Mercury Induces Regional and Cell-Specific Stress Protein Expression in Rat Kidney

Peter L. Goering*,1, Benjamin R. Fisher*,2, Bradley T. Noren{dagger}, Andriana Papaconstantinou*, Jennifer L. Rojko{dagger} and Ronald J. Marler{ddagger}

* Health Sciences Branch, Division of Life Sciences, Office of Science and Technology, Center for Devices and Radiological Health, Food and Drug Administration, Rockville, Maryland 20857; {dagger} Pathology Associates International, Frederick, Maryland 21701; and {ddagger} Covance Laboratories, Vienna, Virginia 22182

Received July 6, 1999; accepted September 2, 1999


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cells respond to physiologic stress by enhancing the expression of specific stress proteins. Heat-shock proteins (hsps) and glucose-regulated proteins (grps) are members of a large superfamily of proteins collectively referred to as stress proteins. This particular stress-protein response has evolved as a cellular strategy to protect, repair, and chaperone other essential cellular proteins. The objective of this study was to evaluate the differential expression of four hsps in the renal cortex and medulla during experimental nephrotoxic injury using HgCl2. Male Sprague-Dawley rats received single injections of HgCl2 (0.25, 0.5, or 1 mg Hg/kg, iv). At 4, 8, 16, or 24 h after exposure, kidneys were removed and processed for histopathologic, immunoblot, and immunohistochemical analyses. Nephrosis was characterized as minimal or mild (cytoplasmic condensation, tubular epithelial degeneration, single cell necrosis) at the lower exposures, and progressed to moderate or severe (nuclear pyknosis, necrotic foci, sloughing of the epithelial casts into tubular lumens) at the highest exposures. Western blots of renal proteins were probed with monoclonal antibodies specific for 4 hsps. In whole kidney, Hg(II) induced a time- and dose-related accumulation of hsp72 and grp94. Accumulation of hsp72 was predominantly localized in the cortex and not medulla, while grp94 accumulated primarily in the medulla but not cortex. The high, constitutive expression of hsp73 did not change as a result of Hg(II) exposure, and it was equally localized in cortex and medulla. Hsp90 was not detected in kidneys of control or Hg-treated rats. Since hsp72 has been shown involved in cellular repair and recovery, and since Hg(II) damage occurs primarily in cortex, we investigated the cell-specific expression of this hsp. Hsp72 accumulated primarily in undamaged distal convoluted tubule epithelia, with less accumulation in undamaged proximal convoluted-tubule epithelia. These results demonstrate that expression of specific stress proteins in rat kidney exhibits regional heterogeneity in response to Hg(II) exposure, and a positive correlation exists between accumulation of some stress proteins and acute renal cell injury. While the role of accumulation of hsps and other stress proteins in vivo prior to or concurrent with nephrotoxicity remains to be completely understood, these stress proteins may be part of a cellular defense response to nephrotoxicants. Conversely, renal tubular epithelial cells that do not or are unable to express stress proteins, such as hsp72, may be more susceptible to nephrotoxicity.

Key Words: mercury; kidney; acute renal injury; heat-shock proteins; hsp72; glucose-regulated proteins; grp94; stress proteins; immunohistochemistry..


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cellular repair and recovery from acute renal injury involves the expression of inducible "stress" genes such as those of epidermal growth factor, c-myc, c-jun, c-fos, prostaglandin synthase, and heat-shock proteins (hsps) associated with cell proliferation and regeneration (Bardella and Comolli, 1994Go; Coimbra et al., 1990Go; Cowley and Gudapaty, 1995Go; Yamamoto et al., 1993Go). The rapid synthesis of hsps exposed to a variety of adverse stimuli represents a transient reprogramming of genetic expression and biological activity that serves to protect sensitive cellular macromolecules from damage and to assist in rapid recovery after cessation of the physiologic stress (for reviews, see Morimoto et al., 1990; Nover, 1991; Welch, 1992). Originally termed the heat-shock response because of enhanced induction following hyperthermia (Ritossa, 1962Go), the signaling mechanism involved in initiation of heat-shock gene transcription is sensitive to a variety of physical and chemical stressors including metals, oxidative stressors, hypoxia, ischemia/reperfusion, and several carcinogens, mutagens, and teratogens (Nover, 1991Go; Schlesinger, 1990Go). Glucose-regulated proteins (grps) are another group of stress proteins closely related to the hsps, and are induced by agents or conditions that interfere with glucose and oxygen utilization, or that perturb intracellular calcium homeostasis. Because of the diverse number of insults that can initiate this stress response, hsps and grps are collectively referred to as stress proteins.

The stress-protein genes share highly conserved sequence homologies among widely divergent organisms, and several of the major stress proteins are members of gene families distinguished primarily based on apparent molecular mass. The constitutive, or cognate, proteins, called "chaperones," e.g., hsp73, play a role in protein biogenesis and maturation, aid in folding of nascent proteins (Gething and Sambrook, 1992Go; Rothman, 1989Go), bind a variety of intracellular receptors to keep them in a non-activated state (Howard et al., 1990Go), and shuttle nascent polypeptides and proteins across intracellular membranes (Beckmann et al., 1990Go; Craig et al., 1994Go; Hightower et al., 1994Go). During adverse conditions such as heat-shock or toxic insult, the stress-inducible stress protein genes, e.g., hsp72 and grp94, are rapidly up-regulated. These stress proteins bind to other critical proteins in the cytoplasm to prevent irreversible denaturation and aggregation, to facilitate disaggregation of denatured proteins, and to aid in the refolding of proteins during recovery after cessation of the toxic insult (Beckmann et al., 1992Go; Hightower, 1991Go; Welch, 1993Go). Several hsps translocate from cytoplasm to nucleus and nucleolus, where they bind periribosomes and other nuclear protein complexes in order to protect them from damage (Arrigo et al., 1988Go; Brown et al., 1993Go; Ellis, 1990; Lindquist, 1986Go; Welch and Feramisco, 1985Go). Elevated levels of hsp72, a widely investigated hsp, have been associated with cytoprotection against a variety of lethal or toxic insults (Johnston and Kucey, 1988Go; Mirkes, 1987Go; Riabowol et al., 1988Go; Tytell et al., 1994Go; Welch and Mizzen, 1988Go), including protection of kidneys from ischemic injury (Chatson et al., 1990Go; Perdrizet et al., 1993Go). Heart and brain cells in transgenic mice that overexpress the inducible form of hsp70 are resistant to ischemia (Plumier et al., 1995Go, 1997Go).

Although the stress protein response appears to be a ubiquitous response found in all cells and tissues studied to date, the specific stress proteins induced are dependent on the toxicant, the magnitude and duration of exposure, and the tissue. For example, we demonstrated that Hg(II) and Cd(II) induce alterations in the de novo synthesis of stress proteins in kidney and liver, respectively (Goering et al., 1992Go, 1993aGo,Goering et al., bGo). The changes in protein synthesis were target organ-specific, i.e., mercuric chloride, a potent nephrotoxicant, produced changes in kidney, but not liver, and cadmium chloride, a well-known hepatotoxicant, produced changes in liver, but not kidney. The metal-induced changes in protein synthesis that we observed preceded the detection of functional and clinical indicators of organ injury (Goering et al., 1992Go, 1993aGo,Goering et al., bGo). Other studies have shown target tissue specificity of the stress-protein response in tissues such as brain (Nowak, 1993Go) and kidney (Hernández-Pando et al., 1995) after exposure to neurotoxicants and nephrotoxicants, respectively.

Since Hg(II) was shown to alter general stress protein synthesis in whole kidney (Goering et al., 1992Go), the first objective of this study was to determine the changes in expression of 4 specific stress proteins in kidney after Hg(II)-exposure. Second, since Hg(II) damage occurs primarily in proximal tubule cells in the cortex, we were interested in determining if these responses are localized in major regions of the kidney, i.e., the cortex and medulla. Third, since hsp72 has been shown to be extensively involved in cellular repair and recovery after toxic insult, we wanted to determine if the hsp72 response is ubiquitous in all cell types within the kidney, or whether the response is target cell-type specific, i.e., localized to the pars recta epithelia comprising the S3 proximal tubule straight segments of the nephron, the primary target cells for Hg(II) (Gritzka and Trump, 1968Go).


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Animal Care and Treatments
All procedures requiring the use of animals were conducted in accordance with the Public Health Service's Guide for the Care and Use of Laboratory Animals. Sprague-Dawley male rats (CD strain; Charles River, Raleigh, NC) weighing between 225–250 g were housed and maintained on a 12 h light/dark cycle at 22–24°C. Animals were housed in polypropylene cages containing heat-treated hardwood chips, with access to food (Purina 5002; certified maximum Hg concentration, 0.2 ppm) and water ad libitum. Rats were anesthetized briefly with halothane and injected in the saphenous vein with 0.25, 0.5, or 1 mg Hg/kg (HgCl2; Sigma Chemical Co., St. Louis, MO) at 4, 8, 16, or 24 h prior to removing kidneys for assay. Controls received an injection of vehicle (0.9% saline) at a volume of 2 ml/kg.

Histopathological Evaluation of Kidneys
A cross-section of kidney from each rat was fixed on a microscope slide and stained with hematoxylin and eosin. In order to compare the relationship between the presence of renal injury and induction of hsps, all slides were read only for the presence or absence of nephrosis. For this study, nephrosis was defined as the presence of tubular epithelial degeneration and/or necrosis. Individual cell necrosis was the criterion for identification of the earliest histopathologic change. The severity of nephrosis was recorded using a grading scale of 0 to 4 which was related to a subjective impression of the extent of cortical tubular involvement, as follows:

This grading scale was adapted from the NTP Technical Report (National Toxicology Program, 1993Go).

Electrophoresis of Tissue Proteins for Immunodetection of Heat-Shock Proteins
Kidneys were processed as described below in order to detect the expression of stress proteins in major regions of the kidney (cortex vs. medulla) after Hg(II) treatment. Experiments involving the immunodetection of stress proteins were repeated in at least 3 sets of rats. Kidneys were excised and placed in ice-cold 0.9% saline. They were then cut in cross-section approximately 2–3 mm thick. Using small dissecting scissors, the outer section representing the renal cortex and outer stripe of the medulla, collectively referred to as "cortex," was separated from the remaining tissue representing the inner stripe of the medulla, inner medulla, and papilla, collectively referred to as "medulla." Sections of whole kidney, renal cortex, and renal medulla were weighed and homogenized separately in ice-cold 10 mM Tris–HCl, pH 7.4 (200-mg tissue/400-ml buffer) using a hand-held glass microhomogenizer (Thomas Scientific Co, Swedesboro, NJ). Tissue homogenates were centrifuged at 16,000 x g for 15 min at 5°C. Aliquots of the supernatants were stored at –70°C. Thawed samples were assayed for total protein using the BCA assay (Sigma Chemical Co., St. Louis, MO) and prepared for gel electrophoresis.

Tissue proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS–PAGE) on pre-cast 12.5% acrylamide minigels (Phast System, Pharmacia/LKB Biotechnology, Piscataway, NJ) as described by Goering et al. (1992). Samples were diluted with 10 mM Tris–HCl and sample buffer (62.5 mM Tris–HCl, pH 8.0, 2.5% SDS, 2% mercaptoethanol, 0.01% bromophenol blue, and 1 mM EDTA) such that each lane on any given gel contained equivalent amounts of total tissue protein. Pre-stained molecular weight standards (GIBCO/BRL Life Technologies, Inc., Gaithersburg, MD) were separated concurrently on each gel.

Immunologic Detection of Heat-Shock Proteins
Following SDS–PAGE separation, proteins were electrophoretically transferred to nitrocellulose membranes using a Pharmacia/LKB 2117 Multiphor II system (Pharmacia/LKB Biotechnology) as described previously (Goering et al., 1992Go). Successful transfer was verified by the transfer of the pre-stained molecular weight markers. Blots were placed in Tris-buffered blocking solution (TBS; 20 mM Tris, 500 mM NaCl, pH 7.5) containing 4% non-fat dry milk for 3 h at room temperature. Blots were washed in blocking solution containing 0.05% Tween 20 (TTBS) and then incubated overnight with individual stress protein primary antibodies. The primary stress protein antibodies were: inducible hsp72 (mouse {alpha}-human IgG1, dilution 1:500; SPA-810; StressGen Biotechnologies, Inc., Vancouver, BC, Canada), constitutive hsp73 (rat {alpha}-hamster IgG2a, diluted 1:500; SPA-815, StressGen), grp94 (rat {alpha}-chicken IgG2a, diluted 1:500; SPA-850, StressGen), and hsp90 (mouse {alpha}-mold IgG1, diluted 1:500; SPA-830, StressGen). These antibodies have been shown to exhibit cross-reactivity with hsps from a variety of cell lines from different mammalian species. Following this step, blots were washed in TTBS and then incubated for 3 h with appropriate affinity-purified secondary antibody (goat {alpha}-mouse IgG, diluted 1:500, BioRad Laboratories, Hercules, CA; or rabbit {alpha}-rat IgG, diluted 1:1000, Sigma Chemical Co.) conjugated with alkaline phosphatase. Detection was performed colorimetrically by immersing the blots in a developing solution containing nitroblue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolyl phosphate (BCIP; BioRad).

Immunohistochemical Localization of hsp72
Methodology.
Kidneys and livers excised from control and Hg-treated rats were fixed overnight at room temperature in Carnoy's fixative (60% ethanol, 10% acetic acid, 30% chloroform) and then stored in 70% ethanol prior to processing. Sections were embedded in paraffin, serially sectioned, and mounted onto poly-L-lysine-coated glass slides. Sections were mounted unstained for immunohistochemical analysis, deparaffinized in xylene, and rehydrated through a decreasing series of ethanol concentrations (100%, 95%, 70%) into Tris-buffered saline (TBS; 10 mM Tris/150 mM NaCl, pH 8.0). Non-specific antibody binding was blocked by incubating the sections with 1% (v/v) normal sheep serum (Sigma Chemical Co.) in TBS. The blocking solution was drained from the slides and the sections incubated overnight with a mouse anti-human hsp72 antibody (SPA-810; StressGen Biotechnologies) diluted 1:500 in TBS + 1% sheep serum. All incubations were performed at room temperature in a humid chamber. Sections were washed for 5 min in TBS and incubated for 2 h with a secondary antibody-detection system consisting of purified goat anti-mouse IgG conjugated with alkaline phosphatase (BioRad Laboratories) at a dilution of 1:200. After rinsing with TBS, sections were incubated for 2 h with a mouse monoclonal anti-alkaline phosphatase antibody conjugated with alkaline phosphatase (Sigma Chemical Co.) at a 1:200 dilution. The slides were rinsed in TBS and incubated for 5 min with the chromogen substrate buffer (100 mM Tris–HCl, pH 9.5, Vector Laboratories, Burlingame, CA) containing levamisole (1 mM; Vector Laboratories) in order to inhibit endogenous alkaline phosphatase. Antibody signal was visualized using the Vector Black Kit II substrate (Vector Laboratories); the counterstain was hematoxylin. Parallel sections were stained with hematoxylin and eosin for histopathologic evaluation. Photomicrographs were made using Kodak Panatomic-X film.

Specificity controls.
Several parallel control immunohistochemistry experiments were conducted in order to demonstrate the specificity of the primary hsp72 antibody-binding response. First, a series of experiments were conducted such that, at each major protocol step, the appropriate vehicle buffers were substituted for each reagent, e.g., primary antibody, secondary antibody, tertiary antibody, or chromogen substrate. Second, the hsp72 primary antibody was substituted with an unrelated monoclonal antibody of the same immunoglobulin class and subclass (mouse anti-dog IgG1; Sigma Chemical Co). This antibody was diluted to the same working dilution and protein concentration as the hsp72 primary antibody. Third, an experiment employing a dilution series of the primary antibody was conducted that resulted in a gradual reduction of signal as a function of decreasing antibody concentration.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Attempts to understand the role of hsps and other stress proteins in response to toxicants should be accompanied by at least a minimal assessment of the degree of cell injury. While Hg(II) nephrotoxicity has been studied extensively, we were interested in understanding the relationship between the presence of stress proteins and the degree of nephropathy. An example of the nephrotic changes (defined here as the presence of epithelial degeneration and/or necrosis) in kidney after treatment with Hg(II) is shown in Figure 1Go, and the time- and dose-dependent nephrotic changes are summarized in Tables 1 and 2GoGo, respectively. The earliest time at which nephrosis was observed in rats given 1 mg Hg/kg was at 4 h after treatment (Table 1Go). These changes, when present, were minimal, occurring in less than 25% of the cortical tubules. A mixture of epithelial degeneration and individual tubular cell necrosis characterized these changes. At 8 h after injection with 1 mg/kg, all rats had evidence of nephrosis, with the changes characterized by degeneration, necrosis, and sloughing in the tubular epithelia. The severity in most of these kidneys was mild (25–50% tubules affected), although higher grades were recorded in a few rats. At 16 h (Fig. 1BGo) and 24 h after injection with 1 mg/kg, all rats exhibited moderate or severe nephrosis. Renal morphology in rats 16 h after treatment with 0.25 mg/kg was indistinguishable from controls (Table 2Go). Nephrosis was present in all rats given 0.5 mg/kg at 16 h post-treatment, with the predominant severity being mild.



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FIG. 1. Representative morphology in kidneys from control rats and from rats 16 h after receiving a single, intravenous injection of 1 mg Hg/kg, iv, as HgCl2. The kidney sections shown from control (A) and Hg-treated (B) rats incorporate a cortical labyrinth at the junction of the cortex, medullary rays, and outer stripe of the medulla. These regions contain both distal and proximal tubules. Kidneys from Hg-treated rats demonstrated tubular dilatation, loss of nuclei, and widespread degeneration and necrosis in the proximal tubular S3 segments, the predominant target cells for Hg in the kidney. Sections stained with hematoxylin/eosin. Scale bar = 15 µm, for both panels. Histopathology results are summarized in Tables 1 and 2GoGo.

 

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TABLE 1 Nephrosis Severity Time Course in Rats Injected with HgCl2
 

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TABLE 2 Nephrosis Severity Dose-Response in Rats Injected with HgCl2
 
Kidneys from control and Hg(II)-treated rats were analyzed for 4 stress proteins: hsp72 (inducible isoform of hsp70), grp94, hsp73 (constitutive isoform of hsp70), and hsp90. Western blots of renal proteins transferred from SDS–PAGE gels were probed with monoclonal antibodies specific for each of the 4 stress proteins (Fig. 2Go). In whole kidneys 16 h after Hg(II) treatment, dose-related changes in the expression of hsp72 and grp94, two readily inducible hsps, were observed. Minimal levels of hsp72 and grp94 were observed in control rats, accumulation of hsp72 was markedly increased at the two highest Hg(II) doses, and accumulation of grp94 was not increased above control levels except at the highest dose. The high, constitutive expression of hsp73 did not change as a result of Hg(II) exposure. Hsp90 was not detected in kidneys of control or Hg-treated rats.



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FIG. 2. Immunochemical analysis of 4 stress proteins in rat kidney from control rats and rats treated with several doses of Hg. Kidneys were excised 16 h after injection of 0.25, 0.5, or 1 mg Hg/kg, iv, as HgCl2. Western blots were probed with monoclonal antibodies specific for hsp72 (inducible isoform of hsp70), grp94, hsp73 (constitutive isoform of hsp70), and hsp90 (arrows) as described in Materials and Methods. Blots shown are representative of experiments repeated in at least 3 sets of rats. Molecular weights of protein standards are indicated (x1000).

 
Since expression of hsp72, grp94, and hsp73 was observed in whole kidney in the dose-response study, the time-dependent expression of these stress proteins was analyzed in whole kidney, and the cortex and medulla regions (Figs. 3–5GoGoGo). In Hg-treated rats, a time-related increase in the accumulation of hsp72 was evident in whole kidney (Fig. 3Go). The renal cortex and corticomedullary region expressed the majority of the hsp72 signal found in the whole kidney (Fig. 3Go, cortex). Minimal hsp72 signal was apparent in the renal medullary tissue (Fig. 3Go, medulla). Negligible hsp72 signal was expressed in control tissues. Similar to hsp72, a time-related increase in expression of grp94 was observed in whole kidneys from Hg-treated rats (Fig.4Go). In contrast to hsp72 where accumulation was limited to the cortex, grp94 accumulated primarily in the medulla, and not cortex, in a time-related manner that paralleled accumulation in the whole kidney. The high, constitutive expression of hsp73 did not change as a function of time after Hg(II) exposure in whole kidney, cortex, or medulla (Fig. 5Go).



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FIG. 3. Immunochemical detection of hsp72 (inducible isoform of hsp70) in rat kidney demonstrating the time-dependent regional expression of hsp72 in kidney of Hg-treated rats. At various times after injection of 1 mg Hg/kg, iv, as HgCl2, kidneys were excised and cortex and medulla regions were separated for analysis as described in Materials and Methods. Western blots were probed with a monoclonal antibody specific for the inducible hsp72 stress protein (arrow). Top—whole kidney, middle—cortex, bottom—medulla. Blots shown are representative of experiments repeated in at least 3 sets of rats. Molecular weights of protein standards are indicated (x1000).

 


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FIG. 4. Immunochemical detection of grp94 in rat kidney, demonstrating the time-dependent regional expression of grp94 in the kidneys of Hg-treated rats. At various times after injection of 1 mg Hg/kg, iv, as HgCl2, kidneys were excised and cortex and medulla regions were separated for analysis as described in Materials and Methods. Western blots were probed with a monoclonal antibody specific for the grp94 stress protein (arrow). Top—whole kidney, middle—cortex, bottom—medulla. Blots shown are representative of experiments repeated in at least 3 sets of rats. Molecular weights of protein standards are indicated (x1000).

 


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FIG. 5. Immunochemical detection of hsp73 (constitutive isoform of hsp70) in rat kidney demonstrating the regional expression of hsp73 in kidney of Hg-treated rats. At various times after injection of 1 mg Hg/kg, iv, as HgCl2, kidneys were excised and cortex and medulla regions were separated for analysis as described in Materials and Methods. Western blots were probed with a monoclonal antibody specific for the constitutive hsp73 stress protein (arrow). Top—whole kidney, middle—cortex, bottom—medulla. Blots shown are representative of experiments repeated in at least 3 sets of rats. Molecular weights of protein standards are indicated (x1000).

 
Since hsp72 has been implicated in cell recovery following injury and was localized predominately in the renal cortex (Fig. 3Go), and tubular cells within this region are the primary site of Hg-induced injury, we examined the cell-type specificity of the hsp72 response using immunohistochemistry (Fig. 6Go). Control kidney revealed a low-grade, diffuse hsp72 immunoreactivity signal that was localized in proximal and distal tubular epithelia but was absent from the glomeruli (Fig. 6AGo). The low-grade, diffuse signal was absent from kidney sections on which buffer was substituted for the primary antibody (Fig. 6EGo) or incubated with an isotype- and concentration-matched, irrelevant primary antibody (negative control). Other investigators have reported similar findings in which specific cell types in some species, including rat and gerbil kidney, exhibited constitutive expression of a protein recognized by the antibody that is selective for the inducible hsp72 (Nowak, 1993Go; Hernández-Pando et al., 1995).



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FIG. 6. Immunolocalization of hsp72 in kidneys of rats exposed to 1 mg Hg/kg, iv, as HgCl2. (Panel A) Control kidney, incubated with anti-hsp72; (panels B, C, and D) Kidney, 16 h after 1 mg Hg/kg, iv, incubated with anti-hsp72; panels B,C, and D represent the same area of the kidney and differ only in magnification; (E) Kidney, 16-h post treatment, incubated with buffer instead of anti-hsp72. Hg-induced accumulation of hsp72 was limited to intact distal tubules and S1 and S2 proximal tubule segments, and residual staining was evident in necrotic debris of S3 segments. At the highest magnification (C), immunoreactive hsp72 predominated in the cytoplasm of distal and proximal convoluted tubules, and in other cells, hsp72 immunoreactivity predominated in the nucleus. Immunohistochemistry procedures are described in Materials and Methods. All sections were probed with a monoclonal antibody specific for inducible hsp72, except in the primary antibody, buffer substitution experiment (E). Data shown are representative of experiments repeated in at least 3 sets of rats. Scale bar in panel E = 40 µm and represents the following scale in other panels: A and C = 20 µm; D = 40 µm; and B = 100 µm.

 
Changes in the renal tubular cell expression of hsp72 were observed after Hg treatment. At 4 h post-treatment, a diffuse, low-grade signal was present in tubules similar to controls. At 8 h post-treatment, foci representing expression of hsp72 were present in 1–5% of the tubules in the cortical labyrinths, and hsp72 was generally limited to the cytoplasmic compartment (data not shown). In contrast to kidneys from control and from the 4- and 8-h treatment groups, kidneys from rats treated 16 h earlier with Hg(II) demonstrated an intense, multifocal expression of inducible hsp72 in the cortex and outer stripe of the medulla, whereas the glomeruli, inner stripe and inner medulla were devoid of immunoreactivity (Fig. 6BGo). These results are compatible with the Western blot analysis of hsp72 in whole kidney, cortex, and medulla (Fig. 3Go). At higher magnification (Fig. 6DGo), intense foci demonstrating inducible hsp72 were localized to the distal tubules and proximal convoluted tubules, i.e., S1 and S2 segments. Some residual activity was apparent in necrotic proximal tubular straight segments (S3) in the outer stripe. This residual activity may have reflected condensation of cytoplasm (thus intensifying the staining), or previously unavailable non-specific binding sites unmasked by the necrosis. At the highest power evaluated (Fig. 6CGo), we observed intense hsp72 reactivity in distal tubules and proximal convoluted tubule S1 and S2 segments. In some of these cells, immunoreactive hsp72 predominated in the cytoplasm, and in other cells, immunoreactive hsp predominated in the nucleus.


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Metals are effective inducers of stress proteins, although the specific stress proteins induced can vary considerably. This is influenced by the type and dose of metal administered and the organism/tissue studied (for review, see Goering and Fisher, 1995; Sanders et al., 1996). Hsps are experimentally induced in kidney by a variety of insults, which may include exposure to nephrotoxic metals such as mercury, iron complexes, and cisplatin. Other such possible insults can include heat shock, ischemia, oxidative stress, inflammation, autoimmune diseases, hypertension, and vasopressin injection (Bardella and Comolli, 1994Go; Emami et al., 1991Go; Fukuda et al., 1996Go; Goering et al., 1992Go, 1993bGo; Lovis et al., 1994Go; Satoh et al., 1994Go; van Why et al., 1992Go, 1994Go). Previous studies from our laboratory demonstrated that expression of several stress proteins, including hsp72, after acute injection of rats with Hg(II), an effective producer of acute renal tubular necrosis, induced alterations in expression of stress proteins in whole kidney, but not in liver (Goering et al., 1992Go). Our initial goal was to expand these earlier findings in order to consider a broader spectrum of stress proteins that may be responsive to Hg(II) early in the pathogenesis of renal injury. Thus, we evaluated the regional expression of 4 stress proteins, including 3 hsps, in response to Hg(II). Our Western blot analyses demonstrated that the synthesis of 2 of the 4 stress proteins was induced in a dose- and time-dependent manner after exposure to Hg(II); hsp72 was expressed almost exclusively in renal cortex (Fig. 3Go) but not renal medulla, and grp94 was induced predominately in the medulla, but not the cortex (Fig. 4Go). Expression of hsp73 was predominant in controls and was unaffected by Hg(II) exposures, and hsp90 was not expressed in control kidneys or kidneys from Hg(II)-treated rats. These results have corroborated other studies that show regional heterogeneity in hsp localization and kinetics during cell injury and recovery in response to other nephrotoxicants. Iron overload, produced by injection of nephrotoxic iron complexes, resulted in accumulation of hsp90 in renal proximal and distal tubules (Fukuda et al., 1996Go). Hsp90 was expressed exclusively in regenerating cells of the S3 segment of proximal tubules damaged by cisplatin-induced acute tubular injury (Satoh et al., 1994Go). After exposure of rats to heat shock or ischemia, immunoreactive hsp72 was initially evident in the renal papilla, where the highest concentrations were observed, and subsequent hsp72 appeared in cortex and medulla (Emami et al., 1991Go).

Since hsp72, a major stress protein induced in cells undergoing stress, was induced in renal cortex, the primary site of acute Hg(II) injury, we chose to examine the cell-type specificity of the hsp72 response. Other studies have shown that induction of hsps in vivo occurred at the site of tissue injury. For example, in a study of cocaine hepatotoxicity in mice, Salminen et al. (1997) demonstrated that only hepatocytes in the portion of the liver lobule with overt histopathologic changes accumulated inducible hsp25 and hsp70. In our study, accumulation of hsp72 and grp94 was enhanced several h prior to or concurrent with the onset of nephrosis. However, concurrent or ensuing overt toxicity is not necessarily a predicate for stress protein induction (Salminen et al., 1998). Thus, our hypothesis for this segment of our study was that hsp72 synthesis would be enhanced in the primary target cells for Hg(II) in the kidney, i.e., the cells constituting the S3 segment of the proximal tubule found primarily in the renal cortex. In contrast to this hypothesis, the immunohistochemistry results demonstrated that hsp72 synthesis occurs predominantly in intact cells of both distal and proximal convoluted (S1 and S2 segments) tubules (Fig. 6Go), cells not considered primary target sites for Hg(II). Thus, these cell types may be more resistant to injury due to the cytoprotective properties of inducible hsps. Alternatively, lack of accumulation of hsp72 in intact S3 proximal tubule segments, perhaps related to the onset of irreversible cell injury before adequate hsps can be synthesized, may explain the sensitivity of these cell types to Hg(II). This conclusion is supported by the study of Fisher et al. (1995) who observed the lack of accumulation of hsp72 in target tissues, i.e., somites, in rat embryos exposed to heat shock. Hsp72, however, did accumulate in tissues spared thermal injury. Thus, the inability to express hsps in specific tissues, in part related to protective responses being overwhelmed by a high dose, may be associated with the susceptibility of these tissues to injury by toxicants.

Although all cells and tissues may appear to be capable of eliciting a stress response, there appears to be a spectrum of responsiveness between different cell types (Nowak, 1993Go). Pharmacokinetic differences in distribution of a toxic agent to specific cell types cannot completely explain these variations in response. For example, tissue differences in expression of hsp72 were observed in rat embryos exposed to heat shock, which theoretically delivers similar thermal doses to all tissues (Fisher et al., 1995Go). Tissue- and stressor-specific differences in hsp expression may be explained, at least in part, by the presence of multiple heat-shock gene trans-acting factors with different patterns of activation (Buckiová and Jelínek, 1995Go). Although different metals may share the ability to induce stress proteins in the same tissue, the patterns of protein expression may be unique to a specific metal (Goering and Fisher, 1995Go). For example, arsenic and gallium induce similar patterns of multiple stress proteins in rat kidney epithelial cells (Aoki et al., 1990Go). In contrast, lead has been shown to induce a single 32-kDa stress protein in rat kidney epithelial cells (Shelton et al., 1986Go). This specificity in response makes it difficult to offer generalizations regarding the specific induction of stress proteins by metals. These observed differences in protein induction might also reflect differences in the mechanisms of action by which specific metals elicit toxicity.

During exposure to environmental insults, nascent hsp72 translocates from the cytoplasm to the nucleus and back to cytoplasm during recovery (Brown et al., 1993Go; Ellis, 1990; Gething and Sambrook, 1992Go; Lindquist, 1986Go; Nover, 1991Go; Sanders et al., 1996Go; Schlesinger, 1990Go, 1994Go; Welch and Mizzen, 1988Go;). Subsequent binding of hsp72 with periribosomes and other nuclear matrix proteins promotes cellular adaptation and recovery by enabling the cell to conserve the functions of the nucleus and nucleolus and to protect macromolecules involved in protein biosynthesis. After exposure to an insult ceases, hsp72 and hsp90 continue to serve a cytoprotective role by participating in the proper refolding of proteins back to their original conformation and "chaperoning" irreversibly damaged proteins into lysosomes for degradation (Chiang et al., 1989Go; Johnston and Kucey, 1988Go; Nover, 1991Go; Riabowol et al., 1988Go; Sanchez and Lindquist, 1990Go). In the present study, evidence for the interorganelle translocation of hsp72 was seen. We observed hsp72 reactivity in both the cytoplasm and nuclei of distal tubules and proximal convoluted tubule S1 and S2 segments in kidneys of rats exposed to Hg(II). In some cells, the hsp72 signal was predominant in cytoplasm relative to the nucleus; in other cells, the signal predominated in the nucleus when compared to staining observed in the cytoplasm. Thus, these microscopic fields may represent a "snapshot" of kidney cells undergoing different phases of hsp72 translocation from cytoplasm to nucleus during cellular injury, and back to cytoplasm during recovery. These results are supported by those of Satoh et al. (1994), who provided evidence for translocation of hsp90 from nucleus to cytoplasm in regenerating S3-segment proximal tubule epithelia, the target cells of cisplatin toxicity in kidney, during the course of cisplatin-induced acute renal failure and recovery in rats.

We have not established in our study whether enhanced hsp72 synthesis in distal tubules and proximal convoluted tubules served to protect these cells from Hg(II) cytotoxicity or aid in recovery; however, some studies have reported a correlation between the accumulation of hsps in renal cells responding to toxic stress and cytoprotection (Borkan et al., 1993Go; Emami et al., 1991Go; Nissim et al., 1992Go). A transient thermotolerance induced in rat kidney collecting-duct cells was temporally related to the appearance and disappearance of hsp72 (Borkan et al., 1993Go). Heat shock-induced synthesis of hsp72 was associated with resistance of kidneys to subsequent ischemic injury (Chatson et al., 1990Go). A progressive increase in hsp90 synthesis in renal tubular cells in response to repeated doses of iron complexes was correlated with increasing resistance to renal injury caused by iron overload-induced oxidative damage (Fukuda et al., 1996Go).

In our study, the presence of hsp72 immunoreactivity in necrotic S3 segments may be due to residual staining of previously viable cells or represents non-specific staining due to the increased availability of antigenic sites in cells undergoing necrosis. A plausible explanation for the appearance of residual hsp72 in these cells is that the cytoprotective properties of stress proteins at low doses of toxic agents may be overwhelmed at higher doses. This hypothesis is supported by the work of Hansen et al. (1988), who demonstrated that stress proteins are produced by non-teratogenic and teratogenic doses of diphenylhydantoin in embryonic target tissues. Further, Fisher et al. (1995) showed hsp72 accumulates in neuroectoderm and neural tube tissues of rat embryos after heat shock. Thermal doses higher than those employed by Fisher et al. (1995) did result in neural damage (Mirkes, 1985Go; Walsh et al., 1989Go; Kimmel et al., 1993Go).

Our previous studies (Goering et al., 1992Go; 1993aGo,bGo) demonstrated that the induction of hsp72 in Hg(II)- and Cd(II)-treated rats was target organ-specific. In the current study, we expanded these investigations in order to study a variety of stress proteins that accumulate in kidney in response to Hg(II), the regional expression of those stress proteins that are responsive, and finally the intrarenal localization of hsp72 in rats with Hg(II)-induced acute renal injury. We demonstrated that, in response to Hg(II) exposure, enhanced synthesis of hsp72 in kidneys of Hg(II)-treated rats is limited to the cortex and regions constituting the corticomedullary junction, while grp94 expression was limited to the medullary region, and not the cortex. In contrast to our expectations, we demonstrated that within the cortical region, inducible hsp72 was localized primarily in distal tubule and proximal convoluted tubule epithelia, cells generally spared early injury by Hg(II). Stress protein expression in rat kidney exhibits regional and cell-specific heterogeneity in response to Hg(II) exposure, and a positive correlation was shown between accumulation of some stress proteins and acute renal cell injury. While the role of stress protein accumulation in vivo prior to or concurrent with nephrotoxicity remains to be fully understood, stress proteins, including several hsps, may be part of a cellular defense response to nephrotoxicants. Conversely, cells unable to express stress proteins at levels high enough to confer protection, or whose capacity to synthesize them may be overwhelmed by the exposure, may be more susceptible to injury and lose potential for recovery. Further knowledge of the roles of stress proteins, including the hsps, in the cellular responses to toxicants will increase our understanding of the fundamental mechanisms of renal cell injury.


    NOTES
 
Statements contained in this paper are the opinions of the authors and do not represent FDA policy.

1 To whom all correspondence should be addressed at CDRH/FDA (HFZ-112), 12709 Twinbrook Parkway, Rockville, Maryland 20857. Fax: 301–594–6775. E-mail: plg{at}cdrh.fda.gov. Back

2 Present address: Covance Laboratories, Vienna, VA. Back


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aoki, Y., Lipsky, M. M., and Fowler, B. A. (1990). Alteration in protein synthesis in primary cultures of rat kidney proximal tubule epithelial cells by exposure to gallium, indium, and arsenite. Toxicol. Appl. Pharmacol. 106, 462–468.[ISI][Medline]

Arrigo, A. P., Suhan, J. P., and Welch, W. J. (1988). Dynamic changes in the structure and intracellular locale of the mammalian low-molecular-weight heat-shock protein. Mol. Cell Biol. 8, 5059–5071.[ISI][Medline]

Bardella, L., and Comolli, R. (1994). Differential expression of c-jun, c- fos, and hsp70 mRNAs after folic acid and ischemia-reperfusion injury: Effect of antioxidant treatment. Exp. Nephrol. 2, 158–165.[ISI][Medline]

Beckmann, R. P., Mizzen, L. A., and Welch, W. J. (1990). Interaction of hsp70 with newly synthesized proteins: Implications for protein folding and assembly. Science 248, 850–854.[ISI][Medline]

Beckmann, R. P., Lovett, M., and Welch, W. J. (1992). Examining the function and regulation of hsp70 in cells subjected to metabolic stress. J. Cell Biol. 117, 1137–1150.[Abstract]

Borkan, S. C., Emami, C. A., and Schwartz, J. H. (1993). Heat stress protein-associated cytoprotection of inner medullary collecting duct cells from rat kidney. Am. J. Physiol. 265, F333–341.[Abstract/Free Full Text]

Brown, C. R., Martin, R. L., Hansen, W. J., Beckmann, R. P., and Welch, W. J. (1993). The constitutive and stress-inducible forms of hsp70 exhibit functional similarities and interact with one another in an ATP-dependent fashion. J. Cell Biol. 120, 1101–1112.[Abstract]

Buckiová, D., and Jelínek, R. (1995). Heat-shock proteins and teratogenesis. Reprod. Toxicol. 9, 501–511.[ISI][Medline]

Chatson, G., Perdrizet, G., Anderson, C., Pleau, M., Berman, M., and Schweizer, R. (1990). Heat shock protects kidneys against warm ischemic injury. Curr. Surg. Nov-Dec, 420–423.

Chiang, H. L., Terlecky, S. R., Plant, C. P., and Dice, J. F. (1989). A role for a 70-kilodalton heat-shock protein in lysosomal degradation of intracellular proteins. Science 246, 382–385.[ISI][Medline]

Coimbra, T. M., Cieslinski, D. A., and Humes, H. D. (1990). Epidermal growth factor accelerates renal repair in mercuric chloride nephrotoxicity. Am. J. Physiol. 259, F438–443.[Abstract/Free Full Text]

Cowley, Jr., B. D., and Gudapaty, S. (1995). Temporal alterations in regional gene expression after nephrotoxic renal injury. J. Lab. Clin. Med. 125, 187–199.[ISI][Medline]

Craig, E. A., Baxter, B. K., Becker, J., Halladay, J., and Ziegelhoffer, T. (1994). Cytosolic hsp70s of Saccharomyces cerevisiae: Roles in protein synthesis, protein translocation, proteolysis, and regulation. In The Biology of Heat-Shock Proteins and Molecular Chaperones (R. I. Morimoto, A. Tissieres, and C. Georgopoulos, Eds.), pp. 31–53. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Ellis, R. J. (1989). The molecular chaperone concept. (Review) Semin. Cell Biol. 1, 1–9.

Emami, A., Schwartz, J., and Borkan, S. (1991). Transient ischemia or heat-shock stress induces a cytoprotective protein in rat kidney. Am. J. Physiol. 262, F479–486.

Fisher, B. R., Heredia, D. J., and Brown, K. (1995). Induction of hsp72 in heat-treated rat embryos: A tissue-specific response. Teratology 52, 90–100.[ISI][Medline]

Fukuda, A., Osawa, T., Oda, H., Tanaka, T., Toyokuni, S., and Uchida, K. (1996). Oxidative stress response in iron-induced acute nephrotoxicity: Enhanced expression of heat-shock protein 90. Biochem. Biophys. Res. Commun. 219, 76–81.[ISI][Medline]

Gething, M. J., and Sambrook, J. (1992). Protein folding in the cell. Nature 355, 33–45.[ISI][Medline]

Goering, P. L., and Fisher, B. R. (1995). Metals and stress proteins. In Handbook of Experimental Pharmacology, Vol. 115: Toxicology of MetalsBiochemical Aspects (R. A. Goyer and M. G. Cherian, Eds.), pp. 229–266. Springer Verlag, New York.

Goering, P. L., Fisher, B. R., Chaudhary, P. P., and Dick, C. A. (1992). Relationship between stress protein induction in rat kidney by mercuric chloride and nephrotoxicity. Toxicol. Appl. Pharmacol. 113, 184–191.[ISI][Medline]

Goering, P. L., Fisher, B. R., and Kish, C. L. (1993a). Stress protein synthesis induced in rat liver by cadmium precedes hepatotoxicity. Toxicol. Appl. Pharmacol. 122, 139–148.[ISI][Medline]

Goering, P. L., Kish, C. L., and Fisher, B. R. (1993b). Stress protein synthesis induced by cadmium-cysteine in rat kidney. Toxicol. 85, 25–39.[ISI][Medline]

Gritzka, T. L., and Trump, B. F. (1968). Renal tubular lesions caused by mercuric chloride. Am. J. Pathol. 52, 1225–1227.[ISI][Medline]

Hansen, D. K., Anson, J. F., Hinson, W. G., and Pipkin, J. L., Jr. (1988). Phenytoin-induced stress protein synthesis in mouse embryonic tissue. Proc. Soc. Exp. Biol. Med. 189, 136–140.[Abstract]

Hernádez-Pando, R., Pedraza-Chaverri, J., Orozco-Estévez, H., Silva-Serna, P., Moreno, I., Rondán-Zárate, A., Elinos, M., Correa-Rotter, R., and Larriva-Sahd, J. (1995). Histological and subcellular distribution of 65 and 70 kDa heat-shock proteins in experimental nephrotoxic injury. Exp. Toxic Pathol. 47, 501–508.[ISI][Medline]

Hightower, L. E. (1991). Heat shock, stress proteins, chaperones, and proteotoxicity. Cell 66, 191–197.[ISI][Medline]

Hightower, L. E., Sadis, S. E., and Takenaka, I. M. (1994). Interaction of vertebrate hsc70 and hsp70 with unfolded proteins and peptides. In The Biology of Heat-shock Proteins and Molecular Chaperones (R. I. Morimoto, A. Tissieres, and C. Georgopoulos, Eds.), pp. 179–209. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

Howard, K. J., Holley, S. J., Yamamoto, K. R., and Distelhorst, C. W. (1990). Mapping the HSP90 binding region of the glucocorticoid receptor. J. Biol. Chem. 265, 11928–11935.[Abstract/Free Full Text]

Johnston, R. N., and Kucey, B. L. (1988). Competitive inhibition of hsp70 gene expression causes thermosensitivity. Science 242, 1551–1554.[ISI][Medline]

Kimmel, G. L., Cuff, J. M., Kimmel, C. A., Heredia, D. J., Tudor, N., and Silverman, P. M. (1993). Embryonic development in vitro, following short-duration exposure to heat. Teratology 47, 243–251.[ISI][Medline]

Lindquist, S. (1986). The heat-shock response. Ann. Rev. Biochem. 55, 1151–1191.[ISI][Medline]

Lovis, C., Mach, F., Donati, Y. R. A., Bonventre, J. V., and Polla, B. S. (1994). Heat shock proteins and the kidney. Renal Failure 16, 179–192.[ISI][Medline]

Mirkes, P. E. (1985). Effects of acute exposure to elevated temperatures on rat embryo growth and development in vitro. Teratology 32, 259–266.[ISI][Medline]

Mirkes, P. E. (1987). Hyperthermia-induced heat-shock response and thermotolerance in postimplantation rat embryos. Dev. Biol. 119, 115–122.[ISI][Medline]

Morimoto, R. I., Tissieres, A., and Georgopoulos, C. (1990). The stress response, function of the proteins, and perspectives. In Stress Proteins in Biology and Medicine (R. I. Morimoto, A. Tissieres, and C. Georgopoulos, Eds.), pp. 1–36. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

National Toxicology Program (1993). Toxicology and carcinogenesis studies of mercuric chloride in F344 rats and B6C3F1 mice. Technical Report No. 408, pp. 36–65. U.S. Dept. of Hlth. Human Services, Natl. Inst. Hlth., Res. Tri. Pk., NC.

Nissim, I., Hardy, M., Pleasure, J., Nissim, I., and States, B. (1992). A mechanism of glycine and alanine cytoprotective action: Stimulation of stress-induced HSP-70 mRNA. Kidney Intl. 42, 775–782.[ISI][Medline]

Nover, L. (1991). Heat Shock Response. CRC Press, Boca Raton, FL.

Nowak, T. S., Jr. (1993). Synthesis of heat-shock/stress proteins during cellular injury. Ann. N Y Acad. Sci. 679, 142–156.[Abstract]

Perdrizet, G. A., Kaneko, H., Buckley, T. M., Fishman, M. S., Pleau, M., Bow, L., and Schweizer, R. T. (1993). Heat shock and recovery protects renal allografts from warm ischemic injury and enhances hsp72 production. Transplantation Proc. 25, 1670–1673.[ISI][Medline]

Plumier, J. C., Krueger, A. M., Currie, R. W., Kontoyiannis, D., Kollias, G., and Pagoulatos, G. N. (1997). Transgenic mice expressing the human inducible Hsp70 have hippocampal neurons resistant to ischemic injury. Cell Stress Chaperones 2, 162–167.[ISI][Medline]

Plumier, J. C., Ross, B. M., Currie, R. W., Angelidis, C. E., Kazlaris, H., Kollias, G., and Pagoulatos, G. N. (1995). Transgenic mice expressing the human heat shock protein 70 have improved post-ischemic yocardial recovery. J. Clin. Invest. 95, 1854–1860.[ISI][Medline]

Riabowol, K. T., Mizzen, L. A., and Welch, W. J. (1988). Heat shock is lethal to fibroblasts microinjected with antibodies against hsp70. Science 242, 433–436.[ISI][Medline]

Ritossa, F. M. (1962). A new puffing pattern induced by heat shock and DNP in Drosophila. Experimentia 18, 571–573.[ISI]

Rothman, J. E. (1989). Polypeptide chain binding proteins: Catalysts of protein folding and related processes in cells. Cell 59, 591–601.[ISI][Medline]

Salminen, W. F., Jr., Roberts, S. M., Fenna, M., and Voellmy, R. (1997). Heat-shock protein induction in murine liver after acute treatment with cocaine. Hepatology 25, 1147–1153.[ISI][Medline]

Salminen, W. F., Jr., Voellmy, R., and Roberts, S. M. (1997). Effect of N-acetylcysteine on heat-shock-protein induction by acetaminophen in mouse liver. J. Pharmacol. Exp. Ther. 286, 519–524.[Abstract/Free Full Text]

Sanchez, Y., and Lindquist, S. L. (1990). HSP104 required for induced thermotolerance. Science 248, 1112–1115.[ISI][Medline]

Sanders, B. M., Goering, P. L., and Jenkins, K. (1996). The role of general and metal-specific cellular responses in protection and repair of metal-induced damage: Stress proteins and metallothioneins. In Toxicology of Metals (L. W. Chang, Ed.), pp. 165–187. CRC/Lewis Publishers, New York.

Satoh, K., Wakui, H., Komatsuda, A., Nakamoto, Y., Miura, A. B., Itoh, H., and Tashima, Y. (1994). Induction and altered localization of 90-kDa heat-shock protein in rat kidneys with cisplatin-induced acute renal failure. Ren. Fail. 16, 313–323.[ISI][Medline]

Schlesinger, M. J. (1990). Heat shock proteins. J. Biol. Chem. 265, 12111–12114.[Free Full Text]

Schlesinger, M. J. (1994). How the cell copes with stress and the function of proteins. Pediatric Res. 36, 1–6.[Abstract]

Shelton, K. R., Todd, J. M., and Egle, P. M. (1986). The induction of stress-related proteins by lead. J. Biol. Chem. 261, 1935–1940.[Abstract/Free Full Text]

Tytell, M., Barbe, M. F., and Brown, I. R. (1994). Induction of heat-shock (stress) protein 70 and its mRNA in the normal and light-damaged rat retina after whole body hyperthermia. J. Neurosci. Res. 38, 19–31.[ISI][Medline]

Van Why, S. K., Hildebrandt, F., Ardito, T., Mann, A. S., Siegel, N. J., and Kashgarian, M. (1992). Induction and intracellular localization of HSP-72 after renal ischemia. Am. J. Physiol. 263, F769–F775.[Abstract/Free Full Text]

Van Why, S. K., Mann, A. S., Thulin, G., Zhu, X.-H., Kashgarian, M., and Siegel, N. J. (1994). Activation of heat-shock transcription factor by graded reductions in renal ATP, in vivo, in the rat. J. Clin. Invest. 94, 1518–1523.[ISI][Medline]

Walsh, D. A., Li, K., Speirs, J., Crowther, C. E., and Edwards, M. J. (1989). Regulation of the inducible heat-shock 71 genes in early neural development of cultured rat embryos. Teratology 40, 321–334.[ISI][Medline]

Welch, W. J. (1992). Mammalian stress response: Cell physiology, structure/function of stress proteins, and implications for medicine and disease. Physiol. Rev. 72, 1063–1081.[Free Full Text]

Welch, W. J. (1993). How cells respond to stress. Sci. Am. 268, 56–64.

Welch, W. J., and Feramisco, J. R. (1985). Disruption of the three cytoskeletal networks in mammalian cells does not affect transcription, translation, or protein translocation changes induced by heat shock. Mol. Cell Biol. 5, 1571–1581.[ISI][Medline]

Welch, W. J., and Mizzen, L. A. (1988). Characterization of the thermotolerant cell: II. Effects on the intercellular distribution of heat-shock protein 70, intermediate filaments, and small nuclear ribonucleoprotein complexes. J. Cell Biol. 106, 1117–1130.[Abstract]

Yamamoto N., Maki, A., Swann, J. D., Berezesky, I. K., and Trump, B. F. (1993). Induction of immediate early and stress genes in rat proximal tubule epithelium following injury: The significance of cytosolic ionized calcium. Ren. Fail. 15, 163–171.[ISI][Medline]





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