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1 Departments of Pharmacology
and 3 Radiation Oncology,
2 Curriculum in Toxicology, The major side
effect of cyclosporin A is severe nephrotoxicity. It is
likely that cyclosporin A causes vasoconstriction leading to
hypoxia-reperfusion injury; therefore, these experiments were designed
to attempt to obtain physical evidence for hypoxia and free radical
production in kidney following cyclosporin A. Rats were treated daily
with cyclosporin A (25 mg/kg ig) for 5 days, and
pimonidazole, a hypoxia marker, was injected 2 h after the last dose of
cyclosporin A. A dose of
reperfusion; hypoxia marker
CYCLOSPORIN A IS WIDELY used clinically as an
immunosuppressive agent after organ transplantation and in the
treatment of several autoimmune diseases. This drug significantly
improves transplant graft survival; however, recipients must maintain
therapy for the rest of their lives. Unfortunately, cyclosporin A has a
number of serious side effects, kidney damage being the most frequent
and severe. Moderate to severe renal dysfunction occurs in about 30%
of patients receiving cyclosporin A, significantly limiting its
clinical application. Nephrotoxicity of cyclosporin A is characterized
by reduced glomerular filtration rates and pathological changes such as
proximal tubular cell swelling and necrosis, infiltration of
macrophages, and interstitial fibrosis (4); however, mechanisms by
which cyclosporin A causes renal injury remain unclear.
Previous studies showed that cyclosporin A causes vasoconstriction in
the kidney (20, 24), which could theoretically lead to a classic
hypoxia-reoxygenation injury involving radicals. Furthermore, the
antioxidant vitamin E reduced cyclosporin A-induced nephrotoxicity
(39); however, physical evidence for hypoxia and free radicals due to
cyclosporin A is lacking. Therefore, the purpose of this study was to
determine whether cyclosporin A treatment causes hypoxia and free
radical production. Nitroimidazole compounds detect hypoxia in a
variety of tissues, such as tumors, liver, and kidney (1, 13, 19), and
pimonidazole, a 2-nitroimidazole, was used here to investigate whether
cyclosporin A causes hypoxia in the kidney. Free radicals were captured
with a spin trapping reagent and adducts detected with electron spin
resonance (ESR).
Glycine has been shown to cause renal vasodilation (7) and protect
cultured proximal tubules from hypoxic injury (35). Moreover, recent
studies in this laboratory showed that dietary glycine totally blocked
cyclosporin A-induced alterations in renal function, such as decreases
in glomerular filtration rate and pathological changes including
proximal tubular dilatation, cell necrosis, and infiltration of
macrophages (34). Accordingly, the hypothesis that glycine minimizes
cyclosporin A-induced nephrotoxicity by preventing hypoxia-reperfusion
injury in the kidney was also evaluated in this study.
Reagents. Cyclosporin A (Sandimmune
oral solution) was the Novartis product (Basel, Switzerland), and
glycine diets were a kind gift of Novartis Nutrition, Minneapolis, MN.
Creatinine analytic kit, deferoxamine mesylate, and
Animals. Male Sprague-Dawley rats
(200-250 g) were fed a semisynthetic powdered diet containing 5%
glycine and 15% casein (glycine diet) or 20% casein (control diet)
for 3 days prior to cyclosporin A and maintained during cyclosporin A
treatment (Table 1). Previous studies
showed that cyclosporin A at doses ranging from 25-50 mg/kg caused
typical nephrotoxicity including reduced glomerular filtration rates,
increased serum creatinine, and pathological changes such as proximal
tubular cell swelling and necrosis, infiltration of macrophages, and
interstitial fibrosis in rats fed standard rat chow diets (3, 4, 33),
and a recent study in this laboratory confirmed these findings (34).
Higher doses of cyclosporin A are needed in rats than humans to cause
renal damage, probably due to lower sensitivity of rats (3, 4, 33). In
this study, cyclosporin A (25 mg/kg dissolved in olive oil at a
concentration of 10 mg/ml) or an equivalent volume of olive oil vehicle
was given by oral gavage daily for 5 days. Diet consumption was around 6 g · 100 g body
wt
ABSTRACT
Top
Abstract
Introduction
Methods
Results
Discussion
References
-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) was injected 3 h after cyclosporin A to trap free
radicals. Cyclosporin A doubled serum creatinine and decreased
glomerular filtration rates by 65% as expected. Pimonidazole adduct
binding in the kidney was increased nearly threefold by cyclosporin A, providing physical evidence for tissue hypoxia. Moreover, cyclosporin A
increased 4-POBN/radical adducts nearly sixfold in the urine but did
not alter levels in the serum. Glycine, which causes vasodilatation and
prevents cyclosporin A toxicity, minimized hypoxia and blocked free
radical production; however, it did not alter cyclosporin A blood
levels. These results demonstrate for the first time that cyclosporin A
causes hypoxia and increases production of a new free radical species
exclusively in the kidney. Therefore, it is concluded that cyclosporin
A causes renal injury by mechanisms involving hypoxia-reoxygenation,
effects which can be prevented effectively by dietary glycine.
INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References
METHODS
Top
Abstract
Introduction
Methods
Results
Discussion
References
-(4-pyridyl-1-oxide)-N-tert-butylnitrone (4-POBN) were obtained from Sigma Chemical, St. Louis, MO. A
bicinchoninic acid assay kit was purchased from Pierce Chemical,
Rockford, IL. Racemic pimonidazole hydrochloride was synthesized
according to published procedures (31) and characterized using standard
chromatographic, elemental analysis, and spectrographic techniques.
Monoclonal and polyclonal antibodies against reduced, protein-bound
pimonidazole were prepared as described elsewhere (28).
1 · day1
in the four groups studied (control, glycine, control + cyclosporin A,
and glycine + cyclosporin A). All animals were given humane care in
compliance with institutional guidelines.
Table 1.
Composition of control and glycine-containing diets
Cyclosporin A blood levels. Four hours
after one dose of cyclosporin A (25 mg/kg ig), blood
samples (0.5 ml) were collected from the tail vein into vacutainers
containing 8.5 mg tripotassium EDTA and stored at
20°C until analysis. Cyclosporin A in the blood was
determined using a RIA assay kit (Cyclo-Trac SP) from Incstar
(Stillwater, MN).
Glomerular filtration rates and serum creatinine. Animals were placed in metabolic cages, and urine was collected daily. Creatinine levels in urine and sera were determined using commercially available kits, and glomerular filtration rates were calculated from the ratio of creatinine in the urine/blood, the volume of urine produced in 24 h, and the body weight (16).
Determination of protein-bound pimonidazole using ELISA and immunohistochemistry. Nitroimidazole compounds, which are reductively activated at low oxygen concentrations (5) and bind to cellular macromolecules, have been used to detect hypoxia in a variety of tissues, such as tumors, liver, and kidney (1, 13, 19). To validate the use of pimonidazole, a 2-nitroimidazole, to detect hypoxia in the kidney, the left renal artery was clamped 10 min after administration of pimonidazole hydrochloride (120 mg/kg iv), whereas the right kidney served as control. Fifteen minutes later, the kidneys were rinsed with normal saline to remove blood, and pimonidazole adduct formation in kidney homogenates was detected by ELISA. Indeed, pimonidazole binding was about threefold greater in clamped kidneys than controls. These data and previous work (19) clearly validate the use of pimonidazole as a hypoxia marker in the kidney.
To investigate whether cyclosporin A causes renal hypoxia, pimonidazole hydrochloride (120 mg/kg ip) was injected 2 h after either one single dose or after the last dose in a 5-day treatment regimen with cyclosporin A. Two hours after administration of pimonidazole, kidneys were perfused briefly with normal saline to remove blood. One kidney was frozen for ELISA, and the contralateral kidney was sectioned and fixed for subsequent immunohistochemical analysis.
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. Paraffin blocks of Formalin-fixed kidney tissue were sectioned at 6 µm, and pimonidazole adducts were detected with a biotin-streptavidin-peroxidase indirect immunostaining method (1). Sections were hydrated and treated briefly with 0.01% protease (pronase E) and exposed to mouse anti-pimonidazole IgG antibody in PBS-Tween for 2 h at 37°C. 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.
Areas in sections immunostained for pimonidazole adducts were quantitated by image analysis. A Universal Imaging Image-1/AT image acquisition and analysis system (Chester, PA) incorporating an Axioskop 50 microscope (Carl Zeiss, Thornwood, NY) was used to capture and analyze the immunostained tissue sections at ×40 magnification. Image analysis was performed using a modification of a technique described previously (1). Contiguous microscope fields (×40) were captured to encompass the entire tissue section. Detection thresholds were set for the red-brown color of the 3,3'-diaminobenzidine chromogen based on an intensely labeled point and a default color threshold range was assigned. The degree of labeling in each section was determined from the area within the color range divided by the total cellular area. Some kidney sections were also stained with hematoxylin and eosin and examined microscopically for histological alterations. To evaluate alterations in glomerular size, five glomeruli were selected randomly in each slide. Diameters of glomeruli and Bowman's capsule were measured microscopically using a micrometer in the ocular lens at high-power magnification (×400) at an axis perpendicular to the vascular pole of the renal corpuscle.
Detection of free radical adducts. To assess free radical formation, the spin-trapping reagent 4-POBN (1 g/kg body wt) was dissolved in 2.0 ml normal saline and injected slowly into the tail vein 3 h after the last dose of cyclosporin A. Urine was collected using metabolic cages for 3 h after injection of 4-POBN, and blood samples were collected 3 h later into 50 µl of 30 mM deferoxamine mesylate to prevent free radical formation ex vivo. Samples were kept on dry ice until analysis. Sera were extracted with a mixture of chloroform:methanol (2:1) (26), and the organic layer of extracts from sera was vacuum distilled to dryness and then taken up in 0.5 ml of chloroform. Samples were placed in a quartz ESR cell and bubbled with nitrogen for 5 min. Urine samples were placed in an aqueous flat cell and bubbled with oxygen for 5 min to eliminate interfering ascorbyl free radical and with nitrogen for 5 min to reduce oxygen-derived line broadening. Free radical adducts were detected with a Bruker ESP 106 ESR spectrometer (Bruker Instrument, Billerica, MA). Instrument conditions were as follows: 20-mW microwave power; 1.007-G modulation amplitude, and 80-G scan range (14). Spectral data were stored on an IBM-compatible computer and were analyzed for ESR hyperfine coupling constants by computer simulation (14). The magnitude of the ESR signal was measured at the low-field line (the first line from left) at identical gains and expressed in arbitrary units (1 unit = 1 cm chart paper).
Statistical analysis. All groups were compared using ANOVA + Student-Newman-Keul's post-hoc test. Differences were considered significant at the P < 0.05 level.
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RESULTS |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Cyclosporin A blood levels. Cyclosporin A was undetectable before administration and reached levels around 270 ng/ml after 4 h in rats fed a control diet. Dietary glycine did not alter blood levels of cyclosporin A.
Effects of cyclosporin A and dietary glycine on renal
function. Serum creatinine was similar in control and
glycine-treated rats but was increased about twofold by 5 days of
cyclosporin A treatment, indicating inhibition of renal function as
expected. Glomerular filtration rate, another indicator of renal
function, was around 0.5 ml · min1 · 100 g body wt1 in controls and
declined gradually with time of cyclosporin A treatment. After 5 days
of treatment, glomerular filtration rates were decreased by ~65%
(Fig. 1). Dietary glycine blocked increases in serum creatinine and decreases in glomerular filtration rates caused
by cyclosporin A (Fig. 1). This effect is apparently specific for
glycine, since dietary valine (5%) did not block decreases glomerular
filtration rates caused by cyclosporin A (data not shown).
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Effects of cyclosporin A and glycine on renal histology. Previous work from this laboratory showed that 4 wk of cyclosporin A (25 mg/kg po) treatment caused kidney pathology including proximal tubular dilatation, cell necrosis, and infiltration of macrophages (34). In contrast, after 5 days of cyclosporin A treatment, no pathological changes were observed in proximal tubules (Fig. 2, B, D, and F ). However, the diameter of glomerular capillaries was decreased and Bowman's space was enlarged, most likely due to vasoconstriction of afferent arterioles or disturbances in tubular absorption (Fig. 2B). To estimate this change, diameters of glomerular capillaries (Fig. 3, inset, X) and Bowman's capsule (Fig. 3, inset, Y) were measured microscopically, and volumes were calculated. The ratio of the diameter of glomerulus/Bowman's capsule was decreased 13% by cyclosporin A (P < 0.05; Fig. 3A), resulting in a significant decrease in volume of glomerular capillaries from 82% to 53% (Fig. 3B). Dietary glycine completely prevented the alterations in glomerular size caused by cyclosporin A (Figs. 2C and 3).
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Effect of cyclosporin A and glycine on hypoxia marker binding. Figure 4 depicts representative images of kidneys after pimonidazole adducts were detected immunohistochemically. In kidneys from vehicle-treated controls and from rats treated with glycine alone, pimonidazole adducts accumulated primarily in the outer medulla; this is probably due to the high oxygen demand of this region, where reabsorption of ions occurs and partial pressure of oxygen is low (16). Treatment with cyclosporin A increased the relative area of pimonidazole binding extending from the outer medulla toward the cortex, a region where proximal tubular cells, which are highly vulnerable to hypoxia, predominate (7). Pimonidazole adducts were detected in ~10% of the kidney area in controls, whereas cyclosporin A significantly increased pimonidazole adduct binding by a factor of 1.6 (Fig. 5). Dietary glycine did not affect binding in kidneys from vehicle-treated animals but prevented the increase caused by cyclosporin A completely (Fig. 5). Indeed, the area of pimonidazole-labeled cells in kidneys from rats fed glycine and treated with cyclosporin A was not significantly different from control values.
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Although immunohistochemistry detects predominantly protein-bound adducts, quantitation of pimonidazole binding with ELISA detects both protein and nonprotein adducts (e.g., GSH adducts). Figure 6 summarizes the effect of treatment with cyclosporin A for one (Fig. 6A) and 5 days (Fig. 6B) on pimonidazole binding in rats fed control or glycine-containing diets. Level of pimonidazole adduct formation in control kidneys was 312 pmol/mg protein; glycine diet alone did not alter this background value. In contrast, one dose of cyclosporin A increased pimonidazole binding slightly but significantly. However, treatment with cyclosporin A for 5 days increased binding threefold. Importantly, the effects of both one dose and 5 days of treatment with cyclosporin A on pimonidazole binding were prevented with dietary glycine.
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Effects of cyclosporin A and glycine on free radical production. Reperfusion subsequent to hypoxia caused by cyclosporin A could theoretically lead to free radical formation. Accordingly, free radicals were captured with the spin-trapping reagent 4-POBN and detected with ESR. Figure 7A depicts a representative ESR spectrum due to free radical adducts in urine from a cyclosporin A-treated rat. A 6-line ESR spectrum due to 4-POBN radical adducts was detected in urine samples from all animals receiving cyclosporin A. Computer simulation of the spectrum (Fig. 7B) revealed a free radical species having hyperfine coupling constants for nitrogen and hydrogen, respectively, of aN = 15.6 G and aH = 2.4 G, values typical of carbon-centered 4-POBN radical adducts in an aqueous solution. No exact match with radical adducts listed in the NIEHS data base was obtained, presenting the possibility that the species trapped is a new free radical (17). Free radical adducts in urine were increased over threefold after one single dose of cyclosporin A (data not shown) but were increased nearly sixfold after treatment for 5 days (Fig. 8). In contrast, different weak radical species based on coupling constants were detected in serum extracts; however, they were not altered by cyclosporin A (data not shown). Therefore, it is concluded that cyclosporin A increases a new free radical exclusively in the kidney. Significantly, increases in free radical production caused by cyclosporin A in the kidney were blocked totally by dietary glycine (Fig. 8).
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DISCUSSION |
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Top
Abstract
Introduction
Methods
Results
Discussion
References
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Cyclosporin A causes hypoxia in the kidney. Previous studies showed that cyclosporin A causes vasoconstriction in the kidney (2, 3, 20, 24). In an elegant study, English et al. (3) detected progressive constriction of afferent glomerular arterioles over 14 days of cyclosporin A treatment, and renal hypoperfusion as reflected by decreased renal plasma flow and increased renal vascular resistance has been detected in both humans and in animals (2). Therefore, cyclosporin A could theoretically cause hypoxia in the kidney, thus leading to cell injury. However, the kidney receives very high rates of blood flow, so whether hypoxia occurs after cyclosporin A treatment has remained, until now, a subject of debate. In the kidney, 2-nitroimidazole adduct binding was increased in vivo when oxygen was decreased (19); moreover, 2-pimonidazole binding was increased threefold when the renal artery was clamped, validating its use of as a hypoxia marker in renal tissue. In this study, renal hypoxia was detected for the first time in conscious rats following cyclosporin A treatment using pimonidazole (Figs. 5 and 6), a 2-nitroimidazole which is reductively activated at low oxygen concentrations (5) and binds to cellular macromolecules; it has been used to detect the hypoxic fraction of tumors (13). Pimonidazole adducts accumulate in vivo in intact, awake animals and measure tissue hypoxia directly at the cellular level; therefore, results from this study demonstrate clearly for the first time that cyclosporin A treatment causes hypoxia in renal cells.
Cyclosporin A causes hypoxia most likely via vasoconstriction when its blood levels increases. Indeed, hypoxia was detected after administration of cyclosporin A using pimonidazole (Figs. 5 and 6). Cyclosporin A reduced glomerular size and significantly diminished glomerular filtration rates (Figs. 1 and 3) in this study, supporting the hypothesis that it causes vasoconstriction. The question of how cyclosporin A causes vasoconstriction is unclear; one possibility is that it increases sympathetic nerve activity (Fig. 9). Consistent with this hypothesis, cyclosporin A increased renal nerve firing (23), and kidney injury was diminished if the nerve was severed (24). Alternatively, cyclosporin A could directly stimulate vascular smooth muscle or mesangial cell contraction (Fig. 9), processes dependent on influx of calcium. Indeed, cyclosporin A has been shown to increase intracellular calcium in these cells (18, 21) and cause vasoconstriction directly in isolated arterial rings (40). Cyclosporin A also increases many vasoactive mediators, such as renin-angiotensin II (24), thromboxanes (29), and endothelins (15), which could contribute to vasoconstriction. Taken together, it is concluded that cyclosporin A causes vasoconstriction leading to hypoxia in the kidney, which is most likely a critical early event in cyclosporin A-induced renal injury. Therefore, these data lead to the hypothesis that glycine reduces cyclosporin A nephrotoxicity by blocking hypoxia (see below).
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Cyclosporin A increases free radical formation. Another important finding of this study is that cyclosporin A increases free radical formation (Fig. 8). Previous studies showed that antioxidants such as vitamin E, ascorbate, lazaroids, and superoxide dismutase/catalase diminished cyclosporin A-induced renal toxicity (36, 39); however, until this study, physical evidence for free radicals after cyclosporin A treatment has been lacking. Here a kidney-specific free radical adduct due to cyclosporin A was detected for the first time. The coupling constants of the radical adduct detected in urine are not similar to any known free radicals (17); therefore, it is possible that a new carbon-centered free radical species was detected here in the urine after cyclosporin A treatment. It is also possible that free radicals were produced systemically but cleared by the kidney; under such circumstances, they would have to be transported to the kidney by blood. However, free radical signal intensity was not different between control and cyclosporin A-treated rats in serum extracts but was increased nearly sixfold in the urine. Therefore, cyclosporin A most likely increases a unique free radical exclusively in the kidney, the major target organ of cyclosporin A toxicity. Since cyclosporin A increased free radical formation dramatically and most likely exclusively in the kidney, these data are consistent with the hypothesis that hypoxia-reoxygenation is a pivotal early event leading to renal cell injury caused by cyclosporin A.
How cyclosporin A augments free radical formation is unknown, but free radicals may be derived directly from cyclosporin A or its metabolites. Consistent with this possibility, cyclosporin A increased malondialdehyde, a product of lipid oxidation, in isolated hepatic microsomes, the major metabolic site for cyclosporin A (10), suggesting that cyclosporin A or its metabolites produce free radical species that attack lipid components, leading to lipid peroxidation. Therefore, it is possible that metabolism of cyclosporin A by renal cytochrome P-450 could directly produce free radicals. Moreover, cyclosporin A inhibits mitochondrial respiration in renal tubular cells (12), which could also lead to free radical production. Alternatively, reperfusion subsequent to vasoconstriction and hypoxia caused by cyclosporin A could increase free radical formation. It is possible that when cyclosporin A blood levels start to decline, blood vessels dilate, and reoxygenation occurs. It is also possible that some vessels dilate while others constrict within the kidney. In this study, free radicals were detected after cyclosporin A treatment. Glycine, which blocks hypoxia, also prevented free radical production (Fig. 6 and 8), supporting the hypothesis that cyclosporin A causes hypoxia-reoxygenation. Consistent with these results, glycine has been shown to improve microcirculation and prevent hypoxia-reperfusion injury in the liver (41).
How does glycine prevent cyclosporin A-induced nephrotoxicity? How glycine prevents cyclosporin A nephrotoxicity is unclear. Previous studies showed that glycine did not block ATP depletion during hypoxia; however, it protected tubular cells during hypoxia and reoxygenation (6, 38). These studies suggested that protection was independent of ATP. It has also been shown that glycine inhibits activation of phospholipase A2 during hypoxia/reoxygenation and increases membrane stability (30), inhibits proteases (25), blocks activation of calpains, and inhibits chloride influx during hypoxia/reoxygenation (37). These mechanisms may contribute to the protective effects of glycine. Another possibility is that glycine influences pharmacokinetics of cyclosporin A; however, it did not alter blood levels or pharmacokinetics of cyclosporin in both rats (RESULTS) and dogs (B. Briner and H. Schneider, 1997; personal communication). Alternatively, glycine could prevent nephrotoxicity by blocking hypoxia-reoxygenation caused by cyclosporin A. In support of this idea, glycine minimized hypoxia-reperfusion injury caused by 45 min of clamping of the renal artery (M. Yin and R. G. Thurman, 1997; unpublished data). Dietary glycine, which prevented pathological changes associated with chronic cyclosporin A treatment (34), significantly diminished pimonidazole adduct formation, an indicator of renal hypoxia, and free radical production in urine following short term treatment in this study (Fig. 5). Therefore, it is likely that glycine works by blocking hypoxia-reoxygenation caused by cyclosporin A. Glycine, an inhibitory amino acid which hyperpolarizes the nerve cell membrane (11), may decrease renal sympathetic nerve firing (Fig. 9). Indeed, dietary glycine blocked the increases in efferent renal nerve activity caused by cyclosporin A (Z. Zhong, N. Moss, and R. G. Thurman, unpublished data). Moreover, glycine blunts increases in intracellular calcium in a variety of cells (8, 9, 22, 27, 32); therefore, it could also directly block contraction of vascular smooth muscle and mesangial cells (Fig. 9), thus preventing vasoconstriction in the kidney. Indeed, previous studies have shown that perfusion with glycine increased renal blood flow (7), and in this study, glycine prevented changes in glomerular size and volume and hypoxia caused by cyclosporin A (Figs. 3 and 5), supporting the hypothesis that it prevents hypoxia-reperfusion by blocking vasoconstriction. Moreover, metabolically active proximal tubular cells are very vulnerable to hypoxia, and glycine has been shown to protect cultured proximal tubule cells from hypoxic injury (35). Moreover, cyclosporin A treatment increased the area of hypoxia which extended from the outer medulla into the cortex, a region where proximal tubular cells predominate. This alteration was also blocked totally by glycine (Fig. 4). Therefore, it is concluded that glycine prevents cyclosporin A nephrotoxicity by minimizing hypoxia-reoxygenation.
Hypoxia and free radical formation precede pathological changes caused by cyclosporin A. Nephrotoxicity is the most frequent and severe side effect of cyclosporin A. Previous studies in this laboratory showed that treatment with cyclosporin A for 28 days caused impairment of renal function and pathological alterations in the kidney including proximal tubular cell swelling, necrosis, infiltration of macrophages, and interstitial fibrosis (34). In contrast, 5 days of treatment with cyclosporin A in this study impaired renal function reflected by decreased glomerular filtration rates (Fig. 1) and increased serum creatinine but did not cause overt pathology (Fig. 2). Interestingly, vasoconstriction (Fig. 3), hypoxia reflected by increased pimonidazole binding (Figs. 5 and 6) and free radical formation increased dramatically (Figs. 7 and 8) after 5 days of cyclosporin A, indicating that hypoxia-reoxygenation precedes pathological changes. Glycine, which prevented renal pathology after chronic cyclosporin A treatment (34), largely blocked both hypoxia and free radical formation after short-term treatment with cyclosporin A (Figs. 6 and 8). Taken together, this study provides, for the first time, physical evidence for cyclosporin A-induced hypoxia and free radical production in the kidney. It follows that cyclosporin A causes renal injury by mechanisms involving hypoxia-reoxygenation. Glycine blocks these alterations, thus effectively preventing cyclosporin A toxicity.
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ACKNOWLEDGEMENTS |
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We thank Dr. Gisbert Weckbecker (Novartis Pharma, Basel, Switzerland) for assistance with cyclosporin A measurements.
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FOOTNOTES |
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This study was supported, in part, by grants from National Institutes of Health.
Preliminary account of this work has been published in abstract form (Pharmacologist 39: 50, 1997).
Address for reprint requests: R. G. Thurman, Laboratory of Hepatobiology and Toxicology, Dept. of Pharmacology, CB 7365, Mary Ellen Jones Bldg., Univ. of North Carolina at Chapel Hill, Chapel Hill, NC 27599-7365.
Received 1 October 1997; accepted in final form 30 July 1998.
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Abstract
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Discussion
References
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r3;
B) of glomerulus
(X)/Bowman's capsule
(Y) (see
inset) are presented.
Inset: diagram of glomerulus and
Bowman's capsule. X, diameter of
glomerulus; Y, diameter of Bowman's
capsule. Values are means ± SE (ANOVA;
n = 4-5 in each group).
a P < 0.05 compared with control.
b P < 0.05 compared with the CsA group.
