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Renal angiomyolipoma: Ultrastructural study
RENAL ANGIOMYOLIPOMA: ARA CHALVARDJIAti, K,4LhIAN EVA
KOVACS,
HORVATH.
STUDY
Xl. 13. 11. D.
Pfr. I>.
Frown the Department Toronto, Canada
of Patholoe,
.4ngioln).olipoma is a benign tmnor of the kidne)’ Lvhich may, but not invariably, occur in patients with tuberous sclerosis. When associated with the tuberous sclerosis complex, the tumor tends to be small, multifocal, usuall>. bilateral, and asymptomatic; whereas, in patients with no stigmata of tuberous sclerosis, it is often large, and symptomatic. I unifocal. luGlateral, The tulnor has been studied in detail by light microscop);. I2 It consists of a variable mixture of spindle c&ells, adipose tissue, and thick-walled blood vessels. To date, to our knowledge, only two articles have described the ultrastructure of angioln)olipoma, and they differ as to the morphogenesis of the tumor.3q” The purpose of this article is to describe the ultrastructural features of renal arrgiomyolipoma and to shed some light on the nature of the spindle cells and on their relationship to the adipose cells and to the ahno1m;l1 thick-walled vessels. The tumor in this case, which occurred as an isolated finding in an otherwise healthy patient, is also compared with that of the 2 other cases with the tuberous sclerosis conlplexc.~‘~” (:asr
ULTRASTR’CJCTtJRAL
Report
.4 twc~nt~-six-fear-old oriental \voman, with no previous complaints, \vas admitted to the hospital because of sudden onset of a sharp, sevt’re pin beneath the left scapula. radiating down the back and above the iliac crest. Her Mood pr~~ssurc \\‘a~110170 m1I1. Hg. and the or+.
St. Illicharl’s
Hospital,
significant finding was left costl.bvc~rtel>ral I!rinal>.sis sho~vrcl 3 to 4 red tenderness. cells per high-power field. Uriile c:ultures werc sterile. Intravenorls pyelograph!,, rt~nal toinogmphy, and arteriography showel:l the upper pole of the left kidne). to t)c* ill\ol\ Cal \vith tumor. Sllrger\, re\realed a IIMSS arising fiwn the upper pole oi the left kidney and adherent to the diaphragm. A left nephrec+oiny was pt’rformrtl. The patient had an unevwtfnl wcwvery. Investigations failed to reveal an!.’ stigma of the tilberous sclerosis complex.
Thc~ specimen consisted of the left kidntb!; Lvith attached perirenal fat anal ipsilateral adrenal gland. A circwmscribed fi7m miss ineasuring 5 11). 4 lI>, 3 cm. occupied the ilpper pole of the kidtIe>. and had a variegated ~.ellow appearmcc on cut section. The tumor was llot encapsrilated hiit \vas distinct froni tlhc acljacerrt renal tissue. There wxs hemcirrhagr in the perirenal tat.
autl
the
;lcll.tYl;ll
glad
\\‘;I5 grtdy
t10r11ral.
FIGURE 1. Light microscopy shows the typica mingling of smooth muscle cells and fat cells. Part of the collagenized tcall of an abnormal blood vessel is also seen
in the right
lower
corner.
(X 250).
thickened hyalinized walls with scanty cellularity and lacked well-defined internal elastic lamina. The tumor had no true capsule. At the periphery of the tumor an occasional atrophic tubule was surrounded by spindle cells but no residual renal structures could be identified in the center. The periodic acid-Schiff (PAS) reaction stained intensely the clear cytoplasm of some of the spindle cells. The PAS positive granules were removed completely by diastase digestion, indicating that they represented cytoplasmic glycogen. Nuclear pleomorphism was slight and mitotic figures were infrequent. Electron
~rkx-oscopic
findings
Small pieces of tumor tissue, taken from various parts of the neoplasm, were fixed in 2.5 per cent glutaraldehyde in Sorensen buffer, postfixed in 1 per cent 0~04 in Millonig buffer, dehydrated in graded ethanol, processed throllgh propylene oxide, and embedded in Epon 812. Thick sections, cut with a Porter-Blum MT-2 ultramicrotome, were stained with toluidine blue and examined with a light microscope. Representative blocks containing various parts of tumor were selected. Ultrathin sections were
718
stained with uranyl acetate and lead citrate and studied with a Philips 300 eIectron lnicroscope. In the parts of tumor which contained no lipid droplets, the cells were mostly fusiform. The nudei were elongated with blunt edges at their poles. They were often irregularIy outlined with In a few nuclei unusual indeep indentations. clusion bodies were observed. The nucleoli were round and dense. The majority of cytoplasmic organelles were concentrated in the perinuclear areas, and large portions of the cytoplasm were occupied by microfilaments. The rough-s~l&ced endoplasmic reticulum (RER) was prominent and was represented by short cisternae studded with ribosomes. Free ribosomes and polysomes were also observed. The smooth-surfaced cndoplasmic reticulum (SER) was scanty. The perinuclear cytoplasm contained numerous mitochondria with slight variations in shape and size. They usually had a light matrix and transverse lamellar cristae. Mitochondrial abnormalities such as swelling and rarefaction were rare. The Golgi zones were conspicuous and were composed of five or six slightly or moderately dilated sacculi and several vesicles. In the Golgi region and also in other areas, groups of membrane-bound dense granules were found. They were spherical or pleomorphic, showing variable electron density. Occasionally, small crystalline structilres, bound by a single unit membrane or lying free in the hyaloplasm, were also detected. The most prominent ultrastructural feature was the presence of large amounts of glycogen, composed of alpha and beta particles, in the organelle-rich areas (Fig. 2A). The glycogen deposits reached extreme proportions in some cells, displacing the organelles to the periphery of the cytoplasm. In and around the glycogen masses, several irregularly shaped spaces were noted, IWUally delineated by a single membrane. Occasionally a connection between these structures and the SER tubules was noted. Large cytoplasmic areas were occupied by fine filamentous structures exhibiting the fine structural features of myofilaments (Fig, 2B). Numerous densities corresponding to attachA thin layer of comment sites were apparent. pact fibrillar substance, located along cell borders, was a characteristic finding. At different intervals, densities composed of condensed fine fibrils were formed which obscured the otherwise distinct cell membrane. Large numbers of pinocytotic vesicles on the cell membranes were easily detected.
UROLOGY / DECELMBER 1978 / VOLUME
XII, NUMBER6
FIGURE 2. Electron micrographs. (A) Spindle-shaped tumor cells separated by wide spaces containing basement membrane-like material; large areas of cytoplasm are filled with glycogen (g), (B) Spindle-shaped tumor cells: microfilaments occupy considerable part of cytoplasm; numerous densities corresponding to attachment sites (arrowheads). (C) Tumor cells containing lipid droplets; bundle of microfilaments (f) is clearly recognizable in cytoplasm. (D) Portion of thick vascular wall: accumulation of collagen underneath endothelium (e) is conspicuous. Note the fibroblast-like cells ($1 with long cytoplasmic projections; smooth muscle cells (m) are also apparent. (Original magnifications, x 7,800, 13,200, 14,700, and 10,000, respectively.) The individual tumor cells were separated by a space varying in width from 100 nm. up to or over 1,000 nm. In this space a layer of fine fibrillar substance of low electron density, resembling basement membrane material, was noted. Occasionally the intercellular space and layer of basement membrane-like material were interrupted by junctions (fascia occludens).
UROLOGY
: DECEMBER1978
The tumor cells containing lipid droplets appeared to show similar ultrastructural characteristics as the cells described previously (Fig. 2C); however, they showed several differences. The amount of intracellular fat was variable between cells. In the cells with only moderate accumulation of lipids, the amount of glycogen was considerably Iess than in lipid-free cells.
/ VOLUME XII,NUMBER 6
719
The areas of RER were reduced as lvell. The and formapresence of SER was more apparent, tion of smooth-wal1ed fingerprints co~npostd of ER membranes with dilatation of peripheral tubules was also noted. The number of dense bodies increased and several meml,rane-bound clear vacuoles were seen containing only a few glycogen particles and some remnants of membranous structures. Cytosegrosomes were common. Mitochondrial swelling occurred as well. Microfilaments were see11 only in a thin peripheral rim of cytoplasm. In several cells, large lipid globules almost completely occupied displacing the organelles. In the cytoplasm, these areas several macrophages were observed. The blood vessels in the tumor had au endothelium markedly varying in thickness, but exhibiting normal features otherwise. Focally, where the basement membrane was ill-defined or even discontinuous, the endothelium was lying directly over the bundles of collagen or the cellular elements of the thick vascular wall (Fig. 2D). The latter consisted of spindle-shaped cells, exhibiting the same appearance as in other parts of the neoplasm, and of fibroblast-like elongated cells with well-developed RER and long cytoplasmic projections. An additional feature of vessels was the invagination of cell processes into the cytoplasm of endothelial cells. Since electron microscopy revealed only a granular matrix, a modest number of membranous profiles, aud a few small mitochondria, the cellular derivation of these inclusions could not be determined with certainty.
the kidney, an organ which normally has little smooth muscle and adipose tissue. However, the embryologic origin of the kidney from mesoderm is compatible with the mesenchymal nature of the tumor. It is not certain whether the thick-walled blood vessels are an integral part of the tumor or whether they represent an epiphenomenon resulting from an effect of the tumor on the normal development of its nutrient vcsscls. The prcsencc of abnormal smooth muscle cells within the vessel walls could be compatible with both explanations. Auother puzzling feature of angiomyolipoma is the intimate admixture of smooth mllscle cells and f:*t cells. Llie were unable to identify a precursor mesenchymal cell although we did observe an occasional cell showing features of both smooth muscle arid kit cells. In the present case, the majority of the tumor cells could he assigned with no difficulty to either a myocytic or lipocytic liue with only slight overlap between the two. anAnother noteworthy feature of giomyolipoma, observed in a previous study,” is the excessive accumulation of glycogeu in the smooth muscle cells. Rhabdomyoma of the heart, auother coluponent of the tuberous sclerosis complex, is also characterized by intracytoplasmic accumulation of glycogen, in this instance in cardiac muscle cells.6 Glycogen acof both ancumulation in inyocytes giomyolipomas and rhabdomyomas suggests a comtnon abnormality in metabolic pathways and possibly a common genetic defect in both these tumors.
Comment Previous light microscopic studies have indicated that the tumor cells, except for the lipomatous areas, are of smooth ~rluscle origin. The fine structural findings of Sun, White, and Bissada’ are consistent with this view. Other investigators, ho\vever, have suggested that angiomyolipomas derive from Schwann cells or are related to glomus tunlors.3.5 This study, although confirming the myoqrtic and Iipocytic origin of the tumor, demonstrates that the COILIponents of the tumor differ ultrastructura11v from normal smooth muscle cells, fat cells, an;1 Hood vessels. The nature and Inorphngenesis of angiomyolipomas are not known. There is 110 satisfactory answer to why this tumor is pecllliar to
720
Toronto,
Ontario, M5B lW8, Canada (DR. CHALVARD JIAN)
KOVACS,
HORVATH.
STUDY
Xl. 13. 11. D.
Pfr. I>.
Frown the Department Toronto, Canada
of Patholoe,
.4ngioln).olipoma is a benign tmnor of the kidne)’ Lvhich may, but not invariably, occur in patients with tuberous sclerosis. When associated with the tuberous sclerosis complex, the tumor tends to be small, multifocal, usuall>. bilateral, and asymptomatic; whereas, in patients with no stigmata of tuberous sclerosis, it is often large, and symptomatic. I unifocal. luGlateral, The tulnor has been studied in detail by light microscop);. I2 It consists of a variable mixture of spindle c&ells, adipose tissue, and thick-walled blood vessels. To date, to our knowledge, only two articles have described the ultrastructure of angioln)olipoma, and they differ as to the morphogenesis of the tumor.3q” The purpose of this article is to describe the ultrastructural features of renal arrgiomyolipoma and to shed some light on the nature of the spindle cells and on their relationship to the adipose cells and to the ahno1m;l1 thick-walled vessels. The tumor in this case, which occurred as an isolated finding in an otherwise healthy patient, is also compared with that of the 2 other cases with the tuberous sclerosis conlplexc.~‘~” (:asr
ULTRASTR’CJCTtJRAL
Report
.4 twc~nt~-six-fear-old oriental \voman, with no previous complaints, \vas admitted to the hospital because of sudden onset of a sharp, sevt’re pin beneath the left scapula. radiating down the back and above the iliac crest. Her Mood pr~~ssurc \\‘a~110170 m1I1. Hg. and the or+.
St. Illicharl’s
Hospital,
significant finding was left costl.bvc~rtel>ral I!rinal>.sis sho~vrcl 3 to 4 red tenderness. cells per high-power field. Uriile c:ultures werc sterile. Intravenorls pyelograph!,, rt~nal toinogmphy, and arteriography showel:l the upper pole of the left kidne). to t)c* ill\ol\ Cal \vith tumor. Sllrger\, re\realed a IIMSS arising fiwn the upper pole oi the left kidney and adherent to the diaphragm. A left nephrec+oiny was pt’rformrtl. The patient had an unevwtfnl wcwvery. Investigations failed to reveal an!.’ stigma of the tilberous sclerosis complex.
Thc~ specimen consisted of the left kidntb!; Lvith attached perirenal fat anal ipsilateral adrenal gland. A circwmscribed fi7m miss ineasuring 5 11). 4 lI>, 3 cm. occupied the ilpper pole of the kidtIe>. and had a variegated ~.ellow appearmcc on cut section. The tumor was llot encapsrilated hiit \vas distinct froni tlhc acljacerrt renal tissue. There wxs hemcirrhagr in the perirenal tat.
autl
the
;lcll.tYl;ll
glad
\\‘;I5 grtdy
t10r11ral.
FIGURE 1. Light microscopy shows the typica mingling of smooth muscle cells and fat cells. Part of the collagenized tcall of an abnormal blood vessel is also seen
in the right
lower
corner.
(X 250).
thickened hyalinized walls with scanty cellularity and lacked well-defined internal elastic lamina. The tumor had no true capsule. At the periphery of the tumor an occasional atrophic tubule was surrounded by spindle cells but no residual renal structures could be identified in the center. The periodic acid-Schiff (PAS) reaction stained intensely the clear cytoplasm of some of the spindle cells. The PAS positive granules were removed completely by diastase digestion, indicating that they represented cytoplasmic glycogen. Nuclear pleomorphism was slight and mitotic figures were infrequent. Electron
~rkx-oscopic
findings
Small pieces of tumor tissue, taken from various parts of the neoplasm, were fixed in 2.5 per cent glutaraldehyde in Sorensen buffer, postfixed in 1 per cent 0~04 in Millonig buffer, dehydrated in graded ethanol, processed throllgh propylene oxide, and embedded in Epon 812. Thick sections, cut with a Porter-Blum MT-2 ultramicrotome, were stained with toluidine blue and examined with a light microscope. Representative blocks containing various parts of tumor were selected. Ultrathin sections were
718
stained with uranyl acetate and lead citrate and studied with a Philips 300 eIectron lnicroscope. In the parts of tumor which contained no lipid droplets, the cells were mostly fusiform. The nudei were elongated with blunt edges at their poles. They were often irregularIy outlined with In a few nuclei unusual indeep indentations. clusion bodies were observed. The nucleoli were round and dense. The majority of cytoplasmic organelles were concentrated in the perinuclear areas, and large portions of the cytoplasm were occupied by microfilaments. The rough-s~l&ced endoplasmic reticulum (RER) was prominent and was represented by short cisternae studded with ribosomes. Free ribosomes and polysomes were also observed. The smooth-surfaced cndoplasmic reticulum (SER) was scanty. The perinuclear cytoplasm contained numerous mitochondria with slight variations in shape and size. They usually had a light matrix and transverse lamellar cristae. Mitochondrial abnormalities such as swelling and rarefaction were rare. The Golgi zones were conspicuous and were composed of five or six slightly or moderately dilated sacculi and several vesicles. In the Golgi region and also in other areas, groups of membrane-bound dense granules were found. They were spherical or pleomorphic, showing variable electron density. Occasionally, small crystalline structilres, bound by a single unit membrane or lying free in the hyaloplasm, were also detected. The most prominent ultrastructural feature was the presence of large amounts of glycogen, composed of alpha and beta particles, in the organelle-rich areas (Fig. 2A). The glycogen deposits reached extreme proportions in some cells, displacing the organelles to the periphery of the cytoplasm. In and around the glycogen masses, several irregularly shaped spaces were noted, IWUally delineated by a single membrane. Occasionally a connection between these structures and the SER tubules was noted. Large cytoplasmic areas were occupied by fine filamentous structures exhibiting the fine structural features of myofilaments (Fig, 2B). Numerous densities corresponding to attachA thin layer of comment sites were apparent. pact fibrillar substance, located along cell borders, was a characteristic finding. At different intervals, densities composed of condensed fine fibrils were formed which obscured the otherwise distinct cell membrane. Large numbers of pinocytotic vesicles on the cell membranes were easily detected.
UROLOGY / DECELMBER 1978 / VOLUME
XII, NUMBER6
FIGURE 2. Electron micrographs. (A) Spindle-shaped tumor cells separated by wide spaces containing basement membrane-like material; large areas of cytoplasm are filled with glycogen (g), (B) Spindle-shaped tumor cells: microfilaments occupy considerable part of cytoplasm; numerous densities corresponding to attachment sites (arrowheads). (C) Tumor cells containing lipid droplets; bundle of microfilaments (f) is clearly recognizable in cytoplasm. (D) Portion of thick vascular wall: accumulation of collagen underneath endothelium (e) is conspicuous. Note the fibroblast-like cells ($1 with long cytoplasmic projections; smooth muscle cells (m) are also apparent. (Original magnifications, x 7,800, 13,200, 14,700, and 10,000, respectively.) The individual tumor cells were separated by a space varying in width from 100 nm. up to or over 1,000 nm. In this space a layer of fine fibrillar substance of low electron density, resembling basement membrane material, was noted. Occasionally the intercellular space and layer of basement membrane-like material were interrupted by junctions (fascia occludens).
UROLOGY
: DECEMBER1978
The tumor cells containing lipid droplets appeared to show similar ultrastructural characteristics as the cells described previously (Fig. 2C); however, they showed several differences. The amount of intracellular fat was variable between cells. In the cells with only moderate accumulation of lipids, the amount of glycogen was considerably Iess than in lipid-free cells.
/ VOLUME XII,NUMBER 6
719
The areas of RER were reduced as lvell. The and formapresence of SER was more apparent, tion of smooth-wal1ed fingerprints co~npostd of ER membranes with dilatation of peripheral tubules was also noted. The number of dense bodies increased and several meml,rane-bound clear vacuoles were seen containing only a few glycogen particles and some remnants of membranous structures. Cytosegrosomes were common. Mitochondrial swelling occurred as well. Microfilaments were see11 only in a thin peripheral rim of cytoplasm. In several cells, large lipid globules almost completely occupied displacing the organelles. In the cytoplasm, these areas several macrophages were observed. The blood vessels in the tumor had au endothelium markedly varying in thickness, but exhibiting normal features otherwise. Focally, where the basement membrane was ill-defined or even discontinuous, the endothelium was lying directly over the bundles of collagen or the cellular elements of the thick vascular wall (Fig. 2D). The latter consisted of spindle-shaped cells, exhibiting the same appearance as in other parts of the neoplasm, and of fibroblast-like elongated cells with well-developed RER and long cytoplasmic projections. An additional feature of vessels was the invagination of cell processes into the cytoplasm of endothelial cells. Since electron microscopy revealed only a granular matrix, a modest number of membranous profiles, aud a few small mitochondria, the cellular derivation of these inclusions could not be determined with certainty.
the kidney, an organ which normally has little smooth muscle and adipose tissue. However, the embryologic origin of the kidney from mesoderm is compatible with the mesenchymal nature of the tumor. It is not certain whether the thick-walled blood vessels are an integral part of the tumor or whether they represent an epiphenomenon resulting from an effect of the tumor on the normal development of its nutrient vcsscls. The prcsencc of abnormal smooth muscle cells within the vessel walls could be compatible with both explanations. Auother puzzling feature of angiomyolipoma is the intimate admixture of smooth mllscle cells and f:*t cells. Llie were unable to identify a precursor mesenchymal cell although we did observe an occasional cell showing features of both smooth muscle arid kit cells. In the present case, the majority of the tumor cells could he assigned with no difficulty to either a myocytic or lipocytic liue with only slight overlap between the two. anAnother noteworthy feature of giomyolipoma, observed in a previous study,” is the excessive accumulation of glycogeu in the smooth muscle cells. Rhabdomyoma of the heart, auother coluponent of the tuberous sclerosis complex, is also characterized by intracytoplasmic accumulation of glycogen, in this instance in cardiac muscle cells.6 Glycogen acof both ancumulation in inyocytes giomyolipomas and rhabdomyomas suggests a comtnon abnormality in metabolic pathways and possibly a common genetic defect in both these tumors.
Comment Previous light microscopic studies have indicated that the tumor cells, except for the lipomatous areas, are of smooth ~rluscle origin. The fine structural findings of Sun, White, and Bissada’ are consistent with this view. Other investigators, ho\vever, have suggested that angiomyolipomas derive from Schwann cells or are related to glomus tunlors.3.5 This study, although confirming the myoqrtic and Iipocytic origin of the tumor, demonstrates that the COILIponents of the tumor differ ultrastructura11v from normal smooth muscle cells, fat cells, an;1 Hood vessels. The nature and Inorphngenesis of angiomyolipomas are not known. There is 110 satisfactory answer to why this tumor is pecllliar to
720
Toronto,
Ontario, M5B lW8, Canada (DR. CHALVARD JIAN)