HO-1 was adaptively upregulated in injured kidneys and persistently overexpressed in transgenic kidneys.

<p><b>(A)</b> Analysis of HO-1 mRNA levels in kidneys from TG and WT mice after UUO. <b>(B)</b> Representative immunofluorescence images of kidney sections co-labeled with HO-1 (red) and E-cadherin (green) in the Sham and UUO groups on day 7. (Magnification, ×200; bars = 250 μm) White stars indicate glomerulus. <b>(C)</b> Graph showing the area of HO-1 staining. <b>(D)</b> Representative immunofluorescence images of kidney sections co-labeled with HO-1 (red) and F4/80 (green) in the WT and TG groups at 7 day after UUO. (Magnification, ×400; bars = 125μm) (E) Representative higher magnifications views of dual immunostaining with HO-1 (red) and F4/80 (green) after UUO. (Magnification, ×1000; bars = 50 μm). (F-H) Line graphs indicating that there were no differences in body weight (F) before surgery or in serum creatinine levels (G) or proteinuria (H) after sham or UUO surgery between the WT and TG groups. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; ^&P<0.05, ^&&P<0.01 vs. TG mice of the Sham group; #P<0.05, ## P<0.01, ### P<0.001 vs. WT mice of the respective groups.</p

HO-1 overexpression regulated cell proliferation and apoptosis after renal fibrosis induced by UUO.

<p><b>(A)</b> Representative images of TUNEL staining (brown) of kidney sections from the Sham and UUO groups on days 7, 10, and 14. (magnification, ×200; bars = 250 μm) <b>(B)</b> Graph indicating the number of apoptotic cells in mice after UUO. <b>(C)</b> Representative Ki67 immunofluorescence staining of kidneys with UUO at days 7 (magnification, ×200; bars = 250 μm). Red arrows indicate Ki67+ epithelial cells, and white arrows indicate Ki67+ tubulointerstitial cells. <b>(D)</b> Graph showing the number of proliferative cells in renal tubule and interstitial compartments. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p

HO-1 overexpression reduced peripheral capillary loss and inhibited the activation and proliferation of renal interstitial myofibroblasts after UUO.

<p><b>(A)</b> Representative immunofluorescence images of CD31-labeled PTCs (red) from the Sham and UUO groups on days 3, 7, 10 and 14 (magnification, ×200; bars = 250 μm). <b>(B)</b> Graph showing the morphometric quantification of CD31-positive staining. <b>(C)</b> Representative immunofluorescence images of kidney sections co-labeled with PDGFRβ (green) and α-SMA (red) in the Sham and UUO groups on days 7 and 10. PDGFRβ is a specific marker of pericytes, fibroblasts and myofibroblasts, and α-SMA labels activated fibroblasts and myofibroblasts (magnification, ×400; bars = 100 μm). <b>(D)</b> Graph displaying the morphometric quantification of the PDGFRβ-positive area per high-power field. <b>(E)</b> Histogram showing the quantification of the α-SMA -positive area per high-power field. <b>(F)</b> Graph showing the morphometric quantification ratios of the double-positive/PDGFR-β-positive areas. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p

HO-1 decreased tubulointerstitial inflammation in the UUO model.

<p><b>(A)</b> Representative immunofluorescence images of F4/80-labeled macrophages (green) from the Sham and UUO groups on days 3, 7, 10 and 14. DAPI labels the nucleus (blue). (Magnification, ×200; bars = 250 μm) <b>(B)</b> Histogram indicating the morphometric quantification of the number of F4/80-positive cells per high-power field. <b>(C-D)</b> Graph showing the mRNA expression of inflammation-related cytokines at day 7 after UUO in the TG and WT groups. <b>(E)</b> Representative ELISA for inflammatory markers (TNF-α, IL-1β, IL-6, IL-4 and IL-10) in kidney tissue homogenates of HO-1 TG mice on the 7^th day after UUO. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p

HO-1 reduced renal tubulointerstitial injury and fibrosis after UUO.

<p><b>(A)</b> Representative light microscopy images of HE-stained kidney sections from the Sham and UUO groups on days 3, 7, 10 and 14 (magnification, ×200; bars = 250 μm). <b>(B)</b> Histogram showing the semi-quantitative determination of tubulointerstitial lesions. <b>(C)</b> Representative light microscopy images of Masson-stained and picrosirius red-stained kidney sections from the Sham and UUO groups on days 3, 7, 10 and 14 (magnification, ×200; bars = 250 μm). <b>(D)</b> Representative light microscopy images of picrosirius red-stained kidney sections from the Sham and UUO groups on days 3, 7, 10 and 14 (magnification, ×200; bars = 250 μm). <b>(E)</b> Histogram showing the area of picrosirius red-positive staining per high-power field in mice of the WT and TG groups after sham or UUO surgery. <b>(F)</b> Representative images of TGF-β1 immunohistochemical staining (brown) of kidney sections from the Sham and UUO groups at 3, 7, 10 and 14 days (magnification, ×200; bars = 250 μm). <b>(G)</b> Graph showing a semi-quantitative index of TGF-β1 staining. <b>(H)</b> Histogram showing TGF-β1 and CTGF mRNA levels at day 10 after UUO. The results are representative of triplicate analyses. <b>(I-J)</b> Representative Western blot (I) and quantitative data (J) for renal TGF-β1 expression 10 days after sham or UUO surgery. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p

HO-1 inhibited the activation of the Wnt/ β-catenin signaling pathway after renal fibrosis induced by UUO.

<p><b>(A-B)</b> Semi-quantitative real time-PCR of transcript levels of Wnt/β-catenin pathway ligands in the whole kidney 7 days after sham or UUO surgery. <b>(C)</b> Histogram showing the quantification of the β-catenin-positive area per high-power field. <b>(D)</b> Representative immunofluorescence images showing the expression and localization patterns of β-catenin, a common downstream mediator of the canonical Wnt signaling pathway, in the kidneys of the Sham and UUO groups at days 3, 7 and 10 (magnification, ×200; bars = 250 μm). Asterisks indicate β-catenin-positive tubulointerstitial cells. <b>(E-F)</b> Representative Western blot (E) and quantitative data (F) are presented for renal β-catenin expression 10 days after sham or UUO surgery. The data are expressed as the mean ± SD. * P<0.05, ** P<0.01, *** P<0.001 vs. WT mice of the Sham group; #P<0.05, ## P<0.01 vs. WT mice of the respective groups.</p