ml · min-1 · g-1 (Fig. 7D, solid histograms, P < 0.01, n = 5), followed by a sustained level of 16 ± 1.2 ml · min-1 · g-1 (P < 0.025). This vasoconstrictor response, which was comparable to that caused by ANG II (Fig. 2), was completely abolished by pretreatment with 3 µM chelerythrine, a protein kinase C (PKC) inhibitor (Fig. 7, C and D). In the presence of chelerythrine, RPF was 22 ± 1.6 ml · min-1 · g-1 under basal conditions and 22 ± 1.4 ml · min-1 · g-1 following the administration of 2 µM TFLLR-NH2 (P > 0.05, n = 5). In contrast to the PKC inhibitor, neither the COX inhibitor ibuprofen (10 µM) nor the tyrosine kinase inhibitors AG 1478 (10 µM) and genistein (15 µM) altered the vasoconstrictor actions of TFLLR-NH2 (data not shown).



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  		Fig. 7. Vasoconstrictor effect of the PAR1-AP TFLLR-NH2 (but not the receptor-inactive reverse peptide RLLFT-NH2) under basal perfusion conditions. A: representative tracing illustrates that the infusion of 0.5 to 2 µM of the PAR1-AP TFLLR-NH2 caused a marked reduction in RPF. B: prior administration of the reverse sequence peptide that cannot activate PAR1 was neither active on its own nor did it prevent the constrictor action of TFLLR-NH2. TFLLR-NH2 action is characterized by a rapid peak reduction followed by a sustained reduction in RPF. C: representative tracing demonstrates that the infusion of a protein kinase C inhibitor, chelerythrine (3 µM), abolished TFLLR-NH2-mediated vasoconstriction. D: averaged data illustrating the TFLLR-NH2-induced peak and sustained vasoconstriction, blocked by chelerythrine. [P < 0.05 among "- chelerythrine" groups (ANOVA)]. *Reduction in RPF in response to TFLLR-NH2 was significant compared with the basal perfusion flow (P < 0.01, n = 5). The difference between RPFs in response to TFLLR-NH2 in the presence of chelerythrine was not significant compared with the basal flows (P > 0.05, paired Student's t-test).

 

Effect of TFLLR-NH2 on GFR. In the studies examining the effects of PAR1 activation on renal hemodynamics, GFR and RPF during the first 10 min following equilibration of the preparation were 0.8 ± 0.1 and 23 ± 1.8 ml · min-1 · g-1, respectively (n = 6). These control values were used to calculate the percent changes in GFR and RPF. The administration of 2 µM TFLLR-NH2 reduced GFR to 10 ± 5% of the control value (P < 0.01; Fig. 8A), and this response was sustained for 20 min (12 ± 8% of basal at 40 min). In contrast, in the absence of TFLLR-NH2, GFR declined slightly to 78 ± 10 and 80 ± 8% of the basal value over the same time periods. TFLLR-NH2 reduced RPF to an initial value of 42 ± 2 and 56 ± 4% of basal (P < 0.05) after 20 min (Fig. 8B). RPF did not change in control preparations that were not treated with TFLLR-NH2. FF was 3.0 ± 0.3, 2.8 ± 0.3, and 2.7 ± 0.4% in the three consecutive 10-min periods of the control study. In the studies in which TFLLR-NH2 was administered, FF was 3.1 ± 0.3% in the initial period and was reduced to 1.5 ± 0.3 and 1.6 ± 0.4% in the two periods following the addition of TFLLR-NH2.



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  		Fig. 8. Effect of TFLLR-NH2 on GFR. A: GFR was estimated from the control group (dashed line) and from the TFLLR-NH2-treated group (solid line) under basal perfusion conditions. In each experiment, GFR and RPF measured during the first 10 min of perfusion were used as control values to calculate the percent change in GFR and RPF upon TFLLR-NH2 treatment (% control). In the TFLLR-NH2-treated group, TFLLR-NH2 was present at the perfusion time 20-40 min. TFLLR-NH2 infusion markedly reduced the GFR compared with that in the control group. * And ** indicate that the reductions in GFR induced by TFLLR-NH2 at the perfusion time 30 and 40 min, respectively, were significantly different compared with the control group at the same time periods (P < 0.01, n = 5, repeated-measures ANOVA). B: RPFs corresponding to the GFR data shown in A demonstrate that infusion of TFLLR-NH2 induced a significant reduction in RPF.

 

Effect of thrombin on RPF. Proteolytic activation of PAR1 with thrombin elicited a reduction in RPF comparable to that caused by TFLLR-NH2 (Fig. 9). The administration of thrombin, at a concentration of 2 U/ml [20 nM, which activates PAR1 selectively in intact tissues and cultured cells (12, 29, 30)], elicited a rapid renal vasoconstriction, as shown by the representative tracing in Fig. 9A. On average, thrombin reduced RPF from 21 ± 0.5 ml · min-1 · g-1 to a nadir of 4 ± 0.6 ml · min-1 · g-1 (Fig. 9C, filled histograms). This peak reduction in RPF was followed by a sustained reduction in RPF (13 ± 0.9 ml · min-1 · g-1, P < 0.05, n = 5; Fig. 9). This effect of thrombin, like that of TFLLR-NH2, was also abolished by pretreatment with 3 µM chelerythrine (Fig. 9B). In the presence of chelerythrine, RPF was 21 ± 1.2 ml · min-1 · g-1 under basal conditions and 22 ± 0.8 ml · min-1 · g-1 following treatment with thrombin (n = 4; Fig. 9C, open histograms).



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  		Fig. 9. Vasoconstrictor effect of thrombin and the inhibition of thrombin-induced vasoconstriction by chelerythrine. A: representative tracing illustrates the rapid peak reduction followed by a sustained reduction in RPF caused by thrombin (2 U/ml). B: representative tracing demonstrates that chelerythrine (3 µM) completely blocked the reduction in RPF caused by thrombin. C: averaged data from 2 series of experiments show thrombin induced a vasoconstrictor effect under basal perfusion conditions (without chelerythrine, filled histograms), comprising a peak and sustained reduction in RPF. When present, chelerythrine (3 µM, open histograms) completely blocked the thrombin-mediated reduction in RPF. *Reduction in RPF induced by thrombin was significant compared with the basal flow (P < 0.01, n = 5). RPF in response to thrombin in the presence of chelerythrine was not significantly different from the basal flow (P > 0.05, n = 0.05).

 

The observation that TFLLR-NH2 and thrombin elicited vasoconstrictor responses that were similar in magnitude and sensitivity to chelerythrine is consistent with the premise that both agents were working via the same mechanism (i.e., activation of PAR1). Nevertheless, it has been suggested that thrombin can also activate PAR4 in cultured cells (11, 12, 30). We therefore evaluated a potential role for PAR4 in thrombin-induced renal vasoconstriction. As depicted in Fig. 10A, RT-PCR analysis revealed the presence of PAR4 mRNA in the cortex and medulla from both fresh and perfused rat kidneys. The oligonucleotide sequence obtained from the PCR products using the PAR4 primers matched precisely with the published rat PAR4 sequence (11). Notwithstanding, while these results demonstrated the renal expression of PAR4 mRNA, the infusion of the PAR4-activating peptide AYPGKF-NH2 (100 µM) neither affected RPF nor desensitized the kidney to the subsequent actions of thrombin on RPF (Fig. 10B). These data indicate that if PAR4 activation were caused by thrombin, this receptor did not elicit the same renal hemodynamic effect as does activation of PAR1.



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  		Fig. 10. Expression of PAR4 mRNA in rat kidney and lack of an effect of the PAR4-AP AYPGKF-NH2 on RPF. A: RT-PCR detection demonstrates that the cortex (lane 1) and medulla from a fresh kidney (left lanes) and the cortex and medulla from a perfused kidney (right lanes) expressed PAR4 mRNA (expected product: 498 bp, top signals). The bottom signals represent the corresponding actin controls (expected product: 243 bp). B: representative tracing shows that the PAR4-activating peptide AYPGKF-NH2 (100 µM) did not affect basal RPF and that the subsequent infusion of thrombin was still able to reduce RPF.

 

Reversal of the TFLLR-NH2-induced vasoconstriction by SLIGRL-NH2. Because PAR1 and PAR2 activation elicited opposite effects on GFR and RPF, we next determined if activation of one receptor subtype would exert a functional antagonism on the actions of the other, thereby exerting a bidirectional regulation of the renal vasculature. A representative tracing demonstrates that SLIGRL-NH2 reversed the vasoconstrictor action of TFLLR-NH2 (Fig. 11A). As shown in Fig. 11B (filled histograms), on average, the application of 2 µM TFLLR-NH2 reduced RPF from 19 ± 1 ml · min-1 · g-1 to a nadir value of 6 ± 1 ml · min-1 · g-1 (P < 0.01, n = 5). This nadir was followed by a sustained RPF at a level of 9 ± 1 ml · min-1 · g-1. The subsequent administration of 10 µM SLIGRL-NH2 increased RPF to 13 ± 1 ml · min-1 · g-1 (shaded histogram, P < 0.05 vs. TFLLR-NH2 alone).



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  		Fig. 11. SLIGRL-NH2 reversed the vasoconstrictor effect caused by TFLLR-NH2. A representative tracing (A) and averaged data (B) demonstrate that infusion of the PAR1-AP TFLLR-NH2 (2 µM) resulted in a maximal peak reduction and a sustained reduction in RPF (B, solid histograms). The PAR2-AP SLIGRL-NH2 (10 µM) partially reversed the reduction in RPF caused by TFLLR-NH2 (B, gray histogram). *Vasodilatory effect of SLIGRL-NH2 was significant compared with the peak and sustained vasoconstriction caused by TFLLR-NH2 (P < 0.025, n = 6).

 


   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Previous reports of high expression levels of PAR1 and PAR2 in mouse and human kidney suggest a potential role of those PARs in the regulation of renal function. The present studies were designed to investigate the role of activation of PARs in regulating renal hemodynamics using the isolated perfused rat kidney as a model system. The main finding of our studies was that activation of PAR1 and PAR2 can play a bidirectional role in regulating rat renal hemodynamics. Our observations can be summarized as follows: 1) activation of PAR1 with the PAR1-AP TFLLR-NH2 (but not the receptor-inactive reverse peptide sequence RLLFT-NH2) resulted in a marked decrease in RPF and GFR; 2) activation of PAR2 with the PAR2-AP SLIGRL-NH2 (but not the partial reverse sequence receptor-inactive peptide LSIGRL-NH2) had no effect on basal RPF but reversed the reduction in RPF and GFR in response to the infusion of ANG II or the PAR-AP TFLLR-NH2; and 3) in keeping with the effects of the PAR1-AP and PAR2-AP, respectively, thrombin caused a vasoconstriction and trypsin induced a vasodilation. Qualitatively and quantitatively, the responses to the proteinases indicated that thrombin was acting exclusively via PAR1, rather than via other thrombin-mediated pathways, and that trypsin was acting primarily, but not exclusively, via PAR2; 4) the vasodilator effects of the PAR2-AP and trypsin were found to be mediated both via NO-dependent guanylyl cyclase activation as well as via other mechanisms, whereas the vasoconstrictor responses to PAR1-AP and thrombin required PKC; 5) RT-PCR analysis confirmed the presence of PAR1, and PAR2 mRNA in the rat kidney, a finding consistent with the pharmacological studies using the PAR-APs and further supporting a potential regulatory role for these PARs in the kidney; and 6) finally, although PAR4 mRNA was detected in the rat kidney by RT-PCR analysis, the PAR4-AP AYPGKF-NH2 failed to affect the RPF, thereby indicating that the effect of thrombin could be attributed to PAR1 but not PAR4 activation.

It has been documented that activation of PAR1 causes an endothelium-dependent relaxation in aorta (21) and coronary vessels in rat and human tissues (16, 27). It has also been demonstrated that activation of PAR1 can elicit vascular constriction either through a direct, endothelium-independent action (16, 17) or via an endothelium-dependent mechanism (25). These studies suggest that activation of PAR1 can induce different effects, either contraction or relaxation, through mechanisms that differ, depending on the specific tissue and experimental conditions. Our studies showed that PAR1 activation with the PAR1-activating peptide or thrombin elicited renal vasoconstriction in the rat kidney. Although thrombin in principle might activate both PAR1 and PAR4, it elicited this effect at a low concentration (2 U/ml), at which it selectively activates PAR1. Furthermore, the PAR4-activating peptide neither mimicked this action nor desensitized the preparation to thrombin. Our results strongly suggest that the renal vasoconstrictor effect of thrombin is mediated by PAR1. The signal transduction pathways activated by PAR1 are not fully resolved but are known to differ, depending on the tissues involved and the responses elicited. PAR1-induced stimulation of DNA synthesis appears to be mediated by phosphatidylinositol 3-kinase and protein kinase B pathways (2). PAR1 activation of platelet aggregation involves requisite roles for both PKC and tyrosine kinase (22). In the work we describe here, the constrictor responses to TFLLR-NH2 and thrombin were abolished by a PKC inhibitor but were not affected by two tyrosine kinase inhibitors, indicating that a PKC pathway independent of a tyrosine kinase pathway is involved in PAR1-mediated renal vasoconstriction.

In the study we report, the administration of the PAR2-activating peptide SLIGRL-NH2 partially reversed the reduction in RPF induced by ANG II and this vasodilation was greatly attenuated by blockade of NO synthesis. These observations are in agreement with the findings that PAR2 activation causes an endothelium-dependent NO-mediated relaxation in various vessel types obtained from a variety of species (1, 7). Unfortunately, it was not possible to determine in a direct manner whether the endothelium was required for the renal vasodilation observed in our perfused kidney preparation. In contrast to L-NAME, ibuprofen, a COX inhibitor, did not significantly attenuate the vasodilatation, suggesting that NO is a key mediator of the renal vasodilator response to PAR2 activation. Nevertheless, a transient vasodilatation was induced by SLIGRL-NH2 in the presence of L-NAME (with or without ODQ), suggesting that a mechanism independent of NO-activated guanylyl cyclase is also involved. In previous studies, we demonstrated that both NO-dependent and -independent mechanisms contribute to the afferent arteriolar dilation evoked by PAR2 activation in the in vitro perfused hydronephrotic rat kidney preparation (28). In the present study, neither ibuprofen nor the P-450 enzyme inhibitor 17-ODYA affected the L-NAME-resistant relaxation caused by PAR2 activation. In addition, elevated extracellular potassium (30 mM) did not prevent the L-NAME-resistant vasodilatation induced by SLIGRL-NH2 in the present study, whereas we found elevated potassium to abolish the L-NAME- and ibuprofen-insensitive response of the afferent arteriole to SLIGRL-NH2 (28). In concert, our results suggest that an unknown endothelium-derived relaxing factor may be involved in the L-NAME-resistant vasodilation induced by SLIGRL-NH2 in the renal circulation and that the determinants of this response may differ in different vascular segments. In keeping with the SLIGRL-NH2-induced vasodilation, the response to trypsin also exhibited both L-NAME-sensitive and -insensitive components, consistent with our interpretation that PAR2 mediates each response. However, we also observed a renal vasoconstrictor effect of trypsin that was revealed by L-NAME treatment (Fig. 5). It is possible that the trypsin-induced vasoconstriction unmasked by L-NAME may involve an action via a mechanism distinct from the activation of PARs. This possibility merits further investigation.

To our knowledge, the present study is the first to assess the effects of PAR1 and PAR2 activation on GFR. PAR1 activation resulted in a marked decrease in GFR, whereas activation of PAR2 caused a reversal of the reduction in GFR in response to ANG II. Although the precise segmental-specific actions of PARs on the renal microcirculation remain to be determined, we previously demonstrated that PAR2 reverses ANG II-induced constriction of the afferent arteriole (28). However, PAR1 and PAR2 have been localized to glomerular mesangial cells in both the mouse and human kidney (5, 31), and the actions of PAR activation on the filtration coefficient and glomerular capillary pressure have not been examined. Decreases in renal blood flow and GFR are early events in inflammatory kidney diseases, including glomerulonephritis and disseminated intravascular coagulation (DIC) (14, 23). Interestingly, the coagulation cascade would also be activated under such conditions and thrombin is an important mediator of this response. Moreover, trypsinogen is reported to be expressed in the renal vasculature of DIC patients, but not in the vasculature of controls (15). It is therefore interesting to speculate that both PAR1 and PAR2 may play an important role in the renal response to inflammation. Future studies assessing the glomerular and microvascular actions of the PARs and the roles of the PARs in the regulation of renal function in both normal and pathophysiological settings are warranted.

In summary, the present results indicate that PAR1 activation, either in response to thrombin-mediated proteolysis or the actions of the peptide sequence derived from the PAR1-tethered ligand, causes a marked renal vasoconstriction and a decrease in GFR. In contrast, PAR2 activation by either trypsin or a specific receptor-activating peptide elicits renal vasodilation, reversing the constrictor actions of both ANG II and PAR1 activation. We conclude that these bidirectional and functionally antagonistic actions of PAR1 and PAR2 activation may play an important role in the regulation of renal hemodynamics.


   ACKNOWLEDGMENTS
 
These studies were supported principally by a grant from the Kidney Foundation of Canada (M. D. Hollenberg), by ancillary funds from the Heart & Stroke Foundation of Canada (M. D. Hollenberg and R. Loutzenhiser), and by the Canadian Institutes of Health Research (CIHR) (M. D. Hollenberg and R. Loutzenhiser) and an Rx&D/Servier CIHR University-Industry grant (M. D. Hollenberg).


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. D. Hollenberg, Dept. of Pharmacology and Therapeutics and Dept. of Medicine, Univ. of Calgary, Faculty of Medicine, 3330 Hospital Drive NW, Calgary, AB, Canada T2N 4N1 (E-mail: mhollenb{at}ucalgary.ca).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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