Electron microscopic study of the cytoskeleton of human podocytes

The ultrastructural study of man's cytoskeleton of podocytes is carried out. Populations of podocytes with two different types of structure of the cytoskeleton in dependence on age (2, 4, 6, 37 and 65 years) is revealed in kidneys. The first type of cytoskeleton of the podocyte is peculiar for children's age and is characterized by branched, high density microfilament network, expressed by system of microtubules and single myofilaments. The intermediate filaments here are either utterly absent or present so feebly they find themselves "disguised" by other strongly developed components of cytoskeleton and revealing them with the help of technique of electron microscope is impossible. In kidneys of adults, and especially of old aged persons podocytes with other type of organization of the cytoskeleton are mainly identified. The distinctive signs of the last are bundle arrangement of microfilaments, plural bundles of intermediate filaments and individual microtubules. This study permits to make a conclusion that during individual development and growing old in kidneys of high animals and man, probably, physiological changes causing morphological reconstruction of cytoskeleton which is accompanied by intensive development of intermediate filaments' system with simultaneous "involution" of microtubules and microfilaments' systems take place.     

Preliminary scanning electron microscope (SEM) observations of the rat renal parenchyma after microdissection]

Rat kidneys weighting between 250 and 300 g. were treated in HCl. A single nephron observed through SEM shows that the above treatment doesn't interfere with the ultrastructural morphology of nephron.     

Ultrastructural changes associated with renin secretion from the juxtaglomerular apparatus of mice

Summary Thin sections and freeze-fracture replicas were used to investigate the ultrastructural changes associated with renin secretion from the juxtaglomerular part of the afferent arteriole of male mice. Adrenalectomized animals in which renin secretion was stimulated by furosemide application and bleeding were also studied. Exocytosis of mature electron-dense granules was found in all experimental groups. Before extrusion, the region of granule facing the cell membrane changed, with vesicular and/or stacked membrane-like profiles and a small local protrusion of the granule membrane appearance of. Concomitantly, punctuate sites of fusion between the cell and granule membranes were observed. Later, unaltered amorphous, and altered membrane-like granule content was released from omega-shaped cavities into the extracellular space. In stimulated animals the alteration and extrusion of several closely apposed granules was reminiscent of compound exocytosis. Coated pits were frequently seen, suggesting specific retrieval of the former granule membrane. The collapsing silhouette of a depleted granule very rarely took the form of a saccule whose narrow membrane-bounded neck was continuous with the extracellular space.Observed were two additional events by which active and inactive renin may be released. Small electron-lucent vacuoles of undetermined origin fused with the cell membrane and, in stimulated kidneys, some epithelioid cell processes disintegrated. However, the interpretation of the related ultrastructural phenomena was uncertain.These studies were supported by the German Research Foundation within the SFB 90 Cardiovasculäres System     

Functional preservation of the mammalian kidney. III. Ultrastructural effects of perfusion with dimethylsulfoxide (DMSO)

Isolated rabbit kidneys were perfused at 37 ° C with a cell-free solution containing 0.0, 1.4, 2.1, and 2.8 m DMSO for 60 min. Electron microscopic examination of cortical structures revealed relatively high sensitivity of proximal tubular cells to DMSO-induced alterations, which included cytoplasmic clarification and disruption of microvilli. Glomeruli exhibited proliferation of rough endoplasmic reticulum, but were otherwise well preserved after perfusion with and without DMSO. Vascular endothelial cells were also intact after perfusion with DMSO, suggesting that DMSO-induced resistance changes are not necessarily associated with degeneration of capillary cells. Perfusion without DMSO resulted in good preservation of all cortical structures. These studies suggest that ultrastructural effects of DMSO must be considered in assessment of damage done in whole kidneys during freeze-preservation studies.     

Attempted canine renal cryopreservation using dimethyl sulphoxide helium perfusion and microwave thawing

Fourteen dog kidneys were perfused with 1.4 dimethyl sulphoxide (Me₂SO) in modified Sacks' solution (MSS), and cooled to −80 °C while being perfused with helium. After holding for 15 min at −80 °C they were thawed by microwave irradiation in a commercial 2450 mHz microwave oven. Some were then reperfused with MSS and transplanted as pelvic autografts or subjected to morphological examination. Others were transplanted without reperfusion. Although the light microscopic appearances were encouraging, electron microscopy revealed ultrastructural damage, and none of the kidneys exhibited any function after transplantation. Reasons are advanced for doubting the wisdom of using Me₂SO, helium perfusion and microwave irradiation in attempts to preserve kidneys.     

TCDD alters the extracellular matrix and basal lamina of the fetal mouse kidney

The teratogenic effects of the dioxin 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) have previously been studied in several species, and hydronephrosis has been reported to be a frequent abnormality in near-term fetuses. C57BL/6N female mice, given 12 micrograms/kg TCDD, P.O., on day 10 of gestation were killed on days 14, 15, and 16; fetal kidneys were collected and prepared for either immunofluorescent localization of several extracellular matrix components (ECM) or transmission electron microscopy (TEM). The TCDD-treated and control kidneys showed the same pattern of staining for fibronectin, but TCDD-treated kidneys displayed a diminished overall intensity. The intensity of laminin and type IV collagen immunofluorescence also appeared to be decreased, and deviations in the pattern of antibody binding were detected for differentiating TCDD-treated nephrons. Binding of the laminin antibody to the basal lamina was decreased in the parietal layer of Bowman's capsules in more advanced stages of differentiation. TEM analysis focused on the basal lamina of the tubules and Bowman's capsule. In TCDD-exposed kidneys, ECM components adjacent to differentiating nephrons were less abundant, and the basal lamina of the developing Bowman's capsules had a diminished lamina densa. The earliest nephrons to develop display these defects and comprise the first functional filtration units of the metanephric kidney. These ultrastructural changes noted in TCDD-exposed nephrons may promote proteinuria, a condition normally observed in the developing kidney when the filtration barrier is immature.     

Effect of hyperoxia on erythropoietin biogenesis in the "endocrine" kidney model

The erythropoietin plasma level and RNA synthesis in both kidneys were studied in rats with the H. Selye "endocrine" kidney under 4-hour hyperoxia. It was shown that a short period of hyperoxia leads to a 2-fold decrease in erythropoietin plasma level and to the fall of RNA synthesis in the "endocrine" and intact kidneys. From the evidence obtained it is concluded that hyperoxia inhibits erythropoietin production in the kidneys. Changes in high-polymeric RNA synthesis suggest that DNA-dependent RNA synthesis is one of the mechanisms of the hormone biogenesis.     

Thermographic studies of phantom and canine kidneys thawed by microwave radiation

Whole organs, such as kidneys, must be thawed quickly and uniformly to prevent damage during thawing due to excessive heating. Electromagnetic heating with microwaves thaws the kidneys quickly but frequently produces "hot spots" with heat damage. To study heat damage, phantom gelatin kidneys with different dielectric constants and canine kidneys perfused with 12.5% glycerol, ethylene glycol, or dimethyl sulfoxide before freezing were microwave thawed, and the interior temperature was measured by thermography. Phantom kidneys were thawed free standing and canine kidneys were either free standing or packed in a gel mixture. Both phantom and canine kidneys were split symmetrically and separated with a sheet of Styrofoam to facilitate immediate separation and evaluation of the halves after thawing (approximately 3 sec). All the phantoms, regardless of dielectric properties, had areas less than 0 degrees C or greater than 37 degrees C after thawing. The free-standing canine kidneys and the gel-packed ethylene glycol-perfused kidneys had frozen areas (less than 0 degrees C) and hot spots (greater than 37 degrees C). However, glycerol- and dimethyl sulfoxide-perfused kidneys packed in gel before thawing had no areas less than 0 degrees C or greater than 37 degrees C. Altering the geometry from a "kidney shape" to a cylindrical shape with increased volume improved the uniformity of thawing and was more effective than altering the dielectric constant over the range evaluated.     

Effect of acute hypoxia on the renin and erythropoietin activities of the blood serum in rats

A comparative study of renin and erythropoietin content in the blood serum of rats with "endocrine" kidneys and changes in their activity following the action of specific erythropoietic stimulus (4-hour hypoxia) has been conducted. The presence of "endocrine" kidneys increased renin and erythropoietin activity in such animals. Acute hypoxia produced further increase in erythropoietin titre in the blood serum, with renin remaining at the same level. Possible differences in the mechanisms of renin and erythropoietin biogenesis in the kidneys are considered.     

Glomerular histopathology of the contralateral kidney in experimental unilateral hydronephrosis

The aim of this study was to examine the structural, ultrastructural and morphometric alterations which take place in the contralateral kidneys of rats with experimental unilateral hydronephrosis. 20 Wistar rats weighing 250 g., affected by a process of unilateral hydronephrosis following the ligature of the ureter, were used; these rats were then killed 40, 50, 60, or 70 days after the ligature. Among the perceived alterations, were immunoglobulin G deposits shown by positive immunoperoxidase reaction and increase in the size of the glomerular and corpuscle from around the fortieth day, and structural alterations that included the pedicles, electrondense deposits in the podocytes and pseudogranular structures in the basal membrane of the capillary.     

Qualitative difference of subcellular localization of tumor-associated carbohydrate (Le(x)) antigens in renal cell carcinoma and normal kidney.

Renal cell carcinomas are immunohistochemically positive for oligosaccharides with the Le(x) determinant (Gal beta 1----4[Fuc alpha 1----3]GlcNAc) and its derivatives, as oncofetal antigens, and their expression is closely related to a better prognosis of the patients. This study was designed to clarify the difference in antigen localization at the ultrastructural level between renal cell carcinoma and normal tissues. In normal kidneys, Le(x) detected by monoclonal antibody (MAb) FH 2 and sialylated extended Le(x) (sialyl Le(x)-i) by MAb FH 6 were identified along the plasma membrane of microvilli of proximal tubule epithelial cells, with occasional immunoreactivity along the basolateral plasma membranes. Intracellular localization was very sparse. Renal cell carcinoma showed localization of Le(x) and sialyl Le(x)-i antigens along the cell membrane and in the cytosol as aggregates or filaments. Immunoreactive materials were also observed in the lumen formed among carcinoma cells. The cytosolic immunoreactivity, not observed in the normal kidney, was regarded as "abnormal cytosolic accumulation" of the antigens. This pattern was more pronounced in clear-cell carcinoma. Pretreatment of specimens with chloroform-methanol, which extracts glycolipids, decreased immunoreactivity in carcinoma tissues, particularly that in the cytosol. The extracts contained substances immunoreactive for MAb FH6. Our study has demonstrated that (a) remarkable changes occur in the ultrastructural localization patterns of sialyl Le(x)-i and Le(x) in renal cell carcinoma and (b) considerable amounts of glycolipids are contained in the substances with sialyl Le(x)-i deposited in the cytosol of clear-cell carcinoma.     

Functional differentiation of the anterior pituitary cells in the fetal pig

Summary The fetal porcine pituitary was investigated by means of ultrastructural immunocytochemistry (1) to identify the first cells synthesizing the adenohypophyseal hormones, (2) to follow their differentiation during fetal development, and (3) to compare their ultrastructural characteristics with those of mature adult cells.The first ACTH-cells, which produced and stored ACTH, -LPH, -MSH, and - and -endorphin in the same granules, were very numerous at day 34 and displayed a uniform morphology. At day 50 and thereafter, until the end of gestation, the ACTH-cells differed in their appearance probably reflecting various stages of differentiation of one cell type. The GH-cells gained rapidly ultrastructural features comparable to those of mature GH-cells. In contrast, in the case of PRL-cells, which appeared only at the end of the gestation period as immature elements containing very small secretory granules, the morphological maturation seemed to take place only after birth. The first cells synthesizing the glycoprotein hormones (LH, LH, FSH and TSH) displayed ultrastructural features of immature cells. At day 50, their ultrastructural organization started to show a different pattern. At the end of gestation, the TSH-cells and the gonadotropic cells displayed the ultrastructural features of mature cells.     

Factors affecting the glycosphingolipid composition of murine tissues

The effects of genetic strain, sex, age, and pathological state on the distribution and concentration of glycosphingolipids in mouse kidneys and livers were studied. The concentrations of glycosphingolipids and phospholipids in the kidneys and livers of different strains were compared. The major glycosphingolipid in the kidneys of male and female BALB/c, C3H/He, C57/BL, A, and C57xA (F(1) hybrid) mice was a sulfatide, monoglycosyl-(3-sulfate) ceramide; monoglycosyl ceramide was the major component in livers. The kidneys of males of all strains contained significant amounts of diglycosyl ceramide, but those of females contained, at most, only traces. Glycosphingolipid concentration in the male kidneys of C57/BL and C57xA (F(1) hybrid) was much higher than in the female and was also much higher than in the male kidneys of C3H/He, BALB/c, and A strains. The kidneys of "old" (36 wk) male and female C3H/He mice contained much higher proportions of monoglycosyl ceramide than 10-12-wk-old adults. The distributions of glycosphingolipids in kidneys of female C3H/He mice with BP8 ascites tumors and male C57xA (F(1) hybrid) mice with EL4 ascites tumors differed from those in the normal mice. An unknown lipid, present in all glycosphingolipid extracts from kidney and liver, was tentatively identified as cholesterol sulfate.     

Electron histochemical study of 1:2-glycol groups in experimental hamster amyloid

Summary Leishmania-induced amyloid of hamster kidneys was studied by the periodic acid — thiocarbohydrazide — osmium tetroxide method which is analogous to the PAS reaction.The amyloid fibrils failed to give a reaction for 12-glycol groups. The ultrastructural distribution of mucosubstances containing 12-glycol groups was found to be localized in the ground substance between the fibrils.The reaction was unaffected by -amylase digestion, and chloroform-methanol extraction of lipids; it was dependant upon periodic acid oxidation.     

Global gene expression profiling in early-stage polycystic kidney disease in the Han:SPRD Cy rat identifies a role for RXR signaling

Han:SPRD Cy is a spontaneous rat model of polycystic kidney disease (PKD) caused by a missense mutation in Pkdr1. Cystogenesis in this model is not clearly understood. In the current study, we performed global gene expression profiling in early-stage PKD cyst development in Cy/Cy kidneys and normal (+/+) kidneys at 3 and 7 days of postnatal age. Expression profiles were determined by microarray analysis, followed by validation with real-time RT-PCR. Genes were selected with over 1.5-fold expression changes compared with age-matched +/+ kidneys for canonical pathway analysis. We found nine pathways in common between 3- and 7-day Cy/Cy kidneys. Three significantly changed pathways were designated "Vitamin D Receptor (VDR)/Retinoid X Receptor (RXR) Activation," "LPS/IL-1-Mediated Inhibition of RXR Function," and "Liver X Receptor (LXR)/RXR Activation." These results suggest that RXR-mediated signaling is significantly altered in developing kidneys with mutated Pkdr1. In gene ontology analysis, the functions of these RXR-related genes were found to be involved in regulating cell proliferation and organ morphogenesis. With real-time RT-PCR analysis, the upregulation of Ptx2, Alox15b, OSP, and PCNA, major markers of cell proliferation associated with the RXR pathway, were confirmed in 3- and 7-day Cy/Cy kidneys compared with 3-day +/+ kidneys. The increased RXR protein was observed in both the nucleus and cytoplasm of cystic epithelial cells in early-stage Cy/Cy kidneys, and the RXR-positive cells were strongly positive for PCNA staining. Taken together, cell proliferation and organ morphogenesis signals transduced by RXR-mediated pathways may have important roles for cystogenesis in early-stage PKD in this Pkdr1-mutated Cy rat.     

Ultrastructural changes caused by yellow fever virus at the level of the kidneys in newborn mice

The author is studying the ultrastructural modifications provoked by the yellow fever virus in the kidneys of baby mice. As a result of the study it has been found that minor changes start appearing as early as the first day and these lead finally to necrosis. The process consists of 5 phases which are the development of endoplasmatic reticulum, the envelopment of the mitochondria by the folds of endoplasmatic reticulum, mitochondrial autophagocytosis, the development of microvilli at the cell surface and the total necrosis of the renal cell.     

Life-long Dietary Restriction Modulates the Expression of Collagens and Collagen-binding Heat Shock Protein 47 in Aged Fischer 344 Rat Kidney

It has been shown that the expression of HSP47 and collagens is substantially increased in the sclerotic/fibrotic process in various organs, including kidney. However, the factors regulating the increased expression of HSP47 are not yet clear. In this study, we examined the effect of dietary restriction for the expression of collagens and collagen-binding HSP47 in the kidneys of 6- and 24-month-old male Fischer 344 (F 344) rats fed ad libitum or 30% diet-restricted. No significant histological alteration was found in the kidneys of 6-month-old fed or diet-restricted rats. Kidneys obtained from 24-month-old freely fed rats showed glomerulosclerosis with marked tubulointerstitial damage including interstitial fibrosis, while in the kidneys of 24-month-old diet-restricted rats, renal damage was remarkably less than those noted in 24-month-old freely fed rat kidneys. Immunohistochemical analysis showed an increased accumulation of type I, type III and type IV collagens in areas of glomerulosclerosis and interstitial fibrosis in old rat kidneys. Dietary restriction significantly reduces renal accumulation of collagens in old age. Aging enhanced expression of HSP47 in 24-month-old freely fed rat kidneys whereas dietary restriction suppressed its expression in 24-month-old diet-restricted rat kidneys. Also, phenotypic alterations of mesangial cells and interstitial cells (immunopositive for -smooth muscle actin), glomerular epithelial cells (immunopositive for desmin) and tubular epithelial cells (immunopositive for vimentin) were seen in 24-month-old freely fed rat kidneys and found to express HSP47. Dietary restriction significantly diminished phenotypically altered renal cells in 24-month-old rat kidneys. Our results suggest that increased expression of HSP47 is associated with age-related renal damage and that diet-restricted alteration of its expression is associated with the modulation of age-associated renal sclerosis/fibrosis.     

Differential diagnosis of histogenetically distinct human epithelial renal tumours with a monoclonal antibody against γ-glutamyltransferase

Summary The localization of membrane-bound -glutamyltransferase with monoclonal antibody (mAb) 138H11 proved to be of value for differential diagnosis of renal cancer, since it correlated with the histogenetic profile of human epithelial renal tumours. Immunoreactive -glutamyltransferase was located in the proximal tubule in all normal human kidneys (15/15) examined thus far by both ultrastructural and immunohistochemical techniques. From 68 epithelial renal cancers tested, 31/31 clear-cell carcinomas and 15/16 chromophilic carcinomas expressed the target epitope of mAb 138H11. In contrast, 0/11 oncocytomas, 0/9 chromophobic carcinomas, and 0/1 Duct-Bellini carcinoma were immunoreactive. These results support a model of histogenesis and classification of epithelial renal tumours, according to which clear-cell and chromophilic renal carcinomas originate from transformed proximal tubule cells, whereas oncocytomas, chromophilic and Duct-Bellini carcinomas originate from cells of the collecting duct.     

Ultrastructure of lymphocytes from patients with paracoccidioidomycosis in the lymphocyte transformation test by phytohemagglutinin

The morphology and ultrastructure of peripheral blood lymphocytes from patients with paracoccidioidomycosis (PCM) and from unaffected individuals (controls) were studied before and after Ficoll-Hypaque separation and at the end of culture, stimulated with phytohemagglutinin. Patient lymphocytes were cultured in medium with autologous plasma (from the patient himself) and with homologous plasma (from an unaffected donor), while donor lymphocytes were cultured in medium with plasma from a patient or with plasma from the donor himself. The Ficoll-Hypaque mixture caused no morphological or ultrastructural changes in the lymphocytes of patients or of unaffected donors. Patient lymphocytes cultured in medium with autologous plasma showed different degrees of cytoplasmic and nuclear alterations, such as organelle dissolution, vacuoles, amorphous masses, deformed nuclei, and absence of nucleoli. Lymphocytes from control individuals cultured in patient plasma also showed ultrastructural alterations, though they were less marked, and a reduced number of blasts. Patient lymphocytes cultured in medium with homologous plasma (from a control individual) showed a morphology similar to that of lymphocytes from control individuals cultured in medium with their own plasma, although with a lower number of blasts. On the basis of the results obtained using that methodology, we draw the following conclusions: (1) separation by Ficoll-Hypaque does not seem to alter the ultrastructure of patient or donor lymphocytes; (2) patients with diffuse PCM and more markedly impaired general condition can exhibit lymphocytes with morphological and ultrastructural alterations capable of affecting their biological systems and functionality; (3) the morphological and ultrastructural abnormalities and the reduced blastogenesis observed in patient lymphocytes cultured in autologous plasma and in control lymphocytes cultured with patient plasma appear to be due to factor(s) present in the plasma of PCM patients.     

Immunohistochemical and microscopic studies on giant cells in tuberous sclerosis

Tuberous sclerosis (TSC) is an autosomal dominant disease, caused by mutations in TSC1 or TSC2 genes, encoding hamartin and tuberin, respectively. The clinical picture of the disease is connected with the formation of hamartomas, mainly in the heart, kidneys and the brain. In three types of brain lesions: cortical tubers, subependymal nodules and subependymal giant-cell astrocytoma (SEGA) characteristic, so-called "giant cells" are found. In the present review we summarise immunohistochemical findings of two types of studies performed on giant cells aiming at establishing the expression of hamartin and tuberin level and determining the presence of neuron- or astrocyte-specific markers. Moreover, we support our argument with the summary of ultrastructural research done with the purpose of demonstrating structures characteristic of neural and/or glial cells. We conclude that giant cells in cortical tubers and SEGAs are the same undifferentiated cells that, depending on individual determination, can show neural or glial features.