Steroid-resistant nephrotic syndrome (SRNS) is a common cause of chronic kidney disease in childhood and has a significant risk of rapid progression to end-stage renal disease. The identification of ...over 50 monogenic causes of SRNS has revealed dysfunction in podocyte-associated proteins in the pathogenesis of proteinuria, highlighting their essential role in glomerular function. Recent technological advances in high-throughput sequencing have enabled indication-driven genetic panel testing for patients with SRNS. The availability of genetic testing, combined with the significant phenotypic variability of monogenic SRNS, poses unique challenges for clinicians when directing genetic testing. This highlights the need for clear clinical guidelines that provide a systematic approach for mutational screening in SRNS. The likelihood of identifying a causative mutation is inversely related to age at disease onset and is increased with a positive family history or the presence of extra-renal manifestations. An unequivocal molecular diagnosis could allow for a personalised treatment approach with weaning of immunosuppressive therapy, avoidance of renal biopsy and provision of accurate, well-informed genetic counselling. Identification of novel causative mutations will continue to unravel the pathogenic mechanisms of glomerular disease and provide new insights into podocyte biology and glomerular function.
The glomerular basement membrane (GBM) is a key component of the glomerular capillary wall and is essential for kidney filtration. The major components of the GBM include laminins, type IV collagen, ...nidogens and heparan sulfate proteoglycans. In addition, the GBM harbours a number of other structural and regulatory components and provides a reservoir for growth factors. New technologies have improved our ability to study the composition and assembly of basement membranes. We now know that the GBM is a complex macromolecular structure that undergoes key transitions during glomerular development. Defects in GBM components are associated with a range of hereditary human diseases such as Alport syndrome, which is caused by defects in the genes COL4A3, COL4A4 and COL4A5, and Pierson syndrome, which is caused by variants in LAMB2. In addition, the GBM is affected by acquired autoimmune disorders and metabolic diseases such as diabetes mellitus. Current treatments for diseases associated with GBM involvement aim to reduce intraglomerular pressure and to treat the underlying cause where possible. As our understanding about the maintenance and turnover of the GBM improves, therapies to replace GBM components or to stimulate GBM repair could translate into new therapies for patients with GBM-associated disease.
The podocytes within the glomeruli of the kidney maintain the filtration barrier by forming interdigitating foot processes with intervening slit diaphragms, disruption in which results in ...proteinuria. Studies into human podocytopathies to date have employed primary or immortalised podocyte cell lines cultured in 2D. Here we compare 3D human glomeruli sieved from induced pluripotent stem cell-derived kidney organoids with conditionally immortalised human podocyte cell lines, revealing improved podocyte-specific gene expression, maintenance in vitro of polarised protein localisation and an improved glomerular basement membrane matrisome compared to 2D cultures. Organoid-derived glomeruli retain marker expression in culture for 96 h, proving amenable to toxicity screening. In addition, 3D organoid glomeruli from a congenital nephrotic syndrome patient with compound heterozygous NPHS1 mutations reveal reduced protein levels of both NEPHRIN and PODOCIN. Hence, human iPSC-derived organoid glomeruli represent an accessible approach to the in vitro modelling of human podocytopathies and screening for podocyte toxicity.
Basement membranes are formed from condensed networks of extracellular matrix (ECM) proteins. These structures underlie all epithelial, mesothelial and endothelial sheets and provide an essential ...structural scaffold. Candidate-based investigations have established that predominant components of basement membranes are laminins, collagen type IV, nidogens and heparan sulphate proteoglycans. More recently, global proteomic approaches have been applied to investigate ECM and these analyses confirm tissue-specific ECM proteomes with a high degree of complexity. The proteomes consist of structural as well as regulatory ECM proteins such as proteases and growth factors. This review is focused on the proteomic analysis of basement membranes and illustrates how this approach can be used to build our understanding of ECM regulation in health and disease.
•Basement membrane protein and mRNA abundance correlate poorly, due to long protein half-lives and regulation by protoelytic degradation.•Basement membranes are frequently embedded within complex structures, are highly crosslinked and insoluble making biochemical analysis challenging.•Mass spectrometry-based proteomics enables global, unbiased quantification of matrix proteins, including basement membranes components.•Exploiting the insolubility of basement membranes, sequential detergent extraction approaches enable excellent coverage of basement membrane proteomes.•Proteomic analysis of basement membranes has been performed in a wide range of tissues and disease contexts, revealing mechanistic insight in cancer metastasis, lung fibrosis and cardiovascular disease.•The global analysis of post-translational modification is also possible with techniques such as glycoproteomics and degradomics.
The glomerular basement membrane (GBM) is a specialized structure with a significant role in maintaining the glomerular filtration barrier. This GBM is formed from the fusion of two basement ...membranes during development and its function in the filtration barrier is achieved by key extracellular matrix components including type IV collagen, laminins, nidogens, and heparan sulfate proteoglycans. The characteristics of specific matrix isoforms such as laminin-521 (α5β2γ1) and the α3α4α5 chain of type IV collagen are essential for the formation of a mature GBM and the restricted tissue distribution of these isoforms makes the GBM a unique structure. Detailed investigation of the GBM has been driven by the identification of inherited abnormalities in matrix proteins and the need to understand pathogenic mechanisms causing severe glomerular disease. A well-described hereditary GBM disease is Alport syndrome, associated with a progressive glomerular disease, hearing loss, and lens defects due to mutations in the genes
, or
. Other proteins associated with inherited diseases of the GBM include laminin β2 in Pierson syndrome and
in nail patella syndrome. The knowledge of these genetic mutations associated with GBM defects has enhanced our understanding of cell-matrix signaling pathways affected in glomerular disease. This review will address current knowledge of GBM-associated abnormalities and related signaling pathways, as well as discussing the advances toward disease-targeted therapies for patients with glomerular disease.
The glomerular filtration barrier is a highly specialized capillary wall comprising fenestrated endothelial cells, podocytes, and an intervening basement membrane. In glomerular disease, this barrier ...loses functional integrity, allowing the passage of macromolecules and cells, and there are associated changes in both cell morphology and the extracellular matrix. Over the past 3 decades, there has been a transformation in our understanding about glomerular disease, fueled by genetic discovery, and this is leading to exciting advances in our knowledge about glomerular biology and pathophysiology. In current clinical practice, a genetic diagnosis already has important implications for management, ranging from estimating the risk of disease recurrence post-transplant to the life-changing advances in the treatment of atypical hemolytic uremic syndrome. Improving our understanding about the mechanistic basis of glomerular disease is required for more effective and personalized therapy options. In this review, we describe genotype and phenotype correlations for genetic disorders of the glomerular filtration barrier, with a particular emphasis on how these gene defects cluster by both their ontology and patterns of glomerular pathology.
Phospholipase A2 receptor 1 (PLA2R) is a target autoantigen in 70% of patients with idiopathic membranous nephropathy. We describe the location of a major epitope in the N-terminal cysteine-rich ...ricin domain of PLA2R that is recognized by 90% of human anti-PLA2R autoantibodies. The epitope was sensitive to reduction and SDS denaturation in the isolated ricin domain and the larger fragment containing the ricin, fibronectin type II, first and second C-type lectin domains (CTLD). However, in nondenaturing conditions the epitope was protected against reduction in larger fragments, including the full-length extracellular region of PLA2R. To determine the composition of the epitope, we isolated immunoreactive tryptic fragments by Western blotting and analyzed them by mass spectrometry. The identified peptides were tested as inhibitors of autoantibody binding to PLA2R by surface plasmon resonance. Two peptides from the ricin domain showed strong inhibition, with a longer sequence covering both peptides (31-mer) producing 85% inhibition of autoantibody binding to PLA2R. Anti-PLA2R antibody directly bound this 31-mer peptide under nondenaturing conditions and binding was sensitive to reduction. Analysis of PLA2R and the PLA2R-anti-PLA2R complex using electron microscopy and homology-based representations allowed us to generate a structural model of this major epitope and its antibody binding site, which is independent of pH-induced conformational change in PLA2R. Identification of this major PLA2R epitope will enable further therapeutic advances for patients with idiopathic membranous nephropathy, including antibody inhibition therapy and immunoadsorption of circulating autoantibodies.
The spatial information carried by light is scrambled when it propagates through a scattering medium, such as frosted glass, biological tissue, turbulent air, or multimode optical fibres. Digital ...optical phase conjugation (DOPC) is a technique that ‘pre-aberrates’ an illuminating wavefront to compensate for scatterer induced distortion. DOPC systems act as phase-conjugate mirrors: they require a camera to holographically record a distorted wavefront emanating from the scatterer and a spatial light modulator (SLM) to synthesize a phase conjugate of the measured wavefront, which is sent back through the scatterer thus creating a time-reversed copy of the original optical field. High-fidelity DOPC can be technically challenging to achieve as it typically requires pixel-perfect alignment between the camera and SLM. Here we describe a DOPC system in which the normally stringent alignment criteria are relaxed. In our system the SLM and camera are placed in-line in the same optical path from the sample, and the SLM is used in an off-axis configuration. This means high-precision alignment can be achieved by measurement of the transmission matrix (TM) mapping optical fields from the SLM to the camera and vice-versa, irrespective of their relative position. The TM also absorbs and removes other aberrations in the optical system, such as the curvature of the SLM and camera chips. Using our system we demonstrate high-fidelity focussing of light through two ground glass diffusers with a peak-intensity to mean-background ratio of ∼700. We provide a step-by-step guide detailing how to align this system and discuss the trade-offs with alternative configurations. We also describe how our setup can be used as a ‘single-pixel camera’ based DOPC system, offering potential for DOPC at wavelengths in which cameras are not available or are prohibitively expensive.
Alport Syndrome in Women and Girls Savige, Judy; Colville, Deb; Rheault, Michelle ...
Clinical journal of the American Society of Nephrology,
09/2016, Letnik:
11, Številka:
9
Journal Article
Recenzirano
Odprti dostop
Alport syndrome is an inherited disease characterized by progressive renal failure, hearing loss, and ocular abnormalities. Inheritance is X-linked (85%) or autosomal recessive (15%). Many renal ...physicians think of Alport syndrome as primarily affecting men. However, twice as many women are affected by the X-linked diseases. Affected women are commonly undiagnosed, but 15%-30% develop renal failure by 60 years and often hearing loss by middle age. Half of their sons and daughters are also affected. Autosomal recessive Alport syndrome is less common, but is often mistaken for X-linked disease. Recessive inheritance is suspected where women develop early-onset renal failure or lenticonus. Their family may be consanguineous. The prognosis for other family members is very different from X-linked disease. Other generations, including parents and offspring, are not affected, and on average only one in four of their siblings inherit the disease. All women with Alport syndrome should have their diagnosis confirmed with genetic testing, even if their renal function is normal, because of their own risk of renal failure and the risk to their offspring. Their mutations indicate the mode of inheritance and the likelihood of disease transmission to their children, and the mutation type suggests the renal prognosis for both X-linked and recessive disease. Women with X-linked Alport syndrome should be tested at least annually for albuminuria and hypertension. The "Expert guidelines for the diagnosis and management of Alport syndrome" recommend treating those with albuminuria with renin-angiotensin-aldosterone system (RAAS) blockade (and adequate birth control because of the teratogenic risks of angiotensin converting enzyme inhibitors), believing that this will delay renal failure. Current recommendations are that women with autosomal recessive Alport syndrome should be treated with RAAS blockade from the time of diagnosis. In addition, women should be offered genetic counseling, informed of their reproductive options, and monitored closely during pregnancy for the development of hypertension.
The glomerulus contains unique cellular and extracellular matrix (ECM) components, which are required for intact barrier function. Studies of the cellular components have helped to build ...understanding of glomerular disease; however, the full composition and regulation of glomerular ECM remains poorly understood. We used mass spectrometry-based proteomics of enriched ECM extracts for a global analysis of human glomerular ECM in vivo and identified a tissue-specific proteome of 144 structural and regulatory ECM proteins. This catalog includes all previously identified glomerular components plus many new and abundant components. Relative protein quantification showed a dominance of collagen IV, collagen I, and laminin isoforms in the glomerular ECM together with abundant collagen VI and TINAGL1. Protein network analysis enabled the creation of a glomerular ECM interactome, which revealed a core of highly connected structural components. More than one half of the glomerular ECM proteome was validated using colocalization studies and data from the Human Protein Atlas. This study yields the greatest number of ECM proteins relative to previous investigations of whole glomerular extracts, highlighting the importance of sample enrichment. It also shows that the composition of glomerular ECM is far more complex than previously appreciated and suggests that many more ECM components may contribute to glomerular development and disease processes. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD000456.
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