Volume 83, Issue 4, Pages 662-673 (April 2013) Selective estrogen receptor modulation attenuates proteinuria-induced renal tubular damage by modulating mitochondrial oxidative status  Yuko Nishi, Minoru Satoh, Hajime Nagasu, Hiroyuki Kadoya, Chieko Ihoriya, Kengo Kidokoro, Tamaki Sasaki, Naoki Kashihara  Kidney International  Volume 83, Issue 4, Pages 662-673 (April 2013) DOI: 10.1038/ki.2012.475 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Pathological changes in the tubulointerstitium and glomeruli (a-d) Masson-trichrome (Masson) staining showing renal tubulointerstitial morphology. Bar=50μm. (e-h) Periodic acid–Schiff (PAS) staining showing glomerular morphology. Bar=50μm. ICGN, ICR-derived glomerulonephritis mice; ICR, ICR mice; OVX, ovariectomized ICGN mice; RAL, OVX mice treated with raloxifene. (i) Tubulointerstitial injury score, (j) tubulointerstitial fibrosis score, and (k) glomerular injury score; n=10 in each group. Data are shown as mean±s.e.m. *P<0.05 vs. ICR. †P<0.05 vs. ICGN. ‡P<0.05 vs. OVX. Kidney International 2013 83, 662-673DOI: (10.1038/ki.2012.475) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Comparison of oxidative lipid accumulation and mitochondrial function and morphology in vivo. (a-d) Immunohistochemical staining for hexanoyl lysine (HEL). Bar=100μm. (e-h) Cytochrome c oxidase (COX) activity staining. Bar=100μm. (i-l) Succinic dehydrogenase (SDH) activity staining. Bar=100μm. (m-p) Transmission electron microscopy (TEM) for the evaluation of mitochondrial morphology. BM, basement membrane; TL, tubular lumen. Bar=4μm. ICGN, ICR-derived glomerulonephritis mice; ICR, ICR mice; OVX, ovariectomized ICGN mice; RAL, OVX mice treated with raloxifene. (q) Positive area of HEL staining. (r) Positive area of COX staining. (s) Positive area of SDH staining. Data are shown as mean±s.e.m. *P<0.05 vs. ICR. †P<0.05 vs. ICGN. ‡P<0.05 vs. OVX. Kidney International 2013 83, 662-673DOI: (10.1038/ki.2012.475) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Changes in mitochondrial redox system determined in vivo. (a) Thioredoxin reductase (TrxRD) activity in the mitochondrial fraction samples of the kidney. (b) Superoxide dismutase (SOD) activity in the mitochondrial fraction samples of the kidney. (c) mRNA expression of TrxRD 2. (d) mRNA expression of SOD 2. ICGN, ICR-derived glomerulonephritis mice; ICR, ICR mice; OVX, ovariectomized ICGN mice; RAL, OVX mice treated with raloxifene. n=10 in each group. Data are shown as mean±s.e.m. *P<0.05 vs. ICR. †P<0.05 vs. ICGN. ‡P<0.05 vs. OVX. Kidney International 2013 83, 662-673DOI: (10.1038/ki.2012.475) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 Expression of mitochondrial β-oxidation-related genes in vivo. mRNA expression of (a) short-chain acyl-coenzyme A dehydrogenase (SCAD), (b) medium-chain acyl-coenzyme A dehydrogenase (MCAD), (c) long-chain acyl-coenzyme A dehydrogenase (LCAD), and (d) very-long-chain acyl-coenzyme A dehydrogenase (VLCAD). ICGN, ICR-derived glomerulonephritis mice; ICR, ICR mice; OVX, ovariectomized ICGN mice; RAL, OVX mice treated with raloxifene. n=10 in each group. Data are shown as mean±s.e.m. *P<0.05 vs. ICR. †P<0.05 vs. ICGN. ‡P<0.05 vs. OVX. Kidney International 2013 83, 662-673DOI: (10.1038/ki.2012.475) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Alteration in tubular macrophage infiltration. (a-d) Immunohistochemical staining for F4/80 in the renal cortex. Bar=50μm. (e) Number of F4/80-positive cells. (f) mRNA expression of monocyte chemotactic protein-1 (MCP-1). (g) mRNA expression of cluster of differentiation molecule 11b (CD11b). ICGN, ICR-derived glomerulonephritis mice; ICR, ICR mice; OVX, ovariectomized ICGN mice; RAL, OVX mice treated with raloxifene. n=10 in each group. Data are shown as mean±s.e.m. *P<0.05 vs. ICR. †P<0.05 vs. ICGN. ‡P<0.05 vs. OVX. Kidney International 2013 83, 662-673DOI: (10.1038/ki.2012.475) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Evaluation of interleukin-18 (IL-18) expression in vivo. (a-d) Immunohistochemical staining for IL-18 in the renal cortex. Bar=50μm. (e) Number of IL-18 positive cells. (f) IL-18 content in kidney tissue. ICGN, ICR-derived glomerulonephritis mice; ICR, ICR mice; OVX, ovariectomized ICGN mice; RAL, OVX mice treated with raloxifene. n=10 in each group. Data are shown as mean±s.e.m. *P<0.05 vs. ICR. †P<0.05 vs. ICGN. ‡P<0.05 vs. OVX. Kidney International 2013 83, 662-673DOI: (10.1038/ki.2012.475) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 Evaluation of inflammasome activation in vivo. (a) mRNA expression of nucleotide-binding domain, leucine-rich-containing family, pyrin domain–containing-3 (NLRP3). (b) mRNA expression of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). (c) mRNA expression of pro-caspase-1 (casp-1). (d) Western blot analysis for renal ASC expression and casp-1 activation. (e) Relative expression of ASC protein expression to GAPDH protein expression. (f) Relative expression of casp-1 protein expression to pro-casp-1 protein expression. ICGN, ICR-derived glomerulonephritis mice; ICR, ICR mice; OVX, ovariectomized ICGN mice; RAL, OVX mice treated with raloxifene. n=10 in each group. Data are shown as mean±s.e.m. *P<0.05 vs. ICR. †P<0.05 vs. ICGN. ‡P<0.05 vs. OVX. Kidney International 2013 83, 662-673DOI: (10.1038/ki.2012.475) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 8 Terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining. (a-d) Pyroptotic cells were detected using TUNEL staining. Bar=50μm. (e) Number of TUNEL-positive cells. ICGN, ICR-derived glomerulonephritis mice; ICR, ICR mice; OVX, ovariectomized ICGN mice; RAL, OVX mice treated with raloxifene. n=10 in each group. Data are shown as mean±s.e.m. *P<0.05 vs. ICR. †P<0.05 vs. ICGN. ‡P<0.05 vs. OVX. Kidney International 2013 83, 662-673DOI: (10.1038/ki.2012.475) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 9 Mitochondrial oxidative stress in albumin-stimulated tubular cells. MitoSOX staining for the evaluation of mitochondrial reactive oxygen species. (a) No treatment (CON), (b) stimulation with fatty acid-bearing human albumin (FA-Alb), (c) stimulation with FA-Alb with raloxifene (FA-Alb+RAL), (d) stimulation with FA-Alb+RAL and ICI 182780 (FA-Alb+RAL+ICI). Bar=10μm. (e) Relative fluorescence intensity of MitoSOX staining. n=6 in each group. Data are shown as mean± s.e.m. *P<0.05 vs. CON. †P<0.05 vs. FA-Alb. Kidney International 2013 83, 662-673DOI: (10.1038/ki.2012.475) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 10 Nuclear factor-κB (NF-κB) activation in albumin-stimulated tubular cells. (a) Western blot analysis for phospho-IκB-α and NF-κB component p65 in the cytosolic fraction. (b) Relative IκB protein expression to CON. (c) Relative NF-κB p65 protein expression related to CON. CON, no treatment; FA-Alb, stimulation with fatty acid-bearing human albumin; FA-Alb+RAL, stimulation with FA-Alb and raloxifene; FA-Alb+RAL+ICI, stimulation with FA-Alb+RAL and ICI 182780. n=6 in each group. Data are shown as mean±s.e.m. *P<0.05 vs. CON. †P<0.05 vs. FA-Alb. Kidney International 2013 83, 662-673DOI: (10.1038/ki.2012.475) Copyright © 2013 International Society of Nephrology Terms and Conditions