chloride excretion in nephrons of rat kidney during alterations of acid-base equilibrium ·...

AMERXCAN JOURNAL OF PHYSIOLOGY Vol. 218, No. I, January 1970. Printed in U.S.A.

Chloride excretion in nephrons of rat

kidney during alterations of acid-base equilibrium

G. MALNIC, M. MELLO AIRES, AND F. LACAZ VIEIRA Department of Physiology, Sa’o Paul0 University Medical School, GO Pa&, Brazil

MALNIC, G., M. MELLO AIRES, AND F. LACAZ VIEIRA. Chlorz’de

excretion in ne@hns of rat kidney during alterations of acid-base epilib- rium. Am. J. Physiol. 218(I): 20-26. 1970.-Chloride and inulin concentrations were measured in cortical nephrons of rats during 5% NaHC03, 0.4 M Na&Q, and acetazolamide infusjon, and N&Cl acidosis. Transtubular potential differences were measured in similar groups. Proximal tubular chloride TF/P ratios were significantly lower than controls in bicarbonate- and acetazolamide-infused animals; chloride conr.en trations were highest in acidotic rats. Tubular concentrat.ions lower than plasma were not found in any of the experimental groups. Significantly reduced TF/P ratios were found in the distal tubule in the same groups as in the proximal tubule. Proximal fluid reabsorption was significantly reduced in acetazolamide-infused rats. Net chloride reabsorption was also reduced in the proximal tubule in this group. Since chloride concentrations remain constant along individual tubular segments, they may be considered to be steady-state levels. Especially along the proximal convolu- tion these are removed from electrochemical equilibrium, at least in the acidotic rats, suggesting that mechanisms other than simple passive transport might be involved in transtubular chloride distribution

chloride transport; acetazo2amide; impermeant anions

SEVERAL MICROPUNCTURE INVESTIGATIONS have shown that chloride handling by proximal and distal tubules of rat nephron cannot be always understood solely in terms of passive transport along electrochemical potential gradients created by active sodium transfer across the tubular epithelium. Data suggestive of active chloride reabsorption in the distal tubule have been obtained in sodium sulfate- infused rats (19). Also, steady-state concentrations in raffinose droplets not compatible with the present concepts of electrolyte electrochemical equilibrium across the proximal tubular epithelium were found in rats submitted to respiratory acidosis (6). The increase in proximal tubular chloride concentrations above plasma values observed in normal rats has been explained in terms of a preferential reabsorption of bicarbonate along this tubular segment (7, 23). Furthermore, a reciprocal relationship has been demon- strated between plasma levels and excretion rates of chloride and bicarbonate in conditions of metabolic alkalosis and acidosis (Zl), and during respiratory alterations of acid-base balance (9, 17, 20). These findings indicate a close relationship between chloride and bicarbonate excretion and reabsorption.

The present study was undertaken to investigate the characteristics of chloride excretion in situations of metabolic alterations of acid-base balance, including infusion of a poorly reabsorbable anion, sulfate, and carbonic anhydrase inhibition. Chloride concentration gradients as well as electrical potential differences were measured along the rat nephron, and fractional reabsorption of this ion was calculated from the simultaneously measured inulin concentration ratios.

METHODS

Male albino rats weighing 250-400 g were used and maintained on a standard laboratory diet containing approximately 1.5 % chloride. One group of rats was maintained on a diet containing 4 % NH&l and on 75 mEq/liter NH&I as drinking water for 3-5 days prior to the experiments. The rats were anesthetized with 30-40 mg of sodium pentobar- bital per kilogram body weight. Control rats received an infusion of isotonic saline at 0.05 ml/min; the group with acutely induced metabolic alkalosis received 5 % NaHC03 at a rate of 0.1 ml/min beginning 1 hr before the collections; the animals with chronic metabolic acidosis received isotonic NH&l at a rate of 0.05 ml/min; another group received 0.4 M NazS04 at 0.1 ml/min, and the last group received a prime of 15 mg/kg acetazolamide (Diamox) and an infusion of 15 mg/kg per hr of this drug in isotonic NaHC03 at a rate of 0.1 ml/min. ,411 animals also received a prime and constant infusion of adequate amounts of inulin to maintain a plasma level of 50-100 mg per 100 ml.

The left kidney was exposed by abdominal incision with a lateral extension, and free-flow collections and localiza- tions of the puncture site were made as described previously

(12) Tianstubular electrical potential differences were meas-

ured on separate groups of rats under similar experimental conditions, by means of glass microelectrodes (12).

Chloride was measured by the microcoulometric method of Ramsay et al. (18) and Windhager and Giebisch (25). Inulin was determined by a modification of the anthrone method (4, 14). pH in blood and urine was measured’ by means of a Metrohm model E 322 compensator, with model EA 520 capillary glass electrode. Plasma CO2 was measured by a manometric Van Slyke apparatus. Blood samples for pH and CO2 measurement were collected from the abdominal aorta at the end of the experiment.

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CHLORIDE EXCRETION AND ACID-B,4SE EQUILIBRIUM 21

The influence of Donnan equilibrium and plasma protein concentrations was evaluated experimentally by ultrafil- tration through cellophane, a mean ultrafiltrate/plasma ratio for chloride of 1.06 ZII 0.009 being obtained in six measurements.

TABLE 2. Free-+I co&ctions in rats with 5% NuHCO3 infusion

LOG % GFR,

ml/kg- min

Inub ChIoride, mEq/liter

Since frequently a different correction coefficient is used, based on a Donnan factor and correction for plasma pro- teins, without allowing for chloride binding to this protein, we chose not to correct our values by this factor. Thus, the equilibrium situation for TF/P values is not 1.00, but 1.06 according to our measurements, or 1.12 according to the conventional calculation ( 19).

U/P TF U TF/P

1 2.56 2.56 2.48

12.3 101.2 23.1 1 .l3 12.3 91.4 23.1 1.06

2 4.73 3.40 2.52 2.52

1.80

2.59 1.89

15.9 106.0 6.4 1 l 12 9.7 100 .o 4.1 1 l 09 9.7 97.5 3.4 1.05 9.7 86.4 3.4 0.94

3 RESULTS

4.55 4.55 2.92

1.23

1.81

10.4 102.0 39.5 1 .09 10*4 104.0 39.5 1.14

8.5 93.5 27.0 1.05

A summary of mean values of glomerular filtration rate (GFR), plasma chloride values, chloride/inulin clearance ratios, blood and urine pH, as well as plasma total CO2 observed in the different experimental groups is presented in Table 1. The respective standard errors are also given.

4

P 52 P 32

P 30 a 50 P 59 P 16

P7 P 62 P 58

P 38

P 36 P 60 P 24

4*55 7.9 104.5 27.0 1.09

5 6.43 1.05 6.43 1.25 6.08 1.19

13.7 109.6 37.5 1.08 13.7 113.7 37,5 1.11 9.1 110.5 23.0 1.09

Data concerning chloride and inulin concentrations along the nephron in control animals, previously published in detail (11, 14), are summarized in Tables 6 and 7, and the range of chloride TF/P as well as U/P ratios with their distribution along the nephron is shown in Fig. 1. Proximal chloride concentrations are considerably higher than the plasma values (mean TF]P = 1.29), also when corrected for Donnan equilibrium and plasma water, when a ratio of 1.06 would be expected. In the distal tubule, on the other hand, TF/P ratios are considerably lower than unity. These data show a distribution similar to that observed by other authors (10, 23, 25). Data for rats undergoing 5 % NaHC03 infusion are shown in Tables 2 and 6. Chloride TF/P ratios along the nephron are shown in Fig. 1, where it can be noted that in both proximal and distal tubules the distribution is distinctly lower than that of normal rats. These data, as well as those of the other experimental groups, have a distribution which is not significantly different from the normal, as verified by the Kolmogorov-Smirnov adherence test (22). Differences between groups could therefore be analyzed by the f test at a significance level of 0.05. This significance level was corrected for repeated comparisons involving the same group of data (e.g., several comparisons with the same control group), using cr/N, where a is the

Mean & SE

1

1.08 0.014

D 73 D 64 D 92

D 23 D 22

D 22 D 26 D 95 D 18

D 27 D 20 D 38

D 25 D 44 D 84

2.68 6.85 2.68 5.10 2.68 7.98

18.2 31.8 3-7 0.33 18.2 19.6 3.7 0*21 18.2 5.8 3.7 0.06

2 4.73 3.40

2.84 2.44

15.9 12.4 6.4 0.13 9.7 4.4 4.1 0.05

3 4.05 4.05 2.92 2.92

3.13 2.95 3.58 3.03

13.7 37.5 50.0 0.38 13.7 22.0 50.0 0.23 8.5 26.0 27.0 0.29 8.5 15.0 27.0 0.17

4 5.22 2.24 4.55 2.51 4.55 3.30

8.1 17.5 42.0 0.18 7.9 14.5 27.0 0.15 7.9 9.5 27.0 0.10

5 5.72 2.61 5.72 2.57 6.08 4.02

9.0 30,o 0.09 19.4 30.0 0.19 31.6 23.0 0.31

Mean 4.13 &SE 0.25

23.2 2.8

0.19 0.026

P = proximal, D = distal.

chosen level of significance and N the number or compare- sons made.

TABLE 1. Summary of experimental conditions of the studied groups -

It was shown in this way that both the mean TF/P chloride ratios of proximal tubule (1.08) and distal tubule (0.19) in NaHCOa-infused rats are significantly lower than control values. Chloride U/P ratios are within the range of control values, despite the presence of an osmotic diuresis, as indicated by an inulin U/P ratio of 11.3 shown in Table 1. This leads to an increased fractional chloride excretion of 2.5 % of the filtered load, compared to a value of 0.11 % in control animals.

Control

NaHC03, 5%

NH&l

NazS04, 0.4 M

Diamox

GfgR”n’/ .

4.22 106.3 0.11 7.36 6.28 20.9 1tO.27 *0*98 ho.034 ztO.015 d10.061 ztl l 20

4.13 94.9 2.49 7.71 8.05 46.8 zto.25 rtO.80 ho.46 ztO.046 ztO.027 zfzl.52

4.16 121,8 2.08 7.04 6.02 9.9 It0.17 rtO.51 &0,061 ho.049 M.36

3.60 96.1 0.31 7.28 5.75 20.0 zko.29 ho.57 ztO.027 ho.032 kO.082 zt1.71

4.12 97.6 0.29 7.31 7.92 27.5 ZkO.21 ho.60 zto.030 zko.031 5+0.024 zt1.54

The experimental group with chronic NH&l acidosis (Table 3) showed a mean proximal chloride TF/P ratio of 1.24, very similar to that obtained in control animals. It should be noted, however, that these rats had a high plasma chloride (see Table 1), and the tubular fluid concentrations indicate that bicarbonate must be almost totally absent from this fluid, chloride being practically the only anion at

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22 MALNIC, MELLO AIRES, AND LACAZ VTETRA

TABLE 3. Free-flow collections in rats with NH&l acidosis PROXIMAL DISTAL U

I - A - * I t - - - ’ I - Cl. 20 40 60 80 “16 20 40 60 80 %

Inulin I

ChIoride, mEq/liter TF/P T s.0

I.5 r Rat GFR,

ml/kg* min TF/P U/P TF U TF/P

. 6

7

8

9

Mean & SE

6

7

P 50 P 20 P 35 P 38

P 25 P 55

P 42 P 60 P 35 P 31

P 50

4.60 4.60

3*75 3.75 3.67 3,67

D 22

D 28 D 20

D 43 D 22 D 28

4.60 4.60

9

Mean 4J6 =t: SE 0.17

143 148 147 144

267 267

96 96 82 82

160 162

149 145 157 152

149

319 319

288 288 272 272

295

79.5

267 26.3 319 267 30 l 4 319

39.7 295 31.8 295 38.7 295

l48,2 298.0 5.03

1.36 1 l 30 0.’

I .24 I.21 1.31 1.27

3.49 1 l 45 1.60 1 l 34

1 l 30 1.82

1.31 2.13 1.20 1.16

I .54

8.35 4.80

11 .4 8.65 3.55

NH4 CL ACIDOSIS

0 0

ds 0

FIG. 2. Chloride TF/P ratios along nephron in rats with NH&L 1.22 acidosis.

I .24 TABLE 4. Free-joru collections in 0.4 M N&04-infused rats 0.019 I

I Win ChIoride, mEq/liter GFR, ml/kg-

mia LOG % 0.65

0.22 0.25

0.32 0.26 0.31

0*34 0,065

;Rat

10

I1

lr)

* , - TFP w/p TF u TF/P

P 35 P 51 P 26

P 52 P 65 P6

121.4 24.7 1.23 132 14.1 1.35 125 4.9 1.27

2.70

2.90

4.18 3.70 3.12

5.80 3.65

2 -39 2.39

2.56 2.14 1.38

1.75 1.98 1.23

1,62

9.86

8.70

7.55 7.78 6.50

16.2 9.35

4.40 4.40

124 3.0 1.22 120 2.6 1.22 112 2.1 1.14

-- P = proxima1, D = distal.

P 50 P 49 P

104 1.8 1.15 109 1.7 1.21 II6 2.2 I .27

PROXIMAL DISTAL I 1 # 13

20 40 60 80% 12-l

20 40 642 80 %

CL

TF/p 14

P 49 P 65

P 57 P 25

116 2.5 1.20 116 4.3 1.21

I .76 111 1.2 1 J6 104 1.2 1 l lO

Mean I l 21 zt SE 0.018

10 17.8 24.7 0.18 18.6 14.1 0.19 10.1 4.9 0.10

11

D 45 D 27 D 24

D 40 D 19 D 93

D 33

D 46 D 36 D 36 D 49

D 78 D 57

2.70

5 % Na NO3 2.90

4.48 2.53 5.85

9.86

8.70

7.55

9.2 28.2 11.7

3.0

2*1

0.09 0*28 0.12

CONTROL RANGE

4.18 6.2 1.8 0.07

FTG. I. Chloride TF/P ratios along nephron in rats with 5ya NaHC03 infusion. Shaded area is control range,

5-80 3.32 16.2 10.0 2-5 0.10 5.80 4.93 16.2 11.2 2.5 0.12 3.65 2.58 9.35 10.7 4*3 0.11 3.00 3.50 7.35 9.0 2.5 O*lO

this site. Distal chloride ratios are also within the range of 14 2.39 4.40 4.6 1.2 0.05

19.9 1.6 0.21 control values. Final urine chloride concentrations, on the other hand, are considerably higher than those found in Mean 3.60 9.08 5,2E 0.13

normal rats, leading to a higher fractional excretion rate. * SE 0.29 0.93 1.3: 0;018

In rats infused with 0.4 M sodium sulfate the reduction of proximal chloride TF/P ratios was not significant, whereas

p = proximaf D > = distal.

in the distal tubule considerably lower ratios were found, a mean proximal chloride TF/P ratio of 1.09, significantly the lowest ratios occurring in final urine (Tables 4 and 6). lower than controls. It is interesting to note that these ratios,

The last group, undergoing acetazolamide infusion, show as well as those from the bicarbonate-infused group, are

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CHLORIDE EXCRETION AND ACID-BA4SE EQUILIBRIUM 23

PROXIMAL DISTAL U

’ 20% 40% 80% 80 % ’ ’ 20 40% 80% aok ’ - aceta~olumide infusion

Rat

J

LQG %

Cl

TF /P

- - Inulin Chloride, mEq/liter GFR,

ml/kg* min TF/P u/p TF U TF/P

17 P 55 2.32 1.63 29.3 111 9.2 1.09 P 16 2.32 1 l 49 29.3 106 9.3 1 l 04 P 60 3.08 2.01 37.6 101 5.2 1 l 03 P 3.08 2.42 37.6 101 5.2 1 l Ol

18 1 l 29 23.4 109 12.8 I .08 1.79 23.4 109 12.8 1 JO

19 5.54 I .85 30.4 118 10.2 1.18 5.54 1.73 30 l 4 113 10.2 1.13 5.00 1.67 30.5 108 8.1 1 l lO

20 4.08 1.41 27.3 119 9.1 1.23 4.08 I .58 27.3 107 9*1 I .12 4.32 1.97 23.5 102 8.0 1 l lO 4.32 I l 54 23.5 102 8.0 1 .lO

21

P 41 P 65

P 46 P 55 P 50

P 50 P 43 P 45 P 26

P 12 P 18 P 16

4.06 I l 13 25.7 106 5.1 1 .I3 4.68 1.48 19.8 100 3.0 1.05 4.68 1 l 91 19.8 93 3.0 1 l Ol

Mean zt SE

17

1.09 0.015

2.32 14.0 29.3 15.2 9.3 0.15 3.08 12.0 37.6 23.1 5.2 0.23

18

3.30

16.7 23,4 3.8 12.8 0.04 He8 23.4 3,l 12.8 0 -03 4.12 17.0 20.6 5.5 0.21

19 5.54 5.07 30.4 11.5 10.2 0.12 5.60 9.96 34.6 18.6 8,4 0.19 5.60 5.37 34.6 30.2 8.4 0.31

20

21

D 80 D 30

D 47 D 30 D 25

D 80 D 83 D 37

D 81 D 77

D 51 D 40 D 30

4.08 4.48

4.20 10.5

4.13 3.74

27.3 9*5 9.1 0.10 27.7 27.2 9.2 0.29

4.06 4.06 3.70

25.7 4.3 5.1 0.05 25.7 23.8 5.1 0.25 18.9 15 3 3,l 0.17

Mean 4.12 27 l 4 8.02 0.16 + SE 0.21 1.03 0.55 0.026

I.5 1.0 I

0 0 0

0

ao 0 ro 0

---II- ------------1-------____1_3__________

0,5 *

0 0.4M Na2 SO4

0 0

0 0 O 0

0 s-0, 0

FIG. 3. Chlaride TF/P ratios in rats with 0,4 M Na&Or infusion.

PROXIMAL DISTAL U b.-

20 % 40% 60% 80% ’ I 20% 40% 60% 80% ’ -

Cl TF

/P

0

0

0 $0 0 Q 0

0 - - - -1 - - - - - - - - - - - -111- - - - - - - - - 1 - - -

DtAMOX

FIG. 4. Chloride TF/P ratios in rats with Diamox infusion.

The distribution of chloride TF/P values along the nephron is shown on Figs. 2, 3, and 4 for NH&l acidosis, sulfate infusion, and Diamox infusion. No definite variation of these ratios along any single nephron segment was de- tected. In the proximal tubule, values below plasma or the ultrafiltrate/plasma ratio are only seldom found, whereas in the distal tubule these ratios were consistently lower than plasma in all groups studied. Inulin TF/P ratios are summarized in Table 7, where averages of proximal values corresponding to locations between 40 and 65 % of tubular length are compared. In the mean corresponding to rats with bicarbonate infusion, five points from three rats not shown in Table 2 are included, since only inulin determina- tions were made. Only the group with Diamox infusion shows values that are significantly lower than controls (P < 0.05). In the distal tubule, on the other hand, Diamox- infused and acidotic rats show ratios within the %range of controls, whereas animals infused with hypertonic bicarbonate and sulfate present much lower values. These two groups also show the lowest inulin U/P ratios due to the osmotic diuresis induced by their infusion.

Distal transtubular potential differences were measured

P = proximal, D = distal.

TABLE 6. Mean chloride TF/P and U/P ratios

Control NaHCO3,

5% NH&I Na&&

0.4 M

Diamox

I .29zto.o37 12 1.08dzO.014 13

1.24*0.019 I.1 1.2ktO.018 13

1 LO9rkO.015 16

0.3OrtO.032 15 0.29+0.103 I1 0.19ztO.026 15 0.26ztO.045 13

0.34ztO.065 6 2.41ztO+O62 4 0.13ztO.018 13 0.051~0.018 14

0.16&0.026 13 0.076+0.008 13

very similar to the ultrafiltrate/plasma ratio of 1.06 to which reference was made. In this group, also in the distal tubule and in final urine, significantIy lowered TF/P ratios of chloride are found (Table 5).

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24 MALNIC, MELLO AIRES, AND LACAZ VIEIRA

TABLE 7. Mean inulin TF/P and u/P ratios

Proximal* Distal Urine

Control 2.4lztO.350 (7) 8.64zk1.51 (14)201.2H8.8 (12) NaHC03,5a/o 1.80+0.146 (8) 3.68~kO.44 (15) II .3&0.90 (13) NH&21 2.06ztO.383 (5) 7.35h1.41 (5) 148.2 (3) Na$SOd, 0.4 M 1.97&O. 140 (6) 3.88+0.47 (7) 9.08+0.93 (10) Diamox 1.69ztO.073 (10) 8.46ztl.32 (12) 27.4&l .03 (13)

Numbers in parentheses are number of observations. *‘Be- tween 40 and 65y0.

TABLE 8. Distal transtubu~ar jmtential dzxerences

Differences, mv

Control -49.0&l .95 (28) NaHCOs, 570 -54.0zt2.37 (21) NH&l -55.0zt2.76 (20) Na2S04, 0.4 M -51.0zt1.70 (43) Diamox -56.7&l .91 (35)

Numbers in parentheses are number of observations.

acetazolamide infusion, both situations known to increase the bicarbonate concentration of tubular fluid considerably (8). The absence of a variation of these ratios along both proximal and distal tubule indicates that the concentrations measured in the tubular lumen are steady-state concentrations. In the presence of a transtubular potential difference (PD) of about -20 mv, chloride concentrations considerably below plasma level would be expected in electrochemical equilibrium. If this PD should be an artifact, as sug- gested by Froemter and Hegel (Z), the TF/P ratios observed in the groups With bicarbonate and acetazolamide infusion would be near equilibrium. The same would be the case for the observations of Kashgarian et al. (5) in sta- tionary microperfusions with isotonic raffinose. In our re- maining groups, however, the observed concentration ratios, significantly greater than 1.06, are still removed from equilibrium.

Even in conditions of maximal bicarbonate or sulfate availability, chloride concentrations do not fall below plasma levels. On the other hand, as can be seen in Table 3, maximal proximal chloride concentrations are found in rats with chronic metabolic acidosis and NH&l infusion.

with glass microelectrodes, and mean values with their standard errors are shown in Table 8,

DISCUSSTON

Our data permit an analysis of net chloride reabsorption along the different nephron segments in the investigated experimental situations. The fractional reabsorption of filtered chloride in these segments, based on chloride and inulin concentrations, are shown in Table 9. All experimental groups show a reduced reabsorption of chloride along the proximal tubule with respect to controls. This reduction is maximal in acetazolamide-infused rats. In several situations the presence of osmotic diuresis may con- tribute to this observation; in the case of acetazolamide a direct action of the drug on NaCl transport at this site may be responsible. This is also indicated by a significantly reduced mean inulin TF/P ratio at the end of the proximal tubule in this group,

The maintenance of a steady-state situation removed from electrochemical equilibrium in the proximal tubule under some of the present experimental conditions could be explained in several ways, In the first place, it is possible that high luminal concentrations are maintained by faster NaHC03 than NaCl reabsorption where an equalization of chloride concentrations is prevented by volume reduction of tubular fluid (7). In this case, the flow of water into the interstitium should be faster than the diffusional flow of chloride in the absence of a measurable concentration gradient. In other words, chloride reabsorption at the same rate of water reabsorption would only be possible if assisted by a concentration gradient of approximately 1.2 to 1. In conditions of bicarbonate or Diamox infusion, water flow itself would be slowed or Cl permeability increased suffi-

TABLE 9. Fractional chloride reabsor@n along nephron

It is interesting to note that the reduced proximal reabsorption is offset in all cases by an increased reabsorption in Henle’s loop, including the straight portion of the proximal tubule. This segment or the ascending limb could be responsible for the enhanced reabsorption.

Percent Reabsorbed

Group I I I I

/ Prox” 1 Loopt 1 Dist I”“‘h”,‘z;g Urine

A portion of proximal NaCl reabsorption thus appears to be linked to carbonic anhydrase; it is possible that this reabsorption mechanism is absent in more distal segments, leading to compensation of decreased proximal reabsorption. However, direct action of acetazolamide on NaCl transport independent of its effect on carbonic anhydrase cannot be ruled out.

--qpP

Control 49.4 43.5 5.7 1.26 NaHCOa, 570 43.2 50.4 3.3 1 *lo NH&l 43.1 51.9 3-6 3.6 NazSOd, 0.4 M 42.0 53.1 2.7 1.67 Diamox 39*1 57.2 2.1 1.44

99.86 97.90 98.50 99.47 99.74

* Up to 65y0 proximal length. of proximal tubule.

TIncIuding straight portion

Distal reabsorption of chloride also appears reduced in several experimental groups, but this may be due to a reduction of the chloride load of this tubular segment.

TABLE 10. Comparison of distal TF/P ratios for Gl calculated by Nernst equation with mean exfierimental values

It had been shown previously (11, 14) that chloride TF/P concentration ratios along the proximal tubule are not changed during the infusion of fursemide, a diuretic that inhibits NaCl transport at this site, nor during 4 % NaCl infusion. In the present series of experiments, a definite reduction of this ratio was seen in rats with bicarbonate and

Group Calculated TF/P Observed TF/P

Control 0*17 0.30 NaHCOx, 5yo 0.14 0.19 NH&l 0.13 0.34 NazSOd, 0.4 M 0.16 0.13 Diamox 0.13 0.16

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CHLORIDE EXCRETION AND ACID-BASE EQUILIBRIUM 25

ciently to permit Cl diffusion with a minimal gradient. The erably higher than those found by Rector and Clapp (19) establishment of a chloride diffusion gradient by this mech- under similar experimental conditions. The cause for this anism could actually represent an additional force leading difference is not clear. to Na reabsorption in the proximal tubule, the passive flow In final urine, both in sulfate- and acetazolamide-infused of chloride driving that of sodium by a frictional ionic in- rats, very low chloride concentrations are found, indicating teraction. However, if volume changes should not be ade- that along the collecting duct, in the presence of a trans- quate to explain the maintenance of these concentrations, tubular potential difference of the order of only - 15 mv as may be the case in zero net volume flow experiments of (24), an active reabsorption of this ion must probably be Kashgarian et al. (6), other mechanisms would have to be invoked. invoked. Some evidence for a nonpassive behavior of chlo- ln summary, the present findings indicate that chloride ride in proximal tubular epithelium is available (1, 3, 15). concentrations along the nephron depend on acid-base A greater unidirectional flow of chloride into the tubular equilibrium, most probably due to a reciprocal relationship lumen than in the opposite direction may be responsible for with bicarbonate reabsorption. Thus, conditions in which the observed concentration levels; it could be due either to bicarbonate reabsorption is maximal lead to maximal tubu- enhanced passive flow of Cl due to ionic interaction with lar chloride concentrations, as was observed in metabolic the flow of Na or H in the same direction, or to an active acidosis. Whenever bicarbonate reabsorption is absolutely transport of chloride. The present data, however, do not or relatively impaired, Cl concentrations are significantly give further insight into this problem. lower, but never fall significantly below plasma levels in the

Chloride concentrations in the distal tubule are similar proximal tubule. In the distal tubule, in the latter condi- to distal sodium values (13, 25). Mean chloride TF/P ratios tions and during sulfate diuresis, significantly reduced Cl were significantly slower than controls in NaHCOz-, concentrations are found. These data seem to indicate a NasSO4- and acetazolamide-infused rats. In Table 10 a lower permeability for both bicarbonate and sulfate than comparison between these ratios and those calculated ac- for chloride along the nephron, especially in the distal cording to the Nernst equation are shown. In most cases tubule. They could imply that chloride levels along the the observed ratios are higher than the calculated ones. nephron depend on the relative rate of reabsorption of this Only in sulfate-infused rats was the observed ratio some- anion with respect to the other anions present in the tubular what lower, but the difference is too small to be of signifi- 1l;men. However, an alternate explanation involving cou- cance. A few potential differences as high as -70 mv are pling between chloride and Na or H movement into the found in this group, corresponding to TF/P ratios of 0.07, lumen or an active component of chloride transfer cannot and only occasional points with lower Cl concentration are be excluded. found in Table 4, indicating that it is not necessary to as- sume the existence of active chloride reabsorption in this experimental group unless the chloride permeability of this

This study was supported by grants from Funda@o de Amparo a

tubular segment should be too low to permit reabsorption Pesquisa do Estado de SZo Paulo.

at the observed rate. Our distal concentrations are consid- Received for publication 3 January 1969.

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