A rotor-stator homogenizer was used to thoroughly disrupt and homogenize tissues

on MMP-9 production, PECs were incubated with DMEM/F12 containing either 5 or 30 mM D-glucose medium for 24 or 48 hrs. The effect of hyperosmolality was assessed in PECs cultured in DMEM/F-12 containing 5 mM D-glucose supplemented with 25 mM mannitol. In another set of experiment, the PECs were cultured in DMEM/F12 medium for 2448 hrs in the absence or presence of 1 or 2 ng/ml TGF-1. The samples were collected and processed as above. Taqman and quantitative real-time PCR analysis Total RNA was prepared from primary PECs by using ultra-pure TRIzol reagent according to the manufacturer’s instructions. MMP-2, MMP-9 and actin gene-specific Taqman probe and primer sets were obtained from Applied Biosystems as MedChemExpress SNDX 275 Assays-on-Demand gene expression products. The Assays-on-Demand identification numbers were Rn02532334_s1 for MMP-2, Rn01423075_g1 for MMP-9, and 4331182 for rat -actin endogenous control. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19769708 Each sample was run in triplicate, and the comparative threshold cycle method was used to quantify fold increase compared with normal controls. Gelatin Zymography The gelatinolytic activity of MMP was examined in rat urine samples and culture supernatants from primary PECs as described previously. Briefly, samples were resolved by electrophoresis in a 10% polyacrylamide gel containing gelatin. After running, the gels were washed four times in renaturing buffer for 15 mins each before incubating for 1624 hrs at 37C in developing buffer. The gels were stained with Coomassie Brilliant Blue R-250 for 30 mins. After washing with a destaining solution, the gelatinolytic activity was visualized as a clear band in the uniformly stained background. The gels were scanned using white light transillumination, and the relative density of each gelatinolytic band was determined using ImageJ software. Immunoblot analysis Rat urine samples, culture supernatants or cell lysates were separated by 10% SDS-PAGE and transferred electrophoretically to nitrocellulose membrane. The blots were incubated with antibodies for MMP-9, nephrin, synaptopodin, or claudin-1. Some membranes were first hybridized with phospho-specific PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19768415 p44/42 or p38 MAPK antibodies, stripped, and then reprobed with an antibody that recognizes total p44/42 MAPK or total p38 MAPK. Detection was accomplished by enhanced chemiluminescence Western blotting. Relative band intensity was measured densitometrically. 4 / 20 Glomerular MMP-9 in Diabetic Nephropathy Statistical analysis Data are expressed as meanSEM. Student’s t test was used for comparison between two groups. Comparisons among multiple groups were performed by one-way ANOVA followed by Newman-Keuls post hoc test. Statistical significance was set at P<0.05. Results Glomerular Gelatinolytic Activity Detected by in Situ Zymography in Relation to Localization of MMP-9 in Rat Kidney Tissues Dysregulation of MMPs expression and activity has been reported in various glomerular diseases including diabetic nephropathy. Using gelatin in situ zymography, we were able to assess net proteolytic activity of MMP gelatinases in the glomeruli of normal and diabetic kidneys. In normal rat kidneys, gelatinolytic activity was identified in individual cells throughout the entire glomerular tuft. In contrast, focally increased gelatinolytic activity was recognized at the periphery of the glomeruli in 20-week-old Zucker diabetic rats. The enhanced local proteolytic activity was frequently associated with morphologic changes in the activated cells, as shown by HE and Masso