- Email: [email protected]
Protective Effect of Alpha 1-Antitrypsin on Renal Ischemia-Reperfusion Injury
Protective Effect of Alpha 1-Antitrypsin on Renal IschemiaReperfusion Injury Kye-Hwa Jeong, Jeong-Hoon Lim, Kyung-Hee Lee, Min-Jung Kim, Hee-Yeon Jung, Ji-Young Choi, Jang-Hee Cho, Sun-Hee Park, Yong-Lim Kim, and Chan-Duck Kim* Division of Nephrology, Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, Daegu, South Korea
ABSTRACT Background. a1-Antitrypsin (AAT) is an important protein in the anti-inflammatory response that functions to regulate the activity of serine proteinases. We aimed to evaluate the protective effect of AAT on ischemia-reperfusion injury (IRI) in a mouse model. Methods. We investigated the effects of AAT in a C57BL/6 mouse model of IRI by dividing them into 4 groups: normal control, sham operated, ischemia-reperfusion (IR), and IR after AAT pretreatment (IR-AAT). In the IR-AAT group, mice were pretreated with AAT (80 mg/kg/d) for 3 days before renal ischemia was induced by clamping the bilateral renal vascular pedicles for 30 minutes. At 24 hours after IRI, biochemistry, histology, inflammatory cytokines, and apoptosis were assayed. Results. Blood urea nitrogen and serum creatinine levels were significantly lower in the IR-AAT group than in the IR group. Neutrophil gelatinase-associated lipocalin and kidney injury molecule 1 protein levels were significantly lower in the IR-AAT group than in the IR group. In addition, there were fewer tubular injuries and less interstitial fibrosis in the IR-AAT group than in the IR group, and the expression levels of transforming growth factor b, interleukin 1b, and interleukin 6 were significantly lower in the IR-AAT group than in the IR group. When compared with the IR group, there were fewer terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assayepositive cells, lower caspase 3 activity and B-cell lymphoma 2-associated X protein (Bax), and higher B-cell lymphoma 2 (Bcle2) in the IR-AAT group. Conclusions. a1-Antitrypsin preserved renal function, attenuated tubular injuries and interstitial fibrosis, and inhibited inflammation and apoptosis after renal IRI. Our results suggest that AAT has protective effects against renal IRI by inhibiting inflammatory and apoptosis pathways.
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ENAL ischemia-reperfusion injury (IRI) induces the innate and adaptive immune responses and causes cell apoptosis, innate autoimmunity, complement activation, platelet aggregation, and microvascular dysfunction, which lead to acute kidney injury (AKI) [1,2]. Ischemiareperfusion injury is an important determinant of high morbidity and mortality in AKI. In kidney transplant, IRI is an inevitable event. Allograft injury starts with the physiological changes associated with brain death or circulatory 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.04.084
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Funding This work was supported by the Korea Health Technology R&D Project of the Korea Health Industry Development Institute (KHIDI), which is funded by the Ministry of Health and Welfare, Republic of Korea (HI13C1232). *Address correspondence to Chan-Duck Kim, MD, PhD, Professor, Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, 130 Dongdeok-ro, Jung-gu, Daegu 41944, South Korea. Tel: þ82-53-200-5560; Fax: þ82-53-426-9464. E-mail: [email protected] ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 2814e2822 (2019)
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Fig 1. In vivo experimental protocol. Mice were divided into 4 groups: the normal control (NC, n ¼ 5), sham-operated (Sham, n ¼ 6), renal ischemia-reperfusion (IR, n ¼ 8), and AAT treatment before renal IR (IR-AAT, n ¼ 8) groups. Abbreviations: AAT, a1-antitrypsin; IP, intraperitoneal; IR, ischemia-reperfusion.
collapse and persists through the peri- and post-transplant periods. After removal from the donor, the allograft is maintained in ischemic conditions before reperfusion at transplant. During this time, complex pathophysiological processes continue, leading to damage after reperfusion from the proinflammatory environment. Paradoxically, reperfusion does not improve the ischemic conditions but increases the ischemic damage by activating several mechanisms, including the innate and the adaptive immune
responses and programmed cell death [3]. These complex processes can manifest clinically as delayed graft function after kidney transplant. Allograft injury due to IRI reduces the allograft survival rate in kidney transplant and increases the risk of chronic kidney disease [4e6]. A wide range of studies on substances with antiinflammatory and antiapoptotic activities have been conducted to assess their effects on the development of IRI. One of these substances, a1-antitrypsin (AAT) protects
Fig 2. Renal function and the levels of NGAL and KIM-1. (A) Blood urea nitrogen (BUN), (B) creatinine (Cr), (C) neutrophil gelatinaseassociated lipocalin (NGAL), and (D) kidney injury molecule-1 (KIM-1). Abbreviations: NC, normal control; IR, ischemia-reperfusion group. *P < .05 vs NC. yP < .01 vs NC. zP < .001 vs NC. §P < .05 vs IR. ǁP < .01 vs IR. ¶P < .001 vs IR.
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Fig 3. Periodic acid-Schiff (PAS) and Masson’s trichrome staining and collagen deposition. (A & E) the normal control (NC), (B & F) sham-operated (Sham), (C & G) IR, (D & H) IR-AAT, and (I) semiquantitative assessment of the area of renal fibrosis. Abbreviations: IR, ischemia-reperfusion group; IR-ATT, ischemia-reperfusion a1-antitrypsin group; PAS, periodic acid-Schiff. *P < .05 vs NC. yP < .01 vs NC. zP < .001 vs NC. §P < .05 vs IR. ǁP < .01 vs IR. ¶P < .001 vs IR.
tissues from the enzymes of inflammatory cells, especially neutrophil elastase and a protease released from activated neutrophils. a1-Antitrypsin inhibits elastase, which can damage tubular endothelial cells, enhance cytokine production, and express adhesion molecules [7e9]. Based on these mechanisms, the purpose of the present study was to identify the protective effect of AAT, which is known to induce an anti-inflammatory reaction by controlling the activity of serine proteinases in IRI in a mouse model of ischemic renal damage. MATERIALS AND METHODS Mice and the IRI Model For all experiments, 8-week-old male C57BL/6 mice weighing 22 to 25 g (Samtako, Osan, Korea) were used. The mice were housed under a 12-hour light/dark cycle with free access to standard chow and water. All mouse care and experimentation were performed in accordance with the guidelines approved by the Animal Care and Use Committee of Kyungpook National University (KNUe2017 e0013). Animals were anesthetized with tribromoethanol (240 mg/kg, 1.2%) during surgery. To induce ischemia, the renal pedicles were exposed through a minimal flank incision and were completely occluded for 30 minutes with a microaneurysm clamp. Following
induction of warm ischemia for 30 minutes, the artery clamp was removed to allow reperfusion. A similar surgical treatment was performed in sham-operated mice, except the renal pedicle clamping was omitted. During the operation, animals were maintained at 36.5 C to 37 C with a temperature-controlled heating device (Harvard Bioscience, Holliston, Mass, United States).
Experimental Groups Mice were randomly divided into 4 groups as follows (Fig 1): the normal control (NC, n ¼ 5), sham-operated (Sham, n ¼ 6), ischemia-reperfusion (IR, n ¼ 8), and ischemia-reperfusion after AAT treatment (IR-AAT, n ¼ 8) groups. The IR group was administered 0.9% normal saline for 3 days and then underwent renal IR. The IR-AAT group was administered AAT (Aralast, Baxter Healthcare, Vienna, Austria) intraperitoneally at 80 mg/kg/d for 3 days and then underwent renal IR. The AAT dosage used in our study was based on previous mouse studies [9,10]. All mice were euthanized by cardiac puncture under anesthesia at 24 hours after reperfusion. Blood and both kidneys were collected for analysis.
Serum Biochemical Analyses After centrifugation of each blood sample, the serum was collected. Blood urea nitrogen (BUN) and creatinine levels were measured
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Fig 4. Immunohistochemical staining and semiquantitative assessment of F4/80 and TNF-a. (A & E) the normal control (NC), (B & F) sham-operated (Sham), (C & G) IR, (D & H) IR-AAT, and (I & J) semiquantitative assessment of F4/80-positive cells and TNF-a-positive areas. Abbreviations: IR, ischemia-reperfusion group; IR-ATT, ischemia-reperfusion a1-antitrypsin group; TNF, tumor necrosis factor. * P < .05 vs NC. yP < .01 vs NC. zP < .001 vs NC. §P < .05 vs IR. ǁP < .01 vs IR. ¶P < .001 vs IR.
with a Hitachi 7600 automated biochemistry analyzer (Hitachi, Tokyo, Japan).
Quantitative Real-Time Polymerase Chain Reaction Total RNA was extracted with TRIzol reagent (Life Technologies, Carlsbad, Calif, United States) from homogenized whole kidneys according to the manufacturer’s instructions. Total RNA (1 mg) was reverse transcribed to complementary DNA with the PrimeScript complementary DNA synthesis kit (Takara, Otsu, Japan). Quantitative real-time polymerase chain reaction (PCR) was performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif, United States) using SYBR Green PCR master mix (Life Technologies). To quantify relative gene expression, the results were analyzed by the comparative Ct method. The primers used in quantitative PCR were as follows: neutrophil gelatinase-associated lipocalin (NGAL) forward 50 -GCC ACT CCA TCT TTC CTG TTGe30 and reverse 50 -GGG AGT GCT GGC CAA ATA AGe30 ; kidney injury moleculee1 (KIMe1) forward 50 CAG GGA AGC CGC AGA AAA Ae30 and reverse 50 -GAT AGC CAC GGT GCT CAC AAe30 ; interleukin (IL) 1b forward 50 -TCG TGC TGT CGG ACC CAT ATe30 and reverse 50 -GGT TCTC CTT GTA CAA AGC TCA TGe30 ; ILe6 forward 50 -CCC ACC AAG AAC GAT AGT CAA TTe30 and reverse 50 -CAC CAG CAT CAG TCC CAA GAe30 ; transforming growth factor (TGF) b forward
50 -GGC TGT GGC CAT CAA GAA TTe30 and reverse 50 - GCA GAG GGA AGA GTC AAA CAT GTe30 ; tumor necrosis factor (TNF) a forward 50 - GAC TAG CCA GGA GGG AGA ACA Ge30 and reverse 50 -CAG TGA GTG AAA GGG ACA GAA CCTe30 ; and b-actin forward 50 -ACC ACC ATG TAC CCA GGC ATTe30 and reverse 50 -CCA CAC AGA GTA CTT GCG CTC Ae30 , which were designed using Primer Express V1.5 software (Applied Biosystems).
Histologic Examination and Immunohistochemical Studies Kidney tissues were fixed with 4% paraformaldehyde (pH 7.4) and embedded in paraffin. The paraffin-embedded tissue was sliced (2 mm) and stained with periodic acid-Schiff and Masson’s trichrome using standard protocols to determine histologic changes and collagen deposition, respectively. Collagen deposition was quantified using the i-Solution DT image software (IMT i-Solution, Vancouver, British Columbia, Canada) in more than 5 randomly selected fields in sections of the cortex and medulla. For immunohistochemistry staining, the 2-mm-thick kidney sections were deparaffinized and rehydrated, and then endogenous peroxidase was inactivated with 3% hydrogen peroxide. The samples were incubated with anti-F4/80 (1:200; Serotec, Oxford, United Kingdom) and anti-TNF-a (1:100; Abcam, Cambridge, United
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Fig 5. The mRNA levels of (A) tumor necrosis factor (TNF) a, (B) transforming growth factor (TGF) b, (C) interleukin (IL) 1b, and (D) IL-6 in kidney tissue. Abbreviations: IR, ischemia-reperfusion group; mRNA, messenger RNA; NC, normal control. *P < .05 vs NC. yP < .01 vs NC. zP < .001 vs NC. §P < .05 vs IR. ǁP < .01 vs IR. ¶P < .001 vs IR. Kingdom) antibodies overnight at 4 C and then detected with the EnVision-HRP kit (Dako, Carpinteria, Calif, United States). Each section was counterstained with Mayer’s hematoxylin and examined under a Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar, Germany) equipped with a CoolSNAP HQ camera (Photometrics, Tucson, Ariz, United States). To quantify F4/80-positive cells, 5 nonoverlapping fields per slide, at 400 magnification, were counted and normalized per mm2 of tissue. Tumor necrosis factor a expression was quantified as a percentage of the total area using Image J software (National Institutes of Health, Bethesda, Md, United States).
Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End-Labeling Assay To detect apoptotic cells, the In Situ Cell Death Detection Fluorescein Kit (Roche, Mannheim, Germany) was used according to the manufacturer’s protocol. Briefly, the 2-mm-thick kidney sections were deparaffinized, rehydrated, and then treated with proteinase K. For fluorescence terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay staining, the sections were incubated with the TUNEL reagent for 30 minutes at room temperature, washed with phosphate-buffered saline 3 times for 5 minutes, and then counterstained with 40 ,6-diamidinoe2phenylindole (Sigma-Aldrich, St. Louis, Mo, United States) for 1 minute for nuclear staining. Finally, the sections were mounted with Prolong Gold antifade reagent (Invitrogen, Carlsbad, Calif, United States) and observed under a confocal microscope (Carl Zeiss, Göttingen, Germany).
Measurement of Caspase 3 Activity Caspase 3 activity in homogenized whole kidney tissue was measured using a colorimetric assay kit (Sigma-Aldrich) according to the manufacturer’s protocol. In brief, the supernatant of kidney homogenates was incubated with a fluorometric caspase 3 substrate and Ac-DEVD-pNA in assay buffer at 37 C for 90 minutes. Caspase 3 activity was measured at 405 nm using an enzyme-linked immunosorbent assay reader. Values are expressed as the percentage of the control.
Western Blot Analysis Homogenized kidney tissue was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, blocked with 10% skimmed milk for 1 hour, and then incubated with primary antibodies against B-cell lymphoma 2 (Bcle2) (1:1000; Cell Signaling Technology, Danvers, Mass, United States), Bcl-2-associated X protein (Bax) (1:1000; Cell Signaling Technology), and b-actin (1:5000, Sigma-Aldrich) overnight at 4 C. After washing, the membrane was incubated with horseradish peroxidase-conjugated IgG secondary antibodies (1:2000; Dako, Glostrup, Denmark) for 1 hour and detected using the ECL Advanced Detection Kit (GE Healthcare, Little Chalfont, United Kingdom). Bands were analyzed using Scion Image software (Scion, Frederick, Md, United States), and b-actin was used as a loading control.
Statistical Analysis All data are presented as means (SEs). Experiments were repeated at least 3 times independently. All statistical analyses were
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Fig 6. TUNEL assay to detect apoptosis in renal cells and counterstaining of nuclei with DAPI. The normal control (NC), sham-operated (Sham), renal ischemia-reperfusion (IR), and AAT treatment before renal ischemia-reperfusion (IR-AAT) groups. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
performed by 1-way analysis of variance with Tukey’s post hoc test using GraphPad Prism 5.01 software (GraphPad Software, La Jolla, Calif, United States). A P value less than .05 was considered statistically significant.
lower levels of both NGAL and KIMe1 than in the IR group (Fig 2).
a1-Antitrypsin Pretreatment Reduced IRI-Induced Renal Injury
RESULTS a1-Antitrypsin Pretreatment Reduced the IRI-Induced Increase in Serum BUN, Creatinine, NGAL, and KIMe1 Levels
The IR group showed significantly higher BUN and creatinine levels than the control groups (NC and Sham). However, BUN and creatinine levels were significantly lower in the IR-AAT group than in the IR group. The NGAL and KIMe1 levels, which have been identified as early predictive biomarkers for AKI, were significantly higher in the IR group than in the control groups (NC and Sham). However, the IR-AAT group showed significantly
Periodic acid-Schiff and Masson’s trichrome staining were carried out to investigate the histologic changes and collagen deposition in the kidneys. Periodic acid-Schiff and Masson’s trichrome staining showed that there was little tubular injury and interstitial fibrosis in the control groups (NC, Fig 3A, E and Sham, Fig 3B, F). After IRI, collagen deposition in the renal interstitium and tubular damage, such as cast formation and brush border loss, were significantly increased in the IR group compared with the control groups (NC and Sham; Fig 3C, G, I). However, AAT pretreatment (IR-AAT group) reduced IRI-induced renal injury (Fig 3D, H, I).
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Fig 7. Caspase 3 activity and Bcl-2/Bax protein expression. (A) Caspase-3 activity was measured by enzyme-linked immunosorbent assay, and (B) renal expression of Bcl-2 and Bax were measured by western blotting. The expression of Bcl-2 and Bax are shown in representative western blots. The density of the western blots was quantified with Scion image software and normalized to b-actin. The normal control (NC), sham operated (Sham), renal ischemia-reperfusion (IR), and AAT treatment before renal IR (IR-AAT) group. Abbreviations: Bax, B-cell leukemia/lymphoma 2-associated X protein; Bcl-2, B-cell chronic lymphocytic leukemia/lymphoma. * P < .05 vs NC. yP < .01 vs NC. zP < .001 vs NC. §P < .05 vs IR. ǁP < .01 vs IR. ¶P < .001 vs IR.
a1-Antitrypsin Pretreatment Reduced the Increased Expression of Inflammatory Markers Following Renal IRI
F4/80 is a macrophage marker that is required for the induction of the efferent CD8þ regulatory T cells required for peripheral tolerance, and TNF-a is a cytokine that is associated with systemic inflammation. The expression levels of F4/80 and TNF-a were higher in the IR group (Fig 4C, G) than in the control groups (NC, Fig 4A, E and Sham, Fig 4B, F). However, the expression levels of F4/80 and TNF-a were significantly lower in the IR-AAT group than in the IR group (Fig 4D, H-J). Quantitative PCR was used to measure the messenger RNA expression levels of inflammatory cytokines, including TNF-a, TGFb, ILe1b, and ILe6. After IRI, the expression levels of TNF-a, TGFb, ILe1b, and ILe6 were significantly higher than those in the control groups (NC and Sham). With AAT pretreatment, the expression levels of TGFb, ILe1b, and ILe6 were significantly lower than those in the IR group. a1-Antitrypsin pretreatment before IRI also showed a tendency to reduce the upregulation of TNF-a expression in the IR group (Fig 5).
a1-Antitrypsin Pretreatment Inhibits IRI-Induced Apoptosis We used the TUNEL assay to determine the effect of AAT on IRI-induced apoptosis. The TUNEL assay revealed that the apoptosis rate was higher in the IR group than in the control groups (NC and Sham), and the apoptosis rate was lower in the IR-AAT group than in the IR group (Fig 6). Caspases act as initiators and effectors of apoptosis, and we measured the activity of caspase 3, which is main effector caspase. Caspase 3 activity was significantly higher in the IR
group than in the control groups (NC and Sham) and lower in the IR-AAT group than in the IR group (Fig 7A). To investigate the downstream pathway involved in IR-related apoptosis, we analyzed Bcle2 and Bax expression by western blotting. This revealed that IRI downregulated the expression of Bcle2 and upregulated the expression of Bax compared with the expression of control groups (NC and Sham). Moreover, Bcle2 and Bax levels were higher and lower, respectively, in the IR-AAT group than in the IR group (Fig 7B).
DISCUSSION
The present study shows that treatment with AAT before IRI reduced renal IR-induced injury in a mouse model. Our study focused on the mechanisms by which AAT protects renal function against IR-induced injury. Therefore, the effects of AAT on inflammation and apoptosis after renal IR were investigated. a1-Antitrypsin treatment before renal IRI reduced IR injury, as shown by the significantly lower levels of BUN, serum creatinine, NGAL, and KIMe1, fewer tubular injuries, and less interstitial fibrosis in the AAT pretreatment group compared with the IR group. With respect to the mechanisms underlying the protective effect of AAT against renal IRI, administration of AAT before renal IRI decreased inflammation, as demonstrated by the reduced expression of TGFb, ILe1b, and ILe6. In addition, apoptosis, as measured by TUNEL assay, caspase 3 activity, and the expression of Bax, was inhibited by AAT pretreatment. Based on these results, AAT protects against
a1-ANTITRYPSIN ON ISCHEMIA-REPERFUSION INJURY IR-induced kidney damage through anti-inflammatory and antiapoptotic pathways. Acute kidney injury due to renal IRI is a common clinical problem. Although BUN and serum creatinine levels are commonly measured to detect AKI, they do not permit the early diagnosis of AKI, since tubular injury precedes a significant rise in BUN and serum creatinine. Therefore, several potential biomarkers have been investigated for detecting tubular injury in AKI patients at earlier stages. Neutrophil gelatinase-associated lipocalin is upregulated and abundantly expressed in the kidney after renal IRI [11]. In renal IRI, NGAL may function to lessen toxicity by reducing apoptosis; KIMe1 is a type 1 transmembrane glycoprotein that is markedly upregulated in the proximal tubular cells in AKI [12]. In our study, we measured NGAL and KIMe1 levels as an early predictive biomarker for renal IRI. Treatment with AAT before renal IRI attenuated the levels of both NGAL and KIMe1, which were significantly increased after renal IRI. The results of our study are similar to those of a previous study [10] in which they investigated the effects of AAT monotherapy during the early and recovery phases of ischemia-induced AKI. They showed that AAT partially preserved renal function and tubular integrity after induction of bilateral kidney IR injury, which was accompanied by a significant decrease in NGAL protein levels in urine and plasma and KIMe1 protein levels in urine. However, AAT treatment did not have a significant effect on renal fibrosis. They suggested that the lack of effect of AAT on renal fibrosis could be explained by early anti-AAT antibody formation in their experimental setting. In contrast, our study showed that AAT treatment before renal IRI decreased tubular injury and interstitial fibrosis, which was confirmed by decreased collagen deposition and TGFb messenger RNA expression. We speculate the reason that we observed a substantial effect of AAT on renal remodeling is because of differences in the AAT administration period and the development of anti-AAT antibodies. In our previous study, we reported that AAT has antifibrotic effects in Madin-Darby canine kidney cells and a unilateral ureter obstruction mouse model through suppression of TGFb/Smad3 signaling [13]. We evaluated the expression of alpha-smooth muscle actin, vimentin, fibronectin, collagen I, and E-cadherin in Madin-Darby canine kidney cells and a mouse model of unilateral ureter obstruction to confirm the antifibrosis effect of AAT. Our results indicated that AAT could be a potential therapeutic agent to inhibit renal fibrosis. However, this study did not evaluate the various inflammatory cytokines that control inflammatory status, such as ILe1b, ILe6, and TNF-a. In the present study, we demonstrated that AAT not only decreases interstitial fibrosis, which is a typical characteristic of chronic inflammation, but also reduces the levels of important inflammatory cytokines, including ILe1b, ILe6, and TNF-a.
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a1-Antitrypsin is known to prevent apoptosis in lung cells and b cells by inhibiting caspase 3 activity and oxidative stress in vitro and in vivo [14,15]. Caspases act as initiators and effectors of apoptosis and are activated by the sequential cleavage of procaspases. Initiator caspases proteolytically activate effectors, such as caspase 3, which in turn cleave other intracellular proteins, eventually leading to cell death. Caspase inhibition has been shown to exert remarkable effects on organ injury by preventing apoptosis in renal IRI [16]. In addition, proapoptotic Bax and antiapoptotic Bcle2 proteins are important molecules in the shared portion of the cell death pathways, and cell viability is largely determined by the interaction between Bcle2 family proteins [17]. In our study, we showed that AAT treatment before renal IRI decreased apoptosis, as demonstrated by the reduced number of TUNEL-positive cells, reduced caspase 3 activity, increased Bcl-2 expression, and decreased Bax expression compared with the levels in untreated mice with IR. In conclusion, pretreatment with AAT before IRI protected renal function and attenuated tubular injury and interstitial fibrosis and also inhibited IRI-induced inflammation and apoptosis after renal IRI. Our results suggest that AAT has a protective effect against renal IRI by inhibiting the pathways of inflammation and apoptosis. However, further animal and clinical studies are needed to clarify the effects of AAT, to investigate the dose-dependent response of AAT, and to discover other agents that might act synergistically with AAT against renal IRI, including organ transplant.
REFERENCES [1] Denecke C, Tullius SG. Innate and adaptive immune responses subsequent to ischemia reperfusion injury in the kidney. Prog Urol 2014;24(Suppl. 1):S13e9. [2] Mehta RL, Kellum JA, Shah SV, Molitoris BA, Ronco C, Warnock DG, et al. Acute Kidney Injury Network: report of an initiative to improve outcomes in acute kidney injury. Crit Care 2007;11:R31. [3] Yellon DM, Hausenloy DJ. Myocardial reperfusion injury. N Engl J Med 2007;357:1121e35. [4] Coca SG, Singanamala S, Parikh CR. Chronic kidney disease after acute kidney injury: a systematic review and meta-analysis. Kidney Int 2012;81:442e8. [5] Chawla LS, Eggers PW, Star RA, Kimmel PL. Acute kidney injury and chronic kidney disease as interconnected syndromes. N Engl J Med 2014;371:58e66. [6] Siedlecki A, Irish W, Brennan DC. Delayed graft function in the kidney transplant. Am J Transplant 2011;11:2279e96. [7] Carell RW. The molecular structure and pathology of alpha 1-antitrypsin. Lung 1990;168(Suppl):530e4. [8] Massi G, Chiarelli C. Alpha 1-antitrypsin: molecular structure and the Pi system. Acta Paediatr 1994;393(Suppl):1e4. [9] Lewis EC. Expanding the clinical indications for alpha(1)antitrypsin therapy. Mol Med 2012;18:957e70. [10] Maicas N, van der Vlag J, Bublitz J, Florquin S, Bakker-van Bebber M, Dinarello CA, et al. Human alphae1-antitrypsin (hAAT) therapy reduces renal dysfunction and acute tubular necrosis in a murine model of bilateral kidney ischemia-reperfusion injury. PLoS One 2017;12:e0168981.
2822 [11] Mishra J, Dent C, Tarabishi R, Mitsnefes MM, Ma Q, Kelly C, et al. Neutrophil gelatinase-associated lipocalin (NGAL) as a biomarker for acute renal injury after cardiac surgery. Lancet 2005;365:1231e8. [12] Han WK, Bailly V, Abichandani R, Thadhani R, Bonventre JV. Kidney injury moleculee1 (KIMe1): a novel biomarker for human renal proximal tubule injury. Kidney Int 2002;62:237e44. [13] Cho JH, Ryu HM, Oh EJ, Yook JM, Ahn JS, Jung HY, et al. Alpha1-antitrypsin attenuates renal fibrosis by inhibiting TGFbeta1-induced epithelial mesenchymal transition. PLoS One 2016;11:e0162186.
JEONG, LIM, LEE ET AL [14] Serban KA, Petrache I. Alphae1 antitrypsin and lung cell apoptosis. Ann Am Thorac Soc 2016;13(Suppl. 2):S146e9. [15] Zhang B, Lu Y, Campbell-Thompson M, Spencer T, Wasserfall C, Atkinson M, et al. Alpha1-antitrypsin protects betacells from apoptosis. Diabetes 2007;56:1316e23. [16] Daemen MA, van ’t Veer C, Denecker G, Heemskerk VH, Wolfs TG, Clauss M, et al. Inhibition of apoptosis induced by ischemia-reperfusion prevents inflammation. J Clin invest 1999;104: 541e9. [17] Gross A, McDonnell JM, Korsmeyer SJ. BCLe2 family members and the mitochondria in apoptosis. Genes Dev 1999;13: 1899e911.
ABSTRACT Background. a1-Antitrypsin (AAT) is an important protein in the anti-inflammatory response that functions to regulate the activity of serine proteinases. We aimed to evaluate the protective effect of AAT on ischemia-reperfusion injury (IRI) in a mouse model. Methods. We investigated the effects of AAT in a C57BL/6 mouse model of IRI by dividing them into 4 groups: normal control, sham operated, ischemia-reperfusion (IR), and IR after AAT pretreatment (IR-AAT). In the IR-AAT group, mice were pretreated with AAT (80 mg/kg/d) for 3 days before renal ischemia was induced by clamping the bilateral renal vascular pedicles for 30 minutes. At 24 hours after IRI, biochemistry, histology, inflammatory cytokines, and apoptosis were assayed. Results. Blood urea nitrogen and serum creatinine levels were significantly lower in the IR-AAT group than in the IR group. Neutrophil gelatinase-associated lipocalin and kidney injury molecule 1 protein levels were significantly lower in the IR-AAT group than in the IR group. In addition, there were fewer tubular injuries and less interstitial fibrosis in the IR-AAT group than in the IR group, and the expression levels of transforming growth factor b, interleukin 1b, and interleukin 6 were significantly lower in the IR-AAT group than in the IR group. When compared with the IR group, there were fewer terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assayepositive cells, lower caspase 3 activity and B-cell lymphoma 2-associated X protein (Bax), and higher B-cell lymphoma 2 (Bcle2) in the IR-AAT group. Conclusions. a1-Antitrypsin preserved renal function, attenuated tubular injuries and interstitial fibrosis, and inhibited inflammation and apoptosis after renal IRI. Our results suggest that AAT has protective effects against renal IRI by inhibiting inflammatory and apoptosis pathways.
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ENAL ischemia-reperfusion injury (IRI) induces the innate and adaptive immune responses and causes cell apoptosis, innate autoimmunity, complement activation, platelet aggregation, and microvascular dysfunction, which lead to acute kidney injury (AKI) [1,2]. Ischemiareperfusion injury is an important determinant of high morbidity and mortality in AKI. In kidney transplant, IRI is an inevitable event. Allograft injury starts with the physiological changes associated with brain death or circulatory 0041-1345/19 https://doi.org/10.1016/j.transproceed.2019.04.084
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Funding This work was supported by the Korea Health Technology R&D Project of the Korea Health Industry Development Institute (KHIDI), which is funded by the Ministry of Health and Welfare, Republic of Korea (HI13C1232). *Address correspondence to Chan-Duck Kim, MD, PhD, Professor, Department of Internal Medicine, School of Medicine, Kyungpook National University, Kyungpook National University Hospital, 130 Dongdeok-ro, Jung-gu, Daegu 41944, South Korea. Tel: þ82-53-200-5560; Fax: þ82-53-426-9464. E-mail: [email protected] ª 2019 Elsevier Inc. All rights reserved. 230 Park Avenue, New York, NY 10169
Transplantation Proceedings, 51, 2814e2822 (2019)
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Fig 1. In vivo experimental protocol. Mice were divided into 4 groups: the normal control (NC, n ¼ 5), sham-operated (Sham, n ¼ 6), renal ischemia-reperfusion (IR, n ¼ 8), and AAT treatment before renal IR (IR-AAT, n ¼ 8) groups. Abbreviations: AAT, a1-antitrypsin; IP, intraperitoneal; IR, ischemia-reperfusion.
collapse and persists through the peri- and post-transplant periods. After removal from the donor, the allograft is maintained in ischemic conditions before reperfusion at transplant. During this time, complex pathophysiological processes continue, leading to damage after reperfusion from the proinflammatory environment. Paradoxically, reperfusion does not improve the ischemic conditions but increases the ischemic damage by activating several mechanisms, including the innate and the adaptive immune
responses and programmed cell death [3]. These complex processes can manifest clinically as delayed graft function after kidney transplant. Allograft injury due to IRI reduces the allograft survival rate in kidney transplant and increases the risk of chronic kidney disease [4e6]. A wide range of studies on substances with antiinflammatory and antiapoptotic activities have been conducted to assess their effects on the development of IRI. One of these substances, a1-antitrypsin (AAT) protects
Fig 2. Renal function and the levels of NGAL and KIM-1. (A) Blood urea nitrogen (BUN), (B) creatinine (Cr), (C) neutrophil gelatinaseassociated lipocalin (NGAL), and (D) kidney injury molecule-1 (KIM-1). Abbreviations: NC, normal control; IR, ischemia-reperfusion group. *P < .05 vs NC. yP < .01 vs NC. zP < .001 vs NC. §P < .05 vs IR. ǁP < .01 vs IR. ¶P < .001 vs IR.
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Fig 3. Periodic acid-Schiff (PAS) and Masson’s trichrome staining and collagen deposition. (A & E) the normal control (NC), (B & F) sham-operated (Sham), (C & G) IR, (D & H) IR-AAT, and (I) semiquantitative assessment of the area of renal fibrosis. Abbreviations: IR, ischemia-reperfusion group; IR-ATT, ischemia-reperfusion a1-antitrypsin group; PAS, periodic acid-Schiff. *P < .05 vs NC. yP < .01 vs NC. zP < .001 vs NC. §P < .05 vs IR. ǁP < .01 vs IR. ¶P < .001 vs IR.
tissues from the enzymes of inflammatory cells, especially neutrophil elastase and a protease released from activated neutrophils. a1-Antitrypsin inhibits elastase, which can damage tubular endothelial cells, enhance cytokine production, and express adhesion molecules [7e9]. Based on these mechanisms, the purpose of the present study was to identify the protective effect of AAT, which is known to induce an anti-inflammatory reaction by controlling the activity of serine proteinases in IRI in a mouse model of ischemic renal damage. MATERIALS AND METHODS Mice and the IRI Model For all experiments, 8-week-old male C57BL/6 mice weighing 22 to 25 g (Samtako, Osan, Korea) were used. The mice were housed under a 12-hour light/dark cycle with free access to standard chow and water. All mouse care and experimentation were performed in accordance with the guidelines approved by the Animal Care and Use Committee of Kyungpook National University (KNUe2017 e0013). Animals were anesthetized with tribromoethanol (240 mg/kg, 1.2%) during surgery. To induce ischemia, the renal pedicles were exposed through a minimal flank incision and were completely occluded for 30 minutes with a microaneurysm clamp. Following
induction of warm ischemia for 30 minutes, the artery clamp was removed to allow reperfusion. A similar surgical treatment was performed in sham-operated mice, except the renal pedicle clamping was omitted. During the operation, animals were maintained at 36.5 C to 37 C with a temperature-controlled heating device (Harvard Bioscience, Holliston, Mass, United States).
Experimental Groups Mice were randomly divided into 4 groups as follows (Fig 1): the normal control (NC, n ¼ 5), sham-operated (Sham, n ¼ 6), ischemia-reperfusion (IR, n ¼ 8), and ischemia-reperfusion after AAT treatment (IR-AAT, n ¼ 8) groups. The IR group was administered 0.9% normal saline for 3 days and then underwent renal IR. The IR-AAT group was administered AAT (Aralast, Baxter Healthcare, Vienna, Austria) intraperitoneally at 80 mg/kg/d for 3 days and then underwent renal IR. The AAT dosage used in our study was based on previous mouse studies [9,10]. All mice were euthanized by cardiac puncture under anesthesia at 24 hours after reperfusion. Blood and both kidneys were collected for analysis.
Serum Biochemical Analyses After centrifugation of each blood sample, the serum was collected. Blood urea nitrogen (BUN) and creatinine levels were measured
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Fig 4. Immunohistochemical staining and semiquantitative assessment of F4/80 and TNF-a. (A & E) the normal control (NC), (B & F) sham-operated (Sham), (C & G) IR, (D & H) IR-AAT, and (I & J) semiquantitative assessment of F4/80-positive cells and TNF-a-positive areas. Abbreviations: IR, ischemia-reperfusion group; IR-ATT, ischemia-reperfusion a1-antitrypsin group; TNF, tumor necrosis factor. * P < .05 vs NC. yP < .01 vs NC. zP < .001 vs NC. §P < .05 vs IR. ǁP < .01 vs IR. ¶P < .001 vs IR.
with a Hitachi 7600 automated biochemistry analyzer (Hitachi, Tokyo, Japan).
Quantitative Real-Time Polymerase Chain Reaction Total RNA was extracted with TRIzol reagent (Life Technologies, Carlsbad, Calif, United States) from homogenized whole kidneys according to the manufacturer’s instructions. Total RNA (1 mg) was reverse transcribed to complementary DNA with the PrimeScript complementary DNA synthesis kit (Takara, Otsu, Japan). Quantitative real-time polymerase chain reaction (PCR) was performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif, United States) using SYBR Green PCR master mix (Life Technologies). To quantify relative gene expression, the results were analyzed by the comparative Ct method. The primers used in quantitative PCR were as follows: neutrophil gelatinase-associated lipocalin (NGAL) forward 50 -GCC ACT CCA TCT TTC CTG TTGe30 and reverse 50 -GGG AGT GCT GGC CAA ATA AGe30 ; kidney injury moleculee1 (KIMe1) forward 50 CAG GGA AGC CGC AGA AAA Ae30 and reverse 50 -GAT AGC CAC GGT GCT CAC AAe30 ; interleukin (IL) 1b forward 50 -TCG TGC TGT CGG ACC CAT ATe30 and reverse 50 -GGT TCTC CTT GTA CAA AGC TCA TGe30 ; ILe6 forward 50 -CCC ACC AAG AAC GAT AGT CAA TTe30 and reverse 50 -CAC CAG CAT CAG TCC CAA GAe30 ; transforming growth factor (TGF) b forward
50 -GGC TGT GGC CAT CAA GAA TTe30 and reverse 50 - GCA GAG GGA AGA GTC AAA CAT GTe30 ; tumor necrosis factor (TNF) a forward 50 - GAC TAG CCA GGA GGG AGA ACA Ge30 and reverse 50 -CAG TGA GTG AAA GGG ACA GAA CCTe30 ; and b-actin forward 50 -ACC ACC ATG TAC CCA GGC ATTe30 and reverse 50 -CCA CAC AGA GTA CTT GCG CTC Ae30 , which were designed using Primer Express V1.5 software (Applied Biosystems).
Histologic Examination and Immunohistochemical Studies Kidney tissues were fixed with 4% paraformaldehyde (pH 7.4) and embedded in paraffin. The paraffin-embedded tissue was sliced (2 mm) and stained with periodic acid-Schiff and Masson’s trichrome using standard protocols to determine histologic changes and collagen deposition, respectively. Collagen deposition was quantified using the i-Solution DT image software (IMT i-Solution, Vancouver, British Columbia, Canada) in more than 5 randomly selected fields in sections of the cortex and medulla. For immunohistochemistry staining, the 2-mm-thick kidney sections were deparaffinized and rehydrated, and then endogenous peroxidase was inactivated with 3% hydrogen peroxide. The samples were incubated with anti-F4/80 (1:200; Serotec, Oxford, United Kingdom) and anti-TNF-a (1:100; Abcam, Cambridge, United
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Fig 5. The mRNA levels of (A) tumor necrosis factor (TNF) a, (B) transforming growth factor (TGF) b, (C) interleukin (IL) 1b, and (D) IL-6 in kidney tissue. Abbreviations: IR, ischemia-reperfusion group; mRNA, messenger RNA; NC, normal control. *P < .05 vs NC. yP < .01 vs NC. zP < .001 vs NC. §P < .05 vs IR. ǁP < .01 vs IR. ¶P < .001 vs IR. Kingdom) antibodies overnight at 4 C and then detected with the EnVision-HRP kit (Dako, Carpinteria, Calif, United States). Each section was counterstained with Mayer’s hematoxylin and examined under a Leica DM IRB inverted microscope (Leica Microsystems, Wetzlar, Germany) equipped with a CoolSNAP HQ camera (Photometrics, Tucson, Ariz, United States). To quantify F4/80-positive cells, 5 nonoverlapping fields per slide, at 400 magnification, were counted and normalized per mm2 of tissue. Tumor necrosis factor a expression was quantified as a percentage of the total area using Image J software (National Institutes of Health, Bethesda, Md, United States).
Terminal Deoxynucleotidyl Transferase-Mediated dUTP Nick End-Labeling Assay To detect apoptotic cells, the In Situ Cell Death Detection Fluorescein Kit (Roche, Mannheim, Germany) was used according to the manufacturer’s protocol. Briefly, the 2-mm-thick kidney sections were deparaffinized, rehydrated, and then treated with proteinase K. For fluorescence terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assay staining, the sections were incubated with the TUNEL reagent for 30 minutes at room temperature, washed with phosphate-buffered saline 3 times for 5 minutes, and then counterstained with 40 ,6-diamidinoe2phenylindole (Sigma-Aldrich, St. Louis, Mo, United States) for 1 minute for nuclear staining. Finally, the sections were mounted with Prolong Gold antifade reagent (Invitrogen, Carlsbad, Calif, United States) and observed under a confocal microscope (Carl Zeiss, Göttingen, Germany).
Measurement of Caspase 3 Activity Caspase 3 activity in homogenized whole kidney tissue was measured using a colorimetric assay kit (Sigma-Aldrich) according to the manufacturer’s protocol. In brief, the supernatant of kidney homogenates was incubated with a fluorometric caspase 3 substrate and Ac-DEVD-pNA in assay buffer at 37 C for 90 minutes. Caspase 3 activity was measured at 405 nm using an enzyme-linked immunosorbent assay reader. Values are expressed as the percentage of the control.
Western Blot Analysis Homogenized kidney tissue was separated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, blocked with 10% skimmed milk for 1 hour, and then incubated with primary antibodies against B-cell lymphoma 2 (Bcle2) (1:1000; Cell Signaling Technology, Danvers, Mass, United States), Bcl-2-associated X protein (Bax) (1:1000; Cell Signaling Technology), and b-actin (1:5000, Sigma-Aldrich) overnight at 4 C. After washing, the membrane was incubated with horseradish peroxidase-conjugated IgG secondary antibodies (1:2000; Dako, Glostrup, Denmark) for 1 hour and detected using the ECL Advanced Detection Kit (GE Healthcare, Little Chalfont, United Kingdom). Bands were analyzed using Scion Image software (Scion, Frederick, Md, United States), and b-actin was used as a loading control.
Statistical Analysis All data are presented as means (SEs). Experiments were repeated at least 3 times independently. All statistical analyses were
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Fig 6. TUNEL assay to detect apoptosis in renal cells and counterstaining of nuclei with DAPI. The normal control (NC), sham-operated (Sham), renal ischemia-reperfusion (IR), and AAT treatment before renal ischemia-reperfusion (IR-AAT) groups. Abbreviations: DAPI, 4’,6-diamidino-2-phenylindole; TUNEL, terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling.
performed by 1-way analysis of variance with Tukey’s post hoc test using GraphPad Prism 5.01 software (GraphPad Software, La Jolla, Calif, United States). A P value less than .05 was considered statistically significant.
lower levels of both NGAL and KIMe1 than in the IR group (Fig 2).
a1-Antitrypsin Pretreatment Reduced IRI-Induced Renal Injury
RESULTS a1-Antitrypsin Pretreatment Reduced the IRI-Induced Increase in Serum BUN, Creatinine, NGAL, and KIMe1 Levels
The IR group showed significantly higher BUN and creatinine levels than the control groups (NC and Sham). However, BUN and creatinine levels were significantly lower in the IR-AAT group than in the IR group. The NGAL and KIMe1 levels, which have been identified as early predictive biomarkers for AKI, were significantly higher in the IR group than in the control groups (NC and Sham). However, the IR-AAT group showed significantly
Periodic acid-Schiff and Masson’s trichrome staining were carried out to investigate the histologic changes and collagen deposition in the kidneys. Periodic acid-Schiff and Masson’s trichrome staining showed that there was little tubular injury and interstitial fibrosis in the control groups (NC, Fig 3A, E and Sham, Fig 3B, F). After IRI, collagen deposition in the renal interstitium and tubular damage, such as cast formation and brush border loss, were significantly increased in the IR group compared with the control groups (NC and Sham; Fig 3C, G, I). However, AAT pretreatment (IR-AAT group) reduced IRI-induced renal injury (Fig 3D, H, I).
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Fig 7. Caspase 3 activity and Bcl-2/Bax protein expression. (A) Caspase-3 activity was measured by enzyme-linked immunosorbent assay, and (B) renal expression of Bcl-2 and Bax were measured by western blotting. The expression of Bcl-2 and Bax are shown in representative western blots. The density of the western blots was quantified with Scion image software and normalized to b-actin. The normal control (NC), sham operated (Sham), renal ischemia-reperfusion (IR), and AAT treatment before renal IR (IR-AAT) group. Abbreviations: Bax, B-cell leukemia/lymphoma 2-associated X protein; Bcl-2, B-cell chronic lymphocytic leukemia/lymphoma. * P < .05 vs NC. yP < .01 vs NC. zP < .001 vs NC. §P < .05 vs IR. ǁP < .01 vs IR. ¶P < .001 vs IR.
a1-Antitrypsin Pretreatment Reduced the Increased Expression of Inflammatory Markers Following Renal IRI
F4/80 is a macrophage marker that is required for the induction of the efferent CD8þ regulatory T cells required for peripheral tolerance, and TNF-a is a cytokine that is associated with systemic inflammation. The expression levels of F4/80 and TNF-a were higher in the IR group (Fig 4C, G) than in the control groups (NC, Fig 4A, E and Sham, Fig 4B, F). However, the expression levels of F4/80 and TNF-a were significantly lower in the IR-AAT group than in the IR group (Fig 4D, H-J). Quantitative PCR was used to measure the messenger RNA expression levels of inflammatory cytokines, including TNF-a, TGFb, ILe1b, and ILe6. After IRI, the expression levels of TNF-a, TGFb, ILe1b, and ILe6 were significantly higher than those in the control groups (NC and Sham). With AAT pretreatment, the expression levels of TGFb, ILe1b, and ILe6 were significantly lower than those in the IR group. a1-Antitrypsin pretreatment before IRI also showed a tendency to reduce the upregulation of TNF-a expression in the IR group (Fig 5).
a1-Antitrypsin Pretreatment Inhibits IRI-Induced Apoptosis We used the TUNEL assay to determine the effect of AAT on IRI-induced apoptosis. The TUNEL assay revealed that the apoptosis rate was higher in the IR group than in the control groups (NC and Sham), and the apoptosis rate was lower in the IR-AAT group than in the IR group (Fig 6). Caspases act as initiators and effectors of apoptosis, and we measured the activity of caspase 3, which is main effector caspase. Caspase 3 activity was significantly higher in the IR
group than in the control groups (NC and Sham) and lower in the IR-AAT group than in the IR group (Fig 7A). To investigate the downstream pathway involved in IR-related apoptosis, we analyzed Bcle2 and Bax expression by western blotting. This revealed that IRI downregulated the expression of Bcle2 and upregulated the expression of Bax compared with the expression of control groups (NC and Sham). Moreover, Bcle2 and Bax levels were higher and lower, respectively, in the IR-AAT group than in the IR group (Fig 7B).
DISCUSSION
The present study shows that treatment with AAT before IRI reduced renal IR-induced injury in a mouse model. Our study focused on the mechanisms by which AAT protects renal function against IR-induced injury. Therefore, the effects of AAT on inflammation and apoptosis after renal IR were investigated. a1-Antitrypsin treatment before renal IRI reduced IR injury, as shown by the significantly lower levels of BUN, serum creatinine, NGAL, and KIMe1, fewer tubular injuries, and less interstitial fibrosis in the AAT pretreatment group compared with the IR group. With respect to the mechanisms underlying the protective effect of AAT against renal IRI, administration of AAT before renal IRI decreased inflammation, as demonstrated by the reduced expression of TGFb, ILe1b, and ILe6. In addition, apoptosis, as measured by TUNEL assay, caspase 3 activity, and the expression of Bax, was inhibited by AAT pretreatment. Based on these results, AAT protects against
a1-ANTITRYPSIN ON ISCHEMIA-REPERFUSION INJURY IR-induced kidney damage through anti-inflammatory and antiapoptotic pathways. Acute kidney injury due to renal IRI is a common clinical problem. Although BUN and serum creatinine levels are commonly measured to detect AKI, they do not permit the early diagnosis of AKI, since tubular injury precedes a significant rise in BUN and serum creatinine. Therefore, several potential biomarkers have been investigated for detecting tubular injury in AKI patients at earlier stages. Neutrophil gelatinase-associated lipocalin is upregulated and abundantly expressed in the kidney after renal IRI [11]. In renal IRI, NGAL may function to lessen toxicity by reducing apoptosis; KIMe1 is a type 1 transmembrane glycoprotein that is markedly upregulated in the proximal tubular cells in AKI [12]. In our study, we measured NGAL and KIMe1 levels as an early predictive biomarker for renal IRI. Treatment with AAT before renal IRI attenuated the levels of both NGAL and KIMe1, which were significantly increased after renal IRI. The results of our study are similar to those of a previous study [10] in which they investigated the effects of AAT monotherapy during the early and recovery phases of ischemia-induced AKI. They showed that AAT partially preserved renal function and tubular integrity after induction of bilateral kidney IR injury, which was accompanied by a significant decrease in NGAL protein levels in urine and plasma and KIMe1 protein levels in urine. However, AAT treatment did not have a significant effect on renal fibrosis. They suggested that the lack of effect of AAT on renal fibrosis could be explained by early anti-AAT antibody formation in their experimental setting. In contrast, our study showed that AAT treatment before renal IRI decreased tubular injury and interstitial fibrosis, which was confirmed by decreased collagen deposition and TGFb messenger RNA expression. We speculate the reason that we observed a substantial effect of AAT on renal remodeling is because of differences in the AAT administration period and the development of anti-AAT antibodies. In our previous study, we reported that AAT has antifibrotic effects in Madin-Darby canine kidney cells and a unilateral ureter obstruction mouse model through suppression of TGFb/Smad3 signaling [13]. We evaluated the expression of alpha-smooth muscle actin, vimentin, fibronectin, collagen I, and E-cadherin in Madin-Darby canine kidney cells and a mouse model of unilateral ureter obstruction to confirm the antifibrosis effect of AAT. Our results indicated that AAT could be a potential therapeutic agent to inhibit renal fibrosis. However, this study did not evaluate the various inflammatory cytokines that control inflammatory status, such as ILe1b, ILe6, and TNF-a. In the present study, we demonstrated that AAT not only decreases interstitial fibrosis, which is a typical characteristic of chronic inflammation, but also reduces the levels of important inflammatory cytokines, including ILe1b, ILe6, and TNF-a.
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a1-Antitrypsin is known to prevent apoptosis in lung cells and b cells by inhibiting caspase 3 activity and oxidative stress in vitro and in vivo [14,15]. Caspases act as initiators and effectors of apoptosis and are activated by the sequential cleavage of procaspases. Initiator caspases proteolytically activate effectors, such as caspase 3, which in turn cleave other intracellular proteins, eventually leading to cell death. Caspase inhibition has been shown to exert remarkable effects on organ injury by preventing apoptosis in renal IRI [16]. In addition, proapoptotic Bax and antiapoptotic Bcle2 proteins are important molecules in the shared portion of the cell death pathways, and cell viability is largely determined by the interaction between Bcle2 family proteins [17]. In our study, we showed that AAT treatment before renal IRI decreased apoptosis, as demonstrated by the reduced number of TUNEL-positive cells, reduced caspase 3 activity, increased Bcl-2 expression, and decreased Bax expression compared with the levels in untreated mice with IR. In conclusion, pretreatment with AAT before IRI protected renal function and attenuated tubular injury and interstitial fibrosis and also inhibited IRI-induced inflammation and apoptosis after renal IRI. Our results suggest that AAT has a protective effect against renal IRI by inhibiting the pathways of inflammation and apoptosis. However, further animal and clinical studies are needed to clarify the effects of AAT, to investigate the dose-dependent response of AAT, and to discover other agents that might act synergistically with AAT against renal IRI, including organ transplant.
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