ESPN 53rd Annual Meeting

ESPN 2021


 
In vitro assays to demonstrate the presence of plasma-derived circulating permeability factors in the pathogenesis of focal segmental glomerulosclerosis
SUSAN VEISSI 1 BART SMEETS 2 JOANNA VAN WIJK 3 FLOOR VELTKAMP 5 RENE CLASSENS 1 THEA VAN DER VELDEN 1 ANNELIES JERONIMUS-KLAASEN 1 WILLIAM MORELLO 4 GIOVANNI MONTINI 4 ANTONIA BOUTS 5 LAMBERTUS P. W. J. VAN DEN HEUVEL 1 MICHIEL SCHREUDER 1

1- DEPARTMENT OF PEDIATRIC NEPHROLOGY, AMALIA CHILDRENS HOSPITAL, RADBOUD UNIVERSITY MEDICAL CENTER, RADBOUD INSTITUTE FOR MOLECULAR LIFE SCIENCES, NIJMEGEN, THE NETHERLANDS
2- DEPARTMENT OF PATHOLOGY, RADBOUD UNIVERSITY MEDICAL CENTER, RADBOUD INSTITUTE FOR MOLECULAR LIFE SCIENCES, NIJMEGEN, THE NETHERLANDS
3- DEPARTMENT OF PEDIATRIC NEPHROLOGY, AMSTERDAM UNIVERSITY MEDICAL CENTER, AMSTERDAM, THE NETHERLANDS
4- DEPARTMENT OF PEDIATRIC NEPHROLOGY, DIALYSIS AND TRANSPLANT UNIT, FOUNDATION IRCCS Cà GRANDA, IRCCS OSPEDALE MAGGIORE POLICLINICO, MILAN, ITALY
5- DEPARTMENT OF PEDIATRIC NEPHROLOGY, AMSTERDAM UMC, UNIVERSITY OF AMSTERDAM, AMSTERDAM, THE NETHERLANDS
 
Introduction:

 

Circulating permeability factors (CPFs) involved in the pathogenesis of idiopathic focal segmental glomerulosclerosis (FSGS) can lead to early recurrence of FSGS and kidney failure after transplantation. Identification of FSGS patients with CPFs is clinically important as it can predict treatment response and prognosis. Currently, kidney biopsy is the gold standard diagnosis. Therefore, there is an increased demand for diagnostic assays to determine the presence of CPFs in the sera of FSGS patients. Using conditionally immortalized human podocytes  as a substrate, we aim to demonstrate the presence of plasma-derived CPFs using series of in vitro assays.

Material and methods:

 

Podocytes and primary glomerular endothelial cells (GMVECs) were incubated with plasma from  biopsy proven FSGS patients in relapse and remission as well as from steroid-resistant nephrotic syndrome (SRNS), minimal change NS (MCNS), membranous nephropathy (MN), a non-renal control patient, and healthy controls. Cell viability, podocyte actin cytoskeleton architecture,  and reactive oxygen species (ROS) formation in the presence or absence of ROS scavenger, dimethylthiourea, were investigated by CCK-8 assay, immunofluorescence staining, and CM-H2DCFDA probing, respectively.

Results:

 

We show that plasma of patients with FSGS causes a series of events in podocytes but not in endothelial cells. These events include actin cytoskeleton rearrangement, excessive formation of ROS, and eventually also cell death. These effects were solely observed in response to plasma of relapse FSGS patients, but not in response to plasma of kidney patients with SRNS, MCNS, MN and healthy controls. The co-presence of dimethylthiourea, abolished these effects.

Conclusions:

 

Altogether, we provide a panel of assays to measure podocyte injury and predict the presence of CPFs in FSGS plasma, providing a new framework for monitoring CPF activity that can be used for diagnostics or disease monitoring purposes. Moreover, our findings suggest that the inhibition of ROS formation or facilitating rapid ROS scavenging may exert beneficial effects in FSGS patients.