guanosine-triphosphate and Diabetes-Mellitus

Diabetic nephropathy (DN) is the major cause of kidney failure, and glomerular podocytes play critical roles in the pathogenesis of DN by maintaining the glomerular structure and filtration barrier. Klotho and Slit-Robo GTP activating protein 2a (SRGAP2a) have been indicated to play protective roles in reducing kidney injury, but whether there is an internal relationship between these two factors is unclear.. In this study, we cultured differentiated rat podocytes in vitro and measured the SRGAP2a expressions by immunofluorescence staining, quantitative real-time PCR (qRT-PCR) and western blotting, after siRNA-mediated transforming growth factor β1 (TGF-β1) silencing, TGF-β1 overexpression and in the presence of a reactive oxygen species (ROS) inhibitor. And we detected the expressions of SRGAP2a, small mother against decapentaplegic (Smad)2/3, phosphorylated-Smad2/3 (p-Smad2/3), Smad7, and NAD(P)H oxidase 4 (NOX4), ROS levels and podocyte cytoskeletal remodelling under high glucose (HG) and exogenous klotho conditions. In addition, we performed haematoxylin-eosin (HE) staining and immunohistochemistry with diabetic rat models to confirm the in vitro results.. The results indicated that SRGAP2a expression was significantly upregulated under siRNA-mediated TGF-β1 silencing conditions or after adding a ROS inhibitor, but significantly downregulated with TGF-β1 overexpression, in the presence of HG. The supplementation of exogenous klotho under HG conditions significantly increased the SRGAP2a expression, remodelled the actin cytoskeleton and altered the expressions of Smad2/3, p-Smad2/3, Smad7 and NOX4 and reduced the ROS generation in podocytes. Moreover, klotho administration protected kidney injury in DN rats.. This study indicated that klotho may modulate the expression of SRGAP2a by regulating the ROS and TGF-β1 signalling pathways and provided theoretical support for klotho protein as a novel therapeutic strategy for treating DN patients.

Topics: Animals; Diabetes Mellitus; Diabetic Nephropathies; Female; Guanosine Triphosphate; Humans; Klotho Proteins; Male; Podocytes; Rats; Reactive Oxygen Species; RNA, Small Interfering; Transforming Growth Factor beta1

Rad is the prototypic member of a new class of Ras-related GTPases. Purification of the GTPase-activating protein (GAP) for Rad revealed nm23, a putative tumor metastasis suppressor and a development gene in Drosophila. Antibodies against nm23 depleted Rad-GAP activity from human skeletal muscle cytosol, and bacterially expressed nm23 reconstituted the activity. The GAP activity of nm23 was specific for Rad, was absent with the S105N putative dominant negative mutant of Rad, and was reduced with mutations of nm23. In the presence of ATP, GDP.Rad was also reconverted to GTP.Rad by the nucleoside diphosphate (NDP) kinase activity of nm23. Simultaneously, Rad regulated nm23 by enhancing its NDP kinase activity and decreasing its autophosphorylation. Melanoma cells transfected with wild-type Rad, but not the S105N-Rad, showed enhanced DNA synthesis in response to serum; this effect was lost with coexpression of nm23. Thus, the interaction of nm23 and Rad provides a potential novel mechanism for bidirectional, bimolecular regulation in which nm23 stimulates both GTP hydrolysis and GTP loading of Rad whereas Rad regulates activity of nm23. This interaction may play important roles in the effects of Rad on glucose metabolism and the effects of nm23 on tumor metastasis and developmental regulation.

Topics: Animals; Diabetes Mellitus; DNA; Enzyme Activation; Genes, Tumor Suppressor; Glucose; GTP Phosphohydrolases; Guanosine Triphosphate; Humans; Immediate-Early Proteins; Models, Biological; Monomeric GTP-Binding Proteins; Neoplasm Metastasis; NM23 Nucleoside Diphosphate Kinases; Nucleoside-Diphosphate Kinase; Protein Binding; ras Proteins; Rats; Rats, Sprague-Dawley; Transcription Factors

Topics: Adenylyl Cyclases; Animals; Calcium; Carbohydrate Metabolism; Cell Membrane; Cyclic AMP; Diabetes Mellitus; Diabetes Mellitus, Experimental; Glucagon; Guanosine Triphosphate; Kinetics; Liver; Magnesium; Male; Ouabain; Phosphoric Diester Hydrolases; Rats; Streptozocin; Theophylline

Topics: Adenosine Triphosphate; Amino Acyl-tRNA Synthetases; Animals; Carbon Isotopes; Diabetes Mellitus; Guanosine Triphosphate; Liver; Male; Microsomes, Liver; Pancreas; Pancreatectomy; Phenylalanine; Poly U; Protein Biosynthesis; Rats; Ribosomes; Subcellular Fractions