Intrarenal urea and electrolyte concentrations as influenced by water diuresis and by hydrochlorothiazide

EUROPEAN

JOURNAL

OF

PHARMACOLOGY

1 (1967)

124-139.

NORTH-HOLLAND

PUBL.

COMP.,

AMSTERDAM

I N T R A R E N A L U R E A A N D ELECTROLYTE C O N C E N T R A T I O N S AS INFLUENCED BY W A T E R DIURESIS A N D BY H Y D R O C H L O R O T H I A Z I D E * F r a n q o i s e R O C H - R A M E L a n d G. P E T E R S I n s t i t u t de P h a r m a c o l o g i c de l' U n i v e r s i t Y , L a u s a n n e , S w i t z e r l a n d

Accepted 22 D e c e m b e r 1966

F r a n ¢ o i s e ROCH-RAMEL and G. P E T E R S , I n t r a r e n a l u r e a and e l e c t r o l y t e c o n c e n t r a t i o n s as i n f l u e n c e d by w a t e r d i u r e s i s and by h y d r o c h l o r o t h i a z i d e , E u r o p e a n J. P h a r m a c o l . 1 (1967) 124-139. C o n c e n t r a t i o n s of u r e a , a m m o n i u m , s o d i u m and p o t a s s i u m a s well a s w a t e r content w e r e m e a s u r e d in the r e n a l papilla, m e d u l l a and c o r t e x , the h e a r t m u s c l e , p l a s m a and u r i n e of r a t s , in the dehydrated state, the n o n - d i u r e t i c state and a f t e r 3 o r 8 h r of s u s t a i n e d b r i s k w a t e r d i u r e s i s . The s a m e m e a s u r e m e n t s w e r e made on r a t s which had r e c e i v e d a n a t r i u r e t i c d o s e of h y d r o c h l o r o t h i a z i d e d u r i n g the 8th h r of w a t e r d i u r e s i s . W a t e r content of r e n a l t i s s u e i n c r e a s e d f r o m c o r t e x to i n n e r m e d u l l a in all conditions. In h y d r a t e d r a t s the w a t e r content of all l a y e r s of r e n a l t i s s u e i n c r e a s e d d u r i n g the f i r s t 3 h r and r e m a i n e d stable t h e r e a f t e r . "Exaltation" of u r e a e x c r e t i o n continued d u r i n g the f i r s t 2 h r of w a t e r diu r e s i s , but s u b s i d e d s u b s e q u e n t l y . U r i n a r y u r e a c o n c e n t r a t i o n s w e r e equal to, o r h i g h e r than, u r e a c o n c e n t r a t i o n s in p a p i l l a r y w a t e r in the dehydrated and in the n o n - d i u r e t i c s t a t e s . After 3 h r of w a t e r d i u r e s i s , u r e a c o n c e n t r a t i o n s d e c r e a s e d in all l a y e r s of r e n a l t i s s u e . The p a p i l l a r y u r e a c o n c e n t r a t i o n r e m a i n e d c o n s i s t e n t l y h i g h e r than the u r i n a r y c o n c e n t r a t i o n . The p a p i l l o - u r i n a r y u r e a g r a d i e n t of app r o x i m a t e l y 80 m m o l / 1 d e c r e a s e d to 19 m m o l / 1 a f t e r 8 h r of w a t e r d i u r e s i s , and d i s a p p e a r e d in continuous w a t e r d i u r e s i s s u s t a i n e d o v e r 30 days. T h e s e data a r e i n t e r p r e t e d a s a c o n s e q u e n c e of s e q u e s t r a t i o n o r t i s s u e binding of u r e a in c o m p a r t m e n t s of different a c c e s s i b i l i t y to w a t e r flowing t h r o u g h p a p i l l a r y and m e d u l l a r y t i s s u e s . C o r t i c a l t i s s u e w a t e r contained m o r e u r e a than p l a s m a , u n d e r all conditions investigated. A p a r t of c o r t i c a l u r e a may be s e q u e s t e r e d in p a r t i c u l a r c o m p a r t m e n t s . The c o t r i c o - p a p i l l a r y s o d i u m c o n c e n t r a t i o n p r o f i l e c o r r e s p o n d e d to a 2.8-5.1 fold c o n c e n t r a t i o n r a t i o for sodiu m in the n o n - d i u r e t i c and in the d e h y d r a t e d s t a t e s and d e c r e a s e d d u r i n g 8 h r of w a t e r d i u r e s i s . The u r i n a r y s o d i u m c o n c e n t r a t i o n c o n s i s t e n t l y r e m a i n e d m u c h l o w e r than r e n a l o r p l a s m a c o n c e n t r a t i o n s . The low s o d i u m c o n c e n t r a t i o n in c o r t i c a l t i s s u e w a t e r a l l o w s an u p p e r l i m i t of a p p r o x i m a t e l y 1/2 to be set f o r the c o n t r i b u t i o n of i n t r a t u b u l a r fluid to total t i s s u e w a t e r . P o t a s s i u m c o n c e n t r a t i o n in p a p i l l a r y t i s s u e w a t e r was equal to o r l o w e r than c o r t i c a l p o t a s s i u m c o n c e n t r a t i o n and did not change s i g n i f i c a n t ly in w a t e r d i u r e s i s . Calculated u r i n a r y o s m o l a l i t i e s w e r e h i g h e r than m e a s u r e d v a l u e s in c o n c e n t r a t e d u r i n e and in p l a s m a , but w e r e equal in dilute u r i n e . The o s m o l a r c o n c e n t r a t i o n p r o f i l e s in r e n a l t i s s u e depended m o r e on u r e a than on salt c o n c e n t r a t i o n s . U r i n a r y a m m o n i u m c o n c e n t r a t i o n w a s h i g h e r than the p a p i l l a r y c o n c e n t r a t i o n in the d e h y d r a t e d and the n o n - d i u r e t i c state, but l o w e r in w a t e r d i u r e s i s . H y d r o e h l o r o t h i a z i d e , in w a t e r d i u r e s i s , c a u s e d a c o n s i d e r a b l e i n c r e a s e in the u r i n a r y s o d i u m c o n c e n t r a t i o n and e x c r e t i o n , and a slight i n c r e a s e in the u r i n a r y p o t a s s i u m e x c r e t i o n , but did not i n c r e a s e u r i n e flow. H y d r o c h l o r o t h i a z i d e did not c a u s e a significant d e c r e a s e in the r e n a l c l e a r a n c e of u r e a und e r t h e s e c i r c u m s t a n c e s , but induced a slight d e p r e s s i o n of p a p i l l a r y u r e a c o n c e n t r a t i o n . Urea: r e n a l handling Corticomedullary gradients Urinary concentrating mechanism S e q u e s t r a t i o n of u r e a T r a n s p a p i l l a r y w a t e r flow 1. I N T R O D U C T I O N A c t i v e t r a n s p o r t of u r e a f r o m f l u i d in t h e c o l l e c t in g d u c t s to m e dullary tissue has been shown to o c c u r in p r o t ein-depleted sheep (Schmidt* Supported by F o n d s National S u i s s e de la R e c h e r c h e Scientifique (Grant No. 2966); p r e l i m i n a r y c o m m u n i cation: P e t e r s and R o e h - R a m e l , 1966.

U r e a e x c r e t i o n in w a t e r d i u r e s i s W a t e r d i u r e s i s and c o r t i c o m e d u l l a r y g r a d i e n t s H y d r o c h l o r o t h i a z i d e in w a t e r d i u r e s i s H y d r o c h l o r o t h i a z i d e on r e n a l u r e a

N i e l s e n a n d O s a k i , 1958; S c h m i d t - N i e l s e n e t a l . , 1958; S c h m i d t - N i e l s e n a n d O ' D e l l , 1959), a n d h a s b e e n s u p p o s e d to a c c o u n t f o r a d e c r e a s e i n urea excretion observed after protein depletion i n o t h e r s p e c i e s of m a m m a l s ( S c h m i d t - N i e l s e n , 1958) a s w e l l a s i n m a n ( M u r d a u g h e t a l . , 1958). In the n o r m a l r a t t u b u l a r m i c r o p u n c t u r e e x p e r i m e n t s s h o w , u n d e r a v a r i e t y of c o n d i t i o n s ( d e hydration, mannitol diuresis, or isotonic saline

INTRARENAL UREA AND SALT diuresis) increasing T F / P values for urea throughout the whole length of the nephron which n e v e r r e a c h the T F / P v a l u e s for inulin ( L a s s i t e r et al., 1961, 1964, 1966; U l l r i c h et al., 1963; Clapp, 1966). T h e s e findings a r e c o m p a t i b l e with the a s s u m p t i o n that all i n t r a r e n a l m o v e m e n t s of u r e a o c c u r p a s s i v e l y along c o n c e n t r a t i o n g r a clients, s et up by p r i m a r y m o v e m e n t s of s a l t and w a t e r . T h e s e p a s s i v e m o v e m e n t s may r e s u l t in an i n t r a m e d u l l a r y c y c le of u r e a f r o m c o l l e c t i n g duct fluid through m e d u l l a r y t is s u e , the d e s c e n d ing and a s c e n d i n g limb of H e n l e ' s loop and the distal tubule back to the c o l l e c t i n g duct (fig. 1). T h i s i n t r a m e d u l l a r y c y c l e may account f o r the fact that under many e x p e r i m e n t a l conditions the f r a c t i o n of f i l t e r e d u r e a e x c r e t e d in the u r i n e ( C u r e a / G F R ) a p p r o a c h e s the f r a c t i o n which e s -

[34] i

/ 43%

Fig. 1. Schematic representation of urea concentrations and movements in the lower nephron of a dehydrated rat. Concentrations in tissue, plasma and urine are taken from the present data; intratubular concentration ratios (TF,/pVs) from Lassiter et al. (1961, 1966). The plan represents a juxtamedullary nephron, it is assumed that the T F / P ' s found in proximal and distal tubules of a superficial nephron also apply to "juxtamedullary" tubules, and that concentration ratios found in the "non-diuretic" animal also apply to the dehydrated state. Concentrations of urea are mean values. Supposed diffusional movements of urea are shown by arrows. Percentages apply to the fraction of filtered urea reaching a given point in the nephron.

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c a p e s p r o x i m a l tubular r e a b s o r p t i o n ( P e t e r s and Hedwall, 1963). While c o m p a t i b l e with an e n t i r e l y diffusional d i s t r i b u t i o n of u r e a , the r e s u l t s of m i c r o p u n c t u r e studies in the r a t do not exclude the p o s s i bility of an uphill t r a n s p o r t f r o m the c o l l e c t i n g ducts to m e d u l l a r y t i ssu e. The o c c u r r e n c e of such an uphill t r a n s p o r t in the r at was s u g g e s t e d by s e v e r a l o b s e r v a t i o n s : u r e a c o n c e n t r a t i o n s in p a p i l l a r y t i s s u e w a t e r g r e a t e r than in final u r i n e w e r e o c c a s i o n a l l y found in p r o t e i n - d e p l e t e d nond i u r e t i c r a t s (Bray and P r e s t o n , 1961; T r u n i n g e r and S c h m i d t - N i e l s e n , 1964) but o c c u r r e g u l a r l y in n o r m a l (Bray and P r e s t o n , 1961; Br ay , 1963) as w e l l as in p r o t e i n - d e p l e t e d r a t s (Bray and P r e s t o n , 1961; Clapp, 1966), a f t e r p e r i o d s of 30-210 min of b r i s k mannitol d i u r e s i s . We found even g r e a t e r d i f f e r e n c e s in n o r m a l r a t s a f t e r 3 hr of r ap i d w a t e r d i u r e s i s . The w al l s of the c o l l e c t i n g ducts cannot be a s s u m e d to be i m p e r m e able to u r e a u n d er t h e s e conditions. Any p o s i t i v e d i f f e r e n c e b et w een u r e a c o n c e n t r a t i o n in p a p i l l a r y t i s s u e w a t e r and in final u r i n e ( p a p i l l a r u r i n a r y gradient), t h e r e f o r e , Would p r o v e a c t i v e t r a n s p o r t a c r o s s the w a l l s of the c o l l e c t i n g ducts i f it o c c u r r e d under steady state conditions. If, on the o t h e r hand, p a p i l l o - u r i n a r y u r e a g r a d i e n t s o c c u r s o m e t i m e a f t e r the induction of mannitol or w a t e r d i u r e s i s , but d e c r e a s e subsequently, they could be due to a delay in the w a s h - o u t of u r e a f r o m m e d u l l a r y t i ssu e. The p r e s e n t e x p e r i m e n t s w e r e d e s i g n e d to decide b e tween t h e s e two p o s s i b i l i t i e s . T h e r e a r e few st u d i es on the influence of thiazide d i u r e t i c s on the e x c r e t i o n and i n t r a r e n a l d i s t r i b u t i o n of u r ea. In d e h y d r a t e d r a t s Kobinger (1964) found a l a r g e d e c r e a s e of u r i n a r y as w e l l as p a p i l l a r y u r e a c o n c e n t r a t i o n s , when u r i n e flow was i n c r e a s e d by the a d m i n i s t r a t i o n of b e n d r o f l u m e t h i a z i d e . Though at v a r i a n c e with the r e s u l t s of o t h er i n v e s t i g a t o r s (Baer et al., 1962; H e l l e r et al., 1965), t h e s e data w e r e i n t e r p r e t e d to indicate enhanced diffusional l o s s e s of u r e a f r o m tubular fluid ( P e t e r s , 1966). We a t t e m p t e d to r e p r o d u c e an analogous effect of h y d r o c h l o r o thiazide in w a t e r d i u r e s i s , in o r d e r to avoid m a j o r ch an g es in u r i n e flow which may invalidate c o n c l u s i o n s drawn f r o m the r e l a t i o n s h i p of m e d u l l a r y and u r i n a r y c o n c e n t r a t i o n s of solutes.

2. METHODS The e x p e r i m e n t a l a n i m a l s w e r e m a l e white r a t s of m i x e d stock weighing 220-290 g, kept on

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F. ROCH-RAMEL and G. PETERS

r a t p e l l e t s containing a p p r o x i m a t e l y 20% p r o t e i n and 50 m E q / k g of sodium. Dehydrated rats w e r e d e p r i v e d of w a t e r at a p p r o x i m a t e l y 28o f o r 68-72 hr with f r e e a c c e s s to dry food. B e f o r e dehydration, the dome of the u r i n a r y b l a d d e r was tied off and r e m o v e d , u n d e r e t h e r a n e s t h e s i a , in o r d e r to r e d u c e dead s p a c e and to f a c i l i t a t e the c o l l e c t i o n of u r i n e , and a polyethylene tube was tied into the j u g u l a r vein and led out through the skin of the neck. The deh y d r a t e d a n i m a l s w e r e t r a n s f e r r e d to r e s t r a i n ing c a g e s d e s c r i b e d e l s e w h e r e ( P e t e r s and Hedwall, 1963; Bonjour, 1966); u r i n e was c o l l e c t e d under m i n e r a l oil while the a n i m a l s r e c e i v e d an infusion of v a s o p r e s s i n (0.5 m U / k g min in 2.7 ~ / k g m i n of 0.9% NaC1). When a sufficient v o l ume of u r i n e had a c c u m u l a t e d , the a n i m a l s w e r e quickly decapitated. Blood was c o l l e c t e d on hepa r i n (0.5 m g / m l ) and rapidly centrifuged. The kidneys and the h e a r t w e r e quickly r e m o v e d and blotted f r e e of blood. The kidneys w e r e d i s s e c t e d unde r a b i n o c u l a r loupe into p a p il la (weighing 1.5 to 5 mg), i n n er medulla, o u te r m e d u l l a ( c o m p r i s i n g the i n n er as well as the o u t e r stripe) and c o r t e x . S a m p l e s obtained f r o m one kidney and half of the h e a r t w e r e weighed, d r i e d for t h r e e days at 105 o, r e w e i g h e d for the d e t e r m i n a t i o n of t i s s u e w a t e r , d i s s o l v e d in c o n c e n t r a t e d n i t r i c acid, and a s s a y e d for sodium and p o t a s s i u m c o n c e n t r a t i o n s a f t e r suitable dilution. T i s s u e s f r o m the o t h er kidney, and the o t h e r half of the heart, w e r e quickly h o m o g e n i z e d in d i s t i l l e d w a t e r , using e i t h e r s m a l l g l a s s h o m o g e n i z e r s o r the m i c r o h o m o g e n i z e r a t t a c h m e n t of a S e r v a l l O m n i - M i x e r . The h o m o g e n a t e s w e r e c o v e r e d with m i n e r a l oil, put in a boiling w a t e r bath f o r 5 min and w e r e then e i t h e r a n a l y z e d i m m e d i a t e l y f o r u r e a and a m m o n i a c o n c e n t r a t i o n s , or f r o z e n and a n a l y z e d l a t e r . T i s s u e h o m o g e n a t e s w e r e a n a l y z e d for w a t e r content, sodium, p o t a s s i u m , u r e a and a m m o n i a c o n c e n t r a t i o n s . The o s m o l a l i t y of t i s s u e w a t e r was computed as (2[Na+] + [g+] + [NH4+]) + [urea] and e x p r e s s e d in m O s m / k g H20. In s a m p l e s of p l a s m a and u r i n e o s m o l a l i t y was m e a s ured directly. "Non-diuretic" r a t s u n d e r w e n t r e s e c t i o n of the u r i n a r y b l a d d e r and w e r e put back into t h e i r n o r m a l c a g e s with f r e e a c c e s s to food and w a t e r . They w e r e r e m o v e d f r o m th e s e c a g e s 24 hr l a t e r ; u r i n e was c o l l e c t e d f o r 30-150 min *. The a n i m a l s w e r e then killed; t i s s u e s a m p l e s w e r e obtained as d e s c r i b e d above. Water diuresis w a s induced in r a t s 24 h r a f t e r r e s e c t i o n of the u r i n a r y bladder, followed by " t r a i n i n g " , i . e . , the a d m i n i s t r a t i o n of 50 m l / k g

of d i s t i l l e d w a t e r by s t o m a c h tube. On the ex p e r i m e n t a l days the r a t s r e c e i v e d another dose of 50 m l / k g of d i s t i l l e d w a t e r by s t o m a c h tube, and w e r e then put in r e s t r a i n i n g cages. W a t e r d i u r e s i s was s u s t a i n e d by the infusion of a 3% solution of glucose, containing 0.05% of NaC1, at a r a t e of 0.2 m l / r a t , min (0.69-0.91 m l / k g , min) through a tail vein. A f t e r an e q u i l i b r a t i o n p e r i o d of 1 hr, u r i n e was c o l l e c t e d u n d er m i n e r a l oil in two p e r i o d s of 1 hr each, during the 2nd and 3rd hr of w a t e r d i u r e s i s . The a n i m a l s w e r e d e c a p i tated at the end of the s e c o n d c o l l e c t i o n period. Blood and t i s s u e s w e r e p r o c e s s e d as d e s c r i b e d above. Prolonged water diuresis was m a i n t a i n e d in the s a m e way f o r 8 hr. U r i n e was c o l l e c t e d d u r ing the 2nd and again during the 8th hr. The r a t s w e r e k i l l e d and t i s s u e s p r e p a r e d f o r a n a l y s i s at the end of the 8th hr. Hydrochlorothiazide w as given during the 8th hr of p r o l o n g e d w a t e r d i u r e s i s in a dose found in p r e l i m i n a r y e x p e r i m e n t s to e x e r t a s u s t a i n e d n a t r i u r e t i c effect f o r 1-2 hr. The drug was d i s s o l v e d rapidly in n o r m a l NaOH solution (0.1 ml / 1 0 mg) and i m m e d i a t e l y diluted to the d e s i r e d volume. A p r i m i n g i n j ect i o n of 5.0 m g / k g in 1.0 m l / k g 0.9% NaC1 was i n j e c t e d i n t r a v e n o u s l y 6 hr and 45 min a f t e r the beginning of w a t e r d i u r e s i s , followed by the infusion of 1.0 m g / k g , min of hyd r o c h l o r o t h i a z i d e added to the hypotonic g l u co se solution infused. U r i n e was c o l l e c t e d during the 8th hr of w a t e r d i u r e s i s . The a n i m a l s w e r e k i l l e d at the end of this hour, i.e., 75 min a f t e r r e c e i v i n g the p r i m i n g i n j ect i o n of h y d r o c h l o r o thiazide.

2.1. Analytical methods C o n c e n t r a t i o n s of sodium and p o t a s s i u m w e r e m e a s u r e d by d i r e c t f l a m e p h o t o m e t r y a f t e r su i t able dilution of d r i e d t i s s u e s d i s s o l v e d in conc e n t r a t e d HNO3, or of n o n - d e p r o t e i n i z e d p l a s m a or urine. Osmolalities of 2.0 to 0.5 ml of u r i n e or p l a s m a w e r e m e a s u r e d as f r e e z i n g point d e p r e s s i o n using a p p a r a t u s d ev el o p ed in this l a b o r a t o r y ** f r o m the o r i g i n a l d e s c r i p t i o n by Bowman et al. (1954). The m a j o r m o d i f i c a t i o n s w e r e the following: 1) r e f r i g e r a t i o n was p r o v i d e d by a P e l l e t i e r b a t t e r y ; 2) t h e r m a l contact between the cold plate and the s a m p l e contained in a 1.5 ml polyethylene tube was e s t a b l i s h e d by liquid m e r cury. When the s a m p l e had f r o z e n , r e f r i g e r a * At room temperature. ** Built by Electronique M~dicale, H5pital Cantonal, 70, Av. B6thusy, 1005 Lausanne, Switzerland.

INTRARENAL UREA AND SALT tion c o u l d be i n t e r r u p t e d by l e t t i n g the m e r c u r y flow b a c k into a r e s e r v o i r ; 3) the s a m p l e w a s r a p i d l y s t i r r e d by two s m a l l w i r e s ; the n e e d to " s e e d " the s a m p l e w i t h AgI w a s thus e l i m i n a t e d ; and 4) the r e s i s t a n c e of the t h e r m i s t o r p r o b e w a s r e a d d i r e c t l y on a s e n s i t i v e a m m e t e r . Urea concentrations w e r e m e a s u r e d in m o s t s a m p l e s by two d i f f e r e n t m e t h o d s , m e a s u r e m e n t s b e i n g r e p e a t e d w h e n the r e s u l t s d i f f e r e d by m o r e than 15%. In a few s a m p l e s , only one m e t h o d w a s u s e d in d u p l i c a t e . The m e t h o d s w e r e considerably modified from original descriptions and w i l l be d e s c r i b e d in d e t a i l . a) Microdiffusion ( m o d i f i e d f r o m W e i s s and L o n g l e y , 1960) followed by reaction of ammonia with phenol and hypochlorite ( m o d i f i e d f r o m F a w c e t t and Scott, 1960): M i c r o d i f f u s i o n w a s c a r r i e d out in 5 m l E r l e n m e y e r f l a s k s f i t t e d with g l a s s s t o p p e r s . T h e b o t t o m of e a c h g l a s s s t o p p e r b o r e a 2 - 3 m m g l a s s r o d t e r m i n a t i n g in a f l a t t e n e d b e a d w h i c h w a s d i p p e d in a 2% s o l u t i o n of b o r i c acid, so that one d r o p of the a c i d r e m a i n e d on the b e a d and s e r v e d to a b s o r b the a m m o n i a l i b e r a t e d by the r e a c t i o n . 20 pl of s t a n d a r d s o lution, p l a s m a , d i l u t e d u r i n e , o r d i l u t e d t i s s u e h o m o g e n a t e c o n t a i n i n g 0 . 0 4 - 0 . 4 ttmol of u r e a , o r 0 . 0 8 - 0 . 8 t t E q of a m m o n i a , + 20 tll of w a t e r w e r e put in the f l a s k : 10 t~l of a s o l u t i o n of u r e a s e in p h o s p h a t e b u f f e r at pH 6.8 w e r e added. ( B u f f e r s o l u t i o n : 6.9 g N a H 2 P O 4 × 2 H 2 0 + 17.9 g N a 2 H P O 4 × 12H20 m a d e up to 100 ml. 1 m g of s o y b e a n u r e a s e - U r e a s e M e r c k 8489 - w a s s u s p e n d e d in 3 m l of w a t e r + 2 m l of p h o s p h a t e b u f f e r ; the m i x t u r e w a s c e n t r i f u g e d and the s u p e r natant used.) The flasks were quickly stoppered and s t i r r e d m a g n e t i c a l l y f o r 30 rain. A p a r a l l e l d e t e r m i n a t i o n of a m m o n i a w a s run w i t h e a c h u r e a d e t e r m i n a t i o n (using the s a m e t e c h n i q u e but o m i t t i n g the u r e a s e ) . A f t e r 30 rain 300 ~1 of a s a t u r a t e d s o l u t i o n of p o t a s s i u m c a r b o n a t e w a s added, the f l a s k w a s s t o p p e r e d a g a i n r a p i d l y and s t i r r e d m a g n e t i c a l l y f o r 3 hr. E a c h g l a s s s t o p p e r w a s then r e m o v e d i n d i v i d u a l l y , and the g l a s s b e a d b e a r i n g the d r o p of b o r i c a c i d w a s c a r e f u l l y r i n s e d with 2.5 m l of c o l o r r e a g e n t A o v e r a t e s t tube. ( R e a g e n t A: 4.888 g p h e n o l + 25.5 m g s o d i u m n i t r o p r u s s i d e , m a d e up to 500 ml.) To the r i n s i n g f l u i d w e r e a d d e d 2.5 m l of r e a g e n t B. ( R e a g e n t B: s o d i u m h y p o c h l o r i t e 11.3 r e t o o l / 1 + NaOH 130 m m o l / l : p r e p a r e d by diluting 4 m l of the " s o l u t i o n a l c a l i n e d ' h y p o c h l o r i t e de s o d i u m P h H V" w i t h 496 m l of 0.44% NaOH.) T h e t e s t t u b e s w e r e t h e n s t o p p e r e d , put in a w a t e r bath at 37o f o r 30 m i n and c o o l e d at r o o m t e m p e r a t u r e f o r 5 min. T h e e x t i n c t i o n w a s r e a d in a s p e c t r o p h o t o m e t e r at 546 nm. E x t i n c t i o n s b e -

127

t w e e n 0.08 and 0.8 m E q of a m m o n i a f o l l o w e d L a m b e r t - B e e r ' s law. T h e c o n c e n t r a t i o n s of u r e a w e r e c a l c u l a t e d a s [total a m m o n i a ] ( s a m p l e treated with urease) - [free ammonia] (sample without u r e a s e ) ] : 2. " F r e e a m m o n i a " e s t i m a t e d in t h i s w a y r e p r e s e n t e d a c t u a l a m m o n i a only in u r i n e (and p e r h a p s in the highly d i l u t e d h o m o g e n a t e s of the p a pilla). In o t h e r n o n - d e p r o t e i n i z e d t i s s u e h o m o g e n a r e s , a s w e l l a s in p l a s m a , t r e a t m e n t w i t h s a t u r a t e d K2CO 3 l i b e r a t e d NH 3 f r o m o t h e r n i t r o g e n containing compounds * b) Enzymatic-spectrophotometric estimation of urea m o d i f i e d f r o m K i r s t e n et al. (1963) and T a l k e and S c h u b e r t (1965): T h e m e t h o d is b a s e d on the r e a c t i o n of a m m o n i u m ions, e v o l v e d f r o m u r e a by u r e a s e w i t h a l p h a - k e t o g l u t a r a t e in the p r e s e n c e of g l u t a m a t e d e h y d r o g e n a s e (GLDH) and of NADH, w h i c h y i e l d s L - g l u t a m a t e , o x i d i z e d NAD and w a t e r . One e q u i v a l e n t of NH4+ is u s e d to o x i d i z e one m o l e of NADH: the d i s a p p e a r a n c e of the s p e c i f i c e x t i n c t i o n of NADH at 340 n m m e a s u r e d the a m o u n t of NH4+ p r e s e n t . S i n c e the s p e c i f i c i t y of the r e a c t i o n is g r e a t ( K i r s t e n et a l . , 1963) i s o l a t i o n of a m m o n i a f r o m blood, u r i n e o r t i s s u e s a m p l e s is s u p e r f l u o u s . 600 ~1 of buffer-substrate-NADH s o l u t i o n (NADH s t o c k s o l u t i o n : 10 m g / m l in 0.1 M t r i s b u f f e r at pH 9.0; k e p t f r o z e n . To p r e p a r e the b u f f e r - s u b s t r a t e NADH s o l u t i o n 0.25 m l of a 0.08 M s o l u t i o n of ~ - k e t o g l u t a r a t e and 0.25 m l of NADH s t o c k s o l u tion a r e a d d e d to 10 m l of 0.2 M t r i s m a l e a t e b u f f e r at pH 8.6) w e r e p l a c e d in the s e m i m i c r o c u v e t t e of a Z e i s s P M Q II s p e c t r o p h o t o m e t e r . W a t e r (blank), s t a n d a r d o r unknown, c o n t a i n i n g 0.01 -0.1 p m o l NH4+ o r u r e a in 5-10 p l ( 1 - 2 0 m m o l / 1 ) w a s added. T h e e x t i n c t i o n w a s r e a d at 340 n m = E 1. 10 t~l of a m m o n i a - f r e e g l u t a m a t e d e h y d r o g e n a s e s o l u t i o n ( G L D H - S , B o e h r i n g e r und S~hne, M a n n h e i m , G e r m a n y ) w e r e added. T h e e x t i n c t i o n w a s r e a d w h e n it b e c a m e s t a b l e , at r o o m t e m p e r a t u r e , a f t e r 15-20 rain (E2). T h e d i f f e r e n c e b e t w e e n the i n i t i a l e x t i n c t i o n and the s e c o n d v t v a l u e ( E 1 - E 2) m i n u s ( E l - E 2 ) o b t a i n e d with a w a t e r blank, i n d i c a t e d the l o s s of NADH due to the p r e s e n c e of f r e e a m m o n i a . 15 t~1 of u r e a s e s o l u t i o n w e r e a d d e d (as d e s c r i b e d above). A f t e r 15 to 20 m i n the e x t i n c t i o n b e c a m e s t a b l e and w a s r e a d (E3). T h e d i f f e r e n c e b e t w e e n s e c o n d and t h i r d e x t i n c t i o n s ( E 3 - E 2 ) m i n u s ( E ~ - E ~ ) of the blank, g a v e the c o n c e n t r a t i o n of u r e a . If only * This fact has no bearing on the reliability of the estimation of urea which, with both methods, depends only on the well-established specificity of urease {Summer, 1951).

F. R O C H - R A M E L and G. P E T E R S

128

Table 1 W a t e r c o n t e n t ( m l / k g f r e s h weight) of r e n a l t i s s u e s and of h e a r t m u s c l e in d i f f e r e n t s t a t e s of h y d r a t i o n . Dehydration + vasopressin (28) Papilla

775 ± 17 -838±7

Inner medulla

806 ± 2 5 J

:: 8 0 6 ± 8 2 .

Outer medulla Cortex

- 734 ± 5

Heart muscle

"Non-diuretic" state (7)

764 ± 10

- 8 3 5 ± 11 ::

! ]

' 795 ± 8

Water diuresis 3 hr (13)

8 hr

870 ** ± 18 --

874 ** ± 16 --

868 * ± 5

***

866*±5

.~.

834 ± 9

842 * ± 8

747 ± 1 -

774 *** ± 5 --

779 *** ± 6 - -

776 ± 20

769 ± 9

783 * ± 3

F i g u r e s a r e m e a n s ± S.E. N u m b e r s of e x p e r i m e n t a l a n i m a l s a r e s h o w n in b r a c k e t s . W h e n e v e r m e a n s in o t h e r s t a t e s of d i u r e s i s d i f f e r s i g n i f i c a n t l y f r o m the m e a n v a l u e in d e h y d r a t e d a n i m a l s , t h i s d i f f e r e n c e is i n d i c a t e d by a s t e r i s k s (* = p < 0.05; ** = p < 0.01; *** = p < 0.001). On the o t h e r hand, t h e s i g n i f i c a n c e of d i f f e r e n c e s of w a t e r c o n t e n t of d i f f e r e n t t i s s u e s in the s a m e s t a t e of h y d r a t i o n ( v e r t i c a l c o l u m n s ) is s h o w n by b r a c k e t s and a s t e r i s k s . urea concentrations w e r e to b e e s t i m a t e d , GLDH w a s a d d e d to t h e b u f f e r - N A D H - s u b s t r a t e mixture; a first extinction was then read 15-20 min after adding the standard or unknown: a second value 15-20 rain after adding urease. The concentrations o f NH 4 a n d o f u r e a w e r e c o m p u t e d a s : t z m o l of N H 3 ( a b s o l u t e ) = E ( s t a n d a r d o r u n known) - E (blank). 0.161 volume of the reaction mixture in ml. 2 . 2 . Expression of results All concentrations are expressed as molalities per kg of tissue water, and were computed on the assumption that in any one animal the water content of different segments of the kidney was the same on both sides. All numeric a l d a t a a r e g i v e n a s m e a n s J= s t a n d a r d e r r o r . Statistically, differences in concentrations of urea or electrolytes i n d i f f e r e n t t i s s u e s of o n e animal were treated as paired values: the means of the differences were compared to z e r o b y t h e t-test. Similarly, concentration ratios were cons i d e r e d a s s i g n i f i c a n t , if t h e i r m e a n v a l u e d i f f e r e d s i g n i f i c a n t l y f r o m 1.0. 2 . 3 . Drugs used Hydrochlorothiazide: s u b s t a n c e *. V a s o p r e s s i n : p r e s s i n e " S a n d o z " **

3.1. Water content of tissues previously

Table 2 U r i n e flow and c l e a r a n c e of u r e a in d e h y d r a t e d and n o n - d i u r e t i c r a t s and in w a t e r d i u r e s i s . V

Curea ( m l / k g min)

(Peters

* Kindly d o n a t e d by Ciba Ltd., B a s l e .

and Hedwall,

0.012±0.001

Curea GFR

1.97±0.23

0.43±0.05

N o n - h y d r a t e d (7) 0.064 ± 0.024 3 . 1 9 ± 1 . 1 8

0.65±0.22

Water diuresis: 2d h r (14) 3d h r (13) 8th h r (9)

0.89±0.15 0.51±0.08 0.53±0.10

D e h y d r a t e d (17) Esidrex R Ciba, pure synthetic lysine-vaso-

3. R E S U L T S As described

1963; Manitius et al., 1960; Saikia, 1965) in dehydrated animals as well as in water diuresis (Saikia, 1965), the water content of papillary tissue was always higher than in cortical tissue ( t a b l e 1). I n t h e d e h y d r a t e d state, the papilla contained less water than the adjacent inner medulla: this difference disappeared in water diuresis. F r o m c o r t e x to i n n e r m e d u l l a t h e w a t e r content rose continuously in dehydration, in the "non-diuretic state" and in water diuresis. In water diuresis, t h e w a t e r c o n t e n t o f a l l l a y e r s of the kidney increased u p to t h e 3 r d h r , b u t d i d not change significantly thereafter. The water u p t a k e p e r 1 0 0 g of d r y s o l i d s a c c o m p a n y i n g t h e passage from the non-diuretic s t a t e to t h e 8 t h h r of water diuresis was largest in the papilla (+ 2 7 9 m l / 1 0 0 g of dry solids), smaller and e q u a l i n t h e i n n e r a n d o u t e r m e d u l l a (+ 140 m l

0.609±0.084 7.13±1.12 0.946±0.079 4.54±0.77 0.692±0.103 4.07±0.82

All v a l u e s a r e m e a n s ± S.E. N u m b e r s of e x p e r i m e n t a l a n i m a l s a r e s h o w n in b r a c k e t s . G F R w a s not m e a s u r e d in t h e s e e x p e r i m e n t s ; the v a l u e s of C i n u l i n u s e d f o r c o m p u t i n g Curea/Cin at a g i v e n r a t e of u r i n e flow w e r e m e a s u r e d s i m u l t a n e o u s l y in t h i s l a b o r a t o r y on

INTRARENAL UREA AND SALT

3.2. Urea In d e h y d r a t e d r a t s , a d e c r e a s e of u r i n e flow of r o u g h l y ~ ( c o m p a r e d to t h e " n o n - h y d r a t e d s t a t e " ) w a s a c c o m p a n i e d by a n a v e r a g e d e c r e a s e of t h e c l e a r a n c e of u r e a ( C u r e a ) by ~ ( t a b l e 2). T h e h i g h v a l u e of C u r e a o b s e r v e d in the 2nd h r of w a t e r d i u r e s i s , w a s u n d o u b t e d l y due to " e x a l t a t i o n " of u r e a e x c r e t i o n f o l l o w i n g any s u d d e n i n c r e a s e in u r i n e flow, p r e v i o u s l y n o t e d in m a n ( C h a s i s e t a l . , 1933), in the dog (Shannon, 1936), in t h e r a b b i t ( E f f e r s ~ e , 1951a, b) a n d in o t h e r s p e c i e s ( S c h m i d t - N i e l s e n , 1958). A s t e a d y s t a t e of u r e a e x c r e t i o n w a s not r e a c h e d b e f o r e t h e 3 r d h r of w a t e r d i u r e s i s a n d t h e n p e r s i s t e d u n t i l t h e 8th h r ( t a b l e 2). In d e h y d r a t e d a n d in n o n - h y d r a t e d r a t s , the c o n c e n t r a t i o n of u r e a in t i s s u e w a t e r i n c r e a s e d 40, r e s p . 20 f o l d f r o m c o r t i c a l to p a p i l l a r y t i s s u e (fig. 2, t a b l e 3). S i n c e the w a t e r c o n t e n t of the p a p i l l a i s l a r g e r t h a n t h a t of t h e c o r t e x , t h e u r e a c o n t e n t p e r u n i t ( u r e a - f r e e ) d r y w e i g h t (Saikia, 1965) i n c r e a s e d s t i l l m o r e s t e e p l y ( t a b l e 4). U r e a c o n c e n t r a t i o n s in c o r t i c a l t i s s u e w a t e r w e r e a p p r o x i m a t e l y 4 t i m e s h i g h e r t h a n in p l a s m a w a t e r ( t a b l e 3), under all conditions studied. T h e a m o u n t of u r e a c o n t a i n e d in t u b u l a r f l u i d c a n n o t a c c o u n t f o r t h e h i g h c o r t i c a l c o n c e n t r a t i o n found. T h e c o n c e n t r a t i o n of u r e a in p a p i l l a r y t i s s u e w a t e r w a s s l i g h t l y s u p e r i o r , o r did not d i f f e r s i g n i f i c a n t l y f r o m t h a t in u r i n e in the d e h y d r a t e d o r in the n o n - h y d r a t e d s t a t e (table 3, fig. 2). In w a t e r d i u r e s i s , u r e a c o n c e n t r a t i o n s d e c r e a s e d in

e-~-,,DEHYDRATED NON-HYDRATED / /

mMol IL [UREA]

o--o 1200 11001000900" 800" 700600500400" 300" 200100"

PLASMA mC~ ' Z)RTEX OUTER INNER ] PAPILLA MEDULLA MEDULLAI

URINE

Fig. 2. Concentration profile of urea in renal tissue water in the dehydrated and "non-diuretic" states. All values shown are means ± S.E. from groups of 7-28 r a t s . Concentrations are given as molalities in tissue water. A s t e r i s k s designate the significance of differe n c e s of concentrations in different fluids: ** = p < 0.01. Full dots show values after 72 hr dehydration and administration of exogenous v a s o p r e s s i n , open c i r c l e s values in non-hydrated animals. / 1 0 0 g d . s . a n d + 145 s m a l l e r in t h e c o r t e x w a t e r c o n t e n t of h e a r t c a n t l y b e t w e e n the 3 r d d r a t i o n ; the t o t a l w a t e r

129

ml/100 g d.s.) and still (+ 57 m l / 1 0 0 g d . s . ) . T h e muscle increased signifia n d t h e 8th h r of o v e r h y u p t a k e w a s s m a l l (15 m l

/100 g d.s.).

Table 3 Urea concentrations in urine, tissue water and plasma water in different states of hydration. Dehydration + vasopressin

"Non-diuretic" state

3 hr

Water d i u r e s i s 8 hr

(28)

(7)

(13)

(10)

Urine

1286 ± 74

329 ± 122

15±3

19±4

Papilla

1197 ± 143

337 ± 68

88 ± 18

38±6

Inner medulla

518 ± 42

238 ± 42

31±4

19+4

Outer medulla

194 ± 13

86 ± 19

29±8

10± 1

Cortex

F 34±2

I

*l* 13± 1

**

21±5

[

19±3

I

7.3 ± 1.2

I

Heart muscle P l a s m a (water) [U]urine - [U]pap

L-8.9 ± 0.7 +101" ± 47

[Ulurine/[Ulpap

1.33"* ~ 0.11

[U]pap - [U]cor t

+1168"** • 148

[U]pap/[U] cort

42*** ± 6

I

5.7 ± 1.2

***

7.8 ± i . i

4.9 ± 0.3

I

5.1 ± 0.5

-113 ± 108

-80** ± 21

0.92 ± 0.49

0.28*** ± 0.20

+317"* ± 68 20* ± 5

+75** ± 19 5.0*** ± 0.7

i

2.8** ± 0.3 2.9 ± 0.2 -18.7"* ± 5.5 0.61" ± 0.13 +30*** ± 6 6.1"* ± 1.4

Values for urine in mmol/1 (molarity); in t i s s u e s in m m o l / l of tissue water (molalities); means * S.E. The s t a t i s tical significance of the d i f f e r e n c e s of gradients f r o m z e r o and of the difference of ratios f r o m 1.0 is shown as * = p < 0.05; * * = p < 0.01; * * * = p < 0.001.

F. ROCH-RAMEL and G. PETERS

130

Table 4 Urea content of renal t i s s u e s in different states of hydration, e x p r e s s e d as m m o l / k g u r e a - f r e e dry solids. Dehydration + vasopressin

"Non-diuretic" state

Water d i u r e s i s 3 hr t 8 hr

Papilla

5489 ± 508

1526 ~- 287

541 ± 124

264 ± 41

Inner medulla

3192 ± 220

1299 ± 220

208 ± 26

123 ± 23

Outer medulla

849 ± 54

341 ± 74

147 ± 38

51±7

Cortex

94±5

Plasma

96~ 8

62 ± 14

65±9

26±4

54±3

68±7

41±3

Means ± S.C. all l a y e r s of k i d n e y t i s s u e (fig. 3, t a b l e 3). T h e d e c r e a s e r e p r e s e n t e d an a c t u a l l o s s of u r e a , a n d not m e r e l y an e f f e c t of d i l u t i o n ( t a b l e 4). T h e f a l l in r e n a l u r e a w a s m o r e p r o n o u n c e d t h a n the s i m u l t a n e o u s f a l l in p l a s m a u r e a c o n c e n t r a t i o n . Renal cortical urea, however, remained consistently higher than plasma urea. W h i l e the u r i n a r y u r e a c o n c e n t r a t i o n a n d the r e n a l c l e a r a n c e of u r e a (table 2) r e m a i n e d s t a b l e a f t e r the 3 r d h r of w a t e r d i u r e s i s , u r e a c o n c e n t r a t i o n s in a l l l a y e r s of r e n a l t i s s u e c o n t i n u e d to f a l l , a l b e i t at a s l o w e r r a t e , b e t w e e n t h e 3 r d a n d t h e 8th h r of w a t e r d i u r e s i s . In c o r t i c a l o r m e d ullary tissue, steady state urea concentrations w e r e r e a c h e d by t h e e n d of t h e 8th h r , but in p a p illary tissue, urea wash-out continued beyond t h i s t i m e . In fig. 3, u r e a c o n c e n t r a t i o n s o b s e r v e d 8 h r a f t e r the b e g i n n i n g of w a t e r d i u r e s i s a r e

~. mMol I L

[Na+]L ~Eq/

WATER DCJRESIS: 3 HOURS

o--.-o WATER DILIRESIS: 8 HOURS A---A WATER DILRESIS: PERMANANT

90-

c o m p a r e d w i t h t h o s e o b s e r v e d in 15 r a t s m a i n t a i n e d in c o n t i n u o u s w a t e r d i u r e s i s f o r 30 d a y s by f e e d i n g t h e a n i m a l s e x c l u s i v e l y on v e r y d i luted c o n d e n s e d milk ( P e t e r s and R o c h - R a m e l , in p r e p a r a t i o n , 1967). U r i n e f l o w s in t h e s e a n i m a l s (0.738 + 0.006 m l / k g min) w e r e c o m p a r a b l e to t h o s e o b s e r v e d in t h e p r e s e n t e x p e r i m e n t s . P a p i l l a r y u r e a c o n c e n t r a t i o n s , a f t e r 30 d a y s of c o n t i n u o u s w a t e r d i u r e s i s , h a d f a l l e n to v e r y low l e v e l s a n d d i d not d i f f e r s i g n i f i c a n t l y f r o m the s i m u l t a n e o u s u r i n a r y u r e a c o n c e n t r a t i o n s . A s a c o n s e q u e n c e of t h e d e l a y e d f a l l of u r e a c o n c e n t r a t i o n in p a p i l l a r y t i s s u e w a t e r , a h i g h l y significant and f a i r l y large p a p i l l o - u r i n a r y u r e a g r a d i e n t w a s o b s e r v e d a f t e r 3 h r of w a t e r d i u r e s i s (fig. 3, t a b l e 3). In t h e f o l l o w i n g 5 h r , t h i s g r a d i e n t d e c r e a s e d c o n s i d e r a b l y , but did not d i s a p p e a r (fig. 3). In r a t s in c o n t i n u o u s w a t e r

400-

80-

360 -

70-

320-

60-

280 -

50-

2/-.0 -

40-

200-

30-

160-

20-

120-

e--,, DEHYDRATED

~o NON-HYDRATED /~ i

80-

10-

40-

i

PLASMA

CORTEX

OUTER I INNER MEDULLA I MEDULLA

PAPILLA

URINE PLASMA

Fig. 3. Concentration profile of urea in renal tissue water in water diuresis. For explanations see legend of fig. 2. Full dots r e p r e s e n t values after 3 hr of b r i s k water allures±s; open c i r c l e s values after 8 hr of water d i u r e s i s . Triangles are values taken f r o m a study (Roch-Ramel and P e t e r s , unpublished) on r a t s maintained in continued brisk water d i u r e s i s for some weeks ( * = p < 0.05; * * = p < 0.01).

CORTEX

ME~C~LA

J ~OU_A

MEDULLAINNER

Fig. 4. Sodium concentration in p l a s m a water, renal tissue water and urine in the dehydrated and non-diuretic states. Concentrations are e x p r e s s e d as molal±ties (mEq/1 of tissue water). All explanations s i m i l a r to those for fig. 2 (*** = p < 0.001).

INTRARENAL UREA AND SALT d i u r e s i s , u r e a c o n c e n t r a t i o n s in f i n a l u r i n e a n d in p a p i l l a r y t i s s u e w a t e r b e c a m e a p p r o x i m a t e l y equal. A f t e r t h e 3 r d h r of w a t e r d i u r e s i s , t h e d i f f e r ence between cortical and plasma urea concent r a t i o n b e c o m e s s m a l l e r b u t e v e n a f t e r 30 d a y s of c o n t i n u o u s w a t e r d i u r e s i s c o r t i c a l t i s s u e w a t e r u r e a (7.5 + 1.5 m m o l / 1 ) r e m a i n e d h i g h e r t h a n p l a s m a w a t e r u r e a (3.7 =~ 0.3 m m o l / 1 ) . U r e a c o n c e n t r a t i o n in h e a r t m u s c l e , on t h e o t h e r h a n d , never differed significantly from plasma urea e i t h e r in d e h y d r a t e d r a t s o r i n w a t e r d i u r e s i s ( t a b l e 3). 3.3. Sodium T h e e x c e l l e n t s o d i u m c o n s e r v i n g a b i l i t y of r a t s w a s r e f l e c t e d b y t h e v e r y low u r i n a r y s o d i um concentrations and the very steep papillourinary sodium gradients in dehydrated as well a s i n h y d r a t e d r a t s (figs. 4 - 6 ) . S o d i u m c o n c e n t r a t i o n in t i s s u e w a t e r ( t a b l e 5), a s w e l l a s s o d i u m c o n t e n t r e l a t e d to u r e a - f r e e dry solids ( t a b l e 6), i n c r e a s e d f r o m c o r t e x to p a p i l l a in a l l t h e c o n d i t i o n s i n v e s t i g a t e d . E v e n in c o n t i n u o u s w a t e r d i u r e s i s (fig. 5) a s i g n i f i c a n t p a p i l l o - c o r tical sodium gradient was maintained. The conc e n t r a t i o n r a t i o of p a p i l l a r y to c o r t i c a l s o d i u m ( t a b l e 5) w a s m u c h s m a l l e r t h a n f o r u r e a ( t a b l e 3). In d e h y d r a t e d a n d n o n - d i u r e t i c r a t s , u r e a was concentrated approximately 8 times more e f f i c i e n t l y t h a n s o d i u m . In w a t e r d i u r e s i s , t h e sodium concentrating mechanism was better preserved than the urea concentrating mecha-

131

nism: yet urea was still concentrated 3-4 times more efficiently than sodium. For each sodium ion gained by papillary tissue water, compared to c o r t i c a l fluid, a p p r o x i m a t e l y 3.3 m o l e c u l e s of u r e a w e r e g a i n e d in d e h y d r a t e d a n i m a l s , 2.3 m o l e c u l e s in t h e n o n - d i u r e t i c s t a t e , 1.4 m o l e c u l e s a f t e r 3 h r of w a t e r d i u r e s i s a n d l e s s t h a n o n e m o l e c u l e of u r e a a f t e r 8 h r of w a t e r d i u r e sis. C o r t i c a l t i s s u e w a t e r c o n t a i n e d only, r o u g h l y , half as much sodium as plasma. While in water d i u r e s i s u r e a c o n c e n t r a t i o n s in t h e p a p i l l a a n d in t h e i n n e r m e d u l l a f a l l r a p i d l y d u r i n g t h e f i r s t 3 h r a n d m u c h m o r e s l o w l y f r o m t h e 3 r d to t h e 8th h r , t h e f a l l i n s o d i u m c o n c e n t r a t i o n f o l l o w e d f i r s t o r d e r k i n e t i c s up to t h e e n d of t h e 8th h r (fig. 6), w h e n a p p a r e n t l y a s t e a d y s t a t e w a s r e a c h e d (fig. 5). A s s h o w n in t a b l e 6, t h i s d e c r e a s e w a s not o n l y due to d i l u t i o n b u t to a n a c t u a l l o s s of t i s s u e s o d i u m . In t h e o u t e r m e d u l l a o r in t h e c o r t e x , s o d i u m c o n c e n t r a t i o n s a n d c o n t e n t s d i d not c h a n g e d u r i n g the f i r s t 3 h r of w a t e r d i u r e s i s , f e l l s l o w l y b e t w e e n t h e 3 r d a n d 8th hr, but, apparently, remained constant therea f t e r in c o n t i n u o u s w a t e r d i u r e s i s (fig. 5). 3.4. P o t a s s i u m W h i l e in d e h y d r a t e d r a t s the u r i n a r y p o t a s s i um concentration was much higher than the sod i u m c o n c e n t r a t i o n ( t a b l e s 5, 7), a p p r o x i m a t e l y e q u a l c o n c e n t r a t i o n s w e r e o b s e r v e d in n o n - h y d r a t e d o r in d i u r e t i c a n i m a l s . Papillary and inner medullary concentrations rrdvk~/t 300200-

o . ~ , , WATER DIURE51S : 3 N3tlRS mEq/L

o - - - o WATER DI.JRE51S : 8 H ( X I ~ A---~ ~E~

~

S

: PERMANENT

T

100-

i

....

180 160 -

40.

•

140 120 -

20

~ -

10080-

0

I

2 4 6 8 HOURS OF V~TER DIURESIS • CON~NTRATIONS IN PAPILLARY TISSUE WATER o ~NTRATIONS IN TISSUE WATER OF INNER MEDULLA -- lanAI .... [N~÷]

604020CORTEX

~ MEDULLA

INNER MEDULLA

PAPILLA

Fig. 5. Sodium concentration in plasma, renal tissue water and urine in water diuresis. For explanations see fig. 3 (*** = p < 0.001).

Fig. 6. Fall of sodium and u r e a concentrations in t i s sue water of papilla and inner medulla during continued water diuresis. Concentrations a r e shown on a logarithmic scale as functions of the duration of water diuresis. The values r e p r e s e n t e d are the same as in figs. 2-5. F o r an interpretation see section 4.

132

F. ROCH-RAMEL

Sodium concentrations

( m m o l / 1 o f w a t e r ) in u r i n e , D e h y d r a t i o n ~vasopressin

~rine

a n d G. P E T E R S

Table 5 tissue water

and plasma

nNon-diuretic"

water

in d i f f e r e n t

state

states

Water diuresis 3 hr

28±6

of hydration.

8 hr

19±7

2.9 ± 0.7

9.2 ± 2.5

'apilla

436 ± 43

218 ± 9

170 ± 2 4

102 ~ 7

nner medulla

2 1 1 ± 10

157 ± 12

1 2 8 ± 16

98±6

)uter medulla

79=e6

1 1 7 ± 12

104 • 8

93 ± 14

',ortex

86=L3

78±3

88 ± 11

65±3

[eart muscle

57±3

56±4

58±3

43±3

1 4 8 :e 3

141 ± 8

201 ±6

'lasma

164 ± 7

Na~]urine - [Na+]pap

-422*** ± 34

-199"** ± 11

- 1 4 1 " * * ± 13

Na+lurine/[Na~-lpap

0.08*** ± 0.02

0.09*** ± 0.03

0.02*** ± 0.01

Na*]pap-

+352*** ± 43

+ 1 4 0 " * * ± 10

[Na+]cor t

5.1"** ± 0.5

Na+]pap/[Na+lcort For explanation

-92*** ± 8 0.11"** ~ 0.03 +38** ± 9

+ 5 3 * * * :e 6 1.6"** ± 0.1

2.8*** ± 0.2

1.6"* ± 0.2

s e e t a b l e 3.

Sodium content of renal tissues

in d i f f e r e n t

Dehydration * vasopressin

states

Table 6 of h y d r a t i o n ,

expressed

as mmol/kg

urea-free

dry solids.

Water diuresis

"Non-diuretic" state

3 hr

8 hr

Papilla

1999 ± 200

989 ± 41

1 2 3 9 ± 175

Inner medulla

1300 ± 62

856 ± 65

852 ± 1 0 7

638 ± 39

Outer medulla

511 ± 52

4 1 2 ± 30

4 7 2 ± 73

422 ± 32

Cortex

238 ± 9

229 ± 8

303 ± 39

2 2 8 ± 11

718 ± 49

Means ± S . E .

Potassium

concentration

Table 7 tissue water and plasma

in urine,

Dehydration vasopressin

+

"Non-diuretic"

water in different

states of hydration.

state

Water diuresis 3 hr

8 hr

Urine

142 ± 13

23±7

2.7 • 0.4

5.4 • 1.1

Papilla

1 3 3 ± 16

6 7 ± 13

143 ± 22

64+ 6

1 0 8 ± 18

66±3

Inner medulla

77±5

78±3

Outer medulla

94±8

96+3

90±7

Cortex

128 ± 4

126 ± 3

109 ± 6

Heart muscle

115 ±4

105 ± 4

104 ± 8

8.7 ± 0.7

8.3 ± 0.7

10.4 ± 0.5

Plasma [K+]urine-

+ 1 1 ± 20

- 4 4 * ± 16

- 1 3 1 " * ± 27

1.36" ± 0.15

0.45** ± 0.12

0.02*** ± 0.01

[K+]pap

[K+lurine/[K+Ipap [K+]pap - [K+]cor t

+5 ± 206

[K+]pap/[K+]cort

1.0 ± 0.1

All values

in m E q / 1 o f t i s s u e w a t e r .

For explanations

-61"*

± 13

0.53** ± 0.10 s e e l e g e n d o f t a b l e 3.

84± 6 93±5 96±5 8.4 ± 0.3 -58*** ± 6 0.09*** + 0.03

+ 3 6 + 21

-29** ± 8

1 . 2 8 ~- 0 . 1 9

0.71" ± 0.08

INTRARENAL UREA AND SALT w e r e e i t h e r i n f e r i o r o r e q u a l to c o r t i c a l p o t a s s i um, which they never exceeded significantly (tab l e 7). In w a t e r d i u r e s i s , t h e p o t a s s i u m c o n t e n t of t h e s e t i s s u e s d i d n o t c h a n g e s i g n i f i c a n t l y . The outer medulla consistently contained less potassium t h a n t h e c o r t e x ( t a b l e 7). In b o t h layers, potassium concentration decreased slowly and p r o g r e s s i v e l y during water diuresis. A s t i l l s m a l l e r a n d s l o w e r f a l l of p o t a s s i u m c o n c e n t r a t i o n d u r i n g w a t e r d i u r e s i s w a s o b s e r v e d in myocardial tissue water. 3.5. A m m o n i u m ions T h e a m m o n i u m c o n c e n t r a t i o n in t h e p a p i l l a w a s l o w e r than in u r i n e in d e h y d r a t e d and in n o n - h y d r a t e d r a t s , but h i g h e r in w a t e r d i u r e s i s ( t a b l e 8). A m m o n i u m c o n c e n t r a t i o n s in o t h e r l a y e r s of r e n a l t i s s u e a r e n o t s h o w n , b e c a u s e tlley a r e o v e r e s t i m a t e d ( s e e m e t h o d s ) . It m a y , h o w e v e r , be s t a t e d i n c o n f i r m a t i o n of o t h e r o b servers' r e s u l t s ( S a i k i a , 1965) t h a t a m m o n i u m

133

c o n c e n t r a t i o n in t i s s u e w a t e r a l w a y s i n c r e a s e d v e r y m a r k e d l y f r o m c o r t e x to p a p i l l a . 3.6. O s m o l a l i t i e s Tissue osmalalities calculated as [Osm] = 2 ( [ S a + ] + [K+] + [NH4+ ]) + [ u r e a ] a r e e r r o n e o u s i n two r e s p e c t s : 1) t h e f o r m u l a d o e s n o t t a k e i n t o a c c o u n t t h e d e c r e a s e of t h e a c t i v i t y c o e f f i c i e n t s of m o n o v a l e n t i o n s w h i c h s i g n i f i c a n t l y i n f l u e n c e s r e s u l t s a b o v e 300 m O s m / k g H 2 0 ; a n d 2) b o i l e d t i s s u e h o m o g e n a t e s , a s u s e d in the p r e s e n t e x p e r i m e n t s , c o n t a i n s u b s t a n c e s , o t h e r t h a n NH4+ , w h i c h l i b e r a t e g a s e o u s NH 3 u p o n t r e a t m e n t w i t h s t r o n g a l k a l i . B o t h s o u r c e s of e r r o r t e n d to s u g g e s t too h i g h v a l u e s of t i s s u l a r o s m o l a l i t y . W i t h t h e s e r e s e r v a t i o n s , it m a y b e s t a t e d t h a t o s m o l a l i t y i n c r e a s e d s t e p w i s e f r o m c o r t e x to p a p i l l a i n d e h y d r a t e d a n d n o n - d i u r e t i c r a t s ( t a b l e 9). In water diuresis, the papillo-cortical osmotic grad i e n t d e c r e a s e d c o n t i n u o u s l y u p to t h e 8 t h h r . The osmolar difference between cortex and me-

Table 8 A m m o n i a in u r i n e and p a p i l l a r y t i s s u e water Dehydration + vasopressin

" N o n - d i u r e t i c " state

Water d i u r e s i s 3 hr

8 hr

Papilla m m o l / k g . H20

145 :e 26

56 ± 16

62 ± 19

42 ± 10

Urine m m o l / l

226 :e 20

84 :e 26

8.7 ± 1.7

6.5 ± 1.3

+67* ~ 32

+23 :e 33

[NH4+]urine - [NH4+]pap [NH4+]V: # E q / k g . m i n

2.9 ± 0.3

5.4 ± 2.2

-40* ± 13

-36** ± 11

8.2 ± 1.8

4.5 ± 1.0

For explanation see "Methods". Table 9 Osmolalities of urine, tissue water and plasma water in different states of hydration.

Dehydration + vasopressin

" N o n - d i u r e t i c " state

Water d i u r e s i s 3 hr

8 hr

Urine

1579 ± 145 2084 ± 112

455 ~ 155 581 ± 173

Papilla

2646 ± 228

1099 ± 1 1 7

Inner m e d u l l a

1251 ± 88

790 ± 61

Outer m e d u l l a

642 ± 30

516 ± 21

Cortex

496 ± 12

468 ± 10

438 ± 33

359 ± 15

Heart muscle

381 ± 9

368 ± 20

355 ± 19

309 ± 15

Plasma

331 ± 13 440 ± 19

323 ± 30

332 ± 6

266 ± 11 304 ± 12

[ O s m ] u r i n e - [Osm]pap [Osmlpap - [ O s m l e o r t

47:e3 44 ± 4

117 ± 18 103 ± 26

873 ± 96

449 ± 28

523 ± 76

372 ± 24

440 ± 51

345 ± 27

-434* ± 212

-746* • 147

-821"** ± 12

-342*** ± 34

+2108"** ± 230

+633*** ± 116

+434*** ± 86

+101" ± 38

Values in m O s m / k g H20; m e a n s ± S.E. Underlined n u m b e r s show o s m o l a l i t i e s calculated f r o m [OSM] = 2{[Na+] + [K+] + [NH4+]) + [urea]; s t r a i g h t n u m b e r s a r e o s m o l a l i t i e s m e a s u r e d a s f r e e z i n g point d e p r e s s i o n s . F o r all explanations s e e legend of table 3.

134

F. ROCH-RAMEL and G. PETERS

d u l l a a l w a y s d i s a p p e a r e d a f t e r 3 h r of w a t e r d i uresis. Renal cortical tissue was concistently hypert o n i c to p l a s m a , w h i l e m y o c a r d i a l t i s s u e w a t e r w a s i s o t o n i c ( t a b l e 9). In p l a s m a a n d in u r i n e , m e a s u r e d o s m o l a l i t i e s a b o v e 300 m O s m / k g H 2 0 w e r e c o n s i s t e n t l y l o w e r t h a n the c a l c u l a t e d v a l u e s ; t h i s d i f f e r e n c e d i s a p p e a r e d in h y p o t o n i c urine. 3.7. Hydrochlorothiazide A s p r e v i o u s l y o b s e r v e d in b r i s k w a t e r d i u r e s i s ( P e t e r s , 1965), t h e t h i a z i d e d i u r e t i c c a u s e d a l a r g e i n c r e a s e in s o d i u m e x c r e t i o n a n d a s m a l l Table 10 Influence of hydrochlorothiazide (5 mg/kg i.v., followed by 1 mg/kg-min) on renal functions in prolonged water diuresis. Controls (9)

Hydrochlorothiazide (7)

V =/21/kg • min

692 ± 103

705 ± 149

[Na+] : mEq/1 [Kq: mEq/1 [Urea]: mmol/1 [Osm] : m O s m / k g . H20

9.2 =e 2.5 5 . 4 ± 1.1 19±4 117 ± 18

49*** ± 5 10.0" ± 2.2 18±4 204** ± 16

Cosm: /Xl/kg. min CH20: p l / k g , min

304 ± 54 388 =~33

Curea: m l / k g , min

4.07 ± 0.82

541"** ± 48 162"** ± 11 3.46 ± 1.13

Values obtained during the 8th hr of water d i u r e s i s . Means ± S.E.

i n c r e a s e in p o t a s s i u m e x c r e t i o n w i t h o u t i n f l u e n c i n g u r i n e flow ( t a b l e 10). T h e r e w a s , t h u s , a h i g h l y s i g n i f i c a n t i n c r e a s e in o s m o l a r c l e a r a n c e a n d a d e c r e a s e in t h e c l e a r a n c e of f r e e w a t e r . U r e a c l e a r a n c e w a s not s i g n i f i c a n t l y i n f l u e n c e d . No c h a n g e s o c c u r r e d in the u r e a o r e l e c t r o l y t e c o n t e n t of r e n a l c o r t i c a l o r m e d u l l a r y t i s s u e u n d e r the i n f l u e n c e of the t h i a z i d e ( t a b l e 11). While the u r i n a r y c o n c e n t r a t i o n r e m a i n e d unc h a n g e d , t h e r e w a s a s l i g h t d e c r e a s e in p a p i l l a r y u r e a c o n c e n t r a t i o n ; though the d i f f e r e n c e b e t w e e n p a p i l l a r y u r e a c o n c e n t r a t i o n in t r e a t e d a n i m a l s a n d in c o n t r o l s w a s not s t a t i s t i c a l l y s i g nificant, the d i f f e r e n c e b e t w e e n the p a p i l l o - c o r t i c a l u r e a g r a d i e n t s r e a c h e d the 5% l e v e l of s i g n i f i c a n c e . A s a r e s u l t of t h i s c h a n g e , t h e u r e a gradient between papillary tissue water and urine disappeared. Papillary sodium concentration also dec r e a s e d s l i g h t l y ; a g a i n , only the d e c r e a s e in the papillo-cortical gradient was statistically significant.

4. DISCUSSION

4.1. Urea In t h e d e h y d r a t e d a n d in the n o n - d i u r e t i c r a t , u r e a c o n c e n t r a t i o n s in p a p i l l a r y w a t e r do not e x c e e d t h o s e i s u r i n e (fig. 2, t a b l e 3; P e t e r s a n d H e d w a l l , 1963; H e l l e r e t a l . , 1965; M a n i t i u s e t a l . , 1960; S a i k i a , 1965); all the u r e a p r e s e n t in t h e p a p i l l a m a y , t h e r e f o r e , b e a s s u m e d to r e a c h

Table 11 Influence of hydrochlorothiazide (5 m g / k g i.v., followed by 1 m g / k g min) on composition of renal tissue in prolonged water diuresis.

[Urea] Papilla Inner medulla Outer medulla Cortex [Urea]urine - [Urea]pap [Urea]pap - [Urea]cor t [Na+] Papilla Inner medulla Outer medulla Cortex [Na+]urine - [Na+]pap [Na+]pap - [Na+]cort

Controls

Hydroehlorothiazide

37.5~5.9 18.9±3.5 9.5±1.3 7.3±1.2 -18.7"*~6.5 +30.3***±5.7

27.9~3.6 15.2±2.4 10.7~2.9 9.2~2.2 -10.3~3.6(N.S.) +18.7"±3.4

102 ± 7 98=~6 79±6 65±3 -92"** ± 8 +38** ~: 8

88 ± 11 80 ± 19 64+ 9 67±6 - 4 0 * ± 10 4-21"* ± 2

P > > > > > <

0.05 < 0.10 0.10 0.10 0.10 0.10 0.05

> > > > < <

0.10 0.10 0.10 0.10 0.01 0.05

Means ± S.E. A s t e r i s k s designate the significance of the mean d i f f e r e n c e s from zero (* = p< 0.05; ** =p < 0.01; *** = p < 0.001). The significance of differences between controls and treated groups is shown in the last column.

INTRARENAL UREA AND SALT this site f r o m fluid contained in the c o l l e c t i n g ducts by p a s s i v e p r o c e s s e s , i.e., m a i n l y by diffusion and p e r h a p s to a s m a l l extent by s o l v e n t drag. Since the s a m e o s m o l a l i t y is thought to p r e v a i l in c e l l u l a r , i n t e r s t i t i a l , i n t r a t u b u l a r and i n t r a v a s c u l a r w a t e r of the p a p il la (Bray, 1960), and since u r e a is the m a j o r solid in this t i s s u e , its d i s t r i b u t i o n in t h e s e c o m p a r t m e n t s may be a s s u m e d to be uniform. T h e r e is, h o w e v e r , no e v i d e n c e i n c o m p a t i b l e with the a s s u m p t i o n of d i f f e r e n t c o m p a r t m e n t s of p a p i l l a r y t i s s u e containing d i f f eren t c o n c e n t r a t i o n s of u r e a . It is f a r m o r e difficult to explain the high c o n c e n t r a t i o n s of u r e a found in m e d u l l a r y and in c o r t i c a l t i s s u e , which w e r e a l s o noticed, though n e v e r d i s c u s s e d , by p r e v i o u s i n v e s t i g a t o r s ( P e t e r s and Hedwall, 1963; Manitius et al., 1960; Saikia, 1965; Yunibhand and Held, 1965; H e l l e r ef al., 1965). The high c o r t i c a l c o n c e n t r a t i o n of u r e a may be thought to be due to the p r e s e n c e of high c o n c e n t r a t i o n s of u r e a in d i s t a l tubular fluid. If, though, the distal T F / P u r e a w e r e 10 i.n the n o n - d i u r e t i c rat, and if the r e m a i n d e r of the c o r t i c a l w a t e r contained 1.1 t i m e s the conc e n t r a t i o n in p l a s m a w a t e r (i.e., if 20% of this fluid was t e r m i n a l p r o x i m a l fluid with a T F / P u r e a of 1.5), not l e s s than 36% of total c o r t i c a l t i s s u e w a t e r would have to be distal tubular fluid in o r d e r to explain the u r e a c o n c e n t r a t i o n found. The sodium c o n c e n t r a t i o n found in c o r t i c a l t i s sue w a t e r is not c o m p a t i b l e with the a s s u m p t i o n that it contains m o r e than 50% of p r o x i m a l plus distal tubular fluid (see below). A c c o r d i n g to m i c r o s c o p i c a p p e a r a n c e s the f r a c t i o n of total tubul a r fluid contained in s u p e r f i c i a l distal tubules is p r o b a b l y l e s s than one third. U r e a c o n c e n t r a tions h i g h e r than in p l a s m a must, t h e r e f o r e , be p r e s e n t in c e l l s ( a n d / o r in the s m a l l i n t e r s t i t i a l spa c e s ) of c o r t i c a l t is s u e . S i m i l a r l y , a f t e r 8 hr of w a t e r d i u r e s i s , a s s u m i n g a d i s t a l tubular T F / P u r e a of 6.9 ( o b s e r v e d at c o m p a r a b l e r a t e s of u r i n e flow in mannitol d i u r e s i s by Clapp, 1966) 24% of c o r t i c a l fluid would have to be d i s tal tubular fluid, if distal tubular fluid alone a c counted for the high u r e a c o n c e n t r a t i o n in c o r t i cal t i s s u e. T h i s , again, is an u n r e a l i s t i c figure. U r e a c o n c e n t r a t i o n s in c o r t i c a l and m e d u l l a r y t i s s u e w a t e r a r e thus h i g h e r than those found by m i c r o p u n c t u r e in tubular and v a s c u l a r s t r u c t u r e s running through t h e s e l a y e r s (fig. 1; L a s s i t e r et al., 1961, 1964, 1966; U l l r i c h et al., 1963; Clapp, 1966) (with the e x c e p ti o n of c o r t i c a l d i s tal tubules and i n n e r m e d u l l a r y c o l l e c t i n g ducts). Two q u e s t i o n s a r i s e f r o m this o b s e r v a t i o n : 1) is the t i s s u e u r e a c o n c e n t r a t e d e i t h e r in c e l l s , o r in the e x t r a c e l l u l a r s p a c e of the r e n a l m e d u l l a

135

and c o r t e x , or is it d i s t r i b u t e d u n i f o r m l y ? 2) what is the s o u r c e of the l a r g e amounts of t i s sue u r e a ? Though the high diffusion c o e f f i c i e n t of u r e a a p p e a r s to a r g u e in f a v o r of a u n i f o r m d i s t r i b u tion, the p r e s e n t date do not allow any a n s w e r to the f i r s t question. Some s p e c u l a t i o n s about the s e c o n d question may, h o w e v e r , be w a r r a n t e d . The high c o n c e n t r a t i o n of u r e a in i n n er m e d u l l a r y t i s s u e may be due to r a p i d diffusion f r o m c o l l e c t i n g ducts as well as f r o m p a p i l l a r y t i ssu e. One a r g u m e n t f a v o r i n g the l a t t e r a s s u m p t i o n may be the r ap i d d i s a p p e a r a n c e of the c o n c e n t r a t i o n d i f f e r e n c e between p ap i l l a and i n n er m e d u l l a in kidneys which a r e not d i s s e c t e d i m m e d i a t e l y a f t e r r e m o v a l (unpublished o b s e r v a tions). It is m o r e difficult to i m a g i n e that the o u t er l a y e r s of the m ed u l l a or the c o r t e x d e r i v e t h e i r u r e a content by diffusion f r o m the inner m e d u l l a and papilla: the s m a l l v o l u m e of the inn e r l a y e r s as well as the t o p o g r a p h i c a l d i s t r i b u tion of t h ese t i s s u e s a r g u e ag ai n st this hypothesis. Outer m e d u l l a r y and c o r t i c a l t i s s u e , or s o m e c i r c u m s c r i b e d p a r t of t h ese t i s s u e s must, therefore, possess a urea concentrating mechanism. This m e c h a n i s m could e i t h e r function through a c t i v e t r a n s p o r t of u r e a into c e l l s lining tubular or v a s c u l a r s t r u c t u r e s , or through i n t r a c e l l u l a r s e q u e s t r a t i o n of u r e a which, in turn, could be due to binding of u r e a to a compound contained in the c e l l s , or to a c t i v e c o n c e n t r a t i o n of u r e a in s u b c e l l u l a r c o m p a r t m e n t s . The hyp o t h e s i s of c o m p a r t m e n t a l i s a t i o n of r e n a l t i s s u e u r e a is s u p p o r t e d by the o b s e r v a t i o n that in kidney s l i c e s containing mainly c o r t i c a l and o u t er m e d u l l a r y t i s s u e , a f r a c t i o n of the u r e a cannot be r e m o v e d by r e p e a t e d washing and does not e q u i l i b r a t e r ap i d l y with u r e a - 1 4 C (O'Dell and S c h m i d t - N i e l s e n , 1961). The f a i r l y l a r g e d i f f e r e n c e b et w een the h i g h e r p a p i l l a r y and the l o w e r u r i n a r y u r e a c o n c e n t r a tions found a f t e r 3 hr of w a t e r d i u r e s i s is r e m i n i s c e n t of s i m i l a r d i f f e r e n c e s found by o t h er o b s e r v e r s a f t e r 30-210 min of mannitol d i u r e s i s (Bray and P r e s t o n , 1961; Clapp, 1966). Since the w e l l - k n o w n phenomenon of exaltation of u r e a e x c r e t i o n following any i n c r e a s e in u r i n e flow (Chasis et al., 1933; Shannun, 1936; E f f e r s ~ e , 1951a, b) m ay be a s s u m e d to be t e r m i n a t e d 3 hr a f t e r the induction of w a t e r d i u r e s i s (table 2), t h e s e o b s e r v a t i o n s s u g g e s t e d t r a n s p o r t of u r e a a g a i n s t a c h e m i c a l gradient, i.e., a c t i v e t r a n s port. Since the w a l l s of the c o l l e c t i n g ducts cannot be a s s u m e d to be i m p e r m e a b l e to u r e a ( S c h m i d t - N i e l s e n , 1958; C r a w f o r d et al., 1959; J a e n i k e , 1964) the a s s u m p t i o n of an a c t i v e t r a n s -

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p o r t m e c h a n i s m would be n e c e s s a r y , if a conc e n t r a t i o n d i f f e r e n c e f o r u r e a b e t w e e n final u r i n e and p a p i l l a r y fluid was m a i n t a i n e d in a steady state. In the p r e s e n t e x p e r i m e n t s a steady state of u r e a d i s t r i b u t i o n a c r o s s the w a l l s of the c o l l e c t i n g ducts was not r e a c h e d within 3 hr; the papillo-urinary gradient decreased considerably a f t e r 8 hr of w a t e r d i u r e s i s and fell to nil in anim a l s in continuous w a t e r d i u r e s i s . S i m i l a r l y , in n o n - d e h y d r a t e d r a t s with h e r e d i t a r y h y p o t h a l a m ic diabetes insipidus ( B r a t t l e b o r o rats) u r e a c o n c e n t r a t i o n in p a p i l l a r y t i s s u e w a t e r was not s u p e r i o r to that in u r i n e (Valtin, 1966). Since the p a p i l l o - u r i n a r y u r e a g r a d i e n t d i s a p p e a r s with continuous w a t e r d i u r e s i s , the high p a p i l l a r y u r e a c o n c e n t r a t i o n in the f i r s t h o u r s of w a t e r d i u r e s i s a p p e a r s to be due to a delay in w a s h - o u t of u r e a f r o m this t i s s u e r a t h e r than to a c t i v e t r a n s p o r t . While sodium w a s h - o u t f r o m p a p i l l a r y t i s s u e w a t e r in w a t e r d i u r e s i s could be d e s c r i b e d by f i r s t o r d e r k i n e t i c s , u r e a w a s h - o u t c o r r e s p o n d e d to at l e a s t two d i f f e r e n t f i r s t o r d e r p r o c e s s e s (fig. 6). The delay in adaptation of p a p i l l a r y to u r i n a r y u r e a c o n c e n t r a t i o n s could be due e i t h e r to a low r a t e of w a t e r flow through p a p i l l a r y t i s s u e in w a t e r d i u r e s i s , or to s o m e type of binding or s e q u e s t r a t i o n of u r e a in p a p i l l a r y c o m p a r t m e n t s not r e a d i l y a c c e s s i b l e to the w a t e r running through the papilla. W a t e r flow through i n n e r m e d u l l a r y and papi l l a r y t i s s u e cannot be a s s u m e d to be low in w a t e r d i u r e s i s . It has been pointed out that the total v o l u m e of w a t e r r e a b s o r b e d in the c o l l e c t ing ducts in w a t e r d i u r e s i s is probably h i g h e r than in the n o n - d i u r e t i c state, in spite of a dec r e a s e in w a t e r p e r m e a b i l i t y of the c o l l e c t i n g ducts ( B e r l i n e r , 1960; B e r l i n e r and Davidson, 1957). Thus, in a n o n - d i u r e t i c rat, with a r a t e of u r i n e flow of 10 /~l/kidney. rain, an e n d - d i s t a l inulin T F / P of 12 and an inulin U / P of 80, the total v o l u m e of w a t e r a b s o r b e d f r o m the c o l l e c t ing ducts and c r o s s i n g m e d u l l a r y t i s s u e on its way to blood v e s s e l s would be 57 t t l / k i d n e y , min. In w a t e r d i u r e s i s at the r a t e of 133 ~ l / k i d n e y . rain, with an e n d - d i s t a l inulin T F / P of 5 and an inulin U / P of 10, not l e s s than 133 # l / k i d n e y . min of w a t e r would have to run through m e d u l l a r y t i s s u e. On the o th e r hand, the change in m e d u l l a r y sodium c o n c e n t r a t i o n o c c u r r i n g in w a t e r d i u r e s i s m u s t e x p r e s s the r e l a t i v e r a t e s of e n t r y of sodium and of w a t e r into m e d u l l a r y t i ssu e. Since p a p i l l a r y and m e d u l l a r y sodium c o n c e n t r a t i o n s fall (figs. 4, 5) and since t h e r e is no r e a s o n to a s s u m e that the amount of sodium d e l i v e r e d to the m e d u l l a f r o m the nephron dec r e a s e s in w a t e r d i u r e s i s ( B e r l i n e r , 1960; M o r e l

and Guinnebault, 1961), the d e l i v e r y of w a t e r m u s t be a s s u m e d to i n c r e a s e . T h e r e is, thus, no r e a s o n to a s s u m e that delayed w a s h - o u t of u r e a in w a t e r d i u r e s i s could be due to the low r a t e of w a t e r flow through p a p i l l a r y and m e d u l l a r y t i s s u e s . F u r t h e r m o r e , a low r a t e of w a t e r flow through m e d u l l a r y t i s s u e would not explain the d i f f e r e n t r a t e s of u r e a w a s h - o u t o b s e r v e d during the f i r s t 3 and the subsequent 5 hr of w a ter diuresis. S e q u e s t r a t i o n of a f r a c t i o n of the m e d u l l a r y and the p a p i l l a r y u r e a in a slowly exchanging c o m p a r t m e n t , t h e r e f o r e , a p p e a r s as the m o s t likely i n t e r p r e t a t i o n of the slow d ecl i n e in t i s s u e u r e a during w a t e r d i u r e s i s . As pointed out above, such s e q u e s t r a t i o n has been shown to o c cu r in yitro, in c o r t i c a l and e x t e r n a l m e d u l l a r y s l i c e s of r e n a l t i s s u e CO'Dell and S c h m i d t - N i e l sen, 1961). Though a p r e c i s e a s s e s s m e n t of the s i z e of the rapidly and the slowly exchanging f r a c t i o n s of p a p i l l a r y and m e d u l l a r y u r e a would n e c e s s i t a t e m e a s u r e m e n t s of t i s s u e u r e a conc e n t r a t i o n s r e p e a t e d at s h o r t i n t e r v a l s a f t e r the induction of w a t e r d i u r e s i s , the data shown in fig. 6 may offer a v e r y rough idea of the f r a c tions and the p r o c e s s e s involved. Thus, in p a p i l l a r y t i s s u e a p p r o x i m a t e l y 58% of the total u r e a a p p e a r e d to be contained in a r a p i d l y exchanging c o m p a r t m e n t (t/2 = 1.4 hr), while a p p r o x i m a t e l y 42% a p p e a r e d to be contained in the slowly e x changing c o m p a r t m e n t (t/2 = 4.3 hr). In the i n n er m e d u l l a 83% of the total u r e a a p p a r e n t l y b e longed to the r ap i d l y exchanging c o m p a r t m e n t (t/2 = 1.0 hr), while only 17% c o n s t i t u t e d the slowly exchanging f r a c t i o n (t/2 = 7.0 hr). While n e i t h e r t h ese rough c a l c u l a t i o n s nor the e x p e r i m e n t a l data exclude the p o s s i b i l i t y that u r e a may be d i s t r i b u t e d into m o r e than two " c o m p a r t m e n t s ", or may be bound to m o r e than two types of compounds in r e n a l t i s s u e , they definitely show that w a s h - o u t of u r e a f r o m r e n a l m e d u l l a r y t i s sue is a slow and c o m p l i c a t e d p r o c e s s . Since it has not been shown that p a p i l l o - u r i n a r y u r e a g r a d i e n t s in o s m o t i c d i u r e s i s , in n o r m a l and p r o t e i n depleted r a t s (Bray and P r e s t o n , 1961; B r a y , 1963; Clapp, 1966), w e r e actually obs e r v e d u n d er steady state conditions, t h e i r o cc u r r e n c e may also be ex p l ai n ed by the p r o p o s e d Mcompartmentalisation ~ of r e n a l m e d u l l a r y u r e a . F i n a l l y " c o m p a r t m e n t a l i s a t i o n " of m e d u l l a r y u r e a r a t h e r than a c t i v e t r a n s p o r t may a l s o ex plain the m i c r o p u n c t u r e o b s e r v a t i o n s of high u r e a c o n c e n t r a t i o n s in p a p i l l a r y v a s a r e c t a p l a s m a as c o m p a r e d to fluid contained in the c o l l e c t ing ducts in n o r m a l and p r o t e i n depleted young r a t s ( L a s s i t e r et al., 1966); the slowly ex -

INTRARENAL UREA AND SALT changing "compartment" could equilibrate more r e a d i l y w i t h the c o n t e n t s of the v a s a r e c t a than w i t h c o l l e c t i n g duct fluid. C o n s i d e r i n g the e s t i m a t e s of the h a l f - l i f e t i m e of both c o m p a r t m e n t s in w a t e r d i u r e s i s , one m i g h t e v e n s p e c u l a t e that the s l o w l y e x c h a n g i n g c o m p a r t m e n t n o r m a l l y e q u i l i b r a t e s w i t h v a s a r e c t a p l a s m a and p e r h a p s with f l u i d in H e n l e ' s loops, w h i l e the r a p i d l y ex~ c h a n g i n g c o m p a r t m e n t t e n d s to e q u i l i b r a t e w i t h c o l l e c t i n g duct fluid. Another less likely explanation for the delayed wash-out of urea in water diuresis would be the local synthesis of urea in medullary tissue. The enzymatic system for the hepatic pathway of urea synthesis is present in renal tissue (Hutchinson et al., 1964a, b). A number of arguments (Lassiter et al., 1966) appear to exclude a major contribution of renal urea synthesis to medullary urea. In view of the small amounts of urea involved, a role of local synthesis cannot, however, be ruled out with certainty. In order to explain our results the hypothesis of local synthesis would have to be completed by the assumption that this process is depressed in water diuresis. A h y p o t h e t i c a l s c h e m e of u r e a m o v e m e n t s a f t e r s e v e r a l h o u r s of w a t e r d i u r e s i s c o m p i l e d f r o m the p r e s e n t d a t a and the m i c r o p u n c t u r e e x p e r i m e n t s of L a s s i t e r et al. (1961, 1964, 1966) is g i v e n in fig. 7. M o v e m e n t s of u r e a f r o m t i s s u e p o o l s into the n e p h r o n w o u l d be l i m i t e d to the d e s c e n d i n g l i m b and the f l e x u r e of H e n l e ' s loop. An a m o u n t of u r e a e q u i v a l e n t to that g a i n e d in t h e s e s i t e s w o u l d h a v e to l e a v e the c o l l e c t i n g d u c t s d u r i n g t h e i r c o u r s e t h r o u g h the o u t e r and i n n e r m e d u l l a , w h i l e no s u c h m o v e m e n t w o u l d be p o s s i b l e in the l o w e r c o l l e c t i n g duct w h e r e the c h e m i c a l g r a d i e n t w o u l d o p p o s e it. In fact, the a m o u n t of u r e a l e a v i n g the u p p e r c o l l e c t i n g duct c o u l d be j u s t s o m e w h a t s m a l l e r than the a m o u n t p e n e t r a t i n g into H e n l e ' s loop and into the v a s a r e c t a : in t h i s way, t i s s u e u r e a c o n c e n t r a t i o n s w o u l d s l o w l y d e c r e a s e to the low v a l u e s obs e r v e d a f t e r c o n t i n u o u s w a t e r d i u r e s i s of m o r e than 8 hr. One would, then, e x p e c t the d i f f e r e n c e in u r e a c o n c e n t r a t i o n s b e t w e e n v a s a r e c t a b l o o d and c o l l e c t i n g d u c t s f l u i d to d i s a p p e a r in c o n t i n u o u s w a t e r d i u r e s i s (and p e r h a p s a l s o in long lasting osmotic diuresis). 4.2. Sodium, potassium and ammonium A l l c h a n g e s in c o r t i c a l p a p i l l a r y u r e a g r a d i e n t s in the p r e s e n t e x p e r i m e n t s w e r e a c c o m p a n i e d by s i m i l a r , though s m a l l e r , c h a n g e s in s o d i u m g r a d i e n t s . T h e r e is, t h e r e f o r e , no r e a son to doubt the p r e v a i l i n g opinion that a l l m o v e m e n t s of u r e a a r e s e c o n d a r y to a c t i v e m o v e m e n t s of s o d i u m ions. If u r e a is s e q u e s t e r e d in d i f f e r e n t c o m p a r t m e n t s , a s s u g g e s t e d a b o v e , the o b s e r v a t i o n that e t h a c r y n i c a c i d , in dogs, a b o l -

137

.

[28]

[19] $ 5O% Fig. 7. Urea concentrations and movements in the lower part of a juxtaglomerular nephron after 8 hr of water diuresis. The values represent concentrations from the present study and T F / P ' s from W.E. Lassiter et al. (1964), assuming that urea T F / P ' s are similar in water diuresis and in saline diuresis. All other assumptions and explanations as in fig. 1.

i s h e s the c o r t i c o p a p i l l a r y s o d i u m g r a d i e n t w i t h out d e s t r o y i n g the u r e a g r a d i e n t ( G o l d b e r g and R a m i r e z , 1965) c o u l d be due to a d e l a y e d w a s h out of u r e a r a t h e r than to i n d e p e n d e n t m o v e ments. In w a t e r d i u r e s i s , s o d i u m c o n c e n t r a t i o n in all m e d u l l a r y l a y e r s d e c r e a s e s p r o g r e s s i v e l y (table 5, fig. 6). D u r i n g the f i r s t 3 hr, t h i s d e c r e a s e is due to i n c r e a s e d w a t e r c o n t e n t : the a c t u a l s o d i u m c o n t e n t of the t i s s u e d o e s not c h a n g e (table 6). D u r i n g the s u b s e q u e n t h o u r s , s o d i u m c o n t e n t of the t i s s u e a l s o d e c r e a s e s . D u r i n g the f i r s t 3 h r of w a t e r d i u r e s i s , i . e . , at a t i m e w h e n the p a p i l l o - u r i n a r y u r e a g r a d i e n t r e a c h e s i t s h i g h e s t v a l u e , the a m o u n t of s o d i u m d e p o s i t e d in m e d u l l a r y t i s s u e p e r unit of t i m e , m u s t h a v e b e e n s t a b l e . A s p o i n t e d out a b o v e , the r a t e of w a t e r flow t h r o u g h m e d u l l a r y t i s s u e m u s t , t h e r e f o r e , be l a r g e r than in the n o n - d i u r e t i c s t a t e . T h e s u b s e q u e n t w a s h - o u t of s o d i u m f r o m the r e n a l m e d u l l a c o u l d be due to d e c r e a s e d d e p o s i t i o n , to i n c r e a s e d r e m o v a l by the m e d u l l a r y c i r c u l a t i o n , or, e v e n m o r e l i k e l y , to a s t i l l l a r g e r i n c r e a s e in w a t e r flow t h r o u g h the t i s s u e

138

F. ROCH-RAMEL and G. PETERS

in r e l a t i o n to s o d i u m d e p o s i t i o n . T h e low s o d i u m c o n c e n t r a t i o n of r e n a l c o r t i c a l t i s s u e a n d t h e p r e s e n c e of a h i g h e r s o d i u m c o n c e n t r a t i o n in t u b u l a r f l u i d p e r m i t a n e s t i m a t i o n of t h e f r a c t i o n of t o t a l t i s s u e w a t e r w h i c h c o u l d b e c o n t a i n e d in tubular fluid: proximal sodium TF/P ratios are e q u a l to 1.0 in a l l c o n d i t i o n s , w h i l e d i s t a l s o d i u m T F / P r a t i o s a r e > 1.0 i n d e h y d r a t e d o r n o n - d i u r e t i c r a t s ( U l l r i c h e t al., 1963). E v e n if c o r t i cal cellular and interstitial water were comp l e t e l y d e v o i d of s o d i u m , l e s s t h a n 50% of t o t a l c o r t i c a l w a t e r c a n t h e r e f o r e b e c o n t a i n e d in t u b u l a r fluid. S i n c e c o r t i c a l c e l l u l a r a n d i n t e r s t i t i a l f l u i d do c o n t a i n s o d i u m , t h e a c t u a l c o n t r i b u t i o n of i n t r a t u b u l a r fluid is probably much s m a l l e r . In w a t e r d i u r e s i s , d i s t a l t u b u l a r T F / P N a i s l e s s t h a n one; a s i m i l a r r e a s o n i n g y i e l d s a maximal c o n t r i b u t i o n of i n t r a t u b u l a r fluid s l i g h t l y in e x c e s s of 50%; a g a i n t h e a c t u a l v a l u e i s p r o b a b l y m u c h l o w e r . It s h o u l d b e r e m e m 1 l b e r e d a t t h i s p o i n t t h a t o n l y a p p r o x i m a t e l y T-~ of c o r t i c a l t u b u l a r f l u i d c a n b e c o n t a i n e d in d i s t a l t u b u l e s : the f r a c t i o n of c o r t i c a l w a t e r c o n t a i n e d in distal tubular fluid, therefore, cannot exceed 12-18% and is probably much smaller. P o t a s s i u m concentration profiles o b s e r v e d in the p r e s e n t investigation were comparable to those of p r e vious o b s e r v e r s (Manitius et al., 1960; Saikia, 1965). The absence of changes in renal tissue p o t a s s i u m conc e n t r a t i o n s during water d i u r e s i s may also be the consequence of a " c o m p a r t m e n t a l i s a t i o n " .

4.3. Hydrochlorothiazide K o b i n g e r (1964) f o u n d a l a r g e d e c r e a s e in urinary as well as in papillary urea concentration after bendroflumethiazide in dehydrated rats; similar results were reported by Goldberg e t al. (1965) in d e h y d r a t e d d o g s , t r e a t e d w i t h e i t h e r h y d r o c h l o r o t h i a z i d e o r e t h a c r y n i c a c i d . In t h e p r e s e n t e x p e r i m e n t s in w a t e r d i u r e s i s , h y d r o c h l o r o t h i a z i d e d i d n o t i n c r e a s e u r i n e flow; a t the same time the urinary urea concentration r e m a i n e d u n c h a n g e d . T h e f a l l in u r i n a r y u r e a concentration after thiazide diuretics, thus, s e e m s to b e due s i m p l y to d i l u t i o n r a t h e r t h a n to a n y c h a n g e in u r e a r e a b s o r p t i o n . A s i n d e h y drated animals hydrochlorothiazide in water diuresis apparently caused a fall in papillary urea c o n c e n t r a t i o n , w h i c h w a s of b o r d e r l i n e s i g n i f i cance, but corroborated a p a r t of K o b i n g e r ' s f i n d i n g s . T h i s d e c r e a s e c o u l d b e due to a n i n f l u e n c e of h y d r o c h l o r o t h i a z i d e on t h e m e c h a n i s m of m e d u l l a r y s e q u e s t r a t i o n of u r e a . A l t e r n a t i v e l y , i t c o u l d d e n o t e a n i n c r e a s e in t h e u r e a p e r m e a b i l i ty of c o l l e c t i n g t u b u l a r w a l l s w h i c h , u n d e r t h e c o n d i t i o n s p r e v a i l i n g d u r i n g t h e 8 h r of w a t e r d i -

u r e s i s , s h o u l d f a v o r d i f f u s i o n of u r e a f r o m p a p i l l a r y t i s s u e to c o l l e c t i n g d u c t fluid.

ACKNOWLEDGEMENTS We gratefully acknowledge the collaboration of Mlle. F.Chomity, and the technical a s s i s t a n c e of Mlles. H. Amstutz, C. Schwenter and M. Gu~ron.

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