Involvement of receptor-bound prorenin in development of nephropathy in diabetic db/db mice  Atsuhiro Ichihara, MD, PhD, FAHA, Mariyo Sakoda, MD, Asako Kurauchi-Mito, MD, Akira Nishiyama, MD, PhD, FAHA, Hiroshi Itoh, MD, PhD  Journal of the American Society of Hypertension  Volume 2, Issue 5, Pages 332-340 (September 2008) DOI: 10.1016/j.jash.2008.04.009 Copyright © 2008 American Society of Hypertension Terms and Conditions

Figure 1 Preparation of the HRP corresponding to the “handle region” of the prosegment of mouse Ren1-prorenin. (A) Amino acid sequences of the prosegment of the mouse Ren1-prorenin and HRP. (B) Mass of prepared mouse HRP. (C) Mouse HRP (1 μM) binds to anti-mouse-HRP antibody (3 nM) but not to anti-mouse-renin antibody (3 nM). (D) Interference of the recombinant mouse Ren1-prorenin (10 nM) binding to anti-mouse-HRP antibody (3 nM) by mouse HRP (1 μM), but not by NH2-MTRLSAE-COOH (positions 30–36 of prorenin prosegment; 1 μM). HRP, handle region peptide. Journal of the American Society of Hypertension 2008 2, 332-340DOI: (10.1016/j.jash.2008.04.009) Copyright © 2008 American Society of Hypertension Terms and Conditions

Figure 2 Kidney sections stained using the periodic acid–Schiff method in db/db mice treated with the HRP or the control peptide and db/m mice treated with HRP or the control peptide at 16 weeks of age. Scale bars: 25 μm. The glomerulosclerosis index was significantly higher in the db/db mice than in the db/m mice, but the elevation was significantly inhibited by HRP treatment. HRP, handle region peptide. *P < .05 for db/db mice vs. db/m mice. †P < .05 for HRP treatment vs. control peptide treatment. Journal of the American Society of Hypertension 2008 2, 332-340DOI: (10.1016/j.jash.2008.04.009) Copyright © 2008 American Society of Hypertension Terms and Conditions

Figure 3 Immunohistochemical stainings with an antibody to the active site of renin in db/db mice treated with the HRP or the control peptide and db/m mice treated with the HRP or the control peptide at 16 weeks of age. Scale bars: 25 μm. Analyses of active-site-of-renin-positive cells showed an elevation in immunoreactivity for the active site of renin in the juxtaglomerular area of db/db mice; this elevation was reduced by treatment with HRP. Immunostaining for the active site of renin was weak in the kidneys of db/m mice and db/m + HRP mice. HRP, handle region peptide. *P < .05 for db/db mice vs. db/m mice. †P < .05 for HRP treatment vs. vehicle treatment. Journal of the American Society of Hypertension 2008 2, 332-340DOI: (10.1016/j.jash.2008.04.009) Copyright © 2008 American Society of Hypertension Terms and Conditions

Figure 4 Kidney prorenin mRNA, the total renin (renin plus prorenin) content, and (pro)renin receptor mRNA levels in db/m mice treated with the HRP or the control peptide and db/db mice treated with HRP or the control peptide at 16 weeks of age (n = 8 in each). The graphs show an elevation in the kidney levels of prorenin mRNA and the total renin content in db/db mice; HRP treatment had no effect on these levels. HRP, handle region peptide. *P < .05 vs. db/m mice. Journal of the American Society of Hypertension 2008 2, 332-340DOI: (10.1016/j.jash.2008.04.009) Copyright © 2008 American Society of Hypertension Terms and Conditions

Figure 5 Kidney Ang I and II levels in db/m mice treated with the HRP or the control peptide and db/db mice treated with HRP or the vehicle at 16 weeks of age (n = 8 in each). The graphs show elevations in kidney Ang I and II levels in db/db mice, and HRP significantly inhibited the elevations in kidney Ang I and II levels in db/db mice. Ang, angiotensin; HRP, handle region peptide. *P < .05 for db/db mice vs. db/m mice. †P < .05 for HRP treatment vs. vehicle treatment. Journal of the American Society of Hypertension 2008 2, 332-340DOI: (10.1016/j.jash.2008.04.009) Copyright © 2008 American Society of Hypertension Terms and Conditions

Figure 6 Kidney mRNA levels of angiotensinogen and ACE in db/m mice treated with the HRP or the control peptide and db/db mice treated with HRP or the control peptide at 16 weeks of age (n = 8 in each). The graph shows an elevation in the kidney angiotensinogen mRNA level and a decrease in the kidney ACE level in db/db mice; HRP had no effect on these levels. ACE, angiotensin-converting enzyme; HRP, handle region peptide. *P < .05 vs. db/m mice. Journal of the American Society of Hypertension 2008 2, 332-340DOI: (10.1016/j.jash.2008.04.009) Copyright © 2008 American Society of Hypertension Terms and Conditions

Figure 7 Immunohistochemistry of phospho-ERK 1/2 in db/db mice treated with HRP or the control peptide and db/m mice treated with HRP or the control peptide at 16 weeks of age. Scale bars: 25 μm. The percentage of phosphor-ERK–positive glomeruli in the cross-section was significantly higher in the glomeruli of the kidneys from db/db mice. HRP treatment significantly inhibited the elevated immunoreactivity of phospho-ERK in the db/db mice. ERK, extracellular-signal–related protein kinase; HRP, handle region peptide; *P < .05 for db/db mice vs. db/m mice. †P < .05 for HRP treatment vs. the control peptide. Journal of the American Society of Hypertension 2008 2, 332-340DOI: (10.1016/j.jash.2008.04.009) Copyright © 2008 American Society of Hypertension Terms and Conditions