Advanced glycation end products regulate extracellular matrix protein and protease expression by human glomerular mesangial cells

Abstract

Abstract. Advanced glycation end products (AGEs) may play a role in the pathogenesis of diabetic nephropathy, by modulating extracellular matrix turnover. AGEs are known to activate specific membrane receptors, including the receptor for AGE (RAGE). In the present study, we analyzed the various receptors for AGEs expressed by human mesangial cells and we studied the effects of glycated albumin and of carboxymethyl lysine on matrix protein and remodelling enzyme synthesis. Membrane RAGE expression was confirmed by FACS analysis. Microarray methods, RT-PCR, and Northern blot analysis were used to detect and confirm specific gene induction. Zymographic analysis and ELISA were used to measure the induction of tPA and PAI-1. We show herein that cultured human mesangial cells express AGE receptor type 1, type 2 and type 3 and RAGE. AGEs (200 μg/ml) induced at least a 2-fold increase in mRNA for 10 genes involved in ECM remodelling, including tPA, PAI-1 and TIMP-3. The increase in tPA synthesis was confirmed by fibrin zymography. The stimulation of PAI-1 synthesis was confirmed by ELISA. AGEs increased PAI-1 mRNA through a signalling pathway involving reactive oxygen species, the MAP kinases ERK-1/ERK-2 and the nuclear transcription factor NF-κB, but not AP-1. Carboxymethyl lysine (CML, 5 μM), which is a RAGE ligand, also stimulated PAI-1 synthesis by mesangial cells. In addition, a blocking anti-RAGE antibody partially inhibited the AGE-stimulated gene expression and decreased the PAI-1 accumulation induced by AGEs and by CML. Inhibition of AGE receptors or neutralization of the protease inhibitors TIMP-3 and PAI-1 could represent an important new therapeutic strategy for diabetic nephropathy.

Contribution of myo-inositol oxygenase in AGE:RAGE-mediated renal tubulointerstitial injury in the context of diabetic nephropathy

“…Another consideration needs to be kept in perspective for designing the AGE ligand as it relates to the ECM, since the latter undergoes notable transformation in the form of thickening of tubular basement membranes (TBMs) in tubulointerstitial in-jury in diabetic nephropathy (20). The major TBM or GBM proteins include type IV collagen and laminin, and the AGEs are known to modulate the turnover and expression of these ECM proteins and of proteases in glomerular cells, resulting in nodular sclerosis-a hallmark characteristic of diabetic nephropathy (5). In any instance, the glycated ligand of interest should have comparable binding affinity with RAGE as that of ECM proteins and S-100B.…”

“…In fact, numerous past studies have used AGE-BSA to investigate the pathobiology of proximal tubules or tubulointerstitial compartment (17,31,36,44,57). The AGE-BSA also has been used to investigate mesangial cell biology, and in addition, its utility to study AGE-induced dysfunctions of endothelial and retinal pigment epithelial cells in vivo has been well described (5,11,50). The effect of AGE:RAGE interaction on cellular functionality was gauged by adherence assays, where it was noted that AGE-BSA increases adhesiveness of cells in a time-dependent manner with no effect on cell viability (Fig.…”

“…Conceivably, as indicated above, the AGE-induced ROS modulate a multitude of signaling pathways, e.g., ERK-1/ ERK-2 and PI3-kinase with the generation of proinflammatory cytokines, while evoking a chronic subclinical microinflammatory response (5,8,20,62). In addition, there is an upregulation of profibrogenic cytokines such as TGF-␤ 1 , with activation of their putative receptors and consequential increased synthesis of ECM, leading eventually to renal fibrosis, i.e., glomerulosclerosis and tubulointerstitial fibrosis (9,21,44).…”

“…A majority of the studies that focus on renal fibrosis relate to the biology of intrinsic glomerular cells, including mesangial cells and podocytes, that are affected by AGEs, although the information concerning tubular cells is somewhat limited (44). Exposure of mesangial cells to AGEs has been shown to increase in the synthesis of ECM proteins, such as fibronectin, laminin, and collagen (5,8,21,43,62). A similar increase in the synthesis of ECM proteins has been observed in glomerular podocytes, which are believed to undergo epithelial-mesenchymal transformation triggered by AGEs (26,27).…”

Mammalian Diaphanous1 signalling in neurovascular complications of diabetes

“…In the study to determine what is the transcriptomic response of mesangial cells to AGE during DN, microarray gene expression analysis revealed that in vitro propagation of human glomerular mesangial cells in a culture medium containing a high concentration of AGE leads to activation of expression of several genes involved in modulation of extracellular matrix turnover, including those encoding tPA, TIMP‐3 and PAI‐1. According to the authors, such changes in gene expression could trigger the inhibition of extracellular matrix remodelling and induction of glomerulosclerosis in kidney cells (Berrou et al, 2009). A role of RAGE/AGE signalling in acute kidney injury was explored in the study of Gerhardt and colleagues (2021), in which by combining single‐nucleus RNA‐seq with tracing of genetically labelled injured proximal tubule cells (PTCs), they revealed notable transcriptional activation of NF‐κB–, TNF‐ and activating protein‐1 (AP‐1)–signalling pathways in the PTCs during transformation after injury.…”