Biology subjects

Kliewe, F.

Publications and source records attributed to Kliewe, F..

4 recordsLinked to original sources

bioRxiv2025

Regulation of the transcriptome, miRNAs, and alternative splicing in a FSGS zebrafish injury model

BackgroundFocal Segmental Glomerulosclerosis (FSGS) is a severe kidney disorder with complex and not yet fully understood pathogenesis. Alternative splicing (AS) - the generation of distinct protein isoforms from the same gene - might play a critical role by the regulation of gene functions and disease development. MethodsTo investigate the role of AS in FSGS, we used a zebrafish model, which mimics key human FSGS features, including foot process effacement, matrix accumulation, podocyte detachment and parietal epithelial cell activation. We performed total RNA sequencing of isolated zebrafish glomeruli and whole larvae, followed by integrative bioinformatic analysis to identify AS events and regulatory miRNAs. ResultsOur data revealed a downregulation of essential podocyte genes (nphs1, nphs2, podxl, wt1) and an inhibition of pathways associated with nephron development and cytoskeletal organization. We also observed increased expression of the transcription factor stat3 and disease-associated miRNAs such as miR-21 and miR-193. AS analysis identified approximately [~]7,000 splicing events, primarily exon skipping ([~]80%), affecting genes such as nphs1, magi2, and ptpro. A total of 136 and 612 alternatively spliced genes were found at 5 and 6 days post-fertilization (dpf), respectively. Isoform switch analysis uncovered 70 genes affected by AS in FSGS, including epb41l5 (linked to podocyte adhesion), fgfr1a (fibroblast growth signaling), and members of the SRSF splicing factor family (e.g., srsf3a). ConclusionsThese findings emphasize the importance of transcriptional and post-transcriptional regulation, including AS, in FSGS pathogenesis. Furthermore, they support the zebrafish model as a valuable system for identifying novel mechanisms and potential therapeutic targets for kidney diseases.

cell biology↗

bioRxiv2024

Alternative splicing in mechanically stretched podocytes as a model of glomerular hypertension

BackgroundAlterations in pre-mRNA splicing play an important role in disease pathophysiology. However, the role of alternative splicing (AS) for podocytes in hypertensive nephropathy (HN) has not been investigated. The purpose of the Sys_CARE project was to identify AS events that play a role in the development and progression of HN. MethodsMurine podocytes were exposed to mechanical stretch, after which proteins and mRNA were analyzed by proteomics, RNA-Seq and several bioinformatic AS tools. ResultsBased on transcriptomics and proteomics analysis we could observe significant changes in gene expression and abundance of proteins under mechanical stretch compared to unstretched conditions. By RNA-Seq, we identified over 3,000 alternative spliced genes after mechanical stretch, including all types of AS events. We found 17 genes that showed an AS event in four different splicing analysis tools. From these, we focused on Myl6, a component of the myosin protein complex, and Shroom3, an actin-binding protein crucial for podocyte function. We found two Shroom3 isoforms that showed significant changes in expression upon mechanical stretch, which was verified by qRT-PCR and in situ hybridization. Furthermore, we observed an expression switch of two Myl6 isoforms after mechanical stretch. This switch is accompanied by a change in a C-terminally located amino acid sequence. ConclusionsIn summary, mechanical stretch of cultured podocytes is an excellent model to simulate hypertensive nephropathy. In depth RNA-Seq analysis disclosed alternative splicing events, such as in Shroom3 and Myl6, which may play a crucial role in the pathophysiology of hypertension-induced nephropathy.

cell biology↗

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↗

bioRxiv2019

The actin binding protein α-adducin modulates desmosomal turnover and plasticity

Intercellular adhesion is essential for tissue integrity and homeostasis. Desmosomes are especially abundant in the epidermis and the myocardium, tissues, which are under constantly changing mechanical stresses. Yet, it is largely unclear whether desmosomal adhesion can be rapidly adapted to changing demands and the mechanisms underlying desmosome turnover are only partially understood. We here show that loss of the actin-binding protein -adducin prevented the ability of cultured keratinocytes or murine epidermis to withstand mechanical stress paralleled with reduced desmosome number. This effect was not caused by decreased levels or impaired adhesive properties of desmosomal molecules but rather by altered desmosome turnover. Mechanistically, reduced cortical actin density in -adducin knockout keratinocytes resulted in increased mobility of the desmosomal adhesion molecule desmoglein 3 (Dsg3) and impaired interactions with E-cadherin, a crucial step in desmosome formation. Accordingly, loss of -adducin prevented increased membrane localization of Dsg3 in response to cyclic stretch or shear stress. Our data demonstrate plasticity of desmosomal molecules in response to mechanical stimuli and unravel a mechanism how the actin cytoskeleton indirectly shapes intercellular adhesion by restricting the mobility of desmosomal molecules.

cell biology↗