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Electron microscopy in diagnostic and experimental renal pathology
186
ROYAL COLLEGE O F PATHOLOGISTS OF AUSTRALIA
Pathology (1978), 10, April
Vitamin D is activated in the bod) b) a two-step process involving 25-hydroxylation in the liver and 1hydroxylation in the kidney to form l.X(OH),D,. Low levels of this metabolite lead to impaired calcium absorption. osteomalacia and secondary hyperparathyroidism. In individual patients either osteomalacia or hyperparathyroid bone disease (osteitis fibrosa cystica) may dominate the clinical, biochemical, radiological and histological pictures. although most patients show evidence of both. I f secondary hyperparathlroidism is severe. serum C a may be normal or elevated. the latter being termed tertiary hyperparathyroidism although this is misleading as the parathyroid tissue remains responsive to serum Ca concentration. Another problem associated with secondary hyperparathyroidisin is ectopic calcification, which is particularly troublesome ifit involves the walls ofarteries. when it can lead to ischaemic lesions, or the kidney when it ma) result in further impairment of renal function. The main treatment consists of prevention of hyperphosphataemia which indirectly causes parathyroid stimulation. judicious use of vitamin D or its metabolites in hypocalcaemia. osteomalacia, or osteitis fibrosa without hypercalcaemia. and subtotal parathyroidectomq wahere necessary for severe hypercalcaemia, ectopic calcification. or osteitis fibrosa with hypercalcaemia. PLASTIC EMBEDDING TECHNIQUES IN THE STUDY PATHOLOGY IN RENAL DISEASE
OF RENAL BIOPSIES AND BONE
XIP~LL J.. M. Depurrnirnr of Atiaroinic~alpa rho log^.. A ustin Hospital. Melbourne The need for better correlation between light and electron microscopic studies of glomerular morphology has necessitated a search for methods beyond traditional paraffin section techniques. Although the use of plastic embedding for light microscopy appeared a logical solution. poor stain penetration and prolonged processing has hindered the introduction of such techniques into service laboratories. The development of low viscosity epoxy resins mith improved powers of penetration and short polymerization times has solved these difficulties. Methods for staining thin (0.4--1 p n ) sections with haematoxylin and eosin. silver methenamine, trichromes and Congo red techniques in addition to the basic aniline dyes used by electron microscopists, have been developed. Slight changes in basement membranes and mesangial matrix and in the various cellular components of the glomerulus can be clear11 detined and deposits may be recognized. Immunolluorescence and electron microscopy have been used to confirm the validity of the method. With modifications, similar techniques can be applied to undecalcified bone in the study of bone changes in renal failure. Processing does not interfere with the use of tetracycline labels as markers and the preparations are suitable for quantitative bone histolog). Bone changes are invariably present in chronic renal failure. Three main components in various combinations, ma!, be defined.Osteitis fibrosa is commonly seen. osteomalacia occurs less frequently and changes in bone mass, osteosclerosis o r osteoporosis. may also be found. Although in general mild and asymptomatic, renal bone disease has become a major problem in patients treated by long-term haemodialysis as these basic patterns of bone changes tend to increase in severity during long-term dialysis. ELECTRON MICROSCOPY IN DIAGNOSTIC AND EXPERIMENTAL RENAL PATHOLOGY
RYAS, C . B. DtJparttiienl of PulhokJgj.. Cnirersitx of Melboitrne. Purkville Although renal diseases can be diagnosed on the basis of light microscopy and immunofluorescence, electron microscopic examination of the renal biopsy probides additional information and. in some patients, is essential for a definitive diagnosis. Thus. EM is necessary for the detection of the extensive glomerular epithelial spreading characteristic of 'minimal change disease' and 'focal glomerulosclerosis and hyalinosis'. In addition, if immunofluorescence is unavailable (or if glomeruli are absent from the material allotted for immunofluorescence), EM is required for the detection of small. sparse subepithelial deposits in early membranous glomerulonephritis, and the irregular mesangial deposits in 'mesangial IgA disease'. Furthermore, ultrastructural studies of glomeruli commonl> aid or confirm the diagnosis of a variety of other conditions, including post-streptococcal ylomerulonephritis 'dense deposit disease'. early diabetic nephropathy. amyloidosis, systemic lupus erythematosus, hereditarl nephritis or gold nephropathy. It is unusual for any of these to be diagnosed on the basis of EM alone. and it is not suggested that EM can supplant light microscopy and immunofluorescence in these areas. In experimental studies, EM has provided information concerning the structure and function of the normal and diseased kidney. More precise analysis of the architecture of nephron components has become possible and the appearances and properties of individual cells can be more critically examined. Ultrastructural studies of the distribution of macromolecular tracers within the glomerulus have proved to be particularly fruitful in elucidating the mechanisms of proteinuria. Thus. it has recently been established that, during normal ultrafiltration across the
ABSTRACTS OF ANNUAL MEETING 1977
187
glomerular capillary wall, plasma proteins are prevented from passing into the urine by a functionally controlled barrier at the level of the endothelial fenestrae. This barrier depends upon the maintenance of normal blood flow; it breaks down, allowing proteinuria to occur, if an imbalance occurs between convective and diffusive forces across the wall. This can result from changes in blood flow or focal loss of the epithelial layer. CLINICOPATHOLOGICAL CONCEPTS AND THE TREATMENT OF GLOMERULAR DISEASE
KINCAID-SMITH, PRISCILLADepartment of Nephrology, Royal Melbourne Hospital, Melbourne While a morphological classification of glomerular disease is the only practical approach at the present time, it is essential in its use to recognize that whereas each biopsy can be fitted into a specific category, serial biopsies in the same patient may change from one category to another. The purpose of a renal biopsy is both in establishing the prognosis and as a guide to treatment. Discussion of recent advances in the understanding of the morphology of glomerular disease and the related clinical management will be confined in five types of lesions. Focal and segmental proliferative glomerulonephritis, focal and segmental hyalinosis and/or sclerosis, mesangiocapillary glomerulonephritis, diffuse endocapillary and extracapillary glomerulonephritis and lupus nephritis. Focal and segmental proliferative glomerulonephritis may or may not be associated with the syndrome of recurrent macroscopic haematuria and may or may not show associated mesangial IgA. Children frequently show minimal changes on light microscopy, whereas adults usually show focal and segmental proliferative lesions with or without crescents. Between active phases only focal and segmental sclerosis may be seen. Progression to renal failure may be observed in an adult whereas children usually have an excellent long-term outcome. The glomerular lesion of focal and segmental hyalinosis may also occur as a secondary phenomenon in various forms of scarring or sclerosis in the glomerulus. The primary form is usually accompanied by the nephrotic syndrome. About 70% of patients develop renal failure over 10 years thus the prognosis is poor. Mesangiocapillary glomerulonephritis has become much more clearly defined. Two distinct morphological types occur, one associated with dense deposits and the other with sub-endothelial deposits. In both the prognosis is poor. The greatest advance in treatment in recent years has been made in that of diffuse crescentic glomerulonephritis which has a very poor prognosis untreated. Dramatic results have been obtained using plasmapheresis. Lupus nephritis will be discussed as an example of a specific disease entity in which many different morphological varieties are seen.
4. VIRUSES IN CANCER
EVALUATION OF CURRENT EVIDENCE OF POSSIBLE ROLE OF VIRUSES IN HUMAN CANCER SABIN, ALBERT B. Medical University of’ South Carolina, Churleston. U.S.A. There is no question that a great variety of DNA viruses, including many strictly human infectious viruses, can transform normal cells to malignancy both in vitro and in vivo, and that a few special RNA animal viruses are the actual cause of some naturally occurring leukaemias in chickens, mice, cats and cattle and of mammary carcinoma in mice. However, despite the most extensive studies on these cancer-producing viruses there is at present no convincing direct or circumstantial evidence for an aetiological role of any virus in any human cancer. Renato Dulbecco discussing the problem ofevidence said: ‘finding a virus or a viral genome in a cancer has no aetiological connotation’. Macfarlane Burnet, in his 1974 book, Intrinsic Mutagenesis, concluded one chapter on human malignant disease with the colourful words; ‘there is no more place for viruses as causes of cancer than for lice as causes of poverty’. Yet even among some who have already arrived at such conclusions there is always hedging: maybe the evidence for the role of the Epstein-Barr virus as an aetiological co-factor in Burkitt’s lymphoma and nasopharyngeal carcinoma, or of HSV-2 (herpes simplex virus-type 2) in uterine cervical carcinoma, if not yet complete is close to being so. The nature of what should be considered as evidence varies for different viruses that deserve the most consideration in the search for understanding the complex aetiology of different human cancers. The following strictly human DNA viruses have been found to be oncogenic under special experimental conditions and are all known to remain (most likely as viral genomes) in certain tissues of many infected persons in a latent or repressed state with a varying frequency of reappearance as fully infectious virus: the adenoviruses, the SV,, (Simian Virus-40) related human BK and JC papovaviruses, and the herpes group of viruses, including herpes simplex (HSV types 1 and 2), cytomegalorirus, and EBV. The aetiology of malignancies experimentally produced by the adenoviruses and the SV,, group can be established by demonstrating: a. a virus-coded nonvirion, intranuclear tumour antigen for which the tumour-
ROYAL COLLEGE O F PATHOLOGISTS OF AUSTRALIA
Pathology (1978), 10, April
Vitamin D is activated in the bod) b) a two-step process involving 25-hydroxylation in the liver and 1hydroxylation in the kidney to form l.X(OH),D,. Low levels of this metabolite lead to impaired calcium absorption. osteomalacia and secondary hyperparathyroidism. In individual patients either osteomalacia or hyperparathyroid bone disease (osteitis fibrosa cystica) may dominate the clinical, biochemical, radiological and histological pictures. although most patients show evidence of both. I f secondary hyperparathlroidism is severe. serum C a may be normal or elevated. the latter being termed tertiary hyperparathyroidism although this is misleading as the parathyroid tissue remains responsive to serum Ca concentration. Another problem associated with secondary hyperparathyroidisin is ectopic calcification, which is particularly troublesome ifit involves the walls ofarteries. when it can lead to ischaemic lesions, or the kidney when it ma) result in further impairment of renal function. The main treatment consists of prevention of hyperphosphataemia which indirectly causes parathyroid stimulation. judicious use of vitamin D or its metabolites in hypocalcaemia. osteomalacia, or osteitis fibrosa without hypercalcaemia. and subtotal parathyroidectomq wahere necessary for severe hypercalcaemia, ectopic calcification. or osteitis fibrosa with hypercalcaemia. PLASTIC EMBEDDING TECHNIQUES IN THE STUDY PATHOLOGY IN RENAL DISEASE
OF RENAL BIOPSIES AND BONE
XIP~LL J.. M. Depurrnirnr of Atiaroinic~alpa rho log^.. A ustin Hospital. Melbourne The need for better correlation between light and electron microscopic studies of glomerular morphology has necessitated a search for methods beyond traditional paraffin section techniques. Although the use of plastic embedding for light microscopy appeared a logical solution. poor stain penetration and prolonged processing has hindered the introduction of such techniques into service laboratories. The development of low viscosity epoxy resins mith improved powers of penetration and short polymerization times has solved these difficulties. Methods for staining thin (0.4--1 p n ) sections with haematoxylin and eosin. silver methenamine, trichromes and Congo red techniques in addition to the basic aniline dyes used by electron microscopists, have been developed. Slight changes in basement membranes and mesangial matrix and in the various cellular components of the glomerulus can be clear11 detined and deposits may be recognized. Immunolluorescence and electron microscopy have been used to confirm the validity of the method. With modifications, similar techniques can be applied to undecalcified bone in the study of bone changes in renal failure. Processing does not interfere with the use of tetracycline labels as markers and the preparations are suitable for quantitative bone histolog). Bone changes are invariably present in chronic renal failure. Three main components in various combinations, ma!, be defined.Osteitis fibrosa is commonly seen. osteomalacia occurs less frequently and changes in bone mass, osteosclerosis o r osteoporosis. may also be found. Although in general mild and asymptomatic, renal bone disease has become a major problem in patients treated by long-term haemodialysis as these basic patterns of bone changes tend to increase in severity during long-term dialysis. ELECTRON MICROSCOPY IN DIAGNOSTIC AND EXPERIMENTAL RENAL PATHOLOGY
RYAS, C . B. DtJparttiienl of PulhokJgj.. Cnirersitx of Melboitrne. Purkville Although renal diseases can be diagnosed on the basis of light microscopy and immunofluorescence, electron microscopic examination of the renal biopsy probides additional information and. in some patients, is essential for a definitive diagnosis. Thus. EM is necessary for the detection of the extensive glomerular epithelial spreading characteristic of 'minimal change disease' and 'focal glomerulosclerosis and hyalinosis'. In addition, if immunofluorescence is unavailable (or if glomeruli are absent from the material allotted for immunofluorescence), EM is required for the detection of small. sparse subepithelial deposits in early membranous glomerulonephritis, and the irregular mesangial deposits in 'mesangial IgA disease'. Furthermore, ultrastructural studies of glomeruli commonl> aid or confirm the diagnosis of a variety of other conditions, including post-streptococcal ylomerulonephritis 'dense deposit disease'. early diabetic nephropathy. amyloidosis, systemic lupus erythematosus, hereditarl nephritis or gold nephropathy. It is unusual for any of these to be diagnosed on the basis of EM alone. and it is not suggested that EM can supplant light microscopy and immunofluorescence in these areas. In experimental studies, EM has provided information concerning the structure and function of the normal and diseased kidney. More precise analysis of the architecture of nephron components has become possible and the appearances and properties of individual cells can be more critically examined. Ultrastructural studies of the distribution of macromolecular tracers within the glomerulus have proved to be particularly fruitful in elucidating the mechanisms of proteinuria. Thus. it has recently been established that, during normal ultrafiltration across the
ABSTRACTS OF ANNUAL MEETING 1977
187
glomerular capillary wall, plasma proteins are prevented from passing into the urine by a functionally controlled barrier at the level of the endothelial fenestrae. This barrier depends upon the maintenance of normal blood flow; it breaks down, allowing proteinuria to occur, if an imbalance occurs between convective and diffusive forces across the wall. This can result from changes in blood flow or focal loss of the epithelial layer. CLINICOPATHOLOGICAL CONCEPTS AND THE TREATMENT OF GLOMERULAR DISEASE
KINCAID-SMITH, PRISCILLADepartment of Nephrology, Royal Melbourne Hospital, Melbourne While a morphological classification of glomerular disease is the only practical approach at the present time, it is essential in its use to recognize that whereas each biopsy can be fitted into a specific category, serial biopsies in the same patient may change from one category to another. The purpose of a renal biopsy is both in establishing the prognosis and as a guide to treatment. Discussion of recent advances in the understanding of the morphology of glomerular disease and the related clinical management will be confined in five types of lesions. Focal and segmental proliferative glomerulonephritis, focal and segmental hyalinosis and/or sclerosis, mesangiocapillary glomerulonephritis, diffuse endocapillary and extracapillary glomerulonephritis and lupus nephritis. Focal and segmental proliferative glomerulonephritis may or may not be associated with the syndrome of recurrent macroscopic haematuria and may or may not show associated mesangial IgA. Children frequently show minimal changes on light microscopy, whereas adults usually show focal and segmental proliferative lesions with or without crescents. Between active phases only focal and segmental sclerosis may be seen. Progression to renal failure may be observed in an adult whereas children usually have an excellent long-term outcome. The glomerular lesion of focal and segmental hyalinosis may also occur as a secondary phenomenon in various forms of scarring or sclerosis in the glomerulus. The primary form is usually accompanied by the nephrotic syndrome. About 70% of patients develop renal failure over 10 years thus the prognosis is poor. Mesangiocapillary glomerulonephritis has become much more clearly defined. Two distinct morphological types occur, one associated with dense deposits and the other with sub-endothelial deposits. In both the prognosis is poor. The greatest advance in treatment in recent years has been made in that of diffuse crescentic glomerulonephritis which has a very poor prognosis untreated. Dramatic results have been obtained using plasmapheresis. Lupus nephritis will be discussed as an example of a specific disease entity in which many different morphological varieties are seen.
4. VIRUSES IN CANCER
EVALUATION OF CURRENT EVIDENCE OF POSSIBLE ROLE OF VIRUSES IN HUMAN CANCER SABIN, ALBERT B. Medical University of’ South Carolina, Churleston. U.S.A. There is no question that a great variety of DNA viruses, including many strictly human infectious viruses, can transform normal cells to malignancy both in vitro and in vivo, and that a few special RNA animal viruses are the actual cause of some naturally occurring leukaemias in chickens, mice, cats and cattle and of mammary carcinoma in mice. However, despite the most extensive studies on these cancer-producing viruses there is at present no convincing direct or circumstantial evidence for an aetiological role of any virus in any human cancer. Renato Dulbecco discussing the problem ofevidence said: ‘finding a virus or a viral genome in a cancer has no aetiological connotation’. Macfarlane Burnet, in his 1974 book, Intrinsic Mutagenesis, concluded one chapter on human malignant disease with the colourful words; ‘there is no more place for viruses as causes of cancer than for lice as causes of poverty’. Yet even among some who have already arrived at such conclusions there is always hedging: maybe the evidence for the role of the Epstein-Barr virus as an aetiological co-factor in Burkitt’s lymphoma and nasopharyngeal carcinoma, or of HSV-2 (herpes simplex virus-type 2) in uterine cervical carcinoma, if not yet complete is close to being so. The nature of what should be considered as evidence varies for different viruses that deserve the most consideration in the search for understanding the complex aetiology of different human cancers. The following strictly human DNA viruses have been found to be oncogenic under special experimental conditions and are all known to remain (most likely as viral genomes) in certain tissues of many infected persons in a latent or repressed state with a varying frequency of reappearance as fully infectious virus: the adenoviruses, the SV,, (Simian Virus-40) related human BK and JC papovaviruses, and the herpes group of viruses, including herpes simplex (HSV types 1 and 2), cytomegalorirus, and EBV. The aetiology of malignancies experimentally produced by the adenoviruses and the SV,, group can be established by demonstrating: a. a virus-coded nonvirion, intranuclear tumour antigen for which the tumour-