Br. J. exp. Path. (1979) 60, 341

MERCURIC CHLORIDE-INDUCED RENAL TUBULAR NECROSIS IN THE RAT B. H. HAAGSAIA AND A. W. POUND

From, the Department of Pathology, University of Queensland, Brisbane, Australia Received for publication January 9, 1979

Summary.-A light microscopic study of the renal tubulonecrotic lesion in rats given a small dose of HgCl2 is described. The changes consist of a rapidly developing vacuolation of the cytoplasm with loss of basophilic staining within 4 h that leads to cell breakdown, fragmentation and dissolution by 48 h. Nuclear changes appear to set in later. Permanent patchy fibrotic lesions were found in the kidneys at 10 days. The animals pass a large amount of urine of low osmolarity, low Na+, K+ and Clfor a period of 3 days accompanied by an increased water intake. Nevertheless there appeared to be no water or ionic imbalance between daily inputs and outputs. Blood urea levels were greatly increased for 3 days, but did not return to normal by the 10th day.

MERCURY COMPOUNDS are well known potent cellular toxins (Robbins, 1974). The main action of mercuric salts, e.g. HgCI2, when systemically administered, is on the kidneys where, above a threshold dose of about 1 mg HgCl2/kg in rats, they cause death of epithelial cells of the proximal convoluted tubules, mainly the pars recta. Increased dosage results in extension of the tubular involvement (Rodin and Crowson, 1962) and high doses, e.g. 6-16 mg HgCl2/kg in rats, which are often lethal, cause necrosis of almost the full length of the pars convoluta and pars recta (Gritzka and Trump, 1968). The fully developed lesion in experimental animals (Rodin and Crowson, 1962; Gritzka and Trump, 1968) and in man (Anderson and Kissane, 1977; Robbins, 1974), is often associated with renal failure (Flamenbaum, 1973). Mercury compounds also produce cell damage in other tissues e.g. in nervous tissue and, when topically administered, in the gut and other exposed tissues (Martindale, 1972; Robbins, 1974). Descriptions of the structural aspects of the development of the renal tubular lesion still leave matters to be resolved. Early changes have been reported 3 h

after small doses (Cuppage and Tate, 1967). With large doses the effects occur more rapidly. The cytochemical basis of the death of the cells is not understood. It is probable that the cells are "dead" within a few hours of dosing and that all that follows to produce the classical picture is autolysis. The mechanism of the renal failure is controversial (Flamenbaum, 1973). It is assumed that the tubular structure is regenerated without scarring but there are grounds for doubting that this is correct (Oliver, 1953). These experiments were designed to provide an account of the changes that take place in the rat kidney over a period ranging from 1 h to 6 months after an s.c. injection of a small but consistently effective dose of mercuric chloride. This paper will describe mainly the light microscopic changes in the tubulo-necrotic stage and some functional studies; ultrastructural aspects mainly will be dealt with elsewhere. METHODS AND MATERIALS Animals. Random-bred, male SpragueDawley rats weighing 250-350 g were obtained from the Medical School Animal House, Univer-

342

H. B. HAAGSMA AND A. W. POUND

sity of Queensland. The animals had free access to a 20% protein pelleted diet (Bunge, Australia, Ltd) and water at all times. Chemicals. A stock solution of HgCl2 was prepared by dissolving 1 g HgCl2 (A.R.) in 11 sterile 0-9% NaCl solution (Abbott Laboratories Pty. Ltd, Sydney). Experimental. It was desired to produce consistently necrosis of the epithelial cells lining approximately the terminal third of the proximal tubule, and yet to have no mortality. A pilot study using doses of 1, 2, 4 and 6 mg/kg determined that a suitable dose was 1-5 mg HgCl2/kg body wt. The larger two doses had a mortality of more than 40 % after 2 or 3 days, and showed more extensive necrosis. Rats were given the calculated dose of HgCl2 s.c. into the loose tissue of the anterior abdominal wall. At intervals of 1, 2, 4, 6, 8, 16, 24, 36 h, 2, 3, 5, 7, 9, 11-18 days and 1, 2, 3 and 6 months after injection, 2 animals were taken for examination of the kidneys. Control rats were given an equivalent dose of sterile 0-9% saline and killed at similar intervals up to 5 days after

injection. Kidney fixation and preparation. Kidneys were perfused in vivo, through the abdominal aorta under Nembutal anaesthesia, with 2-5% glutaraldehyde (TAAB electron microscope grade) in 0-05M sodium cacodylate buffer, pH 7 3, for a period of 1 min using 45-50 ml of fixative solution. The perfusion pressure ranged between 130 and 140 mmHg. Exposure of the kidneys, which caused inadequate results, was avoided. The kidneys were excised, cleared of perirenal fat, the capsule stripped, and the renal pedicle cut flush with the hilus. They were divided longitudinally into 2 hemisections which were immediately placed in a jar of the same fixative solution as above. Very thin slices of tissue were cut from the median faces of the hemisections and prepared separately for electron microscopy. Occasional inperfectly perfused kidneys were discarded. The hemisections of the kidneys were embedded in paraffin by routine histological methods and sections cut at 6 ,um. The following stains were used: haematoxylin and eosin (H. & E.); periodic acid-Schiff (P.A.S.) and sulphated toluidine blue (S.T.B.) to demonstrate the brush border and basement membranes; Mallory stain to demonstrate interstitial fibrosis. Small blocks ( < 1 mm3) were postfixed in 1% OS04 in cacodylate buffer, 01M, pH 7.3, prestained with 5% uranyl acetate and embedded in epon. Sections 1 ,um thick stained with toluidine blue were used for high-resolution studies by light microscopy. Thin sections were taken for electron microscopy and stained with lead acetate (Pound and Haagsma, unpublished). Urine analysis. Urine was aspirated with a sterile syringe from the bladder. After centrifui-

gation at 1500 rpm for 20 min the residue was resuspended in 0-05 ml supernatant and examined on a glass slide under a cover slip. Cell counts were graded arbitrarily from 0 (normala few scattered cells) to 4 (massive content of cells) according to the number of cells in 10 randomly selected high-power fields ( x 400). An estimate of the number of casts was made in the same manner. Ionic concentrations [Na+], [K+] and [Cl-] in the supernates were determined by Auto-Analyser at the Department of Pathology at the Royal Brisbane Hospital. Osmolarities of the supernates were determined by a Vapour Pressure Osmometer (Wescor Model 5100). One urine sample obtained 24 h after administration of HgCl2 was mixed with an equal volume of buffered glutaraldehyde solution immediately after aspiration. After centrifugation the residue was postfixed in 1% OS04, and suspended in 4% agar. The agar gel was diced into small pieces and embedded in epon following the standard technique. Sections 1,um thick were stained with toluidine blue for light microscopy, and thin sections were stained with uranyl acetate-lead acetate (Reynolds, 1963) for electron microscopy. In separate experiments rats were maintained under treatment in a metabolism apparatus (Metabowl, Jensens, Herts, U.K.). Water intakes, food intakes and urinary outputs were determined from time to time for up to 10 days. In a separate experiment blood samples were collected by cardiac puncture at 6- or 12-hourly intervals for 48 h, and then at intervals for 10 days after dosing with HgCl2, for determinations of blood urea. Specimens from 6 rats were tested at each time. Plasma and urinary creatinine concentrations were determined in other animals, but urinary output could not be undertaken in these groups. Ability to concentrate urine was determined in control and HgCl2-treated rats by depriving groups of 5 or 6 of food, or food and water, for 24 h. Osmolarities [Na+], [K+] and Cl-] were determined in samples of urine after centrifugation obtained at the end of each period. In some groups the specimens had to be pooled in equal amounts for technical reasons. RESULTS

General Inflammation and oedema (estimated to be the equivalent of about 3 ml fluid) at the site of injection were observed in all animals. After 2-3 days about 50% of the animals developed ulcers which later healed. Within 21 h after injection the animals became lethargic and the hair stood on end. They ate and drank little

HgC12-INDUCED RENAL TUBULAR NECROSIS

for 12 h, only I the normal daily intake of food in the first 24 h, and 2 the normal intake in the second 24 h (estimated, not weighed). They gained only 04%0 body wt per day or less for the first 3 days, and even sometimes lost a little in the first day, but thereafter gained weight at about the same rate as controls (1 56+ 016 g/100 g/day). Control animals consumed about 14-4 g of the diet per 100 g per day. There was no mortality. Urine was present in the bladder of most rats when killed.

Gross findinys The earliest evidence visible to the naked eye of the development of renal damage was a band of slight pallor in the cortico-medullary zone of the bisected kidneys about 8 h after injection. This rapidly developed by 24 h into a white necrotic band occupying the outer stripe of the medulla with variable extensions into the cortical zones. The band did not alter up to 48 h, was fading on the 5th and 7th days, and disappeared by the 9th day. Up to the 9th day the renal capsule stripped readily leaving a smooth surface. By the 16th day stripping of the capsule revealed irregular depressions up to 05 mm in diameter in the renal surface. These were irregularly scattered and varied in number between animals; they did not increase in number after the 16th day. It was therefore likely that the number of these lesions was determined by an earlier event. Such lesions were not observed in any control animal.

Microscopical findings The histological structure of the normal kidney conformed to accepted descriptions (Rodin and Crowson, 1962; Trump and Bulger, 1968; Jacobsen and Jorgensen, 1973; McDowell et al., 1976). The term "nephron" will be used when referring to the whole renal unit. The term "tubular profile" or "profile" refers to individual cross-sections as seen in histological material. The earliest histological changes following injection of 1-5 mg

343

HgCL2/kg s.c. were observed after 4 h although ultrastructural changes were present within 1 h (Pound and Haagsma, not published). Four hours after dosing. Epithelial cells of the pars recta were swollen and bulged into the lumen, giving the tubular profiles a stellate appearance (Fig. 1). The cytoplasm contained numerous tiny vesicles, and cytoplasmic structures were less prominent than normal, mainly on the luminal side of the nucleus. The nuclei were normal. In affected profiles all the epithelial cells showed similar changes. Considerable variation in intensity of the changes was noted between individual tubular profiles. Six and eight hours after dosing.-At 6 and 8 h after dosing the changes seen at 4 h were accentuated progressively. The changes varied considerably in intensity between profiles but where a profile was involved most of the cells showed similar changes (Fig. 2). The distribution of profiles with the most advanced changes appeared to be that of the profiles showing the changes at the earlier periods. The cytoplasmic vesicles were much more numerous and were more prominent. Some cells, probably those more severely damaged, contained large membranebound blebs devoid of brush borders, projecting into the lumen of the tubules (Fig. 3). In this period cells in the most affected areas were losing their cytoplasmic basophilia in H.E.-stained sections. The nuclei remained within normal limits except in the most severely affected cells in which some peripheral condensation of the chromatin was apparent by 8 h. Sixteen hours after dosing.-The changes were much more intense. Considerable variations in the extent of the tubular involvement and in the intensity of the changes between profiles were observed, but most cells in any one involved profile showed similar changes. In less affected areas the swollen cells bulged into the lumen, and showed varying degrees of coarse subapical vacuolation of the cytoplasm and a fine vacuolation involving

344

B. H. HAAGSMA AND A. W. POUND

- -: 3 U -8 FIG. 1 Photomicrograph of pars recta h after HgCl2. Swelling of cells, fine vacuolation in apical 1

L

-

w

4

;-wwxPr_- iK>x

s1

4

cytoplasm. Paraffin-embedded. H. & E. x 875. FIa. 2 Photomicrograph of proximal tubule from outer stripe of medulla 8 h after 1-5 mg HgCl2/kg s.c. A range of changes is seen in different profiles: normal profile, mild cell swelling, sub-apical cytoplasmic vacuolation, disruption of brush border. Cells in any one profile show similar changes. Epon-embedded. Toluidine blue. x 875. FIG. 3 Photomicrograph of pars recta 8 h after HgCl2. Swollen cells with vacuolated cytoplasm, large cytoplasmic vacuoles forming apical blebs causing disruption and loss of brush borders. Nuclei normal. Epon-embedded. Toluidine blue. x 875. FIG. 4-Photomicrograph of proximal tubule from outer stripe of medulla 16 h after HgC12. Swelling of cells, extensive fine vacuolation of cytoplasm, large sub-apical cytoplasmic blebs leading to disruption of brush border and shedding of cytoplasmic fragments into lumen. Nuclei show early pyknotic changes. Paraffin-embedded. H. & E. x 875.

most of the cytoplasm (Fig. 4). In most severely affected areas the subapical vacuoles formed large blebs which protruded into the tubular lumen. The blebs sometimes occupied up to half the cell volume, and displaced the nucleus to the base of the cells. They often disrupted the overlying brush borders or appeared to have ruptured to release cell content including the nucleus into the lumen. Cytoplasm of all cells in affected areas was not acidophilic in H.E. sections. Nuclear changes varied. In most swollen cells with only small apical vacuoles the

nuclei appeared to be normal, while in cells with gross swelling and vacuolation the nuclear chromatin was clumped and condensed. In other cells with eosinophilic cytoplasm the nuclei were either pyknotic or karyolytic, while the cells remained attached to the basement membrane. Numerous desquamated cells, cell fragments, often membrane-bound, and other debris were present in the lumen (Fig. 4). Cells in other parts of the nephron appeared within normal limits but at this stage granular casts, made up of packed necrotic epithelial cells and cell debris, and hyaline

HgC12-INDUCED RENAL TUBULAR NECROSIS

345

FIG. 5 Photomicrograph of section of cortex 24 h after HgC12. Necrosis of pars recta and distal part of proximal convoluted tubules. Paraffin-embedded. H. & E. x 160. FIG. 6 Photomicrograph of section from outer stripe of medulla 24 h after HgCl2. Shows sharp transition between necrotic and viable zones of convoluted tubule. Note tendency of viable cells to flatten and spread over the basement membrane under the necrotic tissue; sloughing and fragmentation of dead cells into lumen. Paraffin-embedded. H. & E. x 560. FIG.. 7-Photomicrograph of necrotic tubule 24 h after HgC12. Note fragmentation and autolysis of dead cells and surviving flattened cells. Epon-embedded. Toluidine blue. x 2200. FIG. 8 Photomicrograph of regenerating segment of proximal tubule 5 days after HgCl2. Note irregular mass of proliferating epithelium growing into lumen of the tubule. Necrotic cell debris is present above this mass of cells. Paraffin-embedded. H. & E. x 560.

casts were seen in the ascending limbs of the loop of Henle and distal convoluted tubules, but rarely in collecting ducts. Twenty-four hours after dosing.-Most cells in tubules corresponding to the pallid zone, comprising the pars recta, were eosinophilic and were in various stages of dissolution (Fig. 5). The length of tubule involved varied but cells of the more proximal tubules and glomeruli appeared morphologically normal as did those in the loops of Henle, distal convoluted tubules and collecting ducts. The transition from the necrotic zone in the tubule to the viable zone was often quite sharp

(Fig. 6).

The cells were grossly swollen. Extensive vacuolation of the cytoplasm in the apical areas often displaced the nuclei basally and caused disruption and loss of the brush borders. Many vacuoles had ruptured, or cell membranes had undergone dissolution, to release cytoplasmic granular contents including nuclei into the lumen, leaving remnants of cells adherent to the basement membranes. Other cells appeared to break up to form rounded membranebound fragments, some even with a few tufts of microvilli and containing organelles, which were discharged into the lumen (Fig. 7). Many such fragments, dead cells, debris and granular casts were

346

B. H. HAAGSMA AND A. W. POUND

present in the more distal tubules, collecting ducts and in the urine. Nuclei of the affected cells were sometimes pyknotic but in many cells only karyolytic nuclear ghosts or even no nuclear remnants were seen (Figs 5, 6). In extensive areas the processes of dissolution, disruption and shedding of cells resulted in acellular zones with bare basement membranes, but careful scrutiny of P.A.S.- and S.T.B.-stained sections failed to disclose evidence of rupture of basement membranes. Nevertheless a few cells always survived along the tubules in the necrotic zones. These were flattened epithelial cells with no microvilli and very little cytoplasm in which few organelles were present (Fig. 7). The nucleus was flattened and contained a large nucleolus. At this stage a mild inflammatory infiltration of polymorphonuclear leucocytes, monocytes and lymphocytes into the interstitial tissue was usually seen. Thirty-six hours-10 days after dosing. From 24 h on, dissolution and shedding of dead cells proceeded rapidly so that by 36 h there were large areas of bare but intact basement membrane. By 48 h only a few dead cells, or debris consisting of palely staining eosinophilic material devoid of nuclei or containing only an occasional nuclear ghost, remained adherent to the basement membranes. Cell debris was present in the tubules at all levels distal to the necrotic zone, including the collecting ducts to the tip of the renal papilla. By the 3rd day only a few remnants of dead cells remained in the affected zone. From the 36th h a few cells in mitosis were present in the tubular epithelium adjacent to the necrotic zone. From the 2nd day the residual flattened cells in the area immediately adjacent to the surviving tubular epithelium were more prominent and now lined the tubules. Some of these cells contained phagocytosed cell debris from the lumen. By the 3rd day many of these cells were in mitosis and dividing to repopulate the bare basement membranes of the tubules. A few cells in mitosis could also be found in more proximal parts

of the tubules which had not been involved in the necrosis. By the 5th day the regenerating epithelia formed an almost complete lining of the formerly necrotic segments of the nephron. A few cells in mitosis were still present. In the more advanced zones the cells were now cuboidal with a rounded nucleus containing a prominent nucleolus; the cytoplasm contained more organelles and the cytoplasmic basophilia was reduced. Cells were beginning to develop a brush border. In some areas the epithelial proliferation was disorderly and piled up into the lumen of the tubules (Fig. 8). Cell debris was still present in the loops of Henle and collecting ducts. Foci of inflammation were still present but the infiltrate was now predominantly lymphocytic. By the 7th day the tubules were lined by cuboidal cells with a definite brush border. A few mitoses were still present and small areas of epithelial proliferation into the lumen of tubules still remained. At this stage a few scattered collapsed glomeruli were to be seen. By the 9th and 10th days most of the tubules had regenerated; but there were a number of collapsed segments of proximal tubules associated with lymphocytic infiltration and connective tissue proliferation scattered through all levels of the renal cortex. The glomeruli associated with these foci appeared to be collapsed. The areas of intratubular cell proliferation remained. In later stages these foci seem to give origin to the small scars evident on naked eye examination of the kidneys. Urine cell analysis.-Urine from control rats showed a small but consistent number of cells which was assigned the grade 0. The cell count in animals treated with HgCI2 was normal after 6 h but rose precipitously by 16 h (Fig. 9) to a peak at 24 h so that the urine became quite turbid. The count remained high for about 6 days, but with a minimum at 3 days, and thereafter declined steadily to a normal by 9 days. Granular and occasional hyaline casts were seen in the urine on the 2nd day, increased in number on the 3rd day to a

HgC12-INDUCED RENAL TUBULAR NECROSIS

347

membrane-bound fragments of cells and debris.

2.-

0 1 2

4

6

8 10 12 15 DAYS FIcG . 9. Graph showing variation of incidence of cell content in urine, casts in renal tuLbules and casts in urine at various times after injection of HgCl2. *, cellular content of urine; O, incidence of casts in renal tubules; Fl, presence of casts in urine.

level that remained approximately the same until the 7th day and then declined to reach normal levels by the 1]5th day.

Microscopic examination showed the presence of relatively intact dead but partly autolysed cells, fragmented dead cells and debris. Electron microscopy of the 24-h specimen showed dead cells in various stages of dissolution, with rupture and loss of membranes, disorganized organelles, many still within membranes,

Measurement of renal function The functional measurements made are set out in the tables. Water balances (Table I) show a normal daily water intake of 1541 + 1P2 ml/100 g/day, to which must be added 7-5 ml/100 g/day of water metabolically derived from the diet. The daily urinary output was 7-5 + 1P6 ml/100 g/day. These figures give an estimated insensible loss of about 15-3 ml/100 g/day. After a dose of HgCl2 the total fluid intake (including water from the diet) fell about 50%0 in the first 12 h and was still reduced for the 1st 24 h, although the water intake had possibly increased a little. The fluid intake increased greatly in the 2nd 24 h to a peak on the 3rd day, after which it declined steadily until the 10th day. The calculated insensible loss (approximate though it must be) appears to have been reduced in the 1st 24 h, low in the 2nd day and to have returned in the 3rd day to control values. The urinary output in the first 12-h period fell a little but over the first 24-h period was slightly increased, from which it must be concluded that the rate of excretion in the second 12-h period must

TABLE I. Effect of a Dose of HgCl2, 1.5 mg/kg, on Fluid Balances, at Intervals Thereafter

Interval 12 hr 24 h 48 h 72 h 4 days 6 (lays 7 (lays 10 days

Normalt

MIeasuredlt

Calculated approx. valtues

Urine otutput Wrater intake ml per 100 g/day ml per 100 g/day 4 7, 2-6* 8-5, 4-8* 9-6 + 1-9 16-8 + 0-8 22-6+2-2 14-7+2-1 17-0+ 2-6 24-0+ 3-6 9-3 + 0-8 17-6+ 1-7 16-9 + 3-3 8-8 + 2-5 18-3+ 1-1 9-7+3-5 16-2 + 1-3 8-7 + 2-4 7-5+1-6 15-1+1-2

Fluid intake Insensible loss ml per 100 g/day ml per 100 g/day 9-8, 6-1 5-1, 3-5 18-1 8-5 27-6 12-9 31-5 14-5 25-1 15-8 24-4 15-6 25-8 16-1 23-7 15-0 22-6 15-1

Water initake and urinary output per 12 h, only two rats at this time. Four rats at each interval. Control means over 2 days in metabolism cages. Metabolic water from 14-4 g diet/100 g rat/day- 7-5 ml/100 g/day. *

t

Dietary

water

1st 24

h,

I normal

1-25

ml/100 g/day.

Dietary water 2nd 24 h, 2 normal 5-00 ml/100 g/day. Animals given H.gCl2 at 10 a.m. Daily temperature 65-70°.

348

B. H. HAAGSMA AND A. W. POUND

TABLE II.-Biochemical Parameters in Urine and Blood Urea Levels after Injection

of HgC12, 1.5 mg/kg

Time afteIr in jectioll 6h 12 h 18 h 24 h 30 h 36 h 48 h 3 (lays 4 days 6 days 10 days Control

Urinet

Blood urea* mM/l 6-6+ 1-2 7 9+2-0 8-3+2-3 9-7 + 1-4 8-2+0-9 15-0 + 2-0 21 0 + 5 6 16-7+ 5-4 11-6+2 1 9-1 + 1-4 8-9+0-5 6-3 + 1-0

j~~~~~~~

,

Osmolarity mOs/l 1536 1665 752 634 430 460 611 + 62 1094 + 262 1135 1247 + 86 1779+ 119 1874+ 150

l-Cl-]

[Na+]

[K+]

mM/i

mM-1

mM/

146 109 104

12:3

82 54

65

91 126 86 85

62 71 53+5 124+ 19 156 158 + 44 218 + 7 211 + 10

54

62 + 10 73 + 21 90 120+ 16 123 + 12 115+ 19

70 55 70 85

70(+ 13 107+38 140 177+ 45

200 + 22 197+ 69

Six rats at each interval. rats at each interval; where standard (leviationi is not shown the urines were pooled in equal amounts. At the 30-h interval, the mean of two such pools is shown. Plasma: [Na+], 137-7+2-6; [K+], 3 9+0-3; [C1-], 95+ 1-9. Diet contains approximately 077% NaCl. *

t Three

have been increased. The urinary output was increased on the 2nd and 3rd days, was less on the 4th day and slowly returned to normal by the 10th day. These figures imply a significant degree of dehydration on the first day, at least in the first 12 h, and that the polyuria on the 2nd, 3rd and 4th days in fact started in the 2nd 12 h of the first day, although

masked by the dehydration. The ionic constitution of the urine, sampled at the end of each period, varied significantly (Table II). At 6 and 12 h after injection of HgC12 the osmolarity was depressed a little. From 18 to 36 h it was reduced to about 3000 of the control TABLE III. Total Ionic Excretion, mM/ 100 g/day at Intervals after a Dose of HgCl2, 1-5 mg/kg

10 days

Na 0.453* 0-816 0 779 2-108 1-450 1-390 1-896

K 0-398* 0-787 0-911 1-241 0-837 1-056 0-896

0.449* 0-624 1-029 1-819 1-302 1-557 1-740

Controls

1-582

0-862

1-478

12 h 24 h 48 h 3 days 4 days 6 days

*

For the 12-h period.

Cl

values, and after 48 h it rose slowly to normal by the 10th day. These changes are parallelled by changes in [Na+], [K+] and [Cl-]. It seems likely that the values at 12 h are influenced by the dehydration suggested at this stage by the fluid balances (Table I). Estimates of the total daily ionic excretion (urinary output x ionic concentration; Table III) suggest that the daily excretion of Na+ is reduced a little by 12 h, is much reduced in the second 12 h and in the 2nd day, and has returned to normal on the 3rd day; indeed on the 3rd day it may have increased a little. On the other hand the daily excretion of K+ does not appear to change significantly. Excretion of Cl- appears to follow that of Na+. These values should be interpreted in the light of the reductions in food intake on the 1st and 2nd days. The ability to concentrate urine (Table IV) was severely restricted over the first 3 days especially on the 2nd day but was approaching normal by the 6th day. Plasma creatinine levels did not vary significantly from those of control rats (0.04 + 0-02 mM/l) at any stage after injection of HgCl2. Urinary creatinine levels fell steadily to 50%0 of the normal value (6.1 + 1*37 mM/l) in the 24-h specimen,

349

HgC12-INDUCED RENAL TUBULAR NECROSIS

TABLE IV. Effect of Deprivation of Food or W!ater on Urine Concentrating Capacity of Animals given 1P5 mg/kg HgC12 [Na+] Osmolarity [Cl-I [K+] Period

Treatment

Normal dliet No watert No food, Ino wateIrt HgCl2, no food, no

water

HgCI2,

no

water

24 h 24 h 0-24 h 25-48 h 49-72h 121 -144 h 25-48 h

mOs/l 1887 + 352 2595 + 100 2285 + 100 1049 + 117 908 + 165 1472+1:31 1692+ 193 849-283

mM/i

mM/i

mM/i

198 + 64

153+32 * 150 91+3(0 137+ 30 57+5 59 + 16 71 + 12 * 86

242 + 60

*

215

111+ 33 46 + 19 56 + 12 54 + 16 109 + 23 * 63

*

123 + 34 40 + 15 80 + 23 105+5 128 + 37 *

Periodls started at 10 a.m. Specimens of uriine were collecte(d from bladlder at end of each period. 5 or 6 animals to a group. * Specimens of urine pooled in equal amouints. t Veiy small amounts of urine could be collected fiom the blad(lers of the (depiive(I animals.

remained low until the 3rd day and then rose rapidly to normal values by the 6th day. These values, together with the urinary outputs as shown in Table I, suggest that the creatinine clearancerates were unaffected during the first 18 h, but then dropped to approximately 50%0 normal until the 3rd day, after which they returned to normal. Blood urea values (Table I) were probably elevated only 24 h after injection of HgCI2 (PZ- 5 0) but then increased rapidly to a maximum at 48 h. They then declined fairly rapidly but still appeared to be elevated after 10 days compared with the control values of the same animals before injection with HgCl2. DISCUSSION

The tubular necrosis produced in experimental animals by parenteral administration of HgCl2 has been the subject of a number of papers. The extent of the lesion varies with the animal species used and route of administration as well as with dose. Most work has been done with large doses, viz. 4 0 mg/kg or more in rats. but the lowest of these doses entails a 40-60% mortality so that long-term effects have not been usually pursued. The larger doses produce more extensive necrosis of the nephron, as in our pilot study, and, comparing the present results with those obtained after doses in excess of 4 0 mg/kg

(Oliver, MacDowell and Tracy, 1951; Oliver, 1953; Cuppage and Tate, 1968; Gritska and Trump, 1968; McDowell et al., 1976), perhaps allow more rapid development of the extension of the death of the cells. In the present work, using a dose of 1-5 mg HgCl2/kg, the earliest histocytological evidence of cell damage was in the pars recta after 4 h. Cells of affected tubular profiles were swollen and showed a fine vacuolation of the cytoplasm. Cytoplasmic granularity was obscured but the nuclei were normal. By 6 h the changes in the pars recta were greatly accentuated. The cells in the more advanced zones were now becoming eosinophilic and early clumping of the nuclear chromatin had appeared, so that it is clear that these cells were dead. The changes extended and developed very rapidly and by 16 h numerous dead cells and fragments of dead cells were being extruded into the tubules. By 24 h the epithelium of the pars recta was necrotic, and the cells in various stages of disruption and dissolution. Only a few viable flat epitheliEl cells remained from which regeneration of the tubular epithelium started by 36 h. By the 6th day the tubules were lined by cuboidal epithelium with well defined brush borders. Nevertheless numerous areas of collapsed tubules, apparently associated with collapsed glomeruli, and interstitial fibrosis remained

:3t50

B. H. HAAGSMA AND A. W. POUND

permanently (Haagsma and Pound, to be by the 10th day. Nevertheless the Na+, published). Epithelial debris in the tubules K+ and Cl- daily outputs were normal formed casts, first seen in the ascending after the 2nd day, and even on the 1st and limbs of the loops of Henle and, later, 2nd days when they appeared to be reas far down as the collecting ducts in the duced the reduction was probably conpapilla. The tubules above such casts often sistent with the reduced dietary intake. appeared to be dilated, suggesting an Therefore there was no evidence of retenelement of obstruction, but this cannot tion or loss of total body Na+, K+ or Clbe at all efficient in view of the diuresis. and, apart from the failure of the animals Moreover, the role of tubular obstruction to eat and drink normally on the 1st and as a functionally significant factor in the 2nd days with consequent dehydration, production of renal failure, even in the there was no evidence of water imbalance. presence of anuria, has yet to be defined An overall Na loss is usually implied after (Stein, Lifschitz and Barnes, 1978; larger doses. Na+ and K+ intakes signiFlamenbaum, 1973). The appearance of ficantly affect the effects of HgCl2 in cells and casts in the urine appeared to inducing renal failure (Flamenbaum et at., follow after their presence in the tubules 1973; Stein et al., 1978), and it is evident that ionic-balance as well as water-balance (Fig. 9). The sequence of the development of studies are needed to elucidate the issues. cytological damage in the pars recta is These findings differ from those in the consistent with the view that the site of literature. An anuric phase was said to the lesions may be related to concentra- follow a dose of 1.5 mg HgCl2/kg (Cuppage tion of Hg++ in the urine as it passes to and Tate, 1967) but the published data are the distal part of the proximal convoluted meagre. Some workers reported that a tubules (Rodin and Crowson, 1962; Cup- phase of anuria followed 20 h after a larger page and Tate, 1967; Siegel and Bulger, dose of HgCl2, preceded in the first 16 h 1975). It is also consistent with the possi- by a diuresis and followed after 48 h in bility that the involved segment is more the recovery phase by a second diuresis susceptible to lethal damage. In this con- (Oliver, 1953; Bank, Mutz and Aynedjian, nection it may be noted that, after 45 sec 1967), but there are contrary results clamping of the renal artery, damage is (McDowell et al., 1976). An explanation evident in the whole length of the proximal of these differences is obscure. In the tubule but that necrosis followed only in course of an episode of tubular necrosis, it the distal 3; the more proximal 3 could might be expected that tubular resorption recover (Haagsma, unpublished). Such of water should be diminished, leading to variations could be due to intrinsic cell a diuresis and an inability of the animals factors or other factors vascular, for to concentrate urine, and that these example. Variation in rate of absorption features would be maximal during the and excretion of Hg++ could therefore be period of necrosis, as was found in this expected to influence the rate of develop- work. A diuresis was reported during the ment of the cell changes. renal tubular necrosis induced by injecAfter this dose of HgCl2 (1.5 mg/kg) tions of glycerol (Finckh, 1960). However, the animals displayed a pronounced di- diuresis may be induced by anionic meruresis beginning probably in the second curial compounds in man (Weston, 1957) half of the first day and lasting until the and animals (Kessler, Lozano and Pitts, 3rd or 4th day before returning to normal. 1957) without tubular necrosis, presumThe urine was of low osmolarity and [Na+], ably by a biochemical effect on renal [K+] and [Cl-] were all reduced from tubular function. Such a function might within 6 h of dosing. The levels were at a be exerted on tubular epithelium at a minimum when the urinary output was at different level to that in which necrosis a maximum and then returned to normal occurs, even with HgCl2. Electron micro-

HgC12-INDUCED RENAL TUBULAR NECROSIS

scopic findings (McDowell et al., 1976; Zalme et at., 1976) suggest that HgCl2 induces cytological changes of a reversible character in the proximal parts of the proximal convoluted tubules. Functional changes might also occur at all levels of the nephron including the glomeruli, or even, with such a potent cell toxin, in other issues, e.g. pituitary and adrenal, and so influence urinary secretion. Blood urea levels rose rapidly from 18 h and remained high for 3 days before returning to low levels, that is, in general followed a similar course to the diuresis and to the renal tubulonecrosis, but the relationship to these features is obscure. However, it remained elevated for at least 10 days, although the other biochemical parameters returned to normal. As far as our calculations permit, the creatinine clearance rates appeared to be depressed in the same period. The occurrence of foci of permanent damage, even after a small dose of Hg, may be significant in this regard. Investigations of the renal failure as evidenced by measurements of blood urea levels in the first 24 h in rats dosed with 4.7 mg HgCl2/kg (Flamenbaum, 1973; McDowell et al., 1976) have shown that the blood urea level increased, while the creatinine clearance and the glomerular filtration rate decreased, much more rapidly than suggested by our measurements after the smaller dose. The mechanism of the renal failure appears to involve feed-back mechanisms affecting the glomerular blood flow, mediated through changes in the juxtaglomerular apparatus produced by altered [Na+] in the distal convoluted tubules (Flamenbaum, 1973; McDowell et al., 1976). The rate of elevation of the blood-urea and the apparent rate of decrease of the creatinine-clearance values in the current experiments are not inconsistent with this hypothesis at this stage after injection of the smaller dose. The many factors involved in clinical acute tubulonecrosis and acute renal failure (Finckh, Jeremy and Whyte, 1962); for which HgC12 induced kidney lesions have been used as a model, have

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been reviewed (Stein et al., 1978). It is clear that dose effects with HgCl2 play a considerable role in the physiological consequences that need to be considered in experiments to correlate them with the cellular changes produced. This work was supported by the Mayne Bequest Fund and the Tooth Bequest Fund of the University of Queensland. We thank Mr Mansel Thomas of the Department of Pathology, Royal Brisbane Hospital, for the biochemical analyses, and Mrs Vicky Whiting for technical help. REFERENCES ANDERSON, W. A. D. & KISSANE, J. M., Eds. (1977) Pathology, Vol. 1, 7th Edn. St. Louis: The C. V. Mosby Company. BANK, N., MIJTZ, B. F. & AYNEDJIAN, S. (1967) The Role of "Leakage" of Tubular Fluid in Anuria due to Mercury Poisoning. J. Clin. Invest., 46, 695. CUPPAGE, F. E. & TATE, A. (1967) Repair of the Nephron following Injury with Mercuric Chloride. Amer. J. Path., 51, 405. CuPPAGE, F. E. & TATE, A. (1968) Repair of the Nephron in Acute Renal Failure: Comparative Regeneration following Various Forms of Acute Tubular Injury. Path. Microbiol. (Basel), 32, 327. FINCKH, E. S. (1960) The Failure of Experimental Renal Tubulonecrosis to produce Oliguria in the Rat. Aust. Ann. Med., 9, 283. FINCKH, E. S., JEREMY, D. & WHYTE, H. M. (1962) Structural Renal Damage and its Relation to Clinical Features in Acute Oliguric Renal Failure. Quart. J. Med., 31, 429. FLAMENBAIJM, W., KOTCHEN, J. A., NAGLE, R. & McNEIL, J. S. (1973) The Effect of Potassium on the Renin-Angiotensin System and HgCl2induced Acute Renal Failure. Amer. J. Physiol., 224, 105. FLAMENBAUM, W. (1973) Pathophysiology of Acute Renal Failure. Arch. int. Med., 131, 911. GRITZKA, T. L. & TRUMP, B. F. (1968) Renal Tubular Lesions caused by Mercuric Chloride. Electron Microscopic Observations: Degeneration of the Pars Recta. Amer. J. Path., 52, 1225. JACOBSEN, N. 0. & JORGENSEN, F. (1973) Further Enzyme Histochemical Observations on the Segmentation of the Proximal Tubules in the Kidney of the Male Rat. Histochemie, 34, 11. KESSLER, R. H., LOZANO, R. & PITTS, R. F. (1957) A Comparison of the Pharmacological Behaviour of Chloromerodrin, Meralluride, Mersalyl and Mercuric Chloride in the Dog. J. Pharmacol. Exper. Therap., 121, 432. MARTINDALE: The Extra Pharmacopoeia ( 1972) N. WV. Blacow, Ed. 26th Edn. London: The Pharmaceutical Press. p. 1050. McDOWELL, E. M., NAGLE, R. B., ZALME, R. C., MCNEIL, J. S., FLAMENBAUM, W. & TRUMP, B. F. (1976) Studies on the Pathophysiology of Actute

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Renal Failure. I. Correlation of Ultrastructure and Function in the Proximal Tubule of the Rat following Administration of Mercuric Chloride. Virchows Arch. B. Cell Path., 22, 173. OLIVER, J., MAcDOWELL, M. & TRACY, A. (1951) The Pathogenesis of Acute Renal Failure associated with Traumatic and Toxic Injury. Renal Ischaemia, Nephrotoxic Damage and the Ischemuric Episode. J. Clin. Invest., 30, 1305. OLIVER, J. (1953) Correlations of Structure and Function and Mechanisms of Recovery in Early Tubular Necrosis. Amer. J. Med., 15, 537. REYNOLDS, E. S. (1963) The Use of Lead Citrate at high pH as an Electron-opaque Stain in Electron Microscopy. J. Cell. Biol., 17, 108. ROBBINS, S. L. (1974) Pathologic Basis of Disease. Philadelphia, London, Toronto: W. B. Saunders Company. RODIN, A. E. & CROWSON, C. N. (1962) Mercury Nephrotoxicity in the Rat. 2. Investigation of the Intracellular Site of Mercury Nephrotoxicity by Correlated Serial Time Histologic and Histoenzymatic Studies. Amer. J. Path., 41, 485.

SIEGEL, F. L. & BULGER, R. E. (1975) Scanning and Transmission Electron Microscopy of Mercuric Chloride-induced Acute Tubular Necrosis in the Rat Kidney. Virchows Arch. B. Cell Path., 18, 243. STEIN, J. H., LIFSCHITZ, M. D. & BARNES, L. D. (1978) Current Concepts on the Pathophysiology of Acute Renal Failure. Amer. J. Physiol., 234, F 171. TRUMP, B. F. & BULGER, R. E. (1968) Morphology of the Kidney. In Structural Basis of Renal Disease. E. Lovel Becker, Ed. New York, Evanston, London: Hoeber Medical Division. p. 1. WESTON, R. E. (1957) The Mode and Mechanisms of Mercuroid Diuresis in Normal Subjects and Oedematous Patients. Ann. New York Acad. Sci., 65, 576. ZALME, R. C., McDOWELL, E. M., NAGLE, R. B., MCNEIL, J. S., FLAMBENBAUM, W. & TRUMP, B. F. (1976) Studies on the Pathophysiology of Acute Renal Failure. II. A Histochemical Study of The Proximal Tubule of the Rat following Administration of Mercuric Chloride. Virchows Arch. B. Cell Path., 22, 197.