AMDCC Progress Report Duke/UNC/Stanford Unit Program Director Thomas M. Coffman, MD
Susceptibility Mutation Model Development + ApoE-/- +STZ Kidney Phenotype Screen Vascular & Kidney Phenotype Screen Generate ES Cell Lines From Diabetic Mice Transfer mutations to diabetic backgrounds
Strain Comparison Of the 5 strains tested, DBA/2 found to be most susceptible in STZ model
Albumin-to-Creatinine Ratio in Male Mice 500 *p<0.03 AC Ratio 400 300 200 100 +stz control 0 C57BL/6 MRL/Mp BALB/c DBA/2 Strain
Mesangial Volumes in Male Mice ** * *
Model Comparison Advantages of the Akita (Ins2 +/C96Y ) model
16 Week Blood Glucose Values * (*p=0.0018)
Differential Blood Pressure Responses * (*p=0.0018 vs Akita Control)
Increased Proteinuria in Akita Mice * (*p=0.018 vs. STZ)
Mesangial Volumes in Diabetic Mice
C57BL/6 Ins2 +/C96Y Ins2 +/C96Y DBA/2 F 1 (C57BL / 6 x DBA/2) Ins2 +/C96Y Ins2 +/C96Y
Blood Glucose Levels in Mice * (*p=0.011)
Enhanced Proteinuria in F 1 (DBA/2 x C57BL/6)Ins2 +/C96Y Mice * (*p<0.001 vs. B6-Akita)
Mesangial Expansion in F 1 (DBA/2 x C57BL/6)Ins2 +/C96Y Mice * (*p<0.001 vs. B6-Akita)
Studies in Progress Large group of animals for long-term studies Back-crossing of Ins2 +/C96Y onto DBA/2 and other backgrounds
Susceptibility Mutation Model Development + ApoE-/- +STZ Kidney Phenotype Screen Vascular & Kidney Phenotype Screen Generate ES Cell Lines From Diabetic Mice Transfer mutations to diabetic backgrounds
Angiotensinogen Renin ACE2 Angiotensin 1-9 ACE2 Angiotensin 1-7 Angiotensin I Angiotensin II ACE Kinins Inactive Peptides AT 1 Receptor
Angiotensinogen Renin ACE2 Angiotensin 1-9 ACE2 Angiotensin 1-7 Angiotensin I Angiotensin II ACE Kinins Inactive Peptides AT 1 Receptor
Agtr1a Gene Duplication A X K 1 2 x X 3 1 K X B K X 1 K 90 kb gap TK (K)(X) Neo 2 C X K 1 2 X 3 K (X) Neo 1 2 kb 2 diagnostic PCR K X K 1 2 X 3 K X probe probe 7.5 kb 6.0 kb
1300 1200 1100 1000 900 800 700 2-copy 3-copy Agtr1a Genotype * 4-copy Ligand Binding Density (DPM/mm2)
ACR in Agtr1a 4-Copy Mice
Angiotensinogen Renin ACE2 Angiotensin 1-9 ACE2 Angiotensin 1-7 Angiotensin I Angiotensin II ACE Kinins Inactive Peptides AT 1 Receptor
ACE2 mrna Expression heart kidney sk. muscle liver lung placenta brain pancrea spleen thymus prostate testis ovary sm. intestine colon pmn s stomach thyroid spinal cord lymph node trachea adrenal gland bone marrow 9.5 7.5 4.4 2.4 1.35 Donoghue et al, Circ Res 87:1, 2000
ACR in Ace2-Knockout Mice
Angiotensinogen Renin ACE2 Angiotensin 1-9 ACE2 Angiotensin 1-7 Angiotensin I Angiotensin II ACE Kinins Inactive Peptides AT 1 Receptor
Renin Transgenic Mice Single-copy transgene on C57BL/6 background Renin expression driven by albumin promoter
Blood Glucose in Renin Tg Mice
Blood Pressures in Renin Tg Mice
Albuminuria in Renin Tg Mice
Angiotensinogen Renin ACE2 Angiotensin 1-9 ACE2 Angiotensin 1-7 Angiotensin I Angiotensin II ACE Kinins Inactive Peptides AT 1 Receptor
Absence of Bradykinin B2 receptor increases diabetic albuminuria PNAS 101:13302-13305, 2004
Bdkr2 +/+ Ins2 +/+ Bdkr2 -/- Ins2 +/+ Bdkr2 +/+ Ins2 +/C96Y Bdkr2 -/- Ins2 +/C96Y
Completed Screens Strain Diabetes Blood Glucose Blood AC Ratio Pathology Induction Pressure Tp -/- (C57BL/6) STZ x x x (Renal) Tp -/- (BALB/c) STZ x x x (Renal) TIMP3 -/- STZ x x x (Renal) Agtr1a dupl (2/2) STZ x x x (Renal) Bdkr2 -/- STZ x - x (Renal) Bdkr2 -/- x Ins2 Akita genetic x x x ((Renal) Nos3 -/- STZ (x) - (x) (Renal) Apoe -/- (B6) 2mo STZ x x x Renal/Vascular old Apoe -/- (B6) 6mo STZ x x x (Renal)/Vascular old Cbs -/- Apoe -/- (w/o STZ x x x (Renal)/Vascular Met) Cbs -/- Apoe -/- (w STZ x x x (Renal)/Vascular Met) F 1 (DBA/2 x genetic x x (x) (Renal) Ins2 Akita ) Ppar P435L/+ (129xB6 STZ (x) (x) (x) (Renal/Vascular) F1) 129xB6 F1 STZ (x) (x) (x) (Renal Renal)
Susceptibility Mutation Model Development + ApoE-/- +STZ Kidney Phenotype Screen Vascular & Kidney Phenotype Screen Generate ES Cell Lines From Diabetic Mice Transfer mutations to diabetic backgrounds
Diabetes exacerbates the atherosclerotic lesion in ApoE-/- mice 5 ze (x10 5 ) Lesion si 4 3 2 1 100 ± 8 % 178 ± 8 % p=0.0002 0 ApoE -/- Non-DM ApoE -/- DM
Diabetes exacerbates diabetic nephropathy in ApoE-/- mice U-Albumin Excretion (µg/30gbw/d) 100 50 0 B6 ApoE -/- B ApoE -/- 6 mos 3 mos 6 mos # Non-DM * DM # M Non-DM D M Non-DM D Non-diabetic ApoE-/- diabetic ApoE-/-
% Aortic Occlusion 40 30 20 10 r 2 =0.84; p =0.003 0 0 100 200 300 AER (ug/30g BW/d)
-CBS+/-ApoE-/- given STZ -Supplemented with L-methionine (0.5%) or L-methionine (0.5%)/Folate (200µg/L)
Plasma Hcy levels in apoe-/-cbs+/- Non diabetic (Non-DM) and Diabetic (DM) mice at study initiation (2 months old) and conclusion (8 months old). P<0.05, n=6.
Albumin Excretion Rate, AER (ug/30gbw/d) 175 150 125 100 75 50 25 * # # # 0 Non-DM DM Non-DM DM Non-DM DM Non-DM DM B6 ApoE -/- ApoE -/- ApoE -/- CBS +/- 6 mos 3 Mos 6 Mos 6 Mos Figure 2. Albumin Excretion rate (AER) at the end of 3 or 6 months
35 r 2 =0.63; p=0.03 Ao Occlusion (%) 25 15 5 0 50 100 150 200 AER (µg/30g/d) Proximal Aortic occlusion is related to AER in ApoE-/-CBS+/- Mice
New Model Development Ppar P465L/+ mouse line (Tsai et al., J Clin Invest, 2004) Increased B-cell insulin production Hypertension Abdominal fat distribution 3 UTR modification of the CTGF Gene