Effect of glycation of albumin on its binding to renal brush-border membrane vesicles: influence of aging in rats.

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 Km of 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 Km of 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.

Age-related changes in albumin binding by renal brush-border membrane vesicles.

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 (P < 0.05). 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 (P < 0.05). These data indicate that defective receptor-mediated endocytosis could, at least partly, explain the age-dependent rise in urinary albumin excretion.

Glycation of albumin with aging and diabetes in rats: changes in its renal handling.

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) (P < 0.05). 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) (P < 0.05); 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) (P < 0.01). 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 (P < 0.05), whereas the urine percentage decreased from 12.81 +/- 3.97 to 12.64 +/- 2.87 and 2.63 +/- 0.97% (P < 0.05) 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) (P < 0.01). Editing in microproteinuric diabetic rats was lower (0.95 +/- 0.08) than in 3-month-old control rats (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of advanced glycation endproducts in the rat nephron: link with circulating AGEs during aging.

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.

Age-related changes in the plasma membrane proteoglycans of rat kidney glomerular cells.

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 focuses 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.

Basement membrane proteoglycans and anionic sites in the fetal rat kidney during late gestation.

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.

[Generalities on vascular renal aging]. / Généralités sur le vieillissement vasculaire rénal.

Until the last decade, kidney aging was considered to be the result of progressive loss of nephrons associated with development of glomerulosclerosis and thus decrease in glomerular filtration rate, and finally renal deficiency. However this nephropathy can also result of environmental (such as husbandry conditions, diet ...) and genetical factors. So, food restriction can protect glomeruli against hyperfiltration and risks of glomerulosclerosis. Both capillaries and large vessels were modified during aging. Concerning the glomerular capillaries, the main alterations are changes in glomerular hemodynamics and in the composition and structure of the glomerular basement membrane (noticeably thickening and decrease in heparan sulfate proteoglycans and thus in anionic barrier), glycation of both the structural and plasma protein, resulting in increased permeability of the glomerular capillary wall and development of proteinuria.

Differentiation of glomerular filter and tubular reabsorption apparatus during foetal development of the rat kidney.

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 fom 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 one, this proteinuria seeming chiefly due to the immaturity of the glomerular barrier.

[Effect of collagenase on the permeability of the glomerular basement membrane in the rat kidney]. / Action de la collagénase sur la perméabilite de la membrane basale glomérulaire du rein de rat.

Recently, several authors have emphasized the role of negative sites located in th laminae rarae of the glomerular basement membrane (GBM), in restricting glomerular permeability to anionic macromolecules. In this work, we point out that ultrafiltration properties involve integrity of the GBM. Indeed after intravenous perfusion of bacterian collagenase, anionic ferritin permeates the GBM though negative site distribution (as shown by fixation of colloidal iron) is unaffected.

Transplacental effects of gentamicin on endocytosis in rat renal proximal tubule cells.

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.

[Aging and brain circulation. Role of the extracellular matrix of brain microvessels]. / Vieillissement et circulation cérébrale. Rôle de la matrice extracellulaire des microvaisseaux du cerveau.

Maintenance of normal brain activity is dependent among other factors on the maintenance of a functional blood-brain barrier (BBB), localised mainly at the capillary wall of cerebral microcirculation. The modifications of the BBB during aging play an important role in cognitive decline with aging as well as in dementias. A review of the experiments of our laboratory over the last decades is presented, on the interaction of endothelial cells with their basement membranes, both together representing a functional unit of BBB. The action of proteolytic enzymes on the basement membrane increases BBB permeability by increasing the transcellular transport activity of endothelial cells. Flavonoid drugs protect BBB from proteolytic activity by interacting with collagen fibers and protecting sensitive peptide bonds from attack by proteolytic enzymes. These drugs enhance also the resynthesis of degraded basement membranes.

Binding of 125I-labelled albumin by isolated rat renal brush-border membrane vesicles. Evidence for uptake and internalization process.

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. 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 filtration method. 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. Albumin binding was saturable and reversible, time and temperature dependent and the initial rate enhanced by increasing amounts of lysozyme. 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.

[Changes in the glomerular filtration barrier during aging in rats]. / Modifications de la barrière de filtration glomérulaire au cours du vieillissement, chez le rat.

In this work, we analysed histochemical, biochemical and functional modifications of the glomerular basement membrane (GBM), occurring for aging, in the Rat. The results suggest an increase of collagenous components and a decrease of sulfated glucosaminoglycans as a function of age. In other respects, fixed anionic sites of the GBM, disclosed by colloidal iron, are almost exclusively restricted to the laminae rarae in one month-old rats, whereas the marker appears randomly scattered among the lamina densa in 12 month-old animals. These changes could be the cause of increased permeability of the GBM during aging.

Differentiation of the glomerular filtration barrier in the rat fetus: possible role of collagen.

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.

Gentamicin administered during gestation alters glomerular basement membrane development.

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.

[Epigenetic mechanisms of aging: relations between Maillard reactions and radical generation]. / Mécanismes épigénétiques du vieillissement: relàtions entre réactions de Maillard et attaques radicalaires.

Aging is under the influence of both genetic and epigenetic factors. We are particularly interested in these latter mechanisms which play an important role in the aging of most if not all pluricellular organisms. Genetic factors on the contrary appear to differ among the species investigated. We studied two major epigenetic mechanisms: the Maillard reaction or nonenzymatic glycosylation and generation of free radicals. Using a sensitive method of free radical detection based on the degradation of hyaluronan, we could show that Maillard products are able to produce the depolymerization of this glycosaminoglycan. This may be of special importance in tissues rich in hyaluronan as the vitrous of the eye. Its degradation with age, accelerated in diabetes may well depend on such reactions, intensifyed in diabetes and involved in its complications as diabetic retinopathy.

Glomerular alterations in rat neonates after transplacental exposure to gentamicin.

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.

Localization of basement membrane glycoproteins in rat kidney during foetal development.

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.

Effect of feeding on glomerular filtration rate and proteinuria in conscious aging rats.

Food intake increases glomerular filtration and proteinuria in adult rats. That this postprandial hyperfiltration could be age dependent was investigated in 3-, 10-, 20-, and 30-mo-old rats. Glomerular filtration rate and protein excretion were measured in fed or 24 h fasted conscious animals. In the 3-mo-old rats food ingestion increased renal filtration by 45% from 1.17 +/- 0.08 to 1.73 +/- 0.11 ml.min-1.g kidney wt-1 (n = 6). As the animals became older, the differences between fed and fasted periods became smaller: in 30-mo-old rats glomerular filtration rate was 0.85 +/- 0.03 and 1.01 +/- 0.06 ml.min-1.g kidney wt-1 (n = 6) in fasted and fed conditions, respectively. Proteinuria, which was mainly albuminuria, increased slightly with age and was more markedly reduced by acute food restriction in the 30-mo-old than in the 3-mo-old rats. Because the renin-angiotensin system activity decreases with age, its role in postprandial hyperfiltration was assessed by measuring glomerular filtration in 3-mo-old animals whose angiotensin II converting-enzyme activity was chronically inhibited by daily administration of perindopril. In such experimental conditions there was no longer a difference in renal filtration between fed and fasted rats. These data indicate that 1) postprandial increase in glomerular filtration is to some extent related to the renin-angiotensin system activity; 2) short-term reduction of food intake reduces proteinuria even in senescent rats, although the feeding dependence of the glomerular filtration is blunted with age.