suPAR & FSGS

Circulating urokinase receptor as a cause of focal segmental glomerulosclerosisJournal Club

Peter Schrier, MDHofstra NSLIJ Renal Division

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

Outline What is suPAR?

◦ The molecular biology of the urokinase receptor◦ Biologic pathways involving urokinase

Why suspect suPAR?◦ Recent studies with the urokinase receptor◦ History of the search for a circulating permeability

factor as the causitive agent in FSGS The Article

suPAR in FSGS ß3 integrin in FSGS Mouse Models suggesting causation

Discussion

Urokinase (uPA) 411-residue protein encoded by gene PLAU

(Plasminogen Activator, urokinase)◦ 3 domains: serine protease, kringle, growth factor

Prourokinase urokinase via proteolytic clevage

Disulfide bond remains

Urokinase- Fibrinolysis

Urokinase- ECM Degredation

Cox G et al. Thorax 1999;54:169-179

Urokinase Receptor (uPAR) GPI-anchored three

domain glycoprotein Cellular receptor for

urokinase (uPA) Upon cleavage, the

D2D3 fragment has direct chemotactic activity

uPAR/uPA action

uPAR/Integrin Interaction

uPAR/uPA action

Soluble urokinase receptor (suPAR)

Soluble urokinase receptor (suPAR)

Necessary for: Elevated in:

Neutrophil trafficking Stem cell mobilization ECM degredation

Cancers HIV ICU patients Infection Rheumatic diseases Inflamatory states

So why suspect suPAR?

Previous studies

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

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

A vascular permeability factor in lymphocyte culture supernatants from patients with nephrotic syndrome◦ Biomedicine 1975; 23: 7375�

Rats infused with the serum from a patient with recurrent FSGS developed proteinuria◦ Clin Nephrol 1984; 22: 3238�

Plasmapharesis and immunoadsorption were used successfully for inducing remission of proteinuria, presumably by the removal of the causative circulating PF

FSGS circulating permability factor

http://www.medscape.com/viewarticle/505656_4

The material eluted from the protein A column in patients with recurrent FSGS treated with immunoadsorption induced proteinuria when injected into rats◦ N Engl J Med 1994; 330: 714.�

Plasma obtained from the plasmapheresis of patients with recurrent FSGS and treated with 5070% ammonium sulfate to precipitate plasma �proteins such as albumin and immunoglobulins, contained the presumptive circulating PF◦ Transplantation 2001; 73: 366372�

FSGS circulating permability factor

http://www.medscape.com/viewarticle/505656_4

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

suPAR is increased in the serum of subjects with FSGS

suPAR is increased in the serum of subjects with FSGS

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

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

FSGS-associated suPAR fragment is predominantly the 22kDa protein

suPAR is mostly free rather than albumin-bound

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

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

ß3 integrin

suPAR binds to and activates ß3 integrin FSGS serum or

recombinant suPAR

ß3-integrin small molecule inhibitor

suPAR binds to and activates ß3 integrin in the glomerulus

ß3 integrin activation specific for FSGS

In transplant, ß3 integrin activation is specific for recurrent FSGS

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

ß3 integrin activity is significantly elevated in podocytes incubated with recurrent FSGS pretransplantation serum

suPAR concentrations correlate well with podocyte ß3 ingegrin activity

Inhibiting suPAR binding lowers AP5 activity in podocytes

suPAR concentration and podocyte ß3 integrin activity decrease with Plasmapharesis in recurrent FSGS

Those with plasmapharesis-induced remission had lower suPAR levels and ß3 integrin activity

Those without plasmapharesis-induced remission had higher suPAR levels and ß3 integrin activity

Plasmapharesis induces remission in recurrent

FSGSby removing suPAR and decreasing ß3 integrin activation

Mouse ModelsProof of cause and effect

suPAR induces albuminuria in Plaur-/- knockout mice

suPAR induces albuminuria in Plaur-/- knockout mice

suPAR induces albuminuria in Plaur-/- knockout mice

Creation of hybrid WT/Pleur-/-

Creation of hybrid WT/Pleur-/-

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

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

Creation of ß3 integrin binding-deficient mutant for a control

suPAR binds to ß3 integrin and causes proteinuria

suPAR binds to ß3 integrin and causes foot process effacement

But…Foot process effacement ≠ FSGS

Is this FSGS or MCD or something else entirely?

LM shows features of progressive glomerulopathy similar to FSGS

LM shows features of progressive glomerulopathy similar to FSGS

Theoretical Treatments

Can blocking the effects of suPAR lead to disease remission?

uPAR mAb blocks suPAR binding to ß3 integrin and reduces proteinuria

uPAR mAb X 4 wks improved morphology and histopathology scores

uPAR mAb X 4 wks improved morphology and histopathology scores

uPAR mAb X 4 wks improves podocyte foot process structures

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

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

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

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

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