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The accumulation of advanced glycosylation end products (AGEs) is believed to be a factor in the development of aging nephropathy. We have attempted to establish a link between the formation of AGEs and the onset of renal impairment with aging, indicated by albuminuria, using a fluorescence assay and immunohistochemical detection of AGEs in the renal extracellular matrix in rats. The fluorescence of collagenase-digested Type IV collagen from GBM increased with age, from 1.65 ± 0.05 AU/mM OHPro (3 months) and 1.58 ± 0.04 (10 months) to 2.16 ± 0.06 (26 months) ( p>0.001) and 2.53 ± 0.18 (30 months) ( p>0.001). In contrast, the extent of early glycation products significantly decreased from 5.35 ± 0.25 nmol HCHO/nmol OHPro at 3 months to 3.14 ± 0.19 at 10 months ( p>0.001), 3.42 ± 0.38 at 26 months, and 0.74 ± 0.08 at 30 months ( p>0.001). The urinary fluorescence of circulating AGE rose from 2.42 ± 0.15 AU/mg protein (3 months), 1.69 ± 0.07 (10 months), to 4.63 ± 0.35 (26 months) ( p>0.01) and 4.73 ± 0.72 (30 months), while the serum fluorescence increased from 0.39 ± 0.02 AU/mg protein at 3 months and 0.43 ± 0.02 at 10 months to 0.59 ± 0.04 at 26 months ( p>0.001) and 0.54 ± 0.03 at 30 months ( p>0.04). Polyclonal antibodies raised against AGE RNase showed faint areas of AGE immunoreactivity in mesangial areas in the nephrons of young rats. The immunolabeling of Bowman's capsule, the mesangial matrices, and the peripheral loops of glomerular and tubule basement membranes increased with rat age. The increase in circulating AGE peptides parallels the accumulation of AGEs in the nephron, and this parallels the pattern of extracellular matrix deposition, suggesting a close link between AGE accumulation and renal impairment in aging rats. (J Histochem Cytochem 45:1059–1068, 1997)

Aging is associated with the loss of preferential urinary excretion of Amadori-product glycated albumin. We have measured the binding of 125I-labeled glycated albumin to the renal brush-border membrane vesicles from young and old rats to determine whether a specific receptor-mediated endocytosis system may be involved. 125I-Glycated albumin was specifically bound by renal brush-border membrane vesicles in a time- and temperature-dependent manner; the binding was concentration-dependent, saturable and reversible. Scatchard plots gave an apparent dissociation constant Kmof 488 ± 17 nM, and a number of binding sites N of 33.5 ± 3.4 pmol/mg protein/min in membrane vesicles from young (3 months old) rats; the binding of native [125I]albumin, gave a Kmof 1194 ± 200 nM (P < 2%) and N of 82.4 ± 16.3 pmol/mg protein/min (P < 3%). Vesicles from 10-month-old rats had a similar Km (619.6 ± 135.3 nM) and N (21.91 ± 2.98 pmol/mg protein/min), while those from older (30 months old) rats had significantly increased Km (1344 ± 237 nM, P < 3%) and N (81.3 ± 10.9 pmol/mg protein/ min, P < 1%) for 125I-glycated albumin binding. 125I-Glycated HSA was not displaced by unlabeled native HSA in less than 100-fold excess and native [125I]HSA was only displaced by a 10-fold excess of unlabeled glycated HSA. The binding of native [125I]HSA was partly inhibited (85%) by unlabeled glycated HSA. Thus, there appear to be two different binding sites, one for glycated and the other for native albumin, lying close together; and the glycation site on albumin is the discriminatory recognition factor.

Albumin glycation was investigated in old rats to elucidate the link between the preferential excretion of glycated albumin and age-related microalbuminuria. Postprandial blood glucose and the glycated albumin in the serum and urine of 3-, 10- and 30-month-old Wistar rats and in streptozotocin diabetic rats were determined. Blood glucose increased from 1.46 +/- 0.046 g l-1 in 3-month-old rats to 2.08 +/- 0.06 (10 months) and 1.75 +/- 0.23 (30 months) (P0.05). Albumin glycation level in the serum increased from 0.79 +/- 0.07 nmol HCHO/nmol albumin (3 months) to 1.41 +/- 0.14 (10 months) and 1.73 +/- 0.21 (30 months) (P0.05); urinary level increased from 1.63 +/- 0.39 nmol HCHO/nmol albumin (3 months) to 2.92 +/- 0.57 (10 months) and 2.39 +/- 0.36 (30 months) (P0.01). The percent glycated albumin in serum rose from 3.33 +/- 0.64 to 6.81 +/- 0.63 and 6.99 +/- 1.79% of total albumin (P0.05), whereas the urine percentage decreased from 12.81 +/- 3.97 to 12.64 +/- 2.87 and 2.63 +/- 0.97% (P0.05) in 3-, 10- and 30-month-old rats, respectively. Editing decreased with aging from 4.28 +/- 0.83 (3 months) to 1.84 +/- 0.32 (10 months) and 0.52 +/- 0.14 (30 months) (P0.01). Editing in microproteinuric diabetic rats was lower (0.95 +/- 0.08) than in 3-month-old control rats (P0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Mesenchymal cells (fibroblasts, smooth muscle cells) and endothelial cells were shown to interact with elastin fibers. The strong adhesion of elastin fibers to these cells is mediated by a cell membrane complex with a major glycoprotein component of 120 kDa designated as elastonectin. This interaction was studied by transmission electron microscopy (TEM) and immunocytochemical techniques using antibodies raised against the elastin adhesive proteins. When fibroblasts and smooth muscle cells were cultured in presence of elastin fibers, TEM showed an adhesion mechanism that takes place over several sites along the plasma membrane of these cells. Endothelial cells showed a very close association with elastin, emitting "pseudopodia" that embody the fibers. TEM, indirect immunofluorescence, immunoperoxidase, and confocal microscopy showed the presence and localization of cell membrane components synthesized in large quantities when cells were incubated in presence of elastin. Cells without elastin fibers barely revealed the adhesive membrane complex. These results confirm and extend previous findings concerning the presence of an inducible cell membrane complex that mediates the adhesion of elastin fibers to these cell types.

A selective proteinuria occurs with normal aging. We investigated the contribution of a defect in the receptor-mediated endocytosis to the age-related albuminuria by measuring albumin binding by renal brush-border membrane vesicles from young and old female Wistar rats using a filtration method. Old (24 months) rats had a significantly higher proteinuria (13.29 +/- 5.25 mg prot/24 h/100 g bw) than did young (3 months) rats (1.23 +/- 0.55 mg prot/24 h/100 g bw). Scatchard analysis of the kinetic parameters of 125I-albumin binding revealed a decrease in the binding capacity of brush-border membrane vesicles from old rats. The number of binding sites, N (pmol/mg protein/min) was 236.84 +/- 97.50 in old rat preparations and 380.27 +/- 178.36 in young rat vesicles (P0.05). By contrast, Km did not change significantly with age (478.86 +/- 259.29 nM in old rat vesicles and 498.00 +/- 220.36 nM in young rat preparations). Consequently the index of adsorptive endocytosis efficiency (the N/Km ratio) decreased drastically with age from 0.782 +/- 0.238 at 3 months to 0.547 +/- 0.199 at 24 months (P0.05). These data indicate that defective receptor-mediated endocytosis could, at least partly, explain the age-dependent rise in urinary albumin excretion.

Sinefungin, an antifungal and antiparasitic nucleoside antibiotic, is a very potent antileishmanial agent in vitro and in vivo (Bachrach et al. 1980, FEBS Letters121, 287–291; Neal et al. 1985, Transactions of the Royal Society of Tropical Medicine and Hygiene79, 85–122). It was previously shown that this molecule is a competitive inhibitor of AdoMet for transmethylases (Paolantonacci et al. 1986, Molecular and Biochemical Parasitology21, 47–54; Avila et al. 1987, Molecular and Biochemical Parasitology26, 69–76) and that it induces shape changes of Leishmania donovani promastigotes as observed by light microscopy (Lawrence and Robert-Gero 1990; Bulletin de la Societe Francaise de Parasitologie8, 13–18). In the present work the effect of the antibiotic on the ultrastructure was analyzed by electron microscopy. The main changes induced at sublethal concentrations (0.26 μM sinefungin for 16 hr) were progressive rounding, decreased motility, enlargement of the flagellar pocket, and shortening and loss of the external part of the flagellum. The comparison with control cells showed shorter Golgi saccules and fragmentation of the trans-Golgi network into vesicles, indicating a stimulated Golgi apparatus activity. This result, associated with the enlarged flagellar pocket, suggests an unbalanced cytoplasmic exchange between exocytosis and endocytosis. These effects are quite different from those induced by tunicamycin (Dagger et al. 1984, Biology of the Cell50; 173–180) or paromomycin. In addition, other nucleoside and nonnucleoside growth inhibitors failed to induce similar changes. AdoMet antagonized the sinefungin-induced shape changes and ultrastructural modifications but had no effect with respect to other growth inhibitors. This suggests that the sinefungin activity at the cellular level is specifically related to competition with AdoMet. A comparative study of N-methylation and carboxylmethylation of proteins in sinefungin-treated promastigotes showed that the antibiotic preferentially inhibits the latter, catalyzed by protein-O-methyltransferases. These enzymes are known to regulate the function of various proteins involved in secretion. Overall the results suggest that one of the main targets of sinefungin in exponentially growing cells is the protein carboxylmethylation involved in membrane transport.

In a previous in vivo study, we showed that the glomerular cells of rat kidney synthesize both peripheral and integral plasma membrane proteoglycans. The present work focusses on the age-related changes in these cell membrane proteoglycans. The peripheral proteoglycans in “adult control” rats aged 3 months were found to be heparan sulfate, dermatan sulfate, and chondroitin sulfate, with heparan sulfate being the main glycosaminoglycan. The integral membrane proteoglycans contained mainly dermatan sulfate plus less amounts of heparan sulfate. The relative proportions of the glycosaminoglycans in the integral membrane proteoglycans changed between 1 and 3 months. In addition, the degree of sulfation increased in both families of proteoglycans, and this was associated with an increase in glycosaminoglycan synthesis in the peripheral proteoglycans. The nature and relative proportions of the glycosaminoglycans forming the proteoglycans, did not change with age, after 10 months, and neither did the amount of glycosaminoglycans. But, the degree of sulfation of both peripheral and integral membrane proteoglycans decreased. De novo synthesized proteoglycans from 24-month-old rats had a higher overall charge than did those at other ages, owing to the presence of sulfate and carboxylic groups. We conclude that, as for glomerular basement membrane proteoglycans, biochemical alterations affect the glomerular cell membrane proteoglycans with aging.

Alterations of tubules and glomerules have been reported previously in kidneys of rat neonates after aminoglycosides were given to the mother during gestation. Here, we have studied the effects of gentamicin on the development of the glomerular basement membrane (GBM). Pregnant Wistar female rates were treated with gentamicin. Deliveries occurred normally. Using electron microscopy, we looked at the deepest glomerules of the kidneys of 1-day-old neonates: myeloid bodies were found in podocytes, and the GBM appeared thicker and denser than in controls. Anionic ferritin, injected intravenously crossed the GBM in prenatally gentamicin-exposed animals, but not in controls. Furthermore, urine electrophoresis showed the presence of proteins normally found only in the urine of fetuses 2 days before birth. We suggest then, that in utero exposure to gentamicin leads to a delay of renal maturation and that the GBM is altered in juxtamedullary nephrons while it is normally differentiated and functioning in controls. Thus exposure to drugs before birth could be harmful to the GBM.

The mechanism by which proteins that pass through the glomerular basal lamina are taken up by proximal tubule cells is incompletely characterized. Past work has identified the kinetics of albumin binding to renal brush-border membrane. We have now purified and characterized albumin binding protein (ABP) and shown its distribution in renal proximal tubular cells. ABP was purified from rat renal proximal tubular cell brush-border membrane by affinity chromatography with rat serum albumin-Sepharose. The resulting ABP had two apparent molecular masses (55 and 31 kDa) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Antibodies to ABP were raised in rabbits and checked by immunoassay and immunoblotting. Light-microscopic immunohistochemistry showed ABP all along the proximal tubule in the pars convoluta and pars recta. Electron-microscopic immunohistochemistry showed labeling on microvilli and in apical endocytic vacuoles, dense apical tubules, and lysosomes. These results indicate that ABP is involved in proximal tubule endocytosis.

Changes in kidney maturation in utero have been reported after gentamicin administration to pregnant rats. While the proteinuria commonly observed could be related to modifications of the glomerular basement membrane, perturbed renal protein handling could be accounted for by changes in the proximal tubular cells. Therefore, we studied the effect of gentamicin on the renal handling and transport of proteins in proximal tubular cells using the horseradish peroxidase, a fluid-phase marker, as a probe. Gentamicin was administered intraperitoneally to pregnant Wistar rats (75 mg/kg body weight per day) and neonatal kidneys were studied 1 day after birth. In proximal tubular cells of the deep cortical area, containing the fully matured nephrons of neonates, the transport and digestion of reabsorbed peroxidase was considerably reduced compared with controls where peroxidase reached lysosomes after endocytosis. Urinary protein excretion increased in treated animals. We conclude that gentamicin, entering the proximal tubular cells via the endocytic pathway, decreases the tubular reabsorption of proteins, thus increasing urinary protein excretion.

Gentamicin during gestation alters glomerular basement membrane development. A drug-induced nephrotoxicity was described for neonates after gentamicin was given intraperitoneally to pregnant Wistar rats; glomerular alterations and changes in permselectivity were important. We investigated the ultrastructure of the glomerular basement membrane (GBM), the arrangement of anionic sites, and the urinary proteins at two ages, with 1-day- and 12-month-old control and prenatally exposed animals. For neonates, the pattern of glomerular differentiation was similar, anionic sites were made of heparan sulfate proteoglycans, and the GBM had the same total thickness in both groups. After transplacental gentamicin exposure, the lamina densa was larger; the laminae rarae were thinner; the density of anionic sites was increased; the levels of hydroxyproline, sulfate, and hexuronic acid in the kidney were increased; and the immunoelectrophoresis of urinary proteins was abnormal. For adults, prenatal exposure to gentamicin led to altered juxta-medullary glomeruli with a larger GBM and abundant anionic sites, especially in the lamina densa, and to a protein excretion different from that of controls. Thus, gentamicin administered during pregnancy leads to permanent alterations of the GBM with modifications of both the layers and the anionic sites, possibly because of a perturbed protein metabolism. These altered glomeruli are at risk during life and could be the starting point for a kidney disease.

1. 1. In the kidney, filtered proteins are rapidly reabsorbed by the proximal tubule via adsorptive endocytosis. This process starts with the protein binding to the luminal brush-border membrane. 2. 2. The binding of 125I-labelled albumin to rat renal brush-border membrane vesicles and the effect of a low molecular weight protein lysozyme on that binding was assessed by the nitration method. 3. 3. The Scatchard plot revealed a one-component binding-type curve with a dissociation constant Kd of 430.9 nM and 39.6 pmol/mg membrane protein for the number of binding sites. 4. 4. Albumin binding was saturable and reversible, time and temperature dependent and the initial rate enhanced by increasing amounts of lysozyme. 5. 5. The fact that association of albumin with the brush-border membrane vesicles was dependent upon the intravesicular space suggested a double process, binding of the ligand to the membrane surface and its internalization. These data suggest that albumin has a different binding site than that of a low-molecular weight protein lysozyme, with a constant affinity value near physiological loads. That specificity may confer selectivity upon the endocytic uptake process.

Differentiation of the glomerulus and the proximal tubule was studied in the rat foetus, especially with regard to the development of the protein filtration-reabsorption apparatus. Filtration starts several days before full differentiation of the glomerulus, when the glomerular basement membrane consists of a thin lamina alongside the podocyte membrane. Endocytosis is functional from this time, but fusion between endocytic vesicles and lysosome-like bodies occurs 2 days later. Foetal urine electrophoresis shows the presence of many proteins, including high molecular weight ones, this proteinuria seeming chiefly due to the immaturity of the glomerular barrier.

Proteoglycans were studied in developing rat fetal kidney using cytological and biochemical techniques. These compounds were detected with immunoperoxidase in the nephron basement membranes from the earliest stages of differentiation. In the glomerular basement membrane, immunostaining appeared as both diffuse and granular deposits, as long as this membrane consisted of loose material; however, as soon as a three-layered membrane had formed, staining was confined to the laminae rarae as regularly arranged granules. The same pattern of staining was observed during differentiation of the basement membrane of the proximal tubule. In Bowman's capsule, immunostaining appeared as granules, which were sparsely distributed in the developing glomerulus and then regularly lined the stacked laminae when differentiation was complete. In all basement membranes, anionic sites (disclosed by polyethyleneimine) were colocated with immunostained granular deposits. Total glycosaminoglycan content gradually increased from the beginning of metanephros development to birth. During this period, the relative proportions of glycosaminoglycans changed: heparan sulfate increased and hyaluronic acid decreased as differentiation proceeded. The possible relationship between morphological observations and biochemical changes in glycosaminoglycan content is discussed.

The role of collagen in ultrafiltration properties of the glomerular basement membrane (GBM) was tested after a single administration of bacterial collagenase, using native ferritin as a tracer which does not pass through the GBM under physiological conditions. Experiments were performed both in situ and with isolated kidneys. Increased permeability to ferritin occurs 6 hr following enzyme perfusion and becomes patent after 30 hr, numerous tracer molecules appearing in urinary space, without any readily observable changes either in distribution of fixed negative charges (as revealed by colloidal iron and polyethyleneimine) or in structural organization of the glomerulus. Selective permeability of the GBM is progressively restored so that ferritin is almost confined to capillary lumen one month after enzyme injection. We conclude that collagen plays an important part in restricting plasma protein filtration.

Successive steps leading to the development of glomerular ultrafiltration properties were explored in rat fetuses. The appearance of the lamina densa of the glomerular basement membrane (GBM) concurrently with a sharp rise in collagen biosynthesis suggest a prominent role for these events in restricting permeability to plasma proteins. Sieving functions of the glomerular barrier are shown to depend on macromolecular architecture of the GBM, negative-fixed charges of the laminae rarae representing only one factor in maintaining the structure required for selective permeability.

The distribution of basement membrane glycoproteins (type IV collagen, laminin, fibronectin, and proteoglycans) was studied in foetal rat kidney by immunohistochemical techniques using polyclonal antibodies. From the first stages of nephron differentiation, all these glycoproteins were detectable by immunofluorescence in the tubular and glomerular basement membranes and in the mesangial matrix. As differentiation proceeded, labelling of glycoproteins progressively intensified, except for that of fibronectin, which gradually decreased in the glomerular basement membrane (GBM) and was barely observable at full differentiation. With immunoperoxidase staining in electron microscopy, all glycoproteins were seen to be widely dispersed in the spaces between the epithelial and endothelial glomerular cells so long as the GBM remained a loose structure. However, after it became a compact, 3-layered formation, type IV collagen and laminin were distributed throughout the GBM, whereas proteoglycans and anionic sites appeared as 2 rows of granules confined to the laminae rarae.