The effect of water deprivation on the osmotic release of renin

Kidney International, Vol. 22 (1982), pp. 344—347

The effect of water deprivation on the osmotic release of renin JAIME S. CARVALHO The Department of Medicine, Veterans Administration Medical Center, Division of Biological and Medical Sciences, Brown University,

Providence, Rhode Island

In addition to vascular and tubular effects, the increase in

The effect of water deprivation on the osmotic release of renin. The

effect of water deprivation on the osmotic release of renin was evaluated in conscious rats previously prepared with right nephrectomy

and cannulations of left renal artery and lower abdominal aorta. The osmotic signal was a 4-mm intrarenal infusion of 30% crystallized bovine serum albumin. Changes in aortic plasma concentration of renin (PRC) and total protein were followed serially. In normal hydropenic rats an increase in PRC was not detected with the oncotic challenge.

Following a 48-hr period of water deprivation, the same external oncotic signal increased PRC threefold above baseline within 3 mm. It

is concluded that some intrarenal functional or structural change induced by water deprivation sensitizes the renin-secreting mechanism

to colloid osmotic stimuli. It is suggested that this change could be related to the physical conditions of the renal interstitium.

intravascular oncotic pressure during albumin infusion induces an acute inflow of colloid-free interstitial fluid into the peritubular capillaries [4]. The volume changes in the renal interstitium could affect intrarenal mechanisms of renin release [5]. In these experiments, we explored the possibility of changes in renal interstitial volume influencing the osmotic release of

renin by studying the renin response induced by a strong intrarenal osmotic stimulus in normal hydropenic and in 48-hr water-deprived rats. The effects of anesthesia on the renal microvasculature [61

and on the secretion of renin [7] were avoided by using

Effet de Ia restriction hydrique sur le relargage osmotique de Ia refine. L'effet de Ia restriction hydrique sur le relargage osmotique de rénine a

été évalué chez des rats éveillés, préalablement préparés avec une néphrectomie droite et des canulations de l'artère rénale gauche et de l'aorte abdominale inférieure. Le signal osmotique était une perfusion intrarénale de 4 mm de sérum-albumine bovine cristallisée a 30%. Les modifications des concentrations plasmatiques de rénine (PRC) aortique et de protides totaux ont été suivies de facon sériée. Chez des rats

unanesthetized rats previously prepared with cannulas in the left renal artery and lower abdominal aorta. The complicating renin effects from the contralateral kidney were abolished by right nephrectomy. Serial measurements of aortic plasma concentrations of renin and total protein and of aortic hematocrit were performed.

normaux hydropéniques, aucune augmentation de Ia PRC n'a été détectée avec cette charge oncotique. Après one période de 48 heures de restriction hydrique, Ic méme signal oncotique externe augmentait la PRC de trois fois au-dessus de Ia valeur de base en 3 mm. II est conclu qu'une certaine modification fonctionnelle ou structurelle intrarénale induite par Ia restriction hydrique sensibilise Ic mécanisme de sécrétion de Ia rénine aux stimulus osmotiques colloldaux. II est suggéré que cette modification pourrait être reliée aux conditions physiques de l'interstitium renal.

Sprague-Dawley strain that weighed between 240 and 320 g and

Increases in renal plasma colloid osmotic pressure lead to the release of renin [1—3], but the mechanism involved is not well

days before the studies were performed. The renal artery

Methods

Surgical procedures. We used female white rats of the were purchased from Charles River Breeding Laboratories, Wilmington, Massachusetts. All animals were fed a regular diet

of Purina Rat Chow (180 mEq Naikg, Country Best, Agway, Inc., Syracuse, New York) and tap water ad libitum, unless stated otherwise. Under pentobarbital anesthesia, the left renal artery and the lower abdominal aorta were cannulated several

understood. Hall and Guyton performed acute infusions of cannula was made of PE 10 stretched to an external diameter of hyperoncotic solutions into the renal artery of anesthesized approximately 180 zm at the tip. It was filled with a 0.15 M dogs and found no changes in renal perfusion pressure but sodium chloride solution containing 500 U/ml of heparin, marked decreases in urine flow [1]. These investigators postulated a macula densa-mediated mechanism, but a hemodynamic one from autoregulatory vasodilatation could not be ruled out [1]. Fray and Karuza, working in the isolated rat kidney, could not confirm a primary role for a macula densa receptor in the osmotic release of renin [3].

sealed, and marked with black ink at the tip. It was introduced by way of the left common carotid artery. Through a midline

abdominal incision, the tip was directed into the proximal portion of the left renal artery. Details of the cannulation technique can be found elsewhere [8]. To permit repetitive blood sampling during experiments, another plastic cannula was introduced into the lower abdominal aorta through the left

femoral artery, using techniques similar to those described Received for publication July 8, 1981 and in revised form April 2, 1982

previously [7]. The cannulas were passed through a subcutaneous tunnel and exteriorized at the back of the neck. In addition

to the cannulation procedures, the animals underwent right

0085—2538/82/0022—0344 $01.00

nephrectomy. After the surgical procedures, the animals were

© 1982 by the International Society of Nephrology

housed in individual metabolic cages until the day of the 344

345

Oncotic pressure and renin release

experiments, begun at approximately 9 A.M. on the 4th to 5th day after surgery. Experimental protocol. In the first part of the study, normal hydropenic animals were used. Thereafter, all animals underwent a period of 48-hr water deprivation before the experiments. The experimental animals received a brief intrarenal infusion of hyperoncotic albumin in 0.15 M sodium chloride and the control animals received 0.15 M sodium chloride only. On the day of the experiment, the animal was placed carefully in a

Measurement of total renal renin content. The kidney tissue was prepared according to the method of Peters-Haefeli [10].

Briefly, the frozen kidneys were allowed to thaw at room temperature, 200 mg of cortical tissue were taken from each kidney and sonicated in a tube containing 1.8 ml of iced distilled water. The pH was dropped from 6.8 to 2.0 by the addition of 40 p1 of 2 N sulfuric acid, and the sonicated mixture was allowed to

stay on ice for 30 mm. The pH was brought back to 6.8 by adding 40 p1 of 2 N sodium hydroxide, and the mixture was

plastic laboratory cage with the cannulas extending to the

centrifuged at x 1,000 g for 20 mm. The supernatant was diluted

outside and allowed to rest for about 1 hr. Blood (0.8 ml) was sampled through the aortic cannula for baseline measurements of plasma renin (PRC) and total protein concentrations (PPC), hematocrit, BUN, and creatinine. An intrarenal infusion of either 30% crystallized bovine serum albumin in 0.15 M sodium chloride diluent or diluent alone was then started. A 2.5-mi gastight Hamilton syringe and a Harvard infusion pump, model series 940, were used for the infusions. The solutions were delivered through the renal artery cannula at a constant rate of 260 p1/mm for only 4 mm to minimize plasma volume expansion. Serial sampling of 0.3 ml of blood was made at 1, 3, 5, and

500 times with 0.133 M phosphate buffer solution, pH 6.5. A sample of 50 pi was taken for the enzymatic reaction, as for the measurement of plasma renin above described. Plasma protein concentration was measured by the method of Bradford [111, using human albumin standard. Kinetic assays were used for the measurement of plasma urea nitrogen [12] and plasma creatinine [13]. Statistical significance. The paired t test was used to evaluate statistical significance between control and experimental val-

ues. The Student's t test was used to determine statistical significance between groups. Results are expressed as

10 mm after starting the intrarenal infusions for renin and mean protein measurements. Hematocrit also was obtained at the time of last sampling. All solutions infused into the renal artery were 37 to 38°C. The volume of blood removed each time was replaced immediately after sampling with the same volume of blood obtained from a binephrectomized, pentobarbital-anesthetized donor rat. Previous studies have shown no differences in renin secretion with repeated sampling under similar condi-

1 SEM.

Results

Renal artery cannulation and renal function. Of the 24 animals studied, only in two was the tip of the renal artery cannula found in the abdominal aorta. In spite of all efforts to avoid them, small renal infarctions were seen in about 20% of

the animals, possibly as a result of microthrombi from the tions [7]. At the end of the experiment, the position of the cannula tip. Kidney function of these animals, evaluated by the cannula tip was checked and the analyses were not performed if plasma concentrations of urea nitrogen and creatinine, was found outside the renal artery. normal and basal PRC was similar to that of rats without To evaluate the influence of a 48-hr period of water deprivation on the activity of the renin-angiotensin system, plasma and renal cortical renin levels were measured in a separate group of animals previously prepared with indwelling aortic cannulas.

Analytical procedures Measurement of plasma renin. The micromethod used for the determination of PRC in rat plasma by radioimmunoassay of angiotensin I was that previously described [8]. In brief, PRC was measured by incubating 50 p1 of plasma with a fixed, large amount of homologous renin-substrate under conditions of abolished activity of converting enzyme and angiotensinases. The velocity of angiotensin I formation in the course of 2 hr was measured by radioimmunoassay. Pooled plasma from 24-hr nephrectomized female rats was used as a source of substrate. This plasma had a renin substrate concentration equivalent to 2,700 ng/ml of angiotensin I. The radioimmunologicai determination of angiotensin I was performed with the angiotensin I (Asp'-ileu5) standard, code 71/328, kindly provided by the Medical Research Council, National Institute for Biological Standards and Control, London, England. The final dilution of the angiotensin I antibody used was 1:145,000. A MUNROE

infarctions. Since control and experimental animals were affect-

ed equally and the renin response was not different from uninfarcted animals, they were included in these studies. In normal hydropenic animals, the average concentrations for plasma urea nitrogen and creatinine before the intrarenal infusions were 18 2 and 0.9 0.1 mg/dl, respectively, for those infused with albumin and 19 2 and 0.8 0.1 mg/dl for those infused with sodium chloride. Similar values were obtained in 48-hr water-deprived rats. Renin response to hyperoncotic albumin in normal hydropenic rats. (Fig. 1). Mean systemic plasma protein concentration increased from 6.5 0.2 to 7.0 0.2 mg/dl (P < 0.001) within 1 mm of intrarenal hyperoncotic albumin infusion. It increased further to 8.1 0.4 mg/dl within 3 mm and was still elevated (7.1 0.3 mg/dl, P < 0.05) 6 mm after the infusion was discontinued. Aortic levels of PRC were unchanged by the oncotic signal and did not differ from those of control animals infused with 0.9% sodium chloride. At 10 mm after onset of the hyperoncotic albumin infusion, an 8% fall (P < 0.001) in aortic hematocrit was found. No changes in PRC, PPC, or hematocrit were seen in sodium chloride-infused animals.

computer, model 1860, was used to process the radio-

Renin studies in 48-hr water-deprived rats

immunoassay results, using a program based on the logit-log method for the linearization of dose-response curves [9]. Renin concentrations were given in nanograms of angiotensin I gener-

(1) Renin effects of 48-hr water deprivation (Table 1). The challenge imposed on the renin system by a period of 48-hr water deprivation was evaluated in a separate group of six

ated by the renin in 1 ml plasma during 1 hr under our animals. Basal aortic PRC increased about threefold incubation conditions.

(P < 0.01), but renal renin content was unchanged. Similarly,

Carvalho

346

Table 1. Changes in circulating and renal renin induced by a 48-hr

period of water deprivation in rats (N =

Basal plasma renin concentration ng Al/mi/hr Normal hydropenia 48-hr water deprivation

7.6 21.5

0.4a 3.2h

6:

wt 211 to 236 g)

Total renal renin content ng Al/100 mg cortex/hr 50.9

9.4

50.4 + 8.1

a The values are means SEM of a total of 18 samplings taken daily from a group of 6 animals over a period of 48 hr. b p < 0.01, in relation to normal hydropenic values.

8

the mean basal PRC of all 48-hr water-deprived rats studied was 17.3 1.3 ng AI/ml/hr, significantly higher (P < 0.001) than that of all normal hydropenic animals (7.0 0.8 ng A1/ml/hr). (2) Renin response to hyperoncotic albumin (Fig. 2). Three

12

minutes after onset of the intrarenal hyperoncotic albumin

10

infusion, an over twofold increase in PRC was found 2.2, at 0 time; 35.3 6.1 ng Al/mi/hr at 3 mm, (16.2

8

P < 0.02). One minute after the albumin infusion was discontinued, PRC was still significantly elevated. Over the ensuing 5 mm it decreased to levels the same as those before infusion. No PRC changes were seen in control animals injected with 0.9% sodium chloride. Plasma protein concentration reached levels

6

(9.0

control animals infused with sodium chloride, hut no changes in protein concentration were found. Discussion

At least two conditions are required in a study of renin effects of hyperoncotic albumin in the living animal. First, the albumin

N=6

Signal

0.4 mg/dl, P < 0.05, at 3 mm). There was a

9% fall in hematocrit 10 mm after unset of the hyperoncotic albumin infusion. A 7% fall in hematocrit was also seen in the

{ €N=6

4L

higher than those achieved by normal hydropenic animals 0.3 vs. 8.1

—

I

I

I

0

2

4

6

8

10

Time, rn/n

Fig. 1. Effct of 4-mm renal artery infusions of hyperoncotic albumin

(solid lines) and isotonic saline (dashed lines) on aorric plasma renin concentration (PRC, ng A1/ml/hr), aortic plasma protein concentration (PPC, mg/c/I), and aorlic hematocrit (Hct, %). Data were obtained from

normal, hydropenic rats with a right nephrectomy. Symbol: t, P < 0.01.

solution should be infused in high concentration directly into the renal artery for a short period of time to avoid effects of creased. The concomitant increase in plasma volume by the volume expansion. Second, the animal should he in the con- osmotic Donnan effects of infused albumin may have masked scious state to avoid anesthetic effects on the renal vasculature small rises in PRC. The finding by Hall and Guyton in the [6] and the release of renin [7]. Both conditions were met in anesthetized dog of an increased renin activity in renal venous but not in peripheral plasma during intrarenal infusion of an these experiments. Previous studies in the rat have shown that renal blood flow is not appreciably affected by the presence of a number 30-gauge

needle in the initial portion of the left renal artery [141.

albumin solution (25 g/dl) adds support to this possibility [1]. The lack of appreciable PRC response to intrarenal hyperoncotic albumin infusions by normohydrated rats contrasts sharp-

Therefore, the external diameter of the plastic microcannula ly with the brisk increase seen after a 48-hr period of water used was kept under that of a number 30-gauge needle. Under deprivation. In the dog, this dehydration period does not these conditions, the highest albumin concentration that could decrease renal blood flow which is maintained constant by flow freely through the cannula lumen was 30 g/di, the one used autoregulatory mechanisms [17]. If this is true for the rat, the rate of increase of the osmotic stimulus should have been in these experiments. Chronic cannulas in the renal vein of the rat are difficult to similar in the normohydrated and dehydrated animals. By the maintain in good function. This limitation prevents the mea- end of the albumin infusions, however, the osmotic stimulus surement of renin secretion rates in the conscious animal. may have been stronger in the dehydrated animals since sysPlasma renin concentrations must be measured instead. Fortu- temic plasma protein concentrations were higher. Given the nately, the circulating pool of renin is very small and in rats characteristics of the renin release mechanism, it seems unlikely, nevertheless, that the dissimilar PRC response could he under basal conditions it is secreted and degraded every 5 mm [10, 151. Hence, with a circulation time of 6 to 8 sec [161, and explained solely by a difference in the magnitude of the external under our assay conditions, doubling of basal renin secretion stimulus. The basal PRC of water-deprived rats was two to three times rate should affect PRC significantly within 2 mm. Despite a strong osmotic stimulus, the PRC changes in higher than that of normohydrated counterparts, a change not normohydropenic animals were minimal and indistinguishable entirely explainable by a 20 to 30% contraction in plasma from those of saline-infused animals. This lack of PRC rise does volume [18]. Since total renal renin content is unchanged and not mean necessarily thai renin secretion rate was not in- plasma renin substrate levels are not increased by dehydration

347

Oncotic pressure and renin release

gave technical assistance and Mrs. J. Charpentier provided secretarial support.

i

Reprint requests to Dr. J. S. Carvalho, Department of Medicine, Veterans Administration Medical Center, Davis Park, Providence, Rhode Island 02908, USA

References 9

1. HALL JE, GVYTON AC: Changes in renal heniodynamics and renin release caused by increased plasma oncotic pressure. Am J Physiol 231(5): 1550—1556, 1976

2. HUMPHREYS MH, REID IA, UFFERMAN RC, LIEBERMAN RA, EARLY LE: The relationship between sodium excretion and renin secretion by the perfused kidney. Proc Soc Exp Biol Med 150:728— 734, 1975 3. FRAY JCS, KARUZA AS: Influence of raising albumin concentration on renin release in isolated perfused rat kidneys. J Physiol 299:45— 54, 1980 4. KALLSKOG 0, WOLGAST M: Driving forces over the peritubular capillary membrane in the rat kidney during antidiuresis and saline expansion. Ada Physiol Scand 89:116—125, 1973 5. PERSSON AEG, MULLER-SUUR R, SELEN G: Capillary oncotic pressure as a modifier for tubuloglomerular feedback. Am J Physiol 236(2):F97—Fl02, 1979 6. LINAS SL, BERL T, AISENBREY GA, BETTER OS, ANSERSON Ri: The effect of anesthesia on hemodynamics and renal function in the rat. PflOgers Arch 384:135—141, 1980 7. CARVALHO JS, SHAPIRO R, HOPPER P, PAGE LB: Methods for

S

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30 25 20 15

serial study of renin-angiotensin system in the unanesthetized rat.

10

Am J Physiol 228:369—375, 1975 8. CARVALHO JS: Improved techniques for acute and chronic cannula-

Signal

0

I 2

I

I

I

4

6

8

10

Time, mm

Fig. 2. Explanation as in Figure 1. Data were obtained in 48-hr water-

tion of renal artery in the rat. Kidney Int, 22:75—79, 1982 9. RODBARD D, BRIDSON W, RAYFORD PL: Rapid calculation of radioimmunoassay results. J Lab Clin Med 74:770—781, 1969 10. PETERS-HAEFEL1 L: Renal cortical renin activity and renin secretion at rest and in response to hemorrhage. Am J Physiol 221:1331— 1338, 1971

deprived rats with a right nephrectomy. Symbols: *, P < 0.05; t, P <0.01.

11. BRADFORD MM: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 72:248—254, 1976 12. JUNG D, BIGG5 H, ERIKsON J, LEDYARD PU: New colorimetric

[19, 20], the higher PRC must be a reflection of a primary increase in release rate. The basic cause underlying this rate

reaction for end-point, continuous-flow, and kinetic measurement

increase has not been elucidated but studies in conscious sheep have suggested that a primary intrarenal factor is involved [19]. It is unclear whether and in what way this factor participates in the enhanced renin sensitivity to osmotic stimuli.

These studies have shown that a 48-hr period of water deprivation makes the renin releasing structures of conscious rats very sensitive to an acute intrarenal infusion of hyperoncotic albumin. They give no information on the mechanism respon-

of urea. Clin Chem 21:1136—1 140, 1975

13. LARSEN K: Creatinine assay by a reaction-kinetic principle. Clin Chim Acta 41:209—217, 1972 14. FINE LG, LEE H, GOLDSMITH D, WEBER H, BLAUFOX MD: Effects

of catheterization of renal artery on renal function in the rat. J Appi Physiol 37:930—933, 1974

15. POULSEN K: No evidence of active renin-inhibitors in plasma. The

kinetics of the reaction between renin and substrate in non-

pretreated plasma. Scand J Clin Lab invest 27:37—46, 1971 16. SAPIRSTEIN LA: Regional blood flow by fractional distribution of indicators. Am JPhysiol 163:161—168, 1958 17. KIRKEBO A, TYSSEBOTN I: Effect of dehydration on renal blood flow in the dog. Acta Physiol Scand 101:257—263, 1977 18. THIEL G, WILSON DR, ARCE ML, OKEN DE: Glycerol-induced hemoglobinuric acute renal failure in the rat. Nephron 4:276—297,

sible for this sensitivity. Since a primary intrarenal change of water deprivation is a decrease in renal interstitial volume and pressure [4, 21, 22], it is conceivable that the physical conditions of the renal interstitial space are important determinants in 1967 the osmotic release of renin. Other alternative explanations 19. BLAIR-WEST JR, BROOK AH, SIMPSON PA: Renin responses to water restriction and rehydration. J Physiol 226: 1—13, 1972 cannot be dismissed. New experiments where actual interstitial volume and pres- 20. ROSENTHAL J, BOUCHER R, ROJO-ORTEGA JM, GENEST J: Renin activity in aortic tissue of rats. Can J Pliysiol Pharmacol 47:53—56, sure changes can be recorded must be designed to directly 1969 evaluate the above hypothesis. These experiments must be PF, HARGEN5 AR, MILLER SL: Negative pressure in performed under in vivo conditions and not in isolated kidneys 21. SCHOLANDER the interstitial fluid of animals. Science 161:321—328, 1967 to avoid alterations of the physiologic state of the renal intersti- 22. ACKERMAN U: Changes in interstitial pressure during acute interstitium by artificial perfusion systems [23]. tial volume depletion in normally hydrated rats. Pfldgers Arch 369: Acknowledgments This work was supported by the Veterans Administration. Dr. K. Poulsen kindly provided the angiotensin I antibody. Mr. J. Cherkes

245—250, 1977

23. NAKANE H, NAKANE Y, CORVOL P, MENARD J: Sodium balance

and renin regulation in rats: Role of intrinsic renal mechanisms. Kidney mt 17:607—614, 1980