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.

Advanced glycation endproducts (AGEs): Pharmacological inhibition in diabetes.

AGE inhibitors may act by various mechanisms at different steps of advanced glycation endproduct (AGE) formation (depending on oxidative stress and/or carbonyl stress) and AGE-mediated damage: trapping of reactive dicarbonyl species; antioxidant activity by transition metal chelation; other antioxidant activity including free radical scavenging; AGE cross-link breaking; AGE receptor (RAGE) blocking; RAGE signaling blocking; glycemia reduction by anti-diabetic therapy; aldose reductase inhibition; shunting of trioses-P towards the pentose-P pathway by transketolase activation. Most of the inhibitors have several sites of action. Practically one can distinguish drugs specifically developed as AGE inhibitors or AGE breakers; RAGE and receptor signaling blockers; other therapeutic compounds which were found subsequently to possess also AGE inhibitor activity, including dietary antioxidants. Encouraging results obtained in studies of various AGE inhibitors, conducted in vitro and in diabetic animals, are summarized in this review. However most of the clinical trials have been more or less disappointing, in part because of side effects; the long-term therapeutic interest of the most recently developed AGE inhibitors or breakers remains to be demonstrated in diabetes.

Acute effect of the dual angiotensin-converting enzyme and neutral endopeptidase 24-11 inhibitor mixanpril on insulin sensitivity in obese Zucker rat.

The aim of this study was to determine whether acute dual angiotensin-converting enzyme (ACE)/neutral endopeptidase 24-11 (NEP) inhibition could improve whole body insulin-mediated glucose disposal (IMGD) more than ACE inhibition alone and whether this effect was mediated by the kinin-nitric oxide (NO) pathway activation. We therefore compared in anaesthetized obese (fa/fa) Zucker rats (ZOs) the effects of captopril (2 mg kg(-1), i.v.+2 mg kg(-1) h(-1)), retrothiorphan (25 mg kg(-1), i.v. +25 mg kg(-1) h(-1)), a selective NEP inhibitor, and mixanpril (25 mg kg(-1), i.v. +25 mg kg(-1) h(-1)), a dual ACE/NEP inhibitor, on IMGD using hyperinsulinaemic euglycaemic clamp technique. The role of the kinin-NO pathway in the effects of mixanpril was tested using a bradykinin B2 receptor antagonist (Hoe-140, 300 microg kg(-1)) and a NO-synthase inhibitor (N(omega)-nitro-L-arginine methyl ester, L-NAME, 10 mg kg(-1) i.v. +10 mg kg(-1) h(-1)) as pretreatments. Insulin sensitivity index (ISI) was lower in ZO controls than in lean littermates. Increases in ISI were observed in captopril- and retrothiorphan-treated ZOs. In mixanpril-treated ZOs, ISI was further increased, compared to captopril- and retrothiorphan-treated ZOs. In ZOs, Hoe-140 and L-NAME alone did not significantly alter and slightly reduced the ISI respectively. Hoe-140 and L-NAME markedly inhibited the ISI improvement induced by mixanpril. These results show that in obese insulin-resistant Zucker rats, under acute conditions, NEP or ACE inhibition can improve IMGD and that dual ACE/NEP inhibition improves IMGD more effectively than does either single inhibition. This effect is linked to an increased activation of the kinin-NO pathway.

Effects of combined neutral endopeptidase 24-11 and angiotensin-converting enzyme inhibition on femoral vascular conductance in streptozotocin-induced diabetic rats.

1. The successive effects of the angiotensin-converting enzyme inhibitor captopril (CAP, 2 mg kg(-1)+1 mg kg(-1) 30 min(-1) infusion) and the neutral endopeptidase 24-11 inhibitor retrothiorphan (RT, 25 mg kg(-1)+12.5 mg kg(-1) 30 min(-1) infusion) were studied on femoral vascular conductance (FVC) in streptozotocin-induced diabetic (STZ-SD) and control Sprague-Dawley (C-SD) rats. The role of the kinin-nitric oxide (NO) pathway was assessed by (1) using pre-treatments: a bradykinin (BK) B2 receptor antagonist (Hoe-140, 300 microg kg(-1)), a NO-synthase inhibitor (N(omega)-nitro-L-arginine methyl ester, L-NAME, 10 mg kg(-1)), a kininase I inhibitor (DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, MGTA, 10 mg kg(-1)+20 mg kg(-1) 20 min(-1) infusion) and (2) comparing the effects in STZ-induced diabetic (STZ-BN) and control Brown-Norway kininogen-deficient (C-BN) rats. 2. In C-SDs, CAP and CAP+RT increased FVC similarly. In STZ-SDs, FVC and FBF were decreased compared to C-SDs. CAP+RT increased them more effectively than CAP alone. 3. In both C-SDs and STZ-SDs, the femoral bed vasodilatation elicited by CAP was inhibited by Hoe-140 and L-NAME. The FVC increase elicited by CAP+RT was not significantly reduced by Hoe-140 but was inhibited by L-NAME and Hoe-140+MGTA. 4. In C-BNs, the vasodilatator responses to CAP and CAP+RT were abolished and highly reduced, respectively. In STZ-BNs, these responses were abolished. 5. These results show that in STZ-SDs, CAP+RT improve FBF and FVC more effectively than CAP alone. These effects are linked to an increased activation of the kinin-NO pathway. BK could lead to NO production by BK B2 receptor activation and another pathway in which kininase I may be involved.

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 long-term treatment with a purified micronized flavonoid fraction on pancreatic mononuclear cell infiltration in diabetic BB rats.

Bio Breeding (BB) rats develop a genetically determined insulin-dependent diabetes, because of the early destruction of pancreatic beta cells of Langerhans islets, massively infiltrated by inflammatory mononuclear cells. S 5682, registered as Daflon, 500 mg, is a purified micronized flavonoid fraction (90% diosmin, 10% hesperidin), which has been shown to possess antiinflammatory properties, including anti-free radical activity, effects on vascular permeability, venous tone, and perivenous inflammation. We studied the effect of S 5682 on the course of pancreatic insulitis in diabetic BB rats. All the diabetic BB rats were hyperglycemic, with an increase of plasma levels of fructosamine, alpha-1 acid glycoprotein, and fibrinogen, and a dramatic decrease of C-peptide level. These parameters were not modified by S 5682. Pancreas histologic studies showed that in S 5682-treated diabetic BB rats, lymphocytic infiltration of Langerhans islets was less important and frequent than in untreated diabetic BB rats. By quantitative analysis, a highly significant difference was observed for insulitis, as well as perivasculitis, between S 5682-treated and untreated diabetic BB rats. This inhibitory effect of S 5682 on pancreatic mononuclear cell infiltration may be useful for a complementary treatment to decrease the development of insulitis in human insulin-dependent diabetes mellitus.

Effect of endopeptidase-24.11 inhibitors and C-ANP receptor ligand on responses evoked in arterioles of rat cremaster muscle by atrial natriuretic peptide.

1. The present study examined the effect of exogenous atrial natriuretric peptide (ANP), alone or in presence of inhibitors of the two major mechanisms for clearing ANP, metabolism by neutral endopeptidase-24.11 (NEP) and internalization by C-ANP receptors, on arteriolar responses using intravital microscopy on the rat cremaster muscle after intravenous or topical administration of the peptide. 2. Topical application of ANP (3 x 10(-10) to 3 x 10(-8) M) produced a gradual increase in arteriolar diameter. NEP inhibitors, thiorphan (30 mg kg-1, i.v.), kelatorphan (10 mg kg-1, i.v.) and retrothiorphan (25 mg kg-1, i.v.) alone, did not significantly affect vascular tone but caused significant potentiation of the arteriolar responses to topically applied ANP. 3. When given as an i.v. bolus, ANP dilates skeletal arterioles at a high dose (20 micrograms kg-1). At a lower dose (10 micrograms kg-2), ANP alone or with retrothiorphan or the C-ANP receptor ligand C-ANP (4-23) did not produce any arteriolar responses, while after the combined administration of the two inhibitors, an increase in arteriolar diameter was induced. 4. These results indicate that low doses of topically applied ANP dilate rat cremaster arterioles and that the vasodilator responses can be potentiated by NEP inhibition. When given as an i.v. bolus, a high dose of ANP can also dilate skeletal arterioles. However at a lower dose the rapid metabolism of the peptide prevents it from producing its action.

Renal and microvascular effects of an aldose reductase inhibitor in experimental diabetes. Biochemical, functional and ultrastructural studies.

Aldose reductase inhibitors, and particularly sorbinil, have been reported to prevent glomerular basement membrane thickening (GBMT) and albuminuria development in diabetic rats, but contradictory observations have been published. The aim of this study was to answer the following questions (i) is the corrective effect of sorbinil on GBMT, if confirmed, associated with an effect on collagen metabolism alterations? (ii) Is it associated with an effect on microvascular functional alterations? We therefore studied the influence of sorbinil on glucosyl-galactosyl-hydroxylysyl-glucohydrolase activity (GGHG; EC 3.2.1.107 which is involved in the catabolism of collagen disaccharide units), 3- and 4-hydroxyproline content and GBMT by ultrastructural morphometry in the kidney cortex of streptozotocin-diabetic rats after 5 months of disease. In parallel, the effects on albumin renal clearance and another functional alteration, the microvascular response to norepinephrine, were evaluated. We confirmed a corrective effect of sorbinil on both renal albumin clearance and GBMT. In the diabetic rats, sorbinil diminished the 3-hydroxyproline (but not the 4-hydroxyproline) content, whether expressed per mg protein or per total kidney cortex relative to body weight. Sorbinil reduced GGHG activity measured in the dialysed 10,000 g supernatant whether expressed per mg protein or per total kidney cortex; this activity has been shown to be increased in diabetes. Sorbinil also corrected the microvascular response to norepinephrine which is altered in diabetes.

Relation between pancreatic islet cellular infiltration and plasma fibrinogen or alpha 1-acid glycoprotein levels in spontaneously and streptozotocin-diabetic rats: an increase in these protein levels is not necessary for inducing microcirculatory erythrocyte velocity alteration.

Plasma levels of fibrinogen, alpha 1-acid glycoprotein (AG) and albumin, pancreatic insulitis quantitative scores, and erythrocyte velocity in the mesoappendix microvessels were measured in BB diabetic (BBD) and streptozotocin-diabetic rats (WSTZ) in order to answer the following questions: (a) Does hyperfibrinogenemia or increase in AG plasma level occur in BBD and WSTZ rats, and if so, are these alterations secondary to the hyperglycemia or to an inflammatory process such as insulitis? (b) Is there a decrease in microcirculatory flow in the BBD and WSTZ rats, and if so, is it secondary to the hyperfibrinogenemia and/or the hyperglycemia? Insulitis was present in the BBD rats after 5 weeks of disease (with a score of 2.9 +/- 0.1 vs. 1.4 +/- 0.6 in the normoglycemic controls), but absent in WSTZ rats after 5 months of disease (1.2 +/- 0.06 vs. 1.1 +/- 0.06). Increase in fibrinogen and AG plasma levels was observed in the BBD rats only and appears linked to the insulitis. The major acute phase protein AG level is increased in BBD rats already on the first day of appearance of glycosuria. In the WSTZ rats, without insulitis, chronic hyperglycemia alone did not induce an increase in fibrinogen and AG plasma levels. A decreased microcirculatory erythrocyte velocity has been found in both BBD and WSTZ rats. Thus an increase in fibrinogen or AG plasma levels is not necessary for inducing a decrease in erythrocyte velocity. Hyperglycemia is probably the main factor responsible for the decrease in microcirculatory flow in the BBD and WSTZ rats.

Analgesic responses elicited by endogenous enkephalins (protected by mixed peptidase inhibitors) in a variety of morphine-sensitive noxious tests.

It has been suggested that the endogenous opioid peptides, methionine and leucine enkephalin, participate only in naloxone-facilitated antinociceptive responses. To reassess this proposal, analgesic effects resulting from complete inhibition of enkephalin metabolism by intracerebroventricular (i.c.v.) administration of the mixed inhibitor RB 38A (R,S)HONHCOCH2CH(CH2 phi)CONHCH(CH2 phi)COOH) were compared to the effects of morphine (i.c.v.) in various assays commonly used to select analgesics: mouse hot plate-test, tail flick test with mice and rats, electrical stimulation of the tail (TES), paw pressure test with rats, and phenylbenzoquinone-induced writhing test with mice. The ED50s of morphine vs. ED50s of RB 38A in the writhing, hot plate (jumping) and tail flick tests with mice were 0.24 nmol vs. 38 nmol, 1 nmol vs. 36 nmol and 3.2 nmol vs. 285 nmol, respectively. RB 38A (ED30 153 nmol) was only 15 times less active in the tail flick test with rats than morphine and only halve as active in the paw pressure test. Noxious TES in rat was very sensitive to the inhibitory action of endogenous opioids protected by RB 38A, particularly the post-vocalization response which was also shown to be alleviated by antidepressants. All the analgesic effects observed were reversed by naloxone. This first direct evidence of analgesia resulting from peptidase inhibition, in the tail flick test with mice and rats, hot plate (paw lick) and TES shows that the pain suppressive effects of endogenous opioid peptides are not restricted to naloxone-facilitated noxious stimuli but occur more generally, in all morphine-sensitive tests. The differential effects of RB 38A in the various assays is likely to be related to the amount of enkephalins released and to the efficiency of peptidase inactivation in particular brain regions implicated in the control of a given nociceptive input. This mechanism could account for the reduction in side-effects compared to those of morphine following chronic administration of RB 38A.

Kidney sialidase and sialyltransferase activities in spontaneously and experimentally diabetic rats. Influence of insulin and sorbinil treatments.

Kidney cortex sialic acid level, sialidase and sialyltransferase activities have been measured in spontaneously diabetic BB rats and in streptozotocin-diabetic rats (STZ). In untreated diabetic BB rats, at the onset of the disease, sialidase specific activity was found to be increased by 21% when compared with diabetes-resistant BB controls (P less than 0.05) whereas sialyltransferase activity was not significantly modified and bound sialic acid concentration was diminished (P less than 0.05). In diabetic BB rats submitted to a minimal insulin therapy, during 3 months of disease, sialidase activity and sialic acid concentration were similar to those of Wistar age-matched controls. In STZ-diabetic Wistar rats, sialidase specific activity was increased by 76% after 5 months of disease when compared to age-matched Wistar controls (P less than 0.01); in contrast, specific sialyltransferase activity was decreased by 21% (P less than 0.05); these enzymatic alterations were associated with a decrease in bound sialic acid concentration (P less than 0.01); 1 month's insulin therapy, started 4 months after onset of the disease, normalized sialidase activity but had no effect on sialyltransferase activity and sialic acid concentration; treatment with sorbinil prevented cataract development but had no effect on sialidase activity whereas it emphasized the decrease in sialyltransferase activity and sialic acid concentration. The disturbances in the enzyme activities concerned with sialoglycoconjugate metabolism observed in experimental and spontaneous diabetes may be responsible for the decreased bound sialic acid content observed in the rat kidney cortex.

Inhibitors of the enkephalin degrading enzymes. Modulation of activity of hydroxamate containing compounds by modifications of the C-terminal residue.

To further characterize the S'2 subsite of both the neutral endopeptidase (EC 3.4.24.11, NEP) and aminopeptidase N (EC 3.4.11.2, APN), two enzymes physiologically involved in enkephalin metabolism, a new series of hydroxamate inhibitors containing a cyclic amino acid as the P'2 component were synthesized. These amino acids differ by the size of the cycle, the relative position of the functional groups, and their absolute configuration. Highly efficient inhibitors of NEP were obtained whatever the modification on the P'2 component, while for APN inhibition, a cyclic beta-amino acid was preferred. The most active inhibitors contained a trans cyclopentyl beta-amino acid and a cis or a trans cyclohexyl beta-amino acid. When injected intracerebroventricularly in mice, these two latter compounds elicited potent antinociceptive responses on both the jump latency and the fore paw lick times.

New kelatorphan-related inhibitors of enkephalin metabolism: improved antinociceptive properties.

In order to improve the in vivo protection of enkephalins from enzymatic degradation, a new series of inhibitors derived from kelatorphan [HONHCOCH2CH(CH2Ph)CONHCH(CH3)COOH], the first-described complete inhibitor of enkephalin metabolism, were designed by modification of the C-terminal amino acid. The progressive lengthening of the chain of this residue shows that a beta-alanine seems to be the best basic model for the conception of such types of compounds. On the other hand, the methylation of the amide bond, which is well accepted by aminopeptidase N (EC 3.4.11.2) and dipeptidylaminopeptidase, induced a significant loss of affinity for neutral endopeptidase -24.11. Starting from these data, compounds containing a variously substituted beta-alanine residue and corresponding to the general formula HONHCOCH2CH(CH2Ph)CONHCH(R1)CH(R2)COOH were synthesized. All these molecules inhibit neutral endopeptidase -24.11 and dipeptidylaminopeptidase in the nanomolar range, and those containing an aromatic chain (compound 7A, R1 = CH2Ph,R2 = H, and compound 8A, R1 = Ph, R2 = H) inhibit the biologically relevant aminopeptidase N, with IC50's around 10(-8) M. Intracerebroventricular injection in mice of these multienzyme inhibitors produced an efficient and naloxone-reversible analgesic response (hot plate test): compounds 7A and 8A were shown to be more potent than kelatorphan in increasing the jump latency time, in agreement with their in vitro properties, and these new compounds were found to increase the forepaw lick latency, a reflex considered as a typical morphine response.

Enkephalin-degrading enzyme inhibitors. Crucial role of the C-terminal residue on the inhibitory potencies of retro-hydroxamate dipeptides.

The retro-inversion of the amide bond in kelatorphan and analogs, the first series of complete inhibitors of enkephalin metabolism, led to compounds highly efficient only against the neutral endopeptidase 24-11 (NEP). In order to increase the recognition of the aminopeptidase N (APN) and dipeptidylaminopeptidase (DAP), without loss of affinity for NEP, the malonyl group of these retro-inhibitors was replaced by diversely substituted succinyl moieties. All the molecules synthesized are highly efficient NEP inhibitors with Ki's in the 0.2-1 nM range, indicating that NEP possesses a relatively large and not very selective S'2 subsite. In contrast, inhibition of DAP activity is crucially dependent on the size and the position of the substituent in the succinyl moiety. Inhibitory potencies in the nanomolar range are obtained with compounds containing a benzyl group in the alpha-position related to the retro amide bond. Finally, a relatively modest inhibition of APN was observed with Ki's in the 0.5-1 microM range for compounds with benzyl or cyclohexyl group in P'2 position. However, these data demonstrate that efficient and complete inhibition of enkephalin degradation can be obtained with hydroxamate dipeptides containing a retro amide bond. The analgesic potency of the most active inhibitors was measured using the hot plate test in mice. Significant antinociceptive responses were obtained but these effects were rather weaker than those expected from the in vitro inhibitory potencies of these compounds on the three enkephalin-degrading enzymes.

Modification of enzyme activities concerned with collagen metabolism in the heart of spontaneously hypertensive and aortic-constricted rats.

The activities of three enzymes concerned with collagen metabolism 4-prolyl hydroxylase, UDP-glucose: collagen glucosyltransferase and glucosyl-galactosyl-hydroxylysine glucohydrolase and 4-hydroxyproline content have been studied in the cardiac ventricles of spontaneously hypertensive rats (SHR) during prehypertensive, hypertensive and sustained hypertensive stages (respectively 4.5, 12 and 19 weeks of age). They were compared with values observed in age-matched normotensive Wistar Kyoto rats (WKY). The same studies have been performed in parallel on aortic-constricted rats (ACR) 8 days after suprarenal constriction of the abdominal aorta. The most striking finding was a significant increase in cardiac prolyl hydroxylase specific activity in the ACR but not in the SHR. No variation in 4-hydroxyproline concentration was found in the hearts of ACR. In contrast, a decrease in 4-hydroxyproline concentration was found in the hearts of SHR at 19 weeks. Cardiac glucosyltransferase specific activity was significantly elevated only in the SHR at 12 weeks. No variation in glucohydrolase specific activity was detected in the hearts of either SHR or ACR. The cardiac enzyme activities all decreased with age. These data show that the alterations in cardiac collagen metabolism are different in SHR and ACR. The patterns of the alterations found in the heart mirror those observed in the aorta in both models under the same experimental conditions.

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.

Aorta collagen metabolism in spontaneously hypertensive and aortic-constricted rats: variations in enzyme activities concerned with disaccharide unit synthesis and degradation according to blood pressure and age.

Two enzyme activities concerned with collagen disaccharide unit metabolism (UDP-glucose: collagen glucosyltransferase and glucosyl-galactosyl-hydroxylysine glucohydrolase) have been studied in the thoracic aortic wall together with 4-prolyl hydroxylase activity and 4-hydroxyproline content in spontaneously hypertensive rats (SHR) at the prehypertensive, hypertensive and sustained hypertensive stages (respectively 32 days, 12 weeks and 19 weeks of age). They were compared with values observed in age-matched normotensive Wistar Kyoto rats (WKY). The same studies have been performed in parallel on aortic-constricted rats (ACR) 8 days after suprarenal constriction of the abdominal aorta. Negative regressions of all three specific activities as function of age were observed. The most striking difference observed between the SHR and the WKY was the increase of glucosyltransferase specific activity, already found at the prehypertensive stage and continuing thereafter; the glucohydrolase specific activity was increased only during the establishment of hypertension whereas no modification was found with prolyl hydroxylase at any stage. However, a diminution of hydroxyproline concentration was seen at all ages while total hydroxyproline mass remained unaffected. The alterations of the aortic collagen metabolism observed in the ACR recall those seen in the SHR at the prehypertensive stage: the only significant modification was that of glucosyltransferase activity. Correlation was found between glucosyltransferase activity and blood pressure level in the two animal models.

Connective tissue in diabetes mellitus: biochemical alterations of the intercellular matrix with special reference to proteoglycans, collagens and basement membranes.

Diabetes mellitus induces alterations in the metabolism of the macromolecules present in the intercellular matrices and particularly in the basement membranes. These contribute to the morphological changes characteristic of the disease : basement membrane thickening, skin thickening and induration. Accumulation of overglycosylated collagens and diminution of sulfated proteoglycan concentrations are the most generally reported biochemical modifications in human or animal diabetic states. More limited data are available concerning elastin, fibronectin and laminin in diabetes.

Inhibition of the alpha-glucosidase specific for collagen disaccharide units in diabetic rat kidney by in vivo glucose levels: possible contribution to basement membrane thickening.

The activity of the alpha-glucosidase specific for collagen disaccharide units has been measured in kidney cortex homogenates of streptozotocin-diabetic rats under three different conditions: (1) in dialyzed homogenates; (2) in non-dialyzed homogenates; (3) in non-dialyzed homogenates to which glucose was added to compensate for dilution due to homogenization and to reach the glucose concentration determined in kidney cortex (37.5 +/- 2.8 mmol/kg diabetic cortex versus 6.8 +/- 0.3 mmol/kg normal cortex). Under the latter condition, the enzyme activity was markedly decreased in diabetic kidney cortex when compared with that of normal age-matched controls: 4.03 +/- 0.25 versus 6.82 +/- 0.29 units/mg protein (p less than 0.001). Inhibition of enzyme activity was also significant in non-dialyzed diabetic homogenates without additional glucose. In the absence of glucose (in the dialyzed homogenates), it is confirmed that the enzyme activity is elevated in diabetic kidney. The glucose inhibition of the enzyme activity has been shown to be important under in vivo conditions. It may therefore contribute to kidney basement membrane thickening.