Diabetic complications/Basic mechanisms II

POS-004-087 INCREASING DEGRADTION OF GLOMERULAR BASEMENT MEMBRANE (GBM) COLLAGEN IN DIABETIC

NEPHROPATHY M.SATOH, K.SATOH, S.FUKUCHI THE THIRD DEPARTMENT OF INTERNAL MEDICINE, FUKUSHIMA MEDICAL COLLEGE, FUKUSNIMA,

JAPAN

Urinary excretion of hydroxyproline (HYP), hydroxylysine (HYL) and its glycosides were measured in 51 diabetics and 23 normal subjects. Renal biopsy was performed in 4 diabetics. Diabetics were divided into 3 groups; without complications (G-I, n=14), with retinopathy (G-II, n=18) and with nephropathy (G-III, n=lg). Urinary excretion of HYP, HYL and its glycosides were greater in G-II and G-III than in normal subjects. The ratio of glucosylgalactosyl HYL (GIc-GaI-HYL) to galactosyl HYL (GaI-HYL) was also higher in G-II and G-III than in normal subjects. Excretion of HYP, HYL and its glycosides were high in patients with unsatisfactory control of diabetes mellitus. Ratio of glycosylated HYL to HYL decreased and the ratio of GIc-GaI-HYL to GaI-HYL increased in patients improved by insulin injection. While, in patients poorly controlled by insulin injection, the ratio of glycosylated HYL to HYL increased and the ratio of GIc-GaI-HYL to GaI-HYL decreased. Electron microscopic findings of diabetic kidney showed the prominence of microorgans in mesangium which have the role of degradation of GBM. Patients with elevated excretion of glycosylated HYL and low ratio of HYP to glycosylated HYL developed to diabetic microangiopathy within

2 years. Increased excretion of HYP, HYL and its glycosides indicates that the

degradation of type IV collagen in GBM accerelated in diabetes mellitus. It is presumed that measurement of urinary excretion of collagen metabolites is useful to estimate the possibility to developing to microangiopathy and the efficacy of treatment on diabetic microangiopathy and, furthermore, to outlook for its prognosis.

POS-004-088 THE INHIBITION OF ASCORBIC ACID ACTION BY GLUCOSE

E FISHER, S HEFFERNAN, C CAPOGRECO, S McLENNAN, DK YUE, JR TURTLE Department of Medicine, University of Sydney and Department of Endocrinology, Royal Prince Alfred Hospital, Sydney, Australia.

Ascorbic acid (AA) and glucose (G) have been reported to compete for membrane transport. High ambient G concentration in diabetes may interfere with cellular uptake of AA and its action on collagen synthesis. We investigated the possible interaction of G and AA by studying (i) the efficacy of AA in normalizing wound strength in diabetes and (2) the effect of G and AA on collagen synthesis by fibroblast.

Strength of linear wound in normal, diabetic and AA-treated diabetic rats (igm/L drinking water) was tested at 8 weeks by tensiometry (n=6-10/group). Human neonatal fibroblasts at confluence (n=6-8lgroup) were incubated for 24hr in media c~ntaining G (5 or 25mM) ~ AA (0.54mM). The hydroxyproline (OHPro, nmolxl0-) and amino acid (ug) content of the media were determined by HPLC and Fluram (Roche) respectively.

Wound strength is less in diabetic animals and not normalized by AA treatment (Breakin K strength Normal : 70.3+14.4, Diabetic: 36.0+__20.8, Diabetic+AA 27.8+--8.8 Newton). G(25mM) decreased approximately by half the AA-induced rise in fibroblast protein and collagen synthesis.

G(5mM) G(5mM)+AA G(25mM) G(25mM)+AA OHPro 1.2 ! 0.5 9.1 ! 1.8, 1.4+--0.6 4.6~ 1.7, Amino acid 46.1+__21.4 127.2~32.8, 32.0~5.1 58.9+_14.8,

* Different from G alone, ANOVA, p<0.01, mean~SD We conclude that AA does not normalize wound strength in diabetes.

This may be partly due to G inhibiting the effects of AA on collagen synthesis. Supported by the NHMRC, The Kellion and Hoechst Foundations.

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POS-004-089 ISOLIQUIRITIGENIN, A CONSTITUENT OF KAMPO MEDICINES, IS A NOVEL ALDOSE REDUCTASE INHIBITOR.

M TAWATA, K AIDA, H SHINDO, T ONAYA, H SASAKI*, T YAMAGUCHI*, M CHIN*, H MITSUHASHI* Third Department of Internal Medicine, University of Yamanashi Medical School, Yamanashi 409-38, and *Tsumura Laboratory, Ibaragi 300-11, Japan.

Polyol pathway activation has been implicated in the pathogenesis of chronic diabetic complications. Traditionally in Japan, kampo medicines have long been used for alleviation of subjective symptoms of diabetic complications although its mechanism is not known. We have previously reported the existence of aldose reductase inhibitors (ARIs) in several kampo medicines (Planta Medica 53: 131, 1987). In the present study we further purified and identified a potent ARI to be isoliquiritigenin (ISO). In addition we have found a new compound which inhibits platelet aggregation. Boiled water extracts of Glycyrrhizae Radix were repeatedly chromatographed with Sephadex LH-20 and 17 compounds (GUs 1-17) were obtained. GU-17 was found to be ISO with M.W. of 256 and GU-7 has a M.W. of 384. ISO inhibited rat lens aldose reductase with an IC50 of 3.2xi0 -7 M. It also inhibited sorbitol accumulation in human red blood cells with an IC50 of 2x10 -6 M. The potency of ISO was fairly comparable with ONO-2235 which has been reported as a potent ARI. On the other hand, GU-7 inhibited platelet aggregation, 20K- and 40K-dalton protein phospho- rylation induced by thrombin, but not by 12-0-tetradecanoylphorbol-13-acetate. GU-7 alse suppressed intracellular calcium increase induced by thrombin. Further- more, since GU-7 is a phosphodiesterase inhibitor, it is likely that it inhibits platelet aggregation by increasing intracellular cAMP contents. These ARI and anti-platelet activities in kampo medicines may explain the mechanism by which they are beneficial to the chronic diabetic complications.

POS-004-090 EFFECTS OF ALDOSE REDUCTASE INHIBITORS ON PROSTACYCLIN (PGI2) SYNTHESIS BY AORTIC RINGS FROM DIABETIC RATS.

K AIDA, T NOGUCHI, Y KAZAMA, M TAWATA, H SHINDO, T ONAYA, H SASAKI*, T YAMAGUCHI*, M CHIN*, H MITSUHASHI* Third Department of Medicine, University of Yamanashi Medical School, Yamanashi 409-38, and *Tsumura Laboratory, Ibaragi 300-11, Japan.

Several aldose reductase inhibitors(ARIs) have recently been developed and thought to be useful therapeutic agents for diabetic microangiopathy. However, there has been no direct evidence for the relationship between PGI 2 synthesis by vascular tissues and polyol pathway activities. As ARIs, ONO-2235 and a new compound which was obtained from extracts of Glycyrrhizae radix (GU-17) were used in this study. Male Wistar rats were divided into 4 groups. Groups I and 2 were non-diabetic and diabetic controls, respectively. Diabetes mellitus was induced by intraperitoneal injection of streptozotocin. Groups 3 and 4 were diabetic and given ONO-2235 and GU-17 (100 mg/kg/day, orally) for 14 days, respectively. Increased sorbitol contents in lens, nerve, and red blood cells of group 2 rats were significantly decreased by both ARI treatments (groups 3, 4). On the other hand, PGI 2 synthesis (measured as 6-keto-PGF1~) by aortic rings from group 2 rats (3.9+0.3 ng/mg wet wt./30 min) was significantly decreased compared with that of group ~ rats (16.2+1.7), and this decreased PGI 2 synthesizing capacity was sig- nificantly recovered by both ARI administrations (groups 3, 4), although not completely. Furthermore, PGI 2 synthesis by aortic rings was inversely correlated with sorbitol contents in tissues determined in these experiments. These results suggest that some ARIs have beneficial effects on vascular syn- thesis of PGI 2 by inhibiting sorbitol accumulation in tissues of diabetic compli- cations, although the exact mechanism of correlation of these two systems remains to be elucidated.

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POS-004-091 E F F E C T OF ORAL GLUCOSE LOADING ON ERYTHROCYTE SORBITOL AND SORBITOL DEHYDROGENASE IN DIABETIC AND NC~N-DIABETIC SUBJECI'S

H HOSOJIMA, E MIYAUCHI, H OKADA, T MORIMOTO*, S MORIMOTO Divis ion of Endocr inology, D e p a r t m e n t of I n t e r n a l Medic ine , K a n a z a w a M e d i c a l U n i v e r s i t y , I s h i k a w a and SRL*, T o k y o , J a p a n

The accumula t ion of in t raceUular sorbi tol has been shown to be one of important fac-- t o r s r e spons ib le for c a t a r a c t for tnat ion, p e r i p h e r a l n e u r o p a t h y and vascu l a r c o m p l i - c a t i o n s of d i a b e t e s m e l l i t u s . A l t e r a t i o n of so rb i t o l d e h y d r o g e n a s e (SDI-D , an enzyme conver t ing sorb i to l to f ruc tose , may be a t l e a s t in pa r t r e s p o n s i b l e for this s o r b i t o l accumulat ion. We i n v e s t i g a t e d to assess the role of SDH in the i n - c rease of e r y t h r o c y t e so rb i t o l a f t e r 75 g oral glucose l o a d i n g in 15 p a t i e n t s wi th d i a b e t e s m e l l i t u s {DM), 16 p a t i e n t s w i th i m p a i r e d g l u c o s e t o l e r a n c e ( IGT) and a g e - - m a t c h e d 5 n o n - d i a b e t i c s u b j e c t s (C}. E r y t h r o c y t e s o r b i t o l and SDH was m e a s u r e d e n z y m a t i c a l l y and by f l u o r i m e t r i c m e t h o d , respec t ive ly . Mean l e v e l s of r a s ing p l a s m a g l u c o s e , e r y t h r o c y t e so rb i t o l and e r y t h r o c y t e SDH were 185 t ~ / d l , 45.0 nM//g Hb and 163 mu/g Hb in DiM, 1 1 8 ~ / d I , 26.3 nM//g Hb and 204 mu/g lib in IGT, and 99~/d l , 18,2 nM//g Hb and 199.5 nM//gHb in C, respectively. Tow hours a f t e r oral glucose load ing , mean levels of p lasma glucose and e ry th rocy te s o r - b i to l increased to 415 ~ d l and 62 nlVl/gI-lb in DIM. 213~g/dl and 39 nM//g Hb in IGT, and 98r@/dl and 29 aM/gl ib in C, respect ively. Mean levels of SDH two hours a f te r o ra l glucose loading remained unchanged in the th ree groups. T h e s e da ta d e m o n s t r a t e tha t SDH has a minhTlal e f f e c t in a c u t e a c c u m u l a t i o n of e r y t h r o c y t e s o r b i t o l a f t e r g lucose load ing .

POS-004-092 EFFECTS OF VERY MILD DIABETES ON KIDNEY FILTRATION FUNCTION IN RATS.

G. PUGLIESE, R.G. litTON, A. SPEEDY, K. CHANG, E. SANTARELLI, C. KILO, 0.R. WILLIAMSON Department of Pathology, Washington University, St. Louis, MO, USA.

To assess effects of very mild diabetes of 12 weeks duration on renal hemodynamics and f i l t r a t i o n function, we compared 3 groups of male Sprague-Oawley rats i n i t i a l l y weighing 250, g: nondiabetic control (C), severely diabetic (SD; 65 mg/kg bw streptozotocin), and mildly diabetic (MD; 250 mg/kg bw nicotinamide was given 15 min before injecting streptozotocin). Rats pretreated with nicotinamide developed a very mild diabetes characterized by slight elevation of plasma glucose (134 ± lB mg/dl vs l ib ± I0 for C and 511.4 ± 68.7 for SD; P < 0.025 and P < 0.OOl, respectively; mean ± SD of the mean biweekly values for each rat) and glycated hemoglobin (1.56 ± 0.15 nmol HMF/mg protein vs 1.10 ± 0.20 for C and 2.53 ± 0.33 for SD, P < O.001 in both cases) levels, and by reduced insulin response to intravenous glucose tolerance test. Final body weight (47l ± 27 g vs 467 ± 37 for C) and 24 h urine volume (3.3 ± 1.2 mi/24 h/100 g bw vs 2.5 ± l . l for C) were not affected by MD. SD rats showed reduced final body weight (269 ± 46) and increased urine volume (57.1 ± I I .7); in both cases P < 0.O01 compared to C. After 12 weeks of diabetes, albumin clearance (mgplas~/kidney/min) and GFR (g plasma/kidney/min) were assessed by measuring 1311-BSA and 57Co-EDTA kidney clearances (8 rats per group), renal blood flow (ml/min/kidney) was measured by using 15 pm 85Sr-labelled microspheres (5 rats per group) and endogenous albumin and IgG urinary excretion was quantified by radial immunodiffusion assay. Both SO and MD increased 1311-BSA kidney clearance (7.56 ± 1.54 and 5.TO ± 0.33, respectively, vs 3.53 ± O. T3 for C, P < O.OOl in both cases) and GFR (I.62 ± 0.13 and 1.04 ± 0.02 vs 0.85 ± 0.09, P < 0.001 in both cases), although these increases were s~ l l e r in MD (P < 0.Ol vs SD rats). Renal blood flow was increased by SD (7.54 ± 0.66 vs 6.20 ± 0.70 for C, P < 0.025) but not by MD (6.33 ± 0.98). Urinary excretion of albumin and IgG was markedly increased in $0 (2.83 ± 0.73 SEM and 1.39 ± 0.34, respectively vs 0.20 ± 0.04 and 0.08 ± 0.02 for C, P < 0.00% in both cases) as well as in MD rats (1.41 ± 0.52 and 0.49 ± 0.19, P < 0.025 and P < 0.05). These results indicate that even very mild diabetes impairs kidney f i l t ra t ion function and suggest that virtual euglycemia may be required to prevent such functional changes and the associated structural alterations that culminate in renal failure.

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POS-004-093 EFFECTS OF ICI-128,436 ON ERYTHROCYTE SORBITOL AND ERYTHROCYTE MYO-INOSITOL LEVELS IN MEAL LOAD TESTS

N.KAMON and R.TAKEDA The second Department of Internal Medicine, Kanazawa University, Kanazawa, Japan

School of Medicine,

Recent studies in experimental diabetes have suggested that sorbitol accumulation and/or myo-inositol depletion have an important role in the pathogenesis of diabetic complications including neuropathy and that the aldose reductase inhibitor improves both of the abnormalities in the tissue. To estimate the effects of an aldose reductase inhibitor, ICI-128,436, upon sorbitol and myo-~nositol levels in erythrocytes, a meal load test (MLT) was performed twice for each of 6 diabetic patients. In both MLTs, a test meal containing 80 g carbohydrate and 300 mg myo-inositol was given. Only in the second MLT, ICI-128,436 (150, 300 or 600 mg) was administered orally 30 min before the meal loading. Blood glucose levels 150 min after meal load in the Ist and 2nd MLT increased simllarly (134.2 ± 9.7 to 219.2 ± 33.9; 140.0 ± 16.9 to 246.3 ± 39.7 mg/dl, respectively). Erythrocyte sorbitol level increased in the Ist MLT from 23.7 ± 1.2 to 31.3 ± 2.4 and decreased in the 2nd from 24.1 ± 1.4 to 18.7 ± 1.9 nmol/gHb during the 150 min. There were significant differences in erythrocyte sorbitol levels at 150 min between the two MLTs (p<0.01) while not before meal load. Percent inhibition of erythrocyte sorbitol was closely correlated with plasma concentration of ICI-128,436. Erythrocyte myo-inositol levels in both MLTs did not differ before meal load (191.9 ± 15.? vs 184.0 ± 16.2 nmol/gHb) and remained almost unchanged. In conclusion: (I) ICI-128,436 inhibits erythrocyte sorbitol accumulation in vivo. (2) ICI-128,436 may have no essential effects upon erythrocyte myo-inositol level. The data also suggest that erythrocyte myo-inositol level is not a suitable indicator of the tissue myo-inositol metabolism.

POS-004-094 BLATELET Na-~. EXCHANGE RATES IN DIABETIC PATIENTS

T OOYAMA, K YAMADA Depy. of INt. Medicine, Tohoku Kouseinenkin Hoppital

Increase of cytoplasmic pH takes place during platelet activation. This cyto- plasmic alkalinizationiss believed to be mainly brought about by sodium-v::c proton exchange. Amiloride,inhibitor of this N~H~excange,inhibits platelet aggregation. Na'-H~exchange may play an important role in platelet activation. Na-H exchange was measured indlrectly in platelet as the rate of amiloride- sensitive and sodium dependent volume~gain of cells suspended in sodium pro- pionate after Grinstein's method. Protonated propionate can readily~pearmeate the plasma membrane and dissociate to bring about cytoplasmic acidification, which in turn activates the exchange of extracellar Na~for intracellular Hi Thus Na~H*exchange lead to Na:propionate accumulation,which brings about osmotically obliged water and cell swelling.Rate of swelling reflects rate of Nat~exchan~e rate.Cell swelling is measured by Coulter counter. Platelet NE-H exchange rates were corelated with platelet aggregation rate

(ADP 2uM, Collagen lug/ml).nThis exchange rate was increased in diabetic platelet.Enhanced Na-H exchange may be one of the factors whxch cause altered platelet aggregation in diabeti patients.

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POS-004-095 EFFECTS OF PROSTAGLANDIN El, E2, and 12 DERIVATIVES ON PERIPHERAL NEUROPATHY IN DIABETIC RATS (REPORT II): EFFECTS ON CYCLIC AMP AND SORBITOL CONTENTS OF SCIATIC NERVE.

A. KANAZAWA, A. OHNO, A. TANAKA, T. MIWA, A. UEKI, Y. NOTOYA, H. ITOH Third Department of Internal Medicine, Tokyo Medical Collage, Tokyo, Japan.

We found that continuous administration of 0U-1308 (PGEI derivative),CU-83 (PGE2 derivative) or Iloprost (PGI2 derivative) to streptozotocin (STZ) diabetes mellitus (DM) rats improved the nerve conduction velocity (NCV), and PGEI increas- ed the level of cyclic AMP (CAMP) in the sciatic nerve. In this study, we examined variations of CAMP levels in sciatic nerves of STZ DM rats, and changes in the CAMP and sorbitol contents of sciatic nerves which were measur- ed after the NCV of DM rats was improved by 4 weeks administration of (i) 0U-1308 (lO~g/kg), (2) CU-83 (200 ~g/kg), (3) Iloprost (i0 ~g/kg), (4) methylcobalamin (300 ~g/kg) or (5) Monotard insulin (6 units/animal). CAMP and sorbitol were measured by RIA and HPLC, respectively. The CAMP content of the sciatic nerve in untreated DM rats during the 1st to 7th months was decreased by 30-40% compared to the 840~i000 fmol/mg level detected in normal rats (p<O.O1). The 5 drug groups of DM rats showed improvement in the NCV after 4 weeks of medica- tion, but the sorbitol content of the sciatic nerve increased to 1.7~2.0 nmol/mg, which was similar to the 1.8±0.4 nmol/mg level in the untreated DM rats, whereas the normal rats showed a level of only 0.2~0.i nmol/mg. CAMP in the normal rats was 988 ± 132 fmol/mg, whereas the level in the untreated DM rats decreased to 529 ± 56. CAMP was increased to 653~757 fmol/mg by the administration of drugs (i) (2) (3) and (4) (p<0.Ol). The drug (5) group showed no difference from the untreated DM group. These findings suggest that the decrease of CAMP levels content in peripheral nerves plays an important role in relation to diabetic peripheral neuropathy. It is also surmised that the mechanisms of action of drugs (i), (2), (3) and (4), administered to treat DM neuropathy, increase the CAMP in peripheral nerves.

POS-004-096 EFFECT OF PROSTAGLANDIN E~, E2, AND 12 DERIVATIVES ON PERIPHERAL NEUROPATHY IN STREPTOZOTOCIN DIABETIC RATS (REPORT I): EVALUATION USING THE NERVE CONDUCTION VELOCITY

A OHNO, A KANAZAWA, A TANAKA, A UEKI, Y NOTOYA and H ITOH Third Dept. of Internal Medicine, Tokyo Medical College, Tokyo, Japan

[PURPOSE] A comparative study of the effects of the three prostaglandin derivatives (PG deriv.), methylcobalamin and insulin on the nerve conduction velocity (NCV) of a rat tail nerve. [METHOD] After intraperitoneal injection of streptozotocin 60 mg/kg, five groups of male Wistar rats were administered the following agents during the 4th to 8th weeks, (1) OU-1308 (PGE~ deriv.) 10tag/kg, (2) CU-83 (PGE2 deriv.) 200 #g/kg, (3) lloprost (PGI2 deriv.) 10/ag/kg, (4) methylcobalamin 300 gg/kg, (5) Monotard insulin: 6 units/animal [(1), (2): p.o.; (3), (4), (5): i.p.)]. The pre- and post-administration NCVs were compared with the same aged non-DM rats (control group) and untreated DM rats (DM group). [FINDINGS] The NCV in each of the 5 druged groups and the DM group before drug administration was approximately 22 m/sec, wl~ich is clearly less than the 24.9 m/sec of the control group. After the drug administration, the NCV in each of the 5 druged groups improved to 28 m/sec, which is not sig- nificantly different than the control group value of 29.4 m/sec, whereas the DM group NCV remained constant at 23.5 m/sec. (Figure) [CONCLUSION] These findings suggest that the three PG deriv, studied here are useful in the treatment of diabetic neuropathy.

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POS-004-097 INCREASED SERUM CONCENTRATION OF TYPE IV COLLAGEN PEPTIDE IN DIABETICS - A POSSIBLE BIOCHEMICAL MARKER FOR DIABETIC COMPLICATIONS -

O.MATSUMOTO, E.MATSUMOTO ~ , A.OOSHIMA ~ , H.TABATA, T.OHNO, M.TADA, Y.MIYAMOTO, H.BESSHO ~ , H.KIKUOKA ~, K.NANJO ~, K.MIYAMURA H. WAKAYAMA RED CROSS HOSPITAL, THE FIRST DEPARTMENT OF PATHOLOGY ~ AND MEDICINE**~ WAKAYAMA UNIVERSITY OF MEDICAL SCIENCE, WAKAYAMA, JAPAN.

Type IV collagen is a major constituent of basement membrane. In long term diabetes, thickening of glomerular capillary basement membrane is found by light and electron microscopy, suggesting either an increased fibrogenetic activity or decreased degradation. We have developed a sandwich enzyme immunoassay using monoclonal antibodies for serum type IV collagen peptide. The specificity of monoclonal antibodies to type IV collagen is determined by an immunoblotting and inhibition ELISA. Molecular weight of antigen in the serum determined by gel filtration and the immunoas~ay was found to be 750-800 KD. It was found that the assay system is sensitive and reproducible, and in human chronic liver disease the amount of serum type IV collagen peptide is increased concomitant with increased hepatic fibrogenesis. We have measured the amount of serum type IV collagen peptide in diabetics and non-diabetic healthy subject. In diabetics, the serum concentration (131.8-187.2 ng/ml) is significantly elevated (p<O.01) as compared to the value of control healthy subjects (95.3 ng/ml). There is a tendency to display distinct values among the diabetic patients with different treatment. The value of patients with diet, sulfonylureas and insulin treatment is 131.8 ± 8.6, 157.7 ± 22.9 and 187.2 ± 20 ng/ml (M ± SE), respectively. The patients with proteinuria yielded a higher value (164.5 ± 20.7 ng/ml) as compared to those without proteinuria (146.1 ± 12 ng/ml). Although the mechanism of increment of serum collagen peptide in diabetics remains unknown, the immunoassay developed in this study can be utilized for detection and monitoring diabetic complications such as glomerulosclerosis. It has been reported that collagen synthesis is increased in the glomerulus of streptozotocin-induced diabetic rats. By an immunofluorescence with a monoclonal antibody, we have observed nodular deposition of type IV collagen in the glomerulus of the patient with Kimmelstiel-Wilson syndrome.

POS-004-098 INFLUENCES OF DIETARY PROTEIN CONTENT ON DIABETIC NEPHROPATHY IN NOD MICE,

T NAKAO, Y KADOYA, S KIMURA, M EMOTO, M KONDO, T SANKE,K NANJYO, K MIYAMURA, J HIRAOKA*. Departments of Medicine and Anatomy*,Wakayama University of Medical Science, Wakayama, Japan.

To investigate the influences of protein content in diet on diabetic nephropathy, the following studies were performed in NOD mice which are well known as animal model for IDDM. DM and non-DM groups of female NOD mice aged 3-5 months were given low protein diet (14% protein), normal diet (24% protein) and high protein diet (44% protein) for 7 days. Light and transmission electron microscopic techniques were used. Cut sections of kidney frozen with nitrogen liquid were also prepared for scanning electron microscopy. Changes of urinary NAG and LAP levels were determined in the mice after the feeding with above diets. In DM group, diabetic nephropathy grew worse by the intake of high protein diet, and a significant increase in kidney weight and in the ratio of kidney weight to body weight was noted. Electron microscopy revealed enlarged stomata of glomerular endothelial cells, blistering of renal tubular epithelial cells, derangement and edematous chanqes of microvilli in diabetic mice. These morphological changes were found worsened by high protein diet intake. A trend of increase in urinary NAG and decrease in LAP was observed in DM group, and this trend was more pronounced by high protein diet intake. In conclusion, it was demonstrated that the intake of high protein diet after the occurrence of DM in NOD mice resulted in worsening of the morphological and urinary findings on the kidneys.

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POS-004-099 PREVENTION OF GLOMERULAR BASEMENT MEMBRANE THICKENING IN STREPTOZOTOCIN DIABETIC RATS BY ALDOSE REDUCTASE INHIBITOR 'STATIL'.

Y ID0,S YAGIHASHI,M KAMIJO Department of Pathology, University of Hirosaki, Aomori, Japan

Morphometric analysis was performed in the kidney of streptozotocin-induced diabetic rats to investigate the efficacy of ICI 128,436 (Statil) on glomerular changes. Rats of Wistar strain were rendered diabetic with intravenous injection of streptozotocin(50mg/kg). Age- and sex-matched normal rats served as controls. Diabetic duration and 'Statil'-treatment (per os,25mg/kg/day) were for up to 7 months. Body weight and blood sugar levels were regularly monitored. The rats were killed by perfusion with 2.5% glutaraldehyde solution. The wet weights of left kidney extirpated just before the perfusion were also measured. Glomerular volume, mesangial volume density, mesangial volume and frequency of the exudative lesions in the glomerulus were measured under light microscopy. The thickness of glomerular basement membrane were estimated on electron microscopic photographs by orthogonal intercept method. The thickness of glomerular basement membrane of 'Statil'-treated rats was significantly reduced compared with that of non-treated diabetic rats (202~2.3 v.s. 223~1.2nm; p~0.05), but not to the extent of controls (185~1.8 nm). About a 50~ reduction of the frequency of the exudative lesions was also found in 'Statil'-treated group. However, there were no differences in the kidney to body weight ratio, glomerular volume, mesangial volume density and mesangial volume between two groups. The present study demonstrated that the long-term administration of aldose reductase inhibitor could prevent the thickening of glomerular basement membrane but not affect the mesangial lesions.

POS-O04-100 EFFECT OF GLICLAZIDE ON THROMBOXANE B2, PARAMETERS OF HAEMOSTASIS AND FREE RADICAL MEDIATED REACTION PRODUCTS IN NON-INSULIN DEPENDENT DIABETES MELLITUS.

C.M. FLORKOWSKI 1 , M.R. RICHARDSON 1 , C. Le GUEN 2, P.E. JENNINGS 1 , A.F. JONES 2, J. LUNEC 2 , ~.H. BARNETT l- Department of Medicine, East Birmingham Hospital and 2Department of Clinical Chemistry,

Selly Oak Hospital, and University of Birmingham.

Abnormalities of platelet aggregation, increased levels of lipid peroxides and denatured proteins have been implicated in the pathogenesis of diabetic microangiopathy. Seventeen non-insulin dependent diabetics (mean age 65.7) on established sulphonylurea therapy were converted to an equivalent dose of gliclazide. They were studied at baseline and intervals to 36 weeks. Thromboxane B 2 (TXB 2) and beta thron~oglobulin (BTG) were measured by radioimmunoassay. Products of the free radical oxidation of lipids were measured as diene conjugates (DC) by scanning UV spectophotometry and thiobarbituric acid reactivity (TBA) by absorbance. Oxidation products of proteins were represented by fluorescence/UV ratio of immunoglobulin G (IgG) separated from serum by HPLC. Glycaemic control remained stable throughout the study and there were no differences between -4 and 0 week baseline values. TXB_ fell from 734 ± 233 ng/l at 0 weeks to 488 ± 155 at 36 weeks, p<0.001 and BTG from 120 ± 7~ ng/ml to 48 ± 18 at 24 weeks, p<0.005. Lipid peroxides fell between 0 and 36 weeks; DC from 0.71 ± 0.28 to 0.32 ± 0.09, p<0.001 and TBA from 29.6 ± 22.4 to 4.0 ± 22.22, p<0.001. Fluorescent IgG ratio fell from 1.44 ± 0.36 to 0.32 ± 0.07, p<0.001. This provides evidence for a specific action of gliclazide on thromboxane synthesis and platelet aggregation.

This is independent of glycaemic control and may in part be mediated by the observed fall in lipid peroxides.

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POS-O04-101 SERUM Lg G ANTIBODIES TO LIPOTEICHOIC ACID FROM STREPROCOCUS MUTANS AND TO AN EXTRACT FROM

BACTEROIDES GINGIVALIS IN PATIENTS WITH TYPE I DIABETES MELLITUS

K. Monefeldt, T. Tollefsen and K. Dahl-J~rgensen

Dept. of periodont. University of Oslo and Dept. og Pediad. Aker University Hospital, Oslo

Periodontal disease is caused by interactions between dental plaque bacteria and host immune factors. Recent work suggests that high titers of serum antibody reflect the subgingival microflora. Lipoteichoic acid (LTA) is a surface antigen common to most Gram+ dental plaque bacteria. Diabetic patients show increased susceptability to bacterial infections including perio- dontitis. The aim of the present study was to determine the immune responsiveness to two dental plaque derived antigens, (LTA and to an extract of Bacteroides ginglvalls), in the diabetic patients. The preparations were used in ELISA essay for ig G antibodies in serum samples. The results indicate that young diabetic patients (age 17-30) had increased titer against serum LTA compaired to the systemic healthy group. Older diabetic (age 30-70) showed four times increase in serum antibody values to LTA compaired to systemically healthy patients and two times increase compaired to systemically healthy periodontitis group. All diabetic patients show detectable antibodies reactive with our crude B. gingivalls extract. Their mean values were significantly raised above that of the healthy reference group but subnormal compaired to the "normal" periodontitis group. The patients with type I diabetes mellitus show altered immunological response to LTA prepared from strep, mutans cells and to an extract from B. gingivalis. We suggest that this may explain the increased susceptability to periodontitis in diabetic patients.

POS-004-102 DETERMINATION OF HUMAN BASEMENT MEMBRANE ASSOCIATED PROTEO-HEPARAN SULFATE WITH A SPECIFIC ANTIBODY

E SCHLEICHER, E WAGNER, B OLGEMOLLER, KD GERBITZ Klinisch-chemisches Institut und Forschergruppe Diabetes, 8000 M~nchen 40, FRG

Basement membranes have been shown to contain heparansulfate proteoglycans(HSPG). These negatively charged molecules seem to contribute essentially to the permea- bility of basement membranes, notably glomerular basement membranes. There is also experimental evidence indicating that a decreased content of HSPG could be responsible for some of the pathological findings in diabetes. We have therefore raised antibodies against HSPG purified from porcine glomerular basement mem- branes and developed an immunoassay for specific determination of HSPG. The an- tibody was characterized by enzyme immunoassays, immunoprecipitation and immuno- histological methods. It was shown to recognize specifically the core protein of HSPG from porcine, human and murine glomerular basement membrane but it did not recognize HSPG from rabbit or bovine kidney. The antibody did not cross-react with other basement membrane proteins like laminin or fibronectin nor with chon- droitin sulfate, dermatane sulfate, heparin, hyaluronic acid or DNA. Immunohisto- chemical studies on frozen tissue sections from many human organs showed base- ment membrane specificity. The antibody stained glomerular basement membranes and basement membranes for small vessels most prominently. An ELISA was devel- oped for specific determination of HSPG in quantities of ca. I ng. Using this assay production of HSPG by porcine endothelial cells and by rat glomeruli could be measured. The specific antibody against HSPG provides a good tool for the in- vestigation of HSPG content of glomerular basement membranes in diabetes.

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POS-004-103 CLINICAL IMPLICATIONS OF THE MEASUREMENTS OF LAMININ P1 IN SERA AND URINES FROM DIABETIC PATIENTS.

M TAKAHASHI, S KAWAZU, M SUZUKI, K NEGISHI, T WATANABE, S SHUNTOH, S MORITANI T TANIKAWA, N INABA, S KATAYAMA AND J ISHII The Fourth Department of Internal Medicine, Saitama Medical School,Saitama,Japan

Diabetic microangiopathy is characterized by a thickning of capillary basement membrane or increasing of basement membrane-like materials. It is well known that the major components of basement membrane are type IV collagen and laminin, a non-collagenous glycoprotein. Therefore,we attempted to check the levels of serum laminin P1 concentrations in diabetic patients using RIA-laminin P1 Kit(Hoechst Co. Ltd.) In addition, in some cases, urinary laminin P1 was also determined.

In diabetic patients who were diagnosed having retinopathy and/or nephropathy, serum laminin P1 concentrations were significantly higher than those in patients without any diabetic complications or normal subjects. And the serum levels of laminin P1 seemed to increase depending on the severity of diabetic micro- angiopathy. Moreover, we also found the higher urinary laminin P1 values in dia- betic patients with nephropathy, when corrected by simultaneously measured uri- nary creatinine. In turn, urinary laminin P1 levels were within normal limits in patients with liver cirrosis, despite their significantly higher values of serum laminin PI. During the observation periods about one year, the levels of laminin P1 in poorly controlled diabetic patients significantly increased, in the pres- ence or absence of their diabetic complications. The positive relationship be- tween the level of serum laminin P1 and urinary protein excretion was observed.

Therefore, serum laminin P1 concentrations could be used as an indicator to signify the existence, severity and progression of diabetic microangiopathy.

POS-004-104 A NEW DIAGNOSIS AND A NEW IREAIMENT FOR DIABEIlC POLYNEOROPAIHIES

P K~LIRINCER, 0 EBER, W LANGSIEGER, P LIND, P WAKONIC, G KLIHA

Hospi ta l 8armherzlge BrOder Graz-Eggenberg, BergslraBe 27, 8020 Craz, Aus t r i a , Europe

15 pa t ien ts wi th diabetes m e l l l t u s [ I su f f e r i ng From polyneuropathlc syndrome (PN5) as a complz- ca t ion and showing a decrease in motor-conductzon v e l o c i t y oF peroneal nerve b i l a t e r a l l y , have been studied For the s t l m u l a b l h t y o f thezr m lc roc l rcu la tzon wi th a new measuring-design. A Laser method as a means of measurzng blood f low (Laser-Doppler-Flowmetry) was used to evaluate the in- crease oF mlcrovascular blood Flow o f skin during the hyperthermal phase. I t evaluated the degree oF increase oF blood Flow in the m l c r o c l r c u l a t l o n as wel l as the time beginning of hyperthermy to onset in change o f perFuslon ("hyperthermal la tency" ) as compared to a heal thy c o n t r o l l group oF 15 subjects . The hyperthermal latency was most s i g n i f i c a n t l y prolonged in the group o f PNS. Some of the d iabe t i c pa t ien ts were t rea ted wzth a new comb1nation oF Glnkgo-Flavonglycoslds [ lebo- nln )and Folzc acid (Ca lczumFo l lna t ) during a per iod oF 14 days. ]he con t ro l o f hyperthermal latency aFter therapy showed a slgnlFzcant decrease as wel l as a decrease oF c l l n z c a l symptoms could be observed, lhe motorzc-conduct lon v e l o c i t y oF peroneal nerve increased s i g n i f i c a n t l y a f t e r treatment too.

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POS-004-105 ELECTRICAL ANIONIC CHARGES ON RED CELLS ARE REDUCED IN IDD PATIENTS.

P CAVALLO-PERIN, P ESTIVI, G PAGANO Insti tute of Internal Medicine,University of Turin,Turin,ltaly

A specific loss of charges on the glomerular capil lary wall (GCW) is associated to an increased clearance of albumin. A correlation between electrical charges on cel lular mem- branes of red blood cells and electrical charges of the glomerular capil lary wall has been reported in the rat. We performed a chemical test based on the binding of the cationic dye Alcian Blue 8GX (AB) (Levin et al.,Lancet 2:239,1985) in 28 insulin-dependent diabetics, aged 25±7 yr,normotensive,in good metabolic control (HbAIC,8%) and in 19 well-matched heal- thy controls; II diabetic patients were normoalbuminuric (3.7±I.0 SEM ug/min),ll were micro- albuminuric (32.3±6.3) and 6 were macroalbuminuric (791.6±311.3). Each subject repeated the test on two separate occasions. The mean AB binding value was signif icantly (t=test:p < 0.0005) lower in diabetics than in controls: 87±I.6 SEM versus I00.5±I.6 ng/lObred blood cells. A negative correlation between AB binding and albumin excretion rate was significant in microalbuminuric diabetics only (r=0.85, p<O.OOl). AB binding was not signif icantly cor- related with age,duration of diabetes or metabolic control. These results suggest a possible implication of electr ical charges in diabetic microalbuminuria.

POS-O04-106 RAISED URINARY DIGOXIN-LIKE IMMUNOREACTIVITY IN INSULIN-DEPENDENT DIABETIC PATIENTS.

0 GtAMP!ETRO, A. CLERICO, G. OREGORI, S. BERTOLI, M.G. DEL CHICCA, R. MICOOLI, L. CRUSCHELLI, A. LUOOHETTI, O.PENNO, L. Di PALMA, R. ANICHINI, R. NAVALESI. Chair Metabohc Diseases, Inst. Clin. Med. II, C.NR. inst. Clin. Physiol., Plea University, italy.

,~n endogenous factor with activity similar to cardiac glycosides has bean suggested to play a role in the regulation of fluids and electrolytes in animals and human s as well as in the pathogenesis of low-renin hypertension. Hypertension occurs more frequently m ClaOetlcs than in normal population and hypertension of insulin-dependent diabetes mellitus (IDDM) is char,~cter!zed bv a volume expansion and low renin. We evaluated me excretion rate (ER) of digoxin-like immunoreactivity (DLIS), during timed overnight urinary collections, m 9 i normotensive non-diabeti~- and 104 normotensive IDDM pat]ents. DLIS in urines was assayed by a sensitive RIA method. In contro!s, DLIS-ER correlated with urinary excretion rates of creatinine (r=0.57, p<O.OOl), Na+ (r=0.28, p<O.O3) and K+ (r=0.32, :)<0.02), and also with patients' weight (r=0.34, p<O.O01), height (r=0.29, p<O.OI), body mass index (r=0.25, 3,0.02), systoiic blood pressure (r=0.28, p<O.02). The mean DLIS-ER in diabetics was significantly higher than in controls ( 73=41 vs 63+35 pg/min, p<O.02). Within diabetics, men had mean DLIS-ER higher than women (87+41 vs 66+40 pg/min, p~O Of ) Further, diabetics males had DLIS-ER higher than control ones ( 87+41 vs 74±43 pglmin, p<O,04), as did diabetic in con,~a:'i.son with he4~Ithy females (66+40 vs 52±21 pglmin, p<O03). In diabetics DLIS-ER correlated with urinary AER ( r=0.26, p<O.O I ) ane creatinine-ER (r=OAO, p<O.O001 ), Na+ (r=0.28, p<0.02) and K + excretion rates (r=0.30, p<O.OI ) and also with patients' body weight (r=026, p<O.01 ). In multiple regression analysis, only urinary creatinine-ER and AER slgmficantlv contributed to the correlatlon with DLIS-ER in diabetic patients ( r:0.52, p<O.O01 ). Our findings indicate that increased amounts of a substance or a group of substances with activity similar to cardiac glycosides are excre[ed in urine of some IDDM patients. Urinary excretion of DLIS could be dependent on glomerular filtration rate and physico-chem icai properties of glomerular membrane, besides on the body weight

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