Br. J. exp. Path. (1975) 56, 495
HISTOCHEMICAL EXAMINATION OF LYSOSOMAL ENZYMES IN NECROTIC PROXIMAL RENAL TUBULES OF ALBINO RATS L. SZTRIHA, ZS. BOTI AND J. ORMOS From the Department of Pathology, University of Medicine, Szeged, Hungary Received for publication June 11, 1975
Summary.-The lysosomal enzymatic activity of the necrotic proximal tubules was examined by light microscopy and electron microscopy in 24- and 48-h focal renal cortical necrosis induced by administration of oestrogen and posterior pituitary extract in rats. Organelles exhibiting acid phosphatase activity can also be seen in the necrotic cells but these differ in size and structure from the lysosomes of normal cells. The cytoplasmic nonspecific esterase and thioacetic acid hydrolase activities decrease considerably or disappear, although some morphologically damaged, but active, lysosomes can be observed. The role of the lysosomal enzymes is seen not in the development of the necrosis but rather in the breaking down of the already necrotic cell constituents.
BIoCHEMICAL and histochemical studies have led to contradictory results as to the role and behaviour of the lysosomes and the lysosomal acid hydrolases in the course of necrobiosis. Certain examinations indicate that the necrosis is initiated by acid hydrolases released from the lysosomes (Brandes and Anton, 1966; De Duve and Beaufay, 1959; Van Lancker and Holtzer, 1959; Rene, Darden and Parker, 1971). Other authors consider the release of acid hydrolases to be secondary (Buckley, 1972; Goldblatt, Trump and Stowell, 1965; Griffin et al., 1965; Kerr, 1965; Trump, Goldblatt and Stowell, 1965). The majority of researches deal with the lysosomal activity observed in the early stage of the necrobiotic process. Little attention has been paid to the organelles exhibiting acid hydrolase positivity histochemically in the already necrotic cell. Our aim was the histochemical examination of acid phosphatase, thioacetic acid hydrolase and nonspecific esterase activities in 24- and 48-h necrotic proximal renal tubules, at light and electron microscopical level. According to Kovacs et al. (Kovatcs and DTavid 1963; Kovaics, Csernay and DaTvid, 1964; Kovacs et al., 1965) the 35
hormonal treatment applied by us leads, on an ischaemic basis, to focal necrosis of the proximal renal tubules. The histology and the histochemical changes relating to the oxidative enzymes and to alkaline phosphatase are discussed in detail by Horvtath and Kovacs (1968), while the ultrastructural description is given in our earlier papers (Ormos, Elemer and Csapo, 1973; Ormos and Viragh, 1965). MATERIALS AND METHODS
The experiments were performed on 10 male Wistar albino rats, weighing 160-200 g. For 10 days a daily dose of 1 mg oestrogen (oestrone acetate; Hogival, Chinoin) was administered subcutaneously, the injection on the 10th day being accompanied by 10 i.u. (1 ml) posterior pituitary extract (Glanduitrin, Kobanya Pharmaceutical Works). The animals were divided into two groups of 5, in which the kidneys were removed under i.p. hexobarbitone anaesthesia 24 and 48 h respectively after the final injection. Small pieces (1 mm3) were fixed by immersion for 4 h in 3% glutaraldehyde (cacodylate buffer pH 7.2), and then postfixed for 1 h in 1% buffered (pH 7.2) osmium tetroxide. After alcoholic dehydration and embedding in Durcupan ACM, sections prepared with an LKB Ultratome I were contrasted with uranyl acetate and lead citrate (Reynolds, 1963), and then examined with SEM 3-1 and JEM-JEOL 100 B electron microscopes.
496
L. SZTRIHA, ZS. BOTI AND J. ORMOS
Histochemically, the acid phosphatase was detected both light and electron microscopically by the Ericsson and Trump (1965) modification of the G6m6ri method. The thioacetic acid hydrolase was examined with the electron microscope by the method of Bell and Barnett (1965). In the case of both enzyme reactions, after incubation and osmium tetroxide postfixation the material was prepa-red for electron
microscopy as above. For light microscopical detection of the nonspecific esterase, oc-naphthyl acetate was used as substrate and hexazotized pararosaniline as coupling reagent, according to Barka and Anderson (1963). A substrate free incubation solution was employed for purposes of control examinations in all 3 reactions. The histochemical examinations were also carried out on kidneys from untreated rats.
FIG. 1.-Acid phosphatase active lysosomes of a normal proximal tubular cell. G6m6ri's method. x 17,400. FIG. 2.-Thioacetic acid hydrolase activity of a normal proximal tubular cell. Lead sulphide precipitate is localized mainly in lysosomes (L), but it can be seen along the membranes of the cristae of mitochondria (arrowhead) and those of the microvilli as well. Bell and Barrnett's method. x 12,275.
HISTOCHEMICAL EXAMINATION OF LYSOSOMAL ENZYMES
497
:, I
T-. .;
*.
I
4'
-1,.
.*rl .4: # .
FIG. 3.-24-b necrosis. Acid phosphatase, Gom6ri's method. In some of the necrotic tubules the activity has disappeared (T); in other places active granules can be seen in the basal remnants of cells (arrowhead) and in the debris filling the lumen (D). x 245. FIG. 4.-24-h necrosis. Among the remnants of organelles active formation measuring 2 ,um, which contains membranous material and dense granules can be seen. Acid phosphatase, Gomori's method. x 8175. FIG. 5.-48-h necrosis. Active lysosomes containing numerous myelin figures from a cell having contained disintegrated organelles. Acid phosphatase, Gomori's method. x 13,625. FIG. 6. 24-h necrosis. In the debris filling the lumen active formation can be seen (arrowhead). There is a distinct localization of the reaction product. Acid phosphatase, G6m6ri's method. x 8175.
498
L. SZTRIHA, ZS. BOTI AND J. ORMOS
FIa. 7-8.- 24-h necrosis. Active organelles (presumably remnants of lysosomes (arrowhead)) can be found among the disintegrated organelles. The activity of other membranes is strikingly decreased. Thioacetic acid hydrolase, Bell and Barrnett's method. 7. x 16,650. 8. x 14,150. FIG. 9.-48-h necrosis. Nonspecific esterase. The diffuse plasmatic staining of the remnants of tubular epithelium and that of the debris filling the lumen are decreased and thus the granular activity is more visible (asterisk). On the right side of the picture normal tubules and part of a glomerulus (G) are situated. x 330.
HISTOCHEMICAL EXAMINATION OF LYSOSOMAL ENZYMES RESULTS
Histochemistry of normal proximal renal tubules Light microscopically, the acid phosphatase activity appears mainly in the form of granules situated on the basal half of the cells. With the electron microscope it is obvious that the acitivity is localized on the lysosomes (Fig. 1). The thioacetic acid hydrolase activity is most marked in the lysosomes but it also appears in the form of small, electron dense granules along the membranes of the mitochondria, the endoplasmic reticulum, the Golgi zone and the microvilli (Fig. 2). Without the use of various inhibitors, nonspecific esterase activity can be seen in the form of diffuse plasmatic staining (Fig. 9). E600 (diethyl p-nitrophenyl phosphate) inhibits the cytoplasmic reaction (inhibitor sensitive esterase), and thereby the lysosomal staining (inhibitor resistant esterase) becomes visible. Histochemical changes in the necrotic proximal renal tubules Acid phosphatase activity cannot be observed with the light microscope in a considerable proportion of the necrotic tubules. However, many necrotic tubules can be seen which contain granules indicative of activity. These vary in size and there are fewer of them than in the normal proximal tubules. Some of the granules are found in necrotic cells which have not yet separated from the basement membrane, while others are situated in the detritus filling the lumen (Fig. 3). Electron microscopically, acid phosphatase positive formations can be observed in the necrotic cells and in the debris in the lumen. Many of them morphological exhibit characteristic features. Some of them are about 2 ,tm in size and thus exceed the average size of lysosomes. They contain granules of different size and circular membranous figures with various forms (Fig. 4).
499
Others measure about 1 ,tm and contain a number of myelin figures (Fig. 5). In the debris in the lumen the acid phosphatase positivity appears in very varied forms but always seems to be bound to some structures (Fig. 6). The thioacetic acid hydrolase activity is significantly decreased along the disintegrated membranes in the necrotic cells and has mostly disappeared. Some morphologically now barely identifiable organelles, however, do exhibit activity. Based on the enzymatic activity, these must be regarded as residues of the lysosomes (Fig. 7-8). The nonspecific esterase activity decreases in the necrotic cells, but does not disappear. The cytoplasmic staining exhibits a considerable decrease and in many places permits observation of lysosomal activity in the form of granules. The debris accumulating in the lumen is also active (Fig. 9). Casts exhibiting activity are visible in the medulla. DISCUSSION
Biochemical examinations have demonstrated the increase of the unsedimentable acid phosphatase activity in hepatic tissue in the early stages of in vivo ischaemia (De Duve and Beaufay, 1959) and in vitro autolysis (Van Lancker and Holtzer, 1959). These examinations gave rise to the- hypothesis that the release of lysosomal hydrolases initiates the necrosis. However, electron microscopic, histochemical and biochemical examinations of autolysing hepatic cells did not support this assumption (Goldblatt et al., 1965; Griffin et al., 1965; Kerr, 1965; Trump et al., 1965). Increase of unsedimentable acid phosphatase activity in the early stages of the autolysis occurs because the lysosome membrane, having become fragile in the course of the damage, is less resistant to the homogenization. Trump et al. (1965) and Goldblatt et al. (1965) consider that the lysosomal enzymes are released in the later stages of the necrobiotic process and play a part in the
500
L. SZTRIHA, ZS. B6TI AND J. ORMOS
necrosis. Histochemically, it was not possible to detect acid phosphatase activity with either a light microscope or an electron microscope 8 h after the commencement of autolysis. In contrast, acid hydrolase positive organelles can be seen in the 24- and 48-h necrotized cells under our experimental conditions. Our observations are in accord with the findings that, of the various organelles, it is the lysosomes which morphologically and functionally undergo damage the latest on the action of in vivo ischaemia (Bassi and Bernelli-Zazzera, 1964) or of heat (Buckley, 1972). Following a light microscopic histochemical examination of renal tubular necrosis induced by sublimate, Taylor (1965) suggests that the lysosomes may play a detoxicating role in the necrobiosis. The acid phosphatase positive organelle seen in Fig. 4 is morphologically similar to that "lesion " which develops in the acinar cell of the ethionine damaged pancreas (Hovie et al., 1971). Those organelles which are produced from the autophagic vacuoles during the digestive process are of a similar structure (Ericsson, 1969; Ericsson, Trump, and Weibel, 1965). The appearance of myelin figures, too, is indicative of increased catabolic activity (Daems, Wisse and Brederoo, 1969). Thus, it cannot be excluded that the lysosomal acid hydrolases have a role in a certain stage of the necrobiotic process in the breaking down of the previously necrotic cell constituents, and that their detoxicating function is manifested in such a form. REFERENCES BARKA, T. & ANDERSON, P. J. (1963) Histochemistry: Theory, Practice, and Bibliography. New York: Harper-Row. BASSI, M. & BERNELLI-ZAZZERA, A. (1964) Ultrastructural Cytoplasmic Changes of Liver Cells after Reversible and Irreversible Ischaemia. Expl molec Pathol., 3, 332. BELL, M. & BARRNETT, R. J. (1965) The Use of Thiolsubstituted Carboxylic Acids as Histochemical Substrates. J. Hi8tochem. Cytochem., 13, 611. BRANDES, D. & ANTON, E. (1966) The Role of Lysosomes in Cellular Lytic Processes. III. Electron
Histochemical Changes in Mammary Tumors after Treatment with Cytoxan and Vitamin A. Lab. Invest., 15, 987. BUCKLEY, I. K. (1972) A Light and Electron Microscopic Study of Thermally Injured Cultured Cells. Lab. Invest., 26, 201. DAEMS, W. TH., WIssE, E. & BREDEROO, P. (1969) Electron Microscopy of the Vacuolar Apparatus. In Lysosomes in Biology and Pathology. Eds. J. T. Dingle and H. B. Fell. Amsterdam: North-Holland. DE DUVE, C. & BEAUFAY, H. (1959) Tissue Fractionation studies. 10. Influence of Ischaemia on the State of Some Bound Enzymes in Rat Liver. Biochemn. J., 73, 610. ERICSSON, J. L. E. (1969) Mechanism of Cellular Autophagy. In Lysosomes in Biology and Pathology. Eds. J. T. Dingle and H. B. Fell. Amsterdam: North-Holland. ERICSSON, J. L. E. & TRUMP, B. F. (1965) Observations on the Application of Electronmicroscopy of the Lead Phosphate Technique for the Demonstration of Acid Phosphatase. Histochemistry, 4, 470. ERICSSON, J. L. E., TRUMP. B. F. & WEIBEL, J. (1965) Electron Microscopic Studies of the Proximal Tubule of the Rat Kidney. II. Cytosegresomes and Cytosomes: their Relationship to Each Other and to the Lysosome Concept. Lab. Invest., 14, 1341. GOLDBLATT, P. J., TRUMP, B. F. & STOWELL, R. E. (1965) Studies on Necrosis of Mouse Liver in vitro. Alterations in Some Histochemically Demonstrable Hepatocellular Enzymes. Am. J. Path., 47, 183. GRIFFIN, C. S., WARAVDEKAR, V. S., TRUMP, B. F., GOLDBLATT, P. J. & STOWELL, R. E. (1965) Studies on Necrosis of Mouse Liver in vitro. Alterations in Activities of Succinoxidase, Succinic Dehydrogenase, Glutamic Dehydrogenase, Acid Phosphatase, Uricase, Glucose-6-phosphatase, and NAD-pyrophosphorylase. Am. J. Path., 47, 833. HoVIE, A., TAKINO, T., HERMAN, L. & FITZGERALD, P. J. (1971) Pancreas Acinar Cell Regeneration. VIII. Relationship of Acid Phosphatase and fi-glucuronidase to Intracellular Organelles and Ethionine Lesions. Am. J. Path., 63, 299. HORVATH, P. & KovA_Cs, K. (1968) Histochemische Untersuchungen der hormonal induzierten Nierenrindennekrose. Path. europ., 3, 27. KERR, J. F. R. (1965) A Histochemical Study of Hypertrophy and Ischaemic Injury of Rat Liver with Special Reference to Changes in Lysosomes. J. Path. Bact., 90, 419. KovAcs, K., CSERNAY, L. & DAVID, M. A. (1964) Effect of Oestrone on the Renal Response to Posterior Pituitary Extract on Rats. Naturwissenschaften, 51, 64. Kovics, K. & DAVID, M. A. (1963) Effect of Corticotrophin on the Renal Response to Posterior Pituitary Extracts in Rats. Lancet, ii, 417. KovAcs, K., LASZL6, F. A., S6vEiNYI, E. & KoCsIs, (1965) Angiorenographic Studies in Living Rats during the Development of Renal Cortical Necrosis. Br. J. Radiol., 38, 148. VAN LANCKER, J. L. & HOLTZER, R. L. (1959) The Release of Acid Phosphatase and B-glucuronidase from Cytoplasmic Granules in the Early Course of Autolysis. Am. J. Path., 35, 563. ORMos, J. ELEMAR, G. & CsAP6, Zs. (1973) Ultrastructure of the Proximal Convoluted Tubul s
HISTOCHEMICAL EXAMINATION OF LYSOSOMAL ENZYMES during Repair following Hormonally Induced Necrosis in Rat Kidney. Virchows. Arch. Abt. B. Zellpath., 13, 1. ORMOS, J. & VIRAGH, SZ. ( 1965) Das elektronenmikroskopische Bild der experimentellen Nierenrindennekrose. Verh. dt. Ges. Path., 49, 146. RENA, A. A., DARDEN, J. H. & PARKER, J. L. (1971) Radiation induced Ultrastructural and Biochemical Changes in Lysosomes. Lab. Invest., 25, 230. REYNOLDS, E. S. (1963) The Use of Lead Citrate at
501
High pH as an Electronopaque Strain in Electron Microscopy. J. cell Biol., 17, 208. TAYLOR, N. S. (1965) Histochemical Studies of Nephrotoxicity with Sublethal Doses of Mercury in Rats. Am. J. Path., 46, 1. TRUMP, B. F., GOLDBLATT, P. J. & STOWELL, R. E. (1965) Studies of Necrosis in vitro of Mouse Hepatic Parenchimal Cells. Ultrastructural and Cytochemical Alteration of Cytosomes, Cytosegresomes, Multivesicular bodies, and their Relation to the Lysosome Concept. Lab. Invest., 14, 1946.
HISTOCHEMICAL EXAMINATION OF LYSOSOMAL ENZYMES IN NECROTIC PROXIMAL RENAL TUBULES OF ALBINO RATS L. SZTRIHA, ZS. BOTI AND J. ORMOS From the Department of Pathology, University of Medicine, Szeged, Hungary Received for publication June 11, 1975
Summary.-The lysosomal enzymatic activity of the necrotic proximal tubules was examined by light microscopy and electron microscopy in 24- and 48-h focal renal cortical necrosis induced by administration of oestrogen and posterior pituitary extract in rats. Organelles exhibiting acid phosphatase activity can also be seen in the necrotic cells but these differ in size and structure from the lysosomes of normal cells. The cytoplasmic nonspecific esterase and thioacetic acid hydrolase activities decrease considerably or disappear, although some morphologically damaged, but active, lysosomes can be observed. The role of the lysosomal enzymes is seen not in the development of the necrosis but rather in the breaking down of the already necrotic cell constituents.
BIoCHEMICAL and histochemical studies have led to contradictory results as to the role and behaviour of the lysosomes and the lysosomal acid hydrolases in the course of necrobiosis. Certain examinations indicate that the necrosis is initiated by acid hydrolases released from the lysosomes (Brandes and Anton, 1966; De Duve and Beaufay, 1959; Van Lancker and Holtzer, 1959; Rene, Darden and Parker, 1971). Other authors consider the release of acid hydrolases to be secondary (Buckley, 1972; Goldblatt, Trump and Stowell, 1965; Griffin et al., 1965; Kerr, 1965; Trump, Goldblatt and Stowell, 1965). The majority of researches deal with the lysosomal activity observed in the early stage of the necrobiotic process. Little attention has been paid to the organelles exhibiting acid hydrolase positivity histochemically in the already necrotic cell. Our aim was the histochemical examination of acid phosphatase, thioacetic acid hydrolase and nonspecific esterase activities in 24- and 48-h necrotic proximal renal tubules, at light and electron microscopical level. According to Kovacs et al. (Kovatcs and DTavid 1963; Kovaics, Csernay and DaTvid, 1964; Kovacs et al., 1965) the 35
hormonal treatment applied by us leads, on an ischaemic basis, to focal necrosis of the proximal renal tubules. The histology and the histochemical changes relating to the oxidative enzymes and to alkaline phosphatase are discussed in detail by Horvtath and Kovacs (1968), while the ultrastructural description is given in our earlier papers (Ormos, Elemer and Csapo, 1973; Ormos and Viragh, 1965). MATERIALS AND METHODS
The experiments were performed on 10 male Wistar albino rats, weighing 160-200 g. For 10 days a daily dose of 1 mg oestrogen (oestrone acetate; Hogival, Chinoin) was administered subcutaneously, the injection on the 10th day being accompanied by 10 i.u. (1 ml) posterior pituitary extract (Glanduitrin, Kobanya Pharmaceutical Works). The animals were divided into two groups of 5, in which the kidneys were removed under i.p. hexobarbitone anaesthesia 24 and 48 h respectively after the final injection. Small pieces (1 mm3) were fixed by immersion for 4 h in 3% glutaraldehyde (cacodylate buffer pH 7.2), and then postfixed for 1 h in 1% buffered (pH 7.2) osmium tetroxide. After alcoholic dehydration and embedding in Durcupan ACM, sections prepared with an LKB Ultratome I were contrasted with uranyl acetate and lead citrate (Reynolds, 1963), and then examined with SEM 3-1 and JEM-JEOL 100 B electron microscopes.
496
L. SZTRIHA, ZS. BOTI AND J. ORMOS
Histochemically, the acid phosphatase was detected both light and electron microscopically by the Ericsson and Trump (1965) modification of the G6m6ri method. The thioacetic acid hydrolase was examined with the electron microscope by the method of Bell and Barnett (1965). In the case of both enzyme reactions, after incubation and osmium tetroxide postfixation the material was prepa-red for electron
microscopy as above. For light microscopical detection of the nonspecific esterase, oc-naphthyl acetate was used as substrate and hexazotized pararosaniline as coupling reagent, according to Barka and Anderson (1963). A substrate free incubation solution was employed for purposes of control examinations in all 3 reactions. The histochemical examinations were also carried out on kidneys from untreated rats.
FIG. 1.-Acid phosphatase active lysosomes of a normal proximal tubular cell. G6m6ri's method. x 17,400. FIG. 2.-Thioacetic acid hydrolase activity of a normal proximal tubular cell. Lead sulphide precipitate is localized mainly in lysosomes (L), but it can be seen along the membranes of the cristae of mitochondria (arrowhead) and those of the microvilli as well. Bell and Barrnett's method. x 12,275.
HISTOCHEMICAL EXAMINATION OF LYSOSOMAL ENZYMES
497
:, I
T-. .;
*.
I
4'
-1,.
.*rl .4: # .
FIG. 3.-24-b necrosis. Acid phosphatase, Gom6ri's method. In some of the necrotic tubules the activity has disappeared (T); in other places active granules can be seen in the basal remnants of cells (arrowhead) and in the debris filling the lumen (D). x 245. FIG. 4.-24-h necrosis. Among the remnants of organelles active formation measuring 2 ,um, which contains membranous material and dense granules can be seen. Acid phosphatase, Gomori's method. x 8175. FIG. 5.-48-h necrosis. Active lysosomes containing numerous myelin figures from a cell having contained disintegrated organelles. Acid phosphatase, Gomori's method. x 13,625. FIG. 6. 24-h necrosis. In the debris filling the lumen active formation can be seen (arrowhead). There is a distinct localization of the reaction product. Acid phosphatase, G6m6ri's method. x 8175.
498
L. SZTRIHA, ZS. BOTI AND J. ORMOS
FIa. 7-8.- 24-h necrosis. Active organelles (presumably remnants of lysosomes (arrowhead)) can be found among the disintegrated organelles. The activity of other membranes is strikingly decreased. Thioacetic acid hydrolase, Bell and Barrnett's method. 7. x 16,650. 8. x 14,150. FIG. 9.-48-h necrosis. Nonspecific esterase. The diffuse plasmatic staining of the remnants of tubular epithelium and that of the debris filling the lumen are decreased and thus the granular activity is more visible (asterisk). On the right side of the picture normal tubules and part of a glomerulus (G) are situated. x 330.
HISTOCHEMICAL EXAMINATION OF LYSOSOMAL ENZYMES RESULTS
Histochemistry of normal proximal renal tubules Light microscopically, the acid phosphatase activity appears mainly in the form of granules situated on the basal half of the cells. With the electron microscope it is obvious that the acitivity is localized on the lysosomes (Fig. 1). The thioacetic acid hydrolase activity is most marked in the lysosomes but it also appears in the form of small, electron dense granules along the membranes of the mitochondria, the endoplasmic reticulum, the Golgi zone and the microvilli (Fig. 2). Without the use of various inhibitors, nonspecific esterase activity can be seen in the form of diffuse plasmatic staining (Fig. 9). E600 (diethyl p-nitrophenyl phosphate) inhibits the cytoplasmic reaction (inhibitor sensitive esterase), and thereby the lysosomal staining (inhibitor resistant esterase) becomes visible. Histochemical changes in the necrotic proximal renal tubules Acid phosphatase activity cannot be observed with the light microscope in a considerable proportion of the necrotic tubules. However, many necrotic tubules can be seen which contain granules indicative of activity. These vary in size and there are fewer of them than in the normal proximal tubules. Some of the granules are found in necrotic cells which have not yet separated from the basement membrane, while others are situated in the detritus filling the lumen (Fig. 3). Electron microscopically, acid phosphatase positive formations can be observed in the necrotic cells and in the debris in the lumen. Many of them morphological exhibit characteristic features. Some of them are about 2 ,tm in size and thus exceed the average size of lysosomes. They contain granules of different size and circular membranous figures with various forms (Fig. 4).
499
Others measure about 1 ,tm and contain a number of myelin figures (Fig. 5). In the debris in the lumen the acid phosphatase positivity appears in very varied forms but always seems to be bound to some structures (Fig. 6). The thioacetic acid hydrolase activity is significantly decreased along the disintegrated membranes in the necrotic cells and has mostly disappeared. Some morphologically now barely identifiable organelles, however, do exhibit activity. Based on the enzymatic activity, these must be regarded as residues of the lysosomes (Fig. 7-8). The nonspecific esterase activity decreases in the necrotic cells, but does not disappear. The cytoplasmic staining exhibits a considerable decrease and in many places permits observation of lysosomal activity in the form of granules. The debris accumulating in the lumen is also active (Fig. 9). Casts exhibiting activity are visible in the medulla. DISCUSSION
Biochemical examinations have demonstrated the increase of the unsedimentable acid phosphatase activity in hepatic tissue in the early stages of in vivo ischaemia (De Duve and Beaufay, 1959) and in vitro autolysis (Van Lancker and Holtzer, 1959). These examinations gave rise to the- hypothesis that the release of lysosomal hydrolases initiates the necrosis. However, electron microscopic, histochemical and biochemical examinations of autolysing hepatic cells did not support this assumption (Goldblatt et al., 1965; Griffin et al., 1965; Kerr, 1965; Trump et al., 1965). Increase of unsedimentable acid phosphatase activity in the early stages of the autolysis occurs because the lysosome membrane, having become fragile in the course of the damage, is less resistant to the homogenization. Trump et al. (1965) and Goldblatt et al. (1965) consider that the lysosomal enzymes are released in the later stages of the necrobiotic process and play a part in the
500
L. SZTRIHA, ZS. B6TI AND J. ORMOS
necrosis. Histochemically, it was not possible to detect acid phosphatase activity with either a light microscope or an electron microscope 8 h after the commencement of autolysis. In contrast, acid hydrolase positive organelles can be seen in the 24- and 48-h necrotized cells under our experimental conditions. Our observations are in accord with the findings that, of the various organelles, it is the lysosomes which morphologically and functionally undergo damage the latest on the action of in vivo ischaemia (Bassi and Bernelli-Zazzera, 1964) or of heat (Buckley, 1972). Following a light microscopic histochemical examination of renal tubular necrosis induced by sublimate, Taylor (1965) suggests that the lysosomes may play a detoxicating role in the necrobiosis. The acid phosphatase positive organelle seen in Fig. 4 is morphologically similar to that "lesion " which develops in the acinar cell of the ethionine damaged pancreas (Hovie et al., 1971). Those organelles which are produced from the autophagic vacuoles during the digestive process are of a similar structure (Ericsson, 1969; Ericsson, Trump, and Weibel, 1965). The appearance of myelin figures, too, is indicative of increased catabolic activity (Daems, Wisse and Brederoo, 1969). Thus, it cannot be excluded that the lysosomal acid hydrolases have a role in a certain stage of the necrobiotic process in the breaking down of the previously necrotic cell constituents, and that their detoxicating function is manifested in such a form. REFERENCES BARKA, T. & ANDERSON, P. J. (1963) Histochemistry: Theory, Practice, and Bibliography. New York: Harper-Row. BASSI, M. & BERNELLI-ZAZZERA, A. (1964) Ultrastructural Cytoplasmic Changes of Liver Cells after Reversible and Irreversible Ischaemia. Expl molec Pathol., 3, 332. BELL, M. & BARRNETT, R. J. (1965) The Use of Thiolsubstituted Carboxylic Acids as Histochemical Substrates. J. Hi8tochem. Cytochem., 13, 611. BRANDES, D. & ANTON, E. (1966) The Role of Lysosomes in Cellular Lytic Processes. III. Electron
Histochemical Changes in Mammary Tumors after Treatment with Cytoxan and Vitamin A. Lab. Invest., 15, 987. BUCKLEY, I. K. (1972) A Light and Electron Microscopic Study of Thermally Injured Cultured Cells. Lab. Invest., 26, 201. DAEMS, W. TH., WIssE, E. & BREDEROO, P. (1969) Electron Microscopy of the Vacuolar Apparatus. In Lysosomes in Biology and Pathology. Eds. J. T. Dingle and H. B. Fell. Amsterdam: North-Holland. DE DUVE, C. & BEAUFAY, H. (1959) Tissue Fractionation studies. 10. Influence of Ischaemia on the State of Some Bound Enzymes in Rat Liver. Biochemn. J., 73, 610. ERICSSON, J. L. E. (1969) Mechanism of Cellular Autophagy. In Lysosomes in Biology and Pathology. Eds. J. T. Dingle and H. B. Fell. Amsterdam: North-Holland. ERICSSON, J. L. E. & TRUMP, B. F. (1965) Observations on the Application of Electronmicroscopy of the Lead Phosphate Technique for the Demonstration of Acid Phosphatase. Histochemistry, 4, 470. ERICSSON, J. L. E., TRUMP. B. F. & WEIBEL, J. (1965) Electron Microscopic Studies of the Proximal Tubule of the Rat Kidney. II. Cytosegresomes and Cytosomes: their Relationship to Each Other and to the Lysosome Concept. Lab. Invest., 14, 1341. GOLDBLATT, P. J., TRUMP, B. F. & STOWELL, R. E. (1965) Studies on Necrosis of Mouse Liver in vitro. Alterations in Some Histochemically Demonstrable Hepatocellular Enzymes. Am. J. Path., 47, 183. GRIFFIN, C. S., WARAVDEKAR, V. S., TRUMP, B. F., GOLDBLATT, P. J. & STOWELL, R. E. (1965) Studies on Necrosis of Mouse Liver in vitro. Alterations in Activities of Succinoxidase, Succinic Dehydrogenase, Glutamic Dehydrogenase, Acid Phosphatase, Uricase, Glucose-6-phosphatase, and NAD-pyrophosphorylase. Am. J. Path., 47, 833. HoVIE, A., TAKINO, T., HERMAN, L. & FITZGERALD, P. J. (1971) Pancreas Acinar Cell Regeneration. VIII. Relationship of Acid Phosphatase and fi-glucuronidase to Intracellular Organelles and Ethionine Lesions. Am. J. Path., 63, 299. HORVATH, P. & KovA_Cs, K. (1968) Histochemische Untersuchungen der hormonal induzierten Nierenrindennekrose. Path. europ., 3, 27. KERR, J. F. R. (1965) A Histochemical Study of Hypertrophy and Ischaemic Injury of Rat Liver with Special Reference to Changes in Lysosomes. J. Path. Bact., 90, 419. KovAcs, K., CSERNAY, L. & DAVID, M. A. (1964) Effect of Oestrone on the Renal Response to Posterior Pituitary Extract on Rats. Naturwissenschaften, 51, 64. Kovics, K. & DAVID, M. A. (1963) Effect of Corticotrophin on the Renal Response to Posterior Pituitary Extracts in Rats. Lancet, ii, 417. KovAcs, K., LASZL6, F. A., S6vEiNYI, E. & KoCsIs, (1965) Angiorenographic Studies in Living Rats during the Development of Renal Cortical Necrosis. Br. J. Radiol., 38, 148. VAN LANCKER, J. L. & HOLTZER, R. L. (1959) The Release of Acid Phosphatase and B-glucuronidase from Cytoplasmic Granules in the Early Course of Autolysis. Am. J. Path., 35, 563. ORMos, J. ELEMAR, G. & CsAP6, Zs. (1973) Ultrastructure of the Proximal Convoluted Tubul s
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