Volume 84, Issue 5, Pages 911-919 (November 2013) RAGE-mediated interstitial fibrosis in neonatal obstructive nephropathy is independent of NF-κB activation  Mojca Gasparitsch, Anne-Karin Arndt, Felix Pawlitschek, Stephan Oberle, Ursula Keller, Michael Kasper, Angelika Bierhaus, Franz Schaefer, Lutz T. Weber, Bärbel Lange-Sperandio  Kidney International  Volume 84, Issue 5, Pages 911-919 (November 2013) DOI: 10.1038/ki.2013.171 Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 1 Immunohistochemical staining of receptor for advanced glycation end products (RAGE) in the neonatal kidney of WT mice after unilateral ureteral obstruction (UUO) or sham operation (sham). Surgery was performed on the second day of life. RAGE expression is increased in glomeruli and in tubular and interstitial cells of the obstructed neonatal kidney at day 7 of life (a). Sham-operated control kidney (b). UUO-kidney section with control antibody (non-immune goat serum) (c). Bar = 100μm. Magnification × 200. Kidney International 2013 84, 911-919DOI: (10.1038/ki.2013.171) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 2 Morphometric analysis of tubulointerstitial injury after neonatal unilateral ureteral obstruction (UUO) in wild type (WT), RAGE-/-, and RAGE-/- × Icam1-/- mice. Ureteral ligation (UUO) was performed on the second day of life. Renal sections of UUO- and sham-operated kidneys were stained for macrophage infiltration with F4/80 antibody (a), for profibrogenic M2 macrophages (galectin-3) (b), T-cell infiltration (CD3 antibody) (c), proliferation with Ki67 antibody (d), tubular apoptosis (TUNEL) (e), interstitial apoptosis (f), collagen I (g), and interstitial fibrosis (Masson’s Trichrome) (h) at 3, 7, and 14 days of life and analyzed in 12 high-power fields per section (hpfs) at × 400. Changes are indicated as x-fold above sham-operated control. Data are the mean±s.e. (n=8 in each group). *P<0.05. Kidney International 2013 84, 911-919DOI: (10.1038/ki.2013.171) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 3 Representative photomicrographs of obstructed kidneys. Surgery (unilateral ureteral obstruction, UUO) was performed on the second day of life. Renal sections of wild type (WT), RAGE-/-, and RAGE-/- × Icam1-/- mice were stained for macrophage infiltration (F4/80 antibody) (a–c), galectin-3 (galectin-3 antibody) (d–f), T-cell infiltration (CD3 antibody) (g–i), apoptosis (TUNEL) (j–l), and interstitial fibrosis (Masson’s Trichrome (MT)) (m–o) at day 14 of life (12 days after UUO). Interstitial galectin-3, but not macrophage and T cell, infiltration was reduced in knockout mice after obstruction (a–i). RAGE-/- and RAGE-/- × Icam1-/- mice demonstrated less tubular apoptosis at day 14 of life (12 days after UUO, j–l). Renal sections were stained with MT to detect interstitial collagen (blue) in WT (m), RAGE-/- (n), and RAGE-/- × Icam1-/- (o) mice at day 14 of life. Bar = 100μm. Kidney International 2013 84, 911-919DOI: (10.1038/ki.2013.171) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 4 NF-κB activity (electrophoretic mobility shift assay) in obstructed and sham-operated kidneys. Neonatal RAGE-/-and wild-type (WT) mice were subjected to unilateral ureteral obstruction (UUO) or sham operation on the second day of life. Kidneys were collected at days 3, 7, and 14 and processed for EMSA analysis (n=3 in each group). Neonatal RAGE-/- mice demonstrate the same increase in NF-κB activation after UUO than WT mice with obstruction. Kidney International 2013 84, 911-919DOI: (10.1038/ki.2013.171) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 5 Protein expression of NF-κB (p50 subunit) and E-cadherin in unilateral ureteral obstruction (UUO)- and sham-operated kidneys of neonatal wild type (WT), RAGE-/-, and RAGE-/- × Icam1-/-mice. Neonatal mice were subjected to UUO or sham operation on the second day of life. Whole kidneys were processed for western blot analysis as described under Materials and Methods (n=3). Densitometric analysis was performed to quantify protein expression. UUO induced NF-κB p50 subunit expression in WT and RAGE-/-mice (a). The loss of E-cadherin expression after UUO was demonstrated in WT mice but not in RAGE-/-and RAGE-/- × Icam1-/- mice (a and b). *P<0.05. Kidney International 2013 84, 911-919DOI: (10.1038/ki.2013.171) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 6 Snail and transforming growth factor-ß (TGF-ß) are induced by unilateral ureteral obstruction (UUO) and attenuated in RAGE-/- and RAGE-/- × Icam1-/- mice. Protein expression of snail, α-smooth muscle actin (α-SMA), and TGF-ß. Neonatal mice were subjected to UUO or sham operation on the second day of life. Whole kidneys were processed for western blot analysis as described under Materials and Methods at days 7 and 14 of life. Snail was reduced in RAGE-/- × Icam1-/- mice at day 7 of life. TGF-ß expression was reduced in RAGE-/- and RAGE-/- × Icam1-/- mice at day 14 of life. *P<0.05. WT, wild type. Kidney International 2013 84, 911-919DOI: (10.1038/ki.2013.171) Copyright © 2013 International Society of Nephrology Terms and Conditions

Figure 7 Cell cycle arrest at G2/M in neonatal kidneys with unilateral ureteral obstruction (UUO). Protein expression of cyclin B1 and cyclin D1 at day 7 (a) and day 14 of life (b). Neonatal mice were subjected to UUO or sham operation on the second day of life. Whole kidneys were processed for western blot analysis as described under Materials and Methods at days 7 and 14 of life. UUO induced G2/M arrest in wild-type (WT) kidneys at day 7 and day 14 of life. G2/M arrest, as indicated by the cyclin B1/D1 ratio, was reduced in RAGE-/- mice at day 7 and day 14 of life. RAGE-/- × Icam1-/- mice demonstrated a higher ratio but lesser interstitial fibrosis. *P<0.05. Kidney International 2013 84, 911-919DOI: (10.1038/ki.2013.171) Copyright © 2013 International Society of Nephrology Terms and Conditions