The effect of urea, sodium and calcium on microsomal ATPase activity in different parts of the kidney

BIOCHIMICA ET BIOPHYSICA ACTA

133

BBA 7 5 5 9 5

T H E E F F E C T OF UREA, SODIUM AND CALCIUM ON MICROSOMAL ATPase ACTIVITY IN D I F F E R E N T PARTS OF T H E K I D N E Y Y E H U D A GUTMAN AND Y A F F A KATZPER-SHAMIR

Department of Pharmacology, The Hebrew University-Hadassah Medical School, Jerusalem (Israel) (Received November 3rd, 197 o)

SUMMARY

Mg~+-ATPase and (Na + + K+)-dependent ATPase activities were assayed in the microsomal fractions of the cortex, medulla and papilla of the guinea-pig kidney. MgZ+-ATPase in any part of the kidney was unchanged in the presence of 300 mM urea. (Na + + K+)-dependent ATPase activity was unchanged in the cortex, but was significantly inhibited in the medulla and papilla by 3o0 mM urea. Elevated concentrations of Na + (200 mM) depressed ATPase activity in the microsomal fraction derived from the papilla and medulla, but did not significantly affect the activity in the kidney cortex. Ca ~+ (5 mM) caused a significant inhibition of (Na + + K+)-depen dent ATPase in all three parts of the kidney and depressed Mg2+-ATPase activity only in the papilla. Active Na + transport in the kidney has been shown to take place in different locations along the nephron: the proximal convoluted tubulO, 3, the loop of Henle 3, the thick ascending limb l, the distal convoluted tubules ~, 5 and the collecting duct s. It is assumed that sodium transport from the lumen of the nephron outwards subserves different functions in the various portions of the tubules. Thus, reabsorption of Na + in the proximal tubule is related to regulation of Na + balance; active transport along the loop of Henle is essential for the maintenance of the osmotic gradient in the kidney 3 and thus for the mechanism of urine concentration; active transport of Na + along the thick ascending limb enables the production of hypotonic fluid4 and, therefore, is essential for urine dilution. Although active transport of Na + is involved in each of these sites the effect of diuretic drugs, which inhibit Na + transport, is apparently restricted to specific portions of the nephron n,7. Furthermore, the characteristics of Na + transport differ along the nephron: while Na + absorption is isotonic in the proximal tubule 4, 5, it is against a concentration gradient in the thick ascending part of the loop and in the distal tubule x, 4, 5. Many reports have pointed to the relationship between active Na + transport and the activity of a microsomal (Na + + K+)-activated ATPase 8,~. This enzymic activity has also been shown to occur in the kidney a° and is found in all its parts: cortex, medulla and papilla lx-xS. If the same enzyme is involved in Na + transport at the different locations of the nephron it would be of interest to study whether the differences in the characteristics of Na + transport in various parts of the kidney are reflected in differences in the characteristics Of microsomal ATPase from these parts. Biochim. Biopkys. Aaa, 233 (1971) 133-136

134

¥. GUTMAN, Y. KATZPER-SHAMIR

Microsomal ATPase activity is usually assayed in vitro in isotonic media resembling the composition of plasma s . Cells located in the medulla and particularly in the papilla of the kidney are bathed by a hypertonic medium, particularly rich in urea and Na +, in contrast to cells in the cortex which are perfused by an isotonic fluid. We have, therefore, studied the effects of increased Na + concentration and of elevated urea concentration on microsomal ATPase from the kidney cortex, medulla and papilla. Guinea-pigs were killed b y a blow on the head, the kidneys were immediately removed and placed in ice-cold 0.25 M sucrose containing 2 mM EDTA. The kidneys were cut sagitally and then divided into three portions : the papilla (the pale portion, projecting into the pelvis), the medulla (the dark-red portion, which contains the thick ascending part of the loop of Henle) and the cortex (the outermost part of the kidney). The different portions of the kidneys were homogenized in IO vol. of the medium, then centrifuged at IOOO × g for IO min. The supernatant was centrifuged at IOOOO × g for IO min ; the supernatant from this centrifugation was then subiected to lO5 ooo × g for 30 min to sediment the microsomes. The supernatant was discarded and the microsomes were resuspended in homogenizing medium. Mg2+-ATPase activity and (Na + + K+)-dependent ATPase activity were distinguished by incubating in two different media. For total ATPase activity incubation was carried out in a medium containing IOO mM NaC1, IO mM KC1, 4 mM MgC12, 4 mM ATP (sodium salt) and 33 mM Tris buffer (pH 7.2). For Mg2+-ATPase activity NaC1 and KC1 were replaced b y Tris, and the sodium salt of ATP was replaced by Tris-ATP. The difference between the total ATPase activity and the Mg*+-ATPase activity was the (Na ÷ + K÷)-dependent activity. The results thus obtained were identical with the (Na + + K+)-dependent ATPase activity determined b y incubation in the complete electrolyte medium in the presence of I mM ouabain. The incubation was carded out for 20 min at 37 ° with continuous shaking. The reaction was stopped by the addition of i vol. of 5 % trichloroacetic acid. Inorganic phosphate was determined according to BAGINSKI AND ZAK 14, and protein b y the method of LOWRY et al. 15. To study the effect of urea, 300 mM urea (final concentration) was added either to the medium for Mg*+-ATPase or to the medium containing NaC1 and KC1. No significant changes in the activity of Mg*+-ATPase were observed in the presence of urea in any portion of the kidney: cortex, medulla or papilla. However, (Na ÷ + K ÷)dependent ATPase activity was affected by 300 mM urea. Both in the medulla and in the papilla a significant decrease in (Na + + K÷)-dependent ATPase activity was observed (Table I), the inhibition being 12.6 and 36.0 %, respectively. No significant inhibition of (Na + + K+)-dependent ATPase activity was observed in the kidney cortex. To test the effect of increased electrolyte concentration, incubation was carried out in a medium containing a final concentration of 200 mM NaC1 and 20 mM KC1. (The K + concentration was increased in order to keep the ratio Na ÷ : K+ the same as in the control medium.) As seen in Table I, increased electrolyte concentration also inhibited (Na + + K÷)-dependent ATPase activity differently in the various parts of the kidney: no significant effect on cortical ATPase was noted, but there was a significant inhibition in the medulla and papilla (12.2 and 27.9 %, respectively). Ca *+ has been reported to inhibit the activity of (Na + + K+)-dependent ATPase Biochim. Biophys. Acta, 233 (1971) x33-I36

KIDNEY MICROSOMAL A T P a s e

135

from kidney and other tissues TM. However, no comparison was made between the effects of Ca ~+ on different parts of the kidney. It has been observed that hypercalcemia specifically decreases the concentrating capacity of the kidney 17, and inhibits Na + reabsorption TM. We have, therefore, studied the effect of Ca 2+ (final concentration TABLE

I

EFFECTS OF U R E A A N D N a + O N M I C R O S O M A L A T P a s e ACTIVITY IN D I F F E R E N T FARTS OF T H E K I D N E Y A T P a s e activities and the decrease in A T P a s e activities are given in/,moles PI released/rag microsomal protein per h. Results are expressed as m e a n s ~ S.E. n = n u m b e r of experiments. P values are for the differences between the activities in the presence of high urea (or N a +) a n d the corresponding control values of the s a m e preparation. N.S. = difference not statistically significant.

(Na + + K +)dependent A TPase

Decrease of (Na + + K+)dependent ,4 TPase by urea

Inhibition of (Na + + K +)dependent A TPase by urea

Decrease of (Na+ + K +)dependent A TPase by Na +

Inhibition of (Na + + K+)dependent A TPase by Na +

(%) Cortex

(%)

7.7 4- 0. 4

0.34 4- 0.50 (N.S.) n= io

4.4

0.48 :k 0.26 (N.S.) n= 9

6.2

Medulla

14.8 4- 0.9

1.86 4- 0.48 (P < o.oi) n=io

12.6

1.8o 4- o.69 (P < 0.05) n=9

12.2

Papilla

4.2 -L 0.4

1.51 + 0.30 (P < o.o2) n=5

36.0

1.17 4- 0.26 (P < 0.05) n=4

27. 9

TABLE II EFFECT OF Ca 2+ ON MICROSOMAL A T P a s e ACTIVITIES IN DIFFERENT PARTS OF THE KIDNEY A T P a s e a c t i v i t i e s a n d t h e decrease in A T P a s e a c t i v i t i e s a re g i v e n i n / , m o l e s P t r e l e a s e d / m g micros o m a l p r o t e i n p e r h. R e s u l t s are e x p r e s s e d as m e a n s 4- S.E. n = n u m b e r of e x p e r i m e n t s . P v a l u e s ar e for t h e differences b e t w e e n t h e a c t i v i t i e s in t h e p r e s e n c e of 5 m M Ca ~+ a n d t h e c o r r e s p o n d i n g v a l u e s of t h e s a m e p r e p a r a t i o n in t h e absence of Ca *+. N.S. = difference n o t s t a t i s t i c a l l y s i gni fi c a nt .

Mg*+-A TPase (Na + + K +)dependent A TPase

Decrease of Inhibition of Decrease of MgZ+-A TPase Mg2+-A TPase total A TPase by Ca~+ by Ca~+ by Ca2+

(%)

Inhibition of (Na+ + If+). dependent A TPase

by Ca~+ (%) Cortex

8.8 4- 0. 4

7-7 4- 0. 4

N.S.

4.20 ± 0.90 n=

Medulla

13.2 4- 0.9

14.8 i

0.9

N.S.

3

6.68 4- i . i o

Papilla

6. 5 4- 0. 5

4.2 ± 0.4

1.94 4- 0.55

(P < 0.05) n=

5

29.9

3

2.93 4- 0.70

0.02)

(P < n=

45.1

0.05)

(P < n=

54-5

0.05)

(P <

23.5

5

Biochim. Biophys. Acta, 233 (1971) 133-136

136

Y. GUTMAN, Y. KATZPER-SHAMIR

5 mM) on A T P a s e a c t i v i t y , b o t h Mg 2+- a n d (Na + + K + ) - d e p e n d e n t , in t h e different p a r t s of t h e k i d n e y . T a b l e I I shows t h a t Ca *+ i n h i b i t e d (Na + + K + ) - d e p e n d e n t A T P a s e from cortex, m e d u l l a a n d papilla. However, Mg~+-ATPase from t h e p a p i l l a was also significantly i n h i b i t e d b y Ca* +, whereas Mg2+-ATPase from c o r t e x a n d m e d u l l a were unaffected. W e h a v e p r e v i o u s l y r e p o r t e d t h a t (Na + + K + ) - d e p e n d e n t A T P a s e a c t i v i t y in t h e k i d n e y c o r t e x was decreased b y prolonged saline a d m i n i s t r a t i o n , whereas t h e a c t i v i t y of t h i s e n z y m e in t h e m e d u l l a was unaffected, a n d t h a t d u r i n g s t a r v a t i o n (Na + + K + ) - d e p e n d e n t A T P a s e a c t i v i t y in t h e c o r t e x increased w h e r e a s in t h e m e d u l l a it decreased 13. These findings suggested different r e g u l a t i n g m e c h a n i s m s for cortical a n d m e d u l l a r y ATPase. O u r p r e s e n t r e p o r t d e m o n s t r a t e s p r e f e r e n t i a l effects of u r e a a n d i n c r e a s e d s o d i u m c o n c e n t r a t i o n on A T P a s e a c t i v i t y in t h e p a p i l l a a n d medulla. Since t h e p a p i l l a (and medulla) are c h a r a c t e r i z e d b y t h e presence in t h e i n t e r s t i t i a l spaces of high c o n c e n t r a t i o n s of u r e a a n d N a +, our findings m i g h t be i n t e r p r e t e d as a d e m o n s t r a t i o n of a self-regulating m e c h a n i s m : w h e n t h e t o n i c i t y in t h e p a p i l l a increased, t h e active " p u m p i n g " of N a + was decreased b y i n h i b i t i o n of t h e (Na + + K+)d e p e n d e n t ATPase, whereas a decrease in t o n i c i t y would relieve t h e i n h i b i t i o n a n d accelerate t h e " p u m p " . The k i d n e y cortex, which also shows a (Na + + K + ) - d e p e n d e n t A T P a s e a c t i v i t y , is n o t exposed, in vivo, to high c o n c e n t r a t i o n s of N a ÷ or urea, a n d is n o t affected b y changes in t h e i r c o n c e n t r a t i o n s in vitro. T h e possible significance of t h e effect of Ca* + on Mg2+-ATPase in t h e p a p i l l a is n o t clear. I t m a y be of i n t e r e s t , however, t h a t we have f o u n d a positive correlation b e t w e e n Mg2+-ATPase a c t i v i t y in t h e " m e d u l l a " (including m e d u l l a a n d papilla) a n d t h e c o n c e n t r a t i n g a b i l i t y of several species of r o d e n t 19.

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Biochim. Biophys. Acta, 233 (1971) 133-136