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Altered PGE2 production by glomeruli and papilla of rats with hereditary diabetes insipidus
PROSTAGLANDINS
ALTERED PGE 2 PRODUCTION BY GLOMERULI AND PAPILLA OF RATS WITH HEREDITARY DIABETES INSIPIDUS Lise BANKIR, Marie-Marcelle TRINH TRANG TAN Marie-Paule NIVEZ, Jos4e SRAER and Raymond ARDAILLOU I.N.S.E.R.M. Unit4 64, H6pital Tenon 75790 Paris 20 I.N.S.E.R.M. Unit~ 90, H6pital Necker 75730 Paris 15 France ABSTRACT PGE 2 synthesis rate was studied in vitro in isolated glomeruli and in papillary homogenates prepared from kidneys of Brattleboro rats with hereditary diabetes insipidus (DI) (no ADH) and of BrattleborQ heterozygous control rats. Incubations were carried out in isotonic buffer at 37°C in the presence or absence of arachidonic acid for 15, 30, 60 and 90 min. PGE 2 production was measured in the supernatant by specific radioimmunoassay. Results were compared by analysis of variance. PGE 2 production was significantly decreased in the papilla (p < 0.01) and increased in the glomeruli (p < 0.01) of DI rats compared to controls. Stimulation by arachidonic acid was similar in both groups. Chronic ADH deficiency thus modifies the a b i l i t y of the kidney to produce PGE 2 in vitro. The opposite effects observed in glomeruli and papilla suggest a different hormonal control of PGE 2 synthesis in both tissues. INTRODUCTION Prostaglandin (PG) biosynthesis within the renal medulla of the rat is well documented (I, 2). The specifi'c cellular sites of synthesis include m e d u l l a r y interstitial cells (3, 4) and collecting duct cells (4, 5). In addition, recent reports have shown that glomeruli also synthesize PGs (6, 7, 8) and that PGs influence b o t h glomerular filtration rate (9, I0) and glomeruli ultrastructure (Ii). The control of PG p r o d u c t i o n by these different renal structures has not been yet completely clarified. Interactions between PGs and antidiuretic hormone (ADH) Occur both in the toad b l a d d e r and in the m a m m a l i a n kidney (12). Recently, it has been shown that renal excretion of PGE 2 (13, 14) and PGF2e (14) was markedly reduced in Brattleboro rats with hereditary diabetes insipidus (DI rats) (15).
AUGUST 1980 VOL. 20 NO. 2
349
PROSTAGLANDINS
L a c k o f A D H in t h e s e r a t s a c c o u n t s f o r t h i s r e d u c t i o n since administration of this hormone restored renal e x c r e t i o n o f t h e t w o PGs s t u d i e d (13, 14). S i n c e it is admitted that urinary PGs reflect renal PO synthesis (i) it can b e c o n c l u d e d t h a t A D H s t i m u l a t e s r e n a l p r o d u c t i o n of PGE 2 a n d P G F 2 in vivo. B u t n e i t h e r the s i t e of e f f e c t of A D H in the ~ i d n e y n o r t h e s t e p of P G s y n t h e s i s at w h i c h t h i s h o r m o n e a c t s can be d e d u c e d f r o m t h e s e in vivo experiments. The present study was designed to c o m p a r e the in v i t r o r a t e s of PGE 2 s y n t h e s i s b y g l o m e r u l i a n d b y the p a p i l l a f r o m r a t s w i t h o r w i t h o u t A D H d e f i c i e n c y .
MATERIAL AND METHODS Rats M a l e a n d f e m a l e 3-6 m o n t h s o l d B r a t t l e b o r o rats, b r e d in H o s p i t a l N e c k e r (INSERM, U 90, Paris) w e r e u s e d in t h i s study. E a c h e x p e r i m e n t i n c l u d e d one h o m o z y g o u s rat w i t h d i a b e t e s i n s i p i d u s a n d o n e c o n t r o l h e t e r o z y g o u s rat (HZ) of t h e s a m e strain. H Z r a t s h a v e s u b n o r m a l A D H p r o d u c t i o n a n d p l a s m a l e v e l s (16). A g e a n d sex w e r e m a t c h e d and~ in 5 o u t of 7 e x p e r i m e n t s , DI a n d H Z r a t s w e r e l i t t e r m a t e s . A l l s t e p s w e r e c a r r i e d out in p a r a l l e l for H Z a n d DI rats in a r a n d o m i z e d order.
Preparation
of q l o m e r u l i
and p a p i ! l a
U n d e r p e n t o b a r b i t a l a n e s t h e s i a (Nembutal Abbot, 5 m g / 1 0 0 g B W i.p.), a c a n u l a w a s i n s e r t e d into t h e l o w e r aorta. I s o t o n i c c o l d h e p a r i n i z e d s a l i n e (0.16 M NaCl, 3 0 - 5 0 ml) w a s p e r f u s e d u n d e r a p r e s s u r e o f a b o u t i00 m m H g , a f t e r c l a m p i n g the a o r t a a b o v e t h e r e n a l a r t e r i e s and o p e n i n g a r e n a l v e i n o r v e n a c a v a for d r a i nage. B l a n c h e d k i d n e y s w e r e r e m o v e d a n d i m m e r s e d in icec o l d 20 m M T r i s - H C l b u f f e r , p H 7.4 c o n t a i n i n g 125 m M NaCI, i0 m M KCI, i0 ram s o d i u m a c e t a t e a n d 5 m M g l u c o s e . All s u b s e q u e n t s t e p s w e r e c a r r i e d o u t in the same b u f f e r at 0-4°C. G l o m e r u l i w e r e i s o l a t e d as p r e v i o u s l y d e s c r i b e d with minor-modifications (8). K i d n e y c o r t e x w a s m i n c e d to a p a s t e - l i k e c o n s i s t e n c y a n d m i l d l y p r e s s e d on a 106 ~ m s t a i n l e s s s i e v e w i t h a s p a t u l a . T h i s s t e p r e s u l t s in r u p t u r i n g B o w m a n ' s c a p s u l e s a n d p u s h i n g the g l o m e r u l a r t u f t s t o g e t h e r w i t h s o m e t u b u l a r f r a g m e n t s t h r o u g h the o p e n i n g s of the sieve. The t i s s u e c o l l e c t e d u n d e r the s i e v e w a s S u s p e n d e d in a b o u t i0 ml b u f f e r , p o u r e d on a 50 ~m s t a i n l e s s s i e v e a n d w a s h e d t h o r o u g h l y w i t h b u f f e r . The t i s s u e r e c o v e r e d o n the u p p e r f a c e of the s i e v e w a s
350
AUGUST 1980 VOL. 20 NO. 2
PROSTAGLANDINS
then r e s u s p e n d e d in I0 ml b u f f e r and p a s s e d 3 times t h r o u g h a 25 gauge n e e d l e w i t h a s y r i n g e to b r e a k in small p i e c e s the r e m a i n i n g tubular fragments w h i c h were s u b s e q u e n t l y e l i m i n a t e d by a second p a s s a g e on the 50 pm sieve and two s u c c e s s i v e w a s h i n g s and c e n t r i f u g a t i o n s (120 × g for 90 sec.). The pellet c o n s i s t e d in n e a r l y pure isolated, d e e a p s u l a t e d glomeruli w i t h less than 2 % tubular contamination. No afferent or e f f e r e n t a r t e r i o l e s were observed. R e c o v e r y was about 5,000 - 15,000 glomeruli per rat (roughly 0.5-1.5 mg g l o m e r u l a r protein). The glomeruli of HZ and DI rats w e r e p r e p a r e d in p a r a l l e l and kept in 2 ml cold b u f f e r until use. P a p i l l a r y h o m o g e n a t e s from the same rats were p r e p a red a s - ~ o I ~ o w s - Y - T S e - c o n e Z s h a p e d w h i t e p a p i l l a p r o t r u d i n g in the renal pelvis (inner medulla) w a s separated f r o m the r e d - o u t e r medulla, m i n c e d and h o m o g e n i z e d m i l d l y b y 3-5 strokes in a t i s s u e g r i n d e r (Kontes). The r e s u l t i n g suspension was f i l t e r e d t h r o u g h a 25 um stainless sieve to remove large pieces of tissue and was w a s h e d in i0 ml b u f f e r and c e n t r i f u g e d twice (1500 × g for 20 min.). The h o m o g e n i z e d p a p i l l a r y tissue (about 1-2 mg prot. per rat) was kept in 2 ml cold buffer until use.
Incubation A few m i n u t e s b e f o r e incubation, glomeruli and p a p i l l a r y h o m o g e n a t e s were c e n t r i f u g e d (1500 × g for i0 min) and r e s u s p e n d e d in a b u f f e r similar to that used in the p r e p a r a t i o n steps w i t h a d d i t i o n of 1 m M CaCl 2. G l o m e r u l i and p a p i l l a r y h o m o g e n a t e s from both HZ and DI rats w e r e incubated in this buffer, in the a b s e n c e or in the p r e s e n c e of a r a c h i d o n i c acid (A.A.) 5 pg/ml final concentration, This c o n c e n t r a t i o n p r o d u c e d maximal s t i m u l a t i o n in b o t h p a p i l l a r y h o m o g e n a t e s and glomeruli. A.A. (sodium salt) was o b t a i n e d from S i g m a (St Louis, Mo.), kept under nitrogen a t m o s p h e r e at - 2 0 ° C and e x t e m p o r a n e o u s l y d i s s o l v e d in e t h a n o l - w a t e r (7:10, v/v). Incubation was c a r r i e d out in separate 200 ~i aliquotes in 1 ml p l a s t i c tubes at 37°C in room atmosphere under continuous agitation. P r o t e i n c o n c e n t r a t i o n (200-600 ~g/ml) was d e t e r m i n e d a c c o r d i n g to Lowry et al. (17) w i t h m i n o r modifications. I n c u b a t i o n was s t o p p e d after 15, 30, 60 and 90 m i n u t e s by centrifugation at 3000 × g for 2 m i n u t e s at 10°C. S u p e r n a t a n t s of each tube w e r e c o l l e c t e d and frozen u n t i l PGE 2 a s s a y was performed.
PGE 2 a s s a x R a d i o i m m u n o a s s a y of PGE 2 was c a r r i e d out a c c o r d i n g to Dray et al. (18). A n t i - P G E 2 a n t i s e r u m was p u r c h a s e d from I n s t i t u t P a s t e u r (Paris). The i n c u b a t i o n m e d i u m b e i n g
AUGUST 1980 VOL. 20 NO. 2
351
PROSTAGLANDINS
protein-free, PGE 2 was a s s a y e d in the s u p e r n a t a n t w i t h o u t prior extraction. We have shown this direct p r o c e d u r e to provide results similar to those o b t a i n e d after extraction o v e r a wide range of p r o s t a g l a n d i n c o n c e n t r a t i o n s (8). C r o s s - r e a c t i o n of a r a c h i d o n i c acid w i t h PGE 2 a n t i s e r u m was m e a s u r e d in s e p a r a t e tubes in the a b s e n c e of g l o m e r u l i or p a p i l l a and was s u b t r a c t e d w h e n adequate.
Calculations
and s t a t i s t i c a l analysis
PGE 2 c o n c e n t r a t i o n was m e a s u r e d in supernatants of the f o l l o w i n g samples : g l o m e r u l i and p a p i l l a of control HZ and h o m o z y g o u s DI rats, w i t h o u t or w i t h A.A. after 15, 30, 60 and 90 min of incubation. Results w e r e e x p r e s s e d as pg of PGE 2 p r o d u c e d per mg p r o t e i n a f t e r 15, 30, 60 or 90 minutes. PGE 2 p r o d u c t i o n as pg per m g p r o t e i n per m i n u t e d u r i n g the time intervals 0-15, 15-30, 30-60, 60-90 min was c a l c u l a t e d for each experiment. PGE2 c o n c e n t r a t i o n in the s u p e r n a t a n t at time zero just p r i o r to the i n c u b a t i o n was found to be negligible. The results were a n a l y z e d u s i n g analysis of v a r i a n c e with fixed factors w i t h o u t replication (19). In a first analysis, the d e p e n d e n c y of the total p r o d u c t i o n of PGE 2 from 0 to 90 min on the three following factors was considered : i) rat (DI v e r s u s HZ), 2) e x p e r i m e n t a l condition (without or w i t h A.A.), 3) s u c c e s s i v e e x p e r i m e n t s , n° 1 to 7 c o r r e s p o n d i n g to d i f f e r e n t days. G l o m e r u l i and p a p i l l a r y h o m o g e n a t e s w e r e assessed separately. It a p p e a r e d from our results that the t i m e - c o u r s e of PGE 2 p r o d u c t i o n by g l o m e r u l i w i t h o u t A.A. and those by g l o m e r u l i and p a p i l l a w i t h A.A. w e r e b i p h a s i c w i t h an early p h a s e of high synthesis rate and a second phase of lower but sustained p r o d u c t i o n (as shown in Fig. i). For this reason, in a second set of a n a l y s i s of variance, we c o n s i d e r e d s e p a r a t e l y the first 15 m i n and the follow i n g p e r i o d from 15 to 90 min.
RESULTS M e a n b o d y w e i g h t was 305 ± 28 and 249 ± 22 g (mean ± SEM) in HZ and DI rats respectively. DI rats' impaired growth rate has a l r e a d y b e e n reported (15). Mean urine o s m o l a l i t y (urine c o l l e c t e d by b l a d d e r p u n c t u r e just after anesthesia) was 1190 ± 180 and 120 ± 27 m O s m / k g for HZ and DI rats respectively.
352
AUGUST 1980 VOL. 20 NO. 2
PROSTAGLANDINS
PGE2_ssynthesis by p a p i l l a r y h o m o g e n a t e s PGE 2 a c c u m u l a t e d p r o g r e s s i v e l y w i t h time in the incubation m e d i u m of p a p i l l a r y h o m o g e n a t e s f r o m b o t h HZ and DI rats and this a c c u m u l a t i o n was e n h a n c e d by the addition of A°A. (Table I). Nevertheless, in the absence (basal) as w e l l as in the p r e s e n c e of A.A., PGE 2 production was s i g n i f i c a n t l y lower in p a p i l l a r y h o m o g e n a t e s from DI than f r o m HZ rats (Table 1 and T a b l e 3, 0-90 min). In o r d e r to b e t t e r c h a r a c t e r i z e the t i m e - c o u r s e of PGE2 synthesis, we c a l c u l a t e d the m e a n rates of PGE 2 p r o d u c t i o n per minute for the successive p e r i o d s of incub a t i o n (Fig. i, left panel). This c a l c u l a t i o n provides independent values more s u i t a b l e for an analysis of
B
a
s
PAPILLA a l
GLOMERULI Basal
400
=_ E
300 20O
4O
30 2O
100
m
101-
E
=E 1500 ~ .
=Hz
WithAA
3ooI
WithAA
E 2ooi
1000
I00~
5OO 601
300
40~"
100
20~
3'0
°'o
90
rain
o
;s
6'0'
rai9On
Fiqure !. Time-course of PGE 2 p r o d u c t i o n b y p a p i l l a r y h o m o g e n a t e s (left panel) and isolated g l o m e r u l i (right panel). The m e a n p r o d u c t i o n p e r min has b e e n calculated from the time intervals 0-15, 15-30, 30-60 and 60-90 min in basal conditions (top) and w i t h a d d i t i o n of 5 ~g/ml arachidonic acid (bottom). Closed circles = HZ control rats, open circles = DI rats (mean ± I S E M of 7 experiments ; note the scale change in ordinates of the l o w e r graphs).
AUGUST 1980 VOL. 20 NO. 2
353
PROSTAGLANDINS
variance (cumulated values d e p e n d on the values o b t a i n e d at p r e c e e d i n g times). Fig. I shows that the rate of PGE 2 synthesis d e c r e a s e d w i t h time in both DI and HZ p a p i l l a and r e m a i n e d lower in DI rats compared to t h e i r HZ controls at all times, e i t h e r w i t h or w i t h o u t A.A. The difference b e t w e e n HZ and DI was fairly stable in early and late p e r i o d s (Table 2) w i t h DI/HZ ratios averaging 0.60-0.70 and was s i g n i f i c a n t at the 1 % level (Table 3). The s t i m u l a t i o n induced by A.A. was h i g h e r in the e a r l y than in the late p e r i o d (about × 3.3, p < 0 . 0 0 1 ~ a n d x 1.4, p < 0.01 respectively) and was s i m i l a r in both groups of rats (no i n t e r a c t i o n between the Ist and the 2nd factor) (Tables 2 and 3). F i n a l l y we o b s e r v e d a small but s i g n i f i c a n t v a r i a t i o n b e t w e e n e x p e r i m e n t s p e r f o r m e d on d i f f e r e n t days but this factor did not interfere w i t h the o t h e r factors u n d e r s t u d y (no interaction, Table 3).
P G E 2 - s y n t h e s i s b~ i s o l a t e d qlomeruli PGE 2 a c c u m u l a t e d p r o g r e s s i v e l y w i t h time in both HZ and DI g l o m e r u l a r incubates and the PGE 2 p r o d u c t i o n was increased by the a d j o n c t i o n of A.A, (Table I). C o n t r a r y to what was o b s e r v e d in the papilla, the DI rat glomeruli p r o d u c e d s i g n i f i c a n t l y more P G E 2 than the glomeruli of their HZ controls (Table 1 and Table 3, 0-90 min). W h e n PGE 2 synthesis rates per min w e r e c a l c u l a t e d (Fig. I, right panel) it was o b s e r v e d that the m a g n i t u d e of the d i f f e r e n c e b e t w e e n HZ and DI was not constant. In b a s a l conditions for example, the PGE 2 ~ynthesis in the interval 0-15 m i n was o n l y 1.27 fold h i g h e r in DI than in HZ g l o m e r u l i w h i l e it was 2.72 fold h i g h e r in the 15-30 m i n p e r i o d and remained close to 2 in the two later periods. A similar p a t t e r n was o b s e r v e d in the p r e s e n c e o f A.A, (Table 2). Only in the second p h a s e (15-90 min) was the d i f f e r e n c e b e t w e e n both groups of rats s i g n i f i c a n t (Table 3). S t i m u l ~ t i o n b y A.A. was h i g h e r in the first 15 min (about × 6.5) than in the following periods (× 3) and was s i m i l a r in both groups of rats (no interaction) (Table 3). The o b s e r v e d b e t w e e n e x p e r i m e n t v a r i a t i o n did not interfere w i t h the two other factors (Table 3). A l t h o u g h the purpose of this work was not to compare PGE 2 p r o d u c t i o n by d i f f e r e n t renal structures, it is interesting to note that PGE 2 synthesis per mg of p r o t e i n was m a r k e d l y g r e a t e r in the p a p i l l a than in the glomerulio On the contrary, the d e g r e e of s t i m u l a t i o n b y A.A. was h i g h e r for the glomerull than for the papilla.
354
AUGUST 1980 VOL. 20 NO. 2
u~
O
O
< O
00 O
mean SEM
mean SEM
60
90
954 216
801 145
603 120
499 74
HZ
921 156
633 124
DI
1529 273
1276 303
Basal
4911 648
4414 664
4101 707
3494 485
HZ
with
6653 1044
5604 701
4970 698
4264 536
DI
A.A.
19990 2950
16610 2270
9940 1440
6170 1030
HZ
6940 1610
3770 940
DI
13110 2600
10560 2160
Basal
39870 5980
32620 4570
26400 4160
21760 3120
HZ
with
25670 3080
20920 2330
15970 2100
12480 1195
DI
A.A.
Papilla ............................................
1 : P r o d u c t i o n o f P G E 2 b y i s o l a t e d g l o m e r u l i a n d p a p i l l a r y h o m o g e n a t e s w i t h o u t (basal) o r w i t h a r a c h i d o n i c a c i d (A.A.) 5 ~ g / m l a f t e r 15, 30, 60 a n d 90 m i n i n c u b a t i o n . R e s u l t s a r e e x p r e s s e d as p g P G E 2 p e r m g p r o t e i n (mean ± S E M of 7 e x p e r i m e n t s ) . HZ = h e t e r o z y g o u s c o n t r o l r a t s ; DI = r a t s w i t h d i a b e t e s i n s i p i d u s .
mean SEM
30
Table
mean SEM
15
minutes (cumulative)
Glomeruli
....................................
> Z
>
O
U~
33.24
6.24
0-15 m i n
15-90 min
411
186
0-15 min
15-90 min
124
251
145
12.76
42.40
17.67
DI
0.67
0.61
0.65
2.04
1.27
1.65
249
1424
445
20.31
229
-55.27
HZ
181
805
285
34.94
272
74.53
DI
0.73
0.57
0.64
1.72
1.19
1.35
1.34
3.46
2.00
3.25
6.89
5.15
•
1.46
3.21
1.97
2.74
6.45
4.22
Table 2 : Mean PGE 2 p r o d u c t i o n per m i n u t e from 0 to 90 or from 0 to 15 and 15 to 90 m i n u t e s by i s o l a t e d glomeruli and p a p i l l a r y h o m o g e n a t e s from HZ and DI rats. R e s u l t s are e x p r e s s e d as pg PGE 2 p e r m g p r o t e i n per m i n u t e (means of 7 experiments) or as ratios b e t w e e n DI and HZ rats or b e t w e e n A.A. and basal conditions.
223
0-90 m i n
PAPILLA
10.74
.0-90 min
GLOMERULI
HZ
Basal with A.A. A.A./Basal ........................................................................ pg/mg prot°/min D_~I pg/mg prot./min DI HZ HZ HZ DI
Z
O
z o
O
< o
kO
Table
3
12.68
< 0.001
F =
P
240
< 0.001
F =
p
p
16.01
< 0.01
F =
min
p
5.16
< 0.05
F = p
214
NS
3.15
min
< 0.001
F =
F =
0-15
p
7.50
< 0.05
F = p
44.37
< 0.01
15.09
min
< 0.001
F =
p
F =
15-90
p
9.80 < 0.01
F = p
66.21
< 0.01
2%.54
min
< 0.001
F =
p
F =
0-90
p
5.76 < 0.05
F = p
85.19
< 0.01
21.06
min
< 0.001
F =
p
F =
0~15
F
F
F
p
p
p
min
=
=
< 0.05
12.72
< 0.01
17.55
< 0.01
= 20.04
15-90
Papilla ........................................
: Analysis of variance performed on the pooled data of the 7 individual experiments the means of which a r e s h o w n in T a b l e 2. I n t e r a c t i o n s between rat groups and experimental conditions, or between experiment order and either rat groups or experimental conditions are not significant (not s h o w n ) .
Variation between the different experiments (degrees of freedom : 6,6)
Variation due to the experimental conditions : with vs without arachidonic acid (degrees of freedom : 1,6)
Variation due to the rat group = DI vs HZ (degrees of freedom : 1,6)
0-90
Glomeruli ........................................
Z
> Z
>
O
PROSTAGLANDINS
DISCUSSION D e p e n d e n c y of renal P G synthesis on a n t i d i u r e t i c h o r m o n e has r e c e n t l y b e e n e x e m p l i f i e d by the f i n d i n g that urinary PGE 2 and PGF2e e x c r e t i o n was g r e a t l y reduced in B r a t t l e b o r o rats c o m p a r e d to control rats and could be r e s t o r e d by a d m i n i s t r a t i o n of exogenous v a s o p r e s s i n (13, 14) or of its analogue d e - a m i n o - D - a r g i n i n e v a s o p r e s s i n (dDAVP) (14). The p r e s e n t in vitro e x p e r i m e n t s have shown that p a p i l l a from B r a t t l e b o r o DI rats i n d e e d exhibits a reduced PGE 2 synthesis. Unexpectedly, an i n c r e a s e d PGE 2 synthesis was d e m o n s t r a t e d in the glomeruli of the same rats c o m p a r e d to HZ controls. The a c c u m u l a t i o n of PGE 2 in the incubation m e d i u m really c o r r e s p o n d e d to "de novo" synthesis and did not reflect a p r o g r e s s i v e release of p r e e x i s t i n g i n t r a c e l l u lar stores since the c o n c e n t r a t i o n of PGE 2 was m a r k e d l y e n h a n c e d by the a d d i t i o n of its p r e c u r s o r A.A. in b o t h tissues. The synthetic rates of PGE 2 in p a p i l l a and glomeruli d e c r e a s e d p r o g r e s s i v e l y w i t h time, a p p r o a c h i n g an e q u i l i b r i u m after 15-30 minutes. This t i m e - c o u r s e was similar to that p r e v i o u s l y r e p o r t e d for rat g l o m e r u l i (8) and p a p i l l a (20)° Different q u e s t i o n s are b r o u g h t up by our o b s e r v a tions : W h a t are the c e l l u l a r m e c h a n i s m s responsible for the alterations in PGE 2 synthesis o b s e r v e d in DI rats ? How can a single d e f e c t i.e. ADH deficiency, result in opposite effects on glomeruli and p a p i l l a ? W h a t are the p h y s i o l o g i c a l i m p l i c a t i o n s of the altered PGE 2 synthesis p a t t e r n in the k i d n e y of DI rats ? H e t e r o z y g o u s and h o m o z y g o u s B r a t t l e b o r o rats d i f f e r by the absence of v a s o p r e s s i n and b y its c o n s e q u e n c e s including i n a b i l i t y to concentrate urine (15), p o t a s s i u m d e f i c i e n c y (16), and s t i m u l a t e d r e n i n - a n g i o t e n s i n s y s t e m (16, 21). P a p i l l a r y o s m o l a l i t y of c o n t r o l rats is m u c h h i g h e r t h a n that of DI rats and h y p e r t o n i c m e d i u m has been shown to enhance m a r k e d l y P G synthesis by p a p i l l a r y slices in vitro (22). However, d i f f e r e n c e s in p a p i l l a r y o s m o l a l i t y and the o t h e r d i f f e r e n c e s c i t e d above are met only in vivo. In the in vitro c o n d i t i o n s used here, the p r o p e r h o r m o n a l e n v i r o n m e n t of each rat was no l o n g e r maintained. P r e p a r a t i o n of the tissues i n c l u d e d several w a s h i n g s w i t h large v o l u m e s of i s o t o n i c b u f f e r and subsequently, all samples were i n c u b a t e d in the same b u f f e r and in the absence of vasopressin. Moreover, a d d i t i o n of e x o g e n o u s synthetic A%~ or dDAVP (Ferring, Malm~, Sweden), i0 -° M final concentration, did not m o d i f y PGE 2 synthesis in e i t h e r tissue from HZ or DI rats (2 experiments, results not shown). The o b s e r v e d d i f f e r e n c e b e t w e e n DI and
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HZ p r e p a r a t i o n s thus cannot be a t t r i b u t e d to a direct and i m m e d i a t e effect of vasopressin. It can only be a t t r i b u t e d to a chronic m o d i f i c a t i o n in cell function i n d u c e d by the chronic ADH d e f i c i e n c y or b y its functional. consequences. Actually, m o d i f i e d in v i t r o renal PG synthetic rates have also been reported in another chronic rat model, the s p o n t a n e o u s l y h y p e r t e n s i v e rat (23) . P r e c u r s o r a v a i l a b i l i t y is not the f a c t o r m o d i f y i n g PGE 2 synthesis in DI rats since the d i f f e r e n c e b e t w e e n both types of rats p e r s i s t e d in the p r e s e n c e of an excess of A.A. The factors i n v o l v e d thus, m u s t act at a step b e y o n d the d e a c y l a t i o n of phospholipids, probably on c y c l o o x y g e n a s e a c t i v i t y or on one of the enzymes i n t e r v e n i n g in the d e g r a d a t i o n of PGE 2. This is thus different from the direct m o d e of action of the hormonal peptides w h i c h stimulate P G E 2 p r o d u c t i o n t h r o u g h an increase in the a c t i v i t y of p h o s p h o l i p a s e (20). A l t e r n a tively, changes in 9 k e t o - r e d u c t a s e a c t i v i t y could also alter PGE 2 a c c u m u l a t i o n rates. This w o u l d result in inverse changes in PGF2e accumulation. For example, this enzyme a c t i v i t y has been shown to be i n f l u e n c e d by salt intake or r e l a t e d factors (24). S i m u l t a n e o u s m e a s u r e m e n t s of both PGE 2 and PGF2e w o u l d be n e c e s s a r y to c o n f i r m the o c c u r e n c e of this a l t e r a t i o n in DI rats. H o w does the lack of ADH affect p a p i l l a r y and g l o m e rular PGE 2 p r o d u c t i o n in o p p o s i t e ways ? It should be s t r e s s e d that both tissues s t u d i e d are h e t e r o g e n o u s since they include several c e l l u l a r types. Glomeruli comprise at least three d i f f e r e n t types of cells two of which, the m e s a n g i a l and e p i t h e l i a l cells, h a v e been d e m o n s t r a t e d as capable of s y n t h e s i z i n g PGE 2 (25). Similarly, the p a p i l l a contains m a n y different types of cells from vascular, i n t e r s t i t i a l and t u b u l a r origin. It has been shown that at least two of them, the interstitial and the collecting duct cells, p r o d u c e PGs (26, 3, 4, 5). P r o d u c t i o n of PGs by the v a s c u l a r e n d o t h e l i u m in glomerular capillaries and in p a p i l l a r y v a s a recta cannot be r u l e d out since other v a s c u l a r s t r u c t u r e s are known to s y n t h e s i z e PGs (27). As s t a t e d above, A D H d e f i c i e n c y induces several secondary d i s o r d e r s (15, 16, 21). On the other hand, several factors i n c l u d i n g c o n c e n t r a t i o n of various ions and hormones, oxygen, o s m o l a l i t y and pH, have been shown to influence PGE 2 synthesis (28, 20, 2, 21, i). It can be h y p o t h e t i z e d that the p r o d u c t i o n of PGE 2 by each cell type is i n f l u e n c e d to a d i f f e r e n t e x t e n t by each of these factors. For example, ADH, osmolality, oxygen could be m a j o r d e t e r m i n a n t s of m e d u l l a r y synthesis w h i l e a n g i o t e n s i n II could influence p r i m a r i l y g l o m e r u l a r cells.
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The b i p h a s i c t i m e - c o u r s e of PGE 2 s y n t h e s i s rate in glomeruli might be due to d i f f e r e n t s y n t h e t i c patterns in two d i f f e r e n t cell types. That the d i f f e r e n c e between HZ and DI is more m a r k e d in the later phase suggests that PGE 2 synthesis rate might be increased in the cells responsible for the low but sustained PGE 2 production. Changes in the p r o p o r t i o n of the d i f f e r e n t cells in e a c h structure could also a c c o u n t for the d i f f e r e n t net PGE 2 p r o d u c t i o n o b s e r v e d in DI rats c o m p a r e d to HZ controls. As g l o m e r u l a r m e s a n g i a l cells p r o d u c e a b o u t 15 times more PGE 2 per mg protein or per cell than do g l o m e r u l a r epithelial cells (29), a small increase in mesangial versus e p i t h e l i a l cell n u m b e r in glomeruli from DI rats, could affect m a r k e d l y the n e t PGE 2 production, even in the absence of any change in the cellular m e c h a n i s m of PGE 2 synthesis in e i t h e r cell. In the papilla, indirect arguments suggest that collecting duct cells are at least p a r t l y responsible for the PGE 2 synthesis defect o b s e r v e d in DI rats : I/ B o t h arginine v a s o p r e s s i n and a n g i o t e n s i n II stimulate P G synthesis in c u l t u r e d r e n o m e d u l l a r y cells, but to unequal degrees (30). A n g i o t e n s i n b e e i n g 7 times m o r e potent, it can be a s s u ~ e d that the e f f e c t of the lack of ADH on interstitial cells in DI rats is b l u n t e d by the o p p o s i t e effect induced by h i g h r e n i n - a n g i o t e n s i n levels in these rats (16, 21). 2/ ADH, but not dDAVP, stimulates PGE 2 synthesis by interstitial cells in culture (31) w h i l e both ADH and dDAVP can s i m i l a r l y restore a normal PGE 2 urinary excretion in DI rats in vivo (14). Thus, another cell type, likely the c o l l e c t i n g duct cells, is i n v o l v e d (26). Finally, m o r p h o l o g i c alterations of the medulla, i.e. changes in the p r o p o r t i o n of i n t e r s t i t i a l versus collecting duct cell mass (32) due to the h i g h diuresis and a l t e r e d p o t a s s i u m balance (16) m i g h t contribute to changes in net p a p i l l a r y PGE 2 production. W h a t functional consequences can be e x p e c t e d to result from the PGE 2 a l t e r e d synthesis o b s e r v e d in DI rat p a p i l l a and glomeruli ? C i r c u l a t i n g PGE 2 being catabolized in one pass t h r o u g h the lungs it is considered that its role is limited to the cells in which it is s y n t h e s i z e d or to the n e i g h b o u r i n g cells. Thus, the consequences of impaired synthetic rates p r o b a b l y d e p e n d on the p a r t i c u l a r cell type r e s p o n s i b l e for the anomaly in each tissue. As m u l t i c e l l u l a r structures were analyzed in our study, only assumptions can be p r o p o s e d regarding the p h y s i o l o g i c a l i m p l i c a t i o n s of our observations.
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If the d e c r e a s e d p a p i l l a r y PGE 2 synthesis is due to c o l l e c t i n g duct cells, this w i l l have little effect on k i d n e y f u n c t i o n in DI rats as the main role attributed to PGs in these cells is to m o d u l a t e the h y d r o osmotic effects of ADH (12) w h i c h a c t u a l l y does not occur in these rats. The role of PGE 2 p r o d u c e d by medullary interstitial cells is not clear. In some studies, PGE 2 has been r e p o r t e d to have a v a s o d i l a t o r y e f f e c t on deep c o r t i c a l and m e d u l l a r y v a s c u l a t u r e (i). A defect in i n t e r s t i t i a l cell synthetic rate could a f f e c t deep nephron hemodynamics. It is of interest to note that although w h o l e renal filtration rate is not d e c r e a s e d in DI rats, the normal single n e p h r o n f i l t r a t i o n rate h e t e r o g e n e i t y as well as the anatomical n e p h r o n h e t e r o geneity p r e s e n t in rats and all mammals, are a b o l i s h e d in these rats (33). The functional implications of the i n c r e a s e d PGE 2 synthesis in DI rats glomeruli are d i f f i c u l t to appreciate. As for the papilla, they m a y vary a c c o r d i n g to the cell type(s) r e s p o n s i b l e for this change. Actually, very little is k n o w n yet about the role and the regulation of l o c a l l y p r o d u c e d PGs in the glomeruli (25, 29, 34). Inhibition of PG synthesis has r e v e a l e d a role for PGs in m o d u l a t i n g the e f f e c t s of a n g i o t e n s i n on the ultrafiltration c o e f f i c i e n t (9) and also in a f f e c t i n g the u l t r a s t r u c t u r e of epithelial and mesangial cells (ii) and the control of the feed-back m e c h a n i s m (i0). In summary, in vivo studies h a d r e v e a l e d that whole k i d n e y PGE 2 p r o d u c t i o n was m a r k e d l y r e d u c e d i n DI rats (13, 14) ; by studying two different k i d n e y structures in v i t r o we have shown that this defect is the net result of a d e c r e a s e d PGE 2 synthesis by the p a p i l l a and an i n c r e a s e d synthesis by the glomeruli. These changes m i g h t be brought about e i t h e r by m o d i f i c a t i o n s of c y c l o o x y g e n a s e or 9 - k e t o - r e d u c t a s e activities or by m o d i f i c a t i o n s in the p r o p o r t i o n s of the d i f f e r e n t cell types present in each structure. To further progress in our u n d e r s t a n d i n g of these phenomenons, the next logical step w i l l imply w o r k i n g on i s o l a t e d c e l l u l a r populations.
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ACKNOWLEDGMENTS This w o r k was s u p p o r t e d by grants from the "D~14gation G 4 n 4 r a l e ~ la Recherche S c i e n t i f i q u e et Technique" (n ° 77.7.1395 and 79.7.0985). P r e l i m i n a r y r e s u l t s w e r e p u b l i s h e d in a b s t r a c t f o r m : Abstracts of the 12th A n n u a l M e e t i n g of the A m e r i c a n S o c i e t y of Nephrology, K i d n e y Int. 16 : 847, 1979. We thank Mrs Chantal Lajotte for excellent technical assistance. REFERENCES
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TRINH T R A N G TAN, M.M., J.P. G R U N F E L D and L. BANKIR. Lack of n e p h r o n h e t e r o g e n e i t y in B r a t t l e b o r o rats w i t h h e r e d i t a r y d i a b e t e s insipidus : SNGFR, glomerular v o l u m e and p r o x i m a l t u b u l a r length determinations. (Abstract). U p s a l a J. Med. Sciences, supp 26 : 72. 1979.
34.
SCHLONDORFF, D. and V. FOLKERT. P r o s t a g l a n d i n synthesis in glomeruli from rats w i t h unilateral obstruction. (Abstract). K i d n e y Int. 16 : 858. 1979.
Editor: Gabor Kaley Received: 3-11-80 Accepted: 6-20-80
AUGUST 1980 VOL. 20 NO. 2
365
ALTERED PGE 2 PRODUCTION BY GLOMERULI AND PAPILLA OF RATS WITH HEREDITARY DIABETES INSIPIDUS Lise BANKIR, Marie-Marcelle TRINH TRANG TAN Marie-Paule NIVEZ, Jos4e SRAER and Raymond ARDAILLOU I.N.S.E.R.M. Unit4 64, H6pital Tenon 75790 Paris 20 I.N.S.E.R.M. Unit~ 90, H6pital Necker 75730 Paris 15 France ABSTRACT PGE 2 synthesis rate was studied in vitro in isolated glomeruli and in papillary homogenates prepared from kidneys of Brattleboro rats with hereditary diabetes insipidus (DI) (no ADH) and of BrattleborQ heterozygous control rats. Incubations were carried out in isotonic buffer at 37°C in the presence or absence of arachidonic acid for 15, 30, 60 and 90 min. PGE 2 production was measured in the supernatant by specific radioimmunoassay. Results were compared by analysis of variance. PGE 2 production was significantly decreased in the papilla (p < 0.01) and increased in the glomeruli (p < 0.01) of DI rats compared to controls. Stimulation by arachidonic acid was similar in both groups. Chronic ADH deficiency thus modifies the a b i l i t y of the kidney to produce PGE 2 in vitro. The opposite effects observed in glomeruli and papilla suggest a different hormonal control of PGE 2 synthesis in both tissues. INTRODUCTION Prostaglandin (PG) biosynthesis within the renal medulla of the rat is well documented (I, 2). The specifi'c cellular sites of synthesis include m e d u l l a r y interstitial cells (3, 4) and collecting duct cells (4, 5). In addition, recent reports have shown that glomeruli also synthesize PGs (6, 7, 8) and that PGs influence b o t h glomerular filtration rate (9, I0) and glomeruli ultrastructure (Ii). The control of PG p r o d u c t i o n by these different renal structures has not been yet completely clarified. Interactions between PGs and antidiuretic hormone (ADH) Occur both in the toad b l a d d e r and in the m a m m a l i a n kidney (12). Recently, it has been shown that renal excretion of PGE 2 (13, 14) and PGF2e (14) was markedly reduced in Brattleboro rats with hereditary diabetes insipidus (DI rats) (15).
AUGUST 1980 VOL. 20 NO. 2
349
PROSTAGLANDINS
L a c k o f A D H in t h e s e r a t s a c c o u n t s f o r t h i s r e d u c t i o n since administration of this hormone restored renal e x c r e t i o n o f t h e t w o PGs s t u d i e d (13, 14). S i n c e it is admitted that urinary PGs reflect renal PO synthesis (i) it can b e c o n c l u d e d t h a t A D H s t i m u l a t e s r e n a l p r o d u c t i o n of PGE 2 a n d P G F 2 in vivo. B u t n e i t h e r the s i t e of e f f e c t of A D H in the ~ i d n e y n o r t h e s t e p of P G s y n t h e s i s at w h i c h t h i s h o r m o n e a c t s can be d e d u c e d f r o m t h e s e in vivo experiments. The present study was designed to c o m p a r e the in v i t r o r a t e s of PGE 2 s y n t h e s i s b y g l o m e r u l i a n d b y the p a p i l l a f r o m r a t s w i t h o r w i t h o u t A D H d e f i c i e n c y .
MATERIAL AND METHODS Rats M a l e a n d f e m a l e 3-6 m o n t h s o l d B r a t t l e b o r o rats, b r e d in H o s p i t a l N e c k e r (INSERM, U 90, Paris) w e r e u s e d in t h i s study. E a c h e x p e r i m e n t i n c l u d e d one h o m o z y g o u s rat w i t h d i a b e t e s i n s i p i d u s a n d o n e c o n t r o l h e t e r o z y g o u s rat (HZ) of t h e s a m e strain. H Z r a t s h a v e s u b n o r m a l A D H p r o d u c t i o n a n d p l a s m a l e v e l s (16). A g e a n d sex w e r e m a t c h e d and~ in 5 o u t of 7 e x p e r i m e n t s , DI a n d H Z r a t s w e r e l i t t e r m a t e s . A l l s t e p s w e r e c a r r i e d out in p a r a l l e l for H Z a n d DI rats in a r a n d o m i z e d order.
Preparation
of q l o m e r u l i
and p a p i ! l a
U n d e r p e n t o b a r b i t a l a n e s t h e s i a (Nembutal Abbot, 5 m g / 1 0 0 g B W i.p.), a c a n u l a w a s i n s e r t e d into t h e l o w e r aorta. I s o t o n i c c o l d h e p a r i n i z e d s a l i n e (0.16 M NaCl, 3 0 - 5 0 ml) w a s p e r f u s e d u n d e r a p r e s s u r e o f a b o u t i00 m m H g , a f t e r c l a m p i n g the a o r t a a b o v e t h e r e n a l a r t e r i e s and o p e n i n g a r e n a l v e i n o r v e n a c a v a for d r a i nage. B l a n c h e d k i d n e y s w e r e r e m o v e d a n d i m m e r s e d in icec o l d 20 m M T r i s - H C l b u f f e r , p H 7.4 c o n t a i n i n g 125 m M NaCI, i0 m M KCI, i0 ram s o d i u m a c e t a t e a n d 5 m M g l u c o s e . All s u b s e q u e n t s t e p s w e r e c a r r i e d o u t in the same b u f f e r at 0-4°C. G l o m e r u l i w e r e i s o l a t e d as p r e v i o u s l y d e s c r i b e d with minor-modifications (8). K i d n e y c o r t e x w a s m i n c e d to a p a s t e - l i k e c o n s i s t e n c y a n d m i l d l y p r e s s e d on a 106 ~ m s t a i n l e s s s i e v e w i t h a s p a t u l a . T h i s s t e p r e s u l t s in r u p t u r i n g B o w m a n ' s c a p s u l e s a n d p u s h i n g the g l o m e r u l a r t u f t s t o g e t h e r w i t h s o m e t u b u l a r f r a g m e n t s t h r o u g h the o p e n i n g s of the sieve. The t i s s u e c o l l e c t e d u n d e r the s i e v e w a s S u s p e n d e d in a b o u t i0 ml b u f f e r , p o u r e d on a 50 ~m s t a i n l e s s s i e v e a n d w a s h e d t h o r o u g h l y w i t h b u f f e r . The t i s s u e r e c o v e r e d o n the u p p e r f a c e of the s i e v e w a s
350
AUGUST 1980 VOL. 20 NO. 2
PROSTAGLANDINS
then r e s u s p e n d e d in I0 ml b u f f e r and p a s s e d 3 times t h r o u g h a 25 gauge n e e d l e w i t h a s y r i n g e to b r e a k in small p i e c e s the r e m a i n i n g tubular fragments w h i c h were s u b s e q u e n t l y e l i m i n a t e d by a second p a s s a g e on the 50 pm sieve and two s u c c e s s i v e w a s h i n g s and c e n t r i f u g a t i o n s (120 × g for 90 sec.). The pellet c o n s i s t e d in n e a r l y pure isolated, d e e a p s u l a t e d glomeruli w i t h less than 2 % tubular contamination. No afferent or e f f e r e n t a r t e r i o l e s were observed. R e c o v e r y was about 5,000 - 15,000 glomeruli per rat (roughly 0.5-1.5 mg g l o m e r u l a r protein). The glomeruli of HZ and DI rats w e r e p r e p a r e d in p a r a l l e l and kept in 2 ml cold b u f f e r until use. P a p i l l a r y h o m o g e n a t e s from the same rats were p r e p a red a s - ~ o I ~ o w s - Y - T S e - c o n e Z s h a p e d w h i t e p a p i l l a p r o t r u d i n g in the renal pelvis (inner medulla) w a s separated f r o m the r e d - o u t e r medulla, m i n c e d and h o m o g e n i z e d m i l d l y b y 3-5 strokes in a t i s s u e g r i n d e r (Kontes). The r e s u l t i n g suspension was f i l t e r e d t h r o u g h a 25 um stainless sieve to remove large pieces of tissue and was w a s h e d in i0 ml b u f f e r and c e n t r i f u g e d twice (1500 × g for 20 min.). The h o m o g e n i z e d p a p i l l a r y tissue (about 1-2 mg prot. per rat) was kept in 2 ml cold buffer until use.
Incubation A few m i n u t e s b e f o r e incubation, glomeruli and p a p i l l a r y h o m o g e n a t e s were c e n t r i f u g e d (1500 × g for i0 min) and r e s u s p e n d e d in a b u f f e r similar to that used in the p r e p a r a t i o n steps w i t h a d d i t i o n of 1 m M CaCl 2. G l o m e r u l i and p a p i l l a r y h o m o g e n a t e s from both HZ and DI rats w e r e incubated in this buffer, in the a b s e n c e or in the p r e s e n c e of a r a c h i d o n i c acid (A.A.) 5 pg/ml final concentration, This c o n c e n t r a t i o n p r o d u c e d maximal s t i m u l a t i o n in b o t h p a p i l l a r y h o m o g e n a t e s and glomeruli. A.A. (sodium salt) was o b t a i n e d from S i g m a (St Louis, Mo.), kept under nitrogen a t m o s p h e r e at - 2 0 ° C and e x t e m p o r a n e o u s l y d i s s o l v e d in e t h a n o l - w a t e r (7:10, v/v). Incubation was c a r r i e d out in separate 200 ~i aliquotes in 1 ml p l a s t i c tubes at 37°C in room atmosphere under continuous agitation. P r o t e i n c o n c e n t r a t i o n (200-600 ~g/ml) was d e t e r m i n e d a c c o r d i n g to Lowry et al. (17) w i t h m i n o r modifications. I n c u b a t i o n was s t o p p e d after 15, 30, 60 and 90 m i n u t e s by centrifugation at 3000 × g for 2 m i n u t e s at 10°C. S u p e r n a t a n t s of each tube w e r e c o l l e c t e d and frozen u n t i l PGE 2 a s s a y was performed.
PGE 2 a s s a x R a d i o i m m u n o a s s a y of PGE 2 was c a r r i e d out a c c o r d i n g to Dray et al. (18). A n t i - P G E 2 a n t i s e r u m was p u r c h a s e d from I n s t i t u t P a s t e u r (Paris). The i n c u b a t i o n m e d i u m b e i n g
AUGUST 1980 VOL. 20 NO. 2
351
PROSTAGLANDINS
protein-free, PGE 2 was a s s a y e d in the s u p e r n a t a n t w i t h o u t prior extraction. We have shown this direct p r o c e d u r e to provide results similar to those o b t a i n e d after extraction o v e r a wide range of p r o s t a g l a n d i n c o n c e n t r a t i o n s (8). C r o s s - r e a c t i o n of a r a c h i d o n i c acid w i t h PGE 2 a n t i s e r u m was m e a s u r e d in s e p a r a t e tubes in the a b s e n c e of g l o m e r u l i or p a p i l l a and was s u b t r a c t e d w h e n adequate.
Calculations
and s t a t i s t i c a l analysis
PGE 2 c o n c e n t r a t i o n was m e a s u r e d in supernatants of the f o l l o w i n g samples : g l o m e r u l i and p a p i l l a of control HZ and h o m o z y g o u s DI rats, w i t h o u t or w i t h A.A. after 15, 30, 60 and 90 min of incubation. Results w e r e e x p r e s s e d as pg of PGE 2 p r o d u c e d per mg p r o t e i n a f t e r 15, 30, 60 or 90 minutes. PGE 2 p r o d u c t i o n as pg per m g p r o t e i n per m i n u t e d u r i n g the time intervals 0-15, 15-30, 30-60, 60-90 min was c a l c u l a t e d for each experiment. PGE2 c o n c e n t r a t i o n in the s u p e r n a t a n t at time zero just p r i o r to the i n c u b a t i o n was found to be negligible. The results were a n a l y z e d u s i n g analysis of v a r i a n c e with fixed factors w i t h o u t replication (19). In a first analysis, the d e p e n d e n c y of the total p r o d u c t i o n of PGE 2 from 0 to 90 min on the three following factors was considered : i) rat (DI v e r s u s HZ), 2) e x p e r i m e n t a l condition (without or w i t h A.A.), 3) s u c c e s s i v e e x p e r i m e n t s , n° 1 to 7 c o r r e s p o n d i n g to d i f f e r e n t days. G l o m e r u l i and p a p i l l a r y h o m o g e n a t e s w e r e assessed separately. It a p p e a r e d from our results that the t i m e - c o u r s e of PGE 2 p r o d u c t i o n by g l o m e r u l i w i t h o u t A.A. and those by g l o m e r u l i and p a p i l l a w i t h A.A. w e r e b i p h a s i c w i t h an early p h a s e of high synthesis rate and a second phase of lower but sustained p r o d u c t i o n (as shown in Fig. i). For this reason, in a second set of a n a l y s i s of variance, we c o n s i d e r e d s e p a r a t e l y the first 15 m i n and the follow i n g p e r i o d from 15 to 90 min.
RESULTS M e a n b o d y w e i g h t was 305 ± 28 and 249 ± 22 g (mean ± SEM) in HZ and DI rats respectively. DI rats' impaired growth rate has a l r e a d y b e e n reported (15). Mean urine o s m o l a l i t y (urine c o l l e c t e d by b l a d d e r p u n c t u r e just after anesthesia) was 1190 ± 180 and 120 ± 27 m O s m / k g for HZ and DI rats respectively.
352
AUGUST 1980 VOL. 20 NO. 2
PROSTAGLANDINS
PGE2_ssynthesis by p a p i l l a r y h o m o g e n a t e s PGE 2 a c c u m u l a t e d p r o g r e s s i v e l y w i t h time in the incubation m e d i u m of p a p i l l a r y h o m o g e n a t e s f r o m b o t h HZ and DI rats and this a c c u m u l a t i o n was e n h a n c e d by the addition of A°A. (Table I). Nevertheless, in the absence (basal) as w e l l as in the p r e s e n c e of A.A., PGE 2 production was s i g n i f i c a n t l y lower in p a p i l l a r y h o m o g e n a t e s from DI than f r o m HZ rats (Table 1 and T a b l e 3, 0-90 min). In o r d e r to b e t t e r c h a r a c t e r i z e the t i m e - c o u r s e of PGE2 synthesis, we c a l c u l a t e d the m e a n rates of PGE 2 p r o d u c t i o n per minute for the successive p e r i o d s of incub a t i o n (Fig. i, left panel). This c a l c u l a t i o n provides independent values more s u i t a b l e for an analysis of
B
a
s
PAPILLA a l
GLOMERULI Basal
400
=_ E
300 20O
4O
30 2O
100
m
101-
E
=E 1500 ~ .
=Hz
WithAA
3ooI
WithAA
E 2ooi
1000
I00~
5OO 601
300
40~"
100
20~
3'0
°'o
90
rain
o
;s
6'0'
rai9On
Fiqure !. Time-course of PGE 2 p r o d u c t i o n b y p a p i l l a r y h o m o g e n a t e s (left panel) and isolated g l o m e r u l i (right panel). The m e a n p r o d u c t i o n p e r min has b e e n calculated from the time intervals 0-15, 15-30, 30-60 and 60-90 min in basal conditions (top) and w i t h a d d i t i o n of 5 ~g/ml arachidonic acid (bottom). Closed circles = HZ control rats, open circles = DI rats (mean ± I S E M of 7 experiments ; note the scale change in ordinates of the l o w e r graphs).
AUGUST 1980 VOL. 20 NO. 2
353
PROSTAGLANDINS
variance (cumulated values d e p e n d on the values o b t a i n e d at p r e c e e d i n g times). Fig. I shows that the rate of PGE 2 synthesis d e c r e a s e d w i t h time in both DI and HZ p a p i l l a and r e m a i n e d lower in DI rats compared to t h e i r HZ controls at all times, e i t h e r w i t h or w i t h o u t A.A. The difference b e t w e e n HZ and DI was fairly stable in early and late p e r i o d s (Table 2) w i t h DI/HZ ratios averaging 0.60-0.70 and was s i g n i f i c a n t at the 1 % level (Table 3). The s t i m u l a t i o n induced by A.A. was h i g h e r in the e a r l y than in the late p e r i o d (about × 3.3, p < 0 . 0 0 1 ~ a n d x 1.4, p < 0.01 respectively) and was s i m i l a r in both groups of rats (no i n t e r a c t i o n between the Ist and the 2nd factor) (Tables 2 and 3). F i n a l l y we o b s e r v e d a small but s i g n i f i c a n t v a r i a t i o n b e t w e e n e x p e r i m e n t s p e r f o r m e d on d i f f e r e n t days but this factor did not interfere w i t h the o t h e r factors u n d e r s t u d y (no interaction, Table 3).
P G E 2 - s y n t h e s i s b~ i s o l a t e d qlomeruli PGE 2 a c c u m u l a t e d p r o g r e s s i v e l y w i t h time in both HZ and DI g l o m e r u l a r incubates and the PGE 2 p r o d u c t i o n was increased by the a d j o n c t i o n of A.A, (Table I). C o n t r a r y to what was o b s e r v e d in the papilla, the DI rat glomeruli p r o d u c e d s i g n i f i c a n t l y more P G E 2 than the glomeruli of their HZ controls (Table 1 and Table 3, 0-90 min). W h e n PGE 2 synthesis rates per min w e r e c a l c u l a t e d (Fig. I, right panel) it was o b s e r v e d that the m a g n i t u d e of the d i f f e r e n c e b e t w e e n HZ and DI was not constant. In b a s a l conditions for example, the PGE 2 ~ynthesis in the interval 0-15 m i n was o n l y 1.27 fold h i g h e r in DI than in HZ g l o m e r u l i w h i l e it was 2.72 fold h i g h e r in the 15-30 m i n p e r i o d and remained close to 2 in the two later periods. A similar p a t t e r n was o b s e r v e d in the p r e s e n c e o f A.A, (Table 2). Only in the second p h a s e (15-90 min) was the d i f f e r e n c e b e t w e e n both groups of rats s i g n i f i c a n t (Table 3). S t i m u l ~ t i o n b y A.A. was h i g h e r in the first 15 min (about × 6.5) than in the following periods (× 3) and was s i m i l a r in both groups of rats (no interaction) (Table 3). The o b s e r v e d b e t w e e n e x p e r i m e n t v a r i a t i o n did not interfere w i t h the two other factors (Table 3). A l t h o u g h the purpose of this work was not to compare PGE 2 p r o d u c t i o n by d i f f e r e n t renal structures, it is interesting to note that PGE 2 synthesis per mg of p r o t e i n was m a r k e d l y g r e a t e r in the p a p i l l a than in the glomerulio On the contrary, the d e g r e e of s t i m u l a t i o n b y A.A. was h i g h e r for the glomerull than for the papilla.
354
AUGUST 1980 VOL. 20 NO. 2
u~
O
O
< O
00 O
mean SEM
mean SEM
60
90
954 216
801 145
603 120
499 74
HZ
921 156
633 124
DI
1529 273
1276 303
Basal
4911 648
4414 664
4101 707
3494 485
HZ
with
6653 1044
5604 701
4970 698
4264 536
DI
A.A.
19990 2950
16610 2270
9940 1440
6170 1030
HZ
6940 1610
3770 940
DI
13110 2600
10560 2160
Basal
39870 5980
32620 4570
26400 4160
21760 3120
HZ
with
25670 3080
20920 2330
15970 2100
12480 1195
DI
A.A.
Papilla ............................................
1 : P r o d u c t i o n o f P G E 2 b y i s o l a t e d g l o m e r u l i a n d p a p i l l a r y h o m o g e n a t e s w i t h o u t (basal) o r w i t h a r a c h i d o n i c a c i d (A.A.) 5 ~ g / m l a f t e r 15, 30, 60 a n d 90 m i n i n c u b a t i o n . R e s u l t s a r e e x p r e s s e d as p g P G E 2 p e r m g p r o t e i n (mean ± S E M of 7 e x p e r i m e n t s ) . HZ = h e t e r o z y g o u s c o n t r o l r a t s ; DI = r a t s w i t h d i a b e t e s i n s i p i d u s .
mean SEM
30
Table
mean SEM
15
minutes (cumulative)
Glomeruli
....................................
> Z
>
O
U~
33.24
6.24
0-15 m i n
15-90 min
411
186
0-15 min
15-90 min
124
251
145
12.76
42.40
17.67
DI
0.67
0.61
0.65
2.04
1.27
1.65
249
1424
445
20.31
229
-55.27
HZ
181
805
285
34.94
272
74.53
DI
0.73
0.57
0.64
1.72
1.19
1.35
1.34
3.46
2.00
3.25
6.89
5.15
•
1.46
3.21
1.97
2.74
6.45
4.22
Table 2 : Mean PGE 2 p r o d u c t i o n per m i n u t e from 0 to 90 or from 0 to 15 and 15 to 90 m i n u t e s by i s o l a t e d glomeruli and p a p i l l a r y h o m o g e n a t e s from HZ and DI rats. R e s u l t s are e x p r e s s e d as pg PGE 2 p e r m g p r o t e i n per m i n u t e (means of 7 experiments) or as ratios b e t w e e n DI and HZ rats or b e t w e e n A.A. and basal conditions.
223
0-90 m i n
PAPILLA
10.74
.0-90 min
GLOMERULI
HZ
Basal with A.A. A.A./Basal ........................................................................ pg/mg prot°/min D_~I pg/mg prot./min DI HZ HZ HZ DI
Z
O
z o
O
< o
kO
Table
3
12.68
< 0.001
F =
P
240
< 0.001
F =
p
p
16.01
< 0.01
F =
min
p
5.16
< 0.05
F = p
214
NS
3.15
min
< 0.001
F =
F =
0-15
p
7.50
< 0.05
F = p
44.37
< 0.01
15.09
min
< 0.001
F =
p
F =
15-90
p
9.80 < 0.01
F = p
66.21
< 0.01
2%.54
min
< 0.001
F =
p
F =
0-90
p
5.76 < 0.05
F = p
85.19
< 0.01
21.06
min
< 0.001
F =
p
F =
0~15
F
F
F
p
p
p
min
=
=
< 0.05
12.72
< 0.01
17.55
< 0.01
= 20.04
15-90
Papilla ........................................
: Analysis of variance performed on the pooled data of the 7 individual experiments the means of which a r e s h o w n in T a b l e 2. I n t e r a c t i o n s between rat groups and experimental conditions, or between experiment order and either rat groups or experimental conditions are not significant (not s h o w n ) .
Variation between the different experiments (degrees of freedom : 6,6)
Variation due to the experimental conditions : with vs without arachidonic acid (degrees of freedom : 1,6)
Variation due to the rat group = DI vs HZ (degrees of freedom : 1,6)
0-90
Glomeruli ........................................
Z
> Z
>
O
PROSTAGLANDINS
DISCUSSION D e p e n d e n c y of renal P G synthesis on a n t i d i u r e t i c h o r m o n e has r e c e n t l y b e e n e x e m p l i f i e d by the f i n d i n g that urinary PGE 2 and PGF2e e x c r e t i o n was g r e a t l y reduced in B r a t t l e b o r o rats c o m p a r e d to control rats and could be r e s t o r e d by a d m i n i s t r a t i o n of exogenous v a s o p r e s s i n (13, 14) or of its analogue d e - a m i n o - D - a r g i n i n e v a s o p r e s s i n (dDAVP) (14). The p r e s e n t in vitro e x p e r i m e n t s have shown that p a p i l l a from B r a t t l e b o r o DI rats i n d e e d exhibits a reduced PGE 2 synthesis. Unexpectedly, an i n c r e a s e d PGE 2 synthesis was d e m o n s t r a t e d in the glomeruli of the same rats c o m p a r e d to HZ controls. The a c c u m u l a t i o n of PGE 2 in the incubation m e d i u m really c o r r e s p o n d e d to "de novo" synthesis and did not reflect a p r o g r e s s i v e release of p r e e x i s t i n g i n t r a c e l l u lar stores since the c o n c e n t r a t i o n of PGE 2 was m a r k e d l y e n h a n c e d by the a d d i t i o n of its p r e c u r s o r A.A. in b o t h tissues. The synthetic rates of PGE 2 in p a p i l l a and glomeruli d e c r e a s e d p r o g r e s s i v e l y w i t h time, a p p r o a c h i n g an e q u i l i b r i u m after 15-30 minutes. This t i m e - c o u r s e was similar to that p r e v i o u s l y r e p o r t e d for rat g l o m e r u l i (8) and p a p i l l a (20)° Different q u e s t i o n s are b r o u g h t up by our o b s e r v a tions : W h a t are the c e l l u l a r m e c h a n i s m s responsible for the alterations in PGE 2 synthesis o b s e r v e d in DI rats ? How can a single d e f e c t i.e. ADH deficiency, result in opposite effects on glomeruli and p a p i l l a ? W h a t are the p h y s i o l o g i c a l i m p l i c a t i o n s of the altered PGE 2 synthesis p a t t e r n in the k i d n e y of DI rats ? H e t e r o z y g o u s and h o m o z y g o u s B r a t t l e b o r o rats d i f f e r by the absence of v a s o p r e s s i n and b y its c o n s e q u e n c e s including i n a b i l i t y to concentrate urine (15), p o t a s s i u m d e f i c i e n c y (16), and s t i m u l a t e d r e n i n - a n g i o t e n s i n s y s t e m (16, 21). P a p i l l a r y o s m o l a l i t y of c o n t r o l rats is m u c h h i g h e r t h a n that of DI rats and h y p e r t o n i c m e d i u m has been shown to enhance m a r k e d l y P G synthesis by p a p i l l a r y slices in vitro (22). However, d i f f e r e n c e s in p a p i l l a r y o s m o l a l i t y and the o t h e r d i f f e r e n c e s c i t e d above are met only in vivo. In the in vitro c o n d i t i o n s used here, the p r o p e r h o r m o n a l e n v i r o n m e n t of each rat was no l o n g e r maintained. P r e p a r a t i o n of the tissues i n c l u d e d several w a s h i n g s w i t h large v o l u m e s of i s o t o n i c b u f f e r and subsequently, all samples were i n c u b a t e d in the same b u f f e r and in the absence of vasopressin. Moreover, a d d i t i o n of e x o g e n o u s synthetic A%~ or dDAVP (Ferring, Malm~, Sweden), i0 -° M final concentration, did not m o d i f y PGE 2 synthesis in e i t h e r tissue from HZ or DI rats (2 experiments, results not shown). The o b s e r v e d d i f f e r e n c e b e t w e e n DI and
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HZ p r e p a r a t i o n s thus cannot be a t t r i b u t e d to a direct and i m m e d i a t e effect of vasopressin. It can only be a t t r i b u t e d to a chronic m o d i f i c a t i o n in cell function i n d u c e d by the chronic ADH d e f i c i e n c y or b y its functional. consequences. Actually, m o d i f i e d in v i t r o renal PG synthetic rates have also been reported in another chronic rat model, the s p o n t a n e o u s l y h y p e r t e n s i v e rat (23) . P r e c u r s o r a v a i l a b i l i t y is not the f a c t o r m o d i f y i n g PGE 2 synthesis in DI rats since the d i f f e r e n c e b e t w e e n both types of rats p e r s i s t e d in the p r e s e n c e of an excess of A.A. The factors i n v o l v e d thus, m u s t act at a step b e y o n d the d e a c y l a t i o n of phospholipids, probably on c y c l o o x y g e n a s e a c t i v i t y or on one of the enzymes i n t e r v e n i n g in the d e g r a d a t i o n of PGE 2. This is thus different from the direct m o d e of action of the hormonal peptides w h i c h stimulate P G E 2 p r o d u c t i o n t h r o u g h an increase in the a c t i v i t y of p h o s p h o l i p a s e (20). A l t e r n a tively, changes in 9 k e t o - r e d u c t a s e a c t i v i t y could also alter PGE 2 a c c u m u l a t i o n rates. This w o u l d result in inverse changes in PGF2e accumulation. For example, this enzyme a c t i v i t y has been shown to be i n f l u e n c e d by salt intake or r e l a t e d factors (24). S i m u l t a n e o u s m e a s u r e m e n t s of both PGE 2 and PGF2e w o u l d be n e c e s s a r y to c o n f i r m the o c c u r e n c e of this a l t e r a t i o n in DI rats. H o w does the lack of ADH affect p a p i l l a r y and g l o m e rular PGE 2 p r o d u c t i o n in o p p o s i t e ways ? It should be s t r e s s e d that both tissues s t u d i e d are h e t e r o g e n o u s since they include several c e l l u l a r types. Glomeruli comprise at least three d i f f e r e n t types of cells two of which, the m e s a n g i a l and e p i t h e l i a l cells, h a v e been d e m o n s t r a t e d as capable of s y n t h e s i z i n g PGE 2 (25). Similarly, the p a p i l l a contains m a n y different types of cells from vascular, i n t e r s t i t i a l and t u b u l a r origin. It has been shown that at least two of them, the interstitial and the collecting duct cells, p r o d u c e PGs (26, 3, 4, 5). P r o d u c t i o n of PGs by the v a s c u l a r e n d o t h e l i u m in glomerular capillaries and in p a p i l l a r y v a s a recta cannot be r u l e d out since other v a s c u l a r s t r u c t u r e s are known to s y n t h e s i z e PGs (27). As s t a t e d above, A D H d e f i c i e n c y induces several secondary d i s o r d e r s (15, 16, 21). On the other hand, several factors i n c l u d i n g c o n c e n t r a t i o n of various ions and hormones, oxygen, o s m o l a l i t y and pH, have been shown to influence PGE 2 synthesis (28, 20, 2, 21, i). It can be h y p o t h e t i z e d that the p r o d u c t i o n of PGE 2 by each cell type is i n f l u e n c e d to a d i f f e r e n t e x t e n t by each of these factors. For example, ADH, osmolality, oxygen could be m a j o r d e t e r m i n a n t s of m e d u l l a r y synthesis w h i l e a n g i o t e n s i n II could influence p r i m a r i l y g l o m e r u l a r cells.
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The b i p h a s i c t i m e - c o u r s e of PGE 2 s y n t h e s i s rate in glomeruli might be due to d i f f e r e n t s y n t h e t i c patterns in two d i f f e r e n t cell types. That the d i f f e r e n c e between HZ and DI is more m a r k e d in the later phase suggests that PGE 2 synthesis rate might be increased in the cells responsible for the low but sustained PGE 2 production. Changes in the p r o p o r t i o n of the d i f f e r e n t cells in e a c h structure could also a c c o u n t for the d i f f e r e n t net PGE 2 p r o d u c t i o n o b s e r v e d in DI rats c o m p a r e d to HZ controls. As g l o m e r u l a r m e s a n g i a l cells p r o d u c e a b o u t 15 times more PGE 2 per mg protein or per cell than do g l o m e r u l a r epithelial cells (29), a small increase in mesangial versus e p i t h e l i a l cell n u m b e r in glomeruli from DI rats, could affect m a r k e d l y the n e t PGE 2 production, even in the absence of any change in the cellular m e c h a n i s m of PGE 2 synthesis in e i t h e r cell. In the papilla, indirect arguments suggest that collecting duct cells are at least p a r t l y responsible for the PGE 2 synthesis defect o b s e r v e d in DI rats : I/ B o t h arginine v a s o p r e s s i n and a n g i o t e n s i n II stimulate P G synthesis in c u l t u r e d r e n o m e d u l l a r y cells, but to unequal degrees (30). A n g i o t e n s i n b e e i n g 7 times m o r e potent, it can be a s s u ~ e d that the e f f e c t of the lack of ADH on interstitial cells in DI rats is b l u n t e d by the o p p o s i t e effect induced by h i g h r e n i n - a n g i o t e n s i n levels in these rats (16, 21). 2/ ADH, but not dDAVP, stimulates PGE 2 synthesis by interstitial cells in culture (31) w h i l e both ADH and dDAVP can s i m i l a r l y restore a normal PGE 2 urinary excretion in DI rats in vivo (14). Thus, another cell type, likely the c o l l e c t i n g duct cells, is i n v o l v e d (26). Finally, m o r p h o l o g i c alterations of the medulla, i.e. changes in the p r o p o r t i o n of i n t e r s t i t i a l versus collecting duct cell mass (32) due to the h i g h diuresis and a l t e r e d p o t a s s i u m balance (16) m i g h t contribute to changes in net p a p i l l a r y PGE 2 production. W h a t functional consequences can be e x p e c t e d to result from the PGE 2 a l t e r e d synthesis o b s e r v e d in DI rat p a p i l l a and glomeruli ? C i r c u l a t i n g PGE 2 being catabolized in one pass t h r o u g h the lungs it is considered that its role is limited to the cells in which it is s y n t h e s i z e d or to the n e i g h b o u r i n g cells. Thus, the consequences of impaired synthetic rates p r o b a b l y d e p e n d on the p a r t i c u l a r cell type r e s p o n s i b l e for the anomaly in each tissue. As m u l t i c e l l u l a r structures were analyzed in our study, only assumptions can be p r o p o s e d regarding the p h y s i o l o g i c a l i m p l i c a t i o n s of our observations.
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If the d e c r e a s e d p a p i l l a r y PGE 2 synthesis is due to c o l l e c t i n g duct cells, this w i l l have little effect on k i d n e y f u n c t i o n in DI rats as the main role attributed to PGs in these cells is to m o d u l a t e the h y d r o osmotic effects of ADH (12) w h i c h a c t u a l l y does not occur in these rats. The role of PGE 2 p r o d u c e d by medullary interstitial cells is not clear. In some studies, PGE 2 has been r e p o r t e d to have a v a s o d i l a t o r y e f f e c t on deep c o r t i c a l and m e d u l l a r y v a s c u l a t u r e (i). A defect in i n t e r s t i t i a l cell synthetic rate could a f f e c t deep nephron hemodynamics. It is of interest to note that although w h o l e renal filtration rate is not d e c r e a s e d in DI rats, the normal single n e p h r o n f i l t r a t i o n rate h e t e r o g e n e i t y as well as the anatomical n e p h r o n h e t e r o geneity p r e s e n t in rats and all mammals, are a b o l i s h e d in these rats (33). The functional implications of the i n c r e a s e d PGE 2 synthesis in DI rats glomeruli are d i f f i c u l t to appreciate. As for the papilla, they m a y vary a c c o r d i n g to the cell type(s) r e s p o n s i b l e for this change. Actually, very little is k n o w n yet about the role and the regulation of l o c a l l y p r o d u c e d PGs in the glomeruli (25, 29, 34). Inhibition of PG synthesis has r e v e a l e d a role for PGs in m o d u l a t i n g the e f f e c t s of a n g i o t e n s i n on the ultrafiltration c o e f f i c i e n t (9) and also in a f f e c t i n g the u l t r a s t r u c t u r e of epithelial and mesangial cells (ii) and the control of the feed-back m e c h a n i s m (i0). In summary, in vivo studies h a d r e v e a l e d that whole k i d n e y PGE 2 p r o d u c t i o n was m a r k e d l y r e d u c e d i n DI rats (13, 14) ; by studying two different k i d n e y structures in v i t r o we have shown that this defect is the net result of a d e c r e a s e d PGE 2 synthesis by the p a p i l l a and an i n c r e a s e d synthesis by the glomeruli. These changes m i g h t be brought about e i t h e r by m o d i f i c a t i o n s of c y c l o o x y g e n a s e or 9 - k e t o - r e d u c t a s e activities or by m o d i f i c a t i o n s in the p r o p o r t i o n s of the d i f f e r e n t cell types present in each structure. To further progress in our u n d e r s t a n d i n g of these phenomenons, the next logical step w i l l imply w o r k i n g on i s o l a t e d c e l l u l a r populations.
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ACKNOWLEDGMENTS This w o r k was s u p p o r t e d by grants from the "D~14gation G 4 n 4 r a l e ~ la Recherche S c i e n t i f i q u e et Technique" (n ° 77.7.1395 and 79.7.0985). P r e l i m i n a r y r e s u l t s w e r e p u b l i s h e d in a b s t r a c t f o r m : Abstracts of the 12th A n n u a l M e e t i n g of the A m e r i c a n S o c i e t y of Nephrology, K i d n e y Int. 16 : 847, 1979. We thank Mrs Chantal Lajotte for excellent technical assistance. REFERENCES
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Editor: Gabor Kaley Received: 3-11-80 Accepted: 6-20-80
AUGUST 1980 VOL. 20 NO. 2
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