The authors have declared that no competing interests exist.
Conceived and designed the experiments: SMY YCL SMK KFH. Performed the experiments: SMY YCL SMK. Analyzed the data: SMY KFH YCL AC SMK. Contributed reagents/materials/analysis tools: JMC CLH LKC. Wrote the manuscript: SMY KFH AC SMK.
The pathogenesis of focal segmental glomerulosclerosis (FSGS) is considered to be associated with oxidative stress, mononuclear leukocyte recruitment and infiltration, and matrix production and/or matrix degradation, although the exact etiology and pathogenic pathways remain to be determined. Establishment of a pathogenesis-based therapeutic strategy for the disease is clinically warranted. Citral (3,7-dimethyl-2,6-octadienal), a major active compound in
Focal segmental glomerulosclerosis (FSGS) manifests with heavy proteinuria in association with focal, but progressive, glomerular sclerosis in the kidney [
Although the etiology and pathogenesis of FSGS are poorly understood, its pathogenic pathways may involve oxidative stress [
Citral (3,7-dimethyl-2,6-octadienal), a major active compound in
All animal experiments were performed with the ethical approval of the Institutional Animal Care and Use Committee of The National Defense Medical Center, Taiwan and according to the ethical rules in the NIH
Fruits of
A progressive type of mouse FSGS model was used, particularly characterized by EPHLs, a key histopathology index of progression of FSGS, peri-glomerular inflammation, and progressive glomerular hyalinosis/sclerosis [
Renal tissues were formalin-fixed, embedded in paraffin, and sections (4 µm) prepared and stained with hematoxylin and eosin (H&E) for renal histopathology as described previously [
Superoxide anion levels in serum, urine, and kidney tissues were determined as described previously [
Cytoplasmic and nuclear proteins from renal tissues were prepared using a kit (Active Motif, Tokyo, Japan) according to the manufacturer’s instructions and target proteins detected by immunoblotting using goat antibodies against mouse Nrf2 or p47phox (Santa Cruz Biotechnology, Santa Cruz, CA) or rabbit antibodies against mouse NQO1 (Abcam), caspase-3, caspase-8, or caspase-9 (all from Cell Signaling Technology, MA) or Bcl-2 or Bax (both from Santa Cruz Biotechnology), then with HRP-conjugated rabbit anti-goat IgG antibodies or goat anti-rabbit IgG antibodies (both from Santa Cruz Biotechnology) as described previously [
RAW-BlueTM cells (murine macrophages RAW264.7 stably transfected with the NF-κB reporter gene) purchased from InvivoGen (San Diego, CA). LPS (from
The results for animal model are presented as the mean ± SEM. Comparison between two groups was performed using Student’s
As shown in
(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. *
As shown in
(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. *
Since the antioxidant signaling pathway can be activated by reduced production of NAD(P)H oxidase or by activation of Nrf2, we measured protein levels of p47phox, nuclear Nrf2 (activation), and HO-1 in the kidney to evaluate the effects of Citral on this pathway. As shown in
(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. *
The protective effect of Citral on podocytes was evaluated by detecting the glomerular expression of desmin, a marker of podocyte injury, using IHC. As shown in
(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. *
NF-κB activation and the subsequent induction of expression of various proteins, such as MCP-1, are implicated in the development of FSGS [
(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. *
Renal mononuclear leukocyte infiltration is seen in renal tissues of FSGS mice [
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. *
The anti-oxidative and anti-inflammatory activities of Citral were examined using LPS-activated RAW 264.7 macrophages. The LPS-induced increase in ROS production was reduced by incubation with Citral (10 µg/ml) or NAC (10 mM), a potent antioxidant, 30 min before and during LPS stimulation (
(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. *
LPS can induce macrophage activation and the production of pro-inflammatory cytokines by the activation of various signaling pathways, including the mitogen-activated protein kinase (MAPK) signaling pathways [
(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. *
Our study demonstrated that Citral, a purified major active component of
First, we showed that Citral administration inhibited the increase in ROS and NO production and p47phox levels seen in FSGS mice and activated the Nrf2 signaling pathway involving increasing expression of its downstream molecules NQO1 and HO-1 during the early developmental stage of this FSGS model, thus contributing to the beneficial effects of Citral on the treated mice (
Second, oxidative stress and inflammation are common features of chronic kidney disease [
Podocyte injury has been involved in the pathogenesis of FSGS [
In summary, our results suggest that Citral may have renoprotective potential for renal inflammation and fibrosis in FSGS, based on its anti-oxidant, anti-apoptotic and anti-inflammatory effects. Further studies on its systemic side effects are warranted before it can be considered for a preclinical validation.
We thank Bo-Chung Chen for her technical assistance.