Key features of diabetic nephropathy (DN) include the accumulation of extracellular matrix proteins such as collagen 1-␣ 1 and -2 (Col1a1 and -2). Transforming growth factor ␤1 (TGF-␤), a key regulator of these extracellular matrix genes, is increased in mesangial cells (MC) in DN. By microarray profiling, we noted that TGF-␤ increased Col1a2 mRNA in mouse MC (MMC) but also decreased mRNA levels of an E-box repressor, ␦EF1. TGF-␤ treatment or short hairpin RNAs targeting ␦EF1 increased enhancer activity of upstream E-box elements in the Col1a2 gene. TGF-␤ also decreased the expression of Smad-interacting protein 1 (SIP1), another E-box repressor similar to ␦EF1. 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-␤ in MMC. TGF-␤ 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 ␦EF1 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-␤ and Col1a2 levels. These results uncover a role for miRs in the kidney and DN in controlling TGF-␤-induced Col1a2 expression by down-regulating E-box repressors.diabetic nephropathy ͉ mesangial cells ͉ small noncoding RNA ͉ transforming growth factor ␤1 D iabetic nephropathy (DN) is the most common cause of kidney failure in patients with diabetes mellitus. The major characteristics of DN include glomerular basement-membrane thickening, mesangial expansion and hypertrophy, and an accumulation of extracellular matrix (ECM) proteins (1). Evidence shows that transforming growth factor ␤1 (TGF-␤) levels are increased under diabetic conditions in renal cells, including mesangial cells (MC), can up-regulate ECM proteins such as collagens (2, 3), and also can promote MC survival and oxidant stress (4).To date, Smad transcription factors have been shown to be the major effectors of TGF-␤ signaling (5, 6). Collagen 1-␣ 1 and -2 (Col1a1 and -2) and other ECM genes are regulated in MC by TGF-␤ via Smads (7,8). The regulation of collagen by TGF-␤ in MC also is mediated by mitogen-activated protein kinases (MAPKs) such as p38 and ERKs (9-11). However, the molecular mechanisms by which TGF-␤ regulates ECM genes still are not understood fully. The collagen gene has E-box elements in the far upstream enhancer region (12, 13). An E-box repressor, ␦EF1, is a key inhibitor of E-cadherin (14) and E2-box transcription factors such as Nkx2.5 (12). Moreover, it is a known repressor of collagen type 1 and type 2 genes in other cells (12, 13), but its role in MC is ...

Akt kinase is activated by transforming growth factor-beta1 (TGF-β) in diabetic kidneys and plays important roles in fibrosis, hypertrophy and cell survival in glomerular mesangial cells (MC)1–11. However, the mechanisms of Akt activation by TGF-β are not fully understood. Here we show that TGF-β activates Akt in MC by inducing microRNA-216a (miR-216a) and miR-217, both of which target phosphatase and tensin homologue (PTEN). Both these miRs are located within the second intron of a non-coding RNA (RP23-298H6.1-001). The RP23 promoter was activated by TGF-β and also by miR-192 via E-box-regulated mechanisms as shown previously3. Akt activation by these miRs also led to MC survival and hypertrophy similar to TGF-β. These studies reveal a mechanism of Akt activation via PTEN downregulation by two miRs regulated by upstream miR-192 and TGF-β. Due to the diversity of PTEN function12, 13, this miR amplifying circuit may play key roles not only in kidney disorders, but also other diseases.

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 , 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-b, 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. Diabetic nephropathy (DN) is a major microvascular complication of diabetes and the leading cause of ESRD, which can manifest despite tight glycemic control and various therapeutic interventions. 1 There is thus an imperative need to identify additional biomarkers and novel targets for better management of DN, which is clinically manifested as microalbuminuria, proteinuria, and progressive glomerular dysfunction. The key pathologic features of DN include podocyte loss, mesangial cell (MC) hypertrophy, glomerular basement membrane thickening, and tubulointerstitial fibrosis due to the increased deposition of extracellular matrix (ECM) proteins such as collagens and fibronectin (FN). 2-4 TGF-b1 is increased in several renal cells in diabetes, including MCs, and mediates these profibrotic events, hypertrophy, and cell survival. 3,5,6 Therefore, TGF-b has been evaluated as a major target for DN treatment. However, this approach could have drawbacks due to the multifunctional role of TGF-b. Hence, further evaluation of the subtle molecular mechanisms by which TGF-b regulates fibrotic events in renal cells can lead to more effective translational approaches for DN treatment.MicroRNAs (miRNAs) are small noncoding RNAs that are increasingly recognized as critical players in gene regulation and various diseases. [7][8][9][10] Interestingly, a cluster of miRNAs is reported to be highly expressed in the kidney, and recent studies show that key miRNAs are upregulated in the kidneys of diabetic mice.

Diabetic nephropathy (DN), a severe microvascular complication frequently associated with both type 1 and type 2 diabetes mellitus, is a leading cause of renal failure. It can also lead to accelerated cardiovascular disease and macrovascular complications. Currently available therapies have not been fully efficacious in the treatment of DN, suggesting that further understanding of the molecular mechanisms underlying the pathogenesis of DN is necessary for the improved management of this disease. Although key signal transduction and gene regulation mechanisms have been identified, especially those related to the effects of hyperglycaemia, transforming growth factor-β1 and angiotensin II, progress in functional genomics, high-throughput sequencing technology, epigenetics and systems biology approaches have greatly expanded our knowledge base and uncovered new molecular mechanisms and factors involved in DN. These mechanisms include DNA methylation, chromatin histone modifications, novel transcripts and functional noncoding RNAs such as microRNAs and long noncoding RNAs. In this Review, we will discuss the significance of these emerging mechanisms, how they mediate the actions of growth factors to augment the expression of extracellular matrix and inflammatory genes associated with DN, and their potential usefulness as diagnostic biomarkers or novel therapeutic targets for DN.

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. Since 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.

The development and progression of diabetic kidney disease (DKD), a highly prevalent complication of diabetes mellitus, is influenced by both genetic and environmental factors. DKD is an important contributor to the morbidity of patients with diabetes mellitus, indicating a clear need for an improved understanding of disease aetiology to inform the development of more efficacious treatments. DKD is characterized by an accumulation of extracellular matrix, hypertrophy, and fibrosis in kidney glomerular and tubular cells. Increasing evidence shows that genes associated with these features of DKD are regulated not only by classical signalling pathways, but also by epigenetic mechanisms, involving chromatin histone modifications, DNA methylation, and non-coding RNAs. These mechanisms can respond to changes in the environment and importantly, might mediate the persistent long-term expression of DKD-related genes and phenotypes induced by prior glycaemic exposure, despite subsequent glycaemic control, a phenomenon called metabolic memory. Detection of epigenetic events during the early stages of DKD could be valuable for timely diagnosis and prompt treatment to prevent progression to end-stage renal disease. Identification of epigenetic signatures of DKD via epigenome-wide association studies might also inform precision medicine approaches. Here, we highlight the emerging role of epigenetics and epigenomics in DKD, and the translational potential of candidate epigenetic factors and non-coding RNAs as biomarkers and drug targets for DKD.

It is important to find better treatments for diabetic nephropathy (DN), a debilitating renal complication. Targeting early features of DN, including renal extracellular matrix accumulation (ECM) and glomerular hypertrophy, can prevent disease progression. Here we show that a megacluster of nearly 40 microRNAs and their host long non-coding RNA transcript (lnc-MGC) are coordinately increased in the glomeruli of mouse models of DN, and mesangial cells treated with transforming growth factor-β1 (TGF- β1) or high glucose. Lnc-MGC is regulated by an endoplasmic reticulum (ER) stress-related transcription factor, CHOP. Cluster microRNAs and lnc-MGC are decreased in diabetic Chop−/− mice that showed protection from DN. Target genes of megacluster microRNAs have functions related to protein synthesis and ER stress. A chemically modified oligonucleotide targeting lnc-MGC inhibits cluster microRNAs, glomerular ECM and hypertrophy in diabetic mice. Relevance to human DN is also demonstrated. These results demonstrate the translational implications of targeting lnc-MGC for controlling DN progression.

TGF-␤1-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-␤1-induced gene expression in rat mesangial cells under normal and high-glucose (HG) conditions. TGF-␤1 increased the expression of the ECM-associated genes connective tissue growth factor, collagen-␣1 [⌱], 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-␤1 also increased expression of the H3K4 methyltransferase SET7/9 and recruitment to these promoters. SET7/9 gene silencing with siRNAs significantly attenuated TGF-␤1-induced ECM gene expression. Furthermore, a TGF-␤1 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-␤1-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.