Olmesartan Attenuates Kidney Fibrosis in a Murine Model of Alport Syndrome by Suppressing Tubular Expression of TGFβ

Figures

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  Figure 1. Olmesartan ameliorates kidney fibrosis in Col4a3–/– mice. (a) Tissue morphology of kidney from WT, Col4a3–/–, and Col4a3-/-+Olm mice. Images from glomerulus (left) and tubulointerstitium (right) are presented. H&E, hematoxylin, and eosin staining; PAS, periodic acid–Schiff staining; MT, Masson’s trichrome staining. Note that, contrary to kidneys from the other groups, glomerular capillary lumens are barely patent (arrowheads) with shrinkage of mesangium (white asterisk) in Col4a3–/– mice. In Col4a3–/– mice, crescent are packing the Bowman’s space (arrows). Detachment of tubular epithelial cells from basement membrane (red arrowheads) and collagen deposits (white arrows) are also indicated. Scale bars, 50 μm. (b,c) Comparison of expression level for fibrosis markers determined by immunoblotting (b) and qPCR (c) from the kidney of WT, Col4a3–/–, and Col4a3-/-+Olm mice (n = 4 mice/group). β-actin was used as the endogenous control. (d,e) Representative images of immunohistochemical staining for collagen type 1 (d) and αSMA (d) in the kidney of WT, Col4a3–/–, and Col4a3-/-+Olm mice. Images from glomeruli (upper) and tubules (lower) are presented. Scale bars, 50 μm. * P < 0.05, ** P <…

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  Figure 2. Upregulation of TGFβ and activation of its downstreams in Col4a3–/– mice is attenuated by olmesartan. (a) Comparison of protein expression level TGFβ and its downstream determined by immunoblotting from the kidney of WT, Col4a3–/–, and Col4a3-/-+Olm mice (n = 4 mice/group). β-actin was used as the endogenous control. (b) Comparison of mRNA expression-level TGFβ1 determined by qPCR from the kidney WT, Col4a3–/–, and Col4a3-/-+Olm mice (n = 4 mice/group). β-actin was used as the endogenous control. (c) Representative images of immunohistochemical staining for TGFβ in the renal cortex of WT, Col4a3–/–, and Col4a3-/-+Olm mice. Scale bars, 50 μm. * P < 0.05, ** P < 0.01 versus WT mice; # P < 0.05, ## P < 0.01 versus Col4a3–/– mice by one-way ANOVA with Newman–Keuls multiple comparison test.

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  Figure 3. RAS activation in Col4a3–/– mice is counteracted by olmesartan. Comparison of protein expression level of RAS components determined by immunoblotting from the kidney of WT, Col4a3–/–, and Col4a3-/-+Olm mice (n = 4 mice/group). β-actin was used as the endogenous control. * P < 0.05, ** P < 0.01 versus WT mice; # P < 0.05, ## P < 0.01 vs. Col4a3–/– mice by one-way ANOVA with Newman–Keuls multiple comparison test.

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  Figure 4. Olmesartan attenuates enhanced phosphorylation of P38 in Col4a3–/– mice. Comparison of protein expression level of MAPKs determined by immunoblotting from the kidney of WT, Col4a3–/–, and Col4a3-/-+Olm mice (n = 4 mice/group). β-actin was used as the endogenous control. * P < 0.05 versus WT mice; # P < 0.05 versus Col4a3–/– mice by one-way ANOVA with Newman–Keuls multiple comparison test.

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  Figure 5. Olmesartan suppresses apoptosis and inflammation in the kidney of Col4a3–/– mice. (a) Comparison of protein expression level for molecules related to apoptosis determined by immunoblotting from the kidney of WT, Col4a3–/–, and Col4a3-/-+Olm mice (n = 4 mice/group). β-actin was used as the endogenous control. (b) Representative images of TUNEL (upper) and immunohistochemical staining for F4/80 (lower) in the kidney of WT, Col4a3–/–, and Col4a3-/-+Olm mice. (c) Comparison of mRNA expression level for inflammatory markers determined by qPRC from the kidney of WT, Col4a3–/–, and Col4a3-/-+Olm mice (n = 4 mice/group). β-actin was used as the endogenous control. (d) Comparison of protein expression level for CD68 and hemoxygenase-1 (HO-1) determined by immunoblotting from the kidney of WT, Col4a3–/–, and Col4a3-/-+Olm mice (n = 4 mice/group). β-actin was used as the endogenous control. Scale bars, 50 μm. * P < 0.05, ** P < 0.01 versus WT mice; # P < 0.05, ## P < 0.01 versus Col4a3–/– mice by one-way ANOVA with Newman–Keuls multiple comparison test.

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  Figure 6. Stimulation with Ang II activates downstreams of TGFβ in HK-2 cells. Comparison of protein expression level for RAS components, MAPKs, and downstreams of TGFβ in HK-2 after stimulation with vehicles or recombinant human Ang II (rhAng II) (n = 3−4/group). Data are representative more than three independent experiments. * P < 0.05, ** P < 0.01 versus vehicle group by Student’s T-test.

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  Figure 7. Olmesartan alleviates RAS activation in HK-2 cells stimulated with TGFβ. Comparison of protein expression level for RAS components, MAPKs, and downstreams of TGFβ in HK-2 cells. Cells were treated with vehicles, recombinant human TGFβ (rhTGFβ), Olmesartan medoxomil (Olm), or combination of rhTGFβ and Olm. (n = 3−4/group). Data are representative of more than three independent experiments. * P < 0.05, ** P < 0.01 versus vehicle group; # P < 0.05, ## P < 0.01 versus rhTGFβ group by one-way ANOVA with Newman–Keuls multiple comparison test.