Cell surface GRP78 regulates TGFβ1-mediated profibrotic responses via TSP1 in diabetic kidney disease

Introduction: Diabetic kidney disease (DKD) is the leading cause of kidney failure in North America, characterized by glomerular accumulation of extracellular matrix (ECM) proteins. High glucose (HG) induction of glomerular mesangial cell (MC) profibrotic responses plays a central role in its pathogenesis. We previously showed that the endoplasmic reticulum resident GRP78 translocates to the cell surface in response to HG, where it mediates Akt activation and downstream profibrotic responses in MC. Transforming growth factor beta 1 (TGF beta 1) is recognized as a central mediator of HG-induced profibrotic responses, but whether its activation is regulated by cell surface GRP78 (csGRP78) is unknown. TGF beta 1 is stored in the ECM in a latent form, requiring release for biological activity. The matrix glycoprotein thrombospondin 1 (TSP1), known to be increased in DKD and by HG in MC, is an important factor in TGF beta 1 activation. Here we determined whether csGRP78 regulates TSP1 expression and thereby TGF beta 1 activation by HG.Methods: Primary mouse MC were used. TSP1 and TGF beta 1 were assessed using standard molecular biology techniques. Inhibitors of csGRP78 were: 1) vaspin, 2) the C-terminal targeting antibody C38, 3) siRNA downregulation of its transport co-chaperone MTJ-1 to prevent GRP78 translocation to the cell surface, and 4) prevention of csGRP78 activation by its ligand, active alpha 2-macroglobulin (alpha 2M*), with the neutralizing antibody F alpha 2M or an inhibitory peptide.Results: TSP1 transcript and promoter activity were increased by HG, as were cellular and ECM TSP1, and these required PI3K/Akt activity. Inhibition of csGRP78 prevented HG-induced TSP1 upregulation and deposition into the ECM. The HG-induced increase in active TGF beta 1 in the medium was also inhibited, which was associated with reduced intracellular Smad3 activation and signaling. Overexpression of csGRP78 increased TSP-1, and this was further augmented in HG.Discussion: These data support an important role for csGRP78 in regulating HG-induced TSP1 transcriptional induction via PI3K/Akt signaling. Functionally, this enables TGF beta 1 activation in response to HG, with consequent increase in ECM proteins. Means of inhibiting csGRP78 signaling represent a novel approach to preventing fibrosis in DKD.

Automated summary

Diabetic kidney disease (DKD) is characterized by glomerular accumulation of extracellular matrix (ECM) proteins, and high glucose (HG) induction of glomerular mesangial cell (MC) profibrotic responses plays a central role in its pathogenesis. The endoplasmic reticulum resident GRP78 translocates to the cell surface in response to HG and mediates Akt activation and downstream profibrotic responses in MC. This study investigates whether cell surface GRP78 (csGRP78) regulates thrombospondin 1 (TSP1) expression and TGF beta 1 activation in response to HG.