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Background: Variants in genes encoding nuclear pore complex (NPC) proteins are a newly identified cause of paediatric steroid-resistant nephrotic syndrome (SRNS). Recent reports describing NUP93 variants suggest these could be a significant cause of paediatric onset SRNS. We report NUP93 cases in the UK and demonstrate in vivo functional effects of Nup93 depletion in a fly (Drosophila melanogaster) nephrocyte model., Methods: Three hundred thirty-seven paediatric SRNS patients from the National cohort of patients with Nephrotic Syndrome (NephroS) were whole exome and/or whole genome sequenced. Patients were screened for over 70 genes known to be associated with Nephrotic Syndrome (NS). D. melanogaster Nup93 knockdown was achieved by RNA interference using nephrocyte-restricted drivers., Results: Six novel homozygous and compound heterozygous NUP93 variants were detected in 3 sporadic and 2 familial paediatric onset SRNS characterised histologically by focal segmental glomerulosclerosis (FSGS) and progressing to kidney failure by 12 months from clinical diagnosis. Silencing of the two orthologs of human NUP93 expressed in D. melanogaster, Nup93-1, and Nup93-2 resulted in significant signal reduction of up to 82% in adult pericardial nephrocytes with concomitant disruption of NPC protein expression. Additionally, nephrocyte morphology was highly abnormal in Nup93-1 and Nup93-2 silenced flies surviving to adulthood., Conclusion: We expand the spectrum of NUP93 variants detected in paediatric onset SRNS and demonstrate its incidence within a national cohort. Silencing of either D. melanogaster Nup93 ortholog caused a severe nephrocyte phenotype, signaling an important role for the nucleoporin complex in podocyte biology. A higher resolution version of the Graphical abstract is available as Supplementary information., (© 2022. The Author(s).)

The efficacy of B cell depletion therapies in diseases such as nephrotic syndrome and rheumatoid arthritis suggests a broader role in B cells in human disease than previously recognized. In some of these diseases, such as the minimal change disease subtype of nephrotic syndrome, pathogenic antibodies and immune complexes are not involved. We hypothesized that B cells, activated in the kidney, might produce cytokines capable of directly inducing cell injury and proteinuria. To directly test our hypothesis, we targeted a model antigen to the kidney glomerulus and showed that transfer of antigen-specific B cells could induce glomerular injury and proteinuria. This effect was mediated by IL-4, as transfer of IL-4-deficient B cells did not induce proteinuria. Overexpression of IL-4 in mice was sufficient to induce kidney injury and proteinuria and could be attenuated by JAK kinase inhibitors. Since IL-4 is a specific activator of STAT6, we analyzed kidney biopsies and demonstrated STAT6 activation in up to 1 of 3 of minimal change disease patients, suggesting IL-4 or IL-13 exposure in these patients. These data suggest that the role of B cells in nephrotic syndrome could be mediated by cytokines.

Steroid-resistant nephrotic syndrome (SRNS) causes 15% of chronic kidney disease (CKD). Here we show that recessive mutations in FAT1 cause a distinct renal disease entity in four families with a combination of SRNS, tubular ectasia, haematuria and facultative neurological involvement. Loss of FAT1 results in decreased cell adhesion and migration in fibroblasts and podocytes and the decreased migration is partially reversed by a RAC1/CDC42 activator. Podocyte-specific deletion of Fat1 in mice induces abnormal glomerular filtration barrier development, leading to podocyte foot process effacement. Knockdown of Fat1 in renal tubular cells reduces migration, decreases active RAC1 and CDC42, and induces defects in lumen formation. Knockdown of fat1 in zebrafish causes pronephric cysts, which is partially rescued by RAC1/CDC42 activators, confirming a role of the two small GTPases in the pathogenesis. These findings provide new insights into the pathogenesis of SRNS and tubulopathy, linking FAT1 and RAC1/CDC42 to podocyte and tubular cell function.

Increasing evidence implicates activation of NF-kappaB in a variety of glomerular diseases, but the mechanisms involved are unknown. Here, upregulation of NF-kappaB in the podocytes of transgenic mice resulted in glomerulosclerosis and proteinuria. Absence of the podocyte protein nephrin resulted in NF-kappaB activation, suggesting that nephrin negatively regulates the NF-kappaB pathway. Signal transduction assays supported a functional relationship between nephrin and NF-kappaB and suggested the involvement of atypical protein kinase C (aPKCzeta/lambda/iota) as an intermediary. We propose that disruption of the slit diaphragm leads to activation of NF-kappaB; subsequent upregulation of NF-kappaB-driven genes results in glomerular damage mediated by NF-kappaB-dependent pathways. In summary, nephrin may normally limit NF-kappaB activity in the podocyte, suggesting a mechanism by which it might discourage the evolution of glomerular disease.

The leading causes of albuminuria and end-stage renal failure are secondary to abnormalities in the production or cellular action of insulin, including diabetes and hyperinsulinemic metabolic syndrome. The human glomerular podocyte is a critical cell for maintaining the filtration barrier of the kidney and preventing albuminuria. We have recently shown this cell to be insulin sensitive with respect to glucose uptake, with kinetics similar to muscle cells. We now show that the podocyte protein nephrin is essential for this process. Conditionally immortalized podocytes from two different patients with nephrin mutations (natural human nephrin mutant models) were unresponsive to insulin. Knocking nephrin down with siRNA in wild-type podocytes abrogated the insulin response, and stable nephrin transfection of nephrin-deficient podocytes rescued their insulin response. Mechanistically, we show that nephrin allows the GLUT1- and GLUT4-rich vesicles to fuse with the membrane of this cell. Furthermore, we show that the COOH of nephrin interacts with the vesicular SNARE protein VAMP2 in vitro and ex vivo (using yeast-2 hybrid and coimmunoprecipitation studies). This work demonstrates a previously unsuspected role of nephrin in vesicular docking and insulin responsiveness of podocytes.

Microalbuminuria is significant both as the earliest stage of diabetic nephropathy and as an independent cardiovascular risk factor in nondiabetic subjects, in whom it is associated with insulin resistance. The link between disorders of cellular insulin metabolism and albuminuria has been elusive. Here, we report using novel conditionally immortalized human podocytes in vitro and human glomeruli ex vivo that the podocyte, the principal cell responsible for prevention of urinary protein loss, is insulin responsive and able to approximately double its glucose uptake within 15 min of insulin stimulation. Conditionally immortalized human glomerular endothelial cells do not respond to insulin, suggesting that insulin has a specific effect on the podocyte in the glomerular filtration barrier. The insulin response of the podocyte occurs via the facilitative glucose transporters GLUT1 and GLUT4, and this process is dependent on the filamentous actin cytoskeleton. Insulin responsiveness in this key structural component of the glomerular filtration barrier may have central relevance for understanding of diabetic nephropathy and for the association of albuminuria with states of insulin resistance.

Focal segmental glomerulosclerosis (FSGS) is the main pathology underlying steroid-resistant nephrotic syndrome (SRNS) and a leading cause of chronic kidney disease. Monogenic forms of pediatric SRNS are predominantly caused by recessive mutations, while the contribution of de novo variants (DNVs) to this trait is poorly understood. Using exome sequencing (ES) in a proband with FSGS/SRNS, developmental delay, and epilepsy, we discovered a nonsense DNV in TRIM8, which encodes the E3 ubiquitin ligase tripartite motif containing 8. To establish whether TRIM8 variants represent a cause of FSGS, we aggregated exome/genome-sequencing data for 2,501 pediatric FSGS/SRNS-affected individuals and 48,556 control subjects, detecting eight heterozygous TRIM8 truncating variants in affected subjects but none in control subjects (p = 3.28 × 10-11). In all six cases with available parental DNA, we demonstrated de novo inheritance (p = 2.21 × 10-15). Reverse phenotyping revealed neurodevelopmental disease in all eight families. We next analyzed ES from 9,067 individuals with epilepsy, yielding three additional families with truncating TRIM8 variants. Clinical review revealed FSGS in all. All TRIM8 variants cause protein truncation clustering within the last exon between residues 390 and 487 of the 551 amino acid protein, indicating a correlation between this syndrome and loss of the TRIM8 C-terminal region. Wild-type TRIM8 overexpressed in immortalized human podocytes and neuronal cells localized to nuclear bodies, while constructs harboring patient-specific variants mislocalized diffusely to the nucleoplasm. Co-localization studies demonstrated that Gemini and Cajal bodies frequently abut a TRIM8 nuclear body. Truncating TRIM8 DNVs cause a neuro-renal syndrome via aberrant TRIM8 localization, implicating nuclear bodies in FSGS and developmental brain disease.

After decades of primarily morphological study, positional cloning of the NPHS1 gene was the landmark event that established aberrant podocyte genetics as a pivotal cause of malfunction of the glomerular filter. This ended any uncertainty whether genetic mutation plays a significant role in hereditary nephrotic syndromes (NS) and confirmed podocytes as critical players in regulating glomerular protein filtration. Although subsequent sequencing of candidate genes chosen on the basis of podocyte biology had less success, unbiased analysis of genetically informative kindreds and syndromic disease has led to further gene discovery. However, the 45 genes currently associated with human NS explain not more than 20-30% of hereditary and only 10-20% of sporadic cases. It is becoming increasingly clear both from genetic analysis and phenotypic data - including occasional response to immunosuppressive agents and post-transplant disease recurrence in Mendelian disease - that monogenic inheritance of abnormalities in podocyte-specific genes disrupting filter function is only part of the story. Recent advances in genetic screening technology combined with increasingly robust bioinformatics are set to allow identification and characterization of novel disease causing variants and more importantly, disease modifying genes. Emerging data also support a significant but incompletely characterized immunoregulatory component. [ABSTRACT FROM AUTHOR]