Apelin-13 alleviate inflammatory reaction of ischemia reperfusion in rat kidney transplantation via NF-kappa B signaling pathway

Backgroud Kidney transplantation is the optimal treatment for end-stage renal disease, yet acute rejection remains a significant challenge to the survival rates of grafts. Ischemia-reperfusion injury (IRI) can initiate an inflammatory response that severely damages the transplanted kidney. Methods The APJ/apelin system has been shown to play an anti-inflammatory role across various domains, and in this study, we utilized apelin-13 in a rat kidney transplantation model to investigate its effects on IRI-related inflammation. Results Our findings indicate that apelin predominantly localizes in the renal cortex, and following kidney transplantation, there was destruction of tissue structure and an inflammatory response targeting the transplanted kidney. The administration of apelin-13 led to improved kidney function, reduced organizational structure damage, and lower injury and apoptosis indices compared to the control group. Notably, following the administration of apelin-13, the expression levels of pro-inflammatory cytokines, such as TNF-α, IL-6, and IL-1β, were reduced in comparison to the model control group, as determined by immunofluorescence, western blot, and ELISA Manuscript File Click here to view linked References assays. Furthermore, the extent of CD3 T cell infiltration was lower relative to that in the model control group. We specifically examined the classical inflammation signaling pathway, NF-κB. Our results show that, compared to the model group, the administration of apelin-13 reduced the expression of NF-κB signaling pathway proteins, as evidenced by both immunohistochemistry and western blot analyses. Conclusions In conclusion, apelin-13 appears to reduce the inflammatory response to ischemia-reperfusion injury following kidney transplantation, partly through the NF-κB signaling pathway.


Introduction
Chronic Kidney Disease (CKD) represents a significant global public health issue, eventually progressing to End Stage Renal Disease (ESRD), with a prevalence of about 10% [1].Kidney transplantation (KT) is the premier treatment for ESRD.Despite advancements in immunosuppression protocols and the development of immunosuppressants enhancing patient and graft survival rates, acute rejection (AR) continues to pose a significant challenge for clinicians.The incidence of T cellmediated acute rejection within one year is 16%, and the incidence of subclinical rejection is 30% [2].Although AR's impact can be reduced through intensive use of methandrostenolone and immunosuppressive agents, AR still inflicts considerable harm on the renal graft and contributes to an increased risk of chronic active rejection, for which effective treatments are lacking.Therefore, preventing AR and reducing the damage it causes remain paramount objectives.Ischemia-reperfusion, an inevitable process in kidney transplantation, begins with the infusion of refrigerated perfusate into the donor kidney and continues until blood flow is restored.
This process initiates a cascade of damage to the transplanted kidney, known as ischemia-reperfusion injury (IRI).During IRI, a significant release of proinflammatory cytokines from damaged cells occurs, promoting the activation and migration of immune cells such as dendritic cells (DCs), leading to increased T cell infiltration into the transplanted kidney and their differentiation into effector cells [3,4].Activated infiltrating macrophages during IRI produce various inflammatory mediators [5].Concurrently, T cells differentiate into pro-inflammatory Th1 and Th17 cells, which produce inflammatory mediators and chemokines, recruiting and activating additional immune cells, including neutrophils and monocytes, potentially leading to rejection [6,7].
In summary, IRIs significantly contribute to the development of rejection by amplifying the inflammatory response, secreting inflammatory mediators, and recruiting and activating immune cells.APJ, also known as the apelin receptor, is a G protein-coupled receptor identified by O'Dowd in 1993, exhibiting 40% to 50% homology with the angiotensin receptor AT1 [8], and acts as an antagonist to Ang II on the AT1 receptor.The APJ/apelin system comprises the receptor and two endogenous ligands, apelin peptide (apelin) [9](encoded by apln), and elabela/Toddler [10,11] (encoded by apela).apelin-13 is identified as the physiological ligand for APJ [12].Apelin receptor mRNAs are found extensively across various tissues, including the brain, lungs, heart, spinal cord, kidneys, adipose tissue, and skeletal muscle [13][14][15].In the human kidney, the APJ/apelin system is expressed throughout the renal unit, predominantly in the cortex and vasculature [16,17].
Activation of the apelin system in the kidney can improve renal blood flow, diuresis, and reduce inflammation and fibrosis [18].Both in vitro and in vivo, hypoxia triggers apelin expression, with the hypoxia-inducible factor HIF-1α further promoting apelin expression [19][20][21], indicating apelin's capacity for hypoxia resistance.Gong et al. [22] demonstrated the effects of apelin-13 on autophagy, apoptosis, and inflammation.Çelik et al. [23] confirmed that apelin induces macrophage polarization towards M2.Arababadi et al. [24] reviewed apelin's impact on macrophages, while Leeper et al. [25] highlighted its role in inhibiting macrophage inflammation.Numerous studies have investigated the inflammatory aspects of apelin, yet exploration of its effects on inflammation in renal ischemiareperfusion injury is lacking.Our study aims to apply apelin in a rat renal transplantation model to investigate its role in combating inflammation in ischemiareperfusion injury.

Animal and kidney transplantation models
We utilized orthotopic rat kidney transplantation as our experimental model.The rats were anesthetized with 2% sodium pentobarbital (40mg/Kg), after which the left kidney was perfused with 5ml of heparinized lactated Ringer's solution (25u/ml), excised en bloc, and stored in saline solution at 4˚C until transplanted into the recipient rat.The recipient rat underwent anesthesia followed by a left nephrectomy, placement of the donor kidney into the left renal fossa, end-to-end arterial suturing with 10-0 nylon threads, end-to-side venous suturing with 9-0 nylon threads, and securing the ureter into the bladder.The warm-ischemia time was 15s approximately.and the cold-ischemia time was 90min approximately.To focus on IRI post transplantation, SD rats served as both donors and recipients to eliminate immune responses from genotypic differences.
Intraperitoneal injections of [Pyr1]apelin-13 (5ug/Kg) were administered daily.After 2 days, samples were collected, with whole blood centrifuged for 15min at 3000g to obtain supernatants for biochemical parameter analysis.Kidney sections were fixed overnight in paraformaldehyde, then dehydrated, paraffin-embedded, and sliced into 4 um sections for pathological examination.The remaining renal tissue was snap-frozen in liquid nitrogen and stored at -80°C for protein analysis.

Hematoxylin and eosin staining and periodic acid-Schiff staining
Tissue sections of 4 um thickness were deparaffinized and subjected to hematoxylin and eosin (H&E) and periodic acid-Schiff (PAS) staining to evaluate pathological damage, following the reagent manufacturers' instructions.

Immunohistochemical staining
Tissue sections of 4 m thickness were deparaffinized and subsequently underwent histochemical staining.Microwave heating was employed for antigen retrieval, followed by the use of 3% H2O2 to quench endogenous peroxidase activity.Slides were then blocked with 0.1% Triton X-100 for 10 minutes and 0.5% bovine serum albumin for 1 hour, followed by overnight incubation with diluted primary antibodies (1:200).The next day, sections were incubated with secondary antibodies corresponding to the primary antibodies' species (1:200) for 1 hour.3,3'diaminobenzidine (DAB) served as the chromogen, while hematoxylin was used for counterstaining.All specimens were imaged using an optical microscope (Olympus, TKY, Japan), and the OD gray value was calculated using Fiji software 2.1.0.

Verification the concentrate of LPS and apelin-13
To determine the optimal concentration of LPS, a CCK-8 assay kit was utilized.HK-2 cells were seeded in 96-well plates at a density of 5,000 cells per well.After 12 hours, varying concentrations of LPS (0, 0.5, 1, 5, 10, 20 ug/ml) were added to each well.At 6, 12, and 24 hours post-addition, approximately 10% CCK-8 solution was introduced to the wells and incubated for 2 hours.The absorbance of the cells was measured using a 450 nm filter.Subsequently, cell groups were treated with LPS (1 ug/ml) and apelin-13 (0.5, 1.0, 2.0 uM) for 6 hours.

Immunofluorescence staining
The 4 um kidney paraffin sections underwent the same preparatory steps as for immunohistochemical staining up to the incubation with primary antibodies.Similarly, HK-2 cells seeded onto 24-well plates were prepared for LPS and apelin intervention.Post-intervention, cells were fixed for 15 minutes with paraformaldehyde, and subsequent steps paralleled those of immunohistochemical staining until the incubation with primary antibodies.The following day, sections and cells were incubated with a corresponding fluorescent secondary antibody (1:200, Proteintech, Wuhan, China) for 1 hour, then stained with Hochest (1:200, Beyotime, Shanghai, China) for 20 minutes to label nuclei.Fluorescence images were captured using microscopes (K10587, Nikon, TKY, Japan).

Protein extraction and Western blotting
Protease inhibitor and RIPA buffer (1:100) were added to either 50 mg of kidney tissue or cells from 10 cm dishes, followed by lysis and centrifugation at 4˚C at 12,000 rpm for 20 minutes to collect the supernatant.Protein samples were then mixed with 5× buffer at a 4:1 ratio and boiled for 10 minutes for denaturation.Samples were boiled at 100˚C and stored at a low temperature.Equal amounts of protein were separated by 7.5-12.5%SDS-PAGE (EpiZyme, Shanghai, China) and transferred onto PVDF membranes (Millipore, MA, USA).The membranes were blocked with a blot-blocking buffer (NCM, Suzhou, China) for 10 minutes before immunoblotting was performed using antibodies targeting the proteins of interest.After washing thrice with TBST, the membranes were incubated with secondary antibodies from Zhongshan (Beijing, China).Protein bands were visualized using an enhanced chemiluminescence substrate (34075, Thermo Scientific, Waltham, MA, USA) and ECL reagent (Bio-Rad, CA).Western blot experiments were conducted independently at least three times.

Serum inflammatory cytokines detect
The protein levels of TNF-α and IL-1β in the serum were measured using ELISA kits (Jianglai, Shanghai, China), following the manufacturer's instructions.

Statistical analyses
Data are presented as mean ± SEM.Differences between groups were assessed using Student's T-test with GraphPad Prism (GraphPad Software, version 9.5.0,CA, USA).A p value <0.05 was considered statistically significant.Image processing was conducted using Pixelmator Pro 3.5.6software.All experiments were conducted with more than three biological and technical replicates.

Inflammatory reaction after kidney transplantation and the expression of apelin in kidney
In the context of syngeneic transplantation, the experimental groups were designated as syn and syn+apelin.Following renal transplantation, serum creatinine (Scr) and BUN levels in the syn group were elevated compared to the normal group (Fig1A), alongside increased organizational structure damage (Fig1B) and increased inflammatory factor levels (Fig1C), demonstrating the severe impairment of renal function and inflammatory reaction after kidney transplantation.We determined the distribution of apelin expression in normal kidneys.Through immunofluorescence analysis, we discovered that apelin expression predominantly localized to the tubules, with negligible presence in the glomeruli and medulla (Fig1D).And after transplantation, the expression of apelin was decreased (Fig1E).

Apelin-13 alleviates kidney injury after kidney transplantation
The methodology for animal model creation and intervention is depicted in Fig2A.The findings demonstrated that Scr and BUN levels were reduced in the syn+apelin group relative to the syn group (Fig2B).H&E and PAS staining indicated that tissue damage was reduced in the syn+apelin group compared to the syn group (Fig2C).Immunohistochemistry and WB analyses confirmed that the expression levels of kim-1, ngal, and bax were lower in the syn+apelin group than in the syn group, and the ratio of bcl-2/bax was higher in the syn+apelin group (Fig2DE).

Apelin-13 alleviates inflammation reaction after renal transplantation
Our primary focus was on inflammatory factors, hence we assessed their expression in each group.Relative to the syn group, the expression of inflammatory factors in renal tissues was markedly decreased in the syn+apelin group, as evidenced by both immunofluorescence and WB analyses (Fig3AB).ELISA results indicated that serum inflammatory factor levels were lower in the syn+apelin group than in the syn group (Fig3C).Furthermore, the infiltration of CD3 T cells was significantly reduced in the syn+apelin group compared to the syn group (Fig3D).

Apelin-13 inhibits NF-kB protein expression after kidney transplantation
Following ischemia-reperfusion injury associated with kidney transplantation, alterations in inflammatory pathways, specifically the NF-κB pathway, were examined.The syn+apelin group exhibited significantly lower NF-κB expression compared to the syn group, as evidenced by both immunohistochemistry and WB (Fig 4AB ).

Discussion
Numerous studies have addressed organ ischemia-reperfusion injury (IRI).The challenge of alleviating IRI and the accompanying inflammatory response remains critical for clinicians.Our investigation into the effect of apelin on IRI-related inflammation following kidney transplantation reveals that apelin can reduce apoptosis and the inflammatory response through the NF-κB signaling pathway, thus reducing kidney injury and improving function.Initially, we confirmed apelin's expression in the kidney, aligning with prior research [16], noting its primary localization in the cortex and minimal presence in the medulla.We then implemented a kidney transplantation model, selecting SD rats as both donors and recipients to closely mimic the clinical renal transplantation process-including hot ischemia, cold ischemia, and perfusion-while avoiding the influence of genotypic differences.Inflammatory mediators trigger a cascade of reactions that harm the transplanted kidney, leading to apoptosis, immune cell activation, and further damage, potentially culminating in graft loss.Early postreperfusion, allograft endothelial and parenchymal cells release proinflammatory cytokines such as tumor necrosis factor (TNF-α) and interleukin (IL-1β) [26].Our findings indeed showed an increase in inflammatory markers post-transplantation compared to normal conditions.The critical role of IRI in generating proinflammatory cytokines, contributing not only to renal tubular epithelial and endothelial cell injury but also to inducing renal parenchymal inflammatory reactions [27], exacerbates the situation.These cytokines promote chemokine production, leading to leukocyte migration and infiltration, which in turn induces necrosis through various damaging factors, thus perpetuating a destructive cycle.Therefore, addressing IRI-induced inflammation is crucial for preventing acute rejection.
In our exploration of apelin-13's role in reducing IRI-related inflammation in kidney transplantation, we observed significant reductions in SCr and BUN following apelin-13 administration, indicating improved renal function.H&E and PAS staining further revealed that apelin-13 alleviated tissue structure damage.Kidney injury markers such as kim-1 and ngal, commonly utilized to assess kidney damage [28], demonstrated that apelin-13 could reduce tissue injury.The evaluation of apoptosis and anti-apoptosis in the graft through bax and bcl-2 markers revealed apelin-13's capacity for anti-apoptosis, aligning with findings from Shao et al. [29].Notably, bcl-2 levels were reduced in the syn+apelin group, possibly due to reduced apoptosis and consequent reduction in anti-apoptotic response.However, the ratio of bcl-2/bax was higher in the syn+apelin group, suggesting apelin-13's anti-apoptotic effect.Our focus extended to the inflammatory response.TNF-α, IL-6, and IL-1β, classical inflammatory markers, are often employed to gauge inflammation levels.Echoing studies like those by Yuan et al. [30] and Zhang et al. [31], which showed apelin-13's efficacy in reducing LPS-induced inflammation in acute lung injury, we found that apelin-13 similarly reduced inflammation in IRI following renal transplantation.This was evident in graft pathology, protein quantification, and serum levels.
Immunofluorescence indicated that inflammatory markers were predominantly located in renal tubular epithelial cells, with apelin-13 reducing their presence.Western blot analysis visually confirmed this effect, while ELISA results showed decreased serum inflammatory markers.Furthermore, examining CD3 T cell infiltration in the graft, an indicator of inflammation [32], revealed significantly less infiltration in the syn+apelin group compared to the syn group, further validating apelin-13's potential to alleviate inflammatory responses.For the cellular experiments, LPS were selected to induce cell injury and inflammation [33].In our study, apelin-13 significantly reduced the expression of kim-1, bax and classical inflammation factors, in HK-2 cells, thereby demonstrating its capability to reduce cell injury, apoptosis, and inflammation, similar to the findings from our tissue-based experiments.
Finally, we focused on the classical inflammatory pathway NF-κB, which plays a pivotal role in the body's inflammatory response.Its regulation is crucial for maintaining homeostasis [34].An excessive activation of the NF-κB pathway can lead to undesirable outcomes, including inflammation, apoptosis, and necrosis, adversely affecting the transplanted kidney.Our findings indicated a decrease in NF-κB pathway expression in the grafts of the syn+apelin group compared to the syn group.Specifically, NF-κB pathway proteins were predominantly observed in renal tubular epithelial cells, especially within dilated and injured tubules, highlighting the increased expression of NF-κB proteins in injured cells.Moreover, nonphosphorylated NF-κB proteins were mainly located in the cytoplasm, whereas phosphorylated IκBα and NF-κB p65 were found primarily in the nucleus.Apelin-13 also reduced the expression of NF-κB proteins in cellular experiments.However, it's important to acknowledge that our study primarily establishes a correlation between apelin and the NF-κB pathway, without directly proving causality.Future research will delve into their precise relationship more deeply.

Conclusion
Acute rejection following kidney transplantation is intricately linked to the inflammatory response, and a sustained state of inflammation can gravely impact the survival and functionality of the transplanted kidney.Clinicians and researchers have been striving to reduce the negative consequences of these events.In contrast, our investigation into the apelin/APJ system and its association with graft rejection reveals that apelin can reduce the inflammatory response triggered by ischemiareperfusion injury post-transplantation by inhibiting the NF-κB signaling pathway.Our ongoing research will further explore the mechanisms by which apelin/APJ influences the NF-κB pathway.

ARRIVE Guidelines and Ethics Statement
Our study was in accordance with ARRIVE guidelines (Animal Research: Reporting of In Vivo Experiments).

Figure 1 .
Figure 1.Apelin's normal distribution, tissue structure destroy and inflammatory factors detect.A. kidney function; B. HE staining and PAS staining; C. inflammatory factors of TNF-α, IL-1β and IL-6 were detected by western blot.D apelin's distribution in kidney; E. expression of apelin in normal and syn groups.Bar is 100um.

Figure 2 .
Figure 2. The effect of apelin for kidney injury after kidney transplantation.A. flow chart of the study; B. kidney function; C. HE and PAS staining; D. immunohistochemistry of kim-1,ngal, bcland bax proteins; E. kim-1,ngal,bcl-2 and bax protein levels were detected by western blot.Bar is 100um.