Michigan/Chicago unit

Michigan/Chicago unit

Modifications in Mouse Models to Enhance Nephropathy/Neuropathy- GLUT1 overexpression Increased oxidative stress Increased glucose metabolic flux or alteration in GLUT expression profiles that will lead to changes in glucose metabolism Altered signaling that may enhance glucose toxicity

GLUT1tg C57Bl/6J model (GT1S) GLUT1 is increased in glomerular cells in diabetes GLUT1 leads to enhanced PKCα and AR activity, fibronectin synthesis Modified β-actin promoter drives GLUT1 in many tissues: high in mesangial cells Bred onto db/db C57BL/6J Effect of high fat feeding (breeder chow; ca 7% fat)

GLUT1tg C57BL/6J (GT1S) model

Mesangial Matrix Scores in NT and GT1S Transgenic Glomeruli at Age 26 Weeks

Mean Cross-Sectional Glomerular Tuft Areas in NT and GT1S Transgenic Mice at Age 26 Weeks

Type IV Collagen Accumulation in Glomeruli of GT1S Transgenic Mice at Age 26 weeks

FN Staining Scores in Nontransgenic and GT1S Transgenic Glomeruli at Age 26 Weeks

GLUT1 Protein is Increased Approximately 7-Fold in the Hearts of GT1S Transgenic Mice

Nontransgenic Heart at Age 16 weeks QuickTime and a Microsoft Video 1 decompressor are needed to see this picture.

GT1S Transgenic Heart at Age 16 Weeks

Increased LV Mass and Volume in GT1S Transgenic Hearts

GLUT1tg C57Bl/6J db/db model (GT1S) GLUT1 is increased in glomerular cells in diabetes GLUT1 leads to enhanced PKCα and AR activity, fibronectin synthesis Modified β-actin promoter drives GLUT1 in many tissues: high in mesangial cells Bred onto db/db C57BL/6J Effect of high fat feeding (breeder chow; ca 7% fat)

Fasting blood sugar GLUT1 tg C57BL/6J db/db model fasting glucoses mg/dl 450 400 350 300 250 200 150 100 50 0 week 12 week 16 week 20 week 24 C57 db+ Glut1OVTG db+ C57 dbdb Glut1OVTG dbdb mg/dl 400 350 300 250 200 150 100 50 0 Final fasting glucoses week 24 C57 db+ (n=10) Glut1OVTG db+ (n=11) C57 dbdb (n=6) Glut1OVTG dbdb (n=4)

Albuminuria GLUT1 tg C57BL/6J db/db model-24 wk 600.00 500.00 * * ug/mg 400.00 300.00 200.00 db/+, n=10 GT1 db/+, n=12 db/db, n=6 GT1 db/db, n=5 100.00 0.00 *p<0.01 vs. db/+

PAS expansion GLUT1 tg C57BL/6J db/db model-24 wk % of Glomeruli area PAS positive 30.00 25.00 20.00 15.00 10.00 5.00 0.00 * * Glomeruli area 6000.00 5000.00 4000.00 3000.00 2000.00 1000.00 0.00 * * WT//db/+, n=12 GT1//db/+, n=11 WT//db/db, n=7 GT1//db/db, n=4 wt db/db wt db/+ GLUT1 db/db GLUT1 db/+ *p<0.001 vs. db/+ GLUT1

AMDCC Definition of Neuropathy Evidence of clinical loss of sensory function Tail flick and hind paw withdrawal latencies (TF and HP) Electrophysiological evidence of nerve impairment Sensory and motor distal latencies and nerve conduction velocity (NCV) Anatomical evidence of nerve fiber loss Intraepidermal fiber density (IEFD

Sensory Tests GLUT1tg C57Bl/6J dbdb Mice TF HP 10 7.5 8 seconds 6 4 seconds 5.0 2.5 2 0 wt db+ wt dbdb tg db+ tg dbdb 0.0 wt db+ wt dbdb tg db+ tg dbdb No Significant Differences

Nerve Conduction Velocity GLUT1tg C57Bl/6J dbdb Mice TDML TSNCV milliseconds 7 6 5 4 3 2 1 meters/second 30 25 20 15 10 5 0 wt db+ wt dbdb tg db+ tg dbdb SMNCV 0 wt db+ wt dbdb tg db+ tg dbdb 50 meters/second 40 30 20 10 0 wt db+ wt dbdb tg db+ tg dbdb

C57BlKS dbdb * * * P< 0.001

C57BlKS dbdb: Sensory Tests * # * P< 0.001 # P < 0.05

C57BlKS dbdb: Nerve Conductions * * * * P< 0.001

Normal Cutaneous Innervation Assessed with Skin Biopsy

Footpad Analysis

Nphs2 GLUT1tg db/db C57BLKS model GLUT1 is highly expressed in podocytes High glucose and diabetes in humans and rodents lead to podocyte loss Nphs2 promoter drives GLUT1 in podocytes db/m C57BKS eggs injected to allow direct examination on diabetic background GLUT1 in human kidney podocytes

Fasting blood sugar and weights-24 wk Nphs2 GLUT1tg db/db C57BLKS model Fasting Blood Glucose Body Weight 600 60 mg% 500 400 300 200 100 C57BKLS, db/+, n=6 Nphs2 GLUT1 tg, db/+ n=13 C57BKLS, db/db, n=3 Nphs2 GLUT1, db/db, n=5 Grams of Body Weight 50 40 30 20 10 C57BKLS, db/+, n=6 Nphs2 GLUT1, db/+, n=13 C57BKLS, db/db, n=4 Nphs2 GLUT1, db/db, n=5 0 0

Albuminuria-24 wk Nphs2 GLUT1tg db/db C57BLKS model Albumin/creatinine ratio 24 h total Albumin Excreted 2500 1000 900 2000 1500 1000 500 * C57BKLS, db+, n=7 Nphs2 GLUT1 tg, db+, n=14 C57BKLS, db/db, n=4 Nphs2 GLUT1 tg, db/db, n=5 micrograms 800 700 600 500 400 300 200 100 * C57BKLS, db+, n=7 Nphs2 GLUT1, db+, n=14 C57BKLS, db/db,n=4 Nphs2 GLUT1, db/db, n=4 0 week 24--repeat 0 *p<0.001 vs. C57BLKS db/db

GLUT1 and nephrin expression- 6-8 wk Nphs2 GLUT1tg db/db C57BLKS model Isolated Glomerular GLUT1 expression Isolated Glomerular Nephrin Expression % (Glut1/beta-Tubulin) 140.00 120.00 100.00 80.00 60.00 40.00 20.00 0.00 * * % (Nephrin/beta-Tubulin) 120.00 100.00 80.00 60.00 40.00 20.00 0.00 *p<0.05 vs. C57BL6 C57BL/6, n=9 Nphs2 GLUT1 tg db/db, n=8

Summary GLUT1 tg in nondiabetic C57BL6 mice results in enlarged glomeruli, increased proteinuria and mesangial matrix expansion. GLUT1 tg in nondiabetic C57BL6 mice results in cardiac hypertrophy in the absence of hypertension. GLUT1 tg in C57BL6 db/db diabetic mice fed with higher fat chow fails to increase proteinuria and mesangial matrix expansion significantly by 6 months of age. High fat feeding in C57BL/6 db/db mice leads to sustained hyperglycemia and more robust nephropathy than in mice fed a normal fat chow. Similarly, high fat feeding in C57BL/6 db/db mice leads to more robust neuropathy than in mice fed a normal fat chow, but not as robust as in C57BLKS mice. Podocyte specific GLUT1 overexpression unexpectedly leads to reduced proteinuria in C57BLKS db/db mice