ELSEVIER

Advanced Glvcation Endproducts and Diabetic hephropathi Zenji Makita, Katsuyuki Yanagisawa,Satoru Kuwajima, Naruhito Yoshioka, Tatsuya Atsumi, Yuko Hasunuma, and Takao Koike

ABSTRACT

Diabetic nephropathy is currently the single largest cause of endstage renal disease (ESRD) in the United States and many European countries. The primary cause for the development of diabetic complications (including diabetic nephropathy) is persistent exposure to hyperglycemia, although genetic and other incompletely understood factors also play an important role. Although much consideration has been given to the pathogenesis and genetics of the disease itself, the mechanisms by which persistent exposure to hyperglycemia cause biochemical and metabolic alterations have been very sketchily understood. Recently, a growing body of evidence has linked the accumulation of t.he late products of

glucose-protein interaction to a variety of chronic complications, including diabetic nephropathy. The formation of irreversible advanced glycosylation endproducts (AGES) resulting from the spontaneous reaction between glucose and proteins occur most noticeably on long-lived structural proteins. Recent studies demonstrate that the pathogenesis of diabetic nephropathy is caused by the hyperglycemia-accelerated formation of AGES. Also, reactive AGE peptides in the circulation are thought to play a role as a new version of so called middle molecule toxic substances. This evidence is opening a new window for our understanding of the pathogenesis of diabetic nephropathy. (Journal of Diabetes and Its Complications 9;4:265-268, 1995.)

BIOCHEMICAL PROPERTIES OF ADVANCED GLYCOSYLATION ENDPRODUCTS (AGE)

G lucose is known to form chemically revers-

ible early glycosylation products with pro- tein (Schiff bases) at a rate proportional to the glucose concentration. l These Schiff

bases gradually rearrange to form the more stable Amadori products. As these early glycation products do not continue to accumulate on collagen and other stable tissue proteins in chronic diabetes, it is not sur-

Second Department of Medicine, Hokkaido University School of Medicine, Sapporo, Japan

Reprint requests to be sent to: Dr. Zenji Makita, Department of Medicine II, Hokkaido University School of Medicine, Kita-Ku, Kita- 15, Nishi-7, Sapporo 060, Japan.

]ournal of Diabetes and Its Complications 1995; 9:2&i-268 0 Elsevier Science Inc., 1995 655 Avenue of the Americas, New York, NY 10010

prising that their concentration does not correlate with the severity of diabetic complications.’ These early gly- cation products undergo a slow complex series of chemical rearrangements to form irreversible advanced glycation endproducts (AGES) in vivo. Recent studies have indicated that reactive intermediate substances derived from the degradation of glucose, Schiff bases, and Amadori products, also have accelerated the for- mation of AGES in vivo. Figure 1 shows a newly de- scribed pathway for the formation of AGES. AGES al- ter the structural and functional properties of proteins and contribute to many of the pathophysiological changes that accompany longstanding diabetes. AGES accumulate more rapidly in the tissues of diabetic pa- tient due to their higher circulating glucose levels. Be- cause of their highly cross-linking nature, these AGES have increasingly been recognized as factors in the pathogenesis of diabetic complications.‘” Specific re-

10%-8727/95/$9.50 SSDI 1056-8727(95)00051-3

266 MAKITA ET AL. ] Diab Comp 2995; 9:265-268

Intermediate Substances (3DG etc.)

Glucose Amadori \ +- Products

--‘?-?-AGE NHz-R

FIGURE 1 Schemafic representation of the formation of early

reversible and advanced irreversible glycosylufion products (AGE, advanced glycosylufion endproducts).

ceptors for AGES have been identified on macrophage cells.3,4

The structures of AGE candidates have been identi- fied.‘T3 By their nature, AGES are chemically heteroge- neous. AGES cause extensive protein-to-protein cross- linking and induce complex cellular responses through interaction with AGE-specific cell-surface receptors.3‘7 These include the induction of endothelial cell perme- ability,8 monocytelmacrophage chemotactic activity,9 macrophage activation and cytokinelgrowth factor se- cretion,3,4J0 and EDRF inactivation.” Each of these bio- logical responses, if elicited in vivo, would be likely to contribute to the vascular perturbations present in various stages of diabetes. *s3 Direct evidence on the in vivo pathogenic effect of AGES, independent of hyper- glycemia, was determined recently in studies in which the intravenous administration of exogenous AGES to healthy animals induced multiple vascular defects, including vascular permeability, subendothelial and perivascular mononuclear cell infiltration, and defec- tive nitric oxide-dependent vasodilatory responses.Qi3 These studies demonstrated that AGES, when re- leased in the circulation, are highly reactive substances that adhere to vascular tissues, and generate and/or propagate vascular damage.

WHY MAY AGES BE IMPORTANT FOR THE DEVELOPMENT OF DIABETIC NEPHROPATHY?

In contrast to the early glycation products, a significant correlation has been shown between the levels of tis- sue AGES and the presence and severity of diabetic complications by using AGE-specific fluorescence method.2 Recently, we have developed a novel radio receptor assay (RRA) for the measurement of AGES.‘* Using a sensitive RRA, the AGE content of arterial wall samples from diabetic patients are found to be significantly higher than samples from nondiabetic pa- tients.15 Also, diabetic patients with endstage renal disease have a threefold increase in AGES over diabetic patients without end-stage renal disease.15 Moreover, in the circulation, AGE-peptide levels (molecular weight

less then 10 kDa) are found to be dramatically higher in diabetic patients with renal failure. In addition, lev- els of AGE peptide are found to correlate with renal function.15 After hemodialysis, AGE-peptide levels in diabetic patients are found to decline modestly, but not to the extent that creatinine declines. In contrast to hemodialysis, elevated AGE levels decline to the normal range within 4 days of renal transplantation.15

Most recently, we developed an enzyme-linked im- munosorbent assay (ELISA) system to measure AGES by using an AGE-specific antibody.16 By using this AGE-specific ELISA system, we compared AGE-pep tide levels in different modalities in diabetic and nondi- abetic patients. 17,18 High-flux (HF) dialyzer was found to remove AGES better than the conventional cupro- phane (CONV) dialyzer. AGE removal by CAPD was found to be between HF and CONV hemodialysis. Again, renal transplantation is the best treatment for removing AGE from the circulation. The time-course study for the reaccumulation of circulating AGE-pep- tide after HF dialysis shows that AGE-peptide levels returned to 75% of the predialysis levels by 3 h after hemodialysis, similarly to the rise in serum creatinine levels.17 These studies indicate that hemodialysis sys- tems employing HF filter membranes are indeed sig- nificantly more effective at reducing AGE activity than is conventional hemodialysis, albeit for a brief period of time. Unfortunately, AGE levels return to predial- ysis concentrations within 3-5 h. These data could be explained on the basis of a rapid redistribution of AGE- peptide between the extravascular compartment and the intravascular pool.

The molecular weight of AGE-peptide isolated from the low molecular weight fraction of human serum was assessed by Bio-gel P-6 gel filtration.17 AGE-con- taining fractions eluted as a single major peak that contained peptide with an apparent molecular weight between 1350 and 6000 based on the AGE-specific ELISA. In contrast, fluorescence measurements indi- cated that AGE-like material was more widely distrib- uted across a molecular weight range of 180-6000.17 The molecular weight of AGE-peptide ranges between 1350 and 6000, which is within the range of the “mid- dle-molecular-weight uremic toxic substances” reported to resist effective removal by current hemodialysis methods.

In order to confirm the potential toxicity of circu- lating AGE-peptide that were dramatically elevated in the circulation of diabetic patients with ESRD, we studied the reactivity of AGE-peptide. To determine whether or not these AGE-peptides rebind to tissue, we incubated AGE-peptides from hemodialysis patients with collagen. l7 After 14 days, more than 80% of the starting AGE-peptides are bound to the collagen. The studies indeed demonstrate that AGE-peptides as found in the circulation are chemically “reactive” sub-

J Diab Comp 1995; 9:265-268 GLYCATION ENDPRODUCTS AND NEPHROPATHY 267

stances, which can readily cross-link covalently with collagen in vitro.

The data presented here suggest that the accumula- tion of reactive AGE-peptide in the circulation may act as an accelerating factor for the rapid evolution of diabetic complications, principally micro- and macro- angiopathy. They also introduce a potential explana- tion for the rapid progression of diabetic nephropathy once this complication is initiated, as well as a reason that might explain the excessively high morbidity and mortality of diabetic patients with ESRD.

Additional evidence for the toxicity of AGE was shown by immunohistochemical analysis utilizing AGE-specific antibody.19 Dr. Nakayama et a1.19 dem- onstrated immunohistochemical localization of AGE in the kidney from diabetic patients with ESRD by using AGE-specific antiserum. AGE accumulation was demonstrated around the elastic fiber of the arteri- oles from the specimen obtained by renal biopsy from a 69-year-old diabetic woman. Also, AGE was posi- tively stained in the enlarged nodular mesangium lesion of the specimen obtained by renal biopsy from a 44- year-old man with longstanding diabetes and ESRD.

guanidine may be considered one of the best candi- dates for the prevention of diabetic complications. Now, in late phase 2 clinical trials for the early diabetic nephropathy patient in the United States, aminogua- nidine shows great promise. In the treatment of ESRD with diabetes, little is known about uremic toxic sub- stances. Among the many circulating “toxic factors,” the reduction in AGE levels in diabetics with ESRD may provide an important clue to this yet undefined benefit. Also, in our attempts to improve the mortality and morbidity of these patients, knowledge of the missing link between hyperglycemia and diabetic ne- phropathy will be mandatory. These recent findings potentially open new windows for the understanding and treatment of diabetic nephropathy.

REFERENCES

1. Brownlee, M, Cerami A, Vlassara H: Advanced glyco- sylation endproducts in tissue and the biochemical ba- sis of diabetic compilations. N En@] Med 3X3:1315-1321, 1988.

2.

More recently, Dr. Miyata et al. reported a very in- teresting finding that also supports the toxicity of AGE in ESRD.20 B2 Microglobulin &M) is known to be a major constituent of amyloid fibrils in hemodialysis- associated amyloidosis. ” By using two-dimensional electrophoresis and AGE-specific antibody, they could clearly demonstrate that a dominant constituent of the amyloid deposit in hemodialysis-associated asmy- loidosis is acidic l&M, and the acidic BzM is AGE-modi- fied. They could detect AGE-modified (j2M in sera and urine from ESRD patients. They also showed evidence of the existence of AGE-modified B2M from the urine of a long-term hemodialysis patient by using AGE- specific fluorescence. The B2M from long-term hemo- dialysis showed intense florescence that was identical to the AGE-specific fluorescence. In contrast, normal l&M did not show AGE-specific fluorescence. These data indicate that AGE-modified protein play an im- portant role in the pathogenesis of complications re- lated to the hemodialysis treatment.

Monnier VM, Vishwanath V, Frank KE, Elmets CE, Dauchot P, Kohn RR: Relation between complications of type I diabetes mellitus and collagen-linked fluores- cence. N Engl ] Med 314403~408, 1986.

3. Vlassara H, Bucala R, Striker L: Pathogenic effects of advanced glycosylation: biochemical, biological, and clinical implications for diabetes and aging. Lab Invest 70:138-151, 1994.

4. Vlassara H, Brownlee M, Manoque KR, Dinarello CA, Pasagian A: CachectinlTNF and IL-1 induced by glu- cose-modified proteins: role in normal tissue remodel- ing. Science 240:1546-1548, 1988.

5. Yang Z, Makita Z, Horii Y, Brunelle S, Cerami A, Sehaj- pal I’, Suthanthiran M, Vlassara H: Two novel rat liver membrane proteins that bind advanced glycosylation endproducts: Relationship to macrophage receptor for glucose-modified proteins. 1 Exp Med 174:515-524, 1991.

6. Skolnik EY, Yang Z, Makita Z, Radoff S, Kirstein M, Vlassara H: Human and rat mesangial cell receptors for glucose-modified proteins: Potential role in kidney tissue remodelling and diabetic nephropathy. I Exp Med 174:931-939, 1991.

INTERVENTION OF DIABETIC NEPHROPATHY BY AGE INHIBITOR (AMINOGUANIDINE)

Aminoguanidine is a strong AGE inhibitor, and seems likely to be a good candidate for the prevention of diabetic complications .= Currently, much data has ac- cumulated to show the beneficial effects of aminogua- nidine in vitro and in vivo.22-26 For diabetic nephropa- thy, aminoguanidine shows beneficial effects not only morphologically, but functionally as well. Dr. Itakura and his collaborators26 demonstrated that albuminuria in diabetic rats was almost completely inhibited by the administration of aminoguanidine. Clearly, amino-

Imani F, Horii Y, Suthanthiran M, Skolnik EY, Makita Z, Sharma V, Sehajpal I’, Vlassara H: Advanced glyco- sylation endproducts-specific receptors on human and rat T-lymphocytes mediate synthesis of interferon y: Role in tissue remodeling. J Exp Med 178:2165-2172, 1993.

Esposito C, Gerlach H, Brett J, Stern D, Vlassara H: Endotherial receptor-mediated binding of glucose-modi- fied albumin is associated with increased monolayer permeability and modulation of cell surface coagulant properties. I Exp Med 179:1387-1407, 1989.

Kirstein M, Brett J, Radoff S, Ogawa S, Stern D, Vlas- Sara H: Advanced protein glycosylation induces trans- endothelial human monocyte chemotaxis and secretion of platelet-derived growth factor: Role in vascular dis-

268 MAKITA ET AL. 1 Diab Comp 1995; 9:265-268

10.

11.

12.

13.

14.

15.

16.

17.

18.

ease of diabetes and aging. Proc Nat1 Acad Sci USA 87: 9010-9014, 1990.

Kirstein M, Aston C, Hintz R, VIassara H: Receptor- specific induction of insulin-like growth factor I in hu- man monocytes by advanced glycosylation end prod- uct-modified proteins. I Clin Invest 90:439-446, 1992.

BucaIa R, Tracey KJ, Cerami A: Advanced glycosylation products quench nitric oxide and mediate defective en- dothelium-dependent VasodiIatation in experimental diabetes. I Clin Invest 87~432-438, 1991.

VIassara H, Fuh H, Makita Z, Krungkrai S, Cerami A, Bucala R: Endogenous advanced glycosylation endpro- ducts induce complex vascular dysfunction in normal animals: A model for diabetic and aging complications. Proc Nat1 Acad Sci USA 89:12043-12047, 1992.

Fuh H, Yang D, Striker L, Striker G, Vlassara H: In vivo AGE-peptide injection induces kidney enlargement and glomerular hypertrophy in rabbits; Prevention by aminoguanidine. Diabetes 41:9A, 1992.

Radoff S, Makita Z, VIassara H: Radio receptor assay for advanced glycosylation end products. Diabetes 40: 1731-1738, 1991.

Makita Z, Radoff S, Rayfield E, et al.: Advanced glyco- sylation end products in patients with diabetic ne- phropathy. N Engl J Med 325:836-842, 1991.

Makita Z, VIassara H, Cerami A, Bucala R: Immuno- chemical detection of advanced glycosylation end prod- ucts in vivo. J Biol Chem 267:5133-5138, 1992.

MakitaZ, BucaIa R, RayfieldEJ, et al.: Reactive glycosyl- ation endproducts in diabetic uraemia and treatment of renaI failure. Lancet 343:1519-1522, 1994.

Korbet SM, Makita Z, Catherine A, et al.: Advanced glycosylation end products in continuous ambulatory

peritoneal dialysis patients. Am I Kidney Dis 22:588-591, 1993.

19. Nakayama Y, Horii Y, Nishino T, et al.: Immunohisto- chemical localization of advanced glycosylation end- products in coronary atheroma and cardiac tissue in diabetes mellitus. Am J Pathol. 143:X49-1656, 1993.

20. Miyata T, Oda 0, Inagi R, Iida Y, Araki N, Yamada N, Horiuchi S, Taniguchi N, Maeda K, Kinoshita T: Pz-microglobulin modified with advanced glycation end products is a major component of hemodialysis- associated amyloidosis. JClin Invest 92:1243-1252,1993.

21. Gejyo F, Yamada T, Odani S, et al.: A new form of amyloid protein associated with chronic hemodialysis was identified as beta2-microglobulin. Biochem Biophys Res Commun 129:701-706, 1985.

22. Brownlee M, VIassara H, Kooney P, UIrich A, Cerami A: Aminoguanidine prevents diabetes-induced arterial wall protein cross-linking. Science 232:1629-1632,1986.

23. Makita A, Vlassara H, Rayfield E, Cartwright K, Fried- man E, Rodby R, Cerami A, Bucala R: Hemoglobin- AGE: A circulating marker of advanced glycosylation. Scienc 258:651-653, 1992.

24. Bucala R, Makita Z, Vega G, Grungy S, Koschinsky T, Cerami A, VIassara H: Modification of low density lipoprotein by advanced glycation end products con- tributes to the dyslipidemia of diabetes and renal insuf- ficiency. Proc Nat1 Acad Sci USA 91:9441-9445, 1994.

25. BucaIa R, Makita Z, Kochinsky T, Cerami A, VIassara H: Lipid advanced glycosylation: pathway for lipid oxi- dation in vivo. Proc Nat1 Acad Sci USA 90~6434~6438, 1993.

26. Itakura M, Yoshikawa H, Bannai C, Kato M, Kunika K, Kawakami Y, Yamaoka T, Kamejirou Y: Aminogua- nidine decreases urinary albumin and high-molecular- weight proteins in diabetic rats. Life Sci 49:889-897, 1991.