The Effect of Endurance Training on Fibronectin Gene Expression of the Sciatic Nerve in Diabetic Rats

- Diabetic neuropathy can cause disorders in axon transmission, changes in the extracellular matrix, and peripheral nerve damages. However, its mechanism, along with the beneficial effects of exercise on these disorders is not entirely clear. The aim of the current study was to assess changes in fibronectin mRNA gene expression level of the sciatic nerve in rats with streptozotocin-induced diabetes after endurance training. Eighteen male Wistar rats (10 weeks old with 250±20 gr weight) were randomly assigned to three groups, including healthy, induced diabetes and induced diabetes plus endurance training. Induction of diabetes was conducted using an intraperitoneal injection of a single dose of streptozotocin (STZ). Neuropathy was confirmed using the behavioral tests. Rats in induced diabetes plus training group had 8 weeks of moderate and increasing intensity endurance training on the treadmill. The Fibronectin mRNA gene expression level of the sciatic nerve was assessed using Real-time-PCR. Changes in fibronectin protein and myelin thickness were measured by immunohistochemistry and luxol fast blue staining. The mean and standard deviation was used to report descriptive data. Data were entered into SPSS 22. Fibronectin mRNA gene expression level (1.90) of sciatic nerve fibronectin protein and myelin thickness reduced significantly due to diabetes (P<0.05). Eight weeks of endurance training increased fibronectin gene expression of sciatic nerve fibronectin protein and prevented further destruction of myelin, which was statistically significant. The results showed that diabetes leads to changes in the extracellular matrix and the reduction of the sciatic nerve myelin thickness. Endurance training as a non-drug strategy is effective in preventing these damages.

Swiprosin-1 Promotes Mitochondria-Dependent Apoptosis of Glomerular Podocytes via P38 MAPK Pathway in Early-Stage Diabetic Nephropathy

Background/Aims: Podocyte injury, especially podocyte apoptosis, plays a major role in early-stage diabetic nephropathy (DN). Swiprosin-1, also known as EF hand domain containing 2 (EFhd2), is a Ca2+-binding protein in different cell types. However, the function of swiprosin-1 in podocytes remains unknown. Methods: The expression and distribution of swiprosin-1 were investigated in the mouse renal glomerulus and conditionally immortalized mouse podocyte cell line MPC-5. The expression of swiprosin-1 was also detected in streptozotocin (STZ)-treated mice and MPC-5 cells treated with high glucose (HG). Nephrin and podocin were detected by immunohistochemistry and immunofluroscence. Collagen IV, transforming growth factor-β (TGF-β) and fibronectin mRNA expressions were assayed by real-time PCR. Apoptotic proteins and phosphorylation of p38 mitogen-activated protein kinase (MAPK) were detected by immunoblotting. Results: Swiprosin-1 was found to be expressed in podocytes of the mouse glomerulus and MPC-5 cells. Swiprosin-1 expression was increased in STZ-treated mice and MPC-5 cells treated with HG. In Swiprosin-1-/- diabetic mice, kidney/ body weight, urinary albumin, podocyte foot process effacement and glomerular basement membrane thickening were attenuated; the downregulation of nephrin and podocin expression in the glomerulus was inhibited; and the upregulation of collagen IV, TGF-β and fibronectin mRNA expression in the renal cortex was ameliorated as compared with those in diabetic swiprosin-1+/+ mice. In addition, the increased apoptosis of podocytes, proapoptotic protein expression and p38 phosphorylation in Swiprosin-1-/- diabetic mice were inhibited as compared with those in diabetic swiprosin-1+/+ mice. Knockdown of swiprosin-1 in MPC-5 cells reduced the apoptosis of podocytes, proapoptotic protein expression and p38 phosphorylation induced by HG. Targeted knockdown of p38 attenuated the increased apoptosis of MPC-5 cells over-expressing swiprosin-1. Conclusion: Swiprosin-1 expression in podocytes of the mouse glomerulus played a critical role in early-stage DN. Swiprosin-1 deficiency in early DN attenuated mitochondria-dependent podocyte apoptosis induced by hyperglycemia or HG via p38 MAPK signaling pathway.

MiRNA-200b represses transforming growth factor-β1-induced EMT and fibronectin expression in kidney proximal tubular cells

MicroRNAs (miRNAs) comprise of a novel class of endogenous small noncoding RNAs that frequently downregulate the expression of target genes. Recent reports suggest that miRNA-200b prevents epithelial-to-mesenchymal transition (EMT) in cancer cells by targeting the E-box binding transcription factors Zinc finger E-box-binding homeobox 1 (ZEB1) and Zinc finger E-box-binding homeobox 2 (ZEB2). About 35% of active fibroblasts are derived from EMT which is central to the development of progressive renal fibrosis. Hence, this study was designed to assess the effect of miRNA-200b on transforming growth factor-β (TGF-β1)-induced fibrotic responses in renal tubular cells. Morphologically, human kidney-2 cells transfected with miRNA-200b retained their epithelial cell characteristics when exposed to TGF-β1. miRNA-200b significantly increased E-cadherin ( P < 0.001) and reduced fibronectin mRNA and protein expression (both P < 0.01) independent of phospho-Smad2/3 and phospho-p38 and p42/44 signaling. Increased E-cadherin expression was associated with decreased expression of ZEB1 and ZEB2 and repression of fibronectin was mediated through direct targeting of the fibronectin mRNA, demonstrated using pMIR luciferase reporter assay and site-directed mutagenesis. These results suggest that miRNA-200b suppresses TGF-β1-induced EMT via inhibition of ZEB1 and ZEB2 and the extracellular matrix protein fibronectin by directing targeting of its 3′UTR mRNA, independent of pathways directly involved in TGF-β1 signaling.

Ethanol stimulates the expression of fibronectin in lung fibroblasts via kinase-dependent signals that activate CREB

Ethanol renders the lung susceptible to acute lung injury in the setting of insults such as sepsis. The mechanisms mediating this effect are unknown, but activation of tissue remodeling is considered key to this process. We found that chronic ethanol ingestion in rats increased the expression of fibronectin, a matrix glycoprotein implicated in acute lung injury. In cultured NIH/3T3 cells and in primary rat and mouse lung fibroblasts, ethanol induced fibronectin mRNA and protein expression in a dose- and time-dependent fashion. The effect of ethanol was prevented by inhibitors of protein kinase C and mitogen-activated protein kinases and was associated with the phosphorylation and increased DNA binding of the transcription factor cAMP response element binding protein, followed by increased transcription of the fibronectin gene. Fibroblasts were found to express α7 nicotinic acetylcholine receptor (nAChR), and ethanol induction of fibronectin was abolished by α-bungarotoxin and methyllcaconitine, inhibitors of α7 nAChRs. However, ethanol was able to induce fibronectin mRNA and protein in primary lung fibroblasts isolated from α7 nAChR knockout mice. The ethanol-induced fibronectin response was dependent on ethanol metabolism since 4-methylpyrazole, an inhibitor of alcohol dehydrogenase, abolished the effect and acetaldehyde induced it. These observations suggest that ethanol or ethanol metabolites stimulate lung fibroblasts to produce fibronectin by inducing specific signals transmitted via nAChRs independent of the α7-subunit, and this might represent a mechanism by which ethanol renders the lung susceptible to acute lung injury.