Volume 86, Issue 3, Pages 558-569 (September 2014) Opposite role of CD44-standard and CD44-variant-3 in tubular injury and development of renal fibrosis during chronic obstructive nephropathy  Elena Rampanelli, Kasper M.A. Rouschop, Nike Claessen, Gwendoline J.D. Teske, Steven T. Pals, Jaklien C. Leemans, Sandrine Florquin  Kidney International  Volume 86, Issue 3, Pages 558-569 (September 2014) DOI: 10.1038/ki.2014.87 Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 1 CD44 and β-catenin expression after unilateral ureteral obstruction (UUO). (a) Quantification of renal CD44 expression. Results of digital analysis presented as the percentage of positive area per high-power field (HPF, × 20). Mean±s.e.m., n=6, **P<0.01, ***P<0.001. (b) Representative micrographs ( × 20) of immunohistochemical staining for CD44 in wild type (WT), CD44s, and CD44v3 kidneys 3 days after UUO. (c) Q-PCR analysis for CD44s and (d) CD44v3 expression at mRNA levels at day 3 of UUO. Mean±s.e.m., n=6, *P<0.05. (e) Quantification of renal β-catenin expression. Results of digital analysis presented as the percentage of positive area per HPF ( × 20). Mean±s.e.m., n=6, *P<0.05. (f) Representative micrographs ( × 40) of immunostaining for β-catenin in CD44v3 UUO kidneys at 7 days. (g) Representative micrographs ( × 10) of immunohistochemical staining for CD44 in WT, CD44s, and CD44v3 kidneys 7 days after UUO. Kidney International 2014 86, 558-569DOI: (10.1038/ki.2014.87) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 2 Tubular damage upon unilateral ureteral obstruction (UUO). (a) Semiquantitative scoring of tubular injury rated on a 4-point grading scale. Data are expressed as mean±s.e.m., n=6, *P<0.05, ***P<0.001. (b) Representative histological stainings (PAS-D, × 20) of tubular lesions of wild type (WT), CD44s, and CD44v3 kidneys 3 days after UUO. Kidney International 2014 86, 558-569DOI: (10.1038/ki.2014.87) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 3 Tubular epithelial cell (TEC) proliferation and apoptosis after unilateral ureteral obstruction (UUO). (a) Representative micrographs ( × 20) of immunohistochemical staining for BrdU in wild type (WT), CD44s, and CD44v3 kidneys, 7 days after UUO. (b) Quantification of proliferating TECs. Data are expressed as mean±s.e.m., n=6, *P<0.05, **P<0.01,***P<0.001. (c) Representative micrographs ( × 20) of immunohistochemistry staining for active caspase-3 in WT, CD44s, and CD44v3 kidneys 7 days after UUO. (d) Quantification of apoptotic TECs upon UUO. Mean±s.e.m., n=6, *P<0.05, ***P<0.001. Kidney International 2014 86, 558-569DOI: (10.1038/ki.2014.87) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 4 Fibrosis in obstructed kidneys. (a) Representative micrographs ( × 20) of immunostaining for α-SMA in wild type (WT), CD44s, and CD44v3 kidneys 7 days after unilateral ureteral obstruction (UUO). (b) Digital analysis of renal myofibroblast accumulation; data are shown as percentage of positive area per HPF ( × 20). Mean±s.e.m., n=6, *P<0.05. (c) Representative micrograph ( × 40) of double immunostaining for α-SMA (brown) and CD44 (red) in CD44v3 kidneys 7 days after UUO. (d) Hydroxyproline assay to reveal renal collagen accumulation. Mean±s.e.m., n=6, *P<0.05. (e) Digital analysis of Picrosirius Red immunostaining at day 7 UUO; data shown as the percentage of positive area per HPF (x20). Mean±s.e.m., n=6, *P<0.05. (f) Representative micrographs (x10) of picrosirius red (upper panel) and collagen type I (lower panel) immunostaining in WT, CD44s, and CD44v3 kidneys 7 days after UUO. Kidney International 2014 86, 558-569DOI: (10.1038/ki.2014.87) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 5 Hepatocyte growth factor (HGF) in unilateral ureteral obstruction (UUO). (a) Kidney HGF levels determined by ELISA and corrected for the quantity of protein. Mean±s.e.m., n=6, *P<0.05. (b) Western blot analysis of phosphorylated (p-) and total c-Met at day 7 after UUO. Values normalized for β-actin levels. Mean±s.e.m., n=3, *P<0.05. (c) Representative micrographs ( × 20) of immunostainings for phosphorylated (upper panel) and total (lower panel) c-Met in wild type (WT), CD44s, and CD44v3 renal cortex 7 days after obstruction. Kidney International 2014 86, 558-569DOI: (10.1038/ki.2014.87) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 6 Transforming growth factor-β1 (TGF-β1) in unilateral ureteral obstruction (UUO). (a) Total TGF-β1 and (b) activated TGF-β1 levels quantified by ELISA and corrected for the quantity of protein. Mean±s.e.m., n=6. (c) Western blotting for the assessment of phosphorylated and total Smad-3 at day 7 UUO; β-actin used as a loading control. Mean±s.e.m., n=3, *P<0.05. (d) Representative micrographs ( × 20) of immunostainings for phosphorylated Smad-2/3 of CD44s and CD44v3 renal cortex 7 days after obstruction. (e) Western blot assay for TGF-β type I receptor (TGFβR-I) at day 7 UUO. Values corrected for β-actin levels. Mean±s.e.m., n=3, *P<0.05. Kidney International 2014 86, 558-569DOI: (10.1038/ki.2014.87) Copyright © 2014 International Society of Nephrology Terms and Conditions

Figure 7 In vitro assays. (a–c) Immorto TECs cultured for 24h with (black bars) or without (white bars) 100ng/ml Wnt3a or (d, e) 100ng/ml HGF; (f–h) MEFs cultured for 24h with/without 5ng/ml TGF-β1. CD44 KO cells transfected with pAD-CMV2-CD44s/ pAD-CMV2-CD44v3 plasmids. (a, d, f) Q-PCR using primers against complementary DNA encoding the constant region of CD44 (CD44-pan) in WT cells. (b) In-Cell Western assays for β-catenin and (c, e) phospho-AKT in immTECs. (g) Q-PCR analysis for collagen type I gene expression in MEFs. (h) Detection of collagen type I in the supernatant of TGF-β1-stimulated MEFs. (a–h) Values normalized to control (CTR) equal to 1. Mean±s.e.m., n=3, *P<0.05, **P<0.01, ***P<0.001. MEFs, mouse embryonic fibroblasts. Kidney International 2014 86, 558-569DOI: (10.1038/ki.2014.87) Copyright © 2014 International Society of Nephrology Terms and Conditions