Renoprotective Effect of S14G-Humanin on Renal Ischemia/Reperfusion Injury by Activation of STAT3 and ERK 1/2 Signal Transduction Pathways in Rats

Renoprotective Effect of S14G-Humanin on Renal Ischemia/Reperfusion Injury by Activation of STAT3 and ERK 1/2 Signal Transduction Pathways in Rats | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Renoprotective Effect of S14G-Humanin on Renal Ischemia/Reperfusion Injury by Activation of STAT3 and ERK 1/2 Signal Transduction Pathways in Rats Leyla Abueid, Ahmet Faruk Torun, Ezgi Golal, Nuray Acar, Filiz Basralı This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9040130/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Renal ischemia/reperfusion (I/R) injury leads to acute tubular necrosis and renal failure, triggering pathological mechanisms including inflammation, reactive oxygen species generation, apoptosis, and mitochondrial dysfunction. The mitochondrial peptide Humanin (HN), known to possess anti-apoptotic and anti-inflammatory properties, has been shown to counteract oxidative stress and restore mitochondrial function. This study aimed to investigate the effects of HN on renal I/R injury. Sprague-Dawley male rats were divided into four groups (n = 48): 1.Sham, 2.I/R, 3.HN-Sham, 4.HN-I/R. In I/R groups, renal artery ligation was performed for 45 minutes followed by 24-hour reperfusion. Humanin G (HNG) (2 mg/kg, iv) was administered 10 minutes before reperfusion. Urine was collected during reperfusion, and the experiment was terminated by collecting blood and tissue samples. Blood urea nitrogen and serum creatinine levels were elevated in the I/R group and were not affected by HNG treatment. Glutathione levels as well as superoxide dismutase activities, which were diminished in the I/R group, were significantly restored following HNG administration. Myeloperoxidase activity and malondialdehyde levels were significantly decreased in HN-I/R group compared to the I/R group. ATP levels and mitochondrial Complex I activity were significantly increased in the HN-I/R group compared to I/R. The percentage of apoptotic cells, markedly increased in I/R, was significantly reduced in HN-I/R. STAT3 and ERK 1/2 phosphorylation also increased in HN-I/R rats compared to I/R animals. HNG exerts a protective effect against renal I/R injury by attenuating oxidative stress, inflammation, and apoptosis while enhancing antioxidant capacity and mitochondrial function, through STAT3 and/or ERK 1/2 activation. Humanin renal ischemia reperfusion oxidative stress mitochondrial dysfunction Figures Figure 1 Figure 2 Figure 3 Figure 4 Introduction Renal ischemia-reperfusion (I/R) injury, which may occur in conditions such as hemorrhage, shock, sepsis, or transplantation, can lead to acute kidney failure, which is associated with high morbidity and mortality rates [ 9 , 52 , 51 , 36 ]. I/R injury is a process that begins during the ischemic period and continues to increase during reperfusion [ 17 ]. Renal ischemia damages tubular endothelial and epithelial cells, and, if severe, can result in apoptosis and necrosis [ 74 , 77 , 4 , 50 , 54 , 27 ]. During reperfusion, oxygen is resupplied, but this also causes increased formation of reactive oxygen species (ROS) [ 31 , 43 ]. Increased ROS production exacerbates tissue damage through leukocyte activation and migration, lipid peroxidation of membrane lipids, and oxidative damage to intracellular structures, thereby worsening the degree of apoptosis and necrosis [ 64 , 42 , 32 , 41 ]. Mitochondria play a crucial role in ATP synthesis, energy metabolism, regulation of apoptosis, and Ca 2+ homeostasis [ 5 ]. Mitochondrial dysfunction observed in renal I/R injury is characterized by mitochondrial swelling, expansion of cristae and intermembraneus spaces, and a reduction in mitochondrial membrane potential [ 62 , 75 , 61 ]. Damaged mitochondria during I/R inhibit the activity of the electron transport chain (ETZ) enzymes, including complex I, complex II, and ATP synthase, resulting in significant electron leakage, hence the increased ROS production [ 48 , 60 ]. It is well known that the balance between the oxidant and antioxidant systems is maintained under physiological conditions, and this balance is disrupted in I/R injury due to both increased ROS production and a weakened antioxidant system [ 31 , 49 ]. Various antioxidant agents, such as catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH), have been shown to mitigate ROS-mediated injury during renal I/R. Evidence from experimental kidney I/R models indicates that oxidative stress–related tissue damage is strongly linked to diminished GSH levels. [ 4 , 58 , 55 , 39 ]. HN is a mitochondria-derived peptide identified first in amyloid-beta plaques of Alzheimer's patients [ 63 , 25 , 24 ]. HN is known to improve mitochondrial functions, enhance the antioxidant system, and have an anti-apoptotic effect [ 59 , 12 , 21 ]. It has been shown that HN exhibits protective and therapeutic effects against I/R injury in cerebral, myocardial, and intestinal tissues [ 1 , 38 , 81 , 68 ]. Additionally, it has been demonstrated that HN exerts its cytoprotective and anti-apoptotic effects under cellular stress conditions in various cell types [ 23 , 82 ]. Although the intracellular signaling mechanisms underlying the effects of HN have not yet been fully elucidated, current evidence highlights the signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK 1/2) pathways as key mediators of its cytoprotective and anti-apoptotic effects [ 18 , 34 , 26 ]. Various preconditioning and postconditioning interventions that attenuate I/R injury have been shown to activate both STAT3 and ERK1/2 signaling pathways. During reperfusion, levels of proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) rise rapidly, triggering downstream signaling cascades involving pathways such as JAK/STAT3 and ERK1/2 [ 78 , 72 ] ERK1/2, a member of the MAPK pathway, is involved in many cellular processes including cell proliferation, survival, differentiation, and apoptosis, can be activated by ROS and pro-inflammatory mediators, especially during reperfusion [ 79 ]. Although overstimulation of ERK1/2 signaling can contribute to inflammation, a certain level of ERK1/2 activation is crucial for the induction of cell survival, proliferation, and repair signaling during I/R injury, as it is known to be involved in reperfusion injury salvage kinase (RISK) signaling [ 79 , 78 ]. On the other hand, STAT3 activation in intrinsic protective pathways (such as the SAFE pathway) has been associated with improved mitochondrial function and reduced infarct size in cardiac I/R injury models [ 28 ]. This coordinated activation appears to represent an adaptive compensatory response that limits apoptosis, preserves mitochondrial function, and restrains excessive inflammatory signaling [ 15 , 78 ]. A potent humanin analogue, S14G-humanin (HNG), is generated by replacement of Ser14 with glycine, and this substitution potently increases humanin activity [ 25 , 24 ]. The present study used HNG to evaluate its potential protective or therapeutic effects against renal I/R injury in vivo and to identify the potential mechanism/s underlying this protection. Although there are few studies examining the effects of HNG in experimental chronic renal failure models, its acute effects on renal I/R injury, which is accepted as an experimental model of acute kidney injury (AKI) has not yet been investigated. In the present study, we aimed to investigate the effects of acute HNG administration and the underlying mechanisms in a rat model of AKI induced by renal I/R. Material and Methods Animals and Grouping Forty-eight male Sprague-Dawley rats aged 8–10 week, weighing 200–250 g were used in this study. The animals were housed in stainless steel cages with ad libitum access to food and water. They were maintained under a 12-hour light/dark cycle in a controlled environment with a temperature of 22 ± 2°C and relative humidity ranging from 60% to 70%. All experimental procedures involving rats were conducted in accordance with the guidelines established by the Animal Care and Usage Committee of Akdeniz University (Approval No: 2020.08.004-2024.04.007). Rats were randomly divided into four groups: Sham (n = 12), Ischemia/Reperfusion (I/R; n = 12), Humanin-Sham (HN-Sham; n = 12), and HN-I/R (n = 12). The Sham groups did not undergo I/R procedures, whereas the IR groups were subjected to 45 minutes of bilateral renal ischemia followed by a 24-hour reperfusion period. Induction of Renal I/R Injury All rats were anesthetized with intraperitoneal (ip) ketamine/xylazine (90/10 mg/kg). The tail vein of each rat was cannulated with a 24-G cannula for injections. In the sham groups, a midline abdominal incision was made to expose the renal artery and vein without applying the I/R protocol [ 77 ]. In the I/R groups, renal blood flow was interrupted by clamping the renal pedicle with a clamp for 45 min to establish the renal I/R injury model. Upon completion of the ischemic period, the clamps were removed to initiate reperfusion, and the animals were subjected to a 24-hour reperfusion period. In the HN-Sham and HN-I/R groups, the HN analog (HNG) was administered intravenously via the tail vein at a dose of 2 mg/kg, 10 minutes before the reperfusion period. During the reperfusion period, the rats were housed in metabolic cages, and urine samples were collected over 24 hours [ 77 ]. At the end of reperfusion, the animals were euthanized, blood samples and kidney tissues were collected for further analysis. HNG; [S14G]-humanin (amino acid sequence: Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Gly-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Ala) was purchased from GenScript Biotech Corporation and used as a HN analog. Assessment of Renal Function Blood samples (0.5 ml) were collected and centrifuged to obtain supernatants. Serum creatinine (sCr) and blood urea nitrogen (BUN) levels were assessed using an autoanalyzer (Brand: ROCHE COBAS, Model: C502) at the Central Laboratory of Akdeniz University Hospital. Creatinine clearance (ClCr) as an indicator of glomerular filtration rate (GFR) was calculated using the following formula [ 71 ]. ClCr (ml/min) = Urine (u) Cr (mg/dl) × urine flow (ml/min) / serum (s) Cr (mg/dl) Histopathological Assessment The formalin-fixed and paraffin-embedded kidney was cut into 5 µm sections and stained with Haematoxylin and Eosin (H&E) for histological assessment. Histological injury scores were determined by assessing tubular necrosis, loss of the brush border, tubular cast formation, and tubular dilation as described previously [ 13 , 70 ]. The histological damage scores were counted in a blind study by two observers. Apoptosis Assessment Apoptotic cells in rat kidney tissue were evaluated using a Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay Kit (Abcam, Catalog No: ab206386) in accordance with the manufacturer’s instructions. TUNEL-stained sections were evaluated using a light microscope (Zeiss, Oberkochen, Germany) with a special ocular scale. Five randomly selected slides, each with four different fields at 10x10 magnification, were evaluated for the analysis of TUNEL. Positively stained cell numbers in the fields were counted in a blind study by two observers. Mean positive cell numbers were calculated and then were rated to 100. Positively stained apoptotic cell numbers (apoptotic index) in the tissue were indicated as percent (%). Western Blot Analysis The protein concentration of each sample was determined using a BCA assay kit. Equal amounts of protein (20 ug/ml) were loaded into each well and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V and 30 mA for 2 hours. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane using a transfer system (Trans-Blot Turbo, Bio-Rad) at 25 V and 1.3 A for 10 minutes [ 20 ]. The membranes were incubated overnight with primary antibodies for p-STAT3 (Cat. No: 9145T, Cell Signaling) at 1:1000 dilution, p-ERK1/2 (Cat. No: 4370T, Cell Signaling) at 1:1000 dilution, and beta-actin. A Goat anti-rabbit IgG conjugated secondary antibody was used in accordance with the primary antibodies. The membranes were incubated with a chemiluminescent substrate for 5 minutes, and the bands were visualized using an Azure C280 imaging system. Proteins on the p-STAT3 and p-ERK1/2 membranes were then incubated overnight with the beta-actin antibody (4970, Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:5000, served as an internal control. The obtained results were analyzed using Image J software (Image J Version 1st36b; National Institutes of Health, MD, USA) and protein expression was quantified by normalizing the target protein to the reference protein. Assessments of Mitochondrial Functions Intracellular ATP Levels ATP levels in tissue homogenates were quantified using an ATP Assay Kit (Elabscience, Catalog No: E-BC-K157-S) following the manufacturer's instructions and expressed as ATP content per gram of tissue. Complex I Activities Complex I activity in kidney tissue homogenates was determined using a Complex I Activity Assay Kit (Abcam, Catalog No: ab109721), following the manufacturer's protocol. Results were expressed as enzyme activity per milligram of tissue. Assessments of Oxidative Stress Malondialdehyde (MDA) levels, which are indicators of lipid peroxidation, were determined in kidney tissue homogenate by the reaction of MDA with thiobarbituric acid (TBA) (Sigma-Aldrich, T5500) and detected using spectrophotometric methods as described previously [ 11 ]. MDA levels were expressed as nmol MDA/g tissue. Assessments of Antioxidant Status GSH levels, SOD and CAT activity were examined as antioxidant parameters in kidney tissue homogenates. The level of endogenous antioxidant GSH in tissue was measured by modifying the Ellman method, which detects sulfhydryl groups [ 16 , 3 ]. Absorbances were measured on a Shimadzu UV-1601 spectrophotometer (at 412 nm), and the calculated GSH level was expressed as µmol/g tissue. SOD activity was determined using a previously described method [ 44 ]. The superoxide radical generated by riboflavin under fluorescent light (20 W) is converted to hydrogen peroxide (H 2 O 2 ) by SOD and subsequently H 2 O 2 is detected using o-dianisidine hydrochloride. SOD activity was calculated from the difference between reaction absorbance and basal absorbance at 460 nm in kidney tissue and was expressed as U/g tissue [ 47 ]. CAT activity in kidney tissue homogenates was determined using spectrophotometric methods as previously described by Aebi [ 2 ]. Absorbance was measured at 240 nm using a spectrophotometer (Shimadzu UV-1601). CAT activity was calculated based on an extinction coefficient of 0.004 mM⁻¹·mm⁻¹ and expressed as units per gram of tissue (U/g tissue). Assessments of Inflammation Kidney tissue Myeloperoxidase (MPO) activity was evaluated as an indicator of inflammation. MPO activity was determined through the reaction of H 2 O 2 oxidation with o-dianisidine HCl [ 10 ]. Absorbance changes due to the reaction were measured at 460 nm using a spectrophotometer (Shimadzu UV-1601). MPO activity was expressed as units per gram of tissue (U/g tissue). Statistical Analysis Data are presented as mean ± S.E.M. Significant differences were determined by one-way ANOVA followed by Tukey’s post hoc test, and Kruskal–Wallis test followed by Dunn’s multiple comparison post hoc test using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA). Values of < 0.05 were considered statistically significant. Results Effects of HNG Treatment on Renal Functions As shown in Table 1, rats in I/R groups displayed significant deterioration of renal function 24 h after reperfusion, as reflected by increased levels of sCr and BUN with decreased ClCr (an indicator of GFR) compared to those in sham groups. Table 1 Effects of HNG Treatment on Renal Functions Data are presented as mean ± S.E.M. ***p < 0.001 vs. Sham; **p < 0.01 vs. Sham. I/R, ischemia/reperfusion; HN, humanin; ClCr, creatinine clearance; sCr, serum creatinine; BUN, blood urea nitrogen. Effects of HNG Treatment on Histopathological Damage Renal tissues were examined after H&E staining (Figure 1a). Structural changes in the tubules, such as tubular necrosis, atrophy, dilation in the tubules and Bowman’s capsule, loss of the brush border, and cast formation, were observed and histopathologically scored (Figure 1b). The histopathological damage score in the I/R group was significantly higher compared to the Sham group (p<0.001). The histopathological damage score in the HN-I/R group was also significantly higher compared to the HN-Sham group (p<0.01). The damage observed in the HN-I/R group was not significantly different from that in the I/R group. Effects of HNG Treatment on Apoptosis Apoptotic cells in renal tissues were detected immunohistochemically using the TUNEL kit (Figure 1b). Apoptotic cells were counted, and the percentage of apoptotic cells was calculated (Figure 1d). The percentage of apoptotic cells in the I/R group was significantly higher compared to the Sham group (p<0.001). The apoptotic cell density in the HN-I/R group was significantly lower compared to the I/R group (p<0.01). Effects of HNG Treatment on Mitochondrial Functions To evaluate mitochondrial functions, intracellular ATP levels in renal tissue homogenates were measured (Figure 2a). The ATP levels in the I/R group were found to be significantly lower compared to the Sham group (p<0.001). Tissue ATP levels measured in the HN-I/R group were significantly elevated compared to the I/R group (p<0.05). As an indicator of mitochondrial function, Complex I activity levels were measured in renal tissue homogenates (Figure 2b). The Complex I activity in the I/R group was significantly lower compared to the Sham group (p<0.01). However, the Complex I activity levels in the HN-I/R group were significantly higher compared to the I/R group (p<0.05). Effects of HNG Treatment on Oxidative Stress and Antioxidant Status Levels of MDA, which is the product of lipid peroxidation in renal tissue homogenates, are presented in Figure 3a. MDA levels were significantly higher in the I/R group compared to the Sham group (p<0.001). In the HN-I/R group, MDA levels were not significantly different from those in the HN-Sham group. However, tissue MDA levels were significantly lower in the I/R group treated with HNG compared to the untreated group (p<0.05). Renal tissue GSH levels as shown in Figure 3b were significantly reduced in the I/R group compared to the Sham group (p < 0.001). In the HN-I/R group, GSH levels did not differ significantly from those in the HN-Sham group. However, renal GSH levels in the HN-I/R group were significantly higher than those observed in the I/R group. (p<0.01). SOD activity, the most effective endogenous antioxidant enzyme in cellular defense, was measured as an indicator of antioxidant capacity in renal tissue homogenates (Figure 3c). SOD activity in the I/R group was significantly lower compared to the Sham group (p<0.05). In the HN-I/R group, SOD activity was not significantly different from that in the HN-Sham group. However, renal SOD activity in the HN-I/R group treated with HNG prior to reperfusion was significantly higher compared to the I/R group (p<0.05). CAT activity was measured as an antioxidant marker in renal tissue homogenates (Figure 3d). CAT activity in the I/R group was significantly lower compared to the Sham group (p<0.001). Similarly, CAT activity in the HN-I/R group was significantly lower compared to the HN-Sham group (p<0.001). No significant difference was found in CAT activity between the HN-I/R group and the I/R group. Effects of HNG Treatment on Inflammation MPO, as a marker of neutrophil infiltration into the damaged tissue, was assessed to evaluate the inflammatory response, was significantly elevated in the I/R group compared to the Sham group (p<0.001) (Figure 3e). Similarly, MPO activity in the HN-I/R group was significantly higher compared to the HN-Sham group (p<0.001). Moreover, MPO activity in the HN-I/R group was significantly reduced compared to the I/R group, indicating a potential attenuation of inflammatory response (p<0.05). Effects of HNG Treatment on ERK1/2 and STAT3 Phosphorylation Western blot analyses demonstrated significant alterations in ERK1/2 and STAT3 signaling among the experimental groups (Figure 4 a,c). ERK1/2 phosphorylation was increased in the I/R group compared to the Sham group; however, this difference was not statistically significant. In contrast, Phosphorylated ERK 1/2 (pERK1/2) levels were significantly higher in the HN-I/R group compared with both the HN-Sham and I/R groups (p < 0.001). Similarly, Phosphorylated STAT3 (pSTAT3) expression was significantly increased in the I/R group compared to the Sham group (p < 0.01). pSTAT3 levels in the HN-I/R group were markedly elevated compared with both the I/R and HN-Sham groups (p < 0.001) (Figure 4b,d). Beta-actin expression did not differ among groups, confirming equal protein loading. These results indicate that HNG treatment is associated with increased activation of ERK1/2 and STAT3 signaling pathways in renal tissue following I/R injury. Discussion Renal I/R injury, frequently arises from kidney transplantation hemorrhage and nephrectomy, remains a significant unresolved challenge in clinical practice. Renal I/R injury is one of the main causes of AKI which leads to disease with high mortality and morbidity [4,9].During I/R injury, tissue damage initiated during the ischemic phase persists and further exacerbates upon reperfusion [56,32]. The main mechanism of renal IR injury is associated with multiple mediators including oxidative stress, inflammation, and mitochondrial dysfunction activation of adhesion molecules, ultimately resulting in renal tubular damage and apoptosis [69,8,61,75]. At present, strategies targeting apoptosis and oxidative stress may contribute to maintaining renal function and reducing tubular injury [27,22]. In the present study, we aimed to investigate the effects of HN on renal I/R injury and to elucidate the potential mechanisms involved, using HNG as an HN analog. To the best of our knowledge, this is the first study to examine the effects of HNG administration on renal I/R injury. We found that HNG administration attenuated the pathological alteration in renal I/R injury, which was accompanied with reduced inflammation, oxidative stress and apoptosis. Additionally, HNG preserved mitochondrial functions, increased Complex I activity and elevated ATP levels. These findings demonstrate that HNG confers protective effects against renal I/R injury in rats. Furthermore, our data suggest that HNG-mediated renoprotection may involve STAT3 and ERK 1/2 signaling pathways. In I/R-induced AKI, the decline in GFR is accompanied by an increase in circulating nitrogenous waste products, ultimately resulting in impaired renal function [53,43]. Although our findings confirmed the development of renal dysfunction following I/R injury, they also indicated that HNG treatment did not produce a significant improvement in reduced GFR or in the elevated sCr and BUN levels, which represent key markers of impaired renal function. This outcome may be attributed to several factors, including the administration of HNG immediately before reperfusion rather than during the ischemic phase, its use as a single dose, and the absence of repeated dosing. In our study, renal tissue injury was assessed through histopathological examination and apoptosis. It is well known that necrosis is the primary pathway of cell death during ischemia in renal I/R injury [36,73]. The tubular necrosis parameters, which were elevated in the I/R group, did not show a significant reduction following HNG treatment in our study. Several previous studies conducted in different tissues have demonstrated that administration of HNG either prior to or during ischemia leads to a reduction in necrosis parameters [68]. We believe that the most plausible explanation for the discrepancy between our findings and those reported in the literature is the timing of HNG administration. In our study, HNG was administered 10 minutes prior to reperfusion to specifically investigate its effects on reperfusion-induced injury [46]. In contrast, the aforementioned studies utilized HNG as a preconditioning agent administered before the onset of ischemia, thereby focusing on its protective role against ischemia-induced necrosis [68,1]. Accumulating evidence has suggested that apoptosis is critically involved in the pathological process of renal I/R injury [75,27,54]. The present study also demonstrated that I/R caused a significant increase in TUNEL-positive tubular cells, indicating the occurrence of apoptosis. Moreover, the findings of this present study revealed that HNG treatment prior to reperfusion significantly reduced the apoptosis in renal tubular cells. Anti-apoptotic effects of HN have been demonstrated in numerous in vitro and in vivo studies, including I/R injury models [21,29,66,1]. Previous investigation has indicated that HN inhibits apoptosis by increasing the expression of the anti-apoptotic protein Bcl-2 and decreasing the expression of the pro-apoptotic proteins Bax and suppression of caspase-3 activation in I/R injury [21,29,1]. pSTAT3 has been demonstrated to play a direct anti-apoptotic role in I/R injury by increasing the expression of the anti-apoptotic protein Bcl-2 and decreasing the expression of the pro-apoptotic proteins Bax and suppression of caspase-3 activation [80]. In light of these findings, it could be argued that anti-apoptotic properties of HNG is dependant on STAT3 and/or ERK 1/2 phosphorylation , consistent with our results. Disruption of cellular energy homeostasis, particularly due to compromised mitochondrial activity, represents a central mechanism in the pathogenesis of renal I/R injury [61,5]. It is well established that renal I/R injury induces mitochondrial dysfunction, including a reduction in ETC enzymes activity and ATP production [30,40]. This phenomenon plays a crucial role in I/R induced damage in tubular epithelial cells, which are characterized by increased mitochondrial density. Previous studies have suggested that HN increases intracellular ATP levels in various cell types including neurons, muscle cells, and pancreatic β-cells consistent with our findings [33,37,14,76]. The results of the present study also demonstrated that HN treatment restored intracellular ATP levels and Complex I activity, both of which were reduced by I/R injury. pSTAT3 has been shown to enhance ATP production in hepatic I/R injury by increasing the activity of mitochondrial respiratory chain complexes I and II [78]. In our study, the observed increase in ATP levels and complex I activity following HNG treatment may have been mediated by pSTAT3-dependent mechanisms. Increased production of ROS, which are primarily generated due to mitochondrial dysfunction during renal I/R injury significantly contribute to tissue injury [5,48,30] . MDA, as an end product of lipid peroxidation induced by ROS, can directly cause cytotoxic damage [11,19]. On the other hand, antioxidants such as SOD, CAT and GSH possess protective effects against oxidative stress [6]. It was accepted that the imbalance between oxidants and anti-oxidants can reflect the level of ROS metabolism and tissue damage [58]. In the present study, we observed a significant increase in renal MDA and decrease in renal GSH level and SOD activity in I/R rats, while HNG administration down-regulated MDA and upregulated SOD activity and GSH levels. Similarly to our findings, previous studies have suggested that HN suppresses oxidative stress while enhancing the antioxidant defense system in I/R models involving various tissues including cardiac and cerebral tissues as well as in diverse cell types [81,35] One of the proposed mechanisms underlying the anti-oxidative effects of HNG is its ability to directly modulate mitochondrial function [38]. It is proposed that HNG exerts this effect either through mitochondrial colocalization and/or via activation of the STAT3 signaling pathway [18,67] that is consistent with our results (Fugure 4b). Moreower, pSTAT3 has also been reported to inhibit the formation of the mitochondrial permeability transition pore (mPTP), thereby preserving the mitochondrial membrane potential (ΔΨm), reducing the generation of ROS, and suppressing apoptosis [7]. In the present study HNG treatment did not improve CAT activity, which was reduced by I/R injury. During renal I/R, unlike other antioxidant enzymes, both CAT activity and CAT protein synthesis are known to decrease [58]. The inability of HNG to enhance CAT activity observed in our study may be attributed to a reduction in CAT protein levels. Indeed, previous studies employing in experimental I/R models have demonstrated that HN treatment preserves SOD and glutathione peroxidase activities in intestinal and brain tissues following I/R injury. However, CAT activity was not evaluated in those investigations [81,1]. It has been verified that during renal I/R injury, inflammation is one of the most important mechanisms involved in tissue damage [69,56]. On the other hand, HN has been characterized as an anti-inflammatory agent due to its suppressive effects on pro-inflammatory cytokines [81,57]. It has been shown that HN reduces age-related neuroinflammation and suppresses pro-inflammatory cytokines in an intestinal I/R model, in diabetic nephropathy and traumatic brain injury [1,45,65]. In our study, to investigate the inflammatory response associated with renal I/R injury, we assessed MPO activity as a marker of neutrophil infiltration into kidney tissues. Consistent with previous findings, our results also demonstrated that HNG treatment significantly attenuated I/R-induced inflammation in renal tissue. In conclusion, to the best of our knowledge, the present study demonstrated for the first time that HNG has renoprotective effects against AKI induced by renal I/R injury. According to the findings of our study, HNG exerts renoprotective effects, at least partly, by promoting STAT3 and/or ERK 1/2 activation, thereby enhancing antioxidant, anti-apoptotic, and anti-inflammatory responses, as well as regulating mitochondrial functions. These results suggesting that HNG may be a promising agent in the management of AKI. Declarations Acknowledgements We would like to thank the Scientific Research Projects Commission of Akdeniz University and The Scientific and Technological Research Council of Turkey (TÜBİTAK) for their support and funding to this research. Additionally, we would like to express our sincere gratitude to Erol Nizamoğlu for his valuable assistance in designing the methodological approach. Author contribution All authors participated in the construction of this manuscript. All authors approved the fnal version to be published. Funding This research funding provided by Scientific Research Projects Commission of Akdeniz University (Project No: TDK-2021-5549) and The Scientific and Technological Research Council of Turkey (TUBITAK 1002B Project No: 224S404). Ethics approval and consent to participate All experimental procedures involving rats were conducted in accordance with the guidelines established by the Animal Care and Usage Committee of Akdeniz University (Approval No: 2020.08.004-2024.04.007). Consent for publication All authors approved the publication of this manuscript. Competing interests The authors declare no competing interests. Data availability The corresponding author is willing to provide the data and datasets used and/or analyzed during the current study upon reasonable request. References Abozaid ER, Abdel-Kareem RH, Habib MA (2023) A novel beneficial role of humanin on intestinal apoptosis and dysmotility in a rat model of ischemia reperfusion injury. Pflugers Arch 475:655-666. doi:10.1007/s00424-023-02804-0 Aebi H (1984) [13] Catalase in vitro. In: Methods in Enzymology, vol 105. Academic Press, pp 121-126. doi:https://doi.org/10.1016/S0076-6879(84)05016-3 Aykaç G, Uysal M, Yalçin AS, Koçak-Toker N, Sivas A, Oz H (1985) The effect of chronic ethanol ingestion on hepatic lipid peroxide, glutathione, glutathione peroxidase and glutathione transferase in rats. Toxicology 36:71-76. doi:10.1016/0300-483x(85)90008-3 Basile DP, Anderson MD, Sutton TA (2012) Pathophysiology of acute kidney injury. Compr Physiol 2:1303-1353. doi:10.1002/cphy.c110041 Bhargava P, Schnellmann RG (2017) Mitochondrial energetics in the kidney. Nat Rev Nephrol 13:629-646. doi:10.1038/nrneph.2017.107 Blokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress: a review. Annals of botany 91 Spec No:179-194. doi:10.1093/aob/mcf118 Boengler K, Hilfiker-Kleiner D, Heusch G, Schulz R (2010) Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion. Basic research in cardiology 105:771-785. doi:10.1007/s00395-010-0124-1 Bonventre JV, Weinberg JM (2003) Recent advances in the pathophysiology of ischemic acute renal failure. J Am Soc Nephrol 14:2199-2210. doi:10.1097/01.asn.0000079785.13922.f6 Böhmová R, Viklický O (2001) Renal ischemia--reperfusion injury: an inescapable event affecting kidney transplantation outcome. Folia Microbiol (Praha) 46:267-276. doi:10.1007/bf02815613 Bradley PP, Priebat DA, Christensen RD, Rothstein G (1982) Measurement of Cutaneous Inflammation: Estimation of Neutrophil Content with an Enzyme Marker. Journal of Investigative Dermatology 78:206-209. doi:https://doi.org/10.1111/1523-1747.ep12506462 Buege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302-310. doi:10.1016/s0076-6879(78)52032-6 Cai H, Liu Y, Men H, Zheng YJFiE (2021) Protective mechanism of humanin against oxidative stress in aging-related cardiovascular diseases.680 Chen Y-T, Sun C-K, Lin Y-C, Chang L-T, Chen Y-L, Tsai T-H, Chung S-Y, Chua S, Kao Y-H, Yen C-H, Shao P-L, Chang K-C, Leu S, Yip H-K (2011) Adipose-Derived Mesenchymal Stem Cell Protects Kidneys against Ischemia-Reperfusion Injury through Suppressing Oxidative Stress and Inflammatory Reaction. Journal of Translational Medicine 9:51. doi:10.1186/1479-5876-9-51 Cohen A, Lerner-Yardeni J, Meridor D, Kasher R, Nathan I, Parola AH (2015) Humanin Derivatives Inhibit Necrotic Cell Death in Neurons. Mol Med 21:505-514. doi:10.2119/molmed.2015.00073 de Assis JL, Grelle GMRS, Fernandes AM, da Silva Aniceto B, Pompeu P, de Mello FV, Garrett R, Valverde RHF, Einicker-Lamas M (2024) Sphingosine 1-phosphate protective effect on human proximal tubule cells submitted to an in vitro ischemia model: the role of JAK2/STAT3. Journal of Physiology and Biochemistry 80:831-843. doi:10.1007/s13105-024-01038-7 Ellman GL (1959) Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics 82:70-77. doi:https://doi.org/10.1016/0003-9861(59)90090-6 Eltzschig HK, Eckle T (2011) Ischemia and reperfusion--from mechanism to translation. Nat Med 17:1391-1401. doi:10.1038/nm.2507 Gao G, Fan H, Zhang X, Zhang F, Wu H, Qi F, Zhao L, Li Y (2017) Neuroprotective effect of G14-humanin on global cerebral ischemia/reperfusion by activation of SOCS3 – STAT3 – MCL–1 signal transduction pathway in rats. Neurological Research 39:895-903. doi:10.1080/01616412.2017.1352187 Gaweł S, Wardas M, Niedworok E, Wardas P (2004) [Malondialdehyde (MDA) as a lipid peroxidation marker]. Wiadomosci lekarskie (Warsaw, Poland : 1960) 57:453-455 Golal E, Balci CN, Acar N (2025) Dynamic changes in sumoylation related proteins SUMO1, SENP1, and UBC9 during the peri implantation period in mice. Scientific reports 15:21497. doi:10.1038/s41598-025-07677-0 Guo B, Zhai D, Cabezas E, Welsh K, Nouraini S, Satterthwait AC, Reed JCJN (2003) Humanin peptide suppresses apoptosis by interfering with Bax activation. 423:456-461 Han F, Dou M, Wang Y, Xu C, Li Y, Ding X, Xue W, Zheng J, Tian P, Ding C (2020) Cordycepin protects renal ischemia/reperfusion injury through regulating inflammation, apoptosis, and oxidative stress. Acta biochimica et biophysica Sinica 52:125-132. doi:10.1093/abbs/gmz145 Hashimoto Y, Kurita M, Aiso S, Nishimoto I, Matsuoka MJMbotc (2009) Humanin inhibits neuronal cell death by interacting with a cytokine receptor complex or complexes involving CNTF receptor α/WSX-1/gp130. 20:2864-2873 Hashimoto Y, Niikura T, Ito Y, Sudo H, Hata M, Arakawa E, Abe Y, Kita Y, Nishimoto IJJoN (2001) Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer's disease-relevant insults. 21:9235-9245 Hashimoto Y, Niikura T, Tajima H, Yasukawa T, Sudo H, Ito Y, Kita Y, Kawasumi M, Kouyama K, Doyu MJPotNAoS (2001) A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer's disease genes and Aβ. 98:6336-6341 Hashimoto Y, Suzuki H, Aiso S, Niikura T, Nishimoto I, Matsuoka M (2005) Involvement of tyrosine kinases and STAT3 in Humanin-mediated neuroprotection. Life Sci 77:3092-3104. doi:10.1016/j.lfs.2005.03.031 Havasi A, Borkan SCJKi (2011) Apoptosis and acute kidney injury. 80:29-40 Heusch G, Musiolik J, Gedik N, Skyschally A (2011) Mitochondrial STAT3 activation and cardioprotection by ischemic postconditioning in pigs with regional myocardial ischemia/reperfusion. Circulation research 109:1302-1308. doi:10.1161/circresaha.111.255604 Ikonen M, Liu B, Hashimoto Y, Ma L, Lee K-W, Niikura T, Nishimoto I, Cohen PJPotNAoS (2003) Interaction between the Alzheimer's survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis. 100:13042-13047 Ishimoto Y, Inagi R (2015) Mitochondria: a therapeutic target in acute kidney injury. Nephrology Dialysis Transplantation 31:1062-1069. doi:10.1093/ndt/gfv317 %J Nephrology Dialysis Transplantation Johnson KJ, Weinberg JM (1993) Postischemic renal injury due to oxygen radicals. Current Opinion in Nephrology and Hypertension 2 Kanagasundaram NS (2015) Pathophysiology of ischaemic acute kidney injury. Ann Clin Biochem 52:193-205. doi:10.1177/0004563214556820 Kariya S, Hirano M, Furiya Y, Ueno S (2005) Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA. Neuropeptides 39:97-101. doi:https://doi.org/10.1016/j.npep.2004.11.004 Kim S-J, Guerrero N, Wassef G, Xiao J, Mehta HH, Cohen P, Yen KJO (2016) The mitochondrial-derived peptide humanin activates the ERK1/2, AKT, and STAT3 signaling pathways and has age-dependent signaling differences in the hippocampus. 7:46899 Klein LE, Cui L, Gong Z, Su K, Muzumdar R (2013) A humanin analog decreases oxidative stress and preserves mitochondrial integrity in cardiac myoblasts. Biochem Biophys Res Commun 440:197-203. doi:10.1016/j.bbrc.2013.08.055 Kosieradzki M, Rowiński W (2008) Ischemia/Reperfusion Injury in Kidney Transplantation: Mechanisms and Prevention. Transplantation Proceedings 40:3279-3288. doi:https://doi.org/10.1016/j.transproceed.2008.10.004 Kuliawat R, Klein L, Gong Z, Nicoletta-Gentile M, Nemkal A, Cui L, Bastie C, Su K, Huffman D, Surana M, Barzilai N, Fleischer N, Muzumdar R (2013) Potent humanin analog increases glucose-stimulated insulin secretion through enhanced metabolism in the β cell. Faseb j 27:4890-4898. doi:10.1096/fj.13-231092 Kumfu S, Charununtakorn ST, Jaiwongkam T, Chattipakorn N, Chattipakorn SC (2016) Humanin prevents brain mitochondrial dysfunction in a cardiac ischaemia-reperfusion injury model. Exp Physiol 101:697-707. doi:10.1113/ep085749 Lan TY, Dun RL, Yao DS, Wu F, Qian YL, Zhou Y, Zhan TT, Shao MH, Gao JD, Wang C (2022) Effects of resveratrol on renal ischemia-reperfusion injury: A systematic review and meta-analysis. Frontiers in nutrition 9:1064507. doi:10.3389/fnut.2022.1064507 Li L, Zhang L, Cao Y, Chen X, Gong H, Ma Y, Gui Y, Xiang T, Liu J, Huang X (2023) NDUFV1 attenuates renal ischemia-reperfusion injury by improving mitochondrial homeostasis. J Cell Mol Med 27:1341-1352. doi:10.1111/jcmm.17735 Li Z, Ludwig N, Thomas K, Mersmann S, Lehmann M, Vestweber D, Pittet JF, Gomez H, Kellum JA, Rossaint J, Zarbock A (2022) The Pathogenesis of Ischemia-Reperfusion Induced Acute Kidney Injury Depends on Renal Neutrophil Recruitment Whereas Sepsis-Induced AKI Does Not. Frontiers in immunology 13:843782. doi:10.3389/fimmu.2022.843782 Liu J, Han X, Zhou J, Leng Y (2023) Molecular Mechanisms of Ferroptosis and Their Involvement in Acute Kidney Injury. Journal of inflammation research 16:4941-4951. doi:10.2147/jir.S427505 Malek M, Nematbakhsh M (2015) Renal ischemia/reperfusion injury; from pathophysiology to treatment. J Renal Inj Prev 4:20-27. doi:10.12861/jrip.2015.06 Masayasu M, Hiroshi Y (1979) A simplified assay method of superoxide dismutase activity for clinical use. Clinica Chimica Acta 92:337-342. doi:https://doi.org/10.1016/0009-8981(79)90211-0 Moin H, Shafi R, Ishtiaq A, Liaquat A, Majeed S, Zaidi NN (2023) Effectiveness of analog of Humanin in ameliorating streptozotocin-induced diabetic nephropathy in Sprague Dawley rats. Peptides 165:171014. doi:https://doi.org/10.1016/j.peptides.2023.171014 Muzumdar RH, Huffman DM, Calvert JW, Jha S, Weinberg Y, Cui L, Nemkal A, Atzmon G, Klein L, Gundewar SJA, thrombosis,, biology v (2010) Acute humanin therapy attenuates myocardial ischemia and reperfusion injury in mice. 30:1940-1948 Mylroie AA, Collins H, Umbles C, Kyle J (1986) Erythrocyte superoxide dismutase activity and other parameters of copper status in rats ingesting lead acetate. Toxicology and Applied Pharmacology 82:512-520. doi:https://doi.org/10.1016/0041-008X(86)90286-3 Pabla N, Bajwa A (2022) Role of Mitochondrial Therapy for Ischemic-Reperfusion Injury and Acute Kidney Injury. Nephron 146:253-258. doi:10.1159/000520698 Palipoch S (2013) A review of oxidative stress in acute kidney injury: protective role of medicinal plants-derived antioxidants. Afr J Tradit Complement Altern Med 10:88-93. doi:10.4314/ajtcam.v10i4.15 Pefanis A, Ierino FL, Murphy JM, Cowan PJ (2019) Regulated necrosis in kidney ischemia-reperfusion injury. Kidney international 96:291-301. doi:https://doi.org/10.1016/j.kint.2019.02.009 Poston JT, Koyner JL (2019) Sepsis associated acute kidney injury. Bmj 364:k4891. doi:10.1136/bmj.k4891 Requião-Moura LR, Durão Junior MdS, Matos ACCd, Pacheco-Silva AJE (2015) Ischemia and reperfusion injury in renal transplantation: hemodynamic and immunological paradigms. 13:129-135 Ronco C, Bellomo R, Kellum JA (2019) Acute kidney injury. The Lancet 394:1949-1964. doi:https://doi.org/10.1016/S0140-6736(19)32563-2 Saikumar P, Venkatachalam MA Role of apoptosis in hypoxic/ischemic damage in the kidney. In: Seminars in nephrology, 2003. vol 6. Elsevier, pp 511-521 Schneider MP, Sullivan JC, Wach PF, Boesen EI, Yamamoto T, Fukai T, Harrison DG, Pollock DM, Pollock JS (2010) Protective role of extracellular superoxide dismutase in renal ischemia/reperfusion injury. Kidney international 78:374-381. doi:10.1038/ki.2010.141 Sharfuddin AA, Molitoris BA (2011) Pathophysiology of ischemic acute kidney injury. Nature Reviews Nephrology 7:189-200. doi:10.1038/nrneph.2011.16 Shi D, Zhou X, Wang H (2021) S14G-humanin (HNG) protects retinal endothelial cells from UV-B-induced NLRP3 inflammation activation through inhibiting Egr-1. Inflamm Res 70:1141-1150. doi:10.1007/s00011-021-01489-4 Singh I, Gulati S, Orak JK, Singh AK (1993) Expression of antioxidant enzymes in rat kidney during ischemia-reperfusion injury. Mol Cell Biochem 125:97-104. doi:10.1007/bf00936438 Sreekumar PG, Ishikawa K, Spee C, Mehta HH, Wan J, Yen K, Cohen P, Kannan R, Hinton DRJIo, science v (2016) The mitochondrial-derived peptide humanin protects RPE cells from oxidative stress, senescence, and mitochondrial dysfunction. 57:1238-1253 Stanley WC (2004) Myocardial Energy Metabolism During Ischemia and the Mechanisms of Metabolic Therapies. 9:S31-S45. doi:10.1177/107424840400900104 Su L, Zhang J, Gomez H, Kellum JA, Peng Z (2023) Mitochondria ROS and mitophagy in acute kidney injury. Autophagy 19:401-414. doi:10.1080/15548627.2022.2084862 Szeto HHJJotASoN (2017) Pharmacologic approaches to improve mitochondrial function in AKI and CKD. 28:2856-2865 Tajima H, Niikura T, Hashimoto Y, Ito Y, Kita Y, Terashita K, Yamazaki K, Koto A, Aiso S, Nishimoto I (2002) Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer's disease-related insults. Neurosci Lett 324:227-231. doi:10.1016/s0304-3940(02)00199-4 Tanaka S, Tanaka T, Kawakami T, Takano H, Sugahara M, Saito H, Higashijima Y, Yamaguchi J, Inagi R, Nangaku M (2017) Vascular adhesion protein-1 enhances neutrophil infiltration by generation of hydrogen peroxide in renal ischemia/reperfusion injury. Kidney international 92:154-164. doi:10.1016/j.kint.2017.01.014 Thapak P, Ying Z, Palafox-Sanchez V, Zhang G, Yang X, Gomez-Pinilla F (2024) Humanin ameliorates TBI-related cognitive impairment by attenuating mitochondrial dysfunction and inflammation. Biochim Biophys Acta Mol Basis Dis 1870:166937. doi:10.1016/j.bbadis.2023.166937 Thiankhaw K, Chattipakorn K, Chattipakorn SC, Chattipakorn N (2022) Roles of humanin and derivatives on the pathology of neurodegenerative diseases and cognition. Biochim Biophys Acta Gen Subj 1866:130097. doi:10.1016/j.bbagen.2022.130097 Thummasorn S, Apaijai N, Kerdphoo S, Shinlapawittayatorn K, Chattipakorn SC, Chattipakorn NJCt (2016) Humanin exerts cardioprotection against cardiac ischemia/reperfusion injury through attenuation of mitochondrial dysfunction. 34:404-414 Thummasorn S, Shinlapawittayatorn K, Chattipakorn SC, Chattipakorn N (2017) High-dose Humanin analogue applied during ischemia exerts cardioprotection against ischemia/reperfusion injury by reducing mitochondrial dysfunction. Cardiovasc Ther 35. doi:10.1111/1755-5922.12289 Thurman JM (2007) Triggers of inflammation after renal ischemia/reperfusion. Clin Immunol 123:7-13. doi:10.1016/j.clim.2006.09.008 Tighe JR (1977) The pathology of renal ischaemia. J Clin Pathol Suppl (R Coll Pathol) 11:114-124. doi:10.1136/jcp.s3-11.1.114 Tobias GJ, Mc LR, Jr., Hopper J, Jr. (1962) Endogenous creatinine clearance. A valuable clinical test of glomerular filtration and a prognostic guide in chronic renal disease. N Engl J Med 266:317-323. doi:10.1056/nejm196202152660701 Vázquez-Galán YI, Guzmán-Silahua S, Trujillo-Rangel W, Rodríguez-Lara SQ (2025) Role of Ischemia/Reperfusion and Oxidative Stress in Shock State. Cells 14. doi:10.3390/cells14110808 Venkatachalam MA, Weinberg JM, Kriz W, Bidani AK (2015) Failed Tubule Recovery, AKI-CKD Transition, and Kidney Disease Progression. J Am Soc Nephrol 26:1765-1776. doi:10.1681/asn.2015010006 Verma SK, Molitoris BA (2015) Renal endothelial injury and microvascular dysfunction in acute kidney injury. Semin Nephrol 35:96-107. doi:10.1016/j.semnephrol.2015.01.010 Versteilen AMG, Di Maggio F, Leemreis JR, Groeneveld ABJ, Musters RJP, Sipkema P (2004) Molecular Mechanisms of Acute Renal Failure following Ischemia/Reperfusion. 27:1019-1029. doi:10.1177/039139880402701203 Voigt A, Jelinek HF (2016) Humanin: a mitochondrial signaling peptide as a biomarker for impaired fasting glucose-related oxidative stress. Physiol Rep 4. doi:10.14814/phy2.12796 Wilhelm SM, Stowe NT, Robinson AV, Schulak JA (2001) The use of the endothelin receptor antagonist, tezosentan, before or after renal ischemia protects renal function. Transplantation 71:211-216. doi:10.1097/00007890-200101270-00007 Yang H, Zhang P, Wang Q, Cheng K, Zhao Y (2023) The research development of STAT3 in hepatic ischemia-reperfusion injury. Frontiers in immunology 14:1066222. doi:10.3389/fimmu.2023.1066222 Yu B, Zhang Y, Wang T, Guo J, Kong C, Chen Z, Ma X, Qiu T (2023) MAPK Signaling Pathways in Hepatic Ischemia/Reperfusion Injury. Journal of inflammation research 16:1405-1418. doi:10.2147/jir.S396604 Zhang X, Zhou J, Hu Q, Liu Z, Chen Q, Wang W, Zhang H, Zhang Q, Huang Y (2020) The Role of Janus Kinase/Signal Transducer and Activator of Transcription Signalling on Preventing Intestinal Ischemia/Reperfusion Injury with Dexmedetomidine. Journal of nanoscience and nanotechnology 20:3295-3302. doi:10.1166/jnn.2020.16416 Zhao S-T, Huang X-t, Zhang C, Ke Y (2012) Humanin Protects Cortical Neurons from Ischemia and Reperfusion Injury by the Increased Activity of Superoxide Dismutase. Neurochemical Research 37:153-160. doi:10.1007/s11064-011-0593-0 Zuccato CF, Asad AS, Nicola Candia AJ, Gottardo MF, Moreno Ayala MA, Theas MS, Seilicovich A, Candolfi MJEoott (2019) Mitochondrial-derived peptide humanin as therapeutic target in cancer and degenerative diseases. 23:117-126 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9040130","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":601791021,"identity":"9e2341bf-423c-44ae-9f71-032b8ca673fe","order_by":0,"name":"Leyla Abueid","email":"","orcid":"","institution":"Akdeniz University","correspondingAuthor":false,"prefix":"","firstName":"Leyla","middleName":"","lastName":"Abueid","suffix":""},{"id":601791022,"identity":"870761d2-84aa-467d-a2a9-c3a9dc500b12","order_by":1,"name":"Ahmet Faruk Torun","email":"","orcid":"","institution":"Akdeniz University","correspondingAuthor":false,"prefix":"","firstName":"Ahmet","middleName":"Faruk","lastName":"Torun","suffix":""},{"id":601791023,"identity":"2d40e1ca-e6e8-49a6-a52c-a2c3ecac1011","order_by":2,"name":"Ezgi Golal","email":"","orcid":"","institution":"Akdeniz University","correspondingAuthor":false,"prefix":"","firstName":"Ezgi","middleName":"","lastName":"Golal","suffix":""},{"id":601791024,"identity":"993e81d0-0cdd-4328-81ac-810677b80f14","order_by":3,"name":"Nuray Acar","email":"","orcid":"","institution":"Akdeniz University","correspondingAuthor":false,"prefix":"","firstName":"Nuray","middleName":"","lastName":"Acar","suffix":""},{"id":601791025,"identity":"498175b7-925e-4ba4-8c24-8e78eb0e1241","order_by":4,"name":"Filiz Basralı","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA8UlEQVRIiWNgGAWjYBAC9gYgwcPAIAckDBhAiIEhAb8WngMQLcaoWg4QoSWxgQeinggt/GvMHrxtq0vv7zm88XFBQR0DP3uOAfPHPXi0SLwxN5zbxpY742xbsfEMAzYGyZ43BgwHnuHWYi9xxkyat40nt+E8j5k0jwHQeTdygFrwuIwHokUiXf48j/lvHgMJBnuCWvh7QFoMEgzO9pgx8xgYMBhIELSFrUxyzrkEw41njhUDHZYAtPZZwYEzeG05vE3iTVmdvNyZ5I2fef7UyfG3J298UIFHC4NEApoZIAKfBgYGfvzSo2AUjIJRMAoYGABUD0t4SmbIBwAAAABJRU5ErkJggg==","orcid":"","institution":"Akdeniz University","correspondingAuthor":true,"prefix":"","firstName":"Filiz","middleName":"","lastName":"Basralı","suffix":""}],"badges":[],"createdAt":"2026-03-05 12:53:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9040130/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9040130/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":104469064,"identity":"72c483b0-a0f8-4d94-8c1e-34ff19ecf190","added_by":"auto","created_at":"2026-03-12 06:56:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1049109,"visible":true,"origin":"","legend":"\u003cp\u003eSee image above for figure legend.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-9040130/v1/64092d75ea5e6145d8d53a0a.png"},{"id":104469056,"identity":"608e2d3f-be3b-42d5-8e7d-62c27b971da0","added_by":"auto","created_at":"2026-03-12 06:56:42","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":59040,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of HNG on mitochondrial function in renal tissue following I/R injury.(a) Intracellular ATP levels in renal tissue homogenates. (b) Mitochondrial Complex I activity in renal tissue homogenates. Data are presented as mean ± S.E.M. ***p \u0026lt; 0.001 vs. Sham; **p\u0026lt;0.01 vs. Sham; *p \u0026lt; 0.05 vs. I/R. I/R, ischemia/reperfusion; HN, humanin; ATP, adenosine triphosphate\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-9040130/v1/d72cc4b4aeb3f73109072f74.png"},{"id":104469058,"identity":"b8fefe4f-f349-4d93-b148-67b0b485c607","added_by":"auto","created_at":"2026-03-12 06:56:43","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":107488,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of HN on oxidative stress, antioxidant status, and inflammation in renal tissue. (a) Malondialdehyde (MDA) levels. (b) Glutathione (GSH) levels. (c) Superoxide dismutase (SOD) activity. (d) Catalase (CAT) activity. (e) Myeloperoxidase (MPO) activity. Data are presented as mean ± S.E.M. ***p \u0026lt; 0.001 vs. Sham; **p \u0026lt; 0.01 vs. Sham; *p \u0026lt; 0.05 vs. Sham; \u003csup\u003e\u003cstrong\u003e++\u003c/strong\u003e\u003c/sup\u003ep \u0026lt; 0.01 vs. I/R; \u003csup\u003e\u003cstrong\u003e+\u003c/strong\u003e\u003c/sup\u003ep \u0026lt; 0.05 vs. I/R. I/R, ischemia/reperfusion; HN, humanin; MDA, malondialdehyde; GSH, glutathione; SOD, superoxide dismutase; CAT, catalase; MPO, myeloperoxidase\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-9040130/v1/d316362be530ecd055537a0b.png"},{"id":104469067,"identity":"8874aed9-bf50-410c-9480-12134d0e26d0","added_by":"auto","created_at":"2026-03-12 06:56:49","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":112700,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Humanin G Treatment on p-ERK1/2 and p-STAT3 phosphorylation. Following Ischemia-Reperfusion (IR) Injury. (a,b) Representative Western blot bands of p-ERK1/2 and p-STAT3 protein levels in SHAM, IR, HN SHAM, and HN IR groups. Beta Actin (45 kDa) was used as a loading control to ensure equal protein distribution. (c,d) Densitometric analysis of the immunoreactive bands expressed as relative protein levels. Data are presented as mean ± SEM. **p \u0026lt; 0.01, **p \u0026lt; 0.001 vs. SHAM group; +++p \u0026lt; 0.001 vs. IR group\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-9040130/v1/670f437df12f8e663211f59a.png"},{"id":105729232,"identity":"f171e590-27fd-4c6d-85c5-8ff5217a081f","added_by":"auto","created_at":"2026-03-30 11:13:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2219862,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9040130/v1/b5dba34e-dbc7-45ed-b8a7-cc93a31b9e8a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Renoprotective Effect of S14G-Humanin on Renal Ischemia/Reperfusion Injury by Activation of STAT3 and ERK 1/2 Signal Transduction Pathways in Rats","fulltext":[{"header":"Introduction","content":"\u003cp\u003eRenal ischemia-reperfusion (I/R) injury, which may occur in conditions such as hemorrhage, shock, sepsis, or transplantation, can lead to acute kidney failure, which is associated with high morbidity and mortality rates [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. I/R injury is a process that begins during the ischemic period and continues to increase during reperfusion [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Renal ischemia damages tubular endothelial and epithelial cells, and, if severe, can result in apoptosis and necrosis [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. During reperfusion, oxygen is resupplied, but this also causes increased formation of reactive oxygen species (ROS) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. Increased ROS production exacerbates tissue damage through leukocyte activation and migration, lipid peroxidation of membrane lipids, and oxidative damage to intracellular structures, thereby worsening the degree of apoptosis and necrosis [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMitochondria play a crucial role in ATP synthesis, energy metabolism, regulation of apoptosis, and Ca\u003csup\u003e2+\u003c/sup\u003e homeostasis [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Mitochondrial dysfunction observed in renal I/R injury is characterized by mitochondrial swelling, expansion of cristae and intermembraneus spaces, and a reduction in mitochondrial membrane potential [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Damaged mitochondria during I/R inhibit the activity of the electron transport chain (ETZ) enzymes, including complex I, complex II, and ATP synthase, resulting in significant electron leakage, hence the increased ROS production [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. It is well known that the balance between the oxidant and antioxidant systems is maintained under physiological conditions, and this balance is disrupted in I/R injury due to both increased ROS production and a weakened antioxidant system [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. Various antioxidant agents, such as catalase (CAT), superoxide dismutase (SOD), and glutathione (GSH), have been shown to mitigate ROS-mediated injury during renal I/R. Evidence from experimental kidney I/R models indicates that oxidative stress\u0026ndash;related tissue damage is strongly linked to diminished GSH levels. [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHN is a mitochondria-derived peptide identified first in amyloid-beta plaques of Alzheimer's patients [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. HN is known to improve mitochondrial functions, enhance the antioxidant system, and have an anti-apoptotic effect [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. It has been shown that HN exhibits protective and therapeutic effects against I/R injury in cerebral, myocardial, and intestinal tissues [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR81\" class=\"CitationRef\"\u003e81\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Additionally, it has been demonstrated that HN exerts its cytoprotective and anti-apoptotic effects under cellular stress conditions in various cell types [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAlthough the intracellular signaling mechanisms underlying the effects of HN have not yet been fully elucidated, current evidence highlights the signal transducer and activator of transcription 3 (STAT3) and extracellular signal-regulated kinase (ERK 1/2) pathways as key mediators of its cytoprotective and anti-apoptotic effects [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Various preconditioning and postconditioning interventions that attenuate I/R injury have been shown to activate both STAT3 and ERK1/2 signaling pathways. During reperfusion, levels of proinflammatory cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) rise rapidly, triggering downstream signaling cascades involving pathways such as JAK/STAT3 and ERK1/2 [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] ERK1/2, a member of the MAPK pathway, is involved in many cellular processes including cell proliferation, survival, differentiation, and apoptosis, can be activated by ROS and pro-inflammatory mediators, especially during reperfusion [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e]. Although overstimulation of ERK1/2 signaling can contribute to inflammation, a certain level of ERK1/2 activation is crucial for the induction of cell survival, proliferation, and repair signaling during I/R injury, as it is known to be involved in reperfusion injury salvage kinase (RISK) signaling [\u003cspan citationid=\"CR79\" class=\"CitationRef\"\u003e79\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. On the other hand, STAT3 activation in intrinsic protective pathways (such as the SAFE pathway) has been associated with improved mitochondrial function and reduced infarct size in cardiac I/R injury models [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. This coordinated activation appears to represent an adaptive compensatory response that limits apoptosis, preserves mitochondrial function, and restrains excessive inflammatory signaling [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eA potent humanin analogue, S14G-humanin (HNG), is generated by replacement of Ser14 with glycine, and this substitution potently increases humanin activity [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The present study used HNG to evaluate its potential protective or therapeutic effects against renal I/R injury in vivo and to identify the potential mechanism/s underlying this protection. Although there are few studies examining the effects of HNG in experimental chronic renal failure models, its acute effects on renal I/R injury, which is accepted as an experimental model of acute kidney injury (AKI) has not yet been investigated.\u003c/p\u003e \u003cp\u003eIn the present study, we aimed to investigate the effects of acute HNG administration and the underlying mechanisms in a rat model of AKI induced by renal I/R.\u003c/p\u003e"},{"header":"Material and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eAnimals and Grouping\u003c/h2\u003e \u003cp\u003eForty-eight male Sprague-Dawley rats aged 8\u0026ndash;10 week, weighing 200\u0026ndash;250 g were used in this study. The animals were housed in stainless steel cages with ad libitum access to food and water. They were maintained under a 12-hour light/dark cycle in a controlled environment with a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C and relative humidity ranging from 60% to 70%. All experimental procedures involving rats were conducted in accordance with the guidelines established by the Animal Care and Usage Committee of Akdeniz University (Approval No: 2020.08.004-2024.04.007).\u003c/p\u003e \u003cp\u003eRats were randomly divided into four groups: Sham (n\u0026thinsp;=\u0026thinsp;12), Ischemia/Reperfusion (I/R; n\u0026thinsp;=\u0026thinsp;12), Humanin-Sham (HN-Sham; n\u0026thinsp;=\u0026thinsp;12), and HN-I/R (n\u0026thinsp;=\u0026thinsp;12). The Sham groups did not undergo I/R procedures, whereas the IR groups were subjected to 45 minutes of bilateral renal ischemia followed by a 24-hour reperfusion period.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eInduction of Renal I/R Injury\u003c/h3\u003e\n\u003cp\u003eAll rats were anesthetized with intraperitoneal (ip) ketamine/xylazine (90/10 mg/kg). The tail vein of each rat was cannulated with a 24-G cannula for injections. In the sham groups, a midline abdominal incision was made to expose the renal artery and vein without applying the I/R protocol [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the I/R groups, renal blood flow was interrupted by clamping the renal pedicle with a clamp for 45 min to establish the renal I/R injury model. Upon completion of the ischemic period, the clamps were removed to initiate reperfusion, and the animals were subjected to a 24-hour reperfusion period. In the HN-Sham and HN-I/R groups, the HN analog (HNG) was administered intravenously via the tail vein at a dose of 2 mg/kg, 10 minutes before the reperfusion period. During the reperfusion period, the rats were housed in metabolic cages, and urine samples were collected over 24 hours [\u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. At the end of reperfusion, the animals were euthanized, blood samples and kidney tissues were collected for further analysis. HNG; [S14G]-humanin (amino acid sequence: Met-Ala-Pro-Arg-Gly-Phe-Ser-Cys-Leu-Leu-Leu-Leu-Thr-Gly-Glu-Ile-Asp-Leu-Pro-Val-Lys-Arg-Ala) was purchased from GenScript Biotech Corporation and used as a HN analog.\u003c/p\u003e\n\u003ch3\u003eAssessment of Renal Function\u003c/h3\u003e\n\u003cp\u003eBlood samples (0.5 ml) were collected and centrifuged to obtain supernatants. Serum creatinine (sCr) and blood urea nitrogen (BUN) levels were assessed using an autoanalyzer (Brand: ROCHE COBAS, Model: C502) at the Central Laboratory of Akdeniz University Hospital. Creatinine clearance (ClCr) as an indicator of glomerular filtration rate (GFR) was calculated using the following formula [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eClCr (ml/min) = Urine (u) Cr (mg/dl) \u0026times; urine flow (ml/min) / serum (s) Cr (mg/dl)\u003c/p\u003e\n\u003ch3\u003eHistopathological Assessment\u003c/h3\u003e\n\u003cp\u003eThe formalin-fixed and paraffin-embedded kidney was cut into 5 \u0026micro;m sections and stained with Haematoxylin and Eosin (H\u0026amp;E) for histological assessment. Histological injury scores were determined by assessing tubular necrosis, loss of the brush border, tubular cast formation, and tubular dilation as described previously [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. The histological damage scores were counted in a blind study by two observers.\u003c/p\u003e\n\u003ch3\u003eApoptosis Assessment\u003c/h3\u003e\n\u003cp\u003eApoptotic cells in rat kidney tissue were evaluated using a Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay Kit (Abcam, Catalog No: ab206386) in accordance with the manufacturer\u0026rsquo;s instructions. TUNEL-stained sections were evaluated using a light microscope (Zeiss, Oberkochen, Germany) with a special ocular scale. Five randomly selected slides, each with four different fields at 10x10 magnification, were evaluated for the analysis of TUNEL. Positively stained cell numbers in the fields were counted in a blind study by two observers. Mean positive cell numbers were calculated and then were rated to 100. Positively stained apoptotic cell numbers (apoptotic index) in the tissue were indicated as percent (%).\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eWestern Blot Analysis\u003c/h2\u003e \u003cp\u003eThe protein concentration of each sample was determined using a BCA assay kit. Equal amounts of protein (20 ug/ml) were loaded into each well and separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) at 100 V and 30 mA for 2 hours. The separated proteins were then transferred to a polyvinylidene fluoride (PVDF) membrane using a transfer system (Trans-Blot Turbo, Bio-Rad) at 25 V and 1.3 A for 10 minutes [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe membranes were incubated overnight with primary antibodies for p-STAT3 (Cat. No: 9145T, Cell Signaling) at 1:1000 dilution, p-ERK1/2 (Cat. No: 4370T, Cell Signaling) at 1:1000 dilution, and beta-actin. A Goat anti-rabbit IgG conjugated secondary antibody was used in accordance with the primary antibodies. The membranes were incubated with a chemiluminescent substrate for 5 minutes, and the bands were visualized using an Azure C280 imaging system. Proteins on the p-STAT3 and p-ERK1/2 membranes were then incubated overnight with the beta-actin antibody (4970, Cell Signaling Technology, Danvers, MA, USA) at a dilution of 1:5000, served as an internal control. The obtained results were analyzed using Image J software (Image J Version 1st36b; National Institutes of Health, MD, USA) and protein expression was quantified by normalizing the target protein to the reference protein.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAssessments of Mitochondrial Functions\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eIntracellular ATP Levels\u003c/h2\u003e \u003cp\u003eATP levels in tissue homogenates were quantified using an ATP Assay Kit (Elabscience, Catalog No: E-BC-K157-S) following the manufacturer's instructions and expressed as ATP content per gram of tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eComplex I Activities\u003c/h2\u003e \u003cp\u003eComplex I activity in kidney tissue homogenates was determined using a Complex I Activity Assay Kit (Abcam, Catalog No: ab109721), following the manufacturer's protocol. Results were expressed as enzyme activity per milligram of tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eAssessments of Oxidative Stress\u003c/h2\u003e \u003cp\u003eMalondialdehyde (MDA) levels, which are indicators of lipid peroxidation, were determined in kidney tissue homogenate by the reaction of MDA with thiobarbituric acid (TBA) (Sigma-Aldrich, T5500) and detected using spectrophotometric methods as described previously [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. MDA levels were expressed as nmol MDA/g tissue.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eAssessments of Antioxidant Status\u003c/h2\u003e \u003cp\u003eGSH levels, SOD and CAT activity were examined as antioxidant parameters in kidney tissue homogenates.\u003c/p\u003e \u003cp\u003eThe level of endogenous antioxidant GSH in tissue was measured by modifying the Ellman method, which detects sulfhydryl groups [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Absorbances were measured on a Shimadzu UV-1601 spectrophotometer (at 412 nm), and the calculated GSH level was expressed as \u0026micro;mol/g tissue.\u003c/p\u003e \u003cp\u003eSOD activity was determined using a previously described method [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. The superoxide radical generated by riboflavin under fluorescent light (20 W) is converted to hydrogen peroxide (H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e) by SOD and subsequently H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e is detected using o-dianisidine hydrochloride. SOD activity was calculated from the difference between reaction absorbance and basal absorbance at 460 nm in kidney tissue and was expressed as U/g tissue [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCAT activity in kidney tissue homogenates was determined using spectrophotometric methods as previously described by Aebi [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Absorbance was measured at 240 nm using a spectrophotometer (Shimadzu UV-1601). CAT activity was calculated based on an extinction coefficient of 0.004 mM⁻\u0026sup1;\u0026middot;mm⁻\u0026sup1; and expressed as units per gram of tissue (U/g tissue).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eAssessments of Inflammation\u003c/h2\u003e \u003cp\u003eKidney tissue Myeloperoxidase (MPO) activity was evaluated as an indicator of inflammation. MPO activity was determined through the reaction of H\u003csub\u003e2\u003c/sub\u003eO\u003csub\u003e2\u003c/sub\u003e oxidation with o-dianisidine HCl [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Absorbance changes due to the reaction were measured at 460 nm using a spectrophotometer (Shimadzu UV-1601). MPO activity was expressed as units per gram of tissue (U/g tissue).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;S.E.M. Significant differences were determined by one-way ANOVA followed by Tukey\u0026rsquo;s post hoc test, and Kruskal\u0026ndash;Wallis test followed by Dunn\u0026rsquo;s multiple comparison post hoc test using GraphPad Prism 5 software (GraphPad Software Inc., San Diego, CA, USA). Values of \u0026lt;\u0026thinsp;0.05 were considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eEffects of HNG Treatment on Renal Functions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Table 1, rats in I/R groups displayed significant deterioration of renal function 24 h after reperfusion, as reflected by increased levels of sCr and BUN with decreased ClCr (an indicator of GFR) compared to those in sham groups.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e Effects of\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eHNG Treatment on Renal Functions\u003c/p\u003e\n\u003cp\u003e\u003cimg src=\"data:image/png;base64,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\"\u003e\u003c/p\u003e\n\u003cp\u003eData are presented as mean \u0026plusmn; S.E.M. ***p \u0026lt; 0.001 vs. Sham; **p \u0026lt; 0.01 vs. Sham. I/R, ischemia/reperfusion; HN, humanin; ClCr, creatinine clearance; sCr, serum creatinine; BUN, blood urea nitrogen.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of HNG Treatment on Histopathological Damage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRenal tissues were examined after H\u0026amp;E staining (Figure 1a). Structural changes in the tubules, such as tubular necrosis, atrophy, dilation in the tubules and Bowman\u0026rsquo;s capsule, loss of the brush border, and cast formation, were observed and histopathologically scored (Figure 1b). The histopathological damage score in the I/R group was significantly higher compared to the Sham group (p\u0026lt;0.001). The histopathological damage score in the HN-I/R group was also significantly higher compared to the HN-Sham group (p\u0026lt;0.01). The damage observed in the HN-I/R group was not significantly different from that in the I/R group.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of HNG Treatment on Apoptosis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eApoptotic cells in renal tissues were detected immunohistochemically using the TUNEL kit (Figure 1b). Apoptotic cells were counted, and the percentage of apoptotic cells was calculated (Figure 1d). The percentage of apoptotic cells in the I/R group was significantly higher compared to the Sham group (p\u0026lt;0.001). The apoptotic cell density in the HN-I/R group was significantly lower compared to the I/R group (p\u0026lt;0.01).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of HNG Treatment on Mitochondrial Functions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo evaluate mitochondrial functions, intracellular ATP levels in renal tissue homogenates were measured (Figure 2a). The ATP levels in the I/R group were found to be significantly lower compared to the Sham group (p\u0026lt;0.001). Tissue ATP levels measured in the HN-I/R group were significantly elevated compared to the I/R group (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003eAs an indicator of mitochondrial function, Complex I activity levels were measured in renal tissue homogenates (Figure 2b). The Complex I activity in the I/R group was significantly lower compared to the Sham group (p\u0026lt;0.01). However, the Complex I activity levels in the HN-I/R group were significantly higher compared to the I/R group (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of HNG Treatment on Oxidative Stress and Antioxidant Status\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eLevels of MDA, which is the product of lipid peroxidation in renal tissue homogenates, are presented in Figure 3a. MDA levels were significantly higher in the I/R group compared to the Sham group (p\u0026lt;0.001). In the HN-I/R group, MDA levels were not significantly different from those in the HN-Sham group. However, tissue MDA levels were significantly lower in the I/R group treated with HNG compared to the untreated group (p\u0026lt;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eRenal tissue GSH levels as shown in Figure 3b were significantly reduced in the I/R group compared to the Sham group (p \u0026lt; 0.001). In the HN-I/R group, GSH levels did not differ significantly from those in the HN-Sham group. However, renal GSH levels in the HN-I/R group were significantly higher than those observed in the I/R group. (p\u0026lt;0.01).\u003c/p\u003e\n\u003cp\u003eSOD activity, the most effective endogenous antioxidant enzyme in cellular defense, was measured as an indicator of antioxidant capacity in renal tissue homogenates (Figure 3c). SOD activity in the I/R group was significantly lower compared to the Sham group (p\u0026lt;0.05). In the HN-I/R group, SOD activity was not significantly different from that in the HN-Sham group. However, renal SOD activity in the HN-I/R group treated with HNG prior to reperfusion was significantly higher compared to the I/R group (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003eCAT activity was measured as an antioxidant marker in renal tissue homogenates (Figure 3d). CAT activity in the I/R group was significantly lower compared to the Sham group (p\u0026lt;0.001). Similarly, CAT activity in the HN-I/R group was significantly lower compared to the HN-Sham group (p\u0026lt;0.001). No significant difference was found in CAT activity between the HN-I/R group and the I/R group.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of HNG Treatment on Inflammation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMPO, as a marker of neutrophil infiltration into the damaged tissue, was assessed to evaluate the inflammatory response, was significantly elevated in the I/R group compared to the Sham group (p\u0026lt;0.001) (Figure 3e). Similarly, MPO activity in the HN-I/R group was significantly higher compared to the HN-Sham group (p\u0026lt;0.001). Moreover, MPO activity in the HN-I/R group was significantly reduced compared to the I/R group, indicating a potential attenuation of inflammatory response (p\u0026lt;0.05).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffects of HNG Treatment on ERK1/2 and STAT3 Phosphorylation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWestern blot analyses demonstrated significant alterations in ERK1/2 and STAT3 signaling among the experimental groups (Figure 4 a,c). ERK1/2 phosphorylation\u003cs\u003e\u0026nbsp;\u003c/s\u003ewas increased in the I/R group compared to the Sham group; however, this difference was not statistically significant. In contrast, Phosphorylated ERK 1/2 (pERK1/2) levels were significantly higher in the HN-I/R group compared with both the HN-Sham and I/R groups (p \u0026lt; 0.001).\u003c/p\u003e\n\u003cp\u003eSimilarly, Phosphorylated STAT3 (pSTAT3) expression was significantly increased in the I/R group compared to the Sham group (p \u0026lt; 0.01). pSTAT3 levels in the HN-I/R group were markedly elevated compared with both the I/R and HN-Sham groups (p \u0026lt; 0.001) (Figure 4b,d). Beta-actin expression did not differ among groups, confirming equal protein loading. These results indicate that HNG treatment is associated with increased activation of ERK1/2 and STAT3 signaling pathways in renal tissue following I/R injury.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eRenal I/R injury, frequently arises from kidney transplantation hemorrhage and nephrectomy, remains a significant unresolved challenge in clinical practice. Renal I/R injury is one of the main causes of AKI which leads to disease with high mortality and morbidity [4,9].During I/R injury, tissue damage initiated during the ischemic phase persists and further exacerbates upon reperfusion [56,32].\u0026nbsp;The main mechanism of renal IR injury is associated with multiple mediators including oxidative stress, inflammation, and mitochondrial dysfunction activation of adhesion molecules, ultimately resulting in renal tubular damage and apoptosis\u0026nbsp;[69,8,61,75]. At present, strategies targeting apoptosis and oxidative stress may contribute to maintaining renal function and reducing tubular injury\u0026nbsp;[27,22].\u003c/p\u003e\n\u003cp\u003eIn the present study, we aimed to investigate the effects of HN on renal I/R injury and to elucidate the potential mechanisms involved, using HNG as an HN analog. To the best of our knowledge, this is the first study to examine the effects of HNG administration on renal I/R injury. We found that HNG administration attenuated the pathological alteration in renal I/R injury, which was accompanied with reduced inflammation, oxidative stress and apoptosis. \u0026nbsp;Additionally, HNG preserved mitochondrial functions, increased Complex I activity and elevated ATP levels. These findings demonstrate that HNG confers protective effects against renal I/R\u0026nbsp;injury in rats. Furthermore, our data suggest that HNG-mediated renoprotection may involve STAT3 and ERK 1/2 signaling pathways.\u003c/p\u003e\n\u003cp\u003eIn I/R-induced AKI, the decline in GFR is accompanied by an increase in circulating nitrogenous waste products, ultimately resulting in impaired renal function [53,43]. Although our findings confirmed the development of renal dysfunction following I/R injury, they also indicated that HNG treatment did not produce a significant improvement in reduced GFR or in the elevated sCr and BUN levels, which represent key markers of impaired renal function. This outcome may be attributed to several factors, including the administration of HNG immediately before reperfusion rather than during the ischemic phase, its use as a single dose, and the absence of repeated dosing.\u003c/p\u003e\n\u003cp\u003eIn our study, renal tissue injury was assessed through histopathological examination and apoptosis. It is well known that necrosis is the primary pathway of cell death during ischemia in renal I/R injury [36,73]. The tubular necrosis parameters, which were elevated in the I/R group, did not show a significant reduction following HNG treatment in our study. Several previous studies conducted in different tissues have demonstrated that administration of HNG either prior to or during ischemia leads to a reduction in necrosis parameters [68]. We believe that the most plausible explanation for the discrepancy between our findings and those reported in the literature is the timing of HNG administration. In our study, HNG was administered 10 minutes prior to reperfusion to specifically investigate its effects on reperfusion-induced injury [46]. In contrast, the aforementioned studies utilized HNG as a preconditioning agent administered before the onset of ischemia, thereby focusing on its protective role against ischemia-induced necrosis [68,1].\u003c/p\u003e\n\u003cp\u003eAccumulating evidence has suggested that apoptosis is critically involved in the pathological process of renal I/R injury [75,27,54]. The present study also demonstrated that I/R caused a significant increase in TUNEL-positive tubular cells, indicating the occurrence of apoptosis. Moreover, the findings of this present study revealed that HNG treatment prior to reperfusion significantly reduced the apoptosis in renal tubular cells. Anti-apoptotic effects of HN have been demonstrated in numerous \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e studies, including I/R injury models [21,29,66,1]. Previous investigation has indicated that HN inhibits apoptosis by increasing the expression of the anti-apoptotic protein Bcl-2 and decreasing the expression of the pro-apoptotic proteins Bax and suppression of caspase-3 activation in I/R injury [21,29,1]. pSTAT3 has been demonstrated to play a direct anti-apoptotic role in I/R injury by increasing the expression of the anti-apoptotic protein Bcl-2 and decreasing the expression of the pro-apoptotic proteins Bax and suppression of caspase-3 activation [80]. In light of these findings, \u0026nbsp;it could be argued that anti-apoptotic properties of HNG is dependant on STAT3 and/or ERK 1/2 phosphorylation , consistent with our results.\u003c/p\u003e\n\u003cp\u003eDisruption of cellular energy homeostasis, particularly due to compromised mitochondrial activity, represents a central mechanism in the pathogenesis of renal I/R injury [61,5]. It is well established that renal I/R injury induces mitochondrial dysfunction, including a reduction in ETC enzymes activity and ATP production [30,40]. This phenomenon plays a crucial role in I/R induced damage in tubular epithelial cells, which are characterized by increased mitochondrial density. Previous studies have suggested that HN increases intracellular ATP levels in various cell types including neurons, muscle cells, and pancreatic β-cells consistent with our findings [33,37,14,76]. The results of the present study also demonstrated that HN treatment restored intracellular ATP levels and Complex I activity, both of which were reduced by I/R injury. pSTAT3 has been shown to enhance ATP production in hepatic I/R injury by increasing the activity of mitochondrial respiratory chain complexes I and II [78]. In our study, the observed increase in ATP levels and complex I activity following HNG treatment may have been mediated by pSTAT3-dependent mechanisms.\u003c/p\u003e\n\u003cp\u003eIncreased production of ROS, which are primarily generated due to mitochondrial dysfunction during renal I/R injury significantly contribute to tissue injury [5,48,30] . MDA, as an end product of lipid peroxidation induced by ROS, can directly cause cytotoxic damage [11,19]. On the other hand, antioxidants such as SOD, CAT and GSH possess protective effects against oxidative stress [6]. It was accepted that the imbalance between oxidants and anti-oxidants can reflect the level of ROS metabolism and tissue damage [58]. In the present study, we observed a significant increase in renal MDA and decrease in renal GSH level and SOD activity in I/R rats, while HNG administration down-regulated MDA and upregulated SOD activity and GSH levels. Similarly to our findings, previous studies have suggested that HN suppresses oxidative stress while enhancing the antioxidant defense system in I/R models involving various tissues including cardiac and cerebral tissues as well as in diverse cell types [81,35] One of the proposed mechanisms underlying the anti-oxidative effects of HNG is its ability to directly modulate mitochondrial function [38]. It is proposed that HNG exerts this effect either through mitochondrial colocalization and/or via activation of the STAT3 signaling pathway [18,67] that is consistent with our results (Fugure 4b). Moreower,\u0026nbsp;pSTAT3 has also been reported to inhibit the formation of the mitochondrial permeability transition pore (mPTP), thereby preserving the mitochondrial membrane potential (ΔΨm), reducing the generation of ROS, and suppressing apoptosis [7].\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn the present study HNG treatment did not improve CAT activity, which was reduced by I/R injury. During renal I/R, unlike other antioxidant enzymes, both CAT activity and CAT protein synthesis are known to decrease [58]. The inability of HNG to enhance CAT activity observed in our study may be attributed to a reduction in CAT protein levels. Indeed, previous studies employing in experimental I/R models have demonstrated that HN treatment preserves SOD and glutathione peroxidase activities in intestinal and brain tissues following I/R injury. However, CAT activity was not evaluated in those investigations [81,1].\u003c/p\u003e\n\u003cp\u003eIt has been verified that during renal I/R injury, inflammation is one of the most important mechanisms involved in tissue damage [69,56]. On the other hand, HN has been characterized as an anti-inflammatory agent due to its suppressive effects on pro-inflammatory cytokines [81,57]. It has been shown that HN reduces age-related neuroinflammation and suppresses pro-inflammatory cytokines in an intestinal I/R model, in diabetic nephropathy and traumatic brain injury [1,45,65]. In our study, to investigate the inflammatory response associated with renal I/R injury, we assessed MPO activity as a marker of neutrophil infiltration into kidney tissues. Consistent with previous findings, our results also demonstrated that HNG treatment significantly attenuated I/R-induced inflammation in renal tissue.\u003c/p\u003e\n\u003cp\u003eIn conclusion, to the best of our knowledge, the present study demonstrated for the first time that HNG has renoprotective effects against AKI induced by renal I/R injury. According to the findings of our study, HNG exerts renoprotective effects, at least partly, by promoting STAT3 and/or ERK 1/2 activation, thereby enhancing antioxidant, anti-apoptotic, and anti-inflammatory responses, as well as regulating mitochondrial functions. These results suggesting that HNG may be a promising agent in the management of AKI.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe would like to thank the Scientific Research Projects Commission of Akdeniz University and The Scientific and Technological Research Council of Turkey (T\u0026Uuml;BİTAK) for their support and funding to this research. Additionally, we would like to express our sincere gratitude to Erol Nizamoğlu for his valuable assistance in designing the methodological approach.\u003cstrong\u003e\u003cbr\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e All authors participated in the construction of this manuscript. All authors approved the fnal version to be published.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThis research funding provided by Scientific Research Projects Commission of Akdeniz University (Project No: TDK-2021-5549) and The Scientific and Technological Research Council of Turkey (TUBITAK 1002B Project No: 224S404).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e All experimental procedures involving rats were conducted in accordance with the guidelines established by the Animal Care and Usage Committee of Akdeniz University (Approval No: 2020.08.004-2024.04.007).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e All authors approved the publication of this manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe corresponding author is willing to provide the data and datasets used and/or analyzed during the current study upon reasonable request.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAbozaid ER, Abdel-Kareem RH, Habib MA (2023) A novel beneficial role of humanin on intestinal apoptosis and dysmotility in a rat model of ischemia reperfusion injury. Pflugers Arch 475:655-666. doi:10.1007/s00424-023-02804-0\u003c/li\u003e\n \u003cli\u003eAebi H (1984) [13] Catalase in vitro. In: Methods in Enzymology, vol 105. Academic Press, pp 121-126. doi:https://doi.org/10.1016/S0076-6879(84)05016-3\u003c/li\u003e\n \u003cli\u003eAyka\u0026ccedil; G, Uysal M, Yal\u0026ccedil;in AS, Ko\u0026ccedil;ak-Toker N, Sivas A, Oz H (1985) The effect of chronic ethanol ingestion on hepatic lipid peroxide, glutathione, glutathione peroxidase and glutathione transferase in rats. Toxicology 36:71-76. doi:10.1016/0300-483x(85)90008-3\u003c/li\u003e\n \u003cli\u003eBasile DP, Anderson MD, Sutton TA (2012) Pathophysiology of acute kidney injury. Compr Physiol 2:1303-1353. doi:10.1002/cphy.c110041\u003c/li\u003e\n \u003cli\u003eBhargava P, Schnellmann RG (2017) Mitochondrial energetics in the kidney. Nat Rev Nephrol 13:629-646. doi:10.1038/nrneph.2017.107\u003c/li\u003e\n \u003cli\u003eBlokhina O, Virolainen E, Fagerstedt KV (2003) Antioxidants, oxidative damage and oxygen deprivation stress: a review. Annals of botany 91 Spec No:179-194. doi:10.1093/aob/mcf118\u003c/li\u003e\n \u003cli\u003eBoengler K, Hilfiker-Kleiner D, Heusch G, Schulz R (2010) Inhibition of permeability transition pore opening by mitochondrial STAT3 and its role in myocardial ischemia/reperfusion. Basic research in cardiology 105:771-785. doi:10.1007/s00395-010-0124-1\u003c/li\u003e\n \u003cli\u003eBonventre JV, Weinberg JM (2003) Recent advances in the pathophysiology of ischemic acute renal failure. J Am Soc Nephrol 14:2199-2210. doi:10.1097/01.asn.0000079785.13922.f6\u003c/li\u003e\n \u003cli\u003eB\u0026ouml;hmov\u0026aacute; R, Viklick\u0026yacute; O (2001) Renal ischemia--reperfusion injury: an inescapable event affecting kidney transplantation outcome. Folia Microbiol (Praha) 46:267-276. doi:10.1007/bf02815613\u003c/li\u003e\n \u003cli\u003eBradley PP, Priebat DA, Christensen RD, Rothstein G (1982) Measurement of Cutaneous Inflammation: Estimation of Neutrophil Content with an Enzyme Marker. Journal of Investigative Dermatology 78:206-209. doi:https://doi.org/10.1111/1523-1747.ep12506462\u003c/li\u003e\n \u003cli\u003eBuege JA, Aust SD (1978) Microsomal lipid peroxidation. Methods Enzymol 52:302-310. doi:10.1016/s0076-6879(78)52032-6\u003c/li\u003e\n \u003cli\u003eCai H, Liu Y, Men H, Zheng YJFiE (2021) Protective mechanism of humanin against oxidative stress in aging-related cardiovascular diseases.680\u003c/li\u003e\n \u003cli\u003eChen Y-T, Sun C-K, Lin Y-C, Chang L-T, Chen Y-L, Tsai T-H, Chung S-Y, Chua S, Kao Y-H, Yen C-H, Shao P-L, Chang K-C, Leu S, Yip H-K (2011) Adipose-Derived Mesenchymal Stem Cell Protects Kidneys against Ischemia-Reperfusion Injury through Suppressing Oxidative Stress and Inflammatory Reaction. Journal of Translational Medicine 9:51. doi:10.1186/1479-5876-9-51\u003c/li\u003e\n \u003cli\u003eCohen A, Lerner-Yardeni J, Meridor D, Kasher R, Nathan I, Parola AH (2015) Humanin Derivatives Inhibit Necrotic Cell Death in Neurons. Mol Med 21:505-514. doi:10.2119/molmed.2015.00073\u003c/li\u003e\n \u003cli\u003ede Assis JL, Grelle GMRS, Fernandes AM, da Silva Aniceto B, Pompeu P, de Mello FV, Garrett R, Valverde RHF, Einicker-Lamas M (2024) Sphingosine 1-phosphate protective effect on human proximal tubule cells submitted to an in vitro ischemia model: the role of JAK2/STAT3. Journal of Physiology and Biochemistry 80:831-843. doi:10.1007/s13105-024-01038-7\u003c/li\u003e\n \u003cli\u003eEllman GL (1959) Tissue sulfhydryl groups. Archives of Biochemistry and Biophysics 82:70-77. doi:https://doi.org/10.1016/0003-9861(59)90090-6\u003c/li\u003e\n \u003cli\u003eEltzschig HK, Eckle T (2011) Ischemia and reperfusion--from mechanism to translation. Nat Med 17:1391-1401. doi:10.1038/nm.2507\u003c/li\u003e\n \u003cli\u003eGao G, Fan H, Zhang X, Zhang F, Wu H, Qi F, Zhao L, Li Y (2017) Neuroprotective effect of G14-humanin on global cerebral ischemia/reperfusion by activation of SOCS3 \u0026ndash; STAT3 \u0026ndash; MCL\u0026ndash;1 signal transduction pathway in rats. Neurological Research 39:895-903. doi:10.1080/01616412.2017.1352187\u003c/li\u003e\n \u003cli\u003eGaweł S, Wardas M, Niedworok E, Wardas P (2004) [Malondialdehyde (MDA) as a lipid peroxidation marker]. Wiadomosci lekarskie (Warsaw, Poland : 1960) 57:453-455\u003c/li\u003e\n \u003cli\u003eGolal E, Balci CN, Acar N (2025) Dynamic changes in sumoylation related proteins SUMO1, SENP1, and UBC9 during the peri implantation period in mice. Scientific reports 15:21497. doi:10.1038/s41598-025-07677-0\u003c/li\u003e\n \u003cli\u003eGuo B, Zhai D, Cabezas E, Welsh K, Nouraini S, Satterthwait AC, Reed JCJN (2003) Humanin peptide suppresses apoptosis by interfering with Bax activation. 423:456-461\u003c/li\u003e\n \u003cli\u003eHan F, Dou M, Wang Y, Xu C, Li Y, Ding X, Xue W, Zheng J, Tian P, Ding C (2020) Cordycepin protects renal ischemia/reperfusion injury through regulating inflammation, apoptosis, and oxidative stress. Acta biochimica et biophysica Sinica 52:125-132. doi:10.1093/abbs/gmz145\u003c/li\u003e\n \u003cli\u003eHashimoto Y, Kurita M, Aiso S, Nishimoto I, Matsuoka MJMbotc (2009) Humanin inhibits neuronal cell death by interacting with a cytokine receptor complex or complexes involving CNTF receptor \u0026alpha;/WSX-1/gp130. 20:2864-2873\u003c/li\u003e\n \u003cli\u003eHashimoto Y, Niikura T, Ito Y, Sudo H, Hata M, Arakawa E, Abe Y, Kita Y, Nishimoto IJJoN (2001) Detailed characterization of neuroprotection by a rescue factor humanin against various Alzheimer\u0026apos;s disease-relevant insults. 21:9235-9245\u003c/li\u003e\n \u003cli\u003eHashimoto Y, Niikura T, Tajima H, Yasukawa T, Sudo H, Ito Y, Kita Y, Kawasumi M, Kouyama K, Doyu MJPotNAoS (2001) A rescue factor abolishing neuronal cell death by a wide spectrum of familial Alzheimer\u0026apos;s disease genes and A\u0026beta;. 98:6336-6341\u003c/li\u003e\n \u003cli\u003eHashimoto Y, Suzuki H, Aiso S, Niikura T, Nishimoto I, Matsuoka M (2005) Involvement of tyrosine kinases and STAT3 in Humanin-mediated neuroprotection. Life Sci 77:3092-3104. doi:10.1016/j.lfs.2005.03.031\u003c/li\u003e\n \u003cli\u003eHavasi A, Borkan SCJKi (2011) Apoptosis and acute kidney injury. 80:29-40\u003c/li\u003e\n \u003cli\u003eHeusch G, Musiolik J, Gedik N, Skyschally A (2011) Mitochondrial STAT3 activation and cardioprotection by ischemic postconditioning in pigs with regional myocardial ischemia/reperfusion. Circulation research 109:1302-1308. doi:10.1161/circresaha.111.255604\u003c/li\u003e\n \u003cli\u003eIkonen M, Liu B, Hashimoto Y, Ma L, Lee K-W, Niikura T, Nishimoto I, Cohen PJPotNAoS (2003) Interaction between the Alzheimer\u0026apos;s survival peptide humanin and insulin-like growth factor-binding protein 3 regulates cell survival and apoptosis. 100:13042-13047\u003c/li\u003e\n \u003cli\u003eIshimoto Y, Inagi R (2015) Mitochondria: a therapeutic target in acute kidney injury. Nephrology Dialysis Transplantation 31:1062-1069. doi:10.1093/ndt/gfv317 %J Nephrology Dialysis Transplantation\u003c/li\u003e\n \u003cli\u003eJohnson KJ, Weinberg JM (1993) Postischemic renal injury due to oxygen radicals. Current Opinion in Nephrology and Hypertension 2\u003c/li\u003e\n \u003cli\u003eKanagasundaram NS (2015) Pathophysiology of ischaemic acute kidney injury. Ann Clin Biochem 52:193-205. doi:10.1177/0004563214556820\u003c/li\u003e\n \u003cli\u003eKariya S, Hirano M, Furiya Y, Ueno S (2005) Effect of humanin on decreased ATP levels of human lymphocytes harboring A3243G mutant mitochondrial DNA. Neuropeptides 39:97-101. doi:https://doi.org/10.1016/j.npep.2004.11.004\u003c/li\u003e\n \u003cli\u003eKim S-J, Guerrero N, Wassef G, Xiao J, Mehta HH, Cohen P, Yen KJO (2016) The mitochondrial-derived peptide humanin activates the ERK1/2, AKT, and STAT3 signaling pathways and has age-dependent signaling differences in the hippocampus. 7:46899\u003c/li\u003e\n \u003cli\u003eKlein LE, Cui L, Gong Z, Su K, Muzumdar R (2013) A humanin analog decreases oxidative stress and preserves mitochondrial integrity in cardiac myoblasts. Biochem Biophys Res Commun 440:197-203. doi:10.1016/j.bbrc.2013.08.055\u003c/li\u003e\n \u003cli\u003eKosieradzki M, Rowiński W (2008) Ischemia/Reperfusion Injury in Kidney Transplantation: Mechanisms and Prevention. Transplantation Proceedings 40:3279-3288. doi:https://doi.org/10.1016/j.transproceed.2008.10.004\u003c/li\u003e\n \u003cli\u003eKuliawat R, Klein L, Gong Z, Nicoletta-Gentile M, Nemkal A, Cui L, Bastie C, Su K, Huffman D, Surana M, Barzilai N, Fleischer N, Muzumdar R (2013) Potent humanin analog increases glucose-stimulated insulin secretion through enhanced metabolism in the \u0026beta; cell. Faseb j 27:4890-4898. doi:10.1096/fj.13-231092\u003c/li\u003e\n \u003cli\u003eKumfu S, Charununtakorn ST, Jaiwongkam T, Chattipakorn N, Chattipakorn SC (2016) Humanin prevents brain mitochondrial dysfunction in a cardiac ischaemia-reperfusion injury model. Exp Physiol 101:697-707. doi:10.1113/ep085749\u003c/li\u003e\n \u003cli\u003eLan TY, Dun RL, Yao DS, Wu F, Qian YL, Zhou Y, Zhan TT, Shao MH, Gao JD, Wang C (2022) Effects of resveratrol on renal ischemia-reperfusion injury: A systematic review and meta-analysis. Frontiers in nutrition 9:1064507. doi:10.3389/fnut.2022.1064507\u003c/li\u003e\n \u003cli\u003eLi L, Zhang L, Cao Y, Chen X, Gong H, Ma Y, Gui Y, Xiang T, Liu J, Huang X (2023) NDUFV1 attenuates renal ischemia-reperfusion injury by improving mitochondrial homeostasis. J Cell Mol Med 27:1341-1352. doi:10.1111/jcmm.17735\u003c/li\u003e\n \u003cli\u003eLi Z, Ludwig N, Thomas K, Mersmann S, Lehmann M, Vestweber D, Pittet JF, Gomez H, Kellum JA, Rossaint J, Zarbock A (2022) The Pathogenesis of Ischemia-Reperfusion Induced Acute Kidney Injury Depends on Renal Neutrophil Recruitment Whereas Sepsis-Induced AKI Does Not. Frontiers in immunology 13:843782. doi:10.3389/fimmu.2022.843782\u003c/li\u003e\n \u003cli\u003eLiu J, Han X, Zhou J, Leng Y (2023) Molecular Mechanisms of Ferroptosis and Their Involvement in Acute Kidney Injury. Journal of inflammation research 16:4941-4951. doi:10.2147/jir.S427505\u003c/li\u003e\n \u003cli\u003eMalek M, Nematbakhsh M (2015) Renal ischemia/reperfusion injury; from pathophysiology to treatment. J Renal Inj Prev 4:20-27. doi:10.12861/jrip.2015.06\u003c/li\u003e\n \u003cli\u003eMasayasu M, Hiroshi Y (1979) A simplified assay method of superoxide dismutase activity for clinical use. Clinica Chimica Acta 92:337-342. doi:https://doi.org/10.1016/0009-8981(79)90211-0\u003c/li\u003e\n \u003cli\u003eMoin H, Shafi R, Ishtiaq A, Liaquat A, Majeed S, Zaidi NN (2023) Effectiveness of analog of Humanin in ameliorating streptozotocin-induced diabetic nephropathy in Sprague Dawley rats. Peptides 165:171014. doi:https://doi.org/10.1016/j.peptides.2023.171014\u003c/li\u003e\n \u003cli\u003eMuzumdar RH, Huffman DM, Calvert JW, Jha S, Weinberg Y, Cui L, Nemkal A, Atzmon G, Klein L, Gundewar SJA, thrombosis,, biology v (2010) Acute humanin therapy attenuates myocardial ischemia and reperfusion injury in mice. 30:1940-1948\u003c/li\u003e\n \u003cli\u003eMylroie AA, Collins H, Umbles C, Kyle J (1986) Erythrocyte superoxide dismutase activity and other parameters of copper status in rats ingesting lead acetate. Toxicology and Applied Pharmacology 82:512-520. doi:https://doi.org/10.1016/0041-008X(86)90286-3\u003c/li\u003e\n \u003cli\u003ePabla N, Bajwa A (2022) Role of Mitochondrial Therapy for Ischemic-Reperfusion Injury and Acute Kidney Injury. Nephron 146:253-258. doi:10.1159/000520698\u003c/li\u003e\n \u003cli\u003ePalipoch S (2013) A review of oxidative stress in acute kidney injury: protective role of medicinal plants-derived antioxidants. Afr J Tradit Complement Altern Med 10:88-93. doi:10.4314/ajtcam.v10i4.15\u003c/li\u003e\n \u003cli\u003ePefanis A, Ierino FL, Murphy JM, Cowan PJ (2019) Regulated necrosis in kidney ischemia-reperfusion injury. Kidney international 96:291-301. doi:https://doi.org/10.1016/j.kint.2019.02.009\u003c/li\u003e\n \u003cli\u003ePoston JT, Koyner JL (2019) Sepsis associated acute kidney injury. Bmj 364:k4891. doi:10.1136/bmj.k4891\u003c/li\u003e\n \u003cli\u003eRequi\u0026atilde;o-Moura LR, Dur\u0026atilde;o Junior MdS, Matos ACCd, Pacheco-Silva AJE (2015) Ischemia and reperfusion injury in renal transplantation: hemodynamic and immunological paradigms. 13:129-135\u003c/li\u003e\n \u003cli\u003eRonco C, Bellomo R, Kellum JA (2019) Acute kidney injury. The Lancet 394:1949-1964. doi:https://doi.org/10.1016/S0140-6736(19)32563-2\u003c/li\u003e\n \u003cli\u003eSaikumar P, Venkatachalam MA Role of apoptosis in hypoxic/ischemic damage in the kidney. In: Seminars in nephrology, 2003. vol 6. Elsevier, pp 511-521\u003c/li\u003e\n \u003cli\u003eSchneider MP, Sullivan JC, Wach PF, Boesen EI, Yamamoto T, Fukai T, Harrison DG, Pollock DM, Pollock JS (2010) Protective role of extracellular superoxide dismutase in renal ischemia/reperfusion injury. Kidney international 78:374-381. doi:10.1038/ki.2010.141\u003c/li\u003e\n \u003cli\u003eSharfuddin AA, Molitoris BA (2011) Pathophysiology of ischemic acute kidney injury. Nature Reviews Nephrology 7:189-200. doi:10.1038/nrneph.2011.16\u003c/li\u003e\n \u003cli\u003eShi D, Zhou X, Wang H (2021) S14G-humanin (HNG) protects retinal endothelial cells from UV-B-induced NLRP3 inflammation activation through inhibiting Egr-1. Inflamm Res 70:1141-1150. doi:10.1007/s00011-021-01489-4\u003c/li\u003e\n \u003cli\u003eSingh I, Gulati S, Orak JK, Singh AK (1993) Expression of antioxidant enzymes in rat kidney during ischemia-reperfusion injury. Mol Cell Biochem 125:97-104. doi:10.1007/bf00936438\u003c/li\u003e\n \u003cli\u003eSreekumar PG, Ishikawa K, Spee C, Mehta HH, Wan J, Yen K, Cohen P, Kannan R, Hinton DRJIo, science v (2016) The mitochondrial-derived peptide humanin protects RPE cells from oxidative stress, senescence, and mitochondrial dysfunction. 57:1238-1253\u003c/li\u003e\n \u003cli\u003eStanley WC (2004) Myocardial Energy Metabolism During Ischemia and the Mechanisms of Metabolic Therapies. 9:S31-S45. doi:10.1177/107424840400900104\u003c/li\u003e\n \u003cli\u003eSu L, Zhang J, Gomez H, Kellum JA, Peng Z (2023) Mitochondria ROS and mitophagy in acute kidney injury. Autophagy 19:401-414. doi:10.1080/15548627.2022.2084862\u003c/li\u003e\n \u003cli\u003eSzeto HHJJotASoN (2017) Pharmacologic approaches to improve mitochondrial function in AKI and CKD. 28:2856-2865\u003c/li\u003e\n \u003cli\u003eTajima H, Niikura T, Hashimoto Y, Ito Y, Kita Y, Terashita K, Yamazaki K, Koto A, Aiso S, Nishimoto I (2002) Evidence for in vivo production of Humanin peptide, a neuroprotective factor against Alzheimer\u0026apos;s disease-related insults. Neurosci Lett 324:227-231. doi:10.1016/s0304-3940(02)00199-4\u003c/li\u003e\n \u003cli\u003eTanaka S, Tanaka T, Kawakami T, Takano H, Sugahara M, Saito H, Higashijima Y, Yamaguchi J, Inagi R, Nangaku M (2017) Vascular adhesion protein-1 enhances neutrophil infiltration by generation of hydrogen peroxide in renal ischemia/reperfusion injury. Kidney international 92:154-164. doi:10.1016/j.kint.2017.01.014\u003c/li\u003e\n \u003cli\u003eThapak P, Ying Z, Palafox-Sanchez V, Zhang G, Yang X, Gomez-Pinilla F (2024) Humanin ameliorates TBI-related cognitive impairment by attenuating mitochondrial dysfunction and inflammation. Biochim Biophys Acta Mol Basis Dis 1870:166937. doi:10.1016/j.bbadis.2023.166937\u003c/li\u003e\n \u003cli\u003eThiankhaw K, Chattipakorn K, Chattipakorn SC, Chattipakorn N (2022) Roles of humanin and derivatives on the pathology of neurodegenerative diseases and cognition. Biochim Biophys Acta Gen Subj 1866:130097. doi:10.1016/j.bbagen.2022.130097\u003c/li\u003e\n \u003cli\u003eThummasorn S, Apaijai N, Kerdphoo S, Shinlapawittayatorn K, Chattipakorn SC, Chattipakorn NJCt (2016) Humanin exerts cardioprotection against cardiac ischemia/reperfusion injury through attenuation of mitochondrial dysfunction. 34:404-414\u003c/li\u003e\n \u003cli\u003eThummasorn S, Shinlapawittayatorn K, Chattipakorn SC, Chattipakorn N (2017) High-dose Humanin analogue applied during ischemia exerts cardioprotection against ischemia/reperfusion injury by reducing mitochondrial dysfunction. Cardiovasc Ther 35. doi:10.1111/1755-5922.12289\u003c/li\u003e\n \u003cli\u003eThurman JM (2007) Triggers of inflammation after renal ischemia/reperfusion. Clin Immunol 123:7-13. doi:10.1016/j.clim.2006.09.008\u003c/li\u003e\n \u003cli\u003eTighe JR (1977) The pathology of renal ischaemia. J Clin Pathol Suppl (R Coll Pathol) 11:114-124. doi:10.1136/jcp.s3-11.1.114\u003c/li\u003e\n \u003cli\u003eTobias GJ, Mc LR, Jr., Hopper J, Jr. (1962) Endogenous creatinine clearance. A valuable clinical test of glomerular filtration and a prognostic guide in chronic renal disease. N Engl J Med 266:317-323. doi:10.1056/nejm196202152660701\u003c/li\u003e\n \u003cli\u003eV\u0026aacute;zquez-Gal\u0026aacute;n YI, Guzm\u0026aacute;n-Silahua S, Trujillo-Rangel W, Rodr\u0026iacute;guez-Lara SQ (2025) Role of Ischemia/Reperfusion and Oxidative Stress in Shock State. Cells 14. doi:10.3390/cells14110808\u003c/li\u003e\n \u003cli\u003eVenkatachalam MA, Weinberg JM, Kriz W, Bidani AK (2015) Failed Tubule Recovery, AKI-CKD Transition, and Kidney Disease Progression. J Am Soc Nephrol 26:1765-1776. doi:10.1681/asn.2015010006\u003c/li\u003e\n \u003cli\u003eVerma SK, Molitoris BA (2015) Renal endothelial injury and microvascular dysfunction in acute kidney injury. Semin Nephrol 35:96-107. doi:10.1016/j.semnephrol.2015.01.010\u003c/li\u003e\n \u003cli\u003eVersteilen AMG, Di Maggio F, Leemreis JR, Groeneveld ABJ, Musters RJP, Sipkema P (2004) Molecular Mechanisms of Acute Renal Failure following Ischemia/Reperfusion. 27:1019-1029. doi:10.1177/039139880402701203\u003c/li\u003e\n \u003cli\u003eVoigt A, Jelinek HF (2016) Humanin: a mitochondrial signaling peptide as a biomarker for impaired fasting glucose-related oxidative stress. Physiol Rep 4. doi:10.14814/phy2.12796\u003c/li\u003e\n \u003cli\u003eWilhelm SM, Stowe NT, Robinson AV, Schulak JA (2001) The use of the endothelin receptor antagonist, tezosentan, before or after renal ischemia protects renal function. Transplantation 71:211-216. doi:10.1097/00007890-200101270-00007\u003c/li\u003e\n \u003cli\u003eYang H, Zhang P, Wang Q, Cheng K, Zhao Y (2023) The research development of STAT3 in hepatic ischemia-reperfusion injury. Frontiers in immunology 14:1066222. doi:10.3389/fimmu.2023.1066222\u003c/li\u003e\n \u003cli\u003eYu B, Zhang Y, Wang T, Guo J, Kong C, Chen Z, Ma X, Qiu T (2023) MAPK Signaling Pathways in Hepatic Ischemia/Reperfusion Injury. Journal of inflammation research 16:1405-1418. doi:10.2147/jir.S396604\u003c/li\u003e\n \u003cli\u003eZhang X, Zhou J, Hu Q, Liu Z, Chen Q, Wang W, Zhang H, Zhang Q, Huang Y (2020) The Role of Janus Kinase/Signal Transducer and Activator of Transcription Signalling on Preventing Intestinal Ischemia/Reperfusion Injury with Dexmedetomidine. Journal of nanoscience and nanotechnology 20:3295-3302. doi:10.1166/jnn.2020.16416\u003c/li\u003e\n \u003cli\u003eZhao S-T, Huang X-t, Zhang C, Ke Y (2012) Humanin Protects Cortical Neurons from Ischemia and Reperfusion Injury by the Increased Activity of Superoxide Dismutase. Neurochemical Research 37:153-160. doi:10.1007/s11064-011-0593-0\u003c/li\u003e\n \u003cli\u003eZuccato CF, Asad AS, Nicola Candia AJ, Gottardo MF, Moreno Ayala MA, Theas MS, Seilicovich A, Candolfi MJEoott (2019) Mitochondrial-derived peptide humanin as therapeutic target in cancer and degenerative diseases. 23:117-126\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Humanin, renal ischemia reperfusion, oxidative stress, mitochondrial dysfunction","lastPublishedDoi":"10.21203/rs.3.rs-9040130/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9040130/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eRenal ischemia/reperfusion (I/R) injury leads to acute tubular necrosis and renal failure, triggering pathological mechanisms including inflammation, reactive oxygen species generation, apoptosis, and mitochondrial dysfunction. The mitochondrial peptide Humanin (HN), known to possess anti-apoptotic and anti-inflammatory properties, has been shown to counteract oxidative stress and restore mitochondrial function. This study aimed to investigate the effects of HN on renal I/R injury.\u003c/p\u003e \u003cp\u003eSprague-Dawley male rats were divided into four groups (n\u0026thinsp;=\u0026thinsp;48): 1.Sham, 2.I/R, 3.HN-Sham, 4.HN-I/R. In I/R groups, renal artery ligation was performed for 45 minutes followed by 24-hour reperfusion. Humanin G (HNG) (2 mg/kg, iv) was administered 10 minutes before reperfusion. Urine was collected during reperfusion, and the experiment was terminated by collecting blood and tissue samples.\u003c/p\u003e \u003cp\u003eBlood urea nitrogen and serum creatinine levels were elevated in the I/R group and were not affected by HNG treatment. Glutathione levels as well as superoxide dismutase activities, which were diminished in the I/R group, were significantly restored following HNG administration.\u003c/p\u003e \u003cp\u003eMyeloperoxidase activity and malondialdehyde levels were significantly decreased in HN-I/R group compared to the I/R group. ATP levels and mitochondrial Complex I activity were significantly increased in the HN-I/R group compared to I/R. The percentage of apoptotic cells, markedly increased in I/R, was significantly reduced in HN-I/R. STAT3 and ERK 1/2 phosphorylation also increased in HN-I/R rats compared to I/R animals.\u003c/p\u003e \u003cp\u003eHNG exerts a protective effect against renal I/R injury by attenuating oxidative stress, inflammation, and apoptosis while enhancing antioxidant capacity and mitochondrial function, through STAT3 and/or ERK 1/2 activation.\u003c/p\u003e","manuscriptTitle":"Renoprotective Effect of S14G-Humanin on Renal Ischemia/Reperfusion Injury by Activation of STAT3 and ERK 1/2 Signal Transduction Pathways in Rats","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-03-12 06:56:35","doi":"10.21203/rs.3.rs-9040130/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"647225a3-a66a-4285-b62c-ee7d0f777f8c","owner":[],"postedDate":"March 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-03-28T14:10:06+00:00","versionOfRecord":[],"versionCreatedAt":"2026-03-12 06:56:35","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9040130","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9040130","identity":"rs-9040130","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}