Volume 83, Issue 3, Pages 446-454 (March 2013) A human serum albumin–thioredoxin fusion protein prevents experimental contrast- induced nephropathy  Azusa Kodama, Hiroshi Watanabe, Ryota Tanaka, Hisae Tanaka, Victor T G Chuang, Yohei Miyamoto, Qiong Wu, Masayuki Endo, Keisuke Hamasaki, Yu Ishima, Masafumi Fukagawa, Masaki Otagiri, Toru Maruyama  Kidney International  Volume 83, Issue 3, Pages 446-454 (March 2013) DOI: 10.1038/ki.2012.429 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Renoprotective effect of human serum albumin (HSA)–thioredoxin-1 (Trx) on contrast-induced nephropathy. Changes in the levels of (a) serum creatinine (SCr), (b) blood urea nitrogen (BUN), (c) urinary N-acetyl-β-D-glucosaminidase (NAG) activity, and (d) creatinine clearance (CCr) after an intravenous injection of ioversol. Saline, (HSA–Trx) fusion, the mixture of HSA and Trx, or Trx alone was injected 1h before ioversol injection. Each column represents the mean±s.d. (n=7∼13). Kidney International 2013 83, 446-454DOI: (10.1038/ki.2012.429) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Histological assessment of the kidney of control or contrast-induced nephropathy (CIN) rats with or without human serum albumin (HSA)–thioredoxin-1 (Trx) pretreatment. Representative photomicrographs of periodic acid-Schiff (PAS)-stained kidney sections (a–j) and semiquantitative scoring analysis of tubular degeneration are presented (I). (a and f) Control rats, (b and g) rats with ioversol after saline injection, (c and h) rats with ioversol after the mixture of HSA and Trx injection, (d and i) rats with ioversol after Trx alone injection, and (e and j) rats with ioversol after HSA–Trx fusion injection. Saline, HSA–Trx fusion, the mixture of HSA and Trx, or Trx alone was injected 1h before ioversol injection. The marked tubular injuries caused by ioversol (b and g) were diminished as a result of the HSA–Trx treatment (e and j). Representative enlarged tubular injury from rats treated with ioversol after injection with the mixture of HSA and Trx (k). Detachment (arrow) and foamy degeneration (arrow-head) of tubular cells were indicated. Tubular injury (degeneration and detachment of tubular cells) induced by ioversol was significantly suppressed by the HSA–Trx treatment (l). For the semiquantitative analysis of morphological changes, 20 high-magnification (× 200) fields of the cortex and the outer stripe of the outer medulla in rats were randomly selected. The extent of degeneration and detachment of tubular cells was then graded with an arbitrary score of 0–2 as follows: 0, absence; 1, mild; 2, severe. Original magnifications: × 200 (a–e); × 400 (f–j). Data are presented as mean±s.d. (n=4∼5). Statistical analyses were performed using the Tukey multiple comparison. Kidney International 2013 83, 446-454DOI: (10.1038/ki.2012.429) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining of kidneys of control or contrast-induced nephropathy (CIN) rats with or without human serum albumin (HSA)-thioredoxin-1 (Trx) fusion. Representative photomicrographs of TUNEL-stained kidney sections from (a) control rats, (b) rats treated with ioversol after saline injection, (c) rats treated with ioversol after treatment with a mixture of HSA and Trx, (d) rats treated with ioversol after injection with Trx alone, and (e) rats treated with ioversol after HSA–Trx fusion injection. (f) Image analysis of the extent and intensity of TUNEL staining was performed. Whereas CIN increases the number of TUNEL-positive renal tubular cells, the numbers are markedly decreased by HSA–Trx treatment. Kidney International 2013 83, 446-454DOI: (10.1038/ki.2012.429) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 The redox effect of human serum albumin (HSA)–thioredoxin-1 (Trx) examined by immunostaining of the kidney sections for the redox product oxidized derivative of deoxyguanosine (8-OHdG) and quantification of the amount of lipoperoxidation final reaction substance malondialdehyde (MDA). Representative photomicrographs of 8-OHdG-stained kidney sections from (a) control rats, (b) rats treated with ioversol after a saline injection, (c) rats treated with ioversol after injection with a mixture of HSA and Trx, (d) rats treated with ioversol after Trx alone, and (e) rats treated with ioversol after HSA–Trx fusion injection. (f) Image analysis of the extent and intensity of 8-OHdG staining was performed. (g) Changes in the levels of renal MDA levels are shown. HSA–Trx suppressed the number of 8-OHdG-positive renal tubular cells and renal MDA levels. Kidney International 2013 83, 446-454DOI: (10.1038/ki.2012.429) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Effect of human serum albumin (HSA)–thioredoxin-1 (Trx) on cellular ROS levels and the cytotoxicity induced by H2O2 in HK-2 cells. (a) HK-2 cells were incubated in 96-well plates (1 × 104cells/well) in K-SFM medium containing 5ng/ml of human recombinant EGF and 0.05μg/ml bovine pituitary extract at 37°C for 24h and then incubated with 5μmol/l CM-H2DCFCA for 30min in Dulbecco's phosphate-buffered saline (D-PBS). After removing the D-PBS from the wells, the HK-2 cells were incubated with different concentrations of HSA–Trx (0.1, 0.5, 1, 5, 10μmol/l) for 1h and then incubated with 250μmol/l H2O2 for 3h. Fluorescence intensity was measured at an excitation wavelength of 485nm and an emission wavelength of 535nm. (b) HK-2 cells were incubated in 96-well plates (1 × 104cells/well) in K-SFM medium containing 5ng/ml human recombinant EGF and 0.05μg/ml bovine pituitary extract at 37°C for 24h, and then incubated with different concentrations of HSA–Trx (0.1, 0.5, 1, 5, 10μmol/l) for 1h. HK-2 cells were incubated with 250μmol/l H2O2 for 24h and then incubated with the WST-8 solution for 3h at 37°C. The maximum absorption of the strong orange WST-8 formazan was 450nm. The viability of HK-2 cells was quantified by optical density (OD) measurements using a spectrophotometer. Each column represents the mean±s.d. (n=4). MFI, mean fluorescence intensity. Kidney International 2013 83, 446-454DOI: (10.1038/ki.2012.429) Copyright © 2013 International Society of Nephrology Terms and Conditions