Journal Club:SuPAR and FSGS

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

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  • Urokinase is a 411-residue protein, consisting of three domains: the serine protease domain, the kringle domain, and the growth factor domain. Urokinase is synthesized as a zymogen form (prourokinase or single-chain urokinase), and is activated by proteolytic cleavage between L158 and I159. The two resulting chains are kept together by a disulfide bond.uPA= urokinase Plasminogen activator
  • Urokinase, a Plasminogen Activator (uPA)
  • It is linked to vascular diseases and cancers
  • Interacts with vitronectin, the uPAR associated protein (uPARAP) and the integrin family of membrane proteins
  • Formed from the cleavage of the GPI anchor. Serum soluble. Same effect mediated by the same mechanisms as uPAR. The D2D3 subunit is active as well, and does not require urokinase for activity.
  • Motility= remodeling= foot process effacement
  • b- pretransplant suPAR levels in primary FSGS (not transplanted) and then recurrent and non-recurrent FSGSc- 1 yr post transplant suPAR levels in those with and without recurrent FSGS
  • But not other glomerular or proteinuric diseases78 human subjects with FSGS, 25 with MCD, 7 preeclampsia, 16 with membranous nephropathy and 22 healthy subjects
  • (a) Immunoprecipitation (IP) was performed by incubating a monoclonal antibody to uPAR with FSGS sera, followed by immunobloting with a polyclonal antibody to uPAR.
  • (c) FSGS sera was precipitated by the monoclonal antibody to uPAR, and then blotted with the antibody to human albumin. For positive control, 1 ml of normal human sera (lane 1 and 11) was loaded. Whereas a heavy band indicative of albumin was observed in the control sera, no overt signal was found in the lanes (2 to 9) loaded with uPAR precipitates from FSGS sera. Removal of albumin antibody and reblotting with anti-uPAR antibody indicates the presence of uPAR in the precipitates (bottom panel, lane 2 to 9).
  • Bottom: recurrent FSGS serum ---- Rec FSGS serum + uPAR mAb proves it is not all uPAR, but some of the binding & activation is due to suPAR---- + cycloRGDfV B3 ingegrin inhibitor
  • RIGHT side is all AFTER transplant reperfusion (left is before and after, showing the difference)
  • Fluorescence-activated cell sorting (FACS) for ß3 ingegrin activity in cultured human podocytes with serum from: Normal Subjects, Non-recurrent FSGS, Recurrent FSGS
  • Fluorescence-activated cell sorting (FACS) for ß3 ingegrin activity in cultured human podocytes with serum from: Normal Subjects, Non-recurrent FSGS, Recurrent FSGS
  • REC=recurrent FSGSCyclo RGD= ß3 integrin small molecule inhibitoruPAR mAb= monoclonal antibody against membrane bound urokinase receptor
  • ß3 integrin activity was measured by incubating pre- and post-plasmapharesis blood with podocytes and then measuring AP5 signal
  • Plaur-/- are uPAR-knockout mice. This means that they cannot produce uPAR, and therefore suPAR, and theoretically cannot activate ß3 integrin ( that is, if they are the only molecules that can activate ß3 integrin, a fact on which this hypothesis is based). Note: we don’t know how high these doses have to be. Is this like saccharin? (laboratory experiments cause cancer in laboratory animals?!)
  • uPAR-stains for uPAR or suPAR depositionSynpo- stains for podocytes (synaptopodin)Merge is the the two merged together
  • AP5-stains for ß3 integrin activationSynpo- stains for podocytes (synaptopodin)Merge is the the two merged together
  • transplantation mouse model (n = 10)
  • PBS is control injection (phosphate buffer saline)LPS=lipopolysaccharide causes suPAR release by macrophages and causes HUGE increases concentration of suPAR in miceElectron microscope analysis of the PBS (n = 3) or LPS (n = 5) treated hybrid kidney. Trust me, this is normal structure and they had normal function.
  • EP= elecroporation- mouse plasmid (sPlaur WT) produces suPAR (known coding for secreted suPAR with domains DI and DII) in the skin. It is introduced by weekly electroporation and causes elevated serum suPAR levels. sPlaur E134 plasmids have point mutation in suPAR DII preventing binding of suPAR with ß3 integrin, though the concentrations of suPAR are the same with both plasmids. The results are above…
  • Podocyte damage is reflected by relating the length of effaced foot process (FP) to the total length of the glomerular basement membrane (GBM) analyzed.4 weeks of continuous suPAR exposure
  • Light microscopy showed features of a progressive glomerulopathy, (hypercellularity, mesangial expansion, mesangiolysis and occasional tuft adhesionsNo immune-complex deposition was seen in any of the mice analyzed.
  • Blinded, semi-quantitative histopathological scoring “reminiscent of early FSGS”
  • IgG is isotype control antibody
  • After 4 weeks of uPAR mAb Rx
  • Semiquantitative electron-microscopic analysis showed significantly improved podocyte foot process structures in the uPAR-specific antibody treatment group, in contrast to the sPlaurWT mice that received control IgG and that developed foot process effacement
  • Journal Club:SuPAR and FSGS

    1. 1. suPAR & FSGS<br />Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis<br />Journal Club<br />Peter Schrier, MD<br />Hofstra NSLIJ Renal Division<br />
    2. 2. The bold claim…<br />suPAR is the (or a) circulating permeability factor that causes FSGS (or at least most primary FSGS)<br />
    3. 3. Outline<br /><ul><li>What is suPAR?
    4. 4. The molecular biology of the urokinase receptor
    5. 5. Biologic pathways involving urokinase
    6. 6. Why suspect suPAR?
    7. 7. Recent studies with the urokinase receptor
    8. 8. History of the search for a circulating permeability factor as the causitive agent in FSGS
    9. 9. The Article
    10. 10. suPAR in FSGS
    11. 11. ß3integrin in FSGS
    12. 12. Mouse Models suggesting causation
    13. 13. Discussion</li></li></ul><li>Urokinase (uPA)<br />411-residue protein encoded by gene PLAU (Plasminogen Activator, urokinase)<br />3 domains: serine protease, kringle, growth factor<br />Prourokinase urokinase via proteolytic clevage<br />Disulfide bond remains<br />
    14. 14. Urokinase- Fibrinolysis<br />
    15. 15. Urokinase- ECM Degredation<br />Cox G et al. Thorax 1999;54:169-179<br />
    16. 16. Urokinase Receptor (uPAR)<br />GPI-anchored three domain glycoprotein<br />Cellular receptor for urokinase (uPA)<br />Upon cleavage, the D2D3 fragment has direct chemotactic activity<br />
    17. 17. uPAR/uPA action<br />
    18. 18. uPAR/Integrin Interaction<br />
    19. 19. uPAR/uPA action<br />
    20. 20. Soluble urokinase receptor (suPAR)<br />
    21. 21. Soluble urokinase receptor (suPAR)<br />Necessary for:<br />Elevated in:<br />Neutrophil trafficking<br />Stem cell mobilization<br />ECM degredation<br />Cancers<br />HIV<br />ICU patients<br />Infection<br />Rheumatic diseases<br />Inflamatory states<br />
    22. 22. So why suspect suPAR?<br />Previous studies<br />
    23. 23. uPAR induction in humans and rodents causes proteinuric renal diseases<br />uPAR in podocytes is required for effacement and proteinuria<br />uPAR orchestrates podocyte motility<br />uPAR activates avß3 integrin in podocytes<br />Interference with avß3 integrin modifies/improves proteinuria<br />Modification of kidney barrier function by the urokinase receptor<br />NATURE MEDICINE VOLUME 14, NUMBER 1, JANUARY 2008<br />
    24. 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.<br />Modification of kidney barrier function by the urokinase receptor<br />NATURE MEDICINE VOLUME 14, NUMBER 1, JANUARY 2008<br />
    25. 25. <ul><li>A vascular permeability factor in lymphocyte culture supernatants from patients with nephrotic syndrome
    26. 26. Biomedicine 1975; 23: 73–75
    27. 27. Rats infused with the serum from a patient with recurrent FSGS developed proteinuria
    28. 28. ClinNephrol 1984; 22: 32–38
    29. 29. Plasmapharesis and immunoadsorption were used successfully for inducing remission of proteinuria, presumably by the removal of the causative circulating PF</li></ul>FSGS circulating permability factor<br />http://www.medscape.com/viewarticle/505656_4<br />
    30. 30. <ul><li>The material eluted from the protein A column in patients with recurrent FSGS treated with immunoadsorption induced proteinuria when injected into rats
    31. 31. N Engl J Med 1994; 330: 7–14.
    32. 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. 33. Transplantation 2001; 73: 366–372</li></ul>FSGS circulating permability factor<br />http://www.medscape.com/viewarticle/505656_4<br />
    34. 34. On to our article…<br />Circulating urokinase receptor as a cause of focal segmental glomerulosclerosis<br />
    35. 35. suPAR is increased in the serum of subjects with FSGS<br />
    36. 36. suPAR is increased in the serum of subjects with FSGS<br />
    37. 37. suPAR levels are higher 1yr post-transplant in patients with FSGS recurrence<br />
    38. 38. suPAR is increased specifically in FSGS with a cutoff of 3000 pg/ml<br />
    39. 39. FSGS-associated suPAR fragment is predominantly the 22kDa protein<br />
    40. 40. suPAR is mostly free rather than albumin-bound<br />
    41. 41. suPAR in FSGS<br />suPAR is a circulating, free protein found in the majority of patients with primary FSGS<br />
    42. 42. In podocyte membrane<br />Ligand for uPAR<br />Binding leads to activation of ß3 integrin<br />Results in foot-process effacement and proteinuria<br />Activated ß3 integrin detected with AP5 ß3 integrin-specific antibody<br />ß3integrin<br />
    43. 43. suPAR binds to and activates ß3integrin<br />FSGS serum or recombinant suPAR<br />ß3-integrin small molecule inhibitor<br />
    44. 44. suPAR binds to and activates ß3integrin in the glomerulus<br />
    45. 45. ß3integrin activation specific for FSGS<br />
    46. 46. In transplant, ß3integrin activation is specific for recurrent FSGS<br />
    47. 47. Therefore...<br />Increased podocyte ß3 integrin activity is a feature of both native and recurrent FSGS<br />
    48. 48. ß3integrin activity is significantly elevated in podocytes incubated with recurrent FSGS pretransplantation serum<br />
    49. 49. suPAR concentrations correlate well with podocyte ß3ingegrin activity<br />
    50. 50. Inhibiting suPAR binding lowers AP5 activity in podocytes<br />
    51. 51. suPAR concentration and podocyte ß3integrin activity decrease with Plasmapharesis in recurrent FSGS<br />
    52. 52. Those with plasmapharesis-induced remission had lower suPAR levels and ß3integrin activity <br />
    53. 53. Those without plasmapharesis-induced remission had higher suPAR levels and ß3integrin activity <br />
    54. 54. Plasmapharesis induces remission in recurrent FSGS<br />by removing suPAR and decreasing ß3 integrin activation<br />
    55. 55. Mouse Models<br />Proof of cause and effect<br />
    56. 56. suPAR induces albuminuria in Plaur-/- knockout mice<br />
    57. 57. suPAR induces albuminuria in Plaur-/- knockout mice<br />
    58. 58. suPAR induces albuminuria in Plaur-/- knockout mice<br />
    59. 59. Creation of hybrid WT/Pleur-/-<br />
    60. 60. Creation of hybrid WT/Pleur-/-<br />
    61. 61. suPAR generated in native kidney can deposit in Pleur-/- kidney<br />
    62. 62. Therefore…<br />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<br />
    63. 63. Creation of ß3 integrin binding-deficient mutant for a control<br />
    64. 64. suPAR binds to ß3integrin and causes proteinuria<br />
    65. 65. suPAR binds to ß3integrin and causes foot process effacement<br />
    66. 66. But…<br />Foot process effacement ≠ FSGS<br />Is this FSGS or MCD or something else entirely?<br />
    67. 67. LM shows features of progressive glomerulopathy similar to FSGS<br />
    68. 68. LM shows features of progressive glomerulopathy similar to FSGS<br />
    69. 69. Theoretical Treatments<br />Can blocking the effects of suPAR lead to disease remission?<br />
    70. 70. uPARmAb blocks suPAR binding to ß3integrin and reduces proteinuria<br />
    71. 71. uPARmAb X 4 wks improved morphology and histopathology scores<br />
    72. 72. uPARmAb X 4 wks improved morphology and histopathology scores<br />
    73. 73. uPARmAb X 4 wks improves podocyte foot process structures<br />
    74. 74. Therefore…<br />Neutralization of suPAR action can improve suPAR-induced renal injury<br />
    75. 75. So… <br />suPAR is a circulating factor that may cause primary FSGS<br />
    76. 76. Cleary the current term FSGS represents a slew of diseases, pathophysiologically<br />suPAR-mediated glomerulopathy is one such disease<br />suPAR mediates its actions through ß3 integrin binding, which in turn causes podocyte foot process effacement<br />It is likely that uPAR also plays a role in disease-causing ß3 integrin activation<br />Discussion- FSGS<br />
    77. 77. Other molecules may activate ß3 integrin. The role of these interactions in kidney disease and in other diseases needs to be elucidated.<br />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<br />(research causes cancer in laboratory animals)<br />Pathology was noted to be “reminiscent of early FSGS,” which is not quite full blown human FSGS<br />Discussion<br />
    78. 78. A lab cut-off point for suPAR must be found<br />3000 pg/ml worked for this study group<br />It is unlikely that mean and SD in the general population will be sufficient to use suPAR as a biomarker for FSGS<br />This study did not include a large number of other proteinuric disease <br />Non-renal inflammatory and malignant diseases were not investigated to define role of suPAR nor suPAR serum concentration <br />Discussion<br />

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