The Generalized Shwartzman Reaction in Association With E. Coli Enterotoxemia in a Pig

A pig at the age of approx. 10 weeks died after four days of illness. Distinct necrotic changes were found both in the skin and the cortex of the kidneys. The histological examination revealed fibrinoid thrombi in skin vessels. In the kidneys similar thrombi were observed in capillaries of the glomeruli and in their afferent arterioles and in the interlobular arteries. In these vessels there were also a fibrinoid mural necrosis. These changes were in accordance with those expected to occur in the generalized Shwartzman reaction (GSR). The diagnosis of Escherichia coli enterotoxemia was based on the pathomorphological changes in the alimentary tract. The E. coli enterotoxemia was considered the cause of the GSR-changes.     

Renal vascular changes in hydronephrosis in rats: scanning electron microscopy of resin casts of glomerular capillaries

Vascular changes in the hydronephrotic glomeruli of rats were examined using corrosion casts, India ink injection and histological methods. In severe cases, the diameter of the glomerular capillary conglomerations decreased to 40-90 microns as a result of glomerular atrophy. The capillaries of these glomeruli also had reduced diameters, and were strangulated and torn off to varying degrees. The intact capillary network of the renal medulla was destroyed and the normal alignment of capillaries parallel to the tubules was lost. These morphological characteristics may account in part for the difficulty in glomerular capillary blood circulation.     

The formation of the insulo-acinar portal system in the pancreas of human fetuses]

In human fetuses 12-20-week-old peculiarities in creation and development of the pancreatic hemo-microcirculatory bed have been studied in connection of its incretory part formation. The vascular glomerulus begins to form on the 12th-14th week as transformation of capillaries of the exocrinic parenchyma into the glomerular capillaries, which, on their getting out of the glomeruli, turn into vessels. The latter participate in formation of the insulo-acinar portal system. Certain structures have been revealed for adaptation of an elevated volume of the blood stream in the glomeruli place, where the glomerular microvessels pass into the acinar anastomoses. The latter perform shunting of the glomerular capillaries with the arteriole, which brings blood, enriched in insular hormones, to distantly situating acinar cells.     

Hindered transport of macromolecules in isolated glomeruli. I. Diffusion across intact and cell-free capillaries.

The filtrate formed by renal glomerular capillaries must pass through a layer of endothelial cells, the glomerular basement membrane (GBM), and a layer of epithelial cells, arranged in series. To elucidate the relative resistances of the GBM and cell layers to movement of uncharged macromolecules, we measured the diffusional permeabilities of intact and cell-free capillaries to narrow fractions of Ficoll with Stokes-Einstein radii ranging from 3.0 to 6.2 nm. Glomeruli were isolated from rat kidneys, and diffusion of fluorescein-labeled Ficoll across the walls of single capillary loops was monitored with a confocal microscopy technique. In half of the experiments the glomeruli were treated first to remove the cells, leaving skeletons that retained the general shape of the glomerulus and consisted almost entirely of GBM. The diffusional permeability of cell-free capillaries to Ficoll was approximately 10 to 20 times that of intact capillaries, depending on molecular size. Taking into account the blockage of much of the GBM surface by cells, the contribution of the GBM to the diffusional resistance of the intact barrier was calculated to be 13% to 26% of the total, increasing with molecular size. Thus, the GBM contribution, although smaller than that of the cells, was not negligible. The structure that is most likely to be responsible for the cellular part of the diffusional resistance is the slit diaphragm, which spans the filtration slit between epithelial foot processes. A novel hydrodynamic model was developed to relate the diffusional resistance of the slit diaphragm to its structure, which was idealized as a single layer of cylindrical fibers in a ladder-like arrangement.     

Leukocyte recruitment to the inflamed glomerulus: a critical role for platelet-derived P-selectin in the absence of rolling

The renal glomerulus is one of the few sites within the microvasculature in which leukocyte recruitment occurs in capillaries. However, due to the difficulty of directly visualizing the glomerulus, the mechanisms of leukocyte recruitment to glomerular capillaries are poorly understood. To overcome this, we rendered murine kidneys hydronephrotic to allow the visualization of the functional glomerular microvasculature during an inflammatory response. These experiments demonstrated that following infusion of anti-glomerular basement membrane (GBM) Ab, leukocytes became adherent in glomerular capillaries via a process of immediate arrest, without undergoing prior detectable rolling. However, despite the absence of rolling, this recruitment involved nonredundant roles for the P-selectin/P-selectin glycoprotein ligand-1 and beta2 integrin/ICAM-1 pathways, suggesting that a novel form of the multistep leukocyte adhesion cascade occurs in these vessels. Anti-GBM Ab also increased glomerular P-selectin expression and induced a P-selectin-independent increase in platelet accumulation. Moreover, platelet depletion prevented both the increase in glomerular P-selectin, and the leukocyte recruitment induced by anti-GBM Ab. Furthermore, depletion of neutrophils and platelets also prevented the increase in urinary protein excretion induced by anti-GBM Ab, indicating that their accumulation in glomeruli contributed to the development of renal injury. Finally, infusion of wild-type platelets into P-selectin-deficient mice restored the ability of glomeruli in these mice to support leukocyte adhesion. Together, these data indicate that anti-GBM Ab-induced leukocyte adhesion in glomeruli occurs via a novel pathway involving a nonrolling interaction mediated by platelet-derived P-selectin.     

Vascularization of the embryonic kidney. Detection of endothelial cells with Ulex europaeus I lectin

Frozen sections of human fetal kidneys were studied for the appearance of vascular elements to the developing glomeruli using Ulex europaeus (UEA-I) lectin as a marker for endothelial cells. Chains of UEA-I-positive cells, seen to extend from larger vessels, could be observed among the uninduced cells of the nephrogenic mesenchyme. During the S-shaped body stage of nephrogenesis, the cleft of the comma-shaped cluster of epithelial cells was invaded by the UEA-I-positive cells, and during further glomerular development, the UEA-I-positive cells were seen to grow in number, prior to the appearance of visible lumina to the capillaries. The present results show, in addition to revealing the vascularization pattern of developing glomeruli, that endothelial cells obtain some of their characteristic glycoconjugates during early stages of differentiation. Thus, UEA-I seems to be a valuable tool to study in detail the vascularization of various developing human tissues.     

Tandem scanning confocal microscopy (TSCM) of normal and ischemic living kidneys

Tandem Scanning Confocal Microscopy (TSCM) allows one to section optically into and record real-time images of living organs and tissues in a noninvasive fashion. In this paper, we will present some initial TSCM observations of subcapsular nephrons in the living, intact kidneys of Munich-Wistar rats and evaluate the nephron's responses to temporary ischemia and to intravenous infusion of mannitol. The rats were anesthetized with Inactin and a laparotomy performed to expose the kidneys. Using a TSCM equipped with a 20 x water-immersion objective, we optically sectioned through the intact kidney capsule and recorded real-time images of living subcapsular glomeruli and uriniferous tubules. The proximal tubule brush border was highly reflective and allowed us to distinguish between the first and second segments of the proximal tubules as well as the distal tubules. Cellular elements of the blood could be seen passing rapidly through peritubular capillaries and individual glomerular capillary loops. With fluorescent filters in place, intravenously injected carboxyfluorescein was seen to pass through the glomerular capillary loops and then progressively through the different segments of the uriniferous tubules. Ligation of the renal artery resulted in rapid swelling of proximal tubule cells into the tubular lumens, loss of reflectiveness of the microvillous brush borders, and closure of the peritubular capillary spaces. Upon release of the ligature, the proximal tubule lumens again became patent, often opening up abruptly and in a zipper-like fashion down the length of the tubules. Increasing the glomerular filtration rate by intravenous infusion of mannitol resulted in increases in tubular luminal and perimeter dimensions. Mannitol also acted as an effective impermeant osmotic agent and prevented most of the cellular swelling which was otherwise seen in response to renal ischemia.     

Serology and tissue lesions in rabbits immunized with Streptococcus mutans

Rabbits were immunized i.v. or i.d. with sterile suspensions of disrupted Streptococcus mutans strain MT703 or K1R. Indirect immunofluorescence assays indicated that sera from four of 10 rabbits immunized i.d. contained antibodies reactive with monkey and human heart and kidney components; 19 of 24 rabbits immunized i.v. had antibodies reactive with these tissues. Heart-reactive antibodies were also detected by immunoelectrophoresis and indirect radioimmunoassay. These antibodies were absorbed well by cytoplasmic membranes, a whole cell extract, and an alkali extract of S. mutans but only weakly by intact bacteria. Between 6 and 8 weeks after the first i.v. administration of S. mutans vaccines, rabbits developed proteinuria and hematuria with subsequent weight loss and lethargy. Approximately 25% of the animals died from illness between the fifth and sixth month of immunization. In 13 of 15 rabbits, immune deposits of C3 and IgG, IgM, or IgA and fibrinogen were seen in kidneys within the glomeruli, basement membranes of the peritubular capillaries, and in the interstitium. In the heart, deposits were seen along the capillaries of the myocardium. In 8 of 14 rabbits, focal deposits of S. mutans antigen were detected in glomeruli and in the kidney interstitium. The kidneys showed gross pathologic and histopathologic changes. Most kidneys were pale and enlarged. Microscopic examination revealed hypercellularity of the glomeruli, presence of neutrophils, thickening of glomerular and tubular basement membranes, tubular atrophy, edema, and fibrosis of the interstitium. The kidney disease presented features of poststreptococcal glomerulonephritis. Microscopic examination of heart sections revealed mild perivascular infiltration by polymorphonuclear leukocytes and plasma cells in some of the rabbits.     

Ultrastructural changes of the rat renal glomeruli in the early phase of renovascular hypertension

Electron microscopic studies were carried out on rat kidneys during the early phase of "two-kidney" Goldblatt hypertension. Pathologic ultrastructural changes (confluence of foot processes of the podocytes, formation of intercellular juctions between them, etc.) and an enhanced permeability to ferritin were found in the glomeruli of both the ischaemic and the contralateral kidneys. Signs of activation and exhaustion of the lysosomal system appeared in the glomerular epithelial cells of the untouched kidneys. These signs were absent in the kidneys with constricted arteries. The possible significance and pathogenesis of the alteration are discussed in comparison with other pathological conditions showing a similar ultrastructural picture.     

Intranuclear inclusions in the cells of different tissues of the aging rat

Ultrastructure of neuronal and glial nuclei has been studied in various parts of the CNS, in parenchymatous cells of internal organs, in endotheliocytes and pericytes of their blood capillaries in intact rats--mature (6-8-month-old) and old (26-28-month-old), as well as at certain experimental influences. In all the cellular populations studied (most often in neurons), various in their structure, intranuclear inclusions (INI) have been revealed, that are considered as five main types. The number of INI increases sharply at ageing, that is especially noticeable under experimental conditions. Some INI are situated in morphologically preserved, actively working cells, being normal components of the nucleus, others--reflect profound rearrangements of nuclear proteins, disturbances in lipid metabolism up to irreversible destructive processes in the nucleus.     

Disappearance of dextran 70,000 from plasma of hamadryas baboons

Evans Blue (EB), [131I]human albumin and [14C]dextran 70,000 were injected into three water repleted and water depleted hamadryas baboons, in order to evaluate the permeability of their capillary bed under these conditions. The rate of disappearance of dextran from the plasma is significantly higher than that of the other markers, despite its relatively high molecular weight. Half-life values of EB and albumin in water depleted baboons were significantly higher than values in water repleted animals. Inulin and dextran showed no significant difference before or after dehydration. Chromatography of urine and plasma samples on columns of Sephadex G-75 showed that dextran passed the glomerular capillaries and is excreted into the urine with its same original molecular size. The permeability of capillary beds to dextran 70,000 is negligible. The fact that the kidneys excrete dextran means that dextran 70,000 is not a useful marker for calculating the capillary permeability in hamadryas baboons.     

Comparative evaluation of the adaptive mechanisms of the microcirculatory bed during dehydration

In total preparations of the white rat small intestine mesentery, impregnated with silver nitrate, as well as under conditions of vital microscopical observations at certain stages of alimentary experimental dehydration, analogous changes in the microcirculatory bed links have been demonstrated. Comparison of quantitative parameters in decreasing diameters of the microvessels, specific for dehydrated preparations has shown a great importance of these changes in the impregnated preparations at the expense of tissue condensation at their histological treatment. The advantages of a complex application of both methods for estimating dynamics of vascular, intervascular and extravascular changes are proved.     

Extracellular matrix and capillary ingrowth in interspecies chimeric kidneys

The migration of capillaries into mouse embryonic kidneys grafted on quail chorioallantoic membrane (CAM) was analyzed by two monoclonal antibodies against quail endothelial and haematopoietic cells. As shown by immunohistochemistry, the quail chorioallantoic vessels invaded the kidney explant. Initially, the capillaries were detected in the interstitial stroma and, soon thereafter, tightly adjacent to the branches of the ureteric bud. The induced mesenchymal cell condensates, the prospective nephric vesicles, were avascular, but when the early S-shaped body was formed, the capillaries invaded its lower crevice. Finally chimeric glomeruli consisting of mouse podocytes and quail endothelial cells, were formed and, contemporarily, the capillaries ceased to migrate. Within the endothelial-mesangial area of the chimeric glomeruli, all cells expressed the quail-type nuclear structure and were stained by the quail endothelial-specific antibodies. The pattern of migrating capillaries was compared to the distribution of the extracellular matrix (ECM) molecules by double staining with polyclonal antibodies against laminin or fibronectin, and monoclonal quail endothelial-specific antibodies. Initially, the capillaries migrated in a fibronectin-rich matrix, devoid of laminin, but when the epithelial kidney tubules formed, some capillaries attached to the newly formed epithelial basement membrane. At no stage were the capillaries seen to penetrate the epithelial basement membrane. The orderly branching of the ureteric bud, followed by the formation of nephrons and the shift in the ECM, might create pathways for an oriented capillary migration. The fibronectin-rich areas could be a scaffold for the capillary migration, and the attachment to the basement membranes a means for their cessation.     

Vascularization and glomerular ultrastructure in the pig mesonephros

Summary Vascularization of the pig mesonephros was investigated in embryos 5–8 cm in length. Vascular injections with microfil were cleared and dissected; corrosion casts were studied under the scanning electron microscope (SEM). Perfusion-fixed tissue was used for SEM and transmission electron microscope (TEM) studies, including freeze-fracture specimens.The branches of one mesonephric artery carry up to 15 glomeruli. Several glomeruli occupy the same arterial branch, with very short afferent arterioles proper. The efferent vessels, frequently 2–5, leave the extensive vascular pole opposite the entering arteriole and split into peritubular capillaries radiating towards the superficial veins. These capillaries form vascular regions in the shape of flattened pyramids. Along its course, one nephron is supplied by vessels derived from 4–7 glomeruli. The nephrons have less vascular contact than in the definitive kidney.The ultrastructure of the single mesonephric vessels matches the metanephric counterparts. Epithelioid cells with renin granules are common in afferent arterioles, larger arteries, and efferent vessels. The lobulated glomeruli are up to 750 m long and flattened, showing the usual features of podocytes, mesangial cells, and an attenuated endothelium with fenestrations between 50 and 250 m. It partially retains its own basement membrane. There is no proximal mesangium.Supported by Deutsche Forschungsgemeinschaft.     

Passage of microspheres through vessels of normal and split hydronephrotic rat kidneys

Microspheres (15 micron) were injected intracardially in rats. By means of in vivo microscopy of the split hydronephrotic kidney microcirculation, the passage of microspheres through renal blood vessels was analyzed. Additionally, three sets of experiments were conducted, one consisting of rats with normal kidneys, one of rats with hydronephrotic kidneys, and one of rats with hydronephrotic kidneys under vasodilation. In vivo microscopic as well as histological studies show that the passage of 15-micron microspheres is dependent on the postinjection interval. Microspheres are capable of locally dilating preglomerular vessels and moving towards the glomeruli. Therefore, estimates of preglomerular vessel diameters with microsphere experiments ought to be controlled by intravital microscopy.     

Long-Term Responses to Loss of Renal Mass: Glomerular Changes: Adaptation of Glomerular Forces and Flows to Renal Injury

The mechanism of glomerular ultrafiltration in normal kidneys or after renal injury is reviewed. The role of increased glomerular plasma flow in mediating increases of nephron filtration rate is evidenced under experimental conditions resulting in filtration pressure disequilibrium along glomerular capillaries. The increase of nephron filtration in hypertrophied kidneys appears to be due mainly to a rise of glomerular plasma flow and, to a smaller extent, to an increase of glomerular capillary hydrostatic pressure, the ultrafiltration coefficient remaining unchanged. In contrast, in the early phases of experimentally induced nephrotoxic serum nephritis, a decrease of the ultrafiltration coefficient was observed; nephron filtration rate, however, remained within the normal range, as a consequence of a higher hydrostatic pressure in the glomerular capillaries of the nephritic kidneys.     

Cytoskeleton of podocytes in vertebrate animals and human.

Using TEM and immunofluorescence microscope, a study was made on podocytes in vertebrates where an intermediate-sized filament system is replaced by a microtubule system, accompanied by highly developed microfilaments structures. A comparative immunofluorescence study was carried out on cryotome renal sections of plaice (Pleuronectes platessa L.) and rats, using specific antibodies anti-cytokeratins and anti-vimentin. With polyclonal anti-vimentin serum the capillaries of the renal glomeruli showed a bright colour of plaice and only a week one in the rats. Double staining of renal tissue of mongrel rats of different ages (6-7 weeks and 1.8 years old) with antibodies for actin and anti-vimentin polyclonal serum revealed in young rats an intensive fluorescence for actin and a slight fluorescence for the intermediate filaments. Renal glomeruli of old rats demonstrate a strong vimentin-activity and lower actin one. The ultrastructural study of human podocytes showed two different cytoskeleton age-depending types (2, 4, 6, 37 and 65 years old). It is suggested that during individual development and ageing in kidneys of higher animals and human, physiological changes induce morphological cytoskeleton restructuration accompanied by intensive development of intermediate filaments and simultaneous "involution" of microtubules and microfilaments.     

Renal glomerular evolution in Antarctic notothenioid fishes

Light and electron microscopy were used to document the degree of glomerular development in 10 species of Antarctic notothenioid fishes. When combined with results of previous studies, data revealed that 16 of 20 species inhabiting subzero sea water were aglomerular. One subantarctic and two temperate species were pauciglomerular, and an additional temperate species had a moderate number of glomeruli. Renal corpuscles were variable in number and diameter among the pauciglomerular species, and most had few patent glomerular capillaries. Radiolabelled markers indicated that the glomerular filtration rate was low in the pauciglomerular Notothenia angustata , ranging from 0.005 to 0.124 ml h⁻¹ kg⁻¹ in eight specimens. Arterial perfusion of Microfil demonstrated that arteries supplying aglomerular and pauciglomerular kidneys were confined largely to the periphery of the organ, and glomerular capillaries were absent or few in number. As ancestral notothenioids probably had glomerular kidneys, data from 20–25% of the fauna suggest that there has been an evolutionary loss of glomeruli in many species. The pattern of glomerular reduction is consistent with the hypothesis that the selective advantage of aglomerularism is in the urinary conservation of small molecular weight antifreeze glycopeptide compounds that are vital to survival in sub-zero Antarctic waters.     

Studies on esterase histochemistry of amphibian kidney

Summary In the hope that the histochemical picture of the kidney may help to understand its role in excretion and osmoregulation, an effort is here made to study the distribution of esterases in amphibian kidney.The kidneys of adults and tadpoles of the frog, Rana tigrina and the toad, Bufo melanostictus were used for this study. Some of these animals were subjected to dehydration for 3–4 days and to the effect of 150 mM NaCl for 8–12 days before their kidneys were used. The esterases were visualised using tweens, naphthol esters and 5-bromoindoxyl acetate as substrates. These were accompanied by activator/inhibitor studies.Very interesting results were obtained in the distribution of the esterases. Tween esterase and -naphthyl acetate esterase were found in the proximal tubules of the adult frog kidney only while 5-bromoindoxyl acetate esterase was found to be present in all the animals tested. On the other hand, naphthol AS acetate esterase was absent in the tadpole stages of the frog and toad. Further 5-bromoindoxyl acetate esterase and naphthol AS acetate esterase were demonstrated in the glomeruli of frogs and toads subjected to NaCl solution. Activator/ inhibitor studies helped in characteristically differentiating these different esterases.There seems to be a relationship between the distribution of the different esterases and the excretory and osmoregulatory adaptations of these animals which differ in the adult and tadpole stages and in the experimental conditions mentioned. The possible implications of the esterase distribution is discussed in considerable details.U.G.C. Research Scholar.     

Development of embryonic stem cells in recombinant kidneys

Embryonic stem cells (ESC) are self-renewing and can generate all cell types during normal development. Previous studies have begun to explore fates of ESCs and their mesodermal derivatives after injection into explanted intact metanephric kidneys and neonatal kidneys maturing in vivo. Here, we exploited a recently described recombinant organ culture model, mixing fluorescent quantum dot labeled mouse exogenous cells with host metanephric cells. We compared abilities of undifferentiated ESCs with ESC-derived mesodermal or non-mesodermal cells to contribute to tissue compartments within recombinant, chimeric metanephroi. ESC-derived mesodermal cells downregulated Oct4, a marker of undifferentiated cells, and, as assessed by locations of quantum dots, contributed to Wilms’ tumor 1-expressing forming nephrons, synaptopodin-expressing glomeruli, and organic ion-transporting tubular epithelia. Similar results were observed when labeled native metanephric cells were recombined with host cells. In striking contrast, non-mesodermal ESC-derived cells strongly inhibited growth of embryonic kidneys, while undifferentiated ESCs predominantly formed Oct4 expressing colonies between forming nephrons and glomeruli. These findings clarify the conclusion that ESC-derived mesodermal cells have functional nephrogenic potential, supporting the idea that they could potentially replace damaged epithelia in diseased kidneys. On the other hand, undifferentiated ESCs and non-mesodermal precursors derived from ESCs would appear to be less suitable materials for use in kidney cell therapies.