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reperfusion injury through autophagy activation and apoptosis inhibition in rats
Chemico-Biological Interactions 289 (2018) 40–46
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NGAL attenuates renal ischemia/reperfusion injury through autophagy activation and apoptosis inhibition in rats
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Ya-li Zhanga, Shu-kai Qiaob, Rong-ying Wanga,∗, Xiao-nan Guob a b
Department of General Practice, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, 050000, China Department of Hematology, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, 050000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Neutrophil gelatinase-associated lipocalin Autophagy Apoptosis Ischemia-reperfusion Proliferation
Ischemia/reperfusion (I/R) injury is a main cause of acute kidney injury (AKI), and currently lacks effective therapies. This study is to investigate the level of Neutrophil gelatinase-associated lipocalin (NGAL) and autophagy status during renal I/R injury, so as to determine whether the exogenous NGAL protein could exert a protective effect for I/R injury and explore the potential mechanisms. Forty male Wistar rats were randomly divided into the following four groups: Sham, I/R, pre-treated with NGAL before I/R (I/R + pre-N), treated with NGAL after I/R (I/R + post-N). All rats were subjected to clamping the left renal pedicle for 45 min after right nephrectomy, followed by 24 h of reperfusion. Serum creatinine (SCr) and blood urea nitrogen (BUN) were used for renal function, tubular cell apoptosis and autophagy were measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method, histological examination and electron microscope, respectively. The tubular cell proliferation was assessed by the protein expression of proliferating cell nuclear antigen (PCNA). Western blotting was used to quantitate the levels of LC3, Beclin-1, Bcl-2 and Bax in kidney tissues. Exogenous NGAL protein intervention significantly improved renal function, reduced tubular cell apoptosis, increased tubular cell proliferation and promoted autophagy activation after renal I/R injury. Further, the efficacy in pre-N was significantly better than post-N. The mechanisms were involved in the regulation of several autophagy and apoptosis-related genes. Our study demonstrated that exogenous NGAL protein play a protective role during I/R injury, which may offer a novel may for prevention and treatment of renal I/R injury.
1. Introduction Renal ischemia-reperfusion (I/R) injury is the primary cause of acute kidney injury (AKI), which can cause a significant increase in the expression of Neutrophil gelatinase-associated lipocalin (NGAL) in kidneys. NGAL protein accumulation in the blood and urine can be detected in patients with AKI during a few hours [1–4]. These characteristics have made NGAL a promising biomarker of AKI, abundant in the blood and urine [5–7]. Further research is needed to demonstrate the role of NGAL in renal I/R injury or AKI. NGAL is a protein of the lipocalin family which was found to express in different human tissues, such as uterus, prostate, lung, trachea, stomach, colon, and kidney [8]. In a study by Mishra et al. [2], NGAL was highly upregulated in vivo studies of rat renal I/R injury model, which suggested that NGAL may be involved in kidney development [9]. JAYA et al. [10] found that in mice model of I/R injury, purified NGAL showed protection from tubular damage. Autophagy was activated during renal I/R injury, and the suppression of autophagy by chloroquine and 3-MA worsen the injury, autophagy is thought to be a ∗
renoprotective mechanism during I/R injury [11]. This raises the interesting question of whether the induction of NGAL is involved in the protective of renal I/R injury through autophagy activation. In this study, we investigated the effect of recombinant NGAL administration on renal I/R injury in an established rat model. We hypothesized that NGAL represents a protective effect on renal I/R injury through autophagy activation and apoptosis inhibition of the tubule cells. Our results indicate that NGAL may be a novel therapeutic intervention for renal I/R injury. 2. Materials and methods 2.1. Animals Forty adult male Wistar rats weighing 290 ± 10 g were purchased from Laboratory Animal Center of Peking University Health Science Center. Rats were kept under a 12-h light/12-h dark pattern with rodent chow and tap water ad libitum in a temperature-controlled environment (20 °C–22 °C) and relative humidity as 40–50%. All experimental
Corresponding author. Department of General Practice, The Second Hospital of Hebei Medical University, Heping Western Road No. 215, Shijiazhuang, Hebei, 050000, China. E-mail address: [email protected] (R.-y. Wang).
https://doi.org/10.1016/j.cbi.2018.04.018 Received 21 April 2017; Received in revised form 11 April 2018; Accepted 13 April 2018 Available online 25 April 2018 0009-2797/ © 2018 Published by Elsevier B.V.
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followed by nuclei counterstaining with hematoxylin. Both positive and negative controls were included in every step. To assess the expression of PCNA protein, the scoring system contained intensity score (0, negative staining; 1, weak staining; 2, moderate staining; 3, strong staining) and proportion score (0, none; 1, < 10%; 2, 10–50%; 3, 51–80%; 4, > 80%) was used [14]. Results were analyzed in a blinded manner by two independent investigators. Five visions were scored in every section randomly.
protocols were approved by the Animal Care and Use Committee of the Second Hospital of Hebei Medical University. 2.2. Experimental design The rats had free access to food and water and were pre feeding for 3 days to adapt to the environment. The rats were randomly divided into four groups: Sham group (n = 10), rats underwent identical surgical procedures without renal arterial clamp; I/R group (n = 10), rats underwent renal ischemia for 45 min followed by 24 h reperfusion; I/ R + pre-N group (n = 10), rats received i.v. injection of NGAL (20 μg) 15 min before renal artery clamp was applied; I/R + post-N group (n = 10), rats received i.v. injection of NGAL (20 μg) 15 min after renal artery clamp was removed. Rats were anesthetized with 3% sodium pentobarbital (50 mg/kg) by intraperitoneal injection. Right kidney was removed and the left renal artery was exposed and clamped with an arterial clamp for 45 min. After that, the clamp was removed and the kidney was observed for 4–5 min to assure reperfusion was established successfully. After 24 h of reperfusion, the rats were killed after the left kidney removing.
2.7. Western blot analysis The protein levels of LC3, Beclin-1, Bcl-2, and Bax in renal tissues were detected by western blotting. After weighed, renal tissues were homogenized in radio immunoprecipitation assay (RIPA) clearage solution, and then the homogenates were centrifuged at 12,000 g for 10 min at 4 °C. The proteins were collected and quantified using a bicinchoninicacid protein assay kit (Thermo Fisher Scientific, USA). The protein was separated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes by electroelution. The blots were blocked with 5% non-fat drymilk in TBST at 37 °C for 2 h, followed by incubation with primary antibody against LC3, Beclin-1, Bcl-2, Bax and β-actin (Sigma Life Science, USA) at 4 °C overnight. After incubation with the primary antibody, the membranes were washed with TBST and incubated for 1 h at room temperature with horseradish-peroxidase conjugated secondary antibodies (1:10,000). After again several washes with TBST, protein bands were detected by use of an enhanced chemiluminescence system and visualized using the Odyssey imaging system (LI-COR, USA). Target protein/ β-actin ratio was used as quantitative comparison of expression. The experiment was performed three times.
2.3. Detection of renal function Peripheral blood samples were obtained from each group rats and placed in the centrifuge tube, then in a centrifuge to 3000r/min centrifugal for 10min. 500 μl serum was taken for determining the SCr and BUN using automatic biochemical analyzer (Roche) with a kit from an Olympus AU2700 Analyzer (Olympus, Tokyo, Japan). 2.4. Assessment of kidney histologic pathology
2.8. Electronic microscopy
Renal tissue specimens were stained with hematoxylin-eosin (H&E) according to standard procedures. An experienced pathologist blinded to the groups evaluated the specimens under standard light microscope. Ten areas randomly selected from outer medulla of the kidney were examined at × 200 magnification. Pathological scoring ranged from 0 to 5 point based on the percentage of injury area as follows: 0, no damage; 1, injury area < 10%; 2, injury area > 10% but < 25%; 3, injury area > 25% but < 50%; 4, injury area > 50% but < 75%; 5, injury area > 75% [12,13].
The renal samples of about 1-mm3 were fixed with 2.5% glutaraldehyde overnight at 4 °C, followed by treatment with osmium tetroxide, then rinsed, fixed, embedded and sliced. Autophagosome ultrastructure was observed under the transmission electron microscope. 2.9. Statistical analysis The results are presented as mean ± standard deviation (SD). Treatment effects were compared using one-way ANOVA among different groups. All analyses were performed using GraphPad Prism Version 5.0 (San Diego, USA). P < 0.05 was considered statistically significant.
2.5. TUNEL assay The sections were evaluated by TUNEL assay with an In Situ Cell Death Detection Fluorescein Kit (Roche Diagnostics, annheim, Germany) according to the manufacturer's protocol. Paraffin sections were deparaffinized 4-μm, permeabilized with 20 mg/mL proteinase K, then incubated with 3% hydrogen peroxide. Next, we incubated sections with a mixture of nucleotides and TdT enzyme for 1 h at 37 °C, then with the converter conjugated with horseradish peroxidase for 30 min at 37 °C. Cells were regarded as TUNEL-positive when the nuclei were stained green. The final count was expressed as the percentage of total cells detected by visualization with a fluorescence microscope (Carl Zeiss, Jena, Germany). Finally, TUNEL-positive cells were counted in 10 randomly selected fields under × 200 magnification in a blinded manner.
3. Result 3.1. NGAL improves the renal function In order to understand the severity of renal damage in rats after I/R injury, we detected the levels of BUN and SCr in serum (Fig. 1). From this figure, we could see that treatment with NGAL significantly reduced the levels of BUN and SCr when compared with I/R group, and the effect of pre-NGAL is far better than that of post-NGAL. 3.2. NGAL alleviates the renal injury
2.6. Detection of PCNA protein We examined the renal tissue sections stained with H-E and assessed the renal injury scores after I/R injury. Representative photomicrographs of renal pathological changes in rats from each group were shown in Fig. 2A (a-d). Kidneys from the I/R group rats showed worsened histopathologic features with loss of brush border cells, nuclear pyknosis and flattening of the tubular epithelium. The renal injury score was highest in I/R group. NGAL treatment significantly alleviated the degree of renal histologic damage and lowed the renal injury score
Immunohistochemical staining for PCNA in the renal tissue was detected using commercial assay kits. We deparaffinized 4-μm-thick paraffin sections, which were heated in citrate buffer, then incubated with primary antibodies against PCNA (rabbit anti-rat) (1:800; Cell Signaling Technology, Beverly, MA). Subsequently, renal sections were incubated with secondary antibodies for 30 min at 37 °C. Color was developed using the 3,30-diaminobenzidine tetrahydrochloride, then 41
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Fig. 1. Blood urea nitrogen (A) and serum creatinine (B) levels show the effect of NGAL on renal I/R injury. Data are expressed as mean ± SD. ∗P < 0.05, ∗∗ P < 0.01 vs. Sham group and #P < 0.05, ##P < 0.01 vs. I/R group.
NGAL or post-NGAL treated (Fig. 4E-F). Correspondingly, Bax/Bcl-2 ratio was also significantly decreased in NGAL treated rats (Fig. 4G). These data suggested that exogenous NGAL could clearly inhibit the apoptosis of renal tubular epithelial cells after I/R injury.
when compared with I/R group. Importantly, the renal injury scores in I/R + pre-N group also significantly less than that of I/R + post-N group Fig. 2B. 3.3. NGAL attenuates the apoptosis of tubular epithelium cells
3.4. NGAL enhances the proliferation of tubule cells after I/R injury
TUNEL staining was used to investigate the effect of NGAL on apoptosis. Representative fluorescence photomicrographs of apoptotic cells from each group were shown in Fig. 3A(a-d). As shown in this figure, the apoptotic cells in Sham group were rarely observed, but a very large number of apoptotic cells were found in I/R group. The numbers of apoptotic cells in I/R + pre-N and I/R + post-N groups were significantly less than I/R group. Furtherly, the effect of pre-NGAL was obviously superior to that of post-NGAL (Fig. 3B). Previous reports showed that the apoptosis induced by I/R injury might be associated with the intrinsic pathway, therefore, we determinated the protein expressions of anti-apoptotic Bcl-2 and pro-apoptotic Bax in renal tissues using western blotting. Representative immunoblots were shown in Fig. 4B. The results showed that the expression of Bcl-2 was significantly increased and Bax was markly decreased in rats with pre-
The renal tubule cells following I/R injury requires proliferation of surrounding cells. We next tested the hypothesis that exogenous NGAL administration could drive the proliferative response. We evaluated the proliferation of renal tubules cells through the protein expression of PCNA. As shown in Fig. 5A(a-d), the Sham group displayed a few proliferating cells, but there were only a modest increase of proliferation cells in I/R group. Treatment with NGAL promoted proliferation with a significant number of PCNA-positive cells. There was a marked increase in proliferating proximal tubule cells in rats that were pretreated with NGAL than post-treated with NGAL (Fig. 5B).
Fig. 2. Representative images of renal histology with H&E staining (original magnification × 200) (A) and histologic score of tubular damage in all groups (B). The Sham group showed normal renal cortical tissue structure (A-a). Marked changes of acute tubular necrosis with nuclear pyknosis and necrotic cells in the I/R group (A-b). Treated with NGAL expressed an attenuated histopathologic response (A-c and d) and lower histologic score (B) than the I/R group. Data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. Sham group and #P < 0.05, ##P < 0.01 vs. I/R group. 42
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Fig. 3. Representative images of TUNEL staining. Nuclei of TUNEL-positive cells are stained green (original magnification × 200) (A). Quantification of TUNELpositive cells per high-power field (HPF × 200) (B). Treated with NGAL expressed a decreased number of TUNEL-positive cells (B). Data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. Sham group and #P < 0.05, ##P < 0.01 vs. I/R group.
such as retinoids, arachidonic acid, various steroids and iron [17,18]. The gene expression of NGAL has been demonstrated in many human tissues like uterus, prostate, salivary glands, lung, trachea, stomach, colon, and kidney [8]. Previous studies found that NGAL can be thought as an acute phase protein and up-regulated in appendicitis, inflammatory bowel disease and diverticulitis, also observed in human cancers, such as breast, pancreatic and ovarian cancer [19]. Generally, NGAL is considered as transporters and has some different functions, including immune response, cell growth, proliferation and metabolism, iron transportation, and synthesis of prostaglandins [20]. In our previous study (data no published), the extension of I/R injury is seen at 2–48 h after reperfusion, the injury peaks noted at 24 h of reflow, so we selected 24 h of reperfusion as the time point to explore the effect of NGAL on the renal I/R injury. In present study, we found that NGAL could attenuate tubule cells injury by controlling cell apoptosis. Apoptosis has been shown to be the primary mode of cell death during renal I/R injury and mostly mediated by a family of cysteine proteases termed caspases [21–23]. The Bcl-2 family of proteins contain some members (Bcl-2 and Bcl-XL) function as antiapoptotic factors and others (Bax, Bad or Bcl-Xs) promote apoptosis [24], which can influence cellular processes, including cell cycle progression, calcineurin signaling, and transcriptional repression by p53 [25]. Downregulation of the ratio of Bax/Bcl-2 provide protection against cell apoptosis [26]. NGAL treatment resulted in up-regulated expression of Bcl-2 and down-regulated expression of Bax, as a result of inhibiting apoptotic cell death. Support for this notion corrects with the identification of NGAL as an iron-transporting protein [2,27]. NGAL ameliorated kidney injury may be associated with the transport of iron, retinal or other molecules, which needs further studies to explore. The ratio of LC3 I to LC3 II is closely correlated with the extent of autophagosome formation, so LC3II could be a marker of autophagic
3.5. NGAL promotes autophagy activation after I/R injury To further investigate the molecular mechanisms of renoprotective role mediated by exogenous NGAL during the I/R injury, we detected the expressions of autophagy related protein LC3Ⅰ/Ⅱ and Beclin-1 in renal tissues. Representative immunoblots were shown in Fig. 4A. Our finding showed that NGAL treatment could activated the autophagy with an increasing levels of LC3Ⅱ and Beclin-1. The rats treated with pre-NGAL had a more proteins expressions of LC3Ⅱand Beclin-1 than that of the post-NGAL (Fig. 4C-D). Furthermore, the structures of autophagosomal vacuoles in renal tissues of rats from each group were revealed by electron microscopy which was the gold standard for detecting autophagy (Fig. 6A). Some vacuoles could be detected in I/R group, but more features of autophagic vacuoles were found in rats treated with NGAL, which contained cytoplasm or undigested organelles, with normal mitochondria and endoplasmic reticulum. The numbers of autophagic vacuoles were much more in I/R + pre-N group than that of I/R + post-N group (Fig. 6B). These data indicated that exogenous NGAL could play a renoprotective role through autophagy activation. 4. Discussion This study demonstrated the renoprotective effect of NGAL against renal I/R injury in a rat model. The mechanisms appear to be involved in activating autophagy and decreasing apoptosis of the renal tubular epithelial cells. An important clue for NGAL function comes from the specific structural analyses [15]. NGAL is a protein of the lipocalin family, which has a three-dimensional structure, comprises of eight bstrands that form a β-barrel enclosing a calyx [16]. The calyx could bind and transport a wide variety of low-molecular weight chemicals, 43
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Fig. 4. Representative blots of LC3Ⅰ/Ⅱ, Beclin-1, Bax and Bcl-2 detected by western blot, β-actin was used as a loading control (A and B). Densitometric analysis of the bands analyzed and normalized to β-actin (C–G). The protein expression level of the Sham group was set as 1 in each blot, and the signals of other proteins in the same blot were expressed as percentage of the Sham group, higher protein levels of LC3, Beclin-1 and Bcl-2 but lower levels of Bax and Bax/Bcl ratio when treated with NGAL. Data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Sham group, ##P < 0.01, ###P < 0.001 vs. I/R group. 44
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Fig. 5. Representative of immunoblotting images of PCNA (A) (original magnification × 200). Quantification of the percentage of PCNA expression of all groups (B). Compared with the I/R group, the expression of PCNA was higher when treated with NGAL. Data are expressed as mean ± SD. ∗∗P < 0.01 vs. Sham group and # P < 0.05, ##P < 0.01 vs. I/R group.
subclinical ischemic renal injury or subclinical nephrotoxic damage aimed at preventing progression to overt AKI.
activity [28]. Beclin-1 is recognized to play an important role in the recruitment of other autophagic proteins during expansion of the preautophagosomal membrane and structure [29,30]. Our results showed that treatment with NGAL resulted in the higher expression of LC3 and Beclin-1, and induced more numbers of autophagic vacuoles, indicating that autophagy was enhanced by exogenous NGAL. Meanwhile, NGAL also alleviated renal injury with lower BUN and SCr levels and less renal injury scores. Therefore, these data suggested that induction of autophagy might be one of the protective mechanisms of exogenous NGAL during renal I/R injury. In conclusion, our data provide new insight into the mechanism of protective role of NGAL during renal I/R injury. Further research will be necessary to explore the clinical outcomes of adding NGAL to the organ preservation solutions. On the basis of our data that the injection of NGAL is partially sufficient to decrease renal I/R injury and accelerate tubule regeneration, it is also hoped that NGAL may offer new perspectives for the therapeutic possibilities in patients with subtle,
Conflicts of interest The authors declare that they have no potential conflicts of interest. Acknowledgments Ya-Li Zhang was responsible for experimental design and performed research, analysis, and interpretation of the results; drafted the manuscript; and provided final approval of the version to be published. ShuKai Qiao participated in the design of this study and provided general support. All other authors helped perform the analysis with constructive discussions. All authors read and approved the final manuscript. The study was supported by the Natural Science Foundation of
Fig. 6. Representation of high magnification of electron micrographs showing ultrastructural structures (A). Quantification of the number of autophagic vacuoles per 100 μm2 cytoplasm (B). Autophagic vacuoles (black arrows) were found in the I/R group (A-b). More multiple double- or multiple-membrane autophagic vacuoles were identified when treated with NGAL (black arrows) (A-c and d). Data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. Sham group and #P < 0.05, ## P < 0.01 vs. I/R group. 45
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China 81271903 and National Science and Technology Ministry 2012BA137B01.
[12]
Transparency document [13]
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2018.04.018. [14]
Appendix A. Supplementary data [15]
Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.cbi.2018.04.018.
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Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint
NGAL attenuates renal ischemia/reperfusion injury through autophagy activation and apoptosis inhibition in rats
T
Ya-li Zhanga, Shu-kai Qiaob, Rong-ying Wanga,∗, Xiao-nan Guob a b
Department of General Practice, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, 050000, China Department of Hematology, The Second Hospital of Hebei Medical University, Shijiazhuang, Hebei, 050000, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Neutrophil gelatinase-associated lipocalin Autophagy Apoptosis Ischemia-reperfusion Proliferation
Ischemia/reperfusion (I/R) injury is a main cause of acute kidney injury (AKI), and currently lacks effective therapies. This study is to investigate the level of Neutrophil gelatinase-associated lipocalin (NGAL) and autophagy status during renal I/R injury, so as to determine whether the exogenous NGAL protein could exert a protective effect for I/R injury and explore the potential mechanisms. Forty male Wistar rats were randomly divided into the following four groups: Sham, I/R, pre-treated with NGAL before I/R (I/R + pre-N), treated with NGAL after I/R (I/R + post-N). All rats were subjected to clamping the left renal pedicle for 45 min after right nephrectomy, followed by 24 h of reperfusion. Serum creatinine (SCr) and blood urea nitrogen (BUN) were used for renal function, tubular cell apoptosis and autophagy were measured by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) method, histological examination and electron microscope, respectively. The tubular cell proliferation was assessed by the protein expression of proliferating cell nuclear antigen (PCNA). Western blotting was used to quantitate the levels of LC3, Beclin-1, Bcl-2 and Bax in kidney tissues. Exogenous NGAL protein intervention significantly improved renal function, reduced tubular cell apoptosis, increased tubular cell proliferation and promoted autophagy activation after renal I/R injury. Further, the efficacy in pre-N was significantly better than post-N. The mechanisms were involved in the regulation of several autophagy and apoptosis-related genes. Our study demonstrated that exogenous NGAL protein play a protective role during I/R injury, which may offer a novel may for prevention and treatment of renal I/R injury.
1. Introduction Renal ischemia-reperfusion (I/R) injury is the primary cause of acute kidney injury (AKI), which can cause a significant increase in the expression of Neutrophil gelatinase-associated lipocalin (NGAL) in kidneys. NGAL protein accumulation in the blood and urine can be detected in patients with AKI during a few hours [1–4]. These characteristics have made NGAL a promising biomarker of AKI, abundant in the blood and urine [5–7]. Further research is needed to demonstrate the role of NGAL in renal I/R injury or AKI. NGAL is a protein of the lipocalin family which was found to express in different human tissues, such as uterus, prostate, lung, trachea, stomach, colon, and kidney [8]. In a study by Mishra et al. [2], NGAL was highly upregulated in vivo studies of rat renal I/R injury model, which suggested that NGAL may be involved in kidney development [9]. JAYA et al. [10] found that in mice model of I/R injury, purified NGAL showed protection from tubular damage. Autophagy was activated during renal I/R injury, and the suppression of autophagy by chloroquine and 3-MA worsen the injury, autophagy is thought to be a ∗
renoprotective mechanism during I/R injury [11]. This raises the interesting question of whether the induction of NGAL is involved in the protective of renal I/R injury through autophagy activation. In this study, we investigated the effect of recombinant NGAL administration on renal I/R injury in an established rat model. We hypothesized that NGAL represents a protective effect on renal I/R injury through autophagy activation and apoptosis inhibition of the tubule cells. Our results indicate that NGAL may be a novel therapeutic intervention for renal I/R injury. 2. Materials and methods 2.1. Animals Forty adult male Wistar rats weighing 290 ± 10 g were purchased from Laboratory Animal Center of Peking University Health Science Center. Rats were kept under a 12-h light/12-h dark pattern with rodent chow and tap water ad libitum in a temperature-controlled environment (20 °C–22 °C) and relative humidity as 40–50%. All experimental
Corresponding author. Department of General Practice, The Second Hospital of Hebei Medical University, Heping Western Road No. 215, Shijiazhuang, Hebei, 050000, China. E-mail address: [email protected] (R.-y. Wang).
https://doi.org/10.1016/j.cbi.2018.04.018 Received 21 April 2017; Received in revised form 11 April 2018; Accepted 13 April 2018 Available online 25 April 2018 0009-2797/ © 2018 Published by Elsevier B.V.
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followed by nuclei counterstaining with hematoxylin. Both positive and negative controls were included in every step. To assess the expression of PCNA protein, the scoring system contained intensity score (0, negative staining; 1, weak staining; 2, moderate staining; 3, strong staining) and proportion score (0, none; 1, < 10%; 2, 10–50%; 3, 51–80%; 4, > 80%) was used [14]. Results were analyzed in a blinded manner by two independent investigators. Five visions were scored in every section randomly.
protocols were approved by the Animal Care and Use Committee of the Second Hospital of Hebei Medical University. 2.2. Experimental design The rats had free access to food and water and were pre feeding for 3 days to adapt to the environment. The rats were randomly divided into four groups: Sham group (n = 10), rats underwent identical surgical procedures without renal arterial clamp; I/R group (n = 10), rats underwent renal ischemia for 45 min followed by 24 h reperfusion; I/ R + pre-N group (n = 10), rats received i.v. injection of NGAL (20 μg) 15 min before renal artery clamp was applied; I/R + post-N group (n = 10), rats received i.v. injection of NGAL (20 μg) 15 min after renal artery clamp was removed. Rats were anesthetized with 3% sodium pentobarbital (50 mg/kg) by intraperitoneal injection. Right kidney was removed and the left renal artery was exposed and clamped with an arterial clamp for 45 min. After that, the clamp was removed and the kidney was observed for 4–5 min to assure reperfusion was established successfully. After 24 h of reperfusion, the rats were killed after the left kidney removing.
2.7. Western blot analysis The protein levels of LC3, Beclin-1, Bcl-2, and Bax in renal tissues were detected by western blotting. After weighed, renal tissues were homogenized in radio immunoprecipitation assay (RIPA) clearage solution, and then the homogenates were centrifuged at 12,000 g for 10 min at 4 °C. The proteins were collected and quantified using a bicinchoninicacid protein assay kit (Thermo Fisher Scientific, USA). The protein was separated by 15% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membranes by electroelution. The blots were blocked with 5% non-fat drymilk in TBST at 37 °C for 2 h, followed by incubation with primary antibody against LC3, Beclin-1, Bcl-2, Bax and β-actin (Sigma Life Science, USA) at 4 °C overnight. After incubation with the primary antibody, the membranes were washed with TBST and incubated for 1 h at room temperature with horseradish-peroxidase conjugated secondary antibodies (1:10,000). After again several washes with TBST, protein bands were detected by use of an enhanced chemiluminescence system and visualized using the Odyssey imaging system (LI-COR, USA). Target protein/ β-actin ratio was used as quantitative comparison of expression. The experiment was performed three times.
2.3. Detection of renal function Peripheral blood samples were obtained from each group rats and placed in the centrifuge tube, then in a centrifuge to 3000r/min centrifugal for 10min. 500 μl serum was taken for determining the SCr and BUN using automatic biochemical analyzer (Roche) with a kit from an Olympus AU2700 Analyzer (Olympus, Tokyo, Japan). 2.4. Assessment of kidney histologic pathology
2.8. Electronic microscopy
Renal tissue specimens were stained with hematoxylin-eosin (H&E) according to standard procedures. An experienced pathologist blinded to the groups evaluated the specimens under standard light microscope. Ten areas randomly selected from outer medulla of the kidney were examined at × 200 magnification. Pathological scoring ranged from 0 to 5 point based on the percentage of injury area as follows: 0, no damage; 1, injury area < 10%; 2, injury area > 10% but < 25%; 3, injury area > 25% but < 50%; 4, injury area > 50% but < 75%; 5, injury area > 75% [12,13].
The renal samples of about 1-mm3 were fixed with 2.5% glutaraldehyde overnight at 4 °C, followed by treatment with osmium tetroxide, then rinsed, fixed, embedded and sliced. Autophagosome ultrastructure was observed under the transmission electron microscope. 2.9. Statistical analysis The results are presented as mean ± standard deviation (SD). Treatment effects were compared using one-way ANOVA among different groups. All analyses were performed using GraphPad Prism Version 5.0 (San Diego, USA). P < 0.05 was considered statistically significant.
2.5. TUNEL assay The sections were evaluated by TUNEL assay with an In Situ Cell Death Detection Fluorescein Kit (Roche Diagnostics, annheim, Germany) according to the manufacturer's protocol. Paraffin sections were deparaffinized 4-μm, permeabilized with 20 mg/mL proteinase K, then incubated with 3% hydrogen peroxide. Next, we incubated sections with a mixture of nucleotides and TdT enzyme for 1 h at 37 °C, then with the converter conjugated with horseradish peroxidase for 30 min at 37 °C. Cells were regarded as TUNEL-positive when the nuclei were stained green. The final count was expressed as the percentage of total cells detected by visualization with a fluorescence microscope (Carl Zeiss, Jena, Germany). Finally, TUNEL-positive cells were counted in 10 randomly selected fields under × 200 magnification in a blinded manner.
3. Result 3.1. NGAL improves the renal function In order to understand the severity of renal damage in rats after I/R injury, we detected the levels of BUN and SCr in serum (Fig. 1). From this figure, we could see that treatment with NGAL significantly reduced the levels of BUN and SCr when compared with I/R group, and the effect of pre-NGAL is far better than that of post-NGAL. 3.2. NGAL alleviates the renal injury
2.6. Detection of PCNA protein We examined the renal tissue sections stained with H-E and assessed the renal injury scores after I/R injury. Representative photomicrographs of renal pathological changes in rats from each group were shown in Fig. 2A (a-d). Kidneys from the I/R group rats showed worsened histopathologic features with loss of brush border cells, nuclear pyknosis and flattening of the tubular epithelium. The renal injury score was highest in I/R group. NGAL treatment significantly alleviated the degree of renal histologic damage and lowed the renal injury score
Immunohistochemical staining for PCNA in the renal tissue was detected using commercial assay kits. We deparaffinized 4-μm-thick paraffin sections, which were heated in citrate buffer, then incubated with primary antibodies against PCNA (rabbit anti-rat) (1:800; Cell Signaling Technology, Beverly, MA). Subsequently, renal sections were incubated with secondary antibodies for 30 min at 37 °C. Color was developed using the 3,30-diaminobenzidine tetrahydrochloride, then 41
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Fig. 1. Blood urea nitrogen (A) and serum creatinine (B) levels show the effect of NGAL on renal I/R injury. Data are expressed as mean ± SD. ∗P < 0.05, ∗∗ P < 0.01 vs. Sham group and #P < 0.05, ##P < 0.01 vs. I/R group.
NGAL or post-NGAL treated (Fig. 4E-F). Correspondingly, Bax/Bcl-2 ratio was also significantly decreased in NGAL treated rats (Fig. 4G). These data suggested that exogenous NGAL could clearly inhibit the apoptosis of renal tubular epithelial cells after I/R injury.
when compared with I/R group. Importantly, the renal injury scores in I/R + pre-N group also significantly less than that of I/R + post-N group Fig. 2B. 3.3. NGAL attenuates the apoptosis of tubular epithelium cells
3.4. NGAL enhances the proliferation of tubule cells after I/R injury
TUNEL staining was used to investigate the effect of NGAL on apoptosis. Representative fluorescence photomicrographs of apoptotic cells from each group were shown in Fig. 3A(a-d). As shown in this figure, the apoptotic cells in Sham group were rarely observed, but a very large number of apoptotic cells were found in I/R group. The numbers of apoptotic cells in I/R + pre-N and I/R + post-N groups were significantly less than I/R group. Furtherly, the effect of pre-NGAL was obviously superior to that of post-NGAL (Fig. 3B). Previous reports showed that the apoptosis induced by I/R injury might be associated with the intrinsic pathway, therefore, we determinated the protein expressions of anti-apoptotic Bcl-2 and pro-apoptotic Bax in renal tissues using western blotting. Representative immunoblots were shown in Fig. 4B. The results showed that the expression of Bcl-2 was significantly increased and Bax was markly decreased in rats with pre-
The renal tubule cells following I/R injury requires proliferation of surrounding cells. We next tested the hypothesis that exogenous NGAL administration could drive the proliferative response. We evaluated the proliferation of renal tubules cells through the protein expression of PCNA. As shown in Fig. 5A(a-d), the Sham group displayed a few proliferating cells, but there were only a modest increase of proliferation cells in I/R group. Treatment with NGAL promoted proliferation with a significant number of PCNA-positive cells. There was a marked increase in proliferating proximal tubule cells in rats that were pretreated with NGAL than post-treated with NGAL (Fig. 5B).
Fig. 2. Representative images of renal histology with H&E staining (original magnification × 200) (A) and histologic score of tubular damage in all groups (B). The Sham group showed normal renal cortical tissue structure (A-a). Marked changes of acute tubular necrosis with nuclear pyknosis and necrotic cells in the I/R group (A-b). Treated with NGAL expressed an attenuated histopathologic response (A-c and d) and lower histologic score (B) than the I/R group. Data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. Sham group and #P < 0.05, ##P < 0.01 vs. I/R group. 42
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Fig. 3. Representative images of TUNEL staining. Nuclei of TUNEL-positive cells are stained green (original magnification × 200) (A). Quantification of TUNELpositive cells per high-power field (HPF × 200) (B). Treated with NGAL expressed a decreased number of TUNEL-positive cells (B). Data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. Sham group and #P < 0.05, ##P < 0.01 vs. I/R group.
such as retinoids, arachidonic acid, various steroids and iron [17,18]. The gene expression of NGAL has been demonstrated in many human tissues like uterus, prostate, salivary glands, lung, trachea, stomach, colon, and kidney [8]. Previous studies found that NGAL can be thought as an acute phase protein and up-regulated in appendicitis, inflammatory bowel disease and diverticulitis, also observed in human cancers, such as breast, pancreatic and ovarian cancer [19]. Generally, NGAL is considered as transporters and has some different functions, including immune response, cell growth, proliferation and metabolism, iron transportation, and synthesis of prostaglandins [20]. In our previous study (data no published), the extension of I/R injury is seen at 2–48 h after reperfusion, the injury peaks noted at 24 h of reflow, so we selected 24 h of reperfusion as the time point to explore the effect of NGAL on the renal I/R injury. In present study, we found that NGAL could attenuate tubule cells injury by controlling cell apoptosis. Apoptosis has been shown to be the primary mode of cell death during renal I/R injury and mostly mediated by a family of cysteine proteases termed caspases [21–23]. The Bcl-2 family of proteins contain some members (Bcl-2 and Bcl-XL) function as antiapoptotic factors and others (Bax, Bad or Bcl-Xs) promote apoptosis [24], which can influence cellular processes, including cell cycle progression, calcineurin signaling, and transcriptional repression by p53 [25]. Downregulation of the ratio of Bax/Bcl-2 provide protection against cell apoptosis [26]. NGAL treatment resulted in up-regulated expression of Bcl-2 and down-regulated expression of Bax, as a result of inhibiting apoptotic cell death. Support for this notion corrects with the identification of NGAL as an iron-transporting protein [2,27]. NGAL ameliorated kidney injury may be associated with the transport of iron, retinal or other molecules, which needs further studies to explore. The ratio of LC3 I to LC3 II is closely correlated with the extent of autophagosome formation, so LC3II could be a marker of autophagic
3.5. NGAL promotes autophagy activation after I/R injury To further investigate the molecular mechanisms of renoprotective role mediated by exogenous NGAL during the I/R injury, we detected the expressions of autophagy related protein LC3Ⅰ/Ⅱ and Beclin-1 in renal tissues. Representative immunoblots were shown in Fig. 4A. Our finding showed that NGAL treatment could activated the autophagy with an increasing levels of LC3Ⅱ and Beclin-1. The rats treated with pre-NGAL had a more proteins expressions of LC3Ⅱand Beclin-1 than that of the post-NGAL (Fig. 4C-D). Furthermore, the structures of autophagosomal vacuoles in renal tissues of rats from each group were revealed by electron microscopy which was the gold standard for detecting autophagy (Fig. 6A). Some vacuoles could be detected in I/R group, but more features of autophagic vacuoles were found in rats treated with NGAL, which contained cytoplasm or undigested organelles, with normal mitochondria and endoplasmic reticulum. The numbers of autophagic vacuoles were much more in I/R + pre-N group than that of I/R + post-N group (Fig. 6B). These data indicated that exogenous NGAL could play a renoprotective role through autophagy activation. 4. Discussion This study demonstrated the renoprotective effect of NGAL against renal I/R injury in a rat model. The mechanisms appear to be involved in activating autophagy and decreasing apoptosis of the renal tubular epithelial cells. An important clue for NGAL function comes from the specific structural analyses [15]. NGAL is a protein of the lipocalin family, which has a three-dimensional structure, comprises of eight bstrands that form a β-barrel enclosing a calyx [16]. The calyx could bind and transport a wide variety of low-molecular weight chemicals, 43
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Fig. 4. Representative blots of LC3Ⅰ/Ⅱ, Beclin-1, Bax and Bcl-2 detected by western blot, β-actin was used as a loading control (A and B). Densitometric analysis of the bands analyzed and normalized to β-actin (C–G). The protein expression level of the Sham group was set as 1 in each blot, and the signals of other proteins in the same blot were expressed as percentage of the Sham group, higher protein levels of LC3, Beclin-1 and Bcl-2 but lower levels of Bax and Bax/Bcl ratio when treated with NGAL. Data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001 vs. Sham group, ##P < 0.01, ###P < 0.001 vs. I/R group. 44
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Fig. 5. Representative of immunoblotting images of PCNA (A) (original magnification × 200). Quantification of the percentage of PCNA expression of all groups (B). Compared with the I/R group, the expression of PCNA was higher when treated with NGAL. Data are expressed as mean ± SD. ∗∗P < 0.01 vs. Sham group and # P < 0.05, ##P < 0.01 vs. I/R group.
subclinical ischemic renal injury or subclinical nephrotoxic damage aimed at preventing progression to overt AKI.
activity [28]. Beclin-1 is recognized to play an important role in the recruitment of other autophagic proteins during expansion of the preautophagosomal membrane and structure [29,30]. Our results showed that treatment with NGAL resulted in the higher expression of LC3 and Beclin-1, and induced more numbers of autophagic vacuoles, indicating that autophagy was enhanced by exogenous NGAL. Meanwhile, NGAL also alleviated renal injury with lower BUN and SCr levels and less renal injury scores. Therefore, these data suggested that induction of autophagy might be one of the protective mechanisms of exogenous NGAL during renal I/R injury. In conclusion, our data provide new insight into the mechanism of protective role of NGAL during renal I/R injury. Further research will be necessary to explore the clinical outcomes of adding NGAL to the organ preservation solutions. On the basis of our data that the injection of NGAL is partially sufficient to decrease renal I/R injury and accelerate tubule regeneration, it is also hoped that NGAL may offer new perspectives for the therapeutic possibilities in patients with subtle,
Conflicts of interest The authors declare that they have no potential conflicts of interest. Acknowledgments Ya-Li Zhang was responsible for experimental design and performed research, analysis, and interpretation of the results; drafted the manuscript; and provided final approval of the version to be published. ShuKai Qiao participated in the design of this study and provided general support. All other authors helped perform the analysis with constructive discussions. All authors read and approved the final manuscript. The study was supported by the Natural Science Foundation of
Fig. 6. Representation of high magnification of electron micrographs showing ultrastructural structures (A). Quantification of the number of autophagic vacuoles per 100 μm2 cytoplasm (B). Autophagic vacuoles (black arrows) were found in the I/R group (A-b). More multiple double- or multiple-membrane autophagic vacuoles were identified when treated with NGAL (black arrows) (A-c and d). Data are expressed as mean ± SD. ∗P < 0.05, ∗∗P < 0.01 vs. Sham group and #P < 0.05, ## P < 0.01 vs. I/R group. 45
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China 81271903 and National Science and Technology Ministry 2012BA137B01.
[12]
Transparency document [13]
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.cbi.2018.04.018. [14]
Appendix A. Supplementary data [15]
Supplementary data related to this article can be found at http://dx. doi.org/10.1016/j.cbi.2018.04.018.
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