Complete Experimental Procedures
Mice and genotyping
Adult Bmi-1 heterozygote (Bmi-1+/-) mice (129Ola/FVB/N hybrid background) werebackcrossed 10–12 times to the C57BL/6J background and mated to generate Bmi-1 homozygote (Bmi-1-/-) and their wild-type (WT) littermates genotyped by PCR, asdescribed previously(Zhang et al. 2010). Mice deleted specifically for p16Ink4 have been described(Sharpless et al. 2001). P16Ink4+/- mice on the FVB N2 backgroundwere crossed to Bmi-1+/- mice to generate doubleknockout(DKO) mice.This study was carried out in strictaccordance with NIH Guidefor theCare and Use of Laboratory Animals and theguidelines of the Institute for Laboratory Animal Research ofNanjing Medical University. The protocol was approved by theCommittee on the Ethics of Animal Experiments of NanjingMedical University(Permit Number: BK2006576). All efforts weremade to minimizeanimal suffering and to reduce the number ofanimals used(Cao et al. 2012).
Cell line and cell cultures
The HK2 (human renal proximal tubular epithelial cell line)(American Type CultureCollection)was cultured in 1640 medium supplemented with 100 U/ml penicillin, 100µg/ml streptomycin, 2 mM L-glutamine, 50 µg/ml vitamin C,10% fetal bovine serum (Gibco, USA) until80% confluent(Yang et al. 2010).
Small interference RNA-mediated knockdown of human Bmi-1
For small interference RNA (siRNA) experiments, RNA primers complementary to human Bmi-1 were designed and synthesized by theRibobio Co. LtdinChina. HK2 cellswere seeded into 6-well plates at 2×105cells/well or 24-well plates at 5×104 cells/well. Wemaintained the HK2 cells in 1640 medium without antibiotics one daybefore transfection after cells were 50% confluent. Indicated amounts ofsiRNAs (including positive and negative control and siRNA1-3) (usually 50 nM) and Lipofectamine 2000 (Invitrogen,Carlsbad, CA) were diluted separately in the medium. Blank control cellswere treated with Lipofectamine 2000 butnot siRNA. Six hours after transfection, cells were incubated with 1640 medium supplemented with 100 U/ml penicillin, 100µg/ml streptomycin, 2 mM L-glutamine, 50 µg/ml vitamin C,10% fetal bovine serum.After incubation for 72 hours or 96 hours, cells wereharvested and mRNA and proteindetected by RT-PCR, real-time RT-PCR orimmunocytochemistry stain and Western Blot(Bai et al. 2012). Sequences of siRNAsand targeted mRNAs are inSupporting Information 4- Table 1.
Administration of N-acetylcysteineandpyrro-quinoline quinone
For in vivo administration of N-acetylcysteine(NAC), we randomized 3-week-old Bmi-1-/- mice tonormal drinking water or water containing NAC at 1 mg/ml as previouslydescribed. In addition, because Bmi-1-/- mice are frail, the food ofanimals treated or untreated with antioxidant was soaked in NAC-treated orNAC-untreated drinking water and placed inside the cage(Liu et al. 2009).
For in vitro treatment of NAC, HK2 cellswith Bmi-1 siRNA1 were incubated without or withNAC at 1mM (0.163mg/ml) after transfection until changes were detectedat the mRNA levels(Ambrogini et al. 2010).
For in vivo administration of pyrro-quinoline quinone (PQQ), we randomized3-week-old Bmi-1-/- mice toPQQ-supplemented diet (4mg PQQ/kg diet)(BeijingcooperationFeed Co. Ltd., China) or conventional diet as previouslydescribed.In addition, because Bmi-1-/- mice are frail, the food ofanimals was soaked in drinking water and placed inside the cage(Steinberg et al. 1994; Stites et al. 2006).
Preparation of renal sections
Mice were anesthetized with 3% pentobarbital sodium (40 mg/kg) at 5weeks of age.Renal specimens were perfused with 100 ml normal sodium then perfused and fixed with periodate-lysine-paraformaldehyde (PLP) solution(Zhang et al. 2010; Jin et al. 2011)(for histochemistry or immunohistochemistry) or 1% glutaraldehyde(for conventionalelectron microscopy). Kidneys were cut into 2 identical pieces following the coronal axis. Half of kidney samples were postfixed in PLP solution or 1% glutaraldehydeovernight at4C.For histochemistry or immunohistochemistry, somesections were dehydrated in a series of graded ethanol solutions, embedded in paraffin and cut into 5m sections.Renal cortex and medulla were trimmed, dehydrated and embedded inEpon 812(CEM Corporation,Japan)for electron microscopy. Ultrathin sections of 70 nm were counterstainedon copper grids with uranyl acetate and lead citrate,and examined with a Jeol 1200EX electron microscope (Tokyo,Japan)(Cao et al. 2012).Mitochondriawere quantificated from 2 distinct 25µm2 regions per cell from 2 cells peranimal from at least 3 different animals per genotypeasdescribed previously(Chang et al. 2013).
Histochemical or immunohistochemical and immunocytochemistry stains
For histochemical or immunohistochemical staining, serial paraffin sections were deparaffinized and rehydrated to water.
Pre-embedding Senescence-associated-β-gal (SA-β-gal)(Following fix for 2 hours in PLP, samples were washed three times for 30 min in lacZ wash buffer (2mM MgCl2, 0.01% sodium deoxycholate, 0.02% Nonidet-P40 (NP-40) in PBS (PH6.0)). Staining was carried out in 0.5 mg/ml X-gal, 5 mM potassium ferrocyanide, and 5 mM potassium ferricyanide in lacZ wash buffer at 37C overnight with shaking and protection from light),HE and Picrosirius red(Li et al. 2009)for total collagenhistochemistry stains were performed following previously described methods(Jin et al. 2011).
Masson’s trichrome staining was performed with a D026 Masson detection kit from Nanjing Jiancheng Bioengineering Institute inChinaaccording to the manufacturer’sinstructions.
For immunohistochemical staining, serial paraffin sections were deparaffinized, dehydrated, and for antigen retrieval, steamed for 20 minutes in PBS (0.01mM pH7.4) followed by blocking of endogenous peroxidase (3% H2O2) and preincubation with serum. Primary antibodies againstType Ι collagen(Southern Biotech, USA), -SMA (Abcam, USA), Fibronectin (Sigma, USA), E-cadherin (Santa Cruz Biotechnology, USA), Ki67 (Abcam, USA), NF-B-p65 (Cell Signaling Technology, USA), TNF-(Santa Cruz Biotechnology, USA)IL-6(Santa Cruz Biotechnology, USA), γ-H2A.X (Ser139) (Cell Signaling Technology, USA), 8 hydroxyguanosine (8-OHdG) (Abcam, USA), 53Bip1 (Novus Biological, USA) and pCHK2 (Thr68) (Abcam, USA),were used. After washing, sections were incubated with secondary antibody (biotinylated IgG; Sigma, USA), washed and processed using a Vectastain ABC-HRP kit (Vector Laboratories, Inc., USA). Sections were counterstained with hematoxylin and mounted with Biomount medium(Electron Microscopy Sciences).
For immunocytochemistry staining, cells seeded on coverslips were fixed with PLP solution for 45 minutes, blockedfor endogenous peroxidase (3% H2O2) and preincubated with serum. Primary antibodies against-SMA (Abcam, USA)or E-cadherin (Santa CruzBiotechnology, USA)were used. After washing, cells were incubated with secondary antibody (biotinylated IgG; Sigma, USA), washed, and processed using a Vectastain ABC-HRP kit (Vector Laboratories, Inc., USA). Cells were counterstained with hematoxylin and mounted with Biomount medium on slides.
TUNEL Assay
Dewaxed and rehydrated paraffin sections were stained with an In Situ Cell Death Detection Kit (Roche Diagnostics Corp., Switzerland) using a previously described protocol(Jin et al. 2011).
Flow cytometry analysis
Intracellular ROS analysis
Foranalysis of intracellular ROS, total renal cells from 5-week-old mice were incubated with 5 mMDiacetyldichlorofluorescein(DCFDA) (Invitrogen) andplaced in a shaker at 37C for 30 min, followed immediately by flow cytometryanalysis in a FACScalibur flow cytometer (Becton Dickinson, Heidelberg, Germany) and/or fluorescence microscope(Liu et al. 2009).
Surface labeling
Single-cell suspensions of kidneys from 5-week-old mice were analyzed by flow cytometry using mAb targetingCD11b[Phycoerythrin (PE)-conjugated]or CD3e (PE-CyTM7-conjugated) from BD Biosciences. All labeling was performed in glucose sodium potassium buffer containing 1% BSA incubated for 30 min at 4C, then centrifuged and fixed with 2% formalin and analyzed on a FACScalibur flow cytometer. Flow cytometry data wereanalyzed with FlowJo Version 7.6.1 software (TreeStar, USA).
Color doppler flow imaging of kidneys
Mice were anesthetized with 3% pentobarbital sodium (40 mg/kg) and depilated in abdominal region at 5weeks.A high frequency ultrasound imaging system for small animal research (Vevo 2100, Visualsonics, Toronto, Canada) equipped with theMS-400 transducer of24 MHz central frequencywas usedfor detecting peak systolic velocity (PSV) of renal arteries. Renal interlobular arteries were sampled by pulse dopplerfor displaying blood vessels trees in renal cortex. More than three consistent morphological frames were obtained and frozen. Renal interlobular arteries in 3 different regions of renal cortex were detected foreach kidney of each mouse(Stanley et al. 2012; Yang & Chiu 2013).
RNA isolation and Real-time RT-PCR
RNA was isolated from kidneysof 5-week-old mice or HK2 cells using Trizol reagent (Invitrogen Inc., Carlsbad, CA, USA) according to the manufacturer’s protocol. Sample mRNA levels were semi-quantifiedby RT-PCR or quantifiedby real-time RT-PCR as previously described(Jin et al. 2011).PCR primers are inSupporting Information 4- Table 2.
Western Blot
Samples of kidneysfrom 5-week-old mice were dissected and immediately placed into RIPA lysis buffer containing a cocktail of proteinase inhibitors and phosphatase inhibitors(Roche Diagnostics Corp., Switzerland) for protein extraction. Protein extracts were boiled for 5 min in sample buffer, fractionated by SDS gel electrophoresis and transferred to nitrocellulose membranes. Membraneswere blocked for 2 h at 37C with 5% non-fat dry milk in PBS/Tween 20. Blots were incubated overnight at 4C with antibodies against -SMA (Abcam, USA),E-cadherin (Santa Cruz Biotechnology, USA), Akt(Santa Cruz Biotechnology, USA), phospho-Akt (pAkt)(Santa Cruz Biotechnology, USA), Bcl-2 (Santa Cruz Biotechnology, USA), Caspase3 and phospho-Caspase3 (Cell signaling technology, USA), p16 (Santa Cruz Biotechnology, USA),p19 (Santa Cruz Biotechnology, USA),p53 (Cell Signaling Technology, USA),p21 (Santa Cruz Biotechnology, USA),SOD1 (Abcam, USA), SOD2 (Novus Biological, USA), Prdx1 (Santa Cruz Biotechnology, USA), γ-H2A.X (Ser139) (Cell Signaling Technology, USA), CHK2 (Novus Biological, USA), pCHK2 (Novus Biological, USA), TGF-precursor (Abcam, USA)activatedTGF-(Abcam, USA)TGF-RII (Santa Cruz Biotechnology, USA), pSmad2/3 (Santa Cruz Biotechnology, USA), Smad4 (Millipore, USA), angiotensinII(Novus Biological, USA),Bmi-1 (Millipore, USA) or β-actin (Bioworld Technology, USA), followed by incubation for 1h with HRP-conjugated secondary antibody (Sigma, USA). Immunoreactive bands were visualized by enhanced chemiluminescence reagent treatment and exposure tohyperfilm-ECL detection kit (Amersham Pharmacia, New Jersey, USA). Band intensity was measured using Image J version 1.29(National Institutes of Health).
Biochemical measurements
Oxidative Stresslevels
Renal tissues from 5-week-old mice were homogenized in cold saline. Homogenate (10%) was centrifuged at 4000 rpm at 4C for10 min. Supernatant was used for measurements of hydrogen peroxide (H2O2) (A064 H2O2 detection kit),total superoxide dismutase (T-SOD) (A001-1SOD detection kit) catalase (CAT) (A007 CAT detection kit) andmalondialdehyde (MDA) (A003-1MDA detection kit).Detection kits were from Nanjing Jiancheng Bioengineering Institute inChina. Allexaminations were performed according to the manufacturer’sinstructions(Ma et al. 2009).
Urinary Creatinine and Urea Albumin
At 5weeks of age, mice were placed in metabolic cages the day before sacrificing and given water but not food. Urine was collected for 24 hours before sacrificing and used to measure urinary creatinine (UCr) (C011 Cr detection kit) and urinary albumin (UAL) (A028 UAL detection kit) according to the manufacturer’sinstructions (Nanjing Jiancheng Bioengineering Institute, China)(Guo et al. 2013).
Levels of Serum Urea Nitrogen and Serum Creatinine
At 5weeks of age, mice were anesthetized with 3% pentobarbital sodium (40 mg/kg) and depilated in the abdominal region. Blood was taken by suction from the heart with a 1ml syringe. Serum was isolated for measurements of urea nitrogen (SUN) (C013-2 SUN detection kit) and serum creatinine (SCr) (C011 Cr detection kit) according to the manufacturer’sinstructions (Nanjing Jiancheng Bioengineering Institute, China)(Guo et al. 2013).Hominal serum creatinine clearance (SCrCl) was analyzedby the CockcroftGault Equation: SCrCl =[(140-age) (year)* body mass(kg)]/[0.818* SCr(umol/L)](Michels et al. 2010).The SCrCl level of Bmi-1-/- mice relative to the SCrCl level of WT littermates was assessed by the equation: [Bmi-1-/- body mass (g)/WT body mass (g)] *[WT SCr(µmol/L)/Bmi-1-/- SCr(µmol/L)]:1.
Levels of plasma AngiotensinII and Aldosterone
Plasma angiotensinII (Ang II), and aldosterone (ALD) concentrations from 5-week-old mice were determinedusing commercial radioimmunoassay kits according to the manufacturer’sinstructions (Beijing North Institute of Biological Technology, China)(Liu et al. 2010).
Hematocrit measurements
Blood (20 μl) was collected in heparinized microhematocrit capillary tubes and spun at 3,000 g for 5 min at room temperature. Hematocrits were measured by the equation: Hematocrit(%)= [the height of red blood cells (mm)]/ [the height of whole blood (mm)](Dilauro et al. 2010).
Statistical analysis
All analyses were performed using SPSS software (Version 16.0, SPSS Inc., USA). Measurementdata were described as Mean ± SEM fold-changeover control and analyzed by Student’s t-test and one-way ANOVAto compare differences among groups.Qualitative data were described as percentages and analyzed using a chi-square test as indicated. P-valueswere two-sided and less than 0.05 was considered statistically significant.
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