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

Diabetologia, Apr 1983

Summary The activity of the α-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 < 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.

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

Diabetologia Inhibition of the a-Glucosidase Specific for Collagen Disaccharide Units in Diabetic Rat Kidney by in Vivo Glucose Levels: Possible Contribution to Basement Membrane Thickening 0 1Drpartement de Biochimie,Facult6 de Mrdecine Broussais-H6tel-Dieuand 2Laboratoirede Pharmacologie,Facult6 de Pharmacie , Paris , France 1 Dr. M. Sternberg , D6partement de Biochimie Facult6 de M6decine Broussais 45 rue des Saints-P6res 75270 Paris Cedex 06 , France M. Sternberg 1, J. A n d r e I a n d J. P e y r o u x2 Summary. The activity of the a-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___0.3mmol/kg normal cortex). Under the latter condition, the enzyme activity was markedly decreased in diabetic kidney cortex when compared with that of normal agematched controls: 4.03 + 0.25 versus 6.82_+0.29 units/rag protein (p < 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. a-Glucosidase; rat; basement membrane thickening; collagen disaccharide units; streptozotocin-diabetes; glucose inhibition; kidney - 9 Springer-Verlag1983 With long-term streptozotocin diabetic rats, we have observed previously an increased activity o f the a-glucosid a s e specific for collagen disaccharide units (glucosylgalactosyl-hydroxylysine glucohydrolase) in the dialyzed k i d n e y cortex h o m o g e n a t e a n d s u p e m a t a n t [ 1, 2 ]. In the same animals, we have c o n f i r m e d the presence o f k i d n e y glomerular b a s e m e n t thickening [2]. Basement m e m b r a n e collagen is k n o w n to be rich in disaccharide units [ 3 ] and the p r o p o r t i o n o f these m a y be elevated in diabetes [ 4-6 ]. The increased e n z y m e activity o f the dialyzed (glucose-flee) fractions appears to be related to elevated e n z y m e concentration in the kidney, possibly i n d u c e d b y raised substrate levels. On the other hand, we have s h o w n competitive inhibition o f the e n z y m e activity b y glucose, a p r o d u c t o f the reaction with inhibition constant K p = 7.5 x 10 -3 m o l / l [7]. Since glucose freely penetrates the k i d n e y cells [ 8 ], it was o f interest to m e a s u r e the e n z y m e activity in diabetic k i d n e y cortex without dialysis, in the presence o f glucose levels f o u n d in vivo. F o r this p u r p o s e , it was necessary to evaluate the glucose c o n c e n t r a t i o n in kidney cortex. The enzyme activity was t h e n d e t e r m i n e d u n d e r three conditions: (1) in dialyzed k i d n e y cortex h o m o g e nates; (2) in non-dialyzed k i d n e y cortex h o m o g e n a t e s ; (3) in non-dialyzed h o m o g e n a t e s with a d d e d glucose to c o m p e n s a t e for dilution due to homogenization. Significant inhibition o f e n z y m e activity u n d e r conditions f o u n d in vivo could contribute to b a s e m e n t m e m b r a n e thickening since this enzyme is believed to be involved in the catabolism o f b a s e m e n t m e m b r a n e collagen [ 1 ]. Materials and Methods Diabetic Rats Forty male Wistar rats were divided in random order into two groups: one group (weight231_+12g, mean_+SD) was injected iv. with streptozotocin (Sigma,St Louis, Missouri, USA, 55 mg/kg body weight)in NaCI (100mmol/1)/citrate buffer (ammol/1), pH 4.5. The agematched control group (mean weight 223+_14g) was injected with buffer alone. Animals were housed five to a cage and allowed free accessto food (standard pelleted rat chow A 103,UAR Co, Villemoisson, France) and water. Arterialpressure was estimated weeklyby the tail cuff method. Blood was taken from the orbital sinus in non-fasting animals for glucosedetermination everymonth. The mortalityrate of the diabetic rats before sacrifice was 14% (versus 5% in the controis). Ten diabetic and ten control rats were randomly chosen for the experiment.Theserats were sacrified33 weeksafter streptozotocinin T _T 8 g > == o A > D o ~9 Glucose (mmol/I) 0 0 DIALYZED HOMOGENATES Glucose (mmol fl ) 1,7 9.3 NON-DIALYZED HOMOGENATES Glucose(retool/l) 6.8 37.5 HOMOGENATES +GLUCOSE jection. One day before sacrifice they were put in metabolic cages (Paion, Orlrans, France) with water ad libitum for 24 h urine collection. Some hours later and after feeding, blood was taken from the abdominal aorta under ether anaesthesia. The kidneys were removed and frozen in liquid nitrogen ( - 1 9 6 ~ until used. Diabetic and control rats were sacrified alternately. M. Sternberg et aL: CoUagen-Specific a-Glucosidase in Diabetic Kidney Enzymatic Determinations Kidney homogenates: Frozen cortices were put on ice, dissected and homogenized with a Potter homogenizer in three volumes of cold NaC1 (125 mmol/1) phosphate buffer (25 mmol/1), pH 7.0. Glucose concentration was measured in the homogenate and the concentration in the kidney cortex calculated after dilution correction. The homogenate was divided into three parts: one was dialyzed against cold NaCI (125 retool/l) phosphate buffer (25 retool/l) pH 7.0; the second was kept on ice undialyzed; glucose was added to the third, non-dialyzed, part to simulate the calculated glucose concentration in the kidney cortex before dilution. Substrate and product: The substrate Glucosyl-galactosyl-hydroxylysine (Glc-Gal-Hyl) was prepared from bovine kidney cortex basement membranes as described in (1). The product Galactosyl-hydroxylysine (Gal-Hyt) was prepared by mild acid hydrolysis of GlcGal-Hyl. The substrate and the product were radio-labelled by treatment with galactose-oxydase followed by reduction with tritiated potassium borohydride [ 1, 3 ]. The specific activity obtained was 12.7 Ci/ tool for Glc-[3H]Gal-Hyl and 43 Ci/mol for [3H]Gal-Hyl. Assayfor Glc-Gal-Hylglucohydrolaseactivityin thepresenceof glucose:This was effected by a modification of the procedure described previously [71. Briefly, GIc-[3HIGal-Hyl (43 nCi, 12.7 Ci/mol) was dried with unlabelled Glc-Gal-Hyl (185 nmol) in small glass tubes and dissolved in 1 mol/1 sodium phosphate buffer (20 .ul),pH 4.4. The kidney enzyme (100 ~1) was added and the mixture incubated for I h at 37 ~ Substrate and product were separated by high voltage paper electrophoresis. After scanning with a radiochromatogram scanner (Packard, La Grange, Illinois, USA) 0.5 cm wide bands, corresponding to the peaks of radioactivity, were eluted with 2 ml distilled water. The radioactivity was determined in a Packard Tri-Carb liquid scintillation spectrometer; the [3HIGal-Hyl peak was measured and the total amount of Gal-Hyl released calculated after efficiency correction. The mean difference between duplicate results was 5%. Under these assay conditions product release has been shown to be proportional to the enzyme concentration. One unit of enzyme activity corresponds to I nmol of Gal-Hyl released per h at 37 ~ Chemical Determinations Blood, urine and homogenate glucose concentrations were measured by the glucose oxidase technique. The homogenate samples (100 ~tl) were diluted in water (lml) and ZnSO4 (0.12mol/I, 0.5 ml) and Ba(OH)2 (0.12 tool/l, 0.5 ml) added. After centrifugation for 20 rain at 1,500 rev/min, supernatant samples were taken for glucose determination. Total protein concentrations were measured according to Lowry et al. [91. Aliquots of homogenates were solubilized first in 0.1 N NaOH at 37~ for 2h and urines were dialyzed against 0.15 mol/1 NaCI prior to estimation. Creatinine was determined in urine by the Jaff6 reaction [ 10 ]. Statistical Methods Comparison between diabetic and control groups was by the Student's t-test. R e s u l t s Characteristics o f the Diabetic Rats B l o o d g l u c o s e l e v e l s a f t e r 1, 3 a n d 6 m o n t h s o f d i a b e t e s w e r e r e s p e c t i v e l y : 4 6 . 5 + / . 6 ( m e a n + S E M , n = 1 0 ) , 3 7 . 7 _+ 1.3 a n d 30.2 _+ 1.5 m m o l / I v e r s u s 8.4 + 0.2, 8.2 _+ 0.3 a n d 6.6 +__0.3 m m o l / 1 i n t h e c o n t r o l r a t s ( n = 10). P e r s i s t a n t a r t e r i a l h y p e r t e n s i o n w a s o b s e r v e d f r o m d a y 14 of diabetes (23.6 +__0.4 versus 20.5 + 0.1 kPa in the controls; p < 0.001). Cataracts were present in all diabetic rats after 6 weeks of disease. At sacrifice, after 33 weeks of diabetes, the following data were noted in the diabetic rats: reduced body weight (217_+27 versus 501_+10g in the controls, p < 0.001); polyuria (95 _+11 versus 11.7 _+1.6 m l / d a y , p <0.001); elevated creatininuria (21.4_+1.6 versus 14.3 + 1.1 rag/day, p < 0.005); increased proteinuria (38.4 _+10 versus 18.5 _+1.7 m g / d a y , p < 0.05), but protein excretion per mg creatinine was not significantly elevated. Kidney cortex weight was slightly but not significantly increased (2.59 _+0.11 versus 2.38 _+0.11 g). Glucose concentration was similar in the diabetic kidney cortex (37.5_+ 2.8 m m o l / k g ) and plasma (37.2_+ 1.4 mmol/1); it was lower in the normal kidney cortex (6.81 _+0.31 m m o l / k g ) than in the normal plasma (9.03 _+0.23 mmol/l). There was no change in the protein content of the kidney cortex (155 _+6 m g / g ) on dialysis. Enzymatic Determinations As shown in Figure 1, Glc-Gal-Hyl glucohydrolase specific activity was increased in the dialyzed kidney cortex homogenate of the diabetic rats. In contrast, the specific activity of the enzyme was decreased in the corresponding non-dialysed fractions. When the glucose concentration was adjusted to the level in vivo, the specific activity was further diminished. When the enzyme activity was expressed in units per total kidney cortex, similar variations were observed (Fig.Z). Discussion The increase in glucosyl-galactosyl-hydroxylysine glucohydrolase activity in the dialyzed rat kidney cortex previously reported after 19, 23 and 28 weeks of diabetes [ 2 ] is again confirmed by the measurement of elevated activities after 33 weeks of diabetes induced under similar conditions. In the previous experiments, thickening of kidney glomerular basement membrane was confirmed after 28 weeks of diabetes [ 2 ]. The increase in kidney weight observed here, as previously [ 2 ], is moderate compared with other published data [ 11 ]. The greater severity of the diabetes in our experiments, as reflected by the plasma glucose levels, could account for this. The most striking result obtained here is that, in the presence o f glucose at diabetic concentration in vivo, the glucosyl-galactosyl-hydroxylysine glucohydrolase specific activity is markedly diminished in agreement with our initial hypothesis. This decrease could contribute to gIomerular basement membrane thickening since glucosyl-galactosyl-hydroxylysine glucohydrolase is believed to be involved in basement membrane collagen catabolism [ 1 ]. This effect would complement those of M. Sternberg et al.: Collagen-Specific a-Glucosidase in Diabetic Kidney other enzyme modifications reported in long-term diabetic kidneys: increased activity of lysyl-hydroxylase [ 12 ] and glucosyl transferase [ 13 ] catalyzing the disaccharide unit synthesis; elevation of prolyl-hydroxylase activity specific for collagen synthesis [ 12 ]. In streptozotocin-induced diabetes, blood glucose rises very quickly in the first weeks. Renal cells, unlike adipocytes or muscle cells, appear to be freely permeable to glucose [ 4 ]. Glucose concentration has indeed been found to be similar in diabetic plasma and kidney cortex here. Glucose inhibition of glycosyl-galactosylhydroxylysine glucohydrolase activity may therefore contribute to the progressive kidney glomerular basement membrane thickening [ 15 ]. In fact basement membrane thickening seems to develop earlier in kidney glomeruli than in muscle [ 16 ]. Therefore the increased glucosyl-galactosyl-hydroxylysine glucohydrolase concentration observed in glucose-free kidney cortex homogenates may be a secondary effect related to increase in substrate levels, the primary effect being the inhibition of this enzyme by high glucose levels in vivo. Acknowledgements.This work was supported by the following institutions: Drlrgation Grnrrale/t la Recherche Scientifique et Technique, Universit6 Ren6 Descartes and Facult6 de M+decine Broussais - H6tel-Dieu, Paris. We thank G. Gateau, B. Faure, F. M~ge and J. Bourlier for their excellent technical assistance. 1. 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In: Camerini-Davalos RA , Cole HS (eds) Early diabetes . Advance in metabolic disorders. Academic Press, London, Suppl 2 pp 257 - 269 Received: 5 May 1982 and in revised form: 2 December 1982


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M. Sternberg, J. Andre, J. Peyroux. Inhibition of the α-glucosidase specific for collagen disaccharide units in diabetic rat kidney by in vivo glucose levels: possible contribution to basement membrane thickening, Diabetologia, 1983, 286-289, DOI: 10.1007/BF00282715