A flavonoid fraction purified from Rutaceae aurantiae (Daflon(R)) inhibiting AGE formation, reduces urinary albumin clearance and corrects hypoalbuminemia in normotensive and hypertensive diabetic rats.

AIM: Advanced glycation endproducts (AGEs) have been shown to contribute to alteration of glomerular permselectivity to proteins in diabetes. Oxidative stress is required for AGE formation. Therefore we studied the effect of an antioxidant micronized purified flavonoid fraction (MPFF, Daflon(R) 500 mg), on urinary albumin clearance in diabetic rats. METHODS: Hyperglycaemia was induced by streptozotocin 55 mg/kg IM at days 0 and 7 in normotensive Wistar rats (NWR, diabetes duration 5 months) or hypertensive Wistar Kyoto rats (SHR, diabetes duration 2 months). MPFF was administered at 300 mg/kg/day, from day -2 until sacrifice. RESULTS: After 5 months of diabetes in NWR, MPFF reduced albumin clearance from 729±92 to 392±60 nl/min/kg, p<0.01, and restored albuminemia from 20.4±0.9 to 24.0±1 g/l, p<0.05; albumin fractional clearance was significantly diminished in the flavonoid-treated diabetic rats (0.360±0.037‰ versus 1.335±0.430‰ in the diabetic controls, p<0.001); MPFF did not significantly modify blood glucose and plasma fructosamine levels. After 2 months of diabetes in SHR, MPFF reduced albumin clearance from 243±121 to 101±47 nl/min/kg, p<0.05, and restored albuminemia from 21.1±1.6 to 26.7±2.2 g/l (p<0.05); MPFF also decreased plasma fluorescence characteristic of AGEs (p<0.02). Besides hesperetin, a main metabolite of MPFF recovered in plasma, inhibited in vitro the formation of the crosslinking AGE pentosidine in collagen incubated with high glucose (p<0.001). CONCLUSION: Our results confirm the role of glycoxidative stress in diabetic nephropathy. MPFF might be useful as complementary treatment for preventing diabetic microangiopathy.

Stimulating effect of growth hormone on type IV collagen production by endothelial cells cultured in normal and high glucose.

Collagen IV accumulation is characteristic of diabetic angiopathy. To test the possible contribution of GH, we studied its effects on collagen IV production by human umbilical vein endothelial cells at 5.5 and 16.7 mmol/l glucose. GH (100 ng/ml) markedly increased collagen IV level in the culture supernatant and in the insoluble extracellular matrix and cell fraction at both glucose concentrations. This stimulating effect of GH was additional to that of high glucose. It was more pronounced on collagen IV than on total protein synthesis. GH increased free latent gelatinase activity slightly at normal and markedly at high glucose. Using GF109203X, a PKC inhibitor, we observed that high glucose, but not GH, activated PKC. These two factors stimulating collagen IV production appear to work through different pathways, favoring an additivity of their effects. This supports the contribution of high plasma GH in diabetic vascular basement membrane thickening.

Aspirin inhibits the formation of pentosidine, a cross-linking advanced glycation end product, in collagen.

Aspirin showed an inhibitory effect on the formation of pentosidine, a cross-linking advanced glycation endproduct, in collagen incubated with glucose in vitro. IC(50) was evaluated at 10mmol/l. Aspirin might act by metallic ion chelating (as did EDTA and DTPA) and by oxygen radical scavenging. Since aspirin was reported to inhibit retinopathy in diabetic dogs, it could act partly by inhibiting advanced glycation endproduct accumulation in long-lived proteins like collagens.

Cyclic guanosylmonophosphate urinary excretion in parasympathicomimetic or parasympatholytic syndromes induced by reserpine or diphemanil-methylsulfate.

Parasympathetic hyperactivity is found in some infants presenting faint episodes and could be responsible of certain Sudden Infant Death Syndrome cases. Therefore it was interesting to look for a noninvasive biochemical indicator of parasympathetic activity. A parasympaticomimetic syndrome associated with muscarinic receptor stimulation, which has been followed during 48 h, was obtained in the awake rat by reserpine injection (6.25 mg/kg at T0 and T24h), and a model of prolonged parasympatholytic syndrome, by administration of diphemanil-methylsulfate (DPMS), a muscarinic receptor inhibitor, in drinking water (mean daily dosis: 150 mg/kg). Significant bradycardia and tachycardia were respectively observed. In the reserpine-treated rats we found significantly increased cyclic guanosylmonophosphate (cGMP) urinary excretion between T24h and T48h, when compared with vehicle-treated controls (+87% in one experiment, +135% in the other, when expressed in pmol/microg creatinine); norepinephrine urinary excretion between T24h and T48h was decreased (-44%); the increase in cGMP urinary excretion was not significantly modified by the NO-synthase inhibitor, L-nitroarginine-methyl-ester. In the DPMS-treated rats, we observed a significantly decreased cGMP (-20%) and increased norepinephrine urinary excretion (+61%). Thus cGMP excretion varied in opposite directions in the reserpine- and DPMS-treated rats. The link between these modifications in cGMP excretion and muscarinic receptor stimulation or blockade has still to be fully demonstrated. Urinary cGMP excretion could be tested as screening parameter in infants at risk of faint episodes associated with bradycardia.

Effect of an aldose reductase inhibitor on type IV collagen production by human endothelial cells cultured in high glucose.

Diabetic microangiopathy is characterized by a thickening of capillary basement membranes associated with type IV collagen accumulation. An increase in type IV collagen content of the aortic wall is also observed in macroangiopathy. In order to analyse the importance of the polyol pathway in the development of the collagen metabolism alterations seen in diabetic angiopathy and their prevention by aldose reductase inhibitors, we have studied the effects of sorbinil on the high glucose-induced stimulation of type IV collagen biosynthesis in human umbilical vein endothelial cells. Primary cultures were exposed to high glucose (16.7 mmol/l), with and without 0.11 mmol/l sorbinil, for 3 or 6 days after beginning of confluence. We measured the soluble type IV collagen secreted into the culture medium and the insoluble type IV collagen accumulated in the extracellular matrix and cells, by ELISA. We also studied [14C]proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. High glucose decreased the number of cells and increased the amount of type IV collagen in the culture supernatant and in the extracellular matrix and cell fraction. It also increased proline incorporation into the newly synthesized collagenous and total proteins in the culture supernatant and in the extracellular matrix and cell fraction. Sorbinil corrected all these high glucose-induced alterations. The corrective effects of sorbinil on the proliferation and on type IV collagen metabolism of endothelial cells cultured in high glucose may be attributed to prevention of polyol pathway dysregulation.

Production of type IV collagen and 72-kDa gelatinase by human endothelial cells cultured in high glucose. Effects of a protein kinase C inhibitor, GF 109203X.

Since diabetic microangiopathy and macroangiopathy are characterized by type IV collagen accumulation in vascular basement membranes, it was of interest to study type IV collagen production and type IV collagenase secretion by endothelial cells (EC) cultured in high glucose and to evaluate the role of protein kinase C (PKC) activation in the alterations induced by high glucose. Primary cultures of human umbilical vein EC were exposed to high glucose concentration for 3 days at the beginning of confluence. The number of EC decreased with glucose concentration from 5 to 50 mM. At 16.7 mM glucose concentration, the amount of type IV collagen, determined by a two-step ELISA, increased in the culture supernatant and in the insoluble fraction associated with the extracellular matrix and cells; proline incorporation was more markedly elevated in the collagenous than in the total proteins of the culture supernatant and of the extracellular matrix and cell extracts. Gelatin zymography of the culture supernatant showed that EC mainly produce a 72-kDa gelatinase known to degrade type IV collagen. At 16.7 mM glucose concentration, total gelatinase activity per millilitre of culture supernatant was reduced and the 72-kDa gelatinase activity measured on the zymogram scan was lowered. When EC were exposed to 16.7 mM glucose, the specific PKC inhibitor GF 109203X corrected the increases in type IV collagen concentration and in proline incorporation into the collagenous or total proteins present in he culture supernatant or in the extract of the insoluble fraction, including the extracellular matrix and cells. Our results show that soluble and insoluble type IV collagen accumulation by EC cultured at high glucose concentration is not only associated with increased synthesis of the collagenous and total proteins but also with decreased total 72-kDa gelatinase activity in the extracellular fluid. The observed effects of GF 109203X are in favor of the involvement of PKC activation in the type IV collagen accumulation.

[Effects of glycation process on the macromolecular structure of the glomerular basement membranes and on the glomerular functions in aging and diabetes mellitus]. / Effets du processus de glycation sur la structure macromoléculaire des membranes basales glomérulaires et sur les fonctions glomérulaires au cours du vieillissement et du diabète sucré.

Three stages can be distinguished during the glycation process: initiation with the formation of Amadori product; spreading with glyco-oxidation reactions; terminal formation of advanced glycation end products (AGEs). Some AGEs have been isolated and characterized: pyrraline linked to one aminoacid, pentosidine linked to two aminoacids and forming a cross-link between peptidic chains. The AGE-induced cross-links alter the biophysical properties of the proteins with increased stiffness of the fibrous proteins and resistance to proteases. Glycation of the glomerular basement membrane (GBM) macromolecules modifies the architecture of the glomerular filtration barrier. Type IV collagen is the major constituent of the GBM and the mesangial matrix and is a substrate for prolonged glycation, due to its long half-life. In the GBM, AGE level (particularly pentosidine level per mg collagen) increases with age; it is higher in diabetic or uremic patients than in age-matched controls. In insulin-dependent diabetes mellitus, a correlation has been shown between the pentosidine level of skin collagen and the severity of vascular complications. Glycation inhibits the homotypic polymerization interactions between two type IV collagen molecules through their NC1 ends. Glycation also affects the heterotypic interactions between different GBM macromolecules: the affinity of glycated fibronectin for type IV collagen is diminished. Besides, glycation modifies the interactions between type IV collagen and adjacent cells: mesangial and endothelial cells are less adherent on a glycated type IV collagen matrix and their morphology modified. GBM treated with dimethylmalonimidate, which induces cross-links between amines as does advanced glycation, are more permeable to proteins.

[Changes in collagen type IV metabolism in diabetes]. / Etude des altérations du métabolisme du collagène de type IV au cours du diabète.

In Diabetes Mellitus, type IV collagen biosynthesis is increased: the alpha 1(IV) procollagen specific mRNA concentration is elevated, particularly in the kidney, and the type IV collagen protein is accumulating is the thickened basement membranes. Aldose reductase inhibitors like sorbinil do prevent basement membrane thickening and type IV collagen overproduction. The latter seems related to intracellular sorbitol accumulation and also to protein kinase C activation. Autocrine or paracrine TGF beta may be involved in the type IV collagen oversecretion. The secreted type IV collagen is subject to posttranslational alterations, especially glycation which leads to advanced glycation end-products and covalent crosslinks. This decreases collagen extractability and susceptibility to collagenases and favours basement membrane thickening. Disaccharide unit-specific alpha-glucosidase activity is inhibited by glucose (Kp = 7.5 mM). Type IV collagenase activity secreted by endothelial cells cultured at high glucose concentrations appears to be diminished. Therefore type IV collagen catabolism may be decreased in Diabetes Mellitus.

Studies on the catabolism of glycated haemoglobin: Glycated globin is not a substrate for the glucosyl-galactosyl-hydroxylysine glucosidase but for a peptidase activity present in rat kidney and spleen.

Rat kidney and spleen glucosyl-galactosyl-hydroxylysine glucosidase (EC.3.2.1.107) whose specificity for the hydroxylysine-linked disaccharide units present in collagens depends upon the substrate's free amino group was tested for its glycosidase activity on the ketoamine form of glycated [(14)C]Glc-Globin. The most stable preparations of the enzyme from normal and diabetic rat tissues, partially purified by ultracentrifugation and ammonium sulphate fractionation, were used. These glucosidase preparations did not release any significant amount of radioactive neutral hexose. But a radioactive glycopeptide of about 1,400 Da was released from [(14)C]Glc-Globin at a pH optimum of 4.0. It appears to be released by a peptidase activity present in the kidney and spleen of normal and diabetic rats.

Aging and diabetes increase the aggregating potency of rat skin collagen towards normal platelets.

Acid-soluble collagen samples were prepared from individual skins of 24 month old rats (n = 8), 2 month old young controls (n = 8) and from 6 month old streptozotocin-diabetic rats (n = 5) and their age-matched controls (n = 10). Less collagen was obtained by acid extraction and salt precipitations from skins of diabetic and aged rats than from those of their respective controls. The collagen preparations from diabetic and aged rats showed an increased ratio of beta/alpha components. The rate of "in vitro" fibrillogenesis was less for collagen from diabetic rats than from controls. It was not modified for collagens from aged rats. The aggregating potency towards normal human platelets was markedly increased for collagens from aged and diabetic rats: reduced latency time (p less than 0.01) and increased velocity (p less than 0.01) were observed for collagens from aged rats when compared with young rats (16.5 micrograms/ml). Increased velocity (p less than 0.01) was also observed for collagens from diabetic rats (8.25, 11 and 16.5 micrograms/ml), without modification of latency time.

Alteration in sialidase and other glycosidase activities in the kidney of spontaneously hypertensive rats: persistence after preventive treatment with hydralazine.

Because kidney microangiopathy with capillary basement membrane thickening has been reported in spontaneous hypertension, we have studied the activities of three lysosomal glycosidases able to degrade the carbohydrate moieties of basement membrane constituents in the kidney cortex of 12-week-old spontaneously hypertensive rats (SHR) and age-matched normotensive Wistar Kyoto rats (WKY). These activities were also determined in SHR and WKY treated from 6 to 12 weeks of age with hydralazine (mean dose, 18 mg/kg per day in drinking water). Sialidase specific activity on sialyl-alpha 2-3-[3H]lactitol was markedly decreased in the kidney of untreated SHR, 40% activity remaining relative to that found in untreated age-matched WKY (p less than 0.001). beta-Galactosidase specific activity on p-nitrophenyl-beta-D-galactoside was also decreased, 86% activity remaining relative to that found in untreated WKY (p less than 0.001). Glucosyl-galactosyl-hydroxylysyl glucohydrolase specific activity on glucosyl-galactosyl-hydroxylysine was equally diminished, 74% activity remaining relative to that found in untreated age-matched WKY (p less than 0.001). In contrast, the activities of two control glycosidases inactive on the carbohydrate moieties of basement membrane constituents, alpha-glucosidase assayed with p-nitrophenyl-alpha-D-glucoside as substrate and beta-glucosidase assayed with p-nitrophenyl-beta-D-glucoside as substrate, were significantly increased. All the alterations in enzyme activities observed in the kidney of SHR were also present in the long-term treated normotensive SHR. No effect of the hydralazine treatment on the three enzyme activities investigated could be demonstrated in the WKY. Thus the alterations observed in the kidneys of SHR appear to be independent of blood pressure level.