Scarring of the kidney is a major public health concern, directly promoting loss of kidney function. In order to understand the role of microRNA (miRNA) in the progression of kidney scarring in response to injury, we investigated changes in miRNA expression in two kidney fibrosis models, and identified 24 commonly upregulated miRNAs. Among them, miR-21 was highly elevated in both animal models and human transplant kidney nephropathy. Deletion of miR-21 in mice resulted in no overt abnormality. However, miR-21-/- mice suffered far less interstitial fibrosis in response to kidney injury, which was pheno-copied in wild-type mice treated with anti-miR-21 oligonucleotides. Surprisingly, global de-repression of miR-21 target messenger RNAs was only readily detectable in miR-21-/- kidneys after injury. Analysis of gene expression profiles identified groups of genes involved in metabolic pathways that were up-regulated in the absence of miR-21, including the lipid metabolism pathway regulated by Peroxisome proliferator activated receptor-α (Pparα), a direct miR-21 target. Over-expression of Pparα prevented UUO-induced injury and fibrosis. Pparα deficiency abrogated the anti-fibrotic effect of anti-miR21 oligonucleotides. miR-21 also regulates the redox metabolic pathway. The mitochondrial inhibitor of reactive oxygen species generation, Mpv17l, was repressed by miR-21, correlating closely with enhanced oxidative kidney damage. These studies demonstrate that miR-21 contributes to fibrogenesis and epithelial injury in the kidney in two mouse models and is a candidate target for anti-fibrotic therapies.

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Integrins, which connect the cytoskeleton to the extracellular matrix and mediate a variety of signaling cascades, may transduce mechanical stimuli into biochemical signals. We studied integrin- and matrix-dependent activation of extracellular signal-regulated kinase (ERK2), c-Jun NH2-terminal kinase (JNK1), and p38 in response to 4% static biaxial stretch in rat cardiac fibroblasts. ERK2 and JNK1, but not p38, were rapidly activated by stretch when the fibroblasts were allowed to synthesize their own matrices. When the cells were limited to specific matrix substrates, ERK2 and JNK1 were differentially activated: ERK2 was only activated when the cells were plated on fibronectin, while JNK1 was activated when the cells were plated on fibronectin, vitronectin, or laminin. Plating cells on collagen before stretching did not activate either kinase. Adhesion to all matrices was integrin-dependent because it could be blocked by inhibitors of specific integrins. ERK2 activation could be blocked with a combination of anti-alpha4 and -alpha5 antibodies and an arginine-glycine-aspartic acid (RGD) peptide, while the antibodies or peptide used separately failed to block ERK2 activation. This result suggests that at least two integrins, alpha4beta1 and an RGD-directed, non-alpha5beta1 integrin, activate ERK2 in response to mechanical stimulation. Activation of JNK1 could not be blocked with the inhibitors, suggesting that an RGD-independent integrin or integrins other than alpha4beta1 can activate JNK1 in cells adherent to fibronectin. This study demonstrates that integrins act as mechanotransducers, providing insight into potential mechanisms for in vivo responses to mechanical stimuli.

The cardiac fibroblast is the most abundant cell type present in the myocardium and is mainly responsible for the deposition of extracellular matrix (ECM). Important components of cardiac ECM include structural and adhesive proteins such as collagen and fibronectin. Excess deposition of cardiac ECM (fibrosis) has been associated with the pathophysiological mechanical overload of the heart. Therefore, the role of cardiac fibroblasts in "sensing", "integrating" and "responding" to mechanical stimuli is of great interest. The development of in vitro strain apparatuses has allowed scientists to investigate the effects of mechanical stimuli on cardiac fibroblast function. Cardiac fibroblasts express ECM receptors (integrins) which couple mechanical stimuli to functional responses. Mechanical stimulation of cardiac fibroblasts has been shown to result in activation of various signal transduction pathways. The application of defined mechanical stimuli to cultured cardiac fibroblasts has been associated with ECM gene expression, growth factor production, release and/or bioactivity as well as collagenase activity. Ultimately, for fibrosis to develop the overproduction of ECM must overcome any associated increases in collagenase activity. Mechanically induced upregulation of ECM production may follow direct or indirect pathways through the autocrine or paracrine action of growth factors. Given the complex nature of the interstitial milieu of the working heart, additional research is needed to further our understanding of the roles that mechanical stimuli play in excess deposition of myocardial ECM.

Adherent cells assemble fibronectin into a fibrillar matrix on their apical surface. The fibril formation is initiated by fibronectin binding to the integrins α5β1 and αvβ3, and is completed by a process that includes fibronectin self-assembly. We found that a 76– amino acid fragment of fibronectin (III1-C) that forms one of the self-assembly sites caused disassembly of preformed fibronectin matrix without affecting cell adhesion. Treating attached fibroblasts or endothelial cells with III1-C inhibited cell migration and proliferation. Rho-dependent stress fiber formation and Rho-dependent focal contact protein phosphorylation were also inhibited, whereas Cdc42 was activated, leading to actin polymerization into filopodia. ACK (activated Cdc42-binding kinase) and p38 MAPK (mitogen-activated protein kinase), two downstream effectors of Cdc42, were activated, whereas PAK (p21-activated kinase) and JNK/SAPK (c-Jun NH2-terminal kinase/ stress-activated protein kinase) were inhibited. III1-C treatment also modulated activation of JNK and ERK (extracellular signal-regulated kinases) in response to growth factors, and reduced the activity of the cyclin E–cdk2 complex. These results indicate that the absence of fibronectin matrix causes activation of Cdc42, and that fibronectin matrix is required for Rho activation and cell cycle progression.