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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 b y N a n c y S C C h u n g H o n o u r s B S c , T h e U n i v e r s i t y o f T o r o n t o , 2001 A T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F MASTER OF SCIENCE in T H E F A C U L T Y OF G R A D U A T E STUDIES F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s ( D i v i s i o n o f P h a r m a c e u t i c s a n d B i o p h a r m a c e u t i c s ) W e accep t t h i s thes is i s 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 U N I V E R S I T Y O F B R I T I S H 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. Name of Author (please paht) '2^- / o n / : > ( ( ^ \ Date (dd/mm/yyyy) Title of Thesis: ^£-£ivx{ cells Degree: <rf ,C Year: 2 oo^ Department of \:h,s, ,vy^ ,,:tu^( -,SV The University of British Columbia " Vancouver, BC Canada A B S T R A C T Background: Cyc lospor ine A (CsA) is an effective immunosuppressan t d r u g to treat patients w h o have undergone transplantat ion or to treat au to immune diseases. H o w e v e r , the d r u g is l im i t ed by its na r row therapeutic index and usua l ly becomes discont inued due to h i g h nephrotoxici ty. C s A is k n o w n to h igh ly associate w i t h l ipoproteins , especially L D L and increased toxic effects of C s A have been reported i n patients w h o are hypocholesterolemic. A significant reduc t ion i n [ 3 H ] C s A uptake and toxici ty was observed w h e n L L C - P K i cells were treated w i t h increased concentrations of L D L . Purpose: Based o n the experimental and c l in ica l evidence, it is hypothes ized that w h e n the L D L receptor fami ly act ivi ty is decreased u p o n I g G - C 7 treatment, bo th C s A uptake and toxici ty are reduced i n L L C - P K i cells, a renal p r o x i m a l tubule cell l ine. Methods: The appropriateness of L L C - P K i cells as a cel l m o d e l was assessed by conduc t ing dose-response, L D L specific b i n d i n g and compet i t ive studies w i t h D i l -L D L , and Western blot analysis of the L D L receptor. Assay condi t ions w i t h IgG-C7 , a monoc lona l ant ibody to the L D L receptor, were o p t i m i z e d i n c l u d i n g temperature, pre incubat ion t ime and concentrat ion i n L L C - P K i cells. F ina l l y , the effect of IgG-C7 on [ 3 H ] C s A uptake and toxici ty w i t h L D L was determined. Results: Significant results i n both mean percent b o u n d (2.6% ± 0.6% vs. 5.1% ± 1.3%) and mean percent toxici ty (1.8% ± 0.5% vs. 3.2% ± 0.5%) were observed i n the [ 3 H ] C s A alone group i n the presence of I g G - C 7 versus its absence (p<0.05 w i t h unpa i red t-test). H o w e v e r , no significant differences were observed i n the [ 3 H ] C s A -L D L complex or [ 3 H ] C s A w i t h L D L coadd i t ion groups. In add i t ion , L D L was not associated w i t h a significant reduct ion i n both [ 3 H ] C s A bound , uptake and toxicity. Conclusion: These results suggest that C s A m a y be b i n d i n g direct ly to the L D L receptor fami ly independent of its association w i t h L D L a n d thus, e l ic i t ing its toxic effects at the membrane level . This s tudy provides p re l iminary evidence of the family 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 and toxici ty i n L L C - P K i cells. ii T A B L E OF C O N T E N T S A b s t r a c t i i T a b l e o f C o n t e n t s i i i L i s t o f F i g u r e s v i i L i s t o f T a b l e s . . i x L i s t o f A b b r e v i a t i o n s x A c k n o w l e d g e m e n t s x i i C H A P T E R 1: I N T R O D U C T I O N 1 1.1. C s A B a c k g r o u n d 1.1.1. D e s c r i p t i o n o f 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 c l i n i c a l i m p l i c a t i o n s o f C s A . . . 3 1.1.3. P r o p o s e d m e c h a n i s m o f a c t i o n o f C s A 4 1.1.4. A d v e r s e Effec ts 6 1.1.5. N e p h r o t o x i c i t y 6 1.2. L i p o p r o t e i n s a n d t h e i r m e t a b o l i s m 8 1.2.1. C h y l o m i c r o n s 9 1.2.2. 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 10 1.2.3. L o w - d e n s i t y l i p o p r o t e i n 11 1.2.3. H i g h - d e n s i t y l i p o p r o t e i n 13 1.3. 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 i ts f a m i l y m e m b e r s 14 1.3.1. T h e L D L r e c e p t o r 16 1.3.2. M e g a l i n 18 1.4. C y c l o s p o r i n e A a s s o c i a t i o n w i t h l i p o p r o t e i n s 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 the 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 O b j e c t i v e 24 2.2. H y p o t h e s i s 24 2.3. S p e c i f i c A i m s 24 2.4. R a t i o n a l e 24 2.5. S i g n i f i c a n c e o f R e s e a r c h 25. i i i 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 26 3.1 M a t e r i a l s 3.1.1. C e l l s a n d c e l l c u l t u r e 27 3.1.2. L i p o p r o t e i n s , c h e m i c a l s a n d l a b e l e d d r u g 27 3.1.3. A n t i b o d i e s a n d a s say k i t s 28 3.2 T i s s u e C u l t u r e R e a g e n t s a n d M e d i u m 3.2.1. P r e p a r a t i o n o f L L C - P K i m e d i a 29 3.2.2. P r e p a r a t i o n o f H e p G 2 m e d i a 29 3.2.3. P r e p a r a t i o n o f S e r u m - f r e e L L C - P K i a n d H e p G 2 m e d i a 30 3.2.4. P r e p a r a t i o n o f P h o s p h a t e B u f f e r e d S a l i n e ( P B S ) 30 3.2.5. C u l t u r e E s t a b l i s h m e n t o f L L C - P K i a n d H e p G 2 c e l l s f r o m F r o z e n S t o c k 30 3.2.6. S u b c u l t u r i n g o f L L C - P K i ce l l s 31 3.2.7. S u b c u l t u r i n g o f H e p G 2 ce l l s 31 3.2.8. S e e d i n g p l a t e s 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 c e l l s f o r E x p e r i m e n t a t i o n 32 3.3 P r e p a r a t i o n o f E x p e r i m e n t a l R e a g e n t s 3.3.1. C h o l e s t e r o l c o n c e n t r a t i o n d e t e r m i n a t i o n i n L D L 33 3.3.2. S o l u t i o n s w i t h D i l - L D L 34 3.3.3. S o l u t i o n s w i t h I g G - C 7 34 3.3.4. S o l u t i o n s w i t h [ 3 H ] C s A 35 3.4 P r e p a r a t i o n o f R e a g e n t s fo r W e s t e r n B l o t t i n g 3.4.1. S D S - P A G E 35 3.4.2. I m m u n o b l o t a n d C h e m i l u m i n e s c e n c e d e t e c t i o n 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 38 4.1 . D i l - L D L A s s a y D e v e l o p m e n t 4.1.1. P r o t e i n C o n t e n t D e t e r m i n a t i o n 39 4.1.2. F l u o r e s c e n c e D e t e r m i n a t i o n o f D i l 40 4.1.3. D o s e - R e s p o n s e S t u d i e s 41 4.1.4. L D L S p e c i f i c B i n d i n g 42 4.1.5. C o m p e t i t i v e B i n d i n g 42 4.2. W e s t e r n B l o t t i n g P r o c e d u r e f o r the L D L r e c e p t o r 4.2.1. 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 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 gels ) 44 4.2.3. I m m u n o b l o t t i n g a n d D e t e c t i o n 46 iv 4.3. D i l - L D L w i t h I g G - C 7 Assay Deve lopment 4.3.1. Temperature Dependence 48 4.3.2. O p t i m a l Pre incubat ion pe r iod 48 4.3.3. O p t i m a l Concent ra t ion 49 4.4. C s A Time-Course Incubation S tudy 49 4.5. C s A Up take and Toxic i ty w i t h IgG-C7 Assay Deve lopmen t 4.5.1. [ 3 H ] C s A B o u n d and Up take Assay 50 4.5.2. M e m b r a n e - b o u n d and Intracellular [ 3 H ] C s A determina t ion 52 4.5.3. [ 3 H ] C s A Toxic i ty Study - L D H measurement 53 4.6. Scint i l la t ion 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 57 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 57 5.1.2. L D L specific b i n d i n g 60 5.1.3. Compe t i t ive b i n d i n g 64 5.1.4. Wes te rn blot analysis 65 5.2. Effect of I gG-C7 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 pre incubat ion pe r iod 68 5.2.3. O p t i m a l Concent ra t ion 70 5.3. P H I C s A Treatment 5.3.1. [ 3 H ] C s A time-course incubat ion 72 5.4. Effect of I g G - C 7 o n [ 3 H l C s A Up take a n d Toxic i ty 5.4.1. [ 3 H ] C s A uptake - b o u n d and intracel lular 75 5.4.2. [ 3 H ] C s A toxici ty - L D H measurement 79 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 1 2 5 I - L D L 83 6.2. Rationale of L L C - P K i as an appropriate cel l m o d e l 85 v 6.3. U s e o f 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. I n t e r p r e t a t i o n o f r e su l t s i n [ 3 H ] C s A u p t a k e a n d t o x i c i t y a s says 89 6.5. H y p o t h e t i c a l M o d e l 92 6.6. L i m i t a t i o n s 95 6.7. F u t u r e R e s e a r c h 97 6.8. O v e r a l l C o n c l u s i o n s 98 REFERENCES 100 v i LIST OF 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 a c t i o n i n a c t i v a t e d T - c e l l s 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 the 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 o f c e l l - a s s o c i a t e d L D L 59 F i g u r e 7 B : D o s e - d e p e n d e n t m e a s u r e m e n t o f 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 o f 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 ce 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 ce 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 ce l l s 64 F i g u r e 11 : W e s t e r n B l o t a n a l y s i s o f the L D L r e c e p t o r i n L L C - P K i ce l l s 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 o f 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 the 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 o f m A b to the L D L r e c e p t o r 71 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 I g G - C 7 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 [ 3 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 [ 3 H ] C s A i n L D L w i t h I g G - C 7 78 v i i 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 [ 3 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 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 ce l l s v i a L D L r e c e p t o r f a m i l y 93 viii LIST OF 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 2 T a b l e 2: D e n s i t y , S i z e , P h y s i c a l C o m p o s i t i o n a n d F u n c t i o n o f H u m a n P l a s m a L i p o p r o t e i n s 9 T a b l e 3: P h a r m a c o k i n e t i c cha rac 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 I g G - C 7 E x p e r i m e n t s 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 ce 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 [ 3 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 ix LIST OF ABBREVIATIONS 4 A A P 4 - a m i n o a n t i p y r i n e A b s A b s o r b a n c e A C A T A c y l - C o A : c h o l e s t e r o l O - a c y l t r a n s f e r a s e A N O V A A n a l y s i s o f v a r i a n c e A P S A m m o n i u m P e r s u l f a t e A U C A r e a u n d e r the c u r v e B S A B o v i n e S e r u m A l b u m i n B C A B i c i n c h o n i n i c A c i d C E C h o l e s t e r o l esterases C E T P C h o l e s t e r y l ester t r ans fe r p r o t e i n C O C h o l e s t e r o l o x i d a s e s C P M C o u n t s p e r m i n u t e D i l 3 , 3 ' - d i o c t a d e c y l i n d o - c a r b o c y a n i n e d H 2 0 D i s t i l l e d w a t e r E C M E x t r a c e l l u l a r m a t r i x E G F E p i d e r m a l g r o w t h fac to r F B S F e t a l B o v i n e S e r u m H B A H y d r o x y b e n z o i c A c i d H D L H i g h - d e n s i t y l i p o p r o t e i n H L H e p a t i c l i p a s e H M G - C o A 3 - h y d r o x y - 3 - m e t h y l g l u t a r y l - C o A e n z y m e H R P H o r s e r a d i s h P e r o x i d a s e h r s H o u r s I D L 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 I L I n t e r l e u k i n I N T T e t r a z o l i u m sa l t I P A I s o p r o p a n o l L C A T L e c i t h i n : c h o l e s t e r o l a c y l t r a n s f e r a s e L D H L a c t a t e D e h y d r o g e n a s e L D L L o w - d e n s i t y l i p o p r o t e i n L p L L i p o p r o t e i n l i p a s e m A b M o n o c l o n a l A n t i b o d y M M F M y c o p h e n o l a t e m o f e t i l N F A T N u c l e a r - f a c t o r o f a c t i v a t e d T - c e l l s N F A T c N F A T c y t o s o l i c N F A T n N F A T n u c l e u s N P x Y A s p a r a g i n e - P r o l i n e - X - T y r o s i n e P L T P P h o s p h o l i p i d t r ans fe r p r o t e i n P M N P e r i p h e r a l m o n o n u c l e a r P T P r o x i m a l t u b u l e P O D P e r o x i d a s e X P V D F P o l y v i n y l d i f l u o r i d e m e m b r a n e R O S R e a c t i v e o x y g e n spec i e s r p m r o t a t i o n s p e r m i n u t e S D S S o d i u m D o d e c y l S u l f a t e T C R T - c e l l r e c e p t o r T E M E D N , N , N , N - T e t r a - m e t h y l - e t h e l e n e d i a m i n e T G F T r a n s f o r m i n g g r o w t h fac tor V L D L 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 V s s V o l u m e o f d i s t r i b u t i o n at s t eady-s t a t e Y W T D T y r o s i n e - T r y p t o p h a n - T h r e o n i n e - A s p a r t a t e x i A C K N O W L E D G E M E N T S I w o u l d l i k e to t h a n k m y s u p e r v i s o r , D r . K i s h o r W a s a n f o r h i s s u p p o r t , p a t i e n c e a n d m e n t o r s h i p d u r i n g th i s projec t . Y o u h a v e p r o v i d e d a w a r m a n d e n c o u r a g i n g e n v i r o n m e n t fo r 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 f o r 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 o f y o u r l a b . T o m e m b e r s o f m y M S c c o m m i t t e e , 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 R i g g s a n d D r . K a t h l e e n M a c L e o d , y o u r f e e d b a c k a n d s u g g e s t i o n s h a v e b e e n v e r y h e l p f u l to the p r o g r e s s i o n o f t h i s projec t . 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 f o r c o n t r i b u t i n g the c o l o u r f u l f i g u r e s i n t h i s thes i s . N o t o n l y h a v e y o u b e e n s u p p o r t i v e a n d e n c o u r a g i n g d u r i n g m y g r a d u a t e t r a i n i n g , b u t y o u h a v e b e e n a w o n d e r f u l f r i e n d . W i t h m y m a n y d o u b t s a n d w o r r i e s , y o u h a v e h e l p e d m e k e e p f o c u s e d . W o r d s c a n n o t e x p l a i n h o w I v a l u e o u r f r i e n d s h i p . T o C h i a - T i n g , I a m so g r a t e f u l to h a v e b e c o m e s u c h c l o s e f r i e n d s w i t h y o u . Y o u h a v e h e l p e d m e o u t i n m a n y s t ress fu l m o m e n t s a n d h a v e b e e n t he re f o r 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 fo r l i s t e n i n g a n d b e i n g y o u ! T o " C h r i s t o p h e r " , t h a n k y o u f o r y o u r c o m p a n i o n s h i p a n d k i n d n e s s d u r i n g a d i f f i c u l t p e r i o d i n m y l i f e . Y o u h a v e b e e n a g rea t f r i e n d a n d h a v e s h o w n m e a n e w l i g h t . O u r c o n v e r s a t i o n s 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 fo r t h e i r 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 l i f e i n V a n c o u v e r . I a m g r a t e f u l t o h a v e s u c h a c l o s e r e l a t i o n s h i p w i t h b o t h o f y o u . T h a n k s fo r b e i n g the re fo r m e ju s t to l i s t e n . T o m y r e l a t i v e s , A u n t J u l i a , U n c l e V i c t o r , B r a n d o n a n d V i n c e n t : I a m g l a d tha t 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 . I a p p r e c i a t e a l l y o u r h e l p a n d h o s p i t a l i t y y o u h a v e s h o w n m e . T h a n k y o u f o r m a k i n g m y l i fe a b i t eas ie r d u r i n g m y s t u d i e s . T o m y m o t h e r , f a the r a n d s is ter ; E l l e n , a n d m y c l o s e f r i e n d s i n T . O , y o u w e r e o n l y jus t a p h o n e c a l l a w a y b u t y o u r w o r d s o f e n c o u r a g e m e n t h a v e h e l p e d m e t h r o u g h m y g r a d u a t e s t u d i e s . T h a n k y o u f o r l i s t e n i n g a n d f o r y o u r a d v i c e . x i i To a l l members of the lab: Kr i s t ina , y o u have been a great insp i ra t ion to me. Thank y o u for a l l y o u r advice both scientifically and personal ly . Ver i ca , Olena , M o n a , Aga tha and A l i s o n , y o u have w e l c o m e d and embraced me since the first day I m o v e d to Vancouver . Y o u have been of great help a n d good listeners w h e n I needed someone to talk to. Thanks for a l l the fun times a n d the ' s p i k e d ' cheese fondue! Steve, y o u have added a great male influence to a once female dominan t lab! A n d f inal ly , the undergraduate students: Eugene, E r i n , E d w i n , Benny, G i n a and Tiffany; y o u have made the lab an enjoyable place to w o r k , especial ly d u r i n g the summer sessions. Thanks for a l l the jokes, laughter and interest ing conversations! To m y fe l low graduate students: Vince , Irem, M a r i a and L i n d a , thanks for the support , help and encouragement d u r i n g m y t ra ining. I w i l l a lways remember our conversations and the kindness y o u have s h o w n me. x i i i 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 p o l y p e p t i d e 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 the a m i n o a c i d s are 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 a re 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, 31] . T h e m o l e c u l a r s t r u c t u r e o f the 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 1202.6 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 the 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 = 14 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 to a n u m b e r o f i ts 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 o f 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 . M o l e c u l a r F o r m u l a C62H111N11O12 M o l e c u l a r W e i g h t 1202.635 K p ( o c t a n o l / b u f f e r ) 1000 C r y s t a l F o r m ( c r y s t a l l i z a t i o n f r o m ace tone) C o l o r l e s s , p r i s m a t i c C e l l d i m e n s i o n s A = b = 1 3 . 8 3 7 A , C = 4 1 . 2 4 2 A C r y s t a l V o l u m e 7896 A 3 C r y s t a l D e n s i t y 1.042 g m / c m 3 M o l e c u l e s p e r c e l l Z = 4 M e l t i n g p o i n t 1 4 8 ° C to 1 5 1 ° C O p t i c a l r o t a t i o n ( i n c h l o r o f o r m ) - 2 4 4 ° 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 discovered its immunosuppressive activity [14,135]. 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 to i ts 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 n o t o n l y fo r e f f i cacy o f the C s A b u t a l s o f o r a v o i d a n c e o f t o x i c i t y . T h u s m a n a g i n g a n d m a i n t e n a n c e o f the C s A r e g i m e after t r a n s p l a n t a t i o n is u s u a l l y c h a l l e n g i n g a n d c o n f u s i n g s i n c e i t i s v e r y p a t i e n t - d e p e n d e n t . C a r e f u l a t t e n t i o n 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 i n t e r a c t i o n s w h i l e m i n i m i z i n g a d v e r s e 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 i ts T - c e l l r e c e p t o r ( T C R ) , a c a scade o f i n t r a c e l l u l a r e v e n t s o c c u r s i n c l u d i n g a n e l e v a t i o n o f c a l c i u m l e v e l s 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]. A s a r e su l t , c a l m o d u l i n in te rac t s w i t h c y c l o p h i l i n A w h i c h c a n r e g u l a t e c a l c i n e u r i n , a s u p e r f a m i l y o f p r o t e i n 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 the d e p h o s p h o r y l a t i o n o f N F A T (nuc lea r - f ac to r o f a c t i v a t e d T-ce l l s ) f a m i l y m e m b e r s a l l o w i n g i t to t r ans loca t e i n t o the n u c l e u s a n d ac t i va t e g e n e e x p r e s s i o n o f 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 ( IL -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 s t u d i e s r e v e a l tha t 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 genes fo r I L - 2 a n d I L - 4 [43,49,62]. O n c e C s A enters the c e l l , i t 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 su 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 * A c t i v a t e d 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 to 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 he a c t i v a t i o n o f c a l c i n e u r i n 4. A c t i v a t e d c a l c i n e u r i n d e p h o s p h o r y l a t e s c y t o s o l i c 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 the 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 2 + l e v e l s w h i c h c a n a l s o r e g u l a t e c y c l o p h i l i n A 5 family members is inhibited as well as gene expression of cytokines deactivating T-cell 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 LDL 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 tubule noted by vacuo l i za t ion i n the cells, severe atrophy, th ickening of the basal membrane [76] and thrombosis of the in t raglomerular capil laries [116]. H o w e v e r , i n the case of transplant patients, higher C s A doses (10 -15mg/kg /d ) are usual ly required and so the s i tuat ion becomes even more compl ica ted i n renal transplant patients [1]. T G F - p ( transforming g r o w t h factor) has been p roposed as one of the mechanisms of C s A - i n d u c e d nephrotoxici ty . TGF-(3 is k n o w n to st imulate the increase compos i t ion of extracellular matr ix ( E C M ) and to decrease the p roduc t ion of E C M - d e g r a d i n g proteases w h i c h may exp la in the characteristics of interstitial fibrosis i n C s A - i n d u c e d renal toxici ty [13,94]. It has been also reported that C s A can disrupt functional proteins at the cel lular level such as P-glycoproteins [36], ca lc ineur in [45] as w e l l as induce apoptosis [48,53]. In add i t ion , reactive metabolites p roduced by the cytochrome P 450 system i n the l ive r a n d k i d n e y have been proposed by others [68,73,141]. It has been suggested that C s A can increase the synthesis of reactive oxygen species (ROS) [44,81] , thromboxane [90,112] and l i p i d peroxida t ion products [58]in the k idney . F ina l ly , C s A can reduce t ranscr ipt ion of certain genes i n p r o x i m a l tubule cells such as phosphoenolpyruva te carboxykinase i n gluconeogenesis [79]. In addi t ion , i n response to C s A toxici ty, an increase i n the GLUT1 gene expression was observed [26]. Nevertheless, the mechan i sm of C s A -induced nephrotoxic i ty remains uncertain. 7 1.2 Lipoproteins and their metabolism L i p o p r o t e i n s a re a c lass o f 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 o f b o t h l i p i d a n d p r o t e i n s u b g r o u p s . T h e i r m a i n r e s p o n s i b i l i t y i s t o t r a n s p o r t a n u m b e r o f 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 s y s t e m i c 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 e n v i r o n m e n t . L i p o p r o t e i n s a re c h a r a c t e r i z e d b y a n i n s o l u b l e c o r e o f c h o l e s t e r y l 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 s h e l l o f a m p h i p a t h i c p h o s p h o l i p i d s a n d 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]. T h e y d i f f e r i n t h e i r c o n t e n t o f p r o t e i n s a n d l i p i d s a n d are c l a s s i f i e d b a s e d o n t h e i r d e n s i t y i n t o f i v e m a i n ca tegor i e s : c h y l o m i c r o n s , 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 ( V L D L ) , 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 ) , l o w - d e n s i t y l i p o p r o t e i n s ( L D L ) , a n d h i g h - d e n s i t y 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 are 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 i s r e f l ec t ed 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 r a t i o . T h e y are , h o w e v e r , a l s o c l a s s i f i e d b y the a p o l i p o p r o t e i n c o n t e n t l o c a t e d o n t h e i r su r face w h i c h s u b s e q u e n t l y g o v e r n s the u l t i m a t e fate o f the p a r t i c l e . T h u s , l i p i d t r a n s p o r t is r e g u l a t e d 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 r e cep to r s , l i p o l y t i c e n z y m e s a n d t rans fe r p r o t e i n s w h i c h a l l ac t i n c o n c e r t t o 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 Chylomicrons Very-low- Intermediate Low-density High-density density lipoproteins density lipoproteins lipoproteins lipoproteins Abbreviations V L D L IDL L D L HDL Density (g/ml) <0.95 0.95-1.006 1.006-1.019 1.019-1.063 1.063-1.210 Diameter (nm) 75-1200 30-80 25-35 18-25 5-12 Composition (% dry wt) Proteins 1-2 8 19 22 47 triglycerides 86 55 23 6 4 . cholesterol 5 19 38 50 10 phospholipid 7 18 20 22 30 apoproteins A l , A l l A l , A l l , B-48 B-100 B-100 B-100 AIV ci, cn, cm ci, cn, cm ci, cn, cm ci, cn, cm E E E D , E Main function Transport of Transport of Transport of Cholesterol Reverse exogenous endogenous endogenous transport to cholesterol triglyceride triglyceride cholesterol all tissues . transport and cholesterol Table 2: D e n s i t y , S i z e , P h y s i c a l C o m p o s i t i o n a n d F u n c t i o n o f H u m a n P l a s m a L i p o p r o t e i n s . M o d i f i e d f r o m re fe rences [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 en te rs the e n t e r o c y t e a n d b e c o m e s r e p a c k a g e d w i t h a p o B - 4 8 , a p o A I , a p o A I I a n d a d d i t i o n a l l i p i d s i n t o c h y l o m i c r o n s [115,128]. S u b s e q u e n t l y , c h y l o m i c r o n s b e c o m e sec re t ed b y the i n t e s t i n a l m u c o s a t h r o u g h the 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]. A s s u c h , t h e y a c q u i r e c h o l e s t e r y l es ter f a c i l i t a t e d b y c h o l e s t e r y l es ter t ransfer 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 to the n e w l y a c q u i r e d a p o C s , t r i a c y l g l y c e r o l - r i c h c h y l o m i c r o n s a re r e c o g n i z e d b y the e n d o t h e l i a l - b o u n d 9 l i p o p r o t e i n l i p a s e ( L p L ) w i t h i n the c a p i l l a r y b e d s o f s k e l e t a l m u s c l e a n d a d i p o s e t i s sue [108,128]. A s a r e su l t , c h y l o m i c r o n s b e c o m e r a p i d l y h y d r o l y z e d , r e l e a s i n g 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 are 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 sues f o r e n e r g y p r o d u c t i o n a n d s to r age [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 c h y l o m i c r o n r e m n a n t , l a c k s t r i a c y l g l y c e r o l y e t i s 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 p o B - 4 8 a n d a p o E . A s a r e su l t , the r e m n a n t dec reases i ts a f f i n i t y f o r L p L a n d r e t u r n s to c i r c u l a t i o n to be t a k e n u p b y the l i v e r t h r o u g h 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 w h i c h i n v o l v e s r e c o g n i t i o n o f a p o E of the c h y l o m i c r o n r e m n a n t b y r e c e p t o r s o n the su r face 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 a re the m a j o r t r a n s p o r t e r s o f e n d o g e n o u s l y p r o d u c e d t r i a c y l g l y c e r o l f r o m the l i v e r to e x t r a h e p a t i c t i s sue [108,128]. B o t h e x o g e n o u s a n d e n d o g e n o u s l i p i d s a re a s s e m b l e d w i t h a p o B - 1 0 0 a n d o the r 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]. I n b l o o d , ' n a scen t ' V L D L a c q u i r e s 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 f r o m H D L to b e c o m e m a t u r e V L D L p a r t i c l e s [128]. 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 p a r t i c l e s a re h y d r o l y z e d b y L p L i n t o V L D L r e m n a n t s r e l e a s i n g a p o C a n d a p o E f r o m the su r face coa t a n d t r a n s f e r r i n g t h e m b a c k to H D L [108,128]. V L D L r e m n a n t s are f o r m e d w h i c h are 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 ) . T h e s e r e m n a n t p a r t i c l e s a re 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 b e 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 i n t e r a c t i o n w i t h the L D L r e c e p t o r o r the L D L r e c e p t o r -10 related prote in o n hepatocytes [108]. A majority of I D L , however , becomes further catabolized by hepatic lipase ( H L ) to fo rm L D L particles r i ch i n cholesteryl ester w i t h the apoB-100 attached to surface coat [128]. 1.2.3 Low-density lipoprotein (LDL) Low-dens i ty l ipoproteins are the m a i n carriers of unesterified and esterified cholesterol to per ipheral tissues. A product of V L D L catabol ism, this l ipopro te in is s t r ipped of t r iacylglycerols and associated apoprotein, except apoB-100 [108]. A d i ag ram of the L D L particle is s h o w n i n F igure 3. ApoB-100 is essential for recogni t ion of the L D L particle to its receptor and subsequent uptake into cells [108]. The majority of L D L is r emoved by the l iver th rough the L D L receptor and other n o n receptor-mediated pathways [22,108]. The impor tant role of L D L is the del ivery of cholesterol to extrahepatic tissues for u t i l i za t ion i n a number of processes i n c l u d i n g steroid p roduc t ion or membrane synthesis [35]. Intracellular homeostasis of cholesterol concentrations is due to three regulatory elements: a) H M G - C o A (3-hydroxy-3 -methy lg lu ta ry l -CoA) reductase, the ra te- l imi t ing enzyme i n cholesterol biosynthesis; b) A C A T (acy l -CoA: cholesterol O-acyltransferase) w h i c h re-esterifies excess intracel lular cholesterol for storage; and c) expression of L D L receptors o n the cell surface [108]. Fur thermore, the regulat ion of se rum L D L depends o n two factors: a) the quanti ty of V L D L p roduced by the l ive r w h i c h then becomes converted to L D L by H L and b) the p ropor t ion of V L D L remnants (IDL) 11 Figure 3: T h e l o w - d e n s i t y l i p o p r o t e i n ( L D L ) p a r t i c l e c o n s i s t s o f c h o l e s t e r o l a n d c h o l e s t e r y l ester w i t h a n a p o l i p o p r o t e i n B-100 e m b e d d e d i n a p h o s p h o l i p i d s m o n o l a y e r . 12 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 HDL2 and HDL3 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 HDL 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 HDL 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 disk-shaped 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 and P L T P (phospho l ip id transfer protein) facilitate the acquis i t ion of tr iglycerides and more cholesteryl esters into the HDL3 particle [35]. C E T P exchanges cholesteryl esters for tr iglycerides between H D L and t r ig lycer ide-r ich l ipoprote ins such as chylomicrons and V L D L ; meanwhi le P L T P acquires phospho l ip id s f rom the surface of apoB-conta ining l ipoprote ins w h i c h become substrates for L C A T [28,35]. Subsequently, a mature H D L particle is formed w h i c h is ca l led H D L 2 , consis t ing of a core of cholesteryl ester and tr iglycerides w h i c h can be h y d r o l y z e d by hepatic lipase ( H L ) u p o n transport to the l iver [28,34,34]. Release of a p o A I as a result of H L is used 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 macromolecules , m a i n l y the cholesterol-rich l ipopro te in L D L , into cells th rough a process cal led receptor-mediated endocytosis [85] (see F igure 4). This process involves the cel l surface receptor recogniz ing an L D L particle f rom the extracellular membrane ( E C M ) , in te rna l iz ing it th rough clathrin-coated pits and t ransport ing it in t racel lular ly v i a a vesicle [16,85,137]. Subsequently, the vesicle becomes degraded u p o n fusion w i t h the lysosome, releasing l i p ids into the cy top lasm for cell use; meanwhi le 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 our current knowledge of receptor-mediated 14 Plasma Membrane Figure 4: T h e L D L r e c e p t o r p a t h w a y . 1. N o v e l L D L r e c e p t o r s 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 v e s i c l e s a re t r a n s p o r t e d to the c e l l m e m b r a n e 3. L D L r e c e p t o r e x p r e s s i o n o n the c e l l su r face 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 r e c e p t o r 5. I n t e r n a l i z i n g o f r e c e p t o r - l i g a n d c o m p l e x i n t o v e s i c l e s 6. S o r t i n g o f t he L D L r e c e p t o r f r o m the L D L p a r t i c l e v i a f u s i o n w i t h a n e n d o s o m e 7. R e c y c l i n g o f L D L r e c e p t o r s b a c k to the c e l l su r f ace 8. L y s o s o m a l d e g r a d a t i o n o f L D L pa r t i c l e s i n t o a m i n o a c i d s a n d c h o l e s t e r o l 15 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 s t u d i e s o n the L D L r e c e p t o r p a t h w a y c o n d u c t e d b y G o l d s t e i n a n d B r o w n . T h e i r w o r k i n v o l v e d c h a r a c t e r i z i n g the b i o c h e m i s t r y a n d gene t i c s o f the L D L r e c e p t o r a n d d e t e r m i n i n g i t s 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 c h o l e s t e r o l h o m e o s t a s i s i n t r a c e l l u l a r l y . I n h e r i t e d m u t a t i o n s w i t h i n t h i s r e c e p t o r 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 gene t i c d i sease tha t r e s u l t s i n p a t h o l o g i c a l l y e l e v a t e d b l o o d c h o l e s t e r o l a n d p r e m a t u r e c o r o n a r y d i sease [42]. 1.3.1 The LDL receptor T h e L D L r e c e p t o r cons i s t s o f f i v e d i s t i n c t d o m a i n s w i t h i n d i v i d u a l i z e d f u n c t i o n s : a) a l i g a n d b i n d i n g d o m a i n at the N - t e r m i n u s c o n t a i n i n g c o m p l e m e n t -t y p e r epea t s i n v o l v e d i n L D L b i n d i n g ; b) a n e p i d e r m a l g r o w t h fac to r ( E G F ) p r e c u r s o r - h o m o l o g y r epea t s tha t c o n t a i n Y W T D ( T y r o s i n e - T r y p t o p h a n - T h r e o n i n e -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 d o m a i n a c t i n g as a space r ; d ) m e m b r a n e - s p a n n i n g d o m a i n f o r a n c h o r a g e ; a n d e) a 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 the N P x Y ( A s p a r a g i n e - P r o l i n e - X - T y r o s i n e ) m o t i f , 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 r e c e p t o r i s c o n s t r u c t e d f r o m a v a r i e t y o f 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 i ts g e n e h a s b r o a d e n e d i t s 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 r ecep to r s , 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 are n i n e m e m b e r s o f the f a m i l y w h i c h i n c l u d e the L D L r e c e p t o r , L D L r e c e p t o r - r e l a t e d p r o t e i n ( L R P ) , m e g a l i n , V L D L r e c e p t o r , 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-l inked sugar domain NPxY motif tVieqalin LDL Receptor NH, Figure 5: Structural diagram of two members of the LDL receptor family. Adapted from Nykjaier A et al (2002), Trends Cell Biol 12: 273-80 [85]. 17 s e v e n h a v i n g b e e n i d e n t i f i e d i n m a m m a l s . E a c h m e m b e r o f t h i s r e c e p t o r f a m i l y u n d e r g o e s the p r o c e s s o f 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 ; y e t e a c h m e m b e r i s e x p r e s s e d i n a n u m b e r o f d i f f e r en t t i s sues a n d h a s a w i d e r a n g e o f d i f f e r en t l i g a n d s w h i c h are n o t spec i f i c to the 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 [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 , V 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]. A 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 the L D L r e c e p t o r f a m i l y tha t a re 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 c e l l s i s 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 o f the L D L r e c e p t o r s u p e r f a m i l y [55,85]. 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 sues , b u t m a i n l y i n the 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 the 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) as a n a u t o a n t i b o d y tha t c i r c u l a t e d i n ra ts 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 the 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 to a p o B - 1 0 0 [18,119]. M a p p i n g o f i ts 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 i ts a b i l i t y to a l so 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 , a p r o t i n i n a n d R A P [86]. T h e r e f o r e , l i g a n d s o f m e g a l i n i n c l u d e A p o B , A p o E , c l u s t e r i n , p ro t ea se s a n d p ro t e a se i n h i b i t o r s , c a r r i e r p r o t e i n s f o r l i p o p h i l i c v i t a m i n s , p a r a t h y r o i d h o r m o n e , p o l y b a s i c d r u g s ( a m i n o g l y c o s i d e s ) a n d 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 s u g g e s t the i m p o r t a n c e o f t h i s r e c e p t o r i n r e t r i e v a l 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 i s w e l l e s t a b l i s h e d tha t C s A assoc ia tes w i t h l i p o p r o t e i n s [8,114,130]. 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 t r i g l y c e r i d e -c o n t a i n i n g l i p o p r o t e i n s i n the b l o o d [51]. C s A p r e d o m i n a n t l y assoc ia tes 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 has b e e n r e p o r t e d t ha 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 lso 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 ) f a c i l i t a t e d C E a n d T G t rans fe r a c t i v i t y b u t n o t P C [63,131,134]. I n a d d i t i o n , m o d i f i c a t i o n o f the l i p o p r o t e i n su r face c h a r g e w i t h a n i n c r e a s e d n e g a t i v e c h a r g e r e s u l t e d i n g r ea t e r p e r c e n t a g e o f [ 3 H ] C s A r e c o v e r e d w i t h i n t h e L D L s u b t r a c t i o n 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 (P l ) - t r e a t ed r a b b i t p l a s m a t h a n c o n t r o l p l a s m a [133]. It h a s b e e n r e p o r t e d tha t c h a n g e s i n the l i p i d c o n c e n t r a t i o n a n d c o m p o s i t i o n o f p l a s m a l i p o p r o t e i n s c a n a l t e r t he p r o f i l e o f C s A - l i p o p r o t e i n a s s o c i a t i o n , 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 the t o x i c i t y o f C s A [67,127,128]. D e c r e a s e d effects o f C s A h a v e b e e n r e p o r t e d w i t h h y p e r t r i g l y c e r i d e m i a [25,82] a n d i n c r e a s e d t o x i c effects o f C s A w i t h h y p o c h o l e s t e r o l e m i a [24]. A n 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 the d r u g w a s b o u n d to L D L [67] b u t w a s n o t e v i d e n t w h e n the d r u g w a s b o u n d to 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 ra t k i d n e y m o d e l , the i n t e r a c t i o n o f 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 the r e n a l c l e a r a n c e a n d the ex ten t o f r e n a l t i s sue d i s t r i b u t i o n o f C s A [121]. A r ecen t s t u d y r e p o r t e d tha t t he t o t a l s e r u m c h o l e s t e r o l a n d L D L l e v e l s a f fec ted the c l i n i c a l 19 ef f icacy 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 c h o l e s t e r o l c o n c e n t r a t i o n s , L D L c h o l e s t e r o l c o n c e n t r a t i o n s a n d 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 tha t the h i g h e r the c h o l e s t e r o l , the l o w e r the e f f i cacy o f 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 b y Sco 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]. I n p l a s m a , a l m o s t 9 0 % o f C s A is b o u n d to l i p o p r o t e i n s w i t h the r e m a i n i n g b o u n d to p l a s m a p r o t e i n s [114]. 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 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 -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 the r e m a i n i n g b o u n d to e r y t h r o c y t e s [51] . O f the t o t a l a m o u n t o f 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 less 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 i n p l a s m a o f r e n a l t r a n s p l a n t p a t i e n t is i n the r a n g e o f 0.04 to 0.12 [65]. B i o a v a i l a b i l i t y o f the d r u g is v a r i a b l e r a n g i n g f r o m 8% to 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 the 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 re v a r i a b l e w i t h d i f f e r en t f o r m u l a t i o n s o f the d r u g . It h a s b e e n r e p o r t e d t ha t h i g h e r A U C v a l u e s are 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]. E l i m i n a t i o n o f the d r u g i s p r e d o m i n a n t l y d u e to h e p a t i c m e t a b o l i s m r a t h e r t h a n r e n a l e l i m i n a t i o n [30]. T h e 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 state a n d c l e a r a n c e o f c y c l o s p o r i n e a re u s u a l l y h i g h e r i n t r a n s p l a n t p a t i e n t s c o m p a r e d to h e a l t h y i n d i v i d u a l s [30]. 20 Parameter Value Bioavai lab i l i ty 8%-60% Prote in B i n d i n g 90% to l ipoprote ins Clearance 0.15-0.7 L / h / k g Vss (volume of d is t r ibut ion at steady state) 1.8-13.8 L / k g 11/2 P (elimination) 2.9 -15 .8 h Table 3: Pharmacokinet ic characteristics of C s A . M o d i f i e d f rom references [113,114,130]. 1.4.2. Interaction of CsA with LDL receptor A few investigators have suggested that C s A m a y be affecting L D L receptor act ivi ty subsequently causing 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) hypothes ized that L D L not on ly functions as an impor tant carrier of cyclosporine i n p lasma but also can facilitate transport of C s A into cells by the L D L receptor [23]. Unfortunately, no experimental results were reported to p rov ide evidence for this hypothesis. Fur thermore, Sanghv i et al (1989) proposed that cel lular uptake of C s A may occur th rough the L D L receptor by specific and nonspecific pa thways [109]. They observed that w h e n C s A was complexed to L D L , C s A internal iza t ion was higher than the C s A alone group i n h u m a n lymphocytes [109]. A pi lo t s tudy i n nephrotic patients found that both total se rum cholesterol and LDL-cho les te ro l 21 l e v e l s w e r e s i g n i f i c a n t l y c o r r e l a t e d w i t h s u p p r e s s i v e e f f i cacy o f c y c l o s p o r i n e a g a i n s t p e r i p h e r a l b l o o d l y m p h o c y t e ( P B L ) b l a s t o g e n e s i s [51]. T h e a u t h o r s h y p o t h e s i z e d tha t i n c r e a s e d r e n a l graf t f a i l u r e i n r e c i p i e n t s 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 o f c y c l o s p o r i n e e f f icacy t h r o u g h i ts 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]. 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) r e p o r t e d tha t the u p t a k e a n d t o x i c i t y o f C s A w e r e s i g n i f i c a n t l y r e d u c e d w i t h 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 ce l l s [91]. T h e a u t h o r s h y p o t h e s i z e d tha t t h i s effect m a y be a l s o d u e to the d o w n r e g u l a t i o n o f the 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 s u g g e s t e d tha t C s A c o u l d dec rease L D L i n t e r n a l i z a t i o n w i t h o u t a l t e r i n g the a f f i n i t y o f i ts b i n d i n g to the 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 ce l l s [129]. 22 CHAPTER II: Summary of (Research (Project 23 Overall Objective The overa l l objective was to elucidate the role of the L D L receptor i n media t ing the uptake and toxici ty of C s A i n a renal p r o x i m a l tubule cel l l ine, L L C -P K i . 2.1 Hypothesis It was hypothes ized that w h e n L D L receptor act ivi ty was decreased u p o n IgG-C 7 treatment, both C s A uptake and toxici ty were reduced i n L L C - P K i cells. 2.2 Specific Aims There were three specific a ims to test the hypothesis: Aim 1: To determine whether L L C - P K i cells were a suitable cel l m o d e l to s tudy L D L receptor activity. Aim 2: T o establish the assay methods and condi t ions requi red w i t h IgG-C7, a monoc lona l ant ibody to the L D L receptor i n L L C - P K i cells. Aim 3: T o implement these monoc lona l ant ibody assay methods to investigate both C s A uptake and toxici ty i n L L C - P K i cells. 2.3 Rationale In A i m 1, it was cri t ical to determine that L L C - P K i cells were a suitable cell m o d e l to s tudy L D L receptor fami ly act ivi ty before any inferences c o u l d be made w h e n C s A was present. Based on the data generated f rom A i m 1, the appropriate 24 as say 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 w a s e s t a b l i s h e d i n A i m 2 i n c l u d i n g o p t i m a l c o n c e n t r a t i o n a n d p r e i n c u b a t i o n t i m e . F i n a l l y , o n c e the a n t i b o d y a s say 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 ce l l s , the m e t h o d w a s a p p l i e d i n the p r e s e n c e o f C s A to i n v e s t i g a t e 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 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 c a u s e n e p h r o t o x i c i t y i n pa t i en t s 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 a d m i n i s t r a t i o n o f C s A t h e r a p y . T h e e f f i cacy a n d t o x i c i t y o f 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 o f the pa t i en t . I n a d d i t i o n , k n o w l e d g e a b o u t C s A - i n d u c e d n e p h r o t o x i c i t y m a y be a p p l i c a b l e to o t h e r h y d r o p h o b i c d r u g s tha t c ause r e n a l d y s f u n c t i o n . N o v e l d r u g d e l i v e r y s y s t e m s w i t h C s A c o u l d be d e s i g n e d i n o r d e r to a v o i d t o x i c i t y a n d m a i n t a i n i t s e f f i cacy . 25 CHAPTER III: Materials and'^agents 3.1 Materials 3.1.1. Cells and cell culture P i g k idney cells ( L L C - P K i ) and h u m a n hepatoma cells (HepG2) were purchased f rom A m e r i c a n Type Cu l tu re Co l l ec t ion ( A T C C ) (Wanassas, V A , U S A ) . D M E M : H a m ' s F12 (1:1) culture m e d i u m , D M E M culture m e d i u m , Dulbecco 's phosphate buffered saline, penic i l l in-s t reptomycin (10 ,000U/ml pen ic i l l in , ), t r y p s i n - E D T A (0.25% t ryps in , I m M E D T A - 4 N a and 0.05% t ryps in , 0 . 5 m M E D T A - 4 N a ) , s o d i u m pyruvate , and fetal calf se rum ( U S A source) were a l l purchased f rom G i b c o B R L (Grand Island, N Y , U S A ) . Sterile steritop 0.22pm express membrane bottle top filters were purchased f rom M i l l i p o r e (Bedford, M A , U S A ) . Sterile 50ml centrifuge tubes, disposable 10 and 25ml serological pipettes and 100mm petr i dishes were purchased f rom Starstedt (Montrea l , P Q , Canada) . A n g l e -vented neck T-75 sterile cel l culture flasks, sterile 24-wel l surface-treated plates and 0.45 p m med ia glass bottles were purchased f rom V W R (Edmonton , A B , Canada) . 3.1.2. Lipoproteins, Chemicals and Labelled Drug Pur i f i ed low-dens i ty l ipopro te in ( L D L ) , tissue-culture tested bovine se rum a l b u m i n (BSA) , s o d i u m bicarbonate ( N a H C O s ) , s o d i u m hydrox ide ( N a O H ) were purchased f rom S igma C h e m i c a l co. (St. Lou i s , M O , U S A ) . Tr is Base, SDS, glycine, T E M E D , (3-mercaptoethanol, a m m o n i u m persulfate, Tween-20, acetic acid , Ponceau S and protease inhibi tor cocktai l were also purchased f rom S i g m a C h e m i c a l co. (St. 27 L o u i s , M O , U S A ) . A c r y l a m i d e , B i s a c r y l a m i d e , L a e m r n l i s a m p l e bu f fe r a n d K a l e i d o s c o p e ® p r e s t a i n e d p r o t e i n s t a n d a r d s w e r e p u r c h a s e d f r o m B i o r a d ( H e r c u l e s , C A , U S A ) . M e t h a n o l ( H P L C g r a d e ) , i s o p r o p a n o l ( H P L C g r a d e ) a n d P a s t e u r ® p i p e t t e s w e r e p u r c h a s e d f r o m F i s c h e r ( N e p e a n , O N , C a n a d a ) . R a d i o l a b e l e d c y c l o s p o r i n e A ([mebmt-fi-3H]cyc\osporme A , 2 5 0 C i / m l i n e t h a n o l ) , H y p e r f i l m ® E C L a n d c h e m i l u m i n e s c e n c e r eagen t s w e r e p u r c h a s e d f r o m A m e r s h a m P h a r m a c i a B i o t e c h ( O a k v i l l e , O N , C a n a d a ) a n d D i l - L D L ( 3 , 3 ' - d i o c t a d e c y l i n d o c a r b o c y a n i n e - l o w d e n s i t y l i p o p r o t e i n ) w a s 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 ( E u g e n e , O R , U S A ) . 3.1.3. Antibodies and Assay Kits 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 ( I g G - C 7 ) w a s p u r c h a s e d f r o m R e s e a r c h D i a g n o s t i c s Ins t i tu te ( 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 H R P c o n j u g a t e w a s p u r c h a s e d f r o m J a c k s o n I m m u n o R e s e a r c h L a b o r a t o r i e s ( W e s t g r o v e , P A , U S A ) . M E M - P E R ® E u k a r y o t i c 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 R e a g e n t k i t a n d B C A P r o t e i n A s s a y k i t w e r e p u r c h a s e d f r o m P i e r c e B i o t e c h n o l o g y ( R o c k f o r d , I L , U S A ) . C h o l e s t e r o l s t a n d a r d a n d r e a g e n t w a s p u r c h a s e d f r o m T h e r m o D M A ( L o u i s v i l l e , C O , U S A ) . C y t o t o x 9 6 ® 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 w a s p u r c h a s e d f r o m P r o m e g a C o r p o r a t i o n ( M a d i s o n , W l , U S A ) . 28 3.2 Tissue Culture Reagents and Medium 3.2.1 Preparation ofLLC-PKi media S t e r i l e 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 o f d i s t i l l e d w a t e r to D M E M : H a m ' s F 1 2 1:1 p r e p a c k a g e d p o w d e r . A p p r o x i m a t e l y 1.2 g o f N a H C C b w a s d i s s o l v e d i n the m e d i a w h i l e the p H w a s a d j u s t e d to a b o u t 7.3 w i t h 1 M N a O H . T h e m e d i a s o l u t i o n w a s t o p p e d to a f i n a l v o l u m e o f 9 0 0 m l , m i x e d a n d s te r i l e f i l t e r e d t h r o u g h a 0 . 2 2 p m s t e r i t o p f i l t e r i n t o t w o 5 0 0 m l m e d i a bo t t l e s . 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 . T o c o m p l e t e the 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 fe ta 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 S t e r i l e 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 o f d i s t i l l e d w a t e r to D M E M p r e p a c k a g e d p o w d e r . 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 the m e d i a w h i l e the p H w a s a d j u s t e d to a b o u t 7.3 w i t h 1 M N a O H . T h e m e d i a s o l u t i o n w a s t o p p e d to a f i n a l v o l u m e o f 9 0 0 m l , m i x e d a n d s t e r i l e f i l t e r e d t h r o u g h a 0 . 2 2 p m s t e r i t o p f i l t e r i n t o t w o 5 0 0 m l m e d i a bo t t l e s . 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 fo r 40 m i n u t e s . T o c o m p l e t e the 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 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 4 0 m l (5%) o f h e a t - i n a c t i v a t e d F B S w e r e a d d e d a n d s t o r e d at 4 ° C . 29 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 . T o p r e p a r e 450 m l o f s e r u m - f r e e m e d i a , 0 .9g (0.2%) o f B S A a n d 4 . 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 w e r e a d d e d . T h e c o n c e n t r a t i o n o f 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]. R e p e t i t i v e m i x i n g w i t h the p i p e t t o r w a s n e c e s s a r y to e n s u r e f u l l d i s s o l u t i o n o f the m i x t u r e . T h e m e d i a w e r e s t o r e d o v e r n i g h t at 4 ° C p r i o r to e x p e r i m e n t a t i o n . 3.2.4. Preparation of Phosphate Buffered Saline (PBS) S t e r i l e 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 o f d i s t i l l e d w a t e r to p r e p a c k a g e d D u l b e c c o ' s P B S p o w d e r . T h e p H o f the s o l u t i o n w a s a d j u s t e d to 7.3, t o p p e d to 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 bo 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 ce 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 f reezer fo r L L C - P K i ce l l s a n d H e p G 2 ce l l s , r e s p e c t i v e l y , p r i o r to use . T o e s t a b l i s h the c u l t u r e , o n e c y r o v i a l w a s r e m o v e d , p l a c e d i n t o a 5 0 m l c e n t r i f u g e f i l l e d w i t h 70% e t h a n o l a n d t h a w e d i n a 3 7 ° C w a t e r b a t h for a p p r o x i m a t e l y 5 m i n u t e s . O n c e c o m p l e t e , c e l l s w e r e c a r e f u l l y t r a n s f e r r e d to s te r i l e c u l t u r e f l a s k o r 1 0 0 m m p e t r i d i s h c o n t a i n i n g p r e - w a r m e d c o m p l e t e m e d i a a n 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 3.2.6. Subculturing of LLC-PKi cells Once confluence was reached, the m e d i u m was d iscarded f rom the flask and r insed three times w i t h 8 m l of p r ewarmed sterile Dulbecco 's PBS. Wash ings were discarded and cells were treated w i t h 8 m l of p r e w a r m e d t ryps in . Once the cells were dissociated, the mix ture was poured into a sterile 50ml centrifuge tube. The flask was washed three times w i t h sterile Dulbecco 's P B S and the washings transferred to the centrifuge tube. Cel l s were pelleted d o w n at 1000 rotations per minute (rpm) for about 5-8 minutes and the supernatant was decanted. The cel l pellet was resuspended w i t h a v o l u m e of complete m e d i a accord ing to a decided spli t ratio. For example, one w o u l d resuspend i n 8 m l of complete m e d i a for a 1:8 spli t for confluence i n 3 days. One mi l l i l i t e r of the cel l mix ture was added to a new flask w i t h 25ml of complete med ia and placed i n the incubator at 37°C i n humid i f i ed 5% CO2. The m e d i u m was changed every 48 hours un t i l cells were confluent and ready for subcul tur ing. 3.2.7. Subculturing of HepG2 cells Once confluence was reached i n petr i dishes, the m e d i u m was discarded and r insed three times w i t h 7 m l of p r ewarmed sterile Dulbecco 's PBS . Wash ings were discarded and cells were treated w i t h 5 m l of p r e w a r m e d t ryps in . Once the cells were dissociated, the mix ture was poured into a sterile 50ml centrifuge tube. The flask was washed three times w i t h sterile Dulbecco 's P B S a n d the washings were transferred to a centrifuge tube. Cel l s were 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 d e c a n t e d . T h e c e l l p e l l e t 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 o f 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 r a t i o . F o r e x a m p l e , o n e w o u l d r e s u s p e n d i n 10 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 d a y s . 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 o f c o m p l e t e m e d i a . T h e d i s h w a s s h a k e n fo r a c o u p l e o f m i n u t e s i n o r d e r to d i s p e r s e the ce l l s o v e r the g r o w t h a r ea a n d p l a c e d i n the i n c u b a t o r a t 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 c e l l s w e r e c o n f l u e n t a n d r e a d y fo r s u b c u l t u r i n g a g a i n . 3.2.8. Seeding plates and preparing LLC-PKi and HepG2 cells for Experimentation P e l l e t e d ce l l s w e r e r e s u s p e n d e d 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 3.2.4. A n a l i q u o t o f the c e l l 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 c a l c u l a t e a s e e d i n g d e n s i t y o f 7.0 x 1 0 4 c e l l s / c m 2 fo r L L C - P K i c e l l s a n d 5.0 x 1 0 5 c e l l s / c m 2 fo r H e p G 2 ce l l s i n 2 4 - w e l l p l a t e s . T h e ce l l s w e r e a l l o w e d to p r o p a g a t e fo 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 , c e l l s 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 o f 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 . A t t h i s p o i n t , the L D L r e c e p t o r s w e r e u p r e g u l a t e d [40] a n d r e a d y fo r e x p e r i m e n t a t i o n . 32 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 4-aminoantipyrine (4AAP), catalyzed by peroxidase (POD) forms a quinoneimine dye with peak absorbance at 500nm [122]. CE Cholesterol esters + H 2 O —• Cholesterol + Fatty Acids c o Cholesterol + O2 —*• Cholest-4-en-3-one + H2O2 POD 2 H 2 O 2 + HBA + 4AAP Quinoneimine Dye + 4 H 2 O 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 o f 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 c o n c e n t r a t i o n o f 2 0 0 p g / m l c h o l e s t e r o l c o n c e n t r a t i o n w a s u s e d f o r e x p e r i m e n t a t i o n . 3.3.2. Solutions with Dil-LDL D i l - L D L ( 3 , 3 ' - d i o c t a d e c y l i n d o c a r b o c y a n i n e - l o w d e n s i t y l i p o p r o t e i n ) w a s 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 (Ca t#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 a t 4 ° C . P r i o r t o e x p e r i m e n t a t i o n , D i l - L D L w a s c e n t r i f u g e d at 3000 r p m i n a m i c r o c e n t r i f u g e fo r 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 . S u b s e q u e n t l y , the s t o c k 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 at l m g / m l w a s d i l u t e d to a w o r k i n g s o l u t i o n o f 5 0 0 p g / m l i n s e r u m - f r e e m e d i a fo r 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 r e c e p t o r , w a s p u r c h a s e d f r o m R e s e a r c h D i a g n o s t i c Ins 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 fo 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 . A w o r k i n g s o l u t i o n o f 2 5 p g / m l i n s e r u m - f r e e m e d i a w a s u s e d p r i o r to e x p e r i m e n t a t i o n . F o r i m m u n o b l o t t i n g , a 0.2 p g / m l 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 buf fe r w a s p r e p a r e d a n d s t o r e d at 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 s o l u t i o n o f t r i t i a t e d c y c l o s p o r i n e A w a s m a d e b y d i l u t i n g s t o c k [ 3 H ] C s A (spec i f ic a c t i v i t y 7 . 4 8 u C i / p g ) i n s e r u m - f r e e m e d i a to a c o n c e n t r a t i o n o f 3 2 u g / m l . T o p r e p a r e [ 3 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 o f 8 0 0 n g / m l a n d a n L D L c h o l e s t e r o l c o n c e n t r a t i o n o f 2 0 p g / m l , 7 . 5 p l o f [ 3 H ] C s A w o r k i n g s o l u t i o n a n d 3 0 p l o f L D L w o r k i n g s o l u t i o n w e r e m i x e d t oge the r p e r t r e a t m e n t 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 of Reagents for 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 . 5 M p H = 8 . 8 ; T r i s - H C l 0 . 5 M p H = 6 . 8 ; A c r y l a m i d e / B i s ( 30%T, 0 . 8 % C ) ; 1 0 % ( w / v ) S D S . T r i s - H C l 1 . 5 M p H = 8 . 8 w a s p r e p a r e d b y a d d i n g 2 7 . 2 3 g o f T r i s B a s e i n 8 0 m l o f d H 2 0 , a d j u s t i n g the p H w i t h 1 M H C 1 to 8.8 a n d b r i n g i n g the v o l u m e u p to 1 5 0 m l . T r i s - H C l 0 . 5 M p H = 6 . 8 w a s p r e p a r e d b y m i x i n g 6 g o f T r i s B a s e i n 6 0 m l o f d H i O , a d j u s t i n g the p H w i t h 1 M H C 1 to 6.8 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 . 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 2 9 . 2 g o f 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 o f 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 . T h e s o l u t i o n w a s s t o r e d at 4 ° C i n a bo t t l e w r a p p e d i n a l u m i n u m f o i l . 1 0 % S D S w a s p r e p a r e d b y a d d i n g l O g o f S D S w i t h 9 0 m l o f d H 2 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 . T E M E D w a s u s e d f r o m the m a n u f a c t u r e r . 1 0 % A m m o n i u m P e r s u l f a t e ( A P S ) w a s p r e p a r e d 35 fresh by a d d i n g 20mg of A P S i n 200ul of d h b O . A stock so lu t ion of 5 X runn ing buffer (pH=8.3) consis t ing of 15g of Tr is Base, 72g of g lycine and 5g of S D S i n I L of d h b O was prepared and stored at 4°C. D u r i n g an electrophoresis procedure, the 5X solu t ion was d i lu ted to a I X solu t ion i n d h b O . 3.4.2. Immunoblot and Chemiluminescen.ee detection A I X transfer buffer was prepared by a d d i n g 6.6g (25mM) of Tr is Base, 28.8g of glycine, 20ml of 10% S D S solut ion, 400ml (20%) methanol a n d b r ing ing the remain ing v o l u m e to 2L w i t h dPfcO. The so lu t ion was stored at 4°C. 10% Tween-20 so lu t ion was prepared by a d d i n g 10ml Tween-20 i n 90ml of d l rbO. 10X P B S was prepared by m i x i n g 2.03g of s o d i u m phosphate monobasic monohydra te (NaH2P04*H20), 11.49g of s o d i u m phosphate dibasic anhydrous (Na2HPC>4), and 85g of s o d i u m chlor ide. The p H of the so lu t ion was adjusted to 7.3. The remain ing v o l u m e was added w i t h d h k O to I L and f inal ly , stored at r o o m temperature. A liter of w a s h buffer was prepared by 5 m l (0.05%) of 10% Tween-20 and the remain ing v o l u m e was added w i t h d F h O to I L w i t h 1XPBS. The so lu t ion was stored at 4°C. Fifty mi l l i l i te rs of the ant ibody d i l u t i on buffer was prepared w i t h 0.25g (0.5%) B S A , 0.5ml (0.05%) of 10% Tween-20 so lu t ion and I X PBS. A pro te in concentration of 0 . 2 p g / m l of IgG-C7 (the p r imary antibody) was prepared w i t h the ant ibody d i l u t i o n buffer and stored at 4°C for cont inuous use. 10X Ponceau S was made by d i s so lv ing l g of the dye i n 100ml of d H 2 0 . One hund red mi l l i l i t e rs of a 0.1% Ponceau S / 5 % acetic ac id so lu t ion was prepared by d i l u t i n g 10X Ponceau S to I X so lu t ion i n d F b O 36 w i t h 5 m l of acetic ac id . B l o c k i n g buffer was prepared fresh consis t ing of 1.5g (3%) s k i m m i l k p o w d e r and 0.25g (0.5%) B S A , a n d the r ema in ing v o l u m e was topped to 50ml w i t h I X PBS. Developer and fixer solutions were d i lu t ed w i t h tap water according 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 to s u i t c o n d i t i o n s f o r L L C - P K i ce l l s . A f t e r 48 h o u r s p o s t - s e e d i n g , ce l l s 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 (see s e c t i o n 3.2.8). 4.1.1. Protein Content Determination P r o t e i n c o n t e n t w a s d e t e r m i n e d i n l y s e d ce l l s i n o r d e r to n o r m a l i z e the da t a . 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 u t i l i z e s a d e t e r g e n t - c o m p a t i b l e f o r m u l a t i o n f o r the c o l o r i m e t r i c d e t e c t i o n a n d q u a n t i t a t i o n o f t o t a l 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 2 + ) b e c o m i n g r e d u c e d to c u p r o u s i o n s ( C u 1 + ) b y p r o t e i n i n a n a l k a l i n e m e d i u m . S u b s e q u e n t l y , c u p r o u s c a t i o n s a re d e t e c t e d w i t h h i g h s e n s i t i v i t y a n d s e l e c t i v i t y b y a c o l o r i m e t r i c r e a c t i o n 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 o f t w o m o l e c u l e s o f B C A w i t h o n e c u p r o u s i o n f o r m s a p u r p l e - c o l o u r e d w a t e r s o l u b l e p r o d u c t tha t e x h i b i t s m a x i m u m a b s o r b a n c e at 5 6 2 n m [93]. T h e m a c r o m o l e c u l a r s t r u c t u r e o f p r o t e i n , t he n u m b e r o f p e p t i d e b o n d s a n d the p r e s e n c e o f c y s t e i n e , c y s t i n e , 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 be 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 s c h e m a t i c 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 . P r o t e i n ( p e p t i d e b o n d s ) + C u 2 + -> t e t r a d e n t a t e - C u 1 + c o m p l e x C u 1 + + 2 B i c i n c h o n i n i c A c i d ( B C A ) - » B C A - C u 1 + c o m p l e x 39 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 2 m g / m l . 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 ce l l s , 25 p i o f b o t h s t a n d a r d s a n d c e l l s a m p l e s w e r e a l i q u o t e d i n t o a 9 6 - w e l l n o n s t e r i l e m i c r o t i t e r p l a t e . T h e t o t a l 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 r e a g e n t p e r s a m p l e ) T h e 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 b y m i x i n g 50 p a r t s o f R e a g e n t A w i t h 1 p a r t o f 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 the B C A P r o t e i n a s say 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 o f the w o r k i n g r e a g e n t w a s a l i q u o t e d i n t o e a c h w e l l w i t h a m u l t i - c h a n n e l p i p e t t o r a n d the p l a t e 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 , i t 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 the i n c u b a t o r at 3 7 ° C fo r 30 m i n u t e s . A f t e r i n c u b a t i n g , the p l a t e w a s a l l o w e d to c o o l t o r o o m t e m p e r a t u r e a n d the p e a k a b s o r b a n c e w a s r e a d w i t h a M u l t i s k a n A s c e n t p l a t e r e a d e r at 5 6 2 n m . P r o t e i n c o n c e n t r a t i o n s o f e a c h c e l l s a m p l e w e r e d e t e r m i n e d a g a i n s t the s t a n d a r d c u r v e . 4.1.2. Fluorescence Determination ofDil 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 b y f i r s t 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 ce l l s w i t h th ree i c e - c o l d 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 o f 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 fo r 15 m i n u t e s . T h e I P A w a s h i n g s w e r e t r a n s f e r r e d to m i c r o c e n t r i f u g e tubes a n d s p u n f o r 15 m i n u t e s at 3 0 0 0 r p m . T h e p u r p o s e o f the c e n t r i f u g a t i o n s tep w a s to r e m o v e L D L agg rega t e s w h i c h c a n 40 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 ice-cold 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 Dil-LDL 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 One plate of cells was incubated w i t h increasing concentrations of D i l - L D L f rom the range 5 p g / m l to 4 0 p g / m l i n the presence of a constant concentrat ion of 25-fo ld excess of unlabel led L D L to measure non-specific b i n d i n g . Concent ra t ion of unlabel led L D L was determined based o n prote in content as indicated by the manufacturer ( 5mg /ml ) . Ano the r plate of cells was incubated w i t h the same increasing concentrations of D i l - L D L i n the absence of unlabel led L D L to measure total b ind ing . Bo th plates were covered w i t h a l u m i n u m foi l a n d incubated i n the fridge at 4°C for 2 hours. After incubat ion, fluorescence was determined as described i n section 4.1.2. Specific b i n d i n g was calculated as the difference between total and nonspecific b ind ing . 4.1.5. Competitive Binding Cel l s were incubated w i t h a constant concentrat ion of l O p g / m l of D i l - L D L i n the presence of increasing concentrations of unlabel led L D L at 10 p g / m l - 160pg / m l for 2 hours at 37°C covered i n a l u m i n u m foi l . Concentrat ions of unlabel led L D L were based on prote in content as indica ted by the manufacturer (5mg/ml ) . Subsequently, fluorescence was determined as described i n section 4.1.2. 42 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 ce l l s w e r e g r o w n i n T 7 5 f l a s k s a n d 1 0 0 m m p e t r i d i s h e s , r e s p e c t i v e l y , i n c o m p l e t e m e d i a . T w e n t y - f o u r h o u r s p r i o r to 100% c o n f l u e n c e , the m e d i u m w a s c h a n g e d a n d r e p l a c e d w i t h s e r u m - f r e e m e d i a to u p r e g u l a t e the 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 as 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. T h r e e m i l l i l i t e r s o f s t e r i l e D u l b e c c o ' s P B S w a s a d d e d a n d the p e l l e t w a s r e s u s p e n d e d . C e l l s w e r e c o u n t e d u s i n g a h e m o c y t o m e t e r 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 . A p p r o x i m a t e l y 5 x 1 0 6 L L C - P K i ce l l s a n d 8 x l 0 6 H e p G 2 ce l l s p e r 2 m l w e r e a l i q u o t e d i n t o m i c r o c e n t r i f u g e tubes a n d p e l l e t e d a g a i n at l O O O r p m a n d 2 0 0 0 r p m , r e s p e c t i v e l y . T h e s u p e r n a t a n t w a s c a r e f u l l y r e m o v e d . R e a g e n t s 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 as p a r t o f the 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 o f R e a g e n t A w a s a d d e d to the c e l l p e l l e t s t o l y s e the c e l l s , a n d p i p e t t e d u p a n d d o w n i n o r d e r to o b t a i n a h o m o g e n o u s c e l l s u s p e n s i o n . A t t h i s s tage, 5 p l o f p ro t ea se i n h i b i t o r c o c k t a i l w a s a d d e d to the m i x t u r e . T h e c e l l m i x t u r e w a s 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 fo r 10 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 i t w a s p l a c e d o n ice . T w o p a r t s o f 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 p a r t R e a g e n t 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 4 5 0 p l o f R e a g e n t B / C w a s a d d e d to e a c h t u b e o f l y s e d ce l l s 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 fo r 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 p r o t e i n s . A f t e r 30 m i n u t e s , t ubes w e r e c e n t r i f u g e d at 43 10,000 g fo r 3 m i n u t e s w i t h a n E p p e n d o r f 5 4 1 5 D 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 . T h e s u p e r n a t a n t w a s t r a n s f e r r e d to m i c r o c e n t r i f u g e tubes a n d i n c u b a t e d fo r a n a d d i t i o n a l 10 m i n u t e s at 3 7 ° C to sepa ra t e the h y d r o p h o b i c p r o t e i n s f r o m the h y d r o p h i l i c p r o t e i n s t h r o u g h p h a s e p a r t i t i o n i n g . T u b e s w e r e a g a i n c e n t r i f u g e d w i t h the 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 fo r 2 m i n u t e s at 10,000 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 (bo t tom) l a y e r a n d d i s c a r d e d . T h e b o t t o m p h a s e c o n t a i n i n g the m e m b r a n e p r o t e i n s w a s p l a c e d o n ice u n t i l s t o r a g e at - 2 0 ° C . A p r o t e i n a s say w a s c o n d u c t e d i n e a c h c e l l 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 c o n t e n t 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) T h e M I N I - P R O T E A N 3 C e l l a p p a r a t u s w i t h g l a s s p l a t e s a n d the p o w e r 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 ( H e r c u l e s , C A , U S A ) w e r e the e q u i p m e n t u s e d f o r the e l e c t r o p h o r e s i s . G l a s s p l a t e s o f t h i c k n e s s 1 . 0 m m w e r e r i n s e d w i t h d H 2 0 a n d d r i e d w i t h K i m w i p e s ® . 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 ge 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 3 0 % A c r y l a m i d e / B i s 2.5 m l d H 2 0 4.9 m l 1 0 % ( w / v ) S D S 0.1 m l 1 0 % A P S 50 p i T E M E D 5 p i 44 T h e g e l 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 g l a s s p l a t e s u n t i l the c o m b f ron t . T h e g e l 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 f o r i t t o p o l y m e r i z e fo r 1 h o u r o r o v e r n i g h t . 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 ge 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 3 0 % A c r y l a m i d e / B i s 0.6 m l d H 2 0 3.1 m l 1 0 % ( w / v ) S D S 50 p i 10% A P S 25 p i T E M E D 5 p i T h e s t a c k i n g g e l w a s c a r e f u l l y p o u r e d o n t o p o f the r e s o l v i n g g e l w i t h a P a s t e u r ® p i p e t t e . T h e 1 0 - w e l l c o m b w a s i n s e r t e d 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 s a m p l e bu f fe 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 : buf fe r ) . S a m p l e s w e r e b o i l e d at 9 5 ° C f o r 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 f i l t e r p a p e r a n d the g l a s s p l a t e s w e r e a s s e m b l e d i n the e l e c t r o d e a p p a r a t u s . I X r u n n i n g bu f fe r 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 o f p r e s t a i n e d 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 g e l l o a d i n g t i p s . 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 c o n s t a n t a m o u n t o f p r o t e i n w a s n e e d e d f o r e a c h s a m p l e . T h e e l e c t r o d e a p p a r a t u s w a s p l a c e d i n the t a n k a n d c o n n e c t e d to the p o w e r s u p p l y . 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 c o n s t a n t c u r r e n t o f 1 0 m A u n t i l i t r e a c h e d the b o r d e r o f 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 to 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 the d u r a t i o n 45 w a s a b o u t 70 m i n u t e s . S u b s e q u e n t l y , the e l e c t r o d e a s s e m b l y a n d p l a t e s w e r e d i s a s s e m b l e d , a n d the s e p a r a t i n g g e l w a s d i s c a r d e d . T h e t o p r i g h t h a n d o f 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 t r ans fe r b u f f e r r e a d y to p r o c e e d to i m m u n o b l o t t i n g . 4.2.3. Immunoblotting and Detection F i l t e r p a p e r s a n d f ibe r p a d s w e r e s o a k e d i n I X t r ans fe r b u f f e r 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 f o r 20 m i n u t e s , r i n s e d w i t h d F k O a n d s o a k e d i n I X t rans fe r bu f f e r p r i o r to use . F i l t e r p a p e r w a s s l i d c a r e f u l l y 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 f i l t e r p a p e r s a n d f i b e r p a d s i n the casset te w i t h the g e l f a c i n g the a n o d e . B u b b l e s b e t w e e n the g e l 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 o r d e r t o e n s u r e p r o p e r t rans fe r o f p r o t e i n s f r o m the g e l t o the m e m b r a n e . T h e g e l casset tes w e r e l o c k e d a n d p l a c e d i n c o r r e c t o r i e n t a t i o n i n the e l e c t r o d e m o d u l e o f the M I N I - T R A N S B L O T a p p a r a t u s . T h e B i o - i c e c o o l i n g u n i t w a s p l a c e d i n the t a n k w i t h the e l e c t r o d e u n i t . T h e t a n k w a s f i l l e d w i t h I X t ransfer buf fe r , a s t i r b a r w a s a d d e d a n d the e n t i r e u n i t w a s p l a c e d o v e r a s t i r r e r w i t h ice p a c k s i n o r d e r t o k e e p the en t i r e u n i t c o o l . T h e u n i t w a s c o n n e c t e d to the p o w e r s u p p l y a n d t rans fe r se t t ings 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 h o u r s f o l l o w e d b y 3 0 V f o r 3 0 m i n s - l h o u r . A f t e r the t rans fe r , t he 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 fo r p r o p e r t ransfer . O n c e s u c c e s s f u l , 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 bu f fe r 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 t he b l o c k i n g b u f f e r w a s d i s c a r d e d a n d the m e m b r a n e w a s i n c u b a t e d 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 bu f fe r 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 t h e n w a s h e d 3 t i m e s w i t h w a s h buf fer . 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 las t w a s h c y c l e , 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: 4000 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 d i l u t i o n buf fe r . 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 fo r 45 m i n u t e s w i t h s h a k i n g . A f t e r 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 t i m e s w i t h w a s h buf fe r . C h e m i l u m i n e s c e n c e r e a g e n t w a s p r e p a r e d b y m i x i n g e q u a l pa r t s o f R e a g e n t s A a n d B f r o m A m e r s h a m to a t o t a l v o l u m e o f 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 r e a g e n t f o r 1 m i n u t e . T h e m e m b r a n e w a s 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 casset te w i t h the p r o t e i n s f a c i n g u p . 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 b e t w e e n 10-15 s e c o n d s , d e p e n d i n g o n the d e s i r a b l e i n t e n s i t y o f t he b a n d s . T h e f i l m w a s r e m o v e d f r o m the casset te a n d p l a c e d i n the d e v e l o p e r f o r a c o u p l e o f s e c o n d s , 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 f i xe r . T h e f i l m w a s d r i e d f o r 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 fo 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 IgG -C7 Assay Development It w a s n e c e s s a r y to d e t e r m i n e the 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 o f 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 the 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 i n L L C - P K i ce l l s . 47 4.3.1. Temperature Dependence V a r i o u s c o n c e n t r a t i o n s o f I g G - C 7 r a n g i n g f r o m 0.1 to 2.0 u g / m l w e r e 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 a l l o c a t e d fo r c o m p a r i s o n . S u b s e q u e n t l y , m e d i a w a s r e m o v e d a n d l O 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 . P l a t e s w e r e c o v e r e d i n 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 h o u r s i n c u b a t i o n , 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 ec t i ons 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 to n o n -t r e a t m e n t c o n t r o l . % 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 100% 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 o f 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 a l l o c a t e d fo r e a c h t i m e p o i n t . A f t e r 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 fo r a n a d d i t i o n a l 2 h r s a t 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 . 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 c o n c e n t r a t i o n s 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 w e r e 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 a l l o c a t e d fo r c o m p a r i s o n . S u b s e q u e n t l y , m e d i a w a s r e m o v e d , a n d l O 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 a n d i n c u b a t e d f o r a n 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 . 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 4.3.1. T h e o p t i m a l p r e i n c u b a t i o n p e r i o d a n d I g G - C 7 c o n c e n t r a t i o n 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 tha t a m i n i m u m o f 8 0 % 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 w a s o b s e r v e d . 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 tha t the c e l l u l a r c o n t e n t o f I g G - C 7 r e a c h e d s t e a d y state 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 at e q u i l i b r i u m u p to 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 ce l l s [11]. D u r i n g the 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 the ce l l s a t s t e a d y state [11]. T h e r e f o r e , i t 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 f o r [ 3 H ] C s A i n o r d e r to y i e l d the h i g h e s t p e r c e n t u p t a k e o f the d r u g i n L L C - P K i ce l l s t a k i n g i n t o c o n s i d e r a t i o n the 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 o f [ 3 H ] C s A i n s e r u m - f r e e m e d i a at 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 . A p p r o p r i a t e 49 c o n t r o l s w e r e a l l o c a t e d fo r e a c h t i m e p o i n t . U p t a k e o f [ 3 H ] C s A w a s d e t e r m i n e d b y 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 [ 3 H ] C s A w a s a d d e d to l O O m M N a O H u p o n c e l l l y s i s a n d t r a n s f e r r e d 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 t o t a l r a d i o a c t i v i t y o f 8 0 0 n g / m l o f [ 3 H ] C s A . A l l t r e a t m e n t g r o u p s w e r e c o m p a r e d to t h i s [ 3 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 4.5.2). 4.5. C s A uptake and Toxicity with IgG-C7 Assay Development C s A u p t a k e a n d t o x i c i t y w e r e a s ses sed b y q u a n t i f y i n g [ 3 H ] C s A 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 lac ta te d e h y d r o g e n a s e ( L D H ) re lease i n o n e assay m e t h o d . F o r the u p t a k e s t u d i e s o f [ 3 H ] C s A i n t o L L C - P K i ce l l s , 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 the t o x i c i t y a ssays , L D H w a s a c e l l m a r k e r tha t w a s r e l e a s e d 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 la t ter , 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 the 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 [3H]CsA Bound and Uptake Assay C o n f l u e n t ce l l s 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 e n s u r e n o c e l l d e a t h d u e to 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). T h e m e d i u m w a s r e m o v e d a n d a c o n c e n t r a t i o n o f 1 2 . 0 p g / m l I g G - C 7 i n s e rum- f r ee 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 , [ 3 H ] C s A - L D L c o m p l e x i n 50 s e r u m - f r e e m e d i a w a s p r e p a r e d w h e r e 8 0 0 n g / m l o f [ 3 H ] C s A w a s i n c u b a t e d w i t h 2 0 p g / m l L D L c h o l e s t e r o l fo r 1 h o u r at 3 7 ° C (see s e c t i o n 3.3.4). A f t e r p r e i n c u b a t i o n w i t h I g G - C 7 , 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 w e r e a d d e d to the ce l l s 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 300 p i . Treatment 3 H - C s A i n 3 H - C s A - L D L i n V o l u m e of Treat N o 3 2 0 0 0 n g / m l w o r k i n g so lu t ion f ina l cone: 8 0 0 n g / m l (Pi) L D L complex so lu t ion 2 0 0 p g / m l w o r k i n g so lu t i on f ina l cone: 2 0 u g / m l (ul) s e r u m free m e d i a (pi) 1. Serum-free m e d i a con t ro l 0 0 0 300.0 2 L D H pos i t ive con t ro l 0 0 0 300.0 3 L D L con t ro l 0 0 30.0 270.0 . 4 To ta l 3 H - C s A 7.5 0 0 292.5 i n N a O H 5 3 H - C s A 7.5 0 0 292.5 6 m A b + 3 H - C s A 7.5 0 0 292.5 7 3 H - C s A - L D L 0 37.5 0 262.5 8 m A b + 3 H - C s A - L D L 0 37.5 0 . 262.5 9 3 H - C s A + L D L 7.5 0 30.0 262.5 10 m A b + 3 R - C s A + L D L 7.5 0 30.0 262.5 ( ) - indicates 3 H - C s A - L D L complex 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 E x p e r i m e n t s T h u s f o l l o w i n g p r e i n c u b a t i o n w i t h I g G - C 7 , a l l 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 w i t h [ 3 H ] C s A w e r e i n c u b a t e d f o r a n a d d i t i o n a l 6 h r s a t 3 7 ° C . [ 3 H ] C s A c o n t r o l w a s 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 o f [ 3 H ] C s A i n l y s e d ce l l s w i t h l O O m M N a O H a n d b y t r a n s f e r r i n g the en t i r e 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 t o m e a s u r e i ts t o t a l 51 radioact ivi ty . A l l treatment groups were compared to the [ 3 H ] C s A control to measure percent b o u n d and uptake. Furthermore, the supernatant was removed and al iquoted into 1.5 m l microcentrifuge tubes for L D H analysis (see section 4.5.3). Bo th membrane-bound and intracellular [ 3 H ] C s A were de termined as described i n section 4.5.2. 4 . 5 . 2 . Membrane-bound and Intracellular [3H]CsA determination. M e d i a was r emoved and cells were washed three times w i t h ice-cold I X P B S to remove excess radioact ive C s A . Approx ima te ly , 300pl of 0.001% Tr i ton X-100 was added and incubated for 10 minutes at 37°C. The detergent was r emoved and al iquoted into 7ml scint i l la t ion via ls for count ing to determine membrane-bound [ 3 H ] C s A . Subsequently, cells were lysed w i t h 300pl of l O O m M N a O H for 5 minutes. Twenty-f ive microl i ters of lysed cells was a l iquoted into a microt i ter plate for prote in content analysis (see section 4.1.1) and the r ema in ing v o l u m e into 7 m l scint i l la t ion vials . W e l l s were washed a n addi t iona l 2X w i t h l O O m M N a O H and the washings were a l iquoted into the corresponding v i a l to determine radioact ivi ty by scint i l la t ion count ing (see section 4.6). A l l treatment groups were compared to the [ 3 H ] C s A control to measure percent b o u n d and uptake. % B o u n d = C P M / p g cel lular prote in Treatment detergent wash X 100 % C P M / p g cellular prote in [ 3 H ] C s A contro l 52 % 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 1 0 0 % C P M / p g c e l l u l a r p r o t e i n [ 3 H ] C s A c o n t r o l 4.5.3 [3H]CsA Toxicity Study — LDH measurement L a c t a t e d e h y d r o g e n a s e ( L D H ) w a s the c h o s e n m a r k e r to m e a s u r e d r u g -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 la te s tage t o x i c i t y w h e r e d a m a g e to the p l a s m a m e m b r a n e r e s u l t s i n m e m b r a n e p e r m e a b i l i t y a n d l e a k a g e o f L D H i n t o the e x t r a c e l l u l a r f l u i d [4]. T h u s , L D H is 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 ab le c y t o s o l i c e n z y m e [4,96]. In vitro L D H re lease is d e t e c t e d 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]. L D H is 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 a s say 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 o f N A D a n d lac ta te to p y r u v a t e c a t a l y z e d b y L D H . 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 sa l t ( 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 the 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 i s p r o p o r t i o n a l to the n u m b e r o f l y s e d ce l l s . A s c h e m a t i c 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 N A D + + lac ta te - • p y r u v a t e + N A D H Diaphorase N A D H + I N T N A D + + f o r m a z a n ( red) F o r the 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 a d d e d to the 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 to e n s u r e c e l l d e a t h . T h i s L D H p o s i t i v e c o n t r o l r e p r e s e n t s 100% t o x i c i t y i n the a s say . F o l l o w i n g i n c u b a t i o n , the d e t e r g e n t m i x t u r e w a s a l i q u o t e d i n t o m i c r o c e n t r i f u g e t ubes a n d s t o r e d fo r 53 f u r t h e r L D H a n a l y s i s . T h e s u p e r n a t a n t i n b o t h m e d i a a n d L D L c o n t r o l g r o u p s w e r e a l i q u o t e d i n t o m i c r o c e n t r i f u g e tubes as w e l l . A l l L D H m e a s u r e m e n t s w e r e 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 t o c a l c u l a t e p e r c e n t t o x i c i t y o f e a c h t r e a t m e n t g r o u p . % T o x i c i t y = A b s 4 9 2 Treatment/mg c e l l p r o t e i n - A b s 4 9 2 Control/mg c e l l p r o t e i n x 100% A b s 4 9 2 LDH Positive Control/ m g p r o t e i n F i r s t l y , 50 p i o f the s u p e r n a t a n t / m e d i a o f e a c 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 w e r e a d d e d to a n o n - s t e r i l e 9 6 - w e l l m i c r o t i t e r p l a t e . A s e r u m - f r e e m e d i a c o n t r o l g r o u p w a s 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 l l v a l u e s w e r e s u b s t r a c t e d f r o m . T h e p u r p o s e w a s to ad jus t f o r the in t e r f e rence i n a b s o r b a n c e f r o m p h e n o l r e d . S u b s t r a t e 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 o f A s s a y bu f fe r ( t h a w e d s l o w l y o n ice) w i t h a v i a l o f l y o p h i l i z e d subs t ra t e . 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 l i g h t u n t i l r e a d y fo r use . U s i n g a m u l t i - c h a n n e l p i p e t t o r , 5 0 p l o f subs t r a t e m i x w a s a d d e d to e a c h s a m p l e a n d c o n t r o l . T h e p l a t e w a s c o v e r e d w i t h a l u m i n u m f o i l a n d 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 fo 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 o f 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 l a r g e b u b b l e s w e r e r e m o v e d w i t h a p i p e t t e t i p . M a x i m u m a b s o r b a n c e at 4 9 2 n m of 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 p l a t e r e a d e r w i t h i n 1 h r o f a d d i n g s t o p s o l u t i o n . T h e p r o c e d u r e s f o l l o w e d w e r e those d e s c r i b e d i n the C y t o T o x 9 6 ® 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 say k i t [96]. 54 4.6 Scintillation Counting L i q u i d s c i n t i l l a t i o n c o u n t i n g w a s u s e d to q u a n t i f y [ 3 H ] - C s A i n t r a c e l l u l a r l y . S a m p l e s w e r e c o n t a i n e d i n 7 m l s c i n t i l l a t i o n v i a l s c o n t a i n i n g 5 . 4 m l C y t o S c i n t s c i n t i l l a t i o n f l u i d . T h e s a m p l e s w e r e p l a c e d i n a B e c k m a n l i q u i d s c i n t i l l a t i o n c o u n t e r . U p o n the d i s i n t e g r a t i o n o f the t r i t i u m l a b e l , p h o t o n s a re 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 e m i t t i n g l i g h t . T h e s i g n a l w a s d e t e c t e d b y the 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 ee m i n u t e s . 4.7 Statistical Analyses A n u n p a i r e d t-test (Instat; G r a p h p a d ) w a s i m p l e m e n t e d to d e t e r m i n e d i f f e rences 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 b e t w e e n c o n t r o l a n d t r e a t m e n t g r o u p s . R e s u l t s w e r e c o n s i d e r e d s i g n i f i c a n t i f the p r o b a b i l i t y o f the r e s u l t o c c u r r e d b y c h a n c e less t h a n 5% of the t i m e (p<0.05). O n e - w a y a n a l y s i s o f v a r i a n c e ( A N O V A ) (Instat; G r a p h p a d ) w a s u s e d to d e t e r m i n e d i f f e r ences 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 s i g n i f i c a n t i f the p r o b a b i l i t y o f the r e s u l t o c c u r r e d b y c h a n c e less t h a n 5% (p<0.05). S i g n i f i c a n t d i f f e rences w e r e a s se s sed u s i n g T u k e y ' s p o s t - h o c test. A l l d a t a w e r e e x p r e s s e d as 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 CHAPTER V: (ResuCts 56 5.1. Presence and abundance of L D L receptors in L L C - P K i cells 5.1.1 Dose-response Studies T o c l a r i f y the t e r m i n o l o g y , L D L spec i f i c b i n d i n g s i tes refer to the f a m i l y o f L D L r e c e p t o r s tha 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 re the r e c e p t o r s tha t p l a y a r o l e i n c h o l e s t e r o l h o m e o s t a s i s . 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 tha t w a s g e n e r a t e d b y l i n e a r r e g r e s s i o n i n o r d e r to q u a n t i t a t e the a m o u n t o f L D L i n n g / m l f r o m the I P A w a s h i n g s a g a i n s t i ts f l u o r e s c e n c e ( a r b i t r a r y u n i t s ) . C o n c e n t r a t i o n s o f D i l - L D L a d d e d to the c e l l c u l t u r e m e d i u m w e r e i n u n i t s o f p g / m l b a s e d o n p r o t e i n co n t en t . H o w e v e r , after i n c u b a t i o n , D i l - L D L q u a n t i t a t i o n w a s d e t e r m i n e d i n n g / m l s i n c e o n l y a f r a c t i o n o f D i l - L D L w a s b o u n d to the ce l l s . T h e r e f o r e , 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 q u a n t i t a t e D i l - L D L c o n c e n t r a t i o n s i n u n i t s o f n g / m l b a s e d o n p r o t e i n con ten t . 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 nc rea se i n c e l l - a s s o c i a t e d ( F i g u r e 7 A ) a n d m e m b r a n e - b o u n d L D L ( F i g u r e 7B) . T h e t o t a l a m o u n t o f L D L a s s o c i a t e d w a s 4 . 5 n g / p g o f c e l l p r o t e i n a n d the 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 . It i s a s s u m e d tha t c e l l - a s s o c i a t e d L D L d e s c r i b e s L D L tha t i s b o t h b o u n d a n d i n t e r n a l i z e d w i t h i n t he c e l l s y s t e m a n d m e m b r a n e - b o u n d L D L d e s c r i b e s o n l y L D L tha t is b o u n d to the c e l l su r face . B y s u b t r a c t i n g m e m b r a n e - b o u n d L D L ( F i g u r e 7B) f r o m the m e a n v a l u e s o f c e l l -a s s o c i a t e d L D L ( F i g u r e 7 A ) , i n t e r n a l i z e d L D L w a s s e m i - q u a n t i t a t i v e l y d e t e r m i n e d . I n t e r n a l i z e d L D L ( F i g u r e 7 C ) a l s o 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 c o n c e n t r a t i o n o f 4 0 u g / m l a n d s u b s e q u e n t l y dec reases to 1 . 4 n g / u g o f p r o t e i n at 8 0 p g / m l . T h e s e r e s u l t s s u g g e s t the p r e s e n c e o f r e c e p t o r s w h i c h r e c o g n i z e L D L a n d i ts a c t i v i t y r eaches 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 o f 4 0 p g / m l . H o w e v e r , the c u r v e s d o n o t 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 spec i f i c a n d n o n s p e c i f i c b i n d i n g o f D i l - L D L to L L C P K i ce l l s . 600 H 1 1 1 1 1 0 200 400 600 800 1000 D i l - L D L C o n c e n t r a t i o n ( n g / m l ) F igure 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 v e r s u s F l u o r e s c e n c e at 5 2 2 n m / 5 7 8 n m i n I P A . [y=1.283x-54.723; R 2 = 0.9844] 58 40 60 80 Dil-LDL Concentration (ug/mil 100 Figure 7A: 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 LDL at 3 7 ° C . V a l u e s e x p r e s s e d as m e a n ( 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 t r i p l i c a t e s . 40 60 80 Dil-LDL Concentration tug/ml) 100 Figure 7B: m e a s u r e m e n t bound LDL e x p r e s s e d D o s e - d e p e n d e n t o f membrane-at 4°C. V a l u e s ( n g o f as m e a n 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 t r i p l i c a t e s . Figure 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 o f internalized L D L c a l c u l a t e d as the d i f f e rence b e t w e e n F i g u r e 7 A a n d 7 B . V a l u e s e x p r e s s e d as m e a n ( 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 t r i p l i c a t e s . 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 s p e c i f i c b i n d i n g a s s a y i n L L C P K i ce l l s ; m e a n w h i l e , b o t h t o t a l b i n d i n g a n d n o n - s p e c i f i c b i n d i n g d i d n o t r e a c h s a t u r a t i o n (see F i g u r e 8). R e s u l t s i n d i c a t e tha 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 c o n c e n t r a t i o n o f 2 0 p g / m l . 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 at l O p g / m l w h i c h w a s c h o s e n as the i d e a l c o n c e n t r a t i o n f o r a l l s u b s e q u e n t s t u d i e s w i t h D i l - L D L , A t ab le o f the v a l u e s g e n e r a t e d f r o m the s p e c i f i c b i n d i n g assay is s h o w n i n T a b l e 5. T h e s e r e su l t s a re s i m i l a r t o the 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 the c o n c e n t r a t i o n fo 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 ce 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 fo r the spec i f i c b i n d i n g assay . It is k n o w n tha 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 tes [21,56], w h i c h w a s e v i d e n t i n F i g u r e 9. T h e spec i f i c b i n d i n g c u r v e 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 o f D i l - L D L . A t 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 f o r b o t h F i g u r e s 8 a n d 9, t he a m o u n t o f t o t a l b o u n d L D L r e a c h e d a p p r o x i m a t e l y 0.6 n g / p g o f p r o t e i n fo r L L C - P K i c e l l s a n d l . 6 n g / p g o f p r o t e i n fo r H e p G 2 ce l l s . T h e d i f f e r ence i s a p p r o x i m a t e l y a b o u t a 3 - f o l d i nc rea se w h i c h i n d i c a t e s tha t L L C - P K i ce l l s d o n o t e x p r e s s the s a m e n u m b e r o f L D L spec i f i c b i n d i n g s i tes as H e p G 2 ce l l s . T h u s , L L C - P K i ce l l s h a v e l o w L D L spec i f i c b i n d i n g s i tes c o m p a r e d to H e p G 2 ce l l s . 60 1.1 H 1.0 H Dil-LDL Concentration (ug/ml) -•— Non-specific binding (+LDL) *— Total Binding (-LDL) -•— Specific Binding Figure 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 ce l l s . C e l l s w e r e i n c u b a t e d w i t h i n c r e a s i n g [ D i l - L D L ] i n the p r e s e n c e a n d absence o f 2 5 - f o l d excess 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 c e l l u l a r p r o t e i n ± S D i n t r i p l i c a t e s . 61 T r e a t m e n t T o t a l B i n d i n g ( - L D L ) N o n - s p e c i f i c B i n d i n g ( + L D L ) S p e c i f i c B i n d i n g D i l - L D L ( j i g / m l ) L D L ( n g / u g p r o t ) L D L ( n g / p g p r o t ) L D L ( n g / p g p r o t ) 0 0.04+0.02 0.01+0.07 0 . 0 3 ± 0 . 0 5 5 0 . 2 4 ± 0 . 0 3 0.05+0.07 0 . 1 8 ± 0 . 0 3 10 0 . 4 4 ± 0 . 0 5 0.06+0.01 0.38+0.06 20 0 . 6 9 ± 0 . 0 0 0.12+0.03 0 . 5 7 ± 0 . 0 5 40 0 . 9 2 ± 0 . 2 0 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 ce l l s . See 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 ce l l s . A s a p o s i t i v e c o n t r o l , H e p G 2 ce l l s w e r e i n c u b a t e d w i t h i n c r e a s i n g [ D i l - L D L ] i n the p r e s e n c e a n d absence o f 2 5 - f o l d excess o f u n l a b e l l e d L D L fo 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 t r i p l i c a t e s . 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 tha t 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 d o s e - d e p e n d e n t m a n n e r . T h i s p r o v i d e s a d d i t i o n a l e v i d e n c e o f the p r e s e n c e o f r e c e p t o r s tha t r e c o g n i z e L D L i n L L C P K i ce l l s . Y e t , d u e to the s m a l l d i f f e rence i n c h a n g e o f p e r c e n t o f m a x i m u m c e l l - a s s o c i a t e d L D L o v e r the r a n g e o f u n l a b e l l e d L D L ( m e a n 3 2 % to m e a n 17%) , these r e s u l t s f u r t h e r s u g g e s t a l o w a b u n d a n c e o f L D L b i n d i n g si tes i n L L C P K i ce l l s . o 8*1 20 40 60 80 100 120 140 Unlabelled LDL fue/ml) 160 180 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 ce l l s . A c o n s t a n t l O u g / m l o f D i l -L D L w a s i n c u b a t e d i n the p r e s e n c e o f i n c r e a s i n g c o n c e n t r a t i o n s o f L D L (10-1 6 0 u g / m l ) fo r 2 h r s at 3 7 ° C . V a l u e s e x p r e s s e d as p e r c e n t o f m a x i m u m c e l l -a s s o c i a t e d L D L ( t r ea tmen t v s c o n t r o l ) ± S D i n t r i p l i c a t e s . 64 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 f i r s t e s t a b l i s h the a s s a y w i t h the I g G - C 7 ( m A b to the L D L r e c e p t o r ) , i t w a s n e c e s s a r y to d e t e r m i n e t e m p e r a t u r e d e p e n d e n c e o n D i l - L D L b i n d i n g w i t h d i f f e ren t c o n c e n t r a t i o n s o f the m A b . F r o m the r e s u l t s i n F i g u r e 12, a d o s e - d e p e n d e n t i nc rea se 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 con t ra s t , a d o s e - d e p e n d e n t dec rease 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 tha t t he o b s e r v a t i o n a t 3 7 ° C w a s d u e to 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 l i t e r a tu r e , I g G - C 7 h a s a h i g h e r b i n d i n g a f f i n i t y 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 is 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]. A s p r e d i c t e d , the 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 ce l l s i s at 4 ° C . T h u s , w e e s t a b l i s h e d tha t 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 be c o n d u c t e d at 4 ° C . 66 100 -80 -I , , , , 1 0.0 0.5 1.0 1.5 2.0 2.5 mAb concentration (ug/ml) - • - 4°C - • - 37°C Figure 12: Temperature dependence of D i l - L D L b i n d i n g w i t h var ious concentrations of m A b to the L D L receptor. After L L C - P K i cells have been g r o w n for 24 hrs i n se rum free-media, var ious concentrations of I g G - C 7 (mAb) at 0.1, 0.5, 1.0, 2.0 p g / m l were preincubated for 2 hrs at 4°C. Subsequently, 10 p g / m l of D i l -L D L was further incubated at 4°C and 37°C for an add i t iona l 2 hrs. Va lues expressed as mean percent inh ib i t ion versus non-treatment cont ro 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 -t-» C 01 (8 QJ H I C o c (fl 3 tfl > c o •J3 '£ '£ c M 40 30 20 10 H 0.0 0.5 1.0 1.5 Preincubation period (hours') 2.0 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 Once the pre incubat ion pe r iod was determined, a n op t ima l concentrat ion of m A b treatment was decided based o n the cr i ter ion that a m i n i m u m of 80% inh ib i t ion must be observed i n D i l - L D L b ind ing . This cr i te r ion was necessary i n order to see any differences u p o n C s A treatment w h e n I g G - C 7 was present. L L C -P K i cells were preincubated w i t h var ious concentrations of the m A b at 0.1, 0.5, 1.0, 2.0,4.0, 8.0 and 1 2 . 0 p g / m l at 4°C for 1.5 hours p r io r to the add i t i on of l O . O p g / m l for an add i t ion 2 hours at 4°C. Results i n F igure 14 indicate that the op t imal concentrat ion of m A b was at 1 2 . 0 p g / m l where a 90% ± 6 % (mean ± S E M ) inh ib i t ion i n D i l - L D L b i n d i n g was observed. Results were repeatable w i t h three different subcultures i n triplicates. Therefore, the op t ima l concentrat ion to preincubate w i t h the I g G - C 7 i n L L C - P K i cells was at 1 2 . 0 p g / m l based o n pro te in content. Overall, the optimal conditions for IgG-C7 treatment in LLC-PKi cells are a preincubation period of 1.5 hours at 4°C with a concentration of 12.0pg/ml. 70 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 the 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 o f m A b to the L D L r e c e p t o r . L L C - P K i ce l l s w e r e 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 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 a n d 12.0 p g / m l at 4 ° C p r i o r to the 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 t r i p l i c a t e s . 71 5.3. PHI CsA treatment 5 . 3 . 3 . PHlCsA time-course incubation study Peterherych et al (2001) had established that a 24 hour incubat ion at 37°C w i t h [ 3 H ] C s A was sufficient t ime i n order to observe uptake of the d r u g into L L C - P K i cells [91]. H o w e v e r , the stabili ty of I g G - C 7 at 37°C was a concern due to the possibi l i ty of its h i g h metabol ism. IgG-C7 acts as a compet i t ive inhib i tor w i t h L D L for the L D L receptor w i t h higher affinity at 4 degrees [11]. Thus , it undergoes the same endocytic processes as an L D L particle where it becomes recognized by the L D L receptor, in ternal ized into vesicles and degraded into its components [11]. Beisiegel et al (1981) h a d conducted a s tudy to investigate the metabol i sm of radiolabel led IgG-C7 i n monolayer h u m a n fibroblasts cells [11]. They reported that the cellular content of 1 2 5 I g G - C 7 reached a steady state w i t h i n an hour and remained at e q u i l i b r i u m u p to 6 hours (see F igure 15) [11]. W i t h i n the 6 hours , they also observed approximate ly 4 times as m u c h 1 2 5 I g G - C 7 h a d been degraded as was present i n the cells at steady state at 37°C (see F igure 15) [11]. It is necessary to conduct a l l [ 3 H ] C s A uptake experiments at 37°C i n order to s tudy L D L receptor activity. Therefore, a [ 3 H ] C s A time-course incubat ion s tudy was needed to determine that op t ima l t ime pe r iod for [ 3 H ] C s A uptake t ak ing into considerat ion the h i g h metabol i sm of IgG-C7 . 72 In Table 6, [ 3 H ] C s A uptake only reached a m a x i m u m of 8.9% ± 0.4% (mean ± S E M ) w i t h i n 10 hours at 4°C. M e a n w h i l e [ 3 H ] C s A uptake reached a m a x i m u m of 21.2% ± 1.2% w i t h i n 6 hours at 37 °C. After 6 hours, there was no change i n [ 3 H ] C s A uptake at 37 °C; rather, uptake decreased after an incubat ion pe r iod of 10 hours (19.2% ± 1.6%) and a greater decrease after 24 hours (13.3% ± 2.0%). This corresponded to the literature where IgG-C7 remained i n e q u i l i b r i u m u p to 6 hours at 37°C. Therefore, it was dec ided that the op t ima l t ime per iod for [ 3 H ] C s A treatment was 6 hours. This cond i t ion was app l i ed to the uptake and toxici ty studies w i t h [ 3 H ] C s A (see section 5.4). % Uptake of PHICsA H o u r s 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 uptake of [ 3 H] C s A over 24 hour pe r iod at 37°C vs 4°C. A t ime-course incubat ion s tudy w i t h 8 0 0 n g / m l of [ 3 H ] C s A was conducted at bo th 37°C and 4°C i n L L C - P K i cells where intracellular [ 3 H ] C s A was measured over a per iod of 2, 6, 10 and 24 hours. Percent uptake was calculated by c o m p a r i n g mean counts per minute ( C P M ) / p g of cel lular prote in to total [ 3 H ] C s A control . Va lues expressed as percent uptake ± S E M i n n=3 i n triplicates. 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 ce 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 256 :11923 - 31 [11]. C o p y r i g h t p e r m i s s i o n g r a n t e d f r o m A m e r i c a n S o c i e t y o f B i o c h e m i s t r y a n d M o l e c u l a r B i o l o g y © . 74 5.4 E f f e c t o f I g G - C 7 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 5.4.1. f3H]CsA uptake - bound and intracellular T o m e a s u r e m e m b r a n e - b o u n d [ 3 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 t r a n s f e r r e d 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 s c i n t i l l a t i o n c o u n t i n g . 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 : A ) 800 n g / m l o f [ 3 H ] C s A a l o n e ; B ) 800 n g / m l o f [ 3 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 ) 8 0 0 n g / m l o f [ 3 H ] C s A c o a d d e d w i t h 20 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). 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 o f c e l l u l a r p r o t e i n a n d c o m p a r e d to C P M of [ 3 H ] C s A c o n t r o l / p g o f c e l l u l a r p r o t e i n to c a l c u l a t e p e r c e n t 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 p r e s e n c e a n d absence o f 12.0 p g / m l o f m A b fo r 1.5 h o u r s at 4 ° C p r i o r to [ 3 H ] C s A t r e a t m e n t fo r 6 h o u r s at 3 7 ° C . A s i g n i f i c a n t d i f f e r ence w a s o b s e r v e d i n the [ 3 H ] C s A a l o n e g r o u p i n the p r e s e n c e o f m A b (2.6% ± 0.6%) v e r s u s i ts absence (5 .1% ± 1.3%) (p<0.05 w i t h 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 [ 3 H ] C s A -L D L c o m p l e x a n d [ 3 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 , n o s i g n i f i c a n t d i f f e rences 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 [ 3 H ] C s A i n the p r e s e n c e o f L D L , e i t h e r c o m p l e x e d w i t h [ 3 H ] C s A o r c o a d d e d w i t h [ 3 H ] C s A (p<0.05 w i t h A N O V A ) . T h e s e r e s u l t s s u g g e s t tha t I g G - C 7 c o u l d s i g n i f i c a n t l y r e d u c e m e m b r a n e - b o u n d [ 3 H ] C s A i n L L C - P K i ce l l s ; ye t , w h e n L D L w a s p re sen t , t he re w a s n o effect. 75 T o o b s e r v e the effect o f I g G - C 7 o n u p t a k e o f [ 3 H ] C s A , l y s e d ce l l s w i t h i n the s a m e e x p e r i m e n t w e r e t r a n s f e r r e d 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 to 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 [ 3 H ] C s A . V a l u e s w e r e e x p r e s s e d as C P M / p g o f c e l l u l a r p r o t e i n a n d c o m p a r e d to C P M o f [ 3 H ] C s A c o n t r o l / p g o f c e l l u l a r p r o t e i n to 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 s i g n i f i c a n t d i f f e rences w e r e o b s e r v e d i n t he p r e s e n c e o f m A b w i t h i n e a c h t r e a t m e n t 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 [ 3 H ] C s A . O v e r a l l , b o t h I g G - C 7 a n d L D L h a d n o s i g n i f i c a n t r e d u c t i o n o n [ 3 H ] C s A u p t a k e . These results reveal that IgG-C7 affected [ 3 H]CsA binding on the membrane level; yet no effect was observed on [ 3 H]CsA uptake intracellularly. In 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 bound and uptake. 76 7 6H CsA alone CsA-LDL CsA + LDL 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 [ 3 H ] C s A i n L D L w i t h IgG-C7 . A ) C s A alone indicates on ly [ 3 H ] C s A , B) C s A - L D L indicates [ 3 H ] C s A complexed w i t h L D L , and C) C s A + LDL indicates [ 3 H ] C s A coadded w i t h L D L . L L C - P K i cells were preincubated w i t h 12.0 p g / m l for 1.5 hrs at 4 °C pr io r to treatment w i t h 800 n g / m l of [ 3 H ] C s A w i t h and wi thou t L D L for an addi t iona l 6 hours at 37°C. Membrane -bound [ 3 H ] C s A was measured f rom 0.001% T r i t o n X-100 w a s h i n g and compared to total [ 3 H ] C s A control . Values expressed as mean percent b o u n d ± S E M i n n=6 i n triplicates. denotes significance (p<0.05) compared to control w i t h unpa i r ed t-test 7 7 CsA alone CsA-LDL CsA + LDL Absence of mAb Presence of mAb T r e a t m e n t G r o u p s Figure 17: M e a n p e r c e n t u p t a k e o f [ 3 H ] C s A i n L D L w i t h I g G - C 7 . A) C s A alone i n d i c a t e s o n l y [ 3 H ] C s A , B) C s A - L D L i n d i c a t e s [ 3 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 i n d i c a t e s [ 3 H ] C s A c o a d d e d w i t h L D L . L L C - P K i ce l l s 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 f o r 1.5 h r s at 4 ° C p r i o r to t r e a t m e n t w i t h 800 n g / m l o f [ 3 H ] C s A w i t h a n d w i t h o u t L D L f o r a n a d d i t i o n a l 6 h o u r s at 3 7 ° C . I n t r a c e l l u l a r [ 3 H ] C s A w a s m e a s u r e d f r o m l y s e d ce l l s a n d c o m p a r e d to t o t a l [ 3 H ] C s A c o n t r o l . 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 to 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. [3H]CsA toxicity - LDH measurement Within the same assay method as [3H]CsA bound and uptake, [3H]CsA toxicity was determined by measuring LDH 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 [3H]CsA alone; B) 800ng/ml of [3H]CsA complexed with 20pg/ml LDL cholesterol; and C) 800ng/ml of [3H]CsA coadded with 20pg/ml cholesterol of LDL for a period of 6 hours at 37°C (see section 4.5.1). The culture media was removed and analyzed for LDH activity. Values were expressed as absorbance at 492nm /mg of cellular protein adjusted against control groups and compared to absorbance at 492nm/ mg of cellular protein of the LDH positive control to calculate percent toxicity (see section 4.5.2). In Figure 18, a significant difference was observed in the [3H]CsA alone 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 [3H]CsA-LDL complex and [3H]CsA with LDL coaddition groups. In addition, there was no significant effect of LDL on [3H]CsA toxicity either as a drug complex or as a drug coaddition treatment (p<0.05 with one-way ANOVA). The results suggest that IgG-C7 could significantly reduce [3H]CsA toxicity in LLC-PKi cells; yet, when LDL was present, there was no effect. 79 O v e r a l l , t he r e su l t s a re c o n s i s t e n t w i t h the r e s u l t s r e p o r t e d i n s e c t i o n 5.4.1 w h e r e , a s i g n i f i c a n t d i f f e rence 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 [ 3 H ] C s A a l o n e g r o u p i n the p r e s e n c e o f IgG-C7 v e r s u s i ts absence . H o w e v e r , L D L h a d n o affect o n [ 3 H ] C s A b o u n d a n d u p t a k e . 80 CsA CsA-LDL CsA + LDL T r e a t m e n t G r o u p s 82888 Absence of mAb I I Presence of mAb 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 [ 3 H ] C s A i n L D L w i t h I g G - C 7 . A ) C s A a l o n e i n d i c a t e s o n l y [ 3 H ] C s A , B ) C s A - L D L i n d i c a t e s [ 3 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 i n d i c a t e s [ 3 H ] C s A c o a d d e d w i t h L D L . L L C - P K i ce l l s w e r e 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 to t r e a t m e n t w i t h 8 0 0 n g / m l o f [ 3 H ] C s A w i t h a n d w i t h o u t L D L f o r a n a d d i t i o n a l 6 h o u r s at 3 7 ° C . M e d i a w a s 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 to L D H a c t i v i t y i n 1% T r i t o n X - 1 0 0 t r e a t ed ce 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 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 to 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 U5I-LDL P r e v i o u s w o r k i n v o l v i n g L D L r e c e p t o r s h a v e u s e d 1 2 5 I - L D L as a l a b e l e d l i g a n d . 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 w h e n s t u d y i n g r e c e p t o r - m e d i a t e d L D L m e t a b o l i s m s i n c e f i r s t 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 is l i m i t e d b y i ts 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 cos t i n w a s t e m a n a g e m e n t . A n a l t e r n a t i v e to 1 2 5 I - L D L i s 3 , 3 ' - d i o c t a d e c y l i n d o -c a r b o c y a n i n e L D L , D i l - L D L . T h i s fluorescent p r o b e h a s b e e n e x t e n s i v e l y u s e d 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 s t u d i e s o f r e c e p t o r - m e d i a t e d 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 is 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 t r ans fe r to c e l l m e m b r a n e s o r o t h e r u n l a b e l l e d l i p o p r o t e i n s , a n d d o e s n o t 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]. D i l - L D L c o m b i n e d w i t h c o n f o c a l m i c r o s c o p y c a n be u s e d to m o n i t o r the p roces ses o f 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 t h r o u g h 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]. T h e r e f o r e , D i l - L D L w a s c h o s e n as a l a b e l e d l i g a n d 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 i n L L C - P K i ce l l s . O t h e r i n v e s t i g a t o r s h a v e u s e d th i s s a m e fluorescent p r o b e 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 i n g l o m e r u l a r ce l l s [99], H e p G 2 ce l l s [120] a n d P M N s [98,111]. T o da te , h o w e v e r , n o o n e h a s i n v e s t i g a t e d L D L r e c e p t o r a c t i v i t y i n p r o x i m a l t u b u l e ce l l s . I n the l i t e r a t u r e , the re w a s c o n f l i c t i n g 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 o f D i l -L D L . E x c i t a t i o n a n d e m i s s i o n w e r e r e p o r t e d at t w o d i f f e r e n t w a v e l e n g t h s : 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 sue , 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 o f the a s say w a s a s se s sed (see s e c t i o n 4.1.2 fo r p r o c e d u r e ) . I n F i g u r e 19 , s e n s i t i v i t y o f the 83 D i l - L D L a s say w a s i n c r e a s e d w h e n the p r o b e w a s e x c i t e d a n d e m i t t e d 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 tha t D i l -L D L w o u l d be 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 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 n m v e r s u s 5 5 5 / 5 7 1 n m . 84 6.2. Rationale of LLC-PKi as an appropriate cell model O n e o f the m a i n ob jec t ives w a s to i n v e s t i g a t e C s A n e p h r o t o x i c i t y . T h u s , the s e l e c t i o n o f L L C P K i a p p e a r e d to be a s u i t a b l e c e l l m o d e l s i n c e i t d i s p l a y e d s i m i l a r cha rac t e r i s t i c s as p r o x i m a l t u b u l e ce 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 to i n v e s t i g a t e C s A - i n d u c e d c y t o t o x i c i t y [10,20,47,71]. H o w e v e r , c u l t u r e d h u m a n 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 he e a r l i e r s t u d i e s o n the L D L r e c e p t o r [15,40,41]. S i n c e t h e n , a n u m b e r o f o t h e r c e l l m o d e l s h a v e b e e n u s e d to i n v e s t i g a t e L D L r e c e p t o r a c t i v i t y w h i c h i n c l u d e h u m a n h e p a t o c y t e s ( H e p G 2 ) [21,56,100], l y m p h o c y t e s [97], m o n o c y t e s [111], a n d m a c r o p h a g e s [61]. N e v e r t h e l e s s , k i d n e y ce l l s a l s o e x p r e s s e d r e c e p t o r s tha t r e c o g n i z e L D L , e s p e c i a l l y p r o x i m a l t u b u l a r ce 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 tha t the L D L -H R P spec i f i c 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 the p r e s e n c e o f excess u n l a b e l l e d L D L [88]. I n a d d i t i o n , W a s a n et al (1994) d e t e r m i n e d tha t the K d v a l u e o f 1 2 5 I - L D L i n L L C P K i ce l l s i s 0.0538 ( n g / m l ) 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 96 000 b i n d i n g s i t e s / c e l l [132]. D e s p i t e these f i n d i n g s , n o o n e h a s c o m p l e t e d a n y s t u d i e s o n 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 ce l l s ; t h u s i t w a s n e c e s s a r y to c o n f i r m the p r e s e n c e o f these r e c e p t o r s i n t h i s c e l l l i n e a n d i t s a p p r o p r i a t e n e s s i n the s t u d y o f L D L r e c e p t o r a c t i v i t y . F i g u r e s 7 A , 7 B a n d 7 C r e v e a l e d d o s e - r e s p o n s e s t u d i e s o f c e l l - a s s o c i a t e d , m e m b r a n e - b o u n d a n d i n t e r n a l i z e d L D L , r e s p e c t i v e l y . T h e s e r e s u l t s p r o v i d e d f i rs t e v i d e n c e o f the p r e s e n c e o f r e c e p t o r s w h i c h b i n d L D L . H o w e v e r , these r e su l t s a l so 85 i n d i c a t e d b o t h spec i f i c a n d n o n - s p e c i f i c b i n d i n g o f L D L . T h u s , f u r t h e r e x p e r i m e n t s to i n v e s t i g a t e L D L spec i f i c b i n d i n g w e r e n e e d e d a n d r e s u l t s i n F i g u r e 8 c o n f i r m e d tha 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 a b o u t 0 . 5 7 ± 0 . 0 5 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 p e c i f i c a l l y b o u n d to the r e c e p t o r o f in te res t . A c o m p a r i s o n to the spec 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 c o n t r o l (see F i g u r e 9) r e v e a l e d tha t L L C - P K i ce l l s d o n o t e x p r e s s the s a m e a b u n d a n c e o f L D L b i n d i n g s i tes as H e p G 2 a n d tha t L L C - P K i ce l l s h a v e a l o w n u m b e r o f L D L s p e c i f i c b i n d i n g s i tes . A c o m p e t i t i v e s t u d y w h e r e u n l a b e l l e d L D L w a s ab l e 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 o f the p r e s e n c e o f 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 the 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 tes (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 ex t rac t s o f L L C - P K i u s i n g I g G - C 7 as the p r i m a r y a n t i b o d y a l s o r e v e a l e d the p r e s e n c e o f L D L r e c e p t o r s i n the c e l l l i n e o f in te res t (see F i g u r e 11). T h e r e i s n o d o u b t tha t L L C - P K i c e l l s d o e x p r e s s L D L r e c e p t o r s o r s p e c i f i c a l l y , r e c e p t o r s tha t b i n d to L D L s u c h as t he m e m b e r s o f the L D L r e c e p t o r f a m i l y . D e s p i t e the p r e s e n c e o f L D L r e c e p t o r s , L L C - P K i ce l l s e x p r e s s a l o w a b u n d a n c e o f L D L spec i f i c b i n d i n g s i tes c o m p a r e d to H e p G 2 ce l l s . M e g a l i n has b e e n r e p o r t e d to be h i g h l y 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 o f t he k i d n e y [19,125] a n d L L C - P K i ce 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 i s u n c e r t a i n . N e v e r t h e l e s s , i t i s c o n c l u s i v e t ha t L L C - P K i ce l l s m a y n o t be the m o s t i d e a l c e l l l i n e 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 b u t i t i s s t i l l a n a p p r o p r i a t e c e l l m o d e l to i n v e s t i g a t e the r o l e o f the L D L r e c e p t o r o n C s A u p t a k e a n d t o x i c i t y because the s i te 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 he 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 r e cep to r -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 to L D L [11]. T h u s , i t 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 , the b i n d i n g s i te o f I g G - C 7 to the L D L r e c e p t o r i s d i f f e r en t t h a n the L D L b i n d i n g s i te [123]. I g G - C 7 i s d i r e c t e d a g a i n s t the f i r s t c y s t e i n e - r i c h r e p e a t o f the f i r s t d o m a i n o f the L D L r e c e p t o r [123]. B y con t ra s t , L D L b i n d i n g r e q u i r e s c y s t e i n e r epea t s 3-7 i n the l i g a n d b i n d i n g d o m a i n [105]. F u r t h e r m o r e , i n t e r s p e c i e s d i f f e rences b e t w e e n the h u m a n L D L r e c e p t o r a n d p i g L D L r e c e p t o r m a y res t r i c t the use o f I g G - C 7 b u t a p r o t e i n B L A S T a n a l y s i s has c o n f i r m e d tha t the h u m a n a n d p i g L D L r e c e p t o r s h a v e a b o u t a n 8 7 % s e q u e n c e h o m o l o g y o f i t s 818 a m i n o a c i d r e s i d u e s . T h e s t r a t egy tha t 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 r e c e p t o r a c t i v i t y i n L L C - P K i ce l l s w a s the u s e o f 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 s t r a t egy 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 to o b s e r v 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 . I g G - C 7 w a s f i r s t p r e i n c u b a t e d w i t h the ce l l s , the m e d i a r e m o v e d , a n d t h e n C s A t r e a t m e n t 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 o f 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 , i t w a s i m p o r t a n t t o e s t a b l i s h 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 c e l l s w h i c h i n c l u d e d t e m p e r a t u r e , o p t i m a l 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 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 I g G - C 7 w a s a s se s sed a n d r e su l t s r e v e a l tha t at 4 ° C , the re 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 . R e s u l t s 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 l i t e r a t u r e 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 a f f i n i t y a t 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 of 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 ce l l s [11]. S u b s e q u e n t l y , i n F i g u r e 13 , 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 su l t s r e v e a l tha 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 grea tes t 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 w i t h 2 0 % ± 2 5 % . B a s e d o n the 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 at 4 ° C , t he o p t i m a l c o n c e n t r a t i o n w a s n e e d e d . 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 o f m A b w e r e 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 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 . It w a s f o u n d tha 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 90% + 6% ( m e a n ± S E M ) w h i c h m e t the c r i t e r i a tha 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 , i t w a s c o n c l u d e d tha t the o p t i m a l c o n d i t i o n s fo r 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 c o n c e n t r a t i o n . T h e s e a s s a y 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 . A n o t h e r c o n c e r n w i t h I g G - C 7 w a s i ts s t a b i l i t y a t 3 7 ° C u p o n [ 3 H ] C s A t r ea tmen t . P r i o r c o n d i t i o n s o f the 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 to c o n d u c t the assay at 3 7 ° C to o b s e r v e the effect o f L D L r e c e p t o r a c t i v i t y o n [ 3 H ] C s A u p t a k e a n d t o x i c i t y . H o w e v e r , i t w a s a l s o c r u c i a l to a l l o w e n o u g h t i m e fo r 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 tha t a 24 h o u r i n c u b a t i o n at 3 7 ° C w i t h [ 3 H ] C s A w a s su f f i c i en t t i m e i n o r d e r to o b s e r v e u p t a k e o f the d r u g i n t o L L C - P K i c e l l s [91]. Y e t , w i t h i n the 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 tha 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 . B i e s i e g e l et al (1981) r e p o r t e d tha t the c e l l u l a r c o n t e n t o f 1 2 5 I g G - C 7 r e a c h e d a s t e a d y s tate 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 to 6 h o u r s (see F i g u r e 15) [11]. W i t h i n t he 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 the ce l l s at s t e a d y state at 3 7 ° C (see F i g u r e 15) [11]. T h e r e f o r e , 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 sho r t e s t p e r i o d o f t i m e n e e d e d to o b s e r v e m a x i m u m u p t a k e o f [ 3 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 tha t o p t i m a l u p t a k e o f [ 3 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 . T h e s e r e s u l t s w e r e c o n s i s t e n t w i t h the 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 the t i m e p e r i o d f o r [ 3 H ] C s A t r e a t m e n t to a v o i d f u r t h e r d e g r a d a t i o n o f I g G - C 7 . 6.4. Interpretation of results in [3H]CsA uptake and toxicity assay 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 a s s a y m e t h o d s w e r e u s e d to i n v e s t i g a t e the effect o f I g G - C 7 o n [ 3 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 nece s sa ry 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 a s says 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]. I n o r d e r to d o t h i s , a n o t h e r t o x i c i t y m a r k e r w a s c h o s e n . L a c t a t e d e h y d r o g e n a s e ( L D H ) i s a c y t o s o l i c e n z y m e tha t 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 r e s u l t o f d a m a g e to the 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 o f la te t o x i c i t y , L D H re lease i s d e t e c t e d 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]. S i g n i f i c a n t r e s u l t s w e r e r e v e a l e d i n m e a n p e r c e n t o f m e m b r a n e - b o u n d [ 3 H ] C s A i n the p r e s e n c e o f I g G - C 7 v e r s u s i ts absence (see F i g u r e 16). H o w e v e r , n o s i g n i f i c a n c e w a s o b s e r v e d i n the [ 3 H ] C s A - L D L c o m p l e x o r [ 3 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 , n o s i g n i f i c a n c e w a s s h o w n i n the p r e s e n c e o f L D L 89 v e r s u s i ts absence . T h e s e r e su l t s s u g g e s t t ha t p o s s i b l y C s A is i n t e r a c t i n g d i r e c t l y 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 o f i ts a s s o c i a t i o n w i t h L D L . O n the o t h e r h a n d , i t m a y s u g g e s t tha 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 n o n - s p e c i f i c L D L r e c e p t o r . A n o t h e r p o s s i b i l i t y i s t he s t e r i c 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 y i e l d s i g n i f i c a n t d i f f e rences i n m e a n p e r c e n t b o u n d . N o s i g n i f i c a n t r e s u l t s w e r e r e v e a l e d i n m e a n p e r c e n t u p t a k e o f [ 3 H ] C s A a l o n e a n d [ 3 H ] C s A w i t h L D L i n the p r e s e n c e o f I g G - C 7 v e r s u s i t s absence . I n a d d i t i o n , L D L 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 the u p t a k e o f [ 3 H ] C s A (see F i g u r e 17). T h i s is i n c o n s i s t e n t w i t h the r e su l t 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) w h e r e p r e i n c u b a t i o n o f i n c r e a s i n g L D L l e v e l s s i g n i f i c a n t l y r e d u c e d the u p t a k e o f C s A i n L L C - P K i ce l l s [91]. It i s p o s s i b l e tha t C s A is a s s o c i a t e d w i t h L D L a n d t h u s , less a m o u n t o f C s A is b e i n g t a k e n u p b y the ce l l s a n d n o t a f u n c t i o n o f 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 . T h e d i f fe rences i n r e su l t s c a n be e x p l a i n e d b y the d i f f e r en t a s say m e t h o d s as 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 [ 3 H ] C s A a n d L D L i n c o n t r a s t to a 6 - h o u r i n c u b a t i o n w i t h [ 3 H ] C s A a n d L D L i n th i s s t u d y . A s h o r t e r 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 [ 3 H ] C s A u p t a k e . T h e r e f o r e , t he d i f f e r ence i n i n c u b a t i o n t i m e s m a y e x p l a i n the i n c o n s i s t e n c y i n r e su l t s . I n a d d i t i o n , L L C - P K i ce l l s w e r e p r e t r e a t e d w i t h s e rum- f r ee 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 r ecep to r s . S i n c e the c e l l s 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 o f L D L ( 2 0 p g / m l c h o l e s t e r o l ) , the L D L r e c e p t o r s p o s s i b l y h a d p r e f e r e n t i a l u p t a k e o f L D L r a t h e r t h a n C s A . T h i s w o u l d e x p l a i n the i n s i g n i f i c a n t 90 r e s u l t s i n m e a n p e r c e n t b o u n d , u p t a k e a n d t o x i c i t y o f [ 3 H ] C s A w i t h 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 in fe rences f r o m the r e s u l t s w i t h L D L as i t is 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 o f L D L r e c e p t o r s in vitro. F i n a l l y , s i g n i f i c a n t r e s u l t s w e r e r e p o r t e d i n p e r c e n t t o x i c i t y o f [ 3 H ] C s A i n the p r e s e n c e o f I g G - C 7 v e r s u s i ts absence (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 [ 3 H ] C s A - L D L c o m p l e x a n d [ 3 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 , n o s i g n i f i c a n t d i f f e rences f r o m the [ 3 H ] C s A a l o n e g r o u p w e r e r e v e a l e d i n the p r e s e n c e o f L D L . T h e s e r e su l t s s u g g e s t tha t the L D L r e c e p t o r m a y 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 , i t i s o n l y c o n c l u s i v e t ha 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 be e x p l a i n e d b y the n a t u r e o f the t o x i c i t y m a r k e r . L D H is r e l e a s e d i n t o the c u l t u r e m e d i u m u p o n c e l l 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 o f la te 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 s e n s i t i v e m e t h o d to assess C s A r e n a l t o x i c i t y i s 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 [ 3 H ] l e u c i n e as 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). H o w e v e r , i n t h i s s t u d y , i t w a s n e c e s s a r y to c h o o s e th i s 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 o n e a s say m e t h o d . O v e r a l l , s i g n i f i c a n t r e s u l t s i n p e r c e n t [ 3 H ] C s A t o x i c i t y w e r e c o n s i s t e n t w i t h p e r c e n t [ 3 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 the u p t a k e o f [ 3 H ] C s A (see F i g u r e 17). T h e r e w a s n o s i g n i f i c a n t d i f f e rences i n i n t r a c e l l u l a r a m o u n t s o f [ 3 H ] C s A o b s e r v e d i n the p r e s e n c e o f I g G - C 7 v e r s u s i ts absence . H o w e v e r , t h e r e n e e d s t o b e i n t r a c e l l u l a r a m o u n t s o f t h e 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 i n t e r p r e t the p o s s i b i l i t y tha t [ 3 H ] C s A is t a k e n u p b y a n o n - L D L r e c e p t o r p a t h w a y o r a n o n - s p e c i f i c L D L r e c e p t o r n o t r e c o g n i z e d b y I g G -C 7 . It is s p e c u l a t i v e tha t t h i s r e c e p t o r c o u l d be 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 ce l l s [83]. M e g a l i n is a m e m b e r o f the L D L r e c e p t o r f a m i l y a n d i s a l a r g e c e l l su r face r e c e p t o r tha t 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 ce 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 the c y s t e i n e - r i c h r epea t s s h a r e d b y the n a t i v e L D L r e c e p t o r w i t h i n i ts 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 i s d i r e c t e d a g a i n s t the f i r s t c y s t e i n e - r i c h r epea t o f the f i r s t d o m a i n o f t he L D L r e c e p t o r [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 the l i g a n d b i n d i n g d o m a i n o f m e g a l i n is u n c e r t a i n ; y e t i t is a s s u m e d tha t the re i s h i g h h o m o l o g y i n t h i s r e g i o n b e t w e e n the L D L r e c e p t o r a n d m e g a l i n . O v e r a l l , i t i s c o n c l u s i v e f r o m t h i s s t u d y tha t t he L D L r e c e p t o r f a m i l y i s p l a y i n g a r o l e i n b o t h C s A b i n d i n g a n d t o x i c i t y i n L L C - P K i ce l l s . 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 p roces ses o f C s A b i n d i n g a n d e l i c i t i n g i ts t o x i c i t y i n L L C - P K i ce l l s 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 r e c e p t o r . It i s u n s u r e w h e t h e r C s A is 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 the L D L r e c e p t o r b u t t he r e su l t s d o n o t s u p p o r t t h i s h y p o t h e s i s . 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 r e c e p t o r , a n d e l i c i t s d r u g - i n d u c e d t o x i c i t y at the 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 be m e d i a t e d b y 92 CsA o ^ LDL Receptors Receptor- bound CsA 1 CsA-lndu«dMembrane Disruption / fegaiin ? Apical Renal Cell 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 o f the L D L r e c e p t o r f a m i l y w h i c h i s e x p r e s s e d i n L L C - P K i ce l l s [83]. T h e h y p o t h e s i s o f d r u g d i r e c t l y b i n d i n g to t he L D L r e c e p t o r f a m i l y has a l r e a d y b e e n p r o p o s e d b y o t h e r i n v e s t i g a t o r s [32,77,110]. M o e s t r u p et al (1995) s u g g e s t e d tha t m e g a l i n m a y m e d i a t e the u p t a k e o f p o l y b a s i c d r u g s , s p e c i f i c a l l y a m i n o g l y c o s i d e s s u c h as g e n t a m i c i n , a p r o t i n i n a n d p o l y m y x i n B [77]. T h e 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 says i n c u l t u r e d y o l k sac c a r c i n o m a ce l l s o f these 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 as w e l l as in vivo u p t a k e s t u d i e s 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 ra ts [77]. T h e y c o n c l u d e d tha t g p 3 3 0 m a y a c c o u n t f o r r e n a l u p t a k e o f a n u m b e r o f b a s i c m o l e c u l e s t h r o u g h l o w a f f i n i t y c h a r g e i n t e r a c t i o n w i t h g p 3 3 0 [77]. T h e y d i s c u s s e d 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 the m o l e c u l a r m e c h a n i s m o f u p t a k e o f p o l y b a s i c d r u g s c a n p r o v i d e i n s i g h t to i m p r o v e d a n t i b i o t i c t h e r a p y b y a v o i d i n g k i d n e y a n d ear 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 the s u g g e s t i o n tha 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 be r e c o g n i z e d as a d r u g r e c e p t o r [32]. A r ecen 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 m i c e d e m o n s t r a t e d tha t the u p t a k e o f a m i n o g l y c o s i d e s i n t o t he k i d n e y d i r e c t l y c o r r e l a t e d w i t h r e n a l m e g a l i n a c t i v i t y a n d tha t a c c u m u l a t i o n o f t he 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 the r e c e p t o r [110]. T h e a u t h o r s c o n c l u d e d tha t m e g a l i n w a s the o n l y m a j o r p a t h w a y r e s p o n s i b l e fo r r e n a l a m i n o g l y c o s i d e a c c u m u l a t i o n a n d tha t the r e c e p t o r r e p r e s e n t e d a u n i q u e d r u g ta rge t i n p r e v e n t i o n o f n e p h r o t o x i c i t y i n these p a t i e n t s [110]. A n o t h e r s t u d y b y N a g a i et al (2001) a l s o a t t e m p t e d to 94 i n v e s t i g a t e the 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 ra ts to i n v e s t i g a t e t i s sue u r i n a r y e x c r e t i o n o f e n d o g e n o u s m e g a l i n 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 (DBP) 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 tha t 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 s i g n i f i c a n t l y i n c r e a s e d 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 , the a u t h o r s p r o p o s e d tha t m e g a l i n w a s i n v o l v e d i n the 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 tha t the i n t e r a c t i o n 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 c e l l s w e r e e s t a b l i s h e d to be a n a p p r o p r i a t e c e l l m o d e l to i n v e s t i g a t e the r o l e o f the L D L r e c e p t o r o n C s A b i n d i n g a n d t o x i c i t y , i t expresses a l o w a b u n d a n c e o f L D L spec i f i c b i n d i n g si tes . 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 he re a re a l s o o the 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 t h i s s t u d y . T h e s e i n c l u d e H K - 2 c e l l s , 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 i s a l i m i t a t i o n to t h i s s t u d y . It w a s n e c e s s a r y to d e t e r m i n e the 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 [ 3 H] C s A 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 f u r t h e r d e g r a d a t i o n o f I g G - C 7 . T h e r e f o r e , a [ 3 H] 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 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 , s i n c e 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 to 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 is 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 t h i s s t u d y . Y e t , s i g n i f i c a n t r e s u l t s w e r e o b t a i n e d w h i c h s h o w s tha t t h i s i s n o t a m a j o r l i m i t a t i o n . A 2 0 p g / m l c o n c e n t r a t i o n o f L D L , b a s e d o n c h o l e s t e r o l c o n t e n t , w a s c h o s e n i n the u p t a k e a n d t o x i c i t y a s says w i t h [ 3 H ] C s A a n d I g G - C 7 . T h e r a t i o n a l e w a s b a s e d o n s i g n i f i c a n t r e s u l t s o b s e r v e d 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 the s a m e c o n c e n t r a t i o n o f 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 t u b u l e o f h y p e r c h o l e s t e r o l e m i c pa t i en t s [91]. N e v e r t h e l e s s , t h i s c o n c e n t r a t i o n o f L D L m a y h a v e b e e n t o o h i g h f o r these s t u d i e s w h e n i n v e s t i g a t i n g the effect o f L D L r e c e p t o r s o n C s A b i n d i n g a n d t o x i c i t y . 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 the 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 the 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 p r o v i d i n g a n a d d i t i o n a l c o n f o u n d e r . F i n a l l y , the u s e o f I g G - C 7 to m o d i f y L D L r e c e p t o r a c t i v i t y m a y b e a l i m i t a t i o n i n i tself . 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 spec i f i c to t he h u m a n L D L r e c e p t o r [11] y e t 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 r e c e p t o r i n a p i g c e l l l i n e . In t e r spec ies d i f f e rences i n the L D L r e c e p t o r m a y res t r i c t the s p e c i f i c i t y i n b i n d i n g o f I g G - C 7 w h i c h m a y be the r e a s o n w h y s u c h a h i g h c o n c e n t r a t i o n o f 1 2 . 0 p g / m l w a s n e e d e d i n t h i s s t u d y . H o w e v e r , a p r o t e i n B L A S T a n a l y s i s h a s c o n f i r m e d tha t the h u m a n a n d p i g L D L r e c e p t o r s h a v e a b o u t a n 8 7 % s e q u e n c e h o m o l o g y o f i t s 818 a m i n o a c i d r e s i d u e s . I n a d d i t i o n , the m a n u f a c t u r e r o f the a n t i b o d y ( R D I ) c o u l d n o t 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 i t h a s n o t b e e n t e s ted 96 in pigs. They have tested positive in human and bovine species and negative in 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 in mediating the uptake and toxicity of C s A in L L C - P K i cells. Further studies to support the hypothesis would be to conduct similar studies in a different cell lines such as HEK-293, a human embryonic proximal tubule cell line [64,74] or HK-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 wi th cholesterol transport and loading [142-144]. Addit ional in vitro studies could be implemented in CH01dlA7, 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 would provide much stronger evidence of the role of L D L receptor activity in C s A uptake and toxicity. 97 6.8. Overall Conclusions I n t h i s 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 tha t C s A b i n d s d i r e c t l y to the L D L r e c e p t o r a n d c a n e l i c i t C s A - i n d u c e d t o x i c i t y i n L L C - P K i c e l l s , a p i g p r o x i m a l t u b u l e c e l l l i n e . C s A h a s a n a r r o w t h e r a p e u t i c i n d e x a n d i t i s d u e to i t s n e p h r o t o x i c i t y tha t 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 in te rac t s a n d causes t o x i c i t y i n r e n a l ce l l s c o u l d p r o v i d e i m p r o v e d C s A 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 p r o f i l e o f the pa t i en t . 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 are 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 , pa t i en t s w h o are 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 cacy [25, 82]. T h e r e f o r e , pa t i en t s 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 p a t i e n t s 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 o f 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 r e c e p t o r to a v o i d t o x i c i t y ; ye t t a rge t to s i t e - spec i f i c a reas s u c h as T - c e l l s t o m a i n t a i n i ts e f f i cacy . 98 References 99 [1] N e o r a l a n d S a n d i m m u n e ~ C y c l o s p o r i n e . T h i r t y - F i f t h e d i t i o n . 2000. C o m p e n d i u m o f P h a r m a c e u t i c s l a n d S p e c i a l t i e s ( C P S ) . [2] A . T . W e b b , M . P l a n t , D . A . R e a v e l e y , M . O ' D o n n e l , V . A L u c k , B . O ' C o n n o r , M . 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