Accumulation of Advanced Glycation Endproducts in the Rat Nephron: Link with Circulating AGEs During Aging

Abstract

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)

Keywords

AGEs fluorescence immunolocalization aging kidney rat

The nephropathies associated with normal aging probably reflect the effects of vascular lesions. The resulting mesangial expansion (Couser and Stilmant 1975; Bolton and Sturgill 1981; Bell et al. 1984) can progress to nephrosclerosis (Gray et al. 1982). It is generally agreed that age-related albuminuria is due to increased permeability of the glomerular basement membrane (GBM) (Alt et al. 1980; Horbach et al. 1983; Corman and Michel 1987). Morphological changes in the GBM, including thickening (Dodane et al. 1991; Corman et al. 1988), can lead to focal and segmental glomerulosclerosis (Couser and Stilmant 1975; Bolton and Sturgill 1980; Yoshioka et al. 1988). There is still no evidence that these functional and structural changes are due to chemical or other changes in the glomerular extracellular matrix (GECM), especially the basement membrane and mesangial matrix (MM). However, there is a link between the pattern of glo-merular changes that occur in aging kidneys and the age-dependent accumulation of advanced glycosylation endproducts (AGEs) (Sell and Monnier 1990; Makita et al. 1992). AGEs are the late products of the glycation of matrix proteins exposed to glucose and they accumulate slowly in the renal and extrarenal matrices during normal aging (Bucala et al. 1992; Miyata and Monnier 1992) and more rapidly in diabetes as a result of hyperglycemia (Brownlee et al. 1988; Makita et al. 1992; Vlassara et al. 1994a). Glucose tolerance often becomes impaired with aging (Defronzo 1979; Fink et al. 1984), and although hy-perglycemia is not directly involved in aging, several lines of evidence indicate that the accumulation of GECM in aging kidneys is due to the formation of AGE (Miyata and Monnier 1992; Li et al. 1996). Gly-cated basement membrane macromolecules, especially Type IV collagen (Cohen and Wu 1981; Ziyadeh 1993), can affect renal function by altering the permselectiv-ity and resistance to degradation of thickened GBM, which is the hallmark of a glomerular defect.

The present study used the formation of protein-derived fluorescence as a marker of AGE formation in vivo with aging in rat. The distribution of AGEs in the renal extracellular matrix was also monitored immu-nochemically using an AGE-specific polyclonal antibody raised against an AGE immunogen synthesized in vitro, to link its accumulation with age-associated renal impairment.

Materials and Methods

Animals

Studies were performed on male Wistar rats, a strain that does not suffer from age-associated renal disease (Gray et al. 1982). A colony from a germ-free Wistar (WAG/Rij) rat strain purchased from the Institute for Experimental Gerontology TNO Rijswick (The Netherlands) was developed in a specific pathogen-free husbandry. With advancing age, the rats developed discrete but significant proteinuria due to al-buminuria, which is believed to be a hallmark of normal aging (Heudes et al. 1994). The animals were fed a standard rodent diet (DO4; UAR, Villemoisson sur Orge, France) containing 17% protein, 0.71% phosphorus, 0.78% calcium, 0.62% potassium, 0.27% sodium, 0.22% magnesium, and a total of 2.9 kCal/g. Water was provided ad libitum. Rats were placed in metabolic cages and urine (24 hr) was collected over protease inhibitors (1 mM PMSF, 1 mM EDTA, 1 mM N-ethylmaleimide), and 0.01% NaN₃ and di-alyzed against 0.05 M carbonate buffer, pH 9.6, for 24 hr. Blood samples were taken and centrifuged for 10 min at 3000 rpm and the serum dialyzed against the same buffer. Protein content of the samples was determined by the Lowry method (Lowry et al. 1951), and the concentration was adjusted to 1 mg/ml before measuring AGEs by fluorescence [excitation/emission wavelengths (L_Ex/L_Em) of 370/440 nm] (Sell and Monnier 1989) in an LS-3B Fluorescence spectrometer (Perkin-Elmer; Norwalk, CT). Fluorescence data were expressed in absolute units (AU) as non-treated rat serum albumin (RSA), at 1.0 mg/ml corresponding to 1.0 in AU (Na-kayama et al. 1989).

Biochemical Analysis

Preparation of Basement Membrane. Rats were sacrificed by cervical dislocation and the kidneys were quickly removed and decapsulated. The glomeruli were isolated from paired batches of kidney cortex by gentle homogenization and sieving (Krakower and Greenspon 1978). The glomerular basement membrane (GBM) was isolated by the method of Meezan et al. (1976).

Extraction of Type IV Collagen Peptides from GBM. GBM was extracted with guanidine-HCl to obtain a soluble fraction containing mainly noncollagenous proteins and a residue including Type IV collagen (Mohan and Spiro 1986). GBM was suspended (2 mg/ml) in 4 M guanidine-HCl/50 mM Tris-HCl, pH 7.0, containing protease inhibitors, and was stirred at room temperature for 18 hr. The samples were then centrifuged (12,000 rpm for 30 min), the supernatant (guanidine-HCl extract) was discarded, and the Type IV collagen peptides were extracted from the pellet.

The insoluble material remaining after guanidine-HCl extraction was washed twice with 0.1 M CaCl₂/20 mM Tris-HCl (pH 7.4) and digested with Type VII collagenase (0.1 mg/ml; Sigma, St Louis, MO) by shaking at 37C for 20 hr. The digest was centrifuged at 12,000 rpm for 30 min at 4C and the protein and collagen contents of the supernatant were measured. Collagen was assayed by the method of Bergman and Loxley (1970). An aliquot of supernatant was hydrolized with 6 N HCl for 24 hr at 100C in sealed glass tubes. The HCl was evaporated off the residue dissolved in distilled water and the hydroxyproline (OHPro) assayed. The concentration was adjusted to 1 mmol OHPro/ml and the fluorescence measured as an indirect index of AGE concentration (L_Ex/L_Em 370/440). The fluorescence of an enzyme solution (Type VII collagenase 0.1 mg/ml) was used as one arbitrary unit and results were expressed as AU/mM OHPro.

Extent of Collagen Peptide Glycation. The presence of Ama-dori adducts (glucitol-lysine) on long-lived glycoproteins such as collagen should be an indication of lack of AGE accumulation. The extent of glycation (sugar bound to collagen) was therefore determined by measuring the formaldehyde released by periodate oxidation of C₁-hydroxyls in the Amadori product form of the glycated collagen (Ahmed and Furth 1991). Briefly, aliquots of the above supernatant containing Type IV collagen peptides (1–5 μg OHPro in 40 μl water) was incubated with 20 μl 0.1 N HCl and 20 μl 0.05 M NaIO₄ for 30 min at room temperature. Samples were then cooled in ice for 10 min, mixed with 20 μl cold 15% ZnSO₄ and 20 μl 0.7 N NaOH, and centrifuged. A 100-μl aliquot of the supernatant was placed in a microplate well and incubated with 200 μl acetylacetone-ammonium acetate reagent for 1 hr at 37C. The absorbance of the released formaldehyde (1 mol HCHO per mol glucose) converted to a chromophore (DDL) was read at 405 nm in an ELISA plate reader (Metertech 960). A standard curve of 0–40 nmol fructose was used for each assay. Results are expressed as nmol HCHO/nmol OHPro.

Immunochemical Studies

Preparation of Advanced Glycosylated End products (AGEs). Bovine pancreatic ribonuclease A (RNAse A Type I) and rat serum albumin (RSA) were obtained from Sigma. These were selected because they have been found to be good target proteins for AGE; the high AGE-mediated crosslinking activity of RNase should facilitate immunoge-nicity (Eble et al. 1983). The immunogens, AGE-albumin (AGE-RSA) and AGE-ribonuclease (AGE-RNase) were prepared in vitro by incubating RSA or RNase (5 mg/ml) with 0.5 M glucose in PBS (pH 7.4) for 60 days under sterile conditions in the dark at 37C (Makita et al. 1992). The unbound glucose was then removed by extensive dialysis against PBS and the AGEs formed on RSA and RNase were assessed from the fluorescence spectra (L_Ex/L_Em370/440). Native albumin was removed from AGE-RSA by affinity chromatography on Affi-Gel Blue (Travis et al. 1976). Unadsorbed proteins containing AGE-RSA were eluted with five column volumes and concentrated in an Amicon Cell (PM 10 membrane). The AGEs were reduced by incubation with 100 mM sodium borohydride (Sigma) for 30 min at room temperature and the unreacted borohydride was removed by dialysis against PBS. Fluorescence was measured at a protein concentration of 1 mg/ml and expressed in arbitrary units(AU) compared with native RSA or RNase. Reduced AGE-RSA contained 8 AU/mg, and AGE-RNase had 5.3 AU/mg.

Production of Antibodies Against AGEs. Two female New Zealand White rabbits were injected intradermally at multiple sites with 200 μg/ml of AGE-RSA or AGE-RNase emulsified with an equal volume of Freund's complete adjuvant. Booster injections of the same amount of AGE adjuvant were given 2 weeks later, and the presence of antibodies was monitored by Ouchterlony double diffusion. The rabbits were bled 15 days after the second injection. IgGs were isolated on a protein A-Sepharose column and specific antibodies were purified by affinity chromatography on an AGE-CNBr Sepharose CL-4B column. The polyclonal antibodies were titrated in a noncompetitive ELISA system using AGE-RNase or AGE-RSA as the adsorbed antigen. The titer of the anti-AGE antibodies was defined as the antibody dilution giving a 50% maximal L₄₀₅ signal in a micro-ELISA plate reader (Metertech 960) using a peroxidase-conjugated anti-rabbit IgG second antibody. The specificity of the anti-AGE-RNase antibodies was tested using absorbed antigens (AGE-RSA, soluble and structural glycoproteins, fibronectin, fetuin, Type IV collagen, and laminin) in the same noncompetitive ELISA system.

Histochemical and Immunohistochemical Methods. For light microscopy, small pieces of rat kidney cortex were fixed by immersion in formaldehyde buffered for 48–72 hr. The fixed tissue was dehydrated in a graded ethanol series, cleared in toluene, and embedded in paraffin. Sections (5 μm thick) were cut and stained with periodic acid-Schiff's reagent, or AGEs were measured by immunofluorescence. Other samples of kidney were prepared for electron microscopy. Briefly, cubes of renal cortex were fixed in 4% paraformaldehyde in 0.1 M cacodylate buffer pH 7.4 for 24 hr, dehydrated and embedded in LR White. Thick sections (1 μm) were cut, placed on a slide, and stained with methylene blue. Where glomeruli were located by light microscopy, the blocks were trimmed and ultrathin (600 Å) sections cut. Sections were placed on carbon- and formvar-coated nickel grids and processed for protein-A gold immunochemical labeling (Roth et al. 1978).

Immunofluorescence. Sections were deparaffinized by three immersions for 1 min in toluene and rehydrated through a graded ethanol series. Nonspecific protein binding sites were blocked with 10% normal goat serum in Tris-buffered saline (50 mM Tris-HCl/ 150 mM NaCl, pH 7.6), and then prein-cubated with 2% PBS-fish gelatin (Sigma) in a humidity chamber at room temperature for 1 hr for blocking experiments. They were rinsed three times (10 min) with PBS and incubated for 1 hr with anti-AGE antibody diluted 1:20 and rinsed. Sections were incubated for 1 hr with the second antibody, FITC-labeled goat anti-rabbit IgG (Cappel; Cochranville, PA), rinsed, and examined by epifluorescence under the light microscope (Dialux-Leitz 20). The specificity of the immu-nolabeling was assessed in control experiments in which the primary antibody was replaced by normal rabbit serum before labeling and incubation with the second antibody.

Immunogold Electron Microscopy. Ultrathin sections mounted on grids were floated for 5 min on a large drop of 5% BSA in PBS, pH 7.4, transferred to a drop of anti-AGE antibody (1:20) in PBS containing 1% BSA and 0.1% Tween-20, and incubated at room temperature for 2 hr. Sections were given several washes in drops of PBS and incubated with protein A-gold complex (10 nm) (BioCell; Cardiff, UK) diluted (1:80) in PBS containing 0.1% fish gelatin for 45 min at room temperature. The grids were then thoroughly washed in PBS, postfixed with 2% glutaraldehyde, rinsed with distilled water, dried, counterstained with uranyl acetate and lead citrate, and examined in a Philips 201 transmission electron microscope. The specificity of the labeling was checked by incubating sections with normal rabbit serum before incubation with protein A-gold complex. Sections were photographed and printed to a final magnification of x 16,000 or x 40,000.

Statistics. Differences between age groups were assessed by Student's unpaired t-test. Means are ± SE. All p values of less than 0.05 were considered to indicate statistical significance.

Results

RNase was used to produce the immunogen (AGE-RNase). This gave a high titer of polyclonal rabbit antiserum against the immunogen. The antibodies reacted with other AGEs, such as that derived from rat serum albumin (AGE-RSA), but not with a variety of soluble and structural glycoproteins (Figure 1). These results suggest that the antibodies recognized a common immunological epitope formed in vitro from the reaction of glucose with carrier proteins, providing additional evidence for the conclusions of Makita et al. (1992).

AGE peptide fluorescence in the serum and the 24 hr urine samples of rats aged 3, 10, 26, and 30 months and GBM collagen-associated fluorescence were measured at 440 nm with excitation at 370 nm. The findings were correlated with the immunochemical distribution in the glomerulus extracellular matrix.

Circulating AGE Peptide

Aging resulted in a significant increase in circulating AGE (Figure 2). Fluorescence of the AGE peptide in urine increased from 2.42 ± 0.15 AU/mg protein (n = 8) at 3 months and 1.69 ± 0.07 (n = 8) at 10 months to 4.63 ± 0.35 (n = 12) (p>0.01) at 26 months and 4.73 ± 0.72 (n = 10) at 30 months (Figure 2A). The serum fluorescence increased from 0.39 ± 0.02 AU/mg protein (n = 8) and 0.43 ± 0.02 (n = 7) at 3 months and 10 months to 0.59 ± 0.04 (n = 12) at 24 months (p>0.001) and 0.54 ± 0.03 (n = 12) at 30 months (p>0.04) (Figure 2B).

Figure 1

Antiserum dilution curves for anti-RNase antibodies. Polyclonal antibodies were characterized in a noncompetitive ELISA using the following adsorbed antigens: ▪ AGE-RNase; □, AGE-BSA; ♦, laminin; ⋄, fibronectin; ○, Type IV collagen; •, fetunin.

AGEs in GBM Type IV Collagen

There was also an age-associated increase in AGE in Type IV collagen. The collagen fluorescence was stable between 3 (1.65 ± 0.05 AU/mM OHPro; n = 10) and 10 months (1.58 ± 0.04; n = 10) and increased to 2.16 ± 0.06 (n = 11) at 26 months (p>0.001) and 2.53 ± 0.18 (n = 8) at 30 months (p>0.001) (Figure 3).

Extent of GBM Type IV Collagen Glycation

Aging greatly affected the collagen content of glucitol-lysine, the marker of Amadori product. Early glycation products, in contrast to AGE, were decreased in 10-month-old rats, going from 5.35 ± 0.25 nmol HCHO/ nmol OHPro (n = 9) at 3 months to 3.14 ± 0.19 (n = 10) (p>0.001) at 10 months. It was 3.42 ± 0.38 (n = 11) at 26 months and 0.74 ± 0.08 (n = 16) at 30 months (p>0.001) (Figure 4).

Histochemical and Immunochemical Studies

The distribution of glycoprotein, the site of possible glycation in the glomerular matrix, was determined by staining kidney sections from 3-month- and 30-month-old rats (Figure 5) with PAS. Mesangial matrix increased with age, and the Bowman's capsule, glomerular and tubule basement membranes were also thickened by PAS-positive material (Figure 5B).

Figure 2

Effect of age on AGE-peptide fluorescence in urine (A) and serum (B). Values are arbitrary units (AU)/mg protein. (N) number of animals. ^∗∗ p>0.01; ^∗∗∗ p>0.005, significantly different from 3-month-old-rats. ^°° p>0.01; ^°°° p>0.001, significantly different from 10-month-old rats.

Polyclonal antibodies against AGE-RNase gave faint staining for immunofluorescence (Figure 6) in the mesangial areas and the tubule basement membrane of young rats (3 months) (Figure 6A). Labeling increased considerably as the animals aged and the mesangial matrix, Bowman's capsule (BC), and tubule basement membrane (TBM) were heavily stained in 10-month-old rats (Figure 6B). There was also fainter noticeable immunostaining of the glomerular basement membrane (GBM) at 26 months (Figure 6C). A control micrograph showed no labeling (Figure 6D).

Figure 3

Effect of age on collagen-associated fluorescence in GBM Type IV collagen. Values are arbitrary units (AU)/mM hydrox-yproline. (N) number of animals. ^∗∗∗ p>0.005; ^∗∗∗∗ p>0.001, significantly different from 3-month-old rats. ^°°° p>0.001, significantly different from 10-month-old and 26-month-old rats.

The protein A-gold technique indicated the ultra-structural localization of the AGE antigenic sites. Whereas very few gold particles were found over the different structures of the nephron of young (3-month) rats (Figure 7A), mainly over the BC (Figure 7B) and to a lesser extent the GBM, nephrons from older (26-month) rats exhibited denser labeling (Figure 8). Gold particles were uniformly distributed over the mesan-gial matrix, tubule (Figure 8A) and glomerular (Figure 8C) basement membranes, and Bowman's capsule. These data are consistent with the accumulation of AGE in renal extracellular matrices, especially basement membranes, with aging.

Figure 4

Changes in the extent of Type IV collagen early glycation (Amadori adducts) with age. Results are nmol HCHO/nmol hydrox-yproline. (N) number of animals. ^∗∗∗ p>0.005, significantly different from 3-month-old rats. ^°°° p>0.001, significantly different form 10-month-old rats.

Figure 5

Histological sections (PAS staining) of the kidney cortex from a 3-month-old (A) and a 30-month-old (B) rat. Note the significant expansion of the mesangial matrix (double arrowheads) and thickening of Bowman's capsule (double arrows), the GBM loop (arrowheads), and the tubule basement membrane (arrows). Bar = 20 μm.

Discussion

Our results indicate that there is a link between two key manifestations of physiological aging in the rat, renal impairment and the spontaneous biochemical changes called advanced glycation, which are believed to contribute to tissue damage. Although most strains of rats can develop age-related renal diseases (Gray et al. 1982; Baylis 1994), the Wistar WAG/Rij rats used in this study are only slightly affected up to 30 months of age (Dodane et al. 1991) when fed ad libitum. PAS staining showed that the only significant morphological changes that occur are glomerular enlargement with a moderate thickening of the glomerular and tubule basement membranes and expansion of the me-sangial matrix. All of these appear to indicate normal aging. However, these rats also develop albuminuria and glycosuria after 18 months (Dodane et al. 1991). We have shown that the amount of Amadori albumin adducts, an index of changes in glucose metabolism, increases with age (Bakala et al. 1995). The present study shows that there are AGEs in the extracellular matrix of the nephron in young rats, and that the amounts of AGE increase significantly as the animals age, mainly in Bowman's capsule, the mesangial matrix, tubule basement membrane and, to a lesser extent, in the glomerular basement membrane. All these matrices are rich in collagens, mainly Type IV collagen. The AGE-associated fluorescence in Type IV collagen peptides from GBM also increased with age. This is of particular interest because previous studies on Type IV collagen glycation conducted in vitro showed that they undergo structural and functional changes (Haitoglou et al. 1992). However, data on the interstitial collagen from human skin (Dyer et al. 1993) and from mouse tissues (Sanada et al. 1978) indicate an increase in the fluorescence of collagen with age. The increase in nonenzymatically mediated collagen crosslinking (AGE) is associated with a decrease in reducible enzymatic crosslinks. There are two types of collagen crosslinking: crosslinks mediated by lysyl ox-idase and those derived from glycation. The decrease in lysyl oxidase-mediated crosslinks in interstitial collagen is in agreement with the finding that lysyl oxi-dase activity decreases with age (Sanada et al. 1978; Fornieri et al. 1992). These data suggest that the AGE-mediated collagen crosslinks indicated by increased collagen fluorescence in our study are the main cause of the structural alteration in GBM that occurs with aging. The resulting crosslinked and protease-resistant structures promote accumulation of extracellular matrix (ECM) by slowing down their turnover. Because O-glucosylated lysine/OHlysine residues can be N-gly-cated to the same extent as those that do not contain the enzymatically linked carbohydrate units (Garlic et al. 1988), there is no steric hindrance to block the nonenzymatic attachment of glucose to collagen residues by the disaccharide substituent on the oxygen of the vicinal carbon. The sharp decrease in the content of glucitol-lysine/OHlysine in the GBM Type IV collagen from older rats supports this. The rate of formation of Amadori adducts in Type IV collagen may be balanced by their conversion to the more complex AGEs because of the slow turnover of the GBM glyco-proteins. This, in turn, may restrain the formation of ketoamine adducts. Finally, the glycoprotein constituents of basement membranes are particularly suitable targets for changes due to glycation because their turnover is slow, as indicated by the similarities of the PAS staining and the AGE immunodistribution patterns.

Figure 6

Changes in the immunohistochemical distribution of AGEs in the rat nephron with aging: immunofluorescence staining for AGEs is detectable in the mesangial area (double arrowheads), the GBM (arrowheads), Bowman's capsule (double arrows), and TBM (arrows) of a 3-month-old (A) and a 10-month-old (B) rat. There is extensive antibody binding to the Bowman's capsule and TBM, while there were thin ribbons of fluorescence in the peripheral loop GBM and the mesangial matrices of eldrly rats aged 26 months (C). (D) Representative control showing no fluorescence when the primary antibody was replaced with normal rabbit serum. Bar = 40 μm.

Figure 7

Distribution of AGEs over the nephron extracellular matrices of young (3-month-old) rats. Very few gold particles were found over the glomerular basement membrane (GBM). Bowman's capsule (BC), and the tubule basement membrane (TBM) at this age (B,C). The capillary lumen (CL) and the urinary space (US) appeared free of labeling (A). (D) Representative control as described in Materials and Methods. Bars = 0.5 μm.

Figure 8

Effect of age on the density of AGE labeling. Labeling is intense over the different structures of the nephron of old (26-month) rats (A-C). GBM, glomerular basement membrane; BC, Bowman's capsule; TBM, tubule basement membrane; US, urinary space; EC, edothe-lial cell. Bars = 0.5 μm.

The main new finding of this study is the increase in AGE-associated fluorescence in the urine and plasma of rats with aging. The accumulation of AGE peptides in the serum must reflect the degradation of increased amount of AGEs in the matrix and/or decreased removal of AGE peptides. It has been proposed that the AGE peptides produced by the normal catabolism of AGE-containing proteins are released into the circulation to be cleared by the kidney, showing the link between AGE accumulation in tissues and renal dysfunction (Li et al. 1996). Several recent studies have shown that AGE peptides are cleared more slowly than creat-inine, indicating that not all circulating AGE peptides are filtered (Makita et al. 1994; Vlassara et al. 1994b). Therefore, the serum AGE peptide concentration is closely correlated with the severity of nephropathy (Ma-kita et al. 1994; Papanastasiou et al. 1994; Li et al. 1996). Because AGEs and AGE peptides are both chemically and biologically active, the accumulation of AGE in renal tissue with age and the circulating breakdown products can both have adverse biological effects leading to kidney damage (Doi et al. 1992; Vlassara et al. 1994a).

The influence of AGEs on nephropathy caused by aging could perhaps be prevented by blocking preformed AGEs and/or by preventing further glycation. The AGE inhibitor aminoguanidine can interfere with AGE accumulation, and appears to protect against age-related renal impairment in rats (Li et al. 1996). The use of such pharmacological drugs should allow the contribution of AGE accumulation to GBM thickening and its functional impairment to be defined.

In conclusion, AGEs accumulate in renal tissue, as predicted by the increases in plasma and urinary circulating AGE peptides, and this accumulation is associated with the renal impairment that occurs with age, as indicated by the onset of albuminuria. The increase in AGE linked to Type IV collagen is associated, in turn, with a drastic decrease in early glycation products, suggesting that the turnover of GBM glycopro-teins is slowed and that the collagen crosslinks mainly generated by glycation are associated with the age-related changes in GBM structure and function.

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