PhD Scientific Days 2021

CL_IV_L: Clinical Medicine IV. Lectures

The molecular basis of the exceptional dominant transmission of NPHS2-associated nephrotic syndrome

Text of the abstract

Introduction: The most frequently mutated gene in steroid-resistant nephrotic syndrome is NPHS2. The associated nephrotic syndrome is transmitted in an autosomal recessive fashion. Recently, two de novo heterozygous NPHS2 last-exon frameshift mutations (p.L330Pfs*17 and p.L330Vfs*15) were reported in families with autosomal dominant FSGS. The protein encoded by NPHS2, is a component of the glomerular slit diaphragm. We recently showed that it regulates the distance between neighbouring nephrin molecules, the principal components of the slit diaphragm.
Aims: We aimed to determine the molecular basis of the dominant transmission of NPHS2-associated glomerulopathy.
Methods: We coexpressed eGFP-tagged wild type (wt) podocin with haemagglutinin-tagged (HA) wt, p.L330Pfs*17 or p.L330Vfs*15 podocins in cultured podocytes. After 48 hour incubation plasma membrane was stained with CF405M-conjugated wheat germ agglutinin (WGA) and cells were fixed by ethanol. Immunohistochemical staining was performed with primary anti-HA and secondary AlexaFluor568-conjugated antibodies. The ratio of the membrane-localized podocin and the colocalization of the coexpressed variants were assessed by FiJi software on confocal microscopic images. The calculated coefficients were compared pairwise, using Wilcoxon-test with Bonferroni correction.
Results: The GFP-tagged wt podocin strongly colocalized with the HA-tagged truncated or wt podocin variants. In monoexpression the two frameshift variants were localized intracellularly. While wt podocin is membrane-localized when coexpressed with HA-tagged wt podocin, it is retained in intracellular compartments in the presence of HA-tagged L330Pfs*17 and HA-L330Vfs*15 variants.
Conclusion: The dominant podocin variants retain wt podocin in intracellular compartments explaining the exceptional dominant transmission of NPHS2-associated glomerulopathy.
Funding: Supported by the ÚNKP-20-3-I-SE-29 New National Excellence Program of the Ministry for Innovation and Technology from the source of the National Research, Development and Innovation Fund.

University and Doctoral School

Semmelweis University, Károly Rácz Doctoral School of Clinical Medicine