The small GTPase Rho and its effector Rho-kinase are involved in the pathogenesis of diabetic nephropathy. Accumulating evidence shows that hypoxia-inducible factor-1α (HIF-1α) is a key regulator of renal sclerosis under diabetic conditions. However, the interactions of Rho-kinase and HIF-1α in the development of renal dysfunction have not been defined. Here, we assessed whether Rho-kinase blockade attenuates HIF-1α induction and the subsequent fibrotic response using type 2 diabetic mice and cultured mesangial cells. Fasudil, a Rho-kinase inhibitor, reduced urinary albumin excretion, mesangial matrix expansion, and the expression of fibrotic mediators in db/db mice. Mechanistically, HIF-1α accumulation and the expression of its target genes that contribute to diabetic glomerulosclerosis were also prevented by fasudil in the renal cortex. In mesangial cells, Rho/Rho-kinase signaling was activated under hypoxic conditions. Further in vitro studies showed that pharmacological and genetic inhibition of Rho-kinase promoted proteasomal HIF-1α degradation, which subsequently suppressed HIF-1-dependent profibrotic gene expression by upregulation of prolyl hydroxylase 2. Thus, we found a previously unrecognized renoprotective mechanism for the effects of Rho-kinase inhibition and this could be a potential therapeutic target for the treatment of diabetic nephropathy.

Formation of cytoplasmic aggregates in neuronal and glial cells is one of the pathological hallmarks of amyotrophic lateral sclerosis (ALS). Mutations in two genes

The small GTPase Rho and its downstream effector, Rho-associated coiled-coil containing protein kinase (Rho-kinase), regulate a number of cellular processes, including organization of the actin cytoskeleton, cell adhesion, and migration. While pharmacological inhibitors of Rhokinase signaling are known to block renal inflammation, the molecular basis for this effect is unclear. Here, we provide evidence that proinflammatory TNF-␣ promotes mesangial expression of macrophage colony-stimulating factor (M-CSF), a key regulator for the growth and differentiation of mononuclear phagocytes, in a Rhokinase-dependent manner. Consistent with this observation, TNF-␣-mediated renal expression of M-CSF in insulin-resistant db/db mice was downregulated by Rho-kinase inhibition. Small interfering RNAfacilitated knockdown of Rho-kinase isoforms ROCK1 and ROCK2 indicated that both isoforms make comparable contributions to regulation of M-CSF expression in mesangial cells. From a mechanistic standpoint, Western blotting and EMSA showed that Rho-kinase and its downstream target p38 MAPK regulate nuclear translocation of NF-B RelA/p65 and subsequent DNA binding activity, with no significant effects on IB␣ degradation and RelA/p65 phosphorylation. Moreover, we showed that Rho-kinase-mediated cytoskeletal organization is required for the nuclear uptake of RelA/p65. Collectively, these findings identify Rho-kinase as a critical regulator of chemokine expression and macrophage proliferation.

BackgroundAlthough previous reports have revealed the therapeutic potential of stem cell transplantation in diabetic polyneuropathy, the effects of cell transplantation on long-term diabetic polyneuropathy have not been investigated. In this study, we investigated whether the transplantation of dental pulp stem cells (DPSCs) ameliorated long-term diabetic polyneuropathy in streptozotocin (STZ)-induced diabetic rats.MethodsForty-eight weeks after STZ injection, we transplanted DPSCs into the unilateral hindlimb skeletal muscles. Four weeks after DPSC transplantation (i.e., 52 weeks after STZ injection) the effects of DPSC transplantation on diabetic polyneuropathy were assessed.ResultsSTZ-induced diabetic rats showed significant reductions in the sciatic motor/sensory nerve conduction velocity, increases in the current perception threshold, and decreases in capillary density in skeletal muscles and intra-epidermal nerve fiber density compared with normal rats, all of which were ameliorated by DPSC transplantation. Furthermore, sural nerve morphometrical analysis revealed that the transplantation of DPSCs significantly increased the myelin thickness and area. DPSC-conditioned media promoted the neurite outgrowth of dorsal root ganglion neurons and increased the viability and myelin-related protein expression of Schwann cells.ConclusionsThese results indicated that the transplantation of DPSCs contributed to the neurophysiological and neuropathological recovery from a long duration of diabetic polyneuropathy.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-017-0729-5) contains supplementary material, which is available to authorized users.

Establishing stable coculture systems with neuronal and Schwann cell lines has been considered difficult, presumably because of their high proliferative activity and phenotypic differences from primary cultured cells. The present study is aimed at developing methods for myelin formation under coculture of the neural crest-derived pheochromocytoma cell line PC12 and the immortalized adult rat Schwann cell line IFRS1. Prior to coculture, PC12 cells were seeded at low density (3 × 10(2)/cm(2)) and maintained in serum-free medium with N2 supplement, ascorbic acid (50 μg/ml), and nerve growth factor (NGF) (50 ng/ml) for a week. Exposure to such a NGF-rich environment with minimum nutrients accelerated differentiation and neurite extension, but not proliferation, of PC12 cells. When IFRS1 cells were added to NGF-primed PC12 cells, the cell density ratio of PC12 cells to IFRS1 cells was adjusted from 1:50 to 1:100. The cocultured cells were then maintained in serum-free medium with B27 supplement, ascorbic acid (50 μg/ml), NGF (10 ng/ml), and recombinant soluble neuregulin-1 type III (25 ng/ml). Myelin formation was illustrated by light and electron microscopy performed at day 28 of coculture. The stable PC12-IFRS1 coculture system is free of technical and ethical problems arising from the primary culture and can be a valuable tool to study peripheral nerve degeneration and regeneration.

Glucagon-like peptide-1 (GLP-1) is thought to preserve neurons and glia following axonal injury and neurodegenerative disorders. We investigated the neurotrophic and neuroprotective properties of exendin (Ex)-4, a synthetic GLP-1 receptor (GLP-1R) agonist, on adult rat dorsal root ganglion (DRG) neurons and PC12 cells. GLP-1R was predominantly localized on large and small peptidergic neurons in vivo and in vitro, suggesting the involvement of GLP-1 in both the large and small sensory fiber functions. Ex-4 dose-dependently (1 ≤ 10 ≤ 100 nM) promoted neurite outgrowth and neuronal survival at 2 and 7 days in culture, respectively. Treatment with 100 nM Ex-4 restored the reduced neurite outgrowth and viability of DRG neurons caused by the insulin removal from the medium and suppressed the activity of RhoA, an inhibitory regulator for peripheral nerve regeneration, in PC12 cells. Furthermore, these effects were attenuated by co-treatment with phosphatidylinositol-3'-phosphate kinase (PI3K) inhibitor, LY294002. These findings imply that Ex-4 enhances neurite outgrowth and neuronal survival through the activation of PI3K signaling pathway, which negatively regulates RhoA activity. Ex-4 and other GLP-1R agonists may compensate for the reduced insulin effects on neurons, thereby being beneficial for the treatment of diabetic neuropathy.

Amiodarone hydrochloride (AMD), an anti-arrhythmic agent, has been shown to cause peripheral neuropathy; however, its pathogenesis remains unknown. We examined the toxic effects of AMD on an immortalized adult rat Schwann cell line, IFRS1, and cocultures of IFRS1 cells and adult rat dorsal root ganglion neurons or nerve growth factor-primed PC12 cells. Treatment with AMD (1, 5, and 10 μm) induced time- and dose-dependent cell death, accumulation of phospholipids and neutral lipids, upregulation of the expression of gangliosides, and oxidative stress (increased nuclear factor E2-related factor in nuclear extracts and reduced GSH/GSSG ratios) in IFRS1 cells. It also induced the upregulation of LC3-II and p62 expression, with phosphorylation of p62, suggesting that deficient autolysosomal degradation is involved in AMD-induced IFRS1 cell death. Furthermore, treatment of the cocultures with AMD induced detachment of IFRS1 cells from neurite networks in a time- and dose-dependent manner. These findings suggest that AMD-induced lysosomal storage accompanied by enhanced oxidative stress and impaired lysosomal degradation in Schwann cells might be a cause of demyelination in the peripheral nervous system.

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