The luminal surface of blood vessels is covered by a hydrated mesh of sugars and proteins termed the endothelial glycocalyx. The glycocalyx forms a permeability barrier and helps regulate leucocyte migration. Directly detecting glycocalyx damage could provide a major advance in vascular health monitoring. Here we show that red blood cell glycocalyx mirrors the endothelial glycocalyx in health and disease. Using peripherally sampled blood, we confirm that red blood cell glycocalyx measurements predict cardiac and renal endothelial glycocalyx alterations and direct measures of endothelial barrier function in male rats. To investigate the underlying mechanism, we use Azide-Alkyne cycloaddition ('Click' chemistry) to confirm that contact between endothelial and red blood cells results in continual reciprocal transfer of glycocalyx components. These discoveries facilitate real-time monitoring of endothelial damage in patients whilst simultaneously providing a potential explanation as to how red blood cells maintain their glycocalyx during circulation.

Podocyte integrity depends critically on signalling through the insulin receptor and IGF1 receptor , and this study defines their combined importance using dual-receptor knockdown in mice and cultured podocytes. Podocyte-specific reduction of both receptors in transgenic mice caused kidney disease characterised by albuminuria and glomerulosclerosis, with premature death occurring in some animals between 4 and 24 weeks. Receptor-deficient cultured podocytes exhibited >50% cell loss within 7 days. Integrated proteomic and transcriptomic analyses revealed marked depletion of spliceosome-associated proteins and widespread intron retention with premature termination codons, indicating profound disruption of RNA processing. Phospho-proteomic profiling further showed that insulin/IGF1 stimulation induces dynamic post-translational modifications across spliceosomal components and regulatory kinases. Together, these findings uncover a previously unrecognised role for podocyte insulin/IGF1 signalling in maintaining spliceosomal integrity and transcriptional fidelity, establishing this hormonal axis as a key extrinsic regulator of podocyte gene expression.

Amongst genetic causes of nephrotic syndrome (NS), mutations in the PLCΕ1 gene have been described in patients with early onset NS and diffuse mesangial sclerosis (DMS). However, little is known about how these mutations alter podocyte biology although a role in podocyte differentiation has been proposed (Yu S, et al. Exp Mol Med. 52(4):594–603,2020). To explore the role of PLCε in podocytes, A conditionally immortalised human podocyte cell line was generated from a patient with early onset NS, due to a nonsense PLCΕ1 mutation, resulting histologically in diffuse mesangial sclerosis (DMS). In comparison to wild type podocytes, the PLCΕ1 mutant podocyte cell has reduced epithelial features, altered actin cytoskeleton and significantly lower levels of the epithelial marker ZO-1. We demonstrate that PLCε deficiency is associated with functionally impaired TGF-β1 responses with an altered SMAD2/SMAD3 ratio and absent SMAD2 phosphorylation, resulting in loss of the motility response to TGF-β1. We further show that PLCE1 knockdown in wild-type podocytes recapitulates this phenotype by knock-down of PLCΕ1 in wild-type podocytes. This work reveals that disease-causing mutations in PLCΕ1 result in podocyte dedifferentiation and uncovers a novel association between PLCΕ1 deficiency and SMAD2/3 signalling signalling in maintaining podocyte differentiation.

[This corrects the article DOI: 10.1016/j.mtbio.2025.101932.].

Diabetic kidney disease (DKD) is the leading cause of end-stage renal failure, and current interventions fail to directly target the glomerulus, where the disease initiates. Vascular endothelial growth factor (VEGF)C is a key contributor to glomerular endothelial barrier function. In transgenic mice, podocyte-specific overexpression of human VEGFC was protective in early DKD. Here, we investigated the therapeutic potential of a podocyte-targeted VEGFC gene therapy in DKD. We employed an adeno-associated virus (AAV2/9) to drive human VEGFC in human and mouse podocytes. Expressed VEGFC was functional in vitro. In type 1 diabetic mice (induced by streptozotocin), systemic administration of AAV2/9 increased glomerular human VEGFC expression, ameliorating both albuminuria and increased glomerular permeability. Importantly, VEGFC gene therapy also protected the glomerular endothelial glycocalyx, the first barrier to protein in the glomerular filtration barrier. These findings demonstrate that podocyte-directed VEGFC gene delivery can restore glomerular function and protect against early DKD progression. This novel approach represents a promising therapeutic strategy, particularly for patients with type 1 diabetes at risk of DKD, where there is an unmet clinical need.

Podocytopathies encompass a spectrum of glomerular disorders driven by structural and functional impairments in podocytes: specialized cells critical for maintaining the glomerular filtration barrier. Dysregulation of key podocyte components—such as slit-diaphragm proteins (nephrin, podocin), cytoskeletal regulators (ACTN4, TRPC6), and adhesion complexes (integrins, dystroglycan)—leads to proteinuria and progressive glomerulosclerosis. Current therapies often fail to address underlying genetic or molecular defects, particularly in hereditary or refractory cases. Gene therapy has emerged as a transformative approach, leveraging adeno-associated viral (AAV) vectors, CRISPR-based editing, and RNA modulation to correct pathogenic mutations or restore disrupted pathways. Recent advances in capsid engineering, tissue-specific promoters, and delivery strategies have enhanced podocyte targeting while minimizing off-target effects. Preclinical successes, including AAV-mediated rescue of NPHS2-associated nephrotic syndrome and complement modulation in IgA nephropathy, highlight the therapeutic potential. However, challenges such as immune responses, vector biodistribution, and disease heterogeneity remain. This review synthesizes the molecular mechanisms underlying podocytopathies, evaluates current gene-therapy strategies, and discusses translational hurdles and future directions, including patient-derived organoid models and combinatorial therapies. By bridging mechanistic insights with innovative gene-based interventions, this work underscores the promise of precision medicine in revolutionizing the treatment of podocytopathies.

In this systematic review we have sought to summarise the current knowledge concerning biomarkers that can distinguish between steroid-resistant nephrotic syndrome and steroid-sensitive nephrotic syndrome. Additionally, we aim to select biomarkers that have the best evidence-base and should be prioritised for further research. Pub med and web of science databases were searched using "steroid resistant nephrotic syndrome AND biomarker". Papers published between 01/01/2012 and 10/05/2022 were included. Papers that did not compare steroid resistant and steroid sensitive nephrotic syndrome, did not report sensitivity/specificity or area under curve and reviews/letters were excluded. The selected papers were then assessed for bias using the QUADAS-2 tool. The source of the biomarker, cut off, sensitivity/specificity, area under curve and sample size were all extracted. Quality assessment was performed using the BIOCROSS tool. 17 studies were included, comprising 15 case-control studies and 2 cross-sectional studies. Given the rarity of nephrotic syndrome and difficulty in recruiting large cohorts, case-control studies were accepted despite their limitations. We present a range of candidate biomarkers along with scores relating to the quality of the original publications and the risk of bias to inform future investigations. None of the selected papers stated whether the authors were blinded to the patient's disease when assessing the index test in the cohort. Highlighting a key problem in the field that needs to be addressed. These candidate biomarkers must now be tested with much larger sample sizes. Using new biobanks such as the one built by the NURTuRE-INS team will be very helpful in this regard.

Nephrotic syndrome is frequently associated with pathogenic variants in NPHS2 (podocin), including the common and severe R138Q substitution. Using conditionally immortalized human podocytes expressing Myc-tagged podocin variants (G92C, V180M, R138Q, R238S, and R291W), we systematically compared variant-specific defects in plasma-membrane trafficking, detergent-resistant microdomain (DRM) localization, and protein stability. All variants displayed reduced plasma membrane abundance and altered DRM distribution. Among them, R138Q-podocin showed uniquely reduced protein stability. Consistent with previous reports, quantitative proteomics revealed a strong enrichment of endoplasmic reticulum (ER) quality-control and ubiquitin-proteasome components in the R138Q interactome, confirming its identity as an ER-associated degradation substrate. Proteasome inhibition with MG132 stabilized R138Q-podocin and restored its trafficking to both the plasma membrane and DRMs, indicating that impaired stability-rather than an intrinsic trafficking defect-restricts its surface localization. Proteomic profiling further identified caveolin-1, CDCP1, and myosin VI as previously unrecognized podocin interactors. These findings expand the podocin interaction network and suggest potential roles in adhesion-associated membrane organization. Collectively, these results demonstrate that pathogenic podocin variants disrupt podocyte function through distinct mechanisms involving degradation, trafficking, and membrane microdomain association, providing insight into variant-specific disease pathways in nephrotic syndrome. SYNOPSIS: This study examined the trafficking, membrane localization, and stability of disease-associated podocin variants. All variants showed reduced plasma-membrane abundance and altered detergent-resistant microdomain distribution, whereas only R138Q-podocin exhibited marked proteasomal degradation. Quantitative proteomics confirmed the endoplasmic reticulum-associated degradation signature of R138Q and identified caveolin-1, CDCP1, and myosin VI as previously unrecognized podocin interactors. These findings reveal variant-specific mechanisms governing podocin stability and membrane organization.

Polyhydroxyalkanoates (PHAs) are bacteria-derived polymers that are being actively explored for their potential in biomedical engineering applications. These polymers are not only highly biocompatible in nature but also sustainable, produced using renewable substrates, and hence considered future biomaterials. In addition to normal fermentation, PHAs can also be produced through a synthetic biology approach. This study explores a medium chain-length PHA (mcl-PHA) produced by Pseudomonas mendocina CH50 by batch fermentation, fed with glucose as the sole carbon source. The polymer was extensively characterised, and it exhibited an elastomeric property of a typical mcl-PHA with 215 ± 52 % elongation at break. The mcl-PHA also had a low melting point, T m, of around 55 °C, making it processable with various fabrication methods. The extracted mcl-PHA was prepared as a solvent-cast film and tested as a potential cell culture substrate for human glomerular cells, the conditionally immortalised human podocytes (CiHP) and conditionally immortalised human glomerular endothelial cells (CiGEnC). Initial resazurin assay under proliferative conditions showed promising cell metabolic activities of the cells cultured on the mcl-PHA film, comparable with those cultured on tissue culture plastic (TCP). Despite the decreased expression of collagen IV under proliferative conditions, the differentiated co-cultured cells on mcl-PHA had comparable values with cells those grown on TCP. These promising results verified the biocompatibility of the mcl-PHA produced by P. mendocina CH50 and established its potential as a bio-based sustainable alternative in biomedical applications including glomerular tissue engineering.

Abstract Background and Aims Adeno associated vector (AAV) gene therapy has the potential to treat a wide range of kidney diseases via the targeted delivery of genes to kidney cells to repair cellular and renal function. Despite the potential to impact many kidney conditions, kidney AAV gene therapy has proven challenging due to technical and physiological hurdles limiting the ability of AAV to be effectively delivered to podocytes. We have developed a novel AAV gene therapy, PS-001, and local method of administration, to effectively and safely deliver the podocin gene NPHS2 to podocytes. Biallelic NPHS2 mutations are the most common cause of monogenic childhood nephrotic syndrome. Curative efficacy and resolution of disease phenotype was demonstrated in transgenic murine models following PS-001 gene therapy treatment. The potential for translation of this approach to NPHS2 nephropathy patients in the clinic was also demonstrated in pre-clinical studies in Gottingen Minipigs. Method Transgenic murine models representative of NPHS2-driven nephropathy were treated with an AAV gene therapy encoding NPHS2, including a knock in mouse model (Nphs2R140Q/−) which introduces a R140Q mutation in the podocin gene. Mice were assessed for improvement in disease phenotype via analysis of proteinuria, serum albumin, renal histopathology and survival. Mice were treated with an analogue of PS-001, as the PS-001 capsid is not permissive in rodents. Gottingen minipigs were treated with PS-001 and followed for 4 weeks. Clinically relevant doses and a procedure for local administration specifically to the kidney were established. In-life safety assessments from blood and urine were monitored and at study end pig tissues were assessed for podocin biodistribution using multiple assays (qPCR, RT-qPCR, ELISA, RNAScope and IF) and for toxicological effects via histopathological analysis. Results Using our AAV gene therapy platform, which includes a capsid that is highly effective at transducing human podocytes and promoter that drives gene expression specifically in the podocyte, the gene therapy product PS-001 encoding NPHS2 was generated. Using a murine analogue of PS-001, in vivo proof of concept studies in a translationally relevant murine disease model (Nphs2R140Q/−) showed NPHS2 delivery and podocin expression in glomeruli and efficacy in terms of renal function rescue. This was demonstrated by resolution of severe proteinuria (mean ACR ug/mg 6049 ± 2803 vs 192 ± 51 in PS-001 treated animals, n = 11), improved serum albumin (g/L 27.50 ± 3.33 g/L vs 30.75 ± 2.46 in PS-001 treated animals, P = .021) and reduction in glomerulosclerosis (mean sclerosed glomeruli 81% ± 19 vs 18.75% ± 12 in PS-001 treated animals, P < .0001). The methods to translate the approach to the clinical setting using a local administration were established in pigs. Administration of PS-001 resulted in transgene mRNA expression across 89% ± 11% of kidney glomeruli as assessed by RNAScope (n = 3 pigs), and transgene derived podocin could be specifically detected in podocytes as assessed by IF analysis. Using qPCR, RT-PCR and ELISA it was shown that using local administration, podocin gene expression was restricted exclusively to the kidney glomerulus, with gene expression undetectable in off-target organs including the liver. Additional toxicological assessments of renal and other organ function from urine and blood, and post-study histopathological assessments revealed no pre-clinical treatment-related safety concerns. Conclusion We present data showing development of an AAV gene therapy and method of administration for the delivery of transgenes specifically to podocytes. We have shown transgenes can be efficiently targeted to podocytes to replace defective genes or to use the podocyte as a protein factory to modulate glomerular biology. This technology therefore provides an important novel modality for potentially treating a broad range of glomerular disease. PS-001 is being developed as a novel approach to treat steroid resistant nephrotic syndrome caused by mutated NPHS2, and may become the first podocyte targeted gene therapy to enter clinical development.

Insulin signalling to the glomerular podocyte via the insulin receptor (IR) is critical for kidney function. In this study we show that near complete knockout of the closely related insulin-like growth factor I receptor (IGF1R) in podocytes is detrimental, resulting in albuminuria in vivo and podocyte cell death in vitro. In contrast, partial podocyte IGF1R knockdown confers protection against doxorubicin-induced podocyte injury. Proteomic analysis of cultured podocytes revealed that while near complete loss of podocyte IGF1R results in downregulation of mitochondrial respiratory complex I and DNA damage repair proteins, partial IGF1R inhibition promotes respiratory complex expression. This suggests that altered mitochondrial function and resistance to podocyte stress depends on the level of IGF1R suppression, the latter determining whether receptor inhibition is protective or detrimental. Our work suggests that partial suppression of podocyte IGF1R could have therapeutic benefit in treating albuminuric kidney disease.

Chronic kidney disease (CKD) poses a significant global health challenge, projected to become one of the leading causes of death by 2040. Current treatments primarily manage complications and slow progression, highlighting the urgent need for personalized therapies targeting the disease-causing genes. Our increased understanding on the underlying genomic changes that leads to kidney diseases coupled with recent successful gene therapies targeting specific kidney cells have turned gene therapy and genome editing into a promising therapeutic approach for treating kidney disease. This review paper will reflect on different delivery routes and system that can be exploited to target specific kidney cells, and the ways that gene therapy can be used to improve kidney health.

Background Idiopathic nephrotic syndrome (INS) is a heterogenous disease and current classification is based on observational responses to therapies or kidney histology. The National Unified Renal Translational Research Enterprise (NURTuRE)-INS cohort aims to facilitate novel ways of stratifying INS patients to improve disease understanding, therapeutics and design of clinical trials.Methods NURTuRE-INS is a prospective cohort study of children and adults with INS in a linked biorepository. All recruits had at least one sampling visit collecting serum, plasma, urine and blood for RNA and DNA extraction, frozen within 2 hours of collection. Clinical histology slides and biopsy tissue blocks were also collected.Results A total of 739 participants were recruited from 23 centres to NURTuRE-INS, half of whom were diagnosed in childhood [n = 365 (49%)]. The majority were white [n = 525 (71%)] and the median age at recruitment was 32 years (interquartile range 12-54). Steroid-sensitive nephrotic syndrome (SSNS) was the most common clinical diagnosis [n = 518 (70%)]. Of patients diagnosed in childhood who underwent a kidney biopsy, for SSNS (n =103), 76 demonstrated minimal change disease (MCD), whereas for steroid-resistant nephrotic syndrome (n =80), 21 had MCD. Almost all patients diagnosed in adulthood had a kidney biopsy [n = 352 (94%)]; 187 had MCD and 162 had focal segmental glomerulosclerosis.Conclusions NURTuRE-INS is a prospective cohort study with high-quality biosamples and longitudinal data that will assist research into the mechanistic stratification of INS. Samples and data will be available through a Strategic Access and Oversight Committee. Graphical Abstract

Abstract Background and Aims The majority of children with idiopathic nephrotic syndrome (INS) and adults with Focal Segmental Glomerulosclerosis (FSGS) and Minimal Change Disease (MCD) receive glucocorticoid treatment at diagnosis. Only those with a high likelihood of having monogenic disease or a contraindication to steroids might avoid this treatment. About 10% overall will not respond to steroids and we have no reliable way of prospectively identifying these patients. Therefore, some patients will receive a futile treatment which is accompanied by significant side effects. DNA methylation (DNAm) is an epigenetic mechanism meaning that it can induce stable but reversible changes in gene expression without any change in underlying DNA sequence. DNAm has shown great potential as a treatment stratification tool, for example, DNAm data is used in oncology to identify which patients are likely to benefit from alkylating chemotherapy. We investigated whether DNAm can predict initial response to steroids in children and young adults with nephrotic syndrome. Method Three hundred and seventeen patients with INS were selected from the NephroS and NURTuRE cohorts. All patients were diagnosed with INS ≤ 30 years of age and those who underwent a renal biopsy had a histological diagnosis of either FSGS or MCD. Peripheral blood DNAm measurements were generated using the Illumina MethylationEPIC Beadchip (>850, 000 CpG sites). Clinical data was used to label patients by their initial response to steroids (sensitive, n = 156, or resistant, n = 161). Machine learning models were created to predict steroid response from the DNAm data. Models were generated using elastic net following feature filtering, and model hyperparameters were tuned and performance measured within the context of cross validation. To exclude whether cumulative steroid exposure prior to sample collection had impacted our results, the CpG sites in the final model were compared to those identified in a published study examining steroid exposure and DNAm (4). Results The 317 INS patients had a median age at diagnosis of 5 years (IQR 2-10) and a median time between diagnosis and DNAm sample collection of 4 years (IQR 1-10). The steroid resistant group were made up of patients with known monogenic disease (n = 75, 24%) and those without pathogenic variants (n = 86, 27%). Initial response to steroid treatment could be predicted with 65% accuracy and an area under the curve (AUC) of 0.75, (sensitivity 0.65, specificity of 0.66, see Fig. 1) using DNAm levels at 14 CpG sites. There was no overlap between the 14 CpG sites in our prediction model and those that are known to alter with steroid treatment. Conclusion We have demonstrated that peripheral blood cell DNAm profiles are a promising predictor of steroid response in INS. Further work to incorporate genetic data into the prediction models is underway and external validation of the results in a separate cohort of patients is required.

Background Minimal change disease (MCD) and focal segmental glomerulosclerosis (FSGS) are patterns of kidney injury observed in the filtering units of the kidney known as glomeruli. These histological patterns are seen in kidney biopsies from individuals with idiopathic nephrotic syndrome (iNS), which occurs in both children and adults. However, there is some indication that MCD and FSGS are within the same phenotypic spectrum. Methods From the NURTuRE cohort of individuals with NS, we performed laser microdissection and mass spectrometry analysis of kidney biopsy samples to identify proteomic patterns of disease. 56 individuals with iNS segregated by histological pattern (37 MCD and 19 FSGS) across three age groups: early childhood (0–6 years), late childhood (6–18 years) and adult (>18 years). Results We found no distinct clustering of proteomic profiles between MCD and FSGS, but identified global differences in glomerular cell and extracellular matrix composition related to both histological pattern and age. The proteomic data are available via ProteomeXchange with identifier PXD053362. Conclusions The lack of distinct clustering between MCD and FSGS in our study suggests shared biological processes between these injury patterns of iNS, supporting the hypothesis that they are part of the same disease spectrum. The global differences observed in glomerular cell and extracellular matrix composition suggest involvement of diverse biogeological processes as different patterns of iNS manifests in different age groups. This study also demonstrates the feasibility of pooling bioresources, central processing of heterogeneous tissue samples, and developing laser-microdissection and proteomic analysis methodology.

Abstract Background and Aims DNA methylation (DNAm) is an epigenetic mechanism which can therefore induce changes in gene expression without any change to the underlying DNA sequence. Although generally quite stable, DNAm patterns in whole blood do vary across individuals, with DNAm levels associating with age and responding to environmental factors. We aimed to investigate whether peripheral blood cell DNAm associates with level of kidney function in patients with Idiopathic Nephrotic Syndrome (INS). Method Peripheral blood cell DNAm values were generated using the Illumina MethylationEPIC Beadchip (>850,000 CpG sites) for 293 INS patients recruited to the National Unified Renal Translational Research Enterprise–National Study of INS (NURTuRE- INS and NephroS). All patients were younger than 30 years old at INS diagnosis. An Epigenome Wide Association Study (EWAS) was performed using generalised linear models to evaluate associations between DNAm sites and estimated glomerular filtration rate (eGFR). Regions of differentially methylated sites were identified using the ‘dmrff’ R package. Sex chromosomes were excluded from the analyses and all analyses were adjusted for estimated cell type proportions, age, sex and technical variation. A Bonferroni adjusted p value of 5.88 × 10−8 was used as a significance threshold to adjust for multiple tests. Results Of the selected 293 INS patients, 173 (59%) were male, 213 (73%) were white and the median age at INS diagnosis was 4 years old (IQR 2-10). One hundred and forty-three (48%) patients were steroid responsive at diagnosis and the remainder were steroid resistant. Sixty-eight of the steroid resistant patients had pathogenic NS variants. We identified 6 CpG sites associated with eGFR (p < 5.88 × 10−8) in this cohort of INS patients, one replicating a previously reported association. We identified 3 differentially methylated regions, one coinciding with the transcriptional start site of an RNA binding protein which regulates alternative splicing, and another located in the body of a transcription factor regulator containing genetic variants associated with chronic kidney disease. Conclusion We have demonstrated variation in peripheral blood DNAm by level of kidney function in children and young adults with INS. Associations between DNAm and eGFR have previously been demonstrated in diabetic kidney disease and heterogenous chronic kidney disease cohorts, including one of the sites which we have identified in our study. Further work is underway to determine if these differences in DNAm are likely to be a cause or consequence of reduced kidney function, which will help to determine their potential clinical utility.