1. Role of Muc1 in Acute Kidney Injury Rebecca P Hughey Sandra J Gendler and Cathy S Madsen College of Medicine, Mayo Clinic, Scottsdale, AZ Timothy A Sutton and Henry E Mang Indiana University School of Medicine, Indianapolis, IN Sheldon Bastacky, Kenneth R Hallows, Núria M Pastor-Soler, Mohammad M. Al-bataineh and Carol L Kinlough University of Pittsburgh School of Medicine, Pittsburgh, PA

2. INJURY RECOVERY Adapted from Bonventre and Yang 2011  Hypoxia-inducible factor pathway (HIF-1)   -catenin protective pathway → cyclin D1  p53 pathway: apoptosis or growth-arrest-and-repair  EGF receptor A ROLE FOR MUC1 IN ISCHEMIA-REPERFUSION INJURY AND RECOVERY? Most common cause in patients is hypotension or sepsis – occurs in 5-7% of hospitalized patients with 40-60% mortality and contributes to end-stage renal disease. Epithelia damage is found primarily in the S3 segment of the proximal tubule with loss of cytoskeletal integrity, shedding of brush border membrane into the tubule, loss of tight junctions and thereby loss of cell polarity  Reduced oxygen  Altered metabolism  Reactive oxygen species  Edema, inflammation and cell death

3. VHL protein Prolyl hydroxylase HIF-1  -OH  O 2 +  KG CO 2 + succinate The link between HIF-1 activity and metabolism LDHA Glucose Pyruvate Lactate Acetyl-CoA TCA cycle HIF-1  +  PKM2 (-) PDH PDK1 Extracellular Membrane Cytosol enolase NUCLEAR PKM2 transactivates HIF-1 and  -catenin

4. TRANSACTIVATION OF GENES FOR SHIFTING GLUCOSE METABOLISM FROM OXIDATIVE TO GLYCOLYTIC: Glucose transporters, glycolytic enzymes, LDHA, regulatory PDK1 and PKM2 PKM2 PHD3 HIF-1  +  on HRE cyclin D1 CELL SURVIVAL AND REPAIR  -catenin PKM2 PHD3 PKM2 PKM2 transactivates  -catenin PKM2 & PHD3 Transactivate HIF-1 (2) Cross-talk between HIF-1 and  -catenin protective pathways

5. TRANSACTIVATION OF GENES FOR SHIFTING GLUCOSE METABOLISM FROM OXIDATIVE TO GLYCOLYTIC: Glucose transporters, glycolytic enzymes, LDHA, regulatory PDK1 and PKM2 PKM2 PHD3 MUC1 HIF-1  +  on HRE cyclin D1 CELL SURVIVAL AND REPAIR  -catenin MUC1 PKM2 PHD3 MUC1 PKM2 MUC1 & PKM2 transactivate  -catenin MUC1, PKM2 & PHD3 Transactivate HIF-1 Cross-talk between HIF-1 and  -catenin protective pathways

6. N C Autocatalytic cleavage within SEA module membrane (PD/ET/SRPAPGSTAPP/AAHGVTSA) Near perfect tandem repeats Binding sites for numerous kinases,  -catenin and PKM2 N-linked glycans O-linked glycans MUC1 STRUCTURE  MUC1 is a type 1 transmembrane protein  Extensive mucin-like glycosylation on tandem repeats  Autocatalytic cleavage at a SEA module  Cytoplasmic domain conserved across species

7. N C N-linked glycans O-linked glycans membrane MUC1 FUNCTION  MUC1 mutation – Medullary Cystic Kidney Disease (MCKD-1) Kirby et al. 2013, Nature Genetics  MUC1 polymorphism (LSP) correlates with low blood Mg +2 Meyer et al. 2010, PLoS Genetics  Muc1 global KO mice have no overt phenotype  MUC1 overexpression in tumors is a bad prognosis for the patient; in tumor cell lines, MUC1 has pro-survival and anti- apoptotic activities  MUC1 in pancreatic tumors and cell lines induces key enzymes that shift metabolism from oxidative phosphorylation to glycolysis (metabolomics, ChIP, qPCR) Chaika et al 2012 PNAS MUC1 IN THE KIDNEY  Apical expression on polarized epithelial cells (during development)  Found in the distal nephron in adult kidneys; found in the proximal nephron during development and after injury

8. Is Muc1 protective in a mouse model of ischemia-reperfusion injury? Serum creatinine Blood urea nitrogen (BUN) Muc1 knockout mice versus congenic control C57BL/6 19 min clamp and recovery t=0, 4 h, 24 h and 72 h (n=3-6)

9. Muc1 is protective in ischemia-reperfusion injury One kidney was fixed in PFA and slices stained with H&E

10. Wu et al., J. Clin. Invest. 117:2847–2859 (2007) Scoring of tubular damage: 0 none 1≤ 10 211-25% 326-45% 446-75% 5 >76% C57BL/6 Muc1 KO t=0congestedcongestedn=3 t=4h4, 2, 35, 4, 5n=3 t=24h5, 5, 5, 55, 5, 5n=3-4 t=72hrecoveringcalcificationn=5-6 Muc1 is protective in ischemia-reperfusion injury

12. Muc1 is induced by ischemia-reperfusion injury Muc1 knockout mice and congenic C57BL/6 mice 19 min clamp Recovery t=0, 4 h, 24 h or 72 h Homogenize ¼ kidney – 60  g protein per lane for immunoblotting CT2 antibody

13. VHL protein Prolyl hydroxylase HIF-1  -OH  O 2 +  KG CO 2 + succinate The link between HIF-1 activity and metabolism LDHA Glucose Pyruvate Lactate Acetyl-CoA TCA cycle HIF-1  +  PKM2 (-) PDH PDK1 Extracellular Membrane Cytosol enolase NUCLEAR PKM2 transactivates HIF-1 and  -catenin

14. Muc1-dependent induction of glycolytic enzymes during IRI LDHA Enolase LDHA Glucose Pyruvate Lactate Acetyl-CoA TCA cycle HIF-1  +  PKM2 (-) PDH PDK1 Extracellular Membrane Cytosol enolase * *

15. Muc1-dependent changes in PDK1 during IRI PDK1 LDHA Glucose Pyruvate Lactate Acetyl-CoA TCA cycle HIF-1  +  PKM2 (-) PDH PDK1 Extracellular Membrane Cytosol enolase * * *

16. Muc1-dependent changes in PHD3 and PKM2 during IRI PHD3 PKM2 LDHA Glucose Pyruvate Lactate Acetyl-CoA TCA cycle HIF-1  +  PKM2 (-) PDH PDK1 Extracellular Membrane Cytosol enolase * * * * * No significant changes in GLUT1 or GLUT2 *

17. The absence of Muc1 during IRI activates AMPK AMP-activated kinase (AMPK) is an energy sensor that is phosphorylated in response to high AMP/ATP ratios AMPK pAMPK RATIO: pAMPK to AMPK

18. Does Muc1 stimulate the protective  -catenin pathway? LDHA Glucose Pyruvate Lactate Acetyl-CoA TCA cycle HIF-1  +  PKM2 (-) PDH PDK1 Extracellular Membrane Cytosol enolase NUCLEAR PKM2 transactivates HIF-1 and  -catenin MUC1 co-immunoprecipitates with HIF-1 and  -catenin - 115K IB: HIF-1  - 94K IB:  -catenin - 25K IB: Muc1 CT2 IP: - CT EC - CT EC for Muc1 Normoxic Hypoxic HK-2 cells

19. Muc1-dependent induction of  -catenin and downstream cyclin D1  -catenin Cyclin D1

20. Total Akt Phospho-Akt RATIO: pAkt/Akt Akt activation was not altered in the absence of Muc1 during IRI Zhou et al 2012 Kidney International  -catenin deletion in all tubules p53 p-Akt Bax Caspase activation and apoptosis

21. SUMMARY  MUC1 is induced by hypoxia in HK-2 cells: co-IP with HIF-1  and  -catenin  Muc1 is induced by IRI in the mouse kidney  Muc1 is protective in IRI based on sCr, BUN and kidney morphology  The shift to glycolysis in response to IRI is Muc1-dependent by modulating levels of key enzymes: LDHA, enolase, PHD3, PKM2 and PKD1  The activation of AMPK during IRI is enhanced and prolonged in the absence of Muc1, likely due to metabolic/energy stress (AMPK also responds to calcium stress)  IRI produces a Muc1-dependent increase in  -catenin levels with differential effects on downstream targets cyclin D1 and Akt QUESTIONS REMAINING:  Tubule site of Muc1 action? HIF-1  levels and promoter occupancy?  Mechanism of AMPK activation?  Role of inflammation?

22. FUNDING: NIH R01 Dean’s Bridge Funding Dialysis Clinics, Inc. NIDDK O’Brien Kidney Centers (P30) Pittsburgh, Indiana and UTSW Winters Foundation ACKNOWLEDGMENTS Sandra J Gendler and Cathy S Madsen College of Medicine, Mayo Clinic, Scottsdale, AZ Timothy A Sutton and Henry E Mang Indiana University School of Medicine, Indianapolis, IN Sheldon Bastacky, Kenneth R Hallows, Núria M Pastor-Soler, Mohammad M. Al-bataineh and Carol L Kinlough University of Pittsburgh School of Medicine, Pittsburgh, PA

23.  ME-resistant Muc1 heterodimers increase in mouse kidney after cecal ligation and puncture (with Wen and Kellum) Sham 1 2 3 - 50 - 25 With  -ME - 37 - 25 - 37 Without  -ME 1 2 31 2 3 4 CLP 48 hCLP 24 h Dimer < CT - Dimer <