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. 2024 Dec 5;6(2):208–218. doi: 10.34067/KID.0000000665

Involvement of Mineralocorticoid Receptor Activation by High Mobility Group Box 1 and Receptor for Advanced Glycation End Products in the Development of Acute Kidney Injury

Tomoyuki Otsuka 1, Seiji Ueda 1,2,✉, Sho-ichi Yamagishi 3, Hajime Nagasawa 1,2, Teruyuki Okuma 1,2, Keiichi Wakabayashi 1, Takashi Kobayashi 1, Maki Murakoshi 1, Masami Nakata 1, Tomohito Gohda 1, Takanori Matsui 4, Yuichiro Higashimoto 5, Yusuke Suzuki 1
PMCID: PMC11882257  PMID: 39636697

Abstract

Key Points

  • Our study revealed that high mobility group box 1 activates the mineralocorticoid receptor (MR) through the receptor for advanced glycation end products (RAGE) in AKI.

  • MR antagonists and RAGE aptamers inhibited high mobility group box 1–induced Rac1/MR activation and downstream inflammatory molecules in endothelial cells.

  • MR antagonists and RAGE aptamers may represent promising therapeutic strategies for preventing AKI and CKD progression.

Background

Although AKI is associated with an increased risk of CKD, the underlying mechanisms remain unclear. High mobility group box 1 (HMGB1), one of the ligands for the receptor for advanced glycation end products (RAGE), is elevated in patients with AKI. We recently demonstrated that the mineralocorticoid receptor (MR) is activated by the RAGE/Rac1 pathway, contributing to chronic renal damage in hypertensive mice. Therefore, this study investigated the role of the HMGB1/RAGE/MR pathway in AKI and progression to CKD.

Methods

We performed a mouse model of renal ischemia–reperfusion (I/R) with or without MR antagonist (MRA). In vitro experiments were conducted using cultured endothelial cells to examine the interaction between the HMGB1/RAGE and Rac1/MR pathways.

Results

In renal I/R injury mice, renal MR activation was associated with elevated serum HMGB1, renal RAGE, and activated Rac1, all of which were suppressed by MRA. Renal I/R injury led to renal dysfunction, tubulointerstitial injury, and increased expressions of inflammation and fibrosis mediators, which were ameliorated by MRA. In vitro, RAGE aptamer or MRA inhibited HMGB1-induced Rac1/MR activation and upregulation of monocyte chemoattractant protein 1 and NF-κB expressions. Seven days after I/R injury, renal I/R injury mice developed CKD, whereas MRA prevented renal injury progression and decreased the mortality rate. Furthermore, in case of MRA treatment even after I/R injury, attenuated renal dysfunction compared with untreated mice was also observed.

Conclusions

Our findings suggest that HMGB1 may play a crucial role in AKI and CKD development by activating the Rac1/MR pathway through interactions with RAGE.

Keywords: AKI, CKD

Visual Abstract

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Introduction

AKI is a serious complication that can affect 21.6% of hospitalized patients worldwide, and its incidence is increasing.1 Renal ischemia–reperfusion (I/R) is a notable cause of AKI in hypotensive patients due to several clinical complications, including severe heart failure, systemic sepsis, and trauma-induced hypovolemia.2–4 AKI is also known to be associated with an increased risk of CKD, mortality, and cardiovascular complications.5,6 Indeed, a meta-analysis reported an 8.8-fold risk of CKD progression and a 3.1-fold risk of ESKD in patients with AKI.7 Therefore, the close relationship between AKI and CKD has begun to be recognized, and the concept of transition from AKI to CKD has emerged. Although the mechanisms underlying this concept have been extensively studied over the past decade, effective pharmacological interventions for the prevention or delayed progression of AKI-induced CKD remain elusive.

High mobility group box 1 (HMGB1), a nuclear DNA-binding protein that functions as a potent proinflammatory cytokine when released from necrotic or activated cells,8,9 binds to the receptor for advanced glycation end products (RAGE) and toll-like receptor (TLRs), contributing to endothelial and vascular injury.10 The association between HMGB1 and kidney disease is well known, and HMGB1 levels are reported to be elevated in patients with AKI. A meta-analysis in patients with sepsis revealed that elevated serum HMGB1 levels were associated with sepsis severity and mortality.11 In addition, several studies demonstrated that blockade of HMGB1 protected the kidneys in a renal I/R mouse model by downregulating the production of HMGB1-downstream inflammatory molecules.10 HMGB1 levels are also elevated in patients with CKD, with findings suggesting possible involvement in cardiovascular disease and renal fibrosis.12,13 Furthermore, the reported interaction between HMGB1 and RAGE has been observed to contribute to vascular injury during acute limb I/R.14 Given these findings, it is conceivable that HMGB1-mediated RAGE activation may play a key role in acute inflammatory responses and subsequent chronic fibrosis in AKI.

Rac1, a small GTPase belonging to the Rho family, plays a crucial role in various cellular processes, including actin cytoskeleton organization, cell migration, and reactive oxygen species production.15 In the context of kidney disease, Rac1 has been implicated in podocyte injury, proteinuria, and the progression of renal damage.16 Studies have shown that Rac1 activation can lead to increased oxidative stress and inflammation in the kidney, contributing to the pathogenesis of both AKI and CKD.17 Moreover, Rac1 has been found to interact with the mineralocorticoid receptor (MR) pathway, potentially amplifying its detrimental effects on renal function.18 The interplay between Rac1 and MR signaling has emerged as a significant factor in renal pathophysiology, warranting further investigation into potential therapeutic targets.

While the MR has emerged as a therapeutic target in AKI,19,20 the precise mechanisms remain unclear. Our previous study demonstrated that RAGE-mediated Rac1 activation could enhance the MR pathway, resulting in podocyte damage in hypertensive mice.21 Building on this, this study investigated the endogenous levels of HMGB1, RAGE, and Rac1/MR systems in a renal I/R injury mice model, as well as the effect of RAGE aptamer (RAGE-Apt) and MR antagonist (MRA) on the onset and progression of renal damage. We hypothesized that the HMGB1/RAGE pathway is involved in the Rac1/MR pathway, contributing to AKI pathogenesis and CKD progression.

Methods

Animals and Experimental Design

Male C57BL/6J mice were obtained from CLEA Japan (Tokyo, Japan) and maintained in a specific pathogen-free facility at Juntendo University. All animal experiments were approved by the Ethics Review Committee for Animal Experimentation of Juntendo University Faculty of Medicine (document no. 1452). Procedures were conducted in accordance with the animal experimentation guidelines of Juntendo University in Tokyo, Japan, and European Union Directive 2010/63/European Union for animal experiments. We set the symptoms that occur as kidney failure progresses, including feeding or drinking disorders, edema, and rapid breathing and breathing difficulties, as the end points and euthanized the mice when these symptoms appeared.

Male mice weighing 20–25 g were used in the experiments. Esaxerenone, the MRA for this study (Daiichi Sankyo Co., Ltd., Tokyo, Japan), was administered by oral gavage, and methylcellulose (0.5% w/v) was used as the vehicle. In the first set of experiments (the acute study), 24 mice were examined 24 hours after reperfusion in three different groups: sham-operated mice (sham; n=8); mice subjected to 22 minutes of I/R (I/R; n=8); and mice treated with esaxerenone (3 mg/kg per day) 48, 24, and 1 hour before I/R induction (I/R+Esax; n=8). For the second set of experiments (the subacute study), 24 mice were examined 7 days after reperfusion in three different groups: sham-operated mice (sham; n=4); mice subjected to 22 minutes of I/R (I/R; n=9); and mice treated with esaxerenone at 48, 24, and 1 hour before I/R induction and once daily after reperfusion (I/R+Esax; n=11). In the third experiment, mice were divided into two groups: one group was administered the vehicle daily from 24 hours after reperfusion, and the other group was administered esaxerenone 24 hours after reperfusion. The mice were examined on the 7 days after reperfusion.

Renal I/R Injury Model

Renal I/R injury was induced in 8–10-week-old mice. Mice were placed on a 37°C heat pad and anesthetized with isoflurane. Small flank incisions exposed the bilateral kidneys, and the left kidney pedicle was clamped for 22 minutes using a cerebral aneurysm clip (stainless steel micro serrefines, Muromachi, Tokyo, Japan) after right nephrectomy. Microaneurysm clips were then released at appropriate time points (24 hours or 7 days) for reperfusion. Sham-operated mice underwent a similar procedure without renal pedicle clamping.

Renal Function Evaluation

Mice were euthanized 24 hours or 7 days postoperatively, and serum and kidney samples were collected for further analysis. BUN and serum creatinine (Cr) levels were measured using a Fuji Dri-Chem7000V and DRI-CHEM slide system (FUJIFILM, Kanagawa, Japan) according to the manufacturer's protocol.

ELISA

Serum HMGB1 levels were measured using a commercially available sandwich ELISA kit (Shino-Test Corporation, Tokyo, Japan) according to the manufacturer's instructions.

Histological Analysis

For histological assessment, kidneys were fixed in 4% neutral buffered paraformaldehyde, embedded in paraffin, and cut into 4-μm–thick sections. These sections were subsequently dewaxed using standard sequential techniques and underwent periodic acid–Schiff (PAS) and Sirius red staining. A semiquantitative score for acute tubular necrosis was used, as described by Wang et al.22 For each mouse, at least ten fields were examined, scoring the percentages of tubules with cellular necrosis, loss of brush borders, cast formation, vacuolization, and tubule dilation as follows: 0 for none; 1 for <10%; 2 for 11–25%; 3 for 26–45%; 4 for 46–75%; and 5 for >76%. Using these data, the tubulointerstitial fibrosis score was calculated blindly as follows: 0 for <5% of fibrosis; 1 for 5–25%; 2 for 26–50%; 3 for 51–75%; and 4 for 76–100%.

Cluster of differentiation 31 (CD31) immunohistochemistry was performed, as previously described.23 CD31-positive capillary rarefaction was graded semiquantitatively by fluorescence intensity using a score from 0 to 4 as follows: 0 if all peritubular capillaries (PTCs) were homogenously positive (normal findings), 1 if a single CD31-negative segment was detected, 2 if CD31 was negative in up to 25% of the tissue section, 3 if CD31 was negative in 25–50%, and 4 if CD31 staining was completely absent. A higher score, therefore, indicates a greater degree of capillary loss. PTC rarefaction was evaluated in at least ten cortical fields.24

Cell Culture

Human umbilical vein endothelial cells (HUVECs), obtained from ScienCell Research Laboratories (San Diego, CA) were cultured in endothelial cell medium (ScienCell Research Laboratories) with 5% FBS at 37°C, 5% (v/v) CO2, and 95% humidity. Cells were used for experiments at 70–80% confluence, between passages 3 and 7. HUVECs were pretreated with 10 nM esaxerenone, 100 nmol/L RAGE-Apt, control aptamer (Ctrl-Apt), or PBS for 24 hours before HMGB1 (100 ng/ml, R&D Systems, Wiesbaden, Germany) for 30 minutes or 4 hours. Untreated cells were used as controls.

Preparation of DNA-Aptamer–Directed RAGE

RAGE-Apt and Ctrl-Apt were prepared and selected using systematic evolution of ligands by exponential enrichment, as previously described.25,26 The sequences of phosphorothioate-modified aptamers were as follows: RAGE-Apt, 5′-ccT​gAT​ATg​gTg​TcA​ccg​ccg​ccT​TAg​TAT​Tgg​TgT​cTA​c-3′; Ctrl-Apt, 5′-ttc​ggC​ctg​ggG​gcg​gcC​agt​tcG​ggt​ccA​gtc​gcG​gga​g-3′ (phosphorothioate nucleotides were indicated as capital letters). This RAGE-Apt was confirmed as an HMGB1 antagonist, with a Kd of 48.3 nM.27

Western Blot Analysis

Cells and kidney tissues were lyzed using a radioimmunoprecipitation assay buffer containing a protease inhibitor cocktail on ice. Nuclear fractions were extracted using the Nuclear Extraction Kit (ab113474, Abcam) according to the manufacturer's instructions. Proteins were separated using SDS-PAGE and then transferred onto polyvinylidene difluoride membranes. After blocking with 5% BSA or 5% skimmed milk, the membranes were incubated overnight at 4°C with primary antibodies against MR (ab64457, Abcam), RAGE (ab3611, Abcam), TATA box-binding protein (ab63766, Abcam), or glyceraldehyde-3-phosphate dehydrogenase (G8795, Sigma). After three washes with tris-buffered saline and Tween-20, each membrane was incubated with horseradish peroxidase-conjugated secondary antibodies for 1 hour at room temperature. Signals were visualized using the SuperSignal West Dura Chemiluminescent Substrate (Pierce), and protein bands were quantified by densitometry using Fusion FX7 software (Vilber Lourmat).

Rac1 Pull-Down Assay and Western Blotting Analysis

Rac1 activity was assessed using commercially available kits (Cytoskeleton, Inc., Denver). Samples were homogenized in lysis buffer and incubated at 4°C for 60 minutes with the human p21 activated kinase 1 protein-p21 binding domain protein beads. Rac1 content was determined using SDS-PAGE and immunoblotting.28

RNA Extraction and Real-Time PCR

Gene expression analysis was performed using quantitative real-time PCR, as previously described.29 TaqMan probes were used to measure the expressions of serum and glucocorticoid-regulated kinase 1 (Mm00441380_m1), monocyte chemoattractant protein 1 (MCP-1) (Mm00441242_m1, Hs00234140_m1), NF-κB (Mm00476361_m1, Hs00765730_m1), TGF-β (Mm01178820_m1), kidney injury molecule 1 (Kim-1) (Mm00506686_m1), vascular endothelial growth factor (VEGF) (Mm00437306_m1), intercellular adhesion molecule 1 (ICAM-1) (Mm00516023_m1), and vascular cell adhesion molecule 1 (VCAM-1) (Mm01320970_m1). Eukaryotic 18S ribosomal RNA (Mm03928990_g1, Hs99999901_s1) served as the endogenous control. mRNA was quantified using the ΔΔCT method on a 7500 Fast Real-Time PCR system.

Statistical Analyses

Data were expressed as mean with SD. Parameters were compared among groups using one-way or two-way repeated-measures ANOVA, followed by the Bonferroni test. All statistical analyses were performed using GraphPad Prism, Version 9 (GraphPad Inc., San Diego, CA), and statistical significance was set at P < 0.05.

Results

The HMGB1/RAGE Pathway and Rac1/MR Activation in Renal I/R Injury Mice and the Effects of MRA

To investigate the involvement of the endogenous HMGB1/RAGE and Rac1/MR pathways in AKI, serum HMGB1 levels, renal RAGE, guanosine triphosphate (GTP)-bound Rac1, and MR expression were evaluated in renal I/R injury mice. Serum HMGB1 levels were elevated in renal I/R injury mice than that in sham mice (Figure 1A). Similarly, renal RAGE and GTP-bound Rac1 expressions were significantly higher in renal I/R injury mice (Figure 1, B and C). In addition, nuclear MR expression, an indicator of MR activation, was increased in the kidneys of renal I/R injury mice (Figure 1D). Consequently, the expression of serum and glucocorticoid-regulated kinase 1, a downstream molecule of MR signaling, was higher in the kidneys of I/R injury mice than that in sham mice (Figure 1E). Pretreatment with nonsteroidal MRA esaxerenone significantly suppressed all these alterations (Figure 1, A–E). These findings suggest that the endogenous HMGB1/RAGE and Rac1/MR pathways were enhanced in renal I/R injury mice and that MRA can effectively attenuate their activation.

Figure 1.

Figure 1

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HMGB1/RAGE and Rac1/MR pathways were activated in renal I/R injury and their activation was inhibited with MRA treatment. (A) Serum HMGB1 levels were determined using an ELISA kit. Sham, sham-operated mice; I/R, mice subjected to 22 minutes of renal ischemia; I/R+Esax, mice treated with esaxerenone. (B) The upper panel shows a Western blot of RAGE after renal I/R. Western blot analysis was performed on the whole kidney. Upper panel: RAGE; lower panel: GAPDH as an internal control. (C) The upper panel shows a Western blot of GTP-bound Rac1 after renal I/R. Western blot analysis was performed on the whole kidney. Upper panel: GTP-bound Rac1; lower panel: total Rac1 as a control. (D) The upper panel shows a Western blot of MR after renal I/R. Western blot analysis was performed on nuclear proteins extracted from the whole kidney. Upper panel: MR; lower panel: TBP as an internal control. The lower panel graph displays the corresponding densitometric analysis. (E) Sgk1 mRNA levels were quantified using qRT-PCR. Bar=mean±SD. **P < 0.01, ***P < 0.001, and ****P < 0.0001. Esax, Esaxerenone; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GTP, guanosine triphosphate; HMGB1, high mobility group box 1; I/R, ischemia–reperfusion; MR, mineralocorticoid receptor; MRA, mineralocorticoid receptor antagonist; qRT-PCR, quantitative real-time PCR; RAGE, receptor for advanced glycation end products; Sgk1, serum and glucocorticoid-regulated kinase 1; TBP, TATA box-binding protein.

MRA Ameliorated AKI in the Mouse Renal I/R Model

To investigate whether MRA could prevent renal dysfunction and structural in renal I/R injury mice, BUN and serum Cr levels were measured 24 hours after reperfusion. Renal I/R injury mice demonstrated renal dysfunction, as evidenced by an increase in BUN and serum Cr levels, whereas MRA-treated mice showed a blunted response (Figure 2, A and B). Histological examination revealed cellular necrosis, loss of brush borders, cast formation, vacuolization, and tubule dilation in I/R-induced renal injury. Moreover, these changes were significantly ameliorated in MRA-treated mice (Figure 2, C and D).

Figure 2.

Figure 2

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MRA ameliorated renal I/R-induced acute kidney dysfunction and tubular injury. Renal function was assessed by measuring (A) BUN and (B) serum Cr levels. Sham, sham-operated mice; I/R, mice subjected to 22 minutes of renal ischemia; I/R+Esax, mice treated with esaxerenone. (C) The panels depict representative renal histology stained with PAS. (D) The panel shows the quantitative data from the sham, I/R, and I/R+Esax pretreatment groups. The ATN score was quantified using ten images per mouse (magnification ×200). Renal damage included tubular epithelial cell detachment, interstitial edema, and tubular cell casts were scored, wherein high scores indicated a greater degree of renal injury. Groups were compared using one-way ANOVA. Bar=mean±SD. **P < 0.01 and ****P < 0.0001. ATN, acute tubular necrosis; Cr, creatinine; PAS, periodic acid–Schiff.

The levels of downstream inflammatory and fibrosis molecules that are considered to be crucial mediators of inflammation and fibrosis in AKI,30,31 such as MCP-1, NF-κB, and TGF-β, were increased in the kidneys of renal I/R injury mice compared with sham mice and suppressed in MRA-treated mice (Figure 3, A–C). In addition, the kidney mRNA levels of the tubular injury marker Kim-1 were significantly increased in renal I/R injury mice and ameliorated in MRA-treated mice (Figure 3D).

Figure 3.

Figure 3

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MRA ameliorated mRNA expression of inflammatory, fibrosis, and tubular injury marker-related genes. (A) MCP-1, (B) NF-κB, and (C) TGF-β mRNA levels were quantified using qRT-PCR. MCP-1, NF-κB, and TGF-β expression levels were higher in the kidneys of renal I/R injury mice than in those of sham mice. (D) As a marker of tubular injury, Kim-1 mRNA levels were quantified. The mRNA levels of these markers decreased in MRA-pretreated mice. Groups were compared using one-way ANOVA. Bar=mean±SD. *P < 0.05, ***P < 0.001, and ****P < 0.0001. NFkB, nuclear factor kappa B; Kim-1, kidney injury molecule 1; MCP-1, monocyte chemoattractant protein 1; TGF β, transforming growth factor β.

MRA Improved Endothelial Dysfunction in the Renal I/R-Injured Kidney

Given the crucial role of endothelial damage in AKI development,23 the effects of MRA on I/R-induced renal capillary damage were investigated. Capillary density, assessed by CD31 staining, was markedly reduced in I/R-induced renal injury, whereas I/R-induced damage was significantly attenuated with MRA pretreatment (Figure 4, A and B). Likewise, VEGF mRNA expression levels, measured by quantitative real-time PCR, were significantly suppressed in renal I/R injury mice, which were restored in MRA-treated mice (Figure 4C). Subsequently, the levels of adhesion molecules that are implicated in endothelial dysfunction and vascular disease,32 such as ICAM-1 and VCAM-1, were evaluated. Expression levels of ICAM-1 and VCAM-1 were significantly higher in renal I/R injury mice than that in sham mice, which were suppressed in MRA-treated mice (Figure 4, D and E). These findings indicate the potential role of the MR pathway in the endothelial dysfunction of I/R-induced renal injury mice.

Figure 4.

Figure 4

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MRA improved endothelial dysfunction in renal I/R-induced kidneys. (A) The panels depict representative microphotographs of CD31-positive capillaries. Sham, sham-operated mice; I/R, mice subjected to 22 minutes of renal ischemia; I/R+Esax, mice treated with esaxerenone. (B) The panel shows the quantitative data from the sham, I/R, and I/R+Esax pretreatment groups. CD31 staining was performed on frozen kidney sections to assess endothelial injury, capillary rarefaction, and degree of renal injury. CD31-positive capillary rarefaction was assessed semiquantitatively using a score from 0 to 4, wherein higher scores indicated a greater degree of PTC loss. (C) Kidney VEGF mRNA levels were assessed as a marker of kidney endothelial damage using qRT-PCR. (D) ICAM-1 and (E) VCAM-1 mRNA levels were quantified using qRT-PCR. ICAM-1 and VCAM-1 expression levels were higher in the kidneys of renal I/R injury mice than in those of sham mice. The mRNA levels of both markers decreased in MRA-pretreated mice. Bar=mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001. CD31, cluster of differentiation 31; ICAM-1, intercellular adhesion molecule 1; PTC, peritubular capillary; VCAM-1, vascular cell adhesion molecule 1; VEGF, vascular endothelial growth factor.

Effects of MRA on the RAGE/Rac1/MR Pathway in HMGB1-Treated HUVECs

Because experiments with the animal model suggest that MRA could reduce I/R-induced renal dysfunction and structural injury by suppressing the HMGB1/RAGE and Rac1/MR pathways, the direct interaction between these pathways were investigated using HUVECs, and MR nuclear trafficking was evaluated by immunofluorescence staining. Our results showed that HMGB1 supplementation significantly enhanced MR translocation into the nucleus (Figure 5A), whereas the absence of HMGB1 treatment resulted in a predominant MR distribution in the cytoplasm. Immunofluorescence staining revealed that RAGE-Apt and MRA pretreatments suppressed MR nuclear translocation (Figure 5A). In addition, immunoblotting demonstrated that HMGB1 substantially increased GTP-bound Rac1 expression in HUVECs, which was reduced by pretreatment with MRA or RAGE-Apt (Figure 5B).

Figure 5.

Figure 5

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MR-Rac1 pathway in HMGB1-treated HUVECs. (A) MR expression was investigated in HUVECs. HUVECs were pretreated with 10 nM esaxerenone and 100 nmol/L RAGE-Apt, Ctrl-Apt, or PBS for 24 hours before being exposed to HMGB1 (100 ng/ml) for 30 minutes. Untreated cells were used as controls. The MR was stained green with the Alexa-Fluor 488 probe, and cell nuclei were stained blue with DAPI. (B) The upper panel depicts a Western blot of GTP-bound Rac1, which was analyzed with proteins extracted from HUVECs. Upper panel: GTP-bound Rac1; lower panel: total Rac1 as a control. The lower panel displays the corresponding densitometric analysis. Bar=mean±SD. ***P < 0.001 and ****P < 0.0001. Ctrl-Apt, control-aptamer; DAPI, 4′,6-diamidino-2-phenylindole; HUVEC, human umbilical vein endothelial cell; PBS, phosphate-buffered saline; RAGE-Apt, RAGE aptamer.

The effect of MRA on MCP-1 and NF-κB expressions was also studied in HUVECs. HMGB1 supplementation resulted in significantly increased MCP-1 and NF-κB expressions in HUVECs. However, these measurements were lower in MRA-treated HUVECs than in those incubated with HMGB1 (Figure 6, A and B). Similarly, MCP-1 and NF-κB expression levels were reduced with RAGE-Apt pretreatment, but not altered by Ctrl-Apt pretreatment (Figure 6, A and B).

Figure 6.

Figure 6

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Effects of a MRA on the HMGB1/RAGE pathway in HUVECs. (A) MCP-1 and (B) NF-κB mRNA levels were quantified using real-time PCR. HMGB1 supplementation upregulated MCP-1 and NF-κB expression levels in HUVECs. Interestingly, MCP-1 and NF-κB expression levels were reduced by MRA and RAGE-Apt pretreatment, but they were not altered by Ctrl-Apt. Bar=mean±SD. *P < 0.05 and **P < 0.01.

MRA Administration Prevented the AKI-CKD Transition

To investigate whether MRA could prevent AKI transition to CKD in an ischemic AKI model, histological and laboratory parameters were measured 7 days after I/R induction. Untreated mice developed renal dysfunction, evidenced by elevated BUN and serum Cr levels, which was not observed in MRA-treated mice (Figure 7, A and B). PAS staining exhibited cellular necrosis, loss of brush borders, and cast formation in I/R-induced renal injury in mice 24 hours and 7 days after I/R induction, which were improved with MRA treatment (Figure 7, C and D). Histological quantification of the Sirius red–positive area showed severe interstitial fibrosis in I/R injury mice. By contrast, renal fibrosis development was significantly inhibited in MRA-treated mice (Figure 7, E and F). The effect of MRA treatment on the survival rate of renal I/R injury mice was also investigated. Interestingly, renal I/R injury mice that received a vehicle exhibited survival rate of 43% 7 days after I/R injury, whereas a survival rate of 79% was observed in MRA-treated mice (Figure 7G). These results suggest that MRA administration could attenuate the development of chronic renal failure in an ischemic AKI model.

Figure 7.

Figure 7

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MRA treatment prevented the development of CKD after AKI. Seven days after renal ischemia induction, (A) BUN and (B) serum Cr levels were measured as indicators of renal function. (C) The panels depict representative renal histology stained with PAS and (D) Sirius red. (E) The ATN score was quantified from ten fields per mouse of PAS-stained slides (magnification ×200). (F) The fibrosis score was blindly quantified from eight fields per mouse of Sirius red–stained slides (magnification ×200). (G) A Kaplan–Meier plot of overall survival was established during the 7-day observation period. Untreated renal I/R injury mice demonstrated a survival rate of 43% 7 days after I/R injury, while MRA-treated mice exhibited a survival rate of 79%. In the postinjury treatment group, renal function was assessed by measuring (H) BUN and (I) serum Cr levels. I/R, mice administered vehicle daily from 24 hours after reperfusion; I/R+Esax, mice treated with esaxerenone. (J) The panels depict representative renal histology stained with PAS. (K) The panel shows the quantitative data from the I/R and I/R+Esax post-treatment groups. Bar=mean±SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.

To further investigate the timing of MRA administration, we conducted an additional study where MRA was administered after the induction of I/R injury. In this postinjury treatment group, we observed that MRA-treated mice still showed attenuated renal dysfunction compared with untreated mice. Specifically, BUN and serum Cr levels 7 days after I/R injury were lower in the MRA-treated group compared with untreated I/R injury mice (Figure 7, H and I). In mice that were administered MRA after I/R injury, the tubulointerstitial injury in PAS staining were improved compared with untreated mice (Figure 7, J and K). These results collectively suggest that MRA administration could attenuate the development of chronic renal failure in an ischemic AKI model, potentially through mechanisms that extend beyond its effects on initial injury severity.

Discussion

The present study involving renal I/R injury mice showed that renal MR was activated with elevated serum HMGB1, renal RAGE, and GTP-bound Rac1 levels, all of which were suppressed by MRA treatment. Similarly, renal I/R injury mice demonstrated renal dysfunction with tubulointerstitial injury; PTC loss; and increased MCP-1, NF-κB, TGF-β, and Kim-1 expressions, which were also ameliorated by MRA treatment. In vitro examination further showed that RAGE-Apt and MRA significantly inhibited HMGB1-induced Rac1 activation, MR translocation into the nucleus, and upregulation of MCP-1 and NF-κB expressions in HUVECs. Furthermore, 7 days after injury, renal I/R injury mice developed CKD, whereas MRA-treated mice showed no progression and had better mortality rates. In addition, mice administered MRA after I/R injury were observed to have attenuated renal dysfunction compared with untreated mice. Collectively, our results suggest that HMGB1 may play a crucial role in AKI and subsequent CKD development, at least in a part by inhibiting the Rac1/MR pathway through the interaction with RAGE (Figure 8).

Figure 8.

Figure 8

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Synergistic role of the HMGB1/RAGE and Rac1/MR pathways in the AKI environment. The interaction scheme of the HMGB1/RAGE and Rac1/MR pathways is illustrated. HMGB1, which was elevated in AKI, activated Rac1 using RAGE and MR through an aldosterone-independent pathway. In addition, we found that HMGB1 upregulated the expression of inflammation- and fibrosis-related genes, such as MCP-1, NF-κB, and TGF-β, thus contributing to AKI development and CKD progression.

The underlying mechanisms of AKI are multifactorial and interdependent, including hypoperfusion, hypoxia, inflammatory responses, and nephrotoxicity.33 HMGB1 has been implicated in the pathogenesis of AKI as a protein that is released from renal cells, particularly vascular and tubular cells, after the renal I/R injury, with its levels corresponding to tubular apoptosis and inflammatory response.13,34 Consistent with previous findings,13 our study demonstrated elevated HMGB1 levels in renal I/R injury mice. In this study, it was an unexpected result that MRA reduced HMGB1 secretion. There are several possible mechanisms that could explain this phenomenon. One is that MRA treatment may mitigate cell damage in the kidney during I/R injury, and HMGB1 is mainly released from dead or stressed cells, so the release of HMGB1 into the extracellular space decreased.35 Another possibility is that MRA treatment may have reduced the levels of inflammatory mediators, such as MCP-1 and NF-κB, and suppressed the activation of immune cells that secrete HMGB1 as part of the inflammatory cascade.

Despite recent advances in understanding the molecular mechanisms of AKI, effective pharmacological interventions remain limited in clinical trials. Over the past decade, numerous preclinical studies have highlighted the protective effects of MRAs on AKI and CKD progression.19,20,36,37 Although aldosterone excess has been the established mechanism of MR activation, alternative pathways have also been suggested. Shibata et al. recently discovered that Rac1 mediated the ligand-independent MR activation in salt-induced organ damage.38 Our previous and present studies support the role of RAGE-induced Rac1 activation in eliciting podocyte damage and promoting aldosterone-independent MR activation.21 Because MR activation has been reported in diabetes and CKD,39,40 RAGE signaling may represent a key process in aldosterone-independent MR activation and consequent tissue damage.41 Further studies are required to confirm this assumption.

In many tissues, MR activation has been shown to stimulate multiple pathways of inflammation and fibrosis in other organs by the production of plasminogen activator inhibitor-1, TGF-β, interleukin-6, and MCP-1.42 This aligns with our findings of elevated MCP-1, NF-κB, and TGF-β gene expression levels in renal I/R injury mice. Similar findings were observed in our in vitro experiments using HUVECs. Notably, MRA pretreatment significantly reduced interstitial fibrosis and improved survival rates in both experiments. These findings suggest that the HMGB1/RAGE pathway may contribute to chronic tissue damage by prolonged inflammation and that MRA not only ameliorates acute renal injury, but also potentially prevents CKD progression. In clinical practice, patients with heart failure are prone to developing AKI, likely from ischemic changes related to hypoperfusion or hypo-oxygenation of tissues. Many patients with heart failure are treated with MRAs. Spironolactone is mostly held or stopped in the event of acute decompensation of heart failure, and this is also the time when these patients normally develop AKI. Although we did not directly investigate the effects in patients with heart failure, there is emerging evidence from clinical studies that suggests potential renoprotective effects of MRA. The Eplerenone Post–Acute Myocardial Infarction Heart Failure Efficacy and Survival Study trial, which studied eplerenone in patients with heart failure after myocardial infarction, showed a reduction in the incidence of hyperkalemia and renal dysfunction compared with placebo.43 A retrospective analysis by Beldhuis et al. suggested that continuation of MRA during acute heart failure hospitalization was associated with lower risks of worsening renal function and all-cause mortality.44 However, it should be noted that these findings are not definitive, and more targeted clinical research is needed in the future.

Renal microvascular injury plays a crucial role in the development and progression of ischemic AKI.45 PTC loss, in particular, induces hypoxia and inflammatory reactions in tubulointerstitial tissues, thereby promoting renal scarring and fibrosis.46 Our study showed that renal I/R injury induced PTC loss, evidenced by CD31 staining, while MRA pretreatment made attenuated PTC loss, indicating the potential endothelial protective effect of MRA in AKI. Moreover, VEGF expression levels were found to be moderately reduced after I/R injury and restored after MRA treatment, which was consistent with the study by Yuan et al. of an acute nephrotoxic AKI model.47 Although VEGF loss may be attributed to the reduction in proximal tubular contents,48 its preserved expression may also be significant in the postreperfusion repair. Further investigation is also needed to clarify these relationships.

While this study focused on the HMGB1/RAGE pathway in AKI, it is important to note that HMGB1 is also known as a TLR4 ligand, which is potentially involved in the HMGB1/TLR4 pathway of renal I/R injury in mice.10 Further research into the interaction between RAGE and TLRs is warranted to explore this possibility. Furthermore, the mechanism underlying the unexpected inhibition of HMBG1 increase and RAGE overexpression by MRA requires further elucidation. A possible explanation could be the positive feedback mechanism by which RAGE downstream signaling upregulates RAGE expression, as reported in our previous study.49 Although it could not be performed in this study, one of the cleanest ways to prove the involvement of a specific pathway in developing AKI or transmission to CKD is using RAGE knockout mice. This is one of the biggest limitations of our study.

In conclusion, our findings suggest associations between MRA treatment, changes in I/R-induced AKI markers, and potential influences on CKD progression by inhibiting Rac1/MR activation through the suppression of the HMGB1/RAGE pathway. These observations indicate that MRA and RAGE-Apt might be interesting targets for future research into potential therapeutic strategies for I/R-induced AKI and subsequent CKD.

Acknowledgments

We thank Terumi Shibata for their excellent technical assistance. We also thank the Laboratory of Molecular and Biochemical Research, the Laboratory of Biomedical Research Resources, the Laboratory of Morphology and Image Analysis, the Center for Biomedical Research Resources, and the Research Support Center at Juntendo University Graduate School of Medicine for technical assistance.

Disclosures

Disclosure forms, as provided by each author, are available with the online version of the article at http://links.lww.com/KN9/A808.

Funding

K. Wakabayashi: JSPS KAKENHI (JP23K07706).

Author Contributions

Conceptualization: Tomoyuki Otsuka, Yusuke Suzuki, Seiji Ueda, Keiichi Wakabayashi, Sho-ichi Yamagishi.

Data curation: Tomoyuki Otsuka.

Formal analysis: Tomoyuki Otsuka.

Investigation: Tomohito Gohda, Yuichiro Higashimoto, Takashi Kobayashi, Takanori Matsui, Maki Murakoshi, Hajime Nagasawa, Masami Nakata, Teruyuki Okuma, Tomoyuki Otsuka, Keiichi Wakabayashi.

Methodology: Tomoyuki Otsuka, Seiji Ueda.

Project administration: Yusuke Suzuki, Seiji Ueda.

Resources: Yuichiro Higashimoto, Takanori Matsui, Sho-ichi Yamagishi.

Supervision: Yusuke Suzuki, Seiji Ueda, Keiichi Wakabayashi, Sho-ichi Yamagishi.

Validation: Seiji Ueda.

Writing – original draft: Tomoyuki Otsuka, Seiji Ueda, Keiichi Wakabayashi.

Writing – review & editing: Yusuke Suzuki, Seiji Ueda, Sho-ichi Yamagishi.

Data Sharing Statement

All data were generated and analyzed at Juntendo University. Derived data supporting the findings of this study are available from the corresponding author on request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All data were generated and analyzed at Juntendo University. Derived data supporting the findings of this study are available from the corresponding author on request.

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