Key features of diabetic nephropathy (DN) include the accumulation of extracellular matrix proteins such as collagen 1-α 1 and -2 (Col1a1 and -2). Transforming growth factor β1 (TGF-β), a key regulator of these extracellular matrix genes, is increased in mesangial cells (MC) in DN. By microarray profiling, we noted that TGF-β increased Col1a2 mRNA in mouse MC (MMC) but also decreased mRNA levels of an E-box repressor, δEF1. TGF-β treatment or short hairpin RNAs targeting δEF1 increased enhancer activity of upstream E-box elements in the Col1a2 gene. TGF-β also decreased the expression of Smad-interacting protein 1 (SIP1), another E-box repressor similar to δEF1. Interestingly, we noted that SIP1 is a target of microRNA-192 (miR-192), a key miR highly expressed in the kidney. miR-192 levels also were increased by TGF-β in MMC. TGF-β treatment or transfection with miR-192 decreased endogenous SIP1 expression as well as reporter activity of a SIP1 3′ UTR-containing luciferase construct in MMC. Conversely, a miR-192 inhibitor enhanced the luciferase activity, confirming SIP1 to be a miR-192 target. Furthermore, miR-192 synergized with δEF1 short hairpin RNAs to increase Col1a2 E-box-luc activity. Importantly, the in vivo relevance was noted by the observation that miR-192 levels were enhanced significantly in glomeruli isolated from streptozotocin-injected diabetic mice as well as diabetic db/db mice relative to corresponding nondiabetic controls, in parallel with increased TGF-β and Col1a2 levels. These results uncover a role for miRs in the kidney and DN in controlling TGF-β-induced Col1a2 expression by down-regulating E-box repressors.

How TGF-β activates Akt in disease situations is unclear. By acting on the promotor of RP23, a non-coding RNA which encodes two microRNAs, TGF-β signalling downregulates the phospatase PTEN, a target of these microRNAs and an inhibitor of Akt signalling. Akt kinase is activated by transforming growth factor-β1 (TGF-β) in diabetic kidneys, and has important roles in fibrosis, hypertrophy and cell survival in glomerular mesangial cells1,2,3,4,5,6,7,8,9,10,11. However, the mechanisms of Akt activation by TGF-β are not fully understood. Here we show that TGF-β activates Akt in glomerular mesangial cells by inducing the microRNAs (miRNAs) miR-216a and miR-217, both of which target PTEN (phosphatase and tensin homologue), an inhibitor of Akt activation. These miRNAs are located within the second intron of a non-coding RNA (RP23-298H6.1-001). The RP23 promoter was activated by TGF-β and miR-192 through E-box-regulated mechanisms, as shown previously3. Akt activation by these miRs led to glomerular mesangial cell survival and hypertrophy, which were similar to the effects of activation by TGF-β. These studies reveal a mechanism of Akt activation through PTEN downregulation by two miRs, which are regulated by upstream miR-192 and TGF-β. Due to the diversity of PTEN function12,13, this miR-amplifying circuit may have key roles, not only in kidney disorders, but also in other diseases.

TGF-β1 upregulates microRNA-192 (miR-192) in cultured glomerular mesangial cells and in glomeruli from diabetic mice. miR-192 not only increases collagen expression by targeting the E-box repressors Zeb1/2 but also modulates other renal miRNAs, suggesting that it may be a therapeutic target for diabetic nephropathy. We evaluated the efficacy of a locked nucleic acid (LNA)–modified inhibitor of miR-192, designated LNA–anti-miR-192, in mouse models of diabetic nephropathy. LNA–anti-miR-192 significantly reduced levels of miR-192, but not miR-194, in kidneys of both normal and streptozotocin-induced diabetic mice. In the kidneys of diabetic mice, inhibition of miR-192 significantly increased Zeb1/2 and decreased gene expression of collagen, TGF-β, and fibronectin; immunostaining confirmed the downregulation of these mediators of renal fibrosis. Furthermore, LNA–anti-miR-192 attenuated proteinuria in these diabetic mice. In summary, the specific reduction of renal miR-192 decreases renal fibrosis and improves proteinuria, lending support for the possibility of an anti-miRNA–based translational approach to the treatment of diabetic nephropathy.

TGF-β1–induced expression of extracellular matrix (ECM) genes plays a major role in the development of chronic renal diseases such as diabetic nephropathy. Although many key transcription factors are known, mechanisms involving the nuclear chromatin that modulate ECM gene expression remain unclear. Here, we examined the role of epigenetic chromatin marks such as histone H3 lysine methylation (H3Kme) in TGF-β1–induced gene expression in rat mesangial cells under normal and high-glucose (HG) conditions. TGF-β1 increased the expression of the ECM-associated genes connective tissue growth factor, collagen-α1[Ι], and plasminogen activator inhibitor-1. Increased levels of chromatin marks associated with active genes (H3K4me1, H3K4me2, and H3K4me3), and decreased levels of repressive marks (H3K9me2 and H3K9me3) at these gene promoters accompanied these changes in expression. TGF-β1 also increased expression of the H3K4 methyltransferase SET7/9 and recruitment to these promoters. SET7/9 gene silencing with siRNAs significantly attenuated TGF-β1–induced ECM gene expression. Furthermore, a TGF-β1 antibody not only blocked HG-induced ECM gene expression but also reversed HG-induced changes in promoter H3Kme levels and SET7/9 occupancy. Taken together, these results show the functional role of epigenetic chromatin histone H3Kme in TGF-β1–mediated ECM gene expression in mesangial cells under normal and HG conditions. Pharmacologic and other therapies that reverse these modifications could have potential renoprotective effects for diabetic nephropathy.

Enhanced transforming growth factor-β1 (TGF-β1) expression in renal cells promotes fibrosis and hypertrophy during the progression of diabetic nephropathy. The TGF-β1 promoter is positively controlled by the E-box regulators, upstream stimulatory factors (USFs), in response to diabetic (high glucose) conditions; however, it is not clear whether TGF-β1 is autoregulated by itself. As changes in microRNAs (miRNAs) have been implicated in kidney disease, we tested their involvement in this process. TGF-β1 levels were found to be upregulated by microRNA-192 (miR-192) or miR-200b/c in mouse mesangial cells. Amounts of miR-200b/c were increased in glomeruli from type 1 (streptozotocin) and type 2 (db/db) diabetic mice, and in mouse mesangial cells treated with TGF-β1 in vitro. Levels of miR-200b/c were also upregulated by miR-192 in the mesangial cells, suggesting that miR-200b/c are downstream of miR-192. Activity of the TGF-β1 promoter was upregulated by TGF-β1 or miR-192, demonstrating that the miR-192-miR-200 cascade induces TGF-β1 expression. TGF-β1 increased the occupancy of activators USF1 and Tfe3, and decreased that of the repressor Zeb1 on the TGF-β1 promoter E-box binding sites. Inhibitors of miR-192 decreased the expression of miR-200b/c, Col1a2, Col4a1, and TGF-β1 in mouse mesangial cells, and in mouse kidney cortex. Thus, miRNA-regulated circuits may amplify TGF-β1 signaling, accelerating chronic fibrotic diseases such as diabetic nephropathy.