"Pharmaceutical Sciences, Faculty of"@en . "DSpace"@en . "UBCV"@en . "Chung, Nancy S.C."@en . "2009-11-21T20:42:59Z"@en . "2004"@en . "Master of Science - MSc"@en . "University of British Columbia"@en . "Background: Cyclosporine A (CsA) is an effective immunosuppressant drug to treat\r\npatients who have undergone transplantation or to treat autoimmune diseases.\r\nHowever, the drug is limited by its narrow therapeutic index and usually becomes\r\ndiscontinued due to high nephrotoxicity. CsA is known to highly associate with\r\nlipoproteins, especially LDL and increased toxic effects of CsA have been reported\r\nin patients who are hypocholesterolemic. A significant reduction in [3H]CsA uptake\r\nand toxicity was observed when LLC-PK1 cells were treated with increased\r\nconcentrations of LDL.\r\n\r\nPurpose: Based on the experimental and clinical evidence, it is hypothesized that\r\nwhen the LDL receptor family activity is decreased upon IgG-C7 treatment, both\r\nCsA uptake and toxicity are reduced in LLC-PK1 cells, a renal proximal tubule cell\r\nline.\r\n\r\nMethods: The appropriateness of LLC-PK1 cells as a cell model was assessed by\r\nconducting dose-response, LDL specific binding and competitive studies with Dil-\r\nLDL, and Western blot analysis of the LDL receptor. Assay conditions with IgG-C7,\r\na monoclonal antibody to the LDL receptor, were optimized including temperature,\r\npreincubation time and concentration in LLC-PK1 cells. Finally, the effect of IgG-C7\r\non [3H]CsA uptake and toxicity with LDL was determined.\r\n\r\nResults: Significant results in both mean percent bound (2.6% \u00B1 0.6% vs. 5.1% \u00B1\r\n1.3%) and mean percent toxicity (1.8% \u00B1 0.5% vs. 3.2% \u00B1 0.5%) were observed in the\r\n[3H]CsA alone group in the presence of IgG-C7 versus its absence (p<0.05 with\r\nunpaired t-test). However, no significant differences were observed in the [3H]CsA- LDL complex or [3H]CsA with LDL coaddition groups. In addition, LDL was not\r\nassociated with a significant reduction in both [3H]CsA bound, uptake and toxicity.\r\n\r\nConclusion: These results suggest that CsA may be binding directly to the LDL\r\nreceptor family independent of its association with LDL and thus, eliciting its toxic\r\neffects at the membrane level. This study provides preliminary evidence of the\r\nfamily of LDL receptors playing a role in CsA binding and toxicity in LLC-PK1 cells."@en . "https://circle.library.ubc.ca/rest/handle/2429/15474?expand=metadata"@en . "6908334 bytes"@en . "application/pdf"@en . "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 \u00C2\u00A9 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: ^\u00C2\u00A3-\u00C2\u00A3ivx{ cells Degree: 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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\u00C2\u00B0C 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00E2\u0080\u0094\u00E2\u0080\u00A2 Cholesterol + Fatty Acids c o Cholesterol + O2 \u00E2\u0080\u0094*\u00E2\u0080\u00A2 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\u00C2\u00B0C 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00C2\u00B0C. 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\u00C2\u00B0C 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 ) - \u00C2\u00BB 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 \u00C2\u00B0 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\u00C2\u00AE 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\u00C2\u00B0C 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\u00C2\u00B0C, 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\u00C2\u00B0C of increasing Dil-LDL concentrations from 5pg/ml to 40 pg/ml to measure cell-associated LDL. At 37\u00C2\u00B0C, 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\u00C2\u00B0C while the other in the incubator at 37\u00C2\u00B0C. 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\u00C2\u00B0C 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\u00C2\u00B0C 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00AE . 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 \u00C2\u00AE 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 \u00C2\u00B0 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 \u00C2\u00AE ) 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 \u00C2\u00B0 C f o r 2 h o u r s o r o v e r n i g h t at 4 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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\u00C2\u00B0 a n t i b o d y s o l u t i o n at 3 7 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 C a n d 3 7 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 C a n d 3 7 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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\u00C2\u00B0C. 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 \u00E2\u0080\u0094 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 - \u00E2\u0080\u00A2 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 \u00C2\u00B0 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 \u00C2\u00AE 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 \u00C2\u00B1 S D i n t r i p l i c a t e s o r m e a n \u00C2\u00B1 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 \u00C2\u00B0 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 ) \u00C2\u00B1 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\u00C2\u00B0C. 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 ) \u00C2\u00B1 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 ) \u00C2\u00B1 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 \u00C2\u00B0 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) -\u00E2\u0080\u00A2\u00E2\u0080\u0094 Non-specific binding (+LDL) *\u00E2\u0080\u0094 Total Binding (-LDL) -\u00E2\u0080\u00A2\u00E2\u0080\u0094 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 \u00C2\u00B0 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 \u00C2\u00B1 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 \u00C2\u00B1 0 . 0 5 5 0 . 2 4 \u00C2\u00B1 0 . 0 3 0.05+0.07 0 . 1 8 \u00C2\u00B1 0 . 0 3 10 0 . 4 4 \u00C2\u00B1 0 . 0 5 0.06+0.01 0.38+0.06 20 0 . 6 9 \u00C2\u00B1 0 . 0 0 0.12+0.03 0 . 5 7 \u00C2\u00B1 0 . 0 5 40 0 . 9 2 \u00C2\u00B1 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) -\u00E2\u0080\u00A2\u00E2\u0080\u0094 Non-specific binding -\u00E2\u0080\u00A2\u00E2\u0080\u0094 Total binding -T\u00E2\u0080\u0094 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 \u00C2\u00B0 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 \u00C2\u00B1 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 \u00C2\u00B0 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 ) \u00C2\u00B1 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 C . 66 100 -80 -I , , , , 1 0.0 0.5 1.0 1.5 2.0 2.5 mAb concentration (ug/ml) - \u00E2\u0080\u00A2 - 4\u00C2\u00B0C - \u00E2\u0080\u00A2 - 37\u00C2\u00B0C 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\u00C2\u00B0C. Subsequently, 10 p g / m l of D i l -L D L was further incubated at 4\u00C2\u00B0C and 37\u00C2\u00B0C 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 \u00C2\u00B1 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% \u00C2\u00B110% 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% \u00C2\u00B1 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-\u00C2\u00BB C 01 (8 QJ H I C o c (fl 3 tfl > c o \u00E2\u0080\u00A2J3 '\u00C2\u00A3 '\u00C2\u00A3 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\u00C2\u00B0C prior to addition of lOug/ml Dil-LDL for 2 hrs at 4\u00C2\u00B0C. Values expressed as percent inhibition vs non-treatment control \u00C2\u00B1 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\u00C2\u00B0C 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\u00C2\u00B0C. 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% \u00C2\u00B1 6 % (mean \u00C2\u00B1 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\u00C2\u00B0C 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B1 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\u00C2\u00B0C 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\u00C2\u00B0C 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\u00C2\u00B0C (see F igure 15) [11]. It is necessary to conduct a l l [ 3 H ] C s A uptake experiments at 37\u00C2\u00B0C 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% \u00C2\u00B1 0.4% (mean \u00C2\u00B1 S E M ) w i t h i n 10 hours at 4\u00C2\u00B0C. 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% \u00C2\u00B1 1.2% w i t h i n 6 hours at 37 \u00C2\u00B0C. After 6 hours, there was no change i n [ 3 H ] C s A uptake at 37 \u00C2\u00B0C; rather, uptake decreased after an incubat ion pe r iod of 10 hours (19.2% \u00C2\u00B1 1.6%) and a greater decrease after 24 hours (13.3% \u00C2\u00B1 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\u00C2\u00B0C. 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\u00C2\u00B0C 4\u00C2\u00B0C 2 15.49\u00C2\u00B11.33 1.78\u00C2\u00B10.34 6 21.24\u00C2\u00B11.20 1.80\u00C2\u00B10.40 10 19.15\u00C2\u00B11.57 8.88\u00C2\u00B10.39 24 13.28\u00C2\u00B11.95 6.37+1.00 Table 6: Percent uptake of [ 3 H] C s A over 24 hour pe r iod at 37\u00C2\u00B0C vs 4\u00C2\u00B0C. 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\u00C2\u00B0C and 4\u00C2\u00B0C 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 \u00C2\u00B1 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 \u00C2\u00B0 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 \u00C2\u00A9 . 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 \u00C2\u00B0 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 \u00C2\u00B0 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% \u00C2\u00B1 0.6%) v e r s u s i ts absence (5 .1% \u00C2\u00B1 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 \u00C2\u00B0C 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\u00C2\u00B0C. 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 \u00C2\u00B1 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B1 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\u00C2\u00B0C, 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\u00C2\u00B0C (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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B1 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 \u00E2\u0080\u0094\u00E2\u0080\u00A2\u00E2\u0080\u0094 522/578 nm - O \u00E2\u0080\u0094 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 \u00C2\u00B1 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 % \u00C2\u00B1 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 \u00C2\u00B0 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 \u00C2\u00B1 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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 \u00C2\u00B0 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\u00C2\u00ABdMembrane 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 . S e e , a n d E . A . B r o w n (1992) L i p i d a n d l i p o p r o t e i n (a) c o n c e n t r a t i o n s i n r e n a l t r a n s p l a n t pa t i en t s . Nephrol Dial Transpl 7:636-641. [3] A k h l a g h i F a n d T r u l l A K (2002) D i s t r i b u t i o n o f c y c l o s p o r i n i n o r g a n t r a n s p l a n t r e c i p i e n t s . Clin.Pharmacokinet. 41:615-637. [4] A l l e n M J a n d R u s h t o n N . U s e o f C y t o T o x 9 6 ( T M ) A s s a y i n R o u t i n e B i o c o m p a t i b i l i t y T e s t i n g I n V i t r o . P r o m e g a N o t e s M a g a z i n e 45 , 7 .1994 . [5] A l t s c h u h D (2002) C y c l o s p o r i n A as a m o d e l a n t i g e n : i m m u n o c h e m i c a l a n d s t r u c t u r a l s t u d i e s . J.Mol.Recognit. 15:277-285. [6] A m i n K , W a s a n K M , A l b r e c h t R M , a n d H e a t h T D (2002) C e l l a s s o c i a t i o n o f l i p o s o m e s w i t h h i g h f l u i d a n i o n i c p h o s p h o l i p i d c o n t e n t i s m e d i a t e d s p e c i f i c a l l y b y L D L a n d i t s r e c e p t o r , L D L r . / Pharm Sci 91:1233-1244. [7] A r n a d o t t i r M , T h y s e l l H , a n d N i l s s o n - E h l e P (1991) L i p o p r o t e i n l e v e l s a n d p o s t - h e p a r i n l i p a s e a c t i v i t i e s i n k i d n e y t r a n s p l a n t r e c i p i e n t s : c i c l o s p o r i n -v e r s u s n o n - c i c l o s p o r i n - t r e a t e d pa t i en t s . Am ] Nephrol. 11:391-396. [8] A w n i W M a n d S a w c h u k R J (1984) T h e p h a r m a c o k i n e t i c s o f c y c l o s p o r i n e : B l o o d p l a s m a d i s t r i b u t i o n a n d b i n d i n g s t u d i e s . Drug Metab Dispos 13:133-138. [9] B a l l a n t y n e C M , P o d e t E J , P a t s c h W P , H a r a t i Y , A p p e l V , G o t t o M , a n d o u n g JB (1989) Effec ts o f c y c l o s p o r i n e t h e r a p y o n p l a s m a l i p o p r o t e i n s l e v e l s . J Am Med Assoc 262:53-56. [10] B e c k e r G M , G a n d o l f i A J , a n d N a g l e R B (1987) Effec ts o f c y c l o s p o r i n e A o n a k i d n e y e p i t h e l i a l c e l l l i n e ( L L C P K i ) . Res Comm Chem Path Pharm 11:778-783. [11] B e i s i e g e l U , S c h n e i d e r W J , G o l d s t e i n J L , A n d e r s o n R G W , a n d B r o w n M S (1981) M o n o c l o n a l a n t i b o d i e s t o the 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 as p r o b e s f o r s t u d y 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 a n d the gene t i c s o f 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 . / Biol Chem 25:11923-11931. [12] B e n n e t t W M (1990) R e n a l Effects o f c y c l o s p o r i n e . / Am Acad Dermatol 23:1280-1287. [13] B o r d e r W A a n d N o b l e N A (1997) T G F - b e t a i n k i d n e y f i b r o s i s : a t a rge t for gene t h e r a p y . Kidney Int. 51:1388-1396. 100 [14] Bore l JF and K i s Z L (1991) The discovery and deve lopment of cyclosporine (Sandimmune) . Transplant.Proc. 23:1867-1874. [15] B r o w n M S and Golds t e in JL (1975) Regu la t ion of the act ivi ty of the l o w density l ipopro te in receptor i n h u m a n fibroblasts. Cell 6:307-316. [16] B r o w n M S and Golds t e in JL (1986) A Receptor-mediated Pa thway for Choles terol Homeostasis . Science 232:34-47. [17] Chatterjee S, C la rke K , and K w i t e r o v i c h P O (1986) Up take and metabol ism of lactosylceramide o n l o w density l ipoproteins i n cu l tured p r o x i m a l tubular cells f rom n o r m a l and fami l ia l hypercholesterolemic homozygotes . / Biol Chem 261:13480-13486. [18] C h e n Z , Saffitz JE, La tour M A , and Schonfeld G (1999) Truncated apo B-70.5-conta in ing l ipoproteins b i n d to mega l in but not the L D L receptor. / Clin Invest 103:1419-1430. [19] Chris tensen EI , B i r n H , Verrous t P, and Moes t rup S K (1998) M e g a l i n -media ted endocytosis i n renal p r o x i m a l tubule. Ren Fail. 20:191-199. [20] C o l e E (1989) Toxic effects on renal cells i n cul ture \u00E2\u0080\u0094a compar i son of cyclosporine A and its metabolites. Transp Proc 21:943-945. [21] Dasht i N , Wofbauer G , K o r e n E , K n o w l e s B, and A l a u p o v i c P (1984) Ca tabo l i sm of h u m a n l o w density l ipoproteins by h u m a n hepatoma cell l ine hepG2. Biochem Biophys Acta 794:373-394. [22] Dav i s R A and Vance JE (1996) Structure, assembly a n d secretion of l ipoproteins , i n Biochemistry of lipids, lipoproteins and membranes (Vance D E and Vance JE eds) p p 473-493, Elsevier , N e w Y o r k . [23] D e G r o e n P C (1988) Cyc lospor ine , low-dens i ty l ipopro te in , and cholesterol. Mayo Clin Proc 63:1012-1021. [24] D e G r o e n P C , A k s a m i t A J , Rakela J, Forbes G S , a n d K r o m RJ (1987) Cent ra l nervous system toxici ty after l iver transplantation, The role of cyclosporine and cholesterol. N Engl} Med. 317:861-866. [25] D e K i p p e l N , Sennesael J, Lamote J, Ebinger G , and de Keyser J (1992) Cyc lospor ine leukoencephalopathy induced by intravenous l i p i d solut ion. Lancet 339:1114-1115. 101 [26] D o m i n g u e z J H , S o l e i m a n i M , a n d B a t i u k T (2002) S t u d i e s o f r e n a l i n j u r y I V : T h e GLUT1 g e n e p ro t ec t s r e n a l ce l l s f r o m c y c l o s p o r i n e A t o x i c i t y . Kid Int 62:127-136. [27] D u n n C J , W a g s t a f f A J , P e r r y C M , P l o s k e r G L , a n d G o a K L (2001) C y c l o s p o r i n : a n u p d a t e d r e v i e w o f t he 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 , c l i n i c a l e f f i cacy a n d t o l e r a b i l i t y o f a m i c r o e m u l s i o n - b a s e d f o r m u l a t i o n ( n e o r a l ) l i n o r g a n t r a n s p l a n t a t i o n . Drugs 61 :1957-2016 . [28] E i s e n b e r g S (1984) H i g h - d e n s i t y l i p o p r o t e i n m e t a b o l i s m . / Lipid Res 25:1017-1058. [29] E r k a n E , D e L e o n M , a n d D e v a r a j a n P (2001) A l b u m i n o v e r l o a d i n d u c e s a p o p t o s i s i n L L C - P K ( l ) ce l l s . Am J Physiol Renal Physiol 280:F1107-F1114. [30] F a h r A (1993) C y c l o s p o r i n C l i n i c a l P h a r m a c o k i n e t i c s . Clin Pharmacokinet 24:472-495. [31] F a h r A a n d S e e l i g J (2001) L i p o s o m a l f o r m u l a t i o n s o f C y c l o s p o r i n A : a b i o p h y s i c a l a p p r o a c h to p h a r m a c o k i n e t i c s a n d p h a r m a c o d y n a m i c s . Crit Rev.Ther.Drug Carrier Syst. 18:141-172. [32] F a r q u h a r M G (1995) T h e u n f o l d i n g s t o r y o f m e g a l i n (gp330) : n o w r e c o g n i z e d as a d r u g r e c e p t o r . / Clin Invest 96:1184. [33] F a r q u h a r M G , S a i t o A , K e r j a s c h k i D , a n d O r l a n d o R A (1995) T h e H e y m a n n n e p h r i t i s a n t i g e n i c c o m p l e x : m e g a l i n (gp330) a n d R A P . J.Am.Soc.Nephrol. 6:35-47. [34] F i e l d i n g C J a n d F i e l d i n g P E (1995) M o l e c u l a r p h y s i o l o g y o f r e v e r s e c h o l e s t e r o l t r a n s p o r t . / Lipid Res 36:211-228. [35] F i e l d i n g P E a n d F i e l d i n g C J (1996) D y n a m i c s o f l i p o p r o t e i n t r a n s p o r t i n the h u m a n c i r c u l a t o r y s y s t e m , i n Biochemistry of lipids, lipoproteins and membranes ( V a n c e D E a n d V a n c e J E eds) p p 495-516, E l s e v i e r , N e w Y o r k . [36] F o x w e l l B M , M a c k i e A , L i n g V , a n d R y f f e l B (1989) I d e n t i f i c a t i o n o f the m u l t i -d r u g r e s i s t a n c e - r e l a t e d P - g l y c o p r o t e i n as a c y c l o s p o r i n e b i n d i n g p r o t e i n . Mol Pharm 36 :543-546. [37] G a r d i e r A M , M a t h e D , G u e d e n e y X , B a r r e J , B e n v e n u t t i C , N a v a r r o N , V e r n i l l e t L , L o i s a n c e D , C a c h e r a JP , a n d Jaco to t B (1993) Effec ts o f p l a s m a l i p i d l e v e l s o n b l o o d d i s t r i b u t i o n a n d p h a r m a c o k i n e t i c s o f c y c l o s p o r i n e A . Therap Drug Monitoring 15:274-280. 102 [38] G l i e m a n n J (1998) R e c e p t o r s o f the l o w d e n s i t y l i p o p r o t e i n ( L D L ) r e c e p t o r f a m i l y i n m a n . M u l t i p l e f u n c t i o n s o f the l a r g e f a m i l y m e m b e r s v i a i n t e r a c t i o n w i t h c o m p l e x l i g a n d s . Biol.Chem. 379:951-964. [39] G o k a l R , M a n n JI, M o o r e R A , a n d M o r r i s P J (1979) H y p e r l i p i d e m i a f o l l o w i n g r e n a l t r a n s p l a n t a t i o n . Q } Med: 634. [40] G o l d s t e i n J L , B a s u S K , a n d B r o w n M S (1983) 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 o f l o w - d e n s i t y l i p o p r o t e i n i n c u l t u r e d ce l l s . Methods Enzymol 98:241-260. [41] G o l d s t e i n J L a n d B r o w n M S (1974) B i n d i n g a n d d e g r a d a t i o n o f l o w d e n s i t y l i p o p r o t e i n s b y c u l t u r e d h u m a n f i b rob l a s t s : c o m p a r i o s n o f c e l l s f r o m a n o r m a l subject f r o m a p a t i e n t w i t h h o m o z y g o u s 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 . / Biol Chem 249:5153-5162. [42] G o l d s t e i n J L , H o b b s H H , a n d B r o w n M S (1995) 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 , i n The Metabolic and Molecular Basis of Inherited Disease ( S c r i v e r C S , B a e n d e t A L , S l y W , a n d V a l l e D eds) p p 1981-2030, M c G r a w H i l l , N e w Y o r k . [43] G r a n e l l i - P i p e r n o A (1988) I n s i t u h y b r i d i z a t i o n f o r i n t e r l e u k i n 2 a n d i n t e r l e u k i n 2 r e c e p t o r m R N A i n T ce l l s a c t i v a t e d i n t he p r e s e n c e o r absence o f c y c l o s p o r i n e A . J.Exp.Med. 168:1649-1658. [44] G r e e n E L a n d P a l l e r M S (1991) O x y g e n free r a d i c a l s i n a c u t e r e n a l f a i l u r e . Miner.Electrolyte Metab 17:124-132. [45] H a n d s S c h u m a c h e r R E , H a r d i n g M W , R i c e J , D r u g g e R J , a n d S p e i c h e r D W (1984) C y c l o p h i l i n : a spec i f i c c y t o s o l i c b i n d i n g p r o t e i n f o r c y c l o s p o r i n e A . Science 226:544-547. [46] H a n d s c h u m a c h e r R E , H a r d i n g M W , R i c e J , D r u g g R J , a n d S p e i c h e r D W (1984) C y c l o p h i l i n : a spec i f i c c y t o s o l i c b i n d i n g p r o t e i n fo r c y c l o s p o r i n A . Science 226:544-547. [47] H e a l y E , D e m p s e y M , L a l l y C , a n d R y a n M P (1998) A p o p t o s i s a n d nec ros i s : m e c h a n i s m s o f c e l l d e a t h i n d u c e d b y c y c l o s p o r i n e A i n a r e n a l p r o x i m a l t u b u l a r c e l l l i n e . Kidney Int. 54:1955-1966. [48] H e a l y E , D e m p s e y M , L a l l y C , a n d R y a n M P (1998) A p o p t o s i s a n d nec ros i s : m e c h a n i s m s o f c e l l d e a t h i n d u c e d b y c y c l o s p o r i n e A i n r e n a l p r o x i m a l t u b u l a r c e l l l i n e . Kid Int 54:1955-1966. 1 0 3 [49] H e r o l d K C , L a n c k i D W , M o l d w i n R L , a n d F i t c h F W (1986) I m m u n o s u p p r e s s i v e effects o f c y c l o s p o r i n e A o n c l o n e d T ce l l s . J.Immunol. 136:1315-1321. [50] H e r z J (2001) T h e L D L r e c e p t o r gene f a m i l y : ( u n ) e x p e c t e d s i g n a l t r a n s d u c e r s i n the b r a i n . Neuron 29:571-581. [51] H i r a n o T , A k a s h i T , O k a K , Y o s h i d a M , M a t s u n o N , a n d N a g a o T (2001) S e r u m c h o l e s t e r o l l e v e l s a n d k i d n e y t r a n s p l a n t a t i o n o u t c o m e : 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? Transplant 71:659-660. [52] H i r a n o T , K a w a m u r a T , F u k u d a S, K o h s a k a S, Y o s h i k a w a N , Y o s h i d a M , a n d O k a K (2003) P h a r m a c o d y n a m i c s a n d d r u g a c t i o n : i m p l i c a t i o n o f c h o l e s t e r o l i n c y c l o s p o r i n e p h a r m a c o d y n a m i c s i n m i n i m a l c h a n g e n e p h r o t i c s y n d r o m e . Clin Pharm Therap 74:581-590. [53] H o r t e l a n o S, C a s t i l l a M , T o r r e s A N , T e j e d o r A , a n d B o s c a L (2000) P o t e n t i a t i o n b y n i t r i c o x i d e o f c y c l o s p o r i n e A a n d F K 5 0 6 - i n d u c e d a p o p t o s i s i n r e n a l p r o x i m a l t u b u l e ce l l s . J Am Soc Nephrol 11:2315-2323. [54] H u l l R N , C h e r r y W R , a n d W e a v e r G W (1976) T h e o r i g i n a n d cha rac t e r i s t i c s o f a p i g k i d n e y c e l l s t r a i n , L L C P K i . In vitro 12:670-677. [55] H u s s a i n M M , S t r i c k l a n d D K , a n d B a k i l l a h A (1999) T h e m a m m a l i a n 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 f a m i l y . Annu.Rev.Nutr. 19:141-172. [56] I l l i n g w o r t h D R , L i n s e y S, a n d H a g e m e n a s F C (1984) R e g u l a t i o n o f 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 s i n the h u m a n h e p a t o m a c e l l l i n e h e p G 2 . Exp Cell Res 155:518-526. [57] I n n e r a r i t y T L , P i t a s R E , a n d M a h l e y R W (1986) L i p o p r o t e i n - r e c e p t o r In t e r ac t ions . Methods Enzymol 129:542-565. [58] I n s e l m a n n G , H a n n e m a n n J, a n d B a u m a n n K (1990) C y c l o s p o r i n e A i n d u c e d l i p i d p e r o x i d a t i o n a n d i n f l u e n c e o n g l u c o s e - 6 - p h o s p h a t a s e i n r e n a l h e p a t i c a n d r e n a l m i c r o s o m e s . Res Comm. Chem Path Pharm 68:189-203. [59] K a h a n B D (1989) D r u g T h e r a p y : C y c l o s p o r i n e . N.Engl.J.Med. 321:1725-1738. [60] K e r j a s c h k i D a n d F a r q u h a r M G (1983) I m m u n o c y t o c h e m i c a l l o c a l i z a t i o n o f H e y m a n n n e p h r i t i s a n t i g e n ( G P 3 3 0 ) i n g l o m e r u l a r e p i t h e l i a l c e l l s o f n o r m a l L e w i s ra ts . / Exp Med. 157:667. 104 [61] K r i e g e r M a n d H e r z J (1994) S t r u c t u r e s a n d F u n c t i o n s o f M u l t i l i g a n d l i p o p r o t e i n r ecep to r s : M a c r o p h a g e S c a v e n g e r R e c e p t o r s a n d 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 . Anna Rev.Biochem 63 :601-637 . [62] K r o n k e M , L e o n a r d W T , D e p p e r J M , A y r a S K , W o n g - S t a a l F , G a l l o R C , W a l d m a n T A , a n d G r e e n e W C (1984) C y c l o s p o r i n e A i n h i b i t s T - c e l l g r o w t h f ac to r g e n e e x p r e s s i o n a t the l e v e l o f m R N A t r a n s c r i p t i o n . Proc.Natl.Acad.Sci.U.SA 81 :5214-5218. [63] K w o n g M , S i v a k O , K w o n g E H , a n d W a s a n K M (2001) C y c l o s p o r i n e A t rans fe r b e t w e e n h i g h - a n d l o w - d e n s i t y l i p o p r o t e i n s : i n d e p e n d e n t f r o m l i p i d t r ans fe r p r o t e i n I - f ac i l i t a t ed t ransfer o f l i p o p r o t e i n - c o a t e d p h o s p h o l i p i d s b e c a u s e o f h i g h a f f i n i t y o f c y c l o s p o r i n e A f o r the p r o t e i n c o m p o n e n t o f l i p o p r o t e i n . / Pharm Sci 90 :1308-1317. [64] L a r s s o n M , H j a l m G , S a k w e A M , E n g s t r o m A , H o g l u n d A S , L a r s s o n E , R o b i n s o n R C , S u n d b e r g C , a n d R a s k L (2003) S e l e c t i v e i n t e r a c t i o n of m e g a l i n w i t h p o s t s y n a p t i c d e n s i t y - 9 5 ( P S D - 9 5 ) - l i k e m e m b r a n e - a s s o c i a t e d g u a n y l a t e k i n a s e ( M A G U K ) p r o t e i n s . Biochem J 373 :381 -391 . [65] L e g g B a n d R o w l a n d M (1987) C y c l o s p o r i n : m e a s u r e m e n t o f f r a c t i o n u n b o u n d i n p l a s m a . / Pharm Pharmacol 39 :599-603 . [66] L e h e s t e L , R o l i n s k i B , V o r u m H , H i p e r t J , N y k j a i e r A , J a c o b s e n C , A u c o u t u r i e r P , M o s k a u g J O , O t t o A , C h r i s t e n s e n E I , a n d W i l l n o w T E (1999) M e g a l i n K n o c k o u t M i c e as a n A n i m a l M o d e l o f L o w M o l e c u l a r W e i g h t P r o t e i n u r i a . Am J Path 155 :1361-1370 . [67] L e m a i r e M , a r d r i d g e W M , a n d C h a u d h u r i G (1988) I n f l u e n c e o f b l o o d c o m p o n e n t s o n the t i s sue u p t a k e i n d i c e s o f c y c l o s p o r i n e i n ra ts . / Pharm Exp Ther 244 :740-743 . [68] L e u n i s s e n K M , B e u m a n G H , B o s m a n F T , a n d v a n H o o f J P (1988) T h e n e p h r o t o x i c effects o f c y c l o s p o r i n e m e t a b o l i t e s . Transplant Proc 20 :238-239 . [69] L i u J , F a r m e r J D , L a n e W S , F r i e d m a n J , W e i s s m a n I, a n d S c h r e i b e r S L (1991) C a l c i n e u r i n i s a c o m m o n ta rge t o f c y c l o p h i l i n - c y c l o s p o r i n A a n d F K B P - F K 5 0 6 c o m p l e x e s . Cell 66 :807-815 . [70] L o s s l i H R , K e s s l e r H , O s c h k i n a t , W e b e r H P , P e t c h e r T J , a n d W i d m e r A (1985) T h e c o n f o r m a t i o n o f c y c l o s p o r i n e A i n the c r y s t a l a n d s o l u t i o n . Helv Chim Acta 68 :682 . [71] M a s s i c o t F , L a m o u r i A , M a r t i n C , P h a m - H u y C , H e y m a n s F , W a r n e t J M , G o d f r o i d JJ, a n d C l a u d e J R (1996) P r e v e n t a t i v e effects o f t w o P A F -105 antagonists, P M S 536 and P M S 549, on cyc lospor ine- induced L L C - P K I oxidat ive injury. / Lipid Med Cell Sig 15:203-214. [72] M a t s u d a S and K o y a s u S (2000) Mechan i sms of act ion of cyclosporine. Immunopharmacology 47:119-125. [73] M a y e r R D , Be rman S, Cocket t A T , and Maines M D (1989) Differential effects of cyclosporine o n hepatic and renal heme cytochrome P-450 and d r u g metabol ism. Possible Role i n nephrotoxici ty of the d rug . Biochem Pharmacol 38:1001-1007. [74] M c L a r n o n S, H o l d e n D , W a r d D , Jones M , El l io t t A , a n d R icca rd i D (2002) A m i n o g l y c o s i d e antibiotics induce pH-sens i t ive act ivat ion of the ca lc ium-sensing receptor. Biochem Biophys Res Commun. 297:71-77. [75] M i h a t s c h M J , Th ie l G , and Ryffel B (1990) Rena l side-effects of cyclosporine A w i t h special reference to au to immune diseases. Brit} Derm 122:101-115. [76] M i h a t s c h M J , Th ie l G , and Sp ick t in M P et a l (1983) F ind ings i n k idney transplant after treatment w i t h cyclosporine. Transp Proc 15:2821-2835. [77] Moes t rup S K , C u i S, V o r u m H , Bregengard C , Bjorn SE , N o r r i s K , G l i e m a n n J, and Chris tensen EI (1995) Evidence that epi thel ial g lycopro te in 330/ mega l in mediates uptake of polybasic drugs. / Clin Invest 96:1404-1413. [78] M o l e c u l a r Probes. Product Information for Low-dens i t y l ipoproteins ( D i l complex) Ca t # L-3482. 2002. [79] M o r r i s S M Jr, Kepka-Lenhar t D , M c G i l l R L , C u r t h o g y N P , and A d l e r S (1992) Specific d i s rup t ion of renal funct ion and gene t ranscr ipt ion by cyclosporine A . / Biol Chem 267:13768-13771. [80] N a g a i J, Tanaka H , N a k a n i s h i N , M u r a k a m i T, and Takano M (2001) Role of mega l in i n renal h a n d l i n g of aminoglycosides . Am J Physiol Renal Physiol 281:F334-F337. [81] N a t h K A and N o r b y S M (2000) Reactive oxygen species a n d acute renal failure. Am J Med. 109:665-678. [82] Nemuna i t i s J, Deeg H J , and Yee G C (1986) H i g h cyc lospor in levels after bone m a r r o w transplantat ion associated w i t h hyper t r ig lycer idaemia . Lancet 1:744-745. 106 [83] N i e l s e n R , B i r n H , M o e s t r u p S K , N i e l s e n M , V e r r o u s t P , a n d C h r i s t e n s e n E I (1998) C h a r a c t e r i z a t i o n o f a k i d n e y p r o x i m a l t u b u l e c e l l l i n e , L L C - P K I , e x p r e s s i n g e n d o c y t o t i c a c t i v e m e g a l i n . J Am.Soc.Nephrol. 9:1767-1776. [84] N o v a r t i s . N e o r a l a n d S a n d i m m u n e I V P r o d u c t M o n o g r a p h . 2002. [85] N y k j a e r A a n d W i l l n o w T E (2002) T h e 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 gene f a m i l y : a c e l l u l a r S w i s s a r m y k n i f e ? Trends Cell Biol. 12:273-280. [86] O r l a n d o R A , E x n e r M , C z e k a y R P , Y a m a z a k i H , S a i t o A , U l l r i c h R , K e r j a s c h k i D , a n d F a r q u h a r M G (1997) I d e n t i f i c a t i o n o f the s e c o n d c l u s t e r o f l i g a n d -b i n d i n g r epea t s i n m e g a l i n as a s i te f o r r e c e p t o r - l i g a n d i n t e r a c t i o n s . Proc Natl.Acad.Sci U.S.A 94:2368-2373. [87] P a r d r i d g e W M (1979) C a r r i e r - m e d i a t e d t r a n s p o r t o f t h y r o i d h o r m o n e s t h r o u g h the r a t b l o o d - b r a i n b a r r i e r : p r i m a r y r o l e o f a l b u m i n - b o u n d h o r m o n e . Endocrinology 105:605-612. [88] P e g o r a r o A A , G u d e h i t h l u K P , C a b r e r a E , S h a n k a r R , A r r u d a J A , D u n e a G , a n d S i n g h A K (2002) H a n d l i n g o f l o w - d e n s i t y l i p o p r o t e i n b y the r e n a l t u b u l e : r e lease o f f r a g m e n t s d u e to i n c o m p l e t e d e g r a d a t i o n . / Lab Clin.Med. 139:372-378. [89] P e r a n t o n i A a n d B e r m a n JJ (1979) P r o p e r t i e s o f W i l m ' s T u m o r L i n e ( T u W l ) a n d P i g K i d n e y L i n e ( L L C - P K I ) t y p i c a l o f n o r m a l k i d n e y t u b u l a r e p i t h e l i u m . In vitro 15:446-454. [90] P e r i c o N , B e n i g n i A , Z o j a C , D e l a n i n i F , a n d R e m u z z i G (1986) F u n c t i o n a l s i g n i f i c a n c e o f e x a g g e r a t e d r e n a l t h r o m b o x a n e A 2 s y n t h e s i s i n d u c e d b y c y c l o s p o r i n e A . Am } Physiol Renal Physiol 251 :F581-F587 . [91] P e t e h e r y c h K D a n d W a s a n K M (2001) Effects o f p l a s m a l i p o p r o t e i n s o n c y c l o s p o r i n e A t o x i c i t y a n d u p t a k e i n L L C P K i p i g k i d n e y c e l l . / Pharm Sci 90:1395-1406. [92] P i e r c e B i o t e c h n o l o g y Inc . M e m - P E R ( T M ) 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 K i t ( C a t #89826) I n s t r u c t i o n M a n u a l . 2002. [93] P i e r c e B i o t e c h n o l o g y Inc . B C A P r o t e i n A s s a y K i t (Cat#23225) P r o d u c t M a n u a l . 2003. [94] P i n t a v o r n P a n d B a l l e r m a n n B J (1997) T G F - b e t a a n d the e n d o t h e l i u m d u r i n g i m m u n e i n j u r y . Kidney Int. 51:1401-1412. 107 [95] P r i n c e n H M G , M e i j e r P , H o f s t e e B , H a v e k e s L M , K u i p e r s F , a n d V o n k R J (1987) T h e effects o f c y c l o s p o r i n e A ( C s A ) o n L D L - r e c e p t o r a c t i v i t y a n d b i l e a c i d s y n t h e s i s i n h e p a t o c y t e m o n o l a y e r c u l t u r e s a n d i n v i v o i n rat . Hepatology 7:1109. [96] P r o m e g a C o r p o r a t i o n . C y t o T o x 96 ( T M ) 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 I n s t r u c t i o n M a n u a l . 2001 . [97] R a i n e A E , C a r t e r R , M a n n JI, a n d M o r r i s P J (1988) A d v e r s e effect o f c y c l o s p o r i n e o n p l a s m a c h o l e s t e r o l i n r e n a l t r a n s p l a n t pa t i en t s . Nephrol Dial Transpl 3:458-463. [98] R a n g a n a t h a n S, H a t t o r i H , a n d K a s h y a p M L (1995) A r a p i d f l o w c y t o m e t r i c a s say f o r 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 s i n h u m a n p e r i p h e r a l b l o o d m o n o n u c l e a r ce l l s . / Lab Clin.Med. 124:479-486. [99] R a y n e r H C , H o r s b u r g h T , B r o w n S L , L a v e n d e r F L , W i n d e r A F , a n d W a l l s J (1990) 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 o f l o w - d e n s i t y l i p o p r o t e i n b y c u l t u r e d h u m a n g l o m e r u l a r ce l l s . Nephron 55:292-299. [100] R a y y e s O , W a l l m a r k A , a n d F l o r e n C (1996) C y c l o s p o r i n e I n h i b i t s C a t a b o l i s m o f l o w - d e n s i t y l i p o p r o t e i n s i n H e p G 2 c e l l s b y a b o u t 2 5 % . Hepatology 24:613-619. [101] R e s e a r c h D i a g n o s t i c s I n c o r p o r a t e d . P r o d u c t Inse r t 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 the h u m a n L D L r e c e p t o r ( C a t # R D I - P R O 6 1 0 8 7 ) . 2001 . [102] R e y n o l d s G D (1985) M e t a b o l i s m o f l i p o p r o t e i n s l a b e l e d w i t h f l u o r e s c e n t i n d o c a r b o c y a n i n e d y e s fo r i n v i t r o s t u d i e s o f a the rogenes i s . Suro.Synth.Pathol.Res. 4:389-400. [103] R e y n o l d s G D a n d S t . C l a i r R W (1985) A c o m p a r a t i v e m i c r o s c o p i c a n d b i o c h e m i c a l s t u d y o f the u p t a k e o f f l u o r e s c e n t a n d 1251- labe led l i p o p r o t e i n s b y s k i n f i b r o b l a s t , s m o o t h m u s c l e ce l l s a n d p e r i t o n e a l m a c r o p h a g e s i n c u l t u r e . Am.].Pathol. 121:200-211. [104] R o d i c i o J L (2000) C a l c i u m a n t a g o n i s t s a n d r e n a l p r o t e c t i o n f r o m c y c l o s p o r i n e n e p h r o t o x i c i t y : l o n g - t e r m t r i a l i n r e n a l t r a n s p l a n t a t i o n pa t i en t s . J.Cardiovasc.Pharmacol. 35:S7-11. [105] R u s s e l l D W , B r o w n M S , a n d G o l d s t e i n J L (1989) D i f f e r e n t c o m b i n a t i o n s o f c y s t e i n e - r i c h r epea t s m e d i a t e b i n d i n g o f 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 to t w o d i f f e r en t p r o t e i n s . / Biol Chem 264:21682-21688. 108 [106] S a i t o A , P i e t r o m o n a c o S, L o o A K , a n d F a r q u h a r M G (1994) C o m p l e t e c l o n i n g a n d s e q u e n c i n g o f r a t g p 3 3 0 / \" m e g a l i n , \" a d i s t i n c t i v e m e m b e r o f the 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 g e n e f a m i l y . Proc Natl.Acad.Sci U.SA 91:9725-9729. [107] S a i t o A , Y a m a z a k i H , R a d e r K , N a k a t a n i A , U l l r i c h R , K e r j a s c h k i D , O r l a n d o R A , a n d F a r q u h a r M G (1996) M a p p i n g ra t m e g a l i n : the s e c o n d c l u s t e r o f l i g a n d b i n d i n g r epea t s c o n t a i n s a 4 6 - a m i n o a c i d p a t h o g e n i c e p i t o p e i n v o l v e d i n the f o r m a t i o n o f i m m u n e d e p o s i t s i n H e y m a n n n e p h r i t i s . Proc Natl.Acad.Sci U.S.A 93:8601-8605. [108] Sa l t e r A M a n d B r i n d l e y D N (1988) T h e B i o c h e m i s t r y o f L i p o p r o t e i n s . / Inker Metab Dis 11:4-17. [109] S a n g h v i A , W a r t y V S , D i v e n W F , a n d S t a r z i T (1989) R e c e p t o r - m e d i a t e d c e l l u l a r u p t a k e o f c y c l o s p o r i n e A . Transp Proc 21:858-859. [110] S c h m i t z C , H i p e r t J , J a c o b s e n C , B o e n s c h C , C h r i s t e n s e n E I , L u f t F C , a n d W i l l n o w T E (2002) M e g a l i n d e f i c i e n c y offers p r o t e c t i o n f r o m 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 . / Biol Chem 277:618-622. [ I l l ] S c h m i t z G , B r u n i n g T , K o v a c s E , a n d B a r l a g e S (1993) F l u o r e s c e n c e f l o w c y t o m e t r y o f h u m a n l e u k o c y t e s i n the d e t e c t i o n o f L D L r e c e p t o r defects i n the d i f f e r e n t i a l d i a g n o s i s o f h y p e r c h o l e s t e r o l e m i a . Arteriosclerosis and Thrombosis 13:1053-1065. [112] S c h n a b e l F R , W a i t R B , a n d K a h n g K U (1991) T h e r e l a t i o n s h i p o f u r i n a r y t h r o m b o x a n e e x r e t i o n to c y c l o s p o r i n e A n e p h r o t o x i c i t y . Transplantation 51:686-689. [113] Sco t t J P a n d H i g e n b o t t a m T W (1988) A d v e r s e r e a c t i o n s a n d i n t e r a c t i o n s o f c y c l o s p o r i n e . Medical Toxicology 3:107-127. [114] S g o u t a s S, M a c M a h o n W , L o v e A , a n d J e r k u n i c a I (1986) I n t e r a c t i o n o f c y c l o s p o r i n e A w i t h h u m a n l i p o p r o t e i n s . / Pharm Pharmacol 38:583-588. [115] S h e p h a r d J a n d F r u c h a r t J C (1989) L i p o p r o t e i n s i n H e a l t h a n d D i s e a s e : L i p o p r o t e i n n o m e n c l a t u r e a n d the c l a s s i f i c a t i o n o f h y p e r l i p o p r o t e i n e m i a , i n Human Plasma Lipoproteins, Clinical Biochemistry: Principles, Methods, Application ( S h e p h a r d J a n d F r u c h a r t J C eds) p p 1-22, W a l t e r d e G r y t e r , B e r l i n . [116] S h u l m a n H , S t r i k e r G , D e e g J H , K e n n e d y M , S h o r b R , a n d T h o m a s E D (1981) N e p h r o t o x i c i t y o f c y c l o s p o r i n e A after a l l o g r a f t m a r r o w t r a n s p l a n t a t i o n : g l o m e r u l a r t h r o m b o s i s a n d t u b u l a r i n j u r y . N Engl ] Med. 305:1393-1395. 109 [117] S i g m a Inc. Cyc losp r ine A Produc t Insert. 1999. [118] Spencer M W , M u h l f e l d A S , Segerer S, H u d k i n s K L , K i r k E , LeBoeuf R C , and A l p e r s C E (2003) H y p e r g l y c e m i a and h y p e r l i p i d e m i a act synergist ical ly to induce renal disease i n L D L receptor-deficient B A L B mice. Am ] Nephrol 24:20-31. [119] Stefansson S, C h a p p e l l D A , Argraves K M , S t r ick land D K , and Argraves W S (1995) G lycop ro t e in 3 3 0 / L o w Densi ty L i p o p r o t e i n Receptor-related Protein-2 mediates endocytosis of l o w density l ipoprote ins v i a interact ion w i t h apo l ipopro te in B100. / Biol Chem 270:19417-19421. [120] Stephan Z F and Yurachek E C (1993) R a p i d f luorometr ic assay of L D L receptor act ivi ty by Di l - l abe led L D L . Journal of Lipid Research 34:325-330. [121] St rong M L and U e d a C T (1997) Effects of l o w and h i g h densi ty l ipoproteins on renal cyclosporine A and cyclosporine G d i spos i t ion i n the isolated perfused rat k idney . Pharm Res 14:1466-1471. [122] ThermoTrace D M . Infinity Choles terol L i q u i d Stable Reagent Product Insert. 2003. [123] v a n D r i e l IR, Go lds t e in JL, Sudhof T C , a n d B r o w n M S (1987) First cysteine-r i ch repeat i n l i gand-b ind ing d o m a i n of low-dens i ty l i popro te in receptor b inds Ca2+ and monoc lona l antibodies, but not l ipoprote ins . / Biol Chem 262:17443-17449. [124] V a z i r i N , L i a n g K , and A z a d H (2000) Effect of cyclospor ine o n H M G - C o A Reductase, cholesterol 7a-hydroxylase , L D L receptor, H D L receptor, V L D L receptor and l ipopro te in lipase expressions. / Pharm Exp Ther 294:778-783. [125] Ver rous t PJ, B i r n H , N ie l s en R, K o z y r a k i R, and Chr is tensen EI (2002) The tandem endocytic receptors mega l in and c u b i l i n are impor tant proteins i n renal pathology. Kidney Int. 62:745-756. [126] v o n W a r t b u r g A and Traber R (1986) Chemis t ry of the natural cyclosporine metabolites. Prog Allergy 38:28-45. [127] W a s a n K M (1996) M o d i f i c a t i o n i n p lasma l ipopro te in concentrat ion and l i p i d compos i t ion regulate the bio logica l act ivi ty of hyd rophob ic drugs. / Pharm Toxicol Meth 36:1-11. [128] W a s a n K M and Cass idy S M (1998) Role of P la sma l ipoprote ins i n M o d i f y i n g the Bio log ica l A c t i v i t y of H y d r o p h o b i c Drugs . / Pharm Sci 87:411-423. 110 [129] W a s a n K M , D o n n a c h i e E M , S e c c o m b e D W , a n d P r i t c h a r d P H (2002) Ef fec t o f c y c l o s p o r i n e A o n the b i n d i n g a f f i n i t y a n d i n t e r n a l i z a t i o n o f l o w - d e n s i t y l i p o p r o t e i n s i n h u m a n s k i n f i b r o b l a s t s . / Pharm Sci 91:2520-2524. [130] W a s a n K M , P r i t c h a r d P H , R a m a s w a m y M , W o n g W , D o n n a c h i e E M , a n d B r u n n e r L J (1997) D i f f e r e n c e s i n l i p o p r o t e i n 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 m o d i f y the p l a s m a d i s t r i b u t i o n o f c y c l o s p o r i n e . Pharm Res 14:1613-1620. [131] W a s a n K M , R a m a s w a m y M , W o n g W , a n d P r i t c h a r d P H (1998) L i p i d t rans fe r p r o t e i n I f a c i l i t a t e d t r ans fe r o f c y c l o s p o r i n e f r o m l o w - to h i g h - d e n s i t y l i p o p r o t e i n s i s o n l y p a r t i a l l y d e p e n d e n t o n i t s c h o l e s t e r y l ester t ransfer a c t i v i t y . / Pharmacol Exp Ther 284:599-605. [132] W a s a n K M , R o s e n b l u m M G , C h e u n g L , a n d L o p e z - B e r e s t e i n G (1994) I n f l u e n c e o f L i p o p r o t e i n s o n R e n a l C y t o t o x i c i t y a n d A n t i f u n g a l A c t i v i t y o f A m p h o t e r i c i n B . Antimic Agents and Chemo 38:223-227. [133] W a s a n K M a n d S i v a k O (2003) M o d i f i c a t i o n s i n l i p o p r o t e i n su r face c h a r g e a l t e r 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 o w - d e n s i t y l i p o p r o t e i n s . Pharm Res 20:126-129. [134] W a s a n K M , S u b r a m a n i a n R , C h o u J W , R a m a s w a m y M , a n d P r i t c h a r d P H (1999) C y c l o s p o r i n e t rans fe r f r o m l o w - 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 is p a r t i a l l y i n f l u e n c e d b y l i p i d t rans fe r p r o t e i n I t r i g l y c e r i d e t rans fe r a c t i v i t y . Pharm Res 16:1067-1073. [135] W e n g e r R M (1990) S t r u c t u r e s o f c y c l o s p o r i n e a n d i ts m e t a b o l i t e s . Transplant.Proc. 22:1104-1108. [136] W i e l c h e l m a n K , B r a u n R , a n d F i t z p a t r i c k J (1988) I n v e s t i g a t i o n o f the b i c i n c h o n i n i c a c i d p r o t e i n assay : I d e n t i f i c a t i o n o f t he g r o u p s r e s p o n s i b l e fo r c o l o r f o r m a t i o n . Anal Biochem 175:231-237. [137] W i l l n o w T E , N y k j a e r A , a n d H e r z J (1999) L i p o p r o t e i n r e c e p t o r s : n e w ro l e s fo r a n c i e n t p r o t e i n s . Nat.Cell Biol. 1 :E157-E162. [138] W i n e g a r D a , S a l i s b u r y J A , S u n d s e t h S S , a n d H a w k e R L (1996) Effects o f c y c l o s p o r i n e o n c h o l e s t e r o l 2 7 - h y d r o x y l a t i o n a n d L D L r e c e p t o r a c t i v i t y i n H e p G 2 ce l l s . / Lipid Res 37:179-191. [139] Y a m a m o t o T . , D a v i s C G , B r o w n M S , S c h n e i d e r W J , C a s e y M L , G o l d s t e i n J L , a n d R u s s e l l D W (1984) T h e H u m a n L D L r e c e p t o r : a C y s t e i n e - r i c h p r o t e i n w i t h m u l t i p l e A l u s e q u e n c e s i n i ts m R N A . Cell 39:27-38. I l l [140] Yee G C and S a l o m o n D R (1992) Cyc lospor ine i n A p p l i e d Pharmacokinet ics , i n Principles of Therapeutic Drug Monitoring (Evans W E ed) M a r c e l Dekker , N e w Y o r k . [141] Y o s h i m u r a R, Y o s h i m u r a N , Kusunose E , M a t s u i S, H a m a s h i m a T, K i s h i m o t o T, O k a T, Kusunose M , and M a e k a w a M (1989) Role of k idney mic rosomal cytochrome P450 i n cyclosporine induce nephropathy. Transplant Proc 21:934-936. [142] Zager R A , Johnson A C , and H a n s o n S Y (2003) P r o x i m a l tubular cholesterol l oad ing after mi tochondr ia l , but not g lycolyt ic , b lockade. Am ] Physiol Renal Physiol 285:F1092-F1099. [143] Zager R A , Johnson A C , and H a n s o n S Y (2003) Sepsis syndrome stimulates p r o x i m a l tubule cholesterol synthesis and suppresses the SR-B1 cholesterol transporter. Kidney Int. 63:123-133. [144] Zager R A , Johnson A C , H a n s o n S Y , and Shah V O (2003) Acu te tubular injury causes dysregula t ion of cel lular cholesterol transport proteins. Am J Pathol. 163:313-320. [145] Z h e n g G , Bachinsky D R , Stamenkovic I, S t r ick land D K , B r o w n D , Andre s G , and M c C l u s k e y R T (1994) O r g a n Dis t r ibu t ion i n rats of t w o members of the low-dens i ty l ipopro te in receptor gene family , gp330 and L R P / a 2 M R and the receptor-associated prote in ( R A P ) . / Histo Cyto 42:531-542. 112 "@en . "Thesis/Dissertation"@en . "2004-05"@en . "10.14288/1.0091613"@en . "eng"@en . "Pharmaceutical Sciences"@en . "Vancouver : University of British Columbia Library"@en . "University of British Columbia"@en . "For non-commercial purposes only, such as research, private study and education. Additional conditions apply, see Terms of Use https://open.library.ubc.ca/terms_of_use."@en . "Graduate"@en . "The role of the low-density lipoprotein receptor family on Cyclosporine A uptake and toxicity in renal cells"@en . "Text"@en . "http://hdl.handle.net/2429/15474"@en .