AMDCC Progress Report Duke/UNC/Stanford Unit. Program Director Thomas M. Coffman, MD

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1 AMDCC Progress Report Duke/UNC/Stanford Unit Program Director Thomas M. Coffman, MD

2 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

3 Strain Comparison Of the 5 strains tested, DBA/2 found to be most susceptible in STZ model

4

5 Albumin-to-Creatinine Ratio in Male Mice 500 *p<0.03 AC Ratio stz control 0 C57BL/6 MRL/Mp BALB/c DBA/2 Strain

6 Mesangial Volumes in Male Mice ** * *

7 Model Comparison Advantages of the Akita (Ins2 +/C96Y ) model

8 16 Week Blood Glucose Values * (*p=0.0018)

9 Differential Blood Pressure Responses * (*p= vs Akita Control)

10 Increased Proteinuria in Akita Mice * (*p=0.018 vs. STZ)

11 Mesangial Volumes in Diabetic Mice

12 C57BL/6 Ins2 +/C96Y Ins2 +/C96Y DBA/2 F 1 (C57BL / 6 x DBA/2) Ins2 +/C96Y Ins2 +/C96Y

13 Blood Glucose Levels in Mice * (*p=0.011)

14 Enhanced Proteinuria in F 1 (DBA/2 x C57BL/6)Ins2 +/C96Y Mice * (*p<0.001 vs. B6-Akita)

15 Mesangial Expansion in F 1 (DBA/2 x C57BL/6)Ins2 +/C96Y Mice * (*p<0.001 vs. B6-Akita)

16 Studies in Progress Large group of animals for long-term studies Back-crossing of Ins2 +/C96Y onto DBA/2 and other backgrounds

17 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

18 Angiotensinogen Renin ACE2 Angiotensin 1-9 ACE2 Angiotensin 1-7 Angiotensin I Angiotensin II ACE Kinins Inactive Peptides AT 1 Receptor

19 Angiotensinogen Renin ACE2 Angiotensin 1-9 ACE2 Angiotensin 1-7 Angiotensin I Angiotensin II ACE Kinins Inactive Peptides AT 1 Receptor

20 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

21 copy 3-copy Agtr1a Genotype * 4-copy Ligand Binding Density (DPM/mm2)

22 ACR in Agtr1a 4-Copy Mice

23 Angiotensinogen Renin ACE2 Angiotensin 1-9 ACE2 Angiotensin 1-7 Angiotensin I Angiotensin II ACE Kinins Inactive Peptides AT 1 Receptor

24 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 Donoghue et al, Circ Res 87:1, 2000

25 ACR in Ace2-Knockout Mice

26 Angiotensinogen Renin ACE2 Angiotensin 1-9 ACE2 Angiotensin 1-7 Angiotensin I Angiotensin II ACE Kinins Inactive Peptides AT 1 Receptor

27 Renin Transgenic Mice Single-copy transgene on C57BL/6 background Renin expression driven by albumin promoter

28 Blood Glucose in Renin Tg Mice

29 Blood Pressures in Renin Tg Mice

30 Albuminuria in Renin Tg Mice

31 Angiotensinogen Renin ACE2 Angiotensin 1-9 ACE2 Angiotensin 1-7 Angiotensin I Angiotensin II ACE Kinins Inactive Peptides AT 1 Receptor

32 Absence of Bradykinin B2 receptor increases diabetic albuminuria PNAS 101: , 2004

33 Bdkr2 +/+ Ins2 +/+ Bdkr2 -/- Ins2 +/+ Bdkr2 +/+ Ins2 +/C96Y Bdkr2 -/- Ins2 +/C96Y

34 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)

35 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

36 Diabetes exacerbates the atherosclerotic lesion in ApoE-/- mice 5 ze (x10 5 ) Lesion si ± 8 % 178 ± 8 % p= ApoE -/- Non-DM ApoE -/- DM

37 Diabetes exacerbates diabetic nephropathy in ApoE-/- mice U-Albumin Excretion (µg/30gbw/d) 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-/-

38 % Aortic Occlusion r 2 =0.84; p = AER (ug/30g BW/d)

39

40 -CBS+/-ApoE-/- given STZ -Supplemented with L-methionine (0.5%) or L-methionine (0.5%)/Folate (200µg/L)

41 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.

42 Albumin Excretion Rate, AER (ug/30gbw/d) * # # # 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

43 35 r 2 =0.63; p=0.03 Ao Occlusion (%) AER (µg/30g/d) Proximal Aortic occlusion is related to AER in ApoE-/-CBS+/- Mice

44 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

45

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