- Email: [email protected]
Effect of lysophosphatidylcholine on renal hemodynamics and excretory function in anesthetized rats
L i f e S c i e n c e s , Vol. P r i n t e d in t h e U S A
51, pp.
1571-1575
Pergamon
Press
E F F E C T OF L Y S O P H O S P H A T I D Y L C H O L I N E ON R E N A L H E M O D Y N A M I C S A N D E X C R E T O R Y F U N C T I O N IN A N E S T H E T I Z E D R A T S
Rajash
K. H a n d a
and V a r d a m a n M. Buckalew,
Jr.
D e p a r t m e n t of M e d i c i n e / N e p h r o l o g y Wake Forest University Medical Center Winston-Salem, North Carolina 27157-1053 (Received
in final
form S e p t e m b e r
8,
1992)
Summary The e f f e c t of m y r i s t o y l - l y s o p h o s p h a t i d y l c h o l i n e ( m y r i s t o y l - L P C ) on renal h e m o d y n a m i c s , e l e c t r o l y t e and w a t e r e x c r e t i o n was e x a m i n e d o v e r a 90 m i n p e r i o d in s o d i u m p e n t o b a r b i t a l anesthetized male S p r a g u e D a w l e y rats. I n t r a v e n o u s i n f u s i o n of m y r i s t o y l - L P C at 13 ± 3 p m o l / m i n r e s u l t e d in a small fall in s y s t e m i c b l o o d p r e s s u r e , a 13% d e c r e a s e in renal p l a s m a f l o w w i t h o u t s i g n i f i c a n t l y a l t e r i n g g l o m e r u l a r f i l t r a t i o n r a t e and p r o d u c e d a s l i g h t l y g r e a t e r e x c r e t i o n of s o d i u m and w a t e r t h a n v e h i c l e controls. These results suggest t h a t short t e r m m y r i s t o y l - L P C a d m i n i s t r a t i o n can s i g n i f i c a n t l y a l t e r renal f u n c t i o n p r o d u c i n g a w e a k n a t r i u r e s i s and d i u r e s i s w h i c h is not d e p e n d e n t on s y s t e m i c b l o o d p r e s s u r e and renal h e m o d y n a m i c changes. N u m e r o u s i n v e s t i g a t o r s h a v e p r o p o s e d the e x i s t e n c e of an u n i d e n t i f i e d n a t r i u r e t i c h o r m o n e ( s ) in p l a s m a w h i c h m a y r e g u l a t e salt and w a t e r e x c r e t i o n by i n h i b i t i n g Na+-K + A T P a s e (i). L y s o p h o s p h a t i d y l c h o l i n e (LPC) has b e e n shown to e x h i b i t N a + - K + A T P a s e i n h i b i t o r y a c t i v i t y (2) and it has b e e n r e p o r t e d t h a t b o l u s i n t r a v e n o u s a d m i n i s t r a t i o n of LPC i n c r e a s e d s o d i u m and w a t e r e x c r e t i o n in rats (3). P l a s m a level of L P C is e l e v a t e d d u r i n g r a p i d v o l u m e e x p a n s i o n in rats (4), s u g g e s t i n g its p o s s i b l e i n v o l v e m e n t in the renal r e s p o n s e to plasma volume alteration. The aims of the p r e s e n t s t u d y w e r e to i n i t i a l l y c o n f i r m t h a t a b o l u s i n t r a v e n o u s i n j e c t i o n of LPC could p r o d u c e a n a t r i u r e t i c and d i u r e t i c r e s p o n s e in the rat and t h e n to e x a m i n e w h e t h e r the rat k i d n e y could a l t e r e x c r e t o r y f u n c t i o n w h e n e x p o s e d to a c o n s t a n t i n t r a v e n o u s i n f u s i o n of LPC. Also, we d e t e r m i n e d w h e t h e r the c h a n g e s in e x c r e t i o n w e r e due to a l t e r a t i o n s in s y s t e m i c b l o o d p r e s s u r e a n d / o r renal h e m o d y n a m i c s .
Methods General surqery: Male Sprague Dawley rats (350 - 420 g) w e r e a n e s t h e t i z e d w i t h an i n t r a p e r i t o n e a l i n j e c t i o n of s o d i u m p e n t o b a r b i t a l (60 mg/kg) and maintained with periodic supplementary doses administered intravenously (iv). A f t e r t r a c h e o s t o m y , a c a t h e t e r was i n s e r t e d into the c a r o t i d a r t e r y for a r t e r i a l b l o o d s a m p l i n g and s y s t e m i c b l o o d p r e s s u r e recording. The j u g u l a r v e i n was t h e n c a n n u l a t e d for the iv i n f u s i o n of s a l i n e (150 mmol/l) at 0.i m l / m i n and m a i n t a i n e d at t h i s r a t e t h r o u g h o u t the experiment. R e c t a l t e m p e r a t u r e was m a i n t a i n e d at 36 - 38°C w i t h a h e a t i n g pad. Renal function measurements: The left k i d n e y was e x p o s e d v i a a m i d l i n e a b d o m i n a l i n c i s i o n and its u r e t e r c a n n u l a t e d for the c o l l e c t i o n of urine. Renal blood flow (RBF) was measured by placing a non-cannulating e l e c t r o m a g n e t i c f l o w p r o b e a r o u n d the renal a r t e r y and g l o m e r u l a r f i l t r a t i o n r a t e (GFR) e s t i m a t e d by the c l e a r a n c e of i n u l i n u s i n g a p r e v i o u s l y d e s c r i b e d a s s a y p r o c e d u r e (5). P l a s m a and u r i n a r y s o d i u m c o n c e n t r a t i o n s w e r e m e a s u r e d using a Beckman flame photometer. R e n a l f u n c t i o n m e a s u r e m e n t s w e r e b e g u n 2.5 h f o l l o w i n g t h e c o m p l e t i o n of all surgery. S t a n d a r d f o r m u l a e w e r e u s e d for c a l c u l a t i n g r e n a l p l a s m a flow [RPF = RBF x (i - H e m a t o c r i t ) ] , renal v a s c u l a r resistance (RVR = M e a n a r t e r i a l p r e s s u r e / R P F ) , filtration fraction (FF = GFR/RPF), fractional sodium excretion (FEN~ = renal Na c l e a r a n c e / G F R ) and f r a c t i o n a l w a t e r e x c r e t i o n (FE w = u r i n e f l o w r a t e / G F R ) . Copyright
© 1992
0 0 2 4 - 3 2 0 5 / 9 2 $5.00 + .00 P e r g a m o n P r e s s Ltd All r i g h t s
reserved.
1572
LPC and Renal
Function
Vol.
51, No.
20,
1992
E x p e r i m e n t a l protocol: The e x p e r i m e n t a l p r o t o c o l c o n s i s t e d of t h r e e c l e a r a n c e periods. F o l l o w i n g a 30 m i n b a s e l i n e c o n t r o l period, rats r e c e i v e d e i t h e r an iv i n f u s i o n of v e h i c l e (saline c o n t a i n i n g 36 p g / m l fatty acid free b o v i n e s e r u m albumin) or m y r i s t o y l - L P C at 13 ± 3 p m o l / m i n (= 6 ± 2 ng/min). Two 30 m i n e x p e r i m e n t a l c l e a r a n c e p e r i o d s w e r e t h e n t a k e n 40 m i n after the onset of the infusion. Initial studies e x a m i n e d the c h a n g e s in a r t e r i a l b l o o d pressure, a b s o l u t e s o d i u m e x c r e t i o n and u r i n e flow to a b o l u s i n j e c t i o n of m y r i s t o y l LPC. A f t e r g e n e r a l s u r g i c a l p r o c e d u r e s , the left u r e t e r was e x p o s e d by a small a b d o m i n a l i n c i s i o n and t h e n c a t h e t e r i z e d . Using a similar experimental p r o t o c o l as above, a 0.i ml bolus i n j e c t i o n of v e h i c l e or m y r i s t o y l - L P C (2 pmol = 1 ng) was g i v e n iv after the b a s e l i n e c l e a r a n c e p e r i o d and f o l l o w e d after 40 m i n by two e x p e r i m e n t a l c l e a r a n c e periods. No renal h e m o d y n a m i c measurements were performed. P r e p a r a t i o n of solutions: Myristoyl-LPC (L-a-lysophosphatidylcholine, m y r i s t o y l ; Sigma) was stored at -20°C as a 1 mg/ml stock solution, t h a t had b e e n d i s s o l v e d in m e t h a n o l - c h l o r o f o r m (1:2 vol/vol). For a d m i n i s t r a t i o n , a l i q u o t s w e r e d r i e d in glass g r a d u a t e d flasks and t h e n d i s s o l v e d in s a l i n e c o n t a i n i n g 3 6 ~g/ml fatty acid free b o v i n e s e r u m a l b u m i n (BSA). Vehicle c o n s i s t e d of the m e t h a n o l - c h l o r o f o r m m i x t u r e in 36 ~ g / m l B S A - s a l i n e solution. Statistics: For s t a t i s t i c a l analysis, a m e a n v a l u e of the e x p e r i m e n t a l clearance p e r i o d s was c o m p a r e d to the b a s e l i n e value. Coefficient of v a r i a b i l i t y (CV) b e t w e e n first and s e c o n d e x p e r i m e n t a l s a m p l e s w e r e 13% and 9% for e x c r e t o r y r e s p o n s e s in v e h i c l e and L P C i n j e c t e d groups, r e s p e c t i v e l y . CV = 8% and 7% for renal h e m o d y n a m i c responses, 8% and 24% for e x c r e t o r y r e s p o n s e s in v e h i c l e and LPC i n f u s e d groups, r e s p e c t i v e l y . Comparisons within and a c r o s s g r o u p s w e r e a s s e s s e d u s i n g a 2-way f a c t o r i a l a n a l y s i s of v a r i a n c e f o l l o w e d by a s i m p l e m a i n e f f e c t test (CRunch I n t e r a c t i v e S t a t i s t i c a l P a c k a g e (CRISP); C r u n c h S o f t w a r e C o r p o r a t i o n ] . A significant group x treatment i n t e r a c t i o n i n d i c a t e s that the e f f e c t of t r e a t m e n t on a m e a s u r e d p a r a m e t e r d i f f e r s b e t w e e n g r o u p s (6). D a t a are p r e s e n t e d as m e a n s ± SEM. Results
T h e r e was no c h a n g e in s y s t e m i c b l o o d p r e s s u r e or u r i n e flow and a small rise in a b s o l u t e s o d i u m e x c r e t i o n d u r i n g the t i m e c o u r s e of the e x p e r i m e n t in bolus v e h i c l e i n j e c t e d rats. M y r i s t o y l - L P C a d m i n i s t e r e d to rats as a 2 pmol bolus r e s u l t e d in no c h a n g e in b l o o d p r e s s u r e and an i n c r e a s e in s o d i u m and water excretion. The enhanced diuresis was significantly greater in m y r i s t o y l - L P C t r e a t e d rats t h a n v e h i c l e c o n t r o l s (Table i). T a b l e 2 shows the d a t a o b t a i n e d from a n i m a l s c o n t i n u o u s l y i n f u s e d w i t h v e h i c l e or m y r i s t o y l - L P C (13 ± 3 p m o l / m i n ) . B a s e l i n e v a l u e s for m e a s u r e d v a r i a b l e s w e r e s i m i l a r in b o t h g r o u p s e x c e p t for a s i g n i f i c a n t l y s m a l l e r f r a c t i o n a l e x c r e t i o n of s o d i u m in rats p r i o r to m y r i s t o y l - L P C infusion. The reason(s) for this d i f f e r e n c e in b a s e l i n e f r a c t i o n a l s o d i u m e x c r e t i o n is not r e a d i l y apparent. In b o t h g r o u p s t h e r e was a small d e c r e a s e in s y s t e m i c b l o o d pressure and no c h a n g e in a r t e r i a l b l o o d h e m a t o c r i t or renal vascular r e s i s t a n c e d u r i n g the t i m e c o u r s e of the e x p e r i m e n t . T h e r e was no c h a n g e in renal h e m o d y n a m i c s , u r i n e flow or f r a c t i o n a l w a t e r e x c r e t i o n and a small rise in a b s o l u t e s o d i u m e x c r e t i o n (25%) and f r a c t i o n a l s o d i u m e x c r e t i o n (23%) in a n i m a l s i n f u s e d w i t h the BSA vehicle. In a n i m a l s g i v e n m y r i s t o y l - L P C , t h e r e was a 13% fall in renal p l a s m a flow, a t e n d e n c y for g l o m e r u l a r f i l t r a t i o n rate to d e c r e a s e a l t h o u g h this did not reach s t a t i s t i c a l s i g n i f i c a n c e , no c h a n g e in f i l t r a t i o n f r a c t i o n and an i n c r e a s e in u r i n e f l o w of 40%, a b s o l u t e s o d i u m e x c r e t i o n of 84% and f r a c t i o n a l s o d i u m and w a t e r e x c r e t i o n of 113% and 63%, respectively. The i n c r e m e n t in f r a c t i o n a l s o d i u m and w a t e r e x c r e t i o n w i t h m y r i s t o y l - L P C t r e a t m e n t was s i g n i f i c a n t l y g r e a t e r t h a n that o b s e r v e d f o l l o w i n g vehicle treatment. L i n e a r r e g r e s s i o n a n a l y s i s of the c h a n g e s in renal h e m o d y n a m i c s and in s o d i u m e x c r e t i o n and u r i n e flow r a t e in b o t h v e h i c l e and m y r i s t o y l - L P C i n f u s e d rats i n d i c a t e d one s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n b e t w e e n the c h a n g e s in g l o m e r u l a r f i l t r a t i o n rate and u r i n e flow rate in the m y r i s t o y l - L P C i n f u s e d g r o u p (r= 0.742, P<0.05).
Vol.
51,
No.
20,
1992
LPC
TABLE
and Renal
Function
1
Baseline
Experiment
M A P ( m m Hg) Vehicle (n=7) M-LPC (n=7)
124 126
± 6 ± 3
123 120
U N a V (@Eq.min'J.kg -l) Vehicle M-LPC
5.9 6.3
± 1.9 ± 1.8
7.9 10.2
± 1.9" ± 2.2***
U V ( ~ l . m i n * . k g -l) Vehicle M-LPC
24.1 25.4
27.3 38.0
± 5.0 ± 9.8***%
± 6.2 ± 8.0
± 7 ± 6
* = P<0.05 ** = P < 0 . 0 1 *** = P < 0 . O 0 1 from baseline value, * = P<0.05 from vehicle value using 2-way ANOVA; % = P<0.05 $ = P<0.OI indicates a significant group x treatment interaction (i.e. t r e a t m e n t effect on measured response differs between groups). MAP = mean arterial pressure; RVR = renal vascular resistance; RPF = renal plasma flow; GFR = glomerular filtration rate; FF = filtration f r a c t i o n ; U N a V = a b s o l u t e s o d i u m e x c r e t i o n ; FENa = f r a c t i o n a l s o d i u m excretion; UV = urine flow rate; FE W = fractional water excretion; M-LPC = myristoyl-lysophosphatidylcholine.
TABLE
2
Baseline Hematocrit (%) Vehicle (n=7) M-LPC (n=9) M A P ( m m Hg) Vehicle M-LPC R V R (mm H g / m l . m i n l . k g Vehicle M-LPC
Experiment
49 47
± 1 ± 1
47 46
123 118
± 4 ± 2
112 107
± 1 ± 1
± 3** ± 3**
"') ii.2 8.4
± 1.6 ± 0.6
I0.5 8.7
+ 1.4 ± 0.5#
R P F ( m l . m i n l . k g "I) Vehicle M-LPC
12.1 14.4
± 1.4 ± 0.6
11.8 12.6
± 1.4 ± 0.6***$
G F R ( m l . m i n 1 . k g "I) Vehicle M-LPC
3.6 3.9
± 0.7 ± 0.3
3.8 3.4
F F (%) Vehicle M-LPC
0.29 0.27
U N a V (@Eq. m i n "l. k g "j) Vehicle M-LPC
4.0 2.5
± 0.04 ± 0.02
± 0.7 ± 0.5
0.32 0.28
5.0 4.6
± 0.7 ± 0.4
± 0.04 ± 0.03
± 0.8 ± 0.9**
FEN, (%) Vehicle M-LPC
0.79 0.39
± 0.16 ± 0.07*
0.98 0.83
± 0.18" ± 0.09***%
U V ( @ l . m i n l . k g -I) Vehicle M-LPC
21.9 17.9
± 2.7 ± 1.7
24.6 25.2
± 2.9 ± 2.9**
FEw (%) Vehicle M-LPC
0.76 0.46
± 0.17 ± 0.04
0.82 0.76
± 0.18 ± 0.05***$
Abbreviations
as
in Table
I.
1573
1574
LPC and Renal F u n c t i o n
Vol.
51, No.
20,
1992
Discussion R e n a l s o d i u m e x c r e t i o n is t i g h t l y linked to e x t r a c e l l u l a r fluid (ECF) volume regulation. The m e c h a n i s m ( s ) by w h i c h this c o u p l i n g o c c u r s is not fully u n d e r s t o o d . One theory, that ECF v o l u m e e x p a n s i o n leads to the r e l e a s e of h u m o r a l n a t r i u r e t i c factors, has b e e n s u p p o r t e d by the d i s c o v e r y of a t r i a l n a t r i u r e t i c peptide. However, c o n s i d e r a b l e e v i d e n c e s u p p o r t s the e x i s t e n c e of at least one other n a t r i u r e t i c hormone, a f a c t o r w h i c h i n h i b i t s H a + - K + A T P a s e (1). Lysophosphatidylcholine (LPC) c o n s t i t u t e s a m a j o r c o m p o n e n t of p l a s m a N a + - K + A T P a s e i n h i b i t o r y activity, and s t u d i e s have d e m o n s t r a t e d t h a t acute v o l u m e e x p a n s i o n in rats and pigs i n c r e a s e s the p l a s m a c o n c e n t r a t i o n of LPC (4,7). F u r t h e r m o r e , R a u c h and B u c k a l e w r e p o r t e d that the i n t r a v e n o u s b o l u s i n j e c t i o n of s t e r o y l - L P C into the a n e s t h e t i z e d rat p r o d u c e d a small n a t r i u r e t i c and d i u r e t i c r e s p o n s e after a lag p e r i o d of 20 min, w i t h the p e a k n a t r i u r e t i c e f f e c t o c c u r r i n g at 1 h, and l a s t i n g close to 7 h (3). T h e y also d e m o n s t r a t e d that the m y r i s t o y l a n a l o g u e of LPC had the g r e a t e s t p o t e n c y for n a t r i u r e s i s and d i u r e s i s (3). However, t h e y did not m e a s u r e s y s t e m i c b l o o d p r e s s u r e or renal h e m o d y n a m i c s w h i c h are i m p o r t a n t f a c t o r s in the r e g u l a t i o n of renal s o d i u m and w a t e r e x c r e t i o n and could p o t e n t i a l l y c o n t r i b u t e to the o b s e r v e d n a t r i u r e t i c and d i u r e t i c p r o p e r t i e s of LPC. Furthermore, a constant i n f u s i o n of L P C r a t h e r than a bolus i n j e c t i o n m a y g i v e a b e t t e r r e f l e c t i o n of c h a n g e s in renal f u n c t i o n that may o c c u r d u r i n g ECF v o l u m e e x p a n s i o n . The data o b t a i n e d from rats g i v e n a bolus i n j e c t i o n of m y r i s t o y l - L P C c o n f i r m e d p r e v i o u s l y p u b l i s h e d w o r k (3) that t h i s p h o s p h o l i p i d p o s s e s s e s diuretic properties. Although, we did not demonstrate a statistical d i f f e r e n c e in s o d i u m e x c r e t i o n b e t w e e n m y r i s t o y l - L P C and v e h i c l e i n j e c t e d rats, t h e r e was a t r e n d for a g r e a t e r i n c r e a s e in s o d i u m e x c r e t i o n w i t h myristoyl-LPC administration. Since the n a t r i u r e t i c e f f e c t of LPC lasts for s e v e r a l hours (3), a d d i t i o n a l u r i n a r y c o l l e c t i o n s m a y have d e m o n s t r a t e d a s i g n i f i c a n t d i f f e r e n c e in s o d i u m e x c r e t i o n b e t w e e n the two groups. The p r e s e n t study also d e m o n s t r a t e d that the e n h a n c e d d i u r e s i s was u n r e l a t e d to an a l t e r a t i o n in renal p e r f u s i o n p r e s s u r e as i n d i c a t e d by an u n c h a n g e d s y s t e m i c a r t e r i a l b l o o d pressure. Administration of m y r i s t o y l - L P C as a c o n s t a n t i n f u s i o n e l i c i t e d b o t h a n a t r i u r e t i c and d i u r e t i c r e s p o n s e and was s i m i l a r in m a g n i t u d e to that o b s e r v e d w i t h bolus a d m i n i s t r a t i o n . The s i m i l a r s o d i u m and w a t e r r e s p o n s e to LPC a d m i n i s t e r e d as a bolus or i n f u s i o n m a y be r e l a t e d to e i t h e r o p e n i n g of the a b d o m i n a l c a v i t y and g r e a t e r s u r g i c a l t r a u m a in m y r i s t o y l - L P C i n f u s e d rats w h i c h can r e d u c e the n a t r i u r e t i c and d i u r e t i c r e s p o n s e of the kidney, or due to the b i p h a s i c d o s e r e s p o n s e curve for the n a t r i u r e t i c and d i u r e t i c e f f e c t of LPC (3). The e n h a n c e d n a t r i u r e s i s and d i u r e s i s in m y r i s t o y l - L P C i n f u s e d rats c o m p a r e d to c o n t r o l s w e r e not due to a l t e r a t i o n s in b l o o d p r e s s u r e seen by the k i d n e y or renal h e m o d y n a m i c changes. In fact, m y r i s t o y l - L P C s i g n i f i c a n t l y d e c r e a s e d renal p l a s m a flow by 13% and t e n d e d to d e c r e a s e g l o m e r u l a r f i l t r a t i o n rate w h i c h are h e m o d y n a m i c c h a n g e s m o s t l i k e l y to d e c r e a s e s o d i u m and w a t e r e x c r e t i o n . T h e s e renal h e m o d y n a m i c c h a n g e s c o u l d be due to a c o n s t r i c t o r e f f e c t of m y r i s t o y l - L P C on preg l o m e r u l a r b l o o d v e s s e l s since f i l t r a t i o n f r a c t i o n r e m a i n e d u n c h a n g e d . In contrast, s t u d i e s in the rabbit have shown that mg a m o u n t s of LPC can p r o d u c e coronary and renal v a s o d i l a t i o n (8) and this a p p e a r s to be due to an e n d o t h e l i u m - d e p e n d e n t v a s o d i l a t o r y a c t i o n of LPC on v a s c u l a r s m o o t h m u s c l e w h i c h is c y c l i c G M P - d e p e n d e n t , but d i f f e r s from the e n d o t h e l i u m - d e r i v e d r e l a x i n g factor, n i t r i c oxide (9,10). We have not e x a m i n e d w h e t h e r the renal v a s c u l a r c o n s t r i c t o r e f f e c t of LPC in the rat is a d o s e - r e l a t e d or s p e c i e s dependent phenomenon. A l t e r n a t i v e l y , renal p l a s m a flow c o u l d have d e c r e a s e d as a r e s u l t of the fall in s y s t e m i c b l o o d p r e s s u r e as s u g g e s t e d by an unchanged renal v a s c u l a r resistance. Generally a small fall in renal p e r f u s i o n p r e s s u r e w o u l d lead to a u t o r e g u l a t i o n of renal p l a s m a flow as was o b s e r v e d in the v e h i c l e i n f u s e d rats. W h e t h e r LPC i n t e r f e r e s w i t h the a u t o r e g u l a t o r y a b i l i t y of the rat k i d n e y to a fall in b l o o d p r e s s u r e is unknown. Since the alterations in b l o o d pressure and renal hemodynamics a s s o c i a t e d w i t h m y r i s t o y l - L P C i n f u s i o n do not favor n a t r i u r e s i s and d i u r e s i s , the p o s s i b i l i t y arises that LPC m a y have a d i r e c t t u b u l a r a c t i o n to e n h a n c e s o d i u m and w a t e r e x c r e t i o n . This c o n t e n t i o n is s u p p o r t e d by m y r i s t o y l - L P C i n f u s i o n r e s u l t i n g in a g r e a t e r f r a c t i o n a l e x c r e t i o n of s o d i u m and w a t e r t h a n in v e h i c l e t r e a t e d rats, s u g g e s t i n g a r e d u c e d a b i l i t y of the t u b u l e to r e a b s o r b s o d i u m and water. LPC is c a p a b l e of i n h i b i t i n g the s o d i u m p u m p by i n t e r a c t i n g w i t h the N a ÷ - b i n d i n g sites of N a ÷ - K + A T P a s e (ii), and p r e s u m a b l y
Vol.
51, No.
20,
1992
L P C and R e n a l F u n c t i o n
1575
in t h e r e n a l t u b u l e lead to a loss in the s o d i u m g r a d i e n t at the a p i c a l m e m b r a n e for t h e r e a b s o r p t i o n of s o d i u m and water. In a d d i t i o n , L P C m a y regulate protein kinase C (PKC) by stimulating and inhibiting, in a concentration-dependent m a n n e r , p h o s p h o r y l a t i o n of PKC s u b s t r a t e p r o t e i n s (12). I n h i b i t i o n or d o w n r e g u l a t i o n of PKC in the p r o x i m a l t u b u l e w o u l d reduce the activity of such sodium transporters as the apical Na+-H ÷ a n t i p o r t e r w h i c h w o u l d a l s o lead to r e d u c e d s o d i u m and w a t e r r e a b s o r p t i o n . T h e r e is e v i d e n c e t h a t lipids can also i n h i b i t v a s o p r e s s i n - m e d i a t e d water r e a b s o r p t i o n in c o r t i c a l c o l l e c t i n g d u c t s (13). The r e g u l a t i o n of renal e l e c t r o l y t e and w a t e r e x c r e t i o n by lipids a p p e a r s to be p r e s e n t in s e v e r a l species, as L P C and o t h e r r e l a t e d lipids h a v e b e e n r e p o r t e d to i n c r e a s e s o d i u m and e s p e c i a l l y w a t e r e x c r e t i o n in the rat (3, p r e s e n t study), r a b b i t (13), d o g (14) and h u m a n (15). A l t h o u g h , i n t r a v e n o u s l y a d m i n i s t e r e d L P C is r a p i d l y r e m o v e d from p l a s m a by t i s s u e (t~ = 6 - ii min), its m e t a b o l i s m is v e r y s l o w r e q u i r i n g s e v e r a l hours (16). This is c o n s i s t e n t w i t h our o b s e r v a t i o n t h a t a small d o s e of L P C is c a p a b l e of p r o d u c i n g a long-lasting biological response. T h e n a t r i u r e t i c and d i u r e t i c a c t i o n s of L P C are n o v e l and the intracellular mechanisms mediating these responses have yet to be investigated. In conclusion, our results suggest that myristoyl-LPC has weak n a t r i u r e t i c and d i u r e t i c e f f e c t s in the rat and c o u l d c o n c e i v a b l y p a r t i c i p a t e in t h e r e n a l r e s p o n s e to p l a s m a v o l u m e e x p a n s i o n . The m y r i s t o y l - L P C m e d i a t e d i n c r e a s e in s o d i u m and w a t e r e x c r e t i o n a p p e a r s not to be d e p e n d e n t on the c h a n g e s in s y s t e m i c b l o o d p r e s s u r e or renal h e m o d y n a m i c s , and m a y r e f l e c t a t u b u l a r site of a c t i o n of LPC. References
i. 2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15.
16.
H.E. D e W A R D E N E R and E.M. CLARKSON, Physiol. Rev. 65 6 5 9 - 7 5 9 (1985). J.N. KARLI, G.A. KARIKAS, P.K. HATZIPAVLOU, G.M. LEVIS and S.N. M O U L O P O U L O S , L i f e Sci. 2__4 1 8 6 9 - 1 8 7 6 (1979). A.L. R A U C H and V.M. BUCKALEW, Jr., Am. J. Physiol. 254 F 8 2 4 - F 8 2 7 (1988). A.L. R A U C H and V.M. BUCKALEW, Jr., L i f e Sci. 42 1 1 8 9 - 1 1 9 7 (1988) R.K. H A N D A and E.J. JOHNS, Q u a r t e r l y J. Exptl. Physiol. 7_33 9 1 5 - 9 2 9 (1988). B.J. WINER, S t a t i s t i c a l p r i n c i p l e s in e x p e r i m e n t a l design, M c G r a w - H i l l Inc. p 4 3 1 - 5 1 3 (1971). M. TAMURA, T. INAGAMI, T. K I N O S H I T A and H. KUWANO, J. H y p e r t e n . 5 219225 (1987). A. WOLF, T. SAITO, R. D U D E K and R.J. BING, L i p i d s 26 2 2 3 - 2 2 6 (1991). T. SAITO, A. WOLF, N.K. MENON, M. S A E E D and R.J. BING, Proc. Natl. Acad. Sci. U S A 8-5 8 2 4 6 - 8 2 5 0 (1988). N.K. M E N O N and R.J. 8ING, Proc. Soc. Expt. Biol. Med. 196 4 6 1 - 4 6 3 (1991). K. OISHI, B. Z H E N G and J.F. KUO, J. Biol. Chem. 265 70-75 (1990). K. OISHI, R.L. RAYNOR, P.A. C H A R P and J.F. KUO, J. Biol. Chem. 26__~368656871 (1988). A. Y A S U H I R O , H.R. J A C O B S O N and M.D. BREYER, Am. J. Physiol. 257 F 5 2 4 F 5 3 0 (1989). V.M. B U C K A L E W , Jr., J.W. S T R A N D H O Y and R.K. HANDA, J. C a r d i o v a s c . P h a r m a c o l . (in press). P. B E R N A R D I , F. GHEZZI, L. BASTAGLI, R. G R I M A L D I , M. C A V A Z Z A , C. M I N E L L I , F. FONTANA, C. CLO, C. V E N T U R A and M. CAPELLI, Prost. Leuko. Med. 2-2 2 3 5 - 2 4 8 (1986). Y. S T E I N and O. STEIN, B i o c h i m i c a et B i o p h y s i c a A c t a 116 9 5 - 1 0 7 (1966).
51, pp.
1571-1575
Pergamon
Press
E F F E C T OF L Y S O P H O S P H A T I D Y L C H O L I N E ON R E N A L H E M O D Y N A M I C S A N D E X C R E T O R Y F U N C T I O N IN A N E S T H E T I Z E D R A T S
Rajash
K. H a n d a
and V a r d a m a n M. Buckalew,
Jr.
D e p a r t m e n t of M e d i c i n e / N e p h r o l o g y Wake Forest University Medical Center Winston-Salem, North Carolina 27157-1053 (Received
in final
form S e p t e m b e r
8,
1992)
Summary The e f f e c t of m y r i s t o y l - l y s o p h o s p h a t i d y l c h o l i n e ( m y r i s t o y l - L P C ) on renal h e m o d y n a m i c s , e l e c t r o l y t e and w a t e r e x c r e t i o n was e x a m i n e d o v e r a 90 m i n p e r i o d in s o d i u m p e n t o b a r b i t a l anesthetized male S p r a g u e D a w l e y rats. I n t r a v e n o u s i n f u s i o n of m y r i s t o y l - L P C at 13 ± 3 p m o l / m i n r e s u l t e d in a small fall in s y s t e m i c b l o o d p r e s s u r e , a 13% d e c r e a s e in renal p l a s m a f l o w w i t h o u t s i g n i f i c a n t l y a l t e r i n g g l o m e r u l a r f i l t r a t i o n r a t e and p r o d u c e d a s l i g h t l y g r e a t e r e x c r e t i o n of s o d i u m and w a t e r t h a n v e h i c l e controls. These results suggest t h a t short t e r m m y r i s t o y l - L P C a d m i n i s t r a t i o n can s i g n i f i c a n t l y a l t e r renal f u n c t i o n p r o d u c i n g a w e a k n a t r i u r e s i s and d i u r e s i s w h i c h is not d e p e n d e n t on s y s t e m i c b l o o d p r e s s u r e and renal h e m o d y n a m i c changes. N u m e r o u s i n v e s t i g a t o r s h a v e p r o p o s e d the e x i s t e n c e of an u n i d e n t i f i e d n a t r i u r e t i c h o r m o n e ( s ) in p l a s m a w h i c h m a y r e g u l a t e salt and w a t e r e x c r e t i o n by i n h i b i t i n g Na+-K + A T P a s e (i). L y s o p h o s p h a t i d y l c h o l i n e (LPC) has b e e n shown to e x h i b i t N a + - K + A T P a s e i n h i b i t o r y a c t i v i t y (2) and it has b e e n r e p o r t e d t h a t b o l u s i n t r a v e n o u s a d m i n i s t r a t i o n of LPC i n c r e a s e d s o d i u m and w a t e r e x c r e t i o n in rats (3). P l a s m a level of L P C is e l e v a t e d d u r i n g r a p i d v o l u m e e x p a n s i o n in rats (4), s u g g e s t i n g its p o s s i b l e i n v o l v e m e n t in the renal r e s p o n s e to plasma volume alteration. The aims of the p r e s e n t s t u d y w e r e to i n i t i a l l y c o n f i r m t h a t a b o l u s i n t r a v e n o u s i n j e c t i o n of LPC could p r o d u c e a n a t r i u r e t i c and d i u r e t i c r e s p o n s e in the rat and t h e n to e x a m i n e w h e t h e r the rat k i d n e y could a l t e r e x c r e t o r y f u n c t i o n w h e n e x p o s e d to a c o n s t a n t i n t r a v e n o u s i n f u s i o n of LPC. Also, we d e t e r m i n e d w h e t h e r the c h a n g e s in e x c r e t i o n w e r e due to a l t e r a t i o n s in s y s t e m i c b l o o d p r e s s u r e a n d / o r renal h e m o d y n a m i c s .
Methods General surqery: Male Sprague Dawley rats (350 - 420 g) w e r e a n e s t h e t i z e d w i t h an i n t r a p e r i t o n e a l i n j e c t i o n of s o d i u m p e n t o b a r b i t a l (60 mg/kg) and maintained with periodic supplementary doses administered intravenously (iv). A f t e r t r a c h e o s t o m y , a c a t h e t e r was i n s e r t e d into the c a r o t i d a r t e r y for a r t e r i a l b l o o d s a m p l i n g and s y s t e m i c b l o o d p r e s s u r e recording. The j u g u l a r v e i n was t h e n c a n n u l a t e d for the iv i n f u s i o n of s a l i n e (150 mmol/l) at 0.i m l / m i n and m a i n t a i n e d at t h i s r a t e t h r o u g h o u t the experiment. R e c t a l t e m p e r a t u r e was m a i n t a i n e d at 36 - 38°C w i t h a h e a t i n g pad. Renal function measurements: The left k i d n e y was e x p o s e d v i a a m i d l i n e a b d o m i n a l i n c i s i o n and its u r e t e r c a n n u l a t e d for the c o l l e c t i o n of urine. Renal blood flow (RBF) was measured by placing a non-cannulating e l e c t r o m a g n e t i c f l o w p r o b e a r o u n d the renal a r t e r y and g l o m e r u l a r f i l t r a t i o n r a t e (GFR) e s t i m a t e d by the c l e a r a n c e of i n u l i n u s i n g a p r e v i o u s l y d e s c r i b e d a s s a y p r o c e d u r e (5). P l a s m a and u r i n a r y s o d i u m c o n c e n t r a t i o n s w e r e m e a s u r e d using a Beckman flame photometer. R e n a l f u n c t i o n m e a s u r e m e n t s w e r e b e g u n 2.5 h f o l l o w i n g t h e c o m p l e t i o n of all surgery. S t a n d a r d f o r m u l a e w e r e u s e d for c a l c u l a t i n g r e n a l p l a s m a flow [RPF = RBF x (i - H e m a t o c r i t ) ] , renal v a s c u l a r resistance (RVR = M e a n a r t e r i a l p r e s s u r e / R P F ) , filtration fraction (FF = GFR/RPF), fractional sodium excretion (FEN~ = renal Na c l e a r a n c e / G F R ) and f r a c t i o n a l w a t e r e x c r e t i o n (FE w = u r i n e f l o w r a t e / G F R ) . Copyright
© 1992
0 0 2 4 - 3 2 0 5 / 9 2 $5.00 + .00 P e r g a m o n P r e s s Ltd All r i g h t s
reserved.
1572
LPC and Renal
Function
Vol.
51, No.
20,
1992
E x p e r i m e n t a l protocol: The e x p e r i m e n t a l p r o t o c o l c o n s i s t e d of t h r e e c l e a r a n c e periods. F o l l o w i n g a 30 m i n b a s e l i n e c o n t r o l period, rats r e c e i v e d e i t h e r an iv i n f u s i o n of v e h i c l e (saline c o n t a i n i n g 36 p g / m l fatty acid free b o v i n e s e r u m albumin) or m y r i s t o y l - L P C at 13 ± 3 p m o l / m i n (= 6 ± 2 ng/min). Two 30 m i n e x p e r i m e n t a l c l e a r a n c e p e r i o d s w e r e t h e n t a k e n 40 m i n after the onset of the infusion. Initial studies e x a m i n e d the c h a n g e s in a r t e r i a l b l o o d pressure, a b s o l u t e s o d i u m e x c r e t i o n and u r i n e flow to a b o l u s i n j e c t i o n of m y r i s t o y l LPC. A f t e r g e n e r a l s u r g i c a l p r o c e d u r e s , the left u r e t e r was e x p o s e d by a small a b d o m i n a l i n c i s i o n and t h e n c a t h e t e r i z e d . Using a similar experimental p r o t o c o l as above, a 0.i ml bolus i n j e c t i o n of v e h i c l e or m y r i s t o y l - L P C (2 pmol = 1 ng) was g i v e n iv after the b a s e l i n e c l e a r a n c e p e r i o d and f o l l o w e d after 40 m i n by two e x p e r i m e n t a l c l e a r a n c e periods. No renal h e m o d y n a m i c measurements were performed. P r e p a r a t i o n of solutions: Myristoyl-LPC (L-a-lysophosphatidylcholine, m y r i s t o y l ; Sigma) was stored at -20°C as a 1 mg/ml stock solution, t h a t had b e e n d i s s o l v e d in m e t h a n o l - c h l o r o f o r m (1:2 vol/vol). For a d m i n i s t r a t i o n , a l i q u o t s w e r e d r i e d in glass g r a d u a t e d flasks and t h e n d i s s o l v e d in s a l i n e c o n t a i n i n g 3 6 ~g/ml fatty acid free b o v i n e s e r u m a l b u m i n (BSA). Vehicle c o n s i s t e d of the m e t h a n o l - c h l o r o f o r m m i x t u r e in 36 ~ g / m l B S A - s a l i n e solution. Statistics: For s t a t i s t i c a l analysis, a m e a n v a l u e of the e x p e r i m e n t a l clearance p e r i o d s was c o m p a r e d to the b a s e l i n e value. Coefficient of v a r i a b i l i t y (CV) b e t w e e n first and s e c o n d e x p e r i m e n t a l s a m p l e s w e r e 13% and 9% for e x c r e t o r y r e s p o n s e s in v e h i c l e and L P C i n j e c t e d groups, r e s p e c t i v e l y . CV = 8% and 7% for renal h e m o d y n a m i c responses, 8% and 24% for e x c r e t o r y r e s p o n s e s in v e h i c l e and LPC i n f u s e d groups, r e s p e c t i v e l y . Comparisons within and a c r o s s g r o u p s w e r e a s s e s s e d u s i n g a 2-way f a c t o r i a l a n a l y s i s of v a r i a n c e f o l l o w e d by a s i m p l e m a i n e f f e c t test (CRunch I n t e r a c t i v e S t a t i s t i c a l P a c k a g e (CRISP); C r u n c h S o f t w a r e C o r p o r a t i o n ] . A significant group x treatment i n t e r a c t i o n i n d i c a t e s that the e f f e c t of t r e a t m e n t on a m e a s u r e d p a r a m e t e r d i f f e r s b e t w e e n g r o u p s (6). D a t a are p r e s e n t e d as m e a n s ± SEM. Results
T h e r e was no c h a n g e in s y s t e m i c b l o o d p r e s s u r e or u r i n e flow and a small rise in a b s o l u t e s o d i u m e x c r e t i o n d u r i n g the t i m e c o u r s e of the e x p e r i m e n t in bolus v e h i c l e i n j e c t e d rats. M y r i s t o y l - L P C a d m i n i s t e r e d to rats as a 2 pmol bolus r e s u l t e d in no c h a n g e in b l o o d p r e s s u r e and an i n c r e a s e in s o d i u m and water excretion. The enhanced diuresis was significantly greater in m y r i s t o y l - L P C t r e a t e d rats t h a n v e h i c l e c o n t r o l s (Table i). T a b l e 2 shows the d a t a o b t a i n e d from a n i m a l s c o n t i n u o u s l y i n f u s e d w i t h v e h i c l e or m y r i s t o y l - L P C (13 ± 3 p m o l / m i n ) . B a s e l i n e v a l u e s for m e a s u r e d v a r i a b l e s w e r e s i m i l a r in b o t h g r o u p s e x c e p t for a s i g n i f i c a n t l y s m a l l e r f r a c t i o n a l e x c r e t i o n of s o d i u m in rats p r i o r to m y r i s t o y l - L P C infusion. The reason(s) for this d i f f e r e n c e in b a s e l i n e f r a c t i o n a l s o d i u m e x c r e t i o n is not r e a d i l y apparent. In b o t h g r o u p s t h e r e was a small d e c r e a s e in s y s t e m i c b l o o d pressure and no c h a n g e in a r t e r i a l b l o o d h e m a t o c r i t or renal vascular r e s i s t a n c e d u r i n g the t i m e c o u r s e of the e x p e r i m e n t . T h e r e was no c h a n g e in renal h e m o d y n a m i c s , u r i n e flow or f r a c t i o n a l w a t e r e x c r e t i o n and a small rise in a b s o l u t e s o d i u m e x c r e t i o n (25%) and f r a c t i o n a l s o d i u m e x c r e t i o n (23%) in a n i m a l s i n f u s e d w i t h the BSA vehicle. In a n i m a l s g i v e n m y r i s t o y l - L P C , t h e r e was a 13% fall in renal p l a s m a flow, a t e n d e n c y for g l o m e r u l a r f i l t r a t i o n rate to d e c r e a s e a l t h o u g h this did not reach s t a t i s t i c a l s i g n i f i c a n c e , no c h a n g e in f i l t r a t i o n f r a c t i o n and an i n c r e a s e in u r i n e f l o w of 40%, a b s o l u t e s o d i u m e x c r e t i o n of 84% and f r a c t i o n a l s o d i u m and w a t e r e x c r e t i o n of 113% and 63%, respectively. The i n c r e m e n t in f r a c t i o n a l s o d i u m and w a t e r e x c r e t i o n w i t h m y r i s t o y l - L P C t r e a t m e n t was s i g n i f i c a n t l y g r e a t e r t h a n that o b s e r v e d f o l l o w i n g vehicle treatment. L i n e a r r e g r e s s i o n a n a l y s i s of the c h a n g e s in renal h e m o d y n a m i c s and in s o d i u m e x c r e t i o n and u r i n e flow r a t e in b o t h v e h i c l e and m y r i s t o y l - L P C i n f u s e d rats i n d i c a t e d one s i g n i f i c a n t p o s i t i v e c o r r e l a t i o n b e t w e e n the c h a n g e s in g l o m e r u l a r f i l t r a t i o n rate and u r i n e flow rate in the m y r i s t o y l - L P C i n f u s e d g r o u p (r= 0.742, P<0.05).
Vol.
51,
No.
20,
1992
LPC
TABLE
and Renal
Function
1
Baseline
Experiment
M A P ( m m Hg) Vehicle (n=7) M-LPC (n=7)
124 126
± 6 ± 3
123 120
U N a V (@Eq.min'J.kg -l) Vehicle M-LPC
5.9 6.3
± 1.9 ± 1.8
7.9 10.2
± 1.9" ± 2.2***
U V ( ~ l . m i n * . k g -l) Vehicle M-LPC
24.1 25.4
27.3 38.0
± 5.0 ± 9.8***%
± 6.2 ± 8.0
± 7 ± 6
* = P<0.05 ** = P < 0 . 0 1 *** = P < 0 . O 0 1 from baseline value, * = P<0.05 from vehicle value using 2-way ANOVA; % = P<0.05 $ = P<0.OI indicates a significant group x treatment interaction (i.e. t r e a t m e n t effect on measured response differs between groups). MAP = mean arterial pressure; RVR = renal vascular resistance; RPF = renal plasma flow; GFR = glomerular filtration rate; FF = filtration f r a c t i o n ; U N a V = a b s o l u t e s o d i u m e x c r e t i o n ; FENa = f r a c t i o n a l s o d i u m excretion; UV = urine flow rate; FE W = fractional water excretion; M-LPC = myristoyl-lysophosphatidylcholine.
TABLE
2
Baseline Hematocrit (%) Vehicle (n=7) M-LPC (n=9) M A P ( m m Hg) Vehicle M-LPC R V R (mm H g / m l . m i n l . k g Vehicle M-LPC
Experiment
49 47
± 1 ± 1
47 46
123 118
± 4 ± 2
112 107
± 1 ± 1
± 3** ± 3**
"') ii.2 8.4
± 1.6 ± 0.6
I0.5 8.7
+ 1.4 ± 0.5#
R P F ( m l . m i n l . k g "I) Vehicle M-LPC
12.1 14.4
± 1.4 ± 0.6
11.8 12.6
± 1.4 ± 0.6***$
G F R ( m l . m i n 1 . k g "I) Vehicle M-LPC
3.6 3.9
± 0.7 ± 0.3
3.8 3.4
F F (%) Vehicle M-LPC
0.29 0.27
U N a V (@Eq. m i n "l. k g "j) Vehicle M-LPC
4.0 2.5
± 0.04 ± 0.02
± 0.7 ± 0.5
0.32 0.28
5.0 4.6
± 0.7 ± 0.4
± 0.04 ± 0.03
± 0.8 ± 0.9**
FEN, (%) Vehicle M-LPC
0.79 0.39
± 0.16 ± 0.07*
0.98 0.83
± 0.18" ± 0.09***%
U V ( @ l . m i n l . k g -I) Vehicle M-LPC
21.9 17.9
± 2.7 ± 1.7
24.6 25.2
± 2.9 ± 2.9**
FEw (%) Vehicle M-LPC
0.76 0.46
± 0.17 ± 0.04
0.82 0.76
± 0.18 ± 0.05***$
Abbreviations
as
in Table
I.
1573
1574
LPC and Renal F u n c t i o n
Vol.
51, No.
20,
1992
Discussion R e n a l s o d i u m e x c r e t i o n is t i g h t l y linked to e x t r a c e l l u l a r fluid (ECF) volume regulation. The m e c h a n i s m ( s ) by w h i c h this c o u p l i n g o c c u r s is not fully u n d e r s t o o d . One theory, that ECF v o l u m e e x p a n s i o n leads to the r e l e a s e of h u m o r a l n a t r i u r e t i c factors, has b e e n s u p p o r t e d by the d i s c o v e r y of a t r i a l n a t r i u r e t i c peptide. However, c o n s i d e r a b l e e v i d e n c e s u p p o r t s the e x i s t e n c e of at least one other n a t r i u r e t i c hormone, a f a c t o r w h i c h i n h i b i t s H a + - K + A T P a s e (1). Lysophosphatidylcholine (LPC) c o n s t i t u t e s a m a j o r c o m p o n e n t of p l a s m a N a + - K + A T P a s e i n h i b i t o r y activity, and s t u d i e s have d e m o n s t r a t e d t h a t acute v o l u m e e x p a n s i o n in rats and pigs i n c r e a s e s the p l a s m a c o n c e n t r a t i o n of LPC (4,7). F u r t h e r m o r e , R a u c h and B u c k a l e w r e p o r t e d that the i n t r a v e n o u s b o l u s i n j e c t i o n of s t e r o y l - L P C into the a n e s t h e t i z e d rat p r o d u c e d a small n a t r i u r e t i c and d i u r e t i c r e s p o n s e after a lag p e r i o d of 20 min, w i t h the p e a k n a t r i u r e t i c e f f e c t o c c u r r i n g at 1 h, and l a s t i n g close to 7 h (3). T h e y also d e m o n s t r a t e d that the m y r i s t o y l a n a l o g u e of LPC had the g r e a t e s t p o t e n c y for n a t r i u r e s i s and d i u r e s i s (3). However, t h e y did not m e a s u r e s y s t e m i c b l o o d p r e s s u r e or renal h e m o d y n a m i c s w h i c h are i m p o r t a n t f a c t o r s in the r e g u l a t i o n of renal s o d i u m and w a t e r e x c r e t i o n and could p o t e n t i a l l y c o n t r i b u t e to the o b s e r v e d n a t r i u r e t i c and d i u r e t i c p r o p e r t i e s of LPC. Furthermore, a constant i n f u s i o n of L P C r a t h e r than a bolus i n j e c t i o n m a y g i v e a b e t t e r r e f l e c t i o n of c h a n g e s in renal f u n c t i o n that may o c c u r d u r i n g ECF v o l u m e e x p a n s i o n . The data o b t a i n e d from rats g i v e n a bolus i n j e c t i o n of m y r i s t o y l - L P C c o n f i r m e d p r e v i o u s l y p u b l i s h e d w o r k (3) that t h i s p h o s p h o l i p i d p o s s e s s e s diuretic properties. Although, we did not demonstrate a statistical d i f f e r e n c e in s o d i u m e x c r e t i o n b e t w e e n m y r i s t o y l - L P C and v e h i c l e i n j e c t e d rats, t h e r e was a t r e n d for a g r e a t e r i n c r e a s e in s o d i u m e x c r e t i o n w i t h myristoyl-LPC administration. Since the n a t r i u r e t i c e f f e c t of LPC lasts for s e v e r a l hours (3), a d d i t i o n a l u r i n a r y c o l l e c t i o n s m a y have d e m o n s t r a t e d a s i g n i f i c a n t d i f f e r e n c e in s o d i u m e x c r e t i o n b e t w e e n the two groups. The p r e s e n t study also d e m o n s t r a t e d that the e n h a n c e d d i u r e s i s was u n r e l a t e d to an a l t e r a t i o n in renal p e r f u s i o n p r e s s u r e as i n d i c a t e d by an u n c h a n g e d s y s t e m i c a r t e r i a l b l o o d pressure. Administration of m y r i s t o y l - L P C as a c o n s t a n t i n f u s i o n e l i c i t e d b o t h a n a t r i u r e t i c and d i u r e t i c r e s p o n s e and was s i m i l a r in m a g n i t u d e to that o b s e r v e d w i t h bolus a d m i n i s t r a t i o n . The s i m i l a r s o d i u m and w a t e r r e s p o n s e to LPC a d m i n i s t e r e d as a bolus or i n f u s i o n m a y be r e l a t e d to e i t h e r o p e n i n g of the a b d o m i n a l c a v i t y and g r e a t e r s u r g i c a l t r a u m a in m y r i s t o y l - L P C i n f u s e d rats w h i c h can r e d u c e the n a t r i u r e t i c and d i u r e t i c r e s p o n s e of the kidney, or due to the b i p h a s i c d o s e r e s p o n s e curve for the n a t r i u r e t i c and d i u r e t i c e f f e c t of LPC (3). The e n h a n c e d n a t r i u r e s i s and d i u r e s i s in m y r i s t o y l - L P C i n f u s e d rats c o m p a r e d to c o n t r o l s w e r e not due to a l t e r a t i o n s in b l o o d p r e s s u r e seen by the k i d n e y or renal h e m o d y n a m i c changes. In fact, m y r i s t o y l - L P C s i g n i f i c a n t l y d e c r e a s e d renal p l a s m a flow by 13% and t e n d e d to d e c r e a s e g l o m e r u l a r f i l t r a t i o n rate w h i c h are h e m o d y n a m i c c h a n g e s m o s t l i k e l y to d e c r e a s e s o d i u m and w a t e r e x c r e t i o n . T h e s e renal h e m o d y n a m i c c h a n g e s c o u l d be due to a c o n s t r i c t o r e f f e c t of m y r i s t o y l - L P C on preg l o m e r u l a r b l o o d v e s s e l s since f i l t r a t i o n f r a c t i o n r e m a i n e d u n c h a n g e d . In contrast, s t u d i e s in the rabbit have shown that mg a m o u n t s of LPC can p r o d u c e coronary and renal v a s o d i l a t i o n (8) and this a p p e a r s to be due to an e n d o t h e l i u m - d e p e n d e n t v a s o d i l a t o r y a c t i o n of LPC on v a s c u l a r s m o o t h m u s c l e w h i c h is c y c l i c G M P - d e p e n d e n t , but d i f f e r s from the e n d o t h e l i u m - d e r i v e d r e l a x i n g factor, n i t r i c oxide (9,10). We have not e x a m i n e d w h e t h e r the renal v a s c u l a r c o n s t r i c t o r e f f e c t of LPC in the rat is a d o s e - r e l a t e d or s p e c i e s dependent phenomenon. A l t e r n a t i v e l y , renal p l a s m a flow c o u l d have d e c r e a s e d as a r e s u l t of the fall in s y s t e m i c b l o o d p r e s s u r e as s u g g e s t e d by an unchanged renal v a s c u l a r resistance. Generally a small fall in renal p e r f u s i o n p r e s s u r e w o u l d lead to a u t o r e g u l a t i o n of renal p l a s m a flow as was o b s e r v e d in the v e h i c l e i n f u s e d rats. W h e t h e r LPC i n t e r f e r e s w i t h the a u t o r e g u l a t o r y a b i l i t y of the rat k i d n e y to a fall in b l o o d p r e s s u r e is unknown. Since the alterations in b l o o d pressure and renal hemodynamics a s s o c i a t e d w i t h m y r i s t o y l - L P C i n f u s i o n do not favor n a t r i u r e s i s and d i u r e s i s , the p o s s i b i l i t y arises that LPC m a y have a d i r e c t t u b u l a r a c t i o n to e n h a n c e s o d i u m and w a t e r e x c r e t i o n . This c o n t e n t i o n is s u p p o r t e d by m y r i s t o y l - L P C i n f u s i o n r e s u l t i n g in a g r e a t e r f r a c t i o n a l e x c r e t i o n of s o d i u m and w a t e r t h a n in v e h i c l e t r e a t e d rats, s u g g e s t i n g a r e d u c e d a b i l i t y of the t u b u l e to r e a b s o r b s o d i u m and water. LPC is c a p a b l e of i n h i b i t i n g the s o d i u m p u m p by i n t e r a c t i n g w i t h the N a ÷ - b i n d i n g sites of N a ÷ - K + A T P a s e (ii), and p r e s u m a b l y
Vol.
51, No.
20,
1992
L P C and R e n a l F u n c t i o n
1575
in t h e r e n a l t u b u l e lead to a loss in the s o d i u m g r a d i e n t at the a p i c a l m e m b r a n e for t h e r e a b s o r p t i o n of s o d i u m and water. In a d d i t i o n , L P C m a y regulate protein kinase C (PKC) by stimulating and inhibiting, in a concentration-dependent m a n n e r , p h o s p h o r y l a t i o n of PKC s u b s t r a t e p r o t e i n s (12). I n h i b i t i o n or d o w n r e g u l a t i o n of PKC in the p r o x i m a l t u b u l e w o u l d reduce the activity of such sodium transporters as the apical Na+-H ÷ a n t i p o r t e r w h i c h w o u l d a l s o lead to r e d u c e d s o d i u m and w a t e r r e a b s o r p t i o n . T h e r e is e v i d e n c e t h a t lipids can also i n h i b i t v a s o p r e s s i n - m e d i a t e d water r e a b s o r p t i o n in c o r t i c a l c o l l e c t i n g d u c t s (13). The r e g u l a t i o n of renal e l e c t r o l y t e and w a t e r e x c r e t i o n by lipids a p p e a r s to be p r e s e n t in s e v e r a l species, as L P C and o t h e r r e l a t e d lipids h a v e b e e n r e p o r t e d to i n c r e a s e s o d i u m and e s p e c i a l l y w a t e r e x c r e t i o n in the rat (3, p r e s e n t study), r a b b i t (13), d o g (14) and h u m a n (15). A l t h o u g h , i n t r a v e n o u s l y a d m i n i s t e r e d L P C is r a p i d l y r e m o v e d from p l a s m a by t i s s u e (t~ = 6 - ii min), its m e t a b o l i s m is v e r y s l o w r e q u i r i n g s e v e r a l hours (16). This is c o n s i s t e n t w i t h our o b s e r v a t i o n t h a t a small d o s e of L P C is c a p a b l e of p r o d u c i n g a long-lasting biological response. T h e n a t r i u r e t i c and d i u r e t i c a c t i o n s of L P C are n o v e l and the intracellular mechanisms mediating these responses have yet to be investigated. In conclusion, our results suggest that myristoyl-LPC has weak n a t r i u r e t i c and d i u r e t i c e f f e c t s in the rat and c o u l d c o n c e i v a b l y p a r t i c i p a t e in t h e r e n a l r e s p o n s e to p l a s m a v o l u m e e x p a n s i o n . The m y r i s t o y l - L P C m e d i a t e d i n c r e a s e in s o d i u m and w a t e r e x c r e t i o n a p p e a r s not to be d e p e n d e n t on the c h a n g e s in s y s t e m i c b l o o d p r e s s u r e or renal h e m o d y n a m i c s , and m a y r e f l e c t a t u b u l a r site of a c t i o n of LPC. References
i. 2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15.
16.
H.E. D e W A R D E N E R and E.M. CLARKSON, Physiol. Rev. 65 6 5 9 - 7 5 9 (1985). J.N. KARLI, G.A. KARIKAS, P.K. HATZIPAVLOU, G.M. LEVIS and S.N. M O U L O P O U L O S , L i f e Sci. 2__4 1 8 6 9 - 1 8 7 6 (1979). A.L. R A U C H and V.M. BUCKALEW, Jr., Am. J. Physiol. 254 F 8 2 4 - F 8 2 7 (1988). A.L. R A U C H and V.M. BUCKALEW, Jr., L i f e Sci. 42 1 1 8 9 - 1 1 9 7 (1988) R.K. H A N D A and E.J. JOHNS, Q u a r t e r l y J. Exptl. Physiol. 7_33 9 1 5 - 9 2 9 (1988). B.J. WINER, S t a t i s t i c a l p r i n c i p l e s in e x p e r i m e n t a l design, M c G r a w - H i l l Inc. p 4 3 1 - 5 1 3 (1971). M. TAMURA, T. INAGAMI, T. K I N O S H I T A and H. KUWANO, J. H y p e r t e n . 5 219225 (1987). A. WOLF, T. SAITO, R. D U D E K and R.J. BING, L i p i d s 26 2 2 3 - 2 2 6 (1991). T. SAITO, A. WOLF, N.K. MENON, M. S A E E D and R.J. BING, Proc. Natl. Acad. Sci. U S A 8-5 8 2 4 6 - 8 2 5 0 (1988). N.K. M E N O N and R.J. 8ING, Proc. Soc. Expt. Biol. Med. 196 4 6 1 - 4 6 3 (1991). K. OISHI, B. Z H E N G and J.F. KUO, J. Biol. Chem. 265 70-75 (1990). K. OISHI, R.L. RAYNOR, P.A. C H A R P and J.F. KUO, J. Biol. Chem. 26__~368656871 (1988). A. Y A S U H I R O , H.R. J A C O B S O N and M.D. BREYER, Am. J. Physiol. 257 F 5 2 4 F 5 3 0 (1989). V.M. B U C K A L E W , Jr., J.W. S T R A N D H O Y and R.K. HANDA, J. C a r d i o v a s c . P h a r m a c o l . (in press). P. B E R N A R D I , F. GHEZZI, L. BASTAGLI, R. G R I M A L D I , M. C A V A Z Z A , C. M I N E L L I , F. FONTANA, C. CLO, C. V E N T U R A and M. CAPELLI, Prost. Leuko. Med. 2-2 2 3 5 - 2 4 8 (1986). Y. S T E I N and O. STEIN, B i o c h i m i c a et B i o p h y s i c a A c t a 116 9 5 - 1 0 7 (1966).