Activation of Sensory Neurons Reduces Ischemia/Reperfusion-induced Acute Renal Injury in Rats

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.

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 F^1α(6-keto-PGF^1α) 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-PGF^1α; 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.

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.

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.

Endothelial production of PGI²in kidneys was assessed by measuring renal tissue levels of 6-keto-PGF^1α, a stable metabolite of PGI², using the method described previously.9The PGI²concentration was assayed using a rat-specific enzyme immunoassay system (Amersham).

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.

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.

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-PGF^1α, 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.

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).

Renal tissue levels of 6-keto-PGF^1αwere 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-PGF^1α1 h after reperfusion. Renal tissue levels of 6-keto-PGF^1αin 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-PGF^1α1 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.

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.

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.

Iloprost, a stable derivative of PGI², significantly improved ischemia/reperfusion–induced increases in BUN, creatinine, Evans blue leakage, renal tissue levels of myeloperoxidase, CINC, and TNF-α (fig. 4, A and B; 6; and 7, A–C), and decrease in renal cortical blood flow (fig. 5).

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-PGF^1αwere 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-PGF^1αare 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-PGF^1α. 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.