Pfliigers Arch. 346, 31--38 (1974) 9 by Springer-Verlag 1974

Analysis of Cortical Urea Sequestration in Rat Renal Tissue Slices* H . Schad, H. W . R e i n h a r d t , a n d T. A r m s e n Physiologisches Institut der Universit~t, Miinehcn, Germany Received September 10, 1973

Summary. In antidinretie rats plasma urea concentration (P~) was elevated up to 35 mM by intravenous urea loading. In renal cortical tissue (T) water content and unlabeled (u) and 14-C-urea (u*) were determined. Water content of total renal cortex and of superficial and juxtamedullary layers were not significantly different and were not changed by the elevation of Pa. I n the control group (P~ < 10 raM), both T/P~ (3.06 • 0.71; n = 29) and plasma specific activity were significantly higher than values for T/Pu* (2.20 • 0.57; n = 12) and tissue specific activity, respectively. T/P~ and T/Pu* were decreased significantly to the same level (1.68 ~ 0.35; n = 20 and 1.51 i 0.21; n = 7, respectively) at P= > 20 rnM and plasma and tissue specific activity became equal. Deproteination of tissue homogenates did not depress 14-C-activity. Juxtamedullary T/Pu was significantly higher than superficial TIPs. The difference was independent of P~. The data suggest, that renal cortical tissue urea concentration is determined (i) by distal tubular fluid urea concentration, which is inversely correlated with Pu and (ii) by a constant amount of urea in an extraluminal compartment not readily exchanging. The mechanism by which urea concentration in this extraluminal compartment is maintained independent of P~ might be urea neogenesis or urea binding to cellular structures or proteins. Key words: Urea Infusion -- Tissue Analysis -- Urea Sequestration -- Urea Binding -- Urea Neegenesis. T h e u r e a c o n c e n t r a t i o n in r e n a l cortical tissue (Tu) is h i g h e r t h a n in p l a s m a (P~) [9--16,18]. This p h e n o m e n o n h a s been r e l a t e d to a c c u m u l a t i o n o f u r e a in t u b u l a r cells b y some s e q u e s t r a t i n g m e c h a n i s m w i t h a l i m i t e d c a p a c i t y [10,11]. T h e u n d e r l y i n g m e c h a n i s m could be 1. a c t i v e [9] t r a n s p o r t o f u r e a ; in this case l a b e l e d u r e a (u*) should be c o n c e n t r a t e d in t h e r e n a l c o r t e x t o t h e s a m e e x t e n t as endogenous u r e a a n d T / P J should be i d e n t i c a l to T/Pu; 2. u r e a neogenesis [3, 5]; here, t h e specific * Parts of this study were presented at the 35th meeting of the Deutsche Physiologische Gesellschaft [8].

32

H. Schad

et aL

a c t i v i t y of 14-C-urea should be lower i n cortical tissue w a t e r t h a n i n p l a s m a ; 3. urea b i n d i n g [4] ; if urea is b o u n d to some cellular structures or to proteins a n d the exchange r a t e with free urea is v e r y low, T/Pu* should be lower t h a n T/Pu; u p o n elevation of Pu, however, T/Pu* should r e m a i n c o n s t a n t whereas T/Pu should decrease; 4. urea i n t u b u l a r fluid, if urea c o n c e n t r a t i o n i n the t u b u l a r fluid of deep n e p h r o n s was higher t h a n i n superficial t u b u l e s [7] ; s e p a r a t i o n of the j u x t a m e d u l l a r y p o r t i o n from the r e m a i n d e r of cortical tissue should t h e n reveal a T/Pu higher i n this layer t h a n i n the more superficial parts. To d e t e r m i n e t h e "Tin" of t h e sequestrating mechanism, Pu was elevated gradually. U n d e r this c o n d i t i o n the T/Pu should r e m a i n cons t a n t as long as the c a p a c i t y l i m i t is n o t reached a n d it should decrease as soon as t h e c a p a c i t y is exceeded while Pu is f u r t h e r increased. I n a n a t t e m p t to characterize t h e m e c h a n i s m of urea s e q u e s t r a t i o n T/Pu* was c o m p a r e d to T/Pu i n superficial a n d deep cortical layers.

Methods Experiments were performed in female Wistar rats, body weight 200--250 g. The animals were kept on a normM protein diet (Altromin BO 100) and had free access to food and water. Under anaesthesia (80 mg/kg Inaetin~, a thiobarbiturate, i.p.), the trachea was cannulated and a polyethylene catheter inserted into the jugular vein. Via this catheter the animals of the control group received an infusion of 6 g-~ (w/v) glucose in distilled water at a rate of 0.6 ml/h. To increase Pu, solutions of urea in distilled Water [6 g-e/0, 8 g-e/0, 10 g-~ and 12 g-e/0 (w/v)] were infused at a rate of 0.6 ml/h. I n the 14-C-urea experiments, labeled urea (10 ~C/ml) was added to these solutions. A priming injection of 1 ml was followed by a sustaining dose of 0.45 ml/h in order to achieve a constant level of Pu* within 30 rain. Unlabeled urea plasma concentration was varied independently. At the end of a 2 h infusion time, the kidneys were exposed by a midline incision, clamped at the hilns, removed and quickfrozen in a mixture of dry-ice and isopentane (about --70 ~C). Blood samples were taken from the heart. Preparation of cortical tissue slices and separation of these in a superficial and juxtamedullary portion was carried out under a dissection microscope. During this procedure the kidneys were placed on an aluminum plate, and cooled to --5~ to avoid melting of the tissue. Formation of condensation water was prevented by blowing the kidney slices with dry and cooled air at about --5~ Under these conditions it was possible to exactly separate the cortex from the outer medulla. Leakage of blood, tubular and interstitial fluid was observed to be minimal in the frozen state and hence a significant dissipation of urea from the outer medulla into the cortex should not have occurred. The frozen tissue samples were transferred to glass tubes with a ground-in stopper and weighed to the nearest 10 ~zg. Urea was determined in the supernatant fluid after boiling in 2--3 ml of a 5--10~ HC1 solution or after ultrasonation in 3 ml distilled water. Water content was determined in a different slice from the same layer after drying at 95~ to constant weight. Urea concentration was determined in blood and tissue slices. For chemical analysis the monoxime method [2] was applied using a Technicon Auto Analyzer.

Renal Cortical Urea Sequestration

33

Determination of 14-C-urea was carried out in a Liquid-Scintillation-Spectrometer (Packard). The scintillation solution contained PPO (2,5-diphenyloxazole), POPOP (2,2"-p-phenylen-bis[5-phenoloxazole]),naphCalino anddioxan. I n addition, 14-C-urea was determined in deproteinized homogenates, prepared from the same tissue sample. Protein was precipitated with ZnSOa and NaOH. The quenching facf~r was determined by the "internal standard" method. The water content of the tissue slices was calculated as g H20/100 g wet tissue; chemical urea concentration as ~mol/g tissue water, and 14-C-urea content as cpm/g tissue water. Data are given as mean • S.D. The t-test was used for statistical analysis. Results

The p l a s m a urea c o n c e n t r a t i o n of the control group was wi$hin 5 - - 1 0 raM. T h r o u g h i n t r a v e n o u s application of urea, Pu was elevated u p to 35 mlV[. These v a r i a t i o n s i n plasma urea c o n c e n t r a t i o n h a d n o significant effect u p o n the water c o n t e n t of the t o t a l r e n a l cortex a n d o n the superficial or j u x t a m e d u l l a r y p a r t s ; the water c o n t e n t i n superficial tissue slices did n o t significantly differ from t h a t i n j u x t a m e d u l l a r y layers (Table 1). The tissue urea c o n c e n t r a t i o n of the r e n a l cortex i n the control g r o u p was f o u n d t e be higher b y a factor of 3 t h a n the p l a s m a urea con-

Table 1. Renal cortical water content, cortical tissue to plasma urea ratios, and 14-C-urea specific activity in cortical tissue and in plasma of urea loaded and non loaded rats. (Mean -4- S.D.) Control P Water content (g H20 / 100 g wet tissue) Total cortex n.s. Superficial n.s. Juxtamedul. lary n.s.

T/P~ T/Pu.

< 0.005

saT/saP

Pu<10mM

Urea infusion n

P

Pn>g0mM

n

P

79.1

-4- 2.5 (13) n.s.

77.5

:~ 2.6

(7)

n.s.

( 78.8

-4- 2.7 (13) n.s.

76.0

:[: 2.7

(7)

n.s.

3.06 4- 0.71 (29) < 0.0005 1.68 ~- 0.35 (20) 2.20 :t: 0.57 (12) < 0.005 1,51 :[: 0.21 (7)

n.s.

1.16+ ~ 0.09 (13) n.s.

1.13+ ~: 0.07 (7)

0.63+ -V 0.10 (11) <0.0005

0.97 ~ 0.17 (7)

n.s.: P > 0.01; +: significant difference from 1.0; P < 0.0005; *: tracer; P : plasma concentration; T: tissue concentration; u: urea; sa: specific activity; s: superficial; }': juxtamedullary. 3 Pfltigers Arch., VoL 846

84

It. Schad

et al.

T/PuREA 5O

p< 0 . ~

p
30 84

2.0.

1.0 2~

PU.EA [~1

3~

Fig.1. Influence of increasing plasma urea concentration (Purea) on the ratio of renal cortical tissue urea concentration (T) to P~ea. Mean q- S.D. are illustrated by horizontal line and hatched area, respectively

centration. U p o n raising Pu, the cortical Tu increased also, but to a lesser extent t h a n P u ; this resulted in a gradual decrease of the T/P~ ratio from 3.06 4- 0.71 (n -= 29) to 1.68 4- 0.35 (n = 20) while the plasma urea concentration increased from P u < 10 mM to P u > 20 mM (Fig. 1). The T/Pu, however, did not appear to be linearely correlated with P~; rather, it declined asymptotically and approached a minimum of 1.5 at plasma urea levels of about 25 raM. The decrease of T/Pu was seen both in the superficial and in the juxtamedullary cortex. The rate of decrease was similar; the tissue urea concentration in the juxtamedullary portion, however, was always some 10--150/0 higher t h a n in the superficial p a r t (Fig. 2). This difference was rather constant and independent of Pu throughout the entire range of plasma urea variation: The ratio of the simultaneously determined urea concentration in juxtamedullary and superficial cortex (TuI/Tus) was not different at different levels of Pu, and was significantly higher t h a n 1.0 at any plasma urea concentration (Table 1). Similar observations were made for the 14-C-labeled urea experiments. Although the plasma concentration of 14-C-urea was kept constant while Pu was elevated, the cortical T/Pu* decreased significantly ( P < 0.005) from 2.20 4- 0.57 (n = 12) to 1.51 4- 0.21 (n = 7) when P u was elevated from Pu < 10 mM to Pu > 20 mM (Table 1). The T/Pu in

Renal Cortical Urea Sequestration

35

T/PuREA /.,0' 11~I]

|17|

I~'}

2.0-

1,0 ........ I 5-10 [

10 -20

[

20-3S

iPuREA [mM]

~'ig.2. Superficial (o) and j~tamedullary (o) tissue to plasma urea ratio (T/Pure=) at different levels os P~ea. (]Y[ean :~ S.D.)

the control group, however, was significantly (P < 0.0005) higher than the T/Pu* ratio, whereas this difference had disappeared at plasma urea levels above 20 mM (Table 1). Simultaneous measurements of 14-C-urea in protein-free and nondeproteinized homogenates (n = 24) showed no difference between the 14-C-urea concentration in the deproteinized (Tu*~) and non-deproteinized (Tu*n~) tissue. The mean of the ratio Tu*d/Tu*ng was 1.02 • 0.09 and did not significantly differ from unity (P > 0.4). Discussion The urea concentration found to be higher in renal cortical tissue than in plasma [9--16,18] has been assumed to be due to urea sequestration into an extralumina] compartment [9--12]. I f this assumption is true it should not be possible to explain it as a consequence of urea concentration in distal tubular fluid [9,12]. The nature of the sequestering mechanism, however, has not been specified; it should have "Tin" characteristics since cortical T/Pu is decreased in urea loaded rats [10, 11]. To interpret this sequestration, a different handling of urea b y superficial and deep nephrons might be invoked [7]. The experiments of RochRamel et al. [10, 21] suggest an increased urea delivery at the end of juxtamedullary distal tubules in urea loaded rats. The difference, however, between superficial and juxtamedulIary Tu in this series of experiments is small and not suggestive of a difference in tubular urea concen3*

36

H. Schad et al.

tration between deep and superficial nephrons. The higher urea concentration in deep cortical layers compared to those in superficial tissue section could be related to the increasing contribution of medullary rays to total tissue mass from the surface to the outer medulla [6]. Active transport of urea across the tubular cells could also result in a high cortical T/Pu [9]. This, however, has not been verified b y the mieropuncture studies of Ullrich et al. [17]. A n active step might nevertheless be involved in urea accumulation into subcelhilar compartments. Then, T/Pu* should be equal to T/P u. In the present experiments, however, T/P~** was found to be significantly lower than T/Pu, suggesting passive urea accumulation into a compartment which does not readily exchange its contents. This would also explain the decrease of T/Pu following moderate elevation of Pu, assuming a restrained access for the exogenous urea to this compartment. This elevation of Pu, however, was associated with a significant fall in T/Pu*, although Pu* in this situation was found to be constant. The change in Tu* may be explained b y the assumption of a different, urea containing compartment. The uptake characteristics of this compartment, under the condition of elevated Pu, can be described b y a concentrating factor which is inversely related to PuAssuming these two compartments, T/Pu*, at normal Pu, should not reach the level of T/Pu due to the presence of the "non accesSible" compartment (n.a.c.). During elevation of Pu, both T/Pu and T/Pu* should decrease due to the diminished concentrating factor in the concentrating compartment (c.e.), whereas the urea concentration in the n.a.c. would remain constant. With increasing Pu, the n.a.c, would contribute to the T/P~, to a minor degree, and T/P~ should equal T/Pu* as it has been observed in the present experiments. Concerning the localization of these two compartments in the renal cortex, we have an indication for the concentrating compartment only. Armsen et al. [1] have shown a decreasing distal T F / P u with increasing P u which would be in agreement with the required characteristics of the c.e. Therefore, the distal tubules could be identical to this compartment. Under this condition, the cortical T/Pu could be determined b y the distal TF/Pu at a constant level of urea in the n.a.e.; the decrease in T/Pu would then be due mainly to a decrease of distal TF[Pu. The present experiments cannot offer a localization of the n,a.c, nor a ready explanation of the mechanism by which a urea concentration is maintained independent of Pu, The possibility, however, of urea binding to cellular protein should be considered [4]. The exchange rate in this case should be low, since no labeled urea was found to be bound during the 2 h of 14-C-urea infusion,

Renal Cortical Urea Sequestration

37

as deduced from deproteination of the tissue homogenates which showed no reduction in 14-C-activity. A n o t h e r explanation would be urea synthesis [3, 5]. Truniger et al. [15,16] however, were n o t able to find experimental support for this hypothesis. I n tracer studies, the specific a c t i v i t y of 14-C-urea was identical in plasma and tissue water. I n the present control experiments, the specific activity is smaller in cortical tissue water t h a n in plasma (Table 1). Following elevation of Pu, however, tissue and plasma specific activity tend to equalize. Therefore, urea synthesis m u s t in this situation either have c e a s e d or the synthesized urea was a non detectable fraction of t h e total cortical urea as a consequence of increased urea concentration in other compartments.

Acknowledgement. The authors are grateful to Dr. M. Itorster and Dr. A. DSrge for their discussions of these data, References 1. ~ n s e n , T., Schad, H., Reinhardt, H. W. : Die Konzentrierung des Hamstoffs in der Niere. II. )/iikropunktionsuntersuchungen an der Ratte. Pfliigers Arch. 813, 222 (1969) 2. Beale, R. N., Croft, D. : A sensitive method for the colorimetric determination of urea. J. clin. Path. 14, 418 (1961) 3. Carlisky, N.J., Brodsky, W.A., Huang, K. C.: Biosynthesis of urea by the kidney in relation to the mechanism of urea excretion. Fed. Proc. 21, 427 (1962) 4. O'Dell, R. M., Schmidt-Nielsen, B. : Retention of urea by frog and mammalian kidney slices in vitro. J. cell. comp. Physiol. 57, 211 (1961) 5. Kleinman, L. J., Radford, E. P.: Urea synthesis by rat kidney. Physiologist 7, 179 (1964) 6. Kriz, W.: Der architektonische und funktionelle Aufbau der Rattenniere. Z. Zellforsch. 82, 495 (1967) 7. Rabinowitz, L.: In: Urea and the kidney, lO. 340. Editor: B. Schmidt-Nielsen. Excerpta Medica ICS 195 (1970) 8. Reinhardt, H. W., Schad, H., Armsen, T.: Zur Frage der Harnstoffkonzentrationen in den Kompartimenten der I~ierenrinde (Ratte). Tissue slice-study of urea concentration in structures of the renal cortex (rat). Pflfigers Arch. 807, R74 (1969) 9. Roch-Ramel, F., Chom6ty, F., Peters, G.: Urea concentrations in tubular fluid and in renal tissue of nondiuretic rats. Amer. J. Physiol. 216, 429 (1968) 10. Roch-Ramel, F., Di6zi, J., Chom6ty, F., Ylichoud, P., Peters, G. : Disposal of large urea overloads by the rat kidney: a micropuncture study. Amer. J. Physiol. 218, 1524 (1970) 11. Roch-Ramel, F., Di6zi, J., Chom6ty, F., Michoud, P., Peters, G.: Urea concentrations in tubular fluid and in renal tissue of rats overloaded with urea or with saline solutions. In: Urea and the kidney, p. 333. Editor: B. Schmidt-Nielsen. Excerpta 1VfedicaICS 195 (1970) 12. Roch-Ramel, F., Peters, G.: Intrarenal urea and electrolyte concentrations as influenced by water diuresis and by hydrochlorothiazide. Europ. J. Pharmacol. 1, 124 (1967)

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H. Sehad et al.

13. Saikia, T. C.: Composition of the renal cortex and medulla of rats during water diuresis and antidiuresis. Quart. J. exp. Physiol. 50, 146 (1965) 14. Sehmidt-l~ielsen, B., O'Dell, R.: Structure and concentrating mechanism in the mammalian kidney. Amer. J. Physiol. 290, 1119 (1961) 15. Truniger, B., Schmidt-Nielsen, B.: Intrarenal distribution of urea and related compounds: effects of nitrogen intake. Amer. J. Physiol. 2{}7, 971 (1964) 16. Truniger, B., Sehmidt-Nielsen, B.: Intrarenal distribution and transtubular movement of urea and related compounds. In: Urea and the kidney, p. 314. Editor: B. Schmidt-I~ielsen. Excerpta Niediea ICS 195 (1970) 17. Ullrich, K. J., Rumrich, G., Baldamus, C. A.: Mode of urea transport across the mammalian neplu'on. In: Urea and the kidney, p. 175. Editor- B. SchmidtIqidlsen. Excerpta Niedica ICS 195 (1970) 18. Valtin, H. : Sequestration of urea and non-urea solutes in renal tissues of rats with hereditary hypothalamic diabetes insipidus ." effect of vasopressin and de. hydration on the counter current mechanism. J. clin. Invest. 45, 337 (1966) Hubert Schad Physiologisehes Institut der Universit~t Mfinchen D-8000 NIiinehen 2 PettenkoferstraBe 12 Federal Republic of Germany

Ture Armscn I. Medizinische Univ.-Klinik D-80O0 N[finchen 2 ZiemssenstraBe 1 1%deral Republic of Germany Prof. H. W. Reinhardt Freie Universit~t Berlin St~dt. Krankenhans Wcstend Abt. s exp. Anaesthesie D-1000 Berlin 19 Spandauer Datum 130