*2.8. Immunohistochemistry*

Immunohistochemical staining was performed on formalin fixed, para ffin-embedded kidney sections with ED-1 (clone ED-1, Serotec, Oxford, UK), primary antibodies against MSC CD44 (MCA643GA, Serotec, Kidlington, UK), GRP78 (1:500; Santa Cruz Biotechnology, Dallas, TX, USA), LC3-II (1:1000; MBLI Corporation, Woburn, MA, USA), caspase 3 (Epitomics, Burlingame, CA, USA), terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling (TUNEL, BioVision, Milpitas, CA, USA), collagen IV (Abcam, Cambridge, UK) and 4-hydroxynonenal (4HNE, Bioss, Woburn, MA, USA). Briefly, para ffin sections were depara ffinized with xylene and rehydrated in an alcohol series and water. Kidney sections were subjected to antigen retrieval and were blocked with a peroxidase-blocking reagent. Sections were incubated with the primary antibody overnight at 4 ◦C. After washing, the kidney sections were incubated with Envision system-horseradish peroxidase-labeled polymer (Dako, Glostrup, Denmark) for 1 h at room temperature. The sections were visualized with 3,3-diaminobenzidine tetrahydrochloride (Dako, Glostrup, Denmark) and counterstained with hematoxylin. Apoptotic cells in the kidney were identified by TUNEL staining. The TUNEL method for the in situ apoptotic assay was performed according to the method of Gavrieli et al. with minor modifications [22]. The number of positive ED1 and TUNEL stained cells was evaluated by counting stained cells per high power field (×400) in at least 20 randomly selected fields. The percentage of positive stained area in the GRP-78, LC3-II, caspase 3 and collagen VI assays was analyzed by Adobe Photoshop 7.0.1 imaging software (San Jose, CA, USA) analysis.

#### *2.9. Western Blot and Nuclear Extraction*

Western blot analysis was performed on isolated glomeruli to detect the levels of renal anti-oxidant responsive element proteins, and nuclear extractions were done to detect nuclear factor (erythroid-derived 2)-like 2 (Nrf2), nuclear factor kappa B (NF-kB) expressions. Briefly, tissues were grinded to powder in liquid nitrogen. Then the tissue powder was lysed in RIPA Bu ffer (Bio Basic, Amherst, NY, USA) supplemented with a protease inhibitor (Roche, Basel, Switzerland) for 10 min at 4 ◦C. The concentration of protein was measured by a BCA protein assay kit (Thermo Scientific, Waltham, MA, USA). A protein sample (80 μg) was mixed with 1× sample bu ffer and was boiled for 3 min. Protein samples were resolved in 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membrane (Millipore, Billerica, MA, USA). The blot was blocked with Hyblock (Hycell, Taipei, Taiwan) for 1 min, and incubated with primary antibodies overnight at 4

◦C. Detection of signals was performed by Western Lightning plus-ECL (PerkinElmer, Waltham, MA, USA). Nuclear extracts were obtained using the NE-PER nuclear and cytoplasmic extraction reagents (Thermo Scientific, Waltham, MA, USA) according to the manufacturer's instructions.

Primary antibodies included Mn-superoxide dismutase (MnSOD, 1:1000; Enzo Life Sciences, Farmingdale, NY, USA), Cu/Zn-SOD (1:500; Millipore, Billerica, MA, USA), catalase (Assay Designs, Ann Arbor, MI, USA), heme oxygenase 1(BioVision, Milpitas, CA, USA), Nrf2 (Cayman, Ann Arbor, MI, USA), NFkB (Santa Cruz, Dallas, TX, USA), glutamate-cysteine ligase catalytic subunit (GCLC, Abcam, Cambridge, UK), glutamate-cysteine ligase modifier subunit (GCLM, Abcam, Cambridge, UK), glutathione peroxidase 1(GPX1, Abcam, Cambridge, UK), β-actin (1:5000; Sigma-Aldrich, St. Louis, MO, USA), γ-tubulin (Abcam, Cambridge, UK) and Lamin A/C (Abcam, Cambridge, UK) as a control for nuclear extraction. Secondary antibodies included HRP-conjugated goa<sup>t</sup> anti-mouse IgG, HRP-conjugated rabbit anti-goat IgG, and HRP-conjugated goa<sup>t</sup> anti-rabbit IgG (all at 1:10,000; all from Southern Biotech Laboratories, Birmingham, AL, USA).
