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Background: Membranous nephropathy (MN) is amongst the most common causes of adult nephrotic syndrome worldwide. For the majority of patients, it is an autoimmune condition associated with anti-PLA2R autoantibodies. Despite recent advances in our understanding of the condition, much remains unknown and treatment has not changed in two decades. Here we use immunoadsorption therapy to directly remove IgG antibodies from patients with active autoimmune MN to induce remission. Methods: Using Peptide-GAM immunoadsorption we treated 12 patients with autoimmune MN for five consecutive days. Primary outcome was reduction of anti-PLA2R antibodies at day 14. Secondary outcomes were, change in anti- PLA2R level compared to baseline, uPCR, eGFR, EQ5D and adverse events at months 3, 6 and 12. Immune system modelling was carried out using flow cytometry to study cell populations of B cells, PLA2R specific B cells, T cell and monocytes. Results: At week 2 (day 14), median antibody level increased from a baseline of 679 U/mL (IQR 191-1070) to 902 U/mL (IQR 522-2665). Three patients have completed follow up with the first patient becoming antibody negative at week two and remaining negative at last follow up. Using flow cytometry, we demonstrated evidence for a new pathway in the pathogenesis of the disease in the role of natural T Regs and the potential involvement of IgM antibodies. Conclusion: Immunoadsorption therapy to directly remove the pathogenic anti- PLA2R antibody has potential efficacy in autoimmune MN and could avoid the need for toxic medications. We have also described for the first time important new components in the disease pathway to help further our understanding of the underlying mechanisms.

Background: Nephrotic syndrome (NS) occurs when the glomerular filtration barrier becomes abnormally permeable, leading to the clinical triad of proteinuria, massive oedema, and hypoalbuminaemia. Historically, NS has been thought to result from dysregulation of the immune system, although recent evidence suggests the glomerular podocyte plays a central role in disease pathogenesis. Children with NS are generally treated with an empiric course of glucocorticoid (Gc) therapy; a class of steroids which are activating ligands for the glucocorticoid receptor (GR) transcription factor. A major factor limiting the clinical utility of these agents is the marked variation observed in response to treatment. Although Gc-therapy has been the cornerstone of NS management for several decades, the mechanism of action, and target cell, remain poorly understood. Hypothesis and aims: The central hypothesis for this thesis states that glucocorticoids act directly on the podocyte to produce clinically useful effects without involvement of the immune system. Findings: Using a wild-type human podocyte cell line, I demonstrated that the basic GR-signalling mechanism is intact in the podocyte, and that glucocorticoids produce a direct, protective effect on the podocyte without immune cell involvement, by using electrical resistance across a podocyte monolayer as a surrogate marker for barrier integrity. To understand potential mechanisms underpinning this direct effect I defined the podocyte GR cistrome (using a combination of chromatin immunoprecipitation followed by massively parallel DNA sequencing and transcriptomic analysis) as well as total cell proteomics. Subsequent gene ontology analysis revealed that Gc treatment had prominent effects on podocyte motility, and these findings were validated with live-cell imaging. To gain mechanistic insight, I investigated the role of the pro-migratory small GTPase regulator Rac1, and demonstrated that treatment with Gc reduced Rac1 activity. Furthermore, the Rac1 inhibitor EHT 1864 had a direct, protective effect on the podocyte. To create a model to study the role of podocyte GR in vivo I generated a mouse line with a podocyte-specific GR deletion. Impact: Gc exposure produces potentially clinically-relevant effects directly on the podocyte, and Gc-induced podocyte hypomobility may underlie the clinical efficacy of these agents. Future animal studies investigating the consequences of GR deletion in the podocyte and the anti-proteinuric effects of Rac1 inhibition are warranted.

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.

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.

Introduction: The etiology of steroid sensitive nephrotic syndrome (SSNS) is not well understood. Genetic studies have established common single nucleotide polymorphisms (SNPs) that are associated with increased SSNS disease risk. We review previous genetic association studies of SSNS and nominate particular transcriptional regulators and immune cells as potential key players in the etiology of this disease.Methods: A list of SNPs associated with SSNS was compiled from published genome wide association and candidate gene studies. The Regulatory Element Locus Intersection (RELI) tool was used to calculate the enrichment of the overlap between disease risk SNPs and the genomic coordinates of data from a collection of >10,000 chromatin immunoprecipitation (ChIP-seq) experiments.Results: After linkage disequilibrium expansion of the previously reported tag associated SNPs, we identified 192 genetic variants at 8 independent risk loci. Using the RELI algorithm, we identified transcriptional regulators with enriched binding at SSNS risk loci (10-05 < pcorrected < 10-124), including ZNF530, CIITA, CD74, RFX5, and ZNF425. Many of these regulators have well-described roles in the immune response. RNA polymerase II binding in B cells also demonstrated enriched binding at SSNS risk loci (10-37

Chronic kidney disease (CKD) affects over 500 million people worldwide, and despite this significant disease burden, therapeutic innovation in nephrology has lagged. This trend is partly attributed both to an incomplete understanding of the molecular mechanisms of kidney dysfunction and a lack of appropriate in vitro and in vivo models, a reflection of the multifactorial nature CKD and the complex cellular complexity of the kidney. Genetically defined rare diseases can provide insight into disease pathogenesis. We thus, sought to understand the mechanism underlying a rare Coenzyme Q (CoQ)-deficiency-associated podocytopathy. Podocytes are a terminally differentiated, post-mitotic cell type, essential for kidney filter function. An in vitro podocyte model of CoQ deficiency revealed a susceptibility to ROS-mediated injury and a perturbation in polyunsaturated fatty acid metabolism. Single nucleus sequencing from the kidneys of CoQ-deficient mice validated podocyte-specific transcriptomic changes consistent with in vitro findings and revealed a novel, disease-specific parietal epithelial cell population. Our analysis further revealed the BRAF-targeting GDC-0879 as a putative therapeutic strategy. Kidney organoids derived from human induced pluripotent stem cells (iPSCs) are an emerging technology with the potential to both recapitulate the cellular diversity of the kidney and to further our understanding of disease. However, this progress is dependent on understanding the reproducibility and quality of organoids derived from multiple patients-donors. Thus, we generated a 412,358 single cell census of 47 organoid and iPSC samples derived from four cell lines across four time points of differentiation. All cell lines contained representative segments of the developing nephron, but varied in their proportions of cell types and in the presence of off-target populations. Comparison to single cell fetal kidney data revealed that the organoids were most similar to first trimester fetal kidney. While long-term in vitro culture did not affect organoid composition, in vivo transplantation of organoids resulted in reduced off-target populations.

Background: IgM nephropathy is a variant of minimal change disease (MCD) and is the commonest cause of nephrotic syndrome among Thai adults. Although patients with IgM nephropathy generally respond well to treatment with corticosteroids, patients often have a remitting and relapsing course. The pathogenesis is not well understood. It has been proposed that MCD or IgM nephropathy reflects a disorder of T-lymphocytes. Recently, abnormalities in natural regulatory T cells (nTreg) have been implicated in a number of autoimmune diseases. Objective: This is a pilot study to examine the number of Foxp3+CD4+ T cells in patients with MCD or IgM nephropathy. Methods: Eleven patients (2 patients with MDC and 9 patients with IgM nephropathy) and 25 normal control subjects were studied. T cell subpopulations were measured by flow cytometry. Results: Overall, nTreg were identified by the presence of Foxp3+CD4+ T cells. There were no significant differences in number of CD3+, CD4+ T cells population and Foxp3+CD4+ T cells in MCD/IgM patients compared with normal subjects. In a subgroup analysis, there was a significant increase in Foxp3+CD4+ numbers in untreated MCD/IgM patients with active disease compared to controls. Conclusion: These results suggesting correlation between activation of immune system in pathogenesis. Natural regulatory T cell as detected with Foxp3+CD4+ was increased in active MCD/IgM nephropathy patients.

Introduction: Current guidelines differ on screening for latent tuberculosis (LTB) infection prior to starting immunosuppressive therapy. We developed a Markov state-transition model to determine the most “cost-effective” screening strategy prior to initiating steroid treatment in a 5 year old with newly diagnosed idiopathic nephrotic syndrome. Methods: Using data from the published literature, universal purified protein derivative (PPD) testing was compared with targeted screening using a risk-factor questionnaire. A secondary model also included testing with the newer interferon gamma release assays (IGRAs), requiring only a single visit and having greater specificity than the PPD. Results: At an LTB prevalence of 1.1%, universal PPD testing was more costly and less effective than other strategies. Instead, we found that a targeted strategy using a simple risk assessment questionnaire to determine who should receive a PPD cost roughly $44,000 per quality-adjusted life year (QALY) gained compared to a no screening strategy. Conversely, at an LTB prevalence >12.8%, universal PPD became “cost-effective” compared to targeted screening. In the secondary model, targeted screening with a questionnaire followed by IGRA testing was the preferred strategy. Conclusion: Patients who live in areas with a higher prevalence of LTB will likely benefit from universal PPD testing. Once IGRA testing is approved in children, it may become a component of “cost-effective” screening options.