Figure 1.
Urine protein, renal function, renal histopathology, and hyalinosis/sclerosis.
(A) Urine protein time-course study. (B) Serum BUN levels on days 7, 14, and 28. (C) Serum creatinine levels on days 7, 14, and 28. (D) Kidney histopathological evaluation by H&E staining on days 7, 14, and 28. The arrows indicate hyalinosis/sclerosis, and the rectangles EPHLs. (E) Immunohistochemical staining for renal Col-IV. In D and E, the original magnification was 400× and the scoring is shown on the right. In the histograms, the data are the mean±SEM for seven mice per group. *p < 0.05, **p < 0.01, ***p < 0.005. #, not detectable.
Figure 2.
(A-C) Superoxide anion levels in renal tissue (A), serum (B), and urine (C). (D) Kidney in situ ROS production demonstrated by DHE labeling. The arrows indicate positive staining cells. Original magnification, 400×. The scoring is shown on the right. (E, F) NO levels in serum (E) and urine (F). In the histograms, the data are the mean±SEM for seven mice per group. *p < 0.05, **p < 0.01, ***p < 0.005.
Figure 3.
Renal nuclear Nrf2 levels, cytosolic p47phox, NQO1 and HO-1 levels.
(A) Representative Western blot showing levels of cytosolic p47phox and NQO1 and nuclear Nrf2 in kidney tissues. (B-D) Quantification of the p47 phox/β-actin (cytosolic) ratio (B), the Nrf2/β-actin (nuclear) ratio (C), and the NQO1/β-actin (cytosolic) ratio (D). The horizontal dashed lines indicate levels in normal control mice. (E) HO-1 levels in the kidney. In B-E, the data are the mean±SEM for seven mice per group. *p < 0.05, **p < 0.01, ***p < 0.005.
Figure 4.
Podocyte injury and renal apoptosis in the glomerulus and tubule.
(A) Podocyte injury detected in glomeruli by immunohistochemical staining for desmin. The black arrows indicate podocytes. Original magnification, 400×. Scoring of desmin expression in renal tissue is shown on the right. (B) TUNEL staining in renal tissues at day 7, 14, and 28. Original magnification, 400×. The arrows indicate positively stained cells. The scoring is shown on the right. (C) Representative Western blot for the active forms of caspase-3, caspase-9, Bax and Bcl-2, with β-actin as the internal control. (D-F) Active caspase-3/β-actin ratio (D), active caspase-9/β-actin ratio (E), and Bax/Bcl-2 ratio (F). In the histograms, the data are the mean±SEM for seven mice per group. *p < 0.05, **p < 0.01, ***p < 0.005.
Figure 5.
Renal NF-κB activation and MCP-1 expression.
(A) Detection of NF-κB p65 by immunohistochemical staining. Original magnification, 400×. The arrows indicate positively stained cells. The scoring is shown on the right. (B) Western blot of MCP-1 levels in renal tissues, with β-actin as the internal control for cytosolic protein. (C) MCP-1/β-actin ratio. In the histograms, the data are the mean±SEM for seven mice per group. *p<0.05, ***p<0.005.
Figure 6.
Renal T cell and macrophage infiltration.
Detection of (A) CD3+ T cells or (B) F4/80+ monocytes/macrophages by immunohistochemical staining. The arrows indicate positively stained cells. Original magnification, 400×. The scoring is shown on the right. The data are the mean±SEM for seven mice per group. *p < 0.05, **p < 0.01, ***p < 0.005.
Figure 7.
In vitro ROS generation and inflammatory mediator expression.
(A) RAW 264.7 macrophages were incubated for 30 min with or without 10 µg/ml Citral or 10 mM N-acetyl cysteine (NAC), then for 0-4 h with or without addition of 1 µg/ml of LPS. ROS production was measured as the relative fluorescence intensity. (B) RAW 264.7 macrophages were incubated for 30 min with or without the indicated concentrations of Citral, then for 24 h with or without addition of 1 µg/ml of LPS, then NO generation in the culture medium was measured by the Griess reaction. (C) RAW-BlueTM cells were incubated for 30 min with or without the indicated concentration of Citral, then for 24 h with or without addition of 1 µg/ml of LPS, then secreted embryonic alkaline phosphatase activity was measured using QUANTI-BlueTM. (D-F) RAW 264.7 macrophages were incubated for 30 min with or without the Citral, then for 24 h with or without addition of 1 µg/ml of LPS, then IL-6 (D), TNF-α (E), and IL-1β (F) in the culture medium were measured by ELISA. The data are expressed as the mean ± SD for three separate experiments. *p < 0.05, **p < 0.01 compared to the LPS-treated group.
Figure 8.
In vitro MAPK phosphorylation.
(A) RAW 264.7 macrophages were incubated for 30 min with or without 10 µg/ml Citral, then for 0-60 min with or without addition of 1 µg/ml of LPS, then the phosphorylation levels of ERK1/2, JNK1/2, and p38 were measured by Western blotting. In B-D, the results in the phosphorylation levels of ERK1/2 (B), JNK1/2 (C), and p38 (D) are representative of those obtained in three separate experiments and the histogram shows the results for all three experiments expressed as the mean ± SD. *p < 0.05 compared to the corresponding group without Citral.