Figure 4. ANP deficiency in male rats exacerbates glomerular damage but does not aggravate microalbuminuria and renal protein cast formation induced by a HS diet.

(A) Representative cortical images from Masson trichrome–stained kidneys taken at 10× (upper row), and expended regions of interest (bottom row). Blind scores are shown on the right; each point on the graph is an average of 100 scored glomeruli from 1 rat. n = 6, 10, 5, and 11 independent tissues scored for the SS^WT rats on NS and HS, and SS^NPPA–/– rats on NS and HS. (B) Representative images from Masson trichrome–stained renal tissues showing protein casts formation. Shown are images taken at 20×; graph on the right summarizes the percentage of the protein cast area to whole kidney section area; n = 6, 6, 5, and 11 independent tissue scans analyzed for the SS^WT rats on NS and HS, and SS^NPPA–/– rats on NS and HS. (C) Urinary microalbumin excretion (normalized to urine flow). n = 8, 7, 7, and 8 urine samples from independent animals were analyzed for the SS^WT rats on NS and HS, and SS^NPPA–/– rats on NS and HS. (D) Representative staining for megalin in the renal cortex (20×). (E) Western blot analysis of megalin expression in the renal cortex of the SS^WT rats on NS and HS, and SS^NPPA–/– rats on NS and HS; each lane represents an independent renal tissue sample obtained from a different rat. n = 5 per group. (F) Intensity profiles of megalin staining assessed in the cortical proximal tubules of the SS^WT rats and SS^NPPA–/– rats on NS and HS. n = 3 animals per group; at least 8 random images at 40× were analyzed per animal, with n = 8–10 individual tubules measured per image (total n = 172, 238, 217, 229 in SS^WT rats and SS^NPPA–/– rats on NS and HS). (G) Plasma albumin levels obtained from SS^WT rats on NS and HS, and SS^NPPA–/– rats on NS and HS; n = 7, 7, 8, 8. Data were analyzed with 2-way ANOVA or repeated measures ANOVA; if significant, P values are shown on the graphs. Male animals were used. Scale bars: 50 μm.