Biology subjects

Drenic, V.

Publications and source records attributed to Drenic, V..

3 recordsLinked to original sources

bioRxiv2024

The tricellular junction protein ILDR2 in glomerulopathies: insights and implications

The tricellular tight junctions are crucial for the regulation of paracellular flux at tricellular junctions, where tricellulin (MARVELD2) and angulins (ILDR1, ILDR2 or LSR) are localized. The role of ILDR2 in podocytes, specialized epithelial cells in the kidney, is still unknown. We investigated the role of ILDR2 in glomeruli and its influence on blood filtration. Western blots, scRNA-seq and superresolution microscopy showed a strong expression of MARVELD2 and ILDR2 in podocytes that colocalizes with the podocyte-specific claudin CLDN5. Co-immunoprecipitation revealed that ILDR2 directly binds CLDN5. In glomerulopathies, induced by nephrotoxic serum and by DOCA-salt heminephrectomy, ILDR2 was strongly upregulated. Furthermore, Ildr2 knockout mice exhibited glomerular hypertrophy and decreased podocyte density, however, did not develop effacement of podocyte foot processes or proteinuria. LC-MS/MS proteomic analysis of isolated glomeruli showed an increase in matrix proteins such as fibronectin and agrin. This suggests a protective role of ILDR2 in glomerulopathies.

cell biology↗

bioRxiv2023

A super-resolution compatible workflow for highly multiplexed immunofluorescence of routinely processed kidney tissue

Deep insights into the complex cellular and molecular changes occurring during different (patho-)physiological conditions are essential for understanding the interactions and regulation of different proteins. This understanding is crucial for both research and diagnostics. However, the effectiveness of conventional immunofluorescence, an effective tool for visualizing the spatial distribution of cells or proteins, is limited in complex tissues. This is mainly due to challenges such as the spectral overlap of fluorophore wavelengths, a limited range of antibody types, and the inherent variability of samples. Multiplex immunofluorescence imaging offers a solution to these limitations by enabling precise localization of proteins and identification of different cell types in a single tissue sample. In this study, we demonstrate the cyclic staining and de-staining of paraffin kidney sections, making it suitable for routine use and compatible with super-resolution microscopy for podocyte ultrastructural studies. We have further developed a computerized workflow for data processing which is accessible to all researchers through commercially available reagents and open-access image analysis codes. As a proof of principle, we identified CDH2 as a marker for cellular lesions of sclerotic glomeruli in the nephrotoxic serum nephritis mouse model and cross-validated this finding with a human Nephroseq dataset indicating its translatability. In summary, our work represents a significant advance in multiplex imaging, which is crucial for understanding the localization of numerous proteins in a single FFPE kidney section and the compatibility with super-resolution microscopy to study ultrastructural changes of podocytes.

cell biology↗

bioRxiv2021

Human pluripotent stem cell-derived kidney organoids for personalized congenital and idiopathic nephrotic syndrome modeling

Nephrotic syndrome (NS) is characterized by severe proteinuria as a consequence of kidney glomerular injury due to podocyte damage. In vitro models mimicking in vivo podocyte characteristics are a prerequisite to resolve NS pathogenesis. Here, we report human induced pluripotent stem cell derived kidney organoids containing a podocyte population that heads towards adult podocytes and were superior compared to 2D counterparts, based on scRNA sequencing, super-resolution imaging and electron microscopy. In this study, these next-generation podocytes in kidney organoids enabled personalized idiopathic nephrotic syndrome modeling as shown by activated slit diaphragm signaling and podocyte injury following protamine sulfate treatment and exposure to NS plasma containing pathogenic permeability factors. Organoids cultured from cells of a patient with heterozygous NPHS2 mutations showed poor NPHS2 expression and aberrant NPHS1 localization, which was reversible after genetic correction. Repaired organoids displayed increased VEGFA pathway activity and transcription factor activity known to be essential for podocyte physiology, as shown by RNA sequencing. This study shows that organoids are the preferred model of choice to study idiopathic and congenital podocytopathies. Summary StatementKidney organoid podocytes allow personalized nephrotic sydrome modeling,

molecular biology↗