Rho-associated, coiled-coil-containing protein kinase 1 regulates development of diabetic kidney disease via modulation of fatty acid metabolism.

Dysregulation of fatty acid utilization is increasingly recognized as a significant component of diabetic kidney disease. Rho-associated, coiled-coil-containing protein kinase (ROCK) is activated in the diabetic kidney, and studies over the past decade have illuminated ROCK signaling as an essential pathway in diabetic kidney disease. Here, we confirmed the distinct role of ROCK1, an isoform of ROCK, in fatty acid metabolism using glomerular mesangial cells and ROCK1 knockout mice. Mesangial cells with ROCK1 deletion were protected from mitochondrial dysfunction and redox imbalance driven by transforming growth factor ß, a cytokine upregulated in diabetic glomeruli. We found that high-fat diet-induced obese ROCK1 knockout mice exhibited reduced albuminuria and histological abnormalities along with the recovery of impaired fatty acid utilization and mitochondrial fragmentation. Mechanistically, we found that ROCK1 regulates the induction of critical mediators in fatty acid metabolism, including peroxisome proliferator-activated receptor gamma coactivator 1α, carnitine palmitoyltransferase 1, and widespread program-associated cellular metabolism. Thus, our findings highlight ROCK1 as an important regulator of energy homeostasis in mesangial cells in the overall pathogenesis of diabetic kidney disease.

Glycolaldehyde induces sensory neuron death through activation of the c-Jun N-terminal kinase and p-38 MAP kinase pathways.

Glycolaldehyde (GA) is a highly reactive hydroxyaldehyde and one of the glycolytic metabolites producing advanced glycation endproducts (AGEs), but its toxicity toward neurons and Schwann cells remains unclear. In the present study, we found that GA exhibited more potent toxicity than other AGE precursors (glyceraldehyde, glyoxal, methylglyoxal and 3-deoxyglucosone) against immortalized IFRS1 adult rat Schwann cells and ND7/23 neuroblastoma × neonatal rat dorsal root ganglion (DRG) neuron hybrid cells. GA affected adult rat DRG neurons and ND7/23 cells more severely than GA-derived AGEs, and exhibited concentration- and time-dependent toxicity toward ND7/23 cells (10 < 100 < 250 < 500 µM; 6 h < 24 h). Treatment with 500 µM GA significantly up-regulated the phosphorylation of c-jun N-terminal kinase (JNK) and p-38 mitogen-activated kinase (p-38 MAPK) in ND7/23 cells. Furthermore, GA-induced ND7/23 cell death was significantly inhibited due to co-treatment with 10 µM of the JNK inhibitor SP600125 or the p-38 MAPK inhibitor SB239063. These findings suggest the involvement of JNK and p-38 MAPK-signaling pathways in GA-induced neuronal cell death and that enhanced GA production under diabetic conditions might be involved in the pathogenesis of diabetic neuropathy.

ROCK2 regulates TGF-ß-induced expression of CTGF and profibrotic genes via NF-κB and cytoskeleton dynamics in mesangial cells.

The small GTPase Rho and its effector Rho kinase (ROCK) are involved in the pathogenesis of diabetic kidney disease. Rho kinase has two isoforms: ROCK1 and ROCK2. However, it remains unclear which is mainly involved in the progression of diabetic glomerulosclerosis and the regulation of profibrotic mediators. Glomeruli isolated from type 2 diabetic db/db mice demonstrated increased gene expression of transforming growth factor (TGF)-ß and its downstream profibrotic mediators. Chemical inhibition of ROCK suppressed the expression of profibrotic mediators in both isolated glomeruli and cultured mesangial cells. An investigation of mechanisms underlying this observation revealed activated ROCK functions through the phosphorylation of JNK and Erk and the nuclear translocation of NF-κB via actin dynamics. Knockdown by siRNA against ROCK1 and ROCK2 showed that ROCK2 but not ROCK1 controls this fibrotic machinery. Further in vivo experiments showed that ROCK2 activity in the renal cortex of db/db mice was elevated compared with control db/m mice. Importantly, oral administration of ROCK2 inhibitor attenuated renal ROCK2 activity, albuminuria, and glomerular fibrosis in db/db mice. These observations indicate that ROCK2 is a key player in the development of diabetic renal injury. Glomerular ROCK2 may be a potential therapeutic target for the treatment of diabetic kidney disease.

ROCK2 Regulates Monocyte Migration and Cell to Cell Adhesion in Vascular Endothelial Cells.

The small GTPase Rho and its downstream effector, Rho-kinase (ROCK), regulate various cellular functions, including organization of the actin cytoskeleton, cell adhesion and migration. A pro-inflammatory lipid mediator, lysophosphatidic acid (LPA), is a potent activator of the Rho/ROCK signalling pathway and has been shown to induce the expression of chemokines and cell adhesion molecules (CAMs). In the present study, we aimed to elucidate the precise mechanism by which ROCK regulates LPA-induced expressions and functions of chemokines and CAMs. We observed that ROCK blockade reduced LPA-induced phosphorylation of IκBα and inhibited NF-κB RelA/p65 phosphorylation, leading to attenuation of RelA/p65 nuclear translocation. Furthermore, small interfering RNA-mediated ROCK isoform knockdown experiments revealed that LPA induces the expression of monocyte chemoattractant protein-1 (MCP-1) and E-selectin via ROCK2 in human aortic endothelial cells (HAECs). Importantly, we found that ROCK2 but not ROCK1 controls LPA-induced monocytic migration and monocyte adhesion toward endothelial cells. These findings demonstrate that ROCK2 is a key regulator of endothelial inflammation. We conclude that targeting endothelial ROCK2 is potentially effective in attenuation of atherosclerosis.

Establishment of a myelinating co-culture system with a motor neuron-like cell line NSC-34 and an adult rat Schwann cell line IFRS1.

Co-culture models of neurons and Schwann cells have been utilized for the study of myelination and demyelination in the peripheral nervous system; in most of the previous studies, however, these cells were obtained by primary culture with embryonic or neonatal animals. A spontaneously immortalized Schwann cell line IFRS1 from long-term cultures of adult Fischer rat peripheral nerves has been shown to retain fundamental ability to myelinate neurites in co-cultures with adult rat dorsal root ganglion neurons and nerve growth factor-primed PC12 cells. Our current investigation focuses on the establishment of stable co-culture system with IFRS1 cells and NSC-34 motor neuron-like cells. NSC-34 cells were seeded at a low density (2 × 103/cm2) and maintained for 5-7 days in serum-containing medium supplemented with non-essential amino acids and brain-derived neurotrophic factor (BDNF; 10 ng/mL). Upon observation of neurite outgrowth under a phase-contrast microscope, the NSC-34 cells were exposed to an anti-mitotic agent mitomycin C (1 µg/mL) for 12-16 h, then co-cultured with IFRS1 cells (2 × 104/cm2), and maintained in serum-containing medium supplemented with ascorbic acid (50 µg/mL), BDNF (10 ng/mL), and ciliary neurotrophic factor (10 ng/mL). Double immunofluorescence staining carried out at day 28 of the co-culture showed myelin protein (P0 or PMP22)-immunoreactive IFRS1 cells surrounding the ßIII tubulin-immunoreactive neurites. This co-culture system can be a beneficial tool to study the pathogenesis of motor neuron diseases (e.g., amyotrophic lateral sclerosis, Charcot-Marie-Tooth diseases, and immune-mediated demyelinating neuropathies) and novel therapeutic approaches against them.

Rho-Kinase Blockade Attenuates Podocyte Apoptosis by Inhibiting the Notch Signaling Pathway in Diabetic Nephropathy.

Podocyte apoptosis is a key process in the onset of diabetic nephropathy. A significant body of evidence shows that the Notch signaling pathway plays a central role in this process. We found that Rho-kinase mediates transforming growth factor ß (TGF-ß)-induced Notch ligand Jag1 expression. Importantly, TGF-ß-mediated podocyte apoptosis was attenuated by Rho-kinase inhibition. Mechanistically, Rho-kinase regulated Jag1 induction via the extracellular signal-regulated kinase (ERK) 1/2 and c-Jun N-terminal kinase (JNK) but not Smad pathways. Consistently, the Rho-kinase inhibitor fasudil prevented albuminuria and the urinary excretion of nephrin in db/db mice and reduced the prevalence of podocyte apoptosis and Jag1 expression. Finally, the expression of Jag1 and apoptosis markers such as Bax and cyclin-dependent kinase inhibitor 1A (CDKN1A) was decreased in podocytes derived from db/db mice treated with fasudil. The present study provides evidence that Rho-kinase plays a key role in podocyte apoptosis. Rho-kinase is an attractive therapeutic target for diabetic nephropathy.

SGLT2 Inhibitors as a Therapeutic Option for Diabetic Nephropathy.

Diabetic nephropathy (DN) is a major cause of end-stage renal disease (ESRD) worldwide. Glycemic and blood pressure (BP) control are important but not sufficient to attenuate the incidence and progression of DN. Sodium-glucose cotransporter (SGLT) 2 inhibitors are a new class of glucose-lowering agent suggested to exert renoprotective effects in glucose lowering-dependent and independent fashions. Experimental studies have shown that SGLT2 inhibitors attenuate DN in animal models of both type 1 diabetes (T1D) and type 2 diabetes (T2D), indicating a potential renoprotective effect beyond glucose reduction. Renoprotection by SGLT2 inhibitors has been demonstrated in T2D patients with a high cardiovascular risk in randomized controlled trials (RCTs). These favorable effects of SGLT2 inhibitors are explained by several potential mechanisms, including the attenuation of glomerular hyperfiltration, inflammation and oxidative stress. In this review article, we discuss the renoprotective effects of SGLT2 inhibitors by integrating experimental findings with the available clinical data.