Fluoride-associated ultrastructural changes and apoptosis in human renal tubule: a pilot study

The susceptibility of the kidneys to fluoride toxicity can largely be attributed to its anatomy and function. As the filtrate moves along the complex tubular structure of each nephron, it is concentrated in the proximal and distal tubules and collecting duct. It has been frequently observed that the children suffering from renal impairments also have some symptoms of dental and skeletal fluorosis. The findings suggest that fluoride somehow interferes with renal anatomy and physiology, which may lead to renal pathogenesis. The aim of this study was to evaluate the fluoride-associated nephrotoxicity. A total of 156 patients with childhood nephrotic syndrome were screened and it was observed that 32 of them had significantly high levels ( p ≤ 0.05) of fluoride in urine (4.01 ± 1.83 ppm) and serum (0.1 ± 0.013 ppm). On the basis of urinary fluoride concentration, patients were divided into two groups, namely group 1 (G-1) ( n = 32) containing normal urine fluoride (0.61 ± 0.17 ppm) and group 2 (G-2) ( n = 32) having high urine fluoride concentration (4.01 ± 1.83 ppm). Age-matched healthy subjects ( n = 33) having normal levels of urinary fluoride (0.56 ± 0.15 ppm) were included in the study as control (group 0 (G-0)). Kidney biopsies were taken from G-1 and G-2 only, who were subjected to ultrastructural (transmission electron microscopy) and apoptotic (terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling) analysis. Various subcellular ultrastructural changes including nuclear disintegration, chromosome condensation, cytoplasmic ground substance lysis, and endoplasmic reticulum blebbing were observed. Increased levels of apoptosis were observed in high fluoride group (G-2) compared to normal fluoride group (G-1). Various degrees of fluoride-associated damages to the architecture of tubular epithelia, such as cell swelling and lysis, cytoplasmic vacuolation, nuclear condensation, apoptosis, and necrosis, were observed.

The interleukin-1 beta-converting enzyme (ICE) is localized on the external cell surface membranes and in the cytoplasmic ground substance of human monocytes by immuno-electron microscopy.

Interleukin-1 beta (IL-1 beta)-converting enzyme (ICE) is a novel cysteine protease that cleaves the 31-kD inactive cytoplasmic IL-1 beta precursor into active extracellular 17-kD IL-1 beta. The ICE gene product is a 45-kD proenzyme that requires proteolytic processing to activate ICE. Active ICE is a heterodimer consisting of equal amounts of p20 and p10 subunits. Generation of active ICE is affected by the removal of an 11-kD NH2-terminal precursor domain (p11) and an internal 19-amino acid sequence that separates the 20- and 10-kD subunits. Immuno-electron microscopy was performed on human monocytes with immunoglobulins recognizing the active (p20) or precursor (p11) domains of ICE. Elutriated monocytes were stimulated with 50 pM lipopolysaccharide followed by heat-killed Staphylococcus aureus under conditions that induce maximal rates of IL-1 beta secretion. Ultrathin cryosections were cut from fixed frozen pellets of these monocytes and were immunogold labeled with either antibody. Active and precursor domain ICE epitopes were localized in the cytoplasmic ground substance, but they were not detected within the endoplasmic reticulum, the Golgi apparatus, and secretory granules of activated or inactive monocytes. Importantly, numerous ICE p20 epitopes were also observed on the extracellular surfaces of the cell membrane, and were concentrated on the microvilli. Very similar patterns of ICE localization were obtained with unstimulated blood monocytes. In contrast, ICE p11 epitopes were not detected on the surfaces of these monocytes. Likewise, labeling of fixed ultrathin cryosections of monocytes with a biotinylated irreversible ICE inhibitor [Ac-Tyr-Val-Lys(biotin)-Asp-(acyloxy)-methyl-ketone] showed that the compound localized on the outer cell surface as well, and to a lesser extent, within the cytoplasmic ground substance. Furthermore, antipeptide antibodies specific for either the mature or precursor domains of IL-1 beta were both localized upon the cell membrane after stimulation of IL-1 beta secretion. Lipopolysaccaride-primed monocytes that synthesized, but did not secrete IL-1 beta, exhibited only cytoplasmic staining. The data suggests that mature IL-1 beta is generated via cleavage of the 31-kD inactive cytoplasmic IL-1 beta precursor by ICE after association with the plasma membrane during secretion.

Interleukin 1 beta is localized in the cytoplasmic ground substance but is largely absent from the Golgi apparatus and plasma membranes of stimulated human monocytes.

The subcellular location of IL-1 beta was determined using a postsectioning immunoelectron microscopic method on ultrathin frozen sections of human monocytes stimulated with LPS. This methodology permits access of antibody probes to all sectioned intracellular compartments, and their visualization at high resolution. Staining was performed with a rabbit antibody that specifically recognized amino acids 197-215 in the 33-kD IL-1 beta precursor molecule, followed by affinity-purified goat anti-rabbit IgG conjugated to 10 nm colloidal gold particles. Approximately 90% of the IL-1 beta antigens were localized in the ground substance of the cytoplasm at 4 or 20 h after activation, when both intracellular and extracellular accumulation of IL-1 beta was well underway. No significant IL-1 beta staining was observed on the outer cell membrane, nor within the lumens of the endoplasmic reticulum (ER), the Golgi apparatus, or secretory vesicles. In contrast, lysozyme was localized in the ER and dense secretory granules using these methods. Our results suggest that IL-1 beta is not anchored on the plasma membrane, and that its secretion occurs by a novel mechanism that does not use a secretory leader sequence, nor the classical secretory pathway involving the ER and Golgi apparatus.

Probing the structure of cytoplasm.

We have used size-fractionated, fluorescent dextrans to probe the structure of the cytoplasmic ground substance of living Swiss 3T3 cells by fluorescence recovery after photobleaching and video image processing. The data indicate that the cytoplasm of living cells has a fluid phase viscosity four times greater than water and contains structural barriers that restrict free diffusion of dissolved macromolecules in a size-dependent manner. Assuming these structural barriers comprise a filamentous meshwork, the combined fluorescence recovery after photobleaching and imaging data suggest that the average pore size of the meshwork is in the range of 300 to 400 A, but may be as small as 200 A in some cytoplasmic domains.

The structure of cytoplasm in directly frozen cultured cells. I. Filamentous meshworks and the cytoplasmic ground substance.

Cultured fibroblasts or epithelial cells derived from Xenopus laevis embryos were directly frozen, freeze-substituted by an improved method, and then either critical-point-dried and viewed as whole mounts, or embedded and thin sectioned. In thin regions of these cells, where ice crystal artifacts are absent, the cytoplasm consisted of a dense, highly interconnected meshwork of filaments, embedded in a finely granular ground substance. The meshwork in directly frozen, intact cells was compared with that in cells that were lysed (physically, with detergents, or with filipin), or fixed with glutaraldehyde before freezing. Although filaments tended to be less numerous in lysed cells, their overall organization was the same as that in intact cells. However, fixation with glutaraldehyde before freezing distorted the meshwork to variable degrees depending on the osmolarity of the fixation buffer, and also obscured the granular ground substance which is obvious in directly frozen cells. With optimal preparative methods, the cytoplasm of these directly frozen cells is shown to consist of a cytoskeleton composed of discrete interwoven filaments interconnected by numerous finer filaments and a readily extractable granular matrix which presumably represents aggregations of cytoplasmic proteins.

Observations préliminaires sur l'organisation du cytosquelette des cellules végétales. Mise en évidence d'un “réseau microtrabéculaire”

Electron microscopic observations of particularly favourable sections of pericycle and vascular parenchyma cells fixed with glutaraldéhyde have revealed the existence of a network of exceedingly fine filaments in the cytoplasmic ground substance of plant cells. This highly intricated structure to which polysomes seem to be attached interconnects the cytoskeletal fibers and the various other cellular components. It corresponds evidently to the "microtrabecular lattice" described in animal cells. Moreover, this report points out that microfilaments, which are 6- to 8-nm putative actin filaments, contract with microtubules or sheets of agranular reticulum, to form privileged close relationships whose functional significance is discussed.

Ultrastructural immunocytochemical localization of myosin in cultured fibroblastic cells.

Nonmuscle myosin in the cytoplasm of cultured fibroblastic cells has been localized using light and electron microscopic immunocytochemistry. Antibodies to purified fibroblast myosin were produced in goat and rabbit and purified by affinity chromatography. Light microscopic immunofluorescence localization showed patterns similar to those previously published. Electron microscopic localization using the ethyldimethyl aminopropyl carbodiimide-glutaraldehyde-saponin (EGS) fixation-permeabilization procedure and the ferritin bridge localization method produced quantifiable localization in intracellular sites with well-preserved ultrastructural morphology. Myosin was found to be a major component of the cytosol. It was distributed diffusely with no preferential localization on membranous organelles. Myosin was found to be slightly concentrated on the surface of microfilament-containing structures, including the subplasmalemmal microfilament mat and stress fibers, occasionally with an interrupted periodicity. However, no myosin was found in surface ruffles or microvilli. Morphometric quantitation showed that the majority of the cell's myosin was in the cytosol. This location is compatible with myosin being a component of the microtrabecular lattice of the cytoplasmic ground substance. The concentration of myosin in association with microfilaments was only twice that of the cytosol. This interpretation must be somewhat tempered by the possibility that some myosin bound to tightly packed actin may be inaccessible. The significance of this distribution of myosin in cell function is discussed.

Dynein-like Mg2+-ATPase in mitotic spindles isolated from sea urchin embryos (Strongylocentrotus droebachiensis).

Two distinctly different ATPases have been reported to be endogenous to the mitotic apparatus: a Mg2+-ATPase resembling axonemal dynein, and a Ca2+-ATPase postulated to be bound in membranes. To examine the nature of the Mg2+-ATPase, we isolated membrane-free mitotic spindles from Stronglylocentrotus droebachiensis embryos by rapidly lysing these in a calcium-chelating, low-ionic-strength buffer (5 mM EGTA, 0.5 mM MgCl2, 10 mM PIPES, pH 6.8) that contained 1% Nonidet P-40. The fibrous isolated mitotic spindles closely resembled spindles in living cells, both in general morphology and in birefringence. In electron micrographs, the spindles were composed primarily of microtubules, free from membranes and highly extracted of intermicrotubular cytoplasmic ground substance. As analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the pelleted spindles contain 18% tubulin, variable amounts of actin (2-8%), and an unidentified protein of 55 kdaltons in a constant weight ratio to tubulin (1:2.5). The isolated spindles also contained two polypeptides, larger than 300 kdaltons, that comigrated with egg dynein polypeptides, and ATPase activity (0.02 mumol Pi/mg . min) that closely resembled both flagellar and egg dynein. The spindle Mg2+-ATPase showed a ratio of Ca2+-/Mg2+-ATPase = 0.85, had minimal activity in KCl and EDTA, and cleaved GTP at 35% of the rate of ATP. The Mg2+-ATPase was insensitive to ouabain or oligomycin. The spindle Mg2+-ATPase was inhibited by sodium vanadate but, like egg dynein, was less sensitive to vanadate than flagellar dynein. The spindle Mg2+-ATPase does not resemble the mitotic Ca2+-ATPase described by others. We propose that the spindle Mg2+-ATPase is egg dynein. Bound carbohydrate on the two high-molecular-weight polypeptides of both egg dynein and the spindle enzyme suggest that these proteins may normally associate with membranes in the living cell.