Hilaire Bakala

Inhibition of nitric oxide synthase activity by early and advanced glycation end products in cultured rabbit proximal tubular epithelial cells

Nitric oxide (NO) is important in the regulation of renal tubular function. We have investigated whether glycated proteins could impair the NO production by examining the effects of Amadori products (AP-BSA) and advanced glycation end products (AGE-BSA) on primary cultures of rabbit proximal tubular epithelial (PTE) cells. Nitric oxide synthase activity was assessed by measurement of the conversion of L-arginine to L-citrulline and by production of NO, after short-term (30 min) or long-term (1 or 3 days) incubation. Short incubations of PTE cells with either 200 microg/ml AP-BSA or 40 microg/ml AGE-BSA significantly decreased NO production. AP-BSA (3000 microg/ml) inhibited the Ca(2+)-dependent NOS activity even though above 50 microg/ml it increased Ca(2+)-independent NOS activity. In contrast, 40 microg/ml AGE-BSA inhibited both isoforms of NOS. Longer incubations with 200 microg/ml AP-BSA or 250 microg/ml AGE-BSA decreased NO release and inhibited Ca(2+)-dependent and -independent NOS activities. APs did not affect NO release by S-nitroso-N-acetyl-penicillamine (SNAP), while 250 microg/ml AGEs decreased it. After 3 days incubation, glycation products had no effect on the NOS cell content. Cell viability and proliferation were not modified under these experimental conditions, suggesting that the fall in NO production was not due to there being fewer cells. These data indicate that APs and AGEs directly inhibit NOS activity, and additionally that AGEs quench released NO. Thus, both types of glycated proteins alter the production of NO by PTE cells and could participate in the renal tubule dysfunction associated with aging and diabetes.

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 Bowmans 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)

Glycation damage targets glutamate dehydrogenase in the rat liver mitochondrial matrix during aging

Aging is accompanied by gradual cellular dysfunction associated with an accumulation of damaged proteins, particularly via oxidative processes. This cellular dysfunction has been attributed, at least in part, to impairment of mitochondrial function as this organelle is both a major source of oxidants and a target for their damaging effects, which can result in a reduction of energy production, thereby compromising cell function. In the present study, we observed a significant decrease in the respiratory activity of rat liver mitochondria with aging, and an increase in the advanced glycation endproduct‐modified protein level in the mitochondrial matrix. Western blot analysis of the glycated protein pattern after 2D electrophoresis revealed that only a restricted set of proteins was modified. Within this set, we identified, by mass spectrometry, proteins connected with the urea cycle, and especially glutamate dehydrogenase, which is markedly modified in older animals. Moreover, mitochondrial matrix extracts exhibited a significant decrease in glutamate dehydrogenase activity and altered allosteric regulation with age. Therefore, the effect of the glycating agent methylglyoxal on glutamate dehydrogenase activity and its allosteric regulation was analyzed. The treated enzyme showed inactivation with time by altering both catalytic properties and allosteric regulation. Altogether, these results showed that advanced glycation endproduct modifications selectively affect mitochondrial matrix proteins, particularly glutamate dehydrogenase, a crucial enzyme at the interface between tricarboxylic acid and urea cycles. Thus, it is proposed that glycated glutamate dehydrogenase could be used as a biomarker of cellular aging. Furthermore, these results suggest a role for such intracellular glycation in age‐related dysfunction of mitochondria.

Increased level of glycoxidation product Nε-(carboxymethyl)lysine in rat serum and urine proteins with aging: Link with glycoxidative damage accumulation in kidney

Accumulation of carboxymethylated proteins (CML-proteins) is taken as a biomarker of glycoxidative stress which is thought to contribute to the age-related impairment in tissue and cell function. To investigate the occurrence and extent of glycoxidative damage with aging in rat kidney, serum and urine, we have prepared a polyclonal antibody against CML-modified bovine serum albumin. We subsequently used it for immunolocalization and in enzyme-linked immunosorbent assays to evaluate CML-protein content. In the serum, CML-protein level was 1.43+/-0.14 pmol CML/micrograms protein at 3 months and significantly increased by 50% from 10 to 27 months (1.50+/-0.14 pmol CML/micrograms protein vs 2.27+/-0.26 pmol CML/micrograms protein), albumin and transferrin being the main modified proteins. In the urine, CML-protein level was 2.50+/-0.14 pmol CML/micrograms protein at 3 months and markedly increased from 10 months (2.99+/-0.24 pmol CML/micrograms protein) to 27 months (3.76+/-0.25 pmol CML/micrograms protein), with albumin as the main excreted modified protein. Immunolocalization of CML-proteins in kidney provided evidence for an age-dependent increased accumulation in extracellular matrices. Intense staining of the glomerular basement membrane (GBM), Bowmans capsule, and the tubular basement membrane was found. Additionally, the CML content for collagen from GBM was 195.85+/-28.95 pmol CML/microgrms OHPro at 3 months and significantly increased from 10 months (187.61+/-21.99 pmol CML/micrograms OHPro) to 27 months (334.55+/-62.21 pmol CML/micrograms OHPro). These data show that circulating CML-protein level in serum and urine and CML accumulation in nephron extracellular matrices with aging are increasing in parallel. The CML-protein measurement in serum and urine may thus be used as an index for the assessment of age-associated glycoxidative kidney damage.

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)

Cellular signaling, AGE accumulation and gene expression in hepatocytes of lean aging rats fed ad libitum or food-restricted

The effects of food restriction on liver glucagon and vasopressin V1a receptors, on AGE accumulation and on gene expression were investigated in 10- and 30-month-old WAG/Rij female rats fed ad libitum or chronically food-restricted by 30%. The age-related increase in glucagon and vasopressin V1a receptor density, as well as the rise in glucagon-induced cAMP generation was prevented by the restriction. AGE accumulation, characteristic of the aging process, was normalized in food-restricted animals. Gene expression determined with rat Atlas cDNA Expression Arrays containing 1176 cDNA indicates that a few genes exhibited a greater than twofold change in mRNA ratios with age. Most down-regulated genes were related to oxidative metabolism of lipids, and most of the up-regulated genes were concerned with the cell cycle and transcription factors. Chronic food restriction partially prevents these changes in gene expression and induces up- and down-regulation of several mRNAs which are not modified with age in ad libitum fed rats.

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.

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.

Glycation-induced inactivation of aspartate aminotransferase, effect of uric acid

Glycation is common posttranslational modification of proteins impairing their function, which occurs during diabetes mellitus and aging. Beside extracellular glycation of long-lived proteins, intracellular modifications of short-lived proteins by more reactive sugars like fructose are possible. The process includes free oxygen radicals (glycoxidation). In an attempt to reduce glycoxidation and formation of advanced glycation products (AGE), influence of 0.2–1.2 mM uric acid as endogenous antioxidant on glycoxidation of purified pig heart aspartate aminotransferase (AST) by 50 mM and 500 mM D-fructose in vitro was studied. Uric acid at 1.2 mM concentration reduced AST activity decrease and formation of total AGE products caused by incubation in vitro of the enzyme with sugar up to 25 days at 37 °C. The results thus support the hypothesis that uric acid has beneficial effects in controlling protein glycoxidation. The in vitro system AST-fructose proved to be a useful tool for investigation of glycation process. (Mol Cell Biochem 278: 85–92, 2005)

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.