Figure 3: L6H21 pretreatment prevents Ang II-induced fibrosis and signal activation in renal tubular epithelial cells.

The effects of the MD2 inhibitor L6H21 on Ang II-stimulated inflammatory responses in the renal tubular epithelial cell line, NRK-52E, were determined. NRK-52E were pretreated with the vehicle control DMSO (Ctrl) or L6H21 (2.5, 5.0, 10 μM) for 1 h, and stimulated with Ang II (1 μM) for different periods. (A) Representative Western blot analysis for protein markers of fibrosis, Col-1 (collagen 1), Col-4 (collagen 4), MMP-9 (metalloproteinase 9), and TGF-β (transforming growth factor β), GAPDH as loading control; n = 3 independent determinations. (B) The mRNA levels of TGF-β, Col-1 (collagen 1), Col-4, CTGF, and MMP-9 were detected by real-time qPCR; values normalized to house-keeping gene β-actin. (C) Representative Western blot analysis for p-ERK (phosphorylated ERK) and IκB from total cell lysate, GAPDH as loading addingol, ERK = total ERK; NF-κB p65 subunit detected from nuclear cell fraction, lamin B as loading control; n = 3 independent determinations. (D) The mRNA levels of IL-1β, IL-6 and TNF-α were detected by real-time qPCR; values normalized to house-keeping gene β-actin. (E) Representative immunofluorescent distribution of NF-κB p65 subunit (Texas-red streptavidin), top row; same cells counterstained with nuclear stain DAPI, bottom row, 200x magnification; n = 3 independent determinations. Data in B and D are reported as mean ± S.E.M. of n = 3, *P < 0.05, **P < 0.01, ***P < 0.001 versus Ang II-treated group; ^#P < 0.05 and ^##P < 0.01 versus Ctrl. For panels (A,C) the gels were run under the same experimental conditions. Shown are cropped gels/blots (The gels/blots with indicated cropping lines are shown in Supplementary Figure S6).