Prostaglandin I2 (PGI2) produced by endothelial cells improves ischemia/reperfusion-induced acute renal injury by inhibiting leukocyte activation in rats. However, the underlying mechanism(s) of increased PGI2 production is not fully understood. Activation of sensory neurons increases endothelial PGI2 production by releasing calcitonin gene-related peptide (CGRP) in rats with hepatic ischemia or reperfusion. We examined here whether activation of sensory neurons increases PGI2 endothelial production, thereby reducing ischemia/reperfusion-induced acute renal injury.
Anesthetized rats were subjected to 45 min of renal ischemia/reperfusion. Rats were pretreated with CGRP, capsazepine (a vanilloid receptor-1 antagonist), CGRP(8-37) (a CGRP receptor antagonist), or indomethacin (a cyclooxygenase inhibitor), or subjected to denervation of primary sensory nerves before ischemia/reperfusion.
Renal tissue levels of CGRP and 6-keto-prostaglandin F1alpha, a stable metabolite of PGI2, increased after renal ischemia/reperfusion, peaking at 1 h after reperfusion. Overexpression of CGRP was also noted at 1 h after reperfusion. Increases in renal tissue levels of 6-keto-prostaglandin F1alpha at 1 h after reperfusion were significantly inhibited by pretreatment with capsazepine, CGRP(8-37), and indomethacin. Pretreatment with capsazepine, CGRP(8-37), indomethacin, and denervation of primary sensory nerves significantly increased blood urea nitrogen and serum creatinine levels, renal vascular permeability, renal tissue levels of myeloperoxidase activity, cytokine-induced neutrophil chemoattractant, and tumor necrosis factor-alpha, and decreased renal tissue blood flow. However, pretreatment with CGRP significantly improved these changes.
Our results suggest activation of sensory neurons in the pathologic process of ischemia/reperfusion-induced acute renal injury. Such activation reduces acute renal injury by attenuating inflammatory responses through enhanced endothelial PGI2 production.
ISCHEMIA/reperfusion can cause acute renal injury in association with major vascular surgery, cardiopulmonary bypass, renal transplantation, cardiac failure, and septic shock.1,2Tumor necrosis factor-α (TNF-α) plays an important role in the development of ischemia/reperfusion–induced acute renal injury through the activation of neutrophils.3Activated neutrophils adhere to vascular endothelial cells in the vasa recta of the outer medulla2,4,5and damage endothelial cells by releasing various inflammatory mediators, thereby increasing renal vascular permeability.2,4,5These pathologic events result in reduction of renal tissue blood flow.1,6,7These changes contribute to the development of renal dysfunction, reflected by increases in serum levels of blood urea nitrogen (BUN) and creatinine.8,9
Prostaglandin I2(PGI2) is a well-known cytoprotective agent known to increase tissue blood flow and inhibit leukocyte activation.10We have reported that enhanced endothelial release of PGI2reduces ischemia/reperfusion-induced acute renal injury by inhibiting TNF-α production and leukocyte activation in rats.9Capsaicin-sensitive sensory neurons are nociceptive neurons that are activated by a wide variety of noxious physical and chemical stimuli.11Inhibition of sensory neuron activation results in a marked increase in the severity of inflammation; therefore, sensory neurons might be involved in the maintenance of tissue integrity by regulating local inflammatory responses.12Neuropeptides play a pivotal role in mediating communication between the nervous, endocrine, and immune systems.13Calcitonin gene-related peptide (CGRP), a 37-amino acid neuropeptide,14is localized in both the central and peripheral nervous systems, mainly in nociceptive sensory nerves, including capsaicin-sensitive sensory neurons,15as well as in many other tissues, including the kidney.16CGRP is released from sensory neurons on activation of vanilloid receptor-1 by a wide variety of noxious physical and chemical stimuli.11On activation, sensory neurons release CGRP, which is reported to increase endothelial production of PGI2in vitro .17Therefore, various noxious stimuli that activate sensory neurons to release CGRP are capable of reducing tissue damage.12Consistent with these observations, we previously showed that, in hepatic ischemia/reperfusion rats, activation of sensory neurons contributed to the ischemia/reperfusion–induced liver injury by attenuating inflammatory responses.18These observations suggest that CGRP released from sensory neurons could reduce ischemia/reperfusion–induced acute renal injury by inhibiting leukocyte activation through enhanced endothelial PGI2production in the kidney.
The above background suggests that sensory neurons might be activated in the pathologic process leading to ischemia/reperfusion–induced acute renal injury. In the present study, we examined this possibility by analyzing the effects of CGRP; capsazepine, a vanilloid receptor-1 antagonist; CGRP(8–37), a CGRP receptor antagonist; denervation of primary sensory nerves; and indomethacin, a cyclooxygenase inhibitor, on a series of renal responses leading to acute renal injury in rats subjected to renal ischemia/reperfusion.
Materials and Methods
Capsazepine, capsaicin, and indomethacin were purchased from Sigma Chemical (St. Louis, MO), rat a-CGRP and CGRP(8–37) were purchased from the Peptide Institute (Osaka, Japan), and iloprost was kindly supplied by Eizai Co. (Tokyo, Japan). Evans blue dye and myeloperoxidase were purchased from Sigma Chemical. All other reagents were of analytical grade.
Animal Model of Renal Ischemia/Reperfusion
All experimental procedures were approved by the Oita University Animal Care and Use Committee (Yufu, Oita, Japan). The care and handling of the animals used in the present study were in accordance with the guidelines of the National Institutes of Health. Adult male Wistar rats (Kyudo; Kumamoto, Japan) weighing 200–220 g (n = 492) were used throughout the experiments. The rats were anesthetized with an intraperitoneal injection of 50 mg/kg pentobarbital sodium (Abbott Laboratories, North Chicago, IL), followed by 20 mg/kg (intraperitoneal) every hour for 4 h, and were supplemented with an intraperitoneal injection of 2 μg/kg buprenorphine for postoperative analgesia. Renal ischemia/reperfusion injury was induced as described previously.9In brief, to induce ischemia, the right pedicle was clamped with a noncrushing microvascular clamp for 45 min. Ischemia was confirmed visually by blanching of the kidney. Sham-operated animals underwent the same operation, but without clamping. No animal died until sacrifice after renal reperfusion. Renal dysfunction was evaluated by measuring serum levels of BUN and creatinine by standard assays and picric acid reaction.8Blood samples were taken from the abdominal aorta and collected in tubes, and centrifuged at 2,000g for 10 min for examination of BUN and serum creatinine. The kidneys were harvested for histopathological examination. We also measured renal vascular permeability, renal myeloperoxidase activity, renal tissue content of cytokines, renal tissue levels of 6-keto-prostaglandin F1α(6-keto-PGF1α) and CGRP (see Evaluation of Renal Vascular Permeability; Assay of Renal Myeloperoxidase Activity, Cytokine-induced Neutrophil Chemoattractant, and TNF-α; Measurement of Renal Tissue Levels of 6-keto-PGF1α; and Immunohistochemical Staining for CGRP and Plasma and Renal CGRP Levels ). When measurements were completed, animals were anesthetized with an overdose of pentobarbital sodium and sacrificed.
To elucidate whether activation of sensory neurons with subsequent CGRP release promotes endothelial PGI2production in rats with renal ischemia/reperfusion injury, we investigated the effects of subcutaneously administered 0.01 mg/kg CGRP, subcutaneously administered 20 mg/kg capsazepine (a vanilloid receptor-1 antagonist), intravenously administered 0.1 mg/kg CGRP(8–37) (a CGRP receptor antagonist), and subcutaneously administered 5 mg/kg indomethacin (a cyclooxygenase inhibitor), injected 30 min before renal ischemia. Iloprost (a stable derivative of PGI2) was dissolved in saline and infused continuously (100 ng · kg−1· min−1) via the right jugular vein for 3 h after the onset of renal reperfusion, as described previously.9The same volume of saline was administered continuously in control rats. Furthermore, to elucidate effects of CGRP, capsazepine, CGRP(8–37), and indomethacin on the sham-operated animals, we examined the effects of the agents on the sham-operated animals as compared with sham (saline-administered instead of each agent) at specified time points.
Denervation of Primary Sensory Nerves by Capsaicin
The rats received a total dose of 125 mg/kg capsaicin, administered subcutaneously in divided doses over 2 days.19Two weeks after treatment with high-dose capsaicin, the animals were subjected to stress. To determine the effectiveness of the sensory afferent nerve denervation procedure, a drop of 0.001% capsaicin in saline was instilled into either eye of the rats, and their protective wiping movements were observed. Capsaicin-treated rats that showed any wiping movement were excluded from the study. Control animals were injected subcutaneously 1 ml of 10% Tween 20/10% ethanol (10%) with normal saline.
Immunohistochemical Staining for CGRP and Plasma and Renal CGRP Levels
The peroxide-antiperoxide technique was used for immunohistochemical staining of the kidney with anti-CGRP antibody according to the method described previously.19In brief, the unfixed tissue blocks of rat kidneys were frozen in dry ice-cooled optimum cutting temperature compound (Tissue-Tek; Miles Inc., Elkhart, IN). Sections (3–4 μm thick) were mounted on glass slides, immersed in absolute acetone at -20°C for 5 min, rinsed in phosphate-buffered saline 5 times for 5 min each, and then incubated for 20 min with 10% porcine serum in phosphate-buffered saline at room temperature. They were incubated for 1 h at 37°C with rabbit anti-CGRP polyclonal antibody at 1:100 dilution. After five rinses in phosphate-buffered saline, the sections were treated with horseradish peroxidase–conjugated antirabbit immunoglobulin G (MBL, Nagoya, Japan) at 1:2,000 dilution for 1 h at 37°C. Reaction products were developed by immersing the sections in 3′3-diaminobenzidine tetrahydrochloride solution containing 0.03% hydrogen peroxide. The control for immunostaining was performed by nonimmune rabbit serum as the first step in place of primary antiserum, and omission of the first step or use of the first antiserum preabsorbed with an excess of the homologous antigen. Samples were mounted with Entellan onto glass slides, examined, and photographed under a light microscope. Samples were analyzed by investigators who were blinded to the experimental groups.
Renal tissue and plasma CGRP levels were determined as described previously.20In brief, the tissue samples were weighed and then homogenized in 3 ml of 2N acetic acid. The homogenates were placed in a 90°C water bath for 20 min and then centrifuged (4,500g for 10 min at 4°C). CGRP was extracted from the supernatant by using reverse phase C18 columns (Amersham Biosciences, Little Chalfont, England). Columns were prepared by washing with 5 ml of methanol, followed by 10 ml of water before use. The supernatant was applied onto the column, followed by washing with 20 ml of 0.1% trifluoroacetic acid. CGRP was eluted with 3 ml of 60% acetonitrile in 0.1% trifluoroacetic acid, and the solvent was evaporated under a stream of nitrogen gas. The concentration of CGRP was assayed by using a specific enzyme immunoassay kit (SPI-BIO; Massey Cedex, France). The antiserum cross-reacts 100% of rat α- and β-CGRP, based on the data sheet supplied by the manufacturer.
Measurement of Renal Tissue Levels of 6-keto-PGF1α
Endothelial production of PGI2in kidneys was assessed by measuring renal tissue levels of 6-keto-PGF1α, a stable metabolite of PGI2, using the method described previously.9The PGI2concentration was assayed using a rat-specific enzyme immunoassay system (Amersham).
Measurement of Renal Tissue Blood Flow
Renal tissue blood flow was evaluated by measuring renal cortical blood flow monitored by a laser-Doppler flow meter (ALF21N; Advance, Tokyo, Japan) as described previously.8,9The Doppler probe was directed towards the cortex. Renal cortical blood flow was measured from 30 min before ischemia until 3 h after reperfusion.
Evaluation of Renal Vascular Permeability
Renal vascular permeability was assessed by measuring extravasation of Evans blue dye, as described previously.8,9In brief, the dye (25 μg/kg) was injected intravenously 10 min before sacrifice by exsanguination from the abdominal aorta. The kidneys were removed, weighed, and placed in 5 ml of dimethylformamide (Wako, Osaka, Japan) for 7 days. After centrifugation (2,000g for 10 min), the concentration of Evans blue dye extracted in the dimethylformamide was measured by a spectrophotometer (DU-54; Beckman, Irvine, CA) at a wavelength of 610 nm, and compared with the results obtained with standards.
Assay of Renal Myeloperoxidase Activity, Cytokine-induced Neutrophil Chemoattractant, and TNF-α
Accumulation of neutrophils in the kidney was evaluated by measuring renal myeloperoxidase activity, as described previously.8,9The rats were sacrificed at various time points after reperfusion. The kidneys were removed, weighed, and homogenized (Physcotron; Niti-on, Tokyo, Japan) in 10% (weight/volume) 0.05 m phosphate buffer (pH 6.0) containing 0.5% hexadecyltrimethylammonium bromide and sonicated for 20 s. After centrifugation (4,500g for 20 min at 4°C), 0.1 ml of the supernatant was added to 0.55 ml of 0.1 m phosphate buffer (pH 6.0) containing 1.25 mg/ml o-dianisidine and 0.05% hydrogen peroxide. After 5 min, changes in absorbance at 460 nm were measured using a spectrophotometer. The activity of purified myeloperoxidase was used as the standard. Results are expressed as units of myeloperoxidase activity per gram of tissue.
Renal tissue concentrations of cytokine-induced neutrophil chemoattractant (CINC), the rat equivalent of human interleukin 8,21and TNF-α were measured using enzyme-linked immunosorbent assay kits for rat CINC (Amersham) and TNF-α (Genzyme Corp., Cambridge, MA) according to the instructions supplied by the manufacturer.
Data are expressed as mean ± SD. Statistical analysis was performed on a Macintosh computer system with Statview 4.5 statistical analysis software (Abacus Concepts, Inc., Berkeley, CA). Renal CGRP time course data were analyzed with a Student t test to compare the differences between a baseline (Pre) and responses after the injury, and those between the sham and ischemia/reperfusion groups. Renal CGRP, 6-keto-PGF1α, BUN, creatinine, Evans blue leakage, myeloperoxidase, CINC, and TNF-α data were analyzed for statistical significance by means of one-way ANOVA, followed by the Bonferroni-Dunn method for multiple comparison. Renal cortical blood flow data were analyzed with a Student t test for comparisons among five groups at each time point. A P value of less than 0.05 was considered statistically significant.
Changes in CGRP Levels in Kidneys and Plasma, and Renal Immunohistochemical Expression of CGRP in the Kidney of Rats Subjected to Renal Ischemia/Reperfusion
Renal tissue levels of CGRP were significantly increased 1 h after reperfusion, as compared with the preischemia levels (fig. 1A). However, the levels decreased rapidly to the preischemia levels 3 h after reperfusion, and remained unchanged during 3 to 24 h (fig. 1A). Renal tissue levels of CGRP at 3, 6, 12, and 24 h after reperfusion were not significantly different from those in sham-operated animals (fig. 1A). Plasma CGRP levels were below the detection limit before and after renal ischemia/reperfusion (data not shown). Both pretreatment with capsazepine (a vanilloid receptor-1 antagonist) and denervation of primary sensory nerves by capsaicin significantly inhibited ischemia/reperfusion–induced increases in renal tissue levels of CGRP (fig. 1B). Rats subjected to renal ischemia/reperfusion showed overexpression of CGRP in the outer renal medulla 1 h after reperfusion, as compared with the sham-operated rats (fig. 2, A and B).
Effects of CGRP, Capsazepine, CGRP(8–37), Denervation of Primary Sensory Nerves, and Indomethacin on Ischemia/Reperfusion-induced Increases in Renal Tissue Levels of 6-keto-PGF1α
Renal tissue levels of 6-keto-PGF1αwere increased after reperfusion, reaching a peak level 1 h after reperfusion.9To examine whether activation of sensory neurons with subsequent CGRP release contributes to the observed increase in endothelial PGI2production, we tested the effects of CGRP, capsazepine, CGRP(8–37), denervation of primary sensory nerves, and indomethacin on renal tissue levels of 6-keto-PGF1α1 h after reperfusion. Renal tissue levels of 6-keto-PGF1αin rats subjected to renal ischemia/reperfusion were significantly higher 1 h after reperfusion than those in sham-operated rats (fig. 3). Increases in renal tissue levels of 6-keto-PGF1α1 h after reperfusion were significantly inhibited by both pretreatment with capsazepine, CGRP(8–37), and indomethacin and denervation of primary sensory nerves (fig. 3). However, these levels in rats pretreated with CGRP were significantly higher than those in vehicle rats.
Effects of CGRP, Capsazepine, CGRP(8–37), Denervation of Primary Sensory Nerves, and Indomethacin on Renal Dysfunction, Renal Cortical Blood Flow, and Renal Vascular Permeability
Serum levels of BUN and creatinine were significantly increased after renal ischemia/reperfusion, reaching peak levels 24 h after reperfusion.9Renal cortical blood flow decreased to approximately 40% of the preischemic level 1 to 3 h after reperfusion.9Renal vascular permeability, as assessed by Evans blue dye leakage, increased significantly after renal ischemia/reperfusion, reaching a maximum level 6 h after reperfusion.9To examine whether CGRP released from sensory neurons reduces various pathologic events by increasing the endothelial production of PGI2, we investigated the effects of CGRP, capsazepine, CGRP(8–37), denervation of primary sensory nerves, and indomethacin on BUN and serum creatinine 24 h after reperfusion, renal cortical blood flow after reperfusion, and vascular permeability 6 h after reperfusion. Pretreatment with capsazepine, CGRP(8–37), and indomethacin, and denervation of primary sensory nerves exacerbated these pathologic events in rats subjected to renal ischemia/reperfusion (figs. 4A, 4B, 5, and 6). However, pretreatment with CGRP significantly improved ischemia/reperfusion-induced changes.
Effects of CGRP, Capsazepine, CGRP(8–37), Denervation of Primary Sensory Nerves, and Indomethacin on Renal Tissue Levels of Myeloperoxidase Activity, CINC, and TNF-α
Renal tissue levels of myeloperoxidase activity, CINC, and TNF-α were markedly increased after ischemia/reperfusion, reaching peak levels 6 h, 3 h, and 3 h after reperfusion, respectively.9Such increases were significantly enhanced by pretreatment with capsazepine, CGRP(8–37), and indomethacin, and denervation of primary sensory nerves (figs. 7A, 7B, and 7C). However, pretreatment with CGRP significantly improved these increases.
Effects of Iloprost on Ischemia/Reperfusion–induced Acute Renal Injury
Effects of CGRP, Capsazepine, CGRP(8–37), Denervation of Primary Sensory Nerves and Indomethacin in Sham-Operated Rats
Pretreatment with CGRP, capsazepine, CGRP(8–37) or indomethacin, denervation of primary sensory nerves, and vehicle (saline) did not alter BUN, serum creatinine, Evans blue leakage, renal tissue levels of myeloperoxidase, CINC, and TNF-α at 24 h, 24 h, 6 h, 6 h, 3 h, and 3 h after administration of the agents, respectively (data not shown).
In the present study, we showed that ischemia/reperfusion–induced increases in renal tissue levels of 6-keto-PGF1αwere significantly promoted by pretreatment with CGRP and reduced by both pretreatment with capsazepine (a vanilloid receptor-1 antagonist) and CGRP(8–37) (a CGRP receptor antagonist), and denervation of primary sensory nerves in rats. These suggest that such increases in renal tissue levels of 6-keto-PGF1αare a consequence of the sensory neuron activation. Consistent with these observations are our previous reports demonstrating that inhibition of sensory neuron activation inhibits reperfusion-induced increases in endothelial production of PGI2in rats subjected to hepatic ischemia/reperfusion, water-immersion restraint stress, and endotoxin-induced hypotension.18,22,23
TNF-α and histamine stimulate sensory neurons,24,25and renal production of these substances is increased after renal ischemia/reperfusion in rats.26Therefore, it is possible that sensory neurons are activated by these substances under renal ischemia/reperfusion injury in the present study.
Activated leukocytes are critically involved in the development of ischemia/reperfusion–induced acute renal injury.8This notion was confirmed in the present study by the findings of significantly increased renal tissue levels of TNF-α, CINC, and myeloperoxidase, which reflect leukocyte activation, after renal ischemia/reperfusion injury. TNF-α excessively produced by monocytes plays an important role in the development of ischemia/reperfusion–induced acute renal injury by activating neutrophils.3Activated neutrophils damage endothelial cells by releasing various inflammatory mediators such as neutrophil elastase and oxygen radicals, leading to an increase in renal vascular permeability.2,4,5These pathologic events result in reduction of blood flow to the renal medulla and cortex,1,6,7thereby contributing to the development of acute tubular necrosis and reduction of glomerular filtration rate.2
Since PGI2is known to inhibit monocyte TNF-α production by inhibiting its transcription through regulation of nuclear factor-kappaB (NF-κB) activation,27the sensory neuron-mediated increase in the renal endothelial production of PGI2might contribute to reduce ischemia/reperfusion-induced acute renal injury by inhibiting leukocyte activation. Consistent with this hypothesis are observations in the present study demonstrating that pretreatment with iloprost, a stable derivative of PGI2, significantly improved both ischemia/reperfusion–induced increase in TNF-α production and exacerbation of series of pathologic events leading to the ischemia/reperfusion. These observations suggest that CGRP released from sensory neurons reduces ischemia/reperfusion-induced acute renal injury by increasing endothelial PGI2production. Consistent with this notion, changes in renal tissue levels of CGRP during preischemia and after reperfusion appeared to parallel those in renal tissue of 6-keto-PGF1α. Our observations in the present study demonstrated that pretreatment with capsazepine, CGRP(8–37) and indomethacin, and denervation of primary sensory nerves significantly enhanced ischemia/reperfusion-induced decreases in renal cortical blood flow and increases in Evans blue leakage, and renal tissue levels of TNF-α, CINC, and myeloperoxidase and exacerbated a series of pathologic events leading to ischemia/reperfusion–induced renal injury.9Furthermore, pretreatment with CGRP significantly improved these ischemia/ reperfusion–induced changes. These observations implicate endogenous CGRP in prevention of ischemia/reperfusion–induced acute renal injury by inhibiting leukocyte activation in the kidney through increased endothelial PGI2production. Thus, it is possible that increase in endothelial PGI2production induced by sensory neuron activation is a reparative host response to noxious stimuli that evoke tissue injury.
CGRP itself has been shown to inhibit TNF-α production by inhibiting NF-κB activation in macrophages in vitro .28,29In this study, pretreatment with indomethacin significantly promoted ischemia/reperfusion–induced increase in renal tissue levels of TNF-α, suggesting that endogenous CGRP inhibits TNF-α production by promoting endothelial PGI2production in vivo . The limitation of this study was that indomethacin is a nonspecific cyclooxygenase inhibitor. Indomethacin also inhibits activities of cyclooxygenases, thereby inhibiting the production of certain eicosanoids other than PGI2. Therefore, some other eicosanoids might be involved in the effects of CGRP in the present study. Because PGE2as well as PGI2have been shown to inhibit leukocyte activation and these prostaglandins are synthesized from PGE2in endothelial cells,30,31it seems that PGE2mediates the effects of CGRP in the present study. Consistent with this conclusion, we previously showed that CGRP promotes endothelial PGE2production as well as PGI2in water immersion restraint stress–induced gastric mucosal injury in rats.22This possibility should be investigated further using specific cyclooxygenase inhibitors in this model. In addition, there are some other materials, which stimulate endothelial PGI2production. Thrombin is one of these substances.32It would be very interesting to investigate the role of thrombin in this animal model.
In the present study, renal tissue levels of CGRP in rats subjected to 45 min of ischemia/reperfusion were significantly increased 1 h after ischemia/reperfusion and then decreased to the preischemia levels. The renal tissue levels of CGRP were significantly inhibited by pretreatment of capsazepine, a vanilloid receptor-1 antagonist, and denervation of primary sensory nerves. Furthermore, immunohistochemical expression of CGRP was also observed in the kidneys of rats with ischemia/reperfusion injury, as compared with sham-operated rats. These observations suggest that capsaicin-sensitive sensory neurons might be activated in the pathologic process leading to ischemia/reperfusion–induced acute renal injury. Renal tissue levels of CGRP were increased after renal ischemia/reperfusion, whereas plasma levels of CGRP were not detected after the ischemia/reperfusion. These findings suggest that CGRP locally inhibits the induction of TNF-α production by promoting endothelial PGI2production. TNF-α is involved in the development of ischemia/reperfusion-induced acute renal injury by activated neutrophils that adhere to the vascular endothelium, leading to renal vascular endothelial damage, resulting in increased renal vascular permeability.2,3Consistent with this hypothesis are data in this study demonstrating that the effects of renal tissue levels of TNF-α, CINC, and myeloperoxidase appeared to parallel those of renal tissue levels of Evans blue leakage in rats subjected to renal ischemia/reperfusion. These findings suggest that local effects of CGRP in the kidney are critical in preventing ischemia/reperfusion–induced acute renal injury in rats.
Although CGRP plays a pivotal role in preventing ischemia/reperfusion–induced acute renal injury, acute renal injury developed in rats with significantly high levels of endogenous CGRP release. Therefore, CGRP release in response to renal ischemia/reperfusion might not be sufficient to prevent acute renal injury, probably because of depletion of CGRP or inhibition of CGRP release from the nerve ends. In this regard, we showed previously that activation of β2-adrenoceptor on the nerve cells inhibits CGRP release from sensory nerve ends and increases renal tissue blood flow in rats.20In rats with renal ischemia/reperfusion injury, the sympathetic nervous system is activated;33therefore, it is possible that stimulation of β2-adrenoceptors plays a role in the pathology of renal ischemia/reperfusion injury by inhibiting CGRP release. Consistent with this notion, Kurata et al .33reported that enhancement of renal sympathetic activity and its consequent effect on norepinephrine overflow from nerve endings are important events in ischemia/reperfusion-induced acute renal failure. They also demonstrated a significant augmentation of renal sympathetic activity during renal ischemia, and that this change is accompanied by suppression of elevated renal venous norepinephrine levels immediately after reperfusion.33These findings suggest that pharmacologic intervention to modulate sensory nerve function resulting in an increase of CGRP release might be useful in preventing ischemia/reperfusion-induced acute renal injury.
In conclusion, sensory neurons play a critical role in reducing ischemia/reperfusion-induced acute renal injury in rats by attenuating the inflammatory response through promotion of endothelial production of PGI2. In addition, we reported previously that antithrombin, a physiologic serine protease inhibitor, reduced ischemia/reperfusion-induced acute renal injury in rats.9Since antithrombin activates sensory neurons,19we are currently investigating the neuronal mechanisms by which antithrombin enhances endothelial production of PGI2in the same rat model.