Activation of GLP-1 receptors on vascular smooth muscle cells ...

Am J Physiol Renal Physiol 308: F867–F877, 2015. First published February 5, 2015; doi:10.1152/ajprenal.00527.2014.

Activation of GLP-1 receptors on vascular smooth muscle cells reduces the autoregulatory response in afferent arterioles and increases renal blood flow Elisa P. Jensen,1,2 Steen S. Poulsen,1,2 Hannelouise Kissow,1,2 Niels-Henrik Holstein-Rathlou,1 Carolyn F. Deacon,1,2 Boye L. Jensen,3 Jens J. Holst,1,2 and Charlotte M. Sorensen1 1

Department of Biomedical Sciences, Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark; 2NNF Center for Basic Metabolic Research, Panum Institute, University of Copenhagen, Copenhagen, Denmark; and 3Department of Cardiovascular and Renal Research, Institute of Molecular Medicine, University of Southern Denmark, Odense, Denmark

Submitted 22 September 2014; accepted in final form 3 February 2015

Jensen EP, Poulsen SS, Kissow H, Holstein-Rathlou N-H, Deacon CF, Jensen BL, Holst JJ, Sorensen CM. Activation of GLP-1 receptors on vascular smooth muscle cells reduces the autoregulatory response in afferent arterioles and increases renal blood flow. Am J Physiol Renal Physiol 308: F867–F877, 2015. First published February 5, 2015; doi:10.1152/ajprenal.00527.2014.—Glucagon-like peptide (GLP)-1 has a range of extrapancreatic effects, including renal effects. The mechanisms are poorly understood, but GLP-1 receptors have been identified in the kidney. However, the exact cellular localization of the renal receptors is poorly described. The aim of the present study was to localize renal GLP-1 receptors and describe GLP-1-mediated effects on the renal vasculature. We hypothesized that renal GLP-1 receptors are located in the renal microcirculation and that activation of these affects renal autoregulation and increases renal blood flow. In vivo autoradiography using 125I-labeled GLP-1, 125 I-labeled exendin-4 (GLP-1 analog), and 125I-labeled exendin 9 –39 (GLP-1 receptor antagonist) was performed in rodents to localize specific GLP-1 receptor binding. GLP-1-mediated effects on blood pressure, renal blood flow (RBF), heart rate, renin secretion, urinary flow rate, and Na⫹ and K⫹ excretion were investigated in anesthetized rats. Effects of GLP-1 on afferent arterioles were investigated in isolated mouse kidneys. Specific binding of 125I-labeled GLP-1, 125 I-labeled exendin-4, and 125I-labeled exendin 9 –39 was observed in the renal vasculature, including afferent arterioles. Infusion of GLP-1 increased blood pressure, RBF, and urinary flow rate significantly in rats. Heart rate and plasma renin concentrations were unchanged. Exendin 9 –39 inhibited the increase in RBF. In isolated murine kidneys, GLP-1 and exendin-4 significantly reduced the autoregulatory response of afferent arterioles in response to stepwise increases in pressure. We conclude that GLP-1 receptors are located in the renal vasculature, including afferent arterioles. Activation of these receptors reduces the autoregulatory response of afferent arterioles to acute pressure increases and increases RBF in normotensive rats. glucagon-like peptide-1; glucagon-like peptide-1 receptor; kidney; afferent arteriole; renal blood flow GLUCAGON-LIKE PEPTIDE (GLP)-1 is an incretin hormone secreted from the intestinal epithelium, which enhances postprandial insulin release. Newly secreted intact GLP-1 (7–36)amide is rapidly degraded to GLP-1 (9 –36)amide by the ubiquitous enzyme dipeptidyl peptidase-4 (DPP-4) (8) resulting in an in vivo half-life for the active peptide of ⬃1–2 min (9). There is mounting evidence that GLP-1 is a multifunctional peptide affecting numerous tissues, including the kidneys. Several

Address for reprint requests and other correspondence: C. M. Sorensen, Dept. of Biomedical Sciences, Faculty of Health Sciences, Univ. of Copenhagen, Blegdamsvej 3B, Copenhagen DK-2200, Denmark (e-mail: cmehlin @sund.ku.dk). http://www.ajprenal.org

studies (7, 30) have reported GLP-1-mediated effects on renal hemodynamics and Na⫹ handling. Administration of GLP-1 to high-salt diet-fed Dahl salt-sensitive rats was associated with reduced blood pressure, reduced renal and cardiac injury, and improved endothelial function compared with controls. This was accompanied by the attenuated development of proteinand albuminuria, a prevention of the rise of plasma creatinine concentrations, and a reduction in tubular necrosis and renal interstitial fibrosis, along with an increase in water and Na⫹ excretion (45). In healthy normotensive rats, GLP-1 produced dose-dependent increases in renal blood flow (RBF), glomerular filtration rate (GFR), and Na⫹ and water excretion, which were associated with an inhibition of proximal tubular reabsorption (7, 30). This suggests a direct renal vasodilating effect of GLP-1. In other vascular beds, e.g., mesenteric arteries, GLP-1 has been shown to induce vasodilation (1, 17, 20, 31). In a clinical study (19) in obese insulin-resistant humans with glomerular hyperfiltration, GLP-1 reduced GFR, increased water, Na⫹, Cl⫺, and Ca2⫹ excretion, and reduced H⫹ excretion. Thus, it would appear that GLP-1 induces a decrease in GFR in models characterized by glomerular hyperfiltration (19, 45) and an increase in GFR in models with normal glomerular filtration (30, 43). However, a recent study (41) in healthy men showed that GLP-1 did not affect GFR or renal plasma flow (RPF) but increased Na⫹ excretion without affecting urine flow. The effect of GLP-1 on arterial blood pressure and heart rate (HR) also seems to depend on the experimental model. In normotensive animals, GLP-1 increases blood pressure and HR (4, 13), whereas in hypertensive models, GLP-1 receptor activation reduces arterial blood pressure (23, 26, 30). In a clinical study (28) where patients with type 2 diabetes mellitus were treated with GLP-1 receptor agonists, blood pressure was generally reduced. Although how the renal effects of GLP-1 are mediated remains to be fully established, several studies have suggested that GLP-1 receptors are located in the kidney with expression being reported in proximal tubular cells, in glomeruli (1, 7), and in the renal vasculature (12, 27, 35). However, their precise location has been a subject of debate, possibly due to lack of specific GLP-1 receptor antibodies (10). Nevertheless, a recent study (35) in human and monkey tissue showed GLP-1 receptor-specific staining in the renal vasculature with a newly developed GLP-1 receptor antibody. In the present study, we investigated renal GLP-1 receptor expression and examined whether the location of these receptors can explain GLP-1-mediated renal effects.

1931-857X/15 Copyright © 2015 the American Physiological Society

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We hypothesized that the vascular effects of GLP-1 in the kidney are mediated via receptors expressed on renal vascular smooth muscle cells, specifically in the afferent arteriole. The hypothesis was tested using in vivo autoradiography and a newly developed GLP-1 receptor antibody to show the exact location of the GLP-1 receptor within the renal vasculature. Renal effects of GLP-1 were tested in an in vivo model in which measurements of RBF, mean arterial pressure (MAP), urinary flow rate, and Na⫹ and K⫹ excretion were obtained before and after the addition of GLP-1. Furthermore, renal vascular effects of GLP-1 were examined in an in vitro model where changes in afferent arteriolar diameter in response to acute pressure increases were assessed before and after the addition of GLP-1. METHODS

All procedures were approved by the Danish National Animal Experiments Inspectorate. All animals were kept in the animal facility and received tap water and standard chow ad libitum. Autoradiography Surgical procedures. Female Wistar rats (n ⫽ 12) weighing 190 – 210 g and female CD1 mice (n ⫽ 54) weighing 18.6 –27.9 g were purchased from Taconic (Lille Skensved, Denmark) and Charles River (Sulzfeld, Germany), respectively, at least 1 wk before experimental procedures were performed. Rats were anesthetized with a subcutaneous injection of Hypnorm/ Domicum (0.2 mg/kg Fentanyl, 7.5 mg/kg Fluanisone, 3.75 mg/kg Dormicum, Pharmacy Service, SUND, University of Copenhagen, Copenhagen, Denmark). Mice were anesthetized with an intraperitoneal injection of ketamine-xylazine (100:10 mg/kg, Pharmacy Service, SUND, University of Copenhagen, Copenhagen, Denmark). The abdomen was opened by a midline incision, the inferior vena cava was exposed, and 15 mg of the DPP-4 inhibitor valine pyrrolidide (ValPyr; a gift from Novo Nordisk, Bagsværd, Denmark) in 400 ␮l of 0.04 M phosphate buffer (pH 7.5) for rats and 1.5 mg ValPyr in 50 ␮l of 0.04 M phosphate buffer for mice was injected to inhibit DPP-4 activity. One minute later, animals received 125I-labeled peptide (Table 1) dissolved in 0.04 M phosphate buffer with 1% human serum albumin (pH 7.5) into the inferior vena cava. Half of the animals also received a 1,000-fold excess of unlabeled peptide in combination with 125Ilabeled peptide in the same injection to test for specific binding. The

actual amount of 125I-labeled peptides administered was calculated from the specific radioactivity of the tracer (Table 1). Before injection, 10 ␮l of the 125I-labeled peptide stock solution were counted in a ␥-counter to determine the amount of radioactivity injected into each animal (Table 1). Rats were euthanized after either 2 min (n ⫽ 4), 5 min (n ⫽ 4), or 10 min (n ⫽ 4; Table 1). Two minutes before death, the thorax was opened, and the rat was connected to an animal respirator (Harvard Apparatus). The left cardiac ventricle was cannulated, and the right atrium was opened, after which the vascular system was perfused with a constant flow of 0.9% saline with an outlet through the right atrium until all blood was removed. After this, rats were fixed by flushing the vascular system with ice-cold 4% paraformaldehyde. The same procedure was performed on mice except that they were not connected to a respirator and they were euthanized at different time points after the injection of peptides (Table 1). After fixation, kidney and pancreatic tissues were removed and stored in 4% paraformaldehyde before being further processed. Development of autoradiography sections of mice and rat tissue. Pancreatic and kidney tissues were embedded in paraffin, and histological sections of 4 ␮m were cut with a microtome. In a dark room, histological sections of tissues were coated with 43– 45°C Kodak NTB emulsion (VWR, Herlev, Denmark) diluted 1:1 with 43– 45°C water, dried, and stored in light-proof boxes at 5°C for 6 – 8 wk. After 6 – 8 wk, histological sections were developed in a dark room in Kodak D-19 developer (VWR) for 5 min, dipped 10 times in 0.5% acetic acid, and fixed in 30% sodium thiosulfate for 10 min. Sections were then washed once in water for 10 min, washed three times (1 min) in water, and finally washed in 70% ethanol. Sections were stained with hematoxylin and examined with a light microscope. Images were taken with a camera (Zeiss Axioscope 2 plus, Brock & Michelsen, Birkerød, Denmark) connected to the light microscope. Immunohistochemistry Immunostaining for renin-secreting cells. To clarify the relationships between autoradiographic findings and the localization of reninimmunoreactive cells in juxtaglomerular cells of afferent arterioles, some kidney sections exposed to autoradiography from the mouse experiments were stained by immunohistochemistry using a renin antiserum. Sections were scanned, after which they were boiled at 100°C for 30 min in citrate buffer (pH 6) in a microwave oven to remove the film emulsion. Sections were washed in water and preincubated in PBS with 2% BSA (pH 7.5). Thereafter, sections were incubated overnight at 5°C in phosphate buffer with 2% BSA and primary antibody (rabbit anti-renin, in-house antibody, K026EV). For

Table 1. Overview of autoradiography experiments Species

Non-125I-Labeled Peptide Concentration

Euthanization Time, minutes after peptide infusion

Novo

3.1 nmol, Polypeptide (n ⫽ 6)

Novo

1.3 nmol, Polypeptide (n ⫽ 4)

Phoenix

1.3 nmol, Bachem (n ⫽ 8)

Perkin-


Novo


2 min (n ⫽ 4), 5 min (n ⫽ 4), and 10 min (n ⫽ 4) 10 min (n ⫽ 4) and 20 min (n ⫽ 4) 10 min (n ⫽ 4), 20 min (n ⫽ 8), and 40 min (n ⫽ 4) 10 min (n ⫽ 6), 20 min (n ⫽ 6), and 40 min (n ⫽ 6) 10 min (n ⫽ 6) and 20 min (n ⫽ 6)

125

Type of Peptides

I-Labeled Peptide Concentration and Activity

Rat

125

I-GLP-1 ⫹ GLP-1

Mouse

125

I-GLP-1 ⫹ GLP-1

Mouse

125

I-exendin-4 ⫹ exendin-4

Mouse

125

I-exendin 9–39 ⫹ exendin 9–39

Mouse

125

I-GLP-1 ⫹ exendin 9–39

3.1 pmol (7.500.000 counts/min), Nordisk (n ⫽ 6) 1.3 pmol (2.000.000 counts/min), Nordisk (n ⫽ 4) 1.3 pmol (3.000.000 counts/min), Pharmaceuticals (n ⫽ 8) 0.7 pmol (3.000.000 counts/min), Elmer (n ⫽ 9) 0.7 pmol (2.000.000 counts/min), Nordisk (n ⫽ 4)

Shown is an overview of the different groups in the autoradiography experiments, including species, types of peptides (radioactive and nonradioactive peptides) used in combination, concentration (in pmol) and activity (in counts/min) of the radioactive peptide (125I-labeled peptide), including numbers of animals that received only 125I-labeled peptide, concentration (in pmol) of the nonradioactive peptide, including numbers of animals that received the combination of 125 I-labeled peptide and an overload on nonradioactive peptide, and the number of minutes from peptide infusion until euthanization, including numbers of animals euthanized at the different time points. 125I-GLP-1, 125I-labeled glucagon-like peptide (GLP)-1; 125I-exendin-4, 125I-labeled exendin-4; 125I-exendin 9 –39, 125I-labeled exendin 9 –39. AJP-Renal Physiol • doi:10.1152/ajprenal.00527.2014 • www.ajprenal.org


visualization of the immunoreactions, sections were incubated for 40 min with biotinylated goat anti-rabbit immunoglobulins (Vector Laboratories, Burlingame, CA) as the second layer followed by a preformed avidin and biotinylated horseradish peroxidase macromolecular complex (ABC; Vector Laboratories) for 30 min as the third layer. Finally, immunoreactions were developed with diaminobenzidine (DAB) for 15 min, counterstained with hematoxylin, rinsed in water, dehydrated, and mounted. Generation of anti-mouse GLP-1 receptor antibody. GLP-1 receptor knockout (KO) mice were immunized with baby hamster kidney cells stably transfected with the murine (m)GLP-1 receptor, and antibodies were generated using standard hybridoma technology (35). GLP-1 receptor KO mice are on a C57Bl background and were derived from the previously described GLP-1 receptor KO strain (40) custom bred by Novo Nordisk at Taconic. The strain is not commercially available. Immunostaining for the GLP-1 receptor. Paraffin sections of pancreas and kidney tissues from both wild-type (WT) and GLP-1 receptor KO mice were chosen. Sections were deparafinated and rehydrated in double distilled water. Sections were treated with 0.1% pronase in PBS at 37°C for 10 min and rinsed in Tris-buffered saline (TBS). Thereafter, sections were treated with 1% H2O2 in TBS for 15 min, washed in TBS with 0.05% Tween 20 (TBST), blocked with avidin for 10 min (Dako, Glostrup, Denmark), washed with TBST, blocked with biotin for 10 min (Dako), washed with TBST, and preincubated with 3.2 mg/ml poly-L-lysine, 3% BSA, 7% donkey serum, and 3% skimmed milk (Dako) for 30 min. Sections were incubated overnight with primary biotinylated mouse GLP-1 receptor antibody (7F382A, Novo Nordisk). Thereafter, sections were washed three times for 10 min each in TBST followed by treatment with Vectastain ABComplexHRP in TBS for 30 min and washed again for three times for 10 min each. Sections were developed with DAB⫹ (Dako), counterstained with hematoxylin, rinsed in water, dehydrated, and mounted. Mouse In Vitro Perfused Juxtamedullary Nephron Technique A total of 15 C57/Bl6 mice weighing 24.9 ⫾ 1.1 g obtained from Taconic were used for the in vitro experiments. Surgical procedures. Experiments were conducted using the bloodperfused juxtamedullary nephron technique developed by Casellas and Navar (6) and adapted to mice (22). Briefly, in pentobarbital sodium (SAD, 50mg/kg ip)-anesthetized mice, the abdominal descending aorta was cannulated. The kidney was immediately perfused with Tyrode buffer containing an amino acid mixture (MEM amino acid solution, Sigma-Aldrich, Copenhagen, Denmark) and 5% BSA (ICPbio, Auckland, New Zealand) (pH 7.4). The kidneys were then excised, and the cannula was advanced into the left renal artery. A longitudinal slice was made along the kidney to expose the papilla without damaging it, and the papilla was reflected back to reveal the inner cortical surface. Venous tissue on the cortical surface was cut open to gain access to the renal vasculature. Ligatures (Ethilon, Somerville, NJ, 10.0 sutures) were fastened around larger arteries to restrict perfusion to juxtamedullary afferent arterioles of the inner cortical surface. Thereafter, connective tissue was removed. The kidney was perfused with 5% albumin-Tyrode solution. It has previously been shown that the afferent arteriole maintains an autoregulatory response during perfusion with cell-free Tyrode buffer containing albumin (5, 24, 38). The preparation was viewed and recorded using an Olympus BX50WI microscope with a PixelFly digital 12-bit charge-coupled device camera using CamWare software (PCO, Kelheim, Germany). Renal perfusion pressure (RPP) measured by a Statham pressure transducer was acquired with a PowerLab/8SP data-acquisition system (ADinstruments, Colorado Springs, CO). During the experiment, the kidney was superfused with 37°C Tyrode buffer containing 1% albumin.

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Experimental procedures. Afferent arterioles were chosen from the central interlobar artery to prevent tubuloglomerular feedback being accidentally interrupted during preparation of the kidney. Measurements of afferent arteriolar diameter were made ⬃100 ␮m upstream from the glomerulus. After establishment of a perfusion pressure of 95 mmHg, an equilibration period of 15 min was allowed. During this period, the kidney was perfused with Tyrode buffer containing 5% albumin, to which was added 0.1 mM N␻-nitro-L-arginine methyl ester (L-NAME) and 10 ␮M indomethacin (both Sigma-Aldrich). Thereafter, RPP was increased in steps of 20 mmHg from 95 mmHg up to 155 mmHg. Each pressure step lasted 3 min. After the first set of pressure steps, RPP was again decreased to 95 mmHg, and 10 nM GLP-1 [GLP-1 (7–36), Polypeptide, Strasbourg, France] and 0.1 mM ValPyr or 10 nM exendin-4 (GLP-1 receptor agonist with an extended half-life) and 0.1 mM ValPyr were added to the perfusion buffer. Fifteen minutes after the addition of peptide and ValPyr, the pressure steps were repeated. In an additional group, 10 nM GLP-1 and 0.1 mM ValPyr were added during the first set of pressure steps. Thereafter, 10 nM GLP-1, 0.1 mM ValPyr, and 100 nM exendin 9 –39 were added, and, after 15 min, the pressure steps were repeated. Renal Effects of GLP-1 In Vivo in Rats Nineteen Sprague-Dawley rats weighing 339 ⫾ 11 g obtained from Taconic were used for the in vivo experiments. Surgical procedures. Anesthesia was induced by inhalation of 5% isoflurane delivered in 65% N2-35% O2. Rats were placed on a heating table to maintain body temperature at 37°C. A catheter was placed in the left carotid artery to measure arterial blood pressure using a Statham P23-dB pressure transducer (Gould, Oxnard, CA). For continuous infusion of 0.9% saline and muscle relaxant, Nimbex (GlaxoSmithKline, Brøndby, Denmark), both at a rate of 20 ␮l/min, two catheters were placed in the right jugular vein. A tracheal catheter was placed, and rats were connected to a ventilator (tidal volume: 8 ml/kg body wt, frequency: 60 breaths/min). The abdomen was opened by a midline incision, and the abdominal aorta and left kidney were exposed. A catheter was introduced into the left iliac artery and passed through the abdominal aorta and into the left renal artery. The catheter allowed the introduction of drugs directly into the kidney so that direct renal effects with the least possible systemic effects could be observed. To measure RBF, an ultrasonic flow probe (Transonic 1PRB) was placed around the left renal artery. A catheter was placed in the left ureter to ensure free flow of urine and to measure urinary excretion. Rats were left undisturbed for 30 min after the surgical procedures before initiation of the experiment. The intrarenal infusion rate was increased from 10 to 144 ␮l/min when substances were administered to ensure a rapid distribution. Renal arterial plasma concentrations are estimated plasma concentrations, unless otherwise stated, and were calculated assuming a hematocrit of 40% (34) and an average RPF of 3 ml/min. All agents were dissolved in 0.9% saline containing 1% BSA. Experimental procedures. GLP-1-MEDIATED EFFECTS BEFORE AND AFTER BLOCKADE OF GLP-1 RECEPTORS. The acute effect of intrarenal infusions of GLP-1 was investigated before and after blockade of the GLP-1 receptor using exendin 9 –39, a GLP-1 receptor antagonist. Eight rats were used in this series. The sequence of infusions (intrarenal or intravenous) was varied, and rats served as their own controls. Buffer was infused intrarenally for 5 min at a rate of 144 ␮l/min (control period), and urine was collected. A blood sample (⬃0.3 ml) was drawn from the carotid artery at the end of the control period to determine plasma renin concentration. Thereafter, ValPyr was infused (plasma renal concentration: 0.1 mM). Two minutes later, GLP-1 was infused for 5 min (plasma renal concentration: 1 nM). Urine was collected during GLP-1 infusion, and a second blood sample was drawn at the end of this period.

AJP-Renal Physiol • doi:10.1152/ajprenal.00527.2014 • www.ajprenal.org

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After 15 min of equilibration, an intrarenal infusion of exendin 9 –39 was initiated for 10 min (plasma renal concentration: 100 nM). The described GLP-1 protocol was then repeated during continued exendin 9 –39 infusion. The experiments were repeated using intravenous administration of ValPyr, GLP-1, and exendin 9 –39. The amounts of ValPyr, GLP-1, and exendin 9 –39 administered were equal to the amounts given intrarenally. In four additional rats, the effect of 5-min administration of ValPyr on urine flow was examined before and after infusion of GLP-1 and exendin 9 –39. GLP-1-MEDIATED EFFECTS BEFORE AND AFTER INHIBITION OF NOS AND COX. Buffer was infused intrarenally for 2 min at 144 ␮l/min.

Hereafter, ValPyr was infused (plasma renal concentration: 0.1 mM) for 2 min followed by either GLP-1 (n ⫽ 4) or GLP-1 (9 –36)amide (GLP-1 metabolite generated after cleavage of GLP-1 by DPP-4, n ⫽ 3, Polypeptide) infusion (plasma renal concentration: 1 nM) for 5 min. After 15 min, indomethacin (bolus, 5 mg/kg) and L-NAME (10 mg·kg⫺1·h⫺1 for 30 min) were administered intravenously to inhibit cyclooxygenase (COX) and NO synthase (NOS). After 30 min of equilibration, intrarenal infusion of either GLP-1 or GLP-1 (9 – 36)amide was repeated. Analysis Afferent diameter was measured offline using ImageJ (National Institutes of Health, Bethesda, MD). The edges of the arteriole were tracked, and diameter was measured manually every 10 s throughout the experiment. Results are presented as means ⫾ SE of observations from the last minute of every pressure step. MAP and RBF are presented as actual values in the text and as percentages of the baseline values measured 1 min before GLP-1 administration in the figures. Renal vascular resistance (RVR) was calculated from the normalized values of MAP and RBF as follows: RVR ⫽ MAP/RBF. Results are means ⫾ SE. Urine flow was measured gravimetrically. Concentrations of Na⫹ and K⫹ in urine and plasma were measured by flame photometry (IL243 LED flame photometer, Instrumentation Laboratory, Alleroed, Denmark). Arterial plasma renin concentration was measured by the antibody-trapping method of Poulsen and Jørgensen as previously described (33). Concentrations were measured by the rate of ANG I formation from substrate-enriched nephrectomized sheep plasma and standardized in terms of international units per liter by the activity of the World Health Organization International Standard (Ref. No. 68-356, National Institute for Biological Standards and Control, Hertfordshire, UK) of which samples of 0.5 ␮IU were included in every run of the renin assay. The between-assay coefficient of variation was 9.4%. For statistical analysis, SigmaPlot software (SyStat Software, Chicago, IL) was used. Changes in MAP and RBF within and between groups were analyzed using two-way ANOVA for repeated measurements. Differences in urine flow, plasma renin concentration, and HR before and after GLP-1 treatment were analyzed using a paired Student’s t-test. P values of ⬍0.05 were considered significant. RESULTS

Autoradiography Pancreatic tissue served as a positive control in all animals, since it is well documented that GLP-1 receptors are present in islets of Langerhans. As shown in Fig. 1, A–C, grains were distributed all over the islets of Langerhans, demonstrating that this technique can be used to visualize tissue binding of 125 I-labeled GLP-1 (125I-GLP-1), 125I-labeled exendin-4 (125Iexendin-4), and 125I-labeled exendin 9 –39 (125I-exendin 9 –39). Similar patterns were observed in rats and mice. In

groups that received only 125I-labeled peptide, grains were observed in vascular structures in the cortex of the kidney. A strong signal was observed in small arterioles in proximity to glomeruli corresponding to afferent arterioles (Fig. 1, D and E) and in larger vessels in connection to arterioles corresponding to interlobular arteries and occasionally in arcuate arteries (Fig. 1, F and G). Grains seemed to be localized in smooth muscle cells in the wall of the vascular structures. Furthermore, there were grains in the first part of the proximal tubules, both in the lumen and cytoplasm of the cells. In groups receiving both 125I-labeled peptide and an excess of nonradioactive peptide, grains were only observed in the first part of the proximal tubules (Fig. 2, A and B), indicating that the binding in the renal vasculature is specific and probably involves receptors. Grains in the proximal tubules probably represent peptide that has been filtered in the glomeruli and subsequently reabsorbed in the tubuli. Overall, there was a stronger signal in experiments with 125 I-exendin 9 –39 compared with 125I-GLP-1 and 125I-exendin-4 in both the pancreas and kidney as exemplified in the pancreas, as shown in Fig. 1, A–C. Therefore, it seems that 125 I-exendin 9 –39 is superior to 125I- GLP-1 and 125I-exendin-4 in demonstrating possible locations of the GLP-1 receptor. Immunohistochemistry. Immunostaining for renin-producing cells revealed staining in the same segment of small arterioles as grains from the autoradiography experiments, with a small partial colocalization between renin-producing cells and grains (Fig. 2, C and D). GENERATION OF ANTI-MGLP-1R ANTIBODY. Monoclonal antibody clone 7F38A2 was identified as a specific anti-GLP-1 receptor antibody suited for immunhistochemistry on paraffinembedded tissues (unpublished observations). Immunostaining for the GLP-1 receptor was present all over the islets of Langerhans in pancreatic tissue from WT mice (Fig. 2E) but was absent in kidney tissue from GLP-1 receptor KO mice (Fig. 2G). Immunostaining for the GLP-1 receptor revealed staining in the renal vasculature (Fig. 2F), with localization being similar to what was observed in autoradiography experiments. No staining was observed in proximal tubules. GLP-1-mediated effects on the autoregulatory response of the afferent arteriole. The afferent arteriolar diameter at a RPP of 95 mmHg was 22.3 ⫾ 1.0 ␮m after the addition of L-NAME and indomethacin (n ⫽ 5). L-NAME and indomethacin were added to inhibit the major endothelium-derived vasodilators NO and PGI2 since the vasodilating effect of GLP-1 has been suggested to be endothelial cell dependent (1). Stepwise increases in RPP induced a significant reduction in the diameter of afferent arterioles (Fig. 3A). At a RPP of 155 mmHg, the afferent arteriolar diameter was reduced to 19.3 ⫾ 1.0 ␮m (P ⬍ 0.01 compared with the initial diameter). ValPyr and GLP-1 did not affect the baseline diameter of afferent arterioles (22.3 ⫾ 1.6 ␮m) at a RPP of 95 mmHg, and the diameter was unaffected by stepwise increases in RPP after GLP-1 perfusion. With GLP-1 and RPP at 155 mmHg, the diameter of afferent arterioles was 23.1 ⫾ 1.0 ␮m, indicating that GLP-1 significantly reduced the autoregulatory response of afferent arterioles (Fig. 3A) independently of increased NO and prostaglandin production.


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A

B

C

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D

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E

Fig. 1. Microphotographs [hematoxylin and eosin (H&E) stained, scale bars ⫽ 50 or 100 ␮m] of histological sections of the CD1 mouse pancreas (A–C), mouse kidney (D, F, and G), and Wistar rat kidney (E) after autoradiography. Mice and rats were injected with 125I-labeled glucagon-like peptide (GLP)-1 (125I-GLP-1; A, D, and E), 125I-labeled exendin 4 (125I-exendin 4; B), or 125I-labeled exendin 9 –39 (125I-exendin 9 –39; C, F, and G). A–C: all three isotopes bound specific in islets of Langerhans. D: specific 125I-GLP-1 binding in the vasculature indicated with arrows. E: specific 125I-GLP-1 binding in the arteriole in connection with the glomerulus indicated with an arrow. Magnifications: ⫻370 in A, ⫻200 in B, ⫻250 in C, ⫻400 in D, ⫻400 in E, ⫻75 in F, and ⫻75 in G.

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F

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G

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The experiments were repeated using the stable GLP-1 analog exendin-4 (n ⫽ 5). The addition of exendin-4 at a RPP of 95 mmHg did not change the baseline afferent diameter significantly (20.0 ⫾ 1.5 vs. 22.6 ⫾ 1.9 ␮m). Stepwise increases in RPP from 95 to 155 mmHg in the presence of exendin-4 did not affect the diameter of afferent arterioles (22.1 ⫾ 1.9 ␮m; Fig. 3B), demonstrating similar effects of exendin-4 and GLP-1 on the autoregulatory response of afferent arterioles. To verify that the effect was mediated by the GLP-1 receptor, we tested the effect of exendin 9 –39 on the GLP-1-induced effect on afferent autoregulatory responses (n ⫽ 5). During perfusion with GLP-1, afferent arteriolar diameter slightly but nonsignificantly increased from 22.7 ⫾ 1.5 to 23.7 ⫾ 1.5 ␮m (Fig. 3C). After the addition of exendin 9 –39, the afferent arteriolar diameter decreased significantly from 22.9 ⫾ 1.6 to 20.4 ⫾ 1.6 ␮m (P ⬍ 0.05).

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GLP-1-Mediated Effects In Vivo in Rats GLP-1-mediated effects before and after blockade of GLP-1 receptors. MAP was 94 ⫾ 3 mmHg, and RBF was 9.9 ⫾ 0.5 ml/min (n ⫽ 6; Table 2). Pretreatment with buffer followed by ValPyr did not affect MAP or RBF significantly. Intrarenal GLP-1 infusion for 5 min significantly increased MAP to 97 ⫾ 3 mmHg (4 ⫾ 1%) and RBF to 11.2 ⫾ 0.8 ml/min (13.9 ⫾ 1.3%; Fig. 4, A and B). RVR decreased significantly (Fig. 4C). Blockade of GLP-1 receptors with intrarenal infusion of exendin 9 –39 did not affect baseline MAP (92 ⫾ 2 mmHg) or RBF (9.4 ⫾ 0.8 ml/min). When GLP-1 was infused for 5 min during exendin 9 –39 infusion, MAP increased to 95 ⫾ 3 mmHg (3 ⫾ 1%; Fig. 4A). RBF increased to 9.8 ⫾ 0.8 ml/min (4.1 ⫾ 0.8%; Fig. 4B) during combined exendin 9 –39 and GLP-1 infusion. However, the RBF response after GLP-1 receptor blockade was significantly attenuated, and


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A

Fig. 2. Microphotographs (H&E stained, scale bars ⫽ 50 or 100 ␮m) of histological sections of the CD1 mouse kidney (A and B), Wistar rat kidney (C and D), wild-type (WT) mouse pancreas (E), WT mouse kidney (F), and GLP-1 receptor knockout (KO) mouse kidney (G) after autoradiography (A– C), staining for renin-immunoreactive cells (D), and staining for GLP-1 receptor-immunoreactive cells (E–G). Magnifications: ⫻425 in A, ⫻300 in B, ⫻350 in C, ⫻350 in D, ⫻450 in E, ⫻270 in F, and ⫻270 in G. Mice were injected with 125IGLP-1 together with an excess of unlabeled GLP-1. A and B: 125I-GLP-1 bound in the proximal tubules, indicated with arrows, and not in the renal vasculature. C: rats injected solely with 125I-GLP-1 had specific binding in the small arteriole in proximity to the glomerulus. D: the same section as in C (film emulsion removed) stained for renin-immunoreactive cells (comparable sites indicted with arrows). The short arrow indicates a site of colocalization between 125I-GLP-1-specific binding and renin-immunoreactive cells, whereas the long arrow indicates a site with no colocalization between 125 I-GLP-1-specific binding and renin-immunoreactive cells. E–G: there were GLP-1 receptor-immunoreactive cells in the pancreas (E) and in the renal vasculature (F), indicated with arrows, in WT mice, whereas there were no GLP-1 receptor-immunoreactive cells in the renal vasculature of GLP-1 receptor KO mice (G). The arrows indicate an arteriole that lacks GLP-1 receptor-immunoreactive cells.

B

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no significant reduction in RVR was observed (Fig. 4, B and C). The experiments were repeated using intravenous infusion of GLP-1 and exendin 9 –39 through the jugular vein. GLP-1 increased MAP from 93 ⫾ 2 to 96 ⫾ 3 mmHg (3 ⫾ 1%; Fig. 4A) and RBF from 8.9 ⫾ 0.6 to 9.8 ⫾ 0.7 ml/min (9.8 ⫾ 2.2%; Fig. 4B). In addition, RVR was significantly reduced (Fig. 4C). After GLP-1 receptors were blocked with exendin 9 –39, GLP-1 increased MAP to 97 ⫾ 4 mmHg (4 ⫾ 1%; Fig. 4A). Intravenous infusion of both exendin 9 –39 and GLP-1 did not increase RBF [8.9 ⫾ 1.0 to 9.0 ⫾ 1.1 ml/min (0.8 ⫾ 1.4%); Fig. 4B], resulting in an increasing RVR (Fig. 4C). GLP-1 caused a significant increase in urinary flow rate regardless of the administration route (Table 2). Furthermore, excretion of Na⫹ and K⫹ increased significantly during GLP-1 infusion (Table 2). When GLP-1 receptors were blocked with exendin 9 –39, intrarenal GLP-1 infusion still increased urinary flow rate significantly from 16.9 ⫾ 3.9 to 26.5 ⫾ 5.6 ␮l/min (P ⬍ 0.01). GLP-1 infused intravenously increased urinary flow rate from 15.0 ⫾ 2.5 to 25.5 ⫾ 6.6 ␮l/min (P ⫽ 0.07). Infusion of ValPyr alone for 5 min did not change urinary flow rate significantly (13.0 ⫾ 8.1 to 13.2 ⫾ 8.9 ␮l/min).

G

50µ

50µ

GLP-1, either with or without coinfusion of exendin 9 –39, had no effect on plasma renin concentrations or HR (Table 2). GLP-1-mediated effects before and after inhibition of NOS and COX. Baseline MAP was 103 ⫾ 3 mmHg and RBF was 10.3 ⫾ 0.6 ml/min (n ⫽ 4), and neither was affected by the infusion of buffer followed by ValPyr. Intrarenal infusion of GLP-1 for 5 min caused a significant increase in MAP to 107 ⫾ 3 mmHg (4 ⫾ 1%; Fig. 5A), a significant increase in RBF to 11.8 ⫾ 0.7 ml/min (14.4 ⫾ 1.1%; Fig. 5B), and a significant decrease in RVR (Fig. 5C). Treatment with L-NAME and indomethacin increased MAP to 133 ⫾ 6 mmHg and reduced RBF to 8.3 ⫾ 0.1 ml/min. Infusion of buffer and ValPyr did not affect MAP and RBF during L-NAME and indomethacin treatment. Intrarenal infusion of GLP-1 for 5 min increased MAP to 136 ⫾ 5 mmHg (2 ⫾ 1%; Fig. 5A) and RBF to 9.7 ⫾ 0.1 ml/min (16.0 ⫾ 1.3%; Fig. 5B), resulting in a significant decrease in RVR that was not different from the RVR changes seen before treatment with L-NAME and indomethacin. This indicates that the GLP-1-mediated effect on RBF is NOS and COX independent.


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RENAL EFFECTS OF GLP-1 25

A 120

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Fig. 3. Afferent arteriolar diameter (in ␮m) measured in C57/Bl6 mice during acute increases in renal perfusion pressure measured before (black line) and after (grey line) the addition of GLP-1 (A) or exendin-4 (Exen; B) in the perfusion (n ⫽ 5). In C, GLP-1 (grey line) was tested against GLP-1 and exendin 9 –39 (Ex9 –39; black line; n ⫽ 5). L-N, N␻-nitro-L-arginine methyl ester; Indo, indomethacin. *P ⬍ 0.05 vs. 95 mmHg.

The experiments were repeated with intrarenal infusion of the primary metabolite arising from DPP-4-mediated degradation, GLP-1 (9 –36)amide, given before and after treatment with L-NAME and indomethacin (n ⫽ 3). GLP-1 (9 –36)amide did not induce any significant changes in MAP, RBF, or RVR (results not shown).

Fig. 4. Changes over time in mean arterial pressure (MAP; A), renal blood flow (RBF; B), and renal vascular resistance (RVR; C) during infusion of GLP-1 in Sprague-Dawley rats. GLP-1 was infused into the renal artery (black line; n ⫽ 6) or into the jugular vein (black dashed line; n ⫽ 7). Thereafter, the GLP-1 receptor antagonist exendin 9 –39 was administered into the renal artery followed by GLP-1 infusion (grey line; n ⫽ 6) or exendin 9 –39 was administered through the jugular vein followed by GLP-1 infusion (grey dashed line; n ⫽ 7). *P ⬍ 0.05 vs. control; ¤P ⬍ 0.05 vs. the same time after exendin 9 –39. DISCUSSION

The results demonstrate that the GLP-1 receptor is expressed in the renal vasculature, including the afferent arteriole. Acute activation of these receptors significantly reduces the afferent autoregulatory response to acute pressure increases. Further-

Table 2. Physiological status of rats used for intrarenal and intravenous administration of GLP-1 Intrarenal Administration

Mean arterial pressure, mmHg Renal blood flow, ml/min Heart rate, beats/min Plasma renin concentration, mIU/l Urine flow rate, ␮l/min Urinary Na⫹ excretion rate, ␮mol/min Urinary K⫹ excretion rate, ␮mol/min

Intravenous Administration

Control

GLP-1

Control

GLP-1

94 ⫾ 3 9.9 ⫾ 0.5 369 ⫾ 14 15.0 ⫾ 0.9 12.2 ⫾ 3.1 3.1 ⫾ 0.6 1.8 ⫾ 0.1

97 ⫾ 3* 11.2 ⫾ 0.8* 368 ⫾ 13 13.9 ⫾ 1.3 31.5 ⫾ 5.6* 7.6 ⫾ 1.3* 2.5 ⫾ 0.1*

93 ⫾ 2 8.9 ⫾ 0.6 369 ⫾ 13 14.3 ⫾ 1.3 9.8 ⫾ 2.8 2.4 ⫾ 0.9 1.4 ⫾ 0.1

96 ⫾ 3* 9.8 ⫾ 0.7* 369 ⫾ 12 13.8 ⫾ 1.6 17.6 ⫾ 3.2* 4.7 ⫾ 1.1* 2.6 ⫾ 0.3*

Values are means ⫾ SE. *P ⬍ 0.05 vs. control. AJP-Renal Physiol • doi:10.1152/ajprenal.00527.2014 • www.ajprenal.org

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Fig. 5. Changes over time in MAP (A), RBF (B), and RVR (C) during infusion of GLP-1 in Sprague-Dawley rats. GLP-1 was infused into the renal artery (black line; n ⫽ 4). Thereafter, the nitric oxide synthase inhibitor N␻-nitro-Larginine methyl ester and the cyclooxygenase inhibitor indomethacin were administered through the jugular vein followed by intrarenal GLP-1 infusion (grey line; n ⫽ 4). #,P ⬍ 0.05 vs. control.

more, GLP-1 increases RBF and MAP irrespective of the administration route. The GLP-1 receptor antagonist exendin 9 –39 significantly reduced the GLP-1-mediated increase in RBF, whereas the increase in MAP persisted. The increase in RBF does not involve increased production of NO or PGI2 as treatment with L-NAME and indomethacin was without effect. The results suggest that acute activation of the GLP-1 receptor decreases the efficiency of renal autoregulation and thereby decreases the ability of the afferent arteriole to control GFR and RBF. Consequently, the maintenance of Na⫹ homeostasis and protection of glomerular capillaries could be reduced. A great effort has been put into identifying the precise localization of GLP-1 receptors in different tissues, including the kidney, but with conflicting results. One study (39) showed immunostaining of GLP-1 receptors in the luminal membrane of porcine proximal tubular cells as well as intracellular staining in proximity to the nucleus. Others have been unable to find a GLP-1 receptor antibody suitable for immunostaining (32, 36). A newly developed GLP-1 receptor antibody showed staining in the renal vasculature but not in the proximal tubules in monkey and human tissues (35). In some studies, GLP-1

receptor mRNA was reported in the glomerulus and in the initial part of proximal convoluted tubules, with no mRNA expression in other parts of the nephron (7). Others found mRNA in the renal artery and glomerulus but no tubular expression (12). However, mRNA expression does not necessarily correspond to protein expression (18), and mRNA could be present without functional GLP-1 receptor protein expression. We used in vivo autoradiography to locate renal GLP-1 receptors. Specific binding of 125I-GLP-1, 125I-exendin-4, and 125 I-exendin 9 –39 was observed in vascular smooth muscle cells in larger vessels and small arterioles, including afferent arterioles. Furthermore, there were grains in the initial part of proximal tubules on the luminal side but not further down the nephron. When excess nonradioactive peptide was administered, grains still were observed in the initial part of proximal tubules but not in the renal vasculature. This suggests that binding in the renal vasculature is saturable and therefore specific, supporting receptor involvement. The fact that both agonists of the GLP-1 receptor, GLP-1 and exendin-4, and the antagonist, exendin 9 –39, inhibited saturable binding at identical locations further supports that the binding is specific and related to the presence of the GLP-1 receptor. The presence of grains in proximal tubules after excess nonradioactive peptide administration probably reflects reuptake of labeled iodine or labeled peptide fragments filtered in the glomeruli. These grains were primarily localized to the brush border, where scavenger receptors, i.e., megalin/cubilin and oligopeptidases, are located and the cytoplasm of cells. Binding located specifically to the basolateral membrane was never observed. Immunostaining for the GLP-1 receptor showed no staining in proximal tubules. Staining was observed in the renal vasculature at sites similar to those observed in autoradiography experiments. The specificity of previously used antibodies for the GLP-1 receptor has been questioned (32, 36), but the present newly developed GLP-1 receptor antibody showed no staining in renal tissue from GLP-1 receptor KO mice. In support of the vascular localization, GLP-1 receptor mRNA has been recently demonstrated in renal arteries (12) and immunostained in monkey and human renal vasculatures with no staining in proximal tubules (35). Immunostaining for renin revealed specific binding of GLP-1 in the same juxtaglomerular part of afferent arterioles as renin-producing cells, but there was only little colocalization. GLP-1 administered to anesthetized rats had no effect on plasma renin concentration, suggesting that renin release is not regulated by GLP-1. GLP-1 receptor expression on some renin-producing cells could, therefore, simply reflect the plasticity of smooth muscle cells in arterioles. Whether GLP-1 receptors are located in the efferent arteriole is difficult to determine from the present results. Renin-producing cells and the GLP-1 receptor were located in the same arterioles, but some efferent arterioles also contain renin-secreting cells (2). Thus, we cannot exclude the presence of GLP-1 receptors in the efferent arteriole, but the parallel increase in GFR and RBF does suggest preferential afferent vasodilatation. Perfusion of the isolated murine kidney with GLP-1 and the stable GLP-1 receptor analog exendin-4 significantly reduced afferent autoregulatory responses to acute increases in RPP. The effect was inhibited when the GLP-1 receptor antagonist exendin 9 –39 was added. This suggests a vasodilating effect of



GLP-1 on the afferent arteriole. A micropuncture study (43) on Wistar rats indicated that exenatide, a synthetic version of exendin-4, decreased preglomerular resistance. The vasodilatory action of GLP-1 is consistent with a previous study (17) in isolated rat aorta where activation of the GLP-1 receptor increased the formation of cAMP and induced endotheliumindependent relaxation. In addition, GLP-1 dose dependently relaxed rat mesenteric arteries via a GLP-1 receptor-dependent mechanism as the effect was inhibited by exendin 9 –39 (31). GLP-1 also decreases vascular resistance in porcine mesenteric arteries (21). The vasorelaxing effect of GLP-1 and exendin-4 was present after inhibition of NO and PGI2 synthesis, showing that the effect is independent of these endothelium-derived vasodilators. This is in agreement with studies on isolated rat mesenteric arteries and the aorta (17, 31). However, Ban et al. (1) showed that inhibition of NO production abolished the vasodilatory effect of GLP-1 in murine mesenteric arteries. As opposed to the afferent arteriole, in these vessels, exendin-4 did not induce vasodilation. This may reflect differing responses to GLP-1 between vascular beds or possibly a vasodilating effect of the GLP-1 metabolite GLP-1 (9 –36)amide, which we did not find in the renal vasulature. In our hands, the vasorelaxing effect of GLP-1 was present in both rat and murine renal arterioles before and after inhibition of NO and PGI2 production. GLP-1-induced vasorelaxation has been suggested to be mediated by ATP-sensitive K⫹ (KATP) channels (17) activated by PKA. KATP channels have been found in both rat (29) and murine (42) afferent arterioles and may mediate renal vasodilation (25). The renal vasodilating effect of GLP-1 can be demonstrated in vivo as increased RPF and GFR (7, 30, 43) compatible with our observed preglomerular dilatation. In our hands, GLP-1 significantly increased RBF in anesthetized normotensive rats independent of changes in NO and PGI2 synthesis, supporting the assumption that GLP-1 exerts a direct effect on vascular smooth muscle cells. Since GLP-1 did not affect plasma renin levels, it is unlikely that changes in circulating ANG II levels or sympathetic efferent activity were responsible. Exendin 9 –39 significantly decreased the renal vasodilating effect of GLP-1 and abolished the reduction in RVR, suggesting that GLP-1-induced renal vasodilation is a direct GLP-1 receptormediated effect. The inhibitory effect of exendin 9 –39 was strongest in experiments where GLP-1 was administered intravenously, probably due to a higher local GLP-1 concentration during intrarenal infusions. The DPP-4 generated metabolite of GLP-1, GLP-1 (9 – 36)amide, has been claimed to have vasodilatory effects on murine mesenteric arteries (1) and rat aorta rings (17) independent of the GLP-1 receptor. In our study, GLP-1 (9 – 36)amide did not affect RBF or RVR in vivo, which could be due to differences between the investigated vascular beds. Gardiner et al. (13) found no vascular effects of GLP-1 (9 – 36)amide in conscious rats in an investigation of the renal and mesenteric circulation. However, they did report vasoconstriction in response to GLP-1 in the renal and mesenteric circulation, which may relate to the experiments being performed in conscious rather than anesthetized rats. We observed small, but significant, increases in MAP after acute GLP-1 infusions. This was independent of the administration route and pretreatment with exendin 9 –39, suggesting

F875

that the effect may not be caused by the known GLP-1 receptor. We found no change in HR to account for this, indicating that the increase in MAP is not driven by the sympathetic nervous system. Whether the observed increase in blood pressure is due to an increased cardiac output or to an increase in peripheral resistance cannot be determined from the present results. Blood pressure increases after acute intravenous infusion of GLP-1 or exendin-4 have been observed in both conscious (13) and anesthetized normotensive rats (3, 4, 44). In healthy humans, GLP-1 transiently increases HR (41) or both HR and MAP (11). However, in hypertensive animal models (23, 26, 30), GLP-1 has a blood pressure lowering effect. This effect is also apparent in human studies involving patients with type 2 diabetes (for a recent review, see Ref. 28). It is debated whether the natriuretic effect of GLP-1 is due to specific effects on proximal reabsorption or changes in renal hemodynamics. GLP-1 decreased Na⫹/H⫹ exchanger (NHE)3driven Na⫹ reabsorption both in isolated proximal nephron segments and in vivo in rats (7). Li⫹ clearance, a measure for the proximal outflow rate, was increased, suggesting a decrease in the proximal reabsorption rate (7, 30). However, in accordance with another recent study (12), we did not find expression of the GLP-1 receptor in renal tubules. Thus, the acute natriuretic effect of GLP-1 may not be caused by a direct GLP-1 effect on proximal tubular cells. When exendin 9 –39 was administered in the present study, the GLP-1-mediated increase in MAP and the natriuresis were still present, but the increase in RBF was significantly reduced. This suggests that the increased Na⫹ excretion is caused by the increase in MAP (pressure natriuresis) and not via direct GLP-1 receptor-dependent mechanisms in proximal tubules. Renal interstitial hydrostatic pressure has been shown not to increase significantly during GLP-1 infusion (30) even though Li⫹ and Na⫹ excretion increased. Nevertheless, an increase in renal interstitial hydrostatic pressure of only a few mmHg significantly increases Na⫹ excretion (15). This effect is even more pronounced in vasodilated kidneys (16). Inhibition of DPP-4 may also affect proximal Na⫹ reabsorption via NHE3 (14). A natriuretic effect of DPP-4 inhibition was found in GLP-1 receptor KO mice (37), showing the involvement of other mechanisms apart from or in addition to GLP-1. However, after 5-min infusion of ValPyr alone in anesthetized rats, we did not find any change in urine flow. It is possible that the short timeframe of our experiments and differences between inhibitors could explain the divergent results. In conclusion, the present study demonstrates the expression of GLP-1 receptors in vascular smooth muscle cells of the renal vasculature, including afferent arterioles. GLP-1 significantly reduces renal autoregulation in response to acute pressure changes via a GLP-1 receptor-dependent pathway. Furthermore, GLP-1 increases RBF and MAP without changes in HR and increases urinary flow rate and Na⫹ excretion. The increase in RBF is mediated via a GLP-1 receptor-dependent pathway, whereas the increases in MAP and urinary flow could involve a GLP-1 receptor-independent pathway. It is concluded that activation of GLP-1 receptors on vascular smooth muscle cells in afferent arterioles reduces the renal autoregulatory response and increases RBF. Additional experiments are required to further elucidate the acute effect of GLP-1 on MAP and Na⫹ excretion in normotensive animals.


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ACKNOWLEDGMENTS The skillful technical assistance of Cecilia Vallin, Susanne Hansen, Heidi Paulsen, Lise Strange, Charlotte Rasmussen Frost, and Pia G. Mortensen is gratefully acknowledged. The opportunity to use the GLP-1 receptor antibody developed by Morten G. Rasch is gratefully acknowledged.

15.

16.

GRANTS This work was supported by the Danish National Research Foundation, the Danish Heart Foundation, the A.P Møller Foundation for the Advancement of Medical Sciences, and The Novo Nordisk Foundation for Basic Metabolic Research.

17.

18.

DISCLOSURES E. P. Jensen is supported by Novo Nordisk in a PhD fellowship.

19.

AUTHOR CONTRIBUTIONS Author contributions: E.P.J., S.S.P., N.-H.H.-R., C.F.D., J.J.H., and C.M.S. conception and design of research; E.P.J., H.K., and C.M.S. performed experiments; E.P.J., B.L.J., and C.M.S. analyzed data; E.P.J., S.S.P., C.F.D., J.J.H., and C.M.S. interpreted results of experiments; E.P.J., S.S.P., and C.M.S. prepared figures; E.P.J., S.S.P., H.K., N.-H.H.-R., C.F.D., B.L.J., J.J.H., and C.M.S. drafted manuscript; E.P.J., S.S.P., H.K., N.-H.H.-R., C.F.D., B.L.J., J.J.H., and C.M.S. edited and revised manuscript; E.P.J., S.S.P., H.K., N.-H.H.R., C.F.D., B.L.J., J.J.H., and C.M.S. approved final version of manuscript.

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22.

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