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

Abstract

Key Points:

1. 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.

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

3. 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.

Graphical abstract

Figure 1

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.

Figure 2

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.

Figure 3

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.

Figure 4

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.

Figure 5

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.

Figure 6

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.

Figure 7

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.

Figure 8

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.