PURPOSE: An inflammatory mechanism has been suggested to contribute to the progression of diabetic nephropathy. Both retinoid and PPAR-gamma agonist, known anti-inflammatory agents, have been reported to be beneficial in diabetic nephropathy. Because they form heterodimer for transcription within the nucleus, we investigated the effect of a combination treatment with them in streptozotocin (STZ)-induced diabetic rats. METHODS: STZ-induced diabetic rats were treated with retinoid and PPAR-gamma agonist. The effects were determined by measuring urinary monocyte chemoattractant peptide (MCP)-1, proteinuria, and intrarenal ED-1 expression. RESULTS: Blood glucose concentration was higher in diabetic rats than in control rats. Retinoid and PPAR-gamma agonist did not affect blood glucose concentration. Urinary protein excretion (8.6+/-0.69 vs. 22.1 mg/mgCr, p<0.01) and urinary MCP-1 (19.8+/-3.4 vs. 61.5+/-6.1 pg/mgCr, p<0.01) were significantly higher in diabetic rats at four weeks after the induction of diabetes compared with controls. Proteinuria in the group with retinoic acid (16.9+/-1.4, mg/mgCr, p<0.05) and PPAR-gamma agonist (14.6+/-1.5 mg/mgCr, p<0.05) were decreased. Retinoic acid (42.2+/-2.7 pg/mgCr, p<0.05) and PPAR-gamma agonist (40.5+/-pg/ mgCr, p<0.05) significantly suppressed MCP-1 level in diabetic rats. However, combination treatment was not effective to proteinuria and urinary MCP-1 concentration. Urinary protein excretion was significantly correlated with MCP-1 (r=0.9, p<0.01). Immunohistochemistry revealed a significant increase in staining for ED-1 protein in the diabetic kidneys. Both retinoid and PPAR-gamma agonist significantly suppressed intrarenal ED-1 synthesis. However combination treatment didn't show any additional beneficial effects. CONCLUSION: Both retinoic acid and PPAR-gamma agonist suppressed proteinuria and inflammatory changes in diabetic rats. However, there were no additional effects of the combination treatment present. Further research is needed to determine the effect of the combination treatment on diabetic nephropathy.
Animals ; Anti-Inflammatory Agents ; Blood Glucose ; Diabetic Nephropathies* ; Immunohistochemistry ; Inflammation ; Kidney ; Monocytes ; Peroxisome Proliferator-Activated Receptors* ; Peroxisomes* ; Proteinuria ; Rats ; Retinoids ; Streptozocin ; Tretinoin

BACKGROUND: Aldosterone induces renal injury independent of angiotensin II. This harmful effect might be mediated via inflammatory reaction. Aldosterone receptor blockade can retard renal damage in various renal diseases including diabetic nephropathy. However, it is not clear which mechanism is related to the beneficial effect of aldosterone receptor blockade in diabetic nephropathy. Therefore, we investigated whether aldosterone receptor blockade, spironolactone, inhibited inflammatory changes in Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes. METHODS: To determine the inflammatory effects, urinary MCP-1 protein was measured by ELISA, and intrarenal MCP-1 mRNA and ED-1 expression were examined by RT-PCR and immunohistochemistry, respectively. RESULTS: Blood glucose concentration were higher in diabetic rats than in control rats. Urinary protein excretion was significantly higher in diabetic rats compared with controls since twenty weeks, and proteinuria of the diabetic rats was decreased by spironolactone treatment. Urinary excretion of monocyte chemoattractant peptide-1 (MCP-1) was rapidly increased at the early period in diabetic rats. Spironolactone suppressed urinary level of MCP-1 compared to untreated diabetic rats. Immunohistochemistry revealed a significant increase in ED-1 staining in the diabetic kidney, and spironolactone treatment significantly suppressed intrarenal ED-1 expression in diabetic rats. CONCLUSION: Aldosterone receptor blockade, spironolactone, suppressed proteinuria and inflammatory changes in diabetic rats. These results suggest that spironolactone may have an anti-inflammatory effect in diabetic nephropathy.
Aldosterone* ; Angiotensin II ; Animals ; Blood Glucose ; Diabetic Nephropathies* ; Enzyme-Linked Immunosorbent Assay ; Immunohistochemistry ; Inflammation ; Kidney ; Monocytes ; Proteinuria ; Rats ; Receptors, Mineralocorticoid* ; RNA, Messenger ; Spironolactone

Leucine-rich repeat kinase 2 (LRRK2) is a gene that, upon mutation, causes autosomal-dominant familial Parkinson's disease (PD). Yeast two-hybrid screening revealed that Snapin, a SNAP-25 (synaptosomal-associated protein-25) interacting protein, interacts with LRRK2. An in vitro kinase assay exhibited that Snapin is phosphorylated by LRRK2. A glutathione-S-transferase (GST) pull-down assay showed that LRRK2 may interact with Snapin via its Ras-of-complex (ROC) and N-terminal domains, with no significant difference on interaction of Snapin with LRRK2 wild type (WT) or its pathogenic mutants. Further analysis by mutation study revealed that Threonine 117 of Snapin is one of the sites phosphorylated by LRRK2. Furthermore, a Snapin T117D phosphomimetic mutant decreased its interaction with SNAP-25 in the GST pull-down assay. SNAP-25 is a component of the SNARE (Soluble NSF Attachment protein REceptor) complex and is critical for the exocytosis of synaptic vesicles. Incubation of rat brain lysate with recombinant Snapin T117D, but not WT, protein caused decreased interaction of synaptotagmin with the SNARE complex based on a co-immunoprecipitation assay. We further found that LRRK2-dependent phosphorylation of Snapin in the hippocampal neurons resulted in a decrease in the number of readily releasable vesicles and the extent of exocytotic release. Combined, these data suggest that LRRK2 may regulate neurotransmitter release via control of Snapin function by inhibitory phosphorylation.
Amino Acid Sequence ; Animals ; Exocytosis ; Female ; HEK293 Cells ; Humans ; Mice ; Molecular Sequence Data ; Mutant Proteins/metabolism ; Phosphorylation ; Phosphothreonine/metabolism ; Protein Binding ; Protein Interaction Mapping ; Protein Structure, Tertiary ; Protein-Serine-Threonine Kinases/*metabolism ; Qa-SNARE Proteins/metabolism ; Rats ; Rats, Sprague-Dawley ; Synaptosomal-Associated Protein 25/*metabolism ; Synaptotagmins/metabolism ; Vesicle-Associated Membrane Protein 2/metabolism ; Vesicular Transport Proteins/chemistry/*metabolism