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1. D glucose Glucose Degradation Products GDPs pH 2 PD 1 1 1 2 pH GDPs2. 119 A 5 7 o 3000 Ver3 1 3000 Ver3 1 lt gt 3000 Ver3 1 3000 FL3000 Ver3
3. NOS HPMC EMT HPMC PD 46 5 2 o SMA 1 PDE HPMC BMT PDE HPMC
4. 1 3 a Se o Pro3900 LAS SDS 20mA QE TALIS 15 30
5. i neos qm SEE ss SEE SS sR A SS N SS NN i SS Hg m NN N NNNN X yas 107 ee
6. BPB lt gt o 2 1cm EA EKET
7. 61 10 11 12 13 14 15 16 8 epithelial to mesenchymal transition EMT 2007 Williams JD et al Morphologic change in the peritoneal membrane of patients with renal disease J Am Soc Nephrol 2002 13 470 479 Nakamoto H et al New animal models for encapsulating peritoneal sclerosis role of acidic solution Perit Dial Int 2001 21 349 53 Eu 39 1 7 2010 PD 2011 BPS 18 2012 EBM 2000 E 2003 19 503 508 Betjes MGH et al The Mesothelial Cells in CAPD Effluent and Their Relation
8. DOY 111 C3 C 10 PES SDS 30 SDS 15 15 80C 1 SDS 40 SDS 1
9. E 1 i i J 1 j v 0 Bord nete de MWARUSTZZZJWABUSTVTRERPBROECZVg2 ERARE 2 1 2 AAEL Milli Q Milli Q 4 5
10. TMG AAG GLUT a Tocopherol a TTP a Tocopherol Transfer Protein Lg TTP a TTP a Tocopherol Hig 5 7 TMG a TTP 54 Fig 5 7 a TTP open close N helix iik C helix B sheet C oc Tocopherol B sheet g helix oc TTP a Tocopherol
11. ROS 53 TMG 0 054M 0 5uM 5uM 50uM 500uM 5 ROS 0 05uM Fig 4 12 Fig 4 12 Hig 5 6 TMG AAG TMG 45 N Un Fluorescence Intensity 0 100 200 300 400 500 600 TMG uM Fig 5 6 TMG TMG 397 Vitamin E co Tocopherol a Tocopherol TMG
12. 118 A 5 6 Flamingo lt gt Flamingo Invitrogen 161 0491 ues Methanol WAKO 137 0183 Milli Q lt gt Methanol 250mL 5096 S0mL 10 Milli Q 200mL Flamingo x10 78 1 amp 40mL Milli Q 360mL lt gt D o 220mL 1 30 Milli Q250mL 7 7v 1 3 10 200mL 1 1 Milli Q 250mL 1 1
13. D glucose 2000 EPS PP EPS GDPs pH 2000 pH GDPs IEX Table 1 1 ML Table 1 1 GDPs GDPs uM L Two chambered 3 4 DGE 16 1 7 Formaldehyde 3
14. SDS O 2 lcm 19 4uL 18cm 19 4uLx18 cm 349 2uL 1 6 1 2 18cm 58 2uL 174 6uL 349 2uL O D 1 5mL lt
15. 600mL 50 110 A 5 5 SDS lt gt SDS SDS SDS lt gt 3600 14 anatech SDS Ek lt gt SDS Milli Q
16. HERO TAB XE EO JUX UT pH GDPs GDPs Hig 4 6 C E O C E X PD O PD PD PD
17. Milli Q 116 22 20
18. 121 1L 20 iin EX iir EA ERE DARALE V Vie He RE FH OLESTIE ULLA VC T2 FIRA BL LEE FH 0o de i 0D DLE EE CUN XV TRAS b 7 000 00 27 7 A a RR PE X BC EL TOERE O AWRA Oi B eNA T Table B 2 1 JW BJ ZR ER RNase free 73 57 80 ORE 160 170 C 2 4h 180 C 8h Over Night 180 200 C 0 5 1h 2507C 0 5h Table B 2 1 X AREKIN ODECTHRWRALOT X Ju UV x p UV IXEKI AKI UL 7WTAVSI ES ES UI 131 lt gt o7 w T aH x1 S0ml 100ml x 1 2 D UI SLM ipsi dese
19. gt oooo 8bit TIFF 16bit TIFF 8bit BMP oProgenesis PG200 PG220 Progenesis lt gt D Progenesis PG200 PG220 2 Single stain analysis Next s xj Welcome t iple oe s Wizard This wizard wil guide you through th analysis of a set of related 2D gel images Select the type of experiment you are performing Single stan analysis C Cross stam analysis using an internal standard Enter a name for the experiment optional SA 1 To continue click Next coss De ee 122 Add Next
20. 10 APPENDIX 3 6 1 q35mm PDE HPMC PBS 2 PBS ImL S000rpm4C 5 NE 223 Bic tI PBS 4 15000rpm 4 20 3 6 2 Immobiline DryStrio pH4 7NL 18cm Milli Q 1mm
21. HV eer IV Image J D Image J 200 2 Image 8 bit monocro lios 3 Process substract background 100pixels pixel 0 1 83 Image Adjust Threshold apply Analyze Set measurement Mean Grey Value
22. SVCT GLUT o TTP 57 5 5 AAG TMG AAG TMG PD2509 1 Fig 11 Fig 5 12 zten ty 100 a4 o d r lt 932 td T 99 v 1 50 1 2 t gt M P237 i lt 4 E Fig 5 11 CTL vs AAG Red Control Green AAG 250uM Tiid Qu EL WP 100 LEA 2 d L 75 NEL 214 50 i t 2 Vo l E k4 E Fig 5 12 CTL vs TMG Red Control Green TMG 50uM 58 HPMC PDE HPMC Fig 5 10 Fig5 11 Table 1
23. UPP RhoA ROCK cw SMA IOng mL TGF B1 HPMC a SMA Fig 5 2 48 UA N ON Expression of a SMA vs EMT NO 0 EMT HPMC EMICJHPMC 10ng ml TGF f1 E Fig 5 2 10ng mL TGF B1 HESE 7 5 a SMA ii GDPs EMT EE GDPs PD PD PD
24. DCFH ROS DCF 502 nm 520 mm Hig 10 1 2 T Dichlorodihydrofluorescein diacetate DCFH DA D 6883 5IGMA dimethyl sulfoxide DMSO 049 07213 Wako Fluoview FV1000 OLYMPUS FV10 ASW OLYMPUS mage J 79 COOH H CI CI HaCCOO O OCOCH DCFH DA AEn fios esterase hydrolysis CI CI S A on oxidation DCFH DCF EHEHEHHEHNEHEHEHEHENHENHREHEHHEHEHEHHEHEHNEHEHNEHREHENHEHEHEHEHRHEHNHNHNN No isn di CIS R 520 nm 502 nm ROS Fig 10 1 DCFH DA 80 84 E1548 DCFH DA D DCFH DA DCFH DA 20C 2 Stock
25. IPG IEF Type PX 20 TIPG IEF Type PX D 20C 101 10 20 HEUS A RARE U3decAi C 8 24 2 lt gt 3070 00 e
26. o a n a LOO Fig 1 12 oc p 9 LOO a Tocopherol Fig 1 12 a a
27. D glucose HPMC HEN HPMC 19 3 10 APPENDIX 3 1 PDE E HPMC 3 1 1 PDE informed consent 257 4 OO SE TROO PERLES V 2D BER UT 23 v Z DO 3 1 2 HPMC 12 2 71 5
28. ROS ROS Table 1 2 Table 1 2 ROS 2 s eT EO WAIVER PEA Za jn E AN r yo AO D glucose ROS VVT D glucose AV ATP ATP H ROS O02
29. MI AHE x EBD B4 3 25 3 cy 67 10 Appendix A 1 A 1 1 A 1 2 A 1 3 A 2 SA B Gal A 3 ROS A 4 A 5 A 6 B 1 B 2 C 1
30. 1 BSA PBS 1 200 Anti cytokeratin 18 mouse mono antibody Funakoshi Japan anti human smooth muscle actin antibody Dako Glostrup Denmark D iX fi Us 18 f 2c ds Jl UL 4C C overnight 1 BSA PBS 3 1 200 CARLE KHAS Goat anti mouse IeG H L FITC conjugate antibody Millipore MA USA Si Fluoview FV1000 Olympus Tokyo Japan 100 lt Table 3 4 Dyelist HV Laser C A AS 209 Lamp l 2 0us Pixel 1 1 512by 26 3 6
31. a Tocopherol TMG a Tocopherol TMG a Tocopherol Trolox 7 Trolox TMG Fig 5 9 TMG 2 TMG AAG f 2 e d Q 2 3 b P wo de 7 I a D aA I g 5 T Da Daaa L je P Pe BRS gend at AA a 1 v vy S q 3 efo b s 9 7 d 7 9o b S E 9 s n 4 B e p l oM p 4 4 R WV d Y P N o 4
32. CCD 10 le _ EA 2mm WE
33. Next 2 Image 2 Next ee No Next Next 123 Click the Add button to select your images Gel 1 tf C Documents and Setings manat Gel 2 tif C Documents and Settings minai Hemove Use this page lo check the images you are going to analyse You can also tw wwe o nt mm on its image To continue click Next Averaged gels are artificial gels created by a statistical combination of several nomal gels e g to generate a mo RC You can create your averaged gels below by groupmg the images Matching spots across gels allows you to identily common proteins To senio eat ai patte ehe dis vea ea common How would you like to select your reference gel C Auto select one of the gels in this analysis Progenesis PG220 Xx 2 Do you wish to load an analysis protocol Lagi Matching spots acr
34. 8 LED 120 Blue Green SYPRO Ruby Flamingo Pro Q Diamond Pro O Emerald UV 375nm Phospho QUANTI 6 CD SYPRO Ruby 550nm CCD 580nm Flamingo 530nm CCD 580nm Pro Q Diaond Ne 930mm CCD
35. cw cw w Fig 1 13 15 NADH NAD CY 2 C LOO I VAR Jr EOOL LOOH LOOH LH Cell membrane 1 rm IT Fig 1 13 ga E oc C L LOO NAD w Tocopherol monoglucoside TMG Hig 1 14
36. 18 108 1 V 500V 250mA 2 00M 2 SV 700V 2mA 1 00M 3 SV 1000V 2mA 1 00M 4 SV 1500V 2mA 1 00M 3 SV 2000V 2mA 1 00M 0 SV 2500V 2mA 1 00M 7 SV 3000V 2mA 1 00M 8 SV 3500V 2mA 1 00M 9 SV 3500V 2mA oO 18 V C W 3 SV 2 5 Pause SC 2 5 Pause
37. 150mL 200mL 18 Table 3 5 NN mA mA 27 3 6 3 SDS 30 15 10 Tris Glycine 20mA 30 40mA
38. E 20C O Typsin EDTA solution 10x CD 20C D 37C ml 4ml 20C O D Milli Q 121 C 20 D 70 O DEPC D Milli Q KT 2 Milli Q NI o Milli Q 0 1960 DEPC Diethylpyrocarbonate 500ml Q DBPC 500ul X DEPC 37 NHN 2 30 1 io DEPC RNase
39. 3 Betjes PDE HPMC PD Peritoneal Dialysis Glucose Degradation Products GDPs U 08 Bpithelial to Mesenchymal Transition BMT POIBHB3IB4IGS1 PD pH Viability 5 pH Fig 1 EMT pH ZLE 100 mean t SE 80 n 3 m 30 min 60 3 hr m 6hr 40 20 of Control Viability 0 ER Fe Glu Conc EEDA THESES AR HA ERRET 2003 Fig 5 1 pH HPMC Viability 2003 Mo Hig 4 3
40. AAG TMG Lu 3 D3 BOUE Bi FGA HE T GE Pb M1 EMA PEI ERE RA 1 B4 BAA k RRA KE WEA 3
41. HPMC 77 3 706 EPDI 1991 11 22 26 Fig 1 2 5 1 1 4 6 Hig 1 3 1 7 2 2m 50 150um 500um microvilli MV cilium C junctional complex JC lamellar body LB elastic lamina EL mast cell M fibroblast F Pericyte P endothelium E capillary CAP basement membrane BM Hig 1 3 Human Peri
42. Ink4 p16 p15 p18 p19 CDK4 CDK6 p16 CDK G1 MAP Est Est2 23 p16 DNA TGE B1 pl6 SERS OM PDE HPMC a SMA 30 Hig 4 14 HPMC TGF B1 7 HPMC EMT HPMC TGF B1 THL Smad ILK PI3K Akt a SMA RhoA ROCK
43. NS 9 H PBS 3 10 FBS M199 3 A 1 lt 3 I XT EE HERES gt CO 10 0212 Vortex 25cm Cat No MS 21050 75 cm Cat No MS 21250 6 Cat No MS 80060 Q35mm Cat No MS 80240 15ml Cat No MS S6130 50ml Cat No MS 36300 10ml Cat No MS 66100 Dee Cat No MS 93170 amp 74 Medium 199 SIGMA M2154 PBS SIGMA MS537 TRYPSIN EDTA SOLUTION 10xY SIGMA T4174 Fetal Bovine Serum SIGMA Cat No F9423 lt gt
44. lt 300mL 2 gt 250mL 50 BEBE 50mL KRE 10 e Milli Q 200mL 5 30 SDS lt 400mL 2 gt Flamingo x10 40mL Milli Q 360mL 1x 4C 5 SYPRO Ruby Flamingo 97 o 4 240mL 2 160mL 1 40mL 1 80mL 10 4 ioni 3096 Acrylamide 0
45. pH 6 0 B 4 m K4 e n SS A j Y Dy m den LY BM 3 e Fig 1 15 17 1 3 2 n vitro 4
46. CH OH p OO MN O CH OH HE M T a TH CH Fig 1 14 TMG 16 1 3 1 3 1 S Fig 1 15 DNA DNA gt fU ETE Jo Oil
47. BPB Pro3900 Pause 115 15 16 17 18 19 20 2 1 Pro3900 30mA SDS 40mA 45mA An 3 5 3 5 BPB 1cm Pro3900 Stop
48. 150mL 200mL 3300V Pro3900 Pro3900 Menu Opt S G 18cm 3 10 3500V 1 9 Pro3900 Run A lt gt l V 500V 250mA 2 00M 2 SG 3500V 2mA 6 00M 3 SV 3500V 2mA 10 00M 4 SV 3500V 2mA ve
49. 1well 4 lt gt Table 3 3 AS 307 Jak HE Sj ER TR HER HV ss Gain Offset Laser Ee Uses C A Lamp 24 3 4 22 DCF ROS PDE HPMC 1 us 250uM Ascorbic Acid 2 glucoside AAG 5uM Tocopherol monoglucoside TMG AAG 25uM 125uM 250uM TMG 0 0SuM 0 SuM SuM 50u0M 0 1mM 10uM DCFH DA 15 1well 4 IP en
50. D 15000rpm 4 20 lt 1 103 A 5 4 lt gt IPG IEF Type PX 2 GRE Pro3900 Immobiline DryStrip pHA 7NL 18cm BA S E lt gt D IPG IEF Type PX
51. RA 3600 14 anatech 3600 14 anatech IPG IEF Type PX 3650 01 anatech lt gt OD 1 5mL e ETA RISO IET a SU is Pi j Te j j BH E f 4 c E mW f H bd NN p lt j e K y d ad i A Pi d 2T Bi f k EE Se ES f b 1 i a f A JJ A Mia 1 e ws m x c i n NI T RM IPG IEF Type PX
52. seo iiA Mi iir 0 5mm TEMED TEMED 30 An lt gt 12 5 DT T w om 30 Acrylamide 0 8 Bis 16 7mL 66 7mL 100mL 1 5M Tris HCI pH8 8 60mL Water 74 9mL 10 SDS 2 4mL 10 APS 1 1mL TEES iS 1 TEMED 100 A 5 2 lt gt e mmobiline DryStrip pH4 7NL 18cm 17 1233 01 GE
53. Fig 4 7 SA B Gal PP PDE HPMC AE SA B Gal SA B Gal BITS CAPD SA B Gal fig 4 9 HPMC SA B Gal PD DEERLITKESCC ALAAxCuorm PD PKC NADPH oxidase 47 Em o Reactive Oxygen Species ROS
54. NS E 5 T UL enn SDS SDS 05mL d P N Qo 114 10 11 12 13 14 1
55. ON ra O WindowsXP User ID Administrator Password fluoview L FV10 ASW User ID Administrator Password Administrator 87 I EXCHADROSERA D pu All Clear DyeList Selected Dyes 4 23 Alexa Fluor 488 Bleach Stop Nee uer 594 Bleach start stop hy Key a t lt All Clear Sequential Completed ali initialize DyeList Apply 4 CHSI gt Stop Bleach start stop by Key Filter Mode Kalman C Line C Frame 2 e int CC Photon Cnt Analog Sequential ow J 88 II 10 20 40 60
56. 3 EDTA EDTA Att 72 O TA 18 300 400ml 37 800rpm 50G 10 SEE TOMY RX 200 3 Wl 5 niti iei i 30ml 50ml 750 800rpm 10 dept TREL 15mL 750 800rpm 10 73 CD 15mL 10 FBS M199 1mL 035 10 FBS M199 P d 3TC 5 CO 7 10x10 cells cm
57. Hig 4 6 2 C E O 10096 I Transformed 90 L Fibroblast like 80 70 60 50 40 Cobblestone 30 20 Percentage with Phenotypes 10 QTABCEHNOSUVR n 4 2 1718 3 2 5 132 1 3 1 8 Patients Initial 0 Fig 4 6 PDE HPMC 22 4 2 4 2 1 MSC SA B Gal pH 60 B galactosidase SA B Gal pH 6 0 B galactosidase MSC SA B Gal P8 P15 AAG TMG Hig 4 7 xx 10096 n 90 2 80 Q gt 70 6096 E CTL Ca lt BM m AAG 25uM a AAG 250uM A 40 2 TMG 5uM 1 x y
58. rs EOT Aa M au ues DN OFF OFF 2 ON ON D D HV 600 680 8 0us Pixel 512by ja EN AVV M ous I M AcquisitionSetting m imageAcquisilionCortros Mode iem c NS a De Ji LeO D Bleach Bleach start stop by Key 9 lt lt Fast 20 0us Pixel Slow gt gt ee ZR mm pA E di Size Gain La Aspect Ratio 1 evt Sin ye e mi R LE jets n mE A b wm Area Z H E i zj i m B oce E puc Zl z x T o TN 500 80 um 2 9 V iru a te Lator gt i an 20 0 488 20 _Auto wj a Fa TIN 3j Filter Mode E Laser Kalman Line C Frame 2 Analogint lt Pltpton Cnt 45894 100 5 E 488 JE gt 20 0 Moi s54 gt 75 543 JENNI gt 504 63 m gt 20 0 LambdaScan Stan 531 nm mt 613 nm StepSizel 5 nm Nm 2 S Band Width 77 nm xr Aj YU mmm e TD1 HV100 KEGI f Jg uc LIE
59. 1 2 1 4 8 1 Cr Betjes PCHig 1 2
60. Create a new gel Next Gel lt gt Anatech 2012 6 Anatech Anatech 4 gt 4 IPG IEF Type PX Pro3900 SDS SDS PAGE Dual 200K SDS PAGE 37 200 28 200 36 160 Anatech e mail HP e mail Anatech E mail koga m anatech co jp URL http www anatech co p Tel 03 3812 8701 Fax 03 3818 9167 T 113 0034 2 31 15 129
61. PPT DHA D glucose D IR ED GLUT glucose transtorter v5 Rig 1 10 DHA AA ROS ROS ROS AA 13 Ascorbic Acid AA HO H ts SVCT DHA D glucose ra 0 0H HO VH OH Dehydroascorbic Acid Dehydroascorbic Acid Dehydroascorbic Acid H 3 HH DHA Hydrate Bicyclic Hemiketal HO a _ HO de O M Cyclization 0 OHOH Fig 1 10 AA DHA
62. ROS NADPH PKC ROS ROS O gt Os SOD Superoxide dismutase SOD Or Cu Zn SOD IX Glul32 Glu133 Lys136 Thr137 AHD Cu Arg143 O 7 SOD 20 2H SEZRXUAMRULC ROS se Dd RSH r mo 3 00 57 7 SS LO GSH GSSG 2H O gt 2HO Q ROS SOD ROS
63. Mean Grey Value DATHA Set measurement Analyze Analyze particle Size Circulatory shows Display Exclude on Edge Clear Size 50 infinity pixel Circulatory 0 1 0 show outlines Display on Exclude on Edges on Clear on then OK D 4 84 A 4
64. a TTP a TTP Vall82 Leu189 oe Tocopherol 1 Hig 5 8 B Val182 Tyr 117 Se y 82 B4 a e 27 IX 28335 Phe187 292 is 9 305 Leu189 177 C WT Ser140 s lle154 Phe133 t4c CH _ e aca lle194 Val182 og ides de S Leu183 ile179l 2c 1Ser136 Trp163 lle171 Fig 5 8 a TTP a TTP Vall82 Leu189 55 TMG GLUT o TTP 2 AAG TMG
65. Eig 1 5 7 LATER H H ATP synthase UCP Fig 1 5 ROS Ey T NADH Q c Cyt c V HO02 Fig 1 6 OH ROS L H O Fe gt OH Fet OH Fig 1 6 D glucose C Protein Kinase C PKC AGEs 4
66. JJ Medium 199 SIGMA Cat No M2154 PBS SIGMA D8537 BDTA SIGMA T4174 Fetal Bovine Serum SIGMA Cat No F9423 HPMC informed consent 1 D 2 0 9 NaCl solution 5 0 mmol EDTA 5 ml NaCl solution OD 10cm XS TOO TERR 5 0mmol EDTA 5 0ml 0 9 NaCl solution 2L 2 5mmol L
67. 2007 ROS ROS 19 2008 Biesalski HK Vitamin E Requirements in Parenteral Nutrition Gastroenterology 2009 137 5 892 104 Qian J et al Intracellular trafficking of vitamin E in hepatocytes the role of tocopherol transfer protein J Lipid Res 2005 46 10 2072 82 Christopher Min K et al Structure and function of alpha tocopherol transfer implications for vitamin E metabolism and ANED Vitam Horml 2007 76 23 43 Howard AC et al Promotion of plasma membrane repair by Vitamin E Nat Commun 2011 2 597 Luzio M et al Antioxidant Activity of Vitamin E and Trolox Understanding of the Factors that Govern Lipid Peroxidation Studies n Vitro Food Biophys 2009 4 4 312 320 Demir AY et al Proteome analysis of human mesothelial cells during epithelial to mesenchymal transitions induced by shed menstrual effluent Proteomics 2004 4 9 2608 23 Kratochwill K et al Stress Responses and Conditioning Effects in Mesothelial Cells Exposed to Peritoneal Dialysis Fluid J Proteome Res 2009 8 4 1731 47 Lechner M et al A Proteomic View on the Role of Glucose in Peritoneal Dialysis J Proteome Res 2010 9 5
68. 4 5 1 1 AAG 4 5 1 2 TMG xg lt ee 45 5 1 PDE HPMC 5 2 o SMA 5 3 ROS 5 4 AAG TMG 5 5 AAG TMG EN 9 60 Eee P wc 3 5165 V TM SS LLL LQLLLLLLOLOLAQDLID 67 11 APPENDIX e o o o o o 68 A 1 A 1 1 A 1 2 A 1 3 A 2 SA B Gal A 3 ROS A 4 A 5 B 1 B 2 C 1 1 103 Rs ml 1 1 1 1 1 BERRES 30 10
69. IP ATP ROS ROS BUKIM ROS PL ROS DNA DNA DNA DNA p53 p53 p21 42 TGF f1 p16 SA B Gal TGF B1 o SMA Cyclin Dependent Kinase CDK 2
70. 7 AGEs GDPs AGEs ROS 7 PKC Diacylglycerol DAG PKC NADPH NADPH Or NADPH 8 Hig 1 7 ooo Fig 1 7 ARKI NADPH ROS 02 O2 H202 MPO HOCD O2
71. Table 3 3 3 4 3 ROS Qmage Image J 0 Threshold 1 1 4 1 10 APPENDIX 25 3 5 PDE HPMC Cytokeratin 18 a SMA PDE HPMC Cytokeratin 18 a SMA PDE HPMC L PBS
72. he Uc Ae COE BET Ta d BE 2e EO TERT TITO Fig 10 2 d Q FITC Fig 10 2 FITC fluorescein isothiocyanate HO O O COONa Ss Fig 10 3 FITC 85 e TIE Ri Vortex ESSI gt A EE REELT FV1000 Olympus Tokyo Japan pari aceton WAKO methanol WAKO PBS SIGMA Goat anti mouse IgG H L FITC conjugate Cat No AP308F Millipore Propidium iodide PI P4170 25MG SIGMA Anti Cytokeratin 18 Mouse Mono RGE53 Cat No MUBO327S Anti Cytokeratin 18 phospho Ser 52 Human Rabbit Cat No SC 17032 R mouse anti human smooth muscle actin Cat No M085101 DAKO
73. 20C us 3650 01 anatech 3650 08 anatech 3650 03 anatech 3650 04 anatech 3650 10 anatech 3600 11 anatech 3600 12 anatech 3600 13 anatech 3650 18 anatech 3202 05 anatech 3900 01 anatech 17 1233 01 GE 3600 14 anatech TIPG IEF Type PX 104 o 60 90 PREL
74. PDE HPMC Fig 5 5 HPMC PDE HPMC 200mM PDE HPMC HPMC HPMC 0 1mM H 0 0 1mM H505 1mM H505 Fig 5 5 PDE HPMC HPMC ROS 0 1mM 100mM 0027 7 v a AE BR AE T EL E f ED ROS HPMC ROS PDB HPMC ROS THIXEZ 030r 0 200mM ROS PKC ROS EMT 3323572075 X tv S Table 4 1 Hig 4 14 Fig 4 15
75. 1 103 68 A 1 A 1 1 lt gt 50ml TERUMO 30ml Cat No 60 543 001S PH Millex GV MILLIPORE Cat No SLGV02SLS 0 22um Strivex GS MILLIPORE Cat No SVGSB1010 0 22um STERICUP MILLIPORE Cat No SCGVU0SRE 15ml 0 5ml Cat No 72 699 S Medium 199 SIGMA Cat No M2154 Penicillin streptomycin SIGMA Cat No P 0781 Insulin Transferin Selenate SIGMA Cat No I 1844 L Glutamine SIGMA Cat No G 7513 Hydrocortisone SIGMA Cat No H 0888 IN NAOH aq IN HCL aq 080 08065 Wako Fetal Bovine Serum SIGMA Cat No F9423 Diethylpyrocarbonate Tiu O ITS Insulin Transferin Selenate ITS ORI DEPC 50 ml AGUEERL 1 5m 500ul 4 L G L Glutamine 15 ml 5 ml 4C P S Penicillin Streptomycin Solution 5 m 35 m 20 a HC Hydrocortisone HC 4 mg 2 ml ethanol
76. A 2 DCF ROS KOS ESR WE ROS 27 7 DCFHD 123 Dihydrorhodamine 123 ROS ROS 2 7 Dichlorodihydrofluorescein diacetate DCFH DA DCFH DA DCFH DCFH DA
77. sciens ees AUI s 9 117 lt gt co lt gt D 02g 20mL Milli Q 50mL 4 2
78. 3 37C 2 30 X DEPC E RNase 121 40 1 500ml DBPC DBPC o CV SE i bI E mE PS TIO DBPC IKETE ED DEPC 133 Random Primers Primer gt Stock Solution S S 9 RA OO ASIE DEPC lt gt D 1L D 121C
79. 9 1096FBS M 199 Iml 3ml RAZIEL 10 200ul 25Scm 1ml 75cm 37C COS 75 A 2 SA p Gal Senescent Cells Histochemical Staining Kit p galactosidase pH6 B galactosidase Senescent Cells Histochemical Staining Kit Cat No CS0030 Sigma Aldrich Components Fixation Buffer 10x Cat No F1797 15 ml Solution containing 20 formaldehyde 2 glutaraldehyde 70 mM NasHPO4 14 7 mM KH 5PO 1 37 M NaCl and 26 8 mM KCI Reagent B Cat No R5272 1 5 ml 400 mM Potassium Ferricyanide Reagent C Cat No R5147 1 5 ml 400 mM Potassium Ferrocyanide e X gal Solution Cat No X3753 4 ml 40 mg ml e Staining Solution 10x Cat
80. 180 Over Night 132 B 4 lt FBS gt D 20C FBS 500mDZ 37 S 56C 30 STERICUP 50ml 40ml 20 CCD lt ITS gt ITSA RO DEPC 50ml ZAI TREE HC HC 2mg EtOH DEPC 1ml MILLEXGGP DEPC gt D Milli Q ZKZ4Z amp B C 229 7 AREE ROO 500ml 500ml Milli Q DEPC 300ul 0 1 DEPC
81. EAE o 2 6 SDS DTT SmL Milli Q 9 Pro3900 STOP Pro3900 SDS SDS Milli Q 9 1
82. 3068 35 anatech lt gt D 99 D Sn m Lk DF aA T 7 TEK E HHZ omm RROA Fu P s e LR X d m L2 30 MRRENEENEENENENEEREERENNN 99 P enu SUE IT l 2 Il ee
83. a SMA zH 505212925 74 APPIA JA J A TIAA DLA IAS LA DN 3 PD EMT Fig 4 Dialysate glucose N end products AGEs Xidauve sue Epithelial to Mesenchymal Cellular Transition EMT Senescence Fig 5 4 PD EMT 535 fa E 0o JOE DIREA 50 5 3 ROS ROS DCFH 4 ROS Fig 4 10 ROS ROS
84. 2 Ascorbic Acid 2 Glucoside AAG CGTase cyclomaltodextrin glucanotransferase AA cw Fig 1 11 AA 2 C D glucose 338 27 CH OH CH20H ks H C OH G HO OH GO i a Au H O H CGTase b OH H O HO H OH O O Ascorbic Acid AA Ascorbic Acid 2 glucoside AAG Fig l 11 Ascorbic acid 2 glucoside AAG 14 AA AAG AA we AA Glucose 1 2 2 2 a a E
85. E 1 DNA p53 7 D glucose PH 18 2 Human Peritoneal Mesothelial Cells HPMC
86. PD 2000 PD 45 Mo PD 3 EU ATHV 2o 2003 PD EMT HPMC 23 PD PD 4 1 PD Hig 4 5
87. gt lmMAAG AAG 0 0135g 40mL OJ amp TSZKUCIG REL 1mM AAG mM TMG TMG 0 0159g 40mL 1mM TMG lt AAG gt 1 1mM AAG 3 70uL 10 FBS M199 SmL 250uM AAG 10 FBS M199 2 250uM AAG 10 100 25uM 2 5uM AAG lt TMG gt 1 1mM TMG 1 26uL 10 FBS M199 SmL 500uM TMG 10 FBS M199 2 500uM TMG 10 100 50uM SuM AAG 82 III JCf8 V V SA XC AC Ji JG ORI XE 4
88. 3 6 4 Flamingo Flamingo 1 30 Milli Q 3 10 L Flamingo 1 Milli Q 1 3000 Ver3 1 2 Progenesis PG200 PG220 2 StatViewe J 5 0 SAS Control t Control t P P lt 0 05
89. CD 20 20 8 24 102 A 5 3 lt gt Ea FFIBCED UO xs C e ce lt gt D D PBS ImL 1 PBS PBS 1mL 5000rpm 4C 5 D PBS 4
90. ROS ROS 200mM 6 ROS ROS 51 6 1 6 200mM ROUX 200mM ROS PDE HPMC HPMC ROS 0 1mM 1mM
91. SA B Gal o SMA 3 i ROS ROS DNA p53 p21 SA B Gal aA TGF pl p16 SA B Gal TGF B1 a SMA ii GDPs EMT ROS DNA p53 p21 SA B Gal SA B Gal MSC SA B Gal
92. 2 PDE HPMC 3 ROS PDE HPMC AAG TMG AAG SVCT GLUT TMG GLUT TTP EMT AE AAG TMG 60 7
93. AA Ascorbic Acid 3p 723452237 AA AAG SVCT Sodium dependent Vitamin C Transporter 1 IL SVCT 12 SVCT1 SVCT2 2 Na SVCT1 SVCT2 AAG AA SVCT AAG AA 2 GLUT Glucose Transporter AAG SVCT GLUT 2
94. D 121C 20 eS mS 9 x1 x3 4 x 1 xl 20cm x20 121 20 WAZITO o Reaction Tube with Cap PCR Ex FH ODE Z ERCO WAT Z TCE 1210 20 o10ml D20 24 24 2 10ml 3 3 180C C 4 o SLE HIC 7cm
95. DEPC 2 ml 69 0 5 ml 200 ul Jam D L G P S 5 ml L G HC 50 ul 10 ml TERUMO Millex GV Medium 199 x pH pH pH 7 2 pH 7 4 XPH 7 2 pH 0 2 1N HCI aq IN NaOH aq pH lit O Fetal bovine serum D FBS 37C 55 56 CT 30 50 ml TERUMO Strivex GS Stericup 30 ml 25 ml
96. AAG TMG AAG TMG SVCT GLUT TTP SVCT GLUT TTP HPMC AAG TMG TOF MS TGF B1 EMT EMT ARREO RR REIES OREM
97. Fig 4 16 Control Fig 4 17 AAG 250uM 4 gt 7 pl i 250 3 T WP e 150 7 ti ci 3400 E i 75 ELIT P 50 9 B t x i j 6 p 37 2 O Fig 4 18 Red Control Green AAG 250uM 43 4 5 1 2 TMG TMG Fig 4 19 Fig 4 20 Fig 4 21 TOF MS Fig 4 19 Control Fig 4 20 TMG 50uM pl 250 AIT 150 Free i E 5 1 re 100 Mo COLE m Qo s se 3 25 Fig 4 21 Red Control Green TMG 50uM 44 5 5 1 PDE HPMC PDE HPMC Peritoneal Dialysis Effluent derived Human Peritoneal Mesothelial Cells HPMC
98. lt SDS 2 gt e Urea 3 03g e Dithiothreitol DTT 0 05g e 0 5M Tris HCI pH 6 8 0 5mL e 10 w v SDS 2 00mL e 0 1 w v BPB 0 25mL e 50 V Y Glycerol 5 00mL DTT BPB 0 5M Tris HCl pH 6 8 1 1096 w v SDS S0 V y Glycerol 1 lt 2 gt e Urea 3 03g e odoacetamide 0 45g e 0 5M Tris HCI pH 6 8 0 50mL e 10 w v SDS 2 00mL e 0 1 w v BPB 0 25mL e 50 wl v Glycerol 5 00mL BPB 96 lt SDS Tris Glycine gt 10x e Tris 30 3g e Glycine 144g e SDS 10g e Distilled Water to 1000mL 1x e Tris 3 03g e Glycine 14 4g e SDS 1 0g e Distilled Water to 1000mL 1 8L 1 10x 10
99. 121C 40 1 500ml pe NE X DEPC DEPC X DBPC DEPC A 1 2 CO l10 0212 TOMY RX 200 TE TL FS e Vortex TA 18 15ml Cat No MS 56150 50ml Cat No MS 56500 OX XC 10ml Cat No MS 66100 e Q35mm Cat No MS 80240 71
100. S Ascorbic Acid AA ROS AA AA AA AA Fig 1 9 AA 1 DHA DHA 2
101. 2 1 105 1 18uL 1 18uL 1mm 18uL 3 34uL 106
102. A 6 lt gt DONO StatView J 5 0 StatView ex 1 2 3 2 1 qu UN Post hoc gt Tukey Kramer 1 2 e T E Fg DE NAN J 2 REJ r 5 Ef e J 5 2 667 12 247 1 2 1 X 1 3 2 3 S gt 130 B 1 i HS ABS ri HER ER CA Z7 v 7 3 WES UI vig E 2e E FH OO ACER HARE IH 121C 20 50 EOF
103. G SG 5 Pause 0 gt nb 109 lt gt D E FE 15 2 0 0 2mA 15 1mA BPB
104. MSC 5000cells cm2 3ng uL bFGF 10 FBS DMEM o35mm 3 1 3 2 1 Table 3 2 Table 3 2 uus AAG TMG 3M o Contrl 0 0 sw 3 3 PDE HPMC B galactosidase Senescent Associated B Galactosidase SA B Gal Sigma Saint Louis USA Tor pH 6 0 B Galactosidase B Galactosidase X Gal X Gal PDE HPMC PBS PBS Staining Mixture 37C
105. OH 1 2 2 ROS ROS ROS DHEARC Do Cv ROS Superoxide dismutase SOD SOD 4 SOD 2 0 gt Cu Zn SOD Hig 1 8 9 XO Jf e e e e 0 Oy p H 0 eOH H O o SOD g 0 MPO GE HOCI HO LOOH 2GSH oxTRX H O GPX GR o TR De LOH H O GSSG H20 redTRX 2H 0 Fig 1 8 ROS
106. XO SOD Cat MPO GPX GR TR PRX GSH GSSG oxTRX redTRX ROS ROS ROS Ed DNA DNA Table 1 3 10 Table 1 3 E O SOD in O OH O H202 O OH O
107. D 10mM DCFH 20uL M199 20mL 10uM DCFH Control ERO 10uM DCFH 1 08mL 1mM HO 0 12mL 0 1mM H20 10uM DCFH 81 I 24well plate D 24well plate 2 10 FBS 0 4mL well 3 JUL ER TS CHR GE 1x104 cells cm 1Sample Control 24well plates 1well 0 1mM H202 2well D 37C 5 CO confluent AAG TMG 1 I D PBS D 0 1imM HLO 10uM DCFH DA 15 Control 1well DCFH 0 1mM H202 2well 0 1mM H202 10uM DCFH DCF AAG TMG
108. BATO 1 1 30 Hemodialysis HD Peritoneal Dialysis PD 2 ENCREIRE O D glucose pH 3 1 4 5 1 2 1 3 4 1 1 MIDLET
109. fJ SmL ARCAIA SDS PATa YY 15 SDS 112 A 5 6 SDS lt gt 3320 01 anatech 200 3068 39 anatech 3068 34 anatech 3068 35 anatech Pro3900 3900 01 anatech 3202 05 anatech lt gt D D SDS 2L Ke npo
110. 580nm Pro Q Emerald 490nm CCD 520nm Phospho QUANTI CCD 440nm 9 SYPRO Ruby Pro Q Emerald Blue Flamingo Blue Green 2 XLED 550nm Blue Pro Q Diamond Green 2 Phospho QUANTI UV 37Snm SYPRO Ruby 5 15 Flamingo 3 10 Pro Q Diamond 60 7 120 Pro Q Emerald 120 240 Phosp
111. v A R 126 B ET ues Ue Gel 1 Gel 2 Normal Overlaid Gel 2
112. 100 90 80 70 60 50 4 4096 3096 of a SMA positive cells 20 1096 4 ABCEHNOQRT n 16 15 1 3 5 10 1 4 6 2 Fig 4 14 a SMA 0 41 4 4 3 CAPD 12 oc SMA Hig 4 15 EMT 60 n s 40 E of a SMA positive cells e 1096 0 12 13 CAPD months Fig 4 15 CAPD a SMA Tukey Kramer P 0 01 P 0 05 n s Not Significant 42 4 5 4 5 1 4 5 1 1 AAG 500 Hig 4 16 Fig 4 17 Fig 4 18 Da CN 3H n e aU i QC
113. VBF NN E Ee re 168 80 um 2 9 V vy v X 9o v X Laser Laser e 488 v 20 0 Auto Filter Mode C Analog Int C Photon Cnt r 0 Export m 2D View Image0001 Ful Sasen Image Serd This Area of the Image to Cipbosrd Save Ores Shitecr4s UHE spisy m Data Manager Save As AYI Export Pret Select AL ROIS QtA Save in kadosono e ga Cancel Selection m Fip Selector 2305 11 30 06 5 17 06 9 6 06 10 27 DJ Delete ol ROE 05 12 1 06 5 25 06 9 12 1306 11 21 1 Paste ROI F E06 t 17 B06 6 6 C306 9 15 C306 12 5 C306 2 10 1306 6 8 C306 9 26 C306 12 12 306 4 11 306 8 31 C306 9 27 SMActn E06 4 21 IDAY 506 9 5 06 10 3 Show Profe Vert Show Profile Ho wv Show Overlay Add a View Remove Selectec View File name ImageQ001 Save File type JPEG Custom Export Settings Information Folder Jump Bt Depth 24 BksiPpixel ROI Overlay e No Overlay A 4 Ol SO es e4 H ELLE IIT 9 TILECh x 328 279 Int 263 PEG Output Format 1RG5 Color using ed LUT pa sintak i Quality 100 Gray Scale lowest 100 highest otheredefauk 70 zd TILE Ch xyz 378 442 Int0 Z
114. amp 1 amp l 2 SDS lt 93 o SDS PAGE 4 X 7 250kDa d v r di MC 5 a 5 V Pet 25kDa p5 A 5 1 o d HH as H E e Urea 219 00175 Wako e Thiourea 208 01205 Wako e CHAPS 347 04723 Wako e Dithiothreitol DTT 045 08974 Wako e Pharmalyte pH 3 10 17 0456 01 GE e Protease Inhibitor e Triton X 100 T9284 Sigma e Acetic acid 017 00256 Wako e Bromophenol Blue BPB MS8G8844 e Tris 207 06275 Wako e HCI 080 01066 Wako 94 SDS Glycerol Iodoacetamide Glycine Methanol Flamingo x10 Milli Q 199 07141 Wako
115. un 8096 4 70 Transformed ibroblast like Ac 60 L 35 50 mu z Sp 40 p S e 30 z 20 w Cobblestone 10 This study Previous study This study Previous study 1 12 13 Duration of PD month Fig 4 3 CAPD PDE HPMC Cobblestone hke Morphology Fibroblast like Morphology Fig 4 4 PDE HPMC Scale bar 200u m 30 Percentage with Phenotypes Ui o Q o A 3 1 Extraneal Fig 4 5 n 6 n 73 Transformed Fibroblast like Cobblestone Only Neutralized PD fluid amp Neutralized PD fluid Acidic PD fluid Fig 4 5 PDE HPMC 31 Fig 4 5
116. 10 99 98 30 100 e S P2 99 o 310 s o c ET ecu Eun ERE ER ad RR e Boc DES i sanc m o SMA Pt 5 a 34 K 4 18 BE E HH A mE mauu NT Lm n ad p c uu o cur m m EE rnit Nera Ec UE cun ue cgi ua oi i ee LE EE 51 Er ERE ERES DEREN RENE E NE ETT DECENT RTT o 77 50 50 8 7 7 77 83 958 oec mon 2 5 5 m 3 a 1 o BRKE Es mew 1500 zx isoo o mo isoo so ro o so g 1573 2684 2751 174 1558 1802 1520 1572 1761 405 ee erim LL aum li NEQNE E ci Wi Rag INT cg RT n IT ID a WIE i ENT nt NT ci Pe Wa 140 Re rc com 62 65 68 2012 5 28 2012 5 29 2012 6 5 2012 6 12 2012 6 13 2012 6 19 TCR TT 19 EE EI EE E o DEUM p 1395 948 1632 1759 1272 936 1185
117. 15 CO SA B Gal 10 APPENDIX 23 3 4 PDE HPMC ROS 3 4 1 DCF ROS 33657 Uu 7 DCFH DA 2 7 Dichlorodihydrofluorescein diacetate ROS DCFH DA DCFH DCFH ROS DCF 502nm 520nm ROS DCF ROS jt lt gt PDB HPMC K 0 1mM 10uM DCFH DA 1
118. xi The analysis of your gels it now ready lo be pedomed TT esse oo 125 lt gt Progenesis PG200 PG220 A B C C A V EM e SSA Mm oo V Gell AN l 8 cel2 e
119. 24 Anti Oxidative Potential and Cellular senescence of Peritoneal Dialysis Effluent Derived Cells mu mll J 11 1 1 1 1 1 2 pH GDPs 1 1 3 1 1 4 1 2 1 2 1 ROS 1 22 ROS 1 2 2 1 1 2 2 2 1 3 1 3 1 1 3 2 19 e eoe ot on 20 3 1 PDE E HPMC 3 1 1 PDE 3 1 2 HPMC 3 1 3 3 2 MSC 3 2 1 3 3 PDE HPMC
120. 28 4 4 1 4 1 1 3 1 3 1 Hig 4 1 Hig 4 2 2096 T 9 of Adhesion A 0 5 10 Fig 4 1 PDE HPMC PDE HPMC Aout Fig 4 2 PDE HPMC HPMC Scale bar 200u m 29 4 1 2 Midi a addidi ig 4 3 n 23 n 44 n 56 n 15 100 EE Em 90
121. D D 10ml Lc 3 PBS 3m25cm 10ml 75 cm 10ml Lg lt gt D Iml 9ml 10 EARLE TVARI 2 sml 25 cm 10ml 75 cm 37 COs 10 3 FBS 1ml 25 cm 2ml 75 cm O 10ml 15ml CANA TAE 50G 750rpm5 4C dst 07 BE UT CD 10 FBS M 199 37C 5 CO2
122. Gel 1 C E Main Image Depay Choose how to declay the gel in tha Iain Image winda Homa Altez these aels a gale i especiment Pa M arp ta mecher E o Perom automatic wa 2d Cles manual werpng A Clea all wa ping Hl Warping Mode ww RA lt gt 1 yir une epa A Experiment 7 Gel 3 Create Reference Ee SSA 3 PEP Select Gel a gel4 Create Averaged Gel Move Up Move Down Remove Gel Rename Cancel 127 S SSA 3 a SSA 3 Ref gel 3 e gel
123. Table 5 1 2D m comm nw x p Actin 5 3 1 Cytokeratin 8 E 3 C 19 C Cytokeratin 19 19 Heat Shock Protein 27 HSP27 PI Glutathione S transferase a 1 B Actin Cytokeratin CK 8 19 Z CK 8 B Actin CK 19 1 AAG TMG CK 19 TOWO 2 B Actin CK 8 2 CK 19 1 CK 19 ptI CK 19 p 2
124. 3 HSP Heat Shock Protein 27 4 S TOF MS TOE MS AAG TMG EMT 59 6 D PDE HPMC 2 HPMC 2 Extraneal
125. 5 4 AAG TMG AAG TMG ROS Hig 4 11 Fig 4 12 DCFH DA PBS 2 AAG TMG 0 1mM AAG TMG ROS AAG 2 SuM 25uM 125uM 250uM 4 AAG Hig 4 11 AAG AAG
126. 223 3525 AGE 1997 EZ 2000 2004 Wu X et al Upregulation of sodium dependent vitamin C transporter 2 expression in adrenals increases norepinephrine production and aggravates hyperlipidemia in mice with streptozotocin induced diabetes Biochem Pharmacol 2007 74 1020 1028 Seno T et al Functional expression of sodium dependent vitamin C transporter 2 in human endothelial cells J Vasc Res 2004 41 345 351 Tsukaguchi H et al A family of mammalian Na dependent L ascorbic acid transporters Nature 1999 399 70 75 Corpe C P et al 6 Bromo 6 deoxy L ascorbic acid an ascorbate analog specific for Na dependent vitamin C transporter but not glucose transporter pathway J Biol Chem 2005 280 5211 5220 Kc S et al Vitamin C enters mitochondria via facilitative glucose transporter 1 Glut 1 and confers mitochondrial protection against oxidative injury Faseb J 2005 19 1657 1667 Aga H et al Synthesis of 2 O a D Glucopyranosyl L Ascorbic Acid by Cyclomaltodextrin Glucanotransferase from Bacillus stearothemophilus Agric Biol Chem 1991 55 1751 1756 EAA 2006 51 1 10 17 Dimri GP et al A biomarker that 1dentifies senescent human cells in culture and i
127. 348 15um 0504 50 op SaD V StepSize 1 ns um op Clear StartEnd Slices 23 Focus Handle On Escape X 0 334um pix Y 0 334um pix Z 0 700umislice TimeScan Interval FreeRun sec Num 2 3m 89 m Bleach Stop Bleach start stop by Key D 40 Ous Pixel AcquisitionSetting r Size price 1 1 C 4 3 C arbitrary P 1024 by 102 Iz Laser 458 lH 10 0 488 lt 20 0 515 4l gt 75 543 lt j gt 50 4 6334 m gt 20 0 LambdaScan Stan 531 qm End 613 nm StepSizel 5 nm Num 2 S Band Width 77 nm Microsc pL stpo 20 20X NA 75 Ys Set 860 56 um 6o 1130 83 7 oium Go V EE sem o 348 15um Set 0 iv StepSize os nm um op Clear StartEnd Slices 22 Focus Handle On Escape X 0 334um pix Y 0 334um pix Z 0 700um slice r TimeScan terval FreeRun sec nm 7 CX 90 2 lt XY UM LI a a ImageAcquisitionControl lt SU TR C A Lamp
128. GDPs EMT 2 AGEs AGEs 49 Fig 3 3 N m PHE a NADH NAD PIP2 E a Ras Smad2 3 DHAP gt a Glycerol P PKB Akt RhoGEF Mitchondoria Raf Smad2 3 RhoA i ROS 2 MEK1 2 GSK ROCK N p catenin ME ERK1 2 Lef 1 MRTF NENS s er MRTE em nTa ad ID DNA damage Est2 Estl ma B catenm MRTF I DA LE z Cp bs Fig 5 3
129. HPMC APPENDIX NL 2 3 1 3 935mm ZESRISUR CK2 Olumpus Tokyo Japan CES 7 CAMEDIA C 3040 ZOOM 4 PC fig 3 1 Hig 3 2 Digital camera CAMEDIA C 3040 ZOOM Sample USB SmartMedia Reader Wnter MLAUSB 2 Phase contrast microscope CK2 Personal computer Fig 3 1l 1400 1200 vzAePS z hd 5 1000 9 NII E 9 2 600 2 T 7 HE tg 0 U 10 1 CAE aZ cells REF 00 aTOSHEK cd Fig 3 2 22 3 2 MSC MSC
130. d gt 9uL 1000uL 1 3 5mm 5mm D 6 6 9 3 O 1000uL 5 SDS
131. lt gt l APRECI OR 2 R ON aAa NN 1 pH eU 2 Milli Q 3 113 4 RB gInBmBU 19cm 18cm
132. p S b S d od R Fig 5 9 a Tocopherol Trolox P 1 a Tocopherol 2 Trolox cw Tocopherol Trolox 56 AAG SVCT GLUT TMG GLUT co TTP TMG Fig 10 A 19 TMG AAG Non mediated lt i Transporter R diffusion e GIUT g NADH NAD ee DHAP gt a Glycerol P 2 TGF p1 1 Membrane Repair Qm Fig 5 10 AAG TMG
133. 170 150 1500 1700 1500 2500 2000 150 250 200 g 1929 1790 1775 2412 1475 2759 2131 1603 2924 2465 g 214 277 697 38 236 113 401 447 x 10 cells 6039 106 8 1 130 363 513 2 0 59 0 40 1 5396 0 58 0 99 5 34 days A N N 7465 0 76 m N m N m N N ad d N O m o np CO N Oo 10096 99 98 45 9 48 Un N X 96 85 99 91 99 98 99 99 Cytokeratin 18 96 N HG 2 10096 9996 100 In a SMA 18 9 28 2 27 22 16 20 25 41 9 2 70 72 37 Tig A N TER O cm2 i N m DUENE Waa ne Wel le Wt li t t 0 160 0 068 0 249 0 206 0 034 0 053 0 442 0 221 0 390 0 118 0 279 0 395 0 266 0 152 0 186 0 006 0 302 0 270 0 169 0 088 0 255 0 278 0 092 0 002 0 162 0 074 k day ji IET Desmin Vimentin SAHB Gal P 35 1 009g ml id TEE IE 1 136 ue 322 523 26 8 mn man man man man man man mn mn man CAPD month 7 9 o
134. 2472 9 65 61 Lau AT et al A Proteome analysis of the arsenite response in cultured lung cells evidence for in vitro oxidative 1nduced apoptosis Biochem J 2004 382 Pt 2 641 50 62 Hirahara I et al Methylglyoxal induces peritoneal thickening by mesenchymal like mesothelial cells in rats Nephrol Dial Transplant 2009 24 2 437 47 66 9 J
135. 61 1185 2455 2415 73 om qo pue e e zo0 om 5 om D p p DD TEE i i T i YC E Lo BH IN Er ad D cid o ad Rcg Rat NT EE NUT ag Tg Hc NU TT t a E E E MEHR NUT uu Nd UU cal NUT NOT ui UO ci BI a Uca at Tni LOL Th hh ll Th ss ss guess MU og INIT ig UT ug INI i OTT Cog NUIT WI Tc a BU cid 141 m j nl 2 E g w 9 z a F S s fjs HR RA EIE E gt 3 s s ses HEP RHEE E vj JAHORHR I5 sals E 28 j amp s EWE I i pani ll I JOE AO ERU TE OE LEBER TETTE EET PR WOOO ur E O f mi i e Tar O EE hel o lt s XH a ET 3 E amp Is gt 5 missa s d aHa in 5 5 8 e on 8 E 2 3 9 amp e n i 8 in RET pex e NMM 1 OE LEE BE pret UNAU ERE peet UNAN E eeN PE I JODIE LEA EH dh WE CEN ii i PE i il i mid i Ri S TER O cm qu i HR CNN MU ud sadi P l l l D i it ca It Ii RP nl LP El LLL l LE LE LEE LLL
136. D IECIT E Ag 50ml PBS 40ml Vortex l TR OLE D 1 BSA PBS 035 PBS o o Aceton methanol 1 1 rt 5 10 1 BSA PBS 20 1 BSA PBS 1 200 1ml 4 C overnight 10 86 1 BSA PBS 3 1 BSA PBS 1 200 1ml 30 rt D 1 BSA PBS 3 D ON ON 3
137. GDPs 3 3 4 3 Fig 1 1 3 Monosaccharide l HO C H HO C H autoxidation HO C H lt po H 0H H OH O H50 H C 0H H 08 H 0H H C 0H Glucose HOH HOH HOH glucopyranose Glucose 1 2 Ene diol Glucosone getto MN acyclic a 0 C e 8 chron Glycolaldehyde Monosaccharide autoxidation O H20 0 HH 0 H 0 H N X Retroaldol hf condensation 0H 0H Hs Glyseraldehyde C H H p i ui on H H OH HOH Glyoxal 2 3 Enol 3 Deoxyglucosone Methylglyoxal Fie 1 1 GDPs 4 1 1 3 D glucose 24 BEePHEEIEAUCI E 1 3 4
138. P 0 01 P 0 05 n s Not Significant 37 4 3 3 AAG AAG Hig 4 11 2 5uM 25uM 12SuM 250uM 4 AAG Fluorescence Intensity N gt es gt CTL AAG AAG AAG AAG 2 5uM 25uM 125uM 250uM 1 4 3 4 Control AAG 2 5uM AAG 25uM AAG 125uM AAG 250uM Fig 4 11 AAG ROS Scale bar 200um Tukey Kramer P 0 01 P 0 05 38 4 3 4 TMG TMG 0 0SuuM 0 5uM SuM 50 uM 500uM 5 Hig 4 12 TMG 0 0SuM NA un S WA C Rn C Ui Fluorescence Intensity Un N N A Un o O Un GTI TMG TMG TMG TMG TMG 0 005uM 0 5uM LM SOuM 500uM n lt 3
139. o j Oo J Oo 0 Ee ml 2500 1500 2000 1500 1500 2000 isoo 2000 1500 isoo _ 2618 15855 2305 2983 279 2 6 1612 2c0t1 1646 1507 x 10 cells 491 245 079 054 400 136 2700 097 120 days Cytokeratin 18 Pl 10 9 se 100 1006 100 P2 99 10 99 see 109 59 EN a SMA Pl se 4 33 35 9 so 18 2955 PO 1 m V TB o c ERNEEIEE AEN EP MEE ENT e MEE MEE rci cadi cid ad d m ai aiii tu 9 1 50 OO mo GO m 9 1 9 OUA 1 Se0n kd PL oio 026 034 ooo or os nerada om om J A os ox ow om EE Desmn p2 Nimes poe ccce ees inesse G C Soo PO L L LLL LL LI LI LL L 137 lee es 35 36 8 39 woman woman man man man man ma man man ma 68 68 76 249 8 83 449 7 8 49 Go on 2 2 a s s
140. 17018 25 Nacalai 095 02891 Wako 077 00735 Wako 137 01823 Wako 161 0491 Bio Rad lt gt e Urea 1 50g e Thiourea 0 75g e 20 w v CHAPS 0 50mL 0 10g e Dithiothreitol DTT 0 05g e Pharmalyte pH 3 10 pH 0 10mL e Protease Inhibitor Milli Q 5mL DTT 1mL DTT Pharmalyte pH lt 4 gt Milli Q 20mL DTT Urea 7 2g Thiourea 3 04g 20 w v Triton X 100 2mL Dithiothreitol DTT 0 04g Pharmalyte pH 3 10 pH 0 20mL 0 1M acetic acid 0 50g 0 1 w v BPB 0 50mL BPB 95 20 w v Triton X 100 1 0 1M acetic acid 1 F Pharmalyte pH
141. 34 PDE HPMC ROS 3 4 1 DCF ROS 3 4 2 DCF ROS 3 4 3 ROS 3 5 PDE HPMC Cytokeratin 18 a SMA 3 6 3 6 1 3 6 2 3 6 3 3 6 4 Flamingo 3 7 lt 5 29 4 1 4 1 1 4 1 2 4 2 4 2 1 MSC SA B Gal 4 2 2 Senescence Associated B Galactosidase SA B Gal 4 2 3 CAPD 4 3 ROS 4 3 1 DCF ROS 4 3 2 CAPD 4 3 3 AAG 4 3 4 TMG 44 Cytokeratin 18 a SMA 4 4 1 Cytokeratin 18 4 4 2 a SMA 4 4 3 CAPD 4 5 4 5 1
142. 4 B EZUSZUU UCVvZ FCAS Gel 4 Main Image ges Display Warped Affect these gels 5 3 Ref Current gel 2 Zoom to Fit Eg 128 C 1 2 2 Edit Chose reference gel
143. 8 Bis 80mL 1 5M Tris HCI pH8 8 60mL Water 96 3mL 10 SDS 2 4mL 10 APS 1 1mL TEMEDR A ET 1 TEMED 10 12 5 TEMED 1 5M Tris HCl pH 6 8 4C 10 SDS 10 APS 4C 1 30 Milli Q 4C 1 38 98 o lt gt SDS PAGE 3037 00 anatech 3037 00 anatech es T NAE 3022 07 anatech e 3037 00 anatech e 3070 41 anatech e ys DJ mes 3070 38 anatech SDS 3068 40 anatech 3068 34 anatech
144. 9 0 4 Methylglyoxal 8 1 0 2 Glyoxal 7 8 0 3 5 HMF 5 1 0 3 Furfural 1 1 16 3 DG 328 22 Acetaldehyde 60 0 9 D glucose ZiEXEHEWHPUCRE C3 5 EXSTROO polyglucose icodextrin k Z AEWA LL THODA Glucose Glucose pH 2 D glucose D glucose 1960 X
145. No 55818 15 ml e Phosphate Buffered Saline PBS 10x Cat No P3621 60 ml Preparation Instructions 24well plates 10tests plates wells Appendix 1 LN aba O Z 76 X gal Solution 1 37C XX gal Solution 1x Fixation Buffer Fixation Buffer 10x 10 1x Fixation Buffer 20C 24well plates 10tests 3mL 0 3mL x 10well 1 PBS PBS 10x amp 10 f DUJATEZK CART 1x PBS 2 8C 24well plates 10tests 15mL Staining Mixture Prepare just prior to use 3mL 1Owell Staining Solution e300uL Staining Solution 10x 37 5uL Reagent B 37 5uL Reagent C 75uL X gal Solution 2 55mL
146. OH O HOCI 1 2 2 1 irap igi irn 13 Table 1 4 Table 1 4 C B12 m BERE EE S ADRE ii A JV D NS ON TA aE A RD J dE
147. gam a TMG 50uM 30 lt 209 se 20 10 0 P2 PS P19 Fig 4 7 MSC SA B Gal Tukey Kramer P 0 01 P 0 05 33 4 2 2 Senescence Associated B Galactosidase SA B Gal pH 60 B galactosidase SA B Gal PO Table 4 1 SA B Gal PDE HPMC Table 4 1 DD SA B Gal Age CAPD duration SA p Gal 19 072 12 36 ae n 9 27 452 22 89 KEEN n 12 21 06 12 11 n3 26 49 31 17 n 6 4 36 2 11 n 4 12 653 3 83 n 8 8 38 Fig 4 8 SA B Gal Scale Bar 200um 34 4 2 3 CAPD CAPD 12 13 CAPD p lt 0 01 Fig 4 9 kk Un n un n of SA B Gal posi
148. intensity of non spot pixel in a margin around each spot to approximate its background e M ON SD E E T different gels Normally the images are warped to a reference gel before matching is performed v Warp the gel images to the reference gel Match detected spots to spots in the reference gel Add unmatched spots to the reference gel By normalising spot volumes you can accurately compare the measurements ol spots in diferent gels V Normalise spot volumes using this method Total Spot Volume j C Multiply by total area Multiply by 100 Use only spots that we present in al gels To continue click Next Saving the analysis you have just specified as a protocol enables you to apply identical analyses to other images m the future Save an analysis protocol I rcludet Ielerence ge the protocol To continue click Next 3 lt Next You have set up the following experiments SSA1 Add To continue to the analysis stage click Next Back Nee _ Conee Finish
149. solution 343 DCFH DA 9 7k 4 mg DMSO 826 ul L 10 mM DCFH DA 0 5 ml 20 ul 3 Stock solution DCFH DA 50 ml 20 O DMSO 100 uM DCFH DA Stock solution 100 L4 0 22 um HO gt 100mM HO gt 113uL 30 H0 9 887mL 100mM H20 mM H O 1 133mL 30 H0 8 867mL 1M H20 1M HO gt 10uL 10mL 1mM H 0 H20 10uM DCFH
150. to Peritonitis Incidence Perit Dial Int 1991 11 22 26 Castro MF et al CELL POPULATIONS PRESENT IN THE NOCTURNAL PERITONEAL EFFLUENT OF PATIENTS ON CONTINUOUS AMBULATORY PERITONEAL DIALYSIS AND THEIR RELATIONSHIP WITH PERITONEAL FUNCTION AND INCIDENCE OF PERITONITIS Perit Dial Int 1994 14 265 270 Brownlee Biochemistry and molecular cell biology of diabetic complications Nature 2001 414 813 820 Shostak A et al Cultured rat mesothelial cells generate hydrogen peroxide a new player in peritoneal defense J Am Soc Nephrol 1996 7 2371 2378 Lee HB et al Reactivve oxygen species amplify protein kinase C signaling in high glucose induced fibronectin expression by human peritoneal mesothelial cells Kidney Int 2004 65 1170 1179 Nishikawa T et al Normalizing mitochondrial superoxide production blocks three pathways of hyperglycaemic damage Nature 2000 404 787 790 Inoguchi T et al Protein kinase C dependent increase in reactive oxygen species ROS production in vascular tissues of diabetes role of vascular NAD P H oxidase J Am Soc Nephrol 2003 14 s227 s232 2003 FE 1999 62 17 18 19 20 ZI 22 23 24 25 26 21 28 29 30 31 32
151. 12 11 2434 41 Ha H et al High glucose induced PKC activation mediated TGF beta 1 and fibronectin synthesis by peritoneal mesothelial cells Kidney Int 2001 59 463 47 qa 23 FE 2012 Higashi Y et al Characterization of peritoneal dialysis effluent derived cells diagnosis of peritoneal integrity J Artif Organs 2012 JR PE 23 FE 2012 Ksiazek K et al Premature senescence of mesothelial cells 1s associated with non telomeric DNA damage Biochem Biophys Res Commun 2007 362 3 707 11 Ksiazek K et al Mitochondrial dysfunction is a possible cause of accelerated senescence of mesothelial cells exposed to high glucose Biochhem Biophys Res Commun 2008 366 3 793 9 Madan E et al Regulation of glucose metabolism by p53 Emerging new roles for the tumor suppressor Oncotarget 2011 2 12 948 57 Ohtani N et al Opposing effects of Ets and Id proteins on pl6INK4a expression during cellular senescence Nature 2001 409 6823 1067 70 Takahashi A et al Mitogenic signaling and the pl6INK4a Rb pathway
152. 3 L L E DHA GSH DHA AA 12 NADPH H iod P e HO OH NADPH H Ti ed o 2GSH e HO Ascorbic Acid pKa 4 25 ee ak OH OH O O CHCHzOH O O _CHCH2OH Og t lt eS j lt Of HO o Dehydro Ascorbic Acid Ascorbate Radical Ascorbate Anion o c on O C e e O CCH d uo u ocon HO CH D NH Oxalic Acid CH2OH CH OH 2 3 Di Keto L Glonic Acid L Threonic Acid Fig 1 9 AA DHA AA AA AA AA SVCT soudium dependent vitamin C transporter
153. 3 4 1 Control TMG 0 05uM TMG 0 5uM TMG 5uM TMG 50uM TMG 500uM Fig 4 12 TMG ROS Scale bar 200um Tukey Kramer P 0 01 P lt 0 05 39 4 4 Cytokeratin 18 a SMA Cytokeratin 18 oc SMA 4 4 1 Cytokeratin 18 PDE HPMC Cytokeratin 18 98 2 2 2 Fig 4 13 PDB HPMC 120 2 C Y 100 iWopttetet et iE Z soo CC br E 6096 e 4096 pa Q 209 cx 0 A BC EH NOQR T n 17 14 1 3 5 10 1 4 6 2 Fig 4 13 Cytokeratin 18 40 4 4 2 o SMA a SMA PDE HPMC 28 2 10 7 Fig 4 14 PDE HPMC Epithelial to Mesenchymal Transition EBMT
154. 40 134 2013 2 6 A B C E H N O Q R S T U V W X Y Z 17 C E DM j n e sw s 86 O 8 9 o 82 76 969 J J amp amp 8 8 J 8 J 8 9 X GC 4JIJNVX 0 odati QD e e 0 wa Ca pg poppe e e rox people ep ee e Baxter N PD 2 1 5 7 24 25 32 Baxter N PD 2 2 5 5 12 13 16 21 24 25 27 29 30 31 32 33 34 37 40 41 42 43 45 46 Baxter TER O cm C 1 1 135 Wl 4 5 HH7 ms 9 0 2 uH 2010 8 3 2010 8 17 2010 8 31 2010 9 28 2010 10 5 2010 10 19 2010 10 26 2010 11 9 2010 11 16 2010 11 30 83 82 e9 82 7e 49 8 7e 249 8 j CAPD month o j vo j j TD I Ne ml
155. 6 30 84 29 64 1 71 Table 3 1 Table 3 1 PatiensID A B C E H N OJQ R S T UV W Age 83 78 169 62 83 50168 68 71 175 71141173159 CAPD duration 39 71 11 4124 27 4 12 16 37 50 20 1 2 month Amount of 3 15 1500 dialysate 2000 imt me x 150 150 1500 Number of PDE collected DDianeal N PD 2 1 5 Baxter Z Dianeal N PD 2 2 5 Baxter S Extraneal Baxter 20 RR a 7 BE re 77 1 5 0mmol EDTA 5mL NaCl Solution 2 5mmol L TA 18 800rp m 10 30mL 1mL 935mm 10 FBS M199 THELLE 2 3
156. EM EIS xS D glucose Na 132mEq L Ca 3 5mEq L Mg 1 5mEq L CI 96mEq L Lactate 35mEq L THA D glucose pH pH pH EPS pH 2 pH 3 D glucose D glucose GDPs GDPs AGEs
157. Staining Solution 0 2um filter os Procedure 1 2 0 3mL Ix PBS 2 per wellplate 0 3mL well 1x Fixation Buffer 6 7 Staining Mixture 0 3mL Ix PBS 3 per well plate 0 3mL well Staining Mixture 37 w o CO 2hours to overnight DAU 77 8 BLZ RV OC plate Parafilm T PJT 5 pH CO 9 B galactosidase 10 Staining Solution Ix PBS Appendix 1 Relative Volumes for Scale Up Down of Staining Procedure Well Growth Volumes Plate diameter area relative to mm 35 mm plate 78
158. a 5 Oo Oo vo vo ZEE Es ml 1500 1500 1500 2500 1500 1500 1500 1500 1500 2500 1524 1413 1617 3288 1701 1367 2464 1494 1948 2694 10 1o0 104 7 188 146 950 20 434 m 17 u mic 27 2 a 27 gt s o s s TIE on o o urs gt eww sox om F Ww s o w cia Dr Ea cal cag A tokeratin18 Pi nw ae 3e ae a 5 EE EE o mitom NENNEN cn ncn cu RN Rc RR ERR car Ram m 5 gt 3 2 ie zz nsa Lm 8 en e ow ox om os 0 MA 47 48 js 2011 9 13 2011 9 27 2011 10 4 2011 10 11 2011 10 25 2011 11 1 2011 11 8 H5 Bao Na Aa Ban naz A03 77 50 833 ER IEEE DESDE NN DE ESSERE EIER ml 1500 25oo 2500 1500 1500 1500 1500 2500 1500 1141 2924 2768 1801 1e21 2454 176o 1595 2392 1670 373 402 246 288 108 247 815 1305 1565 x 10 cells io Hs daysv n n 15 21 33 12 35 j A H Cytokeratin 18 Pi 100
159. cooperated to enforce irreversible cellular senescence Nat Cell Biol 2006 8 11 1291 7 Takeuchi S et al Intrinsic Cooperation between pl6INK4a and p21Watl1 Cipl in the Onset of Cellular Senescence and Tumor Suppression n Vivo Cancer Res 2010 70 2thre2 9381 90 EAA RR 2006 51 1 10 17 Yamaguchi Y et al THREE DIMENSIONAL INVASION OF EPITHELIAL MESENCHYMAL TRANSITION POSITIVE HUMAN PERITONEAL MESOTHELIAL CELLS INTO COLLAGEN GEL IS 64 48 49 50 51 52 53 54 55 56 57 58 59 60 PROMOTED BY THE CONCENTRATION GRADIENT OF FIBRONECTIN Perit Dial Int 2011 31 4 477 85 Maszi A et al Central role for Rho in TGF betal induced alpha smooth mucle actin expression during epithelial mesenchymal transition Am J Physiol Renal Physiol 2003 284 5 F911 24 Bhowmick NA et al Transforming Growth Factor B1 Mediates Epithelial to Mesenchymal Iransdifferentiation through a RhoA dependent Mechanism Mol Biol Cell 2001 12 1 27 36 Fan L et al Cell Contact dependent Regulation of Epithelial Myofibroblast Transition via the Rho Rho kinase Phospho Myosin Pathway Mol Biol Cell 2007 18 3 1083 97 EUMD 18 FE
160. ho QUANTI 1 fb CL Vig 121 s L7 IBI SU Fal ra Saturation itd LL Cv v2 MESS d AEDT Saturation D ProgenesisPG200 8bit TIFF gt oooo 8bit TIFF 16bit TIFF 8bit BMP BMP BMP
161. n aging skin in vivo Proc Natl Acad Sci USA 1995 92 20 9363 7 2009 81 6 502 510 Parrinello S et al Oxygen sensitivity severely limits the replicative lifespan of murine fibroblast Nat Cell Biol 2003 5 8 741 7 Ksiazek K et al Oxidative stress mediated early senescence contributes to the short replicative life span of human peritoneal mesothelial cells Free Radic Biol Med 2008 45 4 460 467 Ksiazek K et al Oxidative stress contributes to accelerated development of the senescent phenotype in human peritoneal mesothelial cells exposed to high glucose Free Radic Biol Med 2007 42 636 641 Yanez Mo M et al Peritoneal dialysis and epithelial to mesenchymal transition of mesothelial cells N Eng J Med 2003 348 5 403 13 Ksiazek K et al Accelerated senescence of human peritoneal mesothelial cells exposed to high glucose the role of TGF B1 Lab Invest 2007 87 4 345 56 63 34 Bo 36 37 38 39 40 4 42 43 44 45 46 4T Witowski J et al New Insights into the Biology of Peritoneal Mesothelial Cells The Roles of Epithelial to Mesenchymal Transition and Cellular Senescence Nephron Exp Nephrol 2008 108 4 e69 73 Witowski J et al Prolonged Exposure to Glucose Degradation Products Impairs Viability and Function of Human Peritoneal Mesothelial Cells J am Soc Nephrol 2001
162. oom 100 91 V D FV10 ASW WindowsXP Start ShutDown 1ShutDown OFF D OFF AcquisitionSetting Mode 3s QNI 4 OEN lt lt F 20 0usPixel Slow gt gt F Z i TT pi P 20 0us L 21 600ms F 22 334s S2224s s Size ri A poct Rati 6G11 43 ey EE R neH zx p ge E fozatyoa 7 Area 2 A Rotation r 4 F oop lt ves PanY E1 LUC ale Offset 0 10 0 5 ri HV gt 20 0 Bleach Stop Bleach start stop by Key 5 i Filter Mode D eris Line C Frame 2 Analogint Photon Cnt Sequential lt 0 p 9 92 o 0 MP SDS PAGE 2 o
163. oss gels allows you to identify common proteins To arsso MONI Maca Nau os Sad Donc a common How would you like to select your reference gel C Auto select one of the gels in this analysis 3 Gel 1 C Use an extemal shared reference gel w No relerence at To continue click Next Be leo caen Next MID Next VITA I II Eva DF ER RERTA D Next Next Qo 124 xj If you re only interested INDE TEN spots you can tum off spot detection Iv Detect spots Only include spots that meet these criteria Descriptor Takes the most frequently occurring
164. tive cells Un 1 12 13 CAPD duration months Fig 4 9 CAPD SA B Gal Tukey Kramer P 0 01 P 0 05 35 4 3 ROS 4 3 1 DCF ROS Table 4 2 0 1mM DCF PDB HPMC ROS Table 4 2 0 1mM H20 DCF ROS Patients ID aa Fluorescence Intensity i a months 215 E15 3 n5 21 5 17 8 n 9 27 0 n 1 10 72 6 0 n 4 10 8 1 1 n 4 19 3 n 1 22 6 n 1 36 4 3 2 CAPD CAPD 12 13 O CAPD Higs 4 10 CAPD 30 n s N un n n i Fluorescence Intensity y un 1 12 13 CAPD duration months Fig 4 10 CAPD Tukey Kramer
165. toneal Mesothelial Cells HPMC 2 3um 0 08uum Hig 1 4 Ui 4 n Ju gus X zh T ey t y x Is gt E F IMS gnat 4 4 5 rJ e 1 4 yv XI P 1 2 1 2 1 ROS Reactive Oxygen Species ROS O ROS
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