1. Candesartan suppresses chronic renal inflammation by a novel antioxidant action independent of AT1R blockade 
Shan Chen, Yan Ge, Jin Si, Abdalla Rifai, Lance D. Dworkin, Rujun Gong 
Kidney International 
Volume 74, Issue 9, Pages (November 2008)
DOI: /ki
Copyright © 2008 International Society of Nephrology Terms and Conditions

2. Figure 1
Candesartan suppresses TNF-induced mRNA expression and protein production of MCP-1 and RANTES in a dose- and time-dependent fashion. HKCs were treated with TNF-α (2ng/ml), Cand (10−5M), or TNF in combination with Cand in varying concentrations (10−5, 10−7, 10−9M in a, c) for 12h (a, c) or different time intervals as indicated (b, d). After treatment, conditioned media and mRNA were prepared and subjected to ELISA (c, d, e) or semiquantitative (a) and real-time (b) RT-PCR. In (b) and (d, e), *P<0.05 vs TNF alone treated cells at each time point; in (c), *P<0.05 vs all other groups (n=5).
Kidney International  , DOI: ( /ki )
Copyright © 2008 International Society of Nephrology Terms and Conditions

3. Figure 2
Candesartan AT1R antagonizing activity is not required for its suppressive effect on TNF-induced MCP-1 and RANTES expression. HKCs were transfected with AT1R-specific or scrambled siRNA followed by different treatments as indicated in Figure 1 for 12h (b) or 24h (c). (a) Immunocytochemistry demonstrated that AT1R expression (white arrow heads) was not affected in cells transfected with scrambled siRNA, but largely silenced by AT1R-specific siRNA; (b) Immunoblot analysis showed that AT1R abundance was reduced after RNAi, whereas the suppressive effect of Cand (10−5M) on mRNA expression of MCP-1 and RANTES was not affected. (c) The suppressive effect of Cand (10−5M) on protein production of MCP-1 and RANTES was not affected after AT1R knockdown. *P<0.05, #P<0.05 vs TNF alone treated cells transfected with the same siRNA (n=6).
Kidney International  , DOI: ( /ki )
Copyright © 2008 International Society of Nephrology Terms and Conditions

4. Figure 3
Candesartan obliterates TNF-elicited NF-κB activity independently of AT1R blockade. (a) HKCs were co-transfected with siRNA or the 3 × κB luciferase reporter gene construct followed by different treatments as indicated in Figure 2 for 12h. Cells were collected and cell lysates processed for luciferase assay. *P<0.05 vs other groups transfected with the same siRNA (n=6). (b) After different treatments, nuclear extracts were prepared from RNA silenced cells or control cells followed by EMSA by using oligonucleotide probes containing the consensus sequence for the NF-κB-binding site. For supershift assay, anti-p65 antibody or control preimmune immunoglobulin (Ig) was applied. (c) After RNAi, cells were treated as indicated for 3h and then prepared for chromatin immunoprecipitation (ChIP) assay using anti-RelA/p65 antibody. The amount of MCP-1 and RANTES DNA that coprecipitated with transcription factor RelA/p65 was estimated by PCR and agarose gel electrophoresis, followed by densitometry. *P<0.05 vs TNF alone treated cells transfected with the same siRNA (n=6). (d) After RNAi, cells were treated as indicated for 3h and then collected for immunoblot analysis for different molecules, followed by densitometry. *P<0.05 vs TNF alone treated cells transfected with the same siRNA (n=6).
Kidney International  , DOI: ( /ki )
Copyright © 2008 International Society of Nephrology Terms and Conditions

5. Figure 4
ROS are essential and sufficient for activation of NF-κB and induced expression of its target genes. (a) HKCs were labeled with DCF (green) and stimulated with TNF (2ng/ml) for varying intervals followed by fluorometric analysis. Results were determined by fluorometry and presented as percent change of light units from baseline as folds of normal controls (n=6). (b) HKCs were labeled with DCF and treated with TNF (2ng/ml), an ROS scavenger pyrrolidine dithiocarbamate (PDTC, 7.5μM), hydrogen peroxide 100μM or in combination for 15min followed by fluorometric analysis. #P<0.05 vs nontreated cells; *P<0.05 vs TNF alone treated cells (n=6). (c) HKCs received various treatments for 15min followed by GSH and GSSG assay. #P<0.05 vs nontreated cells; *P<0.05 vs TNF alone treated cells (n=6). (d) HKCs were treated for 1h followed by immunoblot analysis of the cell lysates for different molecules. (e) HKCs were treated for 12h followed by mRNA extraction and profiling of MCP-1 and RANTES expression by real-time RT-PCR. #P<0.05 vs nontreated cells; *P<0.05 vs TNF alone treated cells (n=6).
Kidney International  , DOI: ( /ki )
Copyright © 2008 International Society of Nephrology Terms and Conditions

6. Figure 5
Candesartan is a potent antioxidant that suppresses ROS generation and corrects redox imbalance induced by TNF or hydrogen peroxide. (a) HKCs were labeled with DCF and then treated with TNF (2ng/ml) or Cand (10−5M) for 15min before microscopic detection of DCF fluorescence. (b–e) Cells were stimulated with TNF (2ng/ml), or hydrogen peroxide (100μM) and cotreated with ROS scavengers N-acetylcysteine (NAC, 1mM), pyrrolidine dithiocarbamate (PDTC, 7.5μM) or Cand (10−5M or different concentrations of 10−5, 10−7, 10−9M) for 15min (b, d, e) or the indicated time (c) followed by fluorometric analysis (b, c) or GSH and GSSG assay (d, e). In (b, d, e) *P<0.05 vs TNF other groups treated with TNF; #P<0.05 vs other groups treated with hydrogen peroxide (n=6). In (c) *P<0.05 vs TNF alone treated cells (n=6). (f) HKCs were treated with TNF (2ng/ml) or Cand at indicated concentrations for 15min and then labeled with dihydrorhodamine. Final ROS levels were determined by flow cytometry.
Kidney International  , DOI: ( /ki )
Copyright © 2008 International Society of Nephrology Terms and Conditions

7. Figure 6
The antioxidant effect is unique to Cand and does not involve AT1R or PPARγ. (a) HKCs were subjected to siRNA transfection as described in Figure 2, and labeled with DCF before stimulation with TNF (2ng/ml) in the presence or absence of Cand (10−5M) for 15min. Cells were then collected and processed for fluorometry and GSH/GSSG measurement. DCF fluorescence values were relative to nontreated control cells. *P<0.05 vs Cand-treated cells transfected with the same siRNA (n=6). (b) Swiss R3T3 cells, which lack AT1R, were labeled with DCF and stimulated with TNF (2ng/ml) in the presence of Cand or vehicle at indicated concentrations for 15min before fluorometric analysis (n=6). (c) HKCs were labeled with DCF and stimulated for 15min with TNF (2ng/ml) alone or in the presence of Cand, irbesartan (Irbe), or losartan (Los) at different concentrations (10−5, 10−7, 10−9M) or GW9662 (GW; 2μM) before fluorometric analysis. *P<0.05 vs Cand-treated cells (n=6).
Kidney International  , DOI: ( /ki )
Copyright © 2008 International Society of Nephrology Terms and Conditions

8. Figure 7
Candesartan suppresses chronic renal inflammation and tubular NF-κB activity in kidneys from SHR rats in a dose-dependent manner. (a–d) High and ultrahigh dose Cand had equal effect on (a) controlling whole body weight, (b) correcting hypertension (measured at the 14th month), (c) attenuating proteinuria, and (d) improving kidney function (measured at the 14th month) in the SHR rats. *P<0.05 vs Cand-treated groups (n=10). (e–j) Representative immunohistochemistry micrographs depict staining of ED-1-positive monocytes/macrophages (e–g) and nuclear staining of phosphorylated NF-κB p65/RelA in tubular cells (h–j) in SHR kidney after treatment with vehicle (c, h), high dose (f, i) or ultrahigh dose (g, j) of Cand, original magnification × 200 (× 400 for inserts); (k) absolute counting of ED-1-positive cells illustrated in (e–g) in kidney sections from each SHR group or normal rats (Ctrl); #P<0.05 vs all other groups; *P<0.05 vs group H (n=10). (l) Western immunoblot analysis of kidney homogenates for phosphorylated or total NF-κB p65 and relative ratios of the densitometric data for the immunoblot bands. (m) Kidney homogenates were processed for ELISA analysis for MCP-1 and RANTES contents. *P<0.05 vs other groups (n=10). (n) Message RNA was extracted from kidney specimens and subjected to real-time RT-PCR analysis of AT2R mRNA levels. *P<0.05 vs other groups (n=10).
Kidney International  , DOI: ( /ki )
Copyright © 2008 International Society of Nephrology Terms and Conditions

9. Figure 8
Anti-inflammatory and renoprotective effect of Cand is associated with improved renal redox status. (a–c) Exogenous AII challenge test. Response of mean arterial pressure (a), renal plasma flow (b), and inulin clearance (c) to exogenous AII infusion in SHR rats from each group; *P<0.05 vs preinfusion of AII. (d, e) Redox status as reflected by GSH levels and GSH/GSSG ratios in the kidney homogenates in the kidneys from different animal groups; *P<0.05 vs other groups; #P<0.05 vs other groups (n=10). (f, g) GSH/GSSG ratios were correlated significantly with the magnitude of NF-κB activation (Figure 7l) and the extent of renal inflammation (Figure 7k) in the SHR rats; data for different groups are circled and well segregated in this correlation.
Kidney International  , DOI: ( /ki )
Copyright © 2008 International Society of Nephrology Terms and Conditions