ABSTRACT
Diabetic nephropathy (DN) is a progressive kidney disease that is caused by injury to kidney glomeruli. Podocytes are glomerular epithelial cells and play critical roles in the glomerular filtration barrier. Recent studies have shown the importance of regulating the podocyte actin cytoskeleton in early DN. The phosphoinositide 3-kinase (PI3K) inhibitor, wortmannin, simultaneously regulates Rac1 and Cdc42, which destabilize the podocyte actin cytoskeleton during early DN. In this study, in order to evaluate the reno-protective effects of wortmannin in early DN by regulating Rac1 and Cdc42, streptozotocin (STZ)-induced proteinuric renal disease (SPRD) rats were treated with wortmannin. The albuminuria value of the SPRD group was 3.55 +/- 0.56 mg/day, whereas wortmannin group was 1.77 +/- 0.48 mg/day. Also, the albumin to creatinine ratio (ACR) value of the SPRD group was 53.08 +/- 10.82 mg/g, whereas wortmannin group was 20.27 +/- 6.41 mg/g. Changes in the expression level of nephrin, podocin and Rac1/Cdc42, which is related to actin cytoskeleton in podocytes, by wortmannin administration were confirmed by Western blotting. The expression levels of nephrin (79.66 +/- 0.02), podocin (87.81 +/- 0.03) and Rac1/Cdc42 (86.12 +/- 0.02) in the wortmannin group were higher than the expression levels of nephrin (55.32 +/- 0.03), podocin (53.40 +/- 0.06) and Rac1/Cdc42 (54.05 +/- 0.04) in the SPRD group. In addition, expression and localization of nephrin, podocin and desmin were confirmed by immunofluorescence. In summary, we found for the first time that wortmannin has a reno-protective effect on SPRD rats during the early DN. The beneficial effects of wortmannin in SPRD rats indicate that this compound could be used to delay the progression of the disease during the early DN stage.
Subject(s)
Animals , Humans , Rats , Albumins/metabolism , Androstadienes/administration & dosage , Creatinine/blood , Desmin/genetics , Diabetes Mellitus, Experimental/drug therapy , Diabetic Nephropathies/drug therapy , Disease Models, Animal , Intracellular Signaling Peptides and Proteins/genetics , Kidney/pathology , Membrane Proteins/genetics , Phosphatidylinositol 3-Kinases/antagonists & inhibitors , Podocytes/drug effects , Rats, Inbred Strains , cdc42 GTP-Binding Protein/genetics , rac1 GTP-Binding Protein/geneticsABSTRACT
Cdc42 is a member of the Rho family of small GTP-ase and plays an important role in intracellular signaling pathways regulating cell morphology, motility and stimulation of DNA synthesis. We have isolated cDNA encoding Cdc42 from a rat brain cDNA library using PCR-cloning strategy. The sequence of isolated gene revealed an open reading frame of 576 nucleotides with the potential to encode a protein of 191 amino acids with a predicted molecular weight of 21 kD. The resulting sequence was incorporated into the GenBank with accession number, AF205635. Sequence analysis revealed that overall cDNA sequence identity is 96% with human G25K and 52% with rat Chp, a homologue of the GTPase human Cdc42Hs, and having one nucleotide difference from the mouse Cdc42. However, putative protein sequence was identical to the mouse and human brain Cdc42Hs. On expression of the cDNA in COS-7 cells, a protein molecular weight of 21 kD was detected in immunoblotting using anti-human Cdc42 antibodies. Therefore, these results suggest that the cDNA we are reporting is most likely the rat homologue of the GTPase human Cdc42.