ELSEVIER

Localization of Advanced Glycation Endproducts in the Kidne of Experimental Diabetic I? ats Kenichi Shikata, Hirofumi Makino, Hikaru Sugimoto, Masahiko Kushiro, Kosuke Ota, Kenji Akiyama, Norie Araki, Seikoh Horiuchi, and Zensuke Ota

ABSTRACT

Advanced glycation endproducts (AGE) have been proposed as a major mediator in the development of various diabetic complications. In order to evaluate the involvement of AGE in the development of diabetic nephropathy, we examined the localization of AGE in the kidney of the streptozotocin-induced diabetic rats immunohistochemically using a monoclonal antibody directed to AGE. In the diabetic rats,

glomerular hypertrophy, thickening of the glomerular basement membrane, and expansion of mesangial matrix were observed. AGE was detected in expanded mesangial area and glomerular basement membrane in the kidneys of diabetic rats. The present results suggest that AGE may participate in the development of diabetic nephropathy. (Journal of Diabetes and Its Complications 9;4:269-271, 1995.)

INTRODUCTION

G

lucose reacts nonenzymatically with pro- teins to form advanced glycation endpro- ducts (AGE).* Protein modification by AGE is expected to occur in proteins with rela-

tively long half-lives.2 Formation of AGE may contrib- ute to the development of various diabetic complica- tions. In this study, in order to clarify the involvement of AGE in the development of diabetic nephropathy, we examined the localization of AGE in the renal tissue of experimental diabetic rats immunohistochemically.

METHODS

Induction of Diabetes. Five Sprague-Dawley rats, weighing 130 g, were injected intravenously with 65

Third Department of Internal Medicine (K.S., H.M., H.S., M.K., K.O., K.A., Z.O.), Okayama University Medical School, Okayama; and Department of Biochemistry (N.A., S.H.), Kumamoto Univer- sity Medical School, Kumamoto, Japan

Reprint requests to be sent to: Dr. Kenichi Shikata, Third Depart- ment of Internal Medicine, Okayama University Medical School, 2-5-1 Shikata-cho, Okayama, 700, Japan.

Journal of Diabetes and Its Complications 1995; 9:X9-271 0 Elsevier Science Inc., 1995 655 Avenue of the Americas, New York, NY 10010

mglkg streptozotocin (Sigma, St. Louis, USA) in ci- trate buffer, pH 4.5. Five control rats were injected with citrate buffer alone. Blood glucose level and uri- nary protein were assayed. Five experimental and con- trol rats were killed 28 weeks after injection, and the kidneys were harvested. The renal specimens were processed for light and electron microscopic study and immunohistochemical study.

Immunohistochemical Procedure. The distribution of AGE was studied using immunoperoxidase tech- niques. Renal specimens were immediately snap fro- zen and 4pm thick sections were prepared. The slides were incubated with biotin-labeled mouse anti-AGE monoclonal antibody (MAb) for 2 h at 37°C. The slides were washed and then incubated with avidin biotinyl- ated horseradish peroxidase (Vector Lab. CA, USA). The slides were washed, and color reaction was carried out. As an additional control, positively stained tissues were subjected to blocking experiments in which un- labeled anti-AGE MAb was preincubated. Sections preincubated with anti-AGE MAb were uniformly negative.

1056~8727/95/$9.50 SSDI 1056-8727(95)00032-W

270 SHIKATA ET AL. ] Diab Comp 1995; 9:269-271

FIGURE 1 Immunoperoxidase sfaining for advanced glycation endproducts (AGE) with the monoclonal antibody in streptozo- tocin-induced diabetic rat at 28 weeks after injection. Expanded mesangial area and capilla y walls are stained (arrows).

RESULTS

Plasma glucose level and excretion of urinary protein in diabetic rats were significantly higher than in con- trol rats. In normal rats (6 weeks old), AGE was not detected in the glomeruli. In age-matched control rats that were injected with citrate buffer, AGE was de- tected weakly in the mesangium and the capillary wall. In the diabetic rats, significant glomerular hypertro- phy, expansion in the mesangial matrix, and thick- ening of’ the glomerular basement membrane (GBM) were observed. AGE was stained strongly in the ex- panded mesangial area and capillary wall (Figure 1).

DISCUSSION

Concerning the involvement of AGE in the develop- ment of diabetic nephropathy, Mitsuhashi et a1.4 re- ported the increased content of AGE in the renal cortex of streptozotocin-induced diabetic rats in their immu- nochemical study, ‘Miyata and Monniel5 have demon- strated the presence of pyrraline only in the fibrous crescents of the glomeruli or obliterated glomeruli. In our present study, AGE was demonstrated to localize in the expanded mesangial area and in the capillary wall in nonobliterated glomeruli in the kidney of the diabetic rats.

Glomerular extracellular matrix (ECM) is composed of type IV collagen, laminin, proteoglycans, and some other glycoproteins. AGE induces abnormal associa- tion and self-assembly of these molecules. Recently, loosening of the network structure of the tubular base- ment membrane after nonenzymatic glycation in vitro was reported.6 It was reported that nonenzymatic gly-

cation of laminin and type IV collagen significantly reduced the cooperative binding with heparin.’ We demonstrated the decreased density of HS-PG and type IV collagen and increased porosity of the meshwork structure of the GBM in diabetic nephropathy.8-10 Thus, glycation of the GBM is suggested to be responsible for proteinuria in diabetic nephropathy. Recently, Doi et al.” revealed that AGE stimulates the production of ECM by mesangial cells through AGE receptor. Fur- thermore, ECM crosslinked with AGE is more resis- tant to digestion by proteases.12 Thus AGE formation in the glomerulus may result in thickening of the GBM and expansion of mesangial matrix.

The present results suggest that AGE may partici- pate in development of diabetic nephropathy.

ACKNOWLEDGMENT

This was supported by a Grant-in Aid for Scientific Research (C-04671481) from the Ministry of Education, Science and Culture of Japan to Dr. H. Makino.

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