Chao Lu

We recently showed that cell surface translocation of the endoplasmic reticulum–resident protein GRP78, when bound by activated α 2-macroglobulin (α2M*), induces pro-fibrotic responses in glomerular mesangial cells in response to high glucose and regulates activation of the pro-fibrotic cytokine transforming growth factor-β1 (TGF-β1), implicating a pathogenic role in glomerulosclerosis. Interstitial fibrosis, largely mediated by proximal tubular epithelial cells (PTEC) and renal fibroblasts, develops later in kidney disease and correlates with functional decline. Here we investigated whether interstitial fibrosis was mediated by cell surface GRP78 (csGRP78)/α2M*. High glucose and TGF-β1 increased csGRP78 and α2M* in PTEC and renal fibroblasts, and their inhibition prevented fibrotic protein production. Interestingly, for TGF-β1, this depended on inhibition of noncanonical signaling through YAP/TAZ, with Smad3 activation unaffected. In vivo, type 1 diabetic Akita mice overexpressing TGF-β1 were treated with either a neutralizing antibody for csGRP78 (C38) or α2M* (Fα2M) or an inhibitory peptide blocking csGRP78/α2M* interaction, and mice with unilateral ureteral obstruction were treated with Fα2M or inhibitory peptide. Consistently, inhibition by antibody or peptide attenuated fibrosis and pro-fibrotic signaling. These findings show an important role for csGRP78/α2M* in mediating tubulointerstitial fibrosis in both diabetic and nondiabetic kidney disease and support their inhibition as a potential antifibrotic therapeutic intervention.

Up to 40% of patients with diabetes mellitus will develop diabetic kidney disease (DKD), characterized pathologically by the accumulation of extracellular matrix proteins, which leads to the loss of kidney function over time. Our previous studies showed that the pan-protease inhibitor alpha 2-macroglobulin (A2M) is increased in DKD and is a critical regulator of the fibrotic response in glomerular mesangial cells (MC), an initial site of injury during DKD development. How A2M is regulated by high glucose (HG) has not yet been elucidated and is the focus of this investigation. Using serial deletions of the full A2M promoter, we identified the −405 bp region as HG-responsive in MC. Site-directed mutagenesis, siRNA, and ChIP studies showed that the transcription factor, nuclear factor of activated T cells 5 (NFAT5), regulated A2M promoter activity and protein expression in response to HG. Forkhead box P1 (FOXP1) served as a cooperative binding partner for NFAT5, required for A2M upregulation. Lastly, we showed that Smad3, known for its role in kidney fibrosis, regulated A2M promoter activity and protein production independently of HG. The importance of NFAT5, FOXP1, and Smad3 in A2M regulation was confirmed in ex vivo studies using isolated glomeruli. In conclusion, Smad3 is required for basal and HG-induced A2M expression, while NFAT5 and FOXP1 cooperatively regulate increased A2M transcription in response to HG. Inhibition of NFAT5/FOXP1 will be further evaluated as a potential therapeutic strategy to inhibit A2M production and attenuate profibrotic signaling in DKD.