computerized morphometry visitron systems  (VISITRON Inc)

Figure Legend Snippet: Transfer of phosphorothioate ODN against TSP1 inhibits glomerular extracellular matrix accumulation and MC activation. In all figures treated kidneys (solid bars) were compared to nontreated control kidneys (open bars). EDA-fibronectin mRNA, a typical TGF-β-dependent matrix gene, was quantified via quantitative real-time PCR in pooled isolated glomeruli comparing scrambled- with antisense-treated kidneys. Antisense therapy against TSP1 almost completely abolished transcript expression of EDA-fibronectin in isolated glomeruli from nephritic rats on day 7 (A). Glomerular ECM accumulation on day 7 as determined by immunostaining for collagen IV (C and D) or collagen I (G and H) was markedly inhibited by antisense but not by scrambled ODN therapy against TSP1. E: A representative picture of glomerular collagen IV accumulation (dark gray immunostaining) in antisense-treated rats; F shows typical collagen IV expression in scrambled-treated rats on day 7. Glomerular de novo expression of smooth-muscle actin during glomerulonephritis, a marker of MC activation, was decreased by the TSP1 antisense but not by scrambled ODN transfer (B). Matrix accumulation was either evaluated by scoring system (C and G) or by computerized morphometry (B, D, H). * marks significant differences (P < 0.01) of antisense groups versus the control (scrambled) group.

Techniques Used: Activation Assay, Control, Real-time Polymerase Chain Reaction, Isolation, Expressing, Immunostaining, Marker

Figure Legend Snippet: Transfer of phosphorothioate ODN against TSP1, but not scrambled ODN, into glomerulonephritic rats inhibits glomerular TSP1 expression. In all figures treated kidneys (solid bars) were compared to nontreated control kidneys (open bars). On day 2 after disease induction, successful transfer of Cy3-labeled phosphorothioate ODN against TSP1 was demonstrated in almost 100% of glomeruli (A) of the left kidney, but none of the right kidney (not shown). Transfer of antisense oligonucleotides selectively inhibited de novo expression of glomerular TSP1 protein in the perfused left kidney on day 7 by more than 60% compared to the non-perfused right kidney, while gene transfer of scrambled control oligonucleotides did not alter glomerular TSP1 expression (B, evaluated by scoring; C, evaluated by computerized morphometry). Representative examples of unaltered TSP1 expression by scrambled ODNs (D) versus reduced TSP1 expression by antisense ODNs (E) are demonstrated by the extent of dark gray immunostaining within glomeruli. The * marks significant differences (P < 0.01) of antisense groups versus the control (scrambled) group.

Techniques Used: Expressing, Control, Labeling, Immunostaining

Figure Legend Snippet: Transfer of phosphorothioate ODN against TSP1 decreases activation, but not expression of TGF-β in nephritic glomeruli. In all figures treated kidneys (solid bars) were compared to nontreated control kidneys (open bars). Glomerular TGF-β1 or TGF-β2 protein (by brown immunostaining) was not changed in any group of nephritic rats (A and B). In agreement, equal levels of total TGF-β levels were determined using the PAI-1 luciferase assay. Active TGF-β levels were significantly reduced in the antisense-treated kidneys (C). Additionally, active TGF-β in nephritic glomeruli was determined by an antibody specifically recognizing the active form of TGF-β1 (D, gray cytoplasmic staining) and by an antibody specific for the phosphorylated form of the TGF-β signal-transduction molecule Smad 2/3 (H, black nuclear staining; arrowheads indicate examples for P-Smad2/3-positive nuclei). Inhibition of TSP1 expression after antisense ODN therapy but not scrambled ODN therapy was associated with a markedly decreased glomerular TGF-β activity in the left kidney as reflected by immunostaining for active TGF-β1 (E, evaluated by scoring system; F, evaluated by computerized morphometry) and by a marked reduction of glomerular cells showing positive nuclei for the TGF-β signaling molecule phospho-Smad2/3 (G). * marks significant differences (P < 0.01) of antisense groups versus the control (scrambled) group.

Techniques Used: Activation Assay, Expressing, Control, Immunostaining, Luciferase, Staining, Transduction, Inhibition, Activity Assay