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The role of the low-density lipoprotein receptor family on Cyclosporine A uptake and toxicity in renal… Chung, Nancy S.C. 2004

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The Role of the Low-density lipoprotein Receptor Family on Cyclosporine A Uptake and Toxicity in Renal cells by Nancy SC Chung H o n o u r s B S c , T h e U n i v e r s i t y of T o r o n t o ,  2001  A THESIS SUBMITTED IN PARTIAL F U L F I L L M E N T O F T H E REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES Faculty of Pharmaceutical ( D i v i s i o n of Pharmaceutics a n d  Sciences Biopharmaceutics)  W e accept this thesis is c o n f o r m i n g to the r e q u i r e d s t a n d a r d  T H E UNIVERSITY OF BRITISH C O L U M B I A A p r i l 2004 © N a n c y S C h u n g , 2004  Library Authorization  In presenting this thesis in partial fulfillment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  '2^-  Name of Author (please paht)  / o n  /:>((^\  Date (dd/mm/yyyy)  Title of Thesis:  ^£-£ivx{  cells  Degree: Department of  <rf \ h,s, :  ,vy^  ,C  Year: ,,:t ^(  The University of British Columbia Vancouver, B C  Canada  u  -,SV "  2  oo^  ABSTRACT  Background: C y c l o s p o r i n e A ( C s A ) is a n effective i m m u n o s u p p r e s s a n t d r u g to treat patients w h o h a v e u n d e r g o n e t r a n s p l a n t a t i o n or to treat a u t o i m m u n e diseases. H o w e v e r , the d r u g is l i m i t e d b y its n a r r o w therapeutic i n d e x a n d u s u a l l y becomes d i s c o n t i n u e d d u e to h i g h n e p h r o t o x i c i t y . C s A is k n o w n to h i g h l y associate w i t h l i p o p r o t e i n s , especially L D L a n d increased toxic effects of C s A h a v e been r e p o r t e d i n patients w h o are h y p o c h o l e s t e r o l e m i c . A significant r e d u c t i o n i n [ H ] C s A u p t a k e and toxicity w a s o b s e r v e d w h e n L L C - P K i cells w e r e treated w i t h increased concentrations of L D L . 3  Purpose: B a s e d o n the e x p e r i m e n t a l a n d c l i n i c a l e v i d e n c e , it is h y p o t h e s i z e d that w h e n the L D L receptor f a m i l y activity is decreased u p o n I g G - C 7 treatment, b o t h C s A u p t a k e a n d toxicity are r e d u c e d i n L L C - P K i cells, a r e n a l p r o x i m a l t u b u l e cell line. Methods: T h e appropriateness of L L C - P K i cells as a cell m o d e l w a s assessed b y c o n d u c t i n g dose-response, L D L specific b i n d i n g a n d c o m p e t i t i v e studies w i t h D i l L D L , a n d W e s t e r n b l o t analysis of the L D L receptor. A s s a y c o n d i t i o n s w i t h I g G - C 7 , a m o n o c l o n a l a n t i b o d y to the L D L receptor, w e r e o p t i m i z e d i n c l u d i n g temperature, p r e i n c u b a t i o n t i m e a n d c o n c e n t r a t i o n i n L L C - P K i cells. F i n a l l y , the effect of I g G - C 7 o n [ H ] C s A u p t a k e a n d toxicity w i t h L D L w a s d e t e r m i n e d . 3  Results: Significant results i n b o t h m e a n percent b o u n d (2.6% ± 0.6% vs. 5.1% ± 1.3%) a n d m e a n percent toxicity (1.8% ± 0.5% vs. 3.2% ± 0.5%) w e r e o b s e r v e d i n the [ H ] C s A alone g r o u p i n the presence of I g G - C 7 v e r s u s its absence (p<0.05 w i t h u n p a i r e d t-test). H o w e v e r , n o significant differences w e r e o b s e r v e d i n the [ H ] C s A L D L c o m p l e x or [ H ] C s A w i t h L D L c o a d d i t i o n g r o u p s . I n a d d i t i o n , L D L w a s not associated w i t h a significant r e d u c t i o n i n b o t h [ H ] C s A b o u n d , u p t a k e a n d toxicity. 3  3  3  3  Conclusion: These results suggest that C s A m a y be b i n d i n g d i r e c t l y to the L D L receptor f a m i l y i n d e p e n d e n t of its association w i t h L D L a n d thus, e l i c i t i n g its toxic effects at the m e m b r a n e l e v e l . T h i s s t u d y p r o v i d e s p r e l i m i n a r y e v i d e n c e of the f a m i l y of L D L receptors p l a y i n g a role i n C s A b i n d i n g a n d toxicity i n L L C - P K i cells.  ii  TABLE OF CONTENTS Abstract  ii  Table of Contents  iii  List of Figures  vii  List of Tables..  ix  List of A b b r e v i a t i o n s  x  Acknowledgements  xii  C H A P T E R 1: I N T R O D U C T I O N 1.1.  1.2.  1.3.  1.4.  1  C s A Background 1.1.1.  D e s c r i p t i o n of C s A p h y s i o c h e m i c a l p r o p e r t i e s  2  1.1.2.  T h e r a p e u t i c uses a n d clinical i m p l i c a t i o n s of C s A . . .  3  1.1.3.  P r o p o s e d m e c h a n i s m of action of C s A  4  1.1.4.  A d v e r s e Effects  6  1.1.5.  Nephrotoxicity  6  L i p o p r o t e i n s a n d their m e t a b o l i s m  8  1.2.1.  9  Chylomicrons  1.2.2.  Very low-density lipoprotein  10  1.2.3.  Low-density lipoprotein  11  1.2.3.  High-density lipoprotein  13  L o w - d e n s i t y l i p o p r o t e i n r e c e p t o r a n d its f a m i l y m e m b e r s  14  1.3.1.  The L D L receptor  16  1.3.2.  Megalin  18  C y c l o s p o r i n e A association w i t h lipoproteins  19  1.4.1. P h a r m a c o k i n e t i c p a r a m e t e r s  20  1.4.2. I n t e r a c t i o n o f C s A w i t h t h e L D L r e c e p t o r  21  C H A P T E R 2: S U M M A R Y O F R E S E A R C H P R O J E C T  23  2.1.  O v e r a l l Objective  24  2.2.  Hypothesis  24  2.3.  Specific A i m s  24  2.4.  Rationale  24  2.5.  Significance of Research  25.  iii  C H A P T E R 3: M A T E R I A L S A N D R E A G E N T S 3.1  3.2  Materials 3.1.1.  Cells a n d cell culture  3.1.2.  Lipoproteins, chemicals and labeled d r u g  27  3.1.3.  A n t i b o d i e s a n d assay kits  28  27  Tissue Culture Reagents and M e d i u m 3.2.1.  P r e p a r a t i o n of L L C - P K i m e d i a  29  3.2.2.  Preparation of H e p G 2 m e d i a  29  3.2.3.  Preparation of Serum-free L L C - P K i a n d H e p G 2 m e d i a  30  3.2.4.  Preparation of Phosphate Buffered Saline (PBS)  30  3.2.5.  C u l t u r e E s t a b l i s h m e n t of L L C - P K i a n d H e p G 2 cells f r o m F r o z e n Stock  30  3.2.6.  S u b c u l t u r i n g of L L C - P K i cells  31  3.2.7.  S u b c u l t u r i n g of H e p G 2 cells  31  3.2.8.  S e e d i n g plates a n d p r e p a r i n g L L C - P K i a n d H e p G 2 cells for Experimentation  3.3  3.4  32  P r e p a r a t i o n of E x p e r i m e n t a l Reagents 3.3.1.  Cholesterol concentration determination i n L D L  3.3.2.  Solutions w i t h D i l - L D L  34  3.3.3.  Solutions w i t h IgG-C7  34  3.3.4.  Solutions w i t h [ H ] C s A  35  3  4.2.  33  P r e p a r a t i o n of Reagents for W e s t e r n B l o t t i n g 3.4.1.  SDS-PAGE  35  3.4.2.  Immunoblot and Chemiluminescence detection  36  C H A P T E R 4: E X P E R I M E N T A L D E S I G N A N D M E T H O D O L O G Y 4.1.  26  38  D i l - L D L Assay Development 4.1.1.  Protein Content Determination  39  4.1.2.  Fluorescence D e t e r m i n a t i o n of D i l  40  4.1.3.  Dose-Response Studies  41  4.1.4.  L D L Specific B i n d i n g  42  4.1.5.  Competitive Binding  42  W e s t e r n B l o t t i n g P r o c e d u r e for the L D L receptor 4.2.1.  Membrane Protein Extraction  43  4.2.2.  S D S - P A G E (3.5% s t a c k i n g , 7.5% r e s o l v i n g g e l s )  44  4.2.3.  Immunoblotting and Detection  46  iv  4.3.  D i l - L D L w i t h IgG-C7 Assay Development 4.3.1.  Temperature Dependence  48  4.3.2.  O p t i m a l Preincubation period  48  4.3.3.  O p t i m a l Concentration  49  4.4.  C s A Time-Course Incubation Study  4.5.  C s A Uptake and Toxicity w i t h IgG-C7 Assay Development 4.5.1. 4.5.2. 4.5.3.  49  [ H ] C s A Bound and Uptake Assay M e m b r a n e - b o u n d a n d Intracellular [ H ] C s A d e t e r m i n a t i o n [ H ] C s A Toxicity Study - L D H measurement 3  3  3  50 52 53  4.6.  Scintillation C o u n t i n g  55  4.7.  Statistical A n a l y s i s  55  C H A P T E R 5: R E S U L T S 5.1.  5.2.  5.3.  Presence a n d a b u n d a n c e of L D L receptors i n L L C - P K i cells 5.1.1. 5.1.2.  Dose-response studies L D L specific b i n d i n g  57 60  5.1.3.  Competitive binding  64  5.1.4.  W e s t e r n blot analysis  65  Effect of I g G - C 7 o n D i l - L D L b i n d i n g 5.2.1.  Temperature dependence  66  5.2.2.  O p t i m a l preincubation period  68  5.2.3.  Optimal Concentration  70  P H I C s A Treatment 5.3.1.  5.4.  57  [ H ] C s A time-course i n c u b a t i o n 3  72  Effect of I g G - C 7 o n [ H l C s A U p t a k e a n d T o x i c i t y 3  5.4.1.  [ H ] C s A uptake - b o u n d and intracellular  75  5.4.2.  [ H ] C s A toxicity - L D H measurement  79  3  3  C H A P T E R 6: D I S C U S S I O N  82  6.1.  D i l - L D L versus  83  6.2.  R a t i o n a l e of L L C - P K i as a n a p p r o p r i a t e cell m o d e l  1 2 5  I-LDL  v  85  6.3.  U s e of I g G - C 7 , a m o n o c l o n a l a n t i b o d y to L D L r e c e p t o r  87  6.4.  Interpretation of results i n [ H] C s A u p t a k e a n d t o x i c i t y assays  89  6.5.  Hypothetical M o d e l  92  6.6.  Limitations  95  6.7.  Future Research  97  6.8.  Overall Conclusions  98  3  REFERENCES  100  vi  LIST O F FIGURES F i g u r e 1: M o l e c u l a r s t r u c t u r e o f C y c l o s p o r i n e A  3  F i g u r e 2: T h e m e c h a n i s m o f a c t i o n o f C s A i m m u n o s u p p r e s s i v e  action i n  activated T-cells  5  F i g u r e 3: T h e L D L p a r t i c l e  12  F i g u r e 4: T h e L D L r e c e p t o r p a t h w a y  15  F i g u r e 5: S t r u c t u r a l d i a g r a m o f t w o m e m b e r s o f t h e L D L r e c e p t o r f a m i l y  17  F i g u r e 6: S t a n d a r d C u r v e o f D i l - L D L C o n c e n t r a t i o n  58  F i g u r e 7 A : D o s e - d e p e n d e n t m e a s u r e m e n t of cell-associated L D L  59  F i g u r e 7B: D o s e - d e p e n d e n t m e a s u r e m e n t of m e m b r a n e - b o u n d L D L  59  F i g u r e 7 C : D o s e - d e p e n d e n t m e a s u r e m e n t of i n t e r n a l i z e d L D L  59  F i g u r e 8: L D L S p e c i f i c b i n d i n g i n L L C - P K i c e l l s  61  F i g u r e 9: L D L S p e c i f i c b i n d i n g i n H e p G 2 c e l l s  63  F i g u r e 10: C o m p e t i t i v e b i n d i n g s t u d y i n L L C - P K i c e l l s  64  F i g u r e 11: W e s t e r n B l o t analysis of the L D L receptor i n L L C - P K i cells  65  F i g u r e 12: T e m p e r a t u r e d e p e n d e n c e o f D i l - L D L b i n d i n g w i t h v a r i o u s c o n c e n t r a t i o n s of m A b to the L D L r e c e p t o r  67  F i g u r e 13: T i m e - c o u r s e i n c u b a t i o n w i t h m A b f r o m 0.5hr to 1.5hrs  69  F i g u r e 14: P e r c e n t i n h i b i t i o n o f D i l - L D L b i n d i n g i n t h e p r e s e n c e o f v a r i o u s c o n c e n t r a t i o n s of m A b to the L D L receptor F i g u r e 15: T i m e - c o u r s e i n c u b a t i o n o f m e t a b o l i s m o f  1 2 5  71 IgG-C7  74  F i g u r e 16: M e a n p e r c e n t b o u n d o f [ H ] C s A i n L D L w i t h I g G - C 7  77  F i g u r e 17: M e a n p e r c e n t u p t a k e o f [ H ] C s A i n L D L w i t h I g G - C 7  78  3  3  vii  F i g u r e 18: M e a n p e r c e n t t o x i c i t y o f [ H ] C s A i n L D L w i t h I g G - C 7  81  F i g u r e 19: S e n s i t i v i t y o f D i l - L D L A s s a y at 5 2 2 / 5 7 8 n m v e r s u s 5 5 5 / 5 7 1 n m  84  3  F i g u r e 20: H y p o t h e t i c a l m o d e l o f C s A u p t a k e a n d t o x i c i t y i n t o L L C - P K i c e l l s via L D L receptor family  93  viii  LIST O F T A B L E S T a b l e 1: P h y s i c o c h e m i c a l p r o p e r t i e s o f C s A Table  2: D e n s i t y ,  Size, Physical C o m p o s i t i o n  2 and  Function  of H u m a n  Lipoproteins  Plasma 9  T a b l e 3: P h a r m a c o k i n e t i c c h a r a c t e r i s t i c s o f C s A  21  T a b l e 4: T r e a t m e n t a n d C o n t r o l g r o u p s i n C s A U p t a k e a n d T o x i c i t y w i t h IgG-C7 Experiments  51  T a b l e 5: L D L S p e c i f i c B i n d i n g V a l u e s i n L L C - P K i c e l l s  62  T a b l e 6: P e r c e n t u p t a k e o f [ H ] C s A o v e r 24 h o u r p e r i o d at 3 7 ° C v s 4 ° C  73  3  ix  LIST O F A B B R E V I A T I O N S 4 A A P  4-aminoantipyrine  Abs  Absorbance  ACAT  A c y l - C o A : cholesterol  A N O V A  A n a l y s i s of variance  APS  Ammonium  O-acyltransferase  Persulfate  AUC  A r e a u n d e r the c u r v e  BSA  Bovine Serum A l b u m i n  BCA  Bicinchoninic A c i d  CE  C h o l e s t e r o l esterases  CETP  C h o l e s t e r y l ester transfer p r o t e i n  CO  Cholesterol oxidases  CPM  C o u n t s per minute  Dil  3,3'-dioctadecylindo-carbocyanine  dH 0  Distilled water  ECM  Extracellular matrix  EGF  E p i d e r m a l g r o w t h factor  FBS  Fetal Bovine S e r u m  HBA  Hydroxybenzoic Acid  2  HDL  High-density lipoprotein  HL  Hepatic lipase  HMG-CoA  3-hydroxy-3-methylglutaryl-CoA enzyme  HRP  Horseradish Peroxidase  hrs  Hours  IDL  Intermediate-density  IL  Interleukin  INT  T e t r a z o l i u m salt  IPA  Isopropanol  LCAT  Lecithin: cholesterol  LDH  Lactate  LDL  Low-density lipoprotein  LpL  L i p o p r o t e i n lipase  mAb  Monoclonal Antibody  MMF  Mycophenolate mofetil  NFAT  Nuclear-factor of activated T-cells  NFATc  N F A T cytosolic  NFATn  NFAT  NPxY  Asparagine-Proline-X-Tyrosine  PLTP  P h o s p h o l i p i d transfer p r o t e i n  P M N  Peripheral mononuclear  PT  Proximal tubule  POD  Peroxidase  lipoprotein  acyltransferase  Dehydrogenase  nucleus  X  PVDF  Polyvinyl difluoride membrane  ROS  Reactive o x y g e n species  rpm  rotations per  SDS  S o d i u m D o d e c y l Sulfate  TCR  T-cell receptor  TEMED  N, N,N ,  TGF  T r a n s f o r m i n g g r o w t h factor  VLDL  Very low-density lipoprotein  Vss  V o l u m e o f d i s t r i b u t i o n at s t e a d y - s t a t e  YWTD  Tyrosine-Tryptophan-Threonine-Aspartate  minute  N-Tetra-methyl-ethelenediamine  xi  ACKNOWLEDGEMENTS  I w o u l d like to thank m y supervisor, D r . K i s h o r W a s a n for his s u p p o r t , patience a n d mentorship  d u r i n g this project.  Y o u have  provided a w a r m and  encouraging  e n v i r o n m e n t for m e to g r o w p e r s o n a l l y a n d a c a d e m i c a l l y . T h a n k y o u for g i v i n g m e the o p p o r t u n i t y to be p a r t of y o u r lab. To members  of m y M S c committee, D r . J o h n M c N e i l l , D r . J o h n H i l l , D r . W a y n e  Riggs a n d D r . K a t h l e e n M a c L e o d , y o u r feedback a n d suggestions have been v e r y h e l p f u l to the p r o g r e s s i o n of this project. I w o u l d l i k e to a c k n o w l e d g e M i c h e l l e for c o n t r i b u t i n g the c o l o u r f u l figures i n this thesis.  N o t only have y o u been supportive and encouraging d u r i n g m y graduate  training, but y o u have been a wonderful friend. worries, y o u have helped m e keep focused.  With my many  doubts  and  W o r d s cannot explain h o w I value our  friendship. T o C h i a - T i n g , I a m so grateful to h a v e b e c o m e s u c h close f r i e n d s w i t h y o u .  You  h a v e h e l p e d m e out i n m a n y stressful m o m e n t s a n d h a v e b e e n there for m e t h r o u g h e v e r y t h i n g . T h a n k s for l i s t e n i n g a n d b e i n g y o u ! To  "Christopher", thank  y o u for y o u r c o m p a n i o n s h i p  and  kindness  during  a  difficult p e r i o d i n m y life. Y o u h a v e b e e n a great f r i e n d a n d h a v e s h o w n m e a n e w light. O u r conversations w i l l a l w a y s be r e m e m b e r e d . I w o u l d l i k e to t h a n k P a u l i n a a n d A u n t i e G u y l a i n for their u n c o n d i t i o n a l s u p p o r t d u r i n g m y life i n V a n c o u v e r .  I a m grateful to h a v e s u c h a close relationship w i t h  b o t h of y o u . T h a n k s for b e i n g there for m e just to listen. To m y relatives, A u n t Julia, U n c l e Victor, B r a n d o n a n d Vincent:  I a m g l a d that I  t o o k the o p p o r t u n i t y to c o m e to V a n c o u v e r to get to k n o w a l l o f y o u . all y o u r help a n d hospitality y o u have s h o w n me.  I appreciate  T h a n k y o u for m a k i n g m y life a  bit easier d u r i n g m y studies. T o m y m o t h e r , father a n d sister; E l l e n , a n d m y close f r i e n d s i n T . O , y o u w e r e o n l y just a p h o n e call a w a y but y o u r w o r d s of e n c o u r a g e m e n t  have helped me  m y graduate studies. T h a n k y o u for listening a n d for y o u r advice.  xii  through  T o a l l m e m b e r s of the lab: K r i s t i n a , y o u h a v e b e e n a great i n s p i r a t i o n to me. T h a n k y o u for a l l y o u r a d v i c e b o t h scientifically a n d p e r s o n a l l y . V e r i c a , O l e n a , M o n a , A g a t h a a n d A l i s o n , y o u h a v e w e l c o m e d a n d e m b r a c e d m e since the first d a y I m o v e d to V a n c o u v e r . Y o u have been of great h e l p a n d g o o d listeners w h e n I n e e d e d s o m e o n e to talk to. T h a n k s for a l l the f u n times a n d the ' s p i k e d ' cheese fondue! Steve, y o u h a v e a d d e d a great m a l e influence to a once female d o m i n a n t lab! A n d finally, the u n d e r g r a d u a t e students: E u g e n e , E r i n , E d w i n , B e n n y , G i n a a n d Tiffany; y o u h a v e m a d e the lab a n enjoyable place to w o r k , e s p e c i a l l y d u r i n g the s u m m e r sessions. T h a n k s for a l l the jokes, laughter a n d i n t e r e s t i n g conversations! T o m y f e l l o w graduate students: V i n c e , Irem, M a r i a a n d L i n d a , thanks for the support, help a n d encouragement d u r i n g m y training. I w i l l always remember our conversations a n d the k i n d n e s s y o u h a v e s h o w n me.  xiii  CHAPTER I:  1  1.1 B a c k g r o u n d o n C y c l o s p o r i n e A ( C s A )  1.1.1 Description of CsA physiochemical  properties  C y c l o s p o r i n e A ( C s A ) is a h i g h l y l i p o p h i l i c a n d n e u t r a l c y c l i c  polypeptide  w i t h e l e v e n a m i n o a c i d s [3,5,27,31]. S e v e n o f t h e a m i n o a c i d s a r e N - m e t h y l a t e d a n d the r e m a i n i n g f o u r are h y d r o g e n b o n d e d w i t h c a r b o n y l g r o u p s i n o r d e r to f o r m a r i g i d c y c l i c s k e l e t o n [3, 3 1 ] . T h e m o l e c u l a r s t r u c t u r e o f t h e d r u g i s s h o w n i n F i g u r e 1 w i t h a m o l e c u l a r w e i g h t o f 1 2 0 2 . 6 a t o m i c m a s s u n i t s [117]. It i s s o l u b l e i n m e t h a n o l , e t h a n o l , e t h e r a n d c h l o r o f o r m a n d e s s e n t i a l l y i n s o l u b l e i n w a t e r ( S w = 1 4 u g / m l at 2 0 ° C i n a t m o s p h e r i c p r e s s u r e ) d u e t o a n u m b e r o f i t s a l i p h a t i c s i d e c h a i n s [70,126]. A s u m m a r y of the p h y s i c o c h e m i c a l p r o p e r t i e s o f C s A is s h o w n b e l o w . Molecular Formula  C62H111N11O12  Molecular Weight  1202.635  Kp  1000  (octanol/buffer)  Crystal  Form  (crystallization  Colorless, prismatic  from  acetone) Cell dimensions  A=b=13.837 A , C=41.242 A  Crystal V o l u m e  7896 A  Crystal Density  1.042 g m / c m  Molecules per cell  Z = 4  Melting point  1 4 8 ° C to 1 5 1 ° C  Optical rotation (in chloroform)  -244°  3  3  Data from von Wartburg and Traber [126] and Loosli et al. [70]. Adapted from Fahr et al. [31] T a b l e 1: P h y s i c o c h e m i c a l p r o p e r t i e s o f C s A  2  F i g u r e 1: Molecular structure of cyclosporine A The drug was first isolated from the fungal species Tolypocladium inflatum Gams by the company Sandoz which subsequently activity  [14,135].  discovered its immunosuppressive  Further work on the drug revolutionized the field of  transplantation as its use has increased successful organ transplants in patients [14]. Its initial formulation was marketed as Sandimmune® but due to the inconsistent bioavailability of the drug, Neoral® was introduced as a microemulsion [1]. This formulation improved absorption and bioavailability of CsA with less variability among patients [1].  3.3.2 Therapeutic uses and clinical implications of CsA  CsA is an effective immunosuppressant used to treat patients who have undergone organ transplantation especially heart, lung and kidney transplantation as well as to treat certain autoimmune diseases such as uveitis, psoriasis and rheumatoid arthritis [1,84].  It is a part of a class of calcineurin inhibitors and  routinely prescribed in combination with other new immunosuppressive drugs such  3  as m y c o p h e n o l a t e m o f e t i l ( M M F ) , s i r o l i m u s o r a z a t h i o p r i n e a n d p r e d n i s o n e as w e l l as c o r t i c o s t e r o i d s [14,72].  D u e t o i t s n a r r o w t h e r a p e u t i c i n d e x , d r u g m o n i t o r i n g is  i m p o r t a n t not o n l y for efficacy of the C s A b u t also for a v o i d a n c e of toxicity. managing  and  maintenance  of the  C s A regime  after  transplantation  c h a l l e n g i n g a n d c o n f u s i n g since it is v e r y patient - d e p e n d e n t .  Thus  is u s u a l l y  Careful  m u s t be m a d e to d o s i n g , a n d to d r u g - d r u g interactions w h i l e m i n i m i z i n g  attention adverse  effects.  1.1.3. Proposed mechanism of action of CsA W h e n a T - c e l l r e c o g n i z e s a f o r e i g n a n t i g e n t h r o u g h its T - c e l l r e c e p t o r ( T C R ) , a cascade of intracellular events occurs i n c l u d i n g a n elevation of c a l c i u m levels a n d s u b s e q u e n t a c t i v a t i o n o f c a l m o d u l i n [72]. cyclophilin  A  which  can  regulate  A s a result, c a l m o d u l i n interacts  calcineurin,  a  superfamily  of  with  protein  s e r i n e / t h r e o n i n e p h o s p h a t a s e s [72]. C a l c i n e u r i n c a t a l y z e s t h e d e p h o s p h o r y l a t i o n o f N F A T (nuclear-factor of a c t i v a t e d T-cells) f a m i l y m e m b e r s a l l o w i n g it to translocate i n t o the n u c l e u s a n d a c t i v a t e g e n e e x p r e s s i o n of c y t o k i n e s s u c h as i n t e r l e u k i n - 2 ( I L 2) a n d I L - 4 a n d e l i c i t a n i m m u n o l o g i c a l r e s p o n s e [72]. B i o l o g i c a l studies r e v e a l that C s A i n h i b i t s T - c e l l a c t i v a t i o n b y b l o c k i n g the t r a n s c r i p t i o n o f c y t o k i n e g e n e s f o r I L - 2 a n d I L - 4 [43,49,62].  O n c e C s A enters the  cell, it b i n d s to c y c l o p h i l i n A l o c a t e d w i t h i n the c y t o s o l , p r e v e n t i n g c a l c i n e u r i n m e d i a t e d d e p h o s p h o r y l a t i o n [46,69]. A s a r e s u l t , n u c l e a r t r a n s l o c a t i o n o f N F A T  4  Figure 2: T h e m e c h a n i s m o f C s A i m m u n o s u p p r e s s i v e a c t i o n i n a c t i v a t e d T - c e l l s * Activated  1. C s A u p t a k e i n t o a T - c e l l 2. C s A b i n d s t o c y c l o p h i l i n A 3. C s A i n h i b i t s t h e a c t i v a t i o n o f c a l c i n e u r i n 4.  Activated calcineurin dephosphorylates cytosolic N F - A T  5. N F - A T c t r a n s l o c a t e s i n t o t h e n u c l e u s 6. A c t i v a t e d N F - A T n t r a n s c r i b e s I L 2 , I L 4 a n d o t h e r c y t o k i n e g e n e s 7. C a l m o d u l i n i s r e g u l a t e d b y i n c r e a s e d a m o u n t s o f c y t o s o l i c C a also regulate c y c l o p h i l i n A  5  2 +  levels w h i c h can  family members is inhibited as well as gene expression of cytokines deactivating Tcell response [46,72]. A diagram of these processes is shown in Figure 2.  1.1.4 Adverse Effects  The use  of CsA has been limited by its adverse  effects including  nephrotoxicity, hepatotoxicity, neurotoxicity, hypertension, diabetes mellitus and dyslipidemia [1,59]. A number of patients also experience physical changes such as gum hypertrophy, hirsutism, flushing, and mild hand tremors [1]. When patients have undergone kidney transplants, a majority of these patients experience a significant increase in total serum cholesterol levels [39].  The incidence of  hyperlipidemia is much higher after renal transplantation with CsA treatment [39]. It has been reported that long-term administration of CsA elevates plasma total cholesterol and triglyceride concentrations including L D L and VLDL levels in humans [2,9,39].  1.1.5  Nephrotoxicity  CsA-induced renal toxicity is one of the most important side effect which often results in discontinuation of CsA therapy [140].  Acute renal toxicity is  characterized by a rise in serum creatinine levels, a decrease in the glomerular filtration rate [12], inability to concentrate urine and sodium retention [104]. CsA acute nephrotoxicity produces hemodynamic changes and is often reversible with low CsA doses (<5 mg/kg/d) [75,104]. Histologically, the site of acute injury occurs  6  i n the straight segment of the p r o x i m a l t u b u l e n o t e d b y v a c u o l i z a t i o n i n the cells, severe a t r o p h y , t h i c k e n i n g of the basal m e m b r a n e  [76] a n d t h r o m b o s i s of the  i n t r a g l o m e r u l a r capillaries [116]. H o w e v e r , i n the case of transplant patients, h i g h e r C s A doses ( 1 0 - 1 5 m g / k g / d ) are u s u a l l y r e q u i r e d a n d so the s i t u a t i o n becomes e v e n m o r e c o m p l i c a t e d i n r e n a l transplant patients [1]. T G F - p ( t r a n s f o r m i n g g r o w t h factor) has b e e n p r o p o s e d as one of the m e c h a n i s m s of C s A - i n d u c e d n e p h r o t o x i c i t y .  TGF-(3 is k n o w n to stimulate  the  increase c o m p o s i t i o n of extracellular m a t r i x ( E C M ) a n d to decrease the p r o d u c t i o n of E C M - d e g r a d i n g proteases w h i c h m a y e x p l a i n the characteristics of interstitial fibrosis i n C s A - i n d u c e d r e n a l toxicity [13,94]. It has b e e n also r e p o r t e d that C s A can d i s r u p t f u n c t i o n a l proteins  at the c e l l u l a r l e v e l s u c h as P - g l y c o p r o t e i n s [36],  c a l c i n e u r i n [45] as w e l l as i n d u c e apoptosis [48,53]. I n a d d i t i o n , reactive metabolites p r o d u c e d b y the c y t o c h r o m e P 450 s y s t e m i n the l i v e r a n d k i d n e y h a v e p r o p o s e d b y others [68,73,141].  been  It has been suggested that C s A c a n increase the  synthesis of reactive o x y g e n species (ROS) [44,81] , t h r o m b o x a n e [90,112] a n d l i p i d p e r o x i d a t i o n p r o d u c t s [58]in the k i d n e y . F i n a l l y , C s A c a n r e d u c e t r a n s c r i p t i o n of certain genes i n p r o x i m a l t u b u l e cells s u c h as p h o s p h o e n o l p y r u v a t e c a r b o x y k i n a s e i n gluconeogenesis [79]. I n a d d i t i o n , i n response to C s A toxicity, a n increase i n the GLUT1  gene e x p r e s s i o n w a s o b s e r v e d [26]. N e v e r t h e l e s s , the m e c h a n i s m of C s A -  i n d u c e d n e p h r o t o x i c i t y r e m a i n s uncertain.  7  1.2 Lipoproteins and their metabolism L i p o p r o t e i n s are a class of c o m p l e x m a c r o m o l e c u l e s c o n s i s t i n g of b o t h l i p i d and protein subgroups.  T h e i r m a i n r e s p o n s i b i l i t y is to t r a n s p o r t a n u m b e r of w a t e r  i n s o l u b l e n u t r i e n t s t h r o u g h o u t the systemic c i r c u l a t i o n , m a i n l y l i p i d s i n a n a q u e o u s environment.  L i p o p r o t e i n s are characterized  ester a n d t r i g l y c e r i d e s u r r o u n d e d  b y a n insoluble core of cholesteryl  b y a shell of a m p h i p a t h i c  s p e c i a l i z e d p r o t e i n s c a l l e d a p o l i p o p r o t e i n s [115,128]. proteins  and  categories: density  lipids  and  chylomicrons,  lipoproteins  are  classified based  very  (IDL),  low-density  low-density  on  phospholipids  and  T h e y differ i n their content of their  density  lipoproteins  lipoproteins  into  (VLDL),  (LDL),  and  five  main  intermediate high-density  l i p o p r o t e i n s ( H D L ) (see T a b l e 2). L i p o p r o t e i n s a r e c o n v e n t i o n a l l y d e s c r i b e d b y t h e i r d e n s i t y , w h i c h is reflected b y a n i n c r e a s e d p r o t e i n / l i p i d ratio. also  classified  subsequently  by  the  governs  apolipoprotein  content  t h e u l t i m a t e fate o f t h e  located particle.  on  T h e y are, their  however,  surface  which  T h u s , l i p i d t r a n s p o r t is  regulated b y a p o l i p o p r o t e i n s , l i p o p r o t e i n receptors, l i p o l y t i c e n z y m e s a n d transfer p r o t e i n s w h i c h a l l act i n concert to m a i n t a i n c h o l e s t e r o l a n d t r i g l y c e r i d e h o m e o s t a s i s in vivo [115].  8  Characteristics  Abbreviations Density (g/ml) Diameter (nm) Composition (% dry wt) Proteins triglycerides cholesterol phospholipid apoproteins  Main function  Table 2:  Chylomicrons  <0.95 75-1200  1-2 86 5 7 Al, All B-48  Highdensity lipoproteins HDL 1.063-1.210 5-12  Very-lowdensity lipoproteins VLDL 0.95-1.006 30-80  Intermediate density lipoproteins IDL 1.006-1.019 25-35  Low-density lipoproteins  8 55 19 18  19 23 38 20  22 6 50 22  B-100  B-100  B-100  47 4 . 10 30 Al, AIV  Cholesterol transport to all tissues .  LDL 1.019-1.063 18-25  All,  ci, cn, c m  ci, cn, c m ci, cn, c m  ci, cn, c m  E Transport of exogenous triglyceride and cholesterol  E Transport of endogenous triglyceride  D,E Reverse cholesterol transport  E Transport of endogenous cholesterol  Density, Size, Physical C o m p o s i t i o n a n d F u n c t i o n of H u m a n  Plasma  L i p o p r o t e i n s . M o d i f i e d f r o m r e f e r e n c e s [128] a n d [108].  1.2.1 Chylomicrons A f t e r a m e a l , fat i n the f o r m o f c h o l e s t e r o l a n d t r i a c y l g l y c e r o l s enters the enterocyte a n d becomes repackaged l i p i d s i n t o c h y l o m i c r o n s [115,128].  w i t h apoB-48, a p o A I , apoAII a n d additional  Subsequently, c h y l o m i c r o n s become secreted b y  t h e i n t e s t i n a l m u c o s a t h r o u g h t h e m e s e n t e r i c l y m p h i n t o s y s t e m i c c i r c u l a t i o n [22]. As  such,  they  acquire  cholesteryl  ester facilitated  b y cholesteryl  ester  p r o t e i n ( C E T P ) as w e l l as a p o E a n d a p o C s f r o m H D L . D u e t o t h e n e w l y a p o C s , triacylglycerol-rich c h y l o m i c r o n s are recognized b y the  9  transfer acquired  endothelial-bound  l i p o p r o t e i n lipase ( L p L ) w i t h i n the c a p i l l a r y b e d s of skeletal m u s c l e a n d t i s s u e [108,128].  adipose  A s a result, c h y l o m i c r o n s become r a p i d l y h y d r o l y z e d , releasing  free f a t t y - a c i d s , a n d m o n o - a n d d i g l y c e r i d e s w h i c h a r e a b s o r b e d b y n e i g h b o u r i n g t i s s u e s f o r e n e r g y p r o d u c t i o n a n d s t o r a g e [115,128]. T h e r e s i d u a l p a r t i c l e , k n o w n as a chylomicron remnant, apoB-48 a n d apoE. returns  to  lacks t r i a c y l g l y c e r o l yet is e n r i c h e d w i t h c h o l e s t e r o l a n d  A s a result, the r e m n a n t  c i r c u l a t i o n to  be  taken  up  by  d e c r e a s e s its a f f i n i t y f o r L p L a n d  the  liver  through  receptor-mediated  e n d o c y t o s i s w h i c h i n v o l v e s r e c o g n i t i o n of a p o E of the c h y l o m i c r o n r e m n a n t  by  r e c e p t o r s o n t h e s u r f a c e o f h e p a t o c y t e s [108,128].  1.2.2 Very low-density lipoprotein  (VLDL)  V e r y l o w - d e n s i t y l i p o p r o t e i n s are the major t r a n s p o r t e r s of produced exogenous  triacylglycerol from and  endogenous  the  liver  lipids  are  to  extrahepatic  assembled  a p o p r o t e i n s i n t o a ' n a s c e n t ' V L D L p a r t i c l e [108,128].  with  [108,128].  apoB-100  Both  and  other  In b l o o d , 'nascent'  VLDL  acquires c h o l e s t e r y l esters a n d a p o C a n d a p o E t r a n s f e r r e d m a t u r e V L D L p a r t i c l e s [128].  tissue  endogenously  f r o m H D L to  become  S i m i l a r to c h y l o m i c r o n s , m a t u r e V L D L particles are  h y d r o l y z e d b y L p L into V L D L r e m n a n t s releasing a p o C a n d a p o E f r o m the  surface  c o a t a n d t r a n s f e r r i n g t h e m b a c k t o H D L [108,128].  formed  VLDL  r e m n a n t s are  w h i c h a r e a l s o k n o w n as i n t e r m e d i a t e - d e n s i t y l i p o p r o t e i n s ( I D L ) .  These  remnant  particles are r e l a t i v e l y e n r i c h e d i n a p o B - 1 0 0 a n d a p o B a n d c a n be r e m o v e d f r o m c i r c u l a t i o n d i r e c t l y t h r o u g h interaction w i t h the L D L receptor or the L D L receptor-  10  related p r o t e i n o n hepatocytes [108]. A majority of I D L , h o w e v e r , becomes further c a t a b o l i z e d b y hepatic lipase ( H L ) to f o r m L D L particles r i c h i n c h o l e s t e r y l ester w i t h the apoB-100 attached to surface coat [128].  1.2.3 Low-density  lipoprotein  (LDL)  L o w - d e n s i t y l i p o p r o t e i n s are the m a i n carriers of unesterified a n d esterified cholesterol to p e r i p h e r a l tissues.  A p r o d u c t of V L D L c a t a b o l i s m , this l i p o p r o t e i n is  s t r i p p e d of t r i a c y l g l y c e r o l s a n d associated a p o p r o t e i n , except apoB-100 [108]. d i a g r a m of the L D L particle is s h o w n i n F i g u r e 3.  A  A p o B - 1 0 0 is essential for  r e c o g n i t i o n of the L D L particle to its receptor a n d subsequent u p t a k e i n t o cells [108]. T h e majority of L D L is r e m o v e d b y the l i v e r t h r o u g h the L D L receptor a n d other n o n receptor-mediated p a t h w a y s [22,108]. T h e i m p o r t a n t role of L D L is the d e l i v e r y of cholesterol to extrahepatic  tissues for u t i l i z a t i o n i n a n u m b e r  of processes  i n c l u d i n g s t e r o i d p r o d u c t i o n or m e m b r a n e synthesis [35]. Intracellular homeostasis of cholesterol concentrations is d u e to three r e g u l a t o r y elements: a) H M G - C o A (3h y d r o x y - 3 - m e t h y l g l u t a r y l - C o A ) reductase, the r a t e - l i m i t i n g e n z y m e i n cholesterol biosynthesis; b) A C A T ( a c y l - C o A : cholesterol O-acyltransferase) w h i c h re-esterifies excess i n t r a c e l l u l a r cholesterol for storage; a n d c) e x p r e s s i o n of L D L receptors o n the cell surface [108].  F u r t h e r m o r e , the r e g u l a t i o n of s e r u m L D L d e p e n d s o n t w o  factors: a) the q u a n t i t y  of V L D L p r o d u c e d b y the l i v e r w h i c h then  c o n v e r t e d to L D L b y H L a n d b) the p r o p o r t i o n of V L D L r e m n a n t s ( I D L )  11  becomes  Figure 3: cholesteryl  The low-density lipoprotein ( L D L ) particle consists ester  with  an  apolipoprotein  monolayer.  12  B-100  embedded  in  of cholesterol a  and  phospholipids  that are removed by the LDL receptor in the liver. Therefore, the LDL receptor is the critical regulator of serum LDL concentrations both in rate of formation and rate of clearance of LDL particles.  1.2.4 High-density  lipoprotein (HDL)  High-density lipoproteins are a heterogenous class of lipoproteins containing several subclasses of lipoproteins, mainly H D L 2 and H D L 3 which are present in most species [108]. HDL is responsible for removal of cholesterol from peripheral tissues and for subsequent transport to the liver through a process known as 'reverse cholesterol' transport [34].  Sources of HDL apolipoproteins, apoAl and  apoE are found in the intestine and liver, respectively [108].  Nascent H D L is  hypothesized to be formed in the extracellular space by the association of lipid-free apoAI with phospholipids and free cholesterol released from chylomicrons and VLDL during LpL action [28,35,108].  These disk-shaped H D L consist of a  phospholipid bilayer which also contains apoAII and apoE, and are acceptors of free cholesterol transferred from plasma membrane of peripheral tissues [34].  As a  result, these particles become substrates for lecithin: cholesterol acyltransferase (LCAT) which catalyzes the esterification of free cholesterol by transferring a fatty acid from phosphatidylcholine (lecithin) to a cholesterol molecule [35]. As a result, the accumulation of cholesteryl esters forms a lipid core transforming the diskshaped HDL particle into spheres known as HDL3 [28,34,35]. With a high content of cholesteryl esters and absence of triglycerides, lipid transfer proteins such as CETP  13  a n d P L T P ( p h o s p h o l i p i d transfer protein) facilitate the a c q u i s i t i o n of triglycerides a n d m o r e c h o l e s t e r y l esters i n t o the H D L 3 particle [35]. C E T P exchanges cholesteryl esters for triglycerides b e t w e e n H D L a n d t r i g l y c e r i d e - r i c h l i p o p r o t e i n s s u c h as c h y l o m i c r o n s a n d V L D L ; m e a n w h i l e P L T P acquires p h o s p h o l i p i d s f r o m the surface of  apoB-containing lipoproteins  w h i c h become  substrates  for  LCAT  [28,35].  Subsequently, a m a t u r e H D L particle is f o r m e d w h i c h is c a l l e d H D L 2 , c o n s i s t i n g of a core of cholesteryl ester a n d triglycerides w h i c h c a n be h y d r o l y z e d b y hepatic lipase ( H L ) u p o n t r a n s p o r t to the l i v e r [28,34,34].  Release of a p o A I as a result of H L is  u s e d to synthesize n e w H D L particles.  1.3. The Low-density Lipoprotein (LDL) Receptor and its Family Members The  L D L receptor  is  an  endocytic  receptor  that  transports  relevant  m a c r o m o l e c u l e s , m a i n l y the cholesterol-rich l i p o p r o t e i n L D L , i n t o cells t h r o u g h a process c a l l e d receptor-mediated  endocytosis [85] (see F i g u r e 4).  T h i s process  i n v o l v e s the cell surface receptor r e c o g n i z i n g a n L D L particle f r o m the extracellular m e m b r a n e ( E C M ) , i n t e r n a l i z i n g it t h r o u g h clathrin-coated pits a n d t r a n s p o r t i n g it i n t r a c e l l u l a r l y v i a a vesicle [16,85,137]. Subsequently, the vesicle becomes d e g r a d e d u p o n f u s i o n w i t h the l y s o s o m e , releasing l i p i d s i n t o the c y t o p l a s m for cell use; m e a n w h i l e the receptor recycles back to the cell surface to b i n d to another L D L particle [16,85,137]. M u c h of o u r current k n o w l e d g e of r e c e p t o r - m e d i a t e d  14  Plasma Membrane  Figure 4:  The L D L receptor  pathway.  1.  N o v e l L D L receptors s y n t h e s i z e d b y the g o l g i b o d i e s  2.  R e c e p t o r s c o n t a i n e d i n vesicles are t r a n s p o r t e d to the cell  3.  L D L receptor e x p r e s s i o n o n the cell surface  4.  R e c o g n i t i o n o f the L D L p a r t i c l e b y the L D L  5.  Internalizing of receptor-ligand c o m p l e x into vesicles  6.  S o r t i n g of the  L D L receptor  f r o m the  membrane  receptor  L D L particle  via fusion w i t h  endosome 7.  R e c y c l i n g of L D L receptors b a c k to the cell surface  8.  L y s o s o m a l d e g r a d a t i o n of L D L particles into a m i n o acids a n d cholesterol  15  an  e n d o c y t o s i s o r i g i n a t e d f r o m the p i o n e e r i n g studies o n the L D L receptor conducted  by  Goldstein and  Brown.  Their w o r k  pathway  i n v o l v e d characterizing  the  b i o c h e m i s t r y a n d genetics of the L D L receptor a n d d e t e r m i n i n g its i m p o r t a n t r o l e i n m a i n t a i n i n g cholesterol homeostasis intracellularly. Inherited m u t a t i o n s w i t h i n this receptor h a v e b e e n l i n k e d to f a m i l i a l h y p e r c h o l e s t e r o l e m i a , a genetic disease that results i n pathologically elevated b l o o d cholesterol a n d p r e m a t u r e coronary disease [42].  1.3.1 The LDL receptor The functions: type  L D L receptor  consists  of five  distinct domains  with individualized  a) a l i g a n d b i n d i n g d o m a i n at t h e N - t e r m i n u s c o n t a i n i n g  repeats  i n v o l v e d i n L D L b i n d i n g ; b)  an  p r e c u r s o r - h o m o l o g y repeats that contain Y W T D  epidermal  growth  complementfactor  (Tyrosine-Tryptophan-Threonine-  A s p a r t a t e ) m o t i f s r e s p o n s i b l e f o r l i g a n d d i s s o c i a t i o n ; c) a n O - l i n k e d s u g a r acting  as  a  spacer;  d)  membrane-spanning  c y t o p l a s m i c t a i l , a l s o k n o w n as t h e N P x Y  (EGF)  domain  for  anchorage;  domain  and  e)  a  (Asparagine-Proline-X-Tyrosine) motif,  i n v o l v e d i n i n t e r n a l i z a t i o n o f L D L p a r t i c l e s i n t o c o a t e d p i t s [55,139] (see F i g u r e 5). T h e L D L receptor is c o n s t r u c t e d f r o m a v a r i e t y of p r o t e i n ' m o d u l e s ' a n d the c l o n i n g of its g e n e has b r o a d e n e d its k n o w l e d g e to be c o n s i d e r e d a f a m i l y o f L D L receptors, e a c h s h a r i n g s t r u c t u r a l l y s i m i l a r m o t i f s [137]. T h e r e a r e n i n e m e m b e r s o f t h e f a m i l y which  i n c l u d e the  L D L receptor,  L D L receptor-related  protein  (LRP),  megalin,  V L D L receptor, a p o E R 2 a n d s o r L A / L R P l l , L R P l b , M E G F 7 , L R P 5 / 6 ; the f o r m e r  16  Key:  Complement-type repeat E6F precursor homology domain 0-linked sugar domain NPxY motif tVieqalin  LDL Receptor NH,  Figure 5: Structural diagram of two members of the L D L receptor family. Adapted from Nykjaier A et al (2002), Trends Cell Biol 12: 273-80 [85].  17  seven having been identified i n mammals.  E a c h m e m b e r of this receptor  family  u n d e r g o e s the process of r e c e p t o r - m e d i a t e d e n d o c y t o s i s ; yet e a c h m e m b e r is expressed i n a n u m b e r of different tissues a n d has a w i d e r a n g e of different l i g a n d s w h i c h a r e n o t s p e c i f i c t o t h e r e c o g n i t i o n o f t h e L D L p a r t i c l e [38,85,137]. O n l y f i v e o f the m e m b e r s  h a v e b e e n r e c o g n i z e d to b i n d to the l i p o p r o t e i n s ; L D L - R ,  m e g a l i n , L R P a n d a p o E R 2 [38,50,55].  VLDL-R,  A s t r u c t u r a l d i a g r a m of t w o m e m b e r s of the  L D L r e c e p t o r f a m i l y that are e x p r e s s e d i n the p r o x i m a l t u b u l e cells is s h o w n i n F i g u r e 5.  1.3.2 Megalin M e g a l i n , a l s o k n o w n as g p 3 3 0 , i s a 6 0 0 k D a m e m b e r s u p e r f a m i l y [55,85].  of the L D L receptor  It i s a m u l t i l i g a n d r e c e p t o r e x p r e s s e d i n a n u m b e r o f t i s s u e s ,  b u t m a i n l y i n t h e g l o m e r u l u s a n d p r o x i m a l t u b u l e c e l l s o f t h e k i d n e y [55,85,106,145]. It w a s f i r s t i d e n t i f i e d b y S a i t o et al (1994) a s a n a u t o a n t i b o d y t h a t c i r c u l a t e d i n r a t s w i t h H e y m a n n - t y p e a u t o i m m u n e n e p h r i t i s [60,106]. In vitro s t u d i e s s u g g e s t t h e r o l e o f m e g a l i n i n L D L c a t a b o l i s m b y b i n d i n g t o a p o B - 1 0 0 [18,119].  M a p p i n g o f its  l i g a n d b i n d i n g r e g i o n i n d i c a t e its a b i l i t y to also b i n d to a p o E , p - V L D L , l a c t o f e r r i n , aprotinin and  R A P [86].  Therefore,  ligands of m e g a l i n i n c l u d e A p o B ,  ApoE,  clusterin, proteases a n d protease inhibitors, carrier proteins for lipophilic vitamins, parathyroid  hormone,  polybasic  drugs  (aminoglycosides)  and  R A P [19,33,86].  K n o c k o u t m e g a l i n m i c e m o d e l s suggest the i m p o r t a n c e of this receptor i n retrieval o f f i l t e r e d v i t a m i n / c a r r i e r c o m p l e x e s a n d r e a b s o r p t i o n o f p r o t e i n s [66].  18  1.4 Cyclosporine A (CsA) association with lipoproteins It  is w e l l  established  that  C s A associates w i t h l i p o p r o t e i n s  A p p r o x i m a t e l y 50-60% o f b l o o d C s A is b o u n d  to c h o l e s t e r o l - a n d  [8,114,130]. triglyceride-  c o n t a i n i n g l i p o p r o t e i n s i n t h e b l o o d [51]. C s A p r e d o m i n a n t l y a s s o c i a t e s w i t h H D L a n d L D L u p o n i n c u b a t i o n i n p l a s m a [130]. It h a s b e e n r e p o r t e d t h a t c y c l o s p o r i n e A w a s p a r t i a l l y i n f l u e n c e d b y C E T P ( a l s o k n o w n as l i p i d - t r a n s f e r p r o t e i n o r L T P - 1 ) facilitated  C E and  T G transfer  activity but  not  PC  [63,131,134].  In  addition,  m o d i f i c a t i o n of the l i p o p r o t e i n surface charge w i t h a n i n c r e a s e d negative  charge  resulted i n greater percentage of [ H ] C s A recovered w i t h i n the L D L subtraction 3  after  i n c u b a t i o n i n p h o s p h a t i d y l i n o s i t o l (Pl)-treated  plasma  [133].  composition  It h a s b e e n r e p o r t e d of  plasma  rabbit  plasma  than  control  that changes i n the l i p i d c o n c e n t r a t i o n  lipoproteins  can  alter  the  profile  of  and  CsA-lipoprotein  association, a n d t h u s m a y m o d i f y the p h a r m a c o l o g i c a l a c t i v i t y a n d p h a r m a c o k i n e t i c p r o p e r t i e s as w e l l as t h e t o x i c i t y o f C s A [67,127,128]. Decreased [25,82]  and  effects  increased  of C s A h a v e t o x i c effects  been  reported  w i t h hypertriglyceridemia  of C s A w i t h hypocholesterolemia  [24].  An  e n h a n c e d a n t i p r o l i f e r a t i v e effect o f C s A w a s o b s e r v e d w h e n t h e d r u g w a s b o u n d t o L D L [67] b u t w a s n o t e v i d e n t w h e n t h e d r u g w a s b o u n d t o e i t h e r V L D L o r H D L [87].  I n a d d i t i o n , w h e n C s A w a s a d m i n i s t e r e d i n a n i s o l a t e d p e r f u s e d rat k i d n e y  m o d e l , the interaction of the d r u g w i t h H D L a n d L D L s i g n i f i c a n t l y r e d u c e d r e n a l c l e a r a n c e a n d t h e e x t e n t o f r e n a l t i s s u e d i s t r i b u t i o n o f C s A [121].  the  A recent  s t u d y r e p o r t e d that the total s e r u m cholesterol a n d L D L levels affected the clinical  19  e f f i c a c y o f c y c l o s p o r i n e [52]. A s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n b e t w e e n s e r u m t o t a l cholesterol concentrations,  L D L cholesterol concentrations  and  IC50 o f C s A w a s '  o b s e r v e d s u g g e s t i n g that the h i g h e r the cholesterol, the l o w e r the efficacy of the d r u g [52].  1.4.1. Pharmacokinetic  parameters  A s u m m a r y o f s o m e p h a r m a c o k i n e t i c c h a r a c t e r i s t i c s o f C s A as r e p o r t e d S c o t t et al (1988) i s p r e s e n t e d i n T a b l e 3 [113]. bound  to  lipoproteins  with  the  remaining  by  I n p l a s m a , a l m o s t 9 0 % o f C s A is bound  A p p r o x i m a t e l y , 50-60% of b l o o d C s A is b o u n d  to  plasma  proteins  to cholesterol- a n d  [114].  triglyceride-  c o n t a i n i n g l i p o p r o t e i n s a n d t h e r e m a i n i n g b o u n d t o e r y t h r o c y t e s [51] . O f t h e t o t a l a m o u n t of C s A a s s o c i a t e d w i t h l i p o p r o t e i n s , 43-57% is b o u n d to H D L , 25-37% w i t h L D L a n d l e s s t h a n 1 0 % w i t h V L D L [114,130]. T h e u n b o u n d f r a c t i o n o f c y c l o s p o r i n e in  plasma  of  renal  transplant  patient  is  in  the  range  of  0.04  to  0.12  [65].  B i o a v a i l a b i l i t y o f t h e d r u g i s v a r i a b l e r a n g i n g f r o m 8% t o 6 0 % c o n t r i b u t e d b y  the  l i p o p h i l i c i t y o f t h e d r u g [30]. A r e a - u n d e r - t h e - c u r v e ( A U C ) v a l u e s a r e v a r i a b l e w i t h d i f f e r e n t f o r m u l a t i o n s o f t h e d r u g . It h a s b e e n r e p o r t e d t h a t h i g h e r A U C v a l u e s a r e o b s e r v e d i n p a t i e n t s a d m i n i s t e r e d N e o r a l ® v e r s u s S a n d i m m u n e ® [1].  Elimination  of  than  the  drug  elimination cyclosporine  is [30]. are  predominantly The volume usually  due  to  hepatic  metabolism  o f d i s t r i b u t i o n at s t e a d y  higher  in  transplant  i n d i v i d u a l s [30].  20  patients  rather  state a n d compared  renal  clearance to  of  healthy  Parameter  Value  Bioavailability  8%-60%  Protein Binding  90% to l i p o p r o t e i n s  Clearance  0.15-0.7 L / h / k g  V s s ( v o l u m e of d i s t r i b u t i o n at steady  1.8-13.8 L / k g  state) 2.9 - 1 5 . 8 h  11/2 P (elimination)  Table  3:  P h a r m a c o k i n e t i c characteristics  of C s A .  Modified  from  references  [113,114,130].  1.4.2. Interaction of CsA with LDL receptor A f e w investigators h a v e suggested that C s A m a y be affecting L D L receptor activity subsequently  c a u s i n g h y p e r l i p i d e m i a [95,100,124,138].  D e G r o e n (1988)  h y p o t h e s i z e d that L D L not o n l y functions as a n i m p o r t a n t carrier of c y c l o s p o r i n e i n p l a s m a b u t also c a n facilitate transport of C s A into cells b y the L D L receptor [23]. U n f o r t u n a t e l y , n o e x p e r i m e n t a l results w e r e r e p o r t e d to p r o v i d e evidence for this hypothesis. F u r t h e r m o r e , S a n g h v i et al (1989) p r o p o s e d that c e l l u l a r u p t a k e of C s A m a y occur t h r o u g h the L D L receptor b y specific a n d nonspecific p a t h w a y s [109]. T h e y o b s e r v e d that w h e n C s A w a s c o m p l e x e d to L D L , C s A i n t e r n a l i z a t i o n w a s h i g h e r t h a n the C s A alone g r o u p i n h u m a n l y m p h o c y t e s [109].  A pilot study i n  nephrotic patients f o u n d that b o t h total s e r u m cholesterol a n d L D L - c h o l e s t e r o l  21  levels w e r e significantly correlated w i t h s u p p r e s s i v e efficacy of c y c l o s p o r i n e against peripheral b l o o d l y m p h o c y t e (PBL) blastogenesis  [51].  The authors hypothesized  that i n c r e a s e d r e n a l graft f a i l u r e i n recipients w i t h h i g h c h o l e s t e r o l l e v e l s w a s  the  a t t e n u a t i o n of c y c l o s p o r i n e efficacy t h r o u g h its u p t a k e i n t o T - l y m p h o c y t e s t h r o u g h L D L r e c e p t o r d o w n r e g u l a t i o n [51]. reported  that the  uptake  and  F i n a l l y , a s t u d y b y P e t e r h e r y c h et al (2001)  toxicity of C s A were  e l e v a t e d L D L c o n c e n t r a t i o n s i n L L C - P K i c e l l s [91].  significantly reduced  with  T h e authors h y p o t h e s i z e d that  t h i s effect m a y b e a l s o d u e t o t h e d o w n r e g u l a t i o n o f t h e L D L r e c e p t o r s [91]. F u r t h e r in vitro s t u d i e s  suggested  that C s A c o u l d decrease L D L internalization w i t h o u t  a l t e r i n g t h e a f f i n i t y o f i t s b i n d i n g t o t h e L D L r e c e p t o r i n h u m a n f i b r o b l a s t c e l l s [129].  22  CHAPTER II:  Summary of (Research (Project  23  Overall Objective The  o v e r a l l objective w a s to elucidate the role of the L D L receptor i n  m e d i a t i n g the u p t a k e a n d toxicity of C s A i n a r e n a l p r o x i m a l t u b u l e cell line, L L C PKi.  2.1 Hypothesis It w a s h y p o t h e s i z e d that w h e n L D L receptor a c t i v i t y w a s decreased u p o n I g G C 7 treatment, b o t h C s A u p t a k e a n d toxicity w e r e r e d u c e d i n L L C - P K i cells.  2.2 Specific Aims There w e r e three specific a i m s to test the hypothesis: Aim 1: T o d e t e r m i n e w h e t h e r L L C - P K i cells w e r e a suitable cell m o d e l to s t u d y L D L receptor activity. Aim 2:  T o establish the assay m e t h o d s a n d c o n d i t i o n s r e q u i r e d w i t h I g G - C 7 , a  m o n o c l o n a l a n t i b o d y to the L D L receptor i n L L C - P K i cells. Aim 3: T o i m p l e m e n t these m o n o c l o n a l a n t i b o d y assay m e t h o d s to investigate b o t h C s A u p t a k e a n d toxicity i n L L C - P K i cells.  2.3 Rationale In A i m 1, it w a s critical to d e t e r m i n e that L L C - P K i cells w e r e a suitable cell m o d e l to s t u d y L D L receptor f a m i l y activity before a n y inferences c o u l d be m a d e w h e n C s A w a s present.  B a s e d o n the data generated f r o m A i m 1, the a p p r o p r i a t e  24  assay m e t h o d s i n v o l v i n g IgG-C7, a m o n o c l o n a l a n t i b o d y to the L D L r e c e p t o r established  i n A i m 2 including optimal concentration  and  preincubation  was time.  F i n a l l y , once the a n t i b o d y assay c o n d i t i o n s w e r e o p t i m i z e d i n L L C - P K i cells, the m e t h o d w a s a p p l i e d i n the presence of C s A to investigate b o t h C s A u p t a k e  and  t o x i c i t y i n A i m 3.  2.4 Significance of Research U n d e r s t a n d i n g the m e c h a n i s m b y w h i c h C s A c o u l d cause n e p h r o t o x i c i t y i n patients w i t h d y s l i p i d e m i a w o u l d a l l o w i m p r o v e d administration of C s A therapy. T h e efficacy a n d t o x i c i t y of C s A c o u l d be p r e d i c t e d b a s e d o n the l i p i d p r o f i l e of the patient.  In  addition,  knowledge  about  CsA-induced  nephrotoxicity  a p p l i c a b l e to other h y d r o p h o b i c d r u g s that cause r e n a l d y s f u n c t i o n . delivery  systems  w i t h C s A c o u l d be  designed  m a i n t a i n its efficacy.  25  i n order  to  may  Novel  a v o i d toxicity  be drug and  CHAPTER III:  Materials and'^agents  3.1 Materials  3.1.1. Cells and cell culture Pig  kidney  cells  (LLC-PKi)  and  human  hepatoma  cells  (HepG2)  were  p u r c h a s e d f r o m A m e r i c a n T y p e C u l t u r e C o l l e c t i o n ( A T C C ) (Wanassas, V A , U S A ) . D M E M : H a m ' s F 1 2 (1:1) c u l t u r e m e d i u m , D M E M phosphate  buffered  trypsin-EDTA  (0.25%  saline,  culture m e d i u m , Dulbecco's  penicillin-streptomycin (10,000U/ml  trypsin,  I m M EDTA-4Na  and  0.05%  penicillin,  trypsin,  ),  0.5mM  E D T A 4 N a ) , s o d i u m p y r u v a t e , a n d fetal calf s e r u m ( U S A source) w e r e a l l p u r c h a s e d -  from  G i b c o B R L ( G r a n d Island, N Y , U S A ) .  Sterile steritop  0.22pm  express  m e m b r a n e bottle top filters w e r e p u r c h a s e d f r o m M i l l i p o r e ( B e d f o r d , M A , U S A ) . Sterile 5 0 m l centrifuge tubes, disposable 10 a n d 2 5 m l s e r o l o g i c a l pipettes  and  1 0 0 m m p e t r i dishes w e r e p u r c h a s e d f r o m Starstedt ( M o n t r e a l , P Q , C a n a d a ) . A n g l e v e n t e d neck T-75 sterile cell c u l t u r e flasks, sterile 2 4 - w e l l surface-treated plates a n d 0.45 p m m e d i a glass bottles w e r e p u r c h a s e d f r o m V W R ( E d m o n t o n , A B , C a n a d a ) .  3.1.2. Lipoproteins, Chemicals and Labelled Drug P u r i f i e d l o w - d e n s i t y l i p o p r o t e i n ( L D L ) , tissue-culture tested b o v i n e s e r u m a l b u m i n ( B S A ) , s o d i u m bicarbonate ( N a H C O s ) , s o d i u m h y d r o x i d e ( N a O H ) w e r e p u r c h a s e d f r o m S i g m a C h e m i c a l co. (St. L o u i s , M O , U S A ) . T r i s Base, S D S , glycine, T E M E D , (3-mercaptoethanol, a m m o n i u m persulfate, T w e e n - 2 0 , acetic a c i d , P o n c e a u S a n d protease i n h i b i t o r c o c k t a i l w e r e also p u r c h a s e d f r o m S i g m a C h e m i c a l co. (St.  27  Louis,  M O , USA).  Acrylamide,  Bisacrylamide, Laemrnli  sample  buffer  and  K a l e i d o s c o p e ® prestained protein standards were purchased f r o m Biorad (Hercules, CA,  USA).  Methanol (HPLC  pipettes were  purchased  grade),  from  Fischer  isopropanol ( H P L C (Nepean,  c y c l o s p o r i n e A ([mebmt-fi-3H]cyc\osporme  grade) a n d P a s t e u r ®  O N , Canada).  A , 2 5 0 C i / m l i n ethanol),  Radiolabeled Hyperfilm®  E C L a n d chemiluminescence reagents were purchased f r o m A m e r s h a m P h a r m a c i a Biotech (Oakville, O N , Canada) a n d D i l - L D L (3,3'-dioctadecylindocarbocyanine-low density lipoprotein) w a s purchased f r o m M o l e c u l a r Probes (Eugene, O R , U S A ) .  3.1.3. Antibodies and Assay Kits Monoclonal  antibody  to the L D L receptor  (IgG-C7) w a s purchased  from  R e s e a r c h D i a g n o s t i c s I n s t i t u t e ( R D I ) ( F l a n d e r s , N J , U S A ) . A n t i - m o u s e I g G F(ab')2 HRP  conjugate  (Westgrove,  was  purchased  PA, USA).  from  MEM-PER®  Jackson  ImmunoResearch  Eukaryotic Membrane  Laboratories  Protein Extraction  Reagent kit a n d B C A Protein A s s a y k i t were purchased f r o m Pierce Biotechnology (Rockford,  IL, USA).  Cholesterol standard  a n d reagent w a s purchased  from  T h e r m o D M A (Louisville, C O , U S A ) . Cytotox 9 6 ® Non-Radioactive Cytotoxicity Assay K i t was purchased from Promega Corporation (Madison, W l , USA).  28  3.2 Tissue Culture Reagents and M e d i u m  3.2.1  Preparation ofLLC-PKi  media  Sterile m e d i a w e r e p r e p a r e d b y a d d i n g 8 0 0 m L of distilled w a t e r to Ham's  F 1 2 1:1  prepackaged  powder.  A p p r o x i m a t e l y 1.2  g  DMEM:  of N a H C C b  was  d i s s o l v e d i n t h e m e d i a w h i l e t h e p H w a s a d j u s t e d t o a b o u t 7.3 w i t h 1 M N a O H .  The  m e d i a s o l u t i o n w a s t o p p e d to a final v o l u m e of 9 0 0 m l , m i x e d a n d sterile filtered t h r o u g h a 0 . 2 2 p m steritop filter i n t o t w o 5 0 0 m l m e d i a bottles. s t o r e d at 4 ° C a n d c o n s i d e r e d i n c o m p l e t e .  The media  were  T o c o m p l e t e t h e m e d i a , 5 m l (1%) o f  p e n i c i l l i n - s t r e p t o m y c i n a n d 5 0 m l (5%) o f f e t a l b o v i n e s e r u m ( F B S ) w e r e a d d e d a n d s t o r e d at 4 ° C .  3.2.2. Preparation ofHepGl  Media  Sterile m e d i a w e r e p r e p a r e d b y a d d i n g 8 5 0 m L of d i s t i l l e d w a t e r to prepackaged powder.  DMEM  A p p r o x i m a t e l y 2.0 g o f N a H C C > 3 w a s d i s s o l v e d i n t h e m e d i a  w h i l e t h e p H w a s a d j u s t e d t o a b o u t 7.3 w i t h 1 M N a O H .  The media solution was  t o p p e d to a final v o l u m e of 9 0 0 m l , m i x e d a n d sterile filtered t h r o u g h a 0 . 2 2 p m steritop filter i n t o t w o 5 0 0 m l m e d i a bottles.  T h e m e d i a w e r e s t o r e d at 4 ° C a n d  c o n s i d e r e d i n c o m p l e t e . H e a t - i n a c t i v a t e d F B S w a s p r e p a r e d b y h e a t i n g F B S at 5 5 ° C for  40 minutes.  T o c o m p l e t e t h e m e d i a , 5 m l o f s o d i u m p y r u v a t e , 5 m l (1%) o f  penicillin-streptomycin and 40ml  (5%) o f h e a t - i n a c t i v a t e d  s t o r e d at 4 ° C .  29  FBS were added  and  3.2.3. Preparation of Serum-free LLC-PKi  and HepG2 media  I n c o m p l e t e m e d i a w e r e p r e w a r m e d at 3 7 ° C i n a w a t e r b a t h . ml  of  serum-free  media,  streptomycin were added.  0.9g  (0.2%)  of  B S A and  4.5ml  T o p r e p a r e 450  (1%)  of  penicillin-  T h e c o n c e n t r a t i o n of B S A i n the c u l t u r e m e d i u m w a s  b a s e d o n r e s u l t s r e p o r t e d b y E r k a n et al (2001) [29].  Repetitive mixing w i t h  p i p e t t o r w a s necessary to e n s u r e full d i s s o l u t i o n of the m i x t u r e .  the  The media were  s t o r e d o v e r n i g h t at 4 ° C p r i o r t o e x p e r i m e n t a t i o n .  3.2.4. Preparation of Phosphate Buffered Saline (PBS) Sterile P B S w a s p r e p a r e d b y a d d i n g 9 0 0 m l of distilled w a t e r to p r e p a c k a g e d Dulbecco's P B S powder.  T h e p H o f t h e s o l u t i o n w a s a d j u s t e d t o 7.3, t o p p e d t o I L  t h e n s t e r i l e f i l t e r e d t h r o u g h a 0.22 p m s t e r i t o p f i l t e r i n t o m e d i a b o t t l e s a n d s t o r e d at 4°C  3.2.5. Culture Establishment ofLLC-PKi  and HepG2 cells from Frozen Stock  F r o z e n c e l l s f r o m A T C C w e r e s t o r e d i n 1.2 m l c y r o v i a l s s u b m e r s e d i n l i q u i d n i t r o g e n a n d - 7 0 ° C freezer for L L C - P K i cells a n d H e p G 2 cells, r e s p e c t i v e l y , p r i o r to use.  T o establish the  centrifuge  filled  with  culture, one cyrovial w a s 70%  approximately 5 minutes.  ethanol  and  thawed  r e m o v e d , placed into a in  a  37°C  water  bath  50ml for  O n c e complete, cells w e r e carefully transferred to sterile  culture flask or 1 0 0 m m petri d i s h containing p r e - w a r m e d i n c u b a t e d at 3 7 ° C i n h u m i d i f i e d 5% CO2.  30  complete media  and  3.2.6. Subculturing of LLC-PKi cells O n c e confluence w a s reached, the m e d i u m w a s d i s c a r d e d f r o m the flask a n d r i n s e d three times w i t h 8 m l of p r e w a r m e d sterile D u l b e c c o ' s P B S . W a s h i n g s w e r e d i s c a r d e d a n d cells w e r e treated w i t h 8 m l of p r e w a r m e d t r y p s i n . O n c e the cells w e r e dissociated, the m i x t u r e w a s p o u r e d i n t o a sterile 5 0 m l centrifuge tube. flask w a s w a s h e d  three times w i t h sterile D u l b e c c o ' s P B S a n d the  transferred to the centrifuge tube.  The  washings  C e l l s w e r e p e l l e t e d d o w n at 1000 rotations per  m i n u t e (rpm) for about 5-8 m i n u t e s a n d the s u p e r n a t a n t w a s decanted.  T h e cell  pellet w a s r e s u s p e n d e d w i t h a v o l u m e of c o m p l e t e m e d i a a c c o r d i n g to a d e c i d e d split ratio. F o r e x a m p l e , one w o u l d r e s u s p e n d i n 8 m l of c o m p l e t e m e d i a for a 1:8 split for confluence i n 3 days. O n e m i l l i l i t e r of the cell m i x t u r e w a s a d d e d to a n e w flask w i t h 2 5 m l of c o m p l e t e m e d i a a n d p l a c e d i n the i n c u b a t o r at 3 7 ° C i n h u m i d i f i e d 5% C O 2 . T h e m e d i u m w a s c h a n g e d every 48 h o u r s u n t i l cells w e r e confluent a n d r e a d y for s u b c u l t u r i n g .  3.2.7. Subculturing of HepG2 cells O n c e confluence w a s reached i n p e t r i dishes, the m e d i u m w a s d i s c a r d e d a n d r i n s e d three times w i t h 7 m l of p r e w a r m e d sterile D u l b e c c o ' s P B S . W a s h i n g s w e r e d i s c a r d e d a n d cells w e r e treated w i t h 5 m l of p r e w a r m e d t r y p s i n . O n c e the cells w e r e dissociated, the m i x t u r e w a s p o u r e d into a sterile 5 0 m l centrifuge tube.  The  flask w a s w a s h e d three times w i t h sterile D u l b e c c o ' s P B S a n d the w a s h i n g s w e r e transferred to a centrifuge tube. C e l l s w e r e pelleted d o w n at 2000 r p m for about 2-3  31  m i n u t e s a n d the s u p e r n a t a n t w a s decanted.  The cell pellet w a s resuspended w i t h a  v o l u m e of c o m p l e t e m e d i a a c c o r d i n g to a d e c i d e d s p l i t ratio. w o u l d resuspend days.  For example,  one  i n 1 0 m l o f c o m p l e t e m e d i a f o r a 1:10 s p l i t f o r c o n f l u e n c e i n 5  O n e m i l l i l i t e r o f the c e l l m i x t u r e w a s a d d e d to a p e t r i d i s h w i t h 15 m l of  complete media.  T h e d i s h w a s s h a k e n for a c o u p l e of m i n u t e s i n o r d e r to disperse  the cells o v e r the g r o w t h area a n d p l a c e d i n the i n c u b a t o r at 3 7 ° C i n h u m i d i f i e d 5% CO2. T h e m e d i u m w a s c h a n g e d e v e r y 72 h o u r s u n t i l cells w e r e c o n f l u e n t a n d r e a d y for s u b c u l t u r i n g again.  3.2.8. Seeding plates and preparing LLC-PKi Pelleted cells w e r e r e s u s p e n d e d 3.2.4.  and HepG2 cells for  Experimentation  i n c o m p l e t e m e d i a as d e s c r i b e d i n s e c t i o n  A n a l i q u o t of the cell m i x t u r e w a s c o u n t e d b y a h e m o c y t o m e t e r to calculate a  s e e d i n g d e n s i t y o f 7.0 x 1 0 c e l l s / c m 4  H e p G 2 cells i n 24-well plates.  2  f o r L L C - P K i c e l l s a n d 5.0 x 1 0 c e l l s / c m 5  2  for  T h e c e l l s w e r e a l l o w e d t o p r o p a g a t e f o r 24 h o u r s a t  3 7 ° C i n h u m i d i f i e d 5% CO2. A f t e r 24 h o u r s , cells s h o u l d h a v e r e a c h e d a b o u t 60-70% c o n f l u e n c e as v i e w e d u n d e r a n i n v e r t e d m i c r o s c o p e . C e l l s w e r e r e p l a c e d w i t h 1 m l of s e r u m - f r e e m e d i a f o r a n a d d i t i o n a l 24 h o u r s a n d t h e n the m e d i u m w a s r e m o v e d . At  this  point,  the  L D L receptors  were  experimentation.  32  upregulated  [40]  and  ready  for  3.3 Preparation of Experimental Reagents  3.3.3. Cholesterol concentration determination in LDL  Cholesterol content was determined using a colorimetric-based enzyme reaction.  In the presence of cholesterol esterases (CE), cholesterol esters are  enzymatically hydrolyzed to cholesterol and free fatty acids. The free cholesterol, both contained in LDL and converted from cholesterol esters, are then oxidized by cholesterol oxidase (CO) to cholest-4-en-3-one and hydrogen peroxide.  The  hydrogen peroxide product combines with hydroxybenzoic acid (HBA) and 4aminoantipyrine (4AAP), catalyzed by peroxidase (POD) forms a quinoneimine dye with peak absorbance at 500nm [122]. CE  Cholesterol esters + H 2 O —• Cholesterol + Fatty Acids co  Cholesterol + O2 —*• Cholest-4-en-3-one + H2O2 POD 2H2O2  + HBA + 4AAP  Quinoneimine Dye +  4H2O  A standard curve was prepared by serial dilution of a 200mg/dl cholesterol stock solution.  To measure cholesterol content, lOul of both standard and LDL sample  were aliquoted into 10 X 75 mm test tubes with 1 ml of cholesterol reagent. The test tubes were vortexed and placed in a water bath at 37°C for 5 minutes. Following, the absorbance of each sample was determined at 505nm with a Hewlett Packard 8452 Diode Array Spectrophotometer. From the standard curve, the concentration  33  of c h o l e s t e r o l i n the L D L s a m p l e w a s d e t e r m i n e d .  A w o r k i n g concentration of  2 0 0 p g / m l cholesterol concentration w a s u s e d for experimentation.  3.3.2. Solutions with Dil-LDL Dil-LDL  (3,3'-dioctadecylindocarbocyanine-low density  lipoprotein)  was  p u r c h a s e d f r o m M o l e c u l a r P r o b e s (Cat#L-3482) a n d w a s s t o r e d a w a y f r o m l i g h t at 4°C.  Prior  to  experimentation,  Dil-LDL  was  centrifuged  m i c r o c e n t r i f u g e for 3 m i n u t e s to r e m o v e a g g r e g a t i o n . concentration  based  o n protein content  at l m g / m l  at  3000  rpm  S u b s e q u e n t l y , the  was  in  a  stock  d i l u t e d to a w o r k i n g  s o l u t i o n of 5 0 0 p g / m l i n serum-free m e d i a for e x p e r i m e n t a t i o n .  3.3.3. Solutions with IgG-C7 I g G - C 7 , a m o n o c l o n a l a n t i b o d y to the L D L receptor, w a s p u r c h a s e d  from  R e s e a r c h D i a g n o s t i c I n s t i t u t e as a l y o p h i l i z e d p o w d e r . O n c e r e c o n s t i t u t e d w i t h 1 m l of d i s t i l l e d w a t e r , a 50 p g / m l c o n c e n t r a t i o n b a s e d o n p r o t e i n c o n t e n t w a s r e a d y f o r e x p e r i m e n t a t i o n o r s t o r e d at 4 ° C . media  was  used  A w o r k i n g solution of 2 5 p g / m l i n  p r i o r to e x p e r i m e n t a t i o n .  serum-free  F o r i m m u n o b l o t t i n g , a 0.2  pg/ml  s o l u t i o n i n a n t i b o d y d i l u t i o n b u f f e r w a s p r e p a r e d a n d s t o r e d a t 4 ° C (see s e c t i o n 3.4.2).  34  3.3.4. Solutions with PHlCsA A w o r k i n g solution of tritiated cyclosporine A w a s m a d e b y d i l u t i n g stock [ H ] C s A (specific a c t i v i t y 7 . 4 8 u C i / p g ) i n serum-free m e d i a to a c o n c e n t r a t i o n  of  32ug/ml.  T o p r e p a r e [ H ] C s A - L D L c o m p l e x s o l u t i o n at a C s A c o n c e n t r a t i o n  of  800ng/ml  and  3  3  an L D L cholesterol  w o r k i n g solution and  concentration  of 2 0 p g / m l ,  7.5pl of [ H ] C s A 3  30pl of L D L w o r k i n g solution w e r e m i x e d together  per  treatment g r o u p i n a 300 p i w o r k i n g v o l u m e .  3.4. P r e p a r a t i o n o f R e a g e n t s f o r W e s t e r n B l o t t i n g  3.4.1  SDS-PAGE S t o c k s o l u t i o n s u s e d to p r e p a r e a S D S - P A G E w e r e the f o l l o w i n g : T r i s - H C l  1.5M  pH=8.8; T r i s - H C l 0 . 5 M pH=6.8; A c r y l a m i d e / B i s (30%T, 0.8%C); 10%  (w/v)  SDS.  T r i s - H C l 1 . 5 M pH=8.8 w a s p r e p a r e d b y a d d i n g 27.23g of T r i s Base i n 8 0 m l of  d H 2 0 , a d j u s t i n g t h e p H w i t h 1 M H C 1 t o 8.8 a n d b r i n g i n g t h e v o l u m e u p t o 1 5 0 m l . T r i s - H C l 0 . 5 M pH=6.8 w a s p r e p a r e d b y m i x i n g 6g of Tris Base i n 6 0 m l of adjusting  the  p H w i t h 1 M H C 1 t o 6.8 a n d  b r i n g i n g the  volume  up  to  dHiO, 100ml.  A c r y l a m i d e / B i s (30%T, 0.8%C) w a s m a d e b y a d d i n g 29.2g of A c r y l a m i d e w i t h 0.8g of B i s i n 5 0 m l of d H 2 0 a n d b r i n g i n g the v o l u m e u p to 1 0 0 m l . s t o r e d at 4 ° C i n a b o t t l e w r a p p e d  i n a l u m i n u m foil.  The solution  10% S D S w a s prepared  a d d i n g l O g of S D S w i t h 9 0 m l of d H 0 a n d b r i n g i n g v o l u m e u p to 1 0 0 m l . 2  w a s u s e d f r o m the m a n u f a c t u r e r .  was by  TEMED  10% A m m o n i u m Persulfate ( A P S ) w a s p r e p a r e d  35  fresh b y a d d i n g 2 0 m g of A P S i n 200ul of d h b O .  A stock s o l u t i o n of 5 X r u n n i n g  buffer (pH=8.3) c o n s i s t i n g of 15g of T r i s Base, 72g of g l y c i n e a n d 5 g of S D S i n I L of d h b O w a s p r e p a r e d a n d stored at 4 ° C . D u r i n g a n electrophoresis p r o c e d u r e , the 5 X s o l u t i o n w a s d i l u t e d to a I X s o l u t i o n i n d h b O .  3.4.2. Immunoblot and Chemiluminescen.ee  detection  A I X transfer buffer w a s p r e p a r e d b y a d d i n g 6.6g ( 2 5 m M ) of T r i s Base, 28.8g of g l y c i n e , 2 0 m l of 10% S D S s o l u t i o n , 4 0 0 m l (20%) m e t h a n o l a n d b r i n g i n g the r e m a i n i n g v o l u m e to 2 L w i t h dPfcO. T h e s o l u t i o n w a s s t o r e d at 4 ° C . 10% Tween-20 s o l u t i o n w a s p r e p a r e d b y a d d i n g 1 0 m l T w e e n - 2 0 i n 9 0 m l of d l r b O .  10X P B S was  prepared  monohydrate  by  mixing  2.03g  of  sodium  phosphate  monobasic  (NaH2P04*H20), 11.49g of s o d i u m p h o s p h a t e dibasic a n h y d r o u s (Na2HPC>4), a n d 85g of s o d i u m c h l o r i d e . T h e p H of the s o l u t i o n w a s adjusted to 7.3. T h e r e m a i n i n g v o l u m e w a s a d d e d w i t h d h k O to I L a n d f i n a l l y , stored at r o o m temperature. A liter of w a s h buffer w a s p r e p a r e d b y 5 m l (0.05%) of 10% T w e e n - 2 0 a n d the r e m a i n i n g v o l u m e w a s a d d e d w i t h d F h O to I L w i t h 1 X P B S .  T h e s o l u t i o n w a s stored at 4 ° C .  Fifty m i l l i l i t e r s of the a n t i b o d y d i l u t i o n buffer w a s p r e p a r e d w i t h 0.25g (0.5%) B S A , 0.5ml (0.05%) of 10% T w e e n - 2 0 s o l u t i o n a n d I X P B S . A p r o t e i n c o n c e n t r a t i o n of 0 . 2 p g / m l of I g G - C 7 (the p r i m a r y a n t i b o d y ) w a s p r e p a r e d w i t h the a n t i b o d y d i l u t i o n buffer a n d stored at 4 ° C for c o n t i n u o u s use. 1 0 X P o n c e a u S w a s m a d e b y d i s s o l v i n g l g of the d y e i n 1 0 0 m l of d H 0 . 2  O n e h u n d r e d m i l l i l i t e r s of a 0.1% P o n c e a u S / 5 %  acetic a c i d s o l u t i o n w a s p r e p a r e d b y d i l u t i n g 1 0 X P o n c e a u S to I X s o l u t i o n i n d F b O  36  w i t h 5 m l of acetic a c i d . B l o c k i n g buffer w a s p r e p a r e d fresh c o n s i s t i n g of 1.5g (3%) s k i m m i l k p o w d e r a n d 0.25g (0.5%) B S A , a n d the r e m a i n i n g v o l u m e w a s t o p p e d to 5 0 m l w i t h I X P B S . D e v e l o p e r a n d fixer solutions w e r e d i l u t e d w i t h tap water a c c o r d i n g to the manufacturer instructions.  37  CHAPTER IV:  ExperimentaC (Design and''MetftodoCogy  38  4.1 D i l - L D L Assay Development T h e f o l l o w i n g p r o c e d u r e s w e r e a d a p t e d f r o m S t e p h a n et al (1993) [120] a n d G o l d s t e i n et al (1983) [40], a n d m o d i f i e d t o s u i t c o n d i t i o n s f o r L L C - P K i c e l l s . 48 h o u r s p o s t - s e e d i n g , cells w e r e c o n f l u e n t w i t h L D L r e c e p t o r s u p r e g u l a t e d  After (see  s e c t i o n 3.2.8).  4.1.1. Protein Content  Determination  P r o t e i n content w a s d e t e r m i n e d i n l y s e d cells i n o r d e r to n o r m a l i z e the  data.  T h e c h o s e n m e t h o d w a s the B i c i n c h o n i n i c A c i d ( B C A ) P r o t e i n A s s a y w h i c h utilizes a detergent-compatible  f o r m u l a t i o n for the c o l o r i m e t r i c detection a n d  of total p r o t e i n . T h e b i u r e t r e a c t i o n i n v o l v e s c u p r i c i o n s ( C u cuprous  ions (Cu  1 +  ) by protein i n an alkaline m e d i u m .  2 +  quantitation  ) b e c o m i n g r e d u c e d to  Subsequently,  cuprous  cations are detected w i t h h i g h sensitivity a n d selectivity b y a c o l o r i m e t r i c reaction w i t h b i c i n c h o n i n i c a c i d . C h e l a t i o n of t w o m o l e c u l e s of B C A w i t h one c u p r o u s i o n forms a p u r p l e - c o l o u r e d water soluble p r o d u c t that exhibits m a x i m u m  absorbance  at 5 6 2 n m [93].  of  T h e m a c r o m o l e c u l a r structure of protein, the n u m b e r  peptide  b o n d s a n d the presence of cysteine, cystine, t r y p t o p h a n a n d t y r o s i n e are r e p o r t e d to b e r e s p o n s i b l e f o r c o l o u r f o r m a t i o n w i t h B C A [136].  A schematic d i a g r a m of the  r e a c t i o n is s h o w n b e l o w . Protein (peptide bonds) + C u Cu  1 +  2 +  -> t e t r a d e n t a t e - C u  + 2 Bicinchoninic A c i d ( B C A ) -» B C A - C u  39  1 +  1 +  complex complex  A s t a n d a r d c u r v e w i t h B S A w a s p r e p a r e d i n N a O H at the r a n g e f r o m 2 5 p g / m l to 2mg/ml.  T o m e a s u r e p r o t e i n c o n t e n t i n l y s e d cells, 25 p i o f b o t h s t a n d a r d s a n d cell  samples w e r e aliquoted into a 96-well nonsterile microtiter plate.  T h e total v o l u m e  of B C A w o r k i n g r e a g e n t w a s p r e p a r e d a c c o r d i n g to the f o l l o w i n g f o r m u l a : (# o f s t a n d a r d s  + # o f s a m p l e s ) * (# o f r e p l i c a t e s ) * ( v o l u m e o f w o r k i n g  reagent per sample) T h e w o r k i n g reagent w a s p r e p a r e d b y m i x i n g 50 parts of R e a g e n t A w i t h 1 part of R e a g e n t B . B o t h R e a g e n t A a n d B w e r e p a r t o f t h e B C A P r o t e i n a s s a y k i t [93]. T w o h u n d r e d m i c r o l i t r e s of the w o r k i n g reagent w a s a l i q u o t e d i n t o each w e l l w i t h a m u l t i - c h a n n e l pipettor a n d the plate w a s c o v e r e d w i t h p a r a f i l m .  S u b s e q u e n t l y , it  w a s s h a k e n f o r a c o u p l e o f s e c o n d s a n d p l a c e d i n t h e i n c u b a t o r at 3 7 ° C f o r 3 0 minutes.  A f t e r i n c u b a t i n g , the plate w a s a l l o w e d to c o o l to r o o m t e m p e r a t u r e  the p e a k absorbance  was read w i t h a M u l t i s k a n Ascent plate reader  P r o t e i n concentrations of each cell s a m p l e w e r e d e t e r m i n e d against the  and  at 5 6 2 n m . standard  curve.  4.1.2. Fluorescence Determination  ofDil  Fluorescence w a s d e t e r m i n e d b y first r e m o v i n g the m e d i a a n d t h o r o u g h l y w a s h i n g the cells w i t h three ice-cold I X P B S w a s h i n g s . O n e m i l l i l i t e r of i s o p r o p a n o l ( I P A ) w a s a d d e d to e a c h w e l l a n d s h a k e n for 15 m i n u t e s . transferred purpose  The I P A washings were  t o m i c r o c e n t r i f u g e t u b e s a n d s p u n f o r 1 5 m i n u t e s at 3 0 0 0 r p m .  of the  centrifugation step w a s  40  to r e m o v e  L D L aggregates w h i c h  The can  interfere with the fluorescent signal. Once complete, the washings were aliquoted to a 48-well plate and fluorescence was determined at excitation and emission wavelengths, 522nm and 578nm, respectively using the Cytofluor® Multi-well Plate Reader Series 4000 from PerSeptive Biosystems. A standard curve of a range from 25ng/ ml to 800ng/ ml in IPA was generated in order to quantitate the amount of associated and bound LDL with each cell sample (see section 4.1.3 for explanation). Finally, cells were lysed with 1 ml of lOOmM of NaOH and a protein assay was performed on each sample (see section 4.1.1).  4.1.3. Dose-Response Studies  One plate was pre-cooled at 4°C for 30 minutes and then replaced with icecold serum-free media of increasing Dil-LDL concentrations from 5pg/ml to 40 pg/ ml to measure membrane-bound LDL. At 4°C, the cell system is static which allows for quantitation of only LDL bound to the cell membrane. Further, another plate was replaced with pre-warmed serum-free media at 37°C of increasing DilLDL concentrations from 5pg/ml to 40 pg/ml to measure cell-associated LDL. At 37°C, the cell system is non-static and LDL undergoes metabolism. Cell-associated LDL describes LDL that is both bound and internalized within the cell system. Finally, both plates were covered in aluminum foil. The cold plate was placed in the fridge at 4°C while the other in the incubator at 37°C. Both plates were incubated for a period of 2 hours. After 2 hours, fluorescence was determined as described in section 4.1.2.  41  4.1.4. LDL Specific  Binding  O n e plate of cells w a s i n c u b a t e d w i t h i n c r e a s i n g concentrations of D i l - L D L f r o m the range 5 p g / m l to 4 0 p g / m l i n the presence of a constant concentration of 25f o l d excess of u n l a b e l l e d L D L to measure non-specific b i n d i n g . unlabelled L D L was determined manufacturer  (5mg/ml).  C o n c e n t r a t i o n of  based o n p r o t e i n content as i n d i c a t e d b y  A n o t h e r plate of cells w a s i n c u b a t e d w i t h the  the  same  i n c r e a s i n g concentrations of D i l - L D L i n the absence of u n l a b e l l e d L D L to measure total b i n d i n g . B o t h plates w e r e c o v e r e d w i t h a l u m i n u m foil a n d i n c u b a t e d i n the fridge at 4 ° C for 2 hours.  After i n c u b a t i o n , fluorescence  was  determined  as  described i n section 4.1.2. Specific b i n d i n g w a s calculated as the difference b e t w e e n total a n d nonspecific b i n d i n g .  4.1.5. Competitive  Binding  C e l l s w e r e i n c u b a t e d w i t h a constant c o n c e n t r a t i o n of l O p g / m l of D i l - L D L i n the presence of i n c r e a s i n g concentrations of u n l a b e l l e d L D L at 10 p g / m l - 1 6 0 p g / m l for 2 h o u r s at 3 7 ° C c o v e r e d i n a l u m i n u m foil. were  based  on protein  C o n c e n t r a t i o n s of u n l a b e l l e d L D L  content as i n d i c a t e d b y the  manufacturer  Subsequently, fluorescence w a s d e t e r m i n e d as d e s c r i b e d i n section 4.1.2.  42  (5mg/ml).  4.2. Western Blotting Procedure for the L D L receptor  4.2.1. Membrane Protein Extraction B o t h L L C - P K i a n d H e p G 2 cells w e r e g r o w n i n T75 flasks a n d 1 0 0 m m petri dishes,  respectively,  confluence,  the  in  complete  m e d i u m was  media.  changed  Twenty-four  and  hours  replaced w i t h  prior  serum-free  to  100%  media  to  u p r e g u l a t e t h e L D L r e c e p t o r s . C e l l s w e r e h a r v e s t e d a s d e s c r i b e d i n s e c t i o n 3.2.6 a n d 3.2.7.  Three milliliters of sterile D u l b e c c o ' s P B S w a s a d d e d  resuspended. microscope. were  Cells  were  counted  Approximately 5 x 10  6  using a  a n d the pellet  hemocytometer  L L C - P K i cells a n d 8 x l 0  aliquoted into microcentrifuge tubes a n d  6  under  an  was  inverted  H e p G 2 cells per 2 m l  p e l l e t e d a g a i n at l O O O r p m  and  2000rpm, respectively. The supernatant was carefully r e m o v e d . Reagents A , B a n d C w e r e p r o p r i e t o r y s o l u t i o n s a s p a r t o f t h e m e m b r a n e p r o t e i n e x t r a c t i o n k i t [92]. T h e n , 1 5 0 p l of Reagent A w a s a d d e d to the cell pellets to l y s e the cells, a n d p i p e t t e d u p a n d d o w n i n order to o b t a i n a h o m o g e n o u s cell s u s p e n s i o n . protease  inhibitor cocktail  was  added  to  the  mixture.  The  A t this stage, 5 p l of cell  mixture  was  i n c u b a t e d at r o o m t e m p e r a t u r e f o r 1 0 m i n u t e s w i t h o c c a s i o n a l v o r t e x i n g after w h i c h it w a s p l a c e d o n ice. T w o parts of R e a g e n t C w a s d i l u t e d w i t h o n e part Reagent B i n o r d e r to m a k e s u f f i c i e n t v o l u m e to a d d 4 5 0 p l o f the m i x t u r e i n t o e a c h c e l l s a m p l e . T h u s 450pl of Reagent B / C w a s a d d e d to each tube of l y s e d cells a n d v o r t e x e d . T u b e s w e r e i n c u b a t e d o n ice for 30 m i n u t e s , v o r t e x i n g e v e r y 5 m i n u t e s i n o r d e r to s o l u b i l i z e the m e m b r a n e  proteins.  A f t e r 30 m i n u t e s , tubes w e r e c e n t r i f u g e d  43  at  10,000  g  for  3  temperature.  minutes  The  with  supernatant  an  Eppendorf  was  5415D  transferred  to  microcentrifuge  at  room  microcentrifuge tubes  and  i n c u b a t e d f o r a n a d d i t i o n a l 1 0 m i n u t e s at 3 7 ° C t o s e p a r a t e t h e h y d r o p h o b i c p r o t e i n s from  the  hydrophilic  proteins  through  phase  partitioning.  Tubes  were  again  c e n t r i f u g e d w i t h t h e m i c r o c e n t r i f u g e at r o o m t e m p e r a t u r e f o r 2 m i n u t e s at 1 0 , 0 0 0 g . T h e h y d r o p h i l i c l a y e r (top) w a s c a r e f u l l y r e m o v e d f r o m the h y d r o p h o b i c (bottom) layer a n d discarded.  T h e b o t t o m phase c o n t a i n i n g the m e m b r a n e  p l a c e d o n i c e u n t i l s t o r a g e at - 2 0 ° C .  proteins  was  A p r o t e i n assay w a s c o n d u c t e d i n each cell  s a m p l e i n o r d e r to d e t e r m i n e the p r o t e i n content u p o n l o a d i n g i n S D S - P A G E  (see  s e c t i o n 4.1.2).  4.2.2.  SDS-PAGE (3.5% stacking, 7.5% resolving gels) The M I N I - P R O T E A N  3 Cell apparatus w i t h  glass plates  and  the  power  s u p p l y p u r c h a s e d f r o m B i o r a d (Hercules, C A , U S A ) w e r e the e q u i p m e n t u s e d for the electrophoresis.  G l a s s plates of thickness 1 . 0 m m w e r e r i n s e d w i t h d H 0  dried with Kimwipes®.  2  and  M e a n w h i l e , the c a s t i n g a p p a r a t u s w a s a s s e m b l e d a n d n o  l e a k a g e w a s e n s u r e d w i t h d H a O . T w o 7 . 5 % r e s o l v i n g g e l s w e r e p r e p a r e d as f o l l o w s : G e l B u f f e r 1 . 5 M T r i s - H C l p H 8.8  2.5 m l  30% A c r y l a m i d e / B i s  2.5 m l  dH20  4.9 m l  10% ( w / v ) S D S  0.1 m l  10% A P S  50 p i  TEMED  5 pi  44  The gel s o l u t i o n w a s p o u r e d s l o w l y a n d s m o o t h l y b e t w e e n the glass plates u n t i l the c o m b front.  T h e gel w a s o v e r l a y e d w i t h d l - b O i n o r d e r for it to p o l y m e r i z e for 1  h o u r or overnight.  E x c e s s w a t e r w a s b l o t t e d d r y w i t h f i l t e r p a p e r a n d t w o 3.5%  s t a c k i n g g e l s w e r e p r e p a r e d as f o l l o w s : G e l B u f f e r 0 . 5 M T r i s - H C l p H 6.8  1.25 m l  30% A c r y l a m i d e / B i s  0.6 m l  dH 0  3.1 m l  10% ( w / v ) S D S  50 p i  10% A P S  25 p i  2  TEMED The  5 pi  s t a c k i n g gel w a s carefully p o u r e d o n top of the r e s o l v i n g gel w i t h a P a s t e u r ®  pipette.  T h e 1 0 - w e l l c o m b w a s inserted a n d the g e l w a s a l l o w e d to p o l y m e r i z e for  30 m i n u t e s . C e l l s a m p l e s w e r e t h a w e d s l o w l y o n ice a n d d i l u t e d i n L a e m m l i b u f f e r c o n t a i n i n g p - m e r c a p t o e t h a n o l i n a 1:2 r a t i o ( s a m p l e : b u f f e r ) .  sample  Samples were  b o i l e d at 9 5 ° C for 5 m i n u t e s i n a w a t e r b a t h . W e l l s w e r e d r i e d w i t h filter p a p e r a n d the glass plates w e r e a s s e m b l e d i n the electrode a p p a r a t u s .  I X r u n n i n g buffer w a s  a d d e d to b o t h i n n e r a n d t a n k c h a m b e r s . O n c e r e d u c e d , 40 p i o f s a m p l e a n d 10 p i of prestained p r o t e i n s t a n d a r d s ( K a l e i d o s c o p e ® ) w e r e l o a d e d i n t o the w e l l s w i t h gel l o a d i n g tips.  L o a d i n g v o l u m e s v a r i e d w h e n a constant a m o u n t of p r o t e i n w a s  needed for each sample.  T h e electrode a p p a r a t u s w a s p l a c e d i n the tank  c o n n e c t e d to the p o w e r s u p p l y .  and  E l e c t r o p h o r e s i s c o n d i t i o n s w e r e i n i t i a l l y set at a  constant c u r r e n t of 1 0 m A u n t i l it r e a c h e d the b o r d e r of the s e p a r a t i n g g e l w h e r e the c u r r e n t s e t t i n g w a s c h a n g e d t o 2 0 m A . T h e v o l t a g e w a s set at 2 0 0 V a n d t h e d u r a t i o n  45  was  about  70 m i n u t e s .  Subsequently, the electrode  disassembled, a n d the separating gel w a s discarded.  assembly  a n d plates  were  T h e top right h a n d of the  r e s o l v i n g g e l w a s r e m o v e d a n d p l a c e d i n I X transfer buffer r e a d y to p r o c e e d to immunoblotting.  4.2.3. Immunoblotting  and Detection  Filter papers a n d fiber p a d s w e r e s o a k e d i n I X transfer buffer f o r 15 m i n u t e s . P V D F m e m b r a n e w a s s o a k e d i n 100% m e t h a n o l for 20 m i n u t e s , r i n s e d w i t h  dFkO  a n d s o a k e d i n I X transfer buffer p r i o r to use. Filter p a p e r w a s s l i d carefully u n d e r the g e l a n d a s a n d w h i c h w a s f o r m e d b e t w e e n t w o filter p a p e r s a n d fiber p a d s i n the cassette w i t h the g e l f a c i n g the a n o d e .  Bubbles between the gel a n d m e m b r a n e w e r e  l i g h t l y s q u e e z e d i n order to ensure p r o p e r transfer o f proteins f r o m the g e l to the membrane.  T h e g e l cassettes w e r e l o c k e d a n d p l a c e d i n correct o r i e n t a t i o n i n the  electrode m o d u l e of the M I N I - T R A N S B L O T apparatus.  T h e Bio-ice cooling unit  w a s p l a c e d i n the tank w i t h the electrode unit. T h e t a n k w a s f i l l e d w i t h I X transfer buffer, a stir b a r w a s a d d e d a n d the entire u n i t w a s p l a c e d o v e r a stirrer w i t h ice packs i n order to keep the entire u n i t cool.  T h e u n i t w a s connected to the p o w e r  s u p p l y a n d transfer settings w e r e at a c o n s t a n t 70 V a n d 3 5 0 m A f o r a p p r o x i m a t e l y 2 hours followed b y 3 0 V for 30mins-lhour.  After the transfer, the m e m b r a n e w a s  s t a i n e d w i t h P o n c e a u S s o l u t i o n a n d d e s t a i n e d w i t h d F b O as a c o n t r o l for p r o p e r transfer.  O n c e successful, the m e m b r a n e w a s p l a c e d face u p i n a u t i l i t y b o x a n d  b l o c k e d w i t h b l o c k i n g buffer at 3 7 ° C f o r 2 h o u r s o r o v e r n i g h t at 4 ° C . T h e n e x t d a y ,  46  the  blocking  buffer  was  discarded  and  the  membrane  was  incubated  at  4°C  o v e r n i g h t w i t h 0.2 u g / m l p r i m a r y a n t i b o d y ( I g G - C 7 , a n t i - L D L r e c e p t o r ) i n a n t i b o d y d i l u t i o n buffer w h i l e s h a k i n g . T h e m e m b r a n e w a s then w a s h e d 3 times w i t h w a s h buffer.  E a c h w a s h c y c l e c o n s i s t e d o f 25 m l w a s h b u f f e r f o l l o w e d b y a 10 m i n u t e  i n c u b a t i o n at 3 7 ° C .  D u r i n g the last w a s h cycle, s e c o n d a r y a n t i b o d y s o l u t i o n w a s  p r e p a r e d b y d i l u t i n g 1: 4 0 0 0 a n t i - m o u s e I g G H R P c o n j u g a t e i n a n t i b o d y buffer.  dilution  T h e m e m b r a n e w a s i n c u b a t e d i n the 2° a n t i b o d y s o l u t i o n at 3 7 ° C for 45  minutes w i t h shaking. w i t h w a s h buffer.  After i n c u b a t i o n , the m e m b r a n e w a s a g a i n w a s h e d 3 times  C h e m i l u m i n e s c e n c e reagent w a s prepared b y m i x i n g equal parts  of Reagents A a n d B f r o m A m e r s h a m to a total v o l u m e of 10 m l . W i t h a t i m e r , the m e m b r a n e w a s i n c u b a t e d i n the l u m i n a l reagent for 1 m i n u t e .  The membrane  was  q u i c k l y c o v e r e d i n s a r a n w r a p a n d p l a c e d i n the g e l cassette w i t h the p r o t e i n s f a c i n g up.  I n the d a r k r o o m , the H y p e r f i l m E C L w a s e x p o s e d to the m e m b r a n e  10-15 s e c o n d s , d e p e n d i n g o n t h e d e s i r a b l e i n t e n s i t y o f t h e b a n d s .  between  The film  was  r e m o v e d f r o m the cassette a n d p l a c e d i n the d e v e l o p e r for a c o u p l e of seconds, w a s h e d w i t h d L h O , t h e n p l a c e d i n the fixer. T h e f i l m w a s d r i e d for 15 m i n u t e s t h e n s a v e d as a h a r d c o p y f o r f u r t h e r a n a l y s i s .  4 . 3 . D i l - L D L with I g G - C 7 Assay Development It w a s n e c e s s a r y t o d e t e r m i n e t h e o p t i m a l c o n d i t i o n s i n c l u d i n g t e m p e r a t u r e , p r e i n c u b a t i o n p e r i o d a n d c o n c e n t r a t i o n of I g G - C 7 i n o r d e r to s t u d y the i n h i b i t o r y effect o f t h e m o n o c l o n a l a n t i b o d y t o t h e L D L r e c e p t o r i n L L C - P K i c e l l s .  47  4.3.1. Temperature Various  Dependence  concentrations  of I g G - C 7  ranging from  0.1  to  2.0 u g / m l  were  i n c u b a t e d i n s e r u m - f r e e m e d i a f o r 2 h r s at 4 ° C . A n o n - t r e a t m e n t c o n t r o l c o n t a i n i n g n o I g G - C 7 w a s allocated for c o m p a r i s o n . lOpg/ml  of D i l - L D L  i n serum-free  Subsequently, m e d i a was removed and  media was  added.  Plates were  covered  in  a l u m i n u m f o i l a n d f u r t h e r i n c u b a t e d f o r 2 h r s at b o t h 4 ° C a n d 3 7 ° C . A f t e r a t o t a l o f 4 hours  incubation, fluorescence  was determined  as d e s c r i b e d i n s e c t i o n s 4.1.2.  V a l u e s w e r e e x p r e s s e d as p e r c e n t i n h i b i t i o n i n D i l - L D L b i n d i n g c o m p a r e d t o n o n treatment control.  % I n h i b i t i o n = n g L D L / p g c e l l p r o t e i n control - n g L D L / p g c e l l p r o t e i n treatment x 1 0 0 % n g L D L / p g p r o t e i n control  4.3.2. Optimal Preincubation  period  A c o n c e n t r a t i o n o f 2 . 0 p g / m l of I g G - C 7 w a s i n c u b a t e d at v a r i o u s t i m e p e r i o d s o f 0 . 5 , 1 . 0 , 1 . 5 , 2.0, a n d 4.0 h o u r s at 4 ° C . P r o p e r n o n - t r e a t m e n t c o n t r o l s c o n t a i n i n g n o I g G - C 7 w e r e allocated for each t i m e point. After the p r e i n c u b a t i o n p e r i o d , m e d i a w a s r e m o v e d a n d 10 p g / m l o f D i l - L D L i n s e r u m - f r e e  m e d i a w a s a d d e d to b o t h  t r e a t m e n t a n d c o n t r o l g r o u p s , a n d i n c u b a t e d for a n a d d i t i o n a l 2 h r s at 4 ° C . plate w a s c o v e r e d i n a l u m i n u m foil.  The  F l u o r e s c e n c e w a s d e t e r m i n e d as d e s c r i b e d i n  s e c t i o n 4.1.2 a n d v a l u e s w e r e c a l c u l a t e d as d e s c r i b e d i n s e c t i o n 4 . 3 . 1 .  48  4.3.3. Optimal  Concentration  V a r i o u s concentrations  o f I g G - C 7 r a n g i n g f r o m 0.1  to 12.0 u g / m l  were  p r e i n c u b a t e d f o r a p e r i o d o f 1.5 h o u r s at 4 ° C . A n o n - t r e a t m e n t c o n t r o l c o n t a i n i n g n o I g G - C 7 w a s allocated for c o m p a r i s o n . lOpg/ml  of  Dil-LDL  i n serum-free  Subsequently, m e d i a was removed, and  media  was  added  and  incubated  a d d i t i o n a l 2 h o u r s at 4 ° C . T h e p l a t e w a s c o v e r e d i n a l u m i n u m f o i l . was  determined  as  d e s c r i b e d i n 4.3.1.  described  i n s e c t i o n 4.1.2  and  values  were  The optimal preincubation period and IgG-C7  for  an  Fluorescence calculated  as  concentration  w e r e c h o s e n b a s e d o n the c r i t e r i o n that a m i n i m u m of 80% i n h i b i t i o n of D i l - L D L binding was observed.  4.4. C s A T i m e - C o u r s e I n c u b a t i o n S t u d y B e i s i e g e l et al (1981) r e p o r t e d  that the c e l l u l a r content  of I g G - C 7  reached  s t e a d y s t a t e w i t h i n 1 h o u r a n d s u b s e q u e n t l y r e m a i n e d a t e q u i l i b r i u m u p t o 6 h r s at 3 7 ° C i n m o n o l a y e r h u m a n f i b r o b l a s t c e l l s [11]. D u r i n g t h e p e r i o d o f 6 h o u r s , 4 t i m e s as m u c h I g G - C 7 w a s d e g r a d e d  as w a s p r e s e n t i n t h e c e l l s a t s t e a d y  s t a t e [11].  Therefore, it w a s i m p o r t a n t to d e t e r m i n e the o p t i m a l i n c u b a t i o n t i m e n e e d e d  for  [ H ] C s A i n o r d e r to y i e l d the highest percent u p t a k e of the d r u g i n L L C - P K i cells 3  t a k i n g i n t o c o n s i d e r a t i o n t h e m e t a b o l i s m o f I g G - C 7 at 3 7 ° C .  C e l l s w e r e i n c u b a t e d w i t h 800 n g / m l of [ H ] C s A i n s e r u m - f r e e 3  v a r i o u s t i m e p o i n t s : 2, 6, 10 a n d 24 h o u r s a t b o t h 4 ° C a n d 3 7 ° C .  49  media  at  Appropriate  controls w e r e allocated for each time point. U p t a k e of [ H ] C s A w a s d e t e r m i n e d b y 3  i n t r a c e l l u l a r r a d i o a c t i v i t y (see s e c t i o n 4.5.2). I n a d d i t i o n , 8 0 0 n g / m l o f [ H ] C s A w a s 3  a d d e d to l O O m M N a O H u p o n cell lysis a n d transferred to a 7 m l s c i n t i l l a t i o n v i a l i n o r d e r to m e a s u r e total r a d i o a c t i v i t y of 8 0 0 n g / m l of [ H ] C s A .  A l l treatment groups  3  w e r e c o m p a r e d t o t h i s [ H ] C s A c o n t r o l t o d e t e r m i n e p e r c e n t u p t a k e (see s e c t i o n 3  4.5.2).  4.5. C s A uptake and Toxicity with IgG-C7 Assay Development CsA  uptake  and  toxicity  were  assessed  by  quantifying  [ H]CsA 3  i n t e r n a l i z a t i o n , a n d d e t e r m i n i n g lactate d e h y d r o g e n a s e ( L D H ) release i n one assay method.  F o r the  uptake  studies  of [ H ] C s A into L L C - P K i 3  cells, p r o c e d u r e  as  e s t a b l i s h e d b y P e t e r h e r y c h et al (2001) w a s f o l l o w e d w i t h m o d i f i c a t i o n s [91]. F o r t h e toxicity assays, L D H w a s a cell m a r k e r that w a s released i n t o the c u l t u r e m e d i u m after t u b u l a r c e l l d a m a g e .  F o r the latter, the p r o c e d u r e s f o l l o w e d w e r e t a k e n f r o m  t h e m a n u f a c t u r e r ' s i n s t r u c t i o n m a n u a l [96].  4 . 5 . L [ H]CsA 3  Bound and Uptake Assay  C o n f l u e n t cells w e r e e x a m i n e d u n d e r the i n v e r t e d m i c r o s c o p e to ensure c e l l d e a t h d u e t o p r o l o n g e d i n c u b a t i o n i n s e r u m - f r e e m e d i a (see s e c t i o n 3.2.8). m e d i u m w a s r e m o v e d a n d a concentration of 1 2 . 0 p g / m l I g G - C 7 i n  no The  serum-free  m e d i a w a s p r e i n c u b a t e d f o r 1.5 h o u r s at 4 ° C . M e a n w h i l e , [ H ] C s A - L D L c o m p l e x i n 3  50  serum-free  media was prepared  where 8 0 0 n g / m l of [ H ] C s A w a s incubated w i t h 3  2 0 p g / m l L D L c h o l e s t e r o l f o r 1 h o u r at 3 7 ° C (see s e c t i o n 3.3.4). w i t h IgG-C7, treatment and control groups were a d d e d  After  preincubation  to the cells a c c o r d i n g to  T a b l e 4. T h e w o r k i n g v o l u m e i n e a c h w e l l w a s 3 0 0 p i .  Treatment  3H-CsA  in  3H-CsA-  LDL  in  Volume  Treat  32000ng/ml  LDL  200pg/ml  serum  No  working  complex  working  m e d i a (pi)  solution  solution  solution  final  Serum-free  media  free  f i n a l cone:  cone:  20ug/ml  800ng/ml 1.  of  (Pi) 0  0  (ul) 0  300.0  0  0  0  300.0  control 2  LDH  positive  control 3  L D L control  0  0  30.0  270.0  4  Total 3 H - C s A  7.5  0  0  292.5 i n  .  5  3H-CsA  7.5  0  0  292.5  6  mAb + 3H-CsA  7.5  0  0  292.5  7  3H-CsA-LDL  0  37.5  0  262.5  8  mAb + 3H-CsA-LDL  0  37.5  0  .  262.5  7.5  0  30.0  262.5  7.5  0  30.0  262.5  NaOH  9  3H-CsA + L D L  10  mAb  +  3R-CsA  +  LDL () - indicates H - C s A - L D L c o m p l e x 3  T a b l e 4: T r e a t m e n t a n d C o n t r o l g r o u p s i n C s A U p t a k e a n d T o x i c i t y w i t h I g G - C 7 Experiments  Thus following preincubation w i t h IgG-C7, all treatment a n d control groups w i t h [ H]CsA were  [ H ] C s A control  was  d e t e r m i n e d b y a d d i n g 8 0 0 n g / m l of [ H ] C s A i n l y s e d cells w i t h l O O m M N a O H  and  3  incubated  for a n a d d i t i o n a l 6 h r s at 3 7 ° C . 3  by  transferring  3  the entire v o l u m e to a 7 m l s c i n t i l l a t i o n v i a l to m e a s u r e its total  51  radioactivity .  A l l treatment g r o u p s w e r e c o m p a r e d to the [ H ] C s A c o n t r o l to 3  m e a s u r e percent b o u n d a n d uptake.  F u r t h e r m o r e , the s u p e r n a t a n t w a s r e m o v e d  a n d a l i q u o t e d i n t o 1.5 m l microcentrifuge tubes for L D H a n a l y s i s (see section 4.5.3). B o t h m e m b r a n e - b o u n d a n d i n t r a c e l l u l a r [ H ] C s A w e r e d e t e r m i n e d as described i n 3  section 4.5.2.  4.5.2.  Membrane-bound  and Intracellular [ H]CsA  determination.  3  M e d i a w a s r e m o v e d a n d cells w e r e w a s h e d three times w i t h i c e - c o l d I X P B S to r e m o v e excess r a d i o a c t i v e C s A . A p p r o x i m a t e l y , 300pl of 0.001% T r i t o n X-100 w a s a d d e d a n d i n c u b a t e d for 10 m i n u t e s at 37°C. T h e detergent w a s r e m o v e d a n d a l i q u o t e d i n t o 7 m l s c i n t i l l a t i o n v i a l s for c o u n t i n g to d e t e r m i n e  membrane-bound  [ H ] C s A . Subsequently, cells w e r e l y s e d w i t h 300pl of l O O m M N a O H for 5 minutes. 3  T w e n t y - f i v e m i c r o l i t e r s of l y s e d cells w a s a l i q u o t e d i n t o a m i c r o t i t e r plate for p r o t e i n content analysis (see section 4.1.1) a n d the r e m a i n i n g v o l u m e i n t o 7 m l s c i n t i l l a t i o n vials. W e l l s w e r e w a s h e d a n a d d i t i o n a l 2 X w i t h l O O m M N a O H a n d the w a s h i n g s w e r e a l i q u o t e d into the c o r r e s p o n d i n g v i a l to d e t e r m i n e r a d i o a c t i v i t y b y s c i n t i l l a t i o n c o u n t i n g (see section 4.6). A l l treatment g r o u p s w e r e c o m p a r e d to the [ H ] C s A c o n t r o l to m e a s u r e percent b o u n d a n d uptake. 3  % B o u n d = C P M / p g c e l l u l a r p r o t e i n Treatment  detergent wash X  C P M / p g cellular protein [ H ] C s A control 3  52  100 %  % U p t a k e = C P M / p g c e l l u l a r p r o t e i n lysed cells  x  100%  C P M / p g cellular protein [ H ] C s A control 3  4.5.3  [ H]CsA 3  Lactate  Toxicity Study — LDH measurement dehydrogenase  ( L D H ) was  the  chosen  marker  to m e a s u r e  drug-  i n d u c e d c e l l u l a r t o x i c i t y . It i s a m a r k e r o f l a t e s t a g e t o x i c i t y w h e r e d a m a g e t o t h e p l a s m a m e m b r a n e results i n m e m b r a n e e x t r a c e l l u l a r f l u i d [4].  p e r m e a b i l i t y a n d leakage of L D H into the  T h u s , L D H i s r e l e a s e d u p o n c e l l l y s i s as a s t a b l e c y t o s o l i c  e n z y m e [4,96]. In vitro L D H r e l e a s e i s d e t e c t e d i n t h e c u l t u r e m e d i u m a n d p r o v i d e s a n a c c u r a t e m e a s u r e o f c e l l v i a b i l i t y [4]. L D H i s m e a s u r e d w i t h a 3 0 - m i n u t e c o u p l e d e n z y m e assay w h i c h b e g i n s w i t h the r e d u c t i o n of N A D a n d lactate to catalyzed by L D H .  pyruvate  N e x t , r e d u c e d N A D a n d t e t r a z o l i u m salt ( I N T ) c o n v e r t to a r e d  f o r m a z a n p r o d u c t i n t h e p r e s e n c e o f a n e l e c t r o n a c c e p t o r d i a p h o r a s e [4,96]. C o l o u r p r e c i p i t a t i o n is p r o p o r t i o n a l to the n u m b e r of l y s e d cells.  A schematic d i a g r a m of  the c o l o r i m e t r i c r e a c t i o n is s h o w n b e l o w : LDH  NAD  +  + lactate - •  pyruvate +  N A D H  Diaphorase  N A D H  +  INT  NAD  +  +  f o r m a z a n (red)  F o r t h e L D H p o s i t i v e c o n t r o l , m e d i a w a s d i s c a r d e d a n d 1% T r i t o n X - 1 0 0 w a s  added  t o t h e w e l l s a n d i n c u b a t e d f o r a c o u p l e m i n u t e s at 3 7 ° C t o e n s u r e c e l l d e a t h .  This  L D H p o s i t i v e c o n t r o l represents 100% t o x i c i t y i n the assay.  Following incubation,  the  tubes a n d  detergent mixture was  aliquoted into microcentrifuge  53  stored  for  further L D H analysis. The supernatant i n b o t h m e d i a a n d L D L control groups were aliquoted  into  microcentrifuge tubes  as  well.  A l l L D H measurements  were  c o m p a r e d to the L D H p o s i t i v e c o n t r o l to calculate percent toxicity of each treatment group.  % T o x i c i t y = A b s 2 Treatment/mg c e l l p r o t e i n - A b s 9 2 Control/mg c e l l p r o t e i n x 1 0 0 % 4 9  4  A b s 9 2 LDH Positive Control/ m g 4  protein  F i r s t l y , 50 p i of the s u p e r n a t a n t / m e d i a of e a c h treatment a n d c o n t r o l g r o u p s w e r e a d d e d to a non-sterile 9 6 - w e l l m i c r o t i t e r plate. was  a l l o c a t e d as b a c k g r o u n d to w h i c h  A serum-free m e d i a control g r o u p  all values were substracted  from.  p u r p o s e w a s to adjust for the interference i n absorbance f r o m p h e n o l r e d .  The  Substrate  m i x w a s p r e p a r e d b y a d d i n g 12 m l of A s s a y buffer ( t h a w e d s l o w l y o n ice) w i t h a v i a l of l y o p h i l i z e d substrate.  T h e m i x t u r e w a s k e p t o n ice a n d p r o t e c t e d f r o m light  u n t i l r e a d y for use. U s i n g a m u l t i - c h a n n e l pipettor, 5 0 p l of substrate m i x w a s a d d e d to e a c h s a m p l e  and  control.  T h e plate  was  covered with  a l u m i n u m foil  and  i n c u b a t e d at r o o m t e m p e r a t u r e f o r 30 m i n u t e s w i t h s h a k i n g . O n c e c o m p l e t e , 50 p i of S t o p S o l u t i o n w a s a d d e d to e a c h w e l l w i t h the m u l t i - c h a n n e l p i p e t t o r a n d large bubbles were r e m o v e d w i t h a pipette tip.  M a x i m u m a b s o r b a n c e at 4 9 2 n m o f e a c h  w e l l w a s d e t e r m i n e d w i t h a M u l t i s k a n A s c e n t plate reader w i t h i n 1 h r of a d d i n g stop solution.  T h e procedures f o l l o w e d w e r e those described i n the  n o n - r a d i o a c t i v e c y t o t o x i c i t y a s s a y k i t [96].  54  CytoTox96®  4.6  Scintillation Counting L i q u i d scintillation c o u n t i n g w a s u s e d to quantify [ H ] - C s A intracellularly. 3  Samples  were  contained  scintillation fluid. counter.  i n 7ml scintillation vials containing  The samples  were  5.4ml CytoScint  placed i n a B e c k m a n liquid scintillation  U p o n the d i s i n t e g r a t i o n of the t r i t i u m l a b e l , p h o t o n s are p r o d u c e d w h i c h  c o l l i d e w i t h m o l e c u l e s i n the s c i n t i l l a t i o n f l u i d  emitting light.  The signal  was  d e t e c t e d b y t h e c o u n t e r as c o u n t s p e r m i n u t e o v e r t h r e e m i n u t e s .  4.7  Statistical Analyses An  unpaired  t-test  (Instat;  Graphpad)  was  implemented  to  determine  differences i n C s A u p t a k e a n d toxicity w i t h I g G - C 7 b e t w e e n c o n t r o l a n d treatment groups.  Results w e r e c o n s i d e r e d significant if the p r o b a b i l i t y of the result o c c u r r e d  by chance  l e s s t h a n 5 % o f t h e t i m e (p<0.05).  O n e - w a y analysis of  variance  ( A N O V A ) (Instat; G r a p h p a d ) w a s u s e d t o d e t e r m i n e d i f f e r e n c e s i n t r e a t m e n t g r o u p s c o n t a i n i n g L D L c o m p a r e d to the c o n t r o l g r o u p . R e s u l t s w e r e c o n s i d e r e d significant i f t h e p r o b a b i l i t y o f t h e r e s u l t o c c u r r e d b y c h a n c e l e s s t h a n 5 % (p<0.05). d i f f e r e n c e s w e r e a s s e s s e d u s i n g T u k e y ' s p o s t - h o c test.  A l l data were expressed  m e a n ± S D i n t r i p l i c a t e s o r m e a n ± S E M i n n=3 a n d i n n=6 i n s o m e cases.  55  Significant as  CHAPTER V:  (ResuCts  56  5.1. Presence and abundance of L D L receptors i n L L C - P K i cells  5.1.1 Dose-response Studies T o clarify the t e r m i n o l o g y , L D L specific b i n d i n g sites refer to the f a m i l y of L D L r e c e p t o r s t h a t r e c o g n i z e L D L as a l i g a n d a n d L D L r e c e p t o r s a r e t h e  receptors  that p l a y a role i n cholesterol homeostasis. F i g u r e 6 s h o w s the s t a n d a r d c u r v e that w a s generated b y linear regression i n o r d e r t o q u a n t i t a t e t h e a m o u n t o f L D L i n n g / m l f r o m t h e I P A w a s h i n g s a g a i n s t its fluorescence (arbitrary units). medium  were  i n units  C o n c e n t r a t i o n s of D i l - L D L a d d e d to the cell c u l t u r e  of p g / m l  based  on  protein  content.  However,  after  incubation, D i l - L D L quantitation w a s determined i n n g / m l since o n l y a fraction of D i l - L D L w a s b o u n d to the cells. Therefore, the s t a n d a r d c u r v e w a s s c a l e d d o w n to quantitate D i l - L D L concentrations i n units of n g / m l based o n p r o t e i n content. R e s u l t s r e v e a l a d o s e - d e p e n d e n t i n c r e a s e i n c e l l - a s s o c i a t e d ( F i g u r e 7A ) membrane-bound  L D L (Figure 7B).  T h e total a m o u n t  of L D L associated  and was  4 . 5 n g / p g o f c e l l p r o t e i n a n d t h e a m o u n t o f L D L b o u n d w a s 3.1 n g / p g o f c e l l p r o t e i n at a D i l - L D L c o n c e n t r a t i o n o f 8 0 p g / m l . describes  L D L that is b o t h b o u n d a n d  membrane-bound subtracting  It i s a s s u m e d  that cell-associated L D L cell system  and  L D L d e s c r i b e s o n l y L D L t h a t is b o u n d to the c e l l surface.  By  membrane-bound  i n t e r n a l i z e d w i t h i n the  L D L (Figure 7B) f r o m  the  mean  values  of  cell-  associated L D L (Figure 7 A ) , internalized L D L w a s semi-quantitatively determined. I n t e r n a l i z e d L D L ( F i g u r e 7 C ) also f o l l o w s the s a m e d o s e - d e p e n d e n t r e l a t i o n s h i p  57  u n t i l a D i l - L D L concentration of 4 0 u g / m l a n d subsequently decreases to 1 . 4 n g / u g of  p r o t e i n at 8 0 p g / m l .  These results  suggest the  presence of receptors  which  r e c o g n i z e L D L a n d its a c t i v i t y reaches a m a x i m u m at a D i l - L D L c o n c e n t r a t i o n of 4 0 p g / m l . H o w e v e r , the c u r v e s d o not r e a c h s a t u r a t i o n w h i c h i n d i c a t e s b o t h specific a n d n o n s p e c i f i c b i n d i n g of D i l - L D L to L L C P K i cells.  600 H 0  1  1  1  1  200  400  600  800  1 1000  D i l - L D L Concentration (ng/ml)  Figure  6:  Standard  Curve  of D i l - L D L  Concentration  5 2 2 n m / 5 7 8 n m i n I P A . [y=1.283x-54.723; R = 0.9844] 2  58  versus  Fluorescence  at  Figure  7A:  Dose-dependent  measurement as  mean  (ng  cellular  cell-associated  of  LDL a t 3 7 ° C .  Values of  protein)  expressed  LDL/pg  of  ±  in  SD  triplicates.  40  60  80  100  Dil-LDL Concentration (ug/mil  Figure  7B:  Dose-dependent  measurement  bound  of membrane-  LDL at  expressed  as  Values  4°C. mean  (ng  of  L D L / p g of cellular protein) ± S D i n triplicates.  40  60  80  100  Dil-LDL Concentration tug/ml)  Figure 7 C :  Dose-dependent  m e a s u r e m e n t o f internalized L D L c a l c u l a t e d as the difference between Figure 7 A a n d7B. V a l u e s e x p r e s s e d as m e a n ( n g of L D L / p g of cellular protein) ± S D i n triplicates.  40  60  80  Dil-LDL Concentration tug/ml)  59  5.1.2. LDL Specific Binding A s a t u r a t i o n c u r v e w a s g e n e r a t e d f r o m the specific b i n d i n g assay i n L L C P K i cells;  meanwhile,  both  total  s a t u r a t i o n (see F i g u r e 8). concentration  of 2 0 p g / m l .  binding  and  non-specific  did  not  reach  R e s u l t s i n d i c a t e t h a t m a x i m a l b i n d i n g w a s r e a c h e d at a T h u s the c o n c e n t r a t i o n  at h a l f - m a x i m a l b i n d i n g w a s  l O p g / m l w h i c h w a s c h o s e n as t h e i d e a l c o n c e n t r a t i o n with Dil-LDL,  binding  at  for all subsequent studies  A t a b l e o f t h e v a l u e s g e n e r a t e d f r o m t h e s p e c i f i c b i n d i n g a s s a y is  s h o w n i n T a b l e 5. T h e s e r e s u l t s a r e s i m i l a r t o t h e l i t e r a t u r e w h e r e 1 0 p g / m l o f L D L w a s t h e c o n c e n t r a t i o n f o r h a l f - m a x i m a l b i n d i n g at 3 7 ° C i n f i b r o b l a s t c e l l s [40]. F u r t h e r m o r e , a n a d d i t i o n a l s t u d y w a s c o n d u c t e d i n H e p G 2 c e l l s as a p o s i t i v e c o n t r o l for the specific b i n d i n g assay.  It is k n o w n t h a t H e p G 2 c e l l s a b u n d a n t l y  e x p r e s s L D L b i n d i n g s i t e s [21,56], w h i c h w a s binding curve  evident  d i d n o t r e a c h s a t u r a t i o n at 4 0 p g / m l  i n F i g u r e 9.  of D i l - L D L .  The At a  specific Dil-LDL  c o n c e n t r a t i o n o f 2 0 p g / m l f o r b o t h F i g u r e s 8 a n d 9, t h e a m o u n t o f t o t a l b o u n d L D L reached approximately  0.6 n g /  p r o t e i n for H e p G 2 cells.  p g of p r o t e i n for L L C - P K i cells a n d l . 6 n g / p g  T h e difference  is a p p r o x i m a t e l y  of  about a 3-fold increase  w h i c h indicates that L L C - P K i cells d o not express the s a m e n u m b e r of L D L specific b i n d i n g s i t e s as H e p G 2 c e l l s .  T h u s , L L C - P K i cells h a v e l o w L D L specific b i n d i n g  sites c o m p a r e d to H e p G 2 cells.  60  1.1 H 1.0  H  Dil-LDL Concentration (ug/ml)  -•— Non-specific binding (+LDL) * — Total Binding (-LDL) -•— Specific Binding  Figure 8: increasing  L D L Specific B i n d i n g i n L L C - P K i  cells.  Cells were incubated  [ D i l - L D L ] i n the presence a n d absence of 25-fold excess of  with  unlabelled  L D L f o r 2 h r s a t 4 ° C . V a l u e s e x p r e s s e d as n g o f L D L / p g o f c e l l u l a r p r o t e i n ± S D i n triplicates.  61  Treatment Dil-LDL  Total Binding  Non-specific  (-LDL)  Binding (+LDL)  (jig/ml) L D L ( n g / u g prot) L D L ( n g / p g prot)  Specific B i n d i n g L D L ( n g / p g prot)  0  0.04+0.02  0.01+0.07  0.03±0.05  5  0.24±0.03  0.05+0.07  0.18±0.03  10  0.44±0.05  0.06+0.01  0.38+0.06  20  0.69±0.00  0.12+0.03  0.57±0.05  40  0.92±0.20  0.42+0.09  0.50+0.10  Table 5: L D L S p e c i f i c B i n d i n g V a l u e s i n L L C - P K i c e l l s . S e e F i g u r e 8.  62  10  8H  0  10  20  30  40  50  Dil-LDL Concentration (ug/ml)  -•— Non-specific binding -•— Total binding -T— Specific binding  F i g u r e 9: L D L S p e c i f i c B i n d i n g i n H e p G 2 c e l l s . were incubated w i t h increasing  A s a p o s i t i v e c o n t r o l , H e p G 2 cells  [ D i l - L D L ] i n the presence a n d absence of 25-fold  e x c e s s o f u n l a b e l l e d L D L f o r 2 h r s a t 4 ° C . V a l u e s e x p r e s s e d as n g o f L D L / p g o f cellular protein ± S D i n triplicates.  63  5.1.3. Competitive  Binding  R e s u l t s i n F i g u r e 10 r e v e a l that u n l a b e l l e d L D L c a n d i s p l a c e l a b e l l e d L D L i n a dose-dependent manner.  This provides additional evidence  receptors that recognize L D L i n L L C P K i cells.  of the  presence of  Y e t , d u e to the s m a l l difference i n  c h a n g e of percent of m a x i m u m cell-associated L D L o v e r the r a n g e of u n l a b e l l e d LDL  ( m e a n 32% to m e a n 17%), these results further s u g g e s t a l o w a b u n d a n c e of  L D L b i n d i n g sites i n L L C P K i cells.  o 8*1  20  40  60  80  100  120  140  160  180  Unlabelled L D L fue/ml)  F i g u r e 10: C o m p e t i t i v e b i n d i n g s t u d y i n L L C - P K i c e l l s . A c o n s t a n t l O u g / m l o f D i l LDL  was  160ug/ml)  incubated for  2 hrs  i n the at  presence  37°C.  of i n c r e a s i n g  Values expressed  concentrations  as  associated L D L (treatment vs control) ± S D i n triplicates.  64  percent  o f L D L (10-  of m a x i m u m  cell-  5.1.4. Western Blot Analysis  In Figure 11, HepG2 cell membrane sample was loaded in lane 1 as a positive control and LLC-PKi cell membrane sample, the cell line of interest, was loaded in lane 2. At the same horizontal front at 120kDa in lane 1, a band was also visible in lane 2 providing stronger evidence that LLC-PKi cells do express LDL receptors. However, the band intensity was low; thus, twice the amount of protein of LLC-PKi cell membrane sample from lane 2 was added to lane 3. Band intensity was much greater reconfirming the results. Exposure time was kept constant at 15 seconds.  Overall, L L C - P K i cells express L D L receptors but also express low specific binding sites and abundance compared to HepG2 cells.  120kDa 1  2  3  Figure 11: Western Blot analysis of the LDL receptor in LLC-PKi cells. Lane 1 was loaded with HepG2 cell membrane sample; Lane 2 was loaded with LLC-PKi cell membrane sample; Lane 3 was loaded with twice the amount of protein of LLC-PKi cell membrane sample from Lane 2.  65  5.2. Effect of IgG-C7 with D i l - L D L  5.2.1.  Temperature  Dependence  T o first establish the assay w i t h the I g G - C 7 ( m A b to the L D L receptor), it w a s necessary to determine temperature dependence o n D i l - L D L b i n d i n g w i t h different concentrations of the m A b . F r o m the results i n F i g u r e 12, a d o s e - d e p e n d e n t  increase  i n p e r c e n t i n h i b i t i o n w a s o b s e r v e d at 4 ° C ; i n contrast, a d o s e - d e p e n d e n t  decrease  w a s o b s e r v e d at 3 7 ° C .  It i s p o s s i b l e t h a t t h e o b s e r v a t i o n a t 3 7 ° C w a s d u e t o b o t h  I g G - C 7 a n d L D L m e t a b o l i s m . I n the literature, I g G - C 7 has a h i g h e r b i n d i n g affinity at 4 ° C s i n c e h a l f - m a x i m a l b i n d i n g i s a t a c o n c e n t r a t i o n o f I n M v s . 7 5 n M a t 3 7 ° C i n f i b r o b l a s t c e l l s [11]. A s p r e d i c t e d , t h e o p t i m a l t e m p e r a t u r e f o r I g G - C 7 t r e a t m e n t i n L L C - P K i cells is at 4 ° C . T h u s , w e e s t a b l i s h e d that a l l s t u d i e s w i t h I g G - C 7 t r e a t m e n t w o u l d b e c o n d u c t e d at 4 ° C .  66  100  -80 -I 0.0  ,  ,  ,  ,  1  0.5  1.0  1.5  2.0  2.5  mAb concentration (ug/ml) - • - 4°C - • - 37°C  F i g u r e 12: Temperature dependence of D i l - L D L binding with various concentrations of m A b to the L D L receptor. After L L C - P K i cells h a v e been g r o w n for 24 hrs i n s e r u m free-media, v a r i o u s concentrations of I g G - C 7 ( m A b ) at 0.1, 0.5, 1.0, 2.0 p g / m l w e r e p r e i n c u b a t e d for 2 hrs at 4 ° C . S u b s e q u e n t l y , 10 p g / m l of D i l L D L w a s further i n c u b a t e d at 4 ° C a n d 3 7 ° C for a n a d d i t i o n a l 2 hrs. Values expressed as m e a n percent i n h i b i t i o n v e r s u s non-treatment c o n t r o l ± S D i n triplicates.  67  5.2.2. Optimal Preincubation  Period  Beisiegel et al (1981) reported that IgG-C7 reached steady state within 1 hr and subsequently remained at equilibrium up to 6hrs in fibroblast cells [11]. Therefore, a study to investigate an optimal preincubation period was needed to determine percent inhibition of 2.0pg/ml mAb with 10 pg/ml of Dil-LDL. An mAb concentration of 2.0pg/ml was selected to conduct these studies based on the results in Figure 12 where observable inhibition at 20% ±10% was found at a similar concentration.  Preliminary studies with a preincubation period of 1, 2 and 4 hours were conducted and it was decided that a 2 hour window was needed to determine the optimal time period.  Subsequently, 2.0pg/ml mAb was preincubated for 30  minutes, 1 hour and 1.5 hours, and then followed by 10 pg/ml of Dil-LDL treatment.  Results in Figure 13 reveal that a preincubation of 1.5 hours was the  optimal time period with a 20% ± 25% inhibition in Dil-LDL binding. Even with such a high standard deviation, the results were sufficient enough to conclude that the optimal preincubation period for mAb treatment in LLC-PKi cells was at 1.5 hours.  68  50  o  J3  C o u  40  -t-»  C  01 (8 QJ  H  30  I  C  o c (fl 3  tfl  20  >  c o  •J3  '£ '£  c  10  H  M  0.0  0.5  1.0  1.5  2.0  Preincubation period (hours')  Figure 13: Time-course incubation with mAb from 0.5hr to 1.5hrs. LLC-PKi cells were preincubated at different time periods of 0.5, 1.0, and 1.5 hrs with 2.0ug/ml mAb at 4°C prior to addition of lOug/ml Dil-LDL for 2 hrs at 4°C. Values expressed as percent inhibition vs non-treatment control ± SD in triplicates.  69  5.2.3.  Optimal  Concentration  O n c e the p r e i n c u b a t i o n p e r i o d w a s d e t e r m i n e d , a n o p t i m a l c o n c e n t r a t i o n of mAb  treatment w a s  d e c i d e d based  o n the c r i t e r i o n that a m i n i m u m of 80%  i n h i b i t i o n m u s t be o b s e r v e d i n D i l - L D L b i n d i n g . T h i s c r i t e r i o n w a s necessary i n o r d e r to see a n y differences u p o n C s A treatment w h e n I g G - C 7 w a s present. L L C P K i cells w e r e p r e i n c u b a t e d w i t h v a r i o u s concentrations of the m A b at 0.1, 0.5, 1.0, 2.0,4.0, 8.0 a n d 1 2 . 0 p g / m l at 4 ° C for 1.5 h o u r s p r i o r to the a d d i t i o n of l O . O p g / m l for an addition 2 hours  at 4 ° C .  Results i n F i g u r e 14 i n d i c a t e that the  optimal  c o n c e n t r a t i o n of m A b w a s at 1 2 . 0 p g / m l w h e r e a 90% ± 6 % ( m e a n ± S E M ) i n h i b i t i o n i n D i l - L D L b i n d i n g was observed.  Results w e r e repeatable w i t h three different  subcultures i n triplicates. Therefore, the o p t i m a l c o n c e n t r a t i o n to preincubate w i t h the I g G - C 7 i n L L C - P K i cells w a s at 1 2 . 0 p g / m l based o n p r o t e i n content.  Overall, the optimal conditions for IgG-C7 treatment i n LLC-PKi cells are a preincubation period of 1.5 hours at 4°C with a concentration of 12.0pg/ml.  70  Figure  14:  Percent  inhibition  of D i l - L D L  concentrations of m A b to the L D L receptor.  binding  i n the  presence  of v a r i o u s  L L C - P K i cells w e r e p r e i n c u b a t e d for a  p e r i o d o f 1.5 h o u r s w i t h v a r i o u s c o n c e n t r a t i o n s o f m A b o f 0 . 1 , 0 . 5 , 1 . 0 , 2.0, 4 . 0 , 8.0 and  12.0 p g / m l a t 4 ° C p r i o r t o t h e a d d i t i o n o f l O p g / m l o f D i l - L D L f o r 2 h r s at 4 ° C .  V a l u e s e x p r e s s e d as p e r c e n t i n h i b i t i o n v e r s u s n o n - t r e a t m e n t c o n t r o l ± S E M i n n=3 i n triplicates.  71  5.3. PHI C s A t r e a t m e n t  5.3.3.  PHlCsA  time-course incubation study  P e t e r h e r y c h et al (2001) h a d established that a 24 h o u r i n c u b a t i o n at 37°C w i t h [ H ] C s A w a s sufficient t i m e i n o r d e r to observe u p t a k e of the d r u g i n t o L L C - P K i 3  cells [91].  H o w e v e r , the stability of I g G - C 7 at 3 7 ° C w a s a c o n c e r n d u e to the  p o s s i b i l i t y of its h i g h m e t a b o l i s m . I g G - C 7 acts as a c o m p e t i t i v e i n h i b i t o r w i t h L D L for the L D L receptor w i t h h i g h e r affinity at 4 degrees [11]. T h u s , it u n d e r g o e s the same e n d o c y t i c processes as a n L D L particle w h e r e it becomes r e c o g n i z e d b y the L D L receptor, i n t e r n a l i z e d into vesicles a n d d e g r a d e d i n t o its c o m p o n e n t s [11].  Beisiegel et al (1981) h a d c o n d u c t e d a s t u d y to investigate the m e t a b o l i s m of r a d i o l a b e l l e d I g G - C 7 i n m o n o l a y e r h u m a n fibroblasts cells [11]. T h e y r e p o r t e d that the c e l l u l a r content of  1 2 5  I g G - C 7 reached a steady state w i t h i n a n h o u r a n d r e m a i n e d  at e q u i l i b r i u m u p to 6 h o u r s (see F i g u r e 15) [11]. o b s e r v e d a p p r o x i m a t e l y 4 times as m u c h  1 2 5  W i t h i n the 6 h o u r s , they also  I g G - C 7 h a d b e e n d e g r a d e d as w a s  present i n the cells at steady state at 3 7 ° C (see F i g u r e 15) [11].  It is necessary to  c o n d u c t a l l [ H ] C s A u p t a k e experiments at 3 7 ° C i n o r d e r to s t u d y L D L receptor 3  activity.  Therefore,  a  [ H ] C s A time-course 3  incubation study  was  needed  to  determine that o p t i m a l time p e r i o d for [ H ] C s A u p t a k e t a k i n g i n t o c o n s i d e r a t i o n the 3  h i g h m e t a b o l i s m of I g G - C 7 .  72  I n T a b l e 6, [ H ] C s A u p t a k e o n l y reached a m a x i m u m of 8.9% ± 0.4% (mean ± 3  S E M ) w i t h i n 10 h o u r s at 4 ° C . M e a n w h i l e [ H ] C s A u p t a k e r e a c h e d a m a x i m u m of 3  21.2% ± 1.2% w i t h i n 6 h o u r s at 37 °C. After 6 h o u r s , there w a s n o change i n [ H ] C s A 3  u p t a k e at 37 ° C ; rather, u p t a k e decreased after a n i n c u b a t i o n p e r i o d of 10 h o u r s (19.2% ± 1.6%)  a n d a greater  decrease after 24 h o u r s (13.3% ± 2.0%).  This  c o r r e s p o n d e d to the literature w h e r e I g G - C 7 r e m a i n e d i n e q u i l i b r i u m u p to 6 h o u r s at 37°C.  Therefore, it w a s d e c i d e d that the o p t i m a l t i m e p e r i o d for [ H ] C s A 3  treatment w a s 6 h o u r s .  T h i s c o n d i t i o n w a s a p p l i e d to the u p t a k e a n d toxicity  studies w i t h [ H ] C s A (see section 5.4). 3  % Uptake of P H I C s A Hours  37°C  4°C  2  15.49±1.33  1.78±0.34  6  21.24±1.20  1.80±0.40  10  19.15±1.57  8.88±0.39  24  13.28±1.95  6.37+1.00  Table 6: Percent u p t a k e of [ H ] C s A o v e r 24 h o u r p e r i o d at 3 7 ° C v s 4 ° C . A t i m e course i n c u b a t i o n s t u d y w i t h 8 0 0 n g / m l of [ H ] C s A w a s c o n d u c t e d at b o t h 3 7 ° C a n d 4 ° C i n L L C - P K i cells w h e r e i n t r a c e l l u l a r [ H ] C s A w a s m e a s u r e d o v e r a p e r i o d of 2, 6, 10 a n d 24 h o u r s . Percent u p t a k e w a s c a l c u l a t e d b y c o m p a r i n g m e a n counts per m i n u t e ( C P M ) / p g of c e l l u l a r p r o t e i n to total [ H ] C s A c o n t r o l . V a l u e s expressed as percent u p t a k e ± S E M i n n=3 i n triplicates. 3  3  3  3  73  0  2  4  6  Time (hoars)  Figure 15: T i m e - c o u r s e i n c u b a t i o n o f m e t a b o l i s m o f  1 2 5  I g G - C 7 at 3 7 ° C i n m o n o l a y e r  h u m a n f i b r o b l a s t c e l l s . R e f e r e n c e d f r o m B e i s i e g e l et al (1981) J B C 2 5 6 : 1 1 9 2 3 - 3 1 [11]. Copyright  permission  granted  from  American  Molecular Biology ©.  74  Society  of  Biochemistry  and  5.4  5.4.1.  Effect o f IgG-C7 o n P H I C s A U p t a k e a n d T o x i c i t y  f H]CsA  uptake - bound and intracellular  3  T o m e a s u r e m e m b r a n e - b o u n d [ H ] C s A , 0.001% T r i t o n X - 1 0 0 w a s h i n g s w e r e 3  transferred  to s c i n t i l l a t i o n v i a l s a n d r a d i o a c t i v i t y w a s m e a s u r e d b y  counting.  T r e a t m e n t g r o u p s i n c l u d e d the f o l l o w i n g :  scintillation  A ) 800 n g / m l of [ H ] C s A 3  alone; B ) 800 n g / m l of [ H ] C s A c o m p l e x e d w i t h 20 p g / m l L D L c h o l e s t e r o l ; a n d C ) 3  8 0 0 n g / m l o f [ H ] C s A c o a d d e d w i t h 2 0 p g / m l c h o l e s t e r o l o f L D L (see s e c t i o n 4.5.1). 3  V a l u e s w e r e e x p r e s s e d as c o u n t s p e r m i n u t e ( C P M ) / p g  of cellular p r o t e i n a n d  c o m p a r e d to C P M of [ H ] C s A c o n t r o l / p g of cellular p r o t e i n to calculate percent 3  b o u n d (see s e c t i o n 4.5.2). I n F i g u r e 16, e a c h t r e a t m e n t  g r o u p w a s p r e i n c u b a t e d i n the presence  and  a b s e n c e o f 1 2 . 0 p g / m l o f m A b f o r 1.5 h o u r s at 4 ° C p r i o r t o [ H ] C s A t r e a t m e n t f o r 6 3  h o u r s at 3 7 ° C .  A significant difference w a s o b s e r v e d i n the [ H ] C s A alone g r o u p i n 3  t h e p r e s e n c e o f m A b (2.6% ± 0.6%) v e r s u s i t s a b s e n c e ( 5 . 1 % ± 1.3%) (p<0.05  with  u n p a i r e d t-test). M e a n w h i l e , n o s i g n i f i c a n t r e s u l t s w e r e o b s e r v e d i n e i t h e r [ H ] C s A 3  L D L c o m p l e x a n d [ H ] C s A w i t h L D L coaddition groups. In addition, n o significant 3  differences w e r e o b s e r v e d o n m e m b r a n e - b o u n d [ H ] C s A i n the presence of L D L , 3  e i t h e r c o m p l e x e d w i t h [ H ] C s A o r c o a d d e d w i t h [ H ] C s A (p<0.05 w i t h 3  These results  suggest  3  that I g G - C 7 c o u l d  significantly  reduce  membrane-bound  [ H ] C s A i n L L C - P K i c e l l s ; y e t , w h e n L D L w a s p r e s e n t , t h e r e w a s n o effect. 3  75  ANOVA).  T o o b s e r v e t h e effect o f I g G - C 7 o n u p t a k e o f [ H ] C s A , l y s e d c e l l s w i t h i n t h e 3  same  experiment  were  transferred  to scintillation  vials  a n d radioactivity was  m e a s u r e d t o d e t e r m i n e i n t r a c e l l u l a r a m o u n t s o f [ H ] C s A . V a l u e s w e r e e x p r e s s e d as 3  C P M / p g of cellular protein a n dc o m p a r e d to C P M of [ H ] C s A c o n t r o l / p g of cellular 3  p r o t e i n t o c a l c u l a t e p e r c e n t u p t a k e (see s e c t i o n 4.5.2).  I n F i g u r e 17, n o significant  differences w e r e o b s e r v e d i n the presence o f m A b w i t h i n each treatment g r o u p . A l s o , L D L h a d n o s i g n i f i c a n t effect o n u p t a k e o f [ H ] C s A . O v e r a l l , b o t h I g G - C 7 a n d 3  L D L h a dn o significant reduction o n [ H ] C s A uptake. 3  These  results  reveal  that  IgG-C7  affected  [ H]CsA 3  b i n d i n g on  the  membrane level; yet no effect was observed on [ H ] C s A uptake intracellularly. In 3  the presence of L D L as a drug complex or as a drug coaddition treatment, there was no effect on both percent [3H]CsA b o u n d and uptake.  76  7  6H  CsA alone  CsA-LDL  CsA + L D L  Treatment G r o u p s Absence of mAb Presence of mAb  Figure 16: M e a n percent b o u n d of [ H ] C s A i n L D L w i t h I g G - C 7 . A ) C s A alone indicates o n l y [ H ] C s A , B) C s A - L D L indicates [ H ] C s A c o m p l e x e d w i t h L D L , a n d C) C s A + L D L indicates [ H ] C s A c o a d d e d w i t h L D L . L L C - P K i cells w e r e p r e i n c u b a t e d w i t h 12.0 p g / m l for 1.5 hrs at 4 ° C p r i o r to treatment w i t h 800 n g / m l of [ H ] C s A w i t h a n d w i t h o u t L D L for a n a d d i t i o n a l 6 h o u r s at 37°C. M e m b r a n e - b o u n d [ H ] C s A w a s m e a s u r e d f r o m 0.001% T r i t o n X-100 w a s h i n g a n d c o m p a r e d to total [ H ] C s A c o n t r o l . V a l u e s expressed as m e a n percent b o u n d ± S E M i n n=6 i n triplicates. 3  3  3  3  3  3  3  denotes significance (p<0.05) c o m p a r e d to c o n t r o l w i t h u n p a i r e d t-test  77  CsA alone  CsA-LDL  CsA + LDL  Treatment Groups  Absence of mAb Presence of mAb  Figure 17:  A) C s A alone  M e a n percent u p t a k e of [ H ] C s A i n L D L w i t h I g G - C 7 . 3  i n d i c a t e s o n l y [ H ] C s A , B) C s A - L D L i n d i c a t e s [ H ] C s A c o m p l e x e d 3  C)  CsA  +  LDL  with L D L , and  3  indicates  [ H]CsA 3  coadded  with  LDL.  LLC-PKi  cells  were  p r e i n c u b a t e d w i t h 12.0 p g / m l f o r 1.5 h r s at 4 ° C p r i o r t o t r e a t m e n t w i t h 8 0 0 n g / m l o f [ H ] C s A w i t h a n d w i t h o u t L D L for a n a d d i t i o n a l 3  [ H]CsA was 3  6 h o u r s at 3 7 ° C .  Intracellular  m e a s u r e d f r o m l y s e d cells a n d c o m p a r e d to total [ H ] C s A control. 3  V a l u e s e x p r e s s e d as m e a n p e r c e n t u p t a k e ± S E M i n n = 6 i n t r i p l i c a t e s . d e n o t e s s i g n i f i c a n c e (p<0.05) c o m p a r e d t o c o n t r o l w i t h u n p a i r e d t-test  78  5.4.2.  [ H]CsA  toxicity - LDH measurement  3  Within the same assay method as [ H]CsA bound and uptake, [ H]CsA 3  3  toxicity was determined by measuring L D H activity within the culture medium. After preincubation with and without 12.0pg/ml of IgG-C7 for 1.5 hours at 4°C, three treatment groups were added to LLC-PKi cells: A) 800ng/ml of [ H]CsA 3  alone; B) 800ng/ml of [ H]CsA complexed with 20pg/ml LDL cholesterol; and C) 3  800ng/ml of [ H]CsA coadded with 20pg/ml cholesterol of LDL for a period of 6 3  hours at 37°C (see section 4.5.1). The culture media was removed and analyzed for L D H activity.  Values were expressed as absorbance at 492nm / m g of cellular  protein adjusted against control groups and compared to absorbance at 492nm/ mg of cellular protein of the L D H positive control to calculate percent toxicity (see section 4.5.2). In Figure 18, a significant difference was observed in the [ H]CsA alone 3  group in the presence of mAb (1.8% + 0.5%) versus its absence (3.2% + 1.5%) (p<0.05 with unpaired t-test). However, no significant results were revealed in the [ H]CsA3  LDL complex and [ H]CsA with LDL coaddition groups. In addition, there was no 3  significant effect of LDL on [ H]CsA toxicity either as a drug complex or as a drug 3  coaddition treatment (p<0.05 with one-way ANOVA).  The results suggest that IgG-C7 could significantly reduce [ H]CsA toxicity 3  in LLC-PKi cells; yet, when LDL was present, there was no effect.  79  O v e r a l l , the results are consistent w i t h the results r e p o r t e d i n section  5.4.1  w h e r e , a significant difference w a s o b s e r v e d i n m e m b r a n e - b o u n d [ H ] C s A alone 3  g r o u p i n the p r e s e n c e of IgG-C7 v e r s u s its absence. [ H ] C s A b o u n d and uptake. 3  80  H o w e v e r , L D L h a d n o affect o n  CsA  CsA-LDL  CsA + L D L  Treatment Groups  82888 Absence of mAb I I Presence of mAb  F i g u r e 18:  M e a n percent toxicity of [ H ] C s A i n L D L w i t h I g G - C 7 . 3  indicates only [ H ] C s A , B) C s A - L D L indicates [ H ] C s A c o m p l e x e d 3  C)  CsA  +  3  L D L indicates  [ H]CsA 3  coadded  with  LDL.  A ) C s A alone with L D L , and  LLC-PKi  cells  were  p r e i n c u b a t e d w i t h 1 2 . 0 u g / m l f o r 1.5 h r s at 4 ° C p r i o r t o t r e a t m e n t w i t h 8 0 0 n g / m l o f [ H]CsA with and 3  w i t h o u t L D L for a n a d d i t i o n a l  6 h o u r s at 3 7 ° C .  Media  was  r e m o v e d a n d a n a l y z e d f o r L D H a c t i v i t y a n d c o m p a r e d t o L D H a c t i v i t y i n 1% T r i t o n X-100  t r e a t e d c e l l s as 1 0 0 % t o x i c i t y .  V a l u e s e x p r e s s e d as m e a n p e r c e n t t o x i c i t y ±  S E M i n n=6 i n triplicates. * d e n o t e s s i g n i f i c a n c e (p<0.05) c o m p a r e d t o c o n t r o l w i t h u n p a i r e d t-test  81  CHAPTER VI:  ^Discussion  82  6.1.  Dil-LDL  versus  I-LDL  U5  P r e v i o u s w o r k i n v o l v i n g L D L receptors ligand.  have  used  1 2 5  I - L D L as a  labeled  It h a s h i g h s e n s i t i v i t y a n d s t a b i l i t y , a n d c a n b e e a s i l y q u a n t i f i e d  studying receptor-mediated  when  L D L m e t a b o l i s m since first i n t r o d u c e d b y G o l d s t e i n  a n d B r o w n [40,41]. H o w e v e r , i t i s l i m i t e d b y i t s b i o h a z a r d p o t e n t i a l w i t h a h i g h c o s t in  waste  management.  An  carbocyanine L D L , D i l - L D L .  alternative  This  fluorescent  to  1 2 5  I-LDL  is  3,3'-dioctadecylindo-  probe has been extensively used i n  m o r p h o l o g i c a l a n d m i c r o s c o p i c studies of receptor-mediated m e t a b o l i s m of L D L i n m a n y c e l l l i n e s [99,102,103,120]. It i s e a s i l y i n c o r p o r a t e d i n t o l i p o p r o t e i n s , d o e s n o t r e a d i l y transfer to cell m e m b r a n e s  or other unlabelled lipoproteins, a n d does not  affect r e c e p t o r b i n d i n g a c t i v i t y [57]. can  D i l - L D L combined w i t h confocal microscopy  be u s e d to m o n i t o r the processes  of receptor-mediated  b i n d i n g a n d i n t e r n a l i z a t i o n b u t n o t d e g r a d a t i o n [99,120].  endocytosis  through  Therefore, D i l - L D L  c h o s e n as a l a b e l e d l i g a n d t o s t u d y L D L r e c e p t o r a c t i v i t y i n L L C - P K i c e l l s . investigators h a v e u s e d this same  fluorescent  was  Other  p r o b e to s t u d y L D L receptor activity  i n g l o m e r u l a r c e l l s [99], H e p G 2 c e l l s [120] a n d P M N s [98,111]. T o d a t e , h o w e v e r , n o one has investigated L D L receptor activity i n p r o x i m a l t u b u l e cells. In the literature, there w a s conflicting i n f o r m a t i o n o n the a p p l i c a t i o n of D i l LDL.  Excitation  and  emission  were  reported  at  two  different  wavelengths:  5 2 2 / 5 7 8 n m [111,120] v e r s u s 5 5 5 / 5 7 8 n m [78]. T o c l a r i f y t h i s i s s u e , a s t a n d a r d c u r v e f r o m the r a n g e 2 5 n g / m l to 800 n g / m l w a s c o n s t r u c t e d a n d the s e n s i t i v i t y of the a s s a y w a s a s s e s s e d (see s e c t i o n 4.1.2 f o r p r o c e d u r e ) .  83  I n F i g u r e 19, s e n s i t i v i t y of the  Dil-LDL  assay  was  increased  when  the  probe  was  excited  and  emitted  at  w a v e l e n g t h s 5 2 2 n m a n d 57811111, r e s p e c t i v e l y . T h e r e f o r e , i t w a s d e t e r m i n e d t h a t D i l L D L w o u l d b e q u a n t i f i e d at 5 2 2 / 5 7 8 n m .  3000  2500 A  1000  D i l - L D L Concentration (ng/mP —•— 522/578 nm - O — 555/571 nm  F i g u r e 1 9 : S e n s i t i v i t y o f D i l - L D L A s s a y at 5 2 2 / 5 7 n m v e r s u s 5 5 5 / 5 7 1 n m .  84  6.2.  Rationale of LLC-PKi One  as an appropriate cell model  of the m a i n objectives w a s to investigate C s A n e p h r o t o x i c i t y . T h u s , the  selection of L L C P K i a p p e a r e d to be a suitable cell m o d e l since it d i s p l a y e d s i m i l a r c h a r a c t e r i s t i c s as p r o x i m a l t u b u l e c e l l s i n c l u d i n g m o r p h o l o g y a n d f u n c t i o n [54,89]. I n a d d i t i o n , i t i s a w e l l e s t a b l i s h e d in vitro k i d n e y m o d e l w h i c h h a s b e e n u s e d t o investigate  CsA-induced  c y t o t o x i c i t y [10,20,47,71].  However, cultured  human  f i b r o b l a s t c e l l s h a v e b e e n u s e d as c e l l m o d e l s i n t h e e a r l i e r s t u d i e s o n t h e L D L r e c e p t o r [15,40,41].  S i n c e then, a n u m b e r of other cell m o d e l s h a v e b e e n u s e d to  investigate  L D L receptor  [21,56,100],  lymphocytes  activity w h i c h [97],  include h u m a n  monocytes  [111],  and  hepatocytes  (HepG2)  macrophages  [61].  Nevertheless, k i d n e y cells also expressed receptors that recognize L D L , especially p r o x i m a l t u b u l a r c e l l s [17,88,132,145]. P e g o r a r o et al (2002) o b s e r v e d t h a t t h e HRP the  LDL-  specific s t a i n i n g m a i n l y l o c a l i z e d i n the p r o x i m a l t u b u l e s a n d w a s r e d u c e d i n presence  determined  of excess that the  u n l a b e l l e d L D L [88].  K d value  of  1 2 5  I-LDL  I n a d d i t i o n , W a s a n et al  in LLCPKi  c e l l s i s 0.0538  i n d i c a t i n g h i g h - a f f i n i t y b i n d i n g w i t h 96 0 0 0 b i n d i n g s i t e s / c e l l [132].  (1994)  (ng/ml)  D e s p i t e these  findings, n o one has c o m p l e t e d any studies o n L D L receptor activity i n L L C P K i cells; t h u s it w a s necessary to c o n f i r m the presence of these receptors i n this cell line a n d its a p p r o p r i a t e n e s s i n the s t u d y of L D L r e c e p t o r a c t i v i t y . Figures 7 A , 7B a n d 7 C revealed dose-response  studies  of cell-associated,  membrane-bound and internalized L D L , respectively.  T h e s e results p r o v i d e d first  e v i d e n c e of the presence of receptors w h i c h b i n d L D L .  H o w e v e r , these results also  85  indicated b o t h specific a n d non-specific b i n d i n g of L D L .  Thus, further experiments  to investigate L D L specific b i n d i n g w e r e n e e d e d a n d results i n F i g u r e 8 c o n f i r m e d t h a t m a x i m a l b i n d i n g w a s r e a c h e d at a D i l - L D L c o n c e n t r a t i o n o f 2 0 p g / m l w i t h about 0.57±0.05 n g of L D L / p g of cellular p r o t e i n specifically b o u n d to the receptor of interest. control  (see  A c o m p a r i s o n t o t h e s p e c i f i c b i n d i n g c u r v e i n H e p G 2 as a p o s i t i v e Figure  9) r e v e a l e d t h a t  LLC-PKi  cells  do  not  a b u n d a n c e o f L D L b i n d i n g s i t e s as H e p G 2 a n d t h a t L L C - P K i n u m b e r o f L D L specific b i n d i n g sites.  express  the  same  cells h a v e a l o w  A competitive study where unlabelled L D L  w a s able to d i s p l a c e D i l - L D L p r o v i d e d m o r e e v i d e n c e of the presence of L D L r e c e p t o r s b u t a l s o c o n f i r m e d t h e l o w a b u n d a n c e o f L D L b i n d i n g s i t e s (see F i g u r e 10).  W e s t e r n b l o t a n a l y s i s o f c e l l m e m b r a n e e x t r a c t s o f L L C - P K i u s i n g I g G - C 7 as t h e  p r i m a r y a n t i b o d y also r e v e a l e d the presence of L D L receptors i n the cell l i n e of i n t e r e s t (see F i g u r e 11).  T h e r e is n o d o u b t that L L C - P K i  cells d o express L D L  receptors or specifically, receptors that b i n d to L D L s u c h as the m e m b e r s of the L D L receptor family.  D e s p i t e the presence of L D L receptors, L L C - P K i cells express a l o w  a b u n d a n c e o f L D L specific b i n d i n g sites c o m p a r e d to H e p G 2 cells.  Megalin  has  b e e n r e p o r t e d t o b e h i g h l y e x p r e s s e d i n t h e p r o x i m a l t u b u l e o f t h e k i d n e y [19,125] and  L L C - P K i c e l l s e x p r e s s m e g a l i n [83], b u t w h e t h e r L L C - P K i c e l l s h i g h l y e x p r e s s  m e g a l i n is u n c e r t a i n .  N e v e r t h e l e s s , it is c o n c l u s i v e that L L C - P K i cells m a y n o t be  the m o s t i d e a l cell line to s t u d y L D L receptor activity b u t it is still a n a p p r o p r i a t e cell m o d e l to investigate the role of the L D L receptor o n C s A u p t a k e a n d toxicity b e c a u s e t h e s i t e o f C s A - i n d u c e d n e p h r o t o x i c i t y o c c u r s i n t h e p r o x i m a l t u b u l e [104].  86  6.3.  Use ofIgG-C7, a monoclonal antibody to LDL receptor I g G - C 7 , a m o n o c l o n a l a n t i b o d y a g a i n s t the h u m a n L D L r e c e p t o r , is receptor-  b o u n d , i n t e r n a l i z e d a n d d e g r a d e d i n l y s o s o m e s i n a s i m i l a r f a s h i o n t o L D L [11]. T h u s , it acts as a c o m p e t i t i v e i n h i b i t o r to the L D L r e c e p t o r w i t h s l i g h t l y h i g h e r a f f i n i t y t h a n L D L [11]. H o w e v e r , t h e b i n d i n g s i t e o f I g G - C 7 t o t h e L D L r e c e p t o r i s different  t h a n t h e L D L b i n d i n g s i t e [123].  I g G - C 7 is d i r e c t e d a g a i n s t the  first  c y s t e i n e - r i c h r e p e a t o f t h e f i r s t d o m a i n o f t h e L D L r e c e p t o r [123]. B y c o n t r a s t , L D L binding  requires  cysteine  repeats  3-7  in  the  ligand  binding  domain  [105].  F u r t h e r m o r e , interspecies differences b e t w e e n the h u m a n L D L receptor a n d p i g LDL  receptor m a y restrict the use of I g G - C 7 b u t a p r o t e i n B L A S T analysis has  c o n f i r m e d that the h u m a n a n d p i g L D L receptors h a v e a b o u t a n 87%  sequence  h o m o l o g y o f its 818 a m i n o a c i d r e s i d u e s . The  strategy that w a s i m p l e m e n t e d i n o r d e r to m o d i f y L D L receptor activity  i n L L C - P K i cells w a s the use of I g G - C 7 .  T h e r a t i o n a l e b e h i n d t h i s strategy w a s to  i n i t i a l l y i n h i b i t L D L r e c e p t o r a c t i v i t y a n d t o o b s e r v e t h e effect o f I g G - C 7 o n C s A uptake  and  toxicity.  I g G - C 7 w a s first p r e i n c u b a t e d w i t h  the  cells, the  media  r e m o v e d , a n d t h e n C s A treatment w a s a d d e d i n o r d e r to o m i t the p o s s i b i l i t y of I g G C 7 n o n - s p e c i f i c a l l y b i n d i n g to C s A . N e v e r t h e l e s s , it w a s i m p o r t a n t to establish the assay c o n d i t i o n s w i t h I g G - C 7 i n L L C - P K i cells w h i c h i n c l u d e d temperature, o p t i m a l preincubation time a n d concentration.  I n F i g u r e 12, t e m p e r a t u r e  dependence  of  I g G - C 7 w a s a s s e s s e d a n d r e s u l t s r e v e a l t h a t at 4 ° C , t h e r e w a s c o n s i s t e n t i n h i b i t i o n i n D i l - L D L b i n d i n g . Results p a r a l l e l e d w h a t w a s r e p o r t e d i n the literature w h e r e I g G -  87  C 7 h a d a h i g h e r b i n d i n g affinity at 4 ° C w i t h h a l f - m a x i m a l b i n d i n g at a c o n c e n t r a t i o n o f I n M v s . 7 5 n M at 3 7 ° C i n f i b r o b l a s t c e l l s [11]. S u b s e q u e n t l y , i n F i g u r e 1 3 , o p t i m a l p r e i n c u b a t i o n t i m e w a s d e t e r m i n e d a n d r e s u l t s r e v e a l t h a t a p r e i n c u b a t i o n o f 1.5 h o u r s y i e l d e d the greatest percent i n h i b i t i o n i n D i l - L D L b i n d i n g w i t h 20% ± 25%. B a s e d o n t h e c o n d i t i o n s o f a p r e i n c u b a t i o n p e r i o d o f 1.5 h o u r s a t 4 ° C , t h e o p t i m a l concentration was needed.  I n F i g u r e 14, v a r i o u s c o n c e n t r a t i o n s  of m A b  were  p r e i n c u b a t e d to d e t e r m i n e the m a x i m u m percent i n h i b i t i o n i n D i l - L D L b i n d i n g .  It  w a s f o u n d t h a t a m A b c o n c e n t r a t i o n o f 12.0 p g / m l r e s u l t e d i n a y i e l d o f a b o u t 9 0 % + 6% ( m e a n ± S E M ) w h i c h m e t t h e c r i t e r i a t h a t a n 8 0 % i n h i b i t i o n i n D i l - L D L b i n d i n g m u s t be o b s e r v e d . O v e r a l l , it w a s c o n c l u d e d that the o p t i m a l c o n d i t i o n s for I g G - C 7 t r e a t m e n t i n L L C - P K i c e l l s w a s a p r e i n c u b a t i o n p e r i o d o f 1.5 h o u r s at 4 ° C w i t h 12.0 p g / m l concentration.  T h e s e assay c o n d i t i o n s w e r e i m p l e m e n t e d t o i n v e s t i g a t e the  effect o f I g G - C 7 o n C s A u p t a k e a n d t o x i c i t y . Another  concern  with  IgG-C7  was  its  s t a b i l i t y at  37°C  upon  [ H]CsA 3  t r e a t m e n t . P r i o r c o n d i t i o n s o f t h e m A b w e r e at 4 ° C b u t i t w a s n e c e s s a r y t o c o n d u c t t h e a s s a y at 3 7 ° C t o o b s e r v e t h e effect o f L D L r e c e p t o r a c t i v i t y o n [ H ] C s A u p t a k e 3  and  toxicity. H o w e v e r , it w a s also c r u c i a l to a l l o w e n o u g h t i m e for m a x i m u m C s A  u p t a k e at 3 7 ° C . P e t e r h e r y c h et al (2001) h a d e s t a b l i s h e d t h a t a 2 4 h o u r i n c u b a t i o n at 3 7 ° C w i t h [ H ] C s A w a s sufficient t i m e i n o r d e r to observe u p t a k e of the d r u g into 3  L L C - P K i c e l l s [91]. Y e t , w i t h i n t h e s a m e p e r i o d o f t i m e , i t w a s e x p e c t e d t h a t I g G - C 7 w o u l d u n d e r g o d e g r a d a t i o n as i n d i c a t e d i n the l i t e r a t u r e . r e p o r t e d that the cellular content of  1 2 5  B i e s i e g e l et al (1981)  I g G - C 7 r e a c h e d a steady state w i t h i n a n h o u r  88  a n d r e m a i n e d at e q u i l i b r i u m u p t o 6 h o u r s (see F i g u r e 15) [11]. W i t h i n t h e 6 h o u r s , t h e y a l s o o b s e r v e d a p p r o x i m a t e l y 4 t i m e s as m u c h  1 2 5  I g G - C 7 h a d b e e n d e g r a d e d as  w a s p r e s e n t i n t h e c e l l s at s t e a d y s t a t e at 3 7 ° C (see F i g u r e 15) [11].  Therefore, a  s t u d y w a s c o n d u c t e d to d e t e r m i n e the shortest p e r i o d of t i m e n e e d e d to observe m a x i m u m u p t a k e o f [ H ] C s A at 3 7 ° C .  I n T a b l e 6, r e s u l t s r e v e a l e d t h a t o p t i m a l  3  u p t a k e o f [ H ] C s A o c c u r r e d at 3 7 ° C w i t h i n 6 h o u r s . 3  These results w e r e consistent  w i t h t h e l i t e r a t u r e a n d w a s t h u s c h o s e n as t h e t i m e p e r i o d f o r [ H ] C s A t r e a t m e n t t o 3  a v o i d further degradation of I g G - C 7 .  6.4.  Interpretation of results in [ H]CsA  uptake and toxicity assay  3  O n c e the c o n d i t i o n s w e r e o p t i m i z e d , the d e v e l o p e d assay m e t h o d s w e r e u s e d t o i n v e s t i g a t e t h e effect o f I g G - C 7 o n [ H ] C s A u p t a k e a n d t o x i c i t y . It w a s n e c e s s a r y 3  to a p p l y b o t h u p t a k e a n d t o x i c i t y assays i n o n e p r o c e d u r e as a m o d i f i c a t i o n to the m e t h o d s d e v e l o p e d b y P e t e r h e r y c h et al (2001) [91]. toxicity marker was chosen.  Lactate dehydrogenase  I n o r d e r to d o this, another ( L D H ) is a cytosolic e n z y m e  t h a t i s r e l e a s e d u p o n c e l l l y s i s a s a r e s u l t o f d a m a g e t o t h e p l a s m a m e m b r a n e [4,96]. A l t h o u g h a m a r k e r of late toxicity, L D H release is detected i n the c u l t u r e m e d i u m a n d p r o v i d e s a n a c c u r a t e m e a s u r e o f c e l l v i a b i l i t y [4]. Significant  results  were  revealed  in mean  percent  of  membrane-bound  [ H ] C s A i n t h e p r e s e n c e o f I g G - C 7 v e r s u s i t s a b s e n c e (see F i g u r e 16). H o w e v e r , n o 3  significance w a s  observed  coaddition groups.  i n the  [ H ] C s A - L D L c o m p l e x or 3  [ H]CsA with L D L 3  I n a d d i t i o n , n o significance w a s s h o w n i n the presence of L D L  89  v e r s u s its absence.  These results suggest that p o s s i b l y C s A is i n t e r a c t i n g directly  w i t h the L D L r e c e p t o r i n d e p e n d e n t of its a s s o c i a t i o n w i t h L D L .  O n the other h a n d ,  it m a y s u g g e s t t h a t C s A h a s p r e f e r e n t i a l a s s o c i a t i o n w i t h L D L a n d i s i n t e r a c t i n g w i t h a non-specific L D L receptor. A n o t h e r p o s s i b i l i t y is the steric h i n d e r a n c e d u e to p r e i n c u b a t i o n o f I g G - C 7 p r i o r to C s A t r e a t m e n t w h i c h w o u l d  yield  significant  differences i n m e a n percent b o u n d . N o significant results were revealed i n m e a n percent uptake of [ H ] C s A alone 3  and LDL  [ H ] C s A w i t h L D L i n the presence of I g G - C 7 v e r s u s its absence. 3  In addition,  d i d n o t s i g n i f i c a n t l y r e d u c e t h e u p t a k e o f [ H ] C s A (see F i g u r e 17). 3  inconsistent  with  the  results  reported  by  Peterherych  et  al  T h i s is  (2001)  where  p r e i n c u b a t i o n of increasing L D L levels significantly r e d u c e d the u p t a k e of C s A i n L L C - P K i c e l l s [91].  It i s p o s s i b l e t h a t C s A i s a s s o c i a t e d w i t h L D L a n d t h u s , l e s s  a m o u n t of C s A is b e i n g t a k e n u p b y the cells a n d n o t a f u n c t i o n of L D L receptor downregulation.  T h e differences i n results c a n be e x p l a i n e d b y the different assay  m e t h o d s a s r e p o r t e d b y P e t e r h e r y c h et al (2001).  P e t e r h e r y c h et al h a d a 2 4 - h o u r  i n c u b a t i o n w i t h [ H ] C s A a n d L D L i n contrast to a 6-hour i n c u b a t i o n w i t h [ H ] C s A 3  and  L D L i n this study.  [ H ] C s A uptake. 3  3  A shorter i n c u b a t i o n t i m e d i d n o t r e n d e r e n o u g h t i m e for  Therefore, the difference i n i n c u b a t i o n times m a y e x p l a i n  the  inconsistency i n results. I n a d d i t i o n , L L C - P K i cells w e r e pretreated w i t h serum-free m e d i a i n o r d e r to u p r e g u l a t e the L D L receptors.  S i n c e the cells w e r e e x p o s e d to a  h i g h c o n c e n t r a t i o n of L D L ( 2 0 p g / m l cholesterol), the L D L receptors p o s s i b l y h a d preferential u p t a k e of L D L rather t h a n C s A . T h i s w o u l d e x p l a i n the insignificant  90  results  in  mean  percent  bound,  uptake  and  toxicity  of  [ H]CsA 3  with L D L .  N e v e r t h e l e s s , i t i s d i f f i c u l t t o d r a w i n f e r e n c e s f r o m t h e r e s u l t s w i t h L D L as i t i s u n c e r t a i n h o w the p a r t i c l e is r e g u l a t i n g the e x p r e s s i o n a n d a c t i v i t y of L D L  receptors  in vitro. F i n a l l y , significant results w e r e r e p o r t e d i n percent toxicity of [ H ] C s A i n the 3  p r e s e n c e o f I g G - C 7 v e r s u s its a b s e n c e (see F i g u r e 18). N o s i g n i f i c a n t r e d u c t i o n w a s o b s e r v e d i n the [ H ] C s A - L D L c o m p l e x a n d [ H ] C s A w i t h L D L c o a d d i t i o n g r o u p s . 3  3  In a d d i t i o n , n o significant differences f r o m the [ H ] C s A alone g r o u p w e r e revealed 3  i n the presence  of L D L .  These results suggest  that the L D L receptor  may  be  m e d i a t i n g C s A - i n d u c e d t o x i c i t y . H o w e v e r , it is o n l y c o n c l u s i v e t h a t t h i s t o x i c i t y is a m e m b r a n e d i s r u p t i o n effect w h i c h c a n b e e x p l a i n e d b y t h e n a t u r e o f t h e t o x i c i t y m a r k e r . L D H is released i n t o the c u l t u r e m e d i u m u p o n cell m e m b r a n e p e r m e a b i l i t y a n d is a m a r k e r of late t o x i c i t y i n d i c a t i n g c e l l v i a b i l i t y .  A m o r e sensitive m e t h o d to  assess C s A r e n a l t o x i c i t y is to m o n i t o r p r o t e i n s y n t h e s i s u s i n g [ H ] l e u c i n e 3  r e p o r t e d i n m e t h o d s b y P e t e r h e r y c h et al (2001).  as  H o w e v e r , i n this study, it w a s  necessary to c h o o s e this t o x i c i t y m a r k e r to assess b o t h C s A u p t a k e a n d t o x i c i t y i n one assay m e t h o d . O v e r a l l , significant results i n percent [ H] C s A toxicity w e r e consistent w i t h 3  p e r c e n t [ H ] C s A b o u n d (see F i g u r e 16); y e t i n c o n s i s t e n t w i t h r e s u l t s r e p o r t e d i n t h e 3  uptake  o f [ H ] C s A (see  intracellular amounts absence.  F i g u r e 17).  3  There  was  no  significant differences  of [ H ] C s A o b s e r v e d i n the presence 3  in  o f I g G - C 7 v e r s u s its  H o w e v e r , there n e e d s to be i n t r a c e l l u l a r a m o u n t s o f the d r u g i n o r d e r to  91  e l i c i t a t o x i c effect.  O n e c a n interpret the p o s s i b i l i t y that [ H ] C s A is t a k e n u p b y a 3  n o n - L D L receptor p a t h w a y or a non-specific L D L receptor not recognized by I g G C7.  It i s s p e c u l a t i v e t h a t t h i s r e c e p t o r c o u l d b e m e g a l i n w h i c h i s e x p r e s s e d i n L L C -  P K i c e l l s [83].  M e g a l i n is a m e m b e r of the L D L r e c e p t o r f a m i l y a n d is a l a r g e cell  surface r e c e p t o r that is h i g h l y e x p r e s s e d i n the a p i c a l s i d e o f the p r o x i m a l t u b u l e c e l l s [19,125]. M e g a l i n c o n t a i n s f o u r c l u s t e r s o f t h e c y s t e i n e - r i c h r e p e a t s s h a r e d b y t h e n a t i v e L D L r e c e p t o r w i t h i n i t s l i g a n d - b i n d i n g r e g i o n [86,106,107].  I g G - C 7 is  directed against the first cysteine-rich repeat of the first d o m a i n of the L D L  receptor  [123]. W h e t h e r I g G - C 7 c a n a l s o r e c o g n i z e s a n e p i t o p e i n t h e l i g a n d b i n d i n g d o m a i n of m e g a l i n is u n c e r t a i n ; y e t it is a s s u m e d that t h e r e is h i g h h o m o l o g y i n this r e g i o n b e t w e e n the L D L receptor a n d m e g a l i n . O v e r a l l , it is c o n c l u s i v e f r o m  this s t u d y that the L D L receptor family  is  p l a y i n g a role i n b o t h C s A b i n d i n g a n d toxicity i n L L C - P K i cells.  6.5.  Hypothetical Model A h y p o t h e t i c a l m o d e l o f the processes o f C s A b i n d i n g a n d e l i c i t i n g its t o x i c i t y  i n L L C - P K i cells t h r o u g h the L D L r e c e p t o r f a m i l y is p r e s e n t e d i n F i g u r e 20. C s A b i n d s d i r e c t l y to the L D L receptor.  It i s u n s u r e w h e t h e r C s A i s a s s o c i a t i n g w i t h  L D L as a c o m p l e x g r o u p a n d r e c o g n i z e d b y t h e L D L r e c e p t o r b u t t h e r e s u l t s d o n o t support this hypothesis.  N e v e r t h e l e s s , C s A b i n d s to the L D L receptor, a n d elicits  d r u g - i n d u c e d t o x i c i t y at t h e m e m b r a n e l e v e l . U p t a k e o f C s A m a y b e m e d i a t e d b y  92  CsA  o ^ LDL Receptors  Receptor- bound CsA CsA-lndu«dMembrane Disruption  1  /  Renal Cell  fegaiin ?  Apical  Ba so lateral  Plasma Membrane  Figure 20: Hypothetical model of CsA uptake and toxicity into LLC-PKi cells via LDL receptor family  1. CsA binds directly to the LDL receptor 2. CsA elicits toxicity at the membrane level 3. CsA uptake may be mediated by a non-specific LDL receptor pathway such as megalin  93  b i n d i n g to m e g a l i n , a m e m b e r of the L D L receptor f a m i l y w h i c h is expressed i n L L C - P K i c e l l s [83]. The hypothesis of d r u g directly b i n d i n g to the L D L receptor family has already been proposed suggested  b y other investigators  that m e g a l i n m a y mediate  aminoglycosides  such  as g e n t a m i c i n ,  [32,77,110].  M o e s t r u p et al (1995)  the u p t a k e of polybasic drugs, specifically aprotinin  and polymyxin  B [77].  The  i n v e s t i g a t o r s c o n d u c t e d in vitro i n h i b i t i o n a s s a y s i n c u l t u r e d y o l k s a c c a r c i n o m a c e l l s o f t h e s e d r u g s w i t h R A P , a g p 3 3 0 i n h i b i t o r a s w e l l a s in vivo u p t a k e  studies  w i t h m i c r o i n f u s i o n s i n p r o x i m a l c o n v o l u t e d t u b u l e s i n f e m a l e W i s t a r r a t s [77]. T h e y c o n c l u d e d that gp330 m a y account for renal u p t a k e of a n u m b e r of basic molecules through  l o w affinity charge  interaction w i t h gp330  [77].  They  discussed  the  p o t e n t i a l r o l e o f g p 3 3 0 as a d r u g r e c e p t o r a n d h o w t h e m o l e c u l a r m e c h a n i s m o f uptake of polybasic drugs c a n p r o v i d e insight to i m p r o v e d antibiotic therapy b y a v o i d i n g k i d n e y a n d e a r t o x i c i t i e s [77]. F a r q u h a r et al (1995) f u r t h e r s u p p o r t e d t h e s u g g e s t i o n t h a t g p 3 3 0 / m e g a l i n c o u l d p o s s i b l y b e r e c o g n i z e d a s a d r u g r e c e p t o r [32]. A  r e c e n t in vivo s t u d y  b y S c h m i t z et al (2002) i n m e g a l i n - d e f i c i e n t  mice  demonstrated that the uptake of a m i n o g l y c o s i d e s into the k i d n e y directly correlated w i t h renal m e g a l i n activity a n d that a c c u m u l a t i o n of the d r u g i n the k i d n e y w a s e l i m i n a t e d i n m i c e l a c k i n g t h e r e c e p t o r [110].  T h e authors c o n c l u d e d that m e g a l i n  was the only major p a t h w a y responsible for renal a m i n o g l y c o s i d e accumulation a n d that the receptor represented a u n i q u e d r u g target i n p r e v e n t i o n of n e p h r o t o x i c i t y i n these patients  [110].  Another  study  b y N a g a i et al (2001)  94  also  attempted  to  i n v e s t i g a t e t h e m e c h a n i s m o f a m i n o g l y c o s i d e n e p h r o t o x i c i t y [80]. T h e a u t h o r s u s e d m a l e W i s t a r rats to investigate tissue u r i n a r y e x c r e t i o n of e n d o g e n o u s  megalin  l i g a n d s s u c h as v i t a m i n D - b i n d i n g p r o t e i n ( D B P ) a n d c a l c i u m [80]. R e s u l t s r e v e a l e d that a m i n o g l y c o s i d e a d m i n i s t r a t i o n significantly increased b o t h u r i n a r y D B P a n d c a l c i u m [80]. I n c o n c l u s i o n , t h e a u t h o r s p r o p o s e d t h a t m e g a l i n w a s i n v o l v e d i n t h e r e n a l c o r t i c a l a c c u m u l a t i o n o f a m i n o g l y c o s i d e s in vivo a n d  that the  interaction  b e t w e e n a m i n o g l y c o s i d e s a n d c a l c i u m i n the k i d n e y m a y be d u e to the c o m p e t i t i v e b i n d i n g f o r m e g a l i n [80].  6.6.  Limitations A l t h o u g h L L C - P K i cells w e r e established to be a n a p p r o p r i a t e cell m o d e l to  investigate the r o l e of the L D L receptor o n C s A b i n d i n g a n d t o x i c i t y , it expresses a l o w a b u n d a n c e o f L D L specific b i n d i n g sites. L L C - P K i c e l l s h a v e b e e n w i d e l y u s e d as a m o d e l f o r C s A - i n d u c e d n e p h r o t o x i c i t y [10,20,47,71], b u t t h e r e a r e a l s o o t h e r c e l l m o d e l s m o r e a p p r o p r i a t e to this s t u d y . T h e s e i n c l u d e H K - 2 cells, a h u m a n p r o x i m a l t u b u l e c e l l l i n e [142-144] a n d H E K - 2 9 3 , a n h u m a n e m b r y o n i c p r o x i m a l t u b u l e c e l l l i n e w h i c h e x p r e s s a h i g h e r a b u n d a n c e o f L D L r e c e p t o r s [64,74]. T h u s , L L C - P K i is n o t the m o s t i d e a l c e l l m o d e l to s t u d y L D L r e c e p t o r a c t i v i t y w h i c h is a l i m i t a t i o n to this study. It w a s n e c e s s a r y t o d e t e r m i n e t h e o p t i m a l t i m e p e r i o d f o r m a x i m u m [ H ] C s A 3  u p t a k e w i t h o u t c o m p r o m i s i n g further d e g r a d a t i o n of I g G - C 7 . Therefore, a [ H] C s A 3  time-course i n c u b a t i o n s t u d y w a s i m p l e m e n t e d . N e v e r t h e l e s s , since the m o n o c l o n a l  95  a n t i b o d y f u n c t i o n s s i m i l a r t o a L D L p a r t i c l e [11], i t s m e t a b o l i s m i s u n a v o i d a b l e w h i c h m a y be a c o n i o u n d e r to this study.  Yet, significant results were  obtained  w h i c h s h o w s that this is n o t a major l i m i t a t i o n . A 2 0 p g / m l concentration of L D L , based o n cholesterol content, w a s c h o s e n i n the u p t a k e a n d toxicity assays w i t h [ H ] C s A a n d I g G - C 7 . 3  on  significant  results  observed  in  C s A uptake  and  The rationale was based toxicity  with  the  same  c o n c e n t r a t i o n of L D L a n d o n a h y p o t h e t i c a l c o n c e n t r a t i o n o b s e r v e d i n the p r o x i m a l tubule of hypercholesterolemic patients  [91].  Nevertheless, this concentration of  L D L m a y h a v e b e e n t o o h i g h f o r t h e s e s t u d i e s w h e n i n v e s t i g a t i n g t h e effect o f L D L receptors o n C s A b i n d i n g a n d toxicity.  It w a s d i f f i c u l t t o p r e d i c t w h e t h e r t h e L D L  p a r t i c l e w a s s i m u l t a n e o u s l y r e g u l a t i n g t h e e x p r e s s i o n L D L r e c e p t o r s in vitro t h e r e b y providing an additional confounder. F i n a l l y , the use of I g G - C 7 to m o d i f y L D L receptor a c t i v i t y m a y be a l i m i t a t i o n i n i t s e l f . I g G - C 7 i s a m o n o c l o n a l a n t i b o d y s p e c i f i c t o t h e h u m a n L D L r e c e p t o r [11] yet the s t u d y i n v o l v e s o b s e r v i n g the L D L receptor i n a p i g cell line.  Interspecies  differences i n the L D L receptor m a y restrict the specificity i n b i n d i n g of I g G - C 7 w h i c h m a y be the reason w h y s u c h a h i g h concentration of 1 2 . 0 p g / m l w a s  needed  i n this study.  human  H o w e v e r , a p r o t e i n B L A S T analysis has c o n f i r m e d that the  a n d p i g L D L receptors h a v e a b o u t a n 87% s e q u e n c e h o m o l o g y of its 818 a m i n o a c i d residues.  I n a d d i t i o n , the m a n u f a c t u r e r of the a n t i b o d y (RDI) c o u l d not c o n f i r m n o r  d e n y the c r o s s - r e a c t i v i t y o f I g G - C 7 w i t h p i g L D L r e c e p t o r s as it h a s n o t b e e n t e s t e d  96  in pigs. They have tested positive i n human and bovine species and negative i n rat, mouse, hamster, dog and rabbit [101].  6.7.  Future Research  This study provides preliminary evidence for the role of the L D L receptor family i n mediating the uptake and toxicity of C s A i n L L C - P K i cells.  Further  studies to support the hypothesis w o u l d be to conduct similar studies i n a different cell lines such as HEK-293, a human embryonic proximal tubule cell line [64,74] or H K - 2 , a human proximal tubule cell [142-144] rather than L L C - P K i cells, a pig proximal tubule cell line. Both cell lines, HEK-293 and H K - 2 express an abundance of L D L receptors since the former is an embryonic cell line and the latter is associated w i t h cholesterol transport and loading [142-144].  Additional in vitro  studies could be implemented i n C H 0 1 d l A 7 , a mutant form of the Chinese Hamster Ovarian cell line that lacks the L D L receptor [6]. Transfected cell lines with the L D L receptor gene could also be investigated. To study the effect of C s A in vivo, an L D L receptor deficient mice model such as B A L B . L D L R - / - could be used [118]. Finally, to construct L D L receptor gene knockout in vitro and in vivo models w o u l d provide much stronger evidence of the role of L D L receptor activity i n C s A uptake and toxicity.  97  6.8.  Overall  Conclusions  I n this s t u d y , w e h a v e p r o v i d e d e v i d e n c e that C s A b i n d s d i r e c t l y to the L D L receptor a n d c a n elicit C s A - i n d u c e d toxicity i n L L C - P K i cells, a p i g p r o x i m a l tubule cell line. C s A has a n a r r o w therapeutic i n d e x a n d it is d u e to its n e p h r o t o x i c i t y that the d r u g n e e d s to be d i s c o n t i n u e d .  U n d e r s t a n d i n g the m e c h a n i s m b y w h i c h C s A  interacts  renal  and  causes  toxicity  in  cells  could  provide  improved  a d m i n i s t r a t i o n a n d t h e r a p y b a s e d o n the l i p i d profile of the patient.  CsA  B a s e d o n the  c l i n i c a l d a t a , p a t i e n t s w h o a r e h y p o c h o l e s t e r o l e m i c h a v e i n c r e a s e C s A t o x i c i t y [24]. M e a n w h i l e , p a t i e n t s w h o a r e h y p e r t r i g l y c e r i d e m i c h a v e d e c r e a s e d C s A e f f i c a c y [25, 82].  Therefore, patients w h i c h are h y p o l i p i d e m i c w o u l d be a d m i n i s t e r e d a l o w e r  d o s e c o m p a r e d to patients w h o are h y p e r l i p i d e m i c . I n a d d i t i o n , f o r m u l a t i o n of the d r u g c a n be m a n i p u l a t e d i n o r d e r to b y p a s s the L D L receptor to a v o i d t o x i c i t y ; yet t a r g e t t o s i t e - s p e c i f i c a r e a s s u c h as T - c e l l s t o m a i n t a i n i t s e f f i c a c y .  98  References  99  [1]  Neoral  and  Sandimmune  ~  Cyclosporine.  Thirty-Fifth  edition.  2000.  C o m p e n d i u m of Pharmaceuticsl a n d Specialties (CPS). [2]  A.T.Webb,  M.Plant,  D.A.Reaveley,  M.O'Donnel,  M . S e e , a n d E . A . B r o w n (1992) L i p i d  V . A Luck,  B.O'Connor,  a n d l i p o p r o t e i n (a) c o n c e n t r a t i o n s  in  r e n a l t r a n s p l a n t p a t i e n t s . Nephrol Dial Transpl 7 : 6 3 6 - 6 4 1 . [3]  Akhlaghi  F and Trull  A K (2002)  Distribution of cyclosporin i n  t r a n s p l a n t r e c i p i e n t s . Clin.Pharmacokinet. [4]  Allen  M J and Rushton  organ  41:615-637.  N . 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