Nephrotic syndrome (NS) is the second leading cause of pediatric chronic kidney disease. Pathogenic de novo C-terminal truncating variants in the gene TRIM8 (tripartite motif containing 8) causes a syndrome of NS and epilepsy. In contrast, N-terminal truncating variants are observed in gnomAD control subjects, indicating haploinsufficiency does not cause disease. Based on previously reported TRIM8-TRIM8 interactions, we hypothesized that NS-associated TRIM8 variants may impact wildtype TRIM8 through dominant-negative effects. Alternatively, these variants may have gain-of-function mechanisms in the development of NS. MG132, a 26S proteasome inhibitor, was used to modulate wildtype (WT) and patient variant containing (PV) TRIM8 protein levels. Protein-protein interactions were determined using co-immunoprecipitation of WT- and PV-TRIM8 protein upon transfection of immortalized podocytes with tagged cDNA constructs. Ubiquitination of WT and PV TRIM8 protein was measured by immunoprecipitation and western blotting upon overexpression of tagged Ubiquitin and TRIM8 cDNA constructs in immortalized podocytes. MYC-tagged WT and PV TRIM8 half-lives were determined through cycloheximide (CHX) treatment over a five-hour timed trial. CHX, used to inhibit the elongation step in eukaryotic protein translation, was administered to prevent protein synthesis. CHX-chase assay was performed to determine steady state protein stability of PV-TRIM8 relative to WT. Tagged TRIM8 WT protein levels increased upon treatment with MG132 whereas protein levels of tagged TRIM8 PV proteins were not. Flag-tagged TRIM8 PV proteins, but not flag-tagged WT protein, co-immunoprecipitated with MYC-tagged TRIM8 WT protein. MG132 rescued co-immunoprecipitation of FLAG- and MYC-tagged WT TRIM8 proteins. Tagged TRIM8 WT protein undergoes polyubiquitination, which is impaired by TRIM8 patient variants. PV-TRIM8 had a significantly longer half-life when compared to the WT. TRIM8 PV cause reduced TRIM8 polyubiquitination, impaired 26S proteasomal degradation, and increased TRIM8-TRIM8 interactions, conferring a gain-of-function mechanism of disease. / 2026-03-07T00:00:00Z

Nephrotic syndrome (NS) is the second leading cause of chronic kidney disease (CKD) presenting under the age of 30. NS presents in children with edema and severe proteinuria, caused by the effacement of podocyte foot processes within the glomerular filtration barrier. Patients with steroid-resistant NS (SRNS) frequently develop end-stage renal disease (ESRD). Additionally, renal biopsies from these patients often reveal focal segmental glomerulosclerosis (FSGS). Pathogenic mutations in known monogenic disease genes have been found in 11-45% children with FSGS/SRNS. Notably, most Mendelian etiologies exhibit recessive inheritance, while dominant vertical inheritance with incomplete penetrance is observed in the remainder. The role of de novo variants (DNVs) in NS necessitates further investigation. Tripartite motif containing 8, TRIM8, is an E3 ubiquitin ligase. De novo TRIM8 variants were previously implicated in a syndromic disease consisting of neurodevelopmental delay, epilepsy, cerebral atrophy, and nephrotic syndrome. In this study, we recapitulate the patient-specific mutations in inducible overexpression cell lines and in CRISPR/Cas9-generated mouse models. N-terminal MYC or GFP-tagged TRIM8 inducible cell lines were generated and characterized using the pInducer21 system. Western blot and immunofluorescence data show that MYC- and GFP-TRIM8 were induced by doxycycline in immortalized podocyte cell lines. Candidate interactors for TRIM8 from the literature and stratified using kidney single cell mRNA sequencing expression were cloned into mammalian expression vectors. Finally, a Trim8 knockout allele (c. 56_162del; p.H20Qfs*124 and c.367_463+304delins46) was generated and bred to yield an allelic series of wildtype, heterozygous and homozygous animals. These mice exhibited normal survival and did not demonstrate proteinuria through three to four months of life. Overall, further studies are ongoing with regards to the continued monitoring of proteinuria and kidney dysfunction, as well as the potential interactor cloning and cell line characterization. / 2025-02-06T00:00:00Z