Figure 1.

Hyaluronidase (HYAL)-2 demonstrates increased interstitial expression in renal fibrosis in areas of α-smooth muscle actin (SMA)–positive myofibroblasts. Adult male Lewis rats underwent a midline laparotomy and were divided into two groups: sham operation (A) and bilateral ischemia reperfusion injury (IRI) (cross-clamping of both renal pedicles for 45 minutes) (B). At 28 days postoperatively, kidney tissue was retrieved with the animals under terminal anesthesia and stored in formalin. Kidneys were later embedded in paraffin and sections of 4 μm in thickness were cut. Sections were deparaffinized and rehydrated using xylene and reducing concentrations of ethanol. Antigen retrieval was performed in an autoclave using sodium citrate buffer with Tween. After blocking of nonspecific sites, sections were incubated with anti-HYAL2 and α-SMA monoclonal antibodies with appropriate fluorescence-labeled secondary antibodies [HYAL2 Alexa Fluor 555 (red) and α-SMA Alexa Fluor 488 (green)] and DAPI nuclear staining (blue). Sections were analyzed using confocal microscopy. Areas depicted as yellow demonstrate areas of Hyal2 and α-Sma colocalization. Dashed circle indicates a glomerulus; solid circles, tubules; arrowheads, blood vessels; dashed arrows, areas of red blood cell autofluorescence to be disregarded; solid arrows, the renal interstitium between the tubules where fibroblasts and myofibroblasts reside. n = 5 per group. Scale bars = 50 μm. Original magnification, ×400 (A and B, top rows); ×630 (A and B, bottom rows).