Soluble Urokinase-Type Plasminogen Activator Receptor (suPAR) in Focal Segmental Glomerulosclerosis

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© Springer Japan 2016

Kazunari Kaneko (ed.)Molecular Mechanisms in the Pathogenesis of Idiopathic Nephrotic Syndrome10.1007/978-4-431-55270-3_9

9. Soluble Urokinase-Type Plasminogen Activator Receptor (suPAR) in Focal Segmental Glomerulosclerosis

Jochen Reiser  and Nada Alachkar2

Department of Medicine, Rush University Medical Center, 1735 West Harrison Street, Cohn Building, Suite 724, Chicago, IL 60612, USA

Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
Jochen Reiser
Focal and segmental glomerulosclerosis (FSGS) is a histopathological entity that identifies a group of glomerular kidney disorders, which manifest by a certain pattern of sclerosis that involves parts of some glomeruli (focal segmental) on light microscopy. In most cases of FSGS, in particular the primary or idiopathic FSGS, the first site of the damage is the podocyte, which marks the beginning of this disease. However, FSGS can be a secondary process to another injury in the glomeruli, giving the definition of secondary FSGS. A large number of pathogenic factors have been identified, which lead to podocyte injury and, thereafter, to FSGS. Several genetic predispositions and mutations have been confirmed, especially in young patients, causing an early onset of primary FSGS. Acquired causes of FSGS constitute a large list of factors that may directly or indirectly injure the podocyte cells. Identifying these factors in the cases of primary or idiopathic FSGS has been the focus of extensive research investigations. For many decades, researchers speculated the presence of circulating factors to be the pathogenic causes of primary FSGS. These factors are thought to be the cause of FSGS recurrence post-kidney transplantation as well. However, not until recently, these factors are being identified. In 2011, soluble urokinase plasminogen activator receptor (suPAR) was suggested to be a circulating factor leading to primary FSGS and post-transplantation FSGS recurrence.

Focal segmental glomerulosclerosissuPARPermeability factors

9.1 Introduction

Primary or idiopathic focal segmental glomerulosclerosis (FSGS) is the most common cause of end-stage renal disease (ESRD) caused by primary glomerular disease in the United States [1], affecting both children and adults. Furthermore, FSGS recurs very commonly post-kidney transplantation in approximately 30–40 % of adult patients and much higher (80 %) in children [2], in many cases very shortly after transplantation, but can recur at any time.
In addition to our understanding of pathogenesis of FSGS that identified the podocyte cell as the originating site of this disease, circulating soluble urokinase plasminogen activator receptor (suPAR) has become the focus of extensive researches, making it the most accepted pathogenic factor that leads to, what we considered in the past, an idiopathic type of FSGS [3]. Podocyte foot process effacement is considered the first mark of injury and closely correlated with a loss of function in glomerular permeability and the characteristic hallmark of proteinuric glomerular disorders resulting in FSGS. Circulating suPAR is thought to bind to a receptor on the podocyte cell membrane leading to cell injury and death, resulting in glomerular hyalinosis, sclerosis, and chronic kidney disease [3].
In this chapter, we will present the current knowledge of suPAR as a pathogenic factor of primary and recurrent FSGS. We will review the recent data on the source of suPAR, the type of pathological suPAR, and its effect on the podocyte cells. Additionally, we will assess the relevant clinical data that support the suPAR role in this complicated disease.

9.2 Circulating Permeability Factors in FSGS

For couple decades, serum circulating permeability factor/s was proposed to exist in patients with primary FSGS and suggestive of the rapid recurrence of the disease after kidney transplantation. An early data indicated that the circulating permeability factor was a non-immunoglobulin protein with a molecular weight of approximately 30–50 kd [4]. A high level of this type of protein was detected in patients with FSGS recurrence post-kidney transplantation compared with much lower levels in normal subjects. To prove that this protein is the possible permeability factor, the researchers showed that serum obtained from patients with primary FSGS increases the albumin permeability of isolated culture glomeruli. Additionally, patients with recurrent FSGS had higher permeability to albumin compared to normal subjects or patients without recurrences, and plasmapheresis resulted in a significant decrease in permeability effect and proteinuria in patients with FSGS recurrence [5]. In a later study, the investigators confirmed that recurrent FSGS after kidney transplantation was much higher in patients whose glomerular albumin permeability sera was substantially higher compared with those whom sera had less permeability activity [4]. Similar to adults’ data, researchers showed the same findings in pediatric patients; recurrence occurred in most of children whose serum increased the glomerular albumin permeability compared with those with negative effect [6].
However, in spite of this long prediction of the presence of circulating permeability factor/s as the most likely causes of primary or idiopathic FSGS and the reason for this disease to recur after kidney transplantation, these early studies were unable to identify the exact molecular type or the mechanism of action of these factors.

9.3 Soluble Urokinase Plasminogen Activator Receptor (suPAR)

In 2011, Wei and colleagues presented the first work on suPAR and its role in FSGS disease. After an extensive work that lasted for more than a decade, the researchers reached a tipping point in identifying the permeability factor; and serum soluble urokinase receptor (suPAR) was the most evident factor. Applying their work in the laboratory to the clinical setting, the investigators found that serum suPAR was indeed elevated in subjects with primary FSGS, but not in control group with other glomerular diseases such as minimal change disease (MCD) or membranous nephropathy (MN). Furthermore, a significantly higher level of suPAR before transplantation was detected in patients who later on developed recurrence of FSGS after kidney transplantation [3]. In Reiser et al’s previous work, podocyte urokinase receptor was found to play a significant role in glomerular disease [7]. On the cellular level, uPAR is a glycosylphosphatidylinositol (GPI)-anchored three-domain protein, making up a cellular receptor for urokinase that serves as a versatile signaling orchestrator via association with other transmembrane receptors, including integrins [810]. Furthermore, uPAR can be released from the cell membrane forming a soluble molecule (suPAR) by cleavage of the GPI anchor [9]. In the blood circulation or at the cell membrane level, suPAR is further cleaved [11] at the linkage region between domains, releasing three types of fragments: DI, DI–III, and DII DIII; the latter is thought to play the major role in the pathogenesis of FSGS (unpublished data). Consistent with the earlier findings of the permeability factors, suPAR was also found to be a protein ranging between 20 and 50 kD molecular weight, depending on the degree of glycosylation and proteolytic cleavage [10]. However, suPAR is present under physiological conditions in low concentrations in non-FSGS human serum, with a known role in neutrophil trafficking and stem cell mobilization [9].

9.4 Source of suPAR

There has been a great interest in identifying the source of the pathological suPAR in FSGS and the cause of its release in the circulation. Long-standing data showed that uPAR is expressed on various types of cells and interacts with ligand urokinase plasminogen activator (uPA). As a result of inflammatory [12] or other stimulations, uPAR is cleaved from the cell surface, such as the monocytes [12], by protease enzymes leading to the formation of the soluble form of the receptor, suPAR, which can be detected via different assays in blood and urine. In addition to the inflammatory cells, endothelial [13], cancerous [14], and other cells are able to release uPAR from the cell membrane. Therefore, low levels of suPAR existed in normal subjects, in contrast to much higher levels that correlate with pathological conditions and associate with worse diseases’ prognosis [15, 16].
Although high suPAR levels have been documented in different disorders, such as sepsis and cancers, most of these disorders were not associated with proteinuric or FSGS findings. Therefore, it is possible that the source and the pathological types of suPAR are different in each disorder. Ongoing investigations are focusing on various sites of the immune system as the source of suPAR in FSGS animal models as well as in patients; the results so far have been promising.

9.5 suPAR’s Mechanism of Action

In 1996, Harold Chapman’s group reported binding of cell membrane-anchored uPAR to integrin [8]. This paper provided the base for bidirectional signaling of uPAR through cell-surface receptors. Our own experiments provided the data that circulating suPAR can bind to and activate podocyte β(3) integrin [3]. Binding of suPAR to podocyte β(3) integrin is causing its activation. This activation depends on the type of suPAR (domain and glycosylation structure) as well as the species in which this binding occurs. A particular soluble form of suPAR is created by alternative splicing of IMAGE cDNA clone 3158012 resulting in a variant of suPAR containing parts of suPAR domains I and II [3]. Expression of this type of suPAR causes foot processes effacement and progressive injury in mice resembling FSGS-like glomerular changes. The short-term effects of three-domain suPAR are accordingly weaker when infused into wild-type mice, establishing the concept that podocyte injury may result from podocyte β(3) integrin activation based on reaching a threshold [3] which relies on the suPAR variant to sufficiently activate this receptor.
Additionally, using rodent models of glomerular disease suggested that inducible podocyte-specific expression of the constitutively active nuclear factor of activated T cells 1 (NFATc1) which represent a downstream target of calcium signaling may increase podocyte uPAR expression by binding to the urokinase-type plasminogen activator receptor (Plaur) gene promoter (which encodes uPAR). Such an increase in podocyte uPAR expression may favor podocyte cell dynamics/motility via activation of β(3) integrin, all independent of T cells, causing foot process effacement [7, 17]. These changes can be blocked by cyclosporine use and NFAT-siRNA or cell-permeable NFAT inhibitors [18].
Recently, data emerged that linked the podocyte-protective effects of rituximab to the stabilization of podocyte SMPDL3b, a molecule participating in plasma membrane lipid composition [19]. In a follow-up paper, Yoo et al. have shown that SMPDL3b may bind suPAR to allow for modulation of podocyte function in conditions with high or low podocyte SMPDL3b expression [20].
Finally, Kobayashi et al. suggested a PAI-1/uPA complex to mediate uPAR-dependent podocyte β1-integrin endocytosis and introduce a novel mechanism of glomerular injury, leading to progressive podocytopenia [21].

9.6 The Pathological Type/s of suPAR in FSGS

As indicated above, three types of suPAR fragments have been identified, depending on the cleavage sites of the molecule. Clinical data showed that different suPAR sub-domains are associated with different disorders, e.g., levels of suPARI–III and suPARII–III are higher in ovarian cancer [22] and not suPARI; however, data beyond the total suPAR level that would systematically assess the potential involvement of suPAR sub-types that correlate with FSGS pathology is not available yet. Therefore, identifying suPAR sub-domain/s that particularly strongly activate podocyte β(3) integrin and thus may lead to FSGS has been the focus of ongoing work by several investigators. This examination of pathological sub-type/s of suPAR in comparison to the full length of suPAR will be important and will probably explain the differences of phenotypes between different suPAR forms utilized in various animal models [3, 23, 24] and some of the open questions when measuring suPAR with commercial ELISA [25].

9.7 Clinical Data of suPAR in Primary FSGS

Since the discovery of the suPAR’s active role in FSGS, cumulative clinical data have emerged to confirm this role.
Circulating suPAR was investigated in two well-characterized cohorts of children and adults with biopsy-proven primary FSGS: 70 patients from the North America-based FSGS clinical trial (CT) and 94 patients from PodoNet, the Europe-based consortium studying steroid-resistant nephrotic syndrome [26]. The investigators measured the level of circulating suPAR levels in the serum obtained from these cohorts at time of disease diagnosis and after therapy. Serum suPAR levels were elevated in 84.3 % and 55.3 % of patients with FSGS patients in the CT and PodoNet cohorts, respectively, compared with 6 % of controls (P < 0.0001). In multiple regression analysis, the investigators found that lower suPAR levels were associated with higher estimated GFR, male gender, and treatment with mycophenolate mofetil. In the PodoNet cohort, patients with a nephrosis 2, idiopathic, steroid-resistant (podocin) (NPHS2) mutation had higher suPAR levels than those without a mutation [26].
Another study sought to identify the role of suPAR in predicting the response to the main therapy for FSGS steroid. Li and colleagues enrolled 109 patients with biopsy-proven primary FSGS between 2011 and 2013; the patients were treated with prednisone and followed up for 6–24 months. These patients were compared with control groups that consist of 96 healthy volunteers, 20 MCD patients, and 22 patients with MN. Using ELISA methods, suPAR levels were measured in all patients and controls. Patients with FSGS had significantly higher suPAR levels (median, 3512 [interquartile range (IQR), 2232–4231] pg/ml) than healthy controls (median, 1823 [IQR, 1563–2212] pg/ml; P < 0.001), patients with MCD (median, 1678 [IQR, 1476–2182] pg/ml; P < 0.001), and patients with MN (median, 1668 [IQR, 1327–2127] pg/ml; P < 0.001). When the investigators used a level of 3000 pg/ml as a cutoff, they found that suPAR was elevated in 48.6 % of patients with FSGS, in contrast to 5 % of patients with MCD and 4.5 % of those with MN. Additionally, when using a level of 3400 pg/ml as the threshold, the investigators found that suPAR level was independently associated with steroid response in patients with FSGS (odds ratio, 85.02; P = 0.001); patients who were responsive to steroids had significantly higher suPAR levels than nonsensitive patients (median, 3426 [IQR, 2670–5655] pg/ml versus 2523 [IQR, 1977–3460] pg/ml; P = 0.001). Interestingly, patients who had initially suPAR levels ≥ 3400 pg/ml had a significant decrease in these levels (median, 4553 [IQR, 3771–6120] pg/ml), compared to those with levels <3400 pg/ml, in whom the level did not change after therapy [27].
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