Introduction
Septic acute kidney injury (S-AKI) is a common and serious problem in critically ill patients. The mortality rates for S-AKI patients are much higher than those of AKI patients resulting from other etiologies, despite recent advances in treatments [1,2]. Therefore, an optimal therapy for S-AKI remains to be established. Previous studies have shown that renal tubular cells after ischemic injury associated with sepsis were detached from the tubular basement membrane and formed tubular casts, resulting in acute tubular necrosis in S-AKI [3,4]. Recently, it has become increasingly recognized that many pro- and anti-inflammatory mediators rather than ischemic insult in renal tubules play a more important role in the development of sepsis-associated tubular injury, suggesting that inflammation is a key event contributing to the occurrence of AKI in patients with sepsis [5,6]. Meanwhile, recent studies demonstrated that pattern recognition receptors, such as receptor for advanced glycation end products (RAGE), are involved in the development of inflammation in the kidney [7,8]. RAGE is a member of the immunoglobulin (Ig) superfamily, which consists of one Ig-like V-type domain and two Ig-like C-type domains in the extracellular portion, a single transmembrane-spanning domain, and a cytoplasmic tail [9]. Binding of RAGE to not only advanced glycation end products (AGEs), but also high-mobility group B1 (HMGB1), S100/calgranulin, and β2-integrin Mac-1 activates signal transduction pathways and transcription factors, including mitogen-activated protein kinase (MAPK), as well as the downstream activation of nuclear factor kappa B (NF-κB). Activation of these pathways leads to the production of reactive oxygen species (ROS) and pro-inflammatory cytokines, thereby inducing inflammation [9,10].
Soluble RAGE (sRAGE), which is a circulating form of RAGE with no membrane anchor or cytoplasmic portion, is produced endogenously by ectodomain shedding of RAGE or alternative splicing of the RAGE messenger RNA transcript [9]. It acts as a decoy receptor by competitively binding to RAGE and thus plays an antagonistic role to RAGE. Recently, blocking of RAGE signaling using sRAGE has been revealed to be a potential therapeutic candidate for various diseases associated with inflammation [11]. However, the role of blocking RAGE signaling through sRAGE has not been clearly identified in S-AKI. Therefore, we aimed to investigate the therapeutic effects of sRAGE on lipopolysaccharide (LPS)-induced renal tubular cells in vitro and in experimental S-AKI animals in vivo.
Methods
Measurement of serum sRAGE level in human subjects
Blood samples from S-AKI patients undergoing continuous renal replacement therapy (CRRT) enrolled in HICORES study [12], which was a prospective open-label trial to assess the effectiveness of high-dose CRRT in S-AKI, and were collected after obtaining approval from the Institutional Review Board (IRB) of Yonsei University Health System Clinical Trial Center (IRB No. 10-0440; Clinical Trial Registration No. NCT 01191905). Blood samples were also collected from age- and sex-matched healthy controls. All participants also provided written informed consent. sRAGE (Human RAGE Immunoassay; R&D Systems, Inc.) and extracellular newly identified RAGE-binding protein (EN-RAGE; CircuLexTM S100A12/EN-RAGE ELISA kit, CycLex Co., Ltd.) were measured using commercially available enzyme-linked immunosorbent assay (ELISA) kits, following the manufacturer’s protocols.
Animal study
All animal procedures were conducted under protocols approved by the Committee for the Care and Use of Laboratory Animals at Yonsei University, Seoul, Korea. Male 32 C57/BL6 mice, 9 to 10 weeks old, were used in this study. To generate septic animals, cecal ligation and puncture (CLP) were performed as previously reported [13]. Mice were anesthetized with ketamine and xylazine, and then midline laparotomy and cecal ligation were performed at 1 cm from the cecum tip using 4.0 silk, and the cecum was punctured twice by a 21-gauge needle. Eight mice from each of the sham operation and CLP groups were injected intraperitoneally with either diluent or 1 μg/kg of sRAGE 1 hour before the operation. Purified sRAGE protein was purchased from A&R Therapeutics, and the purification protocol was described previously [14]. Mice were sacrificed 24 hours after sham or CLP surgery, blood was collected, and the kidneys were removed for histological evaluation and molecular biological analysis. Blood urea nitrogen (BUN) and serum creatinine concentrations were determined by a Hitachi 747 automatic analyzer (Hitachi). Interleukin-6 (IL-6) level was measured by a mouse IL-6 ELISA kit (R&D Systems) using the manufacturer’s protocol.
Cell culture study
NRK-52E cells, which are immortalized rat tubular epithelial cells, were maintained in Dulbecco’s modified Eagle’s medium (Invitrogen), supplemented with 5% fetal bovine serum, 100 U/mL penicillin, 100 mg/mL streptomycin, and 26 mM NaHCO3 at 37 °C in humidified 5% CO2 in air. Subconfluent NRK-52E cells were serum-restricted for 24 hours, after which the media was replaced by serum-free medium containing 1 μg/mL LPS (Sigma Chemical Co.) with or without 1 μg/mL sRAGE. RAGE small interfering RNA (siRNA) at a concentration of 100 nM was also used in the current study and was transfected using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s protocol. At 24 hours after the media change, the cells were harvested. To investigate the nuclear translocation of NF-κB, nuclear and cytosolic fractions were separated from cultured cells harvested from plates using a commercially available kit (Thermo Fisher Scientific).
Western blot analysis
For Western blotting, 30 μg of protein extracted from the homogenized whole kidney and cultured cells were lysed in sodium dodecyl sulfate (SDS) sample buffer (2% SDS, 10 mM Tris-HCl, pH 6.8, 10% [vol/vol] glycerol), treated with Laemmli sample buffer, heated at 100 °C for 5 minutes, and electrophoresed in an 8% to 12% acrylamide denaturing SDS-polyacrylamide gel. Proteins were transferred to a Hybond-ECL membrane using a Hoeffer semidry blotting apparatus (Hoeffer Instruments), and the membrane was then incubated in blocking buffer A (1× phosphate-buffered saline [PBS], 0.1% Tween-20, and 8% nonfat milk) at room temperature for 1 hour, followed by an overnight incubation at 4 °C in a 1:1,000 dilution of primary antibodies to HMGB1 (Cell Signaling, Inc.), RAGE (Abcam), Bax, Bcl-2 (Santa Cruz Biotechnology, Inc.), cleaved caspase-3, cleaved poly(ADP-ribose) polymerase (PARP), MyD88, phospho-ERK/ERK, phospho-p38/p38 MAPK, phospho-JNK/JNK, phospho-NF-κB/NF-κB (Cell Signaling, Inc.), intercellular adhesion molecule 1 (ICAM-1; R&D Systems), or β-actin (Sigma Chemical Co.). The membrane was then washed once for 15 minutes and twice for 5 minutes in 1× PBS with 0.1% Tween-20. Next, the membrane was incubated in buffer A containing a 1:1,000 dilution of horseradish peroxidase-linked donkey anti-goat IgG (Amersham Life Science, Inc.). The washes were repeated, and the membrane was developed with a chemiluminescent agent (ECL; Amersham Life Science, Inc.). The band densities were measured using TINA image software (Raytest).
Histological examination
Slices of the kidney were fixed in 10% neutral-buffered formalin, processed in the standard manner, and 5-μm-thick sections of paraffin-embedded tissues were utilized for periodic acid-Schiff (PAS) and immunohistochemical (IHC) staining. PAS staining was used for the analysis of tubular injury score by a standard method, as previously reported [15]. Semiquantitative scores for tubular injury, including tubular epithelial cell swelling, loss of brush border, and necrotic tubules, were determined on a scale of 0 to 4+ as follows: 0, normal; 1+, <25% of the tubules; 2+, 25% to 50% of the tubules; 3+, 50% to 75% of the tubules; and 4+, more than 75% of the tubules. The sections of the kidneys were examined for at least 20 tubulointerstitial fields under ×200 magnification per specimen by two investigators in a blinded manner. For IHC staining of HMGB1 and RAGE, slides were deparaffinized, hydrated in ethyl alcohol, and washed in tap water. Antigen retrieval was carried out in 10-mM sodium citrate buffer for 20 minutes using a Black & Decker vegetable steamer. Primary antibodies for HMGB1 and RAGE were diluted to the appropriate concentrations with 2% casein in bovine serum albumin and then added to the slides, followed by overnight incubation at 4 °C. After washing, a secondary antibody was added for 20 minutes, and the slides were washed and incubated with a tertiary rabbit-PAP complex (Dako Denmark A/S) for 20 minutes. Diaminobenzidine was added for 2 minutes, and the slides were counterstained with hematoxylin. A semiquantitative score of staining intensity was determined by examining at least 20 tubulointerstitial fields under ×400 magnification by two investigators in a blinded fashion, using a digital image analyzer (MetaMorph version 4.6r5; Universal Imaging). The staining score was obtained by multiplying the intensity of staining by the percentage of tubulointerstitium staining for that intensity; these numbers were then summed for each experimental animal to yield the staining score [= Σ (intensity of staining) × (% of tubulointerstitium with that intensity)].
IHC staining for macrophages was performed using an antibody for F4/80 (Abcam). The quantification of F4/80-positive cells was expressed as the number of cells per high power field, which was counted in at least 20 randomly chosen sections under ×100 magnification by two investigators in a blinded fashion.
Transferase-mediated dUTP nick end labeling assay and Hoechst 33342 staining
In addition to the changes in the protein expression of apoptosis-related molecules, apoptosis was also identified within the kidney by terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) using a commercially available kit (Chemicon International) and in cultured NRK-52E cells seeded on coverslips by Hoechst 33342 (Molecular Probes) staining. Apoptosis was defined as TUNEL-positive cells within the tubules and the presence of nuclear condensation on Hoechst staining. The percentages of TUNEL-positive tubular cells in formalin-fixed renal tissue and NRK-52E cells with nuclear condensation were determined by examining at least 30 randomly chosen fields of the kidney sections and 300 cells/condition, respectively, at ×100 magnification.
Fluorescence-activated cell sorting analysis
CD4+Foxp3+ regulatory T (Treg) cells were counted by fluorescence-activated cell sorting (FACS) as previously described [16]. Briefly, immune cells from splenocytes in animals were collected in RPMI medium (Invitrogen). T cells were isolated using a CD4 T Cell Isolation Kit II (Miltenyi Biotec). Treg cells were stained with anti-CD4–fluorescein isothiocyanate, and the cells were fixed and permeabilized with FoxP3 staining buffer (eBioscience), followed by staining for FoxP3–phycoerythrin (eBioscience). Flow cytometric analysis of surface staining was conducted with a FACSVERSE (BD Biosciences), and the results were analyzed using FACSuite software (BD Biosciences).
Statistical analysis
All values are expressed as the mean ± standard deviation. Statistical analysis was performed using the IBM SPSS for Windows version 20.0 (IBM SPSS, Inc.). Results were analyzed using the analysis of variance (ANOVA) and Kruskal-Wallis nonparametric test for multiple comparisons. Significant differences by the ANOVA and Kruskal-Wallis test were further confirmed by the Student t test and Mann-Whitney U test, respectively. P-values less than 0.05 were considered to be statistically significant.
Results
Higher sRAGE level is associated with a better survival rate in patients with septic acute kidney injury
sRAGE and EN-RAGE levels were compared between healthy control subjects and patients with S-AKI needing renal replacement therapy. Serum sRAGE concentration was significantly higher in S-AKI patients (1,813.5 ± 2,692.7 pg/mL) compared to 30 healthy control (age, 58.9 ± 8.8 years; 12 males [40.0%]) subjects (667.3 ± 334.2 pg/mL, p < 0.05). In addition, there was a significant difference in serum level of EN-RAGE between S-AKI patients and healthy control subjects (899.2 ± 520.3 ng/mL vs. 193.9 ± 119.9 ng/mL, p < 0.01). Platelet count was significantly higher in the survivor group compared to non-survivors. Meanwhile, lactate and activated partial thromboplastin time were significantly lower in the survivor group. Serum level of sRAGE but not EN-RAGE was significantly higher in survivors relative to non-survivors in S-AKI patients (Table 1). In addition, mortality rates at 90 days were significantly lower in the highest sRAGE group compared to the lower two sRAGE groups (p = 0.03) (Fig. 1).
In vivo
sRAGE improves renal function in cecal ligation and puncture-induced septic mice
The sepsis model was induced by CLP in mice. At the time of sacrifice at 24 hours after CLP, concentrations of BUN, creatinine, and IL-6 in serum were significantly higher in the CLP group (62.4 ± 24.2, 0.44 ± 0.21 mg/dL, and 109.7 ± 114.2 pg/mL, respectively) compared to the control group (10.2 ± 4.2, 0.10 ± 0.02 mg/dL, and 5.7 ± 10.3 pg/mL, respectively; p < 0.05), and sRAGE pretreatment significantly abrogated the increases in BUN, creatinine, and IL-6 levels in CLP mice (17.9 ± 5.7, 0.21 ± 0.11 mg/dL, and 36.5 ± 28.4 pg/mL, respectively; p < 0.05) (Table 2).
Effects of sRAGE on renal HMGB1 and RAGE protein expression in cecal ligation and puncture-induced septic mice
The protein expression levels of renal HMGB1 and RAGE, assessed by Western blot, were significantly increased in the CLP group compared to the control group (p < 0.05), and sRAGE pretreatment significantly ameliorated these increases in the kidneys of CLP mice (p < 0.05) (Fig. 2A). Moreover, IHC staining for HMGB1 and RAGE confirmed the Western blot findings. The staining intensities for HMGB1 and RAGE within the tubulointerstitium were significantly higher in the CLP group compared to control and control + sRAGE mice, and these changes in CLP mice were significantly attenuated by the administration of sRAGE (Fig. 2B).
sRAGE abrogates cecal ligation and puncture-induced activation of MyD88 and MAPK in the kidney and tubulointerstitial inflammation via inhibition of NF-κB activation
Since RAGE is known to activate a number of intracellular signal transduction pathways, including MyD88 and MAPK, we evaluated the impact of sRAGE on these pathways within the kidneys in CLP-induced septic mice. Renal MyD88 protein expression was significantly increased in the CLP group compared to the control group. The protein expressions of phospho-ERK, phospho-p38, and phospho-JNK were also significantly increased in the kidneys of CLP mice relative to control mice. The increases in renal MyD88 protein expression and MAPK activation in the CLP groups were significantly ameliorated by the administration of sRAGE (Fig. 3A). Since RAGE activates NF-κB, which in turn induces and amplifies the inflammation in sepsis, we explored the protein expression of NF-κB and one of its downstream effectors, ICAM-1, in the kidney. NF-κB phosphorylation and ICAM-1 protein expression were significantly increased in the kidney of CLP mice compared to control mice, and sRAGE treatment significantly abrogated the increases in protein expression levels of renal phospho-NF-κB and ICAM-1 in the CLP group (Fig. 3B).
sRAGE ameliorates cecal ligation and puncture-induced renal tubular cell apoptosis and tubulointerstitial injury
Renal cleaved caspase-3 and cleaved PARP protein expression were significantly increased in the CLP group compared to the control group, and these increases in the kidneys of CLP mice were significantly attenuated by the administration of sRAGE (Fig. 4A). Renal tubular cell apoptosis, assessed by TUNEL assay, was also significantly increased in the CLP group relative to the control group, and sRAGE treatment significantly inhibited CLP-induced renal tubular cell apoptosis. PAS staining demonstrated that tubulointerstitial injury was also significantly abrogated in sRAGE-treated CLP mice. Furthermore, the anti-inflammatory effect of sRAGE on CLP-induced renal tubulointerstitial injury was examined by IHC staining for macrophage using F4/80 antibody. The number of infiltrated F4/80-positive cells was significantly higher in the CLP group compared to the control group, and sRAGE treatment significantly ameliorated the number of F4/80-positive cells (Fig. 4B). Taken together, these results indicate that sRAGE exerts a renoprotective effect partly via protecting renal tubular cells from apoptosis in S-AKI. In addition, FACS analysis with splenocytes found that the number of CD4+Foxp3+ Treg cells was increased in the CLP group compared to the control group, and this increase was further augmented by sRAGE pretreatment in CLP mice (Fig. 5).
In vitro
Lipopolysaccharide induces HMGB1, RAGE, MyD88, and MAPK expression and translocation of NF-κB, and sRAGE attenuates lipopolysaccharide-induced RAGE signaling in NRK-52E cells
The protein expression levels of HMGB1 and RAGE were significantly increased in LPS-stimulated NRK-52E cells, and these increases in HMGB1 and RAGE protein expression were significantly attenuated by the administration of sRAGE in tubular cells. Compared to control cells, MyD88, phospho-ERK, phospho-p38, and phospho-JNK protein expression levels were significantly increased in NRK-52E cells exposed to LPS, and these increases were significantly inhibited by sRAGE treatment (Fig. 6A).
To explore the impact of sRAGE on LPS-induced NF-κB activation, nuclear translocation of NF-κB was determined by investigating the changes in protein expression in both cytosolic and nuclear fractions. In addition, ICAM-1 protein expression as a mediator of inflammatory cascades was examined. NF-κB protein expression in the cytosolic fraction was significantly decreased at 12 hours after the administration of 1 μg/mL LPS (p < 0.05), and this decrease was significantly ameliorated by sRAGE treatment (p < 0.05). In contrast, NF-κB expression in the nuclear fraction was significantly increased by LPS (p < 0.05), and sRAGE treatment significantly attenuated this increase (p < 0.05). ICAM-1 protein expression was also upregulated in LPS-stimulated NRK-52E cells, and this increase was significantly inhibited by sRAGE (Fig. 6B).
sRAGE abrogates lipopolysaccharide-induced apoptosis in NRK-52E cells
To clarify whether LPS induces apoptosis and sRAGE protects against LPS-induced apoptosis in NRK-52E cells, the protein expression levels of apoptosis-related molecules were determined. Hoechst 33342 staining was also performed. Compared to control NRK-52E cells, Bax and cleaved caspase-3 protein expression levels were significantly increased in LPS-stimulated cells, and these changes were significantly blocked by sRAGE treatment (Fig. 7A). Apoptotic cells assessed by Hoechst 33342 staining were also significantly increased in LPS-stimulated NRK-52E cells, and sRAGE significantly attenuated this increment in apoptotic cells induced by LPS (Fig. 7B).
Finally, the protective effect of RAGE inhibition on LPS-induced inflammation and apoptosis was verified using 100 nM RAGE siRNA. The increases in ICAM-1 and cleaved caspase-3 protein expression levels in LPS-stimulated cells were significantly abrogated by RAGE siRNA (Fig. 8).
Discussion
Present study showed that sRAGE levels were significantly increased in severe patients with S-AKI, and high sRAGE levels were associated with better survival rates in these patients. In addition, it was demonstrated that sRAGE treatment improved not only renal function but also systemic inflammation in CLP-induced S-AKI animal models. Moreover, administration of sRAGE inhibited NF-κB-mediated tubulointerstitial inflammation and tubular cell apoptosis in S-AKI both in vivo and in vitro. These data suggest that RAGE modulation by sRAGE may serve as a potential therapeutic target for AKI in severe sepsis patients.
RAGE has emerged as a central regulator of systemic inflammation and subsequent tissue damage. Binding of RAGE to various kinds of ligands, such as HMGB1 and AGEs, results in pro-inflammatory gene activation and consequent propagation of an inflammatory cascade; thus, the interactions between RAGE and its ligands are considered to be associated with a range of inflammatory diseases including sepsis [17]. Meanwhile, the extracellular domain of RAGE is cleaved to produce sRAGE, which is secreted into the systemic circulation and exerts protective effects against RAGE-induced inflammatory tissue damage [18]. Previous studies have shown that RAGE accumulated and existed in several variants in patients with reduced renal function [19,20]. Serum levels of sRAGE and EN-RAGE were also increased in patients with renal insufficiency, including end-stage renal disease patients [11]. Furthermore, serum sRAGE concentration was negatively associated with inflammation and oxidative stress in chronic kidney disease (CKD) patients [19]. In the present study, we demonstrated that sRAGE level was significantly increased in patients with S-AKI compared to healthy control subjects. Furthermore, in patients with S-AKI, increased serum concentration of sRAGE had a protective impact on survival in these patients. Based on these findings, it was inferred that sRAGE might be increased by a counter-regulatory mechanism against enhanced RAGE expression to protect from oxidative stress, inflammation, and pro-apoptotic injury in severe patients with S-AKI.
In the past, sepsis-associated renal injury was considered to be a consequence of tubular ischemia and subsequent acute tubular necrosis due to hemodynamic instability. However, recent robust studies have found that non-hemodynamic injuries, including inflammation and coagulation abnormality, played a critical role in the development of renal tubular cell damage in sepsis [21]. Circulating pro- and anti-inflammatory mediators induce the recruitment of inflammatory cells and tubular cell apoptosis. Among them, HMGB1, which is released from injured cells and macrophages, has been suggested to be one of the late-appearing pro-inflammatory cytokines and damage-associated molecular patterns, and it regulates microbial-induced inflammation and LPS-induced cellular and tissue injuries [22,23]. Moreover, it is an important ligand of RAGE, which is activated by HMGB1 and triggers inflammatory processes in sepsis and acute inflammation. A previous study also showed that HMGB1-neutralizing antibody reversed sepsis in mice [24]. Taken together, these findings infer that HMGB1 may play an important role in the pathogenesis of sepsis. The current study demonstrated that HMGB1 and RAGE protein expression levels were significantly increased in the kidneys of sepsis mice and in LPS-treated tubular cells, and that sRAGE ameliorated CLP- and LPS-induced tubulointerstitial inflammation and tubular cell apoptosis both in vivo and in vitro. Interestingly, the increases in RAGE and HMGB1 expression in tubular cells under septic conditions were also significantly attenuated by sRAGE treatment. If RAGE expression was further induced by continuous ligand activation and HMGB1 was shed from injured cells, RAGE and HMGB1 expression could be influenced by sRAGE via mitigation of RAGE-mediated induction of various chemokines and adhesion molecules as well as release of HMGB1 associated with tubular cell apoptosis [22,25]. IIn accordance with the results of the present study, Lee et al. [26]also found that sRAGE abrogated angiotensin-II-induced RAGE expression in the aorta of apolipoprotein E-deficient mice.
HMGB1 exerts pro-inflammatory effects by binding, primarily to RAGE and/or the IL-1 receptor. Recently, it has been revealed that HMGB1 also interacts with toll-like receptor (TLR) 2/4 [26]. In contrast, LPS, a well-known ligand of TLRs, is an endotoxin released by exogenous pathogens in sepsis [14]. Based on the results of previous studies showing that interactions of HMGB1 and LPS with RAGE and TLR led to signal transduction propagation via MyD88 and the MAPK pathway and cytokine production [27], it was suggested that blocking RAGE signaling could ameliorate HMGB1- and LPS-induced inflammatory responses in sepsis. In this study, we demonstrated that MyD88 and MAPKs, including ERK, p38, and JNK, were activated in the kidneys of CLP mice and in LPS-treated tubular cells, and that sRAGE treatment significantly attenuated these changes in renal tubular cells under septic conditions.
Importantly, signaling through RAGE leads to NF-κB activation by nuclear translocation of NF-κB and, in turn, induces the transcription of inflammation-associated genes such as ICAM-1, which is a cell surface glycoprotein that plays a major role in the infiltration process of macrophages and monocytes [28]. Once the macrophages/monocytes are infiltrated and activated, they release lysosomal enzymes, nitric oxide, ROS, tumor necrosis factor-α, IL-1, and transforming growth factor-β and consequently promote renal injury including tubular cell apoptosis [29,30]. The results of the current study found that NF-κB protein expression and nuclear translocation of NF-κB were significantly increased in the kidneys of CLP mice and in LPS-treated NRK-52E cells, along with an increase in ICAM-1 expression. Furthermore, the number of infiltrated macrophages within the renal tubulointerstitium was significantly higher in CLP mice. In addition, administration of sRAGE significantly abrogated the increases in nuclear translocation of NF-κB, ICAM-1 expression, and macrophage infiltration in the kidneys of CLP mice. These findings indicate that sRAGE ameliorated renal tubular injury via its anti-inflammatory effect in sepsis animal models. In vitro, however, tubular cell apoptosis induced by LPS was also significantly attenuated by RAGE inhibition. Taken together, these results indicate that the impact of sRAGE on renal tubular injury is mediated not only by mitigating the infiltration of macrophages/monocytes but also by its direct beneficial effects on renal tubular cells, independent of inflammation.
Various immunocompetent cells have been shown to play a critical role in kidney injury and repair. Among them, Treg cells were previously demonstrated to be involved in immunomodulation in AKI [31]. A recent study found that the number of Treg cells was increased in heat-preconditioned splenocytes and that this increase contributed to the renoprotective effect of heat preconditioning [32]. Tatura et al. [33] also showed that Foxp3+ Treg cells were increased in a murine sepsis model and that the depletion of Treg cells aggravated the severity of sepsis in these mice. In contrast, another study demonstrated that splenectomy reduced the circulating HMGB1 level in a CKD-sepsis animal model [34]. The results of the present study revealed that the number of CD4+CD25+Foxp3+ cells in the spleen was increased and that this increase was further augmented by sRAGE treatment in CLP mice, suggesting that expanded Treg cells by sRAGE exposure may partly contribute to the improvement in sepsis-associated AKI via inhibition of RAGE signaling. Collectively, these findings indicate that sRAGE may also protect against S-AKI through the expansion of immune modulatory cells, including Treg cells, in addition to its anti-inflammatory and direct anti-apoptotic effects on renal tubular cells.
This study has several limitations. Since the effects were observed only in an animal model, the evidence is insufficient to assert that this could be an effective treatment in humans. To consider the clinical application, it is necessary to evaluate the long-term effects of the sepsis model. In this study, we administered the sRAGE prior to the CLP operation. Although it would have been important to evaluate the therapeutic effects after the onset of sepsis, this was not addressed in our experiments and represented a limitation of our study. Once sepsis develops, many mice experience severe renal damage, making recovery difficult. Therefore, it is challenging to definitively claim therapeutic efficacy under such conditions. Further studies assessing the effects of sRAGE after the onset of sepsis are warranted to address this limitation.
In conclusion, the findings of the present study suggest that RAGE plays an important role in S-AKI, and that its inhibition by sRAGE may be a potential therapeutic target for AKI in severe sepsis.
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