37 research outputs found
Explanting Is an Ex Vivo Model of Renal Epithelial-Mesenchymal Transition
Recognised by their
de novo expression of alpha-smooth muscle actin
(SMA), recruitment of myofibroblasts is key to
the pathogenesis of fibrosis in chronic kidney
disease. Increasingly, we realise that
epithelial-mesenchymal transition (EMT) may be an
important source of these cells. In this study
we describe a novel model of renal EMT. Rat
kidney explants were finely diced on
gelatin-coated Petri dishes and cultured in
serum-supplemented media. Morphology and
immunocytochemistry were used to identify
mesenchymal (vimentin+, α-smooth muscle
actin (SMA)+, desmin+), epithelial
(cytokeratin+), and endothelial (RECA+) cells at
various time points. Cell outgrowths were all
epithelial in origin (cytokeratin+) at day 3. By
day 10, 50 ± 12%
(mean ± SE) of cytokeratin+
cells double-labelled for SMA, indicating EMT.
Lectin staining established a proximal tubule
origin. By day 17, cultures consisted only of
myofibroblasts (SMA+/cytokeratin−). Explanting
is a reproducible ex vivo model
of EMT. The ability to modify this change in
phenotype provides a useful tool to study the
regulation and mechanisms of renal
tubulointerstitial fibrosis
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
The bone morphogenetic protein axis is a positive regulator of skeletal muscle mass
Although the canonical transforming growth factor β signaling pathway represses skeletal muscle growth and promotes muscle wasting, a role in muscle for the parallel bone morphogenetic protein (BMP) signaling pathway has not been defined. We report, for the first time, that the BMP pathway is a positive regulator of muscle mass. Increasing the expression of BMP7 or the activity of BMP receptors in muscles induced hypertrophy that was dependent on Smad1/5-mediated activation of mTOR signaling. In agreement, we observed that BMP signaling is augmented in models of muscle growth. Importantly, stimulation of BMP signaling is essential for conservation of muscle mass after disruption of the neuromuscular junction. Inhibiting the phosphorylation of Smad1/5 exacerbated denervation-induced muscle atrophy via an HDAC4-myogenin–dependent process, whereas increased BMP–Smad1/5 activity protected muscles from denervation-induced wasting. Our studies highlight a novel role for the BMP signaling pathway in promoting muscle growth and inhibiting muscle wasting, which may have significant implications for the development of therapeutics for neuromuscular disorders
Transduction of Skeletal Muscles with Common Reporter Genes Can Promote Muscle Fiber Degeneration and Inflammation
Recombinant adeno-associated viral vectors (rAAV vectors) are promising tools for delivering transgenes to skeletal muscle, in order to study the mechanisms that control the muscle phenotype, and to ameliorate diseases that perturb muscle homeostasis. Many studies have employed rAAV vectors carrying reporter genes encoding for β-galactosidase (β-gal), human placental alkaline phosphatase (hPLAP), and green fluorescent protein (GFP) as experimental controls when studying the effects of manipulating other genes. However, it is not clear to what extent these reporter genes can influence signaling and gene expression signatures in skeletal muscle, which may confound the interpretation of results obtained in experimentally manipulated muscles. Herein, we report a strong pro-inflammatory effect of expressing reporter genes in skeletal muscle. Specifically, we show that the administration of rAAV6:hPLAP vectors to the hind limb muscles of mice is associated with dose- and time-dependent macrophage recruitment, and skeletal muscle damage. Dose-dependent expression of hPLAP also led to marked activity of established pro-inflammatory IL-6/Stat3, TNFα, IKKβ and JNK signaling in lysates obtained from homogenized muscles. These effects were independent of promoter type, as expression cassettes featuring hPLAP under the control of constitutive CMV and muscle-specific CK6 promoters both drove cellular responses when matched for vector dose. Importantly, the administration of rAAV6:GFP vectors did not induce muscle damage or inflammation except at the highest doses we examined, and administration of a transgene-null vector (rAAV6:MCS) did not cause damage or inflammation at any of the doses tested, demonstrating that GFP-expressing, or transgene-null vectors may be more suitable as experimental controls. The studies highlight the importance of considering the potential effects of reporter genes when designing experiments that examine gene manipulation in vivo
Intramuscular administration of rAAV6:CMV-hPLAP vectors induces skeletal muscle inflammation and damage in a dose and time-dependent manner.
<p>(a) The TA muscles of mice were injected with either 1×10<sup>8</sup>, 1×10<sup>9</sup> or 1×10<sup>10</sup> genomes of the control vector, or rAA6:CMV-hPLAP and examined 14 days afterwards. TA muscles were dissected and stained with Hematoxylin & Eosin for general morphology, or with NBT/BCIP to determine the expression of human placental alkaline phosphatase (purple). Asterisks identify common features on the serial sections used for morphology and hPLAP activity. (b) A time-course analysis of muscles examined 7, 14, 21 and 28 days after injection of rAAV6 vectors indicates peak times of induction of inflammation in response to rAAV:CMV-hPLAP, as compared to a gene-less vector (rAAV6:CMV-MCS) or rAAV6:Follistatin288.</p
Substitution of rAAV6:CMV-hPLAP with a muscle-specific CK6 promoter does not ameliorate the effects of vector-mediated hPLAP expression on muscle damage and inflammation (a) Designs of expression cassettes packaged into rAAV6:CMV-hPLAP and rAAV6:CK6-hPLAP
<p>Damage and cellular infiltration was not evident in muscles examined 14 days after injection with rAAV6:CK6-hPLAP, but was notable by 28 days post-injection. (c) EMR, IL-6 and IL-1β expression were assessed at 14 and 28 days after administration of rAAV6:CMV-MCS, rAAV6:CMV-hPLAP and rAAV6:CK6-hPLAP vectors. *, p<0.05 vs. control (d) Protein was extracted from muscles and phosphorylation levels of inflammatory mediators Stat3, JNK and IKK-β were determined by Western blot analysis. *, p<0.05 vs. control (e) MyoD and miR-206 expression was examined in muscles collected 14 or 28 days after administration of rAAV6:CMV-MCS, rAAV6:CMV-hPLAP and rAAV6:CK6-hPLAP vectors. *, p<0.05 vs. control.</p
rAAV6 vector-mediated expression of GFP exerts a reduced inflammatory effect in skeletal muscle compared with expression of hPLAP.
<p>(a) rAAV6:CMV-GFP or “gene less” rAAV6:CMV-MCS vectors were injected into the TA muscles of mice at 1×10<sup>9</sup> or 1×10<sup>10</sup> genomes. Muscles examined 14 and 28 days after injection of 1×10<sup>9</sup> rAAV6:CMV-GFP vector genomes demonstrated strong transgene expression without evidence of cellular infiltration, or muscle breakdown. However inflammation was evident in muscles examined 28 days after receiving 1×10<sup>10</sup> vg of rAAV6:CMV-GFP. (b-c) Expression of EMR, ITGAX, IL-1β and IL-6 was not different in muscles examined 14 or 28 days after receiving 1×10<sup>9</sup> vg of rAAV6:CMV-GFP (compared with muscles receiving rAAV6:CMV-MCS) but was elevated in muscles examined 14 or 28 days after receiving 1×10<sup>10</sup> vg of rAAV6:CMV-GFP. *, p<0.05 vs. control.</p
Administration of rAAV6:CMV-hPLAP results in increased expression of pro-inflammatory macrophages markers, and pro-inflammatory signaling pathway activation.
<p>(a) TA muscles were injected with the indicated doses of rAAV6:CMV-hPLAP or rAAV6:CMV-MCS control. At 14 days post-injection, RNA was extracted from muscle tissue and EMR1 and ITGAX expression was analyzed. *, p<0.05 vs. control. (b) EMR expression, as well as other markers of inflammation, IL-1β, IL-6 and TNFα were analyzed over 14, 21 and 28 days after administration of 1×10<sup>9</sup> genomes of rAAV6:CMV-hPLAP. *, p<0.05 vs. control. (c) The upregulation of IL-6 expression also correlates with increased phosphorylation of Stat3. JNK and IKK-β phosphorylation were also assessed by Western blot *, p<0.05 vs. control. (d) MyoD and miR-206 gene expression, as well as MEF-2 protein levels, were analyzed from tissue harvested 14 or 28 days after vector administration. *, p<0.05 vs. control.</p
miR-206 Represses Hypertrophy of Myogenic Cells but Not Muscle Fibers via Inhibition of HDAC4
<div><p>microRNAs regulate the development of myogenic progenitors, and the formation of skeletal muscle fibers. However, the role miRNAs play in controlling the growth and adaptation of post-mitotic musculature is less clear. Here, we show that inhibition of the established pro-myogenic regulator miR-206 can promote hypertrophy and increased protein synthesis in post-mitotic cells of the myogenic lineage. We have previously demonstrated that histone deacetylase 4 (HDAC4) is a target of miR-206 in the regulation of myogenic differentiation. We confirmed that inhibition of miR-206 de-repressed HDAC4 accumulation in cultured myotubes. Importantly, inhibition of HDAC4 activity by valproic acid or sodium butyrate prevented hypertrophy of myogenic cells otherwise induced by inhibition of miR-206. To test the significance of miRNA-206 as a regulator of skeletal muscle mass <i>in vivo</i>, we designed recombinant adeno-associated viral vectors (rAAV6 vectors) expressing miR-206, or a miR-206 “sponge,” featuring repeats of a validated miR-206 target sequence. We observed that over-expression or inhibition of miR-206 in the muscles of mice decreased or increased endogenous HDAC4 levels respectively, but did not alter muscle mass or myofiber size. We subsequently manipulated miR-206 levels in muscles undergoing follistatin-induced hypertrophy or denervation-induced atrophy (models of muscle adaptation where endogenous miR-206 expression is altered). Vector-mediated manipulation of miR-206 activity in these models of cell growth and wasting did not alter gain or loss of muscle mass respectively. Our data demonstrate that although the miR-206/HDAC4 axis operates in skeletal muscle, the post-natal expression of miR-206 is not a key regulator of basal skeletal muscle mass or specific modes of muscle growth and wasting. These studies support a context-dependent role of miR-206 in regulating hypertrophy that may be dispensable for maintaining or modifying the adult skeletal muscle phenotype – an important consideration in relation to the development of therapeutics designed to manipulate microRNA activity in musculature.</p> </div