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Histological and subcellular distribution of 65 and 70 kD heat shock proteins in experimental nephrotoxic injury
Exp Toxic Pathol 1995; 47: 501-508 Gustav Fischer Verlag Jena
Experimental Pathology Laboratory and Department of Nephrology and Mineral Metabolism, Instituto Nacional de la Nutrici6n Salvador Zubinin, Mexico City, *) Facultad de Quimica, National Autonomus University of Mexico, Mexico
Histological and subcellular distribution of 65 and 70 kD heat shock proteins in experimental nephrotoxic injury ROGELIO HERNANDEZ-PANDO, JOSE PEDRAZA-CHAVERRI*), HECTOR ORozco-EsTEVEZ, PALMIRA SILVA-SERNA, IVETTE MORENO, ADRIAN RONDAN-ZARATE, MARTHA ELINOS, RICARDO CORREA-RoTTER and JORGE LARRIVA-SAHD With 3 figures and 1 table Received: January 31,1995; Accepted: April 25, 1995 Address for correspondence: ROGELIO HERNANDEZ-PANDO, M. D., Ph. D. Experimental Pathology Laboratory, Department of Pathology, Instituto Nacional de la Nutrici6n Salvador Zubinin, Vasco Quiroga No. 15, Tlalpan, 14000, Mexico D.F. MEXICO; Tel. 011-(525)-573-12-00 Ext 2185, Fax 011-(525)-655-10-76. Key words: Heat shock; Shock, heat; Nephrotoxic injury; Kidney, tubular necrosis; Proteins, shock; mercury.
Summary The cellular distribution of 65 and 70 kD heat shock proteins (HSPs) was studied in the normal rat kidney and after acute tubular necrosis (ATN) induced by inorganic mercury (HgCl z). In the normal kidney the 65 kD HSP was found in the cytoplasm of podocytes and proximal convoluted tubules, whereas the 70 kD HSP was located in nuclei and cytoplasm of podocytes, cortical convoluted, and collecting tubules. The distribution of both HSPs along ATN changed as a function oftime. In the early phase, before evidence of histological damage, both HSPs were found in the pielocalyceal epithelium and medullary collecting tubules. During the necrotic phase, HSPs coexisted with sites of severe damage (i.e. cortical tubules). With immunoelectron microscopy, damaged cells showed an abundance of 65 kD HSP-I in mitochondria, as well as in chromatin and nucleoli, while 70 kD HSP-I was overexpressed in the cytoplasm, mitochondria, lysosomes, cytoskeleton, chromatin, and nucleoli, and coincided with urinary excretion of HSPs. In the postregenerative phase, the distribution of HSPs was similar to that found in the normal kidney. HSPs of 65 and 70 kD were encountered constitutionally and their immunolabeling is correlated with the magnitude of cell injury.
Introduction Heat shock proteins playa key role in the homeostatic mechanisms of the cell. Under normal circumstances the cell is restrained to a range of functions and structure established by the interaction of its genomic program of differentiation, the availability of metabolic substrates,
constrains of neighboring cells and the limited capacities of its metabolic pathways. This homeostatic "steady state" can be modified by greater physiological demands or some pathologic stimuli which induce a number of adaptation mechanisms that establish a new, but altered steady state in an attempt to preserve the viability of the cell. Among the various adaptative mechanisms, one of the most remarkable is the rapid and coordinated increased synthesis of a group of distinct proteins collectively referred to as heat shock proteins (HSPs). HSPs were initially recognized by their increased quantity after cell exposure to elevated temperature (16). It has recently been clearly established that induction of HSPs follows a wide variety of cell stressing conditions (19). However, in several cell types, some HSPs occur in the absence of a noxious stimulus (19). HSPs have a fairly constant structure among species, from prokaryotes to mammals (21) and have been grouped into a few families according to their molecular weight. Among them, HSP 70 kD and 65 kD are particularly abundant in all organisms examined so far (9). HSPs can assume multiple important functions in the cell, such as assisting in protein folding and assembly by reducing the tendency to aggregate, thus stabilizing proteins and ultimately protecting cells from the deleterious effect(s) of high temperature and other types of noxious agents (10). The present study describes the tissue distribution and subcellular location of HSP 65 and 70 kD in the normal rat kidney and in the experimental model of acute tubular necrosis (ATN) and regeneration induced by intraperitoneal administration of HgCl z.
Exp Toxic Pathol 47 (1995) 6
501
Material and methods Animals and experimental model of A TN Male wistar rats (250--350 grams of body weight) received intraperitoneal injections of a single dose of 1.5 mg/kg HgCl 2 (n = 24) or vehicle (n = 2). This dose of HgCl 2 induces necrosis and regeneration of cortical tubular elements but is rarely lethal (5). Experimental rats were sacrificed at the following time points: 4, 8, 16, 24 hours and 2, 3, 5,7, 9, 15, 22 and 28 days (n = 2). Vehicle injected rats were kept as controls to assess constitutive HSP expression and were sacrificed 24 hours after injection. Urine samples, for biochemical measurements and Western blot analysis of HSPs were collected by metabolic cage collections. Blood was collected at sacrifice and kidneys were removed and fixed by immersion for subsequent histological and immunohistochemical studies (vide infra).
Biochemical studies Blood urea nitrogen (BUN) and serum and urinary creatinine were measured by autoanalyzer (Cratinine analyzer 2 and BUN analyzer 2, Beckman Instruments Co, Fullerton, CA) and creatinine clearance was calculated. Total urinary protein was measured according to the method described by LOWRY et al. (l1).
Renal histology and immunohistochemistry For light microscopy kidney slices were fixed by immersion in absolute ethanol for 2 hours, paraffin embedded, sectioned at 6 /lm, and stained with hematoxylin and eosin for histological evaluation. Immunohistochemical detection of 65 and 70 kD HSPs was performed with monoclonal antibodies (Sigma Co). Before incubation with the primary antibody, endogenous peroxidase activity was inactivated (18), then tissue sections were incubated with each one of the monoclonal antibodies for 3 hours. After rinsing, rabbit anti-mice antibody conjugated with peroxidase was incubated for one hour. Peroxidase was visualized with 3,3-diaminobenzidine and counterstained with hematoxylin.
Immunoelectron microscopy For immunoelectron microscopic studies, tissue samples were obtained from cortex, juxtamedular cortex, medulla and calyces. The tissues were immediately fixed by immersion in 4 % paraformaldehyde dissolved in Sorensen's buffer pH 7.4 for 2 hours at 4 °C. After rinsing, free aldehyde groups were blocked in 0.5 M NH4Cl in phosphate-buffersaline (PBS) for one hour. Tissue samples were dehydrated in graded ethyl alcohols and embedded in LR-White hydrosoluble resin. Thin sections from 70 to 90 nm were placed ion nickel grids. The grids were incubated with the mouse anti-HSP monoclonal antibodies diluted in PBS with 1 % bovine serum albumin and 0.5 % Tween. After repeatedly rinsing with PBS, the grids were incubated with rabbit antimouse IgG and IgM conjugated to 10 nm gold particles (Amersham, UK) diluted in the same buffer. The grids were stained with uranium salts and examined in a Zeiss EM 10 electron microscope. 502
Exp Toxic Pathol 47 (1995) 6
Analysis of urinary proteins by immunoelectrotransference Urinary proteins were separated by SDS-PAGE under reducing conditions in a discontinuous buffer system (8) on slab gels of 10 % acrylamide. Gels were mounted in an electrotransfer chamber (LKB, Sweden) and transferred to nitrocellulose sheets (Bio Rad Laboratories, Richmond, CA). Unbounded sites were blocked with 3 % BSA in PBS Tween 20. Strips were incubated with the anti-HSP monoclonal antibodies for one hour at 37 °C. After rinsing, strips were exposed to rabbit anti-mouse antibody conjugated with peroxidase for 90 minutes and visualized with HP2 and 4-chloro-I-naphthol for 15 minutes.
Results Biochemical parameters As shown in table 1, creatinine clearance had decreased in the experimental group by 16 hours, reaching the lowest value at 3 days and returning to normal values at Day five. BUN had increased by 16 hours, reaching the peak on Day 3, and returned to control values by Day 7. Urinary volume increased on Day 2 in the HgCl2 injected rats, which remained polyuric until Day 7. Urinary protein excretion increased on Day 1 after HgCl 2 injection and returned to normal values by Day 5.
Histopathology of experimental ATN With the light microscope no evidence of cell injury was seen 4 and 8 hours after HgCl2 administration. Tubular necrosis was evident at 16 hours and more prominent in the deep cortex, especially in the pars recta of the proximal convoluted tubules (PRPCT). Tubular cell necrosis was characterized by pyknosis, granular cytoplasmic appearance, acidophilia, and partial epithelial detachment (fig. la). Kidneys from animals sacrificed 24 hours after HgCl 2 administration displayed tubular necrosis predominantly at the middle portion of the cortex, involving mostly proximal convoluted tubules (PCT) , which contained luminal debris and sloughed epithelial cells presenting an advanced stage of cytolysis. The maximum degree of necrosis was observed at 48 hours after HgCl 2 administration, when tubular necrosis was observed within the entire renal cortex (fig. 1b). On Day 2 morphologic features related to the beginning of regeneration were observed, such as segments of tubular necrosis displaying flat and cuboidal basophilic cells with large nuclei and distinct nucleoli and mitoses (fig. Ic). Cortical convoluted tubules were progressively lined by flat epithelial cells on Day 15. On Day 21 and thereafter the histological aspect of the kidney was comparable to that observed in the normal rat kidney.
Table 1. Biochemical parameters of control and HgCl 2-treated rats.
Time
creatinine clearance (mllmin)
BUN (mg/dl)
urinary volume (m1l24 h)
urinary protein (mg/24 h)
Control
1.2 ± 0.1
23 ± 1
13 ± 5
17 ± 6
HgCl z 16 h 24 h 2d 3d 5d 7d 9d 15 d 22 d 28 d
0.7 ± 0.4 0.7 ± 0.3 0.3 ±0.2 0.2 ± 0.1 1.5 ± 0.1 1.5 ± 0.6 1.1 ± 0.1 1.4 ± 0.2 1.7±0.1 1.2 ± 0.1
50 ±7 42 ±7 56 ± 1 102±6 33 ± 2 23 ± 1 25 ±7 22 ± 1 24 ± 3 25 ± 3
7 ± 2* 14 ± 2 48 ±4 32 ± 3 28 ± 9 35 ± 3 21 ± 8 18 ± 12 16 ± 9 19 ± 2
17 ± 2** 30 ±5 150 ± 74 75 ±2 12 ± 2 12 ± 3 23 ± 10 13 ± 3 10 ± 5 12 ± 2
Data are expressed as mean ± SD of 2 determinations. h = hours, d = days. * mll16 h, ** mg/16 h.
Immunohistochemical distribution of 65 and 70 kD HSPs in normal rat kidney and in experimental ATN Control rat kidneys showed 70 kD heat shock protein immunoreactivity (HSP-I) well circumscribed to nuclei and cytoplasm of podocytes and epithelial cells lining proximal and distal convoluted tubules (DCT) and a lesser extent in epithelial cells of collecting tubules. Although the sites immunoreactive to 65 kD HSP are virtually identical to those identified for 70 kD HSP, labeling was more accentuated in podocytes and cells of the PCT, but abscent in their nuclei (fig. Id). The general pattern of distribution ofHSP-I is heterogeneous with a wide variation in the intensity of HSP-I among nephrons and collecting tubules. Four hours after HgCl 2 administration, intense immunoreactivity for both 65 and 70 kD HSPs was present in the cytoplasm and nuclei of the transitional epithelial cells of the pelvis and calyces as well as in the epithelia of medullary collecting tubules (fig. Ie). The pattern of distribution of HSP-I at 8 hours after HgCl 2 administration closely resembled that seen at 4 hours. However in the later observation the PRPCT had marked HSP-I and the intensity was higher than in other portions of the same tubule. At 16 hours, the epithelium of PRPCT showed prominent selective necrosis accompanied by intense nuclear and cytoplasmic HSP-I to both HSPs, but predominantly to that of 70 kD (fig. If). At this point, there was a noticeable reduction of HSP-I in the epithelia of the collecting tubules, calyces, and pelvis. Widespread tubular necrosis involving the middle portion of the cortex was observed 24 hours after HgCl z administration, and the entire cortical area showed marked necrosis at 48 hours.
HSP-I paralleled to the magnitude of tubular cell injury at these times, with maximal HSP-I in the cytoplasm and nucleus of PCT cells (fig. 1g). Three and 5 days after HgCl 2 poisoning, the so-called regenerative phase began. At these time points regenerating epithelial cells were weakly HSP-I, whereas the necrotic ones, which had sloughed and were occupying tubular lumen, had intense HSP-I (fig. Ih). The regenerative phase ends between 7 and 15 days after HgCl 2 administration. At these time points the most intense HSPI is located in the cortex, including both proximal and distal convoluted tubules (DCT). After Day 15 HSP-I intensity and distribution became indistinguishable from that of the control kidney.
Subcellular distribution of 65 and 70 kD HSPs in normal kidney and in experimental ATN In the control kidney, HSP-I to 65 kD in the PCT was found mainly in mitochondria and weakly in the cytoplasm. In the DCT the subcellular distribution was similar, although the intensity of HSP-I was weaker in all its locations. Podocytes, and epithelia of collecting tubules and Henle's loops had the weakest HSP-I, which was predominately free in the cytoplasm. The distribution of 70 kD HSP-I in the control kidney differed from that of 65 kD; 70 kD HSP-I was present in chromatin, nucleoli, cytoplasm and to a lesser extent mitochondria and lysosomes of cells of PCT and DCT. In podocytes, epithelia of pelvis and calyces the HSP-I was weak in the cytoplasmic matrix. Four hours after HgCl 2 administration, 70 and 65 kD HSP-I was intense in the transitional epithelium of pelvis and calyces. Seventy kD HSP was mostly distributed in Exp Toxic Pathol 47 (1995) 6
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Exp Toxic Pathol47 (1995) 6
the cytoplasmic matrix and filaments of the cytoskeleton associated with cell junctions (fig. 2a). In contrast HSP 65 was present in mitochondria (fig. 2b) only when the cell damage was advanced, and when the cytoskeleton showed marked condensation and aggregation 65 kD HSP-I was intense (fig. 2c). The nuclei of the transitional epithelium showed immunoreactivity to both proteins associated with chromatin and nucleoli. As described in light microscopic findings, at 16 hours cellular necrosis of the PRPCT was associated with intense HSP-1. This immunoreactivity was found in the whole renal cortex and reached its maximal intensity at 24 and 48 hrs after HgCl 2 administration. At the ultrastructural level of the PCT, 65 kD HSP-I was present mainly in the mitochondrial matrix (fig. 2d) and appeared de novo associated with chromatin and nucleoli (fig. 2e). At the same stage, HSP-I to 70 kD in the PCT and OCT epithelium showed intense positivity in cytoplasmic matrix (fig. 2f), extending to microvilli of the brush border (fig. 2g) and to a lesser degree in mitochondria and lysosomes as well as in nuclear chromatin and nucleoli (fig. 2h). During the regenerative phase (Days 3 to 15) HSP-I to both HSPs decreased in regenerating epithelial cells, without an appreciable difference in distribution. Fifteen days after the HgCl 2 administration, the point at which the regenerative phase had concluded, the distribution and intensity of HSP-I was identical to that of the normal kidney.
Detection of HSPs in urinary protein by immunoelectrotransference Urine obtained from control rats was negative for HSPs. Both HSPs were detected 48 hours and three days after the chemical injury. In the case of 70 kD HSP, two distinct immunoreactive bands of 70 and 72 kD were detected on Day 2, while only a single and lighter band of 65 kD HSP was observed at this time (fig. 3).
Discussion Heat shock proteins, originally described as inducible after cell exposure to heat (16), have been found in prokaryotic as well as eukaryotic cells with a relatively high degree of homology (3). HSP's can be identified in a wide variety of conditions compromising cell function (19), and ultimately play an important role in cell adaptation. Since acute tubular necrosis induced by HgC~ is considered a suitable experimental model of acute renal failure (17), and since HSPs synthesis is induced by heavy metals, it was of interest to determine the expression and distribution of 65 and 70 HSPs following HgCl 2 intoxication. As a normative foundation we also determined the immunolocalization of HSPs in the normal kidney since an intrinsic expression of these proteins has already been documented in the normal kidney (12). In regard to 70 kD HSP, our immunohistochemical findings do not differ from those previously described for the normal kidney (7). However, our ultrastructural findings provide direct evidence of its presence in the cytoplasm, nuclei and mitochondria, demonstrating the specific loci underlying the overall light microscopic immunoreactivity observed in the present and previous studies. As far as we know the distribution of 65 kD HSP in the normal kidney has not been reported previously. The localization of 65 kD HSP differs from that of 70 kD in few but important aspects. Thus, the immunoreactivity for the 65 kD HSP was primarily confined to PCT and podocytes, while that of the 70 kD HSP had a wider distribution. Another difference was that 65 kD HSP is preferentially located in the cytoplasmic domain. At the ultrastructural level 65 kD HSP is chiefly confined to the mitochondrial matrix, except in podocytes, where this protein is weakly detected in the cytoplasmic matrix. This distribution is consistent with the fact that 65 kD HSP is synthesized in the cytoplasm and then transported to the mitochondria, where it probably facilitates the appropriate assembly of several enzymatic complexes (9).
Fig. 1. Histological and immunohistochemical comparison of normal rat kidney and mercury treated. (a) Light micrograph from the deep cortical area after 16 hours of mercury injection. Necrosis and cell detachment are seen in that epithelium of the straight part of the proximal convoluted tubules H & E staining (x200). (b) Micrograph taken 48 h after mercury injection. Extensive necrosis is seen in the entire renal cortex. H & E staining (x200). (c) Deep cortical area three days after mercury injection. Some mitoses and cells with vesicular nuclei can be observed. H & E staining (x400). (d) Immunohistochemistry to 65 kD HSP in the normal rat kidney, the immunoreactive sites are chiefly located in epithelial cytoplasm of proximal convoluted tubules and visceral layer of Bowman (x400). (e) Localization of 70 kD HSP after four hours of mercury injection. The immunoreactive sites are found without evidence of histological damage in the epithelial of calyces, pelvis and collecting tubules. (f) Immunohistochemistry to 65 kD HSP from the same animal than in figure a, the reaction is primarily seen in the cytoplasm of necrotic and detached cells of the straight part of the proximal convoluted tubules. (x200). (g) Immunohistochemistry to 70 kD HSP in a section from the same block than in figure b in which the immunoreactivity is found in virtually all cells (x200). (b) Micrograph from the same tissue block of figure c, the specimen was incubated with antibody to 65 kD HSP, the immunoreactivity is found in necrotic and detached cells. (x200). Exp Toxic Pathol 47 (1995) 6
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Fig. 3. Demonstration of 65 and 70 kD HSPs in the urine by Western blot analysis. HSP 70 showed two bands of 70 and 72 kD 48 hours after mercury injection (1), a single and lighter band is observed three days after mercury injection (2). HSP 65 kD was detected as the same time period. The molecular mass in kD is indicated in the left. According to a previous immunohistochemical study, the renal distribution of 90 kD HSP is also different from 65 and 70 kD HSPs (13). Ninety kD HSP is preferentially located in the DCT, medullary collecting tubules, and to a lesser extent in Bowman' s epithelium. This difference in distribution may indicate different functions . With respect to HgCl 2-induced ATN, our light microscopic findings regarding the onset of HSP-I in the absence of structural damage, demonstrate HSPs immunohistochemistry as a sensitive mean of assessing the early stages of kidney cell damage. Future studies are required to provide information on the potential usefulness of increases in HSPs in evaluating the degree of kidney cell damage and perhaps as biomarkers of tissue viability. For
example, in our study, four hours after the administration of HgCl2 there was a marked increase in both HSPs, circumscribed to the epithelia of pelvis, calyces, and medullary collecting tubules. This suggests a lower threshold of HSPs response in these cells than in other renal epithelial cells. This is supported by results obtained from other models of kidney damage induced by ischemia and heat stress (3). Our data further indicate that the kidney epithelium is responsible for the overproduction of HSPs in the HgCl 2-induced ATN. The pattern of distribution of HSPs changes during the necrotic phase of A TN. Thus, 16 hours after HgCl 2 administration, the magnitude of HSPs immunoreactivity in the PRPCT coincided with the degree of histological damage of this part of the nephron, which is known to absorb mercury (6). Twenty four and 48 hours after HgCl 2 poisoning tubular necrosis was seen in the entire cortex, and HSPs were augmented in a parallel fashion, with maximal immunoreactivity in those cells displaying severe damage. Since the subcellular localization of 70 kD HSP-I was modified mainly in terms of immunoreactivity as a result of the intoxication, it is plausible to conclude that under some adverse conditions (i.e. ATN) HSPs may exert protective functions within the same cell loci in which they appear under normal circumstances. However, the case of 65 kD HSP, may be different, since in addition to a fairly constant mitochondrial localization, it showed a de novo nuclear expression induced by ATN. Thus, both HSPs were found in the cellular nuclei but under different cirumstances. Seventy kD HSP-I may be present in the nucleus because the protein is translocated and bounds preferentially to the nucleolus, particularly to denatured and/or aggregates of polyribosomes, where it presumably facilitates the "reconstruction" of the damaged nucleolus (20, 14). The same could be true for 65 kD HSP in the case of adapted kidney cells after noxious stimuli. Since HSPs have been considered as biomarkers of cell damage (7), it was of interest to study their presence in urine. Our results demonstrate that HSP may be found in
Fig. 2. Immunoelectron microscopy of the subcellular distribution of 65 and 70 kD HSPs in experimental acute tubular necrosis induced by mercury. (a) Immunoelectron micrograph of 70 kD HSP taken from the pelvic transitional epithelium, 4 hours after mercury injection, notice that the immunoreactivity sites are distributed on the cytoskeletal filaments (arrowheads) associated with cell junctions, weak immunolabeling is seen in the mitochondrial matrix (upper left) (18,OOOx). (b) Micrograph taken from the same specimen as in figure a, the immunoreactivity to 65 kD HSP is confined to mitochondria (18,300x). (c) micrograph of a transitional epithelial cell of the kidney pelvis in accentuated damage, intense immunoreactivity to 65 HSP is seen in aggregates of cytoeskeletal filaments and mitochondria (lower left) (21,OOOx). (d) Micrograph of an epithelial cell of the proximal convoluted tubule, 48 hours after HgCl 2 injection. The immunoreactivity to 65 kD HSP is predominantly confined to mitochondria, being weak in the cytoplasm (18,000x). (e) Micrograph taken from the same specimen than figure d, the immunoreactivity to 65 kD HSP is associated to chromatin and nucleolus (15,OOOx). (f) Immunoelectron micrograph to 70 kD HSP, 48 hours after the HgCl 2 administration, there is intense immunoreactivity in the cytoplasm and weak in mitochondria (15,OOOx). (g) Immunocytochemistry to 70 kD HSP, abundant labeling is observed in the brush border ofthe convoluted proximal tubule, 48 hours after HgCl 2 injection (15,000x). (b) Immunoelectron micrograph to 70 kD HSP, two days after HgCl2 administration, the immunoreactive sites are located in the perinuclear chromatin and nucleolus (upper right) (l7,500x). Exp Toxic Pathol 47 (1995) 6
507
urine, coinciding with extensive structural damage during the necrotic phase, and implies that their presence by either approach, immunohistochemistry or westemblot analysis, could be used as a reliable indicator of renal injury. The exact mechanism(s) by which the cell recognizes a changes in its environmental circumstances and activates HSPs' genetic overexpression is still unclear, although one characteristic shared by many deleterious agents is their ability to promote protein denaturation and/or aggregation (1). Mercury falls in this category, since it binds to sulfhydryl groups of the cell membrane and other proteins causing their denaturation (2). Another inductor mechanism could be the intracellular depletion of thiols and/or increment of lipid peroxidation (4), both of which have also been shown to induce HSPs overexpression. In conclusion, increased synthesis of HSPs in experimental acute renal failure induced by HgCl, bears a close relation with the sites of cell injury. The dtfferential cellular and subcellular distribution of 65 and 70 HSP-I in specific cell types of the kidney may correlate with different functions of these proteins in each cell type.
Acknowledgements: This work was presented in the 25th annual meeting of the American Society of Nephrology on November 15-18, 1992 and partially published in abstract form in J Am Soc Nephrol3: 724, 1992. This work was supported by CONACYT, Grant No. 0079M9106 and by Bristol Myers Squibb.
References 1. ANATHAN J, GOLDBERG AI, VOELLMY R: Abnormal proteins serve as eukaryotic stress signals and trigger the activation of heat shock genes. Science 1986; 232: 522-524. 2. COTRAND R, KUMAR V, ROBBINS S: Pathological Basis of Disease (4th ed). Philadelphia WB Saunders 1989 p. 13. 3. EMAMI A, SCHWARTZ JH, BORKAN S: Transient ischemia or heat stress induces a cytoprotectan protein in the rat kidney. Am J Physiol 1991; 260: 479-485 . 4. GOERING P, FISHER B, CHAUDHARY P, DICK C: Relationship between stress protein induction in rat kidney by mercuric chloride and nephrotoxicity. Toxicol Appl Pharmacol 1992;113: 184-191. 5. HAAGSMA BH, POUND A W: Mercuric chloride induced
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tubular necrosis in the rat kidney: the recovery phase. Br J Exp Patho11980; 61: 229-241. HULTMAN P, ENESTROM S: Localization of mercury in the kidney during experimental acute tubular necrosis studied by the cytochemical silver amplification method. Br J Exp Patho11986; 67: 493-503. KOMA TSUDA A, W AKUI H, IMAI H, et a1.: Renal localization of the constitutive 73-kDa heat shock protein in normal and PAN rats. Kidney Int 1992; 41: 1204-1212. LAEMMLI UK: Change of structural proteins during the assembly of the head of bacteriophage T4. Nature 1970;227: 680-683. LANGER T, NEUPERT W: Heat shock proteins hsp 60 and hsp 70: Their roles in folding, assembly and membrane translocation of proteins. Current Top Microbiol Immunol 1992; 167: 3-30. LINDQUIST S, CRAIG EA: The heat shock proteins. Ann Rev Genet 1988; 22: 631-677. LOWRY OH, ROSENBROUNGH NJ, FARR AL, RANDAL RJ: Protein measurement with the Folin phenol reagent. J BioI Chem 1951; 193: 265-272. LOVIS CH, MACH F, DONATI Y, et a1.: Heat shock proteins and the kidney. Renal Failure 1994; 16: 179-192. MATSUBARA 0, KASUGA T, MARUMO F, et a1.: Localization of 90 kDa heat shock protein in the kidney. Kidney Int 1990; 38: 830-834. PELHAM HRB: HSP-70 accelerates the recovery of nucleolar morphology after heat shock: EMBO J 1984; 3: 3095-3100. PELHAM HRB: Speculation on the functions of the major heat shock and glucose-regulated proteins. Cell 1986; 46: 959-961. RITOSSA F: A new puffing pattern induced by temperature shock and DNP in Drosophila. Experientia 1962; 18: 571-573. STEIN JH, LIFSCHITZ MD, BARNES LD: Current concepts on the pathophysiology of acute renal failure. Am J Physiol1978; 234: FI71-FI81. STERNBER LA: Immunocytochemistry (2nd ed). New Yark Wiley 1979 pp. 104-130. SUBJECK JR, SHYY IT: Stress protein systems of mammalian cell. Am J Physiol1986; 250: CI-C17. WELCH WJ, FERAMISCO J: Nuclear and nucleolar localization of the 72,000 dalton heat shock protein in heat shocked mammalian cells. J BioI Chern 1984; 259: 4501-4513. YOUNG DB, IVANY J, Cox HK, LAMB JR: The 65 kDa antigen of mycobacterium - a common bacterial antigen? Immunol Today 1987; 8: 215-219.
Experimental Pathology Laboratory and Department of Nephrology and Mineral Metabolism, Instituto Nacional de la Nutrici6n Salvador Zubinin, Mexico City, *) Facultad de Quimica, National Autonomus University of Mexico, Mexico
Histological and subcellular distribution of 65 and 70 kD heat shock proteins in experimental nephrotoxic injury ROGELIO HERNANDEZ-PANDO, JOSE PEDRAZA-CHAVERRI*), HECTOR ORozco-EsTEVEZ, PALMIRA SILVA-SERNA, IVETTE MORENO, ADRIAN RONDAN-ZARATE, MARTHA ELINOS, RICARDO CORREA-RoTTER and JORGE LARRIVA-SAHD With 3 figures and 1 table Received: January 31,1995; Accepted: April 25, 1995 Address for correspondence: ROGELIO HERNANDEZ-PANDO, M. D., Ph. D. Experimental Pathology Laboratory, Department of Pathology, Instituto Nacional de la Nutrici6n Salvador Zubinin, Vasco Quiroga No. 15, Tlalpan, 14000, Mexico D.F. MEXICO; Tel. 011-(525)-573-12-00 Ext 2185, Fax 011-(525)-655-10-76. Key words: Heat shock; Shock, heat; Nephrotoxic injury; Kidney, tubular necrosis; Proteins, shock; mercury.
Summary The cellular distribution of 65 and 70 kD heat shock proteins (HSPs) was studied in the normal rat kidney and after acute tubular necrosis (ATN) induced by inorganic mercury (HgCl z). In the normal kidney the 65 kD HSP was found in the cytoplasm of podocytes and proximal convoluted tubules, whereas the 70 kD HSP was located in nuclei and cytoplasm of podocytes, cortical convoluted, and collecting tubules. The distribution of both HSPs along ATN changed as a function oftime. In the early phase, before evidence of histological damage, both HSPs were found in the pielocalyceal epithelium and medullary collecting tubules. During the necrotic phase, HSPs coexisted with sites of severe damage (i.e. cortical tubules). With immunoelectron microscopy, damaged cells showed an abundance of 65 kD HSP-I in mitochondria, as well as in chromatin and nucleoli, while 70 kD HSP-I was overexpressed in the cytoplasm, mitochondria, lysosomes, cytoskeleton, chromatin, and nucleoli, and coincided with urinary excretion of HSPs. In the postregenerative phase, the distribution of HSPs was similar to that found in the normal kidney. HSPs of 65 and 70 kD were encountered constitutionally and their immunolabeling is correlated with the magnitude of cell injury.
Introduction Heat shock proteins playa key role in the homeostatic mechanisms of the cell. Under normal circumstances the cell is restrained to a range of functions and structure established by the interaction of its genomic program of differentiation, the availability of metabolic substrates,
constrains of neighboring cells and the limited capacities of its metabolic pathways. This homeostatic "steady state" can be modified by greater physiological demands or some pathologic stimuli which induce a number of adaptation mechanisms that establish a new, but altered steady state in an attempt to preserve the viability of the cell. Among the various adaptative mechanisms, one of the most remarkable is the rapid and coordinated increased synthesis of a group of distinct proteins collectively referred to as heat shock proteins (HSPs). HSPs were initially recognized by their increased quantity after cell exposure to elevated temperature (16). It has recently been clearly established that induction of HSPs follows a wide variety of cell stressing conditions (19). However, in several cell types, some HSPs occur in the absence of a noxious stimulus (19). HSPs have a fairly constant structure among species, from prokaryotes to mammals (21) and have been grouped into a few families according to their molecular weight. Among them, HSP 70 kD and 65 kD are particularly abundant in all organisms examined so far (9). HSPs can assume multiple important functions in the cell, such as assisting in protein folding and assembly by reducing the tendency to aggregate, thus stabilizing proteins and ultimately protecting cells from the deleterious effect(s) of high temperature and other types of noxious agents (10). The present study describes the tissue distribution and subcellular location of HSP 65 and 70 kD in the normal rat kidney and in the experimental model of acute tubular necrosis (ATN) and regeneration induced by intraperitoneal administration of HgCl z.
Exp Toxic Pathol 47 (1995) 6
501
Material and methods Animals and experimental model of A TN Male wistar rats (250--350 grams of body weight) received intraperitoneal injections of a single dose of 1.5 mg/kg HgCl 2 (n = 24) or vehicle (n = 2). This dose of HgCl 2 induces necrosis and regeneration of cortical tubular elements but is rarely lethal (5). Experimental rats were sacrificed at the following time points: 4, 8, 16, 24 hours and 2, 3, 5,7, 9, 15, 22 and 28 days (n = 2). Vehicle injected rats were kept as controls to assess constitutive HSP expression and were sacrificed 24 hours after injection. Urine samples, for biochemical measurements and Western blot analysis of HSPs were collected by metabolic cage collections. Blood was collected at sacrifice and kidneys were removed and fixed by immersion for subsequent histological and immunohistochemical studies (vide infra).
Biochemical studies Blood urea nitrogen (BUN) and serum and urinary creatinine were measured by autoanalyzer (Cratinine analyzer 2 and BUN analyzer 2, Beckman Instruments Co, Fullerton, CA) and creatinine clearance was calculated. Total urinary protein was measured according to the method described by LOWRY et al. (l1).
Renal histology and immunohistochemistry For light microscopy kidney slices were fixed by immersion in absolute ethanol for 2 hours, paraffin embedded, sectioned at 6 /lm, and stained with hematoxylin and eosin for histological evaluation. Immunohistochemical detection of 65 and 70 kD HSPs was performed with monoclonal antibodies (Sigma Co). Before incubation with the primary antibody, endogenous peroxidase activity was inactivated (18), then tissue sections were incubated with each one of the monoclonal antibodies for 3 hours. After rinsing, rabbit anti-mice antibody conjugated with peroxidase was incubated for one hour. Peroxidase was visualized with 3,3-diaminobenzidine and counterstained with hematoxylin.
Immunoelectron microscopy For immunoelectron microscopic studies, tissue samples were obtained from cortex, juxtamedular cortex, medulla and calyces. The tissues were immediately fixed by immersion in 4 % paraformaldehyde dissolved in Sorensen's buffer pH 7.4 for 2 hours at 4 °C. After rinsing, free aldehyde groups were blocked in 0.5 M NH4Cl in phosphate-buffersaline (PBS) for one hour. Tissue samples were dehydrated in graded ethyl alcohols and embedded in LR-White hydrosoluble resin. Thin sections from 70 to 90 nm were placed ion nickel grids. The grids were incubated with the mouse anti-HSP monoclonal antibodies diluted in PBS with 1 % bovine serum albumin and 0.5 % Tween. After repeatedly rinsing with PBS, the grids were incubated with rabbit antimouse IgG and IgM conjugated to 10 nm gold particles (Amersham, UK) diluted in the same buffer. The grids were stained with uranium salts and examined in a Zeiss EM 10 electron microscope. 502
Exp Toxic Pathol 47 (1995) 6
Analysis of urinary proteins by immunoelectrotransference Urinary proteins were separated by SDS-PAGE under reducing conditions in a discontinuous buffer system (8) on slab gels of 10 % acrylamide. Gels were mounted in an electrotransfer chamber (LKB, Sweden) and transferred to nitrocellulose sheets (Bio Rad Laboratories, Richmond, CA). Unbounded sites were blocked with 3 % BSA in PBS Tween 20. Strips were incubated with the anti-HSP monoclonal antibodies for one hour at 37 °C. After rinsing, strips were exposed to rabbit anti-mouse antibody conjugated with peroxidase for 90 minutes and visualized with HP2 and 4-chloro-I-naphthol for 15 minutes.
Results Biochemical parameters As shown in table 1, creatinine clearance had decreased in the experimental group by 16 hours, reaching the lowest value at 3 days and returning to normal values at Day five. BUN had increased by 16 hours, reaching the peak on Day 3, and returned to control values by Day 7. Urinary volume increased on Day 2 in the HgCl2 injected rats, which remained polyuric until Day 7. Urinary protein excretion increased on Day 1 after HgCl 2 injection and returned to normal values by Day 5.
Histopathology of experimental ATN With the light microscope no evidence of cell injury was seen 4 and 8 hours after HgCl2 administration. Tubular necrosis was evident at 16 hours and more prominent in the deep cortex, especially in the pars recta of the proximal convoluted tubules (PRPCT). Tubular cell necrosis was characterized by pyknosis, granular cytoplasmic appearance, acidophilia, and partial epithelial detachment (fig. la). Kidneys from animals sacrificed 24 hours after HgCl 2 administration displayed tubular necrosis predominantly at the middle portion of the cortex, involving mostly proximal convoluted tubules (PCT) , which contained luminal debris and sloughed epithelial cells presenting an advanced stage of cytolysis. The maximum degree of necrosis was observed at 48 hours after HgCl 2 administration, when tubular necrosis was observed within the entire renal cortex (fig. 1b). On Day 2 morphologic features related to the beginning of regeneration were observed, such as segments of tubular necrosis displaying flat and cuboidal basophilic cells with large nuclei and distinct nucleoli and mitoses (fig. Ic). Cortical convoluted tubules were progressively lined by flat epithelial cells on Day 15. On Day 21 and thereafter the histological aspect of the kidney was comparable to that observed in the normal rat kidney.
Table 1. Biochemical parameters of control and HgCl 2-treated rats.
Time
creatinine clearance (mllmin)
BUN (mg/dl)
urinary volume (m1l24 h)
urinary protein (mg/24 h)
Control
1.2 ± 0.1
23 ± 1
13 ± 5
17 ± 6
HgCl z 16 h 24 h 2d 3d 5d 7d 9d 15 d 22 d 28 d
0.7 ± 0.4 0.7 ± 0.3 0.3 ±0.2 0.2 ± 0.1 1.5 ± 0.1 1.5 ± 0.6 1.1 ± 0.1 1.4 ± 0.2 1.7±0.1 1.2 ± 0.1
50 ±7 42 ±7 56 ± 1 102±6 33 ± 2 23 ± 1 25 ±7 22 ± 1 24 ± 3 25 ± 3
7 ± 2* 14 ± 2 48 ±4 32 ± 3 28 ± 9 35 ± 3 21 ± 8 18 ± 12 16 ± 9 19 ± 2
17 ± 2** 30 ±5 150 ± 74 75 ±2 12 ± 2 12 ± 3 23 ± 10 13 ± 3 10 ± 5 12 ± 2
Data are expressed as mean ± SD of 2 determinations. h = hours, d = days. * mll16 h, ** mg/16 h.
Immunohistochemical distribution of 65 and 70 kD HSPs in normal rat kidney and in experimental ATN Control rat kidneys showed 70 kD heat shock protein immunoreactivity (HSP-I) well circumscribed to nuclei and cytoplasm of podocytes and epithelial cells lining proximal and distal convoluted tubules (DCT) and a lesser extent in epithelial cells of collecting tubules. Although the sites immunoreactive to 65 kD HSP are virtually identical to those identified for 70 kD HSP, labeling was more accentuated in podocytes and cells of the PCT, but abscent in their nuclei (fig. Id). The general pattern of distribution ofHSP-I is heterogeneous with a wide variation in the intensity of HSP-I among nephrons and collecting tubules. Four hours after HgCl 2 administration, intense immunoreactivity for both 65 and 70 kD HSPs was present in the cytoplasm and nuclei of the transitional epithelial cells of the pelvis and calyces as well as in the epithelia of medullary collecting tubules (fig. Ie). The pattern of distribution of HSP-I at 8 hours after HgCl 2 administration closely resembled that seen at 4 hours. However in the later observation the PRPCT had marked HSP-I and the intensity was higher than in other portions of the same tubule. At 16 hours, the epithelium of PRPCT showed prominent selective necrosis accompanied by intense nuclear and cytoplasmic HSP-I to both HSPs, but predominantly to that of 70 kD (fig. If). At this point, there was a noticeable reduction of HSP-I in the epithelia of the collecting tubules, calyces, and pelvis. Widespread tubular necrosis involving the middle portion of the cortex was observed 24 hours after HgCl z administration, and the entire cortical area showed marked necrosis at 48 hours.
HSP-I paralleled to the magnitude of tubular cell injury at these times, with maximal HSP-I in the cytoplasm and nucleus of PCT cells (fig. 1g). Three and 5 days after HgCl 2 poisoning, the so-called regenerative phase began. At these time points regenerating epithelial cells were weakly HSP-I, whereas the necrotic ones, which had sloughed and were occupying tubular lumen, had intense HSP-I (fig. Ih). The regenerative phase ends between 7 and 15 days after HgCl 2 administration. At these time points the most intense HSPI is located in the cortex, including both proximal and distal convoluted tubules (DCT). After Day 15 HSP-I intensity and distribution became indistinguishable from that of the control kidney.
Subcellular distribution of 65 and 70 kD HSPs in normal kidney and in experimental ATN In the control kidney, HSP-I to 65 kD in the PCT was found mainly in mitochondria and weakly in the cytoplasm. In the DCT the subcellular distribution was similar, although the intensity of HSP-I was weaker in all its locations. Podocytes, and epithelia of collecting tubules and Henle's loops had the weakest HSP-I, which was predominately free in the cytoplasm. The distribution of 70 kD HSP-I in the control kidney differed from that of 65 kD; 70 kD HSP-I was present in chromatin, nucleoli, cytoplasm and to a lesser extent mitochondria and lysosomes of cells of PCT and DCT. In podocytes, epithelia of pelvis and calyces the HSP-I was weak in the cytoplasmic matrix. Four hours after HgCl 2 administration, 70 and 65 kD HSP-I was intense in the transitional epithelium of pelvis and calyces. Seventy kD HSP was mostly distributed in Exp Toxic Pathol 47 (1995) 6
503
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Exp Toxic Pathol47 (1995) 6
the cytoplasmic matrix and filaments of the cytoskeleton associated with cell junctions (fig. 2a). In contrast HSP 65 was present in mitochondria (fig. 2b) only when the cell damage was advanced, and when the cytoskeleton showed marked condensation and aggregation 65 kD HSP-I was intense (fig. 2c). The nuclei of the transitional epithelium showed immunoreactivity to both proteins associated with chromatin and nucleoli. As described in light microscopic findings, at 16 hours cellular necrosis of the PRPCT was associated with intense HSP-1. This immunoreactivity was found in the whole renal cortex and reached its maximal intensity at 24 and 48 hrs after HgCl 2 administration. At the ultrastructural level of the PCT, 65 kD HSP-I was present mainly in the mitochondrial matrix (fig. 2d) and appeared de novo associated with chromatin and nucleoli (fig. 2e). At the same stage, HSP-I to 70 kD in the PCT and OCT epithelium showed intense positivity in cytoplasmic matrix (fig. 2f), extending to microvilli of the brush border (fig. 2g) and to a lesser degree in mitochondria and lysosomes as well as in nuclear chromatin and nucleoli (fig. 2h). During the regenerative phase (Days 3 to 15) HSP-I to both HSPs decreased in regenerating epithelial cells, without an appreciable difference in distribution. Fifteen days after the HgCl 2 administration, the point at which the regenerative phase had concluded, the distribution and intensity of HSP-I was identical to that of the normal kidney.
Detection of HSPs in urinary protein by immunoelectrotransference Urine obtained from control rats was negative for HSPs. Both HSPs were detected 48 hours and three days after the chemical injury. In the case of 70 kD HSP, two distinct immunoreactive bands of 70 and 72 kD were detected on Day 2, while only a single and lighter band of 65 kD HSP was observed at this time (fig. 3).
Discussion Heat shock proteins, originally described as inducible after cell exposure to heat (16), have been found in prokaryotic as well as eukaryotic cells with a relatively high degree of homology (3). HSP's can be identified in a wide variety of conditions compromising cell function (19), and ultimately play an important role in cell adaptation. Since acute tubular necrosis induced by HgC~ is considered a suitable experimental model of acute renal failure (17), and since HSPs synthesis is induced by heavy metals, it was of interest to determine the expression and distribution of 65 and 70 HSPs following HgCl 2 intoxication. As a normative foundation we also determined the immunolocalization of HSPs in the normal kidney since an intrinsic expression of these proteins has already been documented in the normal kidney (12). In regard to 70 kD HSP, our immunohistochemical findings do not differ from those previously described for the normal kidney (7). However, our ultrastructural findings provide direct evidence of its presence in the cytoplasm, nuclei and mitochondria, demonstrating the specific loci underlying the overall light microscopic immunoreactivity observed in the present and previous studies. As far as we know the distribution of 65 kD HSP in the normal kidney has not been reported previously. The localization of 65 kD HSP differs from that of 70 kD in few but important aspects. Thus, the immunoreactivity for the 65 kD HSP was primarily confined to PCT and podocytes, while that of the 70 kD HSP had a wider distribution. Another difference was that 65 kD HSP is preferentially located in the cytoplasmic domain. At the ultrastructural level 65 kD HSP is chiefly confined to the mitochondrial matrix, except in podocytes, where this protein is weakly detected in the cytoplasmic matrix. This distribution is consistent with the fact that 65 kD HSP is synthesized in the cytoplasm and then transported to the mitochondria, where it probably facilitates the appropriate assembly of several enzymatic complexes (9).
Fig. 1. Histological and immunohistochemical comparison of normal rat kidney and mercury treated. (a) Light micrograph from the deep cortical area after 16 hours of mercury injection. Necrosis and cell detachment are seen in that epithelium of the straight part of the proximal convoluted tubules H & E staining (x200). (b) Micrograph taken 48 h after mercury injection. Extensive necrosis is seen in the entire renal cortex. H & E staining (x200). (c) Deep cortical area three days after mercury injection. Some mitoses and cells with vesicular nuclei can be observed. H & E staining (x400). (d) Immunohistochemistry to 65 kD HSP in the normal rat kidney, the immunoreactive sites are chiefly located in epithelial cytoplasm of proximal convoluted tubules and visceral layer of Bowman (x400). (e) Localization of 70 kD HSP after four hours of mercury injection. The immunoreactive sites are found without evidence of histological damage in the epithelial of calyces, pelvis and collecting tubules. (f) Immunohistochemistry to 65 kD HSP from the same animal than in figure a, the reaction is primarily seen in the cytoplasm of necrotic and detached cells of the straight part of the proximal convoluted tubules. (x200). (g) Immunohistochemistry to 70 kD HSP in a section from the same block than in figure b in which the immunoreactivity is found in virtually all cells (x200). (b) Micrograph from the same tissue block of figure c, the specimen was incubated with antibody to 65 kD HSP, the immunoreactivity is found in necrotic and detached cells. (x200). Exp Toxic Pathol 47 (1995) 6
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Fig. 3. Demonstration of 65 and 70 kD HSPs in the urine by Western blot analysis. HSP 70 showed two bands of 70 and 72 kD 48 hours after mercury injection (1), a single and lighter band is observed three days after mercury injection (2). HSP 65 kD was detected as the same time period. The molecular mass in kD is indicated in the left. According to a previous immunohistochemical study, the renal distribution of 90 kD HSP is also different from 65 and 70 kD HSPs (13). Ninety kD HSP is preferentially located in the DCT, medullary collecting tubules, and to a lesser extent in Bowman' s epithelium. This difference in distribution may indicate different functions . With respect to HgCl 2-induced ATN, our light microscopic findings regarding the onset of HSP-I in the absence of structural damage, demonstrate HSPs immunohistochemistry as a sensitive mean of assessing the early stages of kidney cell damage. Future studies are required to provide information on the potential usefulness of increases in HSPs in evaluating the degree of kidney cell damage and perhaps as biomarkers of tissue viability. For
example, in our study, four hours after the administration of HgCl2 there was a marked increase in both HSPs, circumscribed to the epithelia of pelvis, calyces, and medullary collecting tubules. This suggests a lower threshold of HSPs response in these cells than in other renal epithelial cells. This is supported by results obtained from other models of kidney damage induced by ischemia and heat stress (3). Our data further indicate that the kidney epithelium is responsible for the overproduction of HSPs in the HgCl 2-induced ATN. The pattern of distribution of HSPs changes during the necrotic phase of A TN. Thus, 16 hours after HgCl 2 administration, the magnitude of HSPs immunoreactivity in the PRPCT coincided with the degree of histological damage of this part of the nephron, which is known to absorb mercury (6). Twenty four and 48 hours after HgCl 2 poisoning tubular necrosis was seen in the entire cortex, and HSPs were augmented in a parallel fashion, with maximal immunoreactivity in those cells displaying severe damage. Since the subcellular localization of 70 kD HSP-I was modified mainly in terms of immunoreactivity as a result of the intoxication, it is plausible to conclude that under some adverse conditions (i.e. ATN) HSPs may exert protective functions within the same cell loci in which they appear under normal circumstances. However, the case of 65 kD HSP, may be different, since in addition to a fairly constant mitochondrial localization, it showed a de novo nuclear expression induced by ATN. Thus, both HSPs were found in the cellular nuclei but under different cirumstances. Seventy kD HSP-I may be present in the nucleus because the protein is translocated and bounds preferentially to the nucleolus, particularly to denatured and/or aggregates of polyribosomes, where it presumably facilitates the "reconstruction" of the damaged nucleolus (20, 14). The same could be true for 65 kD HSP in the case of adapted kidney cells after noxious stimuli. Since HSPs have been considered as biomarkers of cell damage (7), it was of interest to study their presence in urine. Our results demonstrate that HSP may be found in
Fig. 2. Immunoelectron microscopy of the subcellular distribution of 65 and 70 kD HSPs in experimental acute tubular necrosis induced by mercury. (a) Immunoelectron micrograph of 70 kD HSP taken from the pelvic transitional epithelium, 4 hours after mercury injection, notice that the immunoreactivity sites are distributed on the cytoskeletal filaments (arrowheads) associated with cell junctions, weak immunolabeling is seen in the mitochondrial matrix (upper left) (18,OOOx). (b) Micrograph taken from the same specimen as in figure a, the immunoreactivity to 65 kD HSP is confined to mitochondria (18,300x). (c) micrograph of a transitional epithelial cell of the kidney pelvis in accentuated damage, intense immunoreactivity to 65 HSP is seen in aggregates of cytoeskeletal filaments and mitochondria (lower left) (21,OOOx). (d) Micrograph of an epithelial cell of the proximal convoluted tubule, 48 hours after HgCl 2 injection. The immunoreactivity to 65 kD HSP is predominantly confined to mitochondria, being weak in the cytoplasm (18,000x). (e) Micrograph taken from the same specimen than figure d, the immunoreactivity to 65 kD HSP is associated to chromatin and nucleolus (15,OOOx). (f) Immunoelectron micrograph to 70 kD HSP, 48 hours after the HgCl 2 administration, there is intense immunoreactivity in the cytoplasm and weak in mitochondria (15,OOOx). (g) Immunocytochemistry to 70 kD HSP, abundant labeling is observed in the brush border ofthe convoluted proximal tubule, 48 hours after HgCl 2 injection (15,000x). (b) Immunoelectron micrograph to 70 kD HSP, two days after HgCl2 administration, the immunoreactive sites are located in the perinuclear chromatin and nucleolus (upper right) (l7,500x). Exp Toxic Pathol 47 (1995) 6
507
urine, coinciding with extensive structural damage during the necrotic phase, and implies that their presence by either approach, immunohistochemistry or westemblot analysis, could be used as a reliable indicator of renal injury. The exact mechanism(s) by which the cell recognizes a changes in its environmental circumstances and activates HSPs' genetic overexpression is still unclear, although one characteristic shared by many deleterious agents is their ability to promote protein denaturation and/or aggregation (1). Mercury falls in this category, since it binds to sulfhydryl groups of the cell membrane and other proteins causing their denaturation (2). Another inductor mechanism could be the intracellular depletion of thiols and/or increment of lipid peroxidation (4), both of which have also been shown to induce HSPs overexpression. In conclusion, increased synthesis of HSPs in experimental acute renal failure induced by HgCl, bears a close relation with the sites of cell injury. The dtfferential cellular and subcellular distribution of 65 and 70 HSP-I in specific cell types of the kidney may correlate with different functions of these proteins in each cell type.
Acknowledgements: This work was presented in the 25th annual meeting of the American Society of Nephrology on November 15-18, 1992 and partially published in abstract form in J Am Soc Nephrol3: 724, 1992. This work was supported by CONACYT, Grant No. 0079M9106 and by Bristol Myers Squibb.
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