Tatjana Simić Institute of Medical and Clinical Biochemistry,

1. Molecular and functional interpretation of the stress response and polymorphic expression of genes associated with cardiovascular and cancer risk in CKD Tatjana Simić Institute of Medical and Clinical Biochemistry, Faculty of Medicine, University of Belgrade, Serbia

2. Presentation Agenda • New concept of oxidative stress and its mechanisms in CKD • Glutathione transferases – structure and function • GST polymorphisms as endogenous modulator of oxidative stress and prognosis in end-stage renal disease patients • Association of GST polymorphism and risk of urothelial cancer

3. New Definition of Oxidative Stress “a disturbance in the pro-oxidant–antioxidant balance in favor of the former.” . Sies H, 1985 “a disruption of redox signaling and control.” Jones D, Antiox Redox Signal 2006

4. The “Golden mean” of redox homeostasis Ursini F. et al, Redox Biol, 2016

5. Redox sensitive transcription factors and signaling pathways regulated by redox homeostasis Wang T. et al, Breast Cancer, 2014

6. Redox imballance in chronic kidney disease Balkan endemic nephropathy

7. Antioxidants Prooxidants • Diet with low intake of fruit and vegetables (low intake of antioxidant vitamins) • Lower activity of antioxidantenzymes • Increased production of free radicalscaused by inflamation • Increased production of free radicalsduring dialysis Causes of redox imballance in patients withchronicrenal failure

8. Biomarkers of oxidative stress in CRF patients: increased oxidation of albumin andincreased level of lipid peroxidation byproducts • MDA and F2-isoprostanes levels increased in CKD (Gondouin et al., Nephron 2015) (Handelman et al, Kidney International 2001) • Loss of protein thiols and increased content of protein carbonyls, AGEs and nitrotyrosine (Capusa et al, Kidney Blood Press Res. 2012) (Arsov S et al, Clin Chem Lab Med. 2014 ) (Pirrodi et al, Amino Acids 2010) (Galli F, NDT 2007) • Increased concentrations of 8-OH-deoxyguanosine (Sung et al, Oxidative Medicine and Cellular Longevity, 2013) Mimic-Oka et al. Clin Nephrol 1999 Da Silva et al., Biomedicine&Pharmacotherapy 2007 .

9. Activity of extracellular glutathione peroxidase is decreased, SOD activity is increased in CRF patients Glutation peroksidaza (U/L plazme) Klirens kreatinina (ml/min) Mimic-Oka J. et al. Clinical Nephrology, 1999

10. GST is upregulated in peripheral blood cells of ESRD patients GSTP1 GSTM1 J Mimic-Oka Clinical Nephrology 1995 J Mimic Oka Amino Acids 1992 A Noce et al. Cell Death and Disease, 2012

11. Nuclear factor erythroid 2-related factor 2 (Nrf2) controls several different antioxidants pathways including GSTs

12. NRF2 as the master regulator of antioxidant responses Hayes JD. et al, Trends Biochem Sci, 2014; Gorrini C. et al, Nat Rev Drug Discov, 2013

13. Presentation Agenda • New concept of oxidative stress and its mechanisms in CKD • Glutathione transferases – structure and function • GST polymorphisms as endogenous modulator of oxidative stress and prognosis in end-stage renal disease patients • Association of GST polymorphism and risk of urothelial cancer

14. GSTs in detoxification reactions Simic T. et al. Nat Rev Urol, 2009

15. Functions of cytosolic glutathione transferases • Catalytic functions • Conjugation – detoxification of electrophiles • Reduction – antioxidant activity • Dehydroascorbate reductase activity • S-Glutathionylation • Deglutathionylation • Isomerisation – steroid synthesis, tyrosine metabolism • Sulfatase activity • Prostaglandin D synthase activity • Functions with impact on cell survival pathways • Regulation of mitogen activated protein kinases (MAPK) • Mitogenic influence on myeloproliferative pathways • Cellular partner of HPV-16 E7 oncoprotein in preventing cell death Tew and Townsend, Antioxidants & redox signaling, 2013 Wu and Dong, Trends in Pharmacological Sciences, 2012

16. GSTО – new enzyme, new roles 2. Dehydroascorbate reductase activity 1. thioltransferase activity 3. Monomethylarsenate reductase activity GSTO2-2 enzyme is up to 100 fold more active Whitbread, Methods Enzymol, 2005

17. Structure and GSH-binding site of a GST enzyme Wu B, Dong D, Trends Pharmacol Sci. 2012

18. Glutathione transferase superfamily Alpha class: GST A1-1, A2-2, A3-3, A4-4, A5-5 Mu class: GST M1-1, M2-2, M3-3, M4-4, M5-5 Pi class: GST P1-1 Theta class: GST T1-1, T2-2 Cytoslic GST Zeta class: GST Z1-1 Omega class: GST O1-1, O2-2 Sigma class: GST S1-1 Glutathione transferases (GST) Kappa class: GST K1-1 Mitochondria GST Microsomal GST (MAPEG)MGST1, MGST2, MGST3 Wu B, Dong D, Trends Pharmacol Sci. 2012

19. Major types of GST genes and their SNP distributions nsSNP: non-synonymous SNP; sSNP: synonymous SNP; 3′UTR: 3′ untranslated region; 5′UTR: 5′ untranslated region; iSNP: intronic SNP. NCBI database dbSNP (http://www.ncbi.nlm.nih.gov/Projects/SNP) for computational analysis. Functional analysis of nsSNPs by sequence and structural homology based method (SIFT and Polyphen). Yadav et al., Genomics Data, 2014

20. GSTM1 and GSTT1 deletion polymorphisms result in absent enzyme activity • GSTM1 gene at chromosome 1 (1p13.3) • Distribution: GSTM1-active 50%, GSTM1-null 50% • GSTT1 gene at chromosome 22 (22q11.23) • Distribution: GSTT1-active 80%, GSTT1-null 20% http://www.genecards.org/

21. GSTA1 and GSTP1 polymorphisms result in altered enzyme activity • rs 3957356 • GSTA1 gene variants : GSTA1*A and GSTA1*B. • Three SNPs: T-567G, C-69T and G-52A in promoter • Distribution: GSTA1*AA38.7%, GSTA1*AB45.2%, GSTA1*BB 16.1% • rs 1965 (A313G), Ile105Val • Two GSTP1 gene variants : GSTP1*Ileand GSTP1*Val • Distribution: GSTP1*Ile/Ile 34.8%, GSTP1*Ile/Val 48.7%, GSTP1*Val/Val 16.5% http://www.genecards.org/

22. GSTO1 and GSTO2 polymorphisms result in altered enzyme activity • rs4925 (Ala140Asp) GSTO1 gene at chromosome 10q24.3 • Allele distribution: A140-69.8%, D140-30.2% • rs15667 (Asn142Asp) GSTO2 gene at chromosome 10q24.3 • Allele distribution: N142-65.6%, D142-34.1% Mukherjee, Drug Metab Dispos, 2006

23. GST polymorphisms associated diseases (I) Hollman A et al., Int J Env Res Pub He, 2016; Sies H, Packer L, Method Enzymol, 2005

24. GST polymorphisms associated diseases (II)

25. Presentation Agenda • New concept of oxidative stress and its mechanisms in CKD • Glutathione transferases – structure and function • GST polymorphisms as endogenous modulator of oxidative stress and prognosis in end-stage renal disease patients • Association of GST polymorphism and risk of urothelial cancer

26. Clinical characteristics of patients with end stage renal disease (ESRD) and control group

27. Study design • Determination of GSTA1, GSTM1, GSTO1, GSTO2, GSTP1 and GSTT1 gene polymorphism • Determination of 8 oxidative stress byproducts and 2 adhesive molecules • Protein thiols, protein carbonyls, Nitrotyrosine, AOPP • MDA, MDA adducts • Total oxidant status, prooxidant-antioxidant balance • sVCAM-1 and sICAM-1 • Long term follow up of cardiovascular complications, overall and cardiovascular mortality in cohort of 199 patients with end stage renal disease

28. GST polymorphisms and risk of ESRD development Suvakov S, PhD thesis No significance Statistically significant

29. The presence of the GSTM1 null genotype results in a stronger oxidative modification of proteins Suvakov S. et al., NDT 2013

30. Biomarkers of the oxidative lipid damage in patients with ESRD according to GST genotype Suvakov S. et al., NDT 2013

31. Oxidative stress is most pronounced in ESRD patients with combined GSTM1 / T1 null-genotype Suvakov S, PhD thesis

32. The role of adhesion molecules in atherosclerosis sVCAM-1 measured in plasma of ESRD patients sICAM-1 measured in plasma of ESRD patients ** * AA AB BB Active Null leIle IleVal ValVal Active Null AA AB BB Active Null leIle IleVal ValVal Active Null GSTA1 GSTM1 GSTP1 GSTT1 GSTA1 GSTM1 GSTP1 GSTT1 Blakenberg et al, Atherosclerosis 2003

33. Effect of incubation of HUVECs with uremic serum on monocyte adhesion Suvakov S, PhD thesis

34. GSTM1-null genotype is an independent predictor of overall and cardiovascular mortality in hemodialysis patients Suvakov&Simic, 2016, unpublished

35. Patients with GSTP1-low activity genotype had shorter survival compared to GSTP1 active genotype Suvakov et al. BMC Nephrology, 2014 Suvakov&Simic, 2016, unpublished

36. Kaplan-Meier survival curves for overall and cardiovascular survival of ESRD patients dichotomized as above (>64.3µmol/L) or below (<64.3µmol/L) the median levels of AOPP Suvakov&Simic, 2016, unpublished

37. Kaplan-Meier survival curves for overall and cardiovascular survival of ESRD patients dichotomized as above (>649.6 ng/ml) or below (<649.6 ng/ml) the median levels of VCAM-1 Suvakov&Simic, 2016, unpublished

38. Presentation Agenda • New concept of oxidative stress and its mechanisms in CKD • Glutathione transferases – structure and function • GST polymorphisms as endogenous modulator of oxidative stress and prognosis in end-stage renal disease patients • Association of GST polymorphism and risk of urothelial cancer

39. Polymorphic expression of genes associated with cancer risk in CKD Meta analysis of more than 32,000 participants from 6 studies that the risk of urinary tract carcinoma is doubled at dialysis population Wong et al, BMC Cancer 2016

41. Conclusions

42. Prof. dr Jasmina Mimić-Oka Dr Sonja Šuvakov Prof. Dr Tatjana Pekmezović Prof. Dr Ana Savić-Radojević Prof. Dr Marija Plješa-Ercegovac Doc. Dr Marija Matić Dr Tatjana Đukić Dr Vesna Ćorić Msc Tanja Radić Prof. dr Nada Dimković Dr sci. med Tatjana Damjanović Prof. dr Sanja Simić Ogrizović Prof. Francesco Galli Prof. dr Steva Plješa