Figure 1.
Schematic representation of experimental protocol.
Cisplatin (CP) nephrotoxicity was induced in 6-week-old male Sprague-Dawley (SD) rats by intraperitoneal injection of CP on day 0. Groups of animals were administered either erlotinib (CP+E) or vehicle (an equivalent volume of saline) (CP+V) daily by oral gavage from day -1 to day 3. An additional five male SD rats were used as normal controls (NC). At 4 days after CP injection, all rats were sacrificed.
Table 1.
Effects of erlotinib on biochemical parameters of the study groups.
Figure 2.
Light microscopic findings in the study groups.
Representative images of tissues stained with (a–f) PAS or (g–i) Masson trichrome in a NC rat (a, d, g), a CP+V rat (b, e, h), and a CP+E rat (c, f, i). Original magnifications: (a–c) × 100, (d–i) × 400.
Table 2.
Morphological evaluation of luminal hyaline casts and tubulointerstitial damage of the study groups.
Figure 3.
Immunohistochemistry for PCNA, ED1, TUNEL, and caspase-3 in the study groups.
Representative pictures stained for (a–c) PCNA, (d–f) ED1, (g–i) TUNEL, and (j–l) caspase-3 in a NC rat (a, d, g, j), a CP+V rat (b, e, h, k), and a CP+E rat (c, f, i, l). Original magnifications: x 400.
Table 3.
Quantitative evaluation of immunohistochemistry for PCNA, ED1, TUNEL, and Caspase 3 of the study groups.
Figure 4.
Effects of erlotinib on gene expression levels for fibrogenic molecules, proinflammatory cytokines, apoptosis-regulatory molecules, and EGFR ligands in the study groups.
Real-time RT-PCR for genes encoding fibrogenic molecules (a), proinflammatory cytokines (b), apoptosis-regulatory molecules (c), and EGFR ligands including proHB-EGF and TGF-α (d) in each group. The horizontal dotted lines show the expression levels of the NC rats. Data are expressed as mean ± SEM (n = 5, 14, and 14 for the NC rats, the CP+V rats, and the CP+E rats, respectively). The values were normalized to the GAPDH transcript levels and then expressed as relative quantification. Mann–Whitney test: *P<0.05, **P<0.01, NS, not significant vs. CP+V.
Figure 5.
Effects of erlotinib on pro-apoptotic and anti-apoptotic protein in the study groups.
Representative western blot analysis for Bax, Bcl-2 and GAPDH (a). Densitometric analysis of western blot for Bax (b), Bcl-2 (c), and Bax/Bcl2 ratio (d) was performed using an image analyzer in each group. Data are expressed as mean ± SEM (n = 5, 14, and 14 for the NC rats, the CP+V rats, and the CP+E rats, respectively). The values were expressed after normalization to GAPDH expression and depicted as the percentage change from the average of normal controls. Mann-Whitney test: *P<0.01, **P<0.01 vs. NC, #P<0.05 vs. CP+V.
Figure 6.
Inhibitory effects of erlotinib on CP-induced PI3K-Akt and MAPK activation in cultured human proximal tubular cells.
Phosphorylated Akt (a), total Akt (b), phosphorylated MEK1 (c), and total MEK1 (d) were quantified using the Bio-Plex Suspension Array System. Data are mean ± SEM (n = 4). The values were expressed as the percentage of the mean value of the DMSO+Medium. Mann-Whitney test: *P<0.01 vs. DMSO+Medium, #P<0.05, ## P<0.01 vs. DMSO+CP. Abbreviations: DMSO+Medium, cultures of HK-2 cells without the addition of erlotinib or cisplatin; DMSO+CP, cultures of HK-2 cells stimulated by CP without preincubation with erlotinib; Erlotinib+CP, cultures of HK-2 cells stimulated by CP after preincubation with erlotinib.