1. Volume 83, Issue 4, Pages 582-592 (April 2013)
p-Cresyl sulfate causes renal tubular cell damage by inducing oxidative stress by activation of NADPH oxidase 
Hiroshi Watanabe, Yohei Miyamoto, Daisuke Honda, Hisae Tanaka, Qiong Wu, Masayuki Endo, Tsuyoshi Noguchi, Daisuke Kadowaki, Yu Ishima, Shunsuke Kotani, Makoto Nakajima, Keiichiro Kataoka, Shokei Kim-Mitsuyama, Motoko Tanaka, Masafumi Fukagawa, Masaki Otagiri, Toru Maruyama 
Kidney International 
Volume 83, Issue 4, Pages (April 2013)
DOI: /ki
Copyright © 2013 International Society of Nephrology Terms and Conditions

2. Figure 1
Effect of p-cresyl sulfate (PCS) on reactive oxygen species (ROS) production in HK-2 cells. (a) Time course and (b) concentration dependency (for 3h incubation) for PCS-induced ROS production in HK-2 cells. (c) Effect of 4% human serum albumin (HSA) on PCS-induced ROS production in HK-2 cells. (d) Effect of an OAT inhibitor (1000μmol/l probenecid), NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) inhibitor (50μmol/l DPI), PI3K inhibitor (5μmol/l wortmannin), protein kinase C inhibitor (1μmol/l calphostin C), and N-acetyl-L-cysteine (NAC; 1000μmol/l) on PCS-induced ROS production in HK-2 cells. (e) Extracellular H2O2 was determined in the absence or presence of 1000μmol/l probenecid, 50μmol/l diphenylene iodonium (DPI), 5μmol/l wortmannin, 1μmol/l calphostin C, and 1000μmol/l NAC. Results are expressed as percentages of fluorescence intensity compared with control (0μmol/l PCS) (mean±s.d., n=6, *P<0.05 vs. control, **P<0.01 vs. control, ##P <0.01 vs. PCS). MFI, mean fluorescent intensity.
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

3. Figure 2
Effect of p-cresyl sulfate (PCS) on mRNA and protein expression of p22phox and Nox4 in HK-2 cells. Effect of PCS on mRNA expression of (a) p22phox and (b) Nox4 in HK-2 cells was determined in the absence or presence of 1000μmol/l probenecid by quantitative reverse transcriptase–PCR. The mRNA levels corresponding to p22phox and Nox4 were normalized against 18S ribosomal RNA. Effect of PCS on the protein expression of p22phox and Nox4 in HK-2 cells was determined by western blotting. Representative immunoblots with (c) anti-p22phox and (d) anti-Nox4 antibodies after stimulation with different concentrations of PCS are shown. Densitometric analysis of p22phox and Nox4 was normalized against β-actin. The results are shown as the fold in expression as compared with control (0μmol/l PCS) (mean±s.d., n=4, *P<0.05 vs. control, **P<0.01 vs. control, ##P<0.01 vs. PCS).
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

4. Figure 3
Effects of p-cresyl sulfate (PCS) on mRNA expression of transforming growth factor (TGF)-β1, TIMP-1, and Pro-α1 (I) collagen and active TGF-β1 protein secretion in HK-2 cells. The mRNA levels corresponding to (a) TGF-β1, (b) tissue inhibitor of metalloproteinases-1 (TIMP-1), and (c) Pro-α1 (I) collagen were normalized against 18S ribosomal RNA in the absence or presence of 1000μmol/l probenecid. The results are shown as the fold in expression as compared with control (0μmol/l PCS) (mean±s.d., n=4, **P<0.01 vs. control). (d) Active TGF-β1 secretion in HK-2 cells was determined in the absence or presence of 1000μmol/l probenecid, 50μmol/l diphenylene iodonium (DPI), and 1000μmol/l N-acetyl-L-cysteine (NAC; mean±s.d., n=6, **P<0.01 vs. control, ##P<0.01 vs. PCS).
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

5. Figure 4
Effect of p22phox or Nox4 RNAi on the p-cresyl sulfate (PCS)-induced reactive oxygen species (ROS) production and active transforming growth factor (TGF-β1) protein secretion in HK-2 cells. (a) p22phox or Nox4 protein expression in HK-2 cells. Effect of p22phox or Nox4 RNAi on PCS-induced (b) ROS production, (c) H2O2 production, and (d) active TGF-β1 secretion in HK-2 cells (mean±s.d., n=6, **P<0.01 vs. control, ##P<0.01 vs. PCS).
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

6. Figure 5
Effect of p-cresyl sulfate (PCS) on cell viability of HK-2 cells. (a) Effect of the concentration dependency of PCS on cell viability. (b) Effect of 1000μmol/l probenecid and 1000μmol/l N-acetyl-L-cysteine (NAC) on 1000μmol/l PCS-induced inhibition of HK-2 cell viability. (c) Relationship between PCS-induced reactive oxygen species (ROS) production and HK-2 cell viability. PCS-induced ROS production is expressed according to the fluorescence intensities as shown in Figure 1b. The data of Figure 5a were used as the cell viability of HK-2 cells (y=-0.484x , R2=0.9904) (mean±s.d., n=4, **P<0.01 vs. control, ##P<0.01 vs. PCS).
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions

7. Figure 6
Effect of p-cresyl sulfate (PCS) administration on the renal tissue of chronic kidney disease (CKD) rats. (a) Periodic acid–Schiff (PAS) staining, Masson’s trichrome staining, and immunostaining of transforming growth factor (TGF-β1) in the kidney samples (b) Renal tubular degeneration ratio (%): sham, 0.0±0%; CKD, 28.0±12.5%; CKD+PCS, 43.6±12.5%. (c) Renal active TGF-β1 expression, (d) renal superoxide, (e) renal NADPH oxidase (nicotinamide adenine dinucleotide phosphate-oxidase) activity, (f) renal p22phox protein expression, (g) renal Nox4 protein expression, and (h) immunofluorescence staining of renal 8-OHdG (8-hydroxy-2’-deoxygenase) and NO2-Tyr (nitrotyrosine) in the kidney samples. Sham (n=4), CKD (n=11), and CKD+PCS (n=9) rats were measured after 4 weeks of PCS administration, as described in Materials and Methods (mean±s.d., *P<0.05 vs. sham, **P<0.01 vs. sham, #P<0.05 vs. CKD, ##P<0.01 vs. CKD).
Kidney International  , DOI: ( /ki )
Copyright © 2013 International Society of Nephrology Terms and Conditions