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Written by: Anoushka Krishnan, MD
Figures by: Corina Teodosiu, MD
AcademicCME (www.academiccme.com) is accrediting this educational activity for CE and CME for clinician learners. Please go to https://academiccme.com/kicr_blogposts/ to claim credit for participation
Focal segmental glomerulosclerosis (FSGS) is a common cause of nephrotic syndrome (NS) in adults and can lead to kidney failure. The prevalence of FSGS is increasing worldwide, now the leading glomerular cause of kidney failure in the United States.
A predominant feature of FSGS is glomerular podocyte effacement. Podocytes are terminally differentiated cells lining the outer surface of glomerular capillaries, and form part of the glomerular filtration barrier, serving as a size-selective and charge-dependent molecular sieve. Actin proteins form the main cytoskeleton of the foot processes and anchor the podocyte to the basement membrane (Figure1). Effacement occurs when the actin cytoskeleton of the foot processes breakdown. When podocytes are injured, they lose their structure leading to a reduction in barrier functions and subsequent proteinuria.
Whilst the term FSGS refers to a histopathological pattern of glomerular injury observed on light microscopy, the causes can be broadly sub-divided based on etiology into primary causes and secondary causes; genetic, adaptive, virus-associated, medication-associated and APOL1- associated FSGS (Figure 2). Generally, primary FSGS is a diagnosis of exclusion and is presumably caused by a circulating factor capable of crossing the glomerular basement membrane (GBM), which causes widespread podocyte injury. Primary FSGS commonly presents with abrupt-onset proteinuria and florid nephrotic syndrome.
Figure 1. Schematic of podocyte injury and effacement
Figure 2. Primary ans secondary causes of FSGS
The presence of a permeable circulating factor has long been speculated for a number of reasons. FSGS commonly recurs after kidney transplantation in up to 30-40% of recipients with primary FSGS. Recurrence rate may be up to 80% in those with a history of allograft loss due to recurrent disease. Rarely the disease may recur within hours post-transplant with reports suggesting that plasmapheresis (PP) may result in substantial reduction of proteinuria. Additionally, studies have demonstrated that plasma from patients with FSGS can induce proteinuria in rats. In an interesting case that supports the circulating factor theory, FSGS recurred rapidly in a kidney transplant recipient with biopsy demonstrating foot process effacement. When the kidney was removed and transplanted into a second recipient with kidney failure due to diabetes, the transplant functioned well with reversal of the histopathological lesions of FSGS.
The search for a putative circulating permeability factor (CPF) proven to be pathogenic in FSGS has been elusive. The discovery of the soluble form of the urokinase plasminogen activator receptor (suPAR) created a stir in the glomerular world, seemingly akin to the anti-PLA2R antibody in membranous nephropathy. Just over a decade ago, Wei et al demonstrated elevated levels of suPAR in patients with FSGS as compared to patients with other glomerular lesions or healthy controls. In-vitro and animal studies were highly suggestive of a role for suPAR in the pathogenesis of FSGS. Subsequent studies, however, questioned the specific pathogenic role of suPAR in primary FSGS. While increased suPAR levels were noted in association with atherosclerosis, an inverse relationship was noted with estimated glomerular filtration rate (eGFR) and a positive correlation with the urine protein/creatinine ratio, suggesting that suPAR levels could be elevated in other causes of kidney disease. In fact, after adjusting for eGFR and urine protein, suPAR levels did not seem to be an independent predictor of FSGS. Additionally, there were no significant differences in serum suPAR concentrations among patients with primary FSGS, secondary FSGS and minimal change disease in other studies; thus it cannot be used to accurately distinguish between primary and other forms of FSGS. Whilst it is possible that suPAR is one of many hits to the podocyte, its pathogenic role in FSGS is currently controversial. Current opinion is that routine measurements of suPAR do not add value to clinical care. Other potential factors speculated, but not proven, to contribute to recurrent FSGS include heparinase, hemopexin, angiopoietin-like 4 (ANGPTL4), anti-CD40 antibody and cardiotrophin-like cytokine factor 1. More recently, anti-nephrin antibodies have been implicated as a possible permeability factor inciting minimal change disease in a small cohort of patients with proteinuria and perhaps even in FSGS with two case reports being presented at a recent ASN meeting (2021).
So, what is the need to expand research in this specific area? As patients with primary FSGS have a high risk of disease recurrence, which could respond to therapies like plasmapheresis or immunoadsorption, identification of a circulating factor could predict those at higher risk of disease recurrence. This brings us to the current study: Circulating Permeability Factors in Focal Segmental Glomerulosclerosis: In Vitro Detection (Figure 3).
Visual abstract by Abdul Qader, MD
The aim of the study was to explore novel in vitro bioassays that could predict the presence of CPFs in the plasma of patients with suspected CPFs.
Three different patient cohorts were used for this study- the pilot cohort (consisting of two patients with active FSGS, one without FSGS and one in remission), the nephrotic syndrome (NS) control cohort (consisting of patients with steroid resistant nephrotic syndrome, minimal change disease and membranous nephropathy) and the validation cohort (those with recurrent FSGS after kidney transplant).
Inclusion criteria for those patient samples with presumed CPF included patients with native kidneys with steroid resistant nephrotic syndrome (SRNS) with ongoing kidney function decline despite treatments with immunosuppression such as steroids, cyclosporine and rituximab, who were managed with plasmapheresis (PP) with resultant significant improvement in proteinuria and kidney function (to support the presence of a plasma-derived CPF). The study also included a healthy control pool (8 healthy adult volunteers who had no recent infections, chronic illness, immune disorders or prior use of immunosuppressants).
After the first PP session, the first 500 ml of plasma was stored as “active” FSGS plasma. When patients responded to treatment, similarly, 500 ml of plasma was stored from the final PP session and used as “remission” FSGS plasma.
Podocytes and endothelial cells (glomerular microvascular endothelial cells [GMVECs]) were cultured and incubated with plasma from patients and controls.
The following parameters were investigated
a. Cell viability: by the Cell Counting Kit-8 assay (a colorimetric assay to determine cell viability)
b. Podocyte actin cytoskeleton architecture: using immunofluorescence
c. Reactive oxygen species (ROS): using CM-H2DCFDA probing (a commonly used indicator for ROS)
Results:
a. Podocyte specific cell-death: When plasma from patients with active FSGS was incubated with podocytes, a number of morphological changes were observed in the podocytes after 24 hours, including rounding of the cells, membrane blebbing and full cell detachment. A Cell Counting Kit-8 assay confirmed podocyte cell death, particularly in response to patient active FSGS plasma concentrations of 8% or 10% but not to a plasma concentration of 5%. Cell death was not observed in the GMVECs indicating that the CPF-containing plasma of patients with active FSGS specifically targets podocytes. These changes were not noted in controls or in patients who were in remission (validation cohort).
b. Actin cytoskeleton damage: Within 6 hours of exposure to CPF-containing plasma at a concentration of 8%, the podocyte’s actin cytoskeleton was noted to be severely disrupted. Most cells had no clear actin architecture left in contrast to podocytes exposed to the plasma of the control patient or the FSGS patient in remission.
c. Oxidative stress: Podocytes exposed to 8% active FSGS patient plasma with a potential CPF resulted in formation of reactive oxygen species (ROS) resulting in oxidative stress which can ultimately lead to podocyte death. This change was not seen in the primary GMVECs.
d. CPFs mediated podocyte injury: Podocytes were exposed to other NS samples and healthy controls. ROS production and actin cytoskeleton damage were prominent within 24 hours only in the 8% active FSGS plasma group but not in the other NS samples, non-kidney control and healthy control plasma.
e. Role of in vitro bioassays to predict the presence of a putative CPF: Podocytes exposed to 8% active FSGS plasma or 20% plasma from patients with recurrent FSGS resulted in significant actin cytoskeleton degradation.
To conclude, this study showed that plasma from patients with FSGS (who respond to treatment with PP), can induce injury to human podocytes in vitro. This was evidenced by a significant production of ROS, damage to the actin cytoskeleton and podocyte death. These changes were not seen when sera from healthy and disease control patients were used.
While this study was conducted on a small scale, larger sized studies are required to validate these assays, which in future, may provide a non-invasive method to predict the presence of CPFs in FSGS patients.
AcademicCME (www.academiccme.com) is accrediting this educational activity for CE and CME for clinician learners. Please go to https://academiccme.com/kicr_blogposts/ to claim credit for participation
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