C-peptide (CP) has demonstrated unique beneficial effects in diabetic nephropathy (DN), but whether and how CP regulates NF-κB and its coactivator, p300, to suppress inducible iNOS and antagonize DN are unknown. iNOS expression, NF-κB nuclear translocation, colocalization and binding of NF-κB to p300, binding of NF-κB to the inos promoter, and the bound NF-κB, p300, and histone 3 lysine 9 acetylation (H3K9ac) at binding sites were measured in high glucose-stimulated mesangial cells. We evaluated pathologic changes, iNOS expression, NF-κB, and p300 contents in diabetic rats. We found that CP inhibited iNOS expression and notably prevented colocalization and binding of NF-κB and p300. CP prevented NF-κB from binding to the inos promoter, especially at the distal site, and reduced bound NF-κB, p300, and H3K9ac. N-terminal plus middle fragment could mostly mimic the antagonizing effects of CP against the pathologic changes of DN and equally suppresses renal iNOS expression as CP. In conclusion, CP prevented NF-κB from recruiting p300 and binding to the inos promoter, and decreased H3K9ac at the binding sites to suppress iNOS expression and antagonize DN, with the effect region identified as N-terminal plus middle fragment.-Li, Y., Li, X., He, K., Li, B., Liu, K., Qi, J., Wang, H., Wang, Y., Luo, W. C-peptide prevents NF-κB from recruiting p300 and binding to the inos promoter in diabetic nephropathy.

Abstract. Insufficient matrix metalloproteinase (MMP)-9 and MMP-2 is considered to be a contributor of extracellular matrix (ECM) accumulation in diabetic nephropathy (DN). C-peptide can reverse fibrosis, thus exerting a beneficial effect on DN. Whether C-peptide induces MMP-9 and MMP-2 to reverse ECM accumulation is not clear. In the present study, in order to determine ECM metabolism, rat mesangial cells were treated with high glucose (HG) and C-peptide intervention, then the early and late effects of C-peptide on HG-affected MMP-9 and MMP-2 were evaluated. Firstly, it was confirmed that HG mainly suppressed MMP-9 expression levels. Furthermore, C-peptide treatment induced MMP-9 expression at 6 h and suppressed it at 24 h, revealing the early dual effects of C-peptide on MMP-9 expression. Subsequently, significant increase in MMP-9 expression at 72, 96 and 120 h C-peptide treatment was observed. These changes in MMP-9 protein content confirmed its expression changes following late C-peptide treatment. Furthermore, at 96 and 120 h C-peptide treatment reversed the HG-inhibited MMP-9 secretion, further indicating the late induction effect of C-peptide on MMP-9. The present results demonstrated that C-peptide exerted a late induction effect on MMP-9 in HG-stimulated rat mesangial cells, which may be associated with the underlying mechanism of C-peptide's reversal effects on DN. IntroductionDiabetic nephropathy (DN), the leading cause of end stage renal disease, is the major cause of mortality in type 1 diabetes mellitus (DM) and the second most severe complications in type 2 diabetes (1,2). Deposition of extracellular matrix (ECM) in mesangial areas is a feature of DN, and mesangial cells have been proposed to be the determinant of ECM accumulation (3,4). However, the mechanism underlying ECM accumulation in DN is not fully clarified.Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases that can degrade numerous types of ECM components (5,6). Among others, MMP-2 basally expresses while MMP-9 is an inducible enzyme, both of which primarily degrade types-I and -IV collagen and laminin, major components of ECM (7-9). Generally, MMP-2 and MMP-9 are involved in tumor metastasis (6,10). Furthermore, it has been shown that insufficient MMP-2 and MMP-9 may be a contributor of ECM accumulation in DN (11). However, the expression levels of MMP-2 and MMP-9 in DN remain controversial, and even short-and long-term hyperglycemia may exert differential effects (8,(12)(13)(14). As an inducible enzyme, MMP-9 may be more easily affected in patients with DN (15). Therefore, the changes of MMP-2, and particularly MMP-9, for high glucose (HG) stimulation require clarification.C-peptide is the linker between the A-chain and B-chain of insulin. Lack of C-peptide along with insulin is the primary feature of type 1 DM and late stage of type 2 DM (16). C-peptide has been found to have unique beneficial effects on DN, attenuating glomerular and tubular injury (17)(18)(19). Physiological concentration of C-peptide ca...