Phillip Kantharidis

miR-200a Prevents Renal Fibrogenesis Through Repression of TGF-β2 Expression

OBJECTIVE Progressive fibrosis in the diabetic kidney is driven and sustained by a diverse range of profibrotic factors. This study examines the critical role of microRNAs (miRNAs) in the regulation of the key fibrotic mediators, TGF-β1 and TGF-β2. RESEARCH DESIGN AND METHODS Rat proximal-tubular epithelial cells (NRK52E) were treated with TGF-β1 and TGF-β2 for 3 days, and expression of markers of epithelial-to-mesenchymal transition (EMT) and fibrogenesis were assessed by RT-PCR and Western blotting. The expression of miR-141 and miR-200a was also assessed, as was their role as translational repressors of TGF-β signaling. Finally, these pathways were explored in two different mouse models, representing early and advanced diabetic nephropathy. RESULTS Both TGF-β1 and TGF-β2 induced EMT and fibrogenesis in NRK52E cells. TGF-β1 and TGF-β2 also downregulated expression of miR-200a. The importance of these changes was demonstrated by the finding that ectopic expression miR-200a downregulated smad-3 activity and the expression of matrix proteins and prevented TGF-β–dependent EMT. miR-200a also downregulated the expression of TGF-β2, via direct interaction with the 3′ untranslated region of TGF-β2. The renal expression of miR-141 and miR-200a was also reduced in mouse models representing early and advanced kidney disease. CONCLUSIONS miR-200a and miR-141 significantly impact on the development and progression of TGF-β–dependent EMT and fibrosis in vitro and in vivo. These miRNAs appear to be intricately involved in fibrogenesis, both as downstream mediators of TGF-β signaling and as components of feedback regulation, and as such represent important new targets for the prevention of progressive kidney disease in the context of diabetes.

Connective Tissue Growth Factor Plays an Important Role in Advanced Glycation End Product–Induced Tubular Epithelial-to-Mesenchymal Transition: Implications for Diabetic Renal Disease

Epithelial-to-mesenchymal transition (EMT) of tubular cells contributes to the renal accumulation of matrix protein that is associated with diabetic nephropathy. Both TGF-beta1 and advanced glycation end products (AGE) are able to induce EMT in cell culture. This study examined the role of the prosclerotic growth factor connective tissue growth factor (CTGF) as a downstream mediator of these processes. EMT was assessed by the expression of alpha-smooth muscle actin, vimentin, E-cadherin, and matrix proteins and the induction of a myofibroblastic phenotype. CTGF, delivered in an adenovirus or as recombinant human CTGF (250 ng/ml), was shown to induce a partial EMT. This was not blocked by neutralizing anti-TGF-beta1 antibodies, suggesting that this action was TGF-beta1 independent. NRK-52E cells that were exposed to AGE-modified BSA (AGE-BSA; 40 microM) or TGF-beta1 (10 ng/ml) also underwent EMT. This was associated with the induction of CTGF gene and protein expression. Transfection with siRNA to CTGF was able to attenuate EMT-associated phenotypic changes after treatment with AGE or TGF-beta1. These in vitro effects correlate with the in vivo finding of increased CTGF expression in the diabetic kidney, which co-localizes on the tubular epithelium with sites of EMT. In addition, inhibition of AGE accumulation was able to reduce CTGF expression and attenuate renal fibrosis in experimental diabetes. These findings suggest that CTGF represents an important independent mediator of tubular EMT, downstream of the actions of AGE or TGF-beta1. This interaction is likely to play an important role in progressive diabetic nephropathy and strengthens the rationale to consider CTGF as a potential target for the treatment of diabetic nephropathy.

TGF-β regulates miR-206 and miR-29 to control myogenic differentiation through regulation of HDAC4

MicroRNAs (miRs) are emerging as prominent players in the regulation of many biological processes, including myogenic commitment and skeletal muscle formation. Members of the TGF-β family can influence the proliferation and myogenic differentiation of cells, although it is presently not clear what role miRNAs play in the TGF-β-mediated control of myogenic differentiation. Here, we demonstrate in the myogenic C2C12 cell line, and in primary muscle cells, that miR-206 and miR-29-two miRs that act on transcriptional events implicated in muscle differentiation are down-regulated by TGF-β. We further demonstrate that TGF-β treatment of myogenic cells is associated with increased expression of histone deacetylase 4 (HDAC4), a key inhibitor of muscle differentiation that has been identified as a target for regulation by miR-206 and miR-29. We confirmed that increased expression of miR-206 and miR-29 resulted in the translational repression of HDAC4 in the presence or absence of TGF-β via interaction with the HDAC4 3′-untranslated region. Importantly, we found that miR-206 and miR-29 can attenuate the inhibitory actions of TGF-β on myogenic differentiation. Furthermore, we present evidence that the mechanism by which miR-206 and miR-29 can inhibit the TGF-β-mediated up-regulation of HDAC4 is via the inhibition of Smad3 expression, a transducer of TGF-β signaling. These findings identify a novel mechanism of interaction between TGF-β and miR-206 and -29 in the regulation of myogenic differentiation through HDAC4.

E-Cadherin Expression Is Regulated by miR-192/215 by a Mechanism That Is Independent of the Profibrotic Effects of Transforming Growth Factor-β

OBJECTIVE Increased deposition of extracellular matrix (ECM) within the kidney is driven by profibrotic mediators including transforming growth factor-β (TGF-β) and connective tissue growth factor (CTGF). We investigated whether some of their effects may be mediated through changes in expression of certain microRNAs (miRNAs). RESEARCH DESIGN AND METHODS Proximal tubular cells, primary rat mesangial cells, and human podocytes were analyzed for changes in the expression of key genes, ECM proteins, and miRNA after exposure to TGF-β (1–10 ng/μl). Tubular cells were also infected with CTGF-adenovirus. Kidneys from diabetic apoE mice were also analyzed for changes in gene expression and miRNA levels. RESULTS TGF-β treatment was associated with morphologic and phenotypic changes typical of epithelial-mesenchymal transition (EMT) including increased fibrogenesis in all renal cell types and decreased E-cadherin expression in tubular cells. TGF-β treatment also modulated the expression of certain miRNAs, including decreased expression of miR-192/215 in tubular cells, mesangial cells, which are also decreased in diabetic kidney. Ectopic expression of miR-192/215 increased E-cadherin levels via repressed translation of ZEB2 mRNA, in the presence and absence of TGF-β, as demonstrated by a ZEB2 3′-untranslated region luciferase reporter assay. However, ectopic expression of miR-192/215 did not affect the expression of matrix proteins or their induction by TGF-β. In contrast, CTGF increased miR-192/215 levels, causing a decrease in ZEB2, and consequently increased E-cadherin mRNA. CONCLUSIONS These data demonstrate the linking role of miRNA-192/215 and ZEB2 in TGF-β/CTGF–mediated changes in E-cadherin expression. These changes appear to occur independently of augmentation of matrix protein synthesis, suggesting that a multistep EMT program is not necessary for fibrogenesis to occur.

Diabetes Complications: The MicroRNA Perspective

There remains a critical need to better understand the underlying disease mechanisms responsible for diabetes complications in order to develop new and improved therapeutic strategies for these chronic conditions. These complications are broadly classified as microvascular, including neuropathy, nephropathy, and retinopathy, or macrovascular, including cardiovascular and peripheral vascular disease. The risk for developing complications is influenced by many factors including duration of diabetes and genetic factors. Current treatments have resulted in only a partial reduction in this risk, and the management of these conditions remains a major unmet need for those with diabetes. New insights have come from an unlikely ally, the worm C. elegans , in which research has identified a novel family of endogenous, small (∼22 nucleotides), single stranded, noncoding RNA molecules known as microRNAs (miRNAs) as developmental regulators (1,2). These molecules, only identified in humans in the last decade, modulate physiological and pathological processes by the posttranscriptional inhibition of gene expression (3). Many excellent reviews have been written dealing with biogenesis (4) through to their role in development and disease (5), although this work has generally focused on cancer and infection rather than diabetes and its complications (6). The effect of miRNAs is via the incomplete binding of the “seed sequence” at the 5′ end of the miRNA to the complementary target site in the 3′ untranslated region (UTR) of the messenger RNA. Each miRNA species can inhibit the expression of many genes, while each mRNA can be potentially targeted by several miRNAs. Recent studies have demonstrated clear links between altered miRNA expression and certain diabetes complications, as described later in this article. These findings further highlight the complex nature of diabetes and the huge challenge in better understanding the various vascular complications associated with this disorder. Here we discuss some recent studies demonstrating …

Precipitous Release of Methyl-CpG Binding Protein 2 and Histone Deacetylase 1 from the Methylated Human Multidrug Resistance Gene (MDR1) on Activation

ABSTRACT Overexpression of the human multidrug resistance gene 1 (MDR1) is a negative prognostic factor in leukemia. Despite intense efforts to characterize the gene at the molecular level, little is known about the genetic events that switch on gene expression in P-glycoprotein-negative cells. Recent studies have shown that the transcriptional competence of MDR1 is often closely associated with DNA methylation. Chromatin remodeling and modification targeted by the recognition of methylated DNA provide a dominant mechanism for transcriptional repression. Consistent with this epigenetic model, interference with DNA methyltransferase and histone deacetylase activity alone or in combination can reactivate silent genes. In the present study, we used chromatin immunoprecipitation to monitor the molecular events involved in the activation and repression of MDR1. Inhibitors of DNA methyltransferase (5-azacytidine [5aC]) and histone deacetylase (trichostatin A [TSA]) were used to examine gene transcription, promoter methylation status, and the chromatin determinants associated with the MDR1 promoter. We have established that methyl-CpG binding protein 2 (MeCP2) is involved in methylation-dependent silencing of human MDR1 in cells that lack the known transcriptional repressors MBD2 and MBD3. In the repressed state the MDR1 promoter is methylated and assembled into chromatin enriched with MeCP2 and deacetylated histone. TSA induced significant acetylation of histones H3 and H4 but did not activate transcription. 5aC induced DNA demethylation, leading to the release of MeCP2, promoter acetylation, and partial relief of repression. MDR1 expression was significantly increased following combined 5aC and TSA treatments. Inhibition of histone deacetylase is not an overriding mechanism in the reactivation of methylated MDR1. Our results provide us with a clearer understanding of the molecular mechanism necessary for repression of MDR1.

The role of EMT in renal fibrosis.

It is clear that the well-described phenomenon of epithelial–mesenchymal transition (EMT) plays a pivotal role in embryonic development, wound healing, tissue regeneration, organ fibrosis and cancer progression. EMTs have been classified into three subtypes based on the functional consequences and biomarker context in which they are encountered. This review will highlight findings on type II EMT as a direct contributor to the kidney myofibroblast population in the development of renal fibrosis, specifically in diabetic nephropathy, the signalling molecules and the pathways involved in type II EMT and changes in the expression of specific miRNA with the EMT process. These findings have provided new insights into the activation and development of EMT during disease processes and may lead to possible therapeutic interventions to suppress EMTs and potentially reverse organ fibrosis.

The Role of Tubular Epithelial-Mesenchymal Transition in Progressive Kidney Disease

The accumulation of interstitial matrix represents the final common pathway of most forms of kidney disease. Much of this matrix is synthesized by interstitial myofibroblasts, recruited from resident fibroblasts and circulating precursors. In addition, a significant proportion is derived from epithelial-mesenchymal transition (EMT) of tubuloepithelial cells. The importance of EMT has been demonstrated in experimental models, where blockade of EMT attenuates renal fibrosis. Although a number of factors may initiate EMT in the kidney, the most potent is transforming growth factor-β1 (TGF-β1). Moreover, many other prosclerotic factors have effects on EMT indirectly, via induction of TGF-β1. Signaling events in this pathway include activation of Smad/integrin-linked kinase (ILK) and connective tissue growth factor (CTGF). Basement membrane integrity is also a key regulator of EMT. In particular, overexpression of matrix metalloproteinase-2 has a key role in the initiation of EMT, membrane dissolution, and the interstitial transit of transformed mesenchymal cells. Endogenous inhibitors of EMT also play an important counterregulatory role both to prevent EMT and stimulate uncommitted cells to regain their tubular phenotype (mesenchymal-epithelial transition). Such inhibitors represent a potential therapeutic approach, offering a mechanism to slow or even redress established renal fibrosis.

Dedifferentiation of Immortalized Human Podocytes in Response to Transforming Growth Factor-β: A Model for Diabetic Podocytopathy

OBJECTIVE Diabetic nephropathy is associated with dedifferentiation of podocytes, losing the specialized features required for efficient glomerular function and acquiring a number of profibrotic, proinflammatory, and proliferative features. These result from tight junction and cytoskeletal rearrangement, augmented proliferation, and apoptosis. RESEARCH DESIGN AND METHODS Experiments were performed in conditionally immortalized human podocytes developed by transfection with the temperature-sensitive SV40-T gene. Cells were then cultured in the presence of transforming growth factor (TGF)-β1 or angiotensin II in the presence or absence of a selective inhibitor of the TGF-β type I receptor kinase, SB-431542. Gene and protein expression were then examined by real-time RT-PCR and immunofluorescence, and correlated with changes observed in vivo in experimental diabetes. RESULTS Treatment of cells with TGF-β1 resulted in dynamic changes in their morphology, starting with retraction and shortening of foot processes and finishing with the formation of broad and complex tight junctions between adjacent podocytes. This dedifferentiation was also associated with dose- and time-dependent reduction in the expression of glomerular epithelial markers (nephrin, p-cadherin, zonnula occludens-1) and increased expression of mesenchymal markers (α−smooth muscle actin, vimentin, nestin), matrix components (fibronectin, collagen I, and collagen IV α3), cellular proliferation, and apoptosis. The induction of diabetes in mice was also associated with similar changes in morphology, protein expression, and proliferation in glomerular podocytes. CONCLUSIONS In response to TGF-β and other TGF-dependent stimuli, mature podocytes undergo dedifferentiation that leads to effacement of foot processes, morphologic flattening, and increased formation of intercellular tight junctions. This simplification of their phenotype to a more embryonic form is also associated with reentry of mature podocytes into the cell cycle, which results in enhanced proliferation and apoptosis. These “pathoadaptive” changes are seen early in the diabetic glomerulus and ultimately contribute to albuminuria, glomerulosclerosis, and podocytopenia.

miR-21 promotes renal fibrosis in diabetic nephropathy by targeting PTEN and SMAD7.

The cytokine transforming growth factor (TGF)-β1 plays a central role in diabetic nephropathy (DN) with data implicating the miRNA (miR) miR-21 as a key modulator of its prosclerotic actions. In the present study, we demonstrate data indicating that miR-21 up-regulation positively correlates with the severity of fibrosis and rate of decline in renal function in human DN. Furthermore, concomitant analyses of various models of fibrotic renal disease and experimental DN, confirm tubular miR-21 up-regulation. The fibrotic changes associated with increased miR-21 levels are proposed to include the regulation of TGF-β1-mediated mothers against decapentaplegic homolog 3 (SMAD3)- and phosphoinositide 3-kinase (PI3K)-dependent signalling pathways via co-ordinated repression of mothers against decapentaplegic homolog 7 (SMAD7) and phosphatase and tensin homologue (PTEN) respectively. This represents a previously uncharacterized interaction axis between miR-21 and PTEN-SMAD7. Targeting of these proteins by miR-21 resulted in de-repression of the respective pathways as reflected by increases in SMAD3 and V-Akt murine thymoma viral oncogene homolog 1 (AKT) phosphorylation. Many of the changes typically induced by TGF-β1, including phosphorylation of signalling mediators, were further enhanced by miR-21. Collectively, these data present a unified model for a key role for miR-21 in the regulation of renal tubular extracellular matrix (ECM) synthesis and accumulation and provide important insights into the molecular pathways implicated in the progression of DN.