Effect of chlorpropamide on water and urea transport in the inner medullary collecting duct

Kidney International, Vol. 39 (1991), pp. 79—86

Effect of chiorpropamide on water and urea transport in the inner medullary collecting duct ANTONINO S. ROCHA, WONG C. PING, and LUcIA H. KUDO Laboratório de Pesquisa BOsica, Departamenlo de ClInica Médica, Faculdade de Medicina, Universidade de São Paulo, São Paulo, Brasil

Effect of chiorpropamide on water and urea transport in the inner reveals its antidiuretic effect by potentiating the action of ADH medullary collecting duct. The present in vitro microperfusion study present in very small amounts in patients with diabetes insipiexamined whether chlorpropamide (CPM) has a direct effect on hydraulic conductivity (Lp x 10—6 cm/atm . see) and '4C-urea permeability (Pu x iO cm/sec) in the middle and distal inner medullary collecting duct

(IMCD) obtained from acutely water-loaded Wistar rats and rats homozygous for diabetes insipidus (DI). CPM (10 M) added to the bath fluid increased the Lp in the water-loaded Wistar rats from —0.05 0.13 to 6.25 0.74 (p <0.01) and in the DI rats from 0.05 0.01 to 5.95 0.84 (p < 0.01), but had no effect when it was added to the

perfusate. CPM stimulated Lp in a dose-dependent manner with the threshold effect at 10-6 54. However, the addition of CPM (l0— M) to submaximal concentration of VP in the bath fluid did not increase the Lp. Furthermore, CPM was unable to block the inhibitory action of PGE2 on the vasopressin (VP)-stimulated Lp. On the contrary, PGE2 blocked the CPM-stirnulated Lp. CPM (10—a si) in the pentubular fluid was able to cause a significant rise of the Pu from 13.5 0.8 to 17.3 1.0 reversibly, which represented 16% of maximum stimulated effect

dus [9, 10]. There is considerable disagreement in the literature regarding the existence of a direct effect of CPM on osmotic water flow across the toad bladder. Several investigators found a significant increase in osmotic water flow after the addition of CPM in high concentrations (3 to 6 mM) [11, 12], while others found an extremely small or no increase in the presence of low doses of

CPM (1 m or less) [13, 14]. It is conceivable that the discrepancy between these studies may reflect differences in the

sensitivity of different types of toad bladders to CPM and/or differences in the CPM concentrations tested. The antidiuretic action of CPM could be due to an increase in

the activation of adenylate cyclase by arginine vasopressin produced by 50 U/ml of VP. Thus, pharmacological doses of CPM (VP). The hypothesis was based upon studies in which toad added to the peritubular side have a direct effect on terminal IMCD increasing water and urea permeability in the absence of VP, but this bladder or renal medullary cell membranes [8, 15—17] were drug does not potentiate the VP-stimulated water transport in the prepared in the presence of or after treatment with CPM, leading to an increase in renal cAMP concentration. Since there

IMCD. Our results were unable to confirm the hypothesis that CPM potentiates the VP-antidiuresis by the inhibition of PGE2 action in the rat IMCD.

is a VP-sensitive adenylate cyclase in both the mammalian

collecting tubule and the thick ascending limb of Henle's loop [18], the effect of CPM on enhancing VP-stimulated adenylate cyclase could occur at either or both sites. Recent studies have demonstrated that inhibition of prostaChiorpropamide (CPM), a sulfonylurea derivative, has been shown to reduce urine flow in some patients with diabetes glandin synthesis greatly enhances the VP-stimulated water insipidus (DI). Arduino, Ferraz and Rodrigues [1] reported a permeability in the collecting tubules and sodium transport in decrease in urinary volume and an increase in urine osmolality the thick ascending limb of Henle's loop [19, 20]. Since CPM following administration of CPM to patients with diabetes appears to inhibit VP-stimulated prostaglandin E2 (PGE2) syninsipidus of pituitary origin. A number of studies have shown thesis in the toad bladder [12], there is the hypothesis that the that this drug also has an antidiuretic action in water-loaded antidiuretic effect of CPM is due to an inhibition of PGE2 normal subjects [2, 3] and in patients with diabetes mellitus [4]. biosynthesis. Therefore, many points regarding the proposed CPM action The precise mechanism of CPM antidiuresis has not yet been elucidated. Three possible hypothesis have been suggested: on urinary concentration still remain to be clarified. In particfirst, stimulation of ADH release from the posterior pituitary ular, it remains to be determined whether CPM acts on renal gland [51, but some studies [6, 71 found no evidence for such an tubules directly or through some secondary mechanism after its effect; second, ADH-like action and; third, potentiation of the administration in vivo.

antidiuretic effect of suboptimal levels of ADH at the renal

The goal of the present study was to analyze directly the

tubule [8—10]. Studies in Brattleboro DI rats suggest that CPM effect of CPM on hydraulic conductivity (Lp) and urea perme-

ability (Pu) of the inner medullary collecting duct (IMCD) obtained from acutely water-loaded and DI rats, using the in vitro microperfusion technique. This method permitted a measurement of the capacity of CPM to potentiate the VP-stimulated Lp of IMCD and to determine the effect of CPM on Lp in the presence of PGE2 synthesis inhibition.

Received for publication March 28, 1990 and in revised form July 30, 1990 Accepted for publication August 3, 1990

© 1991 by the International Society of Nephrology 79

80

Rocha et a!: Ch!orpropamide effect on water and urea transport

Methods

(cm s atm1) was calculated in each experiment by the following equation [23]:

Isolated inner medullary collecting duct (IMCD) segments were perfused by routine techniques previously described [211. Kidneys from Wistar rats and homozygous rats of the Brattleboro strain (DI rats) weighing 120 to 125 g, fed a standard chow

(Na = 184 mEqlkg), were obtained and tubules were isolated from a small slice immersed in a dish of chilled Ringer/HCO3 buffer, oxygenated and kept at pH 7.4 by bubbling the solution with 5% CO2 and 95% 02. The IMCD segments were dissected without the use of collagenase or other enzymatic agents. The papillary middle third (IMCD2) and inner third (IMCD3) segments were isolated from the terminal two-thirds where the urea permeability is higher [22]. The tubules were mounted on

Lp =

l/RTACb2 {Cb(V1 — V0) + CiV1 [ln(Cb — Ci) V —

in (CbV0 — C1V1)]}

(1)

where Cb and Ci are the osmolalities of the bath fluid and the perfusate, respectively, V1 the perfusion rate, V0 the collection rate, R, the gas constant, and T the absolute temperature, and

A the luminal surface area. This equation is based on the assumption that the reflection coefficient of NaC1 is 1.0 [23]. No

change in the reflection coefficient of NaCI was seen to occur after exposure to VP and CPM, since the sodium bath-to-lumen permeability of IMCD was found to be similar to control values concentric pipettes as described previously [21] and were in the presence of CPM (1.83 0.13 vs. 1.84 0.15 x l0— studied at 37°C. One end of a single segment was drawn by cm/sec) and VP [24]. The computation of the Lp of tubules with suction into the tip of a glass-holding pipette. An inner pipette two branches in parallel showed that the Lp calculated for each was then advanced into the duct lumen. This pipette contained branch does not differ from the Lp calculated by the equation 1 the solution to be perfused through the duct. Perfusion was using the total area as the sum of the surface of each branch and driven by hydrostatic pressure. The opposite end of a single the collected fluid (nllmin) as the sum of the collected volume of tubule or two branches was held in another glass micropipette each segment. Passive urea permeability from lumen-to-bath (Pu) was estimade according to the size of the branches to avoid leak between them. A viscous silicone liquid (Sylgard 184, Dow mated from the disappearance rate of '4C-urea (50 to 100 Corning Corp., Midland, Michigan, USA) was used to form a cpm/nl) from the perfusion solution. When there was no net seal around the branches which completely separates the col- volume absorption, permeability from lumen-to-bath was callected fluid from the bath solution. At intervals, the collected culated from the following equation: fluid was removed by a sampling pipette which had its volume Ci V, measured before the onset of the experiment. The mean dura(2) tion of the collection periods was 10 minutes. Unless indicated otherwise, Ringer/HCO3 buffer (composition in mM: NaCI, 115; where V, is the perfusion rate, A is the inner surface area of the NaHCO3, 25; Na acetate, 10; KCI, 5; CaCI2, 1.0; MgSO4, 1.2; tubule and Ci and Co are the activities of '4C-urea (cpmlnl), in NaH2PO4, 1.2; glucose, 5.5 without urea) was used as the bath the initial perfusion fluid and the collected fluid, respectively. In and perfusion solution. the case in which net volume reabsorption was present, Pu was Net water absorption (J) was measured with '4C-inulin or 3H-inulin dialyzed immediately before the experiments. The computed from the following equation [25]:

Pu=ln—'

dialyzed isotope was added to the perfusion solution to give a V—V0 ln(Ci/Co) l+cru Pu = (3) final concentration of 25 to 100 cpm/nl. No leakage of radioac2 A in (V1/V0) tive inulin into the bath fluid was detected. J,. was calculated as (V-V0/l) where V1 was the perfusion rate, V0 the collection rate where the characters are the same as in equations 1 and 2 and 1 the length of tubule studied (tubule length and inner except that au is the reflection coefficient of urea being considdiameter can be measured to within 0.05 mm with a precali- ered equal to 1.0 [26]. Similarly to what was described above for brated micrometer in one eyepiece of the inverted microscope Lp, we demonstrated that Pu calculated for each branch is used to observe the perfusion). V0 was directly measured by identical to the Pu obtained by equation (2) using the total area using the collection time while V. was calculated by the and the total collected volume as the sum of the area surface equation: V = V0(In0/In), where In0 is 14C or 3H-inulin cpm/nl and the collected volume of each branch. Perfusate and bath fluids were checked for sodium and of the collected fluid and In1 is cpm/nl of the perfusate. Timed tubular samples were collected for analysis under mineral oil by potassium concentration, osmolality and pH with the Na-K aspiration with a calibrated pipette. All measurements were flame photometer, model 143 (Instrumentation Laboratory, obtained 60 to 90 minutes after onset of perfusion of a given Inc., Lexington, Massachusetts, USA), osmometer (Advanced nephron segment. The internal diameter of the perfused seg- Instruments Inc.) and pH meter (Iris 7 Tecnow), respectively. ments varied from 25 to 35 and the tubular length from 1.5 to The bath fluid was changed every five to seven minutes in order to reduce the effect of evaporation and consequently the 3.0 mm. Hydraulic conductivity (Lp) was determined by measuring increase in osmotic gradient. The bath osmolality did not net fluid movement in response to an imposed osmotic gradient change significantly during the seven minute period, from 296 in the absence and in the presence of VP. Net fluid absorption 2 at 0 mm to 294 1 at seven minutes. Timed fluid collections were made with a constant volume was induced by perfusion with a hypotonic solution of 80 to 90 mOsm/kg . H20 (NaC1 50 mM) and a 300 mosm/kg H2O bath constriction pipette which was rinsed four times with 0.5 ml of solution. Rapid perfusion rates of 20 to 40 nI/mm were used to water and 5 ml of scintillation liquid was then pipetted into the avoid osmotic equilibrium between perfusate and bath. Osmo- vial (Aquasol Universal Cocktail, New England Nuclear, Bos-

lalities of perfusate and bath fluid were measured, and Lp ton, Massachusetts, USA). The isotopic concentration was

81

Rocha et a!: Chiorpropamide effect on water and urea transport Table 1. Effect of chiorpropamide added to the bath fluid on hydrosmotic conductivity in IMCD of Wistar rats

Exp

T. length

Area

V

2.3

19.3

28.1

2 3 4 5

2.0 3.0

Mean

2.9 0.3

23.9 25.3 28.9 31.3 25.7 2.1

27.4 26.6 27.6 27.7 27.5 0.2

1

3.3 3.8

Control Osm grad 214 —0.07 214 —0.07 214 —0.08 214 —0.00

J

0.11 —0.02

0.03

Lp

Chiorpropamide 10 Osm V grad

J

206

—0.22 —0.17 —0.17 —0.01

27.8 31.9 30.7

2.29 2.28 2.08

32.1

213

0.43

214 0.2

—0.05

31.6 30.8 0.8

1.42 1.56 1.93a

207 207 207 207 207

0.18

0.2

0.13

M

Lp

V.

8.44 5.69

27.0 27.8 26.4 29.9 27.4 27.7 0.6

7.51

5.14

4.49 6.25a

0.74

Recovery Osm grad

J

Lp —0.25

+0.70 +0.05 +0.09

214 214 214 211 214 213

0.16

0.6

0,52

—0.07 —0.06 —0.19

—0.07 —0.32

+2.48 +0.29 0.43

Abbreviations are: T. length, tubular length (mm); Area, tubular area (x lO cm2); V,, perfusion rate (nI/mm); J.,,, net water absorption mm); Osm grad, osmotic gradient (mOsm/kg H20) and Lp, hydrosmotic conductivity (x 106 cm/atm sec).

(ni/mm a

p < 0.01

Table 2. Effect of chlorpropamide added to the perfusate on hydraulic conductivity in IMCD of Wistar rats

T. length

Area

V,

1

2.5

26.1

2 3 4

2.1

23.5 22.9 28.9 25.3

24.2 24.4 24.3 24.4 24.4 0.0

Exp

Mean

2.5 2.9 2.5 0.2

1.4

J

Control Osm grad

0.01

0.00 0.00 0.03 0.01 0.01

200 200 200 200 200 0.0

Chiorpropamide iO M

Lp

V

J,.

0.32

23.5 23.6 23.7 24.0 23.7

—0.45 —0.06

0.1

0.05 0.03 0.91 0.33 0.25

Osm grad

Recovery Osm grad

J

Lp

V,

—0.47

24.4

0.00

—0.03

24.3

—0.00

0.03

0.11

0.2

0.10

24.4 24.4 24.4 0.0

0.00

—0.12

201 200 200 200 200

0.00 0.04

—0.01

—0.12

—0.04 —0.02

0.01

200 200 200 200 200 0.0

Lp —0.06 —0.01

+0.05 —0.36 —0.09

0.09

Abbreviations are: T. length, tubular length (mm); Area, tubular area (x l0 cm2); V1, perfusion rate (nI/mm); J, net water absorption (ni/mm. mm); Osm grad, osmotic gradient (mOsm/kg. H20) and Lp, hydraulic conductivity (x 106 cm/atm sec).

Effect of CPM on water transport in IMCD obtained from mod. La-l00-C). When 14C-urea and 3H-inulin were determined Wistar rats in the same sample, after background subtraction, counts were The effect of CPM added to the bath fluid (10 M) on corrected for incomplete discrimination between the two chanhydraulic conductivity (Lp x 106 cm/atm . sec) is presented in nels.

determined in a liquid scintillator spectrometer (Beckman,

Table 1. In this group, we measured the effect of CPM on net water absorption of IMCD in the presence of osmotic gradient. CPM added to the bath fluid (10—a M) increased water absorp0.18 nllmin . mm, which 0.03 to 1.93 tion from —0.02 represented an increase in Lp from —0.05 0.13 to 6.25 0.74 (P < 0.01). The reversibility of the effect was observed restoring the initial condition in the third period when the net water absorption and Lp returned to 0.09 0.16 and 0.43 0.52, period are the mean of three to four collections. Statistical respectively. In another group of IMCD, we examined the effect of CPM analysis was performed by paired or nonpaired 1-test and by analysis of variance when appropriate. The data of each tubule (10 M) added to the perfusate on Lp (Table 2). The absence of To avoid the possibility of inulin binding to glass, all glassware in contact with the perfusion solution and collected fluid was siliconized. VP and CPM and PGE2 were purchased from Sigma Chemical (St. Louis, Missouri, USA) and were of analytical grade. The isotopic materials used were provided by Amersham International and New England Nuclear. The results are expressed as mean SEM. The data for each

are the mean of three to four collection periods per tubule,

net water absorption observed in the control period, in the

presence of osmotic gradient, did not alter significantly by the addition of CPM to the perfusate (0.01 0.01 vs. —0.12 0.11 nI/mm. mm). These experiments indicated that CPM (10—a M) Results was able to increase Lp when it was present in the bath fluid but The effect of CPM on water transport of IMCD was analyzed: not in the luminal fluid. To test whether CPM added in a lower concentration to the first, in Wistar rats with minimum urine osmolality (U0sm = 85 6 mOsm/kg. H20) after acute water-load performed by bath fluid could stimulate Lp in the IMCD, we measured the intraperitoneal infusion of hypotonic saline (NaCI 75 mM) in a effect of CPM 108 M, 106 M, and i0 M. Figure 1 illustrates volume equivalent to 20% of body weight two or three hours these results during five consecutive periods in which Lp was prior to sacrifice; second, in rats of the Brattleboro strain with measured in the absence of VP in the bath. CPM at 10_6 M increased Lp from —0.06 0.03 to 2.05 0.20 (P < 0.01), but hereditary DI allowed free access to tap water (U0sm = 195 at 108 M had no effect on Lp (—0.06 0.03 vs. —0.03 0.05). 20 mOsm/kg . HO). depending on the experimental protocol.

82

Rocha et al: Chiorpropamide effect on water and urea transport 8

16

*

3 x 101 VP 12

6 0 a) 0 a)

0)

E

E

CO

Co

E

0

E

0

4

0

3 >< 102M VP 104M CPM

x

*

x 0. -J

* ,/

8

0

-Ja 4

2

3 x 1013i VP

**

0

0

1'

2

Timed control

3

4'

Time, period Control

108M

10—6M

10-4M

CPM

CPM

CPM

Recovery

Fig. 1. Dose response of chiorpropamide (CPM) on hydraulic conduc-

tivity (Lp) of inner medullary collecting duct (IMCD) obtained from Wistar rats. The experiments were carried out according to the indicated sequence. Each point represents one period. Lines connect data < 0.01 points of individual tubules.

Fig. 2. Comparison between the magnitude of chlorpropamide (CPM)

(LI) vasopressin (VP)-stimulated hydraulic conductivity (Lp) (A) and time control experiments (0). Each point represents the mean 5EM of the values obtained in each period. Lines connect data points of each experimental group of IMCD. ((p < 0.01, **p < 0.05

4

To compare the magnitude of the CPM effect with the VP-stimulated water absorption in the presence of osmotic gradient, we measured the net water absorption and Lp during the control period and after 3 x i0', 10— 12, 10_lI M of VP added successively to the bath fluid (N = 5). Figure 2 shows that the stimulated effect of i0" M of CPM on Lp (N = 5) was similar to the effect of 3 x 10_12 M of VP, and the magnitude of Lp produced by 10_6 M of CPM was similar to the action of 3 X

Co

E CO

E

2

X

1

0

a

-J

i0 M of VP. In an attempt to test the hypothesis that CPM increases net VP+ VP VP+ water absorption due to an inhibitory effect on PGEe, we first CON PGE2 PGE2 analyzed directly the effect of CPM on PGE2-inhibited, VPCPM stimulated water permeability in five consecutive periods. Fig- Fig. 3. The effect of chlorpropamide (CPM) on the inhibitory effect of ure 3 shows that Lp increased from 0.01 0.08 to 3.52 0.03 PGE2 on vasopressin-stimulated hydraulic conductivity (Lp) of IMCD by the addition of submaximal doses of vasopressin (3 x 10_12 obtained from Wistar rats. Each IMCD was studied in five consecutive M) and decreased to 2.48 0.03 when PGE2 (l0— M) was added periods: control (CON), in the presence of AVP (3 x 10-12 M), when to VP in the bath, but CPM (10—n M), in combination with PGE2 and VP in the bath, did not change the Lp (2.40 0.04) which

PGE2 was added to VP in the bath, when CPM (l0— M) was associated

to the VP and PGE2 in the bath fluid and finally, when CPM was withdrawn from the bath and VP plus PGE2 remained. Each point represents the mean SEM of the values of four tubules obtained in

remained when CPM was withdrawn. Therefore, this group of experiments demonstrated that CPM was unable to block the each period. * 0.01 inhibitory of PGE2 on VP-stimulated Lp in the IMCD. In the second step, we analyzed the effect of PGE2 on the CPM-stimulated Lp. Each IMCD was studied during four in the control period, showing that PGE2 did not alter the basal consecutive periods: control, in the presence of PGE2 in the water permeability, but it was able to block the stimulating bath fluid, when CPM was added to the PGE2 in the bath, and effect of CPM (l0 M) on water absorption (0.08 0.04 finally when PGE2 was withdrawn remaining CPM in the bath nl/min . mm) and Lp (0.26 0.14). In the last period when this (Table 3). We observed that PGE2 did not change the net water drug was present in the bath in the absence of PGE2, the Lp absorption (0.02 0.03 vs. —0.04 0.02 nI/mm . mm) observed increased to 5.99 0.49.

83

Rocha et al: Chiorpropamide effect on water and urea transport Table 3. Effect of chiorpropamide (10

Exp 1

2 3

4 5 6

Mean

T. length 2.55 2.55 2.75 3.45 2.40 2.80 2.75 0.10

M)

addition to the bath fluid in the presence of prostaglandin E2 on hydrosmotic conductivity in IMCD of Wistar rats Prostaglandin E2 105M

Control Osm

Area

V1

22.9

25.9

24.3

26.5 25.5 26.1

20.6 29.0 20.6 26.3

23.9 1.36

25.9 25.8 25.9 0.1

i,

grad Lp

0.03 0.08 0.07 0.04 0.02 0.13 0.02 0.03

216 216

0.20 0.26 216 0.26 216 0.15 215 0.07 216 —0.14 216 0.13 0.2 0.06

V1

J

25.1

—0.04

25.6 25.6 25.9

—0.13 —0.03

27.1 27.2 26.1

—0.09

0.4

0.02

Osm

grad

Lp

216 216 216 216 216 216 216 0

—0.14 —0.16 —0.08

0.01

+0.01 —0.04

+0.04

Prostag. + Chiorprop. Osm i, grad Lp V1 0.09 25.7 0.04 216

26.9 0.28 25.8 0.00

215

0.07

215 215

0.89 0.02 0.24 0.42

216 215 0.2

26.1

—0.14

26.2 0.12

+0.01

26.0 —0.01 26.1 0.08

—0.08

0.04

0.2 0.04

216

Chlorpropamide 104M Osm grad Lp V1

i

30.0 30.7

1.98

2.03

30.5

31.3

1.69 1.61

29.1

1.45

—0.08

29.0

0.26 0.14

30.1

3.44 2.03 0.29

0.4

207 208 208 207 210 210 208

6.91

0.6

0.49

6.69 7.06 6.03 5.25 3.98

599k

Abbreviations are: T. length, tubular length (mm); Area, tubular area (x l0 cm2); V, perfusion rate (nI/mm); iv, net water absorption (nl/min mm); Osm grad, osmotic gradient (mOsm/kg. H20) and Lp, hydrosmotic conductivity (X 10-6 cm/atm. sec). a

p

0.01

Table 4. Effect of chl orpropamide

(10 M) addition to the bath fluid on hydraulic conductivity o f IMCD obtained from diabetes insipidus rats

J

Control Osm grad

Recovery Osm

Chlorpropamide Osm

T. lenght

Area

V1

Lp

V,

iv

grad

Lp

V1

1

2.1

214 214

3.8 3.3 3.3

31.3

0.11

213

28.9

27.6

32.1

1.43

33.0 29.6

3.69 2.14

193

6.75 4.49 4.48 5.14 8.89

21.0 21.8 27.4 29.9

21.5 23.8 1.6

214 214 214

202 207 207 207

37.7 28.7 2.8

0.00 0.04

0.43 0.01 0.12 0.05 0.01

25.4 25.9 31.6

2.28

2.0

—0.08 —0.07

3

20.8 21.5 27.7

—0.21

2

22.0 23.6

203

5•95a

24.2

1.6

0.41

2.7

0.84

Exp

4 5

Mean

2.9 0.3

0.01 0.03

0.2

—0.06

1.75 1.56

21.1 1.8

v

grad

Lp

—0.05 —0.07

214 214 214

—0.13

211

2.48

213 213

0.26 0.54 0.49

0.05

0.70 0.10 0.15 0.14

0.6

—0.20

0.29

Abbreviations are: T. length, tubular length (mm); Area, tubular area (X iO cm2); V, perfusion rate (nl/min); iv, net water absorption (nl/min. mm); Osm grad, osmotic gradient (mOsm/kg. H20) and Lp, hydrosmotic conductivity (x lO_6 cm/atm. sec). a P < 0.01

Effect of CPM on Lp of IMCD isolated from DI rats To substantiate the observation that CPM stimulated Lp of IMCD in the absence of VP, experiments were performed in Brattleboro homozygous rats with DI in which the plasma concentration of VP is undetectable [28]. Table 4 shows that in five consecutive experiments, CPM l0 M added to the bath was able to increase net water absorption from 0.01 0.03 nl/min. mm to 2.14 0.41 nI/mm mm reversibly in the IMCD of DI rats, which represents an increase in Lp from 0.05 0.01 to 5.95 0.84 (P < 0.01). It is well known that the administration of CPM potentiates the anti-diuretic effect of VP in DI rats [8—10]. However, there

iments, it was observed that CPM (10 M) in the absence of VP was able to increase Pu from 13.5 0.8 to 17.3 1.0 which returned to 13.5 0.7 when this drug was withdrawn from the bath (Table 5). The stimulating effect produced by CPM repre-

sents 16% of the maximum stimulating effect produced by 10— M of VP is illustrated in Figure 5. Discussion

The studies on the mechanism of CPM's antidiuretic action were performed using; first, clearance studies in DI and waterloaded patients and in DI rats [1—5, 29]; second, determination

is some doubt whether CPM is able to increase the VP- of cAMP, adenyl-cyclase and phosphodiesterase activity in

renal medullary tissues of DI rats and toad bladder [8, 12—14, question, we measured the Lp of IMCD of DI rats in the 161; third, measurements of PGE2 urinary excretion and synpresence of CPM, and after a recovery period we determined thesis [15, 301; fourth, in vitro determination of Lp in toad the effect of 3 x 1013 or 3 x l0_12 M of VP. After this, we bladder [11, 12]. However, these studies did not provide us with compared the determined Lp with the values observed when a definitive conclusion about the antidiuretic mechanism of CPM was added to VP in the bath fluid. The data presented in CPM. The most widely held view obtained from these studies Figure 4 clearly show that CPM was able to increase LP appears to be that CPM potentiates the antidiuretic effect of VP. reversibly to values close to the observed with 3 x 1013 M of This conclusion is based mainly on the observations in DI rats VP. However, the addition of CPM to VP in the bath did not where the antidiuretic action of CPM occurs only in the change the Lp, which indicated that this drug did not potentiate presence of submaximal concentrations of VP and in vitro studies in toad bladder where high concentrations of CPM the VP-stimulated water permeability in the IMCD. (10 M) potentiates the effect of VP-stimulated Lp [9, 10, 14]. Effect of chiorpropamide on the '4C-urea lumen-to-bath The present study shows for the first time a direct effect of permeability (Pu x 1O cm/sec) of IMCD CPM on the Lp and Pu in the rat IMCD. CPM added to the bath The effect of CPM on Pu of IMCD obtained from water- fluid stimulated water and urea transport in water-loaded and loaded Wistar rats was determined. In five consecutive exper- DI rats. These effects were entirely reversible. stimulated water transport in the IMCD [29, 301. To answer this

84

Rocha et al: Chiorpropamide effect on water and urea transport

strain, CPM added to the bath fluid increases the Lp of IMCD which was associated with the corticopapillar sodium gradient [18], and the chloride absolute reabsorption in the Henle's loop [31, 321 also stimulated by CPM appears to be sufficient for 8 CPM administration in DI rats to reduce the urinary volume in absence of VP. l) It is important to point out that the direct maximum effect of CPM (l0— M) on IMCD had a similar magnitude to 3 x 10— 12 M of VP (Fig. 2), which corresponds to 30% of the maximum VP-stimulated Lp. This magnitude of the Lp corresponded to maximum VP-stimulated Lp in the rabbit IMCD which has the concentration capacity of 800 to 1200 mOsm/kg water [311. There is general agreement that CPM potentiates VP' s antidiuretic effect in DI rats, which would make it appear likely that one mechanism of CPM's effect in DI rats is through potentia2 tion of the action of small amounts of VP in the renal tubule [9, 101. The action of CPM in potentiating the effect of VP could be due to enhanced antidiuresis or to prolongation of the effective0 ness of VP. In the present study, we analyzed the capacity of CPM (l0 M) to potentiate submaximum effective doses of VP (3 X iO M or 3 x 10— 12 M) on Lp of IMCD. Contrary to what Control CPM Rec VP VP VP + Rec CPM was predicted by the clearance studies, we observed that the combination of CPM with VP did not increase the effect of VP Fig. 4. Absence of potentiated effect of chiorpropamide (CPM) (10-M or 3 x 10—12 on water transport in the IMCD. Thus, the potentiation of VP M) on the arginine vasopressin (VP) (3 X M)-stimulated hydraulic conductivity (Lp) of IMCD obtained from observed in vivo should be through the combination of CPMdiabetes insipidus (DI) rats. Each IMCD was studied during 7 consecstimulated water and urea transport of IMCD with some other utive periods: control, in the presence of CPM in the bath, recovery period, in the presence of 3 x io' M (t) or 3 x 10—12 M () of VP in effects of VP on other tubular segments or renal hemodynam10

the bath, when CPM (10 M) was added to VP in the bath, when CPM was withdrawn and VP remained, and finally in the recovery period

ics. The final cellular events involved in the action of VP on water

(REC). Each point represents the mean of three collections which transport in the IMCD consist of an increase in water permecorrespond one period. Lines connect data points of individual tubules. 0.01

ability in the apical region of the cell. Activation of adenylcyclase and subsequent generation of 3'S' cAMP is necessary for this to occur [33]. To determine whether the action of CPM

on water permeability is prior to or after cAMP formation, several studies were performed in toad bladder and microdisluminal side due to urinary excretion (20%) and peritubular side sected medullary collecting tubules [8, 34—36]. There is no by the medullary circulation. However, in the present study, evidence that CPM sensitizes collecting tubules to the action of high concentrations of CPM (i0— M) in the perfusion fluid were unable to stimulate Lp of IMCD, showing that the tubular VP, at the cAMP generation step. The CPM present in the plasma reaches the IMCD by the

response is side dependent.

PGE2 has been said to be involved in VP-potentiating effects

The possibility that the observed effect of CPM on Lp of of CPM [37, 38]. Ozer and Sharp [12] proposed that CPM IMCD in vitro is also present in vivo could be analyzed by decreases the inhibitory effect of PGE2 on VP-dependent gendetermination of the therapeutic plasma levels of CPM. Me- eration of cAMP. The results of the present study indicate that lander et al [281, measuring the serum CPM concentrations by CPM was unable to block the inhibitory effect of POE2 on selective and sensitive gas chromatography in 60 patients, VP-stimulated Lp of IMCD. This observation rules out the despite dosage variations of 125 to 500 mg daily, observed possibility of CPM antidiuresis being due to a modulating action

plasma variations of 50 to 500 jimollliter, which represents in on the inhibitory effect of PGE2 on VP-stimulated water transour study a bath fluid concentration capable of maximally port in the IMCD. On the contrary, POE2, similarly to what we stimulating Lp and urea permeability in vitro. Therefore, the have described with VP [ 24], inhibited the CPM-stimulated minimum concentration of CPM capable of stimulating Lp effect on water permeability in the rat IMCD. In summary, the present study shows clearly that CPM in (10—6 M) was within the pharmacological range of CPM. Also, we observed that the progressive increase in bath fluid concen- pharmacological doses added to the peritubular side has a direct effect on the IMCD, increasing the water and urea permeability tration of CPM determined a progressive increase in Lp. Due to the possible presence of small concentrations of VP in in the absence of VP and that the antidiuresis produced by CPM the renal tissue, we substantiate the observation of a direct in DI rats treated with submaximal concentrations of VP cannot action of CPM of Lp studying the IMCD of DI rats. Homozy- be explained by potentiation of CPM on the VP-stimulated gous DI rat of the Brattleboro strain has been shown to be water reabsorption in the IMCD. Besides, we did not confirm completely deficient in biologically active vasopressin and its the hypothesis that CPM potentiates the VP antidiuresis by neurohypophysis is devoid of stored VP [271. Also, in this rat inhibition of POE2 action in the IMCD.

85

Rocha et al: Chlorpropamide effect on water and urea transport Table 5. Effect of chiorpropami de added to the bath fluid on ure a permeability in the IMCD of W istar rats Control

Chiorpropamide

T. length

Area

V1

Pu

V1

2 3 4 5 6

3.5 2.7 3.0 3.1

26.8 24.8 20.0 23.7

26.5 27.6 27.5 28.4

3.7 2.5

30.1

28.1

Mean

3.1

28.9 25.7

0.2

1.5

28.4 27.7 0.3

13.0 16.0 14.7 11.2 11.5 14.8 13.5

27.9 27.5 27.7 28.9 28.0 28.3 28.0 0.2

Exp 1

0.8

Pu 17.9 18.9

20.2 13.5 16.0 17.6 17.3a 1.0

Recovery V1

Pu

28.0 27.7 27.4 28.2 27.9

14.5 15.8 14.0 11.5 11.6 13.5 13.5

28.1

27.9 0.1

0.7

Abbreviations are: T. length, tubular length (mm); Area, tubular area (x104 cm2); V,, perfusion rate (nI/mm); Pu, urea permeability (x l0 cmlsec). a P < 0.01

These studies were supported by the LIM HC/FMUSP and funds from Fundacao de Amparo a Pesquisa do Estado de São Paulo.

40 VP

Reprint requests to Antonino S. Rocha, M.D., Faculdade de Medicin Universidade de São Paulo, Av. Dr. Arnaldo, 455-CEP 01246, São Paulo, Brasil.

*

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30

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