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Increased urinary excretion of platelet activating factor in mice with lupus nephritis
Life Sciences, Vol. 48, pp. 1429-1437 Printed in the U.S.A.
INCREASED
URINARY
E X C R E T I O N OF P L A T E L E T A C T I V A T I N G W I T H LUPUS N E P H R I T I S
Pergamon Press
FACTOR
IN
MICE
D a n i e l a M a c c o n i I), M a r i n a Noris I), E m i l i o B e n f e n a t i I) R o b e r t o Q u a g l i a I), G i a n l u c a P a g l i a r i n o I), and G i u s e p p e R e m u z z i I) 2)
1 M a r i o Negri Institute for P h a r m a c o l o g i c a l Research, Via G a v a z z e n i ii, and 2 D i v i s i o n of Nephrology, O s p e d a l i R i u n i t i di Bergamo, 24100 Bergamo, Italy. (Received in final form February I, 1991) Summary P l a t e l e t a c t i v a t i n g factor (PAF) is p r e s e n t in u r i n e from humans and e x p e r i m e n t a l animals in normal conditions. V e r y l i t t l e is k n o w n about c h a n g e s in PAF u r i n a r y e x c r e t i o n under pathologic conditions a n d no d a t a are a v a i l a b l e about the origin of PAF in the urine. In the p r e s e n t study we e x p l o r e d the p o s s i b i l i t y that i m m u n o l o g i c renal d i s e a s e is a s s o c i a t e d w i t h an i n c r e a s e in PAF u r i n a r y e x c r e t i o n u s i n g gas c h r o m a t o g r a p h y - m a s s spectrometry technique. To c l a r i f y the renal or e x t r a r e n a l o r i g i n of u r i n a r y PAF we evaluated whether exogenously administered PAF (l[l',2'-3H]alkyl) is f i l t e r e d t h r o u g h the g l o m e r u l u s and e x c r e t e d in the urine. The r e s u l t s s h o w that: I) u r i n e f r o m m i c e w i t h lupus n e p h r i t i s in the e a r l y p h a s e of the disease contained amounts of PAF c o m p a r a b l e to t h o s e e x c r e t e d in n o r m a l m o u s e urine, 2) PAF levels i n c r e a s e d when a n i m a l s started to d e v e l o p high grade proteinuria, 3) after i n t r a v e n o u s injection of [3H] PAF in n e p h r i t i c mice, a n e g l i g i b l e amount of [3H] ether lipid, c o r r e s p o n d i n g to [3H]l-alkyl -2-acyl-3-phosphocholine (alkyl-2-acyl-GPC), was r e c o v e r e d from the 24 h urine extract. Platelet activating factor (PAF) or l - O - a l k y l - 2 - a c e t y l - s n glycero-3-phosphocholine is a potent lipid m e d i a t o r of i n f l a m m a t i o n with a wide v a r i e t y of b i o l o g i c a l a c t i v i t i e s i n c l u d i n g p l a t e l e t and neutrophil activation and aggregation (1,2) a n d i n c r e a s e d v a s c u l a r (3,4) a n d g l o m e r u l a r (5-7) p e r m e a b i l i t y . PAF f o r m a t i o n has b e e n d e s c r i b e d in s t i m u l a t e d i n f l a m m a t o r y cells (8,9) as well as c u l t u r e d e n d o t h e l i a l cells (i0). R e l e a s e of PAF has b e e n d o c u m e n t e d in the venous effluent of i s o l a t e d perfused rat k i d n e y upon calcium ionophore (ii) or a n t i g e n challenge (12). Isolated glomeruli (13-15), suspensions of r e n o m e d u l l a r y cells (13), a n d cultured m e s a n g i a l cells (14,16) a l s o g e n e r a t e PAF. M o r e o v e r low a m o u n t s of P A F are p r e s e n t in b l o o d from humans (17,18) and experimental animals (18) in normal conditions. Send correspondence for P h a r m a c o l o g i c a l
to: Dr. D a n i e l a Macconi, M a r i o N e g r i I n s t i t u t e Research, Via G a v a z z e n i ii, 24100 Bergamo, Italy 0024-3205/91 $3.00 +.00 Copyright (c) Pergamon Press pie
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Sanchez-Crespo et al. have p r o v i d e d e v i d e n c e that n o r m a l human u r i n e c o n t a i n s n a n o g r a m a m o u n t s of a p h o s p h o l i p i d compound with platelet-activating and h y p o t e n s i v e a c t i v i t y similar to PAF (19). PAF-related bioactivity has a l s o b e e n d e t e c t e d in s a m p l e s of first v o i d e d u r i n e of n e w b o r n s (20). In a d d i t i o n the p r e s e n c e of n a n o g r a m amounts of PAF in normal m o u s e urine, as d e t e r m i n e d by gas chromatography-mass spectrometry (GC-MS) technique, has been recently reported (21). O t h e r studies have suggested t h a t PAF m e d i a t e s renal d a m a g e (22,23). I n c r e a s e d g l o m e r u l a r s y n t h e s i s (24) and release of P A F in t h e circulation (23,25,26) have been d o c u m e n t e d in i m m u n e - m e d i a t e d g l o m e r u l o n e p h r i t i s . If PAF g e n e r a t i o n is i n c r e a s e d in c o n d i t i o n of g l o m e r u l a r i n j u r y and i n f i l t r a t i o n of inflammatory cells, one may w o n d e r w h e t h e r PAF u r i n a r y e x c r e t i o n reflects such changes. This p o s s i b i l i t y is in k e e p i n g w i t h the data of S a n c h e z - C r e s p o et al. s h o w i n g a i0 fold i n c r e a s e in P A F - r e l a t e d b i o a c t i v i t y in urine from p a t i e n t s with s y s t e m i c lupus e r y t h e m a t o s u s (SLE) a n d h e a v y p r o t e i n u r i a (19). H o w e v e r in the a b o v e m e n t i o n e d p a p e r the m a t e r i a l found in the u r i n e and d e f i n e d as PAF has only b e e n c h a r a c t e r i z e d by mean of its b i o l o g i c a l a c t i v i t y on platelets. M o r e o v e r no s t u d i e s have b e e n a d d r e s s e d to c l a r i f y w h e t h e r u r i n a r y PAF was of renal or e x t r a r e n a l origin. The aims of the p r e s e n t s t u d y were: i) to c l a r i f y w h e t h e r immunologic renal d i s e a s e is a s s o c i a t e d w i t h an i n c r e a s e in PAF u r i n a r y excretion, 2) to assess the e x t r a r e n a l or renal o r i g i n of PAF e x c r e t e d in the urine. Materials
and Methods
S t a n d a r d PAF a n d lyso PAF w i t h t h e l - O - h e x a d e c y l chain were obtained from Bachem Feinchemikalien AG (Bubendorf, Switzerland). [3H]PAF (l-[l',2'-3H]hexadecyl-2-acetyl-sn-gly cer°3-phosphocholine, 56.7 Ci/mmol) was from New England Nuclear (Boston, MA). Thin layer c h r o m a t o g r a p h y (TLC) plates, p r e c o a t e d with silica gel 60 w i t h c o n c e n t r a t i n g zone, layer t h i c k n e s s 0.25 mm, and 50% h y d r o f l u o r i c acid (HF) w e r e o b t a i n e d from Merck (Darmstadt, F.R.G.) . P e n t a f l u o r o b e n z o y l chloride (PFBCI) was p u r c h a s e d from J a n s s e n C h i m i c a (Beerse, Belgium). A c e t i c a n h y d r i d e - d 6 was o b t a i n e d from Sigma Chemical Co. (St. Louis, MO) . S i l i c i c a c i d was f r o m Bio-Rad Laboratories (Richmond, CA). High performance liquid chromatography (HPLC) and a n a l y t i c a l grade s o l v e n t s were p u r c h a s e d f r o m Merck. Studies in mice with luPUS nephritis. Female N e w Z e a l a n d Black x New Zealand White (NZBxNZW) F 1 mice spontaneously develop an autoimmune d i s e a s e that c l o s e l y r e s e m b l e s SLE in m a n (27). The d i s e a s e m a n i f e s t s w i t h c i r c u l a t i n g a n t i b o d i e s to n u c l e i c a c i d a n d endogenous antigens, immune complex formation, and persistent proteinuria. Five female NZBxNZW F1 m i c e (Harlan O L A C 1976 LTD Bicester, England), 8-9 weeks of age at the start of e x p e r i m e n t , were used. Monthly, 24 h urine samples were c o l l e c t e d in m e t a b o l i c c a g e s a n d p r o t e i n content, expressed in mg/day, was m e a s u r e d by Coomassie blue G dye-binding assay for p r o t e i n s (28) . U r i n a r y protein content measured in N Z B x N Z W F 1 m i c e at 14 w e e k s of age, b e f o r e the d i s e a s e was m a n i f e s t (27), was w i t h i n 3 mg/day. U r i n a r y p r o t e i n levels e x c e e d i n g 3 m g / d a y were c o n s i d e r e d abnormal. S t a r t i n g from 26 weeks of age, mice randomly developed high grade p r o t e i n u r i a . PAF u r i n a r y e x c r e t i o n was m e a s u r e d by GC-MS in N Z B x N Z W
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F 1 m i c e at 22 weeks of age, w h e n p r o t e i n e x c r e t i o n was w i t h i n the p h y s i o l o g i c range for normal urine, a n d again as soon as the animals developed proteinuria above 3 mg/day. S i n c e the PAF g e n e r a t e d by m e s a n g i a l cells is e x c l u s i v e l y of h e x a d e c y l s p e c i e s (16), w h i c h is also predominant (over 90% of total s y n t h e s i z e d PAF) in s t i m u l a t e d neutrophils (29), PAF C16:0 was analyzed. E x t r a c t i o n a n d P u r i f i c a t i o n of urine. I n t e r n a l s t a n d a r d P A F - d 3 was synthesized and purified as previously described (21). T w e n t y f o u r h urine samples f r o m N Z B x N Z W F 1 m i c e w e r e a d d e d to 5 ng P A F - d 3 a n d e x t r a c t e d a c c o r d i n g to P i n c k a r d et al. (30). Briefly, 1.5 v o l u m e s of m e t h a n o l was a d d e d to urine and, a f t e r g e n t l e m i x i n g for 30 min, the samples were centrifuged. C h l o r o f o r m and w a t e r were then added to obtain a solvent mixture containing chloroform: methanol:water (1:1:0.9, v/v/v). The lower p h a s e was r e m o v e d and t a k e n to d r y n e s s u n d e r a n i t r o g e n stream. T h e r e a f t e r s a m p l e s were purified according to P i n c k a r d et al. (30), w i t h the f o l l o w i n g m o d i f i c a t i o n : the extract was r e s u s p e n d e d in c h l o r o f o r m : m e t h a n o l 9:1 and l o a d e d on a c o l u m n p a c k e d w i t h 0.5 g of s i l i c i c a c i d (100-200 mesh) activated at i00 °C o v e r n i g h t . P A F was e l u t e d using a s e q u e n t i a l s o l v e n t s y s t e m of c h l o r o f o r m (15 ml), a c e t o n e (15 ml), acetone:methanol (i:I, v/v) (15 ml), c h l o r o f o r m : m e t h a n o l (1:4, v/v) (30 ml) . The f o u r t h f r a c t i o n c o n t a i n i n g PAF was t a k e n to d r y n e s s u n d e r a n i t r o g e n stream. R e c o v e r y of e x t r a c t i o n a n d p u r i f i c a t i o n , a s s e s s e d by a d d i t i o n of [3H] PAF to the s a m p l e b e f o r e p r o c e s s i n g , r a n g e d b e t w e e n 80 and 90%. Derivatization of PAF. A f t e r purification, samples were h y d r o l y z e d to l - O - a l k y l - 2 - a c e t y l - g l y c e r o l by the a d d i t i o n of 0.2 ml of 50% HF at 25 °C. A f t e r 4 h, 0.5 ml of h e x a n e was a d d e d a n d the o r g a n i c p h a s e w a s r e m o v e d a n d t a k e n to d r y n e s s u n d e r a n i t r o g e n s t r e a m (31). R e c o v e r y of the reaction, a s s e s s e d by h y d r o l y s i s of [3H] PAF s t a n d a r d a n d s u b s e q u e n t TLC a n a l y s i s of b y p r o d u c t s (32), was a b o u t 60%. F o r d e r i v a t i z a t i o n (33) 50 ~i of d i s t i l l e d P F B C I was added; the system was heated at i00 °C for 40 min. A f t e r the r e a c t i o n , the e x c e s s r e a g e n t was t a k e n to d r y n e s s u n d e r a n i t r o g e n stream. Recovery of d e r i v a t i z a t i o n was generally above 90% as r e p o r t e d b y M a g n i et al. (34) . The r e s i d u e was d i s s o l v e d in h e x a n e (50 ~I) and 2 ~i a l i q u o t s w e r e u s e d for MS analysis. Mass spectrometry, GC-MS analyses, in the n e g a t i v e ion chemical ionization (NICI) mode, were carried out on a V G 7 0 - 2 5 0 m a s s spectrometer (VG A n a l y t i c a l , Manchester, U . K . ) . A D a n i 6500 gas chromatograph (Dani, M o n z a , Italy) was coupled to the mass s p e c t r o m e t e r . A fused silica c a p i l l a r y column CPSil 5 CB, 25 m, 0.32 m m I.D. (Chrompack, M i d d e l b u r g , The N e t h e r l a n d s ) w a s used, w i t h a p r o g r a m m e d t e m p e r a t u r e v a p o r i z e r (PTV) injector. The c h r o m a t o g r a p h i c p r o g r a m was: oven temperature: 140 °C, initial, for 1 min; then from 140 to 280 °C p r o g r a m m e d temperature, 18 °C/min. For chemical ionization MS a m m o n i a w a s u s e d as t h e r e a g e n t gas. S e l e c t e d ion recording (SIR) a n a l y s e s w e r e d o n e m o n i t o r i n g t h e ions at m/z 552 a n d 555 for P A F a n d P A F - d 3 r e s p e c t i v e l y . A c a l i b r a t i o n curve was c o n s t r u c t e d by u s i n g the c a l c u l a t e d p e a k - a r e a r a t i o of PAF to the i n t e r n a l s t a n d a r d P A F - d 3 (400 pg injected). The p e a k area ratio was linearly correlated (r=0.999) w i t h the PAF c o n c e n t r a t i o n in the range of 2-800 pg. Intravenous NZBxNZW F 1 mice
[3~1 P A F were u s e d
administration for the study.
to lupus mice. Seven G r o u p 1 (n=4) r e c e i v e d
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a b o u t 1 ~Ci of [3H] P A F at 22 w e e k s of age; g r o u p 2 (n=3) r e c e i v e d the same d o s e of [3H] PAF at 26-32 w e e k s of age, as s o o n as the a n i m a l s d e v e l o p e d p r o t e i n u r i a e x c e e d i n g 3 m g / d a y . The r a d i o l a b e l e d compound, dissolved in 0.5% e t h a n o l in s a l i n e c o n t a i n i n g 0.25% b o v i n e s e r u m albumin, was i n j e c t e d as a b o l u s into the tail vein. A f t e r the injection, the p l a s t i c syringe a n d n e e d l e w e r e well r i n s e d w i t h 500 ~i of the i n j e c t i o n s o l u t i o n p l u s 500 ~I of e t h a n o l to c o l l e c t u n i n j e c t e d r a d i o l a b e l e d lipid. All a n i m a l s w e r e h o u s e d in m e t a b o l i c cages for c o l l e c t i o n of 24 h u r i n e samples. The urine was e x t r a c t e d as d e s c r i b e d above. The d r i e d r e s i d u e was r e s u s p e n d e d in m e t h a n o l a n d an a l i q u o t u s e d for r a d i o a c t i v i t y determination by liquid scintillation counting. The yield of [3H] e t h e r lipid r e c o v e r e d f r o m the u r i n e e x t r a c t was e x p r e s s e d as p e r c e n t a g e of the total administered dose. S i n c e PAF can a d s o r b to s u r f a c e s , the a d m i n i s t e r e d dose was the d i f f e r e n c e b e t w e e n the t r i t i u m c o n t e n t in the injection solution and that recovered from the syringe and needle. To a s s e s s w h e t h e r the t r i t i a t e d l i p i d in the u r i n e was a c t u a l l y [3H] PAF or its m e t a b o l i t e s , u r i n e e x t r a c t s f r o m m i c e at 22 w e e k s of age or f r o m m i c e w i t h h i g h g r a d e p r o t e i n u r i a w e r e p o o l e d a n d p u r i f i e d b y s i l i c i c a c i d column. P u r i f i e d s a m p l e s w e r e t h e n a n a l y z e d by H P L C by i s o c r a t i c e l u t i o n as p r e v i o u s l y d e s c r i b e d (32). S t a t i s t i c a l a n a l y s i s . R e s u l t s are m e a n ± S.D. Data were a n a l y z e d by Student's t test. P values <0.05 were considered to b e statistically significant. Results
Gas c h r o m a t o a r a D h v - m a s s s p e c t r o m e t r i c a n a l v s i s of u r i n a r v PAF in NZBxNZW F l mice. Fig. 1 s h o w s r e p r e s e n t a t i v e S I R t r a c e s of PAF e x c r e t e d in a 24 h urine sample from N Z B x N Z W F 1 m i c e at 22 weeks of age and of the c o r r e s p o n d i n g d e u t e r a t e d i n t e r n a l standard. The d e t e c t e d M- ions c o r r e s p o n d e d to the m o l e c u l a r w e i g h t of pentafluorobenzoyl d e r i v a t i v e s of n a t i v e P A F (m/z 552) a n d P A F - d 3 (m/z 555). The c h r o m a t o g r a p h i c r e t e n t i o n time of PAF e x c r e t e d in 24 h u r i n e (9.32-9.38 min) was c h e c k e d a n d c o n f i r m e d by the p r e s e n c e of the i n t e r n a l s t a n d a r d P A F - d 3 w h o s e r e t e n t i o n t i m e was one s e c o n d s h o r t e r t h a n t h a t of n a t i v e PAF due to a p r i m a r y i s o t o p e e f f e c t (Fig.l). The S I R t r a c e s of the d e r i v a t i z e d PAF p r e s e n t e d two p e a k s (peaks 1 a n d 2 on the m/z 552 trace, a n d IA a n d 2A on the m/z 555 trace, fig.l) that c o r r e s p o n d to the two p o s i t i o n a l i s o m e r s b e a r i n g the acetyl moiety in t h e s n - 2 or -3 p o s i t i o n resulting from intramolecular shift of the a c e t y l group. In our c h r o m a t o g r a p h i c c o n d i t i o n s , the t w o c h e m i c a l i s o m e r s w e r e s e p a r a t e d . M o r e o v e r the r e l a t i v e a b u n d a n c e of the t w o p o s i t i o n a l i s o m e r s was c o n s t a n t a n d equal for n a t u r a l a n d d e u t e r a t e d PAF. For the q u a n t i t a t i v e a n a l y s i s both PAF peaks were considered. Altogether these characteristics allowed a correct identification of e n d o g e n o u s PAF. In o r d e r to e x c l u d e the p o s s i b l e i n t e r f e r e n c e f r o m the i s o t o p i c c o n t r i b u t i o n of o t h e r ions at m/z 552 a n d 555, we c o n s t a n t l y m o n i t o r e d the ions at m/z 551 a n d 554. In fig.l the t r a c e at m/z 555 p r e s e n t s a d o u b l e t (peaks 3 a n d 4) p r e c e e d i n g the P A F - d 3 peaks. This doublet, at 9.26 min, was p r e s e n t o n l y in b i o l o g i c a l s a m p l e s a n d was d u e t o an i s o t o p i c c o n t r i b u t i o n of a c o m p o u n d w i t h a s i g n a l at m/z 554. The distribution of t h e t w o p e a k s in t h e c h r o m a t o g r a p h i c doublet s u g g e s t s a p h o s p h o l i p i d n a t u r e of such a c o m p o u n d . However, it is u n l i k e l y that acyl P A F or PAF r e l a t e d c o m p o u n d s give a s i g n a l at m/z 554. In o u r c h r o m a t o g r a p h i c conditions the PAF-d 3 and the unknown
Vol. 48, No. 15, 1991
PAF Urinary Excretion in Lupus Mice
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Norm: 132
I00
m/z 552
6O d.
40
>, 2O r-,
o r..
.r4
100
• ,-t
80
o~ c~
60
31 2A
484
I
';
9:15
Norm:
9 : 30
FIG.
9 : 45
i.
10 : O0
10 : 15
10 : 30
Time (min)
S e l e c t e d ion c h r o m a t o g r a m s m o n i t o r i n g the M- ion of t h e pentafluorobenzoyl d e r i v a t i v e s of PAF e x c r e t e d in a 24 h u r i n e sample from N Z B x N Z W F 1 m i c e at 22 weeks of age (m/z 552; n o r m a l i z e d r e s p e c t p e a k 2) and of i n t e r n a l s t a n d a r d P A F - d 3 (m/z 555; n o r m a l i z e d respect p e a k 4).
d o u b l e t w e r e s e p a r a t e d . PAF levels m e a s u r e d by G C - M S in the u r i n e f r o m 5 N Z B x N Z W F 1 m i c e in the e a r l y p h a s e of the d i s e a s e (22 weeks of age) w e r e 1.56±0.54 ng/day. These values were c o m p a r a b l e to those previotr~ly r e p o r t e d (21) for n o r m a l CD-I (ICR) BR m i c e of the same age. At this stage of the d i s e a s e u r i n a r y p r o t e i n e x c r e t i o n in lupus m i c e was less than 3 m g / d a y (range 0 . 6 6 - 1 . 5 1 mg/day), v a l u e s that were a l s o not s i g n i f i c a n t l y d i f f e r e n t from t h o s e r e c o r d e d in h e a l t h y CD-I (ICR) BR m i c e of the same age (1.14±0.37 mg/day, n=5; r a n g e 0 . 7 8 - 1 . 7 2 mg/day) .To i n v e s t i g a t e changes in u r i n a r y PAF later in the c o u r s e of the disease, studies were p e r f o r m e d as soon as the animals d e v e l o p e d p r o t e i n u r i a e x c e e d i n g 3 m g / d a y (at 26 to 42 weeks of age). As s h o w n in Fig.2 a s i g n i f i c a n t i n c r e a s e in PAF u r i n a r y e x c r e t i o n o c c u r r e d in this p h a s e of the d i s e a s e as c o m p a r e d w i t h v a l u e s f o u n d at 22 weeks of age (5.69±1.83 n g / d a y vs 1.56±0.54 ng/day, p<0.01) . Intravenous r3H] PAF a i v e n to n e p h r i t i c m i c e is not e x c r e t e d ~ In o r d e r to a s s e s s w h e t h e r circulating P A F m a y be f i l t e r e d t h r o u g h the g l o m e r u l u s a n d e x c r e t e d in the urine, [3H] P A F was i n t r a v e n o u s l y i n j e c t e d in four 22 w e e k old N Z B x N Z W F 1 m i c e w i t h proteinuria r a n g i n g f r o m 0.95 to 1.56 m g / d a y . T w e n t y f o u r h a f t e r intravenous i n j e c t i o n of [3H] PAF, the y i e l d of [ 3 H ] e t h e r lipid r e c o v e r e d into the o r g a n i c p h a s e a f t e r u r i n e e x t r a c t i o n r e p r e s e n t e d only 0.084±0.031 % of the t o t a l a d m i n i s t e r e d dose. To e v a l u a t e w h e t h e r the i n c r e a s e d u r i n a r y e x c r e t i o n of PAF in lupus m i c e w i t h h e a v y p r o t e i n u r i a d e p e n d s on the a l t e r e d g l o m e r u l a r p e r m s e l e c t i v i t y or r a t h e r r e f l e c t s an i n c r e a s e d renal s y n t h e s i s of the p h o s p h o l i p i d ,
1434
PAF Urinary Excretion in Lupus Mice
Vol. 48, No. 15, 1991
30 • []
A
E
Proteinuria PAF
20'
6
10'
3~
"E
.c_
LL
o n
0
~ 22 weeks
0 26-42 weeks
FIG.
2
P A F l e v e l s m e a s u r e d by G C - M S in u r i n e f r o m 5 N Z B x N Z W F 1 m i c e w h e n p r o t e i n u r i a was w i t h i n or a b o v e the n o r m a l r a n g e V a l u e s are mean_+S.D. * p < 0 . 0 1 vs v a l u e s at 22 w e e k s of age.
[3H] P A F was i n j e c t e d in t h r e e 26 to 32 w e e k o l d N Z B x N Z W F 1 m i c e w i t h p r o t e i n u r i a e x c e e d i n g 3 m g / d a y (range 5 . 6 - 3 8 . 2 m g / d a y ) . In t h i s g r o u p of m i c e the p e r c e n t a g e of r a d i o a c t i v i t y r e c o v e r e d in the u r i n e 24 h a f t e r [3H] P A F injection was still negligible, reaching 0.072_+0.017 % of t h e radioactivity injected. HPLC analysis of purified urine extracts revealed a s i n g l e p e a k of r a d i o a c t i v i t y c o m i g r a t i n g w i t h [3H] a l k y l - 2 - a c y l - G P C in b o t h g r o u p s (Fig.3) .
1800
[3 H]alkyl-2-acyI-GPC
/I
1200 O9 O_ a
0
.
0
= I
= = ~ ~
2
4
= I
; = = = ~ -- =
6 8 10 12 Retention time (min) FIG.
4 __ = - - ~
14
16
3.
HPLC radiochromatogram of the s e m i p u r i f i e d u r i n e e x t r a c t s f r o m 22 w e e k o l d N Z B x N Z W F 1 m i c e i n j e c t e d w i t h [3H] PAF. r e p r e s e n t s t h e r e t e n t i o n t i m e of s t a n d a r d [3H] PAF.
In t h e p r e s e n t s t u d y we u s e d the c o m b i n e d t e c h n i q u e s of G C - M S to a n a l y z e u r i n a r y PAF e x c r e t i o n in N Z B x N Z W F 1 m i c e w h i c h s p o n t a n e o u s l y
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PAF Urinary Excretion in Lupus Mice
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develop an autoimmune disease characterized by persistent proteinuria, progressive immune complex glomerulonephritis and uremia (27). M a s s spectrometric analysis revealed t h a t P A F is e x c r e t e d in the urine from N Z B x N Z W F 1 m i c e in the early phase of the d i s e a s e in a m o u n t s c o m p a r a b l e to t h o s e f o u n d in u r i n e f r o m n o r m a l mice. A s e c o n d f i n d i n g of the p r e s e n t study was that PAF u r i n a r y e x c r e t i o n in lupus m i c e i n c r e a s e d in a later p h a s e of the d i s e a s e (i.e. weeks 26-42) and that such an increase was a s s o c i a t e d with the d e v e l o p m e n t of heavy proteinuria. Our present results are consistent w i t h t h o s e of S a n c h e z - C r e s p o et al. (19) who p r e v i o u s l y reported that urine from a p a t i e n t with SLE and h e a v y p r o t e i n u r i a c o n t a i n e d a P A F - l i k e m o l e c u l e in a c o n c e n t r a t i o n i0 times h i g h e r t h a n normal. The f i n d i n g that in lupus mice the i n c r e a s e d PAF u r i n a r y e x c r e t i o n is a s s o c i a t e d w i t h the d e v e l o p m e n t of h e a v y p r o t e i n u r i a may have some p a t h o - p h y s i o l o g i c a l implications. It is k n o w n t h a t PAF m a y increase glomerular permeability to p r o t e i n s in v a r i o u s systems. C a m u s s i et al. r e p o r t e d that the i n f u s i o n of PAF into the a b d o m i n a l aorta of rabbits induces the release of platelet and polymorphonuclear cell-derived cationic proteins with concomitant loss of fixed a n i o n i c charges of the g l o m e r u l a r c a p i l l a r y wall and development of r e v e r s i b l e p r o t e i n u r i a (5). R e c e n t l y P e r i c o et al. s h o w e d that the a d d i t i o n of PAF to the p e r f u s a t e in a s y s t e m of i s o l a t e d p e r f u s e d rat k i d n e y p r o d u c e d a dose d e p e n d e n t p r o g r e s s i v e increase in u r i n a r y p r o t e i n e x c r e t i o n (7). The a b s e n c e of b l o o d e l e m e n t s in the p e r f u s i o n system, s u g g e s t e d that PAF m a y i n c r e a s e renal v a s c u l a r p e r m e a b i l i t y even i n d e p e n d e n t l y of the p r e s e n c e of i n f l a m m a t o r y cells. M o r e o v e r it has been r e p o r t e d that PAF r e c e p t o r a n t a g o n i s t s m a y s i g n i f i c a n t l y reduce p r o t e i n u r i a in the e x p e r i m e n t a l models of nephrotoxic nephritis (22) and unilateral glomerulonephritis i n d u c e d by in situ f o r m a t i o n of immune c o m p l e x e s (23) . The o r i g i n of u r i n a r y PAF is still ill d e f i n e d and p a r t i c u l a r l y no i n f o r m a t i o n is a v a i l a b l e so far on its e x t r a r e n a l or r e n a l origin. To c l a r i f y w h e t h e r PAF r e l e a s e d in the s y s t e m i c c i r c u l a t i o n is e x c r e t e d in the urine, we a d d e d e x o g e n o u s PAF to the c i r c u l a t i o n of N Z B x N Z W F 1 m i c e at 22 weeks of age by i n t r a v e n o u s i n j e c t i o n of radiolabeled PAF (I-[I',2'-3H] alkyl) . The r e s u l t s show that 24 h after intravenous i n j e c t i o n of r a d i o l a b e l e d PAF o n l y a n e g l i g i b l e amount of r a d i o a c t i v i t y (less than 0.1%) was r e c o v e r e d in the urine. Moreover HPLC analysis of p u r i f i e d urine extract revealed that radioactivity c o n s i s t e d m a i n l y in [3HI a l k y l - 2 - a c y l - G P C . No p e a k c o r r e s p o n d i n g to [3H] PAF was o b s e r v e d (Fig.3) . The p o s s i b i l i t y that the i n c r e a s e d u r i n a r y e x c r e t i o n of PAF is simply a r e f l e c t i o n of p r o t e i n u r i a is ruled out by our f i n d i n g that the y i e l d of [3H] ether lipids r e c o v e r e d in the urine f r o m N Z B x N Z W F 1 mice w i t h h i g h grade p r o t e i n u r i a was still negligible. C o n s i s t e n t w i t h this result is a recent study s h o w i n g that in i s o l a t e d kidneys from nephrotic rats, a l t e r e d g l o m e r u l a r p e r m e a b i l i t y to p r o t e i n s does not a f f e c t the e x c r e t i o n rate of e x o g e n o u s PAF (32). M o r e o v e r in vivo studies have d o c u m e n t e d that in rats i n t r a v e n o u s l y i n j e c t e d [3H] PAF is r a p i d l y c l e a r e d from the b l o o d (35). The d i s a p p e a r a n c e of [3H] PAF has to be a t t r i b u t e d to a r a p i d d i s t r i b u t i o n of the l i p i d w i t h i n a v a r i e t y of t i s s u e s m a i n l y lung, liver, s p l e e n a n d k i d n e y rather t h a n to a r a p i d m e t a b o l i s m of P A F to l y s o P A F by the a c e t y l h y d r o l a s e a c t i v i t y (36).
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Vol. 48, No. 15, 1991
In s u m m a r y we h a v e d o c u m e n t e d that the a m o u n t of PAF e x c r e t e d in the u r i n e f r o m lupus m i c e d u r i n g the e a r l y p h a s e of the d i s e a s e was c o m p a r a b l e to t h a t f o u n d in u r i n e s f r o m n o r m a l mice. P A F u r i n a r y excretion increased when proteinuria increased and the two variables s h o w e d an h i g h d e g r e e of c o r r e l a t i o n . Findings that e x o g e n o u s PAF i n j e c t e d in N Z B x N Z W F 1 m i c e was not e x c r e t e d in the u r i n e e v e n w h e n a d m i n i s t e r e d to a n i m a l s w i t h h i g h g r a d e p r o t e i n u r i a , make unlikely the extrarenal o r i g i n of u r i n a r y P A F a n d r a t h e r s u g g e s t that the levels of PAF d e t e c t a b l e in the u r i n e of lupus m i c e r e f l e c t a r e n a l o r i g i n or d e r i v e f r o m the u r i n a r y t r a c t . F u r t h e r s t u d i e s are n e e d e d to p r o v i d e a d i r e c t e v i d e n c e of the r e n a l s o u r c e of u r i n a r y PAF. Aknowledsements This study was partially supported by grants from Italian National Research Council (CNR, contract n° 88.01878.04) . The a u t h o r s t h a n k Dr. M a r c o G a v i n e l l i a n d E l e n a R i c c a r d i for t h e i r valuable cooperation. References I. 2.
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INCREASED
URINARY
E X C R E T I O N OF P L A T E L E T A C T I V A T I N G W I T H LUPUS N E P H R I T I S
Pergamon Press
FACTOR
IN
MICE
D a n i e l a M a c c o n i I), M a r i n a Noris I), E m i l i o B e n f e n a t i I) R o b e r t o Q u a g l i a I), G i a n l u c a P a g l i a r i n o I), and G i u s e p p e R e m u z z i I) 2)
1 M a r i o Negri Institute for P h a r m a c o l o g i c a l Research, Via G a v a z z e n i ii, and 2 D i v i s i o n of Nephrology, O s p e d a l i R i u n i t i di Bergamo, 24100 Bergamo, Italy. (Received in final form February I, 1991) Summary P l a t e l e t a c t i v a t i n g factor (PAF) is p r e s e n t in u r i n e from humans and e x p e r i m e n t a l animals in normal conditions. V e r y l i t t l e is k n o w n about c h a n g e s in PAF u r i n a r y e x c r e t i o n under pathologic conditions a n d no d a t a are a v a i l a b l e about the origin of PAF in the urine. In the p r e s e n t study we e x p l o r e d the p o s s i b i l i t y that i m m u n o l o g i c renal d i s e a s e is a s s o c i a t e d w i t h an i n c r e a s e in PAF u r i n a r y e x c r e t i o n u s i n g gas c h r o m a t o g r a p h y - m a s s spectrometry technique. To c l a r i f y the renal or e x t r a r e n a l o r i g i n of u r i n a r y PAF we evaluated whether exogenously administered PAF (l[l',2'-3H]alkyl) is f i l t e r e d t h r o u g h the g l o m e r u l u s and e x c r e t e d in the urine. The r e s u l t s s h o w that: I) u r i n e f r o m m i c e w i t h lupus n e p h r i t i s in the e a r l y p h a s e of the disease contained amounts of PAF c o m p a r a b l e to t h o s e e x c r e t e d in n o r m a l m o u s e urine, 2) PAF levels i n c r e a s e d when a n i m a l s started to d e v e l o p high grade proteinuria, 3) after i n t r a v e n o u s injection of [3H] PAF in n e p h r i t i c mice, a n e g l i g i b l e amount of [3H] ether lipid, c o r r e s p o n d i n g to [3H]l-alkyl -2-acyl-3-phosphocholine (alkyl-2-acyl-GPC), was r e c o v e r e d from the 24 h urine extract. Platelet activating factor (PAF) or l - O - a l k y l - 2 - a c e t y l - s n glycero-3-phosphocholine is a potent lipid m e d i a t o r of i n f l a m m a t i o n with a wide v a r i e t y of b i o l o g i c a l a c t i v i t i e s i n c l u d i n g p l a t e l e t and neutrophil activation and aggregation (1,2) a n d i n c r e a s e d v a s c u l a r (3,4) a n d g l o m e r u l a r (5-7) p e r m e a b i l i t y . PAF f o r m a t i o n has b e e n d e s c r i b e d in s t i m u l a t e d i n f l a m m a t o r y cells (8,9) as well as c u l t u r e d e n d o t h e l i a l cells (i0). R e l e a s e of PAF has b e e n d o c u m e n t e d in the venous effluent of i s o l a t e d perfused rat k i d n e y upon calcium ionophore (ii) or a n t i g e n challenge (12). Isolated glomeruli (13-15), suspensions of r e n o m e d u l l a r y cells (13), a n d cultured m e s a n g i a l cells (14,16) a l s o g e n e r a t e PAF. M o r e o v e r low a m o u n t s of P A F are p r e s e n t in b l o o d from humans (17,18) and experimental animals (18) in normal conditions. Send correspondence for P h a r m a c o l o g i c a l
to: Dr. D a n i e l a Macconi, M a r i o N e g r i I n s t i t u t e Research, Via G a v a z z e n i ii, 24100 Bergamo, Italy 0024-3205/91 $3.00 +.00 Copyright (c) Pergamon Press pie
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Sanchez-Crespo et al. have p r o v i d e d e v i d e n c e that n o r m a l human u r i n e c o n t a i n s n a n o g r a m a m o u n t s of a p h o s p h o l i p i d compound with platelet-activating and h y p o t e n s i v e a c t i v i t y similar to PAF (19). PAF-related bioactivity has a l s o b e e n d e t e c t e d in s a m p l e s of first v o i d e d u r i n e of n e w b o r n s (20). In a d d i t i o n the p r e s e n c e of n a n o g r a m amounts of PAF in normal m o u s e urine, as d e t e r m i n e d by gas chromatography-mass spectrometry (GC-MS) technique, has been recently reported (21). O t h e r studies have suggested t h a t PAF m e d i a t e s renal d a m a g e (22,23). I n c r e a s e d g l o m e r u l a r s y n t h e s i s (24) and release of P A F in t h e circulation (23,25,26) have been d o c u m e n t e d in i m m u n e - m e d i a t e d g l o m e r u l o n e p h r i t i s . If PAF g e n e r a t i o n is i n c r e a s e d in c o n d i t i o n of g l o m e r u l a r i n j u r y and i n f i l t r a t i o n of inflammatory cells, one may w o n d e r w h e t h e r PAF u r i n a r y e x c r e t i o n reflects such changes. This p o s s i b i l i t y is in k e e p i n g w i t h the data of S a n c h e z - C r e s p o et al. s h o w i n g a i0 fold i n c r e a s e in P A F - r e l a t e d b i o a c t i v i t y in urine from p a t i e n t s with s y s t e m i c lupus e r y t h e m a t o s u s (SLE) a n d h e a v y p r o t e i n u r i a (19). H o w e v e r in the a b o v e m e n t i o n e d p a p e r the m a t e r i a l found in the u r i n e and d e f i n e d as PAF has only b e e n c h a r a c t e r i z e d by mean of its b i o l o g i c a l a c t i v i t y on platelets. M o r e o v e r no s t u d i e s have b e e n a d d r e s s e d to c l a r i f y w h e t h e r u r i n a r y PAF was of renal or e x t r a r e n a l origin. The aims of the p r e s e n t s t u d y were: i) to c l a r i f y w h e t h e r immunologic renal d i s e a s e is a s s o c i a t e d w i t h an i n c r e a s e in PAF u r i n a r y excretion, 2) to assess the e x t r a r e n a l or renal o r i g i n of PAF e x c r e t e d in the urine. Materials
and Methods
S t a n d a r d PAF a n d lyso PAF w i t h t h e l - O - h e x a d e c y l chain were obtained from Bachem Feinchemikalien AG (Bubendorf, Switzerland). [3H]PAF (l-[l',2'-3H]hexadecyl-2-acetyl-sn-gly cer°3-phosphocholine, 56.7 Ci/mmol) was from New England Nuclear (Boston, MA). Thin layer c h r o m a t o g r a p h y (TLC) plates, p r e c o a t e d with silica gel 60 w i t h c o n c e n t r a t i n g zone, layer t h i c k n e s s 0.25 mm, and 50% h y d r o f l u o r i c acid (HF) w e r e o b t a i n e d from Merck (Darmstadt, F.R.G.) . P e n t a f l u o r o b e n z o y l chloride (PFBCI) was p u r c h a s e d from J a n s s e n C h i m i c a (Beerse, Belgium). A c e t i c a n h y d r i d e - d 6 was o b t a i n e d from Sigma Chemical Co. (St. Louis, MO) . S i l i c i c a c i d was f r o m Bio-Rad Laboratories (Richmond, CA). High performance liquid chromatography (HPLC) and a n a l y t i c a l grade s o l v e n t s were p u r c h a s e d f r o m Merck. Studies in mice with luPUS nephritis. Female N e w Z e a l a n d Black x New Zealand White (NZBxNZW) F 1 mice spontaneously develop an autoimmune d i s e a s e that c l o s e l y r e s e m b l e s SLE in m a n (27). The d i s e a s e m a n i f e s t s w i t h c i r c u l a t i n g a n t i b o d i e s to n u c l e i c a c i d a n d endogenous antigens, immune complex formation, and persistent proteinuria. Five female NZBxNZW F1 m i c e (Harlan O L A C 1976 LTD Bicester, England), 8-9 weeks of age at the start of e x p e r i m e n t , were used. Monthly, 24 h urine samples were c o l l e c t e d in m e t a b o l i c c a g e s a n d p r o t e i n content, expressed in mg/day, was m e a s u r e d by Coomassie blue G dye-binding assay for p r o t e i n s (28) . U r i n a r y protein content measured in N Z B x N Z W F 1 m i c e at 14 w e e k s of age, b e f o r e the d i s e a s e was m a n i f e s t (27), was w i t h i n 3 mg/day. U r i n a r y p r o t e i n levels e x c e e d i n g 3 m g / d a y were c o n s i d e r e d abnormal. S t a r t i n g from 26 weeks of age, mice randomly developed high grade p r o t e i n u r i a . PAF u r i n a r y e x c r e t i o n was m e a s u r e d by GC-MS in N Z B x N Z W
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F 1 m i c e at 22 weeks of age, w h e n p r o t e i n e x c r e t i o n was w i t h i n the p h y s i o l o g i c range for normal urine, a n d again as soon as the animals developed proteinuria above 3 mg/day. S i n c e the PAF g e n e r a t e d by m e s a n g i a l cells is e x c l u s i v e l y of h e x a d e c y l s p e c i e s (16), w h i c h is also predominant (over 90% of total s y n t h e s i z e d PAF) in s t i m u l a t e d neutrophils (29), PAF C16:0 was analyzed. E x t r a c t i o n a n d P u r i f i c a t i o n of urine. I n t e r n a l s t a n d a r d P A F - d 3 was synthesized and purified as previously described (21). T w e n t y f o u r h urine samples f r o m N Z B x N Z W F 1 m i c e w e r e a d d e d to 5 ng P A F - d 3 a n d e x t r a c t e d a c c o r d i n g to P i n c k a r d et al. (30). Briefly, 1.5 v o l u m e s of m e t h a n o l was a d d e d to urine and, a f t e r g e n t l e m i x i n g for 30 min, the samples were centrifuged. C h l o r o f o r m and w a t e r were then added to obtain a solvent mixture containing chloroform: methanol:water (1:1:0.9, v/v/v). The lower p h a s e was r e m o v e d and t a k e n to d r y n e s s u n d e r a n i t r o g e n stream. T h e r e a f t e r s a m p l e s were purified according to P i n c k a r d et al. (30), w i t h the f o l l o w i n g m o d i f i c a t i o n : the extract was r e s u s p e n d e d in c h l o r o f o r m : m e t h a n o l 9:1 and l o a d e d on a c o l u m n p a c k e d w i t h 0.5 g of s i l i c i c a c i d (100-200 mesh) activated at i00 °C o v e r n i g h t . P A F was e l u t e d using a s e q u e n t i a l s o l v e n t s y s t e m of c h l o r o f o r m (15 ml), a c e t o n e (15 ml), acetone:methanol (i:I, v/v) (15 ml), c h l o r o f o r m : m e t h a n o l (1:4, v/v) (30 ml) . The f o u r t h f r a c t i o n c o n t a i n i n g PAF was t a k e n to d r y n e s s u n d e r a n i t r o g e n stream. R e c o v e r y of e x t r a c t i o n a n d p u r i f i c a t i o n , a s s e s s e d by a d d i t i o n of [3H] PAF to the s a m p l e b e f o r e p r o c e s s i n g , r a n g e d b e t w e e n 80 and 90%. Derivatization of PAF. A f t e r purification, samples were h y d r o l y z e d to l - O - a l k y l - 2 - a c e t y l - g l y c e r o l by the a d d i t i o n of 0.2 ml of 50% HF at 25 °C. A f t e r 4 h, 0.5 ml of h e x a n e was a d d e d a n d the o r g a n i c p h a s e w a s r e m o v e d a n d t a k e n to d r y n e s s u n d e r a n i t r o g e n s t r e a m (31). R e c o v e r y of the reaction, a s s e s s e d by h y d r o l y s i s of [3H] PAF s t a n d a r d a n d s u b s e q u e n t TLC a n a l y s i s of b y p r o d u c t s (32), was a b o u t 60%. F o r d e r i v a t i z a t i o n (33) 50 ~i of d i s t i l l e d P F B C I was added; the system was heated at i00 °C for 40 min. A f t e r the r e a c t i o n , the e x c e s s r e a g e n t was t a k e n to d r y n e s s u n d e r a n i t r o g e n stream. Recovery of d e r i v a t i z a t i o n was generally above 90% as r e p o r t e d b y M a g n i et al. (34) . The r e s i d u e was d i s s o l v e d in h e x a n e (50 ~I) and 2 ~i a l i q u o t s w e r e u s e d for MS analysis. Mass spectrometry, GC-MS analyses, in the n e g a t i v e ion chemical ionization (NICI) mode, were carried out on a V G 7 0 - 2 5 0 m a s s spectrometer (VG A n a l y t i c a l , Manchester, U . K . ) . A D a n i 6500 gas chromatograph (Dani, M o n z a , Italy) was coupled to the mass s p e c t r o m e t e r . A fused silica c a p i l l a r y column CPSil 5 CB, 25 m, 0.32 m m I.D. (Chrompack, M i d d e l b u r g , The N e t h e r l a n d s ) w a s used, w i t h a p r o g r a m m e d t e m p e r a t u r e v a p o r i z e r (PTV) injector. The c h r o m a t o g r a p h i c p r o g r a m was: oven temperature: 140 °C, initial, for 1 min; then from 140 to 280 °C p r o g r a m m e d temperature, 18 °C/min. For chemical ionization MS a m m o n i a w a s u s e d as t h e r e a g e n t gas. S e l e c t e d ion recording (SIR) a n a l y s e s w e r e d o n e m o n i t o r i n g t h e ions at m/z 552 a n d 555 for P A F a n d P A F - d 3 r e s p e c t i v e l y . A c a l i b r a t i o n curve was c o n s t r u c t e d by u s i n g the c a l c u l a t e d p e a k - a r e a r a t i o of PAF to the i n t e r n a l s t a n d a r d P A F - d 3 (400 pg injected). The p e a k area ratio was linearly correlated (r=0.999) w i t h the PAF c o n c e n t r a t i o n in the range of 2-800 pg. Intravenous NZBxNZW F 1 mice
[3~1 P A F were u s e d
administration for the study.
to lupus mice. Seven G r o u p 1 (n=4) r e c e i v e d
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a b o u t 1 ~Ci of [3H] P A F at 22 w e e k s of age; g r o u p 2 (n=3) r e c e i v e d the same d o s e of [3H] PAF at 26-32 w e e k s of age, as s o o n as the a n i m a l s d e v e l o p e d p r o t e i n u r i a e x c e e d i n g 3 m g / d a y . The r a d i o l a b e l e d compound, dissolved in 0.5% e t h a n o l in s a l i n e c o n t a i n i n g 0.25% b o v i n e s e r u m albumin, was i n j e c t e d as a b o l u s into the tail vein. A f t e r the injection, the p l a s t i c syringe a n d n e e d l e w e r e well r i n s e d w i t h 500 ~i of the i n j e c t i o n s o l u t i o n p l u s 500 ~I of e t h a n o l to c o l l e c t u n i n j e c t e d r a d i o l a b e l e d lipid. All a n i m a l s w e r e h o u s e d in m e t a b o l i c cages for c o l l e c t i o n of 24 h u r i n e samples. The urine was e x t r a c t e d as d e s c r i b e d above. The d r i e d r e s i d u e was r e s u s p e n d e d in m e t h a n o l a n d an a l i q u o t u s e d for r a d i o a c t i v i t y determination by liquid scintillation counting. The yield of [3H] e t h e r lipid r e c o v e r e d f r o m the u r i n e e x t r a c t was e x p r e s s e d as p e r c e n t a g e of the total administered dose. S i n c e PAF can a d s o r b to s u r f a c e s , the a d m i n i s t e r e d dose was the d i f f e r e n c e b e t w e e n the t r i t i u m c o n t e n t in the injection solution and that recovered from the syringe and needle. To a s s e s s w h e t h e r the t r i t i a t e d l i p i d in the u r i n e was a c t u a l l y [3H] PAF or its m e t a b o l i t e s , u r i n e e x t r a c t s f r o m m i c e at 22 w e e k s of age or f r o m m i c e w i t h h i g h g r a d e p r o t e i n u r i a w e r e p o o l e d a n d p u r i f i e d b y s i l i c i c a c i d column. P u r i f i e d s a m p l e s w e r e t h e n a n a l y z e d by H P L C by i s o c r a t i c e l u t i o n as p r e v i o u s l y d e s c r i b e d (32). S t a t i s t i c a l a n a l y s i s . R e s u l t s are m e a n ± S.D. Data were a n a l y z e d by Student's t test. P values <0.05 were considered to b e statistically significant. Results
Gas c h r o m a t o a r a D h v - m a s s s p e c t r o m e t r i c a n a l v s i s of u r i n a r v PAF in NZBxNZW F l mice. Fig. 1 s h o w s r e p r e s e n t a t i v e S I R t r a c e s of PAF e x c r e t e d in a 24 h urine sample from N Z B x N Z W F 1 m i c e at 22 weeks of age and of the c o r r e s p o n d i n g d e u t e r a t e d i n t e r n a l standard. The d e t e c t e d M- ions c o r r e s p o n d e d to the m o l e c u l a r w e i g h t of pentafluorobenzoyl d e r i v a t i v e s of n a t i v e P A F (m/z 552) a n d P A F - d 3 (m/z 555). The c h r o m a t o g r a p h i c r e t e n t i o n time of PAF e x c r e t e d in 24 h u r i n e (9.32-9.38 min) was c h e c k e d a n d c o n f i r m e d by the p r e s e n c e of the i n t e r n a l s t a n d a r d P A F - d 3 w h o s e r e t e n t i o n t i m e was one s e c o n d s h o r t e r t h a n t h a t of n a t i v e PAF due to a p r i m a r y i s o t o p e e f f e c t (Fig.l). The S I R t r a c e s of the d e r i v a t i z e d PAF p r e s e n t e d two p e a k s (peaks 1 a n d 2 on the m/z 552 trace, a n d IA a n d 2A on the m/z 555 trace, fig.l) that c o r r e s p o n d to the two p o s i t i o n a l i s o m e r s b e a r i n g the acetyl moiety in t h e s n - 2 or -3 p o s i t i o n resulting from intramolecular shift of the a c e t y l group. In our c h r o m a t o g r a p h i c c o n d i t i o n s , the t w o c h e m i c a l i s o m e r s w e r e s e p a r a t e d . M o r e o v e r the r e l a t i v e a b u n d a n c e of the t w o p o s i t i o n a l i s o m e r s was c o n s t a n t a n d equal for n a t u r a l a n d d e u t e r a t e d PAF. For the q u a n t i t a t i v e a n a l y s i s both PAF peaks were considered. Altogether these characteristics allowed a correct identification of e n d o g e n o u s PAF. In o r d e r to e x c l u d e the p o s s i b l e i n t e r f e r e n c e f r o m the i s o t o p i c c o n t r i b u t i o n of o t h e r ions at m/z 552 a n d 555, we c o n s t a n t l y m o n i t o r e d the ions at m/z 551 a n d 554. In fig.l the t r a c e at m/z 555 p r e s e n t s a d o u b l e t (peaks 3 a n d 4) p r e c e e d i n g the P A F - d 3 peaks. This doublet, at 9.26 min, was p r e s e n t o n l y in b i o l o g i c a l s a m p l e s a n d was d u e t o an i s o t o p i c c o n t r i b u t i o n of a c o m p o u n d w i t h a s i g n a l at m/z 554. The distribution of t h e t w o p e a k s in t h e c h r o m a t o g r a p h i c doublet s u g g e s t s a p h o s p h o l i p i d n a t u r e of such a c o m p o u n d . However, it is u n l i k e l y that acyl P A F or PAF r e l a t e d c o m p o u n d s give a s i g n a l at m/z 554. In o u r c h r o m a t o g r a p h i c conditions the PAF-d 3 and the unknown
Vol. 48, No. 15, 1991
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Norm: 132
I00
m/z 552
6O d.
40
>, 2O r-,
o r..
.r4
100
• ,-t
80
o~ c~
60
31 2A
484
I
';
9:15
Norm:
9 : 30
FIG.
9 : 45
i.
10 : O0
10 : 15
10 : 30
Time (min)
S e l e c t e d ion c h r o m a t o g r a m s m o n i t o r i n g the M- ion of t h e pentafluorobenzoyl d e r i v a t i v e s of PAF e x c r e t e d in a 24 h u r i n e sample from N Z B x N Z W F 1 m i c e at 22 weeks of age (m/z 552; n o r m a l i z e d r e s p e c t p e a k 2) and of i n t e r n a l s t a n d a r d P A F - d 3 (m/z 555; n o r m a l i z e d respect p e a k 4).
d o u b l e t w e r e s e p a r a t e d . PAF levels m e a s u r e d by G C - M S in the u r i n e f r o m 5 N Z B x N Z W F 1 m i c e in the e a r l y p h a s e of the d i s e a s e (22 weeks of age) w e r e 1.56±0.54 ng/day. These values were c o m p a r a b l e to those previotr~ly r e p o r t e d (21) for n o r m a l CD-I (ICR) BR m i c e of the same age. At this stage of the d i s e a s e u r i n a r y p r o t e i n e x c r e t i o n in lupus m i c e was less than 3 m g / d a y (range 0 . 6 6 - 1 . 5 1 mg/day), v a l u e s that were a l s o not s i g n i f i c a n t l y d i f f e r e n t from t h o s e r e c o r d e d in h e a l t h y CD-I (ICR) BR m i c e of the same age (1.14±0.37 mg/day, n=5; r a n g e 0 . 7 8 - 1 . 7 2 mg/day) .To i n v e s t i g a t e changes in u r i n a r y PAF later in the c o u r s e of the disease, studies were p e r f o r m e d as soon as the animals d e v e l o p e d p r o t e i n u r i a e x c e e d i n g 3 m g / d a y (at 26 to 42 weeks of age). As s h o w n in Fig.2 a s i g n i f i c a n t i n c r e a s e in PAF u r i n a r y e x c r e t i o n o c c u r r e d in this p h a s e of the d i s e a s e as c o m p a r e d w i t h v a l u e s f o u n d at 22 weeks of age (5.69±1.83 n g / d a y vs 1.56±0.54 ng/day, p<0.01) . Intravenous r3H] PAF a i v e n to n e p h r i t i c m i c e is not e x c r e t e d ~ In o r d e r to a s s e s s w h e t h e r circulating P A F m a y be f i l t e r e d t h r o u g h the g l o m e r u l u s a n d e x c r e t e d in the urine, [3H] P A F was i n t r a v e n o u s l y i n j e c t e d in four 22 w e e k old N Z B x N Z W F 1 m i c e w i t h proteinuria r a n g i n g f r o m 0.95 to 1.56 m g / d a y . T w e n t y f o u r h a f t e r intravenous i n j e c t i o n of [3H] PAF, the y i e l d of [ 3 H ] e t h e r lipid r e c o v e r e d into the o r g a n i c p h a s e a f t e r u r i n e e x t r a c t i o n r e p r e s e n t e d only 0.084±0.031 % of the t o t a l a d m i n i s t e r e d dose. To e v a l u a t e w h e t h e r the i n c r e a s e d u r i n a r y e x c r e t i o n of PAF in lupus m i c e w i t h h e a v y p r o t e i n u r i a d e p e n d s on the a l t e r e d g l o m e r u l a r p e r m s e l e c t i v i t y or r a t h e r r e f l e c t s an i n c r e a s e d renal s y n t h e s i s of the p h o s p h o l i p i d ,
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PAF Urinary Excretion in Lupus Mice
Vol. 48, No. 15, 1991
30 • []
A
E
Proteinuria PAF
20'
6
10'
3~
"E
.c_
LL
o n
0
~ 22 weeks
0 26-42 weeks
FIG.
2
P A F l e v e l s m e a s u r e d by G C - M S in u r i n e f r o m 5 N Z B x N Z W F 1 m i c e w h e n p r o t e i n u r i a was w i t h i n or a b o v e the n o r m a l r a n g e V a l u e s are mean_+S.D. * p < 0 . 0 1 vs v a l u e s at 22 w e e k s of age.
[3H] P A F was i n j e c t e d in t h r e e 26 to 32 w e e k o l d N Z B x N Z W F 1 m i c e w i t h p r o t e i n u r i a e x c e e d i n g 3 m g / d a y (range 5 . 6 - 3 8 . 2 m g / d a y ) . In t h i s g r o u p of m i c e the p e r c e n t a g e of r a d i o a c t i v i t y r e c o v e r e d in the u r i n e 24 h a f t e r [3H] P A F injection was still negligible, reaching 0.072_+0.017 % of t h e radioactivity injected. HPLC analysis of purified urine extracts revealed a s i n g l e p e a k of r a d i o a c t i v i t y c o m i g r a t i n g w i t h [3H] a l k y l - 2 - a c y l - G P C in b o t h g r o u p s (Fig.3) .
1800
[3 H]alkyl-2-acyI-GPC
/I
1200 O9 O_ a
0
.
0
= I
= = ~ ~
2
4
= I
; = = = ~ -- =
6 8 10 12 Retention time (min) FIG.
4 __ = - - ~
14
16
3.
HPLC radiochromatogram of the s e m i p u r i f i e d u r i n e e x t r a c t s f r o m 22 w e e k o l d N Z B x N Z W F 1 m i c e i n j e c t e d w i t h [3H] PAF. r e p r e s e n t s t h e r e t e n t i o n t i m e of s t a n d a r d [3H] PAF.
In t h e p r e s e n t s t u d y we u s e d the c o m b i n e d t e c h n i q u e s of G C - M S to a n a l y z e u r i n a r y PAF e x c r e t i o n in N Z B x N Z W F 1 m i c e w h i c h s p o n t a n e o u s l y
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develop an autoimmune disease characterized by persistent proteinuria, progressive immune complex glomerulonephritis and uremia (27). M a s s spectrometric analysis revealed t h a t P A F is e x c r e t e d in the urine from N Z B x N Z W F 1 m i c e in the early phase of the d i s e a s e in a m o u n t s c o m p a r a b l e to t h o s e f o u n d in u r i n e f r o m n o r m a l mice. A s e c o n d f i n d i n g of the p r e s e n t study was that PAF u r i n a r y e x c r e t i o n in lupus m i c e i n c r e a s e d in a later p h a s e of the d i s e a s e (i.e. weeks 26-42) and that such an increase was a s s o c i a t e d with the d e v e l o p m e n t of heavy proteinuria. Our present results are consistent w i t h t h o s e of S a n c h e z - C r e s p o et al. (19) who p r e v i o u s l y reported that urine from a p a t i e n t with SLE and h e a v y p r o t e i n u r i a c o n t a i n e d a P A F - l i k e m o l e c u l e in a c o n c e n t r a t i o n i0 times h i g h e r t h a n normal. The f i n d i n g that in lupus mice the i n c r e a s e d PAF u r i n a r y e x c r e t i o n is a s s o c i a t e d w i t h the d e v e l o p m e n t of h e a v y p r o t e i n u r i a may have some p a t h o - p h y s i o l o g i c a l implications. It is k n o w n t h a t PAF m a y increase glomerular permeability to p r o t e i n s in v a r i o u s systems. C a m u s s i et al. r e p o r t e d that the i n f u s i o n of PAF into the a b d o m i n a l aorta of rabbits induces the release of platelet and polymorphonuclear cell-derived cationic proteins with concomitant loss of fixed a n i o n i c charges of the g l o m e r u l a r c a p i l l a r y wall and development of r e v e r s i b l e p r o t e i n u r i a (5). R e c e n t l y P e r i c o et al. s h o w e d that the a d d i t i o n of PAF to the p e r f u s a t e in a s y s t e m of i s o l a t e d p e r f u s e d rat k i d n e y p r o d u c e d a dose d e p e n d e n t p r o g r e s s i v e increase in u r i n a r y p r o t e i n e x c r e t i o n (7). The a b s e n c e of b l o o d e l e m e n t s in the p e r f u s i o n system, s u g g e s t e d that PAF m a y i n c r e a s e renal v a s c u l a r p e r m e a b i l i t y even i n d e p e n d e n t l y of the p r e s e n c e of i n f l a m m a t o r y cells. M o r e o v e r it has been r e p o r t e d that PAF r e c e p t o r a n t a g o n i s t s m a y s i g n i f i c a n t l y reduce p r o t e i n u r i a in the e x p e r i m e n t a l models of nephrotoxic nephritis (22) and unilateral glomerulonephritis i n d u c e d by in situ f o r m a t i o n of immune c o m p l e x e s (23) . The o r i g i n of u r i n a r y PAF is still ill d e f i n e d and p a r t i c u l a r l y no i n f o r m a t i o n is a v a i l a b l e so far on its e x t r a r e n a l or r e n a l origin. To c l a r i f y w h e t h e r PAF r e l e a s e d in the s y s t e m i c c i r c u l a t i o n is e x c r e t e d in the urine, we a d d e d e x o g e n o u s PAF to the c i r c u l a t i o n of N Z B x N Z W F 1 m i c e at 22 weeks of age by i n t r a v e n o u s i n j e c t i o n of radiolabeled PAF (I-[I',2'-3H] alkyl) . The r e s u l t s show that 24 h after intravenous i n j e c t i o n of r a d i o l a b e l e d PAF o n l y a n e g l i g i b l e amount of r a d i o a c t i v i t y (less than 0.1%) was r e c o v e r e d in the urine. Moreover HPLC analysis of p u r i f i e d urine extract revealed that radioactivity c o n s i s t e d m a i n l y in [3HI a l k y l - 2 - a c y l - G P C . No p e a k c o r r e s p o n d i n g to [3H] PAF was o b s e r v e d (Fig.3) . The p o s s i b i l i t y that the i n c r e a s e d u r i n a r y e x c r e t i o n of PAF is simply a r e f l e c t i o n of p r o t e i n u r i a is ruled out by our f i n d i n g that the y i e l d of [3H] ether lipids r e c o v e r e d in the urine f r o m N Z B x N Z W F 1 mice w i t h h i g h grade p r o t e i n u r i a was still negligible. C o n s i s t e n t w i t h this result is a recent study s h o w i n g that in i s o l a t e d kidneys from nephrotic rats, a l t e r e d g l o m e r u l a r p e r m e a b i l i t y to p r o t e i n s does not a f f e c t the e x c r e t i o n rate of e x o g e n o u s PAF (32). M o r e o v e r in vivo studies have d o c u m e n t e d that in rats i n t r a v e n o u s l y i n j e c t e d [3H] PAF is r a p i d l y c l e a r e d from the b l o o d (35). The d i s a p p e a r a n c e of [3H] PAF has to be a t t r i b u t e d to a r a p i d d i s t r i b u t i o n of the l i p i d w i t h i n a v a r i e t y of t i s s u e s m a i n l y lung, liver, s p l e e n a n d k i d n e y rather t h a n to a r a p i d m e t a b o l i s m of P A F to l y s o P A F by the a c e t y l h y d r o l a s e a c t i v i t y (36).
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In s u m m a r y we h a v e d o c u m e n t e d that the a m o u n t of PAF e x c r e t e d in the u r i n e f r o m lupus m i c e d u r i n g the e a r l y p h a s e of the d i s e a s e was c o m p a r a b l e to t h a t f o u n d in u r i n e s f r o m n o r m a l mice. P A F u r i n a r y excretion increased when proteinuria increased and the two variables s h o w e d an h i g h d e g r e e of c o r r e l a t i o n . Findings that e x o g e n o u s PAF i n j e c t e d in N Z B x N Z W F 1 m i c e was not e x c r e t e d in the u r i n e e v e n w h e n a d m i n i s t e r e d to a n i m a l s w i t h h i g h g r a d e p r o t e i n u r i a , make unlikely the extrarenal o r i g i n of u r i n a r y P A F a n d r a t h e r s u g g e s t that the levels of PAF d e t e c t a b l e in the u r i n e of lupus m i c e r e f l e c t a r e n a l o r i g i n or d e r i v e f r o m the u r i n a r y t r a c t . F u r t h e r s t u d i e s are n e e d e d to p r o v i d e a d i r e c t e v i d e n c e of the r e n a l s o u r c e of u r i n a r y PAF. Aknowledsements This study was partially supported by grants from Italian National Research Council (CNR, contract n° 88.01878.04) . The a u t h o r s t h a n k Dr. M a r c o G a v i n e l l i a n d E l e n a R i c c a r d i for t h e i r valuable cooperation. References I. 2.
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