British Journal of Urology (1978). 50, 145-152
Functional Obstruction of the Ureter and Renal Pelvis. A Histological and Electron Microscopic Study J. A.
GOSLING and J. S. DIXON
Department of Anatomy, University Medical School, Manchester
Summary- Histological, histochemical and electron microscopic techniques have been used t o compare dilated and normal calibre segments of ureter and renal pelvis in cases of idiopathic hydronephrosis and primary obstructive megaureter. In both conditions a marked increase in collagen and elastic tissue occurs in the wall of the distended segment and this infiltration extends throughout the proximal dilated ureter and renal pelvis. Evidence is presented t o show that in the dilated segment, smooth muscle cells are directly involved in the synthesis of connective tissue elements. These findings support the view that the primary anomaly in idiopathic hydronephrosis and primary obstructive megaureter can be attributed t o a malfunction of smooth muscle cells in the ureter and renal pelvis. However, the possibility remains that the changes in smooth muscle are secondary and have been induced by obstruction and distension caused in these pathological conditions by unknown aetiological factors.
Of the many theories on the aetiology of idiopathic hydronephrosis and primary obstructive megaureter the most recent has been based upon data obtained using the electron miscroscope. Notley (1968) first applied this method in idiopathic hydronephrosis and described a marked increase in the collagen component of the narrowed pelvi-ureteric segment. This was considered the physical cause of the obstruction by preventing distension of the affected part of the ureter during diuresis (Notley, 1971). In a subsequent study of primary obstructive megaureter (Notley, 1972), a similar accumulation of collagen was proposed as the underlying anomaly responsible for the condition. Hanna et d. (1976) used both light and electron microscopic techniques in studies on functional ureteric obstruction and described a similar infiltration of collagen around and between smooth muscle cells in the obstructed segment. In addition these workers described secondary changes, the smooth muscle cells showing evidence of atrophy with disruption or attenuation of nexuses and an excessive accumulation of collagen fibres between the cells. These features were most apparent immediately above the narrowed segment, becoming less obvious in samples obtained from more proximal parts of the dilated ureter and/or pelvis. Received 18 October 1977. Accepted for publication 9 December 1977.
During the course of a study employing radioisotope renography to evaluate the functional activity of the upper urinary tract (O’Reilly et al., 1978) tissue from the ureter and renal pelvis has been examined histochemically and with light and electron microscopy. The results obtained in cases of idiopathic hydronephrosis and primary obstructive megaureter differ in a number of respects from those described previously.
Materials and Methods Material was obtained from 15 patients with idiopathic hydronephrosis and from 6 cases of obstructive megaureter. In the former group, 14 specimens consisted of part of the renal pelvis in continuity with the pelvi-ureteric segment and the proximal portion of the ureter. The other case underwent nephrectomy, providing the kidney, the pelvis and part of the ureter for examination. Of the specimens from cases of primary obstructive megaureter 4 consisted of the narrow segment with a portion of adjacent ureter and 2 comprised kidney, pelvis and ureter .in continuity. A kidney, pelvis and ureter removed for hypernephroma were used for comparison as control. Tissue samples were removed and processed for electron microscopy (see below). Each nephrectomy specimen was subdivided and tissue for electron microscopy removed from the region of attachment of the minor c a k e s to the renal paren-
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chyma, the c a k e s , the renal pelvis, the ureter and the zone of constriction. Samples for electron microscopy from pelvi-ureteric and megaureter specimens were obtained in a manner similar to that described by Hanna et al. (1976). In each case, tissue was removed from the obstructed segment and from sites proximal and distal to this zone. All remaining tissues were processed for light microscopy as described below.
Light Microscopy Tissue blocks were orientated on cryostat tissue holders, rapidly frozen in 2-methylbutaneYcooled in liquid nitrogen and stored at -80°C until required. Specimens were then transferred to a cryostat at - 25°C internal temperature and serial sections, 10 to 20 p in thickness, were prepared. Sections were mounted on glass slides and those required for histological purposes processed using Masson’s trichrome techniaue. The method desEribed by Gomori (1952) was employed for tissue cholinesterases. In selected sections, acetyl- and pseudo~holinesterases were distinguished using the specific inhibitors TIPA (tetraisopropylpyrophosphoramide)and lY5-bis-(4 alhydimethyl - ammonium - phenyl) - pentane- 3 one dibromide. Some sections were processed for tissue catecholamines using a fluorescence method similar to that described by Spriggs et al. (1966). This technique was also used to determine the distribution of autofluorescent elastin within the tissue sections.
Results The light and electron microscopic findings were the same in idiopathic hydronephrosis and in primary obstructive megaureter. The essential features common to both conditions will be described.
Electron Microscopy Tissue for electron microscopy was subdivided into blocks approximately 1 mm cube prior to fixation in either 1To osmium tetroxide in acetateveronal buffer at 4°C and pH 7.3 for 1 h (Palade, 1952), or 5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4°C and p H 7.3 for 2 h (Sabatini et al., 1963). Using the latter, tissues were post-fixed in 1070 osmium tetroxide for Yz h. Following fixation , tissue blocks were dehydrated in ascending concentrations of ethyl alcohol and embedded in epoxy resin with the aid of propylene oxide. Sections cut with glass knives and a Reichert OmU3 ultramicrotome were mounted on copper grids, double stained with alcoholic uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and examined in a Philips EM300 electron microscope.
Light Microscopy The general arrangement of muscle bundles in pelvis and ureter was the same in all tissue examined, whether from wide or normal calibre regions. Muscle bundles were ‘continuous from dilated to normal ureter across the “narrow” zone and no preponderance of circularly orientated muscle was noted in any region. In trichrome preparations the distribution of connective tissue in the normal calibre ureter below the obstructing lesion (Fig. 1) was identical to that in normal ureter. However, an abrupt increase in connective tissue began in the junctional region between the normal calibre and the dilated ureter and extended throughout the proximal part of the ureter and renal pelvis. This additional connective tissue increased the thickness of the lamina propria and extended into the muscle coat between muscle bundles (Fig. 3), separating individual smooth
Fig. 1 Compact smooth muscle bundles obtained from the nondilated segment of ureter in a case of idiopathic hydronephrosis. Note the minimal amount of connective tissue between the cells within each muscle bundle ( ~ 2 5 0 ) .
FUNCTIONAL OBSTRUCTION OF THE URETER A N D RENAL PELVIS
Fig. 2 This adjacent section to that shown in Fig. 1 demonstrates the distribution of non-specific cholinesterase in the smooth muscle coat. Enzyme reaction product is evident in the cells forming each of the muscle bundles. Compare with Fig. 4 ( x 2 5 0 ) .
Fig. 3 Section from the dilated renal pelvis of the case illustrated in Figs. 1 and 2. Large amounts of connective tissue extend into the muscle bundles and separate individual smooth muscle cells from one another. BV, blood vessel ( x 250).
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Fig. 4 In this adjacent section to that shown in Fig. 3, the smooth muscle cells are almost devoid of non-specific cholinesterase activity. Note the blood vessel (BV) also shown in Fig. 3 ( ~ 2 5 0 ) .
muscle cells from’their neighbours. This infiltration occurred throughout all parts of the ureter and renal pelvis above the “narrowed” segment. The smooth muscle of the pelvis and ureter in the control specimen and that of the ureter distal to the dilated segments (Fig. 2) was rich in pseudocholinesterase. In contrast, pseudo-cholinesterase activity was markedly reduced in the smooth muscle within the wall of the dilated ureter and renal pelvis (Fig. 4) and within the muscle bundles infiltrated by collagen. In all specimens, whether of normal or dilated ureter, acetyl-cholinesterase positive nerves and catecholamine containing nerves had a . similar distribution and arrangement. Both acetyl-cholinesterase and catecholamine containing nerves were exceedingly sparse in specimens from the proximal part of the ureter and renal pelvis (Figs. 2 and 4), but became more common in the juxtavesical region. The fluorescence technique was also employed to study the arrangement of autofluorescent elastin fibres. In the normal calibre ureter numerous fibres were observed running between the muscle bundles and extending into the lamina propria and adventitia (Fig. 5 ) . In contrast, a marked increase in elastin fibres and filaments occurred at the site of dilatation and extended
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from this region throughout the proximal portion of the ureter and renal pelvis. In the dilated portion fine elastin filaments were common between the individual cells which comprised the smooth muscle bundles (Fig. 6).
Fig. 5 Using the fluorescence technique, autofluorescent elastic fibres are seen to be mainly confined to the connective tissue separating the compact smooth muscle bundles (SM)in this specimen obtained from a nondilated segment of ureter. ( x 300).
Electron Microscopy The fine structural appearance of the normal calibre ureter below the obstructive lesion was the same as in normal ureter. Muscle bundles composed of closely packed smooth muscle cells were separated from their neighbours by relatively little intervening connective tissue (Fig. 7). Each smooth muscle cell possessed a sarcoplasm packed with longitudinally orientated myofilaments together with numerous electron dense bodies. Mitochondria were scattered at random throughout the sarcoplasm while scant granular reticulum and Golgi membranes tended to cluster at either pole of the single, elongated nucleus. Rows of caveolae interspersed with electron dense regions were situated near the sarcolemma of each smooth muscle cell. Individual smooth muscle cells were surrounded by a lamina except at regions of close approach (Fig. 8) where a gap of 15 nm separated adjacent ceH membranes in the absence
Fig. 6 Sections from dilated portions of the urinary tract show a marked increase in autofluorescent elastic fibres. In this illustration of the renal pelvis in a of primary obstructive megaureter numerous fibres (arrows) are observed infiltrating between individual smooth muscle cells ( x 300).
Fig. 7 An electron micrograph of smooth muscle cells from a nondistended segment of ureter. Each cell is packed with myofilaments and is separated from its neighbours by very few connective tissue elements ( 6500).
FUNCTIONAL OBSTRUCTION OF THE URETER AND RENAL PELVIS
Fig. 8 An electron micrograph of a region of “close approach” between adjacent smooth muscle cells from a nondilated ureter. A gap of I5 nm separates adjacent cell membranes. The basal lamina, BL, does not extend into the region of “close approach” ( X 25,500).
Fig. 9 An electron micrograph from a dilated portion of urinary tract, showing several smooth muscle cells widely separated by connective tissue. Arrows indicate normal regions of close approach between adjacent cells ( x 7400).
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of intervening basal lamina material. Such regions were common between adjacent smooth muscle cells within a muscle bundle. The narrow intercellular spaces contained occasional collagen fibres. Elastic fibres were rare. Groups of axons were rare in the proximal ureter and renal pelvis, but were more common in the juxtavesical region where occasional vesicle-filled axonal varicosities were present in close association with adjacent . smooth muscle cells. In the dilated portions of the ureter and renal pelvis the smooth muscle cells were separated from one another by unusually large amounts of connective tissue (Fig. 9). The latter consisted of bundles of randomly orientated collagen fibres together with numerous pale staining elastic fibres (Fig. 10). Many of these fibres possessed dense microfilaments around the periphery which were in continuity with the basal laminae of adjacent smooth muscle cells. Despite the increased amounts of connective tissue, regions of close approach between adjacent smooth muscle cells were as common as in the normal calibre Ureter. In most sections of dilated ureter and renal pelvis marked changes were present in the fine structure of many of the smooth muscle cells.
Fig. 10 In this illustration, several pale-staining elastic fibres are in continuity with the basal lamina of an adjacent smooth muscle cell ( x 14,400).
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These alterations in fine structure, whilst similar in all samples from the same dilated ureter and renal pelvis, did vary from 1 patient to another. In some cases the smooth muscle cells showed a considerable increase in their complement of granular reticulum and Golgi membranes, particularly in the perinuclear zone (Fig. 11). In these instances the myofilaments were confined to the periphery and some cells also contained numerous membrane-bound dense bodies and lipid droplets (Fig. 11). In other specimens many of the smooth muscle cells contained a marked increase in electron dense bodies which often formed clusters within the sarcoplasm (Fig. 12). In all specimens of dilated ureter and renal pelvis the most common fine structural change within the smooth muscle cells was a marked decrease in the complement of myofilaments; such cells often displayed large electron lucent regions within their sarcoplasm and a reduced number of caveolae. Other cells appeared totally devoid of myofilaments and possessed pyknotic nuclei, randomly scattered small mitochondria and very few surface caveolae (Fig. 13).
Flg. 11 An electron micrograph of part of a smooth muscle cell from a distended renal pelvis, illustrating extensive granular reticulum and Golgi membranes in the perinuclear zone. Lipid droplets (L) are also present ( x 11.500).
BRITISH JOURNAL OF UROLOGY
Fig. 12 Clusters of electron dense bodies surrounded by myofilaments occur in this smooth muscle cell from a sample of renal pelvis ( x 28,800).
Fig. 13 This electron micrograph illustrates the most commonly observed fine structural change in the smooth muscle ce!ls from distended segments, namely a loss of myofilaments and a reduction in surface caveolae. Arrows indicate numerous elastic fibres closely apposed to the cell surface ( x 5400).
f-IJNCTIONALOBSTRUCTION OF THE URETER A N D RENAL PELVIS
Discussion The purpose of this investigation has been to examine and compare the morphology and histochemistry of ureteric smooth muscle in idiopathic hydronephrosis and primary obstructive megaureter. In addition comparisons have been made between the smooth muscle above and below the “narrow” segment. The structural and histo-. chemical changes found in the 2 conditions are essentially similar; only the extent to which the upper urinary tract is dilated differentiates the one from the other. Notley (1971) and Hanna el al. (1 976) also noted this similarity .and-although the details of the structural changes noted in this study vary from their observations-it seems probable that idiopathic hydronephrosis and primary obstructive megaureter have a common underlying cause, acting at different levels in the ureter. Both Notley (1971, 1972) and Hanna et al. (1 976) considered the narrow segment of ureter to be of principal importance in the aetiology of the condition, postulating that the marked increase in collagen which they observed acted as an inelastic collar preventing adequate distension of the affected zone during ureteric peristalsis. Although Hanna and co-workers also described morphological changes proximal to this segment, these were thought to arise in response to overdistension and the primary cause was attributed to the “narrowed” ureter. However, the present study has demonstrated excessive amounts of connective tissue extending along the entire length of the distended proximal ureter and renal pelvis. The increase was clearly evident in all cases, including those in which the whole of the distended segment was available for study. This infiltration of connective tissue continued distally as far as the “narrowed” segment where it ceased abruptly. Indeed, the structure of the nondilated ureter was indistinguishable both histochemically and fine structurally from normal ureter. Neither Notley (1968, 1972) nor Hanna et al. (1976) report observations of the dilated proximal ureter or pelvis as remote from the zone of narrowing as in this study. Furthermore their method of study, electron microscopy, limits the size and amount of tissue that can be satisfactorily examined. The combination of these 2 limiting factors compared with the spread of this investigation may account for the difference in the findings. There is evidence in this investigation that the
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smooth muscle cells of the dilated ureter above the obstruction may be directly involved in the manufacture of elastin and collagen. Fine fibrils of elastin extend from the basal laminae of these cells and aggregate into large elastic fibres. In addition, the deposition of numerous collagen fibres separating individual smooth muscle cells occurs in the absence of an increase in the fibroblast component of the smooth muscle coat. In some muscle cells a marked reduction in the complement of myofilaments was accompanied by an increase in endoplasmic reticulum, such changes indicating that these cells have increased those subcellular organelles which are believed to be involved in active synthesis, seemingly at the expense of their contractile apparatus. Notley (1971) and Hanna et al. (1976) did not comment upon the source of the excessive connective tissue which they observed but the view that smooth muscle is responsible for the manufacture of these components receives support from the work of Ross and Klebanoff (1971) and Gerrity el al. (1975). In this context, the marked difference in tissue cholinesterase between the dilated ureter and renal pelvis and the control provides additional evidence of a change in the functional activity of smooth muscle in idiopathic hydronephrosis and primary obstructive megaureter. It may be that this reduction of enzyme in smooth muscle within the wall of the distended segment is directly related to the manufacture of connective tissue by these cells. In contrast to the findings of Hanna et al. (1976), the present study has substantiated the work of Notley (1971) by demonstrating no alteration in the arrangement and fine structure of regions of close approach between adjacent smooth muscle cells. In addition, the structure and distribution of the nerves which supply the upper urinary tract in cases of primary obstructive megaureter and idiopathic hydronephrosis are similar to those in normal ureter. Thus, on morphological evidence, it seems unlikely that the basic aetiology in these conditions is caused either by failure of electrotonic coupling between smooth muscle cells or by abnormalities in the autonomic innervation. In conclusion, the present study has shown that in primary obstructive megaureter and idiopathic hydronephrosis structural and histochemical changes extend throughout the distended segment of the ureter and renal pelvis. On the basis of these findings it seems unlikely that the
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pathological conditions are caused by a discrete, Gomori, G. (1 952). Microscopic Histochemistry-Principles and Practice, University of Chicago Press, Chicago. inelastic segment of ureter confined to the distal M. K., Jeffs, R. D., Sturgess, J. M. and Barkin, M. end of the dilated portion. Two alternative Hanna, (1976). Ureteral stfucture and ultrastructure. Part I I . hypotheses can be proposed to account for our Congenital ureteropelvic junction obstruction and primary observations. Firstly, a primary anomaly of obstructive megaureter. Journal of Urology, 116, 725-730. ureteric and renal pelvic smooth muscle is present Notley, R. G. (1968). Electron microscopy of the upper ureter and the pelvi-ureteric junction. British Journal of Urology, in both conditions which is reflected in fine 40, 37-52. structural and histochemical changes associated Notley, R. G. (1971). The structural basis for normal and with the manufacture of excessive amounts of abnormal ureteric motility. Annals of the Royal College of Surgeons of England, 49, 250-267. connective tissue. On this basis an intrinsic malfunction of muscle cells is the cause for these Notley, R. G. (1972). Electron microscopy of the primary obstructive megaureter. British Journal of Urology. 44, conditions. Alternatively, the morphological 229-234. features which occur in the wall of the dilated O’Reilly, P. H., Lawson, R. S., Sheilds, R. A., Testa, H. J., ureter and renal pelvis could arise as secondary Charlton-Edwards, E. and Carroll, R. N. P. (1978). Diuresis renography in equivocal urinary tract obstruction. British changes in response to distension, the latter due Journal of Urology. (In press.) to obstruction from as yet indeterminate causes. Palade, G. E. (1952). A study of fixation for electron microIn an effort to resolve these possibilities an addiscopy. Journal of Experimental Medicine, 95, 285-297. tional study is currently in progress to determine Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal the histochemistry and morphology of the disof Cell Biology, 17. 208-212. tended upper urinary tract in cases of obstruction Ross, R. and Klebanoff, S. J. (1971). The smooth muscle produced by known organic causes. cell. 1. I n vivo synthesis of connective tissue proteins. Acknowledgements The authors are indebted to Mr R. J. Barnard of University Hospital of South Manchester, Mr R. Carroll and Mr E. Charlton-Edwards of Manchester Royal Infirmary and Mr W. Richmond of Leigh General Hospital, without whose cooperation and collaboration the study would not have been possible. The expert technical assistance of Miss G. Moore, Mrs S . A. Thompson and Mr R. Ellis is acknowledged. This investigation was supported in part by a grant from the Manchester and North West Region Kidney Research Association.
References Gerrity, R. G., Adams, E. P. and Cliff, W. J. (1975). The aortic tunica media of the developing rat. 11. Incorporation by medial cells of lH-proline into collagen and elastin: Autoradiographic and chemical studies. Laboratory Investigation, 32, 601-609.
Journal of Cell Biology, 50, 159-171. Sabatini, D. D., Bensch, K. and Barrnett, R. J . (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. Journal of Cell Biology, 17, 19-58. Spriggs, T. L. B., Lever, J. D., Rees, P. M. and Graham, J. D. P. (1966). Controlled formaldehyde-catecholamine condensation in cryostat sections to show adrenergic nerves by fluorescence. Stain Technology. 41, 323-327. Watson, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. Journal of Biophysical and Biochemical Cytology, 4, 475-478.
The Authors John A. Gosling, MB, MD, Professor of Anatomy. John S . Dixon. BSc, PhD, Senior Lecturer. Requests for reprints to: Professor J. A. Gosling, Department of Anatomy, Medical School, University of Manchester, Manchester M13 9PT.
Functional Obstruction of the Ureter and Renal Pelvis. A Histological and Electron Microscopic Study J. A.
GOSLING and J. S. DIXON
Department of Anatomy, University Medical School, Manchester
Summary- Histological, histochemical and electron microscopic techniques have been used t o compare dilated and normal calibre segments of ureter and renal pelvis in cases of idiopathic hydronephrosis and primary obstructive megaureter. In both conditions a marked increase in collagen and elastic tissue occurs in the wall of the distended segment and this infiltration extends throughout the proximal dilated ureter and renal pelvis. Evidence is presented t o show that in the dilated segment, smooth muscle cells are directly involved in the synthesis of connective tissue elements. These findings support the view that the primary anomaly in idiopathic hydronephrosis and primary obstructive megaureter can be attributed t o a malfunction of smooth muscle cells in the ureter and renal pelvis. However, the possibility remains that the changes in smooth muscle are secondary and have been induced by obstruction and distension caused in these pathological conditions by unknown aetiological factors.
Of the many theories on the aetiology of idiopathic hydronephrosis and primary obstructive megaureter the most recent has been based upon data obtained using the electron miscroscope. Notley (1968) first applied this method in idiopathic hydronephrosis and described a marked increase in the collagen component of the narrowed pelvi-ureteric segment. This was considered the physical cause of the obstruction by preventing distension of the affected part of the ureter during diuresis (Notley, 1971). In a subsequent study of primary obstructive megaureter (Notley, 1972), a similar accumulation of collagen was proposed as the underlying anomaly responsible for the condition. Hanna et d. (1976) used both light and electron microscopic techniques in studies on functional ureteric obstruction and described a similar infiltration of collagen around and between smooth muscle cells in the obstructed segment. In addition these workers described secondary changes, the smooth muscle cells showing evidence of atrophy with disruption or attenuation of nexuses and an excessive accumulation of collagen fibres between the cells. These features were most apparent immediately above the narrowed segment, becoming less obvious in samples obtained from more proximal parts of the dilated ureter and/or pelvis. Received 18 October 1977. Accepted for publication 9 December 1977.
During the course of a study employing radioisotope renography to evaluate the functional activity of the upper urinary tract (O’Reilly et al., 1978) tissue from the ureter and renal pelvis has been examined histochemically and with light and electron microscopy. The results obtained in cases of idiopathic hydronephrosis and primary obstructive megaureter differ in a number of respects from those described previously.
Materials and Methods Material was obtained from 15 patients with idiopathic hydronephrosis and from 6 cases of obstructive megaureter. In the former group, 14 specimens consisted of part of the renal pelvis in continuity with the pelvi-ureteric segment and the proximal portion of the ureter. The other case underwent nephrectomy, providing the kidney, the pelvis and part of the ureter for examination. Of the specimens from cases of primary obstructive megaureter 4 consisted of the narrow segment with a portion of adjacent ureter and 2 comprised kidney, pelvis and ureter .in continuity. A kidney, pelvis and ureter removed for hypernephroma were used for comparison as control. Tissue samples were removed and processed for electron microscopy (see below). Each nephrectomy specimen was subdivided and tissue for electron microscopy removed from the region of attachment of the minor c a k e s to the renal paren-
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chyma, the c a k e s , the renal pelvis, the ureter and the zone of constriction. Samples for electron microscopy from pelvi-ureteric and megaureter specimens were obtained in a manner similar to that described by Hanna et al. (1976). In each case, tissue was removed from the obstructed segment and from sites proximal and distal to this zone. All remaining tissues were processed for light microscopy as described below.
Light Microscopy Tissue blocks were orientated on cryostat tissue holders, rapidly frozen in 2-methylbutaneYcooled in liquid nitrogen and stored at -80°C until required. Specimens were then transferred to a cryostat at - 25°C internal temperature and serial sections, 10 to 20 p in thickness, were prepared. Sections were mounted on glass slides and those required for histological purposes processed using Masson’s trichrome techniaue. The method desEribed by Gomori (1952) was employed for tissue cholinesterases. In selected sections, acetyl- and pseudo~holinesterases were distinguished using the specific inhibitors TIPA (tetraisopropylpyrophosphoramide)and lY5-bis-(4 alhydimethyl - ammonium - phenyl) - pentane- 3 one dibromide. Some sections were processed for tissue catecholamines using a fluorescence method similar to that described by Spriggs et al. (1966). This technique was also used to determine the distribution of autofluorescent elastin within the tissue sections.
Results The light and electron microscopic findings were the same in idiopathic hydronephrosis and in primary obstructive megaureter. The essential features common to both conditions will be described.
Electron Microscopy Tissue for electron microscopy was subdivided into blocks approximately 1 mm cube prior to fixation in either 1To osmium tetroxide in acetateveronal buffer at 4°C and pH 7.3 for 1 h (Palade, 1952), or 5% glutaraldehyde in 0.1 M sodium cacodylate buffer at 4°C and p H 7.3 for 2 h (Sabatini et al., 1963). Using the latter, tissues were post-fixed in 1070 osmium tetroxide for Yz h. Following fixation , tissue blocks were dehydrated in ascending concentrations of ethyl alcohol and embedded in epoxy resin with the aid of propylene oxide. Sections cut with glass knives and a Reichert OmU3 ultramicrotome were mounted on copper grids, double stained with alcoholic uranyl acetate (Watson, 1958) and lead citrate (Reynolds, 1963) and examined in a Philips EM300 electron microscope.
Light Microscopy The general arrangement of muscle bundles in pelvis and ureter was the same in all tissue examined, whether from wide or normal calibre regions. Muscle bundles were ‘continuous from dilated to normal ureter across the “narrow” zone and no preponderance of circularly orientated muscle was noted in any region. In trichrome preparations the distribution of connective tissue in the normal calibre ureter below the obstructing lesion (Fig. 1) was identical to that in normal ureter. However, an abrupt increase in connective tissue began in the junctional region between the normal calibre and the dilated ureter and extended throughout the proximal part of the ureter and renal pelvis. This additional connective tissue increased the thickness of the lamina propria and extended into the muscle coat between muscle bundles (Fig. 3), separating individual smooth
Fig. 1 Compact smooth muscle bundles obtained from the nondilated segment of ureter in a case of idiopathic hydronephrosis. Note the minimal amount of connective tissue between the cells within each muscle bundle ( ~ 2 5 0 ) .
FUNCTIONAL OBSTRUCTION OF THE URETER A N D RENAL PELVIS
Fig. 2 This adjacent section to that shown in Fig. 1 demonstrates the distribution of non-specific cholinesterase in the smooth muscle coat. Enzyme reaction product is evident in the cells forming each of the muscle bundles. Compare with Fig. 4 ( x 2 5 0 ) .
Fig. 3 Section from the dilated renal pelvis of the case illustrated in Figs. 1 and 2. Large amounts of connective tissue extend into the muscle bundles and separate individual smooth muscle cells from one another. BV, blood vessel ( x 250).
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Fig. 4 In this adjacent section to that shown in Fig. 3, the smooth muscle cells are almost devoid of non-specific cholinesterase activity. Note the blood vessel (BV) also shown in Fig. 3 ( ~ 2 5 0 ) .
muscle cells from’their neighbours. This infiltration occurred throughout all parts of the ureter and renal pelvis above the “narrowed” segment. The smooth muscle of the pelvis and ureter in the control specimen and that of the ureter distal to the dilated segments (Fig. 2) was rich in pseudocholinesterase. In contrast, pseudo-cholinesterase activity was markedly reduced in the smooth muscle within the wall of the dilated ureter and renal pelvis (Fig. 4) and within the muscle bundles infiltrated by collagen. In all specimens, whether of normal or dilated ureter, acetyl-cholinesterase positive nerves and catecholamine containing nerves had a . similar distribution and arrangement. Both acetyl-cholinesterase and catecholamine containing nerves were exceedingly sparse in specimens from the proximal part of the ureter and renal pelvis (Figs. 2 and 4), but became more common in the juxtavesical region. The fluorescence technique was also employed to study the arrangement of autofluorescent elastin fibres. In the normal calibre ureter numerous fibres were observed running between the muscle bundles and extending into the lamina propria and adventitia (Fig. 5 ) . In contrast, a marked increase in elastin fibres and filaments occurred at the site of dilatation and extended
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from this region throughout the proximal portion of the ureter and renal pelvis. In the dilated portion fine elastin filaments were common between the individual cells which comprised the smooth muscle bundles (Fig. 6).
Fig. 5 Using the fluorescence technique, autofluorescent elastic fibres are seen to be mainly confined to the connective tissue separating the compact smooth muscle bundles (SM)in this specimen obtained from a nondilated segment of ureter. ( x 300).
Electron Microscopy The fine structural appearance of the normal calibre ureter below the obstructive lesion was the same as in normal ureter. Muscle bundles composed of closely packed smooth muscle cells were separated from their neighbours by relatively little intervening connective tissue (Fig. 7). Each smooth muscle cell possessed a sarcoplasm packed with longitudinally orientated myofilaments together with numerous electron dense bodies. Mitochondria were scattered at random throughout the sarcoplasm while scant granular reticulum and Golgi membranes tended to cluster at either pole of the single, elongated nucleus. Rows of caveolae interspersed with electron dense regions were situated near the sarcolemma of each smooth muscle cell. Individual smooth muscle cells were surrounded by a lamina except at regions of close approach (Fig. 8) where a gap of 15 nm separated adjacent ceH membranes in the absence
Fig. 6 Sections from dilated portions of the urinary tract show a marked increase in autofluorescent elastic fibres. In this illustration of the renal pelvis in a of primary obstructive megaureter numerous fibres (arrows) are observed infiltrating between individual smooth muscle cells ( x 300).
Fig. 7 An electron micrograph of smooth muscle cells from a nondistended segment of ureter. Each cell is packed with myofilaments and is separated from its neighbours by very few connective tissue elements ( 6500).
FUNCTIONAL OBSTRUCTION OF THE URETER AND RENAL PELVIS
Fig. 8 An electron micrograph of a region of “close approach” between adjacent smooth muscle cells from a nondilated ureter. A gap of I5 nm separates adjacent cell membranes. The basal lamina, BL, does not extend into the region of “close approach” ( X 25,500).
Fig. 9 An electron micrograph from a dilated portion of urinary tract, showing several smooth muscle cells widely separated by connective tissue. Arrows indicate normal regions of close approach between adjacent cells ( x 7400).
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of intervening basal lamina material. Such regions were common between adjacent smooth muscle cells within a muscle bundle. The narrow intercellular spaces contained occasional collagen fibres. Elastic fibres were rare. Groups of axons were rare in the proximal ureter and renal pelvis, but were more common in the juxtavesical region where occasional vesicle-filled axonal varicosities were present in close association with adjacent . smooth muscle cells. In the dilated portions of the ureter and renal pelvis the smooth muscle cells were separated from one another by unusually large amounts of connective tissue (Fig. 9). The latter consisted of bundles of randomly orientated collagen fibres together with numerous pale staining elastic fibres (Fig. 10). Many of these fibres possessed dense microfilaments around the periphery which were in continuity with the basal laminae of adjacent smooth muscle cells. Despite the increased amounts of connective tissue, regions of close approach between adjacent smooth muscle cells were as common as in the normal calibre Ureter. In most sections of dilated ureter and renal pelvis marked changes were present in the fine structure of many of the smooth muscle cells.
Fig. 10 In this illustration, several pale-staining elastic fibres are in continuity with the basal lamina of an adjacent smooth muscle cell ( x 14,400).
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These alterations in fine structure, whilst similar in all samples from the same dilated ureter and renal pelvis, did vary from 1 patient to another. In some cases the smooth muscle cells showed a considerable increase in their complement of granular reticulum and Golgi membranes, particularly in the perinuclear zone (Fig. 11). In these instances the myofilaments were confined to the periphery and some cells also contained numerous membrane-bound dense bodies and lipid droplets (Fig. 11). In other specimens many of the smooth muscle cells contained a marked increase in electron dense bodies which often formed clusters within the sarcoplasm (Fig. 12). In all specimens of dilated ureter and renal pelvis the most common fine structural change within the smooth muscle cells was a marked decrease in the complement of myofilaments; such cells often displayed large electron lucent regions within their sarcoplasm and a reduced number of caveolae. Other cells appeared totally devoid of myofilaments and possessed pyknotic nuclei, randomly scattered small mitochondria and very few surface caveolae (Fig. 13).
Flg. 11 An electron micrograph of part of a smooth muscle cell from a distended renal pelvis, illustrating extensive granular reticulum and Golgi membranes in the perinuclear zone. Lipid droplets (L) are also present ( x 11.500).
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Fig. 12 Clusters of electron dense bodies surrounded by myofilaments occur in this smooth muscle cell from a sample of renal pelvis ( x 28,800).
Fig. 13 This electron micrograph illustrates the most commonly observed fine structural change in the smooth muscle ce!ls from distended segments, namely a loss of myofilaments and a reduction in surface caveolae. Arrows indicate numerous elastic fibres closely apposed to the cell surface ( x 5400).
f-IJNCTIONALOBSTRUCTION OF THE URETER A N D RENAL PELVIS
Discussion The purpose of this investigation has been to examine and compare the morphology and histochemistry of ureteric smooth muscle in idiopathic hydronephrosis and primary obstructive megaureter. In addition comparisons have been made between the smooth muscle above and below the “narrow” segment. The structural and histo-. chemical changes found in the 2 conditions are essentially similar; only the extent to which the upper urinary tract is dilated differentiates the one from the other. Notley (1971) and Hanna el al. (1 976) also noted this similarity .and-although the details of the structural changes noted in this study vary from their observations-it seems probable that idiopathic hydronephrosis and primary obstructive megaureter have a common underlying cause, acting at different levels in the ureter. Both Notley (1971, 1972) and Hanna et al. (1 976) considered the narrow segment of ureter to be of principal importance in the aetiology of the condition, postulating that the marked increase in collagen which they observed acted as an inelastic collar preventing adequate distension of the affected zone during ureteric peristalsis. Although Hanna and co-workers also described morphological changes proximal to this segment, these were thought to arise in response to overdistension and the primary cause was attributed to the “narrowed” ureter. However, the present study has demonstrated excessive amounts of connective tissue extending along the entire length of the distended proximal ureter and renal pelvis. The increase was clearly evident in all cases, including those in which the whole of the distended segment was available for study. This infiltration of connective tissue continued distally as far as the “narrowed” segment where it ceased abruptly. Indeed, the structure of the nondilated ureter was indistinguishable both histochemically and fine structurally from normal ureter. Neither Notley (1968, 1972) nor Hanna et al. (1976) report observations of the dilated proximal ureter or pelvis as remote from the zone of narrowing as in this study. Furthermore their method of study, electron microscopy, limits the size and amount of tissue that can be satisfactorily examined. The combination of these 2 limiting factors compared with the spread of this investigation may account for the difference in the findings. There is evidence in this investigation that the
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smooth muscle cells of the dilated ureter above the obstruction may be directly involved in the manufacture of elastin and collagen. Fine fibrils of elastin extend from the basal laminae of these cells and aggregate into large elastic fibres. In addition, the deposition of numerous collagen fibres separating individual smooth muscle cells occurs in the absence of an increase in the fibroblast component of the smooth muscle coat. In some muscle cells a marked reduction in the complement of myofilaments was accompanied by an increase in endoplasmic reticulum, such changes indicating that these cells have increased those subcellular organelles which are believed to be involved in active synthesis, seemingly at the expense of their contractile apparatus. Notley (1971) and Hanna et al. (1976) did not comment upon the source of the excessive connective tissue which they observed but the view that smooth muscle is responsible for the manufacture of these components receives support from the work of Ross and Klebanoff (1971) and Gerrity el al. (1975). In this context, the marked difference in tissue cholinesterase between the dilated ureter and renal pelvis and the control provides additional evidence of a change in the functional activity of smooth muscle in idiopathic hydronephrosis and primary obstructive megaureter. It may be that this reduction of enzyme in smooth muscle within the wall of the distended segment is directly related to the manufacture of connective tissue by these cells. In contrast to the findings of Hanna et al. (1976), the present study has substantiated the work of Notley (1971) by demonstrating no alteration in the arrangement and fine structure of regions of close approach between adjacent smooth muscle cells. In addition, the structure and distribution of the nerves which supply the upper urinary tract in cases of primary obstructive megaureter and idiopathic hydronephrosis are similar to those in normal ureter. Thus, on morphological evidence, it seems unlikely that the basic aetiology in these conditions is caused either by failure of electrotonic coupling between smooth muscle cells or by abnormalities in the autonomic innervation. In conclusion, the present study has shown that in primary obstructive megaureter and idiopathic hydronephrosis structural and histochemical changes extend throughout the distended segment of the ureter and renal pelvis. On the basis of these findings it seems unlikely that the
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pathological conditions are caused by a discrete, Gomori, G. (1 952). Microscopic Histochemistry-Principles and Practice, University of Chicago Press, Chicago. inelastic segment of ureter confined to the distal M. K., Jeffs, R. D., Sturgess, J. M. and Barkin, M. end of the dilated portion. Two alternative Hanna, (1976). Ureteral stfucture and ultrastructure. Part I I . hypotheses can be proposed to account for our Congenital ureteropelvic junction obstruction and primary observations. Firstly, a primary anomaly of obstructive megaureter. Journal of Urology, 116, 725-730. ureteric and renal pelvic smooth muscle is present Notley, R. G. (1968). Electron microscopy of the upper ureter and the pelvi-ureteric junction. British Journal of Urology, in both conditions which is reflected in fine 40, 37-52. structural and histochemical changes associated Notley, R. G. (1971). The structural basis for normal and with the manufacture of excessive amounts of abnormal ureteric motility. Annals of the Royal College of Surgeons of England, 49, 250-267. connective tissue. On this basis an intrinsic malfunction of muscle cells is the cause for these Notley, R. G. (1972). Electron microscopy of the primary obstructive megaureter. British Journal of Urology. 44, conditions. Alternatively, the morphological 229-234. features which occur in the wall of the dilated O’Reilly, P. H., Lawson, R. S., Sheilds, R. A., Testa, H. J., ureter and renal pelvis could arise as secondary Charlton-Edwards, E. and Carroll, R. N. P. (1978). Diuresis renography in equivocal urinary tract obstruction. British changes in response to distension, the latter due Journal of Urology. (In press.) to obstruction from as yet indeterminate causes. Palade, G. E. (1952). A study of fixation for electron microIn an effort to resolve these possibilities an addiscopy. Journal of Experimental Medicine, 95, 285-297. tional study is currently in progress to determine Reynolds, E. S. (1963). The use of lead citrate at high pH as an electron opaque stain in electron microscopy. Journal the histochemistry and morphology of the disof Cell Biology, 17. 208-212. tended upper urinary tract in cases of obstruction Ross, R. and Klebanoff, S. J. (1971). The smooth muscle produced by known organic causes. cell. 1. I n vivo synthesis of connective tissue proteins. Acknowledgements The authors are indebted to Mr R. J. Barnard of University Hospital of South Manchester, Mr R. Carroll and Mr E. Charlton-Edwards of Manchester Royal Infirmary and Mr W. Richmond of Leigh General Hospital, without whose cooperation and collaboration the study would not have been possible. The expert technical assistance of Miss G. Moore, Mrs S . A. Thompson and Mr R. Ellis is acknowledged. This investigation was supported in part by a grant from the Manchester and North West Region Kidney Research Association.
References Gerrity, R. G., Adams, E. P. and Cliff, W. J. (1975). The aortic tunica media of the developing rat. 11. Incorporation by medial cells of lH-proline into collagen and elastin: Autoradiographic and chemical studies. Laboratory Investigation, 32, 601-609.
Journal of Cell Biology, 50, 159-171. Sabatini, D. D., Bensch, K. and Barrnett, R. J . (1963). Cytochemistry and electron microscopy. The preservation of cellular ultrastructure and enzymatic activity by aldehyde fixation. Journal of Cell Biology, 17, 19-58. Spriggs, T. L. B., Lever, J. D., Rees, P. M. and Graham, J. D. P. (1966). Controlled formaldehyde-catecholamine condensation in cryostat sections to show adrenergic nerves by fluorescence. Stain Technology. 41, 323-327. Watson, M. L. (1958). Staining of tissue sections for electron microscopy with heavy metals. Journal of Biophysical and Biochemical Cytology, 4, 475-478.
The Authors John A. Gosling, MB, MD, Professor of Anatomy. John S . Dixon. BSc, PhD, Senior Lecturer. Requests for reprints to: Professor J. A. Gosling, Department of Anatomy, Medical School, University of Manchester, Manchester M13 9PT.
