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Localization of the thioredoxin system in normal rat kidney
Free Radical Biology & Medicine, Vol. 30, No. 4, pp. 412– 424, 2001 Copyright © 2001 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/01/$–see front matter
PII S0891-5849(00)00486-X
Original Contribution LOCALIZATION OF THE THIOREDOXIN SYSTEM IN NORMAL RAT KIDNEY TERRY D. OBERLEY*†, ERIC VERWIEBE,† WEIXIONG ZHONG,† SANG WON KANG,‡
and
SUE GOO RHEE‡
*Pathology and Laboratory Medicine Service, Veterans Administration Hospital, Madison, WI, USA; †Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI, USA; and ‡Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, MD, USA (Received 31 August 2000; Revised 7 November 2000; Accepted 17 November 2000)
Abstract—Components of the thioredoxin system were localized in normal rat kidney using immunoperoxidase techniques at the light microscopic level and immunogold techniques at the ultrastructural level. Results from both methods were similar. Thioredoxin, thioredoxin reductases, and peroxiredoxins showed cell-type-specific localization, with the same cell types (proximal and distal tubular epithelial, papillary collecting duct, and transitional epithelial cells) previously identified as having high amounts of antioxidant enzyme immunoreactive proteins and oxidative damage products also having high levels of proteins of the thioredoxin system. In addition, peroxiredoxins II and IV were found in high levels in the cytoplasm of red blood cells, identified in kidney blood vessels. While thioredoxin and thioredoxin reductase 1 were found in all subcellular locations in kidney cells, thioredoxin reductase 2 was found predominantly in mitochondria. Thioredoxin reductase 1 was identified in rat plasma, suggesting it is a secreted protein. Peroxiredoxins often had specific subcellular locations, with peroxiredoxins III and V found in mitochondria and peroxiredoxin IV found in lysosomes. Our results emphasize the complex nature of the thioredoxin system, demonstrating unique cell-type and organelle specificity. © 2001 Elsevier Science Inc. Keywords—Thioredoxin, Thioredoxin reductase, Peroxiredoxin, Reactive oxygen species, Kidney, Free radicals
INTRODUCTION
and interleukin-2 receptors [5,6]. Trx can modulate the DNA-binding activity of some transcription factors either directly (TFIIIC [7], BZLF1 [8], and NF-B [9]) or indirectly (AP-1) through the nuclear factor Ref-1, which in turn is reduced by thioredoxin [10]. Thioredoxin has been shown to regulate activity of the p53 tumor supressor protein by redox mechanisms, including coupling of the oxidative stress response and the p53-dependent DNA repair mechanism [11]. Thioredoxin can be secreted by cells [12–14] and stimulates the proliferation of lymphoid cells, fibroblasts, and a variety of human tumor cell lines [15–18]. Thioredoxin is thought to protect against oxidative damage, since it can reduce hydrogen peroxide [19], scavenge free radicals [20], and protect cells against oxidative stress [21]. Two thioredoxins have been identified in mammalian cells by cell fractionation analysis— one located in cytoplasm and nucleus (Trx1) and one located in mitochondria (Trx2) [22]. Reactive oxygen species (ROS), such as hydrogen peroxide, superoxide anion radical, and hydroxyl radical, have been thought of as toxic byproducts of cellular
Thioredoxin (Trx) is a 12-kDa protein, known to be present in many prokaryotes and eukaryotes and appears to be truly ubiquitous in all living cells [1,2]. It is characterized by an active site sequence, Trp-Cys-GlyPro-Cys-Lys, that is conserved throughout evolution. The two cysteine residues provide the sulfhydryl groups involved in Trx-dependent reducing activity. Oxidized thioredoxin, Trx-S2, is reduced to Trx-(SH)2 by the flavoenzyme thioredoxin reductase (TrxR) and NADPH. Mammalian thioredoxin has been implicated in a wide variety of biochemical functions. It can act as a hydrogen donor for ribonucleotide reductase [3] and methionine sulfoxide reductase [2], facilitate refolding of disulfidecontaining proteins [4], and activate the glucocorticoid Address correspondence to: Dr. Terry D. Oberley, William S. Middleton Memorial Veterans Administration Hospital, Pathology and Laboratory Medicine Service, 2500 Overlook Terrace, Room A35, Madison, WI 53705, USA; Tel: (608) 256-1901, ext. 11722; Fax: (608) 280-7087; E-Mail: [email protected]. 412
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oxygen metabolism. However, recent evidence indicates that at low concentrations, ROS mediate regulatory events and are essential participants in cell signaling [23]. Redox signaling is thought to be achieved through the coupling of ROS with oxidation-reduction processes that involve essential thiol groups in proteins, resulting in the modulation of tyrosine or serine-threonine phosphorylation of target proteins [24,25]. The redox state of essential thiol groups is controlled by two cellular redox systems: the thioredoxin (thioredoxin, thioredoxin reductase, and thioredoxin peroxidase) and glutathione (glutathione, glutathione reductase, glutaredoxin, and glutathione peroxidase) systems [26,27]. Several proteins have been identified as components of redox signaling pathways that act downstream of the generation of intracellular ROS [28 –30]. The redox state and activity of Trx are controlled by TrxR, a selenocysteine-containing flavoprotein composed of two identical 57-kDa subunits [31–35]. Three TrxR proteins have been identified to date (TrxR1, TrxR2, and TrxR3); using cell fractionation and biochemical analyses, TrxR1 was localized in the nucleus and cytoplasm, while TrxR2 and TrxR3 were localized in mitochondria. All three mammalian TrxRs conserve the COOH-terminal penultimate selenocysteine residue. Sun et al. [36] found that when cells generated ROS, selenocysteine in Trx1 was oxidized, while Trx1 expression was elevated. These results suggest that the selenocysteine residue in thioredoxin reductases serves as a cellular redox sensor. Peroxiredoxins (Prx) are a large antioxidant gene family that is well conserved from bacteria to humans. Several of the peroxiredoxins were initially identified by some of the diverse functions they perform, for example, enhancement of natural killer cytotoxicity [37], cell proliferation [38], and heme binding [39]. The family name, Prx, was chosen to reflect the fact that this is a family of peroxidases that uses conserved cysteine (Cys) residues for the sites of antioxidation. The Prx family contains six known protein subtypes [40]. In addition to the four Prx family members with two conserved Cys residues (2Cys), there are also two additional Prx families that contain only one conserved Cys residue (1-Cys). Prxs I–V all use thioredoxin as the electron donor, whereas the electron donor for Prx VI has yet to be identified. To date, most studies of the thioredoxin system have relied on biochemical and molecular biology analyses of tissue homogenates. While such studies may be highly informative, they provide data on the average values for all cell types within a tissue and do not allow subcellular characterization unless further cell fractionation studies are performed. To provide information on cellular and subcellular localization of the thioredoxin system, we chose to study the rat kidney, a complex organ composed
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of many cell types. The present study was performed to determine the cellular and subcellular localization of thioredoxin and thioredoxin reductases and peroxidases in various cell types of the kidney. Our studies show that each protein has unique cellular and subcellular localization. Three proteins, thioredoxin reductase 2 and peroxiredoxins III and V were found predominantly in mitochondria, emphasizing the need for protection of mitochondria from oxidant stress. MATERIALS AND METHODS
Animals Male Wistar rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN, USA). They were transferred at 8 weeks of age (200 –250 g) to the Shared Aging Rodent Facility at the Geriatric Research, Education, and Clinical Center (William S. Middleton Veterans Administration Hospital; Madison, WI, USA). For 2 weeks, the rats were housed individually in solid bottom polycarbonate cages with wire tops and fed a nonpurified diet (Lab Rodent Chow 5001; Purina Feeds; Richmond, IN, USA) ad lib. Rats weighing 300 –350 g (10 weeks old) were then used for analysis. For immunoperoxidase analysis at the light microscopic level, the present study examined 6 rats. For immunogold analysis at the ultrastructural level, 2 rats were studied. A portion of both kidneys from each rat was fixed in 10% neutral buffered formalin for light microscopy immunoperoxidase analysis and Carson-Millonig’s fixative for electron microscopy immunogold analysis.
Antibodies Rabbit antibody to thioredoxin was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Western blot analysis of homogenates of human prostate epithelial cell lines (immortalized, nonmalignant, and malignant) showed a single band of molecular weight 12 kDa, indicating that the antibody to E. coli thioredoxin recognized human thioredoxin. It is not known which thioredoxin form the antibody is detecting. Rabbit antibodies to TrxR1 and R2 and Prxs I–VI were obtained from the laboratory of Dr. Sue Goo Rhee, National Institutes of Health. Their specificities have been previously described [41– 44]. Western blot analyses of homogenates of the same human prostate epithelial cell lines using these antibodies showed appropriate bands of the correct molecular weights (thioredoxin reductase: 55–58 kDa; peroxiredoxin I–IV: 22 kDa; peroxiredoxin V: 17 kDa; peroxiredoxin VI: 25 kDa, data not shown).
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Light microscopy immunoperoxidase technique Tissues were fixed in 10% neutral buffered formalin and processed for embedding in paraffin blocks, using routine histology protocols [45]. For immunohistochemistry, deparaffinized and hydrated 4 micron sections were mounted on slides coated with 3-aminopropyltriethoxysilane. Primary antibodies were incubated with tissue sections overnight at 4°C. The immunoperoxidase procedure used a commercial development kit (LSAB, Dako K0690; Dako Corporation; Carpinteria, CA, USA), and metal enhanced diaminobenzidine substrate (Pierce #34065; Pierce; Rockford, IL; USA). A standard antigen retrieval protocol was utilized. Deparaffinized and rehydrated sections were treated with Tris urea solution (Tris, 121.1 g; urea, 50 g; 800 ml water; pH to 9.5 with concentrated hydrochloric acid); this solution was heated to 90°C, and the slides exposed to this heated solution for first 5 min and then 30 min. Optimal staining was obtained at a 1:100 dilution for all antibodies studied. After immunostaining, slides were counterstained with Harris hematoxylin. The specificity of immunostaining was determined by replacing the primary antibody with normal rabbit serum at the same concentration as the primary antiserum or with diluent buffer (Tris-buffered saline: 150 mM sodium chloride buffered with 50 mM Trizma base, pH 7.4). No staining was observed in these control slides. The degree of immunostaining was judged by comparison to normal rabbit serum controls, and labeling was characterized as negative, trace, light, moderate, and heavy. Photomicrographs shown in the figures are representative of all animals studied. Immunogold ultrastructural analysis Immunogold electron microscopy was performed as previously described [45]. All dehydration and infiltration steps with LR White acrylic resin (Electron Microscopy Sciences; Fort Washington, PA, USA) were undertaken at room temperature unless otherwise noted. Tissues were cut into 1 mm3 blocks and fixed in CarsonMillonig’s fixative (4% formaldehyde in 0.16 M monobasic sodium phosphate buffer, pH 7.2) for 1 h. After rinsing for 30 min in 0.1 M phosphate buffer, pH 7.4, the samples were dehydrated by 30 min changes each of 70, 80, and 90% ethanol, then LR White resin/90% ethanol (2/1, v/v). After immersion and infiltration in undiluted LR White resin overnight, the samples were washed with fresh undiluted LR White resin for 1 h. Resin polymerization was thermally induced in sealed gelatin capsules at 45°C for 48 h in the absence of accelerator. Ultrathin sections (70 – 80 nm) were cut and transferred to nickel grids (G300-NI, Electron Microscopy Sciences) for postembedding immunogold procedures. These sections
were rinsed with Tris-buffered saline (TBS: 0.05 M Tris, 0.9% NaCl, pH 7.6) for 10 min and incubated with 2% BSA and 0.2% Tween-20 in TBS for 30 min to block nonspecific antibody sites. Sections were then incubated with primary antibody (all antibodies were used at a dilution of 1:100, except anti-Trx R1 at 1:50 and anti-Trx R2 at 1:25) overnight at 4°C. After washing four times with TBS wash buffer (1:10 dilution of block buffer), for 5 min each, the sections were washed in one change of alkaline TBS (pH 8.2) for 20 min. The grids were incubated with diluted (1:75) gold conjugated goat antirabbit IgG (EM.GAR15, Goldmark) for 90 min at room temperature. The sections were washed in two changes of TBS wash buffer for 10 min each followed by 2 changes of distilled water for 5 min each. Following counterstaining with 4% aqueous uranyl acetate for 10 min, photographs were taken with a Hitachi H-600 transmission electron microscope operated at 75 kV. The specificity of immunostaining was determined by replacing the primary antibody with normal rabbit serum or preimmune serum at the same concentration as the test antiserum or diluent buffer (Trisbuffered saline: 150 mM sodium chloride buffered with 50 mM Trisma base, pH 7.4). No significant immunolabeling was observed in the sections prepared under these control conditions. Distinct cellular and subcellular labeling greater than background was regarded as positive. Labeling of organelles was graded as negative, trace, light, moderate, and heavy. Photomicrographs shown in the figures are representative of each animal studied. RESULTS
Light microscopy analysis of the thioredoxin system in adult rat kidney Thioredoxin. For all proteins of the thioredoxin system studied, major staining was observed in the proximal and distal tubules (Fig. 1), collecting tubules of the papillae, and transitional epithelium (Fig. 2). Other cell types, including glomerular cells (Fig. 1), collecting ducts in the cortex and superficial medulla, and cells of the loops of Henle showed only light or no staining, with some exceptions noted below. Representative cell types that showed significant staining are described below, though glomerular cells are also illustrated to show the relative light staining observed with these cell types (Fig. 1). In addition, it should be mentioned that the ducts of Bellini (terminal collecting ducts in the papilla of the kidney) showed staining patterns (data not shown) similar to transitional epithelium of the renal pelvis described herein (Fig. 2). In all cases, tissues stained with normal rabbit serum instead of primary antibody showed no significant labeling (Fig. 3).
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Fig. 1. Light microscopy immunoperoxidase analysis of cell types in rat outer kidney cortex using antibodies specific to selected proteins of the thioredoxin system. (A) Anti-thioredoxin: Proximal tubules (PT) showed moderate labeling of both nuclei and cytoplasm. Glomeruli (G) demonstrated only trace labeling. (B) Anti-thioredoxin reductase 1: Proximal (PT) and distal (DT) tubular cells exhibited moderate staining of both cytoplasm and nucleus. Glomeruli (G) were notable for moderate staining of endothelial (EN) cytoplasm and nucleus. (C) Anti-thioredoxin reductase 2: Proximal (PT) and distal (DT) tubular cells showed moderate nuclear and cytoplasmic staining. Glomeruli (G) exhibited only trace staining. (D) Anti-peroxiredoxin I: Proximal (PT) tubular cells had moderate cytoplasmic and focal heavy nuclear labeling (arrowhead). Glomeruli (G) showed only trace staining. (E) Anti-peroxiredoxin II: Proximal (PT) tubular cells demonstrated moderate cytoplasmic and nuclear labeling, while distal (DT) tubular cells had heavy cytoplasmic label. Red blood cells (RBC) had heavy cytoplasmic label, while glomeruli (G) exhibited only trace labeling. (F) Anti-peroxiredoxin III: Proximal (PT) tubular cells had moderate nuclear and cytoplasmic labeling, while distal (DT) tubular cells had intense cytoplasmic staining. Glomeruli (G) showed only trace staining. (G) Anti-peroxiredoxin IV: Proximal (PT) tubular cells exhibited prominent granular staining in the cell cytoplasm (curved arrow). Red blood cells (RBC) had heavy cytoplasmic staining, while glomeruli (G) exhibited only trace labeling. (H) Anti-peroxiredoxin V: Proximal (PT) tubular cells showed moderate nuclear and cytoplasmic label, while distal (DT) tubular cell had heavy cytoplasmic labeling. Glomeruli showed only trace staining. (I) Anti-peroxiredoxin VI: Proximal (PT) and distal (DT) tubular cells showed light to moderate nuclear and cytoplasmic staining. Glomeruli exhibited light labeling. For A through I, ⫻ 3500.
Results with antibody to Trx showed moderate labeling of nuclei and cytoplasm of both proximal (Fig. 1A) and distal tubular cells. Glomerular cells demonstrated
only trace staining (Fig. 1A). Nuclei and cytoplasm of transitional epithelium showed light to moderate labeling (Fig. 2A). Normal rabbit serum controls performed at the
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Fig. 2. Light microscopy immunoperoxidase analysis of transitional epithelium of renal pelvis of rat kidney using antibodies specific to selected proteins of the thioredoxin system. (A) Anti-thioredoxin; (B) Anti-thioredoxin reductase 1; (C) Anti-thioredoxin reductase 2; (D) Anti-peroxiredoxin I; (E) Anti-peroxiredoxin II; (F) Anti-peroxiredoxin III; (G) Anti-peroxiredoxin IV; (H) Anti-peroxiredoxin V; (I) Anti-peroxiredoxin VI. Thioredoxin and thioredoxin reductases 1 and 2 showed light to moderate staining of transitional epithelium (TE), while peroxiredoxins showed moderate labeling, except peroxiredoxin V, which showed heavy labeling. In all cases, both nuclei and cytoplasm appear to show staining. For A through I, ⫻ 3500.
same time showed no significant staining of any cell type (Fig. 3). Thioredoxin reductases. TrxR1 showed moderate staining of nuclei and cytoplasm of both proximal and distal tubular epithelial cells (Fig. 1B). Glomerular endothelial cells demonstrated moderate labeling of nuclei and cytoplasm (Fig. 1B). Nucleus and cytoplasm of transitional epithelium also demonstrated light to moderate labeling for TrxR1 (Fig. 2B).
Proximal and distal tubular cells (Fig. 1C) immunostained with TrxR2 had moderate labeling of nuclei and cytoplasm. Glomeruli did not have significant staining (Fig. 1C). Transitional epithelium had heavy nuclear label, but only light to moderate cytoplasmic staining (Fig. 2C). Peroxiredoxins. Prx I exhibited heavy nuclear and moderate cytoplasmic labeling in proximal (Fig. 1D) and distal epithelial cells. Glomeruli showed light staining
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Cytoplasm of distal tubular epithelial cells demonstrated heavy staining when immunolabeled with antibody to Prx V, while proximal tubule epithelial cells showed moderate labeling (Fig. 1H). Glomeruli and red blood cells did not exhibit significant label (Fig. 1H). Transitional epithelium (Fig. 2H) demonstrated heavy immunolabeling of both nuclei and cytoplasm. Prx VI staining was light in nuclei and cytoplasm of kidney proximal and distal tubular epithelial cells, with only trace labeling in glomeruli (Fig. 1I). Transitional epithelium showed moderate nuclear and cytoplasmic staining (Fig. 2I). Subcellular localization of the thioredoxin system in kidney proximal tubular and glomerular cells
Fig. 3. Immunoperoxidase analysis of staining of normal rat kidney using normal rabbit serum in place of primary antibody. (A) Renal cortex: Glomeruli (G) and proximal (PT) tubular cells did not have any labeling. (B) Transitional epithelium: Transitional epithelium (TE) did not exhibit significant labeling. For A and B, ⫻ 5800.
(Fig. 1D). Nuclei and cytoplasm of transitional epithelium had moderate staining (Fig. 2D). Prx II showed heavy labeling of distal tubular cell cytoplasm, while proximal tubular cells exhibited only moderate labeling (Fig. 1E). Glomeruli exhibited only trace labeling, though erythrocytes within glomerular capillaries were strongly positive (Fig. 1E). Transitional epithelium (Fig. 2E) demonstrated moderate nuclear and cytoplasmic staining. Kidney tissues immunostained with antibody to Prx III had heavy cytoplasmic label in distal tubular cells, moderate cytoplasmic label in proximal tubular cells, and only trace labeling in glomeruli (Fig. 1F). Transitional epithelium exhibited moderate staining of nuclei and cytoplasm, except for the most superficial layer, which showed heavy staining (Fig. 2F). Prx IV showed heavy granular localization in kidney proximal tubular cells and diffuse staining of erythrocytes in glomerular capillaries (Fig. 1G). Transitional epithelium exhibited moderate staining of nuclei and cytoplasm (Fig. 2G).
Thioredoxin. Proximal tubular cells showed heavy mitochondrial labeling and moderate nuclear labeling for Trx (Fig. 4A). These same cells showed light labeling of cytoplasm and microvilli. Although glomerular cells usually showed only light or trace staining by light microscopy, immunogold ultrastructural analysis often revealed these cells were actually positive for components of the thioredoxin system. Intrinsic cells of kidney glomeruli are composed of three cell types: endothelial, mesangial, and visceral epithelial cells (podocytes). Each of these glomerular cell types have only sparse and small mitochondria compared to the large and numerous mitochondria present in proximal and distal tubular epithelial cells. Endothelial and mesangial cell nuclei exhibited moderate labeling, while cytoplasm showed only light labeling (Fig. 5A). Glomerular epithelial cells demonstrated light labeling of both nucleus and cytoplasm (not shown). Red blood cells in glomerular capillaries were also evaluated and showed moderate cytoplasmic label. Thioredoxin reductases. TrxR1 demonstrated heavy nuclear and mitochondrial labeling in proximal tubular epithelial cells (Fig. 4B), while cytoplasm and microvilli exhibited light labeling. Glomerular endothelial and mesangial cells showed moderate labeling of nuclei and light labeling of cytoplasm (Fig. 5B). Glomerular epithelial cells showed similar subcellular distribution of immunogold beads as proximal tubular cells, but only light label (not shown). Red blood cells exhibited light cytoplasmic label. TrxR1 was identified in human plasma (Fig. 5B). TrxR2 was predominantly mitochondrial, but showed lighter staining intensity in all kidney cell types examined (proximal tubule, Fig. 4C; glomerulus, Fig. 5C). Normal rabbit serum control of proximal tubular cells (Fig. 4D) or glomerular cells (Fig. 5D) did not exhibit significant labeling. Peroxiredoxins. Prx I was found in moderate amounts in nuclei, cytoplasm, and microvilli of proximal tubular
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Fig. 4. Immunogold ultrastructural analysis of rat kidney proximal tubular epithelial cells using antibodies specific for thioredoxin and thioredoxin reductases. (A) Anti-thioredoxin: Immunogold labeling was found throughout the cell, including the nucleus (N; arrowhead), mitochondria (M; single arrows), and cytoplasm (double arrow). (B) Anti-thioredoxin reductase 1: Immunogold labeling was observed throughout the cell, including the nucleus (N; arrowheads), mitochondria (M; single arrow), and cytoplasm (double arrow). (C) Anti-thioredoxin reductase 2: Immunogold labeling was found predominantly in the mitochondria (M; single arrow), though trace labeling in the nucleus (N; arrowhead) and cytoplasm (double arrow) was present. (D) Normal rabbit serum: No immunogold beads were present. For A through D, ⫻ 14,000.
epithelial cells, while mitochondria demonstrated only light immunogold labeling (Fig. 6A). The nuclei of glomerular mesangial and endothelial cells exhibited moderate labeling (Fig. 7A), while podocyte nuclei showed only a small number of immunogold beads (light labeling; not shown). The cytoplasm in all glomerular cell types showed only light label. Erythrocytes in glomerular capillaries demonstrated only trace labeling (not shown). Label was thus predominantly cytoplasmic and nuclear,
with only light mitochondrial immunogold bead deposition. Prx II was found in a similar distribution to Prx I (proximal tubule, Fig. 6B; glomerulus, Fig. 7B). The only exception was the heavy labeling observed in erythrocyte cytoplasm (Fig. 7B). Prx III was found almost exclusively in mitochondria and lysosomes of proximal tubular epithelial cells (Fig. 6C). Since glomerular cells have only few mitochondria,
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Fig. 5. Immunogold ultrastructural analysis of rat kidney glomerular cells using antibodies specific for thioredoxin and thioredoxin reductases. (A) Anti-thioredoxin: Immunogold labeling was found throughout the glomerular cells. This photomicrograph illustrates moderate labeling of mesangial cell nuclei (N; arrowheads) and light labeling of mesangial cell cytoplasm (double arrow). (B) Anti-thioredoxin reductase 1: Immunogold labeling was found throughout the glomerular cells. This photomicrograph illustrates moderate labeling of an endothelial cell nucleus (N; arrowhead), light labeling of endothelial cytoplasm (double arrow), and significant labeling of plasma (curved arrow). (C) Anti-thioredoxin reductase 2: Only light or trace labeling was observed in glomerular cells. This photomicrograph shows light labeling of an endothelial cell nucleus (N; arrowhead). (D) Normal rabbit serum: No immunogold beads were identified. For A through C, ⫻ 14,000; for D, ⫻ 11,000.
these cells showed only light immunolabel (Fig. 7C). Red blood cells demonstrated trace immunolabeling (not shown). Prx IV showed numerous immunogold beads in lysosomes of proximal tubular cells (Fig. 6D), with only trace, probably nonspecific, deposition of immunogold beads in other subcellular locations. Glomeruli showed light labeling of nucleus and cytoplasm (Fig. 7D). Cyto-
plasm of erythrocytes exhibited moderate labeling (Fig. 7D). Prx V showed moderate mitochondrial labeling in proximal (Fig. 6E) tubular epithelial cells. Glomeruli (Fig. 7E) and red blood cells demonstrated only trace labeling. Prx VI showed only trace labeling in all kidney cell types and subcellular locations (proximal tubule, Fig. 6F;
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Fig. 6. Immunogold ultrastructural analysis of rat kidney proximal tubular epithelial cells using antibodies specific for peroxiredoxins. (A) Anti-peroxiredoxin I: Immunugold label was found throughout the cell, primarily in the nucleus (N; arrowheads) and cytoplasm (double arrows), but also rarely in mitochondria (M; single arrow). (B) Anti-peroxiredoxin II: Immunogold label was found throughout the cell, primarily in the nucleus (N; arrowheads) and cytoplasm (double arrows, including base of microvilli, MV), but also rarely in mitochondria (M; single arrow). (C) Anti-peroxiredoxin III: Immunogold label was found almost exclusively in mitochondria (M; single arrows), with only rare labeling in nucleus (N) and cytoplasm. (D) Anti-peroxiredoxin IV: Immunogold label was found almost exclusively in lysosomes (Ly; curved arrows) and mitochondria (M). (E) Anti-peroxiredoxin V: Immunogold label was found almost exclusively in mitochondria (M; single arrows), with only rare labeling in nucleus and cytoplasm. (F) Anti-peroxiredoxin VI: Light immunogold labeling was identified in mitochondria (M; single arrow) and nucleus (N; arrowhead). For A, ⫻ 17,700; for B through F, ⫻ 12,000.
glomerulus, Fig. 7F). Thus, this protein is not present in significant amounts in these kidney cell types. Samples incubated with normal rabbit serum in place of the primary antibody did not show significant labeling in any of the tissues studied (not shown). DISCUSSION
The present study used well-established immunoperoxidase light microscopy and immunogold electron microscopy techniques to document localization of selected components of the thioredoxin system at the cellular and
subcellular levels. Previous studies in our laboratory have documented localization of antioxidant enzyme systems and oxidative damage products in normal kidney from various species. We have analyzed antioxidant enzymes from several species, including hamster [45], rat [46], mouse [47], and human [48], and in all cases, of the many cell types present in the mammalian kidney, antioxidant enzymes were found to be highest in proximal and distal tubular cells, cells of the distal collecting duct, and cells of the transitional epithelium. Proximal and distal tubular cells have in common large numbers of mitochondria, which are necessary to provide energy for
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Fig. 7. Immunogold ultrastructural analysis of rat kidney glomerular cells using antibodies specific to peroxiredoxins. (A) Anti-peroxiredoxin I: Light labeling was observed in glomerular cells. This photomicrograph demonstrates an endothelial cells with light labeling of nucleus (N; arrowhead) and cytoplasm (double arrow). (B) Anti-peroxiredoxin II: While glomerular cells showed only trace label, red blood cells (RBC) had extensive cytoplasmic label (double arrow). (C) Anti-peroxiredoxin III: Glomerular cells exhibited only light labeling, since glomerular cells have only a few small mitochondria. This photomicrograph illustrates an endothelial cell with mitochondria (M) showing moderate labeling (arrows). The nucleus (N) shows trace, probably nonspecific labeling. (D) Anti-peroxiredoxin IV: Glomerular cells exhibited only light labeling. Red blood cells (RBC) showed extensive labeling. (E) Anti-peroxiredoxin V: Glomerular cells did not demonstrate significant labeling. The nucleus (N) is that of an endothelial cell. (F) Anti-peroxiredoxin VI: Glomerular cells did not exhibit significant labeling. The nucleus (N) is that of an endothelial cell. For A and C through F, ⫻ 12,000; for B, ⫻ 6800.
the numerous functions of these two cell types, including regulation of transport functions involved in the formation of urine. It has been well documented that mitochondria are an abundant source of reactive oxygen species [49]. One of the primary functions of the papillary collecting ducts and transitional epithelium is xenobiotic metabolism, a process that utilizes enzymes that produce reactive oxygen species. Thus, cells that theoretically should be under oxidant stress are in fact protected by antioxidant enzymes. Using immunogold ultrastructural analysis, we have demonstrated that each antioxidant enzyme was present in specific subcellular locations: manganese superoxide
dismutase in mitochondria; copper, zinc superoxide dismutase in nucleus and cytoplasm; catalase in peroxisomes; and glutathione peroxidase in all subcellular locations. These latter results document that each subcellular organelle is under some type of oxidant stress, with specific enzymes present in specific subcellular locations to reduce this oxidant stress. Despite the presence of antioxidant enzymes in metabolically active tissue, some reactive oxygen species must escape this system, since we have been able to demonstrate oxidative damage products in these same metabolically active kidney cell types using morphologic techniques and antibodies to specific oxidative damage products, for ex-
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Protein Thioredoxin Thioredoxin reductase 1 Thioredoxin reductase 2 Peroxiredoxin I Peroxiredoxin II Peroxiredoxin III Peroxiredoxin IV Peroxiredoxin V Peroxiredoxin VI
Immunogold localization (this study)
Cell fractionation and biochemical analyses (reference)
Throughout the cell Throughout the cell; plasma Mitochondria Nucleus and cytoplasm Nucleus and cytoplasm; red cell Mitochondria Lysosomes; red cell Mitochondria Trace label
ample, antibody to 4-hydroxy-2-nonenal modified proteins [50,51]. The present study analyzes localization of the thioredoxin system in rat kidney. This system, as discussed in the Introduction, is crucial in regulating cell redox state of cells, and has both a protective and a physiological regulatory function. Unresolved by the present type of analysis is whether the presence of the thioredoxin system in any specific cell type is primarily protective or regulatory in function. Since the kidney cells studied are primarily stable cell types, i.e., long-lived but with definite cell turnover, it seems likely that the thioredoxin system is serving both functions in these cell types. In most cases, the subcellular localization of proteins of the thioredoxin system were in good agreement with previous reports at the subcellular level using cell fractionation and biochemical analyses (Table 1). However, analysis of whole tissues using biochemical or molecular biology techniques does not allow one to determine which cell types have high levels of proteins of the thioredoxin system, and this is the first study to document the large variability in levels of proteins of the thioredoxin system in various cell types. In general, light and electron microscopy results were concordant. However, we have previously demonstrated the greater sensitivity of immunogold ultrastructural techniques compared to light microscopy immunoperoxidase techniques [51]; cells that show equivocal staining with immunoperoxidase techniques may show definite positive labeling with immunogold techniques. In the present study, glomerular cells were definitely positive with light microscopy immunoperoxidase only with anti-TrxR1 antibody, whereas immunogold electron microscopy techniques demonstrated that several components of the thioredoxin system were actually present in glomerular cells. Two thioredoxins have been identified in mammalian cells by cell fractionation analysis, one located in cytoplasm and nucleus (Trx1), and one localized in mitochondria (Trx2) [22]. Reverse transcriptase-polymerase chain reaction analysis of transcripts encoding Trx1 or
Throughout the cell [22] Nucleus, cytoplasm [54]; plasma [55] Mitochondria [56] Nucleus and cytoplasm [40,44] Cyotplasm [44]; red cell [40] Mitochondria [44] Cytoplasm [40] Mitochondria, peroxisomes, cytosol [43] Unknown
Trx2 in rat tissues showed relatively uniform expression of Trx1 in most tissues studied, with the exception of very low levels in central nervous system tissues [22]. Trx2 was found in all rat tissues studied, but demonstrated more variability than Trx1, with high levels found in cerebellum, heart, skeletal muscle, testis, and kidney [22]. The present study used a commercial antibody to Trx, which demonstrated labeling of proximal and distal tubular epithelial cells, collecting duct cells of the renal papilla, and transitional epithelium, while glomerular cells showed much lighter labeling. All subcellular organelles were labeled, indicating a ubiquitous distribution of thioredoxin within kidney cells. The antibody utilized in the present study was a polyclonal antibody directed against E. coli thioredoxin, and by Western blot analysis this antibody recognized human thioredoxin in human tissue culture cell lines since a single band of molecular weight 12 kDa was identified. It is not known if this cross reactivity was directed against Trx1 or Trx2 or both. Three thioredoxin reductases have been identified in mammalian cells to date (TrxR1, TrxR2, and TrxR3); the distribution of the first two of these thioredoxin reductases was analyzed in the present study. TrxR1 had a similar distribution of label at both cellular and subcellular levels to Trx (throughout the cell), while TrxR2 labeling was predominantly mitochondrial. A previous study used in situ hybridization studies to localize TrxR1 mRNA in rat kidney proximal tubular cells [52], a finding in agreement with the present study. This same study suggested that TrxR1 was secreted by the proximal tubules into the plasma, a finding in agreement with the present ultrastructural analysis. At the subcellular level, TrxR1was found throughout the cell, while cell fractionation studies have demonstrated that TrxR2 and TrxR3 were found largely in mitochondria [36]. Peroxiredoxins have specific subcellular localizations as determined by cell fractionation studies [40]. Prx I is found in the cytosol and nucleus, Prx II in the cytosol, Prx III in the mitochondria, and Prx IV in the cytosol and secreted. Prx V is presumably in the mitochondria and
Thioredoxin system in rat kidney
peroxisomes since it has mitochondrial and peroxisomal targeting signals [53]. The localization of Prx VI is not yet known. Different cell types in the kidney showed unique Prx labeling patterns. Of note, distal tubular cells showed heavy labeling with Prxs II, III, and V. Prx IV labeled proximal tubular cells. Papillary collecting tubules and transitional epithelium showed heavy labeling with antibodies to Prx V. Red blood cells showed significant cytoplasmic labeling with Prxs II and IV. Prx VI did not significantly stain the rat renal cortical cells, but did stain the transitional epithelium. Glomerular cells had low levels of all proteins of the thioredoxin system studied; presumably these cells are primarily protected by serum antioxidants, including extracellular superoxide dismutase and thioredoxin reductase 1. At the subcellular level, Prxs I and II were found throughout the cell, although showing light deposition of gold beads in mitochondria, whereas Prxs III and V were demonstrated as being almost exclusively localized in mitochondria. Prx IV was found primarily in lysosomes, though we can not rule out peroxisomal staining, since these are difficult to identify in LR White– embedded material. Thus, our immunogold ultrastructural results agree well with cell fractionation studies. One caveat in the present study is that we can not rule out crossreactivity of antibodies with other members of the thioredoxin system; as one example, while Prxs III and V were primarily mitochondrial, some nuclear labeling was observed. It can not be ruled out whether this nuclear label is truly due to the presence of Prxs III and V in the nucleus, or the antibody recognizing another Prx. In conclusion, the present study demonstrates significant levels of proteins of the thioredoxin system in rat kidney. We have confirmed many of these results in human kidney (unpublished observations). The results document that kidney cells have multiple systems to deal with oxidant stress. Future studies will be necessary to separate protective from physiologic functions of the thioredoxin system. Acknowledgements — This work was supported by the Veterans Administration Research Service and a grant from the Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School. The authors would like to thank Dr. Larry Oberley for critical reading of the manuscript.
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Prx—peroxiredoxin ROS—reactive oxygen species TBS—Tris-buffered saline Trx—thioredoxin TrxR—thioredoxin reductase
PII S0891-5849(00)00486-X
Original Contribution LOCALIZATION OF THE THIOREDOXIN SYSTEM IN NORMAL RAT KIDNEY TERRY D. OBERLEY*†, ERIC VERWIEBE,† WEIXIONG ZHONG,† SANG WON KANG,‡
and
SUE GOO RHEE‡
*Pathology and Laboratory Medicine Service, Veterans Administration Hospital, Madison, WI, USA; †Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School, Madison, WI, USA; and ‡Laboratory of Cell Signaling, NHLBI, National Institutes of Health, Bethesda, MD, USA (Received 31 August 2000; Revised 7 November 2000; Accepted 17 November 2000)
Abstract—Components of the thioredoxin system were localized in normal rat kidney using immunoperoxidase techniques at the light microscopic level and immunogold techniques at the ultrastructural level. Results from both methods were similar. Thioredoxin, thioredoxin reductases, and peroxiredoxins showed cell-type-specific localization, with the same cell types (proximal and distal tubular epithelial, papillary collecting duct, and transitional epithelial cells) previously identified as having high amounts of antioxidant enzyme immunoreactive proteins and oxidative damage products also having high levels of proteins of the thioredoxin system. In addition, peroxiredoxins II and IV were found in high levels in the cytoplasm of red blood cells, identified in kidney blood vessels. While thioredoxin and thioredoxin reductase 1 were found in all subcellular locations in kidney cells, thioredoxin reductase 2 was found predominantly in mitochondria. Thioredoxin reductase 1 was identified in rat plasma, suggesting it is a secreted protein. Peroxiredoxins often had specific subcellular locations, with peroxiredoxins III and V found in mitochondria and peroxiredoxin IV found in lysosomes. Our results emphasize the complex nature of the thioredoxin system, demonstrating unique cell-type and organelle specificity. © 2001 Elsevier Science Inc. Keywords—Thioredoxin, Thioredoxin reductase, Peroxiredoxin, Reactive oxygen species, Kidney, Free radicals
INTRODUCTION
and interleukin-2 receptors [5,6]. Trx can modulate the DNA-binding activity of some transcription factors either directly (TFIIIC [7], BZLF1 [8], and NF-B [9]) or indirectly (AP-1) through the nuclear factor Ref-1, which in turn is reduced by thioredoxin [10]. Thioredoxin has been shown to regulate activity of the p53 tumor supressor protein by redox mechanisms, including coupling of the oxidative stress response and the p53-dependent DNA repair mechanism [11]. Thioredoxin can be secreted by cells [12–14] and stimulates the proliferation of lymphoid cells, fibroblasts, and a variety of human tumor cell lines [15–18]. Thioredoxin is thought to protect against oxidative damage, since it can reduce hydrogen peroxide [19], scavenge free radicals [20], and protect cells against oxidative stress [21]. Two thioredoxins have been identified in mammalian cells by cell fractionation analysis— one located in cytoplasm and nucleus (Trx1) and one located in mitochondria (Trx2) [22]. Reactive oxygen species (ROS), such as hydrogen peroxide, superoxide anion radical, and hydroxyl radical, have been thought of as toxic byproducts of cellular
Thioredoxin (Trx) is a 12-kDa protein, known to be present in many prokaryotes and eukaryotes and appears to be truly ubiquitous in all living cells [1,2]. It is characterized by an active site sequence, Trp-Cys-GlyPro-Cys-Lys, that is conserved throughout evolution. The two cysteine residues provide the sulfhydryl groups involved in Trx-dependent reducing activity. Oxidized thioredoxin, Trx-S2, is reduced to Trx-(SH)2 by the flavoenzyme thioredoxin reductase (TrxR) and NADPH. Mammalian thioredoxin has been implicated in a wide variety of biochemical functions. It can act as a hydrogen donor for ribonucleotide reductase [3] and methionine sulfoxide reductase [2], facilitate refolding of disulfidecontaining proteins [4], and activate the glucocorticoid Address correspondence to: Dr. Terry D. Oberley, William S. Middleton Memorial Veterans Administration Hospital, Pathology and Laboratory Medicine Service, 2500 Overlook Terrace, Room A35, Madison, WI 53705, USA; Tel: (608) 256-1901, ext. 11722; Fax: (608) 280-7087; E-Mail: [email protected]. 412
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oxygen metabolism. However, recent evidence indicates that at low concentrations, ROS mediate regulatory events and are essential participants in cell signaling [23]. Redox signaling is thought to be achieved through the coupling of ROS with oxidation-reduction processes that involve essential thiol groups in proteins, resulting in the modulation of tyrosine or serine-threonine phosphorylation of target proteins [24,25]. The redox state of essential thiol groups is controlled by two cellular redox systems: the thioredoxin (thioredoxin, thioredoxin reductase, and thioredoxin peroxidase) and glutathione (glutathione, glutathione reductase, glutaredoxin, and glutathione peroxidase) systems [26,27]. Several proteins have been identified as components of redox signaling pathways that act downstream of the generation of intracellular ROS [28 –30]. The redox state and activity of Trx are controlled by TrxR, a selenocysteine-containing flavoprotein composed of two identical 57-kDa subunits [31–35]. Three TrxR proteins have been identified to date (TrxR1, TrxR2, and TrxR3); using cell fractionation and biochemical analyses, TrxR1 was localized in the nucleus and cytoplasm, while TrxR2 and TrxR3 were localized in mitochondria. All three mammalian TrxRs conserve the COOH-terminal penultimate selenocysteine residue. Sun et al. [36] found that when cells generated ROS, selenocysteine in Trx1 was oxidized, while Trx1 expression was elevated. These results suggest that the selenocysteine residue in thioredoxin reductases serves as a cellular redox sensor. Peroxiredoxins (Prx) are a large antioxidant gene family that is well conserved from bacteria to humans. Several of the peroxiredoxins were initially identified by some of the diverse functions they perform, for example, enhancement of natural killer cytotoxicity [37], cell proliferation [38], and heme binding [39]. The family name, Prx, was chosen to reflect the fact that this is a family of peroxidases that uses conserved cysteine (Cys) residues for the sites of antioxidation. The Prx family contains six known protein subtypes [40]. In addition to the four Prx family members with two conserved Cys residues (2Cys), there are also two additional Prx families that contain only one conserved Cys residue (1-Cys). Prxs I–V all use thioredoxin as the electron donor, whereas the electron donor for Prx VI has yet to be identified. To date, most studies of the thioredoxin system have relied on biochemical and molecular biology analyses of tissue homogenates. While such studies may be highly informative, they provide data on the average values for all cell types within a tissue and do not allow subcellular characterization unless further cell fractionation studies are performed. To provide information on cellular and subcellular localization of the thioredoxin system, we chose to study the rat kidney, a complex organ composed
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of many cell types. The present study was performed to determine the cellular and subcellular localization of thioredoxin and thioredoxin reductases and peroxidases in various cell types of the kidney. Our studies show that each protein has unique cellular and subcellular localization. Three proteins, thioredoxin reductase 2 and peroxiredoxins III and V were found predominantly in mitochondria, emphasizing the need for protection of mitochondria from oxidant stress. MATERIALS AND METHODS
Animals Male Wistar rats were purchased from Harlan Sprague-Dawley (Indianapolis, IN, USA). They were transferred at 8 weeks of age (200 –250 g) to the Shared Aging Rodent Facility at the Geriatric Research, Education, and Clinical Center (William S. Middleton Veterans Administration Hospital; Madison, WI, USA). For 2 weeks, the rats were housed individually in solid bottom polycarbonate cages with wire tops and fed a nonpurified diet (Lab Rodent Chow 5001; Purina Feeds; Richmond, IN, USA) ad lib. Rats weighing 300 –350 g (10 weeks old) were then used for analysis. For immunoperoxidase analysis at the light microscopic level, the present study examined 6 rats. For immunogold analysis at the ultrastructural level, 2 rats were studied. A portion of both kidneys from each rat was fixed in 10% neutral buffered formalin for light microscopy immunoperoxidase analysis and Carson-Millonig’s fixative for electron microscopy immunogold analysis.
Antibodies Rabbit antibody to thioredoxin was obtained from Sigma Chemical Co. (St. Louis, MO, USA). Western blot analysis of homogenates of human prostate epithelial cell lines (immortalized, nonmalignant, and malignant) showed a single band of molecular weight 12 kDa, indicating that the antibody to E. coli thioredoxin recognized human thioredoxin. It is not known which thioredoxin form the antibody is detecting. Rabbit antibodies to TrxR1 and R2 and Prxs I–VI were obtained from the laboratory of Dr. Sue Goo Rhee, National Institutes of Health. Their specificities have been previously described [41– 44]. Western blot analyses of homogenates of the same human prostate epithelial cell lines using these antibodies showed appropriate bands of the correct molecular weights (thioredoxin reductase: 55–58 kDa; peroxiredoxin I–IV: 22 kDa; peroxiredoxin V: 17 kDa; peroxiredoxin VI: 25 kDa, data not shown).
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Light microscopy immunoperoxidase technique Tissues were fixed in 10% neutral buffered formalin and processed for embedding in paraffin blocks, using routine histology protocols [45]. For immunohistochemistry, deparaffinized and hydrated 4 micron sections were mounted on slides coated with 3-aminopropyltriethoxysilane. Primary antibodies were incubated with tissue sections overnight at 4°C. The immunoperoxidase procedure used a commercial development kit (LSAB, Dako K0690; Dako Corporation; Carpinteria, CA, USA), and metal enhanced diaminobenzidine substrate (Pierce #34065; Pierce; Rockford, IL; USA). A standard antigen retrieval protocol was utilized. Deparaffinized and rehydrated sections were treated with Tris urea solution (Tris, 121.1 g; urea, 50 g; 800 ml water; pH to 9.5 with concentrated hydrochloric acid); this solution was heated to 90°C, and the slides exposed to this heated solution for first 5 min and then 30 min. Optimal staining was obtained at a 1:100 dilution for all antibodies studied. After immunostaining, slides were counterstained with Harris hematoxylin. The specificity of immunostaining was determined by replacing the primary antibody with normal rabbit serum at the same concentration as the primary antiserum or with diluent buffer (Tris-buffered saline: 150 mM sodium chloride buffered with 50 mM Trizma base, pH 7.4). No staining was observed in these control slides. The degree of immunostaining was judged by comparison to normal rabbit serum controls, and labeling was characterized as negative, trace, light, moderate, and heavy. Photomicrographs shown in the figures are representative of all animals studied. Immunogold ultrastructural analysis Immunogold electron microscopy was performed as previously described [45]. All dehydration and infiltration steps with LR White acrylic resin (Electron Microscopy Sciences; Fort Washington, PA, USA) were undertaken at room temperature unless otherwise noted. Tissues were cut into 1 mm3 blocks and fixed in CarsonMillonig’s fixative (4% formaldehyde in 0.16 M monobasic sodium phosphate buffer, pH 7.2) for 1 h. After rinsing for 30 min in 0.1 M phosphate buffer, pH 7.4, the samples were dehydrated by 30 min changes each of 70, 80, and 90% ethanol, then LR White resin/90% ethanol (2/1, v/v). After immersion and infiltration in undiluted LR White resin overnight, the samples were washed with fresh undiluted LR White resin for 1 h. Resin polymerization was thermally induced in sealed gelatin capsules at 45°C for 48 h in the absence of accelerator. Ultrathin sections (70 – 80 nm) were cut and transferred to nickel grids (G300-NI, Electron Microscopy Sciences) for postembedding immunogold procedures. These sections
were rinsed with Tris-buffered saline (TBS: 0.05 M Tris, 0.9% NaCl, pH 7.6) for 10 min and incubated with 2% BSA and 0.2% Tween-20 in TBS for 30 min to block nonspecific antibody sites. Sections were then incubated with primary antibody (all antibodies were used at a dilution of 1:100, except anti-Trx R1 at 1:50 and anti-Trx R2 at 1:25) overnight at 4°C. After washing four times with TBS wash buffer (1:10 dilution of block buffer), for 5 min each, the sections were washed in one change of alkaline TBS (pH 8.2) for 20 min. The grids were incubated with diluted (1:75) gold conjugated goat antirabbit IgG (EM.GAR15, Goldmark) for 90 min at room temperature. The sections were washed in two changes of TBS wash buffer for 10 min each followed by 2 changes of distilled water for 5 min each. Following counterstaining with 4% aqueous uranyl acetate for 10 min, photographs were taken with a Hitachi H-600 transmission electron microscope operated at 75 kV. The specificity of immunostaining was determined by replacing the primary antibody with normal rabbit serum or preimmune serum at the same concentration as the test antiserum or diluent buffer (Trisbuffered saline: 150 mM sodium chloride buffered with 50 mM Trisma base, pH 7.4). No significant immunolabeling was observed in the sections prepared under these control conditions. Distinct cellular and subcellular labeling greater than background was regarded as positive. Labeling of organelles was graded as negative, trace, light, moderate, and heavy. Photomicrographs shown in the figures are representative of each animal studied. RESULTS
Light microscopy analysis of the thioredoxin system in adult rat kidney Thioredoxin. For all proteins of the thioredoxin system studied, major staining was observed in the proximal and distal tubules (Fig. 1), collecting tubules of the papillae, and transitional epithelium (Fig. 2). Other cell types, including glomerular cells (Fig. 1), collecting ducts in the cortex and superficial medulla, and cells of the loops of Henle showed only light or no staining, with some exceptions noted below. Representative cell types that showed significant staining are described below, though glomerular cells are also illustrated to show the relative light staining observed with these cell types (Fig. 1). In addition, it should be mentioned that the ducts of Bellini (terminal collecting ducts in the papilla of the kidney) showed staining patterns (data not shown) similar to transitional epithelium of the renal pelvis described herein (Fig. 2). In all cases, tissues stained with normal rabbit serum instead of primary antibody showed no significant labeling (Fig. 3).
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Fig. 1. Light microscopy immunoperoxidase analysis of cell types in rat outer kidney cortex using antibodies specific to selected proteins of the thioredoxin system. (A) Anti-thioredoxin: Proximal tubules (PT) showed moderate labeling of both nuclei and cytoplasm. Glomeruli (G) demonstrated only trace labeling. (B) Anti-thioredoxin reductase 1: Proximal (PT) and distal (DT) tubular cells exhibited moderate staining of both cytoplasm and nucleus. Glomeruli (G) were notable for moderate staining of endothelial (EN) cytoplasm and nucleus. (C) Anti-thioredoxin reductase 2: Proximal (PT) and distal (DT) tubular cells showed moderate nuclear and cytoplasmic staining. Glomeruli (G) exhibited only trace staining. (D) Anti-peroxiredoxin I: Proximal (PT) tubular cells had moderate cytoplasmic and focal heavy nuclear labeling (arrowhead). Glomeruli (G) showed only trace staining. (E) Anti-peroxiredoxin II: Proximal (PT) tubular cells demonstrated moderate cytoplasmic and nuclear labeling, while distal (DT) tubular cells had heavy cytoplasmic label. Red blood cells (RBC) had heavy cytoplasmic label, while glomeruli (G) exhibited only trace labeling. (F) Anti-peroxiredoxin III: Proximal (PT) tubular cells had moderate nuclear and cytoplasmic labeling, while distal (DT) tubular cells had intense cytoplasmic staining. Glomeruli (G) showed only trace staining. (G) Anti-peroxiredoxin IV: Proximal (PT) tubular cells exhibited prominent granular staining in the cell cytoplasm (curved arrow). Red blood cells (RBC) had heavy cytoplasmic staining, while glomeruli (G) exhibited only trace labeling. (H) Anti-peroxiredoxin V: Proximal (PT) tubular cells showed moderate nuclear and cytoplasmic label, while distal (DT) tubular cell had heavy cytoplasmic labeling. Glomeruli showed only trace staining. (I) Anti-peroxiredoxin VI: Proximal (PT) and distal (DT) tubular cells showed light to moderate nuclear and cytoplasmic staining. Glomeruli exhibited light labeling. For A through I, ⫻ 3500.
Results with antibody to Trx showed moderate labeling of nuclei and cytoplasm of both proximal (Fig. 1A) and distal tubular cells. Glomerular cells demonstrated
only trace staining (Fig. 1A). Nuclei and cytoplasm of transitional epithelium showed light to moderate labeling (Fig. 2A). Normal rabbit serum controls performed at the
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Fig. 2. Light microscopy immunoperoxidase analysis of transitional epithelium of renal pelvis of rat kidney using antibodies specific to selected proteins of the thioredoxin system. (A) Anti-thioredoxin; (B) Anti-thioredoxin reductase 1; (C) Anti-thioredoxin reductase 2; (D) Anti-peroxiredoxin I; (E) Anti-peroxiredoxin II; (F) Anti-peroxiredoxin III; (G) Anti-peroxiredoxin IV; (H) Anti-peroxiredoxin V; (I) Anti-peroxiredoxin VI. Thioredoxin and thioredoxin reductases 1 and 2 showed light to moderate staining of transitional epithelium (TE), while peroxiredoxins showed moderate labeling, except peroxiredoxin V, which showed heavy labeling. In all cases, both nuclei and cytoplasm appear to show staining. For A through I, ⫻ 3500.
same time showed no significant staining of any cell type (Fig. 3). Thioredoxin reductases. TrxR1 showed moderate staining of nuclei and cytoplasm of both proximal and distal tubular epithelial cells (Fig. 1B). Glomerular endothelial cells demonstrated moderate labeling of nuclei and cytoplasm (Fig. 1B). Nucleus and cytoplasm of transitional epithelium also demonstrated light to moderate labeling for TrxR1 (Fig. 2B).
Proximal and distal tubular cells (Fig. 1C) immunostained with TrxR2 had moderate labeling of nuclei and cytoplasm. Glomeruli did not have significant staining (Fig. 1C). Transitional epithelium had heavy nuclear label, but only light to moderate cytoplasmic staining (Fig. 2C). Peroxiredoxins. Prx I exhibited heavy nuclear and moderate cytoplasmic labeling in proximal (Fig. 1D) and distal epithelial cells. Glomeruli showed light staining
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Cytoplasm of distal tubular epithelial cells demonstrated heavy staining when immunolabeled with antibody to Prx V, while proximal tubule epithelial cells showed moderate labeling (Fig. 1H). Glomeruli and red blood cells did not exhibit significant label (Fig. 1H). Transitional epithelium (Fig. 2H) demonstrated heavy immunolabeling of both nuclei and cytoplasm. Prx VI staining was light in nuclei and cytoplasm of kidney proximal and distal tubular epithelial cells, with only trace labeling in glomeruli (Fig. 1I). Transitional epithelium showed moderate nuclear and cytoplasmic staining (Fig. 2I). Subcellular localization of the thioredoxin system in kidney proximal tubular and glomerular cells
Fig. 3. Immunoperoxidase analysis of staining of normal rat kidney using normal rabbit serum in place of primary antibody. (A) Renal cortex: Glomeruli (G) and proximal (PT) tubular cells did not have any labeling. (B) Transitional epithelium: Transitional epithelium (TE) did not exhibit significant labeling. For A and B, ⫻ 5800.
(Fig. 1D). Nuclei and cytoplasm of transitional epithelium had moderate staining (Fig. 2D). Prx II showed heavy labeling of distal tubular cell cytoplasm, while proximal tubular cells exhibited only moderate labeling (Fig. 1E). Glomeruli exhibited only trace labeling, though erythrocytes within glomerular capillaries were strongly positive (Fig. 1E). Transitional epithelium (Fig. 2E) demonstrated moderate nuclear and cytoplasmic staining. Kidney tissues immunostained with antibody to Prx III had heavy cytoplasmic label in distal tubular cells, moderate cytoplasmic label in proximal tubular cells, and only trace labeling in glomeruli (Fig. 1F). Transitional epithelium exhibited moderate staining of nuclei and cytoplasm, except for the most superficial layer, which showed heavy staining (Fig. 2F). Prx IV showed heavy granular localization in kidney proximal tubular cells and diffuse staining of erythrocytes in glomerular capillaries (Fig. 1G). Transitional epithelium exhibited moderate staining of nuclei and cytoplasm (Fig. 2G).
Thioredoxin. Proximal tubular cells showed heavy mitochondrial labeling and moderate nuclear labeling for Trx (Fig. 4A). These same cells showed light labeling of cytoplasm and microvilli. Although glomerular cells usually showed only light or trace staining by light microscopy, immunogold ultrastructural analysis often revealed these cells were actually positive for components of the thioredoxin system. Intrinsic cells of kidney glomeruli are composed of three cell types: endothelial, mesangial, and visceral epithelial cells (podocytes). Each of these glomerular cell types have only sparse and small mitochondria compared to the large and numerous mitochondria present in proximal and distal tubular epithelial cells. Endothelial and mesangial cell nuclei exhibited moderate labeling, while cytoplasm showed only light labeling (Fig. 5A). Glomerular epithelial cells demonstrated light labeling of both nucleus and cytoplasm (not shown). Red blood cells in glomerular capillaries were also evaluated and showed moderate cytoplasmic label. Thioredoxin reductases. TrxR1 demonstrated heavy nuclear and mitochondrial labeling in proximal tubular epithelial cells (Fig. 4B), while cytoplasm and microvilli exhibited light labeling. Glomerular endothelial and mesangial cells showed moderate labeling of nuclei and light labeling of cytoplasm (Fig. 5B). Glomerular epithelial cells showed similar subcellular distribution of immunogold beads as proximal tubular cells, but only light label (not shown). Red blood cells exhibited light cytoplasmic label. TrxR1 was identified in human plasma (Fig. 5B). TrxR2 was predominantly mitochondrial, but showed lighter staining intensity in all kidney cell types examined (proximal tubule, Fig. 4C; glomerulus, Fig. 5C). Normal rabbit serum control of proximal tubular cells (Fig. 4D) or glomerular cells (Fig. 5D) did not exhibit significant labeling. Peroxiredoxins. Prx I was found in moderate amounts in nuclei, cytoplasm, and microvilli of proximal tubular
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Fig. 4. Immunogold ultrastructural analysis of rat kidney proximal tubular epithelial cells using antibodies specific for thioredoxin and thioredoxin reductases. (A) Anti-thioredoxin: Immunogold labeling was found throughout the cell, including the nucleus (N; arrowhead), mitochondria (M; single arrows), and cytoplasm (double arrow). (B) Anti-thioredoxin reductase 1: Immunogold labeling was observed throughout the cell, including the nucleus (N; arrowheads), mitochondria (M; single arrow), and cytoplasm (double arrow). (C) Anti-thioredoxin reductase 2: Immunogold labeling was found predominantly in the mitochondria (M; single arrow), though trace labeling in the nucleus (N; arrowhead) and cytoplasm (double arrow) was present. (D) Normal rabbit serum: No immunogold beads were present. For A through D, ⫻ 14,000.
epithelial cells, while mitochondria demonstrated only light immunogold labeling (Fig. 6A). The nuclei of glomerular mesangial and endothelial cells exhibited moderate labeling (Fig. 7A), while podocyte nuclei showed only a small number of immunogold beads (light labeling; not shown). The cytoplasm in all glomerular cell types showed only light label. Erythrocytes in glomerular capillaries demonstrated only trace labeling (not shown). Label was thus predominantly cytoplasmic and nuclear,
with only light mitochondrial immunogold bead deposition. Prx II was found in a similar distribution to Prx I (proximal tubule, Fig. 6B; glomerulus, Fig. 7B). The only exception was the heavy labeling observed in erythrocyte cytoplasm (Fig. 7B). Prx III was found almost exclusively in mitochondria and lysosomes of proximal tubular epithelial cells (Fig. 6C). Since glomerular cells have only few mitochondria,
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Fig. 5. Immunogold ultrastructural analysis of rat kidney glomerular cells using antibodies specific for thioredoxin and thioredoxin reductases. (A) Anti-thioredoxin: Immunogold labeling was found throughout the glomerular cells. This photomicrograph illustrates moderate labeling of mesangial cell nuclei (N; arrowheads) and light labeling of mesangial cell cytoplasm (double arrow). (B) Anti-thioredoxin reductase 1: Immunogold labeling was found throughout the glomerular cells. This photomicrograph illustrates moderate labeling of an endothelial cell nucleus (N; arrowhead), light labeling of endothelial cytoplasm (double arrow), and significant labeling of plasma (curved arrow). (C) Anti-thioredoxin reductase 2: Only light or trace labeling was observed in glomerular cells. This photomicrograph shows light labeling of an endothelial cell nucleus (N; arrowhead). (D) Normal rabbit serum: No immunogold beads were identified. For A through C, ⫻ 14,000; for D, ⫻ 11,000.
these cells showed only light immunolabel (Fig. 7C). Red blood cells demonstrated trace immunolabeling (not shown). Prx IV showed numerous immunogold beads in lysosomes of proximal tubular cells (Fig. 6D), with only trace, probably nonspecific, deposition of immunogold beads in other subcellular locations. Glomeruli showed light labeling of nucleus and cytoplasm (Fig. 7D). Cyto-
plasm of erythrocytes exhibited moderate labeling (Fig. 7D). Prx V showed moderate mitochondrial labeling in proximal (Fig. 6E) tubular epithelial cells. Glomeruli (Fig. 7E) and red blood cells demonstrated only trace labeling. Prx VI showed only trace labeling in all kidney cell types and subcellular locations (proximal tubule, Fig. 6F;
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Fig. 6. Immunogold ultrastructural analysis of rat kidney proximal tubular epithelial cells using antibodies specific for peroxiredoxins. (A) Anti-peroxiredoxin I: Immunugold label was found throughout the cell, primarily in the nucleus (N; arrowheads) and cytoplasm (double arrows), but also rarely in mitochondria (M; single arrow). (B) Anti-peroxiredoxin II: Immunogold label was found throughout the cell, primarily in the nucleus (N; arrowheads) and cytoplasm (double arrows, including base of microvilli, MV), but also rarely in mitochondria (M; single arrow). (C) Anti-peroxiredoxin III: Immunogold label was found almost exclusively in mitochondria (M; single arrows), with only rare labeling in nucleus (N) and cytoplasm. (D) Anti-peroxiredoxin IV: Immunogold label was found almost exclusively in lysosomes (Ly; curved arrows) and mitochondria (M). (E) Anti-peroxiredoxin V: Immunogold label was found almost exclusively in mitochondria (M; single arrows), with only rare labeling in nucleus and cytoplasm. (F) Anti-peroxiredoxin VI: Light immunogold labeling was identified in mitochondria (M; single arrow) and nucleus (N; arrowhead). For A, ⫻ 17,700; for B through F, ⫻ 12,000.
glomerulus, Fig. 7F). Thus, this protein is not present in significant amounts in these kidney cell types. Samples incubated with normal rabbit serum in place of the primary antibody did not show significant labeling in any of the tissues studied (not shown). DISCUSSION
The present study used well-established immunoperoxidase light microscopy and immunogold electron microscopy techniques to document localization of selected components of the thioredoxin system at the cellular and
subcellular levels. Previous studies in our laboratory have documented localization of antioxidant enzyme systems and oxidative damage products in normal kidney from various species. We have analyzed antioxidant enzymes from several species, including hamster [45], rat [46], mouse [47], and human [48], and in all cases, of the many cell types present in the mammalian kidney, antioxidant enzymes were found to be highest in proximal and distal tubular cells, cells of the distal collecting duct, and cells of the transitional epithelium. Proximal and distal tubular cells have in common large numbers of mitochondria, which are necessary to provide energy for
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Fig. 7. Immunogold ultrastructural analysis of rat kidney glomerular cells using antibodies specific to peroxiredoxins. (A) Anti-peroxiredoxin I: Light labeling was observed in glomerular cells. This photomicrograph demonstrates an endothelial cells with light labeling of nucleus (N; arrowhead) and cytoplasm (double arrow). (B) Anti-peroxiredoxin II: While glomerular cells showed only trace label, red blood cells (RBC) had extensive cytoplasmic label (double arrow). (C) Anti-peroxiredoxin III: Glomerular cells exhibited only light labeling, since glomerular cells have only a few small mitochondria. This photomicrograph illustrates an endothelial cell with mitochondria (M) showing moderate labeling (arrows). The nucleus (N) shows trace, probably nonspecific labeling. (D) Anti-peroxiredoxin IV: Glomerular cells exhibited only light labeling. Red blood cells (RBC) showed extensive labeling. (E) Anti-peroxiredoxin V: Glomerular cells did not demonstrate significant labeling. The nucleus (N) is that of an endothelial cell. (F) Anti-peroxiredoxin VI: Glomerular cells did not exhibit significant labeling. The nucleus (N) is that of an endothelial cell. For A and C through F, ⫻ 12,000; for B, ⫻ 6800.
the numerous functions of these two cell types, including regulation of transport functions involved in the formation of urine. It has been well documented that mitochondria are an abundant source of reactive oxygen species [49]. One of the primary functions of the papillary collecting ducts and transitional epithelium is xenobiotic metabolism, a process that utilizes enzymes that produce reactive oxygen species. Thus, cells that theoretically should be under oxidant stress are in fact protected by antioxidant enzymes. Using immunogold ultrastructural analysis, we have demonstrated that each antioxidant enzyme was present in specific subcellular locations: manganese superoxide
dismutase in mitochondria; copper, zinc superoxide dismutase in nucleus and cytoplasm; catalase in peroxisomes; and glutathione peroxidase in all subcellular locations. These latter results document that each subcellular organelle is under some type of oxidant stress, with specific enzymes present in specific subcellular locations to reduce this oxidant stress. Despite the presence of antioxidant enzymes in metabolically active tissue, some reactive oxygen species must escape this system, since we have been able to demonstrate oxidative damage products in these same metabolically active kidney cell types using morphologic techniques and antibodies to specific oxidative damage products, for ex-
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T. D. OBERLEY et al. Table 1. Comparison of Ultrastructural and Biochemical Analyses of Subcellular Localization of the Thioredoxin System
Protein Thioredoxin Thioredoxin reductase 1 Thioredoxin reductase 2 Peroxiredoxin I Peroxiredoxin II Peroxiredoxin III Peroxiredoxin IV Peroxiredoxin V Peroxiredoxin VI
Immunogold localization (this study)
Cell fractionation and biochemical analyses (reference)
Throughout the cell Throughout the cell; plasma Mitochondria Nucleus and cytoplasm Nucleus and cytoplasm; red cell Mitochondria Lysosomes; red cell Mitochondria Trace label
ample, antibody to 4-hydroxy-2-nonenal modified proteins [50,51]. The present study analyzes localization of the thioredoxin system in rat kidney. This system, as discussed in the Introduction, is crucial in regulating cell redox state of cells, and has both a protective and a physiological regulatory function. Unresolved by the present type of analysis is whether the presence of the thioredoxin system in any specific cell type is primarily protective or regulatory in function. Since the kidney cells studied are primarily stable cell types, i.e., long-lived but with definite cell turnover, it seems likely that the thioredoxin system is serving both functions in these cell types. In most cases, the subcellular localization of proteins of the thioredoxin system were in good agreement with previous reports at the subcellular level using cell fractionation and biochemical analyses (Table 1). However, analysis of whole tissues using biochemical or molecular biology techniques does not allow one to determine which cell types have high levels of proteins of the thioredoxin system, and this is the first study to document the large variability in levels of proteins of the thioredoxin system in various cell types. In general, light and electron microscopy results were concordant. However, we have previously demonstrated the greater sensitivity of immunogold ultrastructural techniques compared to light microscopy immunoperoxidase techniques [51]; cells that show equivocal staining with immunoperoxidase techniques may show definite positive labeling with immunogold techniques. In the present study, glomerular cells were definitely positive with light microscopy immunoperoxidase only with anti-TrxR1 antibody, whereas immunogold electron microscopy techniques demonstrated that several components of the thioredoxin system were actually present in glomerular cells. Two thioredoxins have been identified in mammalian cells by cell fractionation analysis, one located in cytoplasm and nucleus (Trx1), and one localized in mitochondria (Trx2) [22]. Reverse transcriptase-polymerase chain reaction analysis of transcripts encoding Trx1 or
Throughout the cell [22] Nucleus, cytoplasm [54]; plasma [55] Mitochondria [56] Nucleus and cytoplasm [40,44] Cyotplasm [44]; red cell [40] Mitochondria [44] Cytoplasm [40] Mitochondria, peroxisomes, cytosol [43] Unknown
Trx2 in rat tissues showed relatively uniform expression of Trx1 in most tissues studied, with the exception of very low levels in central nervous system tissues [22]. Trx2 was found in all rat tissues studied, but demonstrated more variability than Trx1, with high levels found in cerebellum, heart, skeletal muscle, testis, and kidney [22]. The present study used a commercial antibody to Trx, which demonstrated labeling of proximal and distal tubular epithelial cells, collecting duct cells of the renal papilla, and transitional epithelium, while glomerular cells showed much lighter labeling. All subcellular organelles were labeled, indicating a ubiquitous distribution of thioredoxin within kidney cells. The antibody utilized in the present study was a polyclonal antibody directed against E. coli thioredoxin, and by Western blot analysis this antibody recognized human thioredoxin in human tissue culture cell lines since a single band of molecular weight 12 kDa was identified. It is not known if this cross reactivity was directed against Trx1 or Trx2 or both. Three thioredoxin reductases have been identified in mammalian cells to date (TrxR1, TrxR2, and TrxR3); the distribution of the first two of these thioredoxin reductases was analyzed in the present study. TrxR1 had a similar distribution of label at both cellular and subcellular levels to Trx (throughout the cell), while TrxR2 labeling was predominantly mitochondrial. A previous study used in situ hybridization studies to localize TrxR1 mRNA in rat kidney proximal tubular cells [52], a finding in agreement with the present study. This same study suggested that TrxR1 was secreted by the proximal tubules into the plasma, a finding in agreement with the present ultrastructural analysis. At the subcellular level, TrxR1was found throughout the cell, while cell fractionation studies have demonstrated that TrxR2 and TrxR3 were found largely in mitochondria [36]. Peroxiredoxins have specific subcellular localizations as determined by cell fractionation studies [40]. Prx I is found in the cytosol and nucleus, Prx II in the cytosol, Prx III in the mitochondria, and Prx IV in the cytosol and secreted. Prx V is presumably in the mitochondria and
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peroxisomes since it has mitochondrial and peroxisomal targeting signals [53]. The localization of Prx VI is not yet known. Different cell types in the kidney showed unique Prx labeling patterns. Of note, distal tubular cells showed heavy labeling with Prxs II, III, and V. Prx IV labeled proximal tubular cells. Papillary collecting tubules and transitional epithelium showed heavy labeling with antibodies to Prx V. Red blood cells showed significant cytoplasmic labeling with Prxs II and IV. Prx VI did not significantly stain the rat renal cortical cells, but did stain the transitional epithelium. Glomerular cells had low levels of all proteins of the thioredoxin system studied; presumably these cells are primarily protected by serum antioxidants, including extracellular superoxide dismutase and thioredoxin reductase 1. At the subcellular level, Prxs I and II were found throughout the cell, although showing light deposition of gold beads in mitochondria, whereas Prxs III and V were demonstrated as being almost exclusively localized in mitochondria. Prx IV was found primarily in lysosomes, though we can not rule out peroxisomal staining, since these are difficult to identify in LR White– embedded material. Thus, our immunogold ultrastructural results agree well with cell fractionation studies. One caveat in the present study is that we can not rule out crossreactivity of antibodies with other members of the thioredoxin system; as one example, while Prxs III and V were primarily mitochondrial, some nuclear labeling was observed. It can not be ruled out whether this nuclear label is truly due to the presence of Prxs III and V in the nucleus, or the antibody recognizing another Prx. In conclusion, the present study demonstrates significant levels of proteins of the thioredoxin system in rat kidney. We have confirmed many of these results in human kidney (unpublished observations). The results document that kidney cells have multiple systems to deal with oxidant stress. Future studies will be necessary to separate protective from physiologic functions of the thioredoxin system. Acknowledgements — This work was supported by the Veterans Administration Research Service and a grant from the Department of Pathology and Laboratory Medicine, University of Wisconsin Medical School. The authors would like to thank Dr. Larry Oberley for critical reading of the manuscript.
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Prx—peroxiredoxin ROS—reactive oxygen species TBS—Tris-buffered saline Trx—thioredoxin TrxR—thioredoxin reductase