Targeting Mechanosensitive Piezo1 Alleviated Renal Fibrosis Through p38MAPK-YAP Pathway

Abstract

Renal fibrosis is the most common pathological manifestation of a wide variety of chronic kidney disease. Increased extracellular matrix (ECM) secretion and enhanced microenvironment stiffening aggravate the progression of renal fibrosis. However, the related mechanisms remain unclear. Here, we evaluated the mechanism by which ECM stiffness aggravates renal fibrosis. In the present study, renal mesangial cells (MCs) were cultured on polyacrylamide hydrogels with different stiffness accurately detected by atomic force microscope (AFM), simulating the in vivo growth microenvironment of MCs in normal kidney and renal fibrosis. A series of in vitro knockdown and activation experiments were performed to establish the signaling pathway responsible for mechanics-induced MCs activation. In addition, an animal model of renal fibrosis was established in mice induced by unilateral ureteral obstruction (UUO). Lentiviral particles containing short hairpin RNA (sh RNA) targeting Piezo1 were used to explore the effect of Piezo1 knockdown on matrix stiffness-induced MCs activation and UUO-induced renal fibrosis. An in vitro experiment demonstrated that elevated ECM stiffness triggered the activation of Piezo1, which increased YAP nuclear translocation through the p38MAPK, and consequently led to increased ECM secretion. Furthermore, these consequences have been verified in the animal model of renal fibrosis induced by UUO and Piezo1 knockdown could alleviate UUO-induced fibrosis and improve renal function in vivo. Collectively, our results for the first time demonstrate enhanced matrix stiffness aggravates the progression of renal fibrosis through the Piezo1-p38MAPK-YAP pathway. Targeting mechanosensitive Piezo1 might be a potential therapeutic strategy for delaying the progression of renal fibrosis.

Keywords: Piezo1; YAP; extracellular matrix secreation; matrix stiffness; renal fibrosis.

FIGURE 1

ECM stiffness activates MCs. MCs were cultured on hydrogels of different stiffness (2 kPa and 50 kPa) for 48 h. (A) Representative Western blot bands show expression levels of the myofibroblast marker α-SMA and ECM-associated proteins include of Collagen I and Fibronectin in cells cultured on soft and stiff hydrogels. The bar graphs show the results of semi-quantitative measurements of Fibronectin (B), Collagen I (C) and α-SMA (D). (n = 6; *p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 2

ECM stiffness activates MCs through YAP. MCs were cultured on hydrogels of different stiffness (2 and 50 kPa) for 48 h. (A) Representative Western blot bands show the protein expression level of YAP. (B) The bar graph shows the result of semi-quantitative measurement of YAP (n = 6; *p < 0.05, **p < 0.01, ***p < 0.001). (C) Representative photomicrographs show fluorescence staining with anti-α-SMA (red) and anti-YAP (green) antibodies in cells cultured on the soft and stiff hydrogels. Scale bar = 25 µm. (D) The percentage of cells with predominantly nuclear YAP staining. (p < 0.001). (E) Western blot bands show expression levels of Fibronectin, Collagen 1, α-SMA and CTGF after which MCs were incubated with Super-TDU at various concentrations on the stiff gel (50 kPa) for 24 h. The bar graph shows the results of semi-quantitative of Fibronectin (F), α-SMA (G) Collagen 1 (H) and CTGF (I) measurements (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 3

ECM stiffness promotes MAPK signaling pathway activation. (A) Representative western blot bands show the protein expression levels of p-p38, p38, p-ERK, ERK, p-JNK and JNK. (B)The bar graph shows the result of semi-quantitative measurement of p-p38/p38. (C) The bar graph shows the result of semi-quantitative measurement of p-ERK/ERK. (D) The bar graph shows the result of semi-quantitative measurement of p-JNK/JNK (n = 6; *p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 4

p38 inhibitor blocked YAP activation and ECM protein expression induced by enhanced hydrogel stiffness. MCs were treated with vehicle or a p38 inhibitor and cultured on hydrogels with different stiffness. (A) Representative Western blot bands showing YAP expression in each group. (B) Semi-quantitative measurement of YAP (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001). (C) Representative photomicrograph showing fluorescence staining of YAP in cells treated with vehicle and a p38 inhibitor grown on the stiff hydrogels. Scale bar = 25 µm. (D) The percentage of cells with predominantly nuclear YAP staining. (***, p < 0.001). (E) Representative western blot bands show the protein expression levels of Fibronectin, Collagen 1 and α-SMA after MCs were treated with vehicle or a p38 inhibitor and cultured on the soft and stiff hydrogels. The bar graph shows the result of semi-quantitative measurement of Fibronectin (F), Collagen I (G) and α-SMA(H) (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 5

Piezo1 knockdown reduced ECM protein levels. (A) Representative western blot bands of Piezo1 in MCs cultured on hydrogels with a stiffness of 2 kPa or 50 kPa for 48 h. (B) The bar graphs show the results of semi-quantitative measurement of Piezo1 (n = 6; *p < 0.05, **p < 0.01, ***p < 0.001). (C) MCs were transfected with shNC or sh Piezo1 and grown on soft and stiff gels. Representative western blot bands of Fibronectin, α-SMA and Collagen I. The bar graph shows the result of semi-quantitative measurement of Fibronectin (D), α-SMA (E) and Collagen I (F). (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 6

Piezo1 knockdown reduced YAP and p-p38 expression. MCs were transfected with shNC or sh Piezo1 and grown on soft and stiff gels. (A) Representative western blot bands of YAP, p-p38 and p38. Semi-quantitative measurement of YAP (B) and p-p38/p38 (n = 3 *p < 0.05, **p < 0.01, ***p < 0.001). (D) Representative photomicrographs show fluorescence staining with anti-YAP after MCs were transfected with shNC or sh Piezo1 and grown on stiff gels. Scale bar = 25 µm. (E) The percentage of cells with predominantly nuclear YAP staining. (p < 0.001).

FIGURE 7

p38 MAPK inhibitor reduced the increased YAP expression and ECM secretion induced by Piezo1 activator. (A) MCs were incubated with Yoda-1 and SB for 48 h on the soft gel. (A) Representative western blot bands represent the protein expression levels of Fibronectin, Collagen 1, α-SMA and YAP. The bar graph shows the result of semi-quantitative measurement of Fibronectin (B), α-SMA (C), Collagen I (D) and YAP (E). (n = 3; *p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 8

Piezo1 knockdown improved renal function in UUO mice. An animal model of renal fibrosis induced by UUO was established and a loss-of-function experiment was carried out by using shRNA of Piezo1 and shNC in UUO mice. shNC refers to empty vector without Piezo1 shRNA. Renal function is assessed by (A) plasma creatinine and (B) BUN levels. (n = 6; *p < 0.05, **p < 0.01, ***p < 0.001). (C) Masson staining reveals the excessive deposition of ECM (blue) in four different treatment groups. (D) The collagenvolume fraction in four different treatment groups (*p < 0.05, **p < 0.01, ***p < 0.001).

FIGURE 9

Piezo1 knockdown reduces the expression of ECM protein levels in UUO mice. An animal model of renal fibrosis induced by UUO was established and a loss-of-function experiment was carried out by using shRNA of Piezo1 and shNC in UUO mice. (A) Representative western blot bands show the protein expression of Fibronectin, Collagen I and α-SMA in the four different treatment groups. The bar graph shows the result of semi-quantitative measurement of Fibronectin (B), Collagen I (C) and α-SMA (D). (n = 4; *p < 0.05, **p < 0.01, ***p < 0.001). (E) Representative photomicrographs show immunohistochemical staining with Fibronectin and α-SMA after Piezo1 knockdown in UUO mice. scale bar indicates 50 μm.

FIGURE 10

Piezo1 knockdown alleviates renal fibrosis through p38MAPK-YAP pathway. An animal model of renal fibrosis induced by UUO was established and a loss-of-function experiment was carried out by using shRNA of Piezo1 and shNC in UUO mice. (A) Representative western blot bands show the protein expression of Piezo1, p-p38, p38 and YAP in four different treatment groups. The bar graph shows the result of semi-quantitative measurement of Piezo1 (B), p-p38/p38 (C) and YAP (D) (n = 4; *p < 0.05, **p < 0.01, ***p < 0.001). (E) Relative mRNA expression of Piezo1 in four different treatment groups. (n = 5, *p < 0.05, **p < 0.01, ***p < 0.001).