Korean J Physiol Pharmacol.  2018 Nov;22(6):661-670. 10.4196/kjpp.2018.22.6.661.

Fimasartan attenuates renal ischemia-reperfusion injury by modulating inflammation-related apoptosis

• Cho JH
• Choi SY
• Ryu HM
• Oh EJ
• Yook JM
• Ahn JS
• Jung HY
• Choi JY
• Park SH
• Kim CD
• Kim YL

• KMID: 2430088
• DOI: http://doi.org/10.4196/kjpp.2018.22.6.661

Abstract

Fimasartan, a new angiotensin II receptor antagonist, reduces myocyte damage and stabilizes atherosclerotic plaque through its anti-inflammatory effect in animal studies. We investigated the protective effects of pretreatment with fimasartan on ischemia-reperfusion injury (IRI) in a mouse model of ischemic renal damage. C57BL/6 mice were pretreated with or without 5 (IR-F5) or 10 (IR-F10) mg/kg/day fimasartan for 3 days. Renal ischemia was induced by clamping bilateral renal vascular pedicles for 30 min. Histology, pro-inflammatory cytokines, and apoptosis assays were evaluated 24 h after IRI. Compared to the untreated group, blood urea nitrogen and serum creatinine levels were significantly lower in the IR-F10 group. IR-F10 kidneys showed less tubular necrosis and interstitial fibrosis than untreated kidneys. The expression of F4/80, a macrophage infiltration marker, and tumor necrosis factor (TNF)-α, decreased in the IR-F10 group. High-dose fimasartan treatment attenuated the upregulation of TNF-α, interleukin (IL)-1β, and IL-6 in ischemic kidneys. Fewer TUNEL positive cells were observed in IR-F10 compared to control mice. Fimasartan caused a significant decrease in caspase-3 activity and the level of Bax, and increased the Bcl-2 level. Fimasartan preserved renal function and tubular architecture from IRI in a mouse ischemic renal injury model. Fimasartan also attenuated upregulation of inflammatory cytokines and decreased apoptosis of renal tubular cells. Our results suggest that fimasartan inhibited the process of tubular injury by preventing apoptosis induced by the inflammatory pathway.

Keyword

Apoptosis; Fimasartan; Inflammatory cytokine; Ischemia reperfusion injury

MeSH Terms

Animals
Apoptosis*
Blood Urea Nitrogen
Caspase 3
Constriction
Creatinine
Cytokines
Fibrosis
In Situ Nick-End Labeling
Interleukin-6
Interleukins
Ischemia
Kidney
Macrophages
Mice
Muscle Cells
Necrosis
Plaque, Atherosclerotic
Receptors, Angiotensin
Reperfusion Injury*
Tumor Necrosis Factor-alpha
Up-Regulation
Caspase 3
Creatinine
Cytokines
Interleukin-6
Interleukins
Receptors, Angiotensin
Tumor Necrosis Factor-alpha

Figure

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  Fig. 1 Experimental protocol and grouping of mice.After a minimal flank incision, renal pedicles were occluded completely for 30 min using a micro aneurysm clamp. Sham operation was performed with an identical surgical method except for clamping of the renal pedicles.

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  Fig. 2 Serum blood urea nitrogen (BUN), creatinine (Cr), and Neutrophil gelatinase-associated lipocalin (NGAL) levels in mice.Ischemia-reperfusion injury (IRI)-induces increases of (A) BUN, (B) Cr and (C) NGAL compared to control groups. Pretreatment with 10 mg/kg/day fimasartan attenuates the increase of BUN and Cr levels after IRI. The increased level of NGAL after IRI also significantly decreased after fimasartan treatment. Data are expressed as means±SEM (normal control, NC, n=5; sham operation, Sham, n=6; sham operation with 10 mg/kg/day fimasartan, Sham-F10, n=6; IR, n=8; IR with 5 mg/kg/day fimasartan, IR-F5, n=8; IR with 10 mg/kg/day fimasartan, IR-F10, n=8).

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  Fig. 3 Periodic acid-Schiff (PAS) and Masson's trichrome staining after ischemia-reperfusion injury (IRI).(A and G) Normal control (NC). (B and H) Sham operation (Sham). (C and I) Sham operation with 10 mg/kg/day fimasartan (Sham-F10). (D and J) IRI (IR). (E and K) IRI with 5 mg/kg/day fimasartan (IR-F5). (F and L) IRI with 10 mg/kg/day fimasartan (IR-F10). (M) Semi-quantitative assessment of the renal fibrosis area. Data are expressed as means±SEM. Tubular damage, loss of the brush border, and cast formation was significantly increased in the IR compared to the NC, Sham, and Sham-F10 groups, whereas the damages were decreased in the fimasartan-treated groups. Arrows indicate injured tubules and loss of brush border, and asterisks point to cast formation (NC, n=5; Sham, n=6; Sham-F10, n=6; IR, n=8; IR-F5, n=8; IR-F10, n=8).

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  Fig. 4 Immunohistochemistry staining after ischemia-reperfusion injury (IRI).(A and G) Normal control (NC). (B and H) Sham operation (Sham). (C and I) Sham operation with 10 mg/kg/day fimasartan (Sham-F10). (D and J) IRI (IR). (E and K) IRI with 5 mg/kg/day fimasartan (IR-F5). (F and L) IRI with 10 mg/kg/day fimasartan (IR-F10). Immunohistochemistry shows increased labeling intensity of F4/80 and tumor necrosis factor (TNF)-α at the interstitial area after IRI (D and J) compared to NC (A and G), Sham (B and H), and Sham-F10 (C and I). Fimasartan treatment reverses the changes in labeling intensities of F4/80 and TNF-α in the IRI groups (F and L). (M and N) Semi-quantitative assessment of F4/80-positive cells and TNF-α-positive areas. Arrows indicate F4/80 positive cells. Data are expressed as means±SEM. (NC, n=5; Sham, n=6; Sham-F10, n=6; IR, n=8; IR with 5 mg/kg/day fimasartan, IR-F5, n=8; IR with 5 mg/kg/day fimasartan, IR-F10, n=8).

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  Fig. 5 Enzyme-linked immunosorbent assay results of serum interleukin (IL)-6 levels.IL-6 is significantly increased after renal ischemia-reperfusion injury (IRI) compared to the control groups. Pretreatment with fimasartan (5 mg/kg/day, IR-F5; 10 mg/kg/day, IR-F10) before IRI attenuates the change in IL-6. Data are expressed as means±SEM (normal control. NC, n=5; sham operation, Sham, n=6; sham operation with 10 mg/kg/day fimasartan, Sham-F10, n=6; IR, n=8; IR-F5, n=8; IR-F10, n=8).

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  Fig. 6 mRNA levels of tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and transforming growth factor (TGF)-β in mouse kidney tissue.Quantitative real-time polymerase chain reaction analyses revealed significant increases in the expression of (A) TNF-α, (B) IL-1β, (C) IL-6, and (D) TGF-β mRNA in the ischemia-reperfusion (IR) compared to control groups. Fimasartan treatment (5 mg/kg/day, IR-F5; 10 mg/kg/day, IR-F10) significantly inhibits the upregulation of TNF-α, IL-1β, IL-6, and TGF-β levels compared to the IR group. Data are expressed as means±SEM (normal control, NC, n=5; sham operation, Sham, n=6; sham operation with 10 mg/kg/day fimasartan, Sham-F10, n=6; IR, n=8; IR-F5, n=8; IR-F10, n=8).

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  Fig. 7 Apoptosis of renal tubular epithelial cells.The TUNEL assay reveals that apoptosis induced in the ischemia-reperfusion (IR) group is significantly increased compared to that in the control groups. Apoptosis of renal tubule cells is decreased in the fimasartan-pretreated groups (5 mg/kg/day, IR-F5; 10 mg/kg/day, IR-F10) compared to the IR group.

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  Fig. 8 Immunohistochemistry reveals fimasartan pretreatment suppresses ischemia-reperfusion injury (IRI)-induced apoptosis in tubular epithelial cells.(A) Normal control. (B) Sham operation. (C) Sham operation with 10 mg/kg/day fimasartan. (D) IRI. (E) IRI with 5 mg/kg/day fimasartan. (F) IRI with 10 mg/kg/day fimasartan. IR significantly increased the number of apoptotic cells compared with that in the sham group, which was significantly reduced by fimasartan. Arrows indicate TUNEL-positive cells in renal tubule.

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  Fig. 9 Activity of caspase-3, and expression of Bcl-2 and Bax in renal tubular cells following ischemia-reperfusion injury (IRI).(A) Caspase-3 activity is significantly increased in the IR compared to the normal control (NC), sham operation (Sham), and sham operation with 10 mg/kg/day fimasartan (Sham-F10) groups. Fimasartan-treatment after IR (5 mg/kg/day fimasartan, IR-F5, and 10 mg/kg/day fimasartan, IR-F10) diminishes the increases of caspase-3. (B) Immunoblotting shows significantly decreased Bcl-2 and increased Bax levels in IR compared to NC, Sham, and Sham-F10. Quantitation of (C) Bcl-2 and (D) Bax levels. Pretreatment with fimasartan reverses the changes in Bcl-2 and Bax expression. Data are expressed as means±SEM.

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