Effect of beta-D-xyloside on the renal glomerular cells. II. Morphological studies.

The effect of p-nitrophenyl-beta-D-xylopyranoside on the renal glomerulus was studied. Rat kidneys were labeled with [35S]sulfate in the presence or absence of beta-xyloside by using an isolated organ perfusion system and were processed subsequently for morphological studies. By using electron microscopy, preferential intracytoplasmic vesiculation of the visceral epithelium was observed in the beta-xyloside-treated kidneys. The vesicles were distributed throughout the cytoplasm, particularly in the vicinity of Golgi apparatus. They were acid-phosphatase negative, devoid of clathrin-coat, and contained osmium-impregnated reaction products. The visceral epithelial foot processes remained firmly attached to the glomerular basement membrane. No loss of cell-surface associated sialoglycoproteins, as evidenced by colloidal iron staining, was observed. No significant change in the morphological features of glomerular endothelial or mesangial cells was noted. By using electron microscopy autoradiography, a significant increase in the number of silver grains over the epithelium, and a decrease in the number over the extracellular matrices was observed. The majority of the grains were either associated with intracytoplasmic vesicles or Golgi apparatus. The mean grain densities (concentration of radiation) increased by 3.6-fold for the epithelium, and decreased by 2.4- and 1.6-fold for the basement membrane and mesangial matrix, respectively. The grain densities over the endothelial and mesangial cells were similar in control and experimental groups. These data indicate that xyloside induces selective alterations in Golgi apparatus of the visceral epithelium and a dramatic imbalance in the de novo synthesized sulfated macromolecules of cellular and extracellular compartments.

Xylosylated-proteoglycan-induced Golgi alterations.

The effect of p-nitrophenyl beta-D-xylopyranoside on the Golgi apparatus and proteoglycans (PG) of the renal glomerulus was investigated in an isolated kidney organ perfusion system and monitored by utilizing [35S]sulfate as the PG precursor. By electron microscopy, a selective intracytoplasmic vesiculization of Golgi apparatus of visceral epithelium was observed in the beta-xyloside-treated kidneys. Electron microscopic autoradiography revealed most grains localized to the intracytoplasmic Golgi-derived vesicles, while very few grains were associated with the extracellular matrix membranes. Biochemically, a 2.3-fold increase in cellular matrix and a reduction by a factor of 1.7 in extracellular matrix of [35S]sulfate incorporation was observed. Besides a larger macromolecular form (Kavg = 0.25; Mr = 130,000), lower molecular weight PGs were recovered in the cellular (Kavg = 0.46, Mr = 30,000) and matrical (Kavg = 0.42, Mr = 45,000) compartments after xyloside treatment. The xyloside treatment increased the incorporated radioactivity, mostly included in free glycosaminoglycans and small PGs, in the media fraction by 3.8-fold. These data indicate that xyloside induces a dramatic imbalance in the de novo-synthesized PGs of cellular and extracellular compartments and that cellular accumulation of xylosylated (sulfated) PGs selectively alters the Golgi apparatus of the glomerular epithelial cell, the cell that actively synthesizes PGs.

Plasma membrane biogenesis in the avian salt gland: a biochemical and quantitative electron microscopic autoradiographic study.

The avian salt gland provides an ideal system for the study of plasma membrane (PM) biogenesis. Feeding ducklings 1% sodium chloride (salt stress) induces the secretory cells of the gland to synthesize large amounts of PM, which forms an extensive basolateral PM domain after 7-9 days of treatment. In the present study, the initial biosynthetic events following salt stress were investigated. In vivo studies using 3H-uridine indicated that increased rates of RNA synthesis could be detected by 2 hr after the beginning of salt stress and continued through at least 12 hr. Under in vitro conditions, increased rates of protein and glycoprotein synthesis (as monitored by 3H-leucine and 3H-fucose incorporation, respectively) were also detected after 2 hr and continued through 7-9 days. Increased levels of Na,K-ATPase, a specific secretory cell PM marker, were detected after 8 hr of treatment as monitored by specific activity and 3H-ouabain binding. Sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis coupled with fluorography indicated that both 3H-leucine and 3H-fucose were incorporated into partially purified preparations of Na,K-ATPase isolated after 12 hr. Light microscopic autoradiographic analysis of pulse-chase experiments indicated that in secretory cells of 12-hr salt-stressed glands, 3H-leucine- and 3H-fucose-labelled products reached the cell periphery by 1-2 hr after the initial pulse. The incorporation of both tritiated precursors was predominantly associated with the secretory cells. Quantitative electron microscopic autoradiography indicated that 3H-leucine is initially taken up by elements of the rough endoplasmic reticulum (RER) and cytoplasm (5 min postpulse), subsequently transported to and concentrated within components of the Golgi apparatus (10 min of chase), and ultimately incorporated into all domains of the plasma membrane of secretory cells by 1-2 hr of chase. The data is consistent with a flow of newly synthesized membrane components from RER to Golgi to plasma membrane and is analogous to the pattern previously found for the synthesis and processing of PM proteins in a wide variety of cell types.

De novo cellular synthesis of sulfated proteoglycans of the developing renal glomerulus in vivo.

The site of cellular synthesis of glomerular proteoglycans was investigated in developing glomeruli of 4- to 5-day-old rats. [35S]Sulfate was administered intravenously and animals were sacrificed 15 min to 12 hr later. The outermost layers of the kidney cortices were utilized for characterization of proteoglycans and electron microscopic autoradiography. Sepharose CL-6B chromatography and cellulose acetate electrophoresis revealed that most (approximately equal to 96%) of the radioactivity was associated with heparan sulfate-proteoglycan synthesized during maturation of glomerular capillaries. Tissue autoradiography revealed the following: (i) during the S-shaped body stage, there is rapid incorporation of [35S]sulfate by mesenchymal cells into the cleft region (site for development of future glomerular extracellular matrices); (ii) during the precapillary stage, mesenchyme-derived cells showed higher incorporation of radioisotope than did epithelial cells; and (iii) during the mature capillary stage, all glomerular cell types (mesangial, endothelial, and epithelial) incorporated [35S]sulfate, incorporation by mesangial cells being the greatest. Radiolabeling was also higher in the mesangial matrix than in the glomerular basement membrane of peripheral capillary loops. Synthesis of a single major species of sulfated glycosaminoglycan by cells of different embryologic origin may be unique to glomerular capillaries.

Quantitative determination of the intracellular fate of internalized plasma membrane in dissociated pituitary prolactin cells utilizing a radioiodinated cationic ferritin probe (CFI*) and electron microscopic autoradiography.

Dissociated anterior pituitary cells derived from estrogen-treated female rats were incubated with radioiodinated cationic ferritin (CFI) for 2 min and subsequently in the absence of CFI for varying periods of time up to 3 hr in order to quantitate, using electron microscopic autoradiography, the distribution of retrieved plasma membrane in these cells. Following a 2-min incubation with CFI, autoradiographic grains were found to be associated almost exclusively with the plasma membrane. With increasing periods of incubation in the absence of CFI, grain-density analysis revealed increasing levels of CFI in multiple intracellular organelles. The levels of CFI were greatest for the lysosomes, intermediate for the mature secretory granules, and least for the Golgi cisternae and immature secretory granules. These findings are consistent with the idea that a portion of the retrieved plasma membrane is degraded in lysosomes and that the remainder is recycled to organelles comprising the secretory pathway to be reutilized in successive waves of the secretory cycle.

Plasma membrane retrieval in neoplastic pancreatic acinar cells.

The dynamics of plasma membrane retrieval has been studied in the pancreatic acinar carcinoma in order to determine if neoplastic cells exhibiting a heterogeneity of cytodifferentiation states retain the capacity to interiorize and recycle plasma membrane. To this end, the plasma membranes of neoplastic pancreatic acinar cells were labeled with radioiodinated cationic ferritin (125I-CF), and the fate of the tracer was monitored by quantitative electron microscopic autoradiography. The undifferentiated granule-deficient cells of the tumor internalized membrane-bound 125I-CF and sequestered it predominantly in lysosomes. By contrast, the differentiated granule-rich cells internalized significantly more membrane-bound 125I-CF, and the tracer was localized in secretory granules and in lysosomes. The data suggest that neoplastic cells retain the capability of retrieving plasma membrane and that the dynamics of the process is correlated with the state of cytodifferentiation of the neoplastic cells.

Distribution of sulfated glycosaminoglycans in the glomerular basement membrane and mesangial matrix.

The relative distribution of heparan sulfate-glycosaminoglycan (HS-GAG) and chondroitin sulfate-glycosaminoglycans (CS-GAG) of the mesangial matrix (MM) and the glomerular basement membrane (GBM), which represent the two glomerular extracellular matrices, was determined by a combination of enzymatic treatments and autoradiographic methods. The kidneys were digested in situ either with heparinase (degrades HS and CS-GAG) or chondroitinase-ABC (degrades CS-GAG). Subsequently, the sulfated GAGs were labeled with a radioiodinated analog of cationic ferritin (CF, pI approximately 7.5). The tissues were then processed for light and electron microscopic autoradiography. The autoradiographic analysis showed that sulfated GAGs are distributed both in the GBM and mesangial matrix. The predominant GAG present in both the matrices is HS-GAG and the CS-GAG is exclusively present in the mesangial matrix. These data indicate that the GBM and mesangial matrix are compositionally different. These differences may be of importance in the establishment of normal glomerular function and organization and in the alteration of that function and organization as a result of various disease processes, especially of those that are immune-complex mediated.

Distribution of de novo synthesized sulfated glycosaminoglycans in the glomerular basement membrane and mesangial matrix.

The distribution of de novo synthesized heparan sulfate-glycosaminoglycan (GAG) and chondroitin sulfate-GAG of the mesangial matrix and the glomerular basement membrane was determined by a combination of biochemical and autoradiographic methods. The de novo synthesized sulfated GAGs were labeled by the ex situ organ perfusion of kidneys with [35S]sulfate for periods of time up to 7 hours. Biochemical analysis of a combined extract of the matrices revealed that both heparan sulfate-GAG and chondroitin sulfate-GAG are present at levels of approximately 90 and 10%, respectively, and that these relative levels do not change with increasing organ perfusion times. Autoradiographic analysis of the de novo synthesized sulfated GAGs in each of the matrices showed that the mesangial matrix incorporated two to three times as much of the sulfated GAGs as the glomerular basement membrane. The predominant GAG in both the matrices was heparan sulfate-GAG and chondroitin sulfate-GAG was exclusively present in the mesangial matrix, thereby indicating that these two matrices are compositionally different. These differences may be of some importance in the establishment of normal glomerular function and organization and in the alteration of that function and organization as a result of various disease processes, possibly those that are immune complex mediated.

Decreased de novo synthesis of glomerular proteoglycans in diabetes: biochemical and autoradiographic evidence.

The experimental model of streptozotocin-induced diabetes in rats was utilized to determine the biosynthetic and biochemical alterations in the proteoglycans of the glomerular extracellular matrices (glomerular basement membrane and mesangial matrix) in diabetic nephropathy. Isolated kidneys from diabetic and control groups of animals were radiolabeled in an organ perfusion apparatus with [35S]sulfate of high specific activity (greater than 1,200 Ci/mmol; 1 Ci = 3.7 x 10(10) Bq) and processed for electron microscopic autoradiography, and the proteoglycans of the glomerular extracellular matrices were characterized. The results indicate that [35S]sulfate incorporation into glomerular extracellular matrices of diabetic animals was 30-40% less than that of the control group; however, no differences in the biochemical properties of the de novo synthesized proteoglycans from either group were observed. The relevance of the decreased de novo synthesis of sulfated proteoglycans of glomerular extracellular matrices is discussed in terms of increased glomerular permeability to plasma proteins and reduction in the glomerular filtration rate.

Clogging of the glomerular basement membrane.

The negative charges of the sulfated glycosaminoglycans (GAGs) of the glomerular basement membrane (GBM) were differentially neutralized by perfusin with high molarity buffers in order to determine whether or not these charges protect the GBM from being clogged by circulating plasma macromolecules. Progressive elimination of the negative charges resulted in clogging of the GBM by perfused native ferritin (NF) and bovine serum albumin as evidenced ultrastructurally by the increase in accumulation of NF in the GBM. In addition, the permeability of the GBM to 125I-insulin, a macromolecule which is normally freely permeable, and the glomerular filtration rate (as determined by [3H]inulin clearance) were markedly reduced after the GBM had been clogged with NF in the presence of high molarity buffer, thereby indicating that clogging severely reduces the ability of the GMB to act as a selective filter. These findings are consistent with the idea that the sulfated GAGs of the GBM serve as anticlogging agents.

Altered glomerular permeability as a result of focal detachment of the visceral epithelium.

Changes in the permeability of the glomerular capillary wall (GCW) to native ferritin (NF), following detachment of the visceral epithelium from the glomerular basement membrane (GBM) were investigated. Detachment was induced by either perfusing kidneys with highly purified neuraminidase or by the induction of nephrosis through administration of puromycin aminonucleoside (PAN). Both experimental treatments resulted in marked glomerular ultrastructural changes which were characterized by focal detachment of the visceral epithelium from the GBM, replacement of the normal pattern of interdigitating foot processes with flattened expanses of continuous epithelium at certain areas of the GCW, and a generalized loss of sialic acid-rich epithelial cell cost in areas where the epithelium was detached as well as where it remained adherent. These changes were more frequent and prominent in the paramesangial regions of the glomeruli. When experimentally treated kidneys were perfused with NF, the tracer leaked into the urinary spaces in those areas of the GBM where the epithelium was detached. By contrast, in those areas of the GCW where the epithelium remained adherent, the tracer localized within the GBM mainly at the level of the lamina rara interna (LRI), and none of it appeared in the urinary spaces. Nephrotic and neuraminidase control kidneys were ultrastructurally normal, NF localizing mainly in the inner layers of the GBM. These data are consistent with the idea that the firm attachment of the epithelial foot processes to the GBM plays a vital role in determining the permselectivity properties of the GCW to plasma macromolecules.

Glycosaminoglycans of the glomerular basement membrane in normal and nephrotic states.

Alterations in the permeability of the glomerular basement membrane (GBM) towards native ferritin (NF) and iodinated albumin (125I-BSA) following removal of the major glycosaminoglycans (GAGs) of the GBM, heparan sulfate (HS) and hyaluronic acid (HA), were assessed utilizing the techniques of routine electron microscopy and autoradiography, respectively. Kidneys were incubated with heparinase (to degrade the GAGs of the GBM) and subsequently perfused with either NF or 125I-BSA. Control kidneys, which were not treated with heparinase, showed a low permeability to both tracers, with NF being confined to the lamina rara interna and 125I-BSA exhibiting a low level of passage into the urinary spaces (as indicated by a low density of autoradiographic grains over the urinary spaces). After heparinase treatment there was an increase in the permeability of the GBM such that both NF and 125I-BSA passed through the GBM in larger quantities and entered the urinary spaces. Perfusion of cationized ferritin (CF) into control kidneys revealed this probe to bind to the HS-rich anionic sites present within the GBM. Treatment with heparinase resulted in an abolition of the CF binding thereby indicating that the sites are composed mainly of HS and that HS plays a key role in establishing the permeability properties of the GBM. The changes in the pattern of distribution and density of the anionic sites of the GBM following induction of nephrosis was also studied. Animals were rendered nephrotic by subcutaneous injections of an aminonucleoside of puromycin and their kidneys subsequently perfused with either CF or cationized cytochrome c. No difference in either the pattern of distribution on density of the anionic sites in the GBM of nephrotic kidneys was observed when compared to nonnephrotic controls; thus indicating that the proteinuria associated with aminonucleoside nephrosis might be due to changes in components of the glomerular capillary wall other than the anionic sites.

Sites of sulfate incorporation into mammotrophs and somatotrophs of the rat pituitary as determined by quantitative electron microscopic autoradiography.

Dispersed pituitary cells were labeled with [35S]sulfate (1 h), followed by a chase incubation (up to 2 h), in order to study sulfate incorporation and transport in anterior pituitary cells. The initial site of incorporation of sulfate, the kinetics of sulfate transport, and the intracellular localization of incorporated sulfate were studied by quantitative electron microscope autoradiography. Analysis of autoradiograms from estrogen-treated female rats revealed that all granulated cell types incorporate sulfate. The labeling index (relative [35S]sulfate incorporation per unit area of cytoplasm) of the various cell types was greatest for mammotrophs, slightly less for corticotrophs, gonadotrophs, and thyrotrophs (grouped together), and least for somatotrophs. Results of grain counts on mammotrophs indicated that initially (end of pulse) approximately 70% of the grains were localized in the Golgi region. After 1 and 2 h of chase, there was a decline in radioactivity in the Golgi region (to approximately 30% of the total grains), but the percentage of the grains associated with mature granules progressively increased (from 13% to approximately 40% of the total). Analysis of the relative grain density (percentage of total grains/percentage of total area) indicated that at the end of a 1-h pulse, [35S]sulfate is most concentrated in immature PRL granules and the Golgi cisternae: during 1-2 of chase it becomes progressively concentrated in mature PRL granules. Findings in somatotrophs (and other cell types) from estrogen-treated female rats or normal males were similar to those in mammotrophs. These results indicate the [35S]sulfate is initially incorporated into the Golgi complex of all anterior pituitary cell types. The majority of the sulfate-labeled macro-molecules are then packaged into immature secretion granules in the Golgi region, which become mature granules. In addition, a considerable amount (approximately 30% in mammotrophs) of the radioactivity remains associated within the Golgi region for up to 2 h post pulse. The incorporation of sulfate into the Golgi complex and its transfer to secretory granule membranes and/or contents thus appears to be a general property of anterior pituitary cells.