Cell surface GRP78 regulates TGFß1-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 ß1 (TGFß1) is recognized as a central mediator of HG-induced profibrotic responses, but whether its activation is regulated by cell surface GRP78 (csGRP78) is unknown. TGFß1 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 TGFß1 activation. Here we determined whether csGRP78 regulates TSP1 expression and thereby TGFß1 activation by HG. Methods: Primary mouse MC were used. TSP1 and TGFß1 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 TGFß1 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 TGFß1 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.

A therapeutic target for CKD: activin A facilitates TGFß1 profibrotic signaling.

BACKGROUND: TGFß1 is a major profibrotic mediator in chronic kidney disease (CKD). Its direct inhibition, however, is limited by adverse effects. Inhibition of activins, also members of the TGFß superfamily, blocks TGFß1 profibrotic effects, but the mechanism underlying this and the specific activin(s) involved are unknown. METHODS: Cells were treated with TGFß1 or activins A/B. Activins were inhibited generally with follistatin, or specifically with neutralizing antibodies or type I receptor downregulation. Cytokine levels, signaling and profibrotic responses were assessed with ELISA, immunofluorescence, immunoblotting and promoter luciferase reporters. Wild-type or TGFß1-overexpressing mice with unilateral ureteral obstruction (UUO) were treated with an activin A neutralizing antibody. RESULTS: In primary mesangial cells, TGFß1 induces secretion primarily of activin A, which enables longer-term profibrotic effects by enhancing Smad3 phosphorylation and transcriptional activity. This results from lack of cell refractoriness to activin A, unlike that for TGFß1, and promotion of TGFß type II receptor expression. Activin A also supports transcription through regulating non-canonical MRTF-A activation. TGFß1 additionally induces secretion of activin A, but not B, from tubular cells, and activin A neutralization prevents the TGFß1 profibrotic response in renal fibroblasts. Fibrosis induced by UUO is inhibited by activin A neutralization in wild-type mice. Worsened fibrosis in TGFß1-overexpressing mice is associated with increased renal activin A expression and is inhibited to wild-type levels with activin A neutralization. CONCLUSIONS: Activin A facilitates TGFß1 profibrotic effects through regulation of both canonical (Smad3) and non-canonical (MRTF-A) signaling, suggesting it may be a novel therapeutic target for preventing fibrosis in CKD.

Integrin ß1/Cell Surface GRP78 Complex Regulates TGFß1 and Its Profibrotic Effects in Response to High Glucose.

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 ß1 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 ß1 (TGFß1). Intriguingly, integrin ß1 also itself promotes csGRP78 translocation. Furthermore, integrin ß1 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 ß1 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.

Activin A and Cell-Surface GRP78 Are Novel Targetable RhoA Activators for Diabetic Kidney Disease.

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.