Pflugers Archiv-european Journal of Physiology, 1990
Different chronic states of mesangial cell contraction were induced by variation of extracellular... more Different chronic states of mesangial cell contraction were induced by variation of extracellular volume in Munich-Wistar rats for 6 days to study the influence of mesangial cells on the geometry of the glomerular tuft. Stereological analysis of superficial glomeruli in volume-expanded rats (VR, treated with enalapril) and volume-reduced rats (VR, treated with indomethacin) revealed a glomerular tuft volume 28.7% smaller, and a capillary luminal volume 32% smaller in VR than in VE rats. The filtration area [defined as glomerular basement membrane (GBM) area facing fenestrated endothelium] was greatly reduced in VR rats (97±16×103 μm2 vs 137±13×103 μm2). The surface density (Sv) of the GBM was higher by approximately 10% in VR rats primarily due to the considerable increase in Sv of the perimesangial GBM subdivision (0.189±0.01 μm2/μm3 vs 0.153±0.01 μm2/μm3), indicating a higher degree of mesangial cell contraction in these animals. Our results suggest (1) that mesangial cell contraction plays a major role in the adaptation of the glomerular tuft to variations in extracellular volume; (2) that the relevance of mesangial cell contraction for the regulation of glomerular haemodynamics appears to be small; and (3) that the reduction in filtration area, although prominent, cannot fully account for the considerable decreases in the ultrafiltration coefficient observed by others in acute and chronic studies.
Glomerular capillaries are perfused at a high hydraulic pressure. Since the capillary mesangium i... more Glomerular capillaries are perfused at a high hydraulic pressure. Since the capillary mesangium interface presents no morphologically apparent pressure barrier, it is suggested that the hydraulic pressure in the mesangium may also be high. This paper analyzes the structures in the glomerular tuft that are capable of counteracting the distending forces exerted on the tuft by the high hydraulic pressure in its center. The skeletal element of the glomerular tuft is the glomerular basement membrane (GBM). The combination of the GBM with the contractile apparatus of mesangial cells represents the main system stabilizing the glomerular tuft. The mesangial cell-GBM connections counteracting the expansion of glomerular capillaries appear less susceptible to injury than those counteracting mesangial expansion.
The mesangial matrix of the rat glomerulus was studied by transmission electron microscopy in spe... more The mesangial matrix of the rat glomerulus was studied by transmission electron microscopy in specimens preserved by a modified technique, which avoids osmium tetroxide but uses tannic acid as a contrasting agent. It can be demonstrated that microfibrils are a major component of the normal glomerular mesangial matrix. They are non-branching tubular structures with a hollow centre, an undefined length and a thickness of ∼15 nm. Microfibrils make up a dense fibrillar network interconnecting mesangial cells and glomerular basement membrane.
Lesions in glomerular architecture include mesangial expansion, capillary ballooning, capillary u... more Lesions in glomerular architecture include mesangial expansion, capillary ballooning, capillary unfolding and microaneurysm formation. Such lesions appear to develop in response to mechanical overextension. A frequent pathway to segmental glomerulosclerosis starts from capillary ballooning and unfolding. Podocytes supporting those deranged capillaries are exposed to increased mechanical stress. This may lead to podocyte injury terminating in detachments from the GBM. Naked GBM areas at peripheral capillary loops allow the attachment of parietal cells to the GBM, i.e. the formation of a tuft adhesion to Bowman's capsule. An adhesion has a strong tendency to progress to segmental sclerosis.
The aim of this study is to investigate the role of the proximal tubule in microalbuminuria in th... more The aim of this study is to investigate the role of the proximal tubule in microalbuminuria in the early stage of diabetic nephropathy. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (50 mg/kg, i.v.). After 2 weeks, albumin delivery in the proximal tubule was measured using micropuncture and the endocytosis process of FITC-labeled albumin was evaluated with immunoelectron microscopy. Albumin was significantly reabsorbed in the proximal convoluted tubule (PCT) of controls (0.39±0.05 ng/min at early PCT to 0.17±0.08 at late PCT, P<0.05), whereas albumin reabsorption was inhibited in diabetic rats (0.27±0.05 to 0.21±0.08). Immunogold study revealed that FITC-albumin was significantly less reabsorbed in endosomes and lysosomes of S1 segments in diabetic rats than in controls (endosome: 1.20±0.10 vs 2.16±0.15 µm–1, P<0.0001; lysosome: 0.26±0.03 vs 0.83±0.07, P<0.0001). The expression of megalin, an endocytosis receptor, was decreased at the apical membrane of PCT in diabetic rats. The lipid peroxidation production in the proximal tubule was significantly increased in diabetic rats. In conclusion, albuminuria in early-stage diabetic rats can be partly explained by a decreased albumin endocytosis with reduced megalin expression and with increased lipid peroxidation in the proximal tubule.
The kidney plays an important role in protein metabolism. The albumin reabsorption in the proxima... more The kidney plays an important role in protein metabolism. The albumin reabsorption in the proximal tubule is disturbed in the early stage of diabetic nephropathy. We evaluated the effects of angiotensin converting enzyme inhibitor (ACEI) and angiotensin III type 1 receptor blocker (ARB) on albumin reabsorption and expression of megalin, an endocytosis receptor for albumin, in proximal tubules of streptozotocin (STZ)-induced diabetic-rats. Diabetic rats at the second week after STZ injection were treated with quinapril (3 mg/kg/day) or candesartan (0.05 mg/kg/day) for 2 weeks. The tubular reabsorption of fluorescein isothiocyanate (FITC)-labeled albumin was evaluated by immunogold electron microscopy, and megalin expression was investigated by immunohistochemistry and Western blotting. Reabsorption of FITC-labeled albumin and megalin expression were prominently inhibited in the proximal convoluted tubules of diabetic rats compared to the controls. Both quinapril and candesartan restored albumin reabsorption in the proximal tubule due to normalization of megalin expression. Urinary albumin excretion was significantly reduced by both ACEI and ARB treatment. Angiotensin II infusion decreased megalin expression and albumin reabsorption in the proximal tubule. In conclusion, angiotensin II blockade restored albumin reabsorption via amelioration of megalin expression in the proximal tubules of early stage diabetic rats.
The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by mea... more The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by means of high resolution scanning electron microscopy. Specimens were taken from kidneys perfused with paraformaldehyde, freeze-fractured and then processed with conductive staining. The fractured surface of glomerular tufts exhibited the inner and outer surface of the GBM uncovered by endothelial and epithelial cells. The lamina densa was composed of densely packed granular material together with scattered fibrils. The laminae rarae interna and externa were composed of a meshwork that showed some structural heterogeneities. The meshwork composing the lamina rara interna contained 5-to 9-nm-thick fibrils, had pores 11–30 nm wide, and was associated with granular material except in those places that corresponded with endothelial fenestrae. The meshwork of the lamina rara externa was made up of 6- to 11-nm-thick fibrils, and had smaller pores under the foot processes (10–24 nm wide) than those near the filtration slits (16–32 nm wide). In addition to the meshwork, the lamina rara interna contained microfibrils that were arranged differently depending on the topography of the capillary wall: scattered fibrils had no predominant orientation at the convex side, circumferential bundles lay at the concave side of the peripheral capillary wall, and had a circumferential arrangement in the paramesangial wall.
Morphological and morphometric studies of glomeruli were carried out in streptozotocin-induced di... more Morphological and morphometric studies of glomeruli were carried out in streptozotocin-induced diabetic rats using improved tissue processing and computerized morphometry. Increased mesangial matrix, occupying the enlarged diabetic mesangium, contained an abundance of dark granular material in addition to the microfibrils which were usually found in the control glomeruli. In the diabetic glomeruli, the lamina densa was thick and heterogeneous showing a dense layer both on its epithelial and endothelial aspects, and the lamina rara externa contained more fibrils than in control rats. Detailed estimation of the absolute values of the various compartments of the diabetic glomeruli by using perfusion-flxed materials and a computer-assisted digitizer revealed that the volume and surface area of the mesangium were increased more extensively than those of the capillary; the enlargement of the mesangial-capillary interface area was the most pronounced among the morphometric changes of the diabetic glomeruli; and that the moderate increase in capillary volume was associated with an increased radius. Our quantitative results showed that capillaries in the diabetic glomeruli had an extensively wider neck which may be the first sign of structural damage to the glomerular tuft.
The structure of the normal pulmonary arteries in the rat was studied with light and electron mic... more The structure of the normal pulmonary arteries in the rat was studied with light and electron microscopy after use of a newly devised technique of perfusion fixation and tissue preparation. We distinguished two main types of artery in the rat lung on the basis of the structure of the media, an elastic artery and a muscular artery. The elastic artery was characterized by an abundance of extracellular matrix in the media and by an oblique arrangement of smooth muscle cells to connect neighboring elastic laminae. It was subdivided into two segments, a classical elastic and a transitional elastic segment. The muscular artery was distinguished by a paucity of extracellular matrix in the media and by a circumferential arrangement of smooth muscle cells (or pericytes) enclosing the lumina, and was subdivided into four segments, a thick muscular, an ordinary muscular, a partially muscular and a nonmuscular segment. The smooth muscle cells in the muscular artery contained well-developed microfilament bundles compared with those in the elastic artery. Structural differences in smooth muscle cells and in extracellular matrix in the media between the elastic and muscular arteries may reflect the functional heterogeneity of pulmonary arteries in response to hypoxic pulmonary vasoconstriction and to vasoactive substances such as endothelium-derived relaxing and hyperpolarizing factors, and endothelin.
Pflugers Archiv-european Journal of Physiology, 1990
Different chronic states of mesangial cell contraction were induced by variation of extracellular... more Different chronic states of mesangial cell contraction were induced by variation of extracellular volume in Munich-Wistar rats for 6 days to study the influence of mesangial cells on the geometry of the glomerular tuft. Stereological analysis of superficial glomeruli in volume-expanded rats (VR, treated with enalapril) and volume-reduced rats (VR, treated with indomethacin) revealed a glomerular tuft volume 28.7% smaller, and a capillary luminal volume 32% smaller in VR than in VE rats. The filtration area [defined as glomerular basement membrane (GBM) area facing fenestrated endothelium] was greatly reduced in VR rats (97±16×103 μm2 vs 137±13×103 μm2). The surface density (Sv) of the GBM was higher by approximately 10% in VR rats primarily due to the considerable increase in Sv of the perimesangial GBM subdivision (0.189±0.01 μm2/μm3 vs 0.153±0.01 μm2/μm3), indicating a higher degree of mesangial cell contraction in these animals. Our results suggest (1) that mesangial cell contraction plays a major role in the adaptation of the glomerular tuft to variations in extracellular volume; (2) that the relevance of mesangial cell contraction for the regulation of glomerular haemodynamics appears to be small; and (3) that the reduction in filtration area, although prominent, cannot fully account for the considerable decreases in the ultrafiltration coefficient observed by others in acute and chronic studies.
Glomerular capillaries are perfused at a high hydraulic pressure. Since the capillary mesangium i... more Glomerular capillaries are perfused at a high hydraulic pressure. Since the capillary mesangium interface presents no morphologically apparent pressure barrier, it is suggested that the hydraulic pressure in the mesangium may also be high. This paper analyzes the structures in the glomerular tuft that are capable of counteracting the distending forces exerted on the tuft by the high hydraulic pressure in its center. The skeletal element of the glomerular tuft is the glomerular basement membrane (GBM). The combination of the GBM with the contractile apparatus of mesangial cells represents the main system stabilizing the glomerular tuft. The mesangial cell-GBM connections counteracting the expansion of glomerular capillaries appear less susceptible to injury than those counteracting mesangial expansion.
The mesangial matrix of the rat glomerulus was studied by transmission electron microscopy in spe... more The mesangial matrix of the rat glomerulus was studied by transmission electron microscopy in specimens preserved by a modified technique, which avoids osmium tetroxide but uses tannic acid as a contrasting agent. It can be demonstrated that microfibrils are a major component of the normal glomerular mesangial matrix. They are non-branching tubular structures with a hollow centre, an undefined length and a thickness of ∼15 nm. Microfibrils make up a dense fibrillar network interconnecting mesangial cells and glomerular basement membrane.
Lesions in glomerular architecture include mesangial expansion, capillary ballooning, capillary u... more Lesions in glomerular architecture include mesangial expansion, capillary ballooning, capillary unfolding and microaneurysm formation. Such lesions appear to develop in response to mechanical overextension. A frequent pathway to segmental glomerulosclerosis starts from capillary ballooning and unfolding. Podocytes supporting those deranged capillaries are exposed to increased mechanical stress. This may lead to podocyte injury terminating in detachments from the GBM. Naked GBM areas at peripheral capillary loops allow the attachment of parietal cells to the GBM, i.e. the formation of a tuft adhesion to Bowman&#39;s capsule. An adhesion has a strong tendency to progress to segmental sclerosis.
The aim of this study is to investigate the role of the proximal tubule in microalbuminuria in th... more The aim of this study is to investigate the role of the proximal tubule in microalbuminuria in the early stage of diabetic nephropathy. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (50 mg/kg, i.v.). After 2 weeks, albumin delivery in the proximal tubule was measured using micropuncture and the endocytosis process of FITC-labeled albumin was evaluated with immunoelectron microscopy. Albumin was significantly reabsorbed in the proximal convoluted tubule (PCT) of controls (0.39±0.05 ng/min at early PCT to 0.17±0.08 at late PCT, P<0.05), whereas albumin reabsorption was inhibited in diabetic rats (0.27±0.05 to 0.21±0.08). Immunogold study revealed that FITC-albumin was significantly less reabsorbed in endosomes and lysosomes of S1 segments in diabetic rats than in controls (endosome: 1.20±0.10 vs 2.16±0.15 µm–1, P<0.0001; lysosome: 0.26±0.03 vs 0.83±0.07, P<0.0001). The expression of megalin, an endocytosis receptor, was decreased at the apical membrane of PCT in diabetic rats. The lipid peroxidation production in the proximal tubule was significantly increased in diabetic rats. In conclusion, albuminuria in early-stage diabetic rats can be partly explained by a decreased albumin endocytosis with reduced megalin expression and with increased lipid peroxidation in the proximal tubule.
The kidney plays an important role in protein metabolism. The albumin reabsorption in the proxima... more The kidney plays an important role in protein metabolism. The albumin reabsorption in the proximal tubule is disturbed in the early stage of diabetic nephropathy. We evaluated the effects of angiotensin converting enzyme inhibitor (ACEI) and angiotensin III type 1 receptor blocker (ARB) on albumin reabsorption and expression of megalin, an endocytosis receptor for albumin, in proximal tubules of streptozotocin (STZ)-induced diabetic-rats. Diabetic rats at the second week after STZ injection were treated with quinapril (3 mg/kg/day) or candesartan (0.05 mg/kg/day) for 2 weeks. The tubular reabsorption of fluorescein isothiocyanate (FITC)-labeled albumin was evaluated by immunogold electron microscopy, and megalin expression was investigated by immunohistochemistry and Western blotting. Reabsorption of FITC-labeled albumin and megalin expression were prominently inhibited in the proximal convoluted tubules of diabetic rats compared to the controls. Both quinapril and candesartan restored albumin reabsorption in the proximal tubule due to normalization of megalin expression. Urinary albumin excretion was significantly reduced by both ACEI and ARB treatment. Angiotensin II infusion decreased megalin expression and albumin reabsorption in the proximal tubule. In conclusion, angiotensin II blockade restored albumin reabsorption via amelioration of megalin expression in the proximal tubules of early stage diabetic rats.
The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by mea... more The fine structure of the glomerular basement membrane (GBM) of the rat kidney was studied by means of high resolution scanning electron microscopy. Specimens were taken from kidneys perfused with paraformaldehyde, freeze-fractured and then processed with conductive staining. The fractured surface of glomerular tufts exhibited the inner and outer surface of the GBM uncovered by endothelial and epithelial cells. The lamina densa was composed of densely packed granular material together with scattered fibrils. The laminae rarae interna and externa were composed of a meshwork that showed some structural heterogeneities. The meshwork composing the lamina rara interna contained 5-to 9-nm-thick fibrils, had pores 11–30 nm wide, and was associated with granular material except in those places that corresponded with endothelial fenestrae. The meshwork of the lamina rara externa was made up of 6- to 11-nm-thick fibrils, and had smaller pores under the foot processes (10–24 nm wide) than those near the filtration slits (16–32 nm wide). In addition to the meshwork, the lamina rara interna contained microfibrils that were arranged differently depending on the topography of the capillary wall: scattered fibrils had no predominant orientation at the convex side, circumferential bundles lay at the concave side of the peripheral capillary wall, and had a circumferential arrangement in the paramesangial wall.
Morphological and morphometric studies of glomeruli were carried out in streptozotocin-induced di... more Morphological and morphometric studies of glomeruli were carried out in streptozotocin-induced diabetic rats using improved tissue processing and computerized morphometry. Increased mesangial matrix, occupying the enlarged diabetic mesangium, contained an abundance of dark granular material in addition to the microfibrils which were usually found in the control glomeruli. In the diabetic glomeruli, the lamina densa was thick and heterogeneous showing a dense layer both on its epithelial and endothelial aspects, and the lamina rara externa contained more fibrils than in control rats. Detailed estimation of the absolute values of the various compartments of the diabetic glomeruli by using perfusion-flxed materials and a computer-assisted digitizer revealed that the volume and surface area of the mesangium were increased more extensively than those of the capillary; the enlargement of the mesangial-capillary interface area was the most pronounced among the morphometric changes of the diabetic glomeruli; and that the moderate increase in capillary volume was associated with an increased radius. Our quantitative results showed that capillaries in the diabetic glomeruli had an extensively wider neck which may be the first sign of structural damage to the glomerular tuft.
The structure of the normal pulmonary arteries in the rat was studied with light and electron mic... more The structure of the normal pulmonary arteries in the rat was studied with light and electron microscopy after use of a newly devised technique of perfusion fixation and tissue preparation. We distinguished two main types of artery in the rat lung on the basis of the structure of the media, an elastic artery and a muscular artery. The elastic artery was characterized by an abundance of extracellular matrix in the media and by an oblique arrangement of smooth muscle cells to connect neighboring elastic laminae. It was subdivided into two segments, a classical elastic and a transitional elastic segment. The muscular artery was distinguished by a paucity of extracellular matrix in the media and by a circumferential arrangement of smooth muscle cells (or pericytes) enclosing the lumina, and was subdivided into four segments, a thick muscular, an ordinary muscular, a partially muscular and a nonmuscular segment. The smooth muscle cells in the muscular artery contained well-developed microfilament bundles compared with those in the elastic artery. Structural differences in smooth muscle cells and in extracellular matrix in the media between the elastic and muscular arteries may reflect the functional heterogeneity of pulmonary arteries in response to hypoxic pulmonary vasoconstriction and to vasoactive substances such as endothelium-derived relaxing and hyperpolarizing factors, and endothelin.
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Papers by Tatsuo Sakai