ABSTRACT
A nonclassical calpain, calpain 6 (CAPN6), can inhibit skeletal muscle differentiation and regeneration. In the present study, the role of CAPN6 in the regulation of the autophagy of myoblasts in vitro was investigated. The underlying molecular events and the CAPN6 level in atrophic skeletal muscle in a rat model of chronic kidney disease (CKD) were also investigated. In vitro, CAPN6 was overexpressed, or knocked down, in rat L6 myoblasts to assess autophagy and related gene expression and colocalization. Subsequently, myoblasts were treated with a mixture of cytokines, and relative gene expression and autophagy were assessed. A rat model of CKD for muscle atrophy was established, and blood chemical level and the expression of CAPN6 in muscle were assessed. The data revealed that the knockdown of CAPN6 in rat myoblasts resulted in increased microtubuleassociated protein 1 light chain 3 (LC3) levels, while its overexpression decreased LC3 levels and impaired autophagy. Additionally, it was observed that the colocalization of mammalian target of rapamycin (mTOR) and lysosomalassociated membrane protein 1 (LAMP1), a lysosomal marker, proteins was increased. In addition, mTOR, Raptor and αtubulin (a marker of microtubules) increased in the CAPN6 overexpression group. However, inflammatory cytokines, such as interleukin (IL)6, tumor necrosis factor (TNF)α, interferon (INF)γ and lipopolysaccharides upregulated CAPN6 expression, inhibited L6 myoblast autophagy and stabilized mTOR activity. Furthermore, the animal model successfully mimicked human disease as regards an increase in body weight, and a reduction in muscle mass, crosssectional area and blood biomarker concentrations; a slight increase in CAPN6 mRNA and protein levels in muscles was observed. Finally, the data of the present study suggested that CAPN6 reduced autophagy via the maintenance of mTOR signaling, which may play a role in CKDrelated muscle atrophy. However, future studies are required to determine whether CAPN6 may be used as an intervention target for CKDrelated skeletal muscle atrophy.
Subject(s)
Autophagy/physiology , Calpain/metabolism , Myoblasts/metabolism , Renal Insufficiency, Chronic/metabolism , Animals , Apoptosis/physiology , Cell Differentiation/physiology , Cytokines/metabolism , Disease Models, Animal , Humans , Inflammation/metabolism , Male , Microtubules/metabolism , Muscle Development/physiology , Muscle, Skeletal/metabolism , Muscular Atrophy/drug therapy , Muscular Atrophy/metabolism , Rats , Rats, Sprague-Dawley , Signal Transduction/physiologyABSTRACT
It has been reported that butyrate played an protect role in diabetic kidney disease (DKD) while the mechanism was still not clear. Transforming growth factor-ß1 (TGF-ß1) is the initial factor which triggers the profibrotic signaling cascades. P311 is an RNA-binding protein, which could stimulate TGF-ß1 translation in several cell types. In our study, we found that supplementary of butyrate alleviated fibrosis and suppressed the expression of TGF-ß1 and P311 in the kidney of db/db mice as well as high glucose (HG)-induced SV40-MES-13 cells. Overexpression of P311 offset the inhibition of butyrate on TGF-ß1 in SV40-MES-13 cells. To make clear the mechanism of butyrate in regulating P311, microRNAs (miRNAs) of the SV40-MES-13 cells were sequenced. We found that miR-7a-5p was significantly decreased in the HG-induced SV40-MES-13 cells and the kidney of db/db mice, while giving butyrate reversed this change. Besides, miR-7a-5p could specifically target the 3' UTR of P311's mRNA and suppressed the expression of P311 in the SV40-MES-13 cells. Giving miR-7a-5p inhibitor blocked the inhibition of butyrate on P311 and TGF-ß1. Introducing the miR-7a-5p agomir into db/db mice alleviated renal fibrosis and inhibit the expression of P311 and TGF-ß1. In conclusion, butyrate alleviated DKD by mediating the miR-7a-5p/P311/TGF-ß1 pathway.
