Increased L- and H-ferritin, FPN1, and MRP2 expression and fibrosis in HO-1^−/− kidneys reflected the shift of heme-iron recycling. (A) Both L and H ferritin mRNA expression levels were increased in kidney of HO-1^−/− mice. L ferritin increased 4.1-fold (P < .01) and H ferritin increased 3.5-fold (P < .01) as revealed by quantitative RT-PCR. Animals 8 months of age (n = 4 in each group) were used to generate data that are represented in panels A, C, and D. Error bars represent SD of the mean. (B) Immunofluorescence analysis for L-ferritin indicated that protein levels were also increased in the HO-1^−/− kidney. Most ferritin protein was found in proximal tubules and in some glomeruli. This pattern replicated the iron localization pattern revealed by the Perls Prussian blue staining and suggested that most accumulated iron in kidney was stored in the form of ferritin. (C-D) mRNA expression levels of MRP2, MRP3 normalized to actin-β, and FPN1 normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression were obtained by quantitative RT-PCR. (C) A 2.7-fold increase (P < .001) in expression of the apical membrane exporter of conjugated bilirubin, MRP2, in HO-1^−/− kidney suggested that heme degradation occurred in the polarized cells of the proximal tubules. Levels of MRP3, a widely expressed bilirubin exporter of nonpolarized cells, including macrophages, remained unchanged. mRNA levels of MRP2 and MRP3 remained unchanged in liver. (D) A 4-fold increase (P < .01) in FPN1 mRNA expression was observed in kidney, which probably served to return iron from catabolized heme to the circulation. (E) Fibrotic damage occurred to the kidney of HO-1^−/− mice as a result of heme iron recycling, as seen on Masson trichrome–stained paraffin-embedded tissue sections, where fibrosis developed in aging HO-1^−/− mice at much higher levels than in WT animals. Arrows indicate collagen fibers, which are colored in blue.