Autometallographic Localization of Inorganic Mercury in the Kidneys of Rats: Effect of Unilateral Nephrectomy and Compensatory Renal Growth

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1 EXPERIMENTAL AND MOLECULAR PATHOLOGY 54, (191) Autometallographic Localization of Inorganic Mercury in the Kidneys of Rats: Effect of Unilateral Nephrectomy and Compensatory Renal Growth RUDOLFS K. ZALUPS Division of Basic Medical Sciences, Mercer University School of Medicine, Macon, Georgia Received May 9, 1990, and in revised form August 13, 1990 The histochemical technique of autometallography was used in the present study to demonstrate the zonal and tubular localization of inorganic mercury in the kidneys of unilaterally nephrectomized (NPX) and sham-operated (SO) rats given either a nontoxic 0.5 pmol/kg or a toxic 2.5 p,mol/kg dose of mercuric chloride 10 days after surgery. Deposits were found in the cortex and outer stripe of the outer medulla in both groups of rats given either dose of mercuric chloride. The deposits were localized exclusively in the convoluted and straight portion of the proximal tubule. Forty eight hours after the administration of the 0.5 pmol/kg dose of mercuric chloride, there were significantly more deposits in the renal outer stripe of the NPX rats than in the renal outer stripe of the SO rats. The number of deposits in the renal outer stripe of the NPX and SO rats given the 2.5 pmol/kg dose of mercuric chloride was similar after 24 hr, but was greater than the corresponding rats given the nontoxic dose. These findings suggest that the proximal tubule (particularly the pars recta) is the primary site for the accumulation of inorganic mercury in the kidney. They also suggest that, in the rat. there is enhanced accumulation of inorganic mercury in the pars recta of proximal tubules in the outer stripe of the renal outer medulla when a nontoxic dose of inorganic mercury is given after unilateral nephrectomy or when a toxic dose of mercuric chloride is administered. D 1991 Academic press, Inc. INTRODUCTION The kidney is the most susceptible organ to the toxic effects of inorganic mercury. This susceptibility may be related to the fact that the kidney accumulates more mercury than any other organ. As much as 50 percent or more of the administered dose of inorganic mercury accumulates in the total renal mass of a rat within 24 hr (Zalups and Diamond, 1987a). Recent findings indicate that, in the rat, the renal accumulation of a nontoxic dose of inorganic mercury increases significantly after unilateral nephrectomy and compensatory renal growth (Zalups et al., 1987). Much of this enhanced accumulation can be attributed to an increased accumulation of the metal in the renal outer medulla. Other recent findings show that unilateral nephrectomy and compensatory renal growth increase the susceptibility of the rat to the nephrotoxic effects of mercuric chloride (Zalups and Diamond, 1987b). Morphological studies have demonstrated that the nephropathy induced by inorganic mercury consists primarily of cellular and tubular necrosis in the pars recta (straight) segment of the proximal tubule. The pars recta is localized in the outer stripe of the outer medulla and in the medullary rays of the cortex. Since the accumulation of mercury in the renal outer medulla is enhanced after unilateral nephrectomy, it is possible that the accumulation of inorganic mercury in the pars recta of proximal tubules is also enhanced. This hypothesis is premised, however, on the assumption that the pars recta of the proximal tubule accumulates inorganic mercury. It has been demonstrated that the S, and S, segments (which are parts of the pars recta) of the proximal tubules in the kidneys of BALB/c mice accumulate inorganic mercury following a toxic dose of mercuric chloride (Hultman et al., 1985; Hultman and /91 $3.00 Copyright by Academic Press, Inc. All rights of reproduction in any form reserved. 10

2 HISTOCHEMICAL LOCALIZATION OF RENAL MERCURY 11 Enestrom, 1986). Therefore, it would seem likely that the pars recta of proximal tubules in the kidneys of rats accumulate inorganic mercury. Very little is known about which segments of the rat nephron accumulate and transport inorganic mercury. One way to better characterize the renal accumulation of mercury in the rat is to localize the intrarenal sites in which inorganic mercury accumulates using morphological and histochemical methods. The photoemulsion histochemical technique (Choi, 1984), also known as autometallographic technique (Danscher, 1984), has been shown to demonstrate the presence of inorganic mercury in renal tissue (Hultman et al., 1985; Hultman and Ene- Strom, 1986; Danscher and Rungby, 1986). With this histochemical method, the present study will test whether the enhanced renal accumulation of inorganic mercury that occurs in the rat after unilateral nephrectomy and a period of compensatory renal growth is due to enhanced accumulation of the inorganic mercury in the pars recta of proximal tubules. Moreover, determinations will be made as to which segments of the rat nephron are principally involved in the renal accumulation of inorganic mercury. MATERIALS AND METHODS Male Sprague-Dawley rats weighing g (Harlan Sprague-Dawley, Indianapolis, IN) were used in the present study. After receiving the rats, the animals were allowed to acclimate to their new surroundings for several days. Following the period allowed for acclimation, the rats were assigned randomly to one of two surgical groups. Each group consisted of 16 animals. One group of animals underwent unilateral nephrectomy, while the other group underwent a sham operation. All operations were performed on the animals after they were anesthetized with sodium pentobarbital(50 mg/kg). For the animals that underwent the unilateral nephrectomy, the following protocol was used: a 2.5cm flank incision was made on the right side of the body with a #I1 scalpel blade, beginning at the lateral border of the erector spinae muscles and continuing along the angle of the twelfth rib. The skin, underlying fascia, and abdominal muscles, were incised to expose the right kidney situated in the retroperitoneal space. Once the right kidney was located, it was exteriorized from the abdomen using blunt dissection. A 2.0 silk suture was used to ligate the renal artery, renal vein and ureter of the right kidney. After the ligature was tied, the right kidney was excised distal to the ligature. The abdominal muscles were then sewn together with 4.0 silk suture and the skin was closed with 9-mm stainless steel surgical clips. The same procedures were performed on the sham-operated rats, except that the right kidney was not removed and a ligature was not tied around the structures entering and exiting the hilus of the kidney. The uninephrectomized (NPX) and sham-operated (SO) rats were given 10 days to recover from surgery. The lo-day period was also given to allow for the completion of the rapid phase of compensatory renal growth in the NPX rats. Following the period allowed for recovery, the NPX and SO rats were subdivided into three groups. One group of NPX (n = 6) and SO rats (n = 6) was given a nontoxic 0.5 pmol/kg dose of mercuric chloride, while the second group of NPX (n = 6) and SO (n = 6) rats received a nephrotoxic 2.5 PmoVkg dose of mercuric chloride. The third group of NPX (n = 4) and SO (n = 4) rats did not receive a dose of mercuric chloride. Instead they received an injection of vehicle (0.9% sodium chloride; w/v; 2 ml/kg). The doses of mercuric chloride were delivered in

3 12 RUDOLFS K. ZALUPS a vehicle of 0.9% (w/v) sodium chloride at a volume of 2 ml/kg. Forty eight hours after the 0.0 and 0.5 kmol/kg doses of mercuric chloride were given, the animals receiving these doses were prepared surgically in the in viva perfusion and fixation of the left kidney. The rats given the 2.5 p,mol/kg dose of mercuric chloride were treated similarly 24 hr after the dose of mercuric chloride was administered. The procedure used to perfuse the left kidney is described below. Once each animal was anesthetized with an overdose of sodium pentobarbital(lo0 mg/kg) the abdominal cavity was opened along the mid-line from the pubis to the xyphoid process. The organs in the abdominal cavity were moved aside to expose the abdominal aorta and vena cava. The 4.0 silk ligatures were tied loosely around the aorta. One ligature was placed around the aorta at the level between the left and right renal artery. The other two ligatures were placed below the left renal artery. With the ligatures in place, a small vascular clamp was placed around the aorta just above the ligature that was closest to the left renal artery from below. A small, 45 degree incision was made in the aorta just below the ligature that was furthest away from the left renal artery in a caudal direction. The incision was made with a pair of iris scissors. Then the beveled tip of a piece of polyethylene tubing (PE 190, i.d. = 1.19 mm, o.d. = 1.70 mm) attached to a set of perfusion syringes was inserted about 1.5 cm into the abdominal aorta. The polyethylene tubing was secured in place with the two ligatures beneath the left renal artery. With the tubing in place, the vascular clamp was removed. Then the ligature situated between the right and left renal artery was tied firmly and a small incision was made in the left renal vein with a pair of sharpened small surgical scissors. Just after the renal vein was cut, the perfusion of the left kidney was started. First, the left kidney was cleared of blood quickly at a constant pressure of 120 mm Hg with a warm (37 C) phosphate buffer containing 4.3 g/liter sodium dihydrogen phosphate (NaH,PO,), 14.8 g/liter disodium monohydrogen phosphate (Na,HPO,), and 1000 units/liter sodium heparin. After the blood was cleared from the kidneys, the kidneys were fixed in siru at a constant pressure of 120 mm Hg with a fixative containing 4% formaldehyde and 1% glutaraldehyde buffered with 11.6 g/liter NaH,PO, and 2.7 g/liter sodium hydroxide (NaOH). Approximately 60 ml of fixative was perfused through each left kidney. The total osmolality of the fixative was 1120 mosm. When the perfusion was completed the left kidney was removed and a 3-4 mm midtransverse slice of the organ was obtained. The slices were further fixed for 24 hr in cold tixative (4 C). Subsequent to fixation, the slices of renal tissue were dehydrated in a graded ascending series of ethanol and embedded in plastic (Historesin, LKB, Gaithersburg, MD). Sections (2 km in thickness) of the plastic blocks were obtained using glass knives and an LKB 2218 Historange microtome. The sections were mounted onto acid-cleaned and 5% gelatinsubbed glass slides and were allowed to dry for 24 hr. The slides were then dipped into Kodak Nuclear Tract Emulsion (Cat No , Rochester, NY) in the dark and then packed in black, light-proof boxes for 3 days at 4 C. After the third day of exposure, the slides were removed from the black boxes and were developed in Kodak D- 19 developer (1: 1 dilution) for 10 min and then fixed in Kodak fixer for 10 min. One-half of the autoradiographed sections obtained from each animal were left unstained. The remaining sections were stained in 0.25% toluidine blue in 1% sodium borate. The above histochemical method was performed in accordance to the method established by Rodier er al. (1988). All the sections were viewed under a Nikon Optiphot light microscope. The

4 HISTOCHEMICAL LOCALIZATION OF RENAL MERCURY 13 stained sections were viewed with brightfield optics and the unstained sections were viewed with darkfield optics. Unstained sections of renal tissue from both the NPX and SO rats given the 0.5 kmol/kg dose of mercuric chloride were analyzed with an SMI Microcomp Image Analysis System (Southern Micro Instruments, Atlanta, GA) using darkfield optics. This analytical system allowed for the densitometric quantitation of deposits in the renal tissue. Four microscopic fields, each consisting of 207,840 pm*, of renal cortex and outer stripe of the outer medulla were analyzed at random for each animal. Background subtraction was implemented for each field analyzed. The average number of square micrometers of four microscopic fields of the cortex and outer stripe of the outer medulla tilled by the grains was measured and expressed numerically as square micrometers of grains/field. Statistical differences between means for the average number of square micrometers/field of cortex and outer stripe of the outer medulla occupied by grains in the specimens from the NPX and SO rats were evaluated with the unpaired Student t test for two independent samples. Statistical differences between means for the cortex and outer stripe, with respect to the number of square micrometers/field occupied by grains, for each group of rats was evaluated with the paired Student t test. Differences between means were regarded statistically significant when P < RESULTS With darkfield optics, deposits of presumed inorganic mercury were seen in all of the unstained sections of the left kidneys from the NPX and SO rats given the 0.5 pmol/kg dose of mercuric chloride. The deposits were localized in both the cortex and the outer stripe of the outer medulla. They appeared to be situated within tubular epithelial cells. In the sections from the SO rats, there appeared to be a somewhat greater number of deposits in the outer stripe of the outer medulla than in the tubules in the cortex (Fig. 1). This observation was confirmed by densitometric analysis of the unstained sections. The average (*SD) portion of the visual microscopic field of the cortex (207,840 pm ) occupied by deposits was 15,526? 2200 pm2. This value is statistically different (P < 0.05), on the basis of paired analysis, from the value of 20, Fm2, which is the mean portion of the visual microscopic field of the outer stripe of the outer medulla occupied by deposits. A similar pattern of distribution of deposits was found in the sections of left kidney obtained from the NPX rats given the 0.5 kmol/kg dose of mercuric chloride (Fig. 2). That is, more deposits were observed in the outer stripe of the outer medulla than in the cortex. However, one striking difference was observed between the sections obtained from the NPX and SO rats. The number of deposits in the outer stripe of the outer medulla of the renal tissue from the NPX rats was significantly greater than the number of deposits in the outer stripe of the outer medulla of the renal tissue from the SO rats. The average fraction of the visual microscopic field of the outer stripe of the outer medulla occupied by deposits was calculated to be 40, ,286 Frn*. This value is more than twice that for the outer stripe of the renal outer medulla from the SO rats. In the sections of renal tissue from the NPX rats, the density of deposits was so great in many of the tubules situated in the outer stripe that it appeared that the entire volume of epithelial cells in these tubules had been replaced by a cast of fused deposits. In the cortex, the average fraction of the visual microscopic field occupied by de-

5 14 RUDOLFS K. ZALUPS FIG. 1. Darktield view of a low-power field of cortex (A) and outer stripe of the outer medulla (B) of a kidney from a sham-operated (SO) rat given a 0.5 PmoYkg dose of mercuric chloride 10 days after surgery and then sacrificed 48 hr later. The tissue was processed for autometallography. Deposits appear to be in the epithelium of tubules situated in the cortex and outer stripe of the outer medulla. There appear to be slightly more deposits within the outer stripe of the outer medulla than in the cortex. Final printed magnification for both A and B is 90x.

6 HISTOCHEMICAL LOCALIZATION OF RENAL MERCURY 15 FIG. 2. Darkfield view of a low-power field of cortex (A) and outer stripe of the outer medulla (B) of a kidney from a unilaterally nephrectomized (NPX) rat given a 0.5 pmol/kg dose of mercuric chloride 10 days after surgery and then sacrificed 48 hr later. The tissue was processed for autometallography. Numerous deposits are present in the tubules situated in the cortex and outer stripe of the outer medulla. The greatest concentration of deposits is in the epithelium of the tubules in the outer stripe of the outer medulla. Final printed magnification for both A and B is 90 x.

7 16 RUDOLFS K. ZALUPS posits was 21,013? 7362 pm*. This was not significantly different, based on unpaired analysis, from the value obtained for the renal cortex of the SO rats. With brightfield optics and the toluidine blue stained sections it was possible to determine specifically in which segments of the nephron the deposits were localized. In general, deposits were found exclusively in the proximal tubule in the sections of the left kidney from both the NPX and the SO rats. Deposits were for the most part absent from any of the distal segments of the nephron. The deposits were found in both the convoluted and the straight portions of the proximal tubule. In the convoluted segments (S, and the first part of S,) of the proximal tubule, which are located solely in the cortex, the deposits were black in color and appeared to be similar in shape and size to secondary lysosomes normally found in the SZ segments of the proximal tubule of the rat (Fig. 3A). The deposits were generally situated in the central portion of the cytoplasm of the epithelial cells in the proximal convoluted tubule. Very rarely were the deposits seen in nuclei. The deposits found in the straight portion of the proximal tubule (last portion of the S, and entire S,) were also black in color, but were smaller in size than the deposits found in the convoluted segments (Fig. 3B). In the straight portions of the proximal tubule in the outer stripe of the outer medulla the number of deposits in each epithelial cell was much greater in number than that in the straight portion of the proximal tubule situated in the cortex. Although the location of the deposits was similar in the kidneys from both the NPX and the SO rats, there was an obvious difference in the distribution of deposits between the two groups of rats. The most obvious difference was found in the segments of the proximal tubule found in the outer stripe of the outer medulla. In the NPX rats, the number of deposits found in the epithelial cells of the S, segment of the proximal tubule situated in the outer stripe was much greater than that in the epithelial cells of the same segment of the nephron in the renal outer stripe of the SO rats. Differences in the number of deposits per cell in the segments of the proximal tubule in the cortex between the NPX and SO rats were not as easily discernable. It should be noted that no cellular necrosis was observed in the renal tissue of the NPX and SO rats 48 hr after the animals were given the 0.5 kmol/kg dose of mercuric chloride. For the most part, the renal tissue from both the NPX and the SO rats given the 0.5 pmol/kg dose of mercuric chloride appeared to be normal. Some shedding of the brush border and vacuolization was noticed in occasional proximal tubules in a few of the kidneys. However, no consistent pattern or trend was observed. It is believed that some of this occasional degeneration may have been due to some changes in hydrostatic pressure and/or anoxia associated with the perfusion of the kidneys. Deposits of presumed inorganic mercury were also found in the unstained sections of left kidney from the NPX and SO rats given the 2.5 pmol/kg dose of mercuric chloride. As was the case with the 0.5 pmol/kg dose of mercuric chloride, the deposits were found in the cortex and the outer stripe of the outer medulla. In the sections of left kidney from both the NPX and SO rats, the number and density of deposits appeared to be greater in the epithelium of the tubules in the outer stripe of the outer medulla than in the epithelium of the tubules in the cortex. Severe renal injury was discernable with darkfield optics in the unstained sec-

8 FIG. 3. Brightfield view of a high-power field of cortex (A) and outer stripe of the outer medulla (B) of a kidney from a unilaterally nephrectomized (NPX) rat given a 0.5 pmol/kg dose of mercuric chloride 10 days after surgery and then sacrificed 48 hr later. The tissue was processed for autometallography. Subsequently the section was stained with 0.25% toluidine blue in 1% borax. In the cortex (A), black deposits are seen within the epithelial cells of the proximal tubules. The deposits are found primarily in the central regions of the cells. There are no deposits in any of the distal segments of the nephron (D) present in the field of view. In the outer stripe of the outer medulla (B), deposits are present within the epithelial cells of the pars recta (S,) segment of proximal tubules. The deposits appear to be smaller in size and more numerous than those in the epithelial cells of the convoluted portions of the proximal tubule seen in Fig. 3A. The distal segments of the nephron (D) appear to be free of deposits. Final printed magnification for both A and B is 441 X.

9 18 RUDOLFS K. ZALUPS tions of left kidney from both the NPX and the SO rats. Most of the injury occurred in the outer stripe of the outer medulla. The damage was characterized by profiles of basal regions of renal tubules with the absence of the entire epithelium within the tubules. In other words, the epithelium had been shed away from the basement membrane of the tubule. It was possible to identify which segments of the nephron in the kidneys of the NPX and SO rats given the 2.5 pmol/kg dose of mercuric chloride had deposits in them using brightfield optics and the toluidine blue stained sections. Deposits of presumed inorganic mercury were found only in the proximal tubule. The shape and size of the deposits in the convoluted and straight portions of the proximal tubules were very similar to those seen in the convoluted and straight portions of proximal tubules in the kidneys from the NPX and SO rats given the 0.5 pmol/kg dose of mercuric chloride. With brightfield optics, it was confirmed that tubular necrosis in the straight portion of the proximal tubules in the outer stripe of the outer medulla of the kidneys from the NPX and SO rats given the 2.5 pmol/kg dose of mercuric chloride was severe. Some tubular necrosis also occurred in the straight portion of proximal tubules in the medullary rays of the cortex. No obvious differences were seen in the severity of the cellular and tubular necrosis between the NPX and the SO rats. In the epithelial cells of the pars recta of proximal tubules that appeared to be in the process of undergoing cellular necrosis or had undergone cellular necrosis, deposits were sparse or absent. It appeared that the deposits were being released from the necrotic epithelial cells. No signs of loss of deposits or cellular necrosis were observed in the convoluted segments of the proximal tubule. No signs of any deposits were observed in the sections of renal tissue from the NPX and SO rats that did not receive any inorganic mercury. This was true regardless of whether the sections were viewed with darkfield or brightfield optics. DISCUSSION The histochemical technique of autometallography (Danscher, 1984) was used in the present study in an attempt to demonstrate the intrarenal distribution and localization of inorganic mercury in NPX and SO rats given a nontoxic 0.5 pmol/kg or a toxic 2.5 pmol/kg dose of mercuric chloride. Deposits were found in sections of kidneys obtained from both NPX and SO rats 48 hr after the animals received the 0.5 p,mol/kg dose of mercuric chloride using darkfield optics. The deposits were found in the tubular epithelium of segments of the nephron situated in the renal cortex and the outer stripe of the outer medulla. More deposits were present in the outer stripe of the outer medulla than in the cortex in both groups of rats. The most striking feature, however, was the large number of deposits in the outer stripe of the outer medulla of the kidneys from the NPX rats. The number of deposits was significantly greater than that in the same region of the kidney from the SO rats. In a recent metabolic study (Zalups et al., 1987), the renal accumulation of inorganic mercury was studied in NPX and SO rats by gamma-counting renal tissue that was obtained from the rats 48 hr after they received a 0.5 p,mol/kg dose of mercuric chloride. In both the NPX and the SO rats of that study, the accumulation of mercury in the cortex and outer medulla was quite substantial, while very little mercury accumulated in the inner medulla. These findings are consis-

10 HISTOCHEMICAL LOCALIZATION OF RENAL MERCURY 19 tent with the localization of deposits seen with darkfield optics in the present study. The most prominent finding in the metabolic study was that there was increased accumulation of inorganic mercury in the renal outer medulla of the NPX rats. The observations and calculations made in the present study are also in agreement with that finding. If the deposits seen with brightfield optics in the present study truly represent deposits of inorganic mercury, the increased accumulation of mercury in the renal outer medulla of NPX rats appears to be due to an increased accumulation of mercury in the outer stripe of the outer medulla, since deposits are not found in the inner stripe of the outer medulla. In addition, it seems that the increased accumulation of mercury that occurs in the renal outer medulla of NPX rats is due solely to increased accumulation of mercury in the pars recta (straight) segment of the proximal tubule. There are two reasons for this conclusion. One is that deposits were not found in other segments of the nephron in the outer medulla. Second, there was a greater number of deposits within each epithelial cell of the pars recta of proximal tubules in the outer stripe of the outer medulla of the kidneys from the NPX rats than that from the SO rats. Increased accumulation of mercury in the pars recta of proximal tubules in the outer stripe of the outer medulla following unilateral nephrectomy and a period of compensatory renal growth would correlate well with the pathological findings of another recent study (Zalups and Diamond, 1987b). In that study it was found that there is increased damage to the pars recta of proximal tubules in NPX rats given low toxic doses of mercuric chloride. In NPX and SO rats that are given a nephrotoxic 2.5 kmol/kg dose of mercuric chloride, the pattern and distribution for the accumulation of mercury is similar (Zalups and Diamond, 1987b). In both groups of rats there is enhanced accumulation of mercury in the renal outer medulla. The findings from the present study are consistent with those findings. In the present study, darkfield and brightfield optics revealed a large number of deposits in the cortex and outer stripe of the outer medulla in the kidneys of both groups of rats. Moreover, there were more deposits in the outer stripe than in the cortex. The deposits, as viewed with brightfield optics, were localized in all segments of the proximal tubule, but tended to be localized preferentially in the S, and S, segments. The S, segments in the outer stripe of the outer medulla appeared to contain the greatest concentration of deposits. It has also been demonstrated with the silver amplification technique (Danscher and Moller-Madsen, 1985) (which is for the most part the same as the autometallographic technique used in the present study), that deposits of presumed inorganic mercury accumulate exclusively in the renal proximal tubule of BALB/c mice treated with mercuric chloride (Hultman et al., 1985). Investigators found that 30 min after treating BALB/c mice with a nephrotoxic 9 mg/kg dose of mercuric chloride, coarse granular deposits were seen within epithelial cells of all the segments of the proximal tubule in the kidneys of the mice. The granules were most abundant in the S, and S, segments of the proximal tubule. No deposits were found in the distal tubules and collecting ducts. Similar findings were obtained by the same group of investigators in a later study (Hultman and Enestrom, 1986). They also found that deposits, presumably of mercury, were present in the epithelial cells of the proximal tubules days after treatment with mercuric chloride,

11 20 RUDOLFS K. ZALUPS even as long as 17 days after treatment. The findings from these two studies are consistent with observations and findings of the present study. The deposits found in the kidneys of the NPX and SO rats most likely represent deposits of inorganic mercury. The reason for this conclusion is threefold. First of all, there is a very close correlation between the results obtained with the autometallographic histochemical technique and the results obtained with the technique of gamma-counting radiolabeled mercury in samples of renal tissue with respect to the zonal localization of mercury within the kidneys of NPX and SO rats. Second, deposits were not seen with darkfield or brightfield optics in the renal tissue from the NPX and SO rats not given any mercuric chloride. Third, evidence from a couple of studies indicates that the silver amplification technique (which is for the most part the same as the autometallographic technique used in the present study) specifically stains mercuric sullides and selenides in tissues of animals treated with mercury (Danscher and Moller-Madsen, 1985; Hultman and Enestrom, 1983). An important point that needs to be mentioned, however, is that this technique will, under other conditions, also stain metallic gold and silver as well as the sulfides and selenides of gold, silver, and zinc (Danscher et al., 1987). It should also be pointed out that questions have arisen as to the effectiveness of this method for demonstrating the presence of organic forms of mercury in tissue (Rodier et al., 1988; Danscher and Moller-Madsen. 1985; Magos et al., 1985). The deposits observed in the S, and S, segments of proximal tubules appeared to be similar in structure to the secondary lysosomes found in the S2 segment of the proximal tubule. In tissue fractionation studies, it has been shown that the lysosome accumulates inorganic mercury (Ellis and Fang, 1967; Madsen and Hansen, 1980; Weinberg et al., 1982). Electron microscopy has revealed silver deposits in the lysosomes of renal proximal tubules of BALB/c mice treated with a 3.0 mg/kg dose of mercuric chloride (Hultman et al., 1985; Hultman and Ene- Strom, 1986). Thus, some of the deposits found in the renal proximal tubules of the NPX and SO rats may in fact have been within secondary lysosomes. It was not possible to demonstrate any evidence for the accumulation of mercury in segments of the nephron distal to the proximal tubule. This does not mean that mercury does not accumulate in these segments. Inorganic mercury may be transported by, or may accumulate in, distal segments of the nephron. However, the amount of mercury that is present within the epithelial cells of distal segments may not be enough to be detected by the autometallographic technique. Other more sensitive techniques need to be employed to better understand the handling of inorganic mercury throughout the entire nephron. The findings from the present study indicate that the enhanced accumulation of inorganic mercury that occurs in the renal outer medulla of the nephrectomized rat (Zalups and Diamond, 1987b; Zalups et al., 1987) is probably due to an increase in the accumulation of mercury in the pars recta of proximal tubules in the outer stripe of the outer medulla. The reason why this increased accumulation of mercury occurs is unknown. It is known that the remnant kidney undergoes numerous structural, functional, and biochemical changes. Some of these changes include increased single nephron glomerular filtration rate, increased renal blood flow that is associated with a redistribution of blood flow, increased transport of water and solutes, and changes in numerous metabolic processes (Bricker and Fine, 1981). Any one of these changes alone or in combination could cause the

12 HISTOCHEMICAL LOCALIZATION OF RENAL MERCURY 21 increased accumulation of mercury in the remnant kidney. Only further research will uncover the mechanism for this phenomenon. For the time being, the findings in the present study are significant ones, for they have uncovered a possible relationship between the renal accumulation of inorganic mercury and the nephropathy induced by inorganic mercury in NPX and SO rats. ACKNOWLEDGMENTS The author thanks Ms. Barbara Kates from the department of obstetrics and gynecology at the University of Rochester Medical Center for her technical assistance in carrying out the autometallographic procedure used in this study. The author also thanks Mr. Frederik H. Schmidt from the department of anatomy at Emory University School of Medicine for his assistance in performing the computerized densitometric analysis in this study. This research was supported by National Institutes of Health Grant ESO5157. REFERENCES BRICKER, N. S., and FINE, L. G. (1981). The renal response to progressive nephron loss. In The Kidney (B. M. Brenner, and F. C. Rector, Jr., Eds.), 2nd ed., pp W.B. Saunders Co., Philadelphia. CHOI, B. H. (1984). Cellular and subcellular demonstration of mercury in situ by modified sulfidesilver technique and photoemulsion histochemistry. Exp. Mol. Pathol. 40, DANSCHER, G. (1984). A new technique for light and electron microscopic visualization of metals in biological tissues (gold, silver, metal-sulfides, and metal selenides). Histochemisrry 81, DANSCHER, G., and MOLLER-MADSEN, B. (1985). Silver amplification of mercury sulfide and selenide. A histochemical method for light and electron microscopic localization of mercury in tissue. J. Histochem. Cytochem. 33, DANSCHER, G., and RUNGBY, J. (1986). Differentiation of histochemically visualized mercury and silver. Histochem. J. 18, DANSCHER, G., RYTTER NORGAARD, J. O., and BAARTRUP, E. (1987). Autometallography: Tissue metals demonstrated by a silver enhancement kit. Hisrochemistry 86, ELLIS, R. W., and FANG, S. C. (1967). Elimination, tissue accumulation, and cellular incorporation of mercury in rats receiving an oral dose of 203Hg-labelled phenylmercuric acetate and mercuric acetate. Toxicof. Appl. Pharmacol. 11, HULTMAN, P., and ENESTROM, S. (1983). Experimental Hg-nephropathy: Correlation between histochemistry and analytical EM. J. Ultrastruct. Res. 85, 118%1190. HULTMAN, P., and ENESTROM, S. (1986). Localization of mercury in the kidney during experimental acute tubular necrosis studied by the cytochemical silver amplification method. Brit. J. Exp. Puthol. 67, HULTMAN, P., ENESTROM, S., and VON SCHENCK, H. (1985). Renal handling of inorganic mercury in mice. Virchows Arch. B 49, MADSEN, K. M. and HANSEN, J. C. (1980). Subcellular distribution of mercury in the rat kidney cortex after exposure to mercuric chloride. Toxicol. Appl. Pharmacol. 54, MAGOS, L., BROWN, A. W., SPARROW, S.. BAILEY, E., SNOWDEN, R. J., and SKIPP, W. R. (1985). The comparative toxicology of ethyl- and methylmercury. Arch Toxicol. 57, RODIER, P. M., KATES, B., and SIMONS, R. (1988). Mercury localization in mouse kidney over time: Autoradiography versus silver staining. Toxicol. Appl. Pharmacol. 92, WEINBERG, J. M., HARDING, P. G., and HUMES, H. D. (1982). Mitochondrial bioenergetics during the initiation of mercuric chloride-induced renal injury. J. Biol. Chem. 257, ZALUPS, R. K., and DIAMOND, G. L. (1987a). Intrarenal distribution of mercury in the rat: Effect of administered dose of mercuric chloride. Bull. Environ. Contam. Toxicol. 38, ZALUPS, R. K., and DIAMOND, G. L. (1987b). Mercuric chloride-induced nephrotoxicity in the rat following unilateral nephrectomy and compensatory renal growth. Virchows Arch. B 53, ZALUPS, R. K., KLOTZBACH, J. M., and DIAMOND, G. L. (1987). Enhanced accumulation of injected inorganic mercury in renal outer medulla after unilateral nephrectomy. Toxicol. App/. Pharmacol. 89,