Abstract. We have previously identified three distinctive amino acid sequences from type IV collagen which specifically bound to heparin and also inhibited

We examined the effects of in vitro nonenzymatic glycosylation (NEG) of the extracellular matrix (ECM) on various aspects of the adhesive interaction between the ECM and human kidney mesangial (HKM) cells, and also on the ability of HKM cells to spread and proliferate. Isolated type IV collagen (tIV) or intact complexes of glomerular basement membrane and mesangial matrix (GBM/MM) were used as substrates following control incubation or modest glycation. We observed that HKM cells adhered less effectively to glycated tIV at early time intervals and were delayed in attaining maximal levels of adhesion compared to cells interacting with control tIV. The nature of the adhesive interaction was also different since antibodies which blocked the function of beta 1-containing integrins more effectively inhibited adhesion between HKM cells and glycated tIV than cells and control tIV. HKM cells interacting with glycated tIV also demonstrated less cell surface microspike and ruffle formation at five minutes after plating, less extensive spreading throughout the examined time intervals (> or = 90 min after plating), and slightly increased cell numbers 5 to 10 days after plating when compared to cells interacting with control tIV. However, increased cell numbers were not observed when HKM cells were grown on glycated GBM/MM. Similar changes in response to glycated substrate were observed when HKM cells were grown in either 5 or 25 mM glucose. In conclusion, relatively modest glycation of the ECM alone was sufficient to result in specific changes in HKM cell behavior in vitro.(ABSTRACT TRUNCATED AT 250 WORDS)

We examined structural changes in bovine kidney tubular basement membrane (TBM) following in vitro nonenzymatic glycosylation (NEG). Isolated TBM was incubated for 2 wk at 370C in the absence of sugar or in the presence of either glucose or ribitol under conditions that minimized degradation and oxidative damage. NEG and crosslink formation in glycated TBM were confirmed by decreased solubility, increased amounts of low mobility material by SDS-PAGE, and increased specific fluorescence compared to controls. Morphological analysis using high resolution, low voltage scanning electron microscopy (LV-SEM) revealed a complex three-dimensional meshwork of interconnecting strands with intervening openings. Glycated TBM underwent distinct morphological changes, including a 58% increase in the amount of image surface area occupied by openings. This was due to an apparent increase in the number of large openings (diameters > 12.5 nm), whereas the number of small openings (diameters < 12.5 nm) remained unchanged. These findings corroborate earlier physiological studies, which established that the loss of glomerular permselectivity seen in patients with diabetic nephropathy is due to the formation of large pores in the kidney filtration barrier of which the BM is a major component. We conclude that NEG and crosslink formation among BM components lead to modifications of BM ultrastructure, which could play a role in loss of barrier function in diabetic microangiopathy and nephropathy. (J. Clin. Invest. 1993. 92:3045-3052.)

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