Topological analysis of the three-dimensional structure of the human renal glomerulus using a computer-aided reconstruction system

A quantitative topological analysis of human renal glomerular capillaries reconstructed from serial sections was performed with the aid of a computer system. The peripheral portions of five glomeruli from each of the subcapsular, midcortical, and juxtamedullary zones were reconstructed, each portion corresponding to about one-sixth the diameter of a glomerulus. Statistically, there was no significant difference in the cycle rank (number of independent cycles) of the glomerular capillary network per unit volume of glomerular capillaries or Bowman's capsule among the three renal zones. It was proved that, irrespective of the location of the glomerulus, the glomerular capillary network possessed a larger cycle rank as the volume of the glomerular capillaries or Bowman's capsule increased. This finding strongly suggests that the mean cycle rank per single glomerulus is largest in the juxtamedullary zone.     

Glomerular hemodynamics and vascular structure in uremia: A network analysis of glomerular path lengths and maximal blood transit times computed for a microvascular model reconstructed from subserial ultrathin sections

The dimensions of individual capillary segments were determined from a glomerular model constructed on the basis of electron micrographs of subserial ultrathin sections of kidney tissue from a rat made chronically uremic by subtotal nephrectomy. Hemodynamic calculations used computer programs for node pressure analysis and for the determination of path lengths and transit times from afferent to efferent arteriole. The afferent arteriole divided into five primary capillary segments. Three of these carried 84% of the flow through 632 paths, which accounted for 54% of the capillary endothelial area. Their mean path length was 520.97 μm, mean of segment numbers per path 17.66, and mean transit time 0.50 sec. The remaining two capillary segments carried 16% of the flow and led to paths accounting for the remaining 46% of the capillary area. The number of paths from the afferent arteriole through these two segments was 21,244. Mean path length from the initial node through these two segments was 852.18 μm, mean of segment numbers per path 29.62, and mean transit time 20.76 sec. The most striking difference between the two sets of paths was in transit time. This asymmetry would tend to reduce the filtration surface and the K_f (hydraulic conductance × filtering surface area) by causing early filtration pressure equilibrium in much of the capillary network, and suggests an intrinsic glomerular aspect of chronic renal insufficiency.     

Glomerular size and structure in diabetes mellitus

The volume of the glomerular tuft and its components was estimated in juvenile diabetics at onset and after one to six year's duration of the disease. An enlargement of the glomerular tuft was demonstrated in the newly diagnosed diabetics. In these patients the volume of both the individual capillary lumina and of the individual glomerular cells was enlarged, whereas the number of cells was unchanged. Although a partial normalization is seen in diabetics after a few years' disease, such patients still have an elevated capillary volume. The possible relationship between these findings and the well-documented elevation of the glomerular filtration rate in diabetics is discussed. It is suggested that the mechanism behind the morphological and functional changes is either a pressure-induced unfolding of the capillary wall or an increase of the capillary wall area, but the present study permits no distinction between these possibilities.     

Dirofilariasis. IV. Glomerulonephropathy induced by Dirofilaria immitis infection

The kidneys of dogs chronically infected with Dirofilaria immitis were studied by light, electron, and immunofluorescent microscopy. The glomeruli of dogs with high microfilaremia showed a moderately increased mesangium and thickened glomerular basement membrane. There was a deposition of electron-dense particles which were continuously distributed in the lamina rara interna and lamina densa of the glomerular basement membrane. Immunofluorescent microscopy showed intense IgG deposits in a uniform linear pattern along the glomerular basement membrane. This linear deposition of IgG appeared to correspond to continuous distribution of electron-dense particles in the basement membrane. Microfilariae present in the glomerular capillary lumens were connected with the capillary endothelial cells by narrow cytoplasmic bands. The interaction of the worm's internal substances with the glomerular capillaries through these cytoplasmic bands may deposit worm antigen on the glomerular capillary wall before antigen-antibody interaction takes place. These findings suggest that the glomerulopathy seen in dogs infected with D. immitis is caused by the in situ formation of immune complexes in the glomerular basement membrane.     

Fast accumulation of basement membrane material and the rate of morphological changes in acute experimental diabetic glomerular hypertrophy

Summary Renal glomerular structures have been studied during the initial phases of renal and glomerular hypertrophy in streptozotocin diabetic rats. — After four days of diabetes significant increases were found in the following absolute structural quantities: the tuft volume (25%), the surface area (42%) and volume (46%) of peripheral basement membrane, and capillary luminal volume (29%). No further changes in these quantities took place over the succeeding 47 days. The geometry of the capillaries, however, changed over this period: at 4 days the total capillary length had increased significantly (32%) with an unchanged average cross sectional area, whereas at 47 days the length had reverted to normal, but average cross sectional area had increased (30%). The increased surface of filtering area may be the morphological counterpart of increased glomerular filtration rate. The changes in glomerular volume were thus accompanied by changes in glomerular structural composition which after a few weeks returned to normal, in balance with the increased volume.     

Lumenal narrowing and endothelial cell swelling in skeletal muscle capillaries during hemorrhagic shock

Capillary lumenal narrowing in hemorrhagic shock was investigated using intravital microscopy to visualize capillaries directly in the rabbit tenuissimus muscle after a rapid single-withdrawal of 40% of the blood volume. The ensuing 1 hr shock period in the animals was marked by a protracted systemic hypotension and metabolic acidosis. Changes in capillary lumen diameter were inferred by changes in the width and length of red blood cells (RBCs) as they traversed the capillary. By the end of the shock period, RBC flux from nine experiments decreased over 60% with a -24.3 +/- 9.3% and +22.8 +/- 6.4% change in RBC width and length, respectively. The narrowed capillary lumen resulting from endothelial cell swelling elevates hydraulic resistance which may hinder resuscitation efforts to restore shock-impaired flow.     

Fluid Shear Attenuates Endothelial Pseudopodia Formation into the Capillary Lumen

Objective: Endothelial cells have the ability to undergo morphological shape changes, including projection of cytoplasmic pseudopodia into the capillary lumen. These cytoplasmic projections significantly influence the hemodynamic resistance to blood flow. To examine mechanotransduction mechanisms, we investigated in vivo the hemodynamic conditions in capillaries that control endothelial pseudopod formation. Materials and Methods: Capillaries in rat skeletal muscle were fixed under carefully controlled perfusion conditions. The formation of endothelial pseudopodia were observed in cross‐sections with electron microscopy and quantified with differential interference contrast microscopy under physiological, stasis, and reperfusion flow conditions. Results: Application of physiological levels of fluid flow prevents capillary endothelium to project pseudopodia into the capillary lumen. Reduction of fluid flow to near zero promotes the incidence of pseudopod projection from 5% to 55% of capillaries. After capillary pseudopodia have formed under static conditions, about one‐half retract upon restoration of fluid flow. The presence of red blood cells in the capillary lumen prevents pseudopod formation. Conclusions: The results suggest that there is a mechanism that serves to control cytoplasmic projections in capillary endothelium that is under the control of hemodynamic fluid stress. Investigation of pseudopodia growth on endothelial cells may be significant in understanding capillary obstruction in cardiovascular diseases.     

Coronary capillaries during normal and pathological growth

The adaptive capacity of the myocardium with respect to its capillary concentration and distribution has been measured morphometrically during the hypertrophic growth occurring physologically after birth and as a result of induced overload in the adult. In particular, the growth of the capillary network of the left ventricle was examined in rats from one to 150 days of age and in rats with spontaneous hypertension, aortic stenosis, two-kidney one-clip renal hypertension and myocardial infarction. The following quantitative structural parameters of the capillary microvasculature were analyzed: 1. Capillary luminal volume density; 2. Capillary luminal surface density; 3. The average diffusion distance for oxygen; and 4. The aggregate capillary length in the whole ventricle. The major conclusions of the present study are: 1. The postnatal growth of the heart is characterized by lengthening of the whole capillary network that is linearly related to the aging process; 2. The rate of capillary proliferation, measured by changes in capillary density, is greater in the first month of age; 3. In contrast to postnatal development, lengthening of the capillary microvasculature is not a consistent adaptive mechanism of induced cardiac hypertrophy; 4. Capillary luminal volume and surface densities and the diffusion distance for oxygen are essentially maintained in spontaneous, mechanical and renal hypertension; and 5. Cardiac hypertrophy in acute and healed myocardial infarction results in alterations of the capillary properties implicated in tissue oxygenation that may constitute the morphological counterpart of the greater vulnerability to ischemic episodes of the hypertrophied myocardium after infarction.     

Three-dimensional microstructural abnormality of the coronary capillary network after myocardial reperfusion--comparison between 'reflow' and 'no-reflow'.

BACKGROUND: The 3-dimensional (D) structural abnormalities of the coronary capillary network and capillary volume changes after myocardial reperfusion were investigated using confocal laser scanning microscopy. METHODS AND RESULTS: Using open-chest anesthetized rats' hearts, the left anterior descending artery (LAD) was occluded for 7 min followed by reperfusion. The hearts were divided into 2 groups: (1) reperfused area stained well by intravenous indocyanine green after reperfusion (Reflow), and (2) lack of staining in the reperfused area (No-reflow). The entire coronary microvasculature was filled with contrast medium using a Langendorff's perfusion system. Capillaries were observed 3-dimensionally in the control and reperfused areas in both the Reflow and No-reflow group and the capillary volume fraction was computed from the 3-D images. The reperfused area in both groups showed decreased capillary diameter with waving and shrinkage configuration. In the No-reflow group, marked interruption of capillary network was observed. In the Reflow group the capillary volume fraction was significantly reduced in the reperfused area compared with the control area (14.8+/-4.1% vs 22.2+/-5.6%, p<0.05). Capillary volume fraction was further decreased in the No-reflow group compared with Reflow (5.3+/-1.4% vs 14.8+/-4.1%, p<0.01). CONCLUSION: The morphological changes in coronary capillaries after reperfusion were characterized by waving and shrinkage, which resulted in a reduction in capillary volume.     

Capillary tortuosity and degree of contraction or extension of skeletal muscles

The effect of muscle contraction, and extension, on capillary anisotropy was investigated in rat m. soleus fixed by vascular perfusion at sarcomere lengths ranging from 1.62 micron (tetanizing stimulation of sciatic nerve) to 2.85 micron (ankle joint maximally flexed. Capillary length density and tortuosity were estimated by morphometry using two sets of sections (0 and 90 degrees to the fiber axis). Capillary orientation distribution was evaluated from a series of sections taken at 0 to 90 degrees (by steps of 5-10 degrees) to the fiber axis in six preparations (sarcomere length range, 1.62-2.85 micron; capillary length density, 900-2000 mm-2). The Fisher axial distribution provided a good fit for modeling capillary orientation distribution in each case. For a comparable capillary length density per volume of muscle fiber (approximately equal to 2000 mm-2), the degree of orientation of capillary segments parallel to the fiber axis was two and four times larger in extended m. soleus than in the muscles fixed at 1.98- and 1.62-micron sarcomere lengths, respectively. In preparations fixed at 2.85, 1.98, and 1.62 micron, capillary length density per volume of muscle fiber was, respectively, 14, 44, and 65% larger than revealed by capillary counts per sectional area of muscle fiber on transverse section only, an often used parameter to compare capillarity in different muscles.     

Decreased nailfold capillary density in Raynaud's phenomenon: a reflection of immunologically mediated local and systemic vascular disease?

Nailfold capillary patterns were studied in 107 patients with Raynaud's phenomenon (RP), including patients wih (n = 39) and without (n = 68) connective tissue disease (CTD). Capillary density was decreased in patients with sclerodactyly, digital ulcers, tuft resorption, and telangiectasia, compared with patients without these symptoms. In addition, an inverse relationship was found between the severity of RP at first presentation (as graded by photoelectric plethysmography during cooling) and the capillary density in patients with CTD (r = -0.45; p less than 0.05). In the total group of patients nailfold capillary density was inversely related to organ system involvement (r = -0.52; p less than 0.01). Decreased nailfold capillary density was observed, in particular, in patients with oesophageal hypomotility and in patients with chest x-rays compatible with interstitial fibrosis. As to factors supposedly involved in the pathogenesis of vascular changes in CTD, the presence of autoantibodies, increased levels of circulating immune complexes, and increased levels of acute phase reactants were all associated with a decreased number of nailfold capillaries. We conclude that loss of nailfold capillaries as observed by microscopy is a reflection of local and systemic vascular disease.     

Tridimensional ultrastructure of glomerular capillary endothelium revealed by high-resolution scanning electron microscopy

Recent advances in specimen preparation techniques and scanning electron microscope (SEM) design have permitted ultrastructural examination of the glomerular capillary wall in three dimensions using high-resolution scanning electron microscopy (HRSEM). Specimens in which the cytosol and cytoskeleton have been extracted, but cell membranes nuclear structures and organelles left in place, were studied using a Hitachi SEM with a resolution of approximately 3 nm. Each HRSEM micrograph displayed a depth of field and information content equivalent to 15-30 consecutive, ultrathin, transmission electron microscope (TEM) sections viewed simultaneously in perfect serial alignment. The results have confirmed previous ultrastructural observations obtained by use of TEM and, in addition, have revealed new ultrastructural features of the normal rat glomerulus. A morphometric analysis of glomerular endothelium carried out using the HRSEM micrographs revealed that the endothelial cell processes, which lie between the fenestrae, are nearly circular in cross section and that they, as well as the fenestrae, have a diameter of approximately 60 nm. The potential functional role of the fenestrae in controlling access to the underlying basement membrane requires further study.     

Glomerular hemodynamics and vascular structure. The pattern and dimensions of a single rat glomerular capillary network reconstructed from Ultrathin sections.

This paper presents a quantitative study of a model of a rat glomerulus reconstructed from electron micrographs of subserial ultrathin sections. The entire capillary network is graphed, and the diameters and lengths of the capillary segments of the network are recorded, along with the computed resistance of each capillary segment to blood flow. These observations in turn are interpreted in relation to the theory of hydrodynamic flow through cylinders with permeable walls: Bounds are calculated for the pressure drops between the nodes of the network and the efferent arteriole, given the afferent and efferent blood flow. When recently published estimates of single-nephron afferent and efferent plasma flow of the Munich-Wistar rat were used in these calculations, a minimum pressure drop between afferent and efferent arteriole of 2.92 mm Hg was computed. This corroborates studies which suggest that filtration equilibrium is attained in the Munich-Wistar rat.     

Structural changes in the diabetic kidney

Diabetic glomerulopathy is characterized by a very slow development of basement membrane (BM) accumulation, manifested as thickening of the peripheral BM and increased volume of the mesangial BM-like material (BMLM) with mesangial expansion. The initiation of the process is probably at the onset of diabetes since the BM thickening is detectable after a few years. The BM accumulations at the two sites (PBM and BMLM) in the glomerular tuft are considered as two different expressions of a fundamental BM abnormality. The two locations present different conditions for quantitation, may have a different biochemical make-up, and immediate functional implications of the abnormalities may differ as well. In the long run, however, the two in concert lead to the ultimate solidification of the glomerular tuft with loss of capillary surface. The end-stage is glomerular closure, with elimination of glomerular function. A very close correlation has been found between the total remnant surface area of the glomerular capillaries and the level of GFR. Along with the classical changes of the diabetic glomerulopathy, changes in glomerular size are detectable. In early diabetes during the stages of glomerular hyperfunction, hypertrophy develops acutely at the onset of diabetes, leading to an increase in capillary surface corresponding to the increase in filtration rate. In the advanced stages when glomerular closure involves a proportion of the nephrons compensatory hypertrophy develops, thereby probably helping to preserve capillary surface for a period of time. The exact mechanisms that may influence these developments are not known, but underlying them all are the metabolic abnormalities of diabetes.     

Clinicopathologic and histologic evaluation of Dirofilaria immitis-induced nephropathy in dogs

Twelve beagles were infected with 200 Dirofilaria immitis infective larvae to study glomerular lesions associated with filariasis. All developed high serum levels of antibodies to dirofilarial antigens and became persistently microfilaremic. The dogs were killed at various times between 398 and 562 days post-infection and renal lesions were examined by light, electron, and immunofluorescent microscopy and antibody elution techniques. A membranoproliferative glomerulonephritis was observed in all dogs. Immunofluorescence was positive in all; predominantly in a fine granular pattern along the glomerular capillary wall. Ultrastructural examination showed intramembranous globular electron-dense deposits and a linear band of fine electron-dense particles in all dogs. Antibody elution studies demonstrated antibody reactive to dirofilarial antigens. In a subsequent experiment, an aqueous-soluble antigen prepared from adult female D. immitis was infused into the renal arteries of 5 heartworm-naive dogs. Immunofluorescent examination of the infused kidneys showed dirofilarial antigen present on the glomerular capillary wall in a fine granular pattern indicating there was adherence of the antigen to the capillary wall. These observations support the hypothesis of in situ immune complex formation as part of the pathogenesis of glomerulonephritis associated with dirofilariasis.     

Pressure induced deformation of the interstitial route across synovium and its relation to hydraulic conductance

The ease with which fluid passes across the synovial lining (i.e., the lining's hydraulic conductance) is enhanced when intraarticular fluid pressure (IAP) is raised acutely to pathological levels in rabbit knees. A structural basis for this pathophysiological change was sought by morphometry of synovial sections from rabbit knees fixed in situ at less than or equal to 5 cm H2O and 25 cm H2O IAP. Light and electron microscopy showed that the main structural changes induced in areolar synovium by raising IAP to 25 cm H2O were (1) a reduction in synovial thickness to 56% control value; (2) an increase in the area of interstitium exposed at the synovial surface (3) an increased proximity of the synovial capillaries to the joint lumen, the mean distance of capillaries from the surface falling from 8.9 +/- 0.5 microns (n = 391) to 3.3 +/- 0.3 microns (n = 92: p less than 0.001). The capillary profiles showed slight compression under 25 cm H2O IAP, but no collapse. The ratio of interstitial area to thickness is the geometric factor governing hydraulic conductance. The maximum change in interstitial area/thickness was 6.8 times for the blood-joint barrier. A change of this magnitude accounts partly (but not fully) for the experimentally observed conductance changes; and it highlights the importance of capillary depth as a factor governing exchange in joints.     

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??Abstract??Electron microscopy plays an important role in renal biopsy for pathological diagnosis.Most of renal biopsy cases entirely depend on the electron microscopy to confirm a final pathological diagnosis.Furthermore??many renal biopsy cases are diagnosed by electron microscopy combined with other specific examinations.The value of electron microscopy in diagnosis of hereditary glomerular disease is especially high??and diagnosis of the thin basement membrane nephropathy (TBMN) depends entirely on electron microscopy.Many other inherited glomerular diseases??e.g.Alport syndrome??Fabry disease??collagen ?? glomerulopathy??and so on all had specific ultrastructural changes.Therefore??patients highly suspected with hereditary glomerular diseases should be ensured to obtain enough tissue for electron microscopy examination.     

Influence of vascular and transpulmonary pressures on the functional morphology of the pulmonary microcirculation

The effects of airway and vascular pressures on pulmonary capillary morphology were examined by rapidly freezing in situ dog lungs under Zone 1, 2, and 3 conditions at transpulmonary pressures (P_tp) of 5, 15, and 25 cm H₂O. The tissue was preserved using a freeze substitution technique and analyzed by transmission electron microscopy. Capillary and septal wall widths were measured on random sections, and an estimate of the thickness of the blood:gas barrier was made. In addition, the depths of any folds penetrating into the capillary lumen were also measured. Under Zone 2 and 3 conditions, increasing the perfusing pressure increased the mean capillary width, but at higher P_tp, this effect was reduced. Mean fold depth was reduced as P_tp. was increased under both Zone 2 and 3 conditions. At low P_tp (5cm H₂O), mean fold depth was unresponsive to changes in perfusing pressure, but decreased significantly as perfusing pressure increased when the P_tp, was 15 or 25 cm H₂O. Wall thickness was unaffected by change in P_tp. Under Zone 2 conditions, wall thickness was independent of perfusing pressure. However, under Zone 3 conditions, at P_tp of 15 and 25 cm H₂O, there was a significant decrease in wall thickness as perfusing pressure was increased. Presumably, this decrease in wall thickness is due to distention of pulmonary capillaries at medium and high inflation pressures.     

Capillary geometrical changes with fiber shortening in rat myocardium.

Capillary-to-fiber geometrical relations constitute an integral component of peripheral gas exchange. Determination of capillary length and surface area density necessitates quantification of capillary orientation (i.e., tortuosity and branching). In skeletal muscle, capillary tortuosity increases in a curvilinear fashion at reduced sarcomere length, and this compensates for decreased capillary density as fiber cross-sectional area increases. To investigate these relations in myocardium, rat hearts were glutaraldehyde perfusion-fixed in calcium- or barium-induced "systole" to provide varying degrees of fiber shortening. Morphometric techniques were used to analyze capillary geometry in subepicardium (EPI) and subendocardium (ENDO) using 1-micron sections cut transverse and longitudinal to the muscle fiber axis. Capillary density on transverse and longitudinal sections, capillary diameter, fiber cross-sectional area, and sarcomere length were determined in each region. Capillary surface density was computed, and values were related to sarcomere length and compared with published data for diastolic hearts. Sarcomere length in systole ranged from 2.06 +/- 0.03 to 1.35 +/- 0.02 microns (EPI) and from 1.93 +/- 0.04 to 1.44 +/- 0.04 microns (ENDO). Fiber cross-sectional area (EPI, 344 +/- 13 microns2; ENDO, 343 +/- 12 microns2) was significantly larger, and capillary density on transverse sections was significantly smaller (EPI, 4,105 +/- 318 mm-2; ENDO, 4,145 +/- 267 mm-2) than in hearts arrested in diastole. Compared with skeletal muscle, capillary tortuosity was substantially less increased by fiber shortening. Capillary tortuosity and branching did not differ between EPI and ENDO and contributed a maximum of 33% (range, 13-33%) to capillary length density and surface area at a sarcomere length of 1.45 +/- 0.04 microns. Compared with diastolic hearts, capillary length density decreased on average by 19.6% (EPI) and 17.7% (ENDO); similarly, capillary surface density decreased 19.9% (EPI) and 13.7% (ENDO). We conclude that, with fiber shortening in the heart, fiber cross-sectional area increases and capillary numerical density decreases as predicted from reduced sarcomere length. Combined with the minimal geometrical changes of the capillary bed at shorter fiber lengths, this results in a lower capillary length and surface area per fiber volume in systole. Consequently, the structural potential for O2 diffusion into myocytes is determined, in part, by fiber length.     

Capillary Microscopy in Eosinophilic Fasciitis

Capillary microscopy was performed on 19 patients with eosinophilic fasciitis. These patients were compared with 13 individuals with progressive systemic sclerosis (scleroderma). Capillary patterns were normal in 16 of 19 (84%) eosinophilic fasciitis patients; 3 exhibited either borderline or nonspecific changes, and none showed a definite scleroderma pattern. In contrast, characteristic nailfold capillary changes, consisting of both dilatation and loss of capillaries, were present in 11 of 13 (85%) scleroderma patients; the remaining 2 showed scleroderma-type abnormalities of only 1 finger and were, therefore, classified as borderline. These results suggest that capillary microscopy may help to distinguish these 2 disorders.