Protective Effects of Glutathione, Glycine, Or Alanine in an in Vitro Model of Renal Anoxia1

Protective Effects of Glutathione, Glycine, or Alanine in an In Vitro Model of Renal Anoxia1

Mark S. PalIer2 and Marsha Paften

R ecent investigations into the pathobiobogy of is- MS. Paller, M. Patten, Department of Medicine, Uni- chemic renal cell injuny have evaluated the im- versity of Minnesota, Minneapolis, MN portance of cellular glutathione (GSH) in limiting (J. Am. Soc. Nephrol. 1992; 2:1338-1344) damage and of exogenous GSH on gbycine as thera- peutic maneuvers to limit the extent of ischemic injury. GSH has received attention because of its activity as an antioxidant agent. GSH acts directly as ABSTRACT a hydroxyb radical scavenger, and it is also a sub- Both glutathione and glycine provide some protec- strate for the hydropenoxide-metabolizing enzyme tion against ischemic renal injury in a variety of ex- glutathione penoxidase (1 ). During renal ischemia, perimental models. However, results have been in- GSH is oxidized as well as catabobized (2-5). Inhibi- consistent and there may also be model heteroge- tion of the GSH peroxidase system resulted in greater neity. The effects of glutathione, glycine, and alanine free radical-mediated lipid peroxidation and renal dysfunction after ischemia (4,6). Experimental ma- in a cell culture model of renal anoxia/reoxygena- neuvens to increase endogenous GSH stores, such as tion injury were tested. When primary cultures of rat hypothyroidism, renal artery stenosis, on infusion of proximal tubule epithelial cells were subjected to 60 GSH. provided significant protection against is- mm of anoxia and 30 mm of reoxygenation, glutathi- chemic injury, whereas intentional depletion of cel- one (2 mM) essentially eliminated lethal cell injury bular GSH stones exacerbated renal ischemic injury

as determined by lactate dehydrogenase release. (7-9). In the study by Scaduto et al. , GSH mono- Glycine or alanine, on the other hand, provided only ethylesten was not protective during ischemia: how- partial protection. Glutamate did not protect, al- ever, the GSH analog apparently had intrinsic neph- though cysteine did. The glutathione synthesis inhib- rotoxicity unlike GSH (10). The above-cited studies itor buthionine sulfoximine blocked the protective were performed with an in viva model of renal ischemia. Studies in freshly isolated rabbit proximal tu- effect of exogenous glutathione, and the glutathione bube suspensions have confirmed the beneficial ef- transport inhibitor probenecid partially blocked glu- fect of GSH to protect against oxidant injury (1 1 ), as tathione protection. A combination of glycine, glu- well as against hypoxic or anoxic injury (5, 1 2), but tamate, plus cysteine also protected against anoxia/ have suggested a mechanism distinct from the an- reoxygenation injury. The studies suggest that both tioxidant effects of GSH in the latter studies. In this glutathione degradation with intracellular resyn- model, the beneficial effects of GSH could be repro- thesis and transport of intact glutathione into the cell duced by the administration of glycine, one of the are involved in the protection afforded by exoge- constituent amino acids of the tnipeptide GSH (12- nous glutathione. These results are different from 1 5). Exogenous GSH was metabolized to glycine, and those obtained in other experimental models of renal it was the glycine that apparently provided protection ischemia, such as freshly isolated proximal tubules, ( 1 3, 1 4). Protection by glycine was independent of tubule cell ATP bevel and may have been due to an because the protective effects of glutathione were effect independent of glycine metabolism ( 1 2, 1 3). Al- not derived solely from glycine generation. These anine was also found to protect anoxic proximal tu- studies also suggest the need for caution in extra- bules (16). Glycine and alanine have also been dem- polating results from one model of renal anoxic injury onstrated to protect the isolated perfused kidney to another. against the tubular injury that is characteristic of Key Words: Renal epithellal cells. antioxidant. alanine. buth- that preparation (17). Baines et at. suggested that ionine sulfoximine. probenecid amino acids such as alanine and glycine might protect by virtue of physical effects on protein tertiary structure (17). I Received August 7. 1991. Accepted October 30. 1991. 2 correspondence to Dr. M. S. Poller. University of Minnesota. Box 736 UMHC, There are currently five favored models for study- Minneapolis, MN 55455. ing ischemic renal injury: whole animal studies em- 1046-6673/0208- 1338$03.OO/0 pboying (1 ) renal artery occlusion or (2) intrarenal Journal of the American Society of Nephrology Copyright © 1992 by the American Society of Nephrology norepinephrine infusion, (3) the isolated perfused

I 338 Volume 2 - Number 8 ‘ 1992 Paller and Patten

kidney, (4) freshly isolated proximal tubule segments and in homogenized cells (Teflon-glass motorized subjected to anoxia or hypoxia and reoxygenation, mortar and pestle) by monitoring the reduction of and (5) cultured renal epitheliab cells subjected to nicotinamide adenine dinucleotide (NAD) in the pres- anoxia and reoxygenation. The ability of GSH, gly- ence of lactate at 339 nm. The two values were cine, or abanine to protect against anoxic injury has summed to obtain total LDH. not been reported in the cultured cell model. We, To test the protective effects of GSH or glycine, therefore, undertook these studies to compare the these substances were added to the incubation media results with those of the other in vitro models. to obtain a final concentration of 2 mM 2 h before the cells were exposed to 60 mm of anoxia and 30 mm of reoxygenation. In other studies, the ability of METHODS 2 mM L-alanine to protect cells against anoxia and Cell Culture reoxygenation was tested. In addition, groups of con- Rat renal proximal tubule segments were isolated tnol cells subjected to incubation in 95% ain-5% CO2 rather than to anoxia and reoxygenation for 90 mm by the method of Gesek et at. , which employs colla- genase digestion of the renal cortex followed by Per- were exposed to either gbycine or GSH. cobb density gradient centnifugation (18). This proce- Studies to investigate the mechanism of the protec- dure yielded a preparation consisting primarily of tive effect of GSH were also performed. The efficacy proximal tubule fragments (>95%) with approxi- of the amino acid components of GSH other than mately 90% viability by vital dye exclusion. Culture gbycine (glutamate and cystemne) was tested individ- medium was RPMI 1 640, which contains amino acids ually and in combination and was compared with and vitamins, 1 1 mM glucose, 1 mM Ca(NO3)2, 5.4 that of the intact tnipeptide. In other studies, the mM KC1, 0.4 mM MgSO4, 103 mM NaCb, 5.6 mM effect of the inhibition of GSH synthesis was exam- Na2HPO4, 23.8 mM NaHCO3, 10 mM N-2-hydroxy- med by incubating cells in 1 0 mM buthionine sulfox- ethylpiperazine-N’-2-ethanesulfonic acid (HEPES), imine (Sigma) for 60 mm before the addition of GSH 10% fetal calf serum, 100 tU/mL of penicillin, and or the amino acid combination. The role of -y-gluta- 1 00 cg/mL of streptomycin to which 1 0 ng/mL of myltranspeptidase in mediating extracellular GSH epidermal growth factor, 5 g/mL of transfennin, 5 degradation before amino acid uptake and resyn- cg/mL of insulin, and i0 M dexamethasone (final thesis to GSH was tested by exposing cells to the y- concentrations) were added. Tubule fragments were glutamyltranspeptidase inhibitor acivicin (0.25 mM: suspended in culture medium and plated onto colla- Sigma) 20 mm before the addition of GSH. The robe of the uptake of intact GSH in protection was tested gen gel-coated (Type I; Sigma Chemical Co. , St. Louis, MO) plastic 12-multiwell (4.5 cm2) plates. Culture by treating the cells with the GSH transporter inhib- medium was changed every other day. Primary cub- itor probenecid (1 mM) S mm before GSH addition (2). tures were used for all studies. The proximal origin Because the primary endpoint for cell injury was of the cultured cells was supported by preliminany the release of cellular LDH, it was important to as- studies, which showed the expression of the proximal certain that GSH would not interfere with the assay. tubule brush border enzymes alkaline phosphatase Therefore, studies were performed to ascertain and ‘y-gbutamyltranspeptidase and the formation of whether the addition of 2 mM GSH would affect the domes, evidence of vectorial transport (19). measurement of LDH in samples. It was found that 2 mM GSH had no effect on the measurement of LDH. Data are reported as mean ± SE. Statistical com- Anoxic Cell Injury panisons between two groups were made by using an unpaired t test. Multiple group comparisons were Cells were studied in a subconfluent stage, 3 days made by using the Bonfenroni modification of the t after initial plating. Forty-eight hours before study, test. the culture medium was switched to a glucose-free formulation of the usual culture medium to enhance cell susceptibility to anoxia/reoxygenation injury. RESULTS Two hours before study, the medium was changed to In cultured proximal tubule epithelial cells not ex- a glucose-free medium without added growth factors. posed to anoxia, neither glycine nor GSH had an Cell plates were placed in an airtight glass chamber effect on LDH release, which was 5.5 ± 0.7% of total under a continuous flow of humidified gas and were cellular LDH in control cells (N = 8), 5.9 ± 1 . 1 % in

maintained at 37#{176}Cand cells were exposed to anoxia glycmne-treated cells (N = 7), and 6.7 ± 0.6% in GSH-

(95% N2-S% CO2) for 60 mm followed by reoxygena- treated cells (N = 7). On the other hand, GSH had a tion (95% 02-5% CO2) for 30 mm. Cell injury was strikingly protective effect against anoxic injury, re- quantitated as the percentage of total cellular lactate ducing LDH release to nonmoxic levels (Figure 1). dehydrogenase (LDH) released into the culture me- Glycmne had a modest, but statistically significant, dium. LDH was measured in the incubation medium protective effect against anoxic injury of cultured

Journal of the American Society of Nephrology I 339 Glutathione and Glycine in Renal Anoxia

C TABLE I . Comparison of the effects of GSH and glycine over a range of concentrations between C C 1.0 and 10 mM#{176} LDH release 40 I ii (% total) Specific LDH Treatment Release 30 (%)

C p<0.05 20 None 42.9 ± 8.3 GSH 1.0mM 4.1±3.4 10 2.0mM 2.3 ±5.8 5.0mM 0±1.7 10mM 6.4±3.5 Glycine Anoxia Anoxla Anoxia 1.0mM 17.1±4.7 + + 2.0mM 21.9±13.5 Glyclne GSH 5.0mM 8.9±6.1

Figure 1 . Effect of GSH (2 mM) or glycine (2 mM) on anoxia/ 10mM 17.9±9.2 reoxygenation injury as measured by the release of LDH in renal proximal tubule epithelial cells. a To facilitate comparison of cells derived from multiple batches, the results are expressed as specific LDH release. the fraction of cellular LDH released during anoxia/reoxygenation minus the amount released during normoxic incubation of cells from the same batch (8.2 ± I .0%). epithelial cells, reducing LDH release by 39%. Table At each concentration. GSH was more protective than was glycine (P < 0.05). 1 compares the effects of both GSH and glycmne in the protection against injury over a concentration range of 1 to 10 mM. Both agents were maximally effective in concentrations of S mM or less. At no DISCUSSION concentration did the effectiveness of glycmne equal that of GSH (all P values <0.05). Similarly, alanine The purpose of this study was to compare the effi- produced a significant degree of protection against cacy of GSH, glycmne, and alanine In an in vitro model injury by anoxia/reoxygenation in proximal tubule of renal ischemic injury rather than to Investigate epithelial cells (Figure 2). However, alanine, bike gly- the mechanisms of the protective effect. In vivo stud- cine, was only partially protective and was clearly ies have suggested that GSH acts by bolstering an- less protective than was GSH in this particular model tioxidant protective mechanisms (4,6,8,9). An alter- of renal ischemic injury. native explanation, that GSH merely supplies sub- The protective effects of GSH did not appear to be strate for glycmne generation, is not supported by this solely because of its acting as a source of its constit- study because glycine and alanine were bess effective uent amino acids. Glutamate, when provided singly than was GSH in providing protection against pos- in the same concentration, did not provide protection, tanoxic renal cell injury. unlike the intact tnipeptide (Table 2). Cystemne alone The protective effects of glycine in vitro have been did protect, although numerically less well than did extensively investigated, yet no clear-cut mechanism GSH or the combination of amino acids (Table 2). has been defined, despite a number of exciting pos- Intracellular GSH can derive from the synthesis of sibilities (1 1-17,20). The studies presented here in the peptide (the amino acids can be obtained from cultured renal epithebial cells demonstrate that only the extracellubar degradation of GSH by -y-glutamyl- a portion of the protective effect of GSH is a conse- transpeptidase) or by the transport of intact GSH. quence of its metabolism to glycine. The data shown Inhibition of ‘y-glutamyltranspeptidase with acivicin in Table 1 argue against this lesser effectiveness of did not block the protective effect of GSH (Table 3). glycmne as being because of the greater availability of On the other hand, inhibition of GSH synthesis with glycine when delivered to the cell as GSH, because buthionine sulfoximine blocked the protective effect even 1 0 mM glycine was less effective than was 1 of GSH (Table 4). GSH synthesis inhibition also mM GSH, a 1 0-fold difference in concentration. This blocked the protective effect of the combination of finding is strikingly different from the observations

amino acids (Table 2). Buthionine sulfoximine, even of isolated proximal tubules by Weinberg et at. , who in this relatively high concentration, did not affect noted equivalent protection by GSH and glycmne in basal on anoxia/reoxygenation-induced LDH release. that in vitro model (14,20). Likewise, in our studies The blockade of the transport of intact GSH by pro- of cultured renal cells subjected to anoxia and reox- benecid also partially inhibited the beneficial effects ygenation, 2 mM alanine provided less beneficial of exogenous GSH (Table 5). effect than did GSH. On the other hand, abanine was

I 340 Volume 2 . Number 8 . 1992 5. * ... . . : . .. - #{149} . Paller and Patten

C C

I, C C LDH release 50 (% total)

30 * p < 0.05

20 10 I (n.10) (n-16) (n16)

Control Anoxla Anoxla Anoxla + + Alanlne GSH Figure 2. Effect of i-alanine (2 mM) or GSH (2 mM) on anoxia/reoxygenation injury in renal proximal tubule epithelial cells.

TABLE 2. Effects of individual amino acids, a TABLE 3. Effects of inhibition of GSH synthesis with combination of amino acids, or GSH on anoxia/ buthionine sulfoximine on GSH-mediated protection reoxygenation injurya in anoxia/reoxygenation injurya

LDH Release LDH Release Treatment Treatment ( /0) ( /o)

None (N= 28) 38.1 ± 2.9 None (N= 10) 29.6 ± 1.4 GSH 2.0 mM (N= 19) 12.4 ± 1.8b GSH2.OmM(N=8) 11.0± 1,7’ Glutamate 2.0 mM (N= 13) 26.1 ± 3.3 BSO (N= 13) 34.1 ± 2.2 Cysteine 2.0 mM (N= 13) 20.4 ± 34b GSH plus BSO (N= 13) 31.3 ± 3.8

Glutamate, cysteine, plus glycine 16.6 ± 19b 2.0 mM (N= 21) 0 Results are expressed as the fraction of cellular LDH released during anoxia/reoxygenation. LDH release by normoxic cells was 171 ± 3.3% Glutamate, cysteine, glycine, 34.8 ± 3.6 in this particular experiment. The numbers in parentheses are the plus BSO number of cell wells examined. Only GSH provided significant protection against anoxia/reoxygenation injury; buthionine sulfoximine (BSO) a Results are expressed as the fraction of cellular LDH released during blocked the protective effects of GSH. anoxia/reoxygenation. LDH release by normoxic cells was 14.8 ± 2.O% b p< versus no treatment. in this particular experiment. The numbers in parentheses are the number of cell wells examined (these were obtained from I 2 plates from three different batches of cells). GSH and the combination of GSH from enhancing cellular stores if this pathway amino acids provided significant protection against anoxia/reoxygenation injury. Cysteine also protected against injury. The GSH synthesis is dominant. Renal epithebial cells also have a baso- inhibitor buthionine sulfoximine (BSO) completely blocked the protec- lateral membrane Na-dependent transporter that tive effect of glutamate, cysteine. plus glycine. recognizes the ‘y-glutamyl residue of GSH to transport b p < 0.05 versus no treatment. it intact into the cell interior. Investigators have shown that this pathway contributes significantly to found to protect isolated rabbit proximal tubules net renal GSH uptake (2,22). Hagen and colleagues (13,16). showed that this pathway was primarily responsible Exogenous GSH can supplant intracellular stones for the protection exogenous GSH provided to freshly in two ways. -y-Glutamybtranspeptidase, a brush bor- isolated renal proximal tubule epithelial cells against den enzyme, degrades extraceblular GSH to its con- oxidative injury due to t-butyl hydropenoxide (2). stituent amino acids, which are then taken up by the In our studies, buthionine sulfoximine blocked the cell. ‘y-Glutamylcystemne synthase and GSH synthase protective effects of GSH, again demonstrating the then resynthesize GSH within the cell (2 1). The for- importance of intact GSH rather than its constituents mer enzyme is selectively inhibited by buthionine such as glycine. We cannot explain why acivicin sulfoximine, which effectively prevents extracellular (which presumably acts on the same biochemical

Journal of the American Society of Nephrology I 341 Glutathione and Glycine in Renal Anoxia .; . . . .. , \.. .. . ...... .:

pathway at a more proximal step) did not also atten- afforded by the amino acid combination. Enhanced uate the effects of GSH. The combination of amino GSH synthesis may also be the explanation for the acids also provided protection against anoxia/neoxy- protective effect of cystemne, the availability of which genation injury, demonstrating that delivery of the limits GSH synthesis (23,24). On the other hand, individual amino acids to the cell can support GSH probenecid, a blocker of basobateral GSH transport, synthesis. This Is further bobstened by the finding attenuated the protective effect of GSH, a finding that buthionine sulfoximine blocked the protection similar to that of Hagen et at. (2). Overall, these studies suggest that both pathways for GSH uptake were important in protecting these cells from anoxia/ TABLE 4. Effects of inhibition of ‘y- reoxygenation injury, although basobateral transport glutamyltranspeptidase with acivicin on GSH- of intact peptide is probably less important quanti- mediated protection in anoxia/reoxygenation injury0 tatively. Notably, in every experiment, GSH was more protective than was any component or similar amino LDH Release acid, such as alanine. Treatment (%) These findings suggest that there may be consid- enable differences in the cellular biology of anoxic injury depending upon the model studied. These stud- None(N= 6) 47.9 ± 8.0 GSH 2.0 mM(N= 6) 30.0 ± 2.4b ies contrast with published work in other models and Acivicin (N= 12) 40.1 ± 4.5 demonstrate variable effects of GSH versus glycine GSH plus acivicin (N= 12) 23.0 ± 2.2k’ in various renal ischemic models. The findings presented here and those previously published are sum- a Results are expressed as the fraction of cellular LDH released during manized in Table 6. Because all of the reported studies anoxia/reoxygenation. LDH release by normoxic cells was 16.8 ± 3.2% in this particular experiment. The numbers in parentheses are the have been diligently performed, it is unlikely that number of cell wells examined. GSH provided significant protection experimental error alone accounts for the differences against anoxia/reoxygenation injury; acivicin did not block the pro- in results among the models. Therefore, these con- tective effects of GSH. b p< versus no treatment. trasting data point to the likelihood that some cellular metabolic processes may be different among the different experimental models and thus condition the TABLE 5. Effects of inhibition of GSH uptake with responses to various maneuvers. Rather than at- probenecid on GSH-mediated protection in anoxia/ tempting to select one model as being more appropni- reoxygenation injury0 ate for study than another, these findings suggest LDH Release that attempts to characterize the differences in met- Treatment (%) abobic function and responses to anoxia in the different models will provide a greater understanding of the cellular response to ischemia. Insight as to the None(N= 9) 61.4 ± 3.3 heterogeneous response to protective agents has GSH 2.0 mM (N= 9) 6.2 ± 1.2’ great applicability and importance to clinical medi- Probenecid (N= 9) 47.8 ± 2.9k’ GSH plus probenecid (N= 9) 22.4 ± 2.7bc cine. If model heterogeneity is a generalized phenome- 0 Results are expressed as the fraction of cellular LDH released during non in studies of renal ischemia, this suggests a anoxia/reoxygenation. LDH release by normoxic cells was 19.8 ± 4.4% possible explanation for the observations that ther- in this particular experiment. The numbers in parentheses are the number of cell wells examined. GSH provided significant protection apeutic maneuvers other than GSH on glycine have against anoxia/reoxygenation injury; probenecid partially blocked the also demonstrated efficacy in some models but in not protective effects of GSH. b p< versus no treatment. others. For instance, despite a voluminous literature C p< 0.05 versus GSH. suggesting a beneficial effect of oxygen free radical

TABLE 6. Effects of some protective agents in several models of renal ischemia

Model

Protective Agent . Isolated Isolated Perfused Cultured Epithelial In Vivo . Tubules Kidney Cells

GSH Yes Yes Yes Yes Glycine Yes Yes Partial Superoxide dismutase or allopurinol#{176} Yes No Variable Yes

a Inhibitors of superoxide radical.

I 342 Volume 2 . Number 8 . 1992 . . , . . .: Paller and Patten

scavengers to protect against renal ischemic injury 9. Paller MS: Renal work, glutathione and suscep- tibibity to free radical-mediated postischemic in- in vivo (4,6,8,9,25-28), a limited number of studies jury. Kidney Int 1988:33:843-849. employing freshly isolated tubules have failed to 1 0. Scaduto RC Jr. Gattone VH II, Grotyohann LW, demonstrate protection (29-32). Free radical scav- Wertz J, Martin LF: Effect of an altered gluta- engers have also been demonstrated to protect cub- thione content on renal ischemic injury. Am J tured renal epitheliab cells against anoxia and reox- Physiob 1988;255:F91 1-F921. 1 1 . Messana JM, Cieslinski DA, O’Connor RP, ygenation (19). It is of interest that both of the latter Humes HD: Glutathione protects against exoge- preparations have a relatively limited duration of nous oxidant injury to rabbit renal proximal tu- viability when studied under the usual experimental bules. Am J Physiol 1988;2SS:F874-F884. conditions (33), and this feature may suggest why 1 2. Weinberg JM, Davis JA, Abarzua M, Kiani T: Relationship between cell adenosine tniphos- responses to anoxia are at times different than the phate and glutathione content and protection by In vivo responses. It may also explain why these two glycine against hypoxic proximal tubule cell in- models, in particular, are more responsive to the juny. J Lab Clin Med 1989:113:612-622. protective effects of gbycine and alanine. On the other 1 3. Mandel U, Schnellmann RG, Jacobs WR: In- hand, these preparations have allowed expenimen- tracellular glutathione in the protection from anoxic injury in renal proximal tubules. J Clin tation into the mechanisms of cell injury in a detailed Invest 1990:8:316-324. manner that is not possible when employing intact 1 4. Weinberg JM, Davis JA, Abarzua M, Rajan T: animals and, therefore, deserve continued use. The Cytoprotective effects of glycine and gbutathione cell culture model of renal anoxic injury presented against hypoxic injury to renal tubules. J Clin Invest 1987:80:1446-1454. here also menits more intensive characterization and 1 5. Weinberg JM, Davis JA, Abarzua M, Smith RK, utilization. As these studies point out, however, cau- Kunkel R: Ouabain-induced lethal proximal tu- tion should be applied when extrapolating findings bule cell injury is prevented by glycine. Am J from one experimental model to another. Physiol 1 990:258:F346-F355. 1 6. Garza-Quintero R, Ortega-Lopez J, Stein JH, Venkatachalam MA: Abanine protects rabbit proximal tubules against anoxic injury in vitro. ACKNOWLEDGMENTS Am J Physiol 1990:258:F1075-F1083. 1 7. Baines AD, Shaikh N, Ho P: Mechanisms of These studies were supported. in part. by an American Heart Asso- perfused kidney cytoprotection by abanine and elation-Winthrop Pharmaceuticals Grant-in-Aid. gbycine. Am J Physiob 1990:2S9:F80-F87. 1 8. Gesek FA, Wolff DW, Strandhoy JW: Improved separation method for rat proximal and distal REFERENCES renal tubules. Am J Physiol 1987:253: F358-F365. 1 . Chance B, Boveris A, Nakase Y, Sies H: Hydro- 1 9. Paller MS, Neumann TV: Reactive oxygen spe- peroxide metabolism: An overview. In: Sies H. cies and rat renal epitheliab cells during hypoxia Wendeb A, Eds. Functions of Glutathione in Liven and reoxygenation. Kidney Int 1991:40:1041- and Kidney, Berlin: Springer-Verlag: 1978: 1049. 95-106. 20. Weinberg JM, Davis JA, Abarzua M, Kiani T, 2. Hagen TM, Aw TY, Jones DP: Glutathione up- Kunkel R: Protection by gbycine of proximal tu- take and protection against oxidative injury in bules from injury due to inhibitors of mitochon- isolated kidney cells. Kidney Int 1988:34: driab ATP production. Am J Physiol 1990:258: 74-81. Cl 127-Cl 140.

3. Linas SL, Shanley PF, White CW, Parker NP, 2 1 . Griffith OW, Meister A: Glutathioine:interorgan

Repine JE: 02 metabolite-mediated injury In per- translocation , turnover, and metabolism . Proc fused kidneys is reflected by consumption of Natl Acad Sci USA 1979:76:5606-56 10. DMTU and glutathione. Am J Physiob 1987: 22. Rankin BB, Wells W, Curthoys NP: Rat renal 253:F692-F701. penitubular transport and metabolism of plasma 4. McCoy RN, Hill KE, Ayon MA, Stein JH, Burk 1355]glutathione. Am J Physiol 1985:249: RF: Oxidant stress following renal ischemia: F 1 98-F204. Changes in the glutathione redox ratio. Kidney 23. Williamson JM, Boettcher B, Meister A: Intra- Int 1988:33:812-817. cellular cysteine delivery system that protects S. Slusser SO, Grotyohann LW, Martin LF, Sea- against toxicity by promoting glutathione syn- duto RC Jr: Glutathione catabolism by the is- thesis. Proc Natl Acad Sci USA 1982:79: chemic rat kidney. Am J Physiol 1990;258: 6246-6249. F1547-F1SS3. 24. Meister A, Anderson ME: Glutathione. Annu 6. Nath KA, Pailer MS: Dietary deficiency of an- Rev Biochem 1983:2:711-760. tioxidants exacerbates ischemic injury in the rat 25. Hansson R, Jonsson 0, Lundstam S, Petters- kidney. Kidney Int 1990:38:1109-1117. son S, Schersten T, Waldenstrom J: Effects of 7. Brezis M, Rosen 5, Silva P, Epstein FH: Selec- free radical scavengers on renal circulation after tive glutathione depletion on function and struc- ischemia in the rabbit. Clin Sci 1983:65: ture of the isolated perfused rat kidney. Kidney 605-610. Int 1983:24:178-184. 26. Ouriel K, Smedira NG, Ricotta JJ: Protection of 8. Paller MS: Hypothyroidism protects against free the kidney after temporary ischemia: Free radi- radical damage in ischemic acute renal failure. cal scavengers. J Vasc Surg 198S:2:49-S3. Kidney Int 1986:29:1162-1166. 27. Paller MS. Hoidal JR, Ferris TF: Oxygen free

Journal of the American Society of Nephrology I 343 .... . . . :...... #{149}.:. .. . Glutathione and Glycine in Renal Anoxia . . . . ...... . . ..- .

radicals in ischemic acute renal failure in the acute renal failure. Am J Physiol 1988:255: rat. J Clin Invest 1984:74:1156-1164. F450-F460. 28. Baker GL, Cony RL, Autor AP: Oxygen free 3 1 . White M, Hunt D, Humes HD, Weinberg JM: radical induced damage in kidneys subjected to Effects of allopuninol on ischemic injury to iso- warm ischemia and reperfusion: Protective ef- lated tubules [Abstract]. Kidney Int 1985:27: fect of superoxide dismutase. Ann Sung 1985: 239. 202:628-641. 32. Doctor RB, Mandel U: Minimal role of xanthine 29. Borkan SC, Schwartz JH: Role of oxygen free oxidase and oxygen free radicals in rat renal radical species in in vitro models of proximal tubular reoxygenation injury. J Am Soc Nephrol tubular ischemia. Am J Physiol 1989:257: 1991 ; 1:959-969. Fl 14-F125. 33. Maack T: Physiological evaluation of the isolated 30. Gamelin LM, Zager RA: Evidence against oxi- perfused rat kidney. Am J Physiol 1980:238: dant injury as a critical mediator of postischemic F71-F78.

I 344 Volume 2 . Number 8 . 1992