Biology subjects

Grahammer, F.

Publications and source records attributed to Grahammer, F..

6 recordsLinked to original sources

bioRxiv2026

Cryo-electron tomographic analysis of anti-nephrin-mediated podocytopathy

Nephrotic kidney diseases are characterized by proteinuria caused by dysfunction of the glomerular filtration barrier. Here, we perform an ultrastructural analysis of anti-nephrin-mediated podocytopathy by visualizing the near-native slit diaphragm in situ across defined stages of disease progression using cryo-electron tomography. Our analysis shows that at early disease stages punctuate cell-cell membrane approximations appear, with basal membrane apposition between adjacent podocyte foot processes, while the initially planar, fishnet slit diaphragm progressively bends into a dome-shaped configuration towards the urinary space. At later stages, slit diaphragm disappearance coincides with the appearance of endosomal and vesicular structures and extensive cytoskeletal reorganization. These cryo-electron tomography-based observations provide a structural framework for understanding how antibody binding to nephrin translates into podocyte architectural failure.

immunology↗

bioRxiv2024

Localization of Albumin with Correlative Super Resolution Light- and Electron Microscopy in the Kidney

The functioning of vertebrate life relies on renal filtration of surplus fluid and elimination of low-molecular-weight waste products, while keeping serum proteins in the blood. In disease, however, there is leak of serum proteins and tracing them to identify the leaking position within tissue with a nanometer resolution poses a significant challenge. Correlative microscopy integrates the specificity of fluorescent protein labeling into high-resolution electron micrographs. Using chemical tagging of albumin with synthetic fluorophores we achieve protein-specific labeling that preserve their post-embedding fluorescence after high-pressure freezing and freeze-substitution of murine kidney tissue. Using advanced registration techniques for super-resolution correlative light and electron microscopy, we can localize the labeled albumin with a high precision in the x-y plane of electron micrographs and cartograph its distribution. Thereby we can quantify the albumin concentration and measure a linear reduction gradient across the kidney filtration barrier. Our study shows the feasibility of combining different microscopy contrasts for tracing fluorescently labeled protein markers with super resolution in various tissue samples and opens new perspectives for correlative imaging in volume electron microscopy.

biophysics↗

bioRxiv2023

The molecular architecture of the kidney slit diaphragm

Vertebrate life depends on renal function to filter excess fluid and remove low-molecular-weight waste products. An essential component of the kidney filtration barrier is the slit diaphragm (SD), a specialized cell-cell junction between podocytes. Although the constituents of the SD are largely known, its molecular organization remains elusive. Here, we use super-resolution correlative light and electron microscopy to quantify a linear rate of reduction in albumin concentration across the filtration barrier. Next, we use cryo-electron tomography of vitreous lamellae from high-pressure frozen native glomeruli to analyze the molecular architecture of the SD. The resulting densities resemble a fishnet pattern. Fitting of Nephrin and Neph1, the main constituents of the SD, results in a complex interaction pattern with multiple contact sites between the molecules. Using molecular dynamics flexible fitting, we construct a blueprint of the SD, where we describe all interactions. Our architectural understanding of the SD reconciles previous findings and provides a mechanistic framework for the development of novel therapies to treat kidney dysfunction.

cell biology↗

bioRxiv2022

Single-cell transcriptomics reveals a mechanosensitive injury signaling pathway in early diabetic nephropathy

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease and histopathologic glomerular lesions are among the earliest structural alterations of DN. However, the signaling pathways that initiate these glomerular alterations are incompletely understood. To delineate the cellular and molecular basis for DN initiation, we performed single-cell and bulk RNA sequencing of renal cells from type 2 diabetes mice (BTBR ob/ob) at the early stage of DN. Analysis of differentially expressed genes revealed glucose-independent responses in glomerular cell types. The gene regulatory network upstream of glomerular cell programs suggested the activation of mechanosensitive transcriptional pathway MRTF-SRF predominantly taking place in mesangial cells. Importantly, activation of MRTF-SRF transcriptional pathway was also identified in DN glomeruli in independent patient cohort datasets. Furthermore, ex vivo kidney perfusion suggested that the regulation of MRTF-SRF is a common mechanism in response to glomerular hyperfiltration. Overall, our study presents a comprehensive single-cell transcriptomic landscape of early DN, highlighting mechanosensitive signaling pathways as novel targets of diabetic glomerulopathy.

molecular biology↗

bioRxiv2021

Synuclein alpha accumulation mediates podocyte injury in Fabry nephropathy

Current therapies for Fabry disease are based on reversing intra-cellular accumulation of globotriaosylceramide (Gb3) by enzyme replacement therapy (ERT) or chaperone-mediated stabilization of the defective enzyme, thereby alleviating lysosome dysfunction. However, their effect in the reversal of endorgan damage, like kidney injury and chronic kidney disease remains unclear. First, ultrastructural analysis of serial human kidney biopsies showed that longterm use of ERT reduced Gb3 accumulation in podocytes but did not reverse podocyte injury. Then, a CRISPR/CAS9-mediated -Galactosidase knockout podocyte cell line confirmed ERT-mediated reversal of Gb3 accumulation without resolution of lysosomal dysfunction. Transcriptome-based connectivity mapping and SILAC-based quantitative proteomics identified alpha-synuclein (SNCA) accumulation as a key event mediating podocyte injury. Genetic and pharmacological inhibition of SNCA improved lysosomal structure and function in Fabry podocytes, exceeding the benefits of ERT. Together, this work reconceptualizes Fabry-associated cell injury beyond Gb3 accumulation, and introduces SNCA modulation as a potential intervention, especially for patients with Fabry nephropathy.

molecular biology↗

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↗