Peter Schrier, MD discusses the role of SUPAR in FSGS, a recent journal club discussion.

1. suPAR & FSGS Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis Journal Club Peter Schrier, MD Hofstra NSLIJ Renal Division

2. The bold claim… suPAR is the (or a) circulating permeability factor that causes FSGS (or at least most primary FSGS)

4. The molecular biology of the urokinase receptor

5. Biologic pathways involving urokinase

6. Why suspect suPAR?

7. Recent studies with the urokinase receptor

8. History of the search for a circulating permeability factor as the causitive agent in FSGS

9. The Article

10. suPAR in FSGS

11. ß3integrin in FSGS

12. Mouse Models suggesting causation

14. Urokinase- Fibrinolysis

15. Urokinase- ECM Degredation Cox G et al. Thorax 1999;54:169-179

16. Urokinase Receptor (uPAR) GPI-anchored three domain glycoprotein Cellular receptor for urokinase (uPA) Upon cleavage, the D2D3 fragment has direct chemotactic activity

17. uPAR/uPA action

18. uPAR/Integrin Interaction

19. uPAR/uPA action

20. Soluble urokinase receptor (suPAR)

21. Soluble urokinase receptor (suPAR) Necessary for: Elevated in: Neutrophil trafficking Stem cell mobilization ECM degredation Cancers HIV ICU patients Infection Rheumatic diseases Inflamatory states

22. So why suspect suPAR? Previous studies

23. uPAR induction in humans and rodents causes proteinuric renal diseases uPAR in podocytes is required for effacement and proteinuria uPAR orchestrates podocyte motility uPAR activates avß3 integrin in podocytes Interference with avß3 integrin modifies/improves proteinuria Modification of kidney barrier function by the urokinase receptor NATURE MEDICINE VOLUME 14, NUMBER 1, JANUARY 2008

24. The functional value of active avß3 integrin as a downstream effector for increased podocyte motility, which in this case equals foot process effacement and proteinuria, is an attractive outlook for integrin or uPAR inhibitor studies in humans for proteinuric renal diseases. Modification of kidney barrier function by the urokinase receptor NATURE MEDICINE VOLUME 14, NUMBER 1, JANUARY 2008

26. Biomedicine 1975; 23: 73–75

27. Rats infused with the serum from a patient with recurrent FSGS developed proteinuria

28. ClinNephrol 1984; 22: 32–38

29. Plasmapharesis and immunoadsorption were used successfully for inducing remission of proteinuria, presumably by the removal of the causative circulating PFFSGS circulating permability factor http://www.medscape.com/viewarticle/505656_4

31. N Engl J Med 1994; 330: 7–14.

32. Plasma obtained from the plasmapheresis of patients with recurrent FSGS and treated with 50–70% ammonium sulfate to precipitate plasma proteins such as albumin and immunoglobulins, contained the presumptive circulating PF

33. Transplantation 2001; 73: 366–372FSGS circulating permability factor http://www.medscape.com/viewarticle/505656_4

34. On to our article… Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis

35. suPAR is increased in the serum of subjects with FSGS

36. suPAR is increased in the serum of subjects with FSGS

37. suPAR levels are higher 1yr post-transplant in patients with FSGS recurrence

38. suPAR is increased specifically in FSGS with a cutoff of 3000 pg/ml

39. FSGS-associated suPAR fragment is predominantly the 22kDa protein

40. suPAR is mostly free rather than albumin-bound

41. suPAR in FSGS suPAR is a circulating, free protein found in the majority of patients with primary FSGS

42. In podocyte membrane Ligand for uPAR Binding leads to activation of ß3 integrin Results in foot-process effacement and proteinuria Activated ß3 integrin detected with AP5 ß3 integrin-specific antibody ß3integrin

43. suPAR binds to and activates ß3integrin FSGS serum or recombinant suPAR ß3-integrin small molecule inhibitor

44. suPAR binds to and activates ß3integrin in the glomerulus

45. ß3integrin activation specific for FSGS

46. In transplant, ß3integrin activation is specific for recurrent FSGS

47. Therefore... Increased podocyte ß3 integrin activity is a feature of both native and recurrent FSGS

48. ß3integrin activity is significantly elevated in podocytes incubated with recurrent FSGS pretransplantation serum

49. suPAR concentrations correlate well with podocyte ß3ingegrin activity

50. Inhibiting suPAR binding lowers AP5 activity in podocytes

51. suPAR concentration and podocyte ß3integrin activity decrease with Plasmapharesis in recurrent FSGS

52. Those with plasmapharesis-induced remission had lower suPAR levels and ß3integrin activity

53. Those without plasmapharesis-induced remission had higher suPAR levels and ß3integrin activity

54. Plasmapharesis induces remission in recurrent FSGS by removing suPAR and decreasing ß3 integrin activation

55. Mouse Models Proof of cause and effect

56. suPAR induces albuminuria in Plaur-/- knockout mice

57. suPAR induces albuminuria in Plaur-/- knockout mice

58. suPAR induces albuminuria in Plaur-/- knockout mice

59. Creation of hybrid WT/Pleur-/-

60. Creation of hybrid WT/Pleur-/-

61. suPAR generated in native kidney can deposit in Pleur-/- kidney

62. Therefore… Post-transplant recurrent FSGS can be can be caused by a circulating factor (suPAR) created in native kidneys that activates ß3 integrin in the transplanted kidney

63. Creation of ß3 integrin binding-deficient mutant for a control

64. suPAR binds to ß3integrin and causes proteinuria

65. suPAR binds to ß3integrin and causes foot process effacement

66. But… Foot process effacement ≠ FSGS Is this FSGS or MCD or something else entirely?

67. LM shows features of progressive glomerulopathy similar to FSGS

68. LM shows features of progressive glomerulopathy similar to FSGS

69. Theoretical Treatments Can blocking the effects of suPAR lead to disease remission?

70. uPARmAb blocks suPAR binding to ß3integrin and reduces proteinuria

71. uPARmAb X 4 wks improved morphology and histopathology scores

72. uPARmAb X 4 wks improved morphology and histopathology scores

73. uPARmAb X 4 wks improves podocyte foot process structures

74. Therefore… Neutralization of suPAR action can improve suPAR-induced renal injury

75. So… suPAR is a circulating factor that may cause primary FSGS

76. Cleary the current term FSGS represents a slew of diseases, pathophysiologically suPAR-mediated glomerulopathy is one such disease suPAR mediates its actions through ß3 integrin binding, which in turn causes podocyte foot process effacement It is likely that uPAR also plays a role in disease-causing ß3 integrin activation Discussion- FSGS

77. Other molecules may activate ß3 integrin. The role of these interactions in kidney disease and in other diseases needs to be elucidated. While mice are good models of human renal disease, murine serum suPAR was not measured, and the relationship to human suPAR concentrations is not certain (research causes cancer in laboratory animals) Pathology was noted to be “reminiscent of early FSGS,” which is not quite full blown human FSGS Discussion

78. A lab cut-off point for suPAR must be found 3000 pg/ml worked for this study group It is unlikely that mean and SD in the general population will be sufficient to use suPAR as a biomarker for FSGS This study did not include a large number of other proteinuric disease Non-renal inflammatory and malignant diseases were not investigated to define role of suPAR nor suPAR serum concentration Discussion