Effect of bradykinin on the renal medullary osmotic gradient in water diuresis

European Journal o f Pharmacology, 45 ( 1 9 7 7 ) 1 7 3 - - 1 8 3 © E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

173

E F F E C T OF B R A D Y K I N I N ON THE R E N A L M E D U L L A R Y OSMOTIC GRADIENT IN WATER DIURESIS L Y N N R. WILLIS

Departments of Pharmacology and Medicine, Indiana University School o f Medicine, 11 O0 West Michigan Street, Indianapolis, Indiana 46202, U.S.A. Received 14 F e b r u a r y 1 9 7 7 , revised MS received 10 May 1 9 7 7 , a c c e p t e d 7 J u n e 1977

L.R. WILLIS, Effect o f bradykinin on the renal medullary osmotic gradient in water diuresis, E u r o p e a n J. Pharmacol. 45 ( 1 9 7 7 ) 1 7 3 - - 1 8 3 . T h e e f f e c t o f b r a d y k i n i n o n t h e renal m e d u l l a r y o s m o t i c g r a d i e n t was e v a l u a t e d in a n e s t h e t i z e d dogs w h i c h were u n d e r g o i n g w a t e r diuresis a n d w h i c h received a unilateral renal arterial i n f u s i o n o f b r a d y k i n i n . T h e e f f e c t o f the p e p t i d e o n t h e m e d u l l a r y o s m o t i c g r a d i e n t was d e t e r m i n e d b y analysis of m e d u l l a r y tissue e l e c t r o l y t e a n d urea c o n c e n t r a t i o n s a n d b y analysis of c h a n g e s in urine o s m o l a l i t y i n d u c e d b y vasopressin. B r a d y k i n i n decreased t h e t o t a l o s m o l a l i t y per kg H 2 0 in tissue f r o m i n n e r m e d u l l a a n d papilla (--18.7 * 6% a n d - - 1 9 . 3 * 8%) a n d increased f r a c t i o n a l w a t e r e x c r e t i o n (3.8 ~ 1.3%), F u r t h e r m o r e , a direct r e l a t i o n s h i p b e t w e e n c h a n g e s in free w a t e r clearance a n d c h a n g e s in papillary tissue o s m o l a l i t y was f o u n d . Finally, t h e increase in u r i n e o s m o l a l i t y a f t e r A D H was significantly less in v a s o d i l a t e d t h a n in c o n t r o l kidneys. These results i n d i c a t e t h a t b r a d y k i n i n can d i m i n i s h t h e m e d u l l a r y o s m o t i c g r a d i e n t d u r i n g w a t e r diuresis in t h e dog. T h u s , a b r a d y k i n i n - i n d u c e d increase in free w a t e r clearance m a y be a c c o u n t e d for b y o t h e r t h a n an i n h i b i t i o n of p r o x i m a l t u b u l a r s o d i u m r e a b s o r p t i o n . Proximal tubule reabsorption

Free w a t e r clearance

1. Introduction The vasodilator peptide, bradykinin, increases urine flow, free water clearance, and urinary sodium excretion in dogs undergoing a water diuresis (Willis et al., 1969; Massry and Ahumada, 1972). These results have been interpreted to indicate that the peptide inhibits sodium and water reabsorption by the proximal tubule and that this effect is responsible for the natriuresis. In contrast, the results of recent micropuncture studies suggest that bradykinin lacks an inhibitory effect on reabsorption by this nephron segment (Dirks and Seely, 1967; Stein et al., 1972; Schneider et al., 1973). There has been speculation, based on the results of volume expansion studies in the dog, that renal vasodilator drugs may diminish the medullary osmotic gradient (Early and

Vasodilators

Natriuresis

M e d u l l a r y tissue o s m o l a l i t y

Friedler, 1965). Such an effect of acetylcholine in hydropenic dogs has been reported by Vander (1964). However, evidence for a similar effect of bradykinin in water diuresis is lacking. During a water diuresis, the existence of a medullary osmotic gradient, albeit diminished, has been demonstrated in dogs (Jamison et al., 1971). A reduction of the gradient would be expected to cause a greater volume of water to enter the distal nephron and thereby increase urine flow and free water clearance independently of reduced volume reabsorption by the proximal tubule (Early and Friedler, 1965). In the present study, the effects of bradykinin on the medullary osmotic gradient in dogs undergoing water diuresis have been evaluated. The results support the hypothesis that bradykinin increases free water clearance, at least in part, by a reduction of the medullary osmotic gra-

174

L.R. WILLIS

dient, and th ey serve to reconcile the conflicting interpretation of earlier clearance and m i c r o p u n c t u r e studies.

2. Materials and m e t h o d s

2.1. Tissue analysis experiments 2.1.1. diuresis

Surgery

and

induction of water

The effect of bradykinin on the renal medullary osmotic gradient was studied by d e t e r m i n a t i o n of tissue osmolality in a group o f seven dogs (Group 1) undergoing a water diuresis. Each dog was given an oral load of tap water (600 ml) 16 h and 1 h prior to the onset o f anesthesia. Following the intravenous injection o f pentobarbital (30 mg/kg), a t r a c h e o t o m y was p e r f o r m e d and catheters were placed in b o t h jugular veins and a femoral vein and artery for infusion of fluid, sampling of blood and recording of bl ood pressure, respectively. Immediately after placement o f the jugular vein catheters, an infusion into one jugular vein of 3% dextrose in 0.3% saline was started at a rate of 10 ml/ min. After a priming injection, a saline solution containing inulin and PAH was infused into the other jugular vein at the rate of 1 ml/min for the duration of the experiment. Then, b o t h ureters were cannulated through a mid-line incision and a flank incision exposed the left kidney. A curved 23-gauge needle was inserted into the left renal artery close to the aorta. Patency o f the needle was maintained by the continuous infusion of 0.9% sodium chloride solution at a rate of 1 ml/min. During the experimental period, a solution of bradykinin (0.75 pg/kg/min) in isotonic saline was substituted for this infusion. When one liter o f the dextrose solution had been infused, the rate of infusion was reduced t o less than 0.5 ml/min for 30 min. Following this, the infusion rate was raised by increments over another 30 min period until the original rate of 10 ml/min was re-attained. This p r o ced u r e was necessary to avoid appar-

ent water intoxication and virtual cessation of urine flow. Urine flow always began to increase and urine osmolality to decline immediately when the dextrose infusion was increased after the 30-min delay. Measurements of urine osmolality were obtained as 5- to 15-min intervals until urine osmolality remained stable for 15 to 20 rain.

2.1.2. Experimental protocol Control bilateral urine collections were made during three lO-min clearance periods; blood was drawn at the m i dpoi nt of each period. At the conclusion of the third collection, the renal arterial infusion of bradykinin was started. At the end of one lO-min period rapid bilateral n e p h r e c t o m y was performed.

2.1.3. Tissue preparation and analysis Samples of renal tissue were obtained for analysis according to a procedure described by H o o k and Williamson {1965). Each kidney was halved, exposing the papillary ridge. A thin strip of this ridge and succeeding strips of inner and outer medulla and portions of out er cortex were removed, cut into 50--100 mg pieces and weighed. Tissue dry weight was determined and the samples from each kidney were digested overnight in c o n c e n t r a t e d nitric acid and were analyzed for sodium and potassium c o n t e n t by flame p h o t o m e t r y . A n o t h e r set of duplicate samples from each area of the kidney was analyzed for urea c o n t e n t as follows: the dried tissue samples were weighed and transferred to flasks containing 2 ml of water. The flasks were sealed and heated in a boiling water bath for 20 min. The c o n c e n t r a t i o n of urea in the supernatants was determined colorimetrically with a commercial urea nitrogen kit (Harleco).

2.2. Vasopressin-infusion control experiments 2.2.1. Non-bradykinin controls A second group o f six dogs (Group 2) was given an initial oral water load, prepared surgically, and infused with dextrose--saline solution as described above. When urine osmolal-

BRADYKININ AND RENAL OSMOTIC GRADIENT

175

ity and flow rate had stabilized, three 10-min control urine collections were made, following which 20 to 50 mU of aqueous vasopressin were administered intravenously by infusion. Urine and blood samples were collected from each kidney for at least four 15-min periods during vasopressin infusion.

Data for each clearance variable were averaged for each dog during control and bradykinin-infusion periods. All means were analyzed statistically with the Student's t-test for paired data. The 0.05 probability level was used as the criterion for statistical significance.

2.2.2. Bradykinin-infusion controls A third group of four dogs (Group 3) was anesthetized and prepared for water diuresis as described above. A 23-gauge curved needle, attached to polyethylene tubing was inserted into the left renal artery also as described above. After a stabilization period of 1 h, three 10-min control urine samples were collected after which bradykinin in 0.9% saline was infused into the renal artery at a rate of 0.75 gg/kg/min. Urine was collected bilaterally during two 5-min collection periods after which ADH was administered intravenously as described above. Urine was collected during five additional 10-min periods after which the bradykinin infusion was stopped but the ADH infusion was continued. Urine was collected during four additional 10-min periods.

2.3. Urine, plasma and data analysis Blood samples were collected at the midpoint of each urine collection period in all experiments. Inulin and PAH concentrations in plasma and urine were determined by the methods of Fjeldbo and Stamey (1968), and Harvey and Brothers (1962), respectively. Urine and plasma osmolalities were determined by measurement of freezing point depression. Sodium and potassium concentration in urine and plasma was determined by flame p h o t o m e t r y . The results of the renal tissue analysis for electrolyte and urea content were expressed as total osmolality = urea + 2(Na ÷ + K÷), and values were averaged for the respective kidney regions for each dog. Statistical comparisons of tissue osmolality were made between the bradykinin-infused and the non-infused contralateral kidneys.

3. Results

3.1. Tissue analysis experiments The effects of unilateral renal arterial infusion of bradykinin on the medullary osmotic gradient during a water diuresis were evaluated in Group 1. Prior to the infusion of bradykinin, mean arterial blood pressure was 125 + 3 mm Hg, plasma osmolality was 273 + 3 mOsm/1 and plasma sodium concentration was 136 + 3 mEq/1. None of these variables was significantly altered by bradykinin. In table 1 are summarized the urinary excretion data obtained from both kidneys prior to and during the infusion of the peptide. Characteristically, bradykinin induced statistically significant unilateral increases in urine flow, sodium excretion and the clearance of PAH. Glomerular filtration rate was not significantly altered. That the effects of bradykinin were restricted to the experimental kidneys of the dogs in this group is suggested by the fact that there was no change in systemic arterial pressure during bradykinin infusion. However, statistically significant changes were produced in the urinary excretion patterns of the control kidneys. Notable among these were small but significant reductions in urine flow and in the clearances of inulin and PAH. Sodium excretion was not significantly altered. With the exception of glomerular filtration rate, the mean changes in the measured excretory functions of the experimental kidneys were significantly different from those of the control kidneys. Observations of free water clearance obtained from the same group of hydrated dogs are also presented in table 1. In the

176

L.R. W I L L I S

TABLE 1 S u m m a r y o f u r i n a r y e x c r e t i o n a n d free w a t e r c l e a r a n c e d a t a ( g r o u p 1). n = 7 d o g s ; v a l u e s are m e a n s or m e a n d i f f e r e n c e s ±S.E.M. Pre = p r e - b r a d y k i n i n i n f u s i o n p e r i o d s . B K = b r a d y k i n i n infusion periods. Experimental kidney Pre

BK

Difference _

G l o m e r u l a r f i l t r a t i o n rate (ml/min)

50

PAHclearance (ml/min)

130

Urine flow rate (ml/min)

±

5

46

±11

4.8+

195

0.7

+

4

--4

±

_+ 24

65

±21 '

7.7 ±

1.0

Urinary sodium excretion (pEq/min)

82

± 25

295

± 104

Urine osmolality (mOsm/kg H20)

88

± 13

108

±

Osmolar clearance (ml/min) % Fractional water excretion

1.5 ±

0.3

6.5 ±

0.7

.

14

2.9± 213

_ _

3

1.0 1

± 87 ~

20

±

5 ~

3.3 ±

0.3

1.8 ±

0.7 1

10.1 _+

1.3

3.6 ±

1.3 ~

_

_

_ _

Control kidney Pre

BK

Difference -

G l o m e r u l a r f i l t r a t i o n rate (ml/min) PAHclearance (ml/min) U r i n e f l o w rate (ml/min)

51 142

±

4

47

+_ 4

--4

± 12

127

±13

--15

4.5 ±

0.4

2.7 ±

0.5

±

4 '

- - 1 . 8 _+ 0.4 ~

Urinary sodium excretion (pEq/min)

78

± 25

61

± 19

--17

Urine osmolality (mOsm/kg H20 )

88

± 15

132

± 22

44

±

7

± 11 1

Osmolar clearance (ml/min)

1.5 ±

0.3

1.3 ±

0.3

--0.2 ±

0.1

% Fractional water excretion

6

0.4

3

0.8

--3.0 ±

0.9 ~

±

±

-

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I Denotes a statistically significant difference.

experimental kidneys, bradykinin infusion was associated with significant increases in urinary osmolality, sodium excretion rate and fractional water excretion ( C H : o - G F R -~102). The changes in fractional water excretion ranged f r o m --0.9 to 9.6%. The osmolality o f urine f r o m control kidneys was signifi-

cantly elevated, b u t no change in osmolar clearance was observed; fractional water excretion was reduced indicating more net water reabsorption. A comparison of total osmolalities of renal tissue samples of bot h experimental and control kidneys in the same group of dogs under-

BRADYKININ

AND RENAL

OSMOTIC GRADIENT

177

TABLE 2 T h e e f f e c t o f b r a d y k i n i n o n t o t a l o s m o l a l i t y o f r e n a l t i s s u e s a m p l e s ( G r o u p 1). n = 7 d o g s ; v a l u e s are m e a n s _+ S . E . M . E K = b r a d y k i n i n - t r e a t e d k i d n e y ; C K = c o n t r a l a t e r a l c o n t r o l k i d n e y ; EK -- CK CK x l 0 0 = p e r c e n t a g e c h a n g e in o s m o l a l i t y o f E K c o m p a r e d t o C K .

Cortex Outer medulla Inner medulla Papilla

EK osmolality (mOsm/kg H20 )

CK osmolality (mOsm/kg H20)

EK

297 449 432 439

311±15 519 ± 55 529 + 59 578 ± 72

--3.8_+2.9 - - 6 . 1 _+ 4.7 - - 1 8 . 7 _+ 5 . 8 1 --19.3 ± 8.0 2

± 8 ± 35 + 32 + 38

CK ×100 CK d i f f e r e n c e (%) -

-

~ Denotes a statistically significant difference. 2 0.1 > p > 0 . 0 5 . TABLE 3 E f f e c t o f A D H in d o g s u n d e r g o i n g w a t e r d i u r e s i s ( G r o u p 2). V a l u e s are m e a n s o r m e a n d i f f e r e n c e s ± S . E . M . Left kidney Control Inulin clearance (ml/min) PAH clearance (ml/min) Urine osmolality (mOsm/1) Osmolar clearance (ml/min)

49 132 70

ADH _+2

43

Difference +

5

--6

+

5

+

6

+7

122

± 10

--10

_+8

641

+ 56

571

0.86 ± 0.08

0.91±

0.15

+_ 6 0 1

0.05+

Plasma osmolality (mOsm/1)

268

_+ 4

267

+

4

--1

Arterial blood pressure (mm Hg)

128

+_ 6

128

+

7

0

0.11 +_ 1 ±

1

Right kidney ADH

Control Inulin clearance (ml/min) PAH clearance (ml/min) Urine osmolality (mOsm/1) Osmolar clearance (ml/min)

Difference

± 4

41

133

_+ 9

123

+

8

--10

68

_+ 7

659

+_ 6 6

591

0 . 8 6 _+ 0 . 0 9

I D e n o t e s a s t a t i s t i c a l l y s i g n i f i c a n t d i f f e r e n c e ; n = 6.

_+ 5

--5

46

1 . 0 4 _+ 0 . 2 3

+_ 7 +

4

+_ 7 0

0.19 +

0.17

178

L.R. WILLIS I

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PERCENTAGE DIFFERENCE TISSUE (~MOLALITY,

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IN PAPILLARY E vs C

Fig. 1. R e l a t i o n s h i p o f c h a n g e s in o s m o l a l i t y o f r e n a l papilla (control osmolality minus experimental osmol a l i t y ) t o c h a n g e s in f r a c t i o n a l w a t e r e x c r e t i o n ( e x p e r i m e n t a l m i n u s c o n t r o l ) in d o g s u n d e r g o i n g w a t e r d i u r e s i s . F r a c t i o n a l w a t e r e x c r e t i o n = (CH20 × 1 0 0 ) / GFR;r significant at p < 0.05.

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going water diuresis is presented in table 2. Expressed in terms of absolute osmolality, or as the percent difference from the control kidney, osmolalities of inner medulla and papilla tissues of the experimental kidneys were each reduced to a similar degree by bradykinin (--18.7 + 5.6% and --19.8 + 8.0%), respectively. The mean change in inner medullary osmolality was statistically significant; that in papillary osmolality was only marginally significant {0.1 > p > 0.05). Fig. 1 illustrates the relationship between the bradykinin-induced changes in tissue osmolality and the corresponding changes in fractional water excretion. A significant correlation between changes in the medullary osmotic gradient and changes in fractional water excretion was observed when differences between control and experimental kidneys were compared. No significant correlation between changes in tissue osmolality and urinary sodium excretion was apparent.

3.2. Vasopressin-infusion control experiments 3.2.1. Non-bradykinin controls To determine whether the medullary tissue

L E F T KIDNEY 600 - -

400 --

200

o

--

~ WATER DIURESIS

I WATER DIURESIS +

ADH Fig. 2. P e a k u r i n e o s m o l a l i t y p r i o r t o a n d f o l l o w i n g the administration of antidiuretic hormone to 6 dogs d u r i n g w a t e r d i u r e s i s ( G r o u p 2).

osmolality of control kidneys was representative of that in the experimental kidneys prior to the infusion of bradykinin, a second series of experiments was performed ( G r o u p 2 ) . Antidiuretic doses of aqueous vasopressin were administered to a second group of dogs which were undergoing a water diuresis. The

BRADYKININ AND RENAL OSMOTIC GRADIENT

179

results o f this e x p e r i m e n t are summarized in table 3 and fig. 2. Clearly, the response to ADH in this study indicates the presence of a medullary c o n c e n t r a t i o n gradient during a water diuresis. Further, the urine osmolalities measured in this control study were similar to the contralateral control kidney tissue osmolalities measured in the previous bradykinin infusion study.

bradykinin and ADH infusion. Immediately prior to the infusion of bradykinin, the osmolality of urine from left and right kidneys averaged less than 90 mOsm/kg H20. During the infusion of the peptide alone, the osmolality of urine from the infused kidney remained below 100 mOsm/kg H20, whereas that of the contralateral kidney rose slightly. This elevation of control kidney urine osmolality was likely the result of a reduction in the rate of urine flow by this kidney, which presumably allowed additional time for water reabsorption to occur. After the administration of ADH, the osmolality of urine from the control kidney rose to an average peak of 450 + 60 mOsm/kg H~O during the continued infusion of bradykinin into the experimental kidney. In contrast, the osmolality of urine from the bradykinin-treated kidney reached a peak value of only 185 -+ 15 mOsm/

3.2.2. Bradykinin-infusion controls To assess the effectiveness of ADH to cause an increase in urine osmolality during the infusion o f bradykinin, the third series of experiments was c o n d u c t e d (Group 3). Bradykinin infusion into the left renal artery was associated with marked vasodilatation as adjudged by a visible increase in the size of the infused kidney, and a natriuresis. Fig. 3 illustrates the change in urine osmolality during

|

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I

I

I

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BRADYKININ

I

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IN L E F T

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500~

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400~

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HORMONE

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[

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......

.....

300~

200~

100 ~

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50

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60

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70

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80

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90

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100

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110

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120

( M I N U T E S )

Fig. 3. Effect of antidiuretic hormone on peak osmolality of urine from bradykinin-treated kidneys (~, ~) and contralateral control kidneys (©. . . . . . ©) during water diuresis in 4 dogs (Group 3). Brackets denote ±S.E.M.

180

L.R. W I L L I S |

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[ BRADYKININ IN LEFT RENAL ARTERY ]

16-

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ANTIDIURETIC

HORMONE

INFUSION

]

14-

12-

10-

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V ~-~-~xi00 6CH20

~

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4-

2- COsm ~

xl00

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30

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TIME

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( M I N U T E S )

Fig. 4. E f f e c t of a n t i d i u r e t i c h o r m o n e o n f r a c t i o n a l w a t e r and o s m o l a r e x c r e t i o n by b r a d y k i n i n - t r e a t e d k i d n e y s d u r i n g w a t e r diuresis in 4 dogs ( G r o u p 3). B r a c k e t s d e n o t e -+S.E.M.

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r~ 4--

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2~ ~

I

0

x 100

I

10

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I

50

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60

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70

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80

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90

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100

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110

( M I N U T E S )

Fig. 5. Effect o f a n t i d i u r e t i e h o r m o n e o f f r a c t i o n a l w a t e r a n d o s m o l a r e x c r e t i o n b y u n t r e a t e d c o n t r o l k i d n e y s d u r i n g w a t e r diuresis in 4 dogs ( G r o u p 3). B r a c k e t s d e n o t e +_S.E.M.

BRADYKININ AND RENAL OSMOTIC GRADIENT

181

kg H20 under the influence of ADH, a value which was significantly less than that of the contralateral urine. Interestingly, during the continued infusion of bradykinin, the osmolality of urine from the infused kidney declined to a low average value of 110-+ 20 mOsm/kg H20 (fig. 3), and then rose to a level of 240 mOsm/kg H20 when the bradykinin infusion was stopped. This value was still significantly below that of the contralateral kidneys, however. Figs. 4 and 5 illustrate the effects of bradykinin and ADH on fractional urine, water and osmolar excretion in the experimental and control kidneys, respectively. The infusion of bradykinin into the left renal artery was associated with a significant (nearly twofold) increase in fractional water excretion (fig. 4). This effect was reversed by ADH, but mean fractional water excretion remained positive t h r o u g h o u t the experiment. In contrast, the infusion of bradykinin into the left renal artery had no significant effect on fractional water excretion by the contralateral, non-infused kidney (fig. 5). However, ADH produced a significant antidiuresis in this kidney, complete with the excretion of negative free water.

ship between decreases in papillary tissue osmolality and increases in free water clearance (fig. 1). The design of the present experiments prohibited the determination of medullary tissue osmolality in the experimental kidneys prior to the unilateral infusion of bradykinin. Therefore, the medullary osmotic gradient of the non-infused contralateral kidneys was assumed to be representative of the pre-infusion state of the gradient in the experimental kidneys; Groups 2 and 3 were studied to test the validity of this assumption. After administration of antidiuretic hormone to the dogs of Group 2, the osmolality of urine from the left and right kidneys reached identical peak values (peak urine osmolality in the presence of ADH has been shown to correlate with papillary tissue osmolality [Gottschalk and Mylle, 1959]). Furthermore, there was close agreement between the peak urine osmolality values from kidneys of Group 2 dogs and the papillary tissue osmolality values from the kidneys of Group 1 dogs. Thus, the assumption seems valid. On the other hand, since some reduction in clearance functions of the contralateral non-infused kidneys was observed (table 1), it is possible that the medullary gradient of the control kidneys to which the experimental kidneys were compared may have been enhanced. To assess that possibility, the third series of experiments was conducted. If the osmotic gradient of the contralateral kidneys had indeed been enhanced by treatment of the experimental kidney with bradykinin, the osmolality of urine from the control kidneys of Group 3 should have been significantly greater than that for Group 2. Since this was clearly not the outcome of these experiments (fig. 3 vs. fig. 2), the use of the contralateral kidneys in the tissue analysis experiments (Group 1) as pre-infusion controls for the experimental kidneys seems justified. The notion that bradykinin may not inhibit sodium reabsorption by the proximal tubule is not new. Reports from at least three micropuncture laboratories attest to this possibility

4. Discussion The results of the present studies offer evidence in support of the hypothesis that an increase in free water clearance associated with bradykinin-induced renal vasodilatation can be accounted for by a drug-induced reduction of the medullary tissue osmotic gradient. In these studies the condition of the osmotic gradient was assessed both by tissue analysis and by measurement of peak urine osmolality during infusion of antidiuretic hormone. Whether assessed by either method, the renal medullary osmotic gradient of bradykinin-treated kidneys was consistently less than that of contralateral control kidneys. In addition, the hypothesis is further supported by the observation of a direct relation-

182 (Dirks and Seely, 1967; Stein et al., 1972; and Schneider et al., 1973). On the ot he r hand, Stein et al. (1972) have suggested t hat bradykinin may have an indirect inhibitory effect on sodium reabsorption by t he proximal tubules o f the inner c o r t e x which are inaccessible to m i c r o p u n c t u r e . This suggestion is based on the assumption t hat changes in peritubular capillary protein c o n c e n t r a t i o n (associated with altered filtration fraction), necessitate parallel changes in proximal tubular sodium reabsorption (Brenner and T r o y , 1971). However, the e x t e n t to which this relationship represents cause and effect has been questioned (Ott et al., 1974). The question remains, then, w h e t h e r bradykinin inhibits volume reabsorption by inner cortical proximal tubules. The present data support, b u t do n o t prove, the n o t i o n t ha t bradykinin lacks an inhibitory effect on proximal tubules o f the inner as well as the out e r cortex, since a statistically significant correlation was observed in the present studies b e t w e e n increases in free water excretion and reduction o f the medullary osmotic gradient. Had free water excretion been related, instead, to depression o f proximal tubular reabsorption, increases in free water excretion should have been i n d e p e n d e n t of the osmolality df medullary tissue. F u r t h e r m o r e , Schneider et al. (1973) have suggested t hat bradykinin lacks an inhibitory effect on inner as well as superficial cortical proximal tubules. This suggestion was based on t he finding that inhibition of reabsorption by surface proximal tubules is associated with enhanced phosphaturia. These authors observed t hat bradykinin, which did n o t detectably reduce proximal tubular reabsorption by surface nephrons, likewise, did n o t p roduc e phosphaturia. There are at least t w o possible explanations for the mechanism by which medullary tonicity might have been diminished during renal vasodilatation with bradykinin. One possibility is th at an increase in medullary blood flow may have washed out the solute gradient. Alternatively, inhibition of tubular reabsorption would have made more water available

L.R. WILLIS for reabsorption across the wall of the collecting duct t hereby diminishing the gradient by a process of dilution. The evidence cited in the preceding paragraphs argues against the possibility t hat a reduction in reabsorption by the proximal tubule cont ri but ed significantly to the latter alternative. However, bradykinin may have increased fluid delivery into the collecting duct by inhibiting sodium reabsorption in cortical portions of the distal tubule. Subsequent reabsorption of this increased a m o u n t of water across the collecting duct might, then, have diluted the medullary gradient. The present results support an explanation for the decrease in medullary tonicity based on an increase in medullary blood flow a n d / o r an increase in delivery of fluid to the collecting duct from distal portions of the nephron.

Acknowledgements This work was supported by HL 14159 (Specialized Center of Research in Hypertension), and by grants from the American Heart Association and the American Heart Association, Indiana Affiliate.

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