Advanced Glycation End Products in Diabetic Kidney Disease

1. Advanced Glycation End Products in Diabetic Kidney Disease Grishma Parikh, MD Mount Sinai School of Medine 5thAnnual Friedman Fellows Symposium 11/17/2012

2. AGEs • Advanced Glycation End products (AGEs) • Formed by non-enzymatic glycosylation of proteins and lipids • Potent inducers of inflammation and ROS • Source: • Exogenous : Food and Cigarettes • Endogenous : Normal metabolism and Aging • Excess: Linked to major chronic diseases

3. Formation of AGEs (Oxidants) HbA1c AGEs Proteins Lipids RNA/DNA RO· A Glucose + B HO· ROS MG C CML Dry heat AGEs toxicity is due both to the release of ROS and alterations of proteins and lipids. H.Vlassara:Annals of Medicine, 2008 3 3 3 Striker

4. ROS AGEs Regulate Oxidant Stress Food Intake Exogenous Oxidants (AGEs) NAPDH ox, PKC, Mitochondrial Oxidant Stress etc. Glucose, Fatty Acids ROS, AGEs, NOS E.R. Stress Inflammation, Proliferation, Apoptosis or Cell Death Koschinsky, T., Vlassara et al. PNAS 1997 Diet is a major source of excess oxidants (AGEs).

5. What is the Source of AGEs?

6. AGE Formation Depends on Temperature of Cooking Regular diet (U/mg) Low AGE diet (U/mg) Beef: broiled STEWED 2000 5367 Chicken:broiled 5245 1011 STEWED Salmon: broiled 1348 SUSHI 502 MASHED Potato:fried 1522 17 Oxidant (AGEs) Content of Common Foods A 50% Reduction may be sufficient clinically. This is not “starvation”, it is French cooking!!

7. What is the effect of restriction of AGEs in diet?

8. Diabetic Subjects: AGE-Restricted Diet (x 4-6 wks) Reduces Inflammatory Markers hsCRP - TNF 1 α VCAM 100 50 10 25 50 0 % from baseline 0 0 - 10 - 25 - 50 ** * ** - 50 - 20 H.Vlassara, Proc.Nat.Acad.Sc: ‘O2 8 8 Striker et al.

9. Type 2 Diabetes Trial: AGE Restriction and Insulin Resistance (x4 mos) Plasma Insulin (μU/ml) Leptin (ng/ml) 25 40 20 * 30 * 15 20 10 10 5 0 0 Pre- Post- Pre- Post- AGE-Restriction Reduces Plasma Insulin and Leptin Vlassara group: Db Care, 2011

10. Type 2 Diabetes Trial: AGE Restriction (4 mo) AGER1 ADIPONECTIN SIRT-1 * 250 * * 12 500 200 10 400 8 150 300 6 100 200 4 50 100 2 0 0 0 NL NL NL pre pre post post pre post AGE-Restriction Normalizesanti-oxidant defenses in T2D: i.e. AGER1, SIRT-1 and Adiponectin Vlassara group: Db Care, 2011

11. Renal Function and AGEs • The kidneys are a primary site for metabolism and excretion of oxidants • Decreased renal function: increased OS and inflammation (TNFα) due to reduced anti-oxidant defenses Striker et al.

12. Renal Function and Serum AGEs eGFRCrvs. Aging (years) eGFRCr vs. Serum CML r = - 0.447 p = 0.0001 r = - 0.647 p = 0.0001 60 200 50 150 40 30 100 20 50 10 0 0 20 40 60 80 100 0 50 100 150 200 (years) eGFRCr • AGEs and Renal Function are Linked • Oral AGEs Influence Systemic AGEs at All Levels of Renal Function Uribarri, J, Vlassara , H, JAGS, 2009

13. Serum Urine 30 High AGE 60 25 Low AGE 20 sAGE (u/ml) 15 30 10 5 0 0 20 40 0 20 40 hours hours 30 25 20 sAGE (u/ml) 15 10 5 0 0 20 40 hours Serum/Urine AGEs in Diabetes after Meals NORMAL : GFR= 100, μ/alb DIABETIC GFR= 84, proteinuria 60 30 0 20 40 hours AGE excretion can be reduced prior to clinically significant changes in GFR T. Koshinsky. H.Vlassara PNAS 1997

14. Urine MG (nmoles/24 hr) Urine CML (nmoles/24 hr) 20,000.0 400.00 *p<0.05 * p<0.025 16,000.0 300.00 12,000.0 200.00 8,000.0 100.00 4,000.0 0.0 0.00 1st 5th visit 1 5thvisit AGE-Restriction Effect on Oxidant Excretion (MG) and/or filtration (CML) in T2D AGE Restriction Restores Urinary Excretion of AGEs (active and filtration) This also applies to Normal Adults!! Vlassara group, 2009

15. 8-iso AGER1 TNFα RAGE VCAM-1 sCML sMG -100 -50 0 50 100 % Change T2 Diabetic Nephropathy and AGE restriction (4 mo) AGE-Restricted Diet Restores Anti-ox (AGER1) Levels, Promotes Renal Elimination of AGEs, and Reduces OS/Inflammation (i.e.TNFα) Lowering oxidant intake allows kidneys to excrete AGEs, which reduces OS and Inflammation. Vlassara et al: PNAS 2002; JCEM, 2009; Diab Care 2-12

16. AGE-Restricted Diet Prevents Diabetic Nephropathy in NOD Mice Standard Diet AGE-Restricted Restriction of AGEs – not nutrients or blood glucose – can Prevent CKD in T1D-Susceptible Mice Peppa, M.: Diabetes J., 2005

17. Ways to Decrease AGE Uptake • Change AGEs in food • Western diet is rich in AGEs and NOT EVERYONE CAN FOLLOW THE DIET • Sequester AGEs in the gut and eliminate them in the stool • Sevelamer is a non-absorbable polymer that was found to reduce HbA1c and lipids, similar to effects seen in patients treated with a low-oxidant diet. This suggested it might remove AGEs

18. Binding of AGEs in vitro: Sevelamer Sequesters AGEs in a pH-dependent manner Sevelamer 100 * 80 % Bound 125 I-AGE-BSA 60 40 20 0 AGE AGE + pH=1 AGE + pH=7.0

19. Therefore we proposed a Pilot Cross-over Study in 20 T2D patients with CKD Stages 2-4 with the following Hypotheses: 1) Sevelamer carbonate treatment would result in a 20% decrease in serum AGEs within 8 weeks. 2) Renvela treatment would decrease serum inflam/ROS markers by 10% within 8 weeks. No Medications were changed, all participants were on drugs to control BP, many were on statins, insulin, etc..

20. SEVELAMER Lowers OS and Inflammation and Restores AGER1, SIRT1 and FGF23 mRNA in T2D with CKD SEVELAMER CaC03 (CONTROL) * SIRT1 AGER1 * 100 75 50 AGER1 TNFα sMG 8-iso sCML SIRT1 25 0 -25 * * * * * -50 FGF23 TNFα sMG 8-iso sCML -75 -100 % Change Vlassara group, CJASN, 2011

21. SEVELAMER CaC03 20 Total Chol LDL-Chol 10 HbA1c Trig 0 % Change -10 * * -20 * * HbA1c Trig Total Chol LDL-Chol METABOLIC CHANGES: Sevelamer Carbonate Lowers HbA1c and Plasma Lipids in T2D with CKD Vlassara group, CJASN, 2011

22. Sevelamer lowered serum (and cellular) AGE levels, by sequestering AGEs in the gut Sevelamer lowered markers of Oxidative Stress and inflammation and decreased FGF23 Sevelamer lowered HgbA1c, and lipids (t-chol, triglycerides, even though most were on statins) These data suggest that Sevelamer reduces AGEs as one primary target A longer/larger study is now being conducted to validate these results Conclusions Pilot Trial Striker et al.

23. Proposed New Study of Sevelamer: Examine the Effects of Sevelamer Carbonate onCardio-Metabolic Risk Factors and Selected Aspects of Kidney Function in Type 2 Diabetics with Stage 2-4 CKD Hypothesis: Sevelamer carbonate (Renvela) will lower inflammation and oxidative stress in the body by absorbing AGEs in the gut and removing them from the body. Striker et al.

24. Study Design • 6 month prospective, comparative study • 120 patients randomized to receive, in parallel, either: • Sevelamer carbonate (1600 mg tid) • Calcium carbonate (1200 mg tid) • Endpoints • Primary: HbA1c and AGEs • Secondary: markers of glucose metabolism, inflammation, oxidative stress Dietary AGE content is assessed

25. Recruitment • MSSM: • 89 randomized • Beth Israel: • 49 randomized • Sponsor: Genzyme-Sanofi • Industry sponsored (investigator-initiated)

26. Summary AGEs elevate levels of inflammatory markers and oxidative stress The diet is a major source of oxidants (AGEs) in DM. The kidneys are the major sites of disposal of oxidants, (and AGEs), so treatment/prevention of CKD is critical Major internal and external sources of ROS/Infl and AGEs can be managed with current medications Sevelamer reduce oxidants (AGEs) and improves anti-oxidants, lipids, FGF23 and glucose control in T2D Striker et al.

29. Many Thanks to Team AGE MSSM Beth Israel New York Endocrinology: Leonid Poretsky, Augustin Busta, Carla Romero, Yun Feng Nephrology: Nikolai Harbord • Gary Striker • Helen Vlassara • Nephrology: Jaime Uribarri, John He, Shirisha Guthikonda • Endocrinology: Ronald Tamler STUDY COORDINATORS AND MANY OTHER COLLABORATTORS…………