Understanding the Pathogenesis of Nephrotic Syndrome - Studying Signaling Pathways in Response to Nephrotic Plasma

Abstract

Recurrence of steroid-resistant nephrotic syndrome (SRNS) is presumed to be due to an unknown “circulating factor”, the identity of which has so far remained elusive. Our previous work suggests a signaling role for protease-activated receptor-1 (PAR-1), leading to impaired podocyte function. The signaling pathways downstream of PAR-1 in podocytes are unknown and could reveal novel mechanistic insights into the disease. Conditionally immortalized human podocytes (ciPods) were treated with PAR-1 agonist peptide, transforming growth factor-beta 1 (TGF-β1), or nephrotic plasma, in the absence or presence of four different PAR-1 antagonists, TGF-β1 or transient receptor potential canonical type 6 (TRPC6) receptor inhibitors. PAR-1 agonist, TGF-β1, and recurrent nephrotic plasma, but not paired remission plasma, induced the phosphorylation of vasodilator-stimulated phosphoprotein (VASP), c-Jun N-terminal Kinase (JNK), and proteins involved in pro-fibrotic pathways. These changes were inhibited by PAR-1 inhibitors, but not by TGF-β1 inhibition. Four PAR-1 inhibitors demonstrated distinct antagonistic properties. Furthermore, this study suggested the involvement of TRPC6 either as a receptor for the circulating factor or be a part of PAR-1 downstream pathways. The phosphorylation of VASP and JNK in a new 3D spheroid model (GlomSpheres) and from stem-cell-derived kidney organoids corroborated the finding from a 2D ciPods model. Functionally, recurrent nephrotic plasma-induced podocyte motility, and podocyte loss from spheroids both of which were also selectively rescued by PAR-1 inhibitors. Treatment of kidney organoids with recurrent nephrotic plasma-induced VASP and pro-fibrotic phosphorylation in podocytes and the loss of podocyte-specific markers. This work proposes that the circulating factor acts as a pro-fibrotic effector by activating PAR-1, leading to increased podocyte injury. A greater understanding of these signaling pathways will lead to the identification of novel therapeutic targets for this disease.

Date of Award	22 Mar 2022
Original language	English
Awarding Institution	University of Bristol
Supervisor	Moin Saleem (Supervisor) & Gavin I Welsh (Supervisor)