Figure 6.

In vivo approach to evaluate the effect of chronic hrPR infusion with or without COX-2 inhibition on the expression of collagen I, fibronectin, CTGF, TGF-β, and PAI-I in inner medullary CDs. Immunofluorescence performed in mouse kidney sections showing specific labeling of (Pro)renin receptor in the CDs. (A). DAPI staining for nuclei. (B). Anti-PRR. (C) Merge image. (D). 10× digital zoom of merged image showing apical distribution of PRR (red arrows). L indicates tubular lumen. (E). In vivo methodology. Human recombinant prorenin was infused at a rate of 100 ng/min via an osmotic minipump for 36 h. COX-2 inhibitor NS-398 was administered at 10 mg/kg by oral gavage every 6 h. Sham-operated mice and administered methyl cellulose solution were used as controls. At the end of the study, mice were euthanized by conscious decapitation and renal tissues collected to perform immunofluorescence and Western blots in freshly isolated inner medullary CDs. (F). Representative image of the resulting suspension of freshly isolated inner medullary CDs. (G). Inner medullary CDs were fixed, blocked, and stained with anti-fibronectin or anti-collagen I antibody and detected with secondary antibody Alexa Fluor 488 conjugated to anti-rabbit IgG. (H). Measurements of fluorescence intensity in 10 fields from each processed kidney and expressed as fluorescence intensity versus total number of CD in each field.