Effect of acute and chronic glutathione depletion on renal function in the rat

Camp.

Pergamon 0742-8413(95)00042-9

Effect of acute and chronic renal function in the rat Sandra

L. Garber,

Eiochem.

Physiol. Vol. IIIC. No. 2, pp. 237-241. 1995 Copyright 0 1995 Elsevier Science Ltd Printed in Great Britain. All rights reserved 0742.8413195 $9.50+0.00

glutathione

Jose A. L. Arruda

depletion

on

and George Dunea

Section of Nephrology, Cook County Hospital, The Hektoen Institute for Medical Research, The University of Illinois at Chicago and the WSVAMC, Chicago, IL 60612, U.S.A. Renal function was evaluated in normal and acid-loaded rats following acute and chronic depletion of glutathione (GSH) by buthionine sulfoximine (BSO). Creatinine clearance and fractional excretion of electrolytes were normal. There was no acidification or concentration defect detected in animals with acute or chronic GSH depletion. Key words: Glutathione; Comp. Biochem.

Physiol.

GSH; IIIC,

Urinary 237-241,

acidification;

Rat; BSO; Kidney;

Renal

function.

1995.

Introduction The tri-peptide glutathione (GSH) is an electron scavenger that protects cell membranes against oxidative damage by free radicals, superoxides, and peroxides. It may also help maintain renal function and structure. Indeed, some patients with hereditary GSH synthetase deficiency also have a metabolic acidosis, the mechanism of which has not been studied (Prchal et al., 1983). In the laboratory, the effect of GSH depletion on renal function has received much attention (Torres et al., 1986, 1987, 1989; Rodriguez et al., 1991). In the rat, acute GSH depletion was found to decrease GFR; increase Na +, K + and water excretion; decrease urinary concentrating ability; and cause a moderate acidosis interpreted as consistent with a distal tubular acidification defect (Rodriguez et al., 1991). In these studies, acute GSH depletion was achieved by using diethyl maleate (DEM). It is difficult, however, to determine the exact cause of such impaired urinary acidification because DEM, a non-specific depletor of GSH, is known to itself

Correspondence to: Sandra L. Garber. Ph.D.. Section of Nephrology, Cook County Hospital, 1835 W. Harrison Street, Chicago, IL 60612, U.S.A. Tel. (312), 633-7267: Fax (312) 633-8471. Received 19 September 1994; revised 24 January 1995; accepted 31 January 1995. 237

induce a Fanconi syndrome as well as having other non-specific effects resulting from an alteration in high energy phosphate intermediates (Pacanis et al., 1975). The GSH depletor buthionine sulfoximine (BSO) would seem to be a better agent for lowering GSH because it acts specifically on GSH synthesis (Meister, 1988). Acute GSH depletion induced by BSO in rats has been shown not to cause changes in urinary volume or GFR (Scaduto et al., 1988). However, the effect of acute or chronic GSH depletion induced by BSO on urinary acidification has not been studied. Because of this, we have investigated the role of BSO induced GSH depletion on urinary acidification and renal function in the rat.

Material and Methods Male weighing Sprague-Dawley rats, 200-250 g, were used in all the experiments. They were maintained on a 12 : 12 hr light : dark cycle and received food and water ad libitum, except as noted below. One day before ending the experiment, the rats were placed in metabolism cages and balance studies conducted as previously reported (Garber et al., 1995) except that urine was collected for 2 hr. Following this, the rats were anesthetized with sodium

238

S. L. Garber

pentobarbital (50 mg/kg ip). A blood sample was obtained from the inferior vena cava and the kidneys rapidly removed, decapsulated, and weighed. Protocol 1. The efSect of acute and chronic GSH depletion on acid excretion and renal,function in non-acidotic animals Group I-Control (N = 8). Animals were injected intraperitoneally (ip) with phosphate buffered saline (PBS) 5 ml/kg body weight. Group 2-Acute GSH depletion (‘N = 12). Animals received a single injection ip of BSO, 2 mmol/kg body wt as a 5 ml/kg solution in PBS and were sacrificed 2 hr later. Group 3-Chronic GSH depletion (‘N = 10). On day 0, rats were injected with BSO (dosage as above). Drinking water was replaced with a solution of 20 mmoi BSO for the next 8 days. A modification of this regimen has shown this to be an adequate model for GSH depletion in mice (Griffith and Meister, 1985). On day 7, rats were given a second injection of BSO as above and then placed in metabolic cages overnight. The next day, fluids were removed and a 2 hr urine specimen was collected prior to sacrifice. Protocol depletion chloride

2. Effect of acute and chronic GSH on animals loaded with ammonium

In order to assess the combined effect of GSH depletion and acid loading, groups of animals were given 1.5% ammonium chloride as the sole drinking fluid for 4 days. Group I-Control (N = II). Ammonium chloride for 4 days and injection of PBS. Group Z-Acidosis + acute GSH depletion (N = 9). At the end of 4 days of ammonium chloride, rats were injected ip with 2 mmoi/kg BSO, and urine was collected for the next 2 hr. Group 3-Acidosis + chronic GSH depletion (‘N = 10). On day 0, animals were injected with BSO as above. Tap water was replaced with a 20 mmol solution of BSO. On day 4, 1.5% ammonium chloride was added to the BSO solution, and this regimen was continued until day 8. On day 7, animals received a second injection of BSO, and animal handling and urine collection were as above. Analytical methods. GSH levels in tissue homogenates were determined by the method of Ellman (1959) after deproteinization with 4% SSA. Plasma and urine osmolalities were measured with a vapor pressure osmometer (Wescor, Inc.). Blood and urine electrolytes, creatinine and phosphate were assayed by standard clinical methods. Ammonia was determined by the Berthelot reaction.

rr a/.

Titratable acidity was calculated by the Henderson-Hasselbalch equation using a pKa of 5.0 for creatinine and 6.8 for phosphate. Net acid excretion (NAE) was expressed as the sum of titratable acids plus ammonia (Arruda et al., 1979). Results were corrected for body weight where appropriate. Interactions within groups were analyzed by ANOVA. Where appropriate, the data were further analyzed by an independent t-test. The statistical package used was Minitab (State College, PA). A P value of less than or equal to 0.05 was considered statistically significant. Data are expressed as the mean f SE.

Results The results of the initial studies are presented in Table 1, which shows the effect of acute or chronic GSH depletion on urinary acidification in non-acid loaded animals. Acute depletion of GSH reduced the GSH content in the kidney by 51% and in the liver by 59% as compared with controls. These results are comparable with those reported by Meister et al. (1988). Although it is possible to induce a greater degree of GSH depletion, we chose not to do so because this leads to a decrease in renal function which affects urinary acidification. Table 1 shows that acute GSH depletion was not associated with a significant change in creatinine clearance or fractional excretion of NA, K and P as compared with controls. Also not different were urine pH, titratable acid, ammonia and net acid excretion (NAE) between control and acute GSH depleted animals. Urine osmolality and volume were also not significantly different. Thus acute depletion of GSH content in the kidney by 50% was not associated with any detectable change in renal function. We next evaluated the effect of chronic GSH depletion, induced for 8 days prior to urine collection. The inclusion of BSO in the drinking water did not significantly change normal fluid consumption. During this period fluid intake was 54 f 5 ml/24 hr, equivalent to 1.08 mmol of BSO, compared with 51 f 6 ml/24 hr in the control. This protocol resulted in a 30% depletion of GSH content in the kidney and 32% in the liver. Creatinine clearance, fractional excretion of Na, K and P, urine osmolality and pH again were not different between control and chronic GSH depleted animals. Ammonia excretion, however, was decreased by 48%, and titratable acidity was also slightly decreased. The results are shown on the left panel of Fig. 1. To further evaluate the effect of acute and chronic GSH depletion on urinary acidification

12.6 k 1.6 12.0 * 1.3 14.4 * 1.5

7.4 * I.1 12.6? f 1.2 6.9 + 0.9

*Corrected per IOOg body weight. tIndicates P 2 0.05 compared with control.

Control (N = II) Acute GSH depletion (N=9) Chronic GSH depletion (N = IO)

cl,“I p I/min

*Corrected per IOOg body weight. ?-Indicates P I 0.05 compared with control

Control (N = 8) Acute GSH depletion (N = 12) Chronic GSH depletion (N = 10)

u““I

pl/min

5.90 & 0.03 6.03? f 0.02 5.84 f 0.01

uPH

6.18 kO.12 6.08 + 0.06 6.21 + 0.09

uPH 0.50 + 0.05 0.51 i 0.08 0.21t & 0.04

1661 k 76 1288t k 76 I888 f 85

mOsm/kgHzO

“bl”

u 2.83 f 0.52 4.15 * 0.41 3.98 * 0.74

pmol/min*

NH,

Table 2. Effect of glutathione

740 + 72 764 * 69 1033 + 120

pmol/min*

NH,

1. Effect of elutathione

clowl mOsm/kgHzO

Table

0.25 $- 0.03 0.31 f 0.05 0.33 + 0.03

TA pmol/min*

depletion

0.30 t 0.05 0.32 * 0.04 0.26t f 0.04

3.08 f 0.53 4.46 * 0.40 4.30 + 0.75

NAE pmol/min*

Excretion

on renal function

0.80 k 0.08 0.83 i 0.1 I 0.47t * 0.07

NAE ~mol/min*

Excretion

on renal function

TA pmol/min*

depletion

0.52 * 0.09 0.57 f 0.06 0.77 * 0.09

CL ml/min*

in acidotic

0.79 + 0.12 0.65 f 0.09 0.72 * 0.09

CL ml/min*

in non-acidotic

rats

0.2 + 0.1 0.4.t + 0.1 0.2 * 0.1

FE,, %

0.5 +0.1 0.6 + 0.1 0.8 + 0.1

FE,, %

rats

15 *4 12 *2 10 *2

FE, %

24 +3 27 k6 25 +3

FE, %

25 +6 26 &6 19 *1

%

FE,

19 +3 20 21 17 &3

FE, %

Ug

GSH kidney

6.59 + 0.75 5.80 f 0.45 1.47t +0.12

GSH kidney

6.26 + 0.54 3.17t & 0.56 4.36t + 0.64

pglg

S. L. Garber et al.

240

6-

Without

Control

Fig. acid;

Acid Loaded

Acid

Acute GSH Chronic GSH Depletion Depletion

Control

Acute GSH Depletion

Chronic GSH Depletion

I. The effect of acute (left) and chronic (right) GSH depletion on urinary acidification. NH,:

ammonia

excretion;

NAE:

net acid

excretion. *P 5 0.05.

the animals were made chronically acidotic by adding ammonium chloride in the drinking water for 4 days before acute GSH depletion. To achieve a comparable degree of acidification in the chronic GSH depleted animals as compared with the control group, ammonium chloride was introduced on the last 4 days of the protocol. We have shown that the protocol of administration of ammonium chloride for 4 days results in steady state metabolic acidosis with plasma total CO, levels of 16mEq/l and. maximal increase in ammonia and net acid excretion by the end of the 4th day (Arruda et al., 1979). As can be seen in Table 2 and in the right panel of Fig. 1, ammonium chloride loaded control rats had a 5-fold increase in ammonia excretion and NAE compared with non-acid loaded animals (Table 1 and Fig. 1, left panel). As shown in Table 2 and the right panel of Fig. 1, GSH depletion did not interfere with the adaptive increase in NH, excretion or NAE chronically acidotic animals. There was no difference in creatinine clearance or the fractional excretion of K and P. The fractional excretion of Na was slightly increased.

Discussion Several investigators have shown that glutathione depletion, when achieved with a variety of chemical agents, exerts various effects on renal function. Of particular interest is the induction of a urinary acidification defect, because metabolic acidosis has been reported to

Results

are expressed

TA: titratable as the mean + SE.

occur in some patients with hereditary GSH synthetase deficiency. In the laboratory, acute depletion of GSH with diethyl malate (DEM) has been reported to cause a distal acidification defect (Rodriguez et al., 1991) but the findings are difficult to interpret because DEM itself may induce a proximal acidification defect. To resolve this issue, we induced a moderate GSH depletion with BSO, a reproducible model, and we clearly showed that acute and chronic GSH depletion by BSO is not associated with changes in creatinine clearance, which is well known to influence the rate of acid excretion. We deliberately chose to induce a moderate degree of GSH depletion because greater depletion is associated with changes in GFR that would lead to uninterpretable results. The results of our studies, summarized in Fig. 1, indicate that acute or chronic GSH depletion did not cause a defect in urinary acidification when acidification was maximally stimulated with ammonium chloride (right panel). In non-acid loaded animals, acute GSH depletion also did not alter urinary acidification, whereas chronic GSH depletion caused a small but significant decrease in acidification. It is unlikely that these changes are physiologically meaningful, since, in animals that were acidloaded, chronic GSH depletion did not decrease net acid excretion. It is noteworthy, however, that in chronically acidotic animals, BSO given for 8 days causes a significant increase in the degree of GSH depletion. The mechanism responsible for this difference is not clear but it could be related to the fact that mitochondrial

Renal

effect of GSH

metabolism is enhanced by chronic acidosis leading to an enhanced uptake of glutamine. It is also possible that chronic acidosis is associated with enhanced uptake of GSH by the mitochondria and consequently enhances oxidation of this compound. This enhanced oxidation coupled with inhibition of synthesis of GSH by BSO could then lead to the increased depletion seen in this group as compared with the non-acidotic animals (Griffith and Meister, 1985). In summary, our results do not support a major role for GSH in modulating either normal acid excretion or the adaptive increase in acid excretion seen during acidosis. We attribute the difference between our studies and previous studies to our using a specific inhibitory agent to cause GSH depletion instead of DEM, which has non-specific effects on renal function. It should be noted, however, that patients with GSH synthetase deficiency may have a depletion of GSH level to 10% of the control in the red blood cells (Prchal et al., 1983). Therefore, our results do not exclude the fact that greater degrees of GSH depletion may have an effect on urinary acidification. Such depletion, however, is associated with alterations in GFR, making it impossible to decide if such an effect is simply due to hemodynamic changes or to a direct effect of GSH on acidification. Acknok~ledgemenrs-The Mirochnik and Shekika assistance.

authors wish to thank Yelena Ramsey for their expert technical

depletion

241

References Arruda J. A. L., Sabatini S., Mehta P. K., Sodhi B. and Baranowski R. (1979) Functional characterization of drug-induced experimental papillary necrosis. Kid. Inr. 15, 2644215. Ellman G. L. (1959) Tissue sulfhydryl groups. Arch. Biochem. Biophys. 82, 70-77. Garber S. L., Salmassi, J., Arruda J. A. L. and Dunea G. (1995) Xanthopterin induced renal dysfunction: A mode1 of crystal nephropathy. Nephron 69, 71-78. Griffith 0. W. and Meister A. (1985) Origin and turnover of mitochondrial glutathione. Proc. natn. Acad. Sci. U.S.A. 82, 46684672. Meister A. (1988) Glutathione metabolism and its selective modification. J. biol. Chem. 263, 17,205S17,208. Pacanis A., Rogulski J.. Ledochowski H. and Aneielski S. (1975) On themechanism of maleate action on rat kidney mitochondria. Effect on substrate-level phosphorylation. Acta Biochim. Pal. 22, I-10. Prchal J. T., Crist W. M., Roper M. and Wellner V. P. (1983) Hemolytic anemia, recurrent metabolic acidosis, and incomplete albinism associated with glutathione synthetase deficiency. Blood 62, 754757. Rodriquez J. V., Torres A. M. and Elias M. (1991) Effect of glutathione depletion on urinary acidification in the rat. Biochem. Med. Met. Biol. 45, 3 IO-3 18. Scaduto R. C., Gattone V. H., Grotyohann L. W., Wertz J. and Martin L. F. (1988) Effect of an altered glutathione content on renal ischemic injury. Am. J. Physiol. 255, F91 llF921. Torres A. M., Rodriguez J. V. and Elias M. (1987) Urinary concentrating defect in glutathione-depleted rats. Can. J. Physiol. Pharmac. 65, 1461-1466. Torres A. M., Rodriguez J. V. and Elias M. (1989) Rat kidney function related to tissue glutathione levels. Effects of different glutathione depletors. Comp. Biochem. Physiol. 94C, 58 I-583. Torres A. M., Rodriguez J. V., Ochoa J. E. and Elias M. (1986)Rat kidney function related to tissue glutathione levels. Biochem. Pharmac. 35, 3355-3358.