Biology subjects

Butt, L.

Publications and source records attributed to Butt, L..

6 recordsLinked to original sources

bioRxiv2024

The Regulation of MAGI2 and its Diagnostic Application in Podocytopathies

Podocyte dysfunction is central to various glomerular diseases, necessitating reliable biomarkers for early detection and diagnosis. This study investigates the regulatory mechanisms of membrane-associated guanylate kinase inverted 2 (MAGI2) and its potential as a biomarker for podocytopathies. The expression of the gene coding for the scaffolding protein MAGI2 was examined across four species and demonstrated to be conserved within the podocyte filtration slit. In vitro and in vivo studies using isolated glomeruli and mammalian animal models of glomerular disease, including DOCA-salt hypertension, nephrotoxic serum nephritis, and puromycin aminonucleoside nephropathy, demonstrated significant downregulation of MAGI2 in injured podocytes. This downregulation was also conserved in a zebrafish model of focal and segmental glomerulosclerosis (FSGS), and the podocyte-specific MAGI2 ortholog Magi2a was reduced post podocyte injury. CRISPR/Cas9-generated zebrafish mutants for magi2a exhibited marked glomerular filtration barrier defects and downregulation of nephrin, underscoring MAGI2s critical role in podocyte function. Human biopsy analyses revealed differential MAGI2 expression: it was increased in minimal change disease (MCD) patients but significantly decreased in primary, but not secondary FSGS cases. As MAGI2 localization did not change in disease states it is an alternative marker for super-resolution microscopy-based morphometry of the filtration slit, correlating with nephrin-based measurements. These findings highlight the potential of MAGI2 as a sensitive biomarker for podocyte injury and its diagnostic utility in differentiating between primary FSGS and MCD.

cell biology↗

bioRxiv2023

Mechanical forces at the kidney filtration barrier govern spatial orientation of podocyte processes on capillaries

Mammalian kidneys filter enormous volumes of water and small solutes, a filtration driven by the very high hydrostatic pressure in glomerular capillaries. Interdigitating cellular processes of podocytes form the slits for fluid filtration. They are connected by the membrane-like slit diaphragm cell junction containing a mechanosensitive ion channel complex and allow filtration while counteracting hydrostatic pressure. Using high-resolution microscopy, we show that filtration-slit-generating secondary processes preferentially align along the capillaries longitudinal axis while primary processes are preferably perpendicular to the longitudinal direction. The preferential orientation requires maturation in development and is lost in disease states. We demonstrate that loss of proper orientation might contribute to impaired filtration by collapsing of the filtration slits and reducing the mechanical stability of podocyte processes. Together, these data suggest that podocytes sense mechanical strain to utilize circumferential hoop stress balancing the massive mechanical strain generated from fluid flow over the filtration slit.

cell biology↗

bioRxiv2021

Three-dimensional Super-resolved Imaging of Paraffin-embedded Kidney Samples

Diseases of the glomeruli, the renal filtration units, are a leading cause of progressive kidney disease. Assessment of the ultrastructure of podocytes at the glomerular filtration barrier is essential for diagnosing diverse disease entities, providing insight into the disease pathogenesis as well as monitoring treatment responses. New technologies, including superresolved nanoscopy and expansion microscopy, as well as new sample preparation techniques, are starting to revolutionize imaging of biopsy specimens. However, our previous approaches for simple and fast three-dimensional imaging of optically cleared samples are to date not compatible with formalin fixed paraffin-embedded (FFPE) tissue, impeding application in clinical routine. Here we provide protocols that circumvent these limitations and allow for three dimensional STED and confocal imaging of FFPE kidney tissue with similar staining and image quality as compared to our previous approaches. This would increase the feasibility to implement these protocols in clinical routines, as FFPE is the gold standard method for storage of patient samples.

pathology↗

bioRxiv2021

Deep learning-based segmentation and quantification of podocyte foot process morphology

The kidneys constantly filter enormous amounts of fluid, with almost complete retention of albumin and other macromolecules in the plasma. Diseases of podocytes at the kidney filtration barrier reduce the intrinsic permeability of the capillary wall resulting in albuminuria. However, direct quantitative assessment of the underlying morphological changes has previously not been possible. Here we developed a deep learning-based approach for segmentation of foot processes in images acquired with optical microscopy. Our method - Automatic Morphological Analysis of Podocytes (AMAP) - accurately segments foot processes and robustly quantifies their morphology. It also robustly determined morphometric parameters, at a Pearson correlation of r > 0.71 with a previously published semi-automated approach, across a large set of mouse tissue samples. The artificial intelligence algorithm wasWe applied the analysis to a set of human kidney disease conditions allowing comprehensive quantification of various underlying morphometric parameters. These results confirmed that when podocytes are injured, they take on a more simplified architecture and the slit diaphragm length is significantly shortened, resulting in a reduction in the filtration slit area and a loss of the buttress force of podocytes which increases the permeability of the glomerular basement membrane to albumin.

pathology↗

bioRxiv2018

An optimized electroporation approach for efficient CRISPR/Cas9 genome editing in murine zygotes

Electroporation of zygotes represents a rapid alternative to the elaborate pronuclear injection procedure for CRISPR/Cas9-mediated genome editing in mice. However, current protocols for electroporation either require the investment in specialized electroporators or corrosive pre-treatment of zygotes which compromises embryo viability. Here, we describe an easily adaptable approach for the introduction of specific mutations in C57BL/6N mice by electroporation of intact zygotes using a common electroporator with synthetic CRISPR/Cas9 components and minimal technical requirement. Direct comparison to conventional pronuclear injection demonstrates significantly reduced physical damage and thus improved embryo development with successful genome editing in up to 100% of living offspring. Hence, our novel approach for Easy Electroporation of Zygotes (EEZy) allows highly efficient generation of CRISPR/Cas9 transgenic mice while reducing the numbers of animals required.

genetics↗

bioRxiv2017

Individual nephron proteomes connect morphology and function in proteinuric kidney disease

In diseases of many parenchymatous organs, heterogenous detoriation of individual functional units determines the clinical prognosis. However, the molecular characterization of these subunits remains a technological challenge that needs to be addressed in order to better understand pathological mechanisms. Sclerotic and proteinuric glomerular kidney disease is a frequent and heterogeneous disease which affects a fraction of nephrons, glomeruli and draining tubules, to variable extents, and for which no treatment exists. Here, we developed and applied an antibody-independent methodology to investigate heterogeneity of individual nephron segment proteomes from mice with proteinuric kidney disease. This \"one-segment-one-proteome-approach\" defines mechanistic connections between upstream (glomerular) and downstream (tubular) nephron segment populations. In single glomeruli from two different mouse models of sclerotic glomerular disease, we identified a coherent protein expression module consisting of extracellular matrix protein deposition (reflecting glomerular sclerosis), glomerular albumin (reflecting proteinuria) and LAMP1, a lysosomal protein. This module was associated with a loss of podocyte marker proteins. In an attempt to target this protein co-expression module, genetic ablation of LAMP1-correlated lysosomal proteases in mice could ameliorate glomerular damage. Furthermore, individual glomeruli from patients with genetic sclerotic and non-sclerotic proteinuric diseases demonstrated increased abundance of lysosomal proteins, in combination with a decreased abundance of the mutated gene products. Therefore, increased glomerular lysosomal load is a conserved key mechanism in proteinuric kidney diseases, and the technology applied here can be implemented to address heterogeneous pathophysiology in a variety of diseases at a sub-biopsy scale

physiology↗