Life Sciences 117 (2014) 40–45
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C-type natriuretic peptide ameliorates ischemia/reperfusion-induced acute kidney injury by inhibiting apoptosis and oxidative stress in rats Xiunan Jin a, Youchen Zhang b, Xiangdan Li b, Jun Zhang b, Dongyuan Xu b,⁎ a b
Department of Urology, Affiliated Hospital of Yanbian University, Yanji (133000), Jilin Province, China Department of Anatomy, Medical College of Yanbian University, Yanji (133000), Jilin Province, China
a r t i c l e
i n f o
Article history: Received 10 April 2014 Accepted 20 September 2014 Available online 2 October 2014 Keywords: CNP cGMP Acute kidney injury Apoptotic change Oxidative stress Chemical compounds studied in this article: C-type natriuretic peptide (PubChem CID: 16179407)
a b s t r a c t Aims: Although atrial natriuretic peptide has been shown to attenuate ischemia–reperfusion (IR)-induced kidney injury, the effect of natriuretic peptide receptor (NPR)-B activation on IR-induced acute kidney injury is not well documented. The purpose of the present study was to identify the effect of C-type natriuretic peptide (CNP), a selective activator of NPR-B, on the IR-induced acute kidney injury and its mechanisms involved. Main methods: Unilaterally nephrectomized rats were insulted by IR in their remnant kidney, and they were randomly divided into three groups: sham, vehicle + IR, and CNP + IR groups. CNP (0.2 μg/kg/min) was administered intravenously at the start of a 45-min renal ischemia for 2 h. Rats were then killed 24 h after I/R, and the blood and tissue samples were collected to assess renal function, histology, TUNEL assay, and Western blot analysis of kidney Bax and Bcl-2 expressions. Key findings: The levels of blood urea nitrogen and serum creatinine were significantly increased in rats after IR compared with vehicle-treated rats. IR elevated apoptosis, Bcl-2/Bax ratio, TUNEL positivity, oxidative stress parameters, malondialdehyde concentration, and superoxide dismutase activity. IR also induced epithelial desquamation of the proximal tubules and glomerular shrinkage. CNP significantly attenuated the IR-induced increase in BUN and serum creatinine. Furthermore, CNP restored the suppressed renal cyclic guanosine 3′ 5′-monophosphate levels caused by IR insult. Significance: Study findings suggest that CNP could ameliorate IR-induced acute kidney injury through inhibition of apoptotic and oxidative stress pathways, possibly through NPR-B-cGMP signaling. © 2014 Elsevier Inc. All rights reserved.
Introduction Ischemia–reperfusion (IR) injury, which occurs as a result of a major surgery commonly results in acute kidney injury. Multiple factors tend to be involved in IR injury, which include apoptotic and necrotic cell death of renal tubular epithelium and microvasculature (Padanilam, 2003). Acute kidney injury is one of the main causes of chronic renal failure; therefore, it is important to prevent renal IR injury, which is accompanied by renal microvascular and tubular cell injuries caused by inflammation, oxidative stress, and/or apoptosis (Devarajan, 2006; Di Paola et al., 2013). Impaired renal medullary blood flow could also contribute to the pathophysiological changes of renal IR injury (Regner and Roman, 2012); hence, it is imperative to maintain renal blood flow and prevent inflammatory, oxidative, and apoptotic reactions. Atrial natriuretic peptide (ANP) has been shown to protect the kidney from IR injury or acute renal failure (Chujo et al., 2010; Nakamoto et al., 1987; Sear, 2005; Vesely, 2003) by increasing glomerular filtration rate and renal blood flow (Caron and Kramp, 1999). ANP is also known to improve such injuries by inhibiting oxidative stress (De Vito et al., 2010; ⁎ Corresponding author. Tel.: +86 433 2435113; fax: +86 433 2435104. E-mail address: [email protected] (D. Xu).
http://dx.doi.org/10.1016/j.lfs.2014.09.023 0024-3205/© 2014 Elsevier Inc. All rights reserved.
Ogawa et al., 2012) and preserving renal medullary blood flow (Chujo et al., 2010; Dean et al., 1994; Sear, 2005). ANP is synthesized and stored in atrial myocytes and released into the blood stream in response to mechanical, humoral, and neural stimulations (de Bold, 1985; Ruskoaho, 1992). ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) are members of the family of cardiac natriuretic peptides. ANP has natriuretic, diuretic, vasorelaxant, antihypertensive, and anti-renin–angiotensin–aldosterone properties. In addition, ANP has anti-inflammatory, antiproliferative, antioxidative, and metabolic properties (De Vito et al., 2010; Kiemer and Vollmar, 2001; Moro and Lafontan, 2013; Sangawa et al., 2004; Vesely, 2009). Natriuretic peptides exert variable biological effects mainly through activation of guanylyl cyclase-coupled natriuretic peptide receptor (NPR)-A/B and non-guanylyl cyclase-coupled NPR-C receptor. While NPR-A receptor binds ANP and BNP, NPR-B receptor selectively binds CNP, the circulating hormone. In contrast to ANP and BNP, CNP is considered to be an autocrine/ paracrine regulator of peripheral tissues. Since the CNP-NPR-B system is not prominent in the regulation of systemic arterial blood pressure and regulation of renal water and electrolyte balance (Lopez et al., 1997; Wei et al., 1993), the CNP-NPR-B signaling pathway is mainly suggested to be involved in the local modulation including vascular
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regeneration (Kuhn, 2004). CNP is distributed in many peripheral tissues including endothelial cells (Stingo et al., 1992; Suga et al., 1993) and kidney (Dean et al., 1994; Mattingly et al., 1994; Suzuki et al., 1993; Terada et al., 1994). Furthermore, the CNP-selective NPR-B is widely distributed in renal tubules and microvasculatures (Dean et al., 1996; Terada et al., 1994). However, the effect of CNP on the IR injury is not explored much (Vesely, 2003). Hence, the present study aimed to identify the effects of CNP on renal IR injury and explore the mechanisms involved. Materials and methods Animal preparation Male Wistar rats (260–300 g) were obtained from the Yanbian University Laboratory Animal Center. Rats were kept in a temperaturecontrolled and air-conditioned conventional animal house with a 12–12 h light–dark cycle and free access to food and water. All surgical procedures and experimental protocols were approved by the Animal Care and Use Committee of Yanbian University Faculty of Medicine. The procedures were performed according to the recommendations of the Institutional Animal Care Committee. Experimental protocols Animals were randomly allocated to three groups: sham, vehicle + IR, and CNP + IR groups. Sham-operated animals (n = 8) underwent identical surgical procedures to rats with IR, except for the left renal vessel occlusion with a vascular clip. Vehicle (saline 0.03 ml/min/kg body weight) was administered in the same manner as the CNP + IR rats using peristaltic pump. Animals in vehicle + IR group (n = 8) underwent renal ischemia for 45-min followed by reperfusion. Vehicle was administered in the same manner as the CNP + IR rats. In CNP + IR group (n = 6), continuous infusion of CNP was performed at a rate of 0.2 μg/kg/min intravenously, starting immediately with ischemia and continuing until 2 h. Animals were then returned to their cages and allowed access to water ad libitum. At 24 h after the insult, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and then killed by cervical decapitation. Blood and tissue samples were obtained. In preliminary experiments, the dose of CNP 0.2 μg/kg/min maintained higher mean arterial blood pressure compared with the dose of 0.1 and 1 μg/kg/min. As a result, 0.2 μg/kg/min of CNP was chosen for all subsequent experiments. Renal IR Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Right femoral vein and artery were catheterized for infusion and monitoring the changes in blood pressure, respectively. Right kidney was removed, and the left kidney and renal vessels were exposed through dorsal incision (about 2 cm). The left renal artery and vein were occluded with a vascular clip for 45 min and then released as reported previously (Takada et al., 1997). Preparation of blood and tissue samples Blood was allowed to clot at room temperature and then centrifuged to obtain serum. Blood samples were stored at −70 °C until used. The left kidney was excised immediately and divided into portions and stored at −70 °C for subsequent protein analysis; the other tissue sample was fixed in 10% buffered formaldehyde at room temperature and then embedded in paraffin for light microscopy and terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine, 5′-triphosphate nick end labeling (TUNEL) assay.
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Assessment of renal function Blood urea nitrogen (BUN) and creatinine levels were measured for glomerular function. The levels were assessed using a standard autoanalyzer (enzymatic method) at the hospital's Central Laboratory. Light microscopic examination Tissue samples of the kidney embedded in paraffin were cut into 4-μm sections and mounted on glass slides. The sections were then deparaffinized with xylene. Histological evaluations were performed by hematoxylin and eosin staining, and pathological changes were examined under a light microscope. The loss of brush-border membrane, vacuolation, and desquamation of epithelial cells were evaluated in renal tubules at 24 h after IR injury. Two independent renal pathologists examined five different fields of the outer medullary area in every slide. Consensus was arrived after discussion when there were any discrepancies. TUNEL assay TUNEL assay was performed using TUNEL apoptosis assay kit (Nanjing Biobox Biotech Co Ltd., Nanjing, China) per manufacturer's instructions. Ten high power (× 400) fields were randomly selected in a section. The numbers of apoptotic cells, defined by chromatin condensation or nuclear fragmentation, were counted. The apoptotic index was calculated as follows: apoptotic index (%) = (number of positive cells/total number of cells) × 100. Western blot analysis of kidney Bax and Bcl-2 expressions Protein was extracted; and Bcl-2, Bax, and β-actin were identified using Western blotting. The expression of Bcl-2 and Bax was normalized against β-actin expression. Kidney sections stored at −70 °C were lysed in radioimmunoprecipitation assay buffer (50 mM Tris-hydrochloride, pH 7.6, 150 mM sodium chloride, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholic acid, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride) for 30 min on ice prior to centrifugation at 12,000 rpm for 30 min at 4 °C. The protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Proteins were separated by SDS– polyacrylamide gel electrophoresis using a 6–15% acrylamide resolving gel and were transferred to nitrocellulose membranes. Membranes were blocked in Tris-buffered saline-T (0.1% Tween-20) containing 5% milk maintained for 1 h at room temperature, followed by incubation with primary antibody at 4 °C overnight; and they were immunoblotted with the polyclonal antibodies against rabbit Bcl-2 (Cell Signaling Technology, Beverly, MA, USA), Bax, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody. Blots were subsequently probed with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Santa Cruz Biotechnology) at 1:1000–5000 dilutions. Immunoreactive bands were visualized by enhanced chemiluminescence, and densitometry was performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Measurement of renal superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels SOD and MDA levels were assayed using respective commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The SOD activity was measured through the inhibition of nitroblue tetrazolium reduction by oxygen, which was generated by the xanthine/xanthine oxidase system. One SOD activity unit was defined as the enzyme amount causing 50% inhibition in a 1-ml reaction solution per milligram of tissue protein, and the result was expressed as U/mg protein.
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The MDA concentration of the homogenate was measured using thiobarbituric acid method. The amount of lipid peroxide measured by the production of MDA, which in combination with thiobarbituric acid formed a pink chromogen compound with an absorbance of 532 nm. The result was expressed as nmol/mg protein.
cGMP measurement Frozen whole kidney tissue was homogenized at 4 °C in 1 ml of 5% trichloroacetic acid. Total cGMP levels were measured by cGMP enzyme-linked immunosorbent assay detection kit (R&D Systems, Minneapolis, MN, USA) per manufacturer's instructions. Values are presented as pmol/mg of cGMP protein.
Statistical analysis Statistical analyses were performed using SPSS 12.0. Data were expressed as mean ± SEM. Analysis of variance was used for multiple comparisons among all groups followed by post-hoc tests using least significance difference method. For the histopathological renal changes, the Mann–Whitney U test was used to assess the statistical significance of difference between two groups in total severity score. In all tests, P b 0.05 was considered statistically significant.
Results Effects of CNP on IR injury: Renal function and histology BUN and creatinine levels were significantly elevated in IR rats treated with vehicle at 24 h after the insult compared with time-matched shamoperated rats (Fig. 1A and B). The increased levels of BUN and creatinine indicated an acute kidney injury by IR insult. In contrast, intravenous infusion of CNP significantly attenuated the IR-induced increase in BUN and creatinine levels (P b 0.05, Fig. 1). Histological examination revealed that IR insult resulted in the loss of brush border, vacuolation, and desquamation of epithelial cells of the proximal tubules and glomerular shrinkage in vehicle-treated IR rats (Fig. 2). However, treatment with CNP attenuated the IR-induced acute changes in glomeruli and renal tubules (Fig. 2). These findings indicated that CNP treatment could ameliorate IR-induced acute kidney injury.
Antiapoptotic effect of CNP in IR injury: Expression of Bcl-2/Bax and TUNEL-positive cells To identify the effects of CNP on the IR-induced apoptotic changes in renal tissues, Bcl-2 protein and Bax protein expressions were measured in kidney samples at 24 h after IR insult. The level of Bax expression was elevated in the kidneys of IR rats compared with the kidneys of the sham rats (Fig. 3A). Pretreatment with CNP prevented the elevation of Bax in the kidneys of IR rats. No significant difference between CNPand vehicle-treated IR rats was observed in the levels of Bcl-2, an antiapoptotic factor. However, the Bax/Bcl-2 ratio, an index of apoptotic signaling, was elevated in the kidneys of IR rats compared with those of the sham-treated rats (Fig. 3B, P b 0.05). Pretreatment with CNP prevented the elevation of the Bax/Bcl-2 ratio in the kidneys of IR rats. To further confirm the apoptotic changes, numbers of TUNELpositive cells were measured. The number of TUNEL-positive cells increased in the kidneys of IR rats compared with those in the sham group (Fig. 4A and B). CNP administration attenuated the increase in the number of TUNEL-positive cells (Fig. 4B, P b 0.05). Antioxidative stress effect of CNP in IR injury: MDA concentration and SOD activity in renal tissues The levels of MDA in the renal tissues were increased in IR rats compared with those in the sham group (Fig. 5A, P b 0.01). The SOD activity was suppressed in the IR rats (Fig. 5B, P b 0.01). CNP treatment restored the changes in the MDA levels and SOD activity (Fig. 5A and B, both P b 0.05). Effects of CNP on the levels of cGMP in the kidney in IR injury cGMP accumulation was measured in the kidney at 24 h of IR insult, and IR was found to decrease the cGMP levels. CNP treatment slightly but significantly restored the suppressed levels of cGMP accumulation in the kidney from IR rats at 24 h after IR insult (Fig. 6). Discussion The present study attempted to identify the effect of CNP, a selective activator of NPR-B, on the IR-induced acute kidney injury and its mechanisms involved. The study findings showed that CNP could ameliorate IR-induced acute kidney injury through antiapoptotic and antioxidative
Fig. 1. Effects of ischemia–reperfusion (IR) injury on the levels of blood urea nitrogen (A) and creatinine (B) at 24 h after IR and modulation by C-type natriuretic peptide (CNP) for 2 h in rats. Number of experiments: sham, n = 8; vehicle (V) + IR, n = 8; CNP + IR, n = 6. *P b 0.05, **P b 0.01. CNP (0.2 μg/kg/min) was administered intravenously for 2 h.
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Fig. 2. Histological findings of ischemia–reperfusion (IR) kidneys treated with C-type natriuretic peptide (CNP) at 24 h after IR injury (H-E staining, original magnification ×400). Vehicle (V)-treated IR kidneys showed marked injury, with sloughing of tubular epithelial cells and loss of the brush border of proximal tubules (thick arrow, proximal tubule; thin arrow, distal tubule), and glomerular shrinkages (arrow head). These changes were reduced by CNP treatment. [Color reproduction on the Web.]
activities. CNP infusion for 2 h could improve impairment of renal vascular function and glomerular and tubular microstructure damage at 24 h after IR injury. The effects may be closely related to the cGMP signaling through guanylyl cyclase-coupled NPR-B pathway. ANP has been known to protect renal IR injury (Ambwani et al., 2009; Koga et al., 2012; Nakamoto et al., 1987). ANP has natriuretic, diuretic, vasorelaxant, antihypertensive, and anti-renin–angiotensin– aldosterone properties. In addition, ANP has anti-inflammatory, antiproliferative, antioxidative, and metabolic properties (De Vito et al., 2010; Kiemer and Vollmar, 2001; Moro and Lafontan, 2013; Sangawa et al., 2004; Vesely, 2009). ANP could inhibit oxidative stress through NPR-A-cGMP signaling (De Vito et al., 2010; Ogawa et al., 2012). Overexpression of cGMP-dependent protein kinase I could protect against IR-induced kidney injury by inhibiting inflammation and tubular cell apoptosis (Li et al., 2012). Similarly, NO-cGMP (Choi et al., 2009) and NPR-cGMP signaling pathways (De Vito et al., 2010; Ogawa et al., 2012) are also known to protect IR injury by inhibiting apoptosis and oxidative stress. Apoptosis is one of the mechanisms of cell death in renal IR injury (Choi et al., 2009; Waller et al., 2007). CNP has been shown to exert a protective effect against cardiac IR injury (Hobbs et al., 2004). CNP has anti-inflammatory (Scotland et al., 2005) and antiapoptotic properties (Ma et al., 2010). NPR-B is widely expressed in the structures of the kidney like glomeruli, vasa recta bundles, arcuate artey, outer and inner collecting duct cells, and thick ascending limb of Henle (Dean et al., 1996; Terada et al., 1994). Hence, CNP is expected to promote renal cell survival from IR injury. Furthermore, the thick ascending limb of Henle is known to be most susceptible to ischemic injury (Sear, 2005). The presence of CNP and CNP-selective
NPR-B (Dean et al., 1994; Terada et al., 1994) and cGMP generation by CNP in the isolated nephron segment (Terada et al., 1994) suggest the function of CNP-NPR-B-cGMP system in the thick ascending limb of Henle. The CNP-NPR-B signaling pathway expressed in the microstructures, vascular, tubular, and inner and outer renal medulla may act an important role in the protection against apoptosis, oxidative stress (present study), and suppression of medullary blood flow caused by renal IR injury. This notion provides a rationale for CNP, a local autocrine/paracrine modulator, to function in acute renal IR injury without systemic side effects. Because NPR-B is expressed in the renal microstructures, vasa recta bundles, arcuate artery, glomerulus, medullary collecting duct cells, and Henle's loop (Dean et al., 1996; Terada et al., 1994), which are critical for the regulation of renal medullary hemodynamics (O'Connor and Cowley, 2012), this receptor could be one of the targets for CNP in the kidney. Therefore, in addition to the antiapoptotic and antioxidative actions, CNP may improve the IR-induced acute renal injury by limiting the suppression of medullary blood flow. Impaired medullary blood flow could contribute to the pathophysiological changes of renal IR injury (Regner and Roman, 2012). In addition, renal medulla is considered as a site vulnerable to hypoxia due to its unique architecture of the renal vasculature (Eckardt et al., 2005) and high oxygen demand for tubular salt reabsorption (Sear, 2005). Guanylyl cyclase-coupled NPR-B-cGMP signaling induces variable biological effects through its downstream pathways: cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels. NPR-B is the receptor for CNP (Koller et al., 1991). Both NPR-B and its selective activator CNP are expressed
Fig. 3. Ischemia–reperfusion (IR)-induced changes in Bax and Bcl-2 expression in the kidney. (A) Representative photomicrographs of Bax and Bcl-2 immunostaining of renal tissues from IR rats at 24 h after the insult. (B) Densitometric analysis of the ratio of Bax/Bcl-2 immunoblots. Number of experiments: sham, n = 6; vehicle (V) + IR, n = 6; CNP + IR, n = 6. *P b 0.05.
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Fig. 4. Effect of C-type natriuretic peptide (CNP) on the apoptosis of ischemia–reperfusion (IR)-induced kidney injury detected by TUNEL fluorometric staing. (A) Findings showing an attenuation of IR-induced apoptotic changes. DAPI, 4′,6 diamidino-2-phenylindole positive cells; TUNEL, TUNEL positive cells. White arrows indicate chromatin condensation or nuclear fragmentation. TUNEL positivity in red arrow or arrow head. (B) Summarized results for CNP protection against IR-induced apoptotic changes. Number of experiments: sham, n = 6; vehicle (V) + IR, n = 6; CNP + IR, n = 6. *P b 0.05, **P b 0.01. [Color reproduction on the Web (A).]
in the renal microvasculature and tubular cells (Dean et al., 1994; Terada et al., 1994) and CNP increases cGMP levels in the glomeruli, arcuate arteries, and nephron segments (Terada et al., 1994). Although no specific antagonist for the guanylyl cyclase activity of either NPR-A or NPR-B is yet available, desensitization of the guanylyl cyclase activity of NPR-B leads to a reduction in cGMP production by CNP (Potter et al., 2006). Also, an inhibitor of NPR-A and NPR-B, HS-142-1, attenuated CNP-induced activation of cGMP production in the heart and kidney tissues, and cultured vascular endothelial cells (Komatsu et al., 1996; Lee et al., 2000; Lewko et al., 2004) and its biological effects (Komatsu et al., 1996; Lee et al., 2000). In the present study, CNP protects against IR-induced acute kidney injury through activation of antioxidative and antiapoptotic process. Treatment with CNP for 2 h simultaneously with I/R insult (for 45 min) resulted in a mild but significant increase, compared to the vehicle-treated I/R group, in the levels of cGMP of the kidney at 24 h of the insult. The levels of cGMP in the early phase of the insult may be much higher than those in the late phase. Taken
together, it is suggested that CNP-NPR-B-cGMP signaling is involved in the intrarenal protection against IR-induced acute kidney injury through antioxidative and antiapoptotic process.
Conclusion In summary, the study results demonstrate that CNP protects against impaired renal microvasculature and tubular cells caused by IR insult by inhibiting apoptosis and oxidative stress and possibly improving medullary blood flow through activation of NPR-B-cGMP signaling. The results also implicate the effectiveness of CNP in preventing the acute kidney injury.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Fig. 5. Effects of ischemia–reperfusion (IR) injury on the oxidative stress. Renal malondialdehyde (A) and superoxide dismutase (B) levels and its modification by C-type natriuretic peptide (CNP). Number of experiments: sham, n = 6; vehicle (V) + IR, n = 6; CNP + IR, n = 6. *P b 0.05, **P b 0.01.
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Fig. 6. Effects of ischemia–reperfusion (IR) injury on the renal cyclic guanosine 3′ 5′-monophosphate levels and its modification by C-type natriuretic peptide (CNP). Number of experiments: sham, n = 6; vehicle (V) + IR, n = 6; CNP + IR, n = 6. *P b 0.05, **P b 0.01.
Acknowledgements This research was supported by the National Natural Science Foundation of China (81160022). The authors thank Dr. Kyung Woo Cho for his many valuable comments for the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.lfs.2014.09.023. References Ambwani J, Ubhrani D, Saad R, Dunning J. Could atrial natriuretic peptide be a useful drug therapy for high-risk patients after cardiac surgery? Interact Cardiovasc Thorac Surg 2009;8:474–8. Caron N, Kramp R. Measurement of changes in glomerular filtration rate induced by atrial natriuretic peptide in the rat kidney. Exp Physiol 1999;84:689–96. Choi DE, Jeong JY, Lim BJ, Chung S, Chang YK, Lee SJ, et al. Pretreatment of sildenafil attenuates ischemia–reperfusion renal injury in rats. Am J Physiol Renal Physiol 2009;297: F362–70. Chujo K, Ueno M, Asaga T, Sakamoto H, Shirakami G, Ueki M. Atrial natriuretic peptide enhances recovery from ischemia/reperfusion-induced renal injury in rats. J Biosci Bioeng 2010;109:526–30. de Bold AJ. Atrial natriuretic factor: a hormone produced by the heart. Science 1985;230: 767–70. De Vito P, Incerpi S, Pedersen JZ, Luly P. Atrial natriuretic peptide and oxidative stress. Peptides 2010;31:1412–9. Dean AD, Vehaskari VM, Greenwald JE. Synthesis and localization of C-type natriuretic peptide in mammalian kidney. Am J Physiol 1994;266:F491–6. Dean AD, Vehaskari VM, Ritter D, Greenwald JE. Distribution and regulation of guanylyl cyclase type B in the rat nephron. Am J Physiol 1996;270:F311–8. Devarajan P. Update on mechanisms of ischemic acute kidney injury. J Am Soc Nephrol 2006;17:1503–20. Di Paola R, Genovese T, Impellizzeri D, Ahmad A, Cuzzocrea S, Esposito E. The renal injury and inflammation caused by ischemia–reperfusion are reduced by genetic inhibition of TNF-alphaR1: a comparison with infliximab treatment. Eur J Pharmacol 2013;700: 134–46. Eckardt KU, Bernhardt WM, Weidemann A, Warnecke C, Rosenberger C, Wiesener MS, et al. Role of hypoxia in the pathogenesis of renal disease. Kidney Int Suppl 2005:S46–51. Hobbs A, Foster P, Prescott C, Scotland R, Ahluwalia A. Natriuretic peptide receptor-C regulates coronary blood flow and prevents myocardial ischemia/reperfusion injury: novel cardioprotective role for endothelium-derived C-type natriuretic peptide. Circulation 2004;110:1231–5.
45
Kiemer AK, Vollmar AM. The atrial natriuretic peptide regulates the production of inflammatory mediators in macrophages. Ann Rheum Dis 2001;60(Suppl. 3):iii68–70. Koga H, Hagiwara S, Kusaka J, Matsumoto S, Nishida T, Yokoi I, et al. Human atrial natriuretic peptide attenuates renal ischemia–reperfusion injury. J Surg Res 2012;173: 348–53. Koller KJ, Lowe DG, Bennett GL, Minamino N, Kangawa K, Matsuo H, et al. Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP). Science 1991;252:120–3. Komatsu Y, Itoh H, Suga S, Ogawa Y, Hama N, Kishimoto I, et al. Regulation of endothelial production of C-type natriuretic peptide in coculture with vascular smooth muscle cells. Role of the vascular natriuretic peptide system in vascular growth inhibition. Circ Res 1996;78:606–14. Kuhn M. Molecular physiology of natriuretic peptide signalling. Basic Res Cardiol 2004; 99:76–82. Lee SJ, Kim SZ, Cui X, Kim SH, Lee KS, Chung YJ, et al. C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling. Am J Physiol Heart Circ Physiol 2000;278:H208–21. Lewko B, Endlich N, Kriz W, Stepinski J, Endlich K. C-type natriuretic peptide as a podocyte hormone and modulation of its cGMP production by glucose and mechanical stress. Kidney Int 2004;66:1001–8. Li Y, Tong X, Maimaitiyiming H, Clemons K, Cao JM, Wang S. Overexpression of cGMPdependent protein kinase I (PKG-I) attenuates ischemia–reperfusion-induced kidney injury. Am J Physiol Renal Physiol 2012;302:F561–70. Lopez MJ, Garbers DL, Kuhn M. The guanylyl cyclase-deficient mouse defines differential pathways of natriuretic peptide signaling. J Biol Chem 1997;272:23064–8. Ma J, Yu W, Wang Y, Cao G, Cai S, Chen X, et al. Neuroprotective effects of C-type natriuretic peptide on rat retinal ganglion cells. Investig Ophthalmol Vis Sci 2010;51: 3544–53. Mattingly MT, Brandt RR, Heublein DM, Wei CM, Nir A, Burnett Jr JC. Presence of C-type natriuretic peptide in human kidney and urine. Kidney Int 1994;46:744–7. Moro C, Lafontan M. Natriuretic peptides and cGMP signaling control of energy homeostasis. Am J Physiol Heart Circ Physiol 2013;304:H358–68. Nakamoto M, Shapiro JI, Shanley PF, Chan L, Schrier RW. In vitro and in vivo protective effect of atriopeptin III on ischemic acute renal failure. J Clin Investig 1987;80: 698–705. O'Connor PM, Cowley Jr AW. Medullary thick ascending limb buffer vasoconstriction of renal outer-medullary vasa recta in salt-resistant but not salt-sensitive rats. Hypertension 2012;60:965–72. Ogawa Y, Mukoyama M, Yokoi H, Kasahara M, Mori K, Kato Y, et al. Natriuretic peptide receptor guanylyl cyclase-A protects podocytes from aldosterone-induced glomerular injury. J Am Soc Nephrol 2012;23:1198–209. Padanilam BJ. Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. Am J Physiol Renal Physiol 2003;284:F608–27. Potter LR, Abbey-Hosch S, Dickey DM. Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr Rev 2006;27: 47–72. Regner KR, Roman RJ. Role of medullary blood flow in the pathogenesis of renal ischemia– reperfusion injury. Curr Opin Nephrol Hypertens 2012;21:33–8. Ruskoaho H. Atrial natriuretic peptide: synthesis, release, and metabolism. Pharmacol Rev 1992;44:479–602. Sangawa K, Nakanishi K, Ishino K, Inoue M, Kawada M, Sano S. Atrial natriuretic peptide protects against ischemia–reperfusion injury in the isolated rat heart. Ann Thorac Surg 2004;77:233–7. Scotland RS, Cohen M, Foster P, Lovell M, Mathur A, Ahluwalia A, et al. C-type natriuretic peptide inhibits leukocyte recruitment and platelet-leukocyte interactions via suppression of P-selectin expression. Proc Natl Acad Sci U S A 2005;102:14452–7. Sear JW. Kidney dysfunction in the postoperative period. Br J Anaesth 2005;95:20–32. Stingo AJ, Clavell AL, Heublein DM, Wei CM, Pittelkow MR, Burnett Jr JC. Presence of C-type natriuretic peptide in cultured human endothelial cells and plasma. Am J Physiol 1992; 263:H1318–21. Suga S, Itoh H, Komatsu Y, Ogawa Y, Hama N, Yoshimasa T, et al. Cytokine-induced C-type natriuretic peptide (CNP) secretion from vascular endothelial cells–evidence for CNP as a novel autocrine/paracrine regulator from endothelial cells. Endocrinology 1993; 133:3038–41. Suzuki E, Hirata Y, Hayakawa H, Omata M, Kojima M, Kangawa K, et al. Evidence for C-type natriuretic peptide production in the rat kidney. Biochem Biophys Res Commun 1993; 192:532–8. Takada M, Nadeau KC, Shaw GD, Marquette KA, Tilney NL. The cytokine-adhesion molecule cascade in ischemia/reperfusion injury of the rat kidney. Inhibition by a soluble P-selectin ligand. J Clin Investig 1997;99:2682–90. Terada Y, Tomita K, Nonoguchi H, Yang T, Marumo F. PCR localization of C-type natriuretic peptide and B-type receptor mRNAs in rat nephron segments. Am J Physiol 1994; 267:F215–22. Vesely DL. Natriuretic peptides and acute renal failure. Am J Physiol Renal Physiol 2003; 285:F167–77. Vesely DL. Cardiac and renal hormones: anticancer effects in vitro and in vivo. J Investig Med 2009;57:22–8. Waller HL, Harper SJ, Hosgood SA, Bagul A, Kay MD, Kaushik M, et al. Differential expression of cytoprotective and apoptotic genes in an ischaemia-reperfusion isolated organ perfusion model of the transplanted kidney. Transpl Int 2007;20:625–31. Wei CM, Aarhus LL, Miller VM, Burnett Jr JC. Action of C-type natriuretic peptide in isolated canine arteries and veins. Am J Physiol 1993;264:H71–3.
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C-type natriuretic peptide ameliorates ischemia/reperfusion-induced acute kidney injury by inhibiting apoptosis and oxidative stress in rats Xiunan Jin a, Youchen Zhang b, Xiangdan Li b, Jun Zhang b, Dongyuan Xu b,⁎ a b
Department of Urology, Affiliated Hospital of Yanbian University, Yanji (133000), Jilin Province, China Department of Anatomy, Medical College of Yanbian University, Yanji (133000), Jilin Province, China
a r t i c l e
i n f o
Article history: Received 10 April 2014 Accepted 20 September 2014 Available online 2 October 2014 Keywords: CNP cGMP Acute kidney injury Apoptotic change Oxidative stress Chemical compounds studied in this article: C-type natriuretic peptide (PubChem CID: 16179407)
a b s t r a c t Aims: Although atrial natriuretic peptide has been shown to attenuate ischemia–reperfusion (IR)-induced kidney injury, the effect of natriuretic peptide receptor (NPR)-B activation on IR-induced acute kidney injury is not well documented. The purpose of the present study was to identify the effect of C-type natriuretic peptide (CNP), a selective activator of NPR-B, on the IR-induced acute kidney injury and its mechanisms involved. Main methods: Unilaterally nephrectomized rats were insulted by IR in their remnant kidney, and they were randomly divided into three groups: sham, vehicle + IR, and CNP + IR groups. CNP (0.2 μg/kg/min) was administered intravenously at the start of a 45-min renal ischemia for 2 h. Rats were then killed 24 h after I/R, and the blood and tissue samples were collected to assess renal function, histology, TUNEL assay, and Western blot analysis of kidney Bax and Bcl-2 expressions. Key findings: The levels of blood urea nitrogen and serum creatinine were significantly increased in rats after IR compared with vehicle-treated rats. IR elevated apoptosis, Bcl-2/Bax ratio, TUNEL positivity, oxidative stress parameters, malondialdehyde concentration, and superoxide dismutase activity. IR also induced epithelial desquamation of the proximal tubules and glomerular shrinkage. CNP significantly attenuated the IR-induced increase in BUN and serum creatinine. Furthermore, CNP restored the suppressed renal cyclic guanosine 3′ 5′-monophosphate levels caused by IR insult. Significance: Study findings suggest that CNP could ameliorate IR-induced acute kidney injury through inhibition of apoptotic and oxidative stress pathways, possibly through NPR-B-cGMP signaling. © 2014 Elsevier Inc. All rights reserved.
Introduction Ischemia–reperfusion (IR) injury, which occurs as a result of a major surgery commonly results in acute kidney injury. Multiple factors tend to be involved in IR injury, which include apoptotic and necrotic cell death of renal tubular epithelium and microvasculature (Padanilam, 2003). Acute kidney injury is one of the main causes of chronic renal failure; therefore, it is important to prevent renal IR injury, which is accompanied by renal microvascular and tubular cell injuries caused by inflammation, oxidative stress, and/or apoptosis (Devarajan, 2006; Di Paola et al., 2013). Impaired renal medullary blood flow could also contribute to the pathophysiological changes of renal IR injury (Regner and Roman, 2012); hence, it is imperative to maintain renal blood flow and prevent inflammatory, oxidative, and apoptotic reactions. Atrial natriuretic peptide (ANP) has been shown to protect the kidney from IR injury or acute renal failure (Chujo et al., 2010; Nakamoto et al., 1987; Sear, 2005; Vesely, 2003) by increasing glomerular filtration rate and renal blood flow (Caron and Kramp, 1999). ANP is also known to improve such injuries by inhibiting oxidative stress (De Vito et al., 2010; ⁎ Corresponding author. Tel.: +86 433 2435113; fax: +86 433 2435104. E-mail address: [email protected] (D. Xu).
http://dx.doi.org/10.1016/j.lfs.2014.09.023 0024-3205/© 2014 Elsevier Inc. All rights reserved.
Ogawa et al., 2012) and preserving renal medullary blood flow (Chujo et al., 2010; Dean et al., 1994; Sear, 2005). ANP is synthesized and stored in atrial myocytes and released into the blood stream in response to mechanical, humoral, and neural stimulations (de Bold, 1985; Ruskoaho, 1992). ANP, brain natriuretic peptide (BNP), and C-type natriuretic peptide (CNP) are members of the family of cardiac natriuretic peptides. ANP has natriuretic, diuretic, vasorelaxant, antihypertensive, and anti-renin–angiotensin–aldosterone properties. In addition, ANP has anti-inflammatory, antiproliferative, antioxidative, and metabolic properties (De Vito et al., 2010; Kiemer and Vollmar, 2001; Moro and Lafontan, 2013; Sangawa et al., 2004; Vesely, 2009). Natriuretic peptides exert variable biological effects mainly through activation of guanylyl cyclase-coupled natriuretic peptide receptor (NPR)-A/B and non-guanylyl cyclase-coupled NPR-C receptor. While NPR-A receptor binds ANP and BNP, NPR-B receptor selectively binds CNP, the circulating hormone. In contrast to ANP and BNP, CNP is considered to be an autocrine/ paracrine regulator of peripheral tissues. Since the CNP-NPR-B system is not prominent in the regulation of systemic arterial blood pressure and regulation of renal water and electrolyte balance (Lopez et al., 1997; Wei et al., 1993), the CNP-NPR-B signaling pathway is mainly suggested to be involved in the local modulation including vascular
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regeneration (Kuhn, 2004). CNP is distributed in many peripheral tissues including endothelial cells (Stingo et al., 1992; Suga et al., 1993) and kidney (Dean et al., 1994; Mattingly et al., 1994; Suzuki et al., 1993; Terada et al., 1994). Furthermore, the CNP-selective NPR-B is widely distributed in renal tubules and microvasculatures (Dean et al., 1996; Terada et al., 1994). However, the effect of CNP on the IR injury is not explored much (Vesely, 2003). Hence, the present study aimed to identify the effects of CNP on renal IR injury and explore the mechanisms involved. Materials and methods Animal preparation Male Wistar rats (260–300 g) were obtained from the Yanbian University Laboratory Animal Center. Rats were kept in a temperaturecontrolled and air-conditioned conventional animal house with a 12–12 h light–dark cycle and free access to food and water. All surgical procedures and experimental protocols were approved by the Animal Care and Use Committee of Yanbian University Faculty of Medicine. The procedures were performed according to the recommendations of the Institutional Animal Care Committee. Experimental protocols Animals were randomly allocated to three groups: sham, vehicle + IR, and CNP + IR groups. Sham-operated animals (n = 8) underwent identical surgical procedures to rats with IR, except for the left renal vessel occlusion with a vascular clip. Vehicle (saline 0.03 ml/min/kg body weight) was administered in the same manner as the CNP + IR rats using peristaltic pump. Animals in vehicle + IR group (n = 8) underwent renal ischemia for 45-min followed by reperfusion. Vehicle was administered in the same manner as the CNP + IR rats. In CNP + IR group (n = 6), continuous infusion of CNP was performed at a rate of 0.2 μg/kg/min intravenously, starting immediately with ischemia and continuing until 2 h. Animals were then returned to their cages and allowed access to water ad libitum. At 24 h after the insult, rats were anesthetized with sodium pentobarbital (50 mg/kg, i.p.) and then killed by cervical decapitation. Blood and tissue samples were obtained. In preliminary experiments, the dose of CNP 0.2 μg/kg/min maintained higher mean arterial blood pressure compared with the dose of 0.1 and 1 μg/kg/min. As a result, 0.2 μg/kg/min of CNP was chosen for all subsequent experiments. Renal IR Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital (50 mg/kg). Right femoral vein and artery were catheterized for infusion and monitoring the changes in blood pressure, respectively. Right kidney was removed, and the left kidney and renal vessels were exposed through dorsal incision (about 2 cm). The left renal artery and vein were occluded with a vascular clip for 45 min and then released as reported previously (Takada et al., 1997). Preparation of blood and tissue samples Blood was allowed to clot at room temperature and then centrifuged to obtain serum. Blood samples were stored at −70 °C until used. The left kidney was excised immediately and divided into portions and stored at −70 °C for subsequent protein analysis; the other tissue sample was fixed in 10% buffered formaldehyde at room temperature and then embedded in paraffin for light microscopy and terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine, 5′-triphosphate nick end labeling (TUNEL) assay.
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Assessment of renal function Blood urea nitrogen (BUN) and creatinine levels were measured for glomerular function. The levels were assessed using a standard autoanalyzer (enzymatic method) at the hospital's Central Laboratory. Light microscopic examination Tissue samples of the kidney embedded in paraffin were cut into 4-μm sections and mounted on glass slides. The sections were then deparaffinized with xylene. Histological evaluations were performed by hematoxylin and eosin staining, and pathological changes were examined under a light microscope. The loss of brush-border membrane, vacuolation, and desquamation of epithelial cells were evaluated in renal tubules at 24 h after IR injury. Two independent renal pathologists examined five different fields of the outer medullary area in every slide. Consensus was arrived after discussion when there were any discrepancies. TUNEL assay TUNEL assay was performed using TUNEL apoptosis assay kit (Nanjing Biobox Biotech Co Ltd., Nanjing, China) per manufacturer's instructions. Ten high power (× 400) fields were randomly selected in a section. The numbers of apoptotic cells, defined by chromatin condensation or nuclear fragmentation, were counted. The apoptotic index was calculated as follows: apoptotic index (%) = (number of positive cells/total number of cells) × 100. Western blot analysis of kidney Bax and Bcl-2 expressions Protein was extracted; and Bcl-2, Bax, and β-actin were identified using Western blotting. The expression of Bcl-2 and Bax was normalized against β-actin expression. Kidney sections stored at −70 °C were lysed in radioimmunoprecipitation assay buffer (50 mM Tris-hydrochloride, pH 7.6, 150 mM sodium chloride, 1% NP-40, 0.1% sodium dodecyl sulfate (SDS), 0.5% deoxycholic acid, 1 μg/ml leupeptin, 1 μg/ml aprotinin, and 0.5 mM phenylmethylsulfonyl fluoride) for 30 min on ice prior to centrifugation at 12,000 rpm for 30 min at 4 °C. The protein concentrations were determined using a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Rockford, IL, USA). Proteins were separated by SDS– polyacrylamide gel electrophoresis using a 6–15% acrylamide resolving gel and were transferred to nitrocellulose membranes. Membranes were blocked in Tris-buffered saline-T (0.1% Tween-20) containing 5% milk maintained for 1 h at room temperature, followed by incubation with primary antibody at 4 °C overnight; and they were immunoblotted with the polyclonal antibodies against rabbit Bcl-2 (Cell Signaling Technology, Beverly, MA, USA), Bax, and β-actin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibody. Blots were subsequently probed with horseradish peroxidase-conjugated anti-rabbit immunoglobulin G (Santa Cruz Biotechnology) at 1:1000–5000 dilutions. Immunoreactive bands were visualized by enhanced chemiluminescence, and densitometry was performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA). Measurement of renal superoxide dismutase (SOD) activity and malondialdehyde (MDA) levels SOD and MDA levels were assayed using respective commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The SOD activity was measured through the inhibition of nitroblue tetrazolium reduction by oxygen, which was generated by the xanthine/xanthine oxidase system. One SOD activity unit was defined as the enzyme amount causing 50% inhibition in a 1-ml reaction solution per milligram of tissue protein, and the result was expressed as U/mg protein.
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The MDA concentration of the homogenate was measured using thiobarbituric acid method. The amount of lipid peroxide measured by the production of MDA, which in combination with thiobarbituric acid formed a pink chromogen compound with an absorbance of 532 nm. The result was expressed as nmol/mg protein.
cGMP measurement Frozen whole kidney tissue was homogenized at 4 °C in 1 ml of 5% trichloroacetic acid. Total cGMP levels were measured by cGMP enzyme-linked immunosorbent assay detection kit (R&D Systems, Minneapolis, MN, USA) per manufacturer's instructions. Values are presented as pmol/mg of cGMP protein.
Statistical analysis Statistical analyses were performed using SPSS 12.0. Data were expressed as mean ± SEM. Analysis of variance was used for multiple comparisons among all groups followed by post-hoc tests using least significance difference method. For the histopathological renal changes, the Mann–Whitney U test was used to assess the statistical significance of difference between two groups in total severity score. In all tests, P b 0.05 was considered statistically significant.
Results Effects of CNP on IR injury: Renal function and histology BUN and creatinine levels were significantly elevated in IR rats treated with vehicle at 24 h after the insult compared with time-matched shamoperated rats (Fig. 1A and B). The increased levels of BUN and creatinine indicated an acute kidney injury by IR insult. In contrast, intravenous infusion of CNP significantly attenuated the IR-induced increase in BUN and creatinine levels (P b 0.05, Fig. 1). Histological examination revealed that IR insult resulted in the loss of brush border, vacuolation, and desquamation of epithelial cells of the proximal tubules and glomerular shrinkage in vehicle-treated IR rats (Fig. 2). However, treatment with CNP attenuated the IR-induced acute changes in glomeruli and renal tubules (Fig. 2). These findings indicated that CNP treatment could ameliorate IR-induced acute kidney injury.
Antiapoptotic effect of CNP in IR injury: Expression of Bcl-2/Bax and TUNEL-positive cells To identify the effects of CNP on the IR-induced apoptotic changes in renal tissues, Bcl-2 protein and Bax protein expressions were measured in kidney samples at 24 h after IR insult. The level of Bax expression was elevated in the kidneys of IR rats compared with the kidneys of the sham rats (Fig. 3A). Pretreatment with CNP prevented the elevation of Bax in the kidneys of IR rats. No significant difference between CNPand vehicle-treated IR rats was observed in the levels of Bcl-2, an antiapoptotic factor. However, the Bax/Bcl-2 ratio, an index of apoptotic signaling, was elevated in the kidneys of IR rats compared with those of the sham-treated rats (Fig. 3B, P b 0.05). Pretreatment with CNP prevented the elevation of the Bax/Bcl-2 ratio in the kidneys of IR rats. To further confirm the apoptotic changes, numbers of TUNELpositive cells were measured. The number of TUNEL-positive cells increased in the kidneys of IR rats compared with those in the sham group (Fig. 4A and B). CNP administration attenuated the increase in the number of TUNEL-positive cells (Fig. 4B, P b 0.05). Antioxidative stress effect of CNP in IR injury: MDA concentration and SOD activity in renal tissues The levels of MDA in the renal tissues were increased in IR rats compared with those in the sham group (Fig. 5A, P b 0.01). The SOD activity was suppressed in the IR rats (Fig. 5B, P b 0.01). CNP treatment restored the changes in the MDA levels and SOD activity (Fig. 5A and B, both P b 0.05). Effects of CNP on the levels of cGMP in the kidney in IR injury cGMP accumulation was measured in the kidney at 24 h of IR insult, and IR was found to decrease the cGMP levels. CNP treatment slightly but significantly restored the suppressed levels of cGMP accumulation in the kidney from IR rats at 24 h after IR insult (Fig. 6). Discussion The present study attempted to identify the effect of CNP, a selective activator of NPR-B, on the IR-induced acute kidney injury and its mechanisms involved. The study findings showed that CNP could ameliorate IR-induced acute kidney injury through antiapoptotic and antioxidative
Fig. 1. Effects of ischemia–reperfusion (IR) injury on the levels of blood urea nitrogen (A) and creatinine (B) at 24 h after IR and modulation by C-type natriuretic peptide (CNP) for 2 h in rats. Number of experiments: sham, n = 8; vehicle (V) + IR, n = 8; CNP + IR, n = 6. *P b 0.05, **P b 0.01. CNP (0.2 μg/kg/min) was administered intravenously for 2 h.
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Fig. 2. Histological findings of ischemia–reperfusion (IR) kidneys treated with C-type natriuretic peptide (CNP) at 24 h after IR injury (H-E staining, original magnification ×400). Vehicle (V)-treated IR kidneys showed marked injury, with sloughing of tubular epithelial cells and loss of the brush border of proximal tubules (thick arrow, proximal tubule; thin arrow, distal tubule), and glomerular shrinkages (arrow head). These changes were reduced by CNP treatment. [Color reproduction on the Web.]
activities. CNP infusion for 2 h could improve impairment of renal vascular function and glomerular and tubular microstructure damage at 24 h after IR injury. The effects may be closely related to the cGMP signaling through guanylyl cyclase-coupled NPR-B pathway. ANP has been known to protect renal IR injury (Ambwani et al., 2009; Koga et al., 2012; Nakamoto et al., 1987). ANP has natriuretic, diuretic, vasorelaxant, antihypertensive, and anti-renin–angiotensin– aldosterone properties. In addition, ANP has anti-inflammatory, antiproliferative, antioxidative, and metabolic properties (De Vito et al., 2010; Kiemer and Vollmar, 2001; Moro and Lafontan, 2013; Sangawa et al., 2004; Vesely, 2009). ANP could inhibit oxidative stress through NPR-A-cGMP signaling (De Vito et al., 2010; Ogawa et al., 2012). Overexpression of cGMP-dependent protein kinase I could protect against IR-induced kidney injury by inhibiting inflammation and tubular cell apoptosis (Li et al., 2012). Similarly, NO-cGMP (Choi et al., 2009) and NPR-cGMP signaling pathways (De Vito et al., 2010; Ogawa et al., 2012) are also known to protect IR injury by inhibiting apoptosis and oxidative stress. Apoptosis is one of the mechanisms of cell death in renal IR injury (Choi et al., 2009; Waller et al., 2007). CNP has been shown to exert a protective effect against cardiac IR injury (Hobbs et al., 2004). CNP has anti-inflammatory (Scotland et al., 2005) and antiapoptotic properties (Ma et al., 2010). NPR-B is widely expressed in the structures of the kidney like glomeruli, vasa recta bundles, arcuate artey, outer and inner collecting duct cells, and thick ascending limb of Henle (Dean et al., 1996; Terada et al., 1994). Hence, CNP is expected to promote renal cell survival from IR injury. Furthermore, the thick ascending limb of Henle is known to be most susceptible to ischemic injury (Sear, 2005). The presence of CNP and CNP-selective
NPR-B (Dean et al., 1994; Terada et al., 1994) and cGMP generation by CNP in the isolated nephron segment (Terada et al., 1994) suggest the function of CNP-NPR-B-cGMP system in the thick ascending limb of Henle. The CNP-NPR-B signaling pathway expressed in the microstructures, vascular, tubular, and inner and outer renal medulla may act an important role in the protection against apoptosis, oxidative stress (present study), and suppression of medullary blood flow caused by renal IR injury. This notion provides a rationale for CNP, a local autocrine/paracrine modulator, to function in acute renal IR injury without systemic side effects. Because NPR-B is expressed in the renal microstructures, vasa recta bundles, arcuate artery, glomerulus, medullary collecting duct cells, and Henle's loop (Dean et al., 1996; Terada et al., 1994), which are critical for the regulation of renal medullary hemodynamics (O'Connor and Cowley, 2012), this receptor could be one of the targets for CNP in the kidney. Therefore, in addition to the antiapoptotic and antioxidative actions, CNP may improve the IR-induced acute renal injury by limiting the suppression of medullary blood flow. Impaired medullary blood flow could contribute to the pathophysiological changes of renal IR injury (Regner and Roman, 2012). In addition, renal medulla is considered as a site vulnerable to hypoxia due to its unique architecture of the renal vasculature (Eckardt et al., 2005) and high oxygen demand for tubular salt reabsorption (Sear, 2005). Guanylyl cyclase-coupled NPR-B-cGMP signaling induces variable biological effects through its downstream pathways: cGMP-dependent protein kinases, cGMP-regulated phosphodiesterases, and cyclic nucleotide-gated ion channels. NPR-B is the receptor for CNP (Koller et al., 1991). Both NPR-B and its selective activator CNP are expressed
Fig. 3. Ischemia–reperfusion (IR)-induced changes in Bax and Bcl-2 expression in the kidney. (A) Representative photomicrographs of Bax and Bcl-2 immunostaining of renal tissues from IR rats at 24 h after the insult. (B) Densitometric analysis of the ratio of Bax/Bcl-2 immunoblots. Number of experiments: sham, n = 6; vehicle (V) + IR, n = 6; CNP + IR, n = 6. *P b 0.05.
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Fig. 4. Effect of C-type natriuretic peptide (CNP) on the apoptosis of ischemia–reperfusion (IR)-induced kidney injury detected by TUNEL fluorometric staing. (A) Findings showing an attenuation of IR-induced apoptotic changes. DAPI, 4′,6 diamidino-2-phenylindole positive cells; TUNEL, TUNEL positive cells. White arrows indicate chromatin condensation or nuclear fragmentation. TUNEL positivity in red arrow or arrow head. (B) Summarized results for CNP protection against IR-induced apoptotic changes. Number of experiments: sham, n = 6; vehicle (V) + IR, n = 6; CNP + IR, n = 6. *P b 0.05, **P b 0.01. [Color reproduction on the Web (A).]
in the renal microvasculature and tubular cells (Dean et al., 1994; Terada et al., 1994) and CNP increases cGMP levels in the glomeruli, arcuate arteries, and nephron segments (Terada et al., 1994). Although no specific antagonist for the guanylyl cyclase activity of either NPR-A or NPR-B is yet available, desensitization of the guanylyl cyclase activity of NPR-B leads to a reduction in cGMP production by CNP (Potter et al., 2006). Also, an inhibitor of NPR-A and NPR-B, HS-142-1, attenuated CNP-induced activation of cGMP production in the heart and kidney tissues, and cultured vascular endothelial cells (Komatsu et al., 1996; Lee et al., 2000; Lewko et al., 2004) and its biological effects (Komatsu et al., 1996; Lee et al., 2000). In the present study, CNP protects against IR-induced acute kidney injury through activation of antioxidative and antiapoptotic process. Treatment with CNP for 2 h simultaneously with I/R insult (for 45 min) resulted in a mild but significant increase, compared to the vehicle-treated I/R group, in the levels of cGMP of the kidney at 24 h of the insult. The levels of cGMP in the early phase of the insult may be much higher than those in the late phase. Taken
together, it is suggested that CNP-NPR-B-cGMP signaling is involved in the intrarenal protection against IR-induced acute kidney injury through antioxidative and antiapoptotic process.
Conclusion In summary, the study results demonstrate that CNP protects against impaired renal microvasculature and tubular cells caused by IR insult by inhibiting apoptosis and oxidative stress and possibly improving medullary blood flow through activation of NPR-B-cGMP signaling. The results also implicate the effectiveness of CNP in preventing the acute kidney injury.
Conflict of interest statement The authors declare that there are no conflicts of interest.
Fig. 5. Effects of ischemia–reperfusion (IR) injury on the oxidative stress. Renal malondialdehyde (A) and superoxide dismutase (B) levels and its modification by C-type natriuretic peptide (CNP). Number of experiments: sham, n = 6; vehicle (V) + IR, n = 6; CNP + IR, n = 6. *P b 0.05, **P b 0.01.
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Fig. 6. Effects of ischemia–reperfusion (IR) injury on the renal cyclic guanosine 3′ 5′-monophosphate levels and its modification by C-type natriuretic peptide (CNP). Number of experiments: sham, n = 6; vehicle (V) + IR, n = 6; CNP + IR, n = 6. *P b 0.05, **P b 0.01.
Acknowledgements This research was supported by the National Natural Science Foundation of China (81160022). The authors thank Dr. Kyung Woo Cho for his many valuable comments for the manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.lfs.2014.09.023. References Ambwani J, Ubhrani D, Saad R, Dunning J. Could atrial natriuretic peptide be a useful drug therapy for high-risk patients after cardiac surgery? Interact Cardiovasc Thorac Surg 2009;8:474–8. Caron N, Kramp R. Measurement of changes in glomerular filtration rate induced by atrial natriuretic peptide in the rat kidney. Exp Physiol 1999;84:689–96. Choi DE, Jeong JY, Lim BJ, Chung S, Chang YK, Lee SJ, et al. Pretreatment of sildenafil attenuates ischemia–reperfusion renal injury in rats. Am J Physiol Renal Physiol 2009;297: F362–70. Chujo K, Ueno M, Asaga T, Sakamoto H, Shirakami G, Ueki M. Atrial natriuretic peptide enhances recovery from ischemia/reperfusion-induced renal injury in rats. J Biosci Bioeng 2010;109:526–30. de Bold AJ. Atrial natriuretic factor: a hormone produced by the heart. Science 1985;230: 767–70. De Vito P, Incerpi S, Pedersen JZ, Luly P. Atrial natriuretic peptide and oxidative stress. Peptides 2010;31:1412–9. Dean AD, Vehaskari VM, Greenwald JE. Synthesis and localization of C-type natriuretic peptide in mammalian kidney. Am J Physiol 1994;266:F491–6. Dean AD, Vehaskari VM, Ritter D, Greenwald JE. Distribution and regulation of guanylyl cyclase type B in the rat nephron. Am J Physiol 1996;270:F311–8. Devarajan P. Update on mechanisms of ischemic acute kidney injury. J Am Soc Nephrol 2006;17:1503–20. Di Paola R, Genovese T, Impellizzeri D, Ahmad A, Cuzzocrea S, Esposito E. The renal injury and inflammation caused by ischemia–reperfusion are reduced by genetic inhibition of TNF-alphaR1: a comparison with infliximab treatment. Eur J Pharmacol 2013;700: 134–46. Eckardt KU, Bernhardt WM, Weidemann A, Warnecke C, Rosenberger C, Wiesener MS, et al. Role of hypoxia in the pathogenesis of renal disease. Kidney Int Suppl 2005:S46–51. Hobbs A, Foster P, Prescott C, Scotland R, Ahluwalia A. Natriuretic peptide receptor-C regulates coronary blood flow and prevents myocardial ischemia/reperfusion injury: novel cardioprotective role for endothelium-derived C-type natriuretic peptide. Circulation 2004;110:1231–5.
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Kiemer AK, Vollmar AM. The atrial natriuretic peptide regulates the production of inflammatory mediators in macrophages. Ann Rheum Dis 2001;60(Suppl. 3):iii68–70. Koga H, Hagiwara S, Kusaka J, Matsumoto S, Nishida T, Yokoi I, et al. Human atrial natriuretic peptide attenuates renal ischemia–reperfusion injury. J Surg Res 2012;173: 348–53. Koller KJ, Lowe DG, Bennett GL, Minamino N, Kangawa K, Matsuo H, et al. Selective activation of the B natriuretic peptide receptor by C-type natriuretic peptide (CNP). Science 1991;252:120–3. Komatsu Y, Itoh H, Suga S, Ogawa Y, Hama N, Kishimoto I, et al. Regulation of endothelial production of C-type natriuretic peptide in coculture with vascular smooth muscle cells. Role of the vascular natriuretic peptide system in vascular growth inhibition. Circ Res 1996;78:606–14. Kuhn M. Molecular physiology of natriuretic peptide signalling. Basic Res Cardiol 2004; 99:76–82. Lee SJ, Kim SZ, Cui X, Kim SH, Lee KS, Chung YJ, et al. C-type natriuretic peptide inhibits ANP secretion and atrial dynamics in perfused atria: NPR-B-cGMP signaling. Am J Physiol Heart Circ Physiol 2000;278:H208–21. Lewko B, Endlich N, Kriz W, Stepinski J, Endlich K. C-type natriuretic peptide as a podocyte hormone and modulation of its cGMP production by glucose and mechanical stress. Kidney Int 2004;66:1001–8. Li Y, Tong X, Maimaitiyiming H, Clemons K, Cao JM, Wang S. Overexpression of cGMPdependent protein kinase I (PKG-I) attenuates ischemia–reperfusion-induced kidney injury. Am J Physiol Renal Physiol 2012;302:F561–70. Lopez MJ, Garbers DL, Kuhn M. The guanylyl cyclase-deficient mouse defines differential pathways of natriuretic peptide signaling. J Biol Chem 1997;272:23064–8. Ma J, Yu W, Wang Y, Cao G, Cai S, Chen X, et al. Neuroprotective effects of C-type natriuretic peptide on rat retinal ganglion cells. Investig Ophthalmol Vis Sci 2010;51: 3544–53. Mattingly MT, Brandt RR, Heublein DM, Wei CM, Nir A, Burnett Jr JC. Presence of C-type natriuretic peptide in human kidney and urine. Kidney Int 1994;46:744–7. Moro C, Lafontan M. Natriuretic peptides and cGMP signaling control of energy homeostasis. Am J Physiol Heart Circ Physiol 2013;304:H358–68. Nakamoto M, Shapiro JI, Shanley PF, Chan L, Schrier RW. In vitro and in vivo protective effect of atriopeptin III on ischemic acute renal failure. J Clin Investig 1987;80: 698–705. O'Connor PM, Cowley Jr AW. Medullary thick ascending limb buffer vasoconstriction of renal outer-medullary vasa recta in salt-resistant but not salt-sensitive rats. Hypertension 2012;60:965–72. Ogawa Y, Mukoyama M, Yokoi H, Kasahara M, Mori K, Kato Y, et al. Natriuretic peptide receptor guanylyl cyclase-A protects podocytes from aldosterone-induced glomerular injury. J Am Soc Nephrol 2012;23:1198–209. Padanilam BJ. Cell death induced by acute renal injury: a perspective on the contributions of apoptosis and necrosis. Am J Physiol Renal Physiol 2003;284:F608–27. Potter LR, Abbey-Hosch S, Dickey DM. Natriuretic peptides, their receptors, and cyclic guanosine monophosphate-dependent signaling functions. Endocr Rev 2006;27: 47–72. Regner KR, Roman RJ. Role of medullary blood flow in the pathogenesis of renal ischemia– reperfusion injury. Curr Opin Nephrol Hypertens 2012;21:33–8. Ruskoaho H. Atrial natriuretic peptide: synthesis, release, and metabolism. Pharmacol Rev 1992;44:479–602. Sangawa K, Nakanishi K, Ishino K, Inoue M, Kawada M, Sano S. Atrial natriuretic peptide protects against ischemia–reperfusion injury in the isolated rat heart. Ann Thorac Surg 2004;77:233–7. Scotland RS, Cohen M, Foster P, Lovell M, Mathur A, Ahluwalia A, et al. C-type natriuretic peptide inhibits leukocyte recruitment and platelet-leukocyte interactions via suppression of P-selectin expression. Proc Natl Acad Sci U S A 2005;102:14452–7. Sear JW. Kidney dysfunction in the postoperative period. Br J Anaesth 2005;95:20–32. Stingo AJ, Clavell AL, Heublein DM, Wei CM, Pittelkow MR, Burnett Jr JC. Presence of C-type natriuretic peptide in cultured human endothelial cells and plasma. Am J Physiol 1992; 263:H1318–21. Suga S, Itoh H, Komatsu Y, Ogawa Y, Hama N, Yoshimasa T, et al. Cytokine-induced C-type natriuretic peptide (CNP) secretion from vascular endothelial cells–evidence for CNP as a novel autocrine/paracrine regulator from endothelial cells. Endocrinology 1993; 133:3038–41. Suzuki E, Hirata Y, Hayakawa H, Omata M, Kojima M, Kangawa K, et al. Evidence for C-type natriuretic peptide production in the rat kidney. Biochem Biophys Res Commun 1993; 192:532–8. Takada M, Nadeau KC, Shaw GD, Marquette KA, Tilney NL. The cytokine-adhesion molecule cascade in ischemia/reperfusion injury of the rat kidney. Inhibition by a soluble P-selectin ligand. J Clin Investig 1997;99:2682–90. Terada Y, Tomita K, Nonoguchi H, Yang T, Marumo F. PCR localization of C-type natriuretic peptide and B-type receptor mRNAs in rat nephron segments. Am J Physiol 1994; 267:F215–22. Vesely DL. Natriuretic peptides and acute renal failure. Am J Physiol Renal Physiol 2003; 285:F167–77. Vesely DL. Cardiac and renal hormones: anticancer effects in vitro and in vivo. J Investig Med 2009;57:22–8. Waller HL, Harper SJ, Hosgood SA, Bagul A, Kay MD, Kaushik M, et al. Differential expression of cytoprotective and apoptotic genes in an ischaemia-reperfusion isolated organ perfusion model of the transplanted kidney. Transpl Int 2007;20:625–31. Wei CM, Aarhus LL, Miller VM, Burnett Jr JC. Action of C-type natriuretic peptide in isolated canine arteries and veins. Am J Physiol 1993;264:H71–3.
