Fig 1.
Mefunidone attenuated tubulointerstitial injury and extracellular matrix (ECM) deposition in the obstructive kidneys after UUO at 14 days.
A. Mefunidone attenuated tubulointerstitial injury in the obstructive kidneys (HE staining of rats’ kidney sections, ×200). The column section indicated the interstitial injury score of the obstructed kidneys in each group. B. Mefunidone attenuated the deposition of ECM in the obstructive kidneys (Masson staining of rats’ kidney sections, ×200). The column section indicated the interstitial fibrosis score of the obstructed kidneys in each group. C. Mefunidone attenuated the deposition of ECM in the obstructive kidneys (Sirius red staining under polarized light; in green, yellow, or orange light, ×200). The column section indicated the Sirius red staining positive area of the obstructed kidneys in each group. All data were presented as means±SD, n = 5. (*p<0.05 compared to the sham group; #p<0.05 compared to the UUO group; ※p<0.05 compared to the PFD group.)
Fig 2.
Mefunidone decreased collagen I, collagen III, α-smooth muscle actin (α-SMA) and fibronectin (FN) expression in the obstructive kidneys after UUO at 14 days.
A. Mefunidone affected collagen I (left) and collagen III (right) protein expression in the obstructed kidneys stained by immunohistochemistry (×200). B. The histogram was the semi-quantitative analysis result of collagen I and collagen III protein expression in the kidneys. C. The histogram was the relative mRNA expression of collagen I and collagen III in the kidneys. D. Mefunidone decreased the protein expressions of α-SMA and FN in the obstructed kidneys as shown by Western blot. The column section indicated the relative expression level of the obstructive kidneys in each group. All data were presented as means±SD, n = 5. (*p<0.05 compared to the sham group; #p<0.05 compared to the UUO group.)
Fig 3.
Mefunidone inhibited TGF-β1-induced NRK-49F cell proliferation and α-SMA protein expression.
A. Mefunidone and pirfenidone (PFD) decreased NRK-49F cell viability in a dose-dependent manner measured by bromodeoxyuridine (BrdU) incorporation. The effect of Mefunidone and PDF on cell death of NRK-49F was assessed by LDH Cytotoxicity Assay Kit. B. Mefunidone reduced α-SMA protein expression in NRK-49F cells shown by Western blot analysis. The column section indicated the relative expression level of each group. The data were presented as means±SD, n = 3. (*p<0.05 compared to the control group; #p<0.05 compared to the TGF-β1 group.)
Fig 4.
Mefunidone attenuated leukocyte infiltration in the obstructive kidneys after UUO at 3, 7 or 14 days.
Leukocytes were stained as CD3 by immunohistochemistry in the obstructive kidneys (×200). The column section indicated the numbers of CD3 positive cells/HP of each group. The data were presented as means±SD, n = 5. (*p<0.05 compared to the sham group; #p<0.05 compared to the UUO group.)
Fig 5.
Mefunidone attenuated macrophage infiltration in the obstructive kidneys after UUO at 3, 7 or 14 days.
Macrophages were stained as CD68 by immunohistochemistry in the obstructive kidneys (×200). The column section indicated the numbers of CD68 positive cells/HP of each group. The data were presented as means±SD, n = 5. (*p<0.05 compared to the sham group; #p<0.05 compared to the UUO group.)
Fig 6.
Mefunidone inhibited cytokine release and affected ERK1/2, NF-κB and STAT3 signaling pathways in HK-2 cells and macrophages.
A. Mefunidone inhibited cytokine release in HK-2 cells culture supernatants using enzyme-linked immunosorbent assay (ELISA). B. Mefunidone inhibited cytokine release in macrophages culture supernatants using ELISA. C and E. Mefunidone affected the phosphorylation of ERK1/2, NF-κB and STAT3 stimulated with TNF-α in HK-2 cells. The inhibitor was PD98059 (10−6μM), BAY (10μM) or AG490 (150μM) respectively. The columnar section was the optical density of the gels tested by Western blot. D and G. Mefunidone affected the phosphorylation of ERK1/2, NF-κB and STAT3 stimulated with LPS in macrophages. The inhibitor was PD98059 (10−6μM), BAY (10μM) or AG490 (150μM) respectively. The columnar section was the optical density of the gels tested by Western blot. F. Assays of luciferase activity were performed and pNF-κB-luciferase activity was normalized to pRL-SV40 luciferase activity in HK-2 cells. The data were presented as means±SD, n = 3. (*p<0.05 compared to the control group; #p<0.05 compared to the TNF-α or LPS group; ※p<0.05 compared to the DXM group.)