High levels of tissue advanced glycation end products (AGEs) that result from the spontaneous modification of proteins by glucose occur In diabetes and aging. To address the potential pathogenic role of AGEs in the glomerulosclerosis of diabetes or nephrosclerosls of aging, doses of AGE-modified rat albumin (25 mg per kg per day, i.v.) sufficient to elevate circulating AGE levels to the range of diabetic serum were admired daily to healthy rats alone or in combination with the AGE inhibitor ae. After 5 months, the AGE content of renal tissues in AGE-treated rats rose to 50% above controls (P < 0.025), whereas serum contained 2.8-fold greater AGE levels (P < 0.025). Light and electron microscopy of kidneys from AGE-treated rats revealed a more than 50% increase in glomerular volume compared to controls (P < 0.001), I t periodic add/Schiff reagent-positive deposits, basement membrane widening, and mesanga extraceflular matrix increase and indicated significant glomerulosclerosis compared to untreated (P < 0.002) or albumin-treated controls (P < 0.002). These changes were associated with si nt loss of protein (P < 0.005) and albumin (P < 0.002) in the urine of AGE-treated rats compared to controls. Cotreatment with aminoguanidine markedly limited both the structural and fuctional defects. These in vivo data demonstrate that AGEs influence glomerular structure and function in a manner leading to glomerulosclerosis. (8), while in normal SJL mice AGEs enhance the expression of alIV collagen and laminin B1 mRNAs within the glomerulus (9). However, the long-term impact of cumulative AGE deposition on the structure and function of the kidney has remained obscure.In this report, we present evidence indicating that prolonged in vivo exposure of normal renal tissues to AGEmodified homologous serum albumin induces marked renal lesions and that certain aspects ofthis pathology are inhibited by the coadministration of aminoguanidine. METHODSPreparation of Advanced Glycation Products. Rat serum albumin (RSA) (Sigma) was passed over an Affi-Gel Blue column (Bio-Rad), a heparin-Sepharose CL-6B column (Pharmacia), and an endotoxin-binding affinity column (Detoxigel, Pierce) to remove possible contaminants (8, 9). RSA modified by AGEs was prepared as described (8,9). AGE levels were measured by an AGE-specific ELISA (10) (AGE-rat albumin, 62 AGE units/mg of protein; unmodified rat albumin, 1.2 units/mg). Each reagent contained endotoxin (E-Toxate, Sigma) at <0.2 ng/ml. Animal Studies. Male Sprague-Dawley rats (150 g) aged 3 months (n = 50) (Charles River Breeding Laboratories) were used in these studies, which were conducted in accordance with The Picower Institute Laboratory Animal Center guidelines. After a 2-week adaptation period, rats were given tail vein injections with AGE-modified or native RSA (25 mg per kg per day) or with AGE-RSA followed by infusions of aminoguanidine hydrochloride (100 mg per kg per day, i.v.) for 5 months. Serum samples were collected at the end ofthe treatment period from all groups for seru...

Renal disease is one of the most common and severe complications of diabetes mellitus. The hallmark of the disease, glomerulosclerosis, is characterized by an accumulation of extracellular matrix in the mesangial areas, leading to progressive obliteration of the vascular spaces. The role of the metabolic derangements of diabetes mellitus in the development of these lesions is incompletely understood. One of the consequences of hyperglycemia is the formation of advanced glycosylation end products (AGEs), which result from a series of rearrangements secondary to nonenzymatic reaction of glucose with proteins. Specific receptors for proteins modified by AGEs, present in several cell types, were recently described in human and rat mesangial cells. Furthermore, exposure of mesangial cells to AGEs was followed by an increase in fibronectin production. In the present study we show evidence that mouse mesangial cells exhibit an increase in collagen type IV mRNA and peptide synthesis after exposure to AGEs. Antibodies to AGE receptors prevent this increase, indicating that the response is AGE-receptor-mediated. In addition, anti-platelet-derived growth factor abrogates the AGE response, suggesting that platelet-derived growth factor acts as an intermediate factor. Transcription assay reveals that the elevated mRNA levels are due to an increase in the transcription rate, rather than to an increase in the stability of the message. Finally, the mRNAs coding for laminin and heparan sulfate proteoglycan are also increased after exposure to AGE, whereas glyceraldehyde 3-phosphate dehydrogenase mRNA levels remain constant. The increase in extracellular matrix mRNAs seen in the current study suggests that AGE formation in vivo may be one of the metabolic events leading to the development of diabetic glomerulosclerosis.

Several lines of evidence suggest that the excessive accumulation of extracellular matrix in the glomeruli of diabetic kidneys may be due to reactive intermediates forming between glucose and matrix proteins called advanced glycation end products (AGEs). Normal mice received AGEmodified mouse serum albumin i.p. for 4 weeks, and glomerular extracellular matrix, growth factor mRNA levels, and morphology were examined. We found that AGE induced an increase in glomerular extraceflular matrix al(IV) collagen, laminin Bi, and transforming growth factor Pis mRNA levels, as measured by competitive PCR, as well as glomerular hypertrophy. The AGE response was specific because the coadministration of an AGE inhibitor, aminoguanidine, reduced all these changes. We conclude that AGEs affected expression of genes implicated in diabetic kidney disease and may play a major role in nephropathy.reactive AGEs, thus preventing AGE-protein cross-linking (14). Among other effects, nG was shown to reduce AGE content of aortic tissue in long-term-diabetic rats (15) and attenuate the glomerular lesions in diabetic rats (16), suggesting that AGEs participate in the development of these lesions. When the response(s) of cultured renal mesangial cells to AGE-albumin was examined, we found that the in vitro expression and secretion of several ECM components were up-regulated through surface receptors (17, 18). Furthermore, normal rats chronically injected with AGE developed vascular dysfunction and glomerular lesions (19,20), which were markedly reduced in those cotreated with nG.

The culture of glomerular cells has represented an important tool in the understanding of individual glomerular cell functions. However, the complexity of the glomerulus has made it difficult to obtain pure cell populations. It has also been difficult to culture glomerular endothelial cells, even as mixed cell populations. At present there are no established glomerular cell lines from any source. We have established permanent cell lines of cloned glomerular epithelial, mesangial, and endothelial cells from a line of mice transgenic for the early region of simian virus 40 (SV40). These mice appear normal at birth but by three to four months of age have sclerosis affecting a variable percentage of their glomeruli. The cells maintain features characteristic of their normal counterparts despite their transformed phenotype. These cell lines could be useful tools in understanding the pathogenesis of glomerulosclerosis in this transgenic mouse model and in studying those features of normal glomerular cell biology which are not altered by a transformed phenotype.