ARTICLE |
Correspondence to: Hilaire Bakala, Laboratoire de Biologie Cellulaire, T23-33 1er étage, Case 7128, Université Paris 7, 2 Place Jussieu, 75251 Paris Cedex 05, France.
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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)
Key Words: AGEs, fluorescence, immunolocalization, aging, kidney, rat
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The nephropathies associated with normal aging probably reflect the effects of vascular lesions. The resulting mesangial expansion (
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 immunochemically 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 (
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 (
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 (
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
Extent of Collagen Peptide Glycation.
The presence of Amadori 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 (
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 immunogenicity (
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 (
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 preincubated 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 immunolabeling 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
|
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).
|
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).
|
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).
|
The protein A-gold technique indicated the ultrastructural 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 mesangial 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.
|
|
![]() |
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 (
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 (
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 (
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 glycoproteins is slowed and that the collagen crosslinks mainly generated by glycation are associated with the age-related changes in GBM structure and function.
![]() |
Literature Cited |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Ahmed N, Furth AJ (1991) A microassay for protein glycation based on the periodate method. Anal Biochem 192:109-111 [Medline]
Alt J, Hackbarth H, Deerberg F, Stolte H (1980) Proteinuria in rats in relation to age dependent renal changes. Lab Anim 14:95-101 [Medline]
Bakala H, Verbeke P, Perichon M, Corman B, Schaeverbeke J (1995) Glycation of albumin with aging and diabetes in rats: changes in its renal handling. Mech Ageing Dev 78:63-71 [Medline]
Baylis C (1994) Age-dependent glomerular damage in the rat. Dissociation between glomerular injury and both glomerular hypertension and hypertrophy. Male gender as a primary risk factor. J Clin Invest 94:1823-1829 [Medline]
Bell RH, Börjesson BA, Wolf PL, Fernandez-Cruz L, Brimm JE, Lee S, Sayers MJ, Orloff MJ (1984) Quantitative morphological studies of aging changes in the kidney of the Lewis rat. Renal Physiol (Basel) 7:176-184
Bergman J, Loxley R (1970) The determination of hydroxyproline in urine hydrolysates. Clin Chim Acta 27:347-349 [Medline]
Bolton WK, Sturgill BC (1980) Spontaneous glomerular sclerosis in aging Sprague-Dawley rats. II: Ultrastructural studies. Am J Pathol 98:339-356 [Abstract]
Bolton W, Sturgill B (1981) Ultrastructure of the aging kidney. In Johnson JE, ed. Aging and Cell Structure. Vol 1. New York, Plenum Press, 215-250
Brownlee M, Cerami A, Vlassara H (1988) Advanced glycosylation end products in tissue and the biochemical basis of diabetic complications. N Engl J Med 318:1315-1321 [Medline]
Bucala R, Vlassara H, Cerami A (1992) Advanced glycosylation end products. In Harding JJ, Crabba JJC, eds. Post-translational Modifications of Proteins. Boca Raton, FL, CRC Press, 2:53-79
Cohen MP, Wu VY (1981) Identification of specific amino acids in diabetic glomerular basement membrane collagen subject to non-enzymatic glucosylation in vivo. Biochem Biophys Res Commun 100:1549-1554 [Medline]
Corman B, Chami-Khazraji S, Schaeverbeke J, Michel JB (1988) Effect of feeding on glomerular filtration rate and proteinuria in conscious aging rats. Am J Physiol 255:F250-F256
Corman B, Michel JB (1987) Glomerular filtration, renal blood flow, and solute excretion in conscious aging rats. Am J Physiol 253:R555-R560
Couser WG, Stilmant MM (1975) Mesangial lesions and focal glomerular sclerosis in the aging rat. Lab Invest 33:491-501 [Medline]
Defronzo RA (1979) Glucose intolerance and aging: evidence for tissue insensivity to insulin. Diabetes 28:1095-1101 [Medline]
Dodane V, Chevalier J, Bariety J, Pratz J, Corman B (1991) Longitudinal study of solute excretion and glomerular ultrastructure in an experimental model of aging rats free of kidney disease. Lab Invest 64:377-391 [Medline]
Doi T, Vlassara H, Kirstein M, Yamada Y, Striker GE, Striker LJ (1992) Receptor specific increase in extracellular matrix production in mouse mesangial cells by advanced glycation end products is mediated via platelet derived growth factor. Proc Natl Acad Sci USA 89:2873-2877 [Abstract]
Dyer DG, Dunn A, Thorpe SR, Bailie KE, Lyons TJ, McCane DR, Baynes JW (1993) Accumulation of Maillard reaction products in skin collagen in diabetes and aging. J Clin Invest 91:2463-2469 [Medline]
Eble AS, Thorpe SR, Baynes JW (1983) Nonenzymatic glycosylation and glucose-dependent cross linking protein. J Biol Chem 258:9406-9412
Fink RI, Kolterman OG, Olefsky JM (1984) The physiological significance of glucose intolerance of aging. J Gerontol 39:273-278 [Medline]
Fornieri C, Quaglino D, Mori G (1992) Role of the extracellular matrix in age-related modifications of the rat aorta. Arterioscler Thromb 12:1008-1016 [Abstract]
Garlic RL, Bunn HF, Spiro RG (1988) Nonenzymatic glycation of basement membranes from human glomeruli and bovine sources. Diabetes 37:1144-1150 [Abstract]
Gray JE, Van Zwieten MJ, Hollander CF (1982) Early light microscopic changes of chronic progressive nephrosis in several strains of aging laboratory rats. J Gerontol 37:142-150 [Medline]
Haitoglou CS, Tsilibary EC, Brownlee M, Charonis AS (1992) The effect of non-enzymatic glucosylation on self assembly and cell binding properties of Type IV collagen. J Biol Chem 267:12404-12407
Heudes D, Michel O, Chevalier J, Scalbert E, Ezan E, Bariety J, Zimmerman A, Corman B (1994) Effect of chronic ANG I-converting enzyme inhibition on aging processes. I. Kidney structure and function. Am J Physiol 266:R1038-R1051
Horbach GJ, Yap SH, Van Bezooijen CF (1983) Age-related changes in albumin elimination in female WAG/Rij rats. Biochem J 216:309-315 [Medline]
Krakower CA, Greenspon SA (1978) The isolation of basements. In Kefalides NA, ed. Biology and Chemistry of Basement Membranes. New York, Academic Press, 1-16
Li YM, Steffes M, Donnelly T, Liu C, Fuh H, Basgen J, Bucala R, Vlassara H (1996) Prevention of cardiovascular and renal pathology of aging by the advanced glycation inhibitor aminoguanidine. Proc Natl Acad Sci USA 93:3902-3907
Lowry OH, Rosenbrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265-275
Makita Z, Bucala R, Rayfield EJ, Friedman EA, Kaufman AM, Korbert SM, Barth RH, Winston JA, Fuh H, Manogue KR, Cerami A, Vlassara H (1994) Reactive glycosylation end products in diabetic uraemic and treatment of renal failure. Lancet 343:1519-1522 [Medline]
Makita Z, Vlassara H, Cerami A, Bucala R (1992) Immunochemical detection of advanced glycosylation end products in vivo. J Biol Chem 267:5133-5138
Meezan E, Hjelle JT, Brendel K, Carlson EC (1976) A simple, versatile, non-disruptive method for the isolation of morphologically and chemically pure basement membranes from several tissues. Life Sci 17:1721-1732
Miyata S, Monnier V (1992) Immunohistochemical detection of advanced glycosylation end products in diabetic tissues using monoclonal antibody to pyrraline. J Clin Invest 89:1102-1112 [Medline]
Mohan PS, Spiro RG (1986) Macromolecular organization of basement membranes: characterization and comparison of glomerular basement membrane and lens capsule components by immunochemichal and lectin affinity procedures. J Biol Chem 261:4328-4336
Nakayama H, Taneda S, Kuwajima S, Aoki S, Kuroda Y, Misawa K, Nakagawa S (1989) Production and characterization of antibodies to advanced glycation products on proteins. Biochem Biophys Res Commun 162:740-745 [Medline]
Papanastasiou P, Grass L, Rodela H, Patrikarea A, Oreopoulos D, Diamandis EP (1994) Immunological quantification of advanced glycation end products in the serum of patients on hemodialysis or CAPD. Kidney Int 46:216-222 [Medline]
Roth J, Bendayan M, Orcil L (1978) Ultrastructural localization of intracellular antigens by use of protein A gold complex. J Histochem Cytochem 26:1074-1081 [Abstract]
Sanada H, Shikata J, Hamamoto H, Ueba YY, Takeda Y (1978) Changes in collagen cross-linking and lysyl oxidase by estrogen. Biochim Biophys Acta 541:4308-4313
Sell DR, Monnier VM (1989) Isolation, purification and partial characterization of novel fluorophores from aging human insoluble collagen-rich tissue. Conn Tissue Res 19:77-92 [Medline]
Sell DR, Monnier VM (1990) End stage renal disease and diabetes catalyze the formation of a pentose-derived crosslink from aging human collagen. J Clin Invest 85:380-384 [Medline]
Travis J, Bowen J, Tewksbury D, Johnson D, Pannell R (1976) Isolation of albumin from whole human plasma and fractionation of albumin-depleted plasma. Biochem J 157:301-306 [Medline]
Vlassara H, Bucala R, Striker L (1994a) Pathogenic effects of advanced glycosylation: biochemical, biologic, and clinical implications for diabetes and aging. J Lab Invest 70:138-158 [Medline]
Vlassara H, Striker LJ, Teichberg S, Fuh H, Ming LY, Steffes M (1994b) Advanced glycation end products induce glomerular sclerosis and albuminuria in normal rats. Proc Natl Acad Sci USA 91:11704-11708
Yoshioka T, Shiraga H, Yoshida Y, Fogo H, Glick A, Deen W, Hoyer J, Ichikawa I (1988) Intact nephrons as the primary origin of proteinura in chronic renal disease. J Clin Invest 82:1614-1623 [Medline]
Ziyadeh FN (1993) The extracellular matrix in diabetic nephropathy. Am J Kidney Dis 22:736-744 [Medline]