Protective effect of glycine on renal injury induced by ischemia-reperfusion in vivo

Ming Yin1, Zhi Zhong1, Henry D. Connor1, Hartwig Bunzendahl2, William F. Finn3, Ivan Rusyn1, Xiangli Li1, James A. Raleigh4, Ronald P. Mason5, and Ronald G. Thurman1,dagger

Departments of 1 Pharmacology, 2 Surgery, 3 Nephrology, and 4 Radiation Oncology, School of Medicine, University of North Carolina, Chapel Hill 27599; and 5 Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Science, Research Triangle Park, North Carolina 27709


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Although glycine prevents renal tubular cell injury in vitro, its effect in vivo is not clear. The purpose of this study was to investigate whether a bolus injection of glycine given before reperfusion plus continuous dietary supplementation afterward would reduce renal injury caused by ischemia-reperfusion. Female Sprague-Dawley rats received a semisynthetic powdered diet containing 5% glycine and 15% casein (glycine group) or 20% casein (control group). Two days later, renal ischemia was produced by cross-clamping the left renal vessels for 15 min, followed by reperfusion. The right kidney was removed before reperfusion. The postischemic glomerular filtration rate (GFR) showed that renal function was less impaired and recovered more quickly in rats receiving glycine. For example, at day 7, GFR in controls (0.31 ± 0.03 ml · min-1 · 100 g-1) was about one-half that of glycine-treated rats (0.61 ± 0.06 ml · min-1 · 100 g-1, P < 0.05). Furthermore, tubular injury and cast formation observed in controls was minimized by glycine (pathology score, 3.2 ± 0.4 vs. 1.0 ± 0.4, P < 0.05). Urinary lactate dehydrogenase (LDH) concentration was elevated by ischemia-reperfusion in the control group (260 ± 22 U/l), but values were significantly lower by about fourfold (60 ± 30 U/l) in glycine-fed rats. Similarly, free radical production in urine was significantly lower in glycine-treated animals. Importantly, on postischemic day 1, binding of pimonidazole, an in vivo hypoxia marker, was increased in the outer medulla in controls; however, this phenomenon was prevented by glycine. Two weeks later, mild leukocyte infiltration and interstitial fibrosis were still observed in controls, but not in kidneys from glycine-treated rats. In conclusion, these results indicate that administration of glycine indeed reduces mild ischemia-reperfusion injury in the kidney in vivo, in part by decreasing initial damage and preventing chronic hypoxia.

kidney; hypoxia; rat


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ISCHEMIA IN THE KIDNEY LEADS to a series of complex cellular and molecular events that are incompletely understood (7, 30). Ischemia and early reperfusion deplete renal glycine (3), and the effects of glycine on anoxia-induced tubular damage have been studied in vitro (30, 31). It is well recognized that glycine retards the development of lethal hypoxia-induced damage in tubular cells (11, 32, 33). This protective effect appears to be independent of preservation of cellular ATP (32) and fatty acid accumulation (34). In a study in isolated perfused rat kidney, glycine protected against hypoxic injury to the medullary thick ascending limb and slowed functional deterioration in a dose-dependent manner (12). On the other hand, glycine failed to protect rat kidney against ischemia-reperfusion injury induced by 30-60 min of vascular clamping in vivo (13, 35). In that study, a single intravenous infusion of glycine was used before and during ischemic insults, and postischemic renal function was monitored. Thus the effects of glycine remain unclear.

Ischemia-reperfusion injury to the kidney is characterized by profound depression of the glomerular filtration rate and may end in acute renal failure or delayed recovery. Oxygen-derived free radicals are responsible for ischemia-reperfusion damage in the kidney (22). The postischemic kidney usually undergoes a series of complicated pathophysiological changes, including inflammation, regeneration, apoptosis and interstitial fibrosis (19, 27). Therefore, it is hypothesized that a protective agent such as glycine, administered not only before the ischemic insult but also during the following postischemic days, would be beneficial. The most severe damage after ischemia-reperfusion is seen in the outer medulla, a region with marginal oxygenation even under normal circumstances (27). Glycine given as a dietary supplement reduces cyclosporin A-induced hypoxia in the outer medulla and diminishes free radical production (40). Therefore, in this study, the effects of dietary administration of glycine on renal ischemia-reperfusion injury were investigated in vivo. Here, rats received not only a bolus injection of glycine before induction of renal ischemia but also a diet containing 5% glycine postoperatively. Under these conditions, glycine protects the kidney against injury induced by brief ischemia, in part, by reducing both free radical production early after reperfusion as well as chronic hypoxia in the outer medulla during recovery.


    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Animals and diets. Adult female Sprague-Dawley rats (190-230 g) were housed four to a cage in a facility approved by the Association for the Accreditation and Assessment of Laboratory Animal Care International. Two days before surgery, rats were randomly assigned to two experimental groups and fed a semisynthetic powdered diet [AIN-93G (24), Teklad test diets, Madison, WI] containing 5% glycine and 15% casein (glycine group) or 20% casein (control group). After surgery, each rat continued to receive its assigned diet throughout the entire 2-wk experimental period. All animals received humane care in compliance with guidelines approved by the Institutional Animal Care and Use Committee of the University of North Carolina at Chapel Hill.

Renal ischemia and reperfusion. Animals underwent anesthesia by ether inhalation and were placed under a lamp to maintain body temperature. As described previously (37), a midline laparotomy was performed and the left kidney was dissected free from the surrounding tissue. A nontraumatic vascular clamp was placed across the renal pedicle to induce ischemia, and the abdomen was closed temporarily during the ischemic period. Five minutes before the end of the ischemic period, a bolus injection of glycine (100 mg/kg) was given intravenously to rats receiving the glycine diet, whereas saline was given to animals fed the control diet. A contralateral nephrectomy was then performed, and reperfusion of the left kidney was achieved by releasing the vascular clamp. The abdominal wall was closed with double-layer sutures, and the animals were allowed to recover. Two weeks later, the animals were reanesthetized and the ischemically injured left kidneys were excised and cut into two parts. One part was fixed in 4% buffered formaldehyde and embedded in paraffin, and the other part was frozen and stored at -80°C. In a pilot study, glycine protected the kidney against ischemia-reperfusion injury induced by 15 min of warm ischemia, but not 30 or 45 min; therefore, 15 min of renal ischemia was used in this study.

Glomerular filtration rates. Rats were placed in metabolic cages with free access to water and chow, and 24-h urine samples were collected. Blood samples were taken from the tail vein on days 1, 3, 7, and 14 after reperfusion, and creatinine levels in urine and serum were determined using a commercially available kit (Sigma Diagnostic, St. Louis, MO). Glomerular filtration rates (GFRs) corrected for body weight were calculated from the ratio of creatinine in the urine/blood and the volume of urine produced in 24 h (18). Usually, inulin clearance is used to measure GFR, where inulin is infused intravenously and inulin in urine and blood is measured as described elsewhere (5). In previous experiments, GFRs calculated from inulin clearance and creatinine clearance were nearly identical under these conditions (40). Therefore, creatinine clearance was used here to allow evaluation of postischemic renal function at multiple time points.

Measurement of glycine concentration in blood. Blood samples were collected at necropsy, and serum was stored at -80°C until measurement. Concentrations of glycine in serum were determined as described by Ohmori et al. (21). Briefly, glycine was extracted and benzoylated, and the resulting hippuric acid was extracted and dried. Subsequently, the concentration of a colored conjugate of hippuric acid was determined spectrophotometrically at 458 nm.

Histology. Renal tissue fixed in formalin was processed by dehydration and embedded in paraffin. Sections were cut at 5 µm and stained with hematoxylin and eosin for histological assessment. Examination and scoring of the sections were performed on a blinded basis. In the assessment of kidneys harvested 2 h after reperfusion, the severity of renal damage in terms of morphological changes was scored with a grading system of 0-3, where 0 was normal, 1 was mild, 2 was moderate and 3 was severe (28). Mean values in five high-power fields were used for statistical analysis.

Detection of lactate dehydrogenase in urine. After reperfusion, urine was collected through a catheter placed in the urinary bladder for 2 h, and samples were kept at -80°C until analysis. Lactate dehydrogenase (LDH) was measured using standard enzymatic techniques (4).

Determination of protein-bound pimonidazole using immunohistochemistry and ELISA. Nitroimidazole compounds, which are reductively activated at low oxygen concentration (8) and bind to cellular macromolecules, have been used to detect hypoxia in vivo in a variety of tissues, including kidney (2, 23, 40). Pimonidazole hydrochloride (120 mg/kg ip) was injected 24 h after reperfusion. Two hours after administration of pimonidazole, kidneys were perfused briefly with normal saline to remove blood. One part of the kidney was frozen for ELISA, and another part was sectioned and fixed for subsequent immunohistochemical analysis. Paraffin blocks of formalin-fixed kidney tissue were sectioned at 5 µm, and pimonidazole adducts were detected with the biotin-streptavidin-peroxidase indirect immunostaining method (1). The sections were hydrated and treated briefly with 0.01% protease (pronase E) and exposed to mouse anti-pimonidazole IgG antibody in PBS-Tween for 30 min at room temperature. Rat adsorbed horse anti-mouse antibody was then applied to the sections for 30 min. Once the antibody-biotin-peroxidase complex was formed, 3, 3'-diaminobenzidine chromogen was added as the peroxidase substrate. After the immunostaining procedure was completed, a counterstain of hematoxylin was applied, followed by mounting with crystal mount solution.

Pimonidazole-protein adducts were measured in tissue homogenates with a competitive ELISA procedure described elsewhere (1). Protein levels in tissue homogenates were determined with the bicinchoninic acid assay using a commercially available kit (Pierce, Rockford, IL).

Detection of free radical adducts in urine. The spin-trapping reagent alpha -(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN; 1 g/kg body wt) was dissolved in 2 ml of saline and injected slowly into the tail vein 24 h after reperfusion. Two hours after the administration of 4-POBN, urine was collected through a catheter placed in the urinary bladder for 2 h, and samples were kept at -80°C until electron paramagnetic resonance (ESR) analysis. Samples were placed in an aqueous flat cell and bubbled with oxygen in the presence of an ascorbate oxidase paddle for 5 min to eliminate interfering ascorbyl free radicals and with nitrogen for 5 min to reduce oxygen-derived line broadening. Free radical adducts were detected with a Bruker ESP 300 ESR spectrometer (Bruker Instrument, Billerica, MA). Instrument conditions were as follows: 20-mW microwave power; 1.0-G modulation amplitude, and 80-G scan range (17).

Statistics. All results were expressed as means ± SE with n = 10 rats/group for survival studies and n = 6 rats for acute studies, i.e., measurements of LDH, detection of free radicals, and binding of pimonidazole. Statistical differences between means were determined using ANOVA or Fisher's Exact where appropriate. A P value of <0.05 was selected before the study to determine statistical differences between groups.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Effects of glycine on postischemic renal function. Serum creatinine and GFRs during the first 2 wk after ischemia and reperfusion are shown in Fig. 1. Preoperative serum creatinine was ~0.7 mg/dl (Fig. 1A, day 0; no difference between the 2 groups). The creatinine levels in rats fed the control diet increased to 2.5 mg/dl at day 1 and did not recover to normal at 2 wk. Serum creatinine in rats receiving the glycine diet was also elevated, but to a lesser extent than in controls (P < 0.05 at days 1, 3, and 7, respectively). GFR at day 0 (i.e., normal GFR measured before ischemia) was ~0.64 ml · min-1 · 100 g-1 and declined 24 h after reperfusion to about one-half of preischemic values in animals fed the control diet (Fig. 1B). However, GFRs in rats treated with glycine declined to only one-third of preischemic values. During the 2-wk postischemic period, GFRs of the control group stayed at relatively low levels (0.31-0.41 ml · min-1 · 100 g-1). Importantly, GFRs in rats treated with glycine recovered significantly faster and returned to normal 3 days after ischemia. On day 7, GFRs in rats receiving glycine were about twofold higher than those in rats fed the control diet.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 1.   Effects of glycine on glomerular filtration rates (GFR) after ischemia and reperfusion. The left kidney was subjected to 15 min of ischemia, and the right kidney was removed. Urine and blood samples were collected, and GFR was determined as described in MATERIALS AND METHODS. Values are means ± SE (n = >=  8 rats/group). *P < 0.05 vs. the control group by 1-way repeated-measures ANOVA.

Effects of glycine on renal morphology immediately after ischemia. Representative photomicrographs of kidneys 2 h after ischemia-reperfusion are shown in Fig. 2. Kidneys from control rats showed postischemic renal injury (Fig. 2A), including tubular cell swelling, loss of brush border, and cast formation. In contrast, only mild tubular injury was observed in kidneys from rats treated with glycine (Fig. 2B). Nonischemic kidneys appeared normal (sham-operated group score = 0; data not shown). The extent of renal injury in the kidneys of rats fed the control diet increased dramatically (Fig. 3). Kidneys from glycine-fed rats (score of 0.55 ± 0.23), however, exhibited over threefold less injury than kidneys from the control group (1.80 ± 0.25, P < 0.05).


View larger version (151K):
[in this window]
[in a new window]
 
Fig. 2.   Representative photomicrographs of kidneys from rats in control or glycine-treated groups after ischemia-reperfusion. Animals were subjected to 15 min of ischemia and treated as described in MATERIALS AND METHODS. A: kidney 2 h after reperfusion from a rat fed control diet. B: kidney from glycine-treated rat 2 h after reperfusion. C: kidney 2 wk after ischemia and reperfusion from rat fed control diet. D: kidney from glycine-treated rat 2 wk after ischemia and reperfusion. Arrows and arrowheads, tubular injury and cast formation, respectively. Magnification: ×100.



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 3.   Effects of glycine on renal histology 2 h after ischemia and reperfusion. Animals were subjected to 15 min of ischemia. Pathology was scored as described in MATERIALS AND METHODS. Values are means ± SE (n = >=  8 rats/group). *P < 0.05 vs. control groups by Wilcoxon signed rank test.

Renal histology and blood concentration of glycine 2 wk after ischemia-reperfusion. Representative photomicrographs of kidneys from rats treated with either control or glycine-containing diets for 2 wk after ischemia-reperfusion are shown in Fig. 2, C and D, respectively. Increased inflammation and mild interstitial fibrosis were observed in kidneys from rats receiving the control diet (Fig. 2C); however, kidneys from glycine-treated rats showed an almost normal renal architecture (Fig. 2D). Serum concentrations of glycine were determined from blood samples collected at necropsy. The blood concentration of glycine from glycine-fed rats was 0.62 ± 0.05 mmol/l, which was nearly fourfold higher than values of animals fed the control diet (0.17 ± 0.03 mmol/l, P < 0.05).

Effects of glycine on urinary release of LDH. Urinary release of LDH was used as a marker for tubular cell injury (38, 39). LDH release in urine from sham-operated nonischemic rats was minimal (Fig. 4). However, LDH levels 2 h after reperfusion increased dramatically to ~260 U/l in urine from rats fed the control diet. In rats receiving glycine, values were about fivefold lower (P < 0.05).


View larger version (10K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of glycine on urinary lactate dehydrogenase (LDH) release after ischemia and reperfusion. Rats were subjected to 15 min of ischemia. Urine samples were collected via a catheter placed in the urinary bladder during the first 2 h postischemia, and LDH was determined as described in MATERIALS AND METHODS. Values are means ± SE (n = >=  6 rats/group). aP < 0.05 vs. the sham-operated group. bP < 0.05 compared with the control group by 1-way ANOVA with the use of Tukey's post hoc test.

Effects of glycine on free radical release in urine. Reperfusion subsequent to renal ischemia could lead to free radical formation. Accordingly, free radicals were trapped with the spin-trapping reagent 4-POBN and detected with ESR. Figure 5, A-D, shows representative ESR spectra resulting from free radical adducts in urine collected during the first 2 h of reperfusion. A six-line ESR spectrum due to radical adducts was detected in urine samples from both control and glycine groups. However, free radical signals from urine of rats in the control group were dramatically larger than those from glycine-treated rats (P < 0.05; see Fig. 5, bottom), indicating that glycine reduces free radicals.


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 5.   Electron paramagnetic resonance (ESR) spectra of radical adducts detected in urine samples from rats with ischemia-reperfusion-induced renal injury. The spin trapping reagent alpha -(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN; 1 g/kg body wt) was injected intravenously 10 min before ischemia. Urine samples were collected through a catheter placed in the urinary bladder during the first 2 h of reperfusion after 15 min of ischemia. Free radical adducts in urine were detected as described in MATERIALS AND METHODS. A-D: typical spectra. A: sham-operated rats receiving saline. B: rats fed control diet. C: simulation of spectrum B. D: rats fed glycine. Bottom: values are means ± SE (n = >=  4 rats/group). aP < 0.05 vs. sham-operated group. bP < 0.05 compared with control group by 1-way ANOVA Tukey's post hoc test.

Effects of glycine on chronic hypoxia. Binding of pimonidazole has been used as an in vivo hypoxia marker (1, 40). Figure 6 depicts representative images of kidneys after pimonidazole adducts were detected immunohistochemically. In kidneys from the sham-operated group, pimonidazole adducts accumulated mainly in the outer medulla (data not shown), similar to animals treated with glycine (Fig. 6, B and D). In the postischemic kidneys of rats given the control diet, an increased area of pimonidazole binding from the outer medulla toward the cortex was observed (Fig. 6, A and C). In this region, proximal tubular cells, which are metabolically active and highly sensitive to hypoxia, predominate (14).


View larger version (58K):
[in this window]
[in a new window]
 
Fig. 6.   Representative images of pimonidazole binding in the kidney 24 h after ischemia. Pimonidazole (120 mg/kg ip) was injected 24 h after ischemia-reperfusion. Two hours later, kidneys were perfused briefly with saline to remove blood and fixed with 1% paraformaldehyde, and sections were stained immunohistochemically (1) A: sham-operated kidney. B: kidney from a rat fed control diet. C: kidney from a rat fed glycine diet. Areas with dark brown staining denote where binding of pimonidazole occurred.

Pimonidazole binding was also quantified using ELISA to detect both protein and nonprotein adducts (Fig. 7). The level of pimonidazole adducts in sham-operated nonischemic kidneys was 377 pmol/mg protein. Fifteen minutes of ischemia with 24 h of reperfusion increased binding twofold in kidneys from rats fed the control diet. In contrast, levels of pimonidazole adducts in the ischemically injured kidney from glycine-treated rats were similar to values from nonischemic kidneys. This indicates that increased hypoxia exists in the outer medulla during recovery from ischemia-reperfusion injury and that this phenomenon is prevented by glycine.


View larger version (14K):
[in this window]
[in a new window]
 
Fig. 7.   Quantitation of pimonidazole binding. Pimonidazole (120 mg/kg ip) was injected 24 h after ischemia-reperfusion. Two hours later, kidneys were perfused briefly with saline to remove blood and frozen at -80°C until assay. Pimonidazole adduct formation in kidney homogenates was quantified using an ELISA as described in MATERIALS AND METHODS. Values are means ± SE (n = 3-6 rats/group). aP < 0.05 compared with sham-operated nonischemic kidneys. bP < 0.05 compared with controls.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Glycine reduces renal ischemia-reperfusion injury in the kidney. The present study shows that glycine protects against kidney injury induced by a brief period of ischemia in vivo. This finding is supported by three observations. First, in rats subjected to a brief period of ischemia (15 min), GFRs declined less and recovered faster when rats were treated with glycine (Fig. 1). However, animals not receiving glycine (control group) survived with relatively poor renal function during the first 2 wk. Second, after brief ischemia and reperfusion, obvious tubular injury and cast formation were observed in controls, but not in glycine-treated rats (Figs. 2 and 3). Meanwhile, urinary release of LDH was minimized by glycine (Fig. 4). Third, early after reperfusion, free radical production dramatically increased in rats fed the control diet, but not in glycine-treated rats (Fig. 5). Therefore, an increased blood level of glycine during a short period of ischemia alleviates postischemic renal injury.

The pathogenesis of ischemia-reperfusion involves reactive oxygen species (20, 22, 26), and the results of the present study support the harmful role of oxidants. Importantly, glycine minimized free radical production and tubular damage early after reperfusion (Figs. 2-5). Oxygen free radicals can directly trigger the expression of adhesion molecules and activation of leukocytes (9). Enhancement of inflammatory cytokine production (e.g., interleukin-1 and tumor necrosis factor-alpha ), which increases expression of cell adhesion molecules such as intracellular adhesion molecule-1, allows neutrophils to adhere and accumulate in peritubular vessels. This phenomenon causes capillary plugging and obstruction (15, 16, 36), which, in turn, leads to tissue destruction and reduced GFR. Therefore, reduction of free radical production by glycine during the early phase (i.e., 2 h) of ischemia-reperfusion minimizes tissue injury, leading to faster recovery (Figs. 1 and 5). Because no inflammatory reaction was observed in kidneys 2 h after reperfusion, tubular cells are the likely source of free radicals.

Glycine reduces chronic hypoxia in postischemic kidney. Chronic ischemia is an important cause of end-stage renal disease (27) and represents a persistent but limited insult; unfortunately, information addressing this problem is limited. The present study shows that increased hypoxia in the outer medulla exists 24 h after reperfusion in vivo (Figs. 6 and 7). Tubular epithelial cells are characterized by high rates of solute transport activity supported by mitochondrial respiration. Thus these cells would be expected to be sensitive to decreased oxygen availability. Moreover, tubular cells are injured during the initial ischemia-reperfusion insult (Figs. 2-4), and repair and regeneration of renal tissue would be impaired in chronic hypoxia. The exact mechanisms responsible for the reduction of chronic hypoxia by glycine remain unclear. However, diminished initial injury early after reperfusion by glycine may reduce later chronic hypoxia, because reduced tubular damage should minimize microcirculatory disturbances (37). Moreover, continuous administration of glycine through dietary supplementation after ischemia-reperfusion would maintain its therapeutic effects during the recovery period.

Glycine did not attenuate severe renal damage caused by longer periods of ischemia (30- and 45-min ischemia; data not shown). This is consistent with previous studies in which glycine was ineffective in protection against renal injury induced by longer periods of ischemia (13, 35). On the other hand, inhibition of intracellular adhesion molecule-1, platelet-activating factor, and thromboxane synthetase provided benefits against ischemic insult in models with 60 min of ischemia (6, 10, 29). The reasons for these differences are not clear; however, glycine is still clinically relevant, because it also attenuates chronic hypoxia.

Clinical implications. The results of the present study show that the inflammatory reaction in the ischemically injured kidney was still observable 2 wk after the initial ischemic insult in kidneys from control rats (Fig. 2C). Moreover, increased pimonidazole binding in the outer medulla 24 h after reperfusion indicates the existence of chronic hypoxia. These phenomena suggest that dietary glycine might eliminate chronic hypoxia, possibly by reducing injury early after reperfusion. In this study, the beneficial effects of glycine on renal injury may be attributable to both increased blood levels of glycine in the kidney during ischemia-reperfusion and/or its continuous supply in the diet during the recovery period. Moreover, glycine has been given long term to patients without toxic side effects (25), and administration of glycine through diet is simple. Therefore, glycine, a nontoxic amino acid, might be a useful treatment during recovery from acute postischemic renal failure. Taken together, the data presented here support the postulate that a glycine-rich diet may be a promising approach for treatment of disorders where ischemia-reperfusion injury to the kidney occurs.


    FOOTNOTES

dagger  Deceased 14 July 2001.

Address for reprint requests and other correspondence: Z. Zhong, Dept. of Pharmacology, CB#7365, Mary Ellen Jones Bldg., Univ. of North Carolina, Chapel Hill, NC 27599-7365 (E-mail: zzhong{at}med.unc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajprenal.00011.2001


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Arteel, GE, Raleigh JA, Bradford BU, and Thurman RG. Acute alcohol produces hypoxia directly in rat liver tissue in vivo: role of Kupffer cells. Am J Physiol Gastrointest Liver Physiol 271: G494-G500, 1996[Abstract/Free Full Text].

2.   Arteel, GE, Thurman RG, Yates JA, and Raleigh JA. Evidence that hypoxia markers detect oxygen gradients in liver: pimonidazole and retrograde perfusion of rat liver. Br J Cancer 72: 889-895, 1995[ISI][Medline].

3.   Beck, FX, Ohno A, Dorge A, and Thurau K. Ischemia-induced changes in cell element composition and osmolyte contents of outer medulla. Kidney Int 48: 449-457, 1995[ISI][Medline].

4.   Bergmeyer, HU. Methods of Enzymatic Analysis. New York: Academic, 1988.

5.   Davidson, W, and Sackner MA. Simplification of the anthrone method for the determination of inulin in clearance studies. J Lab Clin Med 62: 351-356, 1963[ISI].

6.   Dragun, D, Tullius SG, Park JK, Maasch C, Lukitsch I, Lippoldt A, Gross V, Luft FC, and Haller H. ICAM-1 antisense oligodesoxynucleotides prevent reperfusion injury and enhance immediate graft function in renal transplantation. Kidney Int 54: 590-602, 1998[ISI][Medline].

7.   Edelstein, CL, Ling H, and Schrier RW. The nature of renal cell injury. Kidney Int 51: 1341-1351, 1997[ISI][Medline].

8.   Franko, AJ, and Chapman JD. Binding of 14C-misonidazole to hypoxic cells in V79 spheroids. Br J Cancer 45: 694-699, 1982[ISI][Medline].

9.   Fraticelli, A, Serrano CV, Jr, Bochner BS, and Capogrossi MCZJL Hydrogen peroxide and superoxide modulate leukocyte adhesion molecule expression and leukocyte endothelial adhesion. Biochim Biophys Acta 1310: 251-259, 1996[ISI][Medline].

10.   Garvin, PJ, Niehoff ML, Robinson SM, Heisler T, Salinas-Madrigal L, Contis J, and Solomon H. Evaluation of the thromboxane A2 synthetase inhibitor OKY-046 in a warm ischemia-reperfusion rat model. Transplantation 61: 1429-1434, 1996[ISI][Medline].

11.   Garza-Quintero, R, Weinberg JM, Ortega-Lopez J, Davis JA, and Venkatachalam MA. Conservation of structure in ATP-depleted proximal tubules: role of calcium, polyphosphoinositides, and glycine. Am J Physiol Renal Fluid Electrolyte Physiol 265: F605-F623, 1993[Abstract/Free Full Text].

12.   Heyman, S, Spokes K, Rosen S, and Epstein FH. Mechanism of glycine protection in hypoxic injury: analogies with glycine receptor. Kidney Int 42: 41-45, 1992[ISI][Medline].

13.   Heyman, SN, Brezis M, Epstein FH, Spokes K, and Rosen S. Effect of glycine and hypertrophy on renal outer medullary hypoxic injury in ischemia reflow and contrast nephropathy. Am J Kidney Dis 19: 578-586, 1992[ISI][Medline].

14.   Heyman, SN, Brezis M, Epstein FH, Spokes K, and Rosen S. Effect of glycine and hypertrophy on renal outer medullary hypoxic injury in ischemia reflow and contrast nephropathy. Am J Kidney Dis 19: 578-586, 1992[ISI][Medline].

15.   Humes, HD, and Liu S. Cellular and molecular basis of renal repair in acute renal failure. J Lab Clin Med 124: 749-754, 1994[ISI][Medline].

16.   Kelly, KJ, Williams WW, Colvin RB, Meehan SM, Springer TA, Gutierrez-Ramos J, and Bonventre JV. Intercellular adhesion molecule-1-deficient mice are protected against ischemic renal injury. J Clin Invest 97: 1056-1063, 1996[Abstract/Free Full Text].

17.   Knecht, KT, Bradford BU, Mason RP, and Thurman RG. In vivo formation of a free radical metabolite of ethanol. Mol Pharmacol 38: 26-30, 1990[Abstract].

18.   Laiken, ND, and Fanestil DD. Body fluids and renal function. In: Physiological Basis of Medical Practice, edited by West JB.. Baltimore, MD: Williams & Wilkins, 1985, p. 438-543.

19.   Linas, SL, Shanley PF, Whittenburg D, Berger E, and Repine JE. Neutrophils accentuate ischemia-reperfusion injury in isolated perfused rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 255: F728-F735, 1988[Abstract/Free Full Text].

20.   McCord, JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 312: 159-163, 1985[Abstract].

21.   Ohmori, S, Ikeda M, Watanabe Y, and Hirota K. A simple and specific determination of glycine in biological samples. Anal Biochem 90: 662-670, 1978[ISI][Medline].

22.   Paller, MS, Hoidal JR, and Ferris TF. Oxygen free radicals in ischemic acute renal failure in the rat. J Clin Invest 74: 1156-1164, 1984[ISI][Medline].

23.   Raleigh, JA, and Koch CJ. Importance of thiols in the reductive binding of 2-nitroimidazoles to macromolecules. Biochem Pharmacol 40: 2457-2464, 1990[ISI][Medline].

24.   Reeves, PG. Component of the AIN-93 diets as improvements in the AIN-76A diet. J Nutr 127, Suppl 5: 838S-841S, 1997[Medline].

25.   Rosse, RB, Theut SK, Banay-Schwartz M, Leighton M, Scarella E, Cohen CG, and Deutsch SI. Glycine adjuvant therapy to conventional neuroleptic treatment in schizophrenia: an open-label, pilot study. Clin Neuropharmacol 12: 416-424, 1989[ISI][Medline].

26.   Roy, RS, and McCord JM. Superoxide and ischemia: conversion of xanthine dehydrogenase to xanthine oxidase. In: Oxy Radicals and Their Scavenger Systems: Cellular and Medical Aspects, edited by Greenwald RA, and Cohen G.. New York: Elsevier, 1983, vol. 2, p. 145-153.

27.   Shanley, PF. The pathology of chronic renal ischemia. Semin Nephrol 16: 21-32, 1996[ISI][Medline].

28.   Solez, K, Morel-Maroger L, and Sraer JD. The morphology of "acute tubular necrosis" in man: analysis of 57 renal biopsies and a comparison with the glycerol model. Medicine (Baltimore) 58: 362-376, 1979[ISI][Medline].

29.   Torras, J, Cruzado JM, Riera M, Condom E, Duque N, Herrero I, Merlos M, Espinosa L, Lloberas N, Egido J, and Grinyo JM. Long-term protective effect of UR-12670 after warm renal ischemia in uninephrectomized rats. Kidney Int 56: 1798-1808, 1999[ISI][Medline].

30.   Weinberg, JM. The cell biology of ischemic renal injury. Kidney Int 39: 476-500, 1991[ISI][Medline].

31.   Weinberg, JM. Glutathione and glycine in acute renal failure. Ren Fail 14: 311-319, 1992[ISI][Medline].

32.   Weinberg, JM, Davis JA, Abarzua M, and Kiani T. Relationship between cell adenosine triphosphate and glutathione content and protection by glycine against hypoxic proximal tubule cell injury. J Lab Clin Med 113: 612-622, 1989[ISI][Medline].

33.   Weinberg, JM, Davis JA, Abarzua M, and Rajan T. Cytoprotective effects of glycine and glutathione against hypoxic injury to renal tubules. J Clin Invest 80: 1446-1454, 1987[ISI][Medline].

34.   Weinberg, JM, Venkatachalam MA, Goldberg H, Roeser NF, and Davis JA. Modulation by Gly, Ca, and acidosis of injury-associated unesterified fatty acid accumulation in proximal tubule cells. Am J Physiol Renal Fluid Electrolyte Physiol 268: F110-F121, 1995[Abstract/Free Full Text].

35.   Wetzels, JFM, Yu L, Shanley PF, Burke TJ, and Schrier RW. Infusion of glycine does not attenuate in vivo ischemic acute renal failure in the rat. J Lab Clin Med 121: 263-267, 1993[ISI][Medline].

36.   Yin, M, Buurman WA, Daemen JHC, Janssen MA, and Kootstra G. PAF antagonist TCV-309 reduces PMN infiltration and enhances early function of 24h preserved rat kidneys with long warm ischemia. Transplantation 61: 1443-1446, 1996[ISI][Medline].

37.   Yin, M, Kurvers HAJM, Tangelder GJ, Booster MH, Buurman WA, and Kootstra G. Ischemia-reperfusion injury of rat kidney relates more to cortical tubular than to microcirculatory disturbances. Ren Fail 18: 211-223, 1996[ISI][Medline].

38.   Zager, RA, Iwata M, Burkhart KM, and Schimpf BA. Post-ischemic acute renal failure protects proximal tubules from O2 deprivation injury, possibly by inducing uremia. Kidney Int 45: 1760-1768, 1994[ISI][Medline].

39.   Zalups, RK. Reductions in renal mass and the nephropathy induced by mercury. Toxicol Appl Pharmacol 143: 366-379, 1997[ISI][Medline].

40.   Zhong, Z, Arteel GE, Connor H, Yin M, Frankenberg Mv, Stachlewitz RF, Raleigh JA, Mason RP, and Thurman RG. Cyclosporin A increases hypoxia and free radical production in the rat kidney: prevention by dietary glycine. Am J Physiol Renal Physiol 275: F595-F604, 1998[Abstract/Free Full Text].


Am J Physiol Renal Fluid Electrolyte Physiol 282(3):F417-F423