* 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;
Pathology Associates International, Frederick, Maryland 21701; and
Covance Laboratories, Vienna, Virginia 22182
Received July 6, 1999; accepted September 2, 1999
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ABSTRACT |
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Key Words: mercury; kidney; acute renal injury; heat-shock proteins; hsp72; glucose-regulated proteins; grp94; stress proteins; immunohistochemistry..
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INTRODUCTION |
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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, 1992; Rothman, 1989
), bind a variety of intracellular receptors to keep them in a non-activated state (Howard et al., 1990
), and shuttle nascent polypeptides and proteins across intracellular membranes (Beckmann et al., 1990
; Craig et al., 1994
; Hightower et al., 1994
). 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., 1992
; Hightower, 1991
; Welch, 1993
). 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., 1988
; Brown et al., 1993
; Ellis, 1990; Lindquist, 1986
; Welch and Feramisco, 1985
). Elevated levels of hsp72, a widely investigated hsp, have been associated with cytoprotection against a variety of lethal or toxic insults (Johnston and Kucey, 1988
; Mirkes, 1987
; Riabowol et al., 1988
; Tytell et al., 1994
; Welch and Mizzen, 1988
), including protection of kidneys from ischemic injury (Chatson et al., 1990
; Perdrizet et al., 1993
). Heart and brain cells in transgenic mice that overexpress the inducible form of hsp70 are resistant to ischemia (Plumier et al., 1995
, 1997
).
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., 1992, 1993a
,Goering et al., b
). 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., 1992
, 1993a
,Goering et al., b
). Other studies have shown target tissue specificity of the stress-protein response in tissues such as brain (Nowak, 1993
) 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., 1992), 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, 1968
).
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MATERIALS AND METHODS |
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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, 1993).
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 23 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 TrisHCl, 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 (SDSPAGE) 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 TrisHCl and sample buffer (62.5 mM TrisHCl, 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 SDSPAGE 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., 1992). 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
-human IgG1, dilution 1:500; SPA-810; StressGen Biotechnologies, Inc., Vancouver, BC, Canada), constitutive hsp73 (rat
-hamster IgG2a, diluted 1:500; SPA-815, StressGen), grp94 (rat
-chicken IgG2a, diluted 1:500; SPA-850, StressGen), and hsp90 (mouse
-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
-mouse IgG, diluted 1:500, BioRad Laboratories, Hercules, CA; or rabbit
-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 TrisHCl, 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.
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RESULTS |
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DISCUSSION |
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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. 6), 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, 1993). 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., 1995
). 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, 1995
). 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, 1995
). For example, arsenic and gallium induce similar patterns of multiple stress proteins in rat kidney epithelial cells (Aoki et al., 1990
). In contrast, lead has been shown to induce a single 32-kDa stress protein in rat kidney epithelial cells (Shelton et al., 1986
). 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., 1993; Ellis, 1990; Gething and Sambrook, 1992
; Lindquist, 1986
; Nover, 1991
; Sanders et al., 1996
; Schlesinger, 1990
, 1994
; Welch and Mizzen, 1988
;). 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., 1989
; Johnston and Kucey, 1988
; Nover, 1991
; Riabowol et al., 1988
; Sanchez and Lindquist, 1990
). 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., 1993; Emami et al., 1991
; Nissim et al., 1992
). A transient thermotolerance induced in rat kidney collecting-duct cells was temporally related to the appearance and disappearance of hsp72 (Borkan et al., 1993
). Heat shock-induced synthesis of hsp72 was associated with resistance of kidneys to subsequent ischemic injury (Chatson et al., 1990
). 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., 1996
).
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, 1985; Walsh et al., 1989
; Kimmel et al., 1993
).
Our previous studies (Goering et al., 1992; 1993a
,b
) 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.
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NOTES |
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1 To whom all correspondence should be addressed at CDRH/FDA (HFZ-112), 12709 Twinbrook Parkway, Rockville, Maryland 20857. Fax: 3015946775. E-mail: plg{at}cdrh.fda.gov.
2 Present address: Covance Laboratories, Vienna, VA.
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