Diabetic kidney disease (DKD) is caused by the overproduction of extracellular matrix proteins (ECM) by glomerular mesangial cells (MCs). We previously showed that high glucose (HG) induces cell surface translocation of GRP78 (csGRP78), mediating PI3K/Akt activation and downstream ECM production. Activated alpha 2-macroglobulin (α2M*) is a ligand known to initiate this signaling cascade. Importantly, increased α2M was observed in diabetic patients’ serum, saliva, and glomeruli. Primary MCs were used to assess HG responses. The role of α2M* was assessed using siRNA, a neutralizing antibody and inhibitory peptide. Kidneys from type 1 diabetic Akita and CD1 mice and human DKD patients were stained for α2M/α2M*. α2M transcript and protein were significantly increased with HG in vitro and in vivo in diabetic kidneys. A similar increase in α2M* was seen in media and kidneys, where it localized to the mesangium. No appreciable α2M* was seen in normal kidneys. Knockdown or neutralization of α2M/α2M* inhibited HG-induced profibrotic signaling (Akt activation) and matrix/cytokine upregulation (collagen IV, fibronectin, CTGF, and TGFβ1). In patients with established DKD, urinary α2M* and TGFβ1 levels were correlated. These data reveal an important role for α2M* in the pathogenesis of DKD and support further investigation as a potential novel therapeutic target.

Diabetic kidney disease (DKD) is the leading cause of kidney failure worldwide. Characterized by overproduction and accumulation of extracellular matrix (ECM) proteins, glomerular sclerosis is its earliest manifestation. High glucose (HG) plays a central role by increasing matrix production by glomerular mesangial cells (MC). We previously showed that HG induces translocation of GRP78 from the endoplasmic reticulum to the cell surface (csGRP78), where it acts as a signaling molecule to promote intracellular profibrotic FAK/Akt activation. Here, we identify integrin β1 as a key transmembrane signaling partner for csGRP78. We show that it is required for csGRP78-regulated FAK/Akt activation in response to HG, as well as downstream production, secretion and activity of the well characterized profibrotic cytokine transforming growth factor β1 (TGFβ1). Intriguingly, integrin β1 also itself promotes csGRP78 translocation. Furthermore, integrin β1 effects on cytoskeletal organization are not required for its function in csGRP78 translocation and signaling. These data together support an important pathologic role for csGRP78/integrin β1 in mediating key profibrotic responses to HG in kidney cells. Inhibition of their interaction will be further evaluated as a therapeutic target to limit fibrosis progression in DKD.

Diabetic kidney disease (DKD) is the leading cause of kidney failure. RhoA/Rho-associated protein kinase (ROCK) signaling is a recognized mediator of its pathogenesis, largely through mediating the profibrotic response. While RhoA activation is not feasible due to the central role it plays in normal physiology, ROCK inhibition has been found to be effective in attenuating DKD in preclinical models. However, this has not been evaluated in clinical studies as of yet. Alternate means of inhibiting RhoA/ROCK signaling involve the identification of disease-specific activators. This report presents evidence showing the activation of RhoA/ROCK signaling both in vitro in glomerular mesangial cells and in vivo in diabetic kidneys by two recently described novel pathogenic mediators of fibrosis in DKD, activins and cell-surface GRP78. Neither are present in normal kidneys. Activin inhibition with follistatin and neutralization of cell-surface GRP78 using a specific antibody blocked RhoA activation in mesangial cells and in diabetic kidneys. These data identify two novel RhoA/ROCK activators in diabetic kidneys that can be evaluated for their efficacy in inhibiting the progression of DKD.

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 β1 (TGFβ1) is recognized as a central mediator of HG-induced profibrotic responses, but whether its activation is regulated by cell surface GRP78 (csGRP78) is unknown. TGFβ1 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 TGFβ1 activation. Here we determined whether csGRP78 regulates TSP1 expression and thereby TGFβ1 activation by HG.Methods: Primary mouse MC were used. TSP1 and TGFβ1 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 α2-macroglobulin (α2M*), with the neutralizing antibody Fα2M 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 TGFβ1 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 TGFβ1 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.