Biology subjects

Amann, K.

Publications and source records attributed to Amann, K..

4 recordsLinked to original sources

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↗

bioRxiv2022

The extracellular matrix protein fibronectin modulates metanephric kidney development

Complex interactions of the branching ureteric bud and surrounding mesenchymal cells during metanephric kidney development determine the final number of nephrons. Alterations that result in impaired nephron endowment predispose to arterial hypertension and chronic kidney disease. In the kidney, extracellular matrix (ECM) proteins are usually regarded as acellular scaffolds or as the common histological end-point of chronic kidney diseases. Only little is known about their physiological role in kidney development. The ECM protein fibronectin is expressed at early time points of kidney growth. Therefore, we were interested in the characterization of its expression and role in kidney development. In mouse, fibronectin was expressed during all stages of kidney development with significant changes over time. At embryonic day (E) 12.0 and E13.5 fibronectin lined the ureteric bud epithelium, which was less pronounced at E16.5 and then switched to a glomerular expression in the postnatal and adult kidneys. Similar results were obtained in human kidneys. Deletion of fibronectin at E13.5 in cultured metanephric mouse kidneys resulted in reduced kidney sizes, impaired branching morphogenesis and decreased glomerulogenesis. In line with these findings, fibronectin deletion led to reduced cell proliferation of the branching epithelial cells. Fibronectin colocalized with alpha8 integrin and fibronectin loss caused a reduction in alpha8 integrin-dependent formation of glial cell line-derived factor and expression of Wnt11, both of which are essentially involved in promoting ureteric bud branching. In conclusion, the ECM protein fibronectin acts as a regulator of metanephric kidney development and is a determinant of nephron number.

developmental biology↗

bioRxiv2021

Reconstitution of T cell immunity against EBV in the immunocompromised host by adoptive transfer of peptide-stimulated T cells after allogeneic stem cell transplantation

Reconstitution of T cell repertoire after allogeneic stem cell transplantation is a long and often incomplete process. As a result, reactivation of Epstein-Barr virus (EBV) is a frequent complication that may be treated by adoptive transfer of donor-derived EBV-specific T cells. We generated donor-derived EBV-specific T cells by peptide stimulation and adoptively transferred them to a patient with angioimmunoblastic T-cell lymphoma (AITL), who had developed persisting high titers of EBV concomitant to relapse after transplantation. T cell receptor beta (TCR{beta}) deep sequencing showed that the T cell repertoire of the patient early after transplantation (day 60) was strongly reduced and only very low numbers of EBV-specific T cells were detectable. Manufacturing and in vitro expansion of donor-derived EBV-specific T cells resulted in enrichment of EBV epitope-specific, HLA-restricted T cells. Monitoring after adoptive transfer revealed that the dominant TCR sequences from peptide-stimulated T cells persisted long-term and established an EBV-specific TCR clonotype repertoire in the host, with many of the EBV-specific TCRs present in the donor. This reconstituted repertoire was associated with immunological control of EBV and with lack of further AITL relapse. Author summaryA characteristic feature of all herpesviruses is their persistence in the hosts body after primary infection. Hence, the hosts immune system is confronted with the problem to control these viruses life-long. Well-known representative of the herpesvirus group are the classic Herpes-Simplex Virus (HSV-1) and Varicella Zoster Virus (VZV, causing chicken pox); a less known representative is Epstein Barr Virus (EBV, causing mononucleosis). When the immune system is severely compromised, for example after stem cell transplantation from a foreign (allogeneic) donor, these viruses can reappear, as they are already in the hosts body. Especially EBV cause life-threatening complications after stem cell transplantation and only reinforcement of the hosts immune system can reestablish viral control. Here we show that ex vivo manufactured EBV-specific T cells can reestablish long-term control of EBV and that these cells persist in the hosts body over months. These results give us a better understanding of viral immune reconstitution post-transplant and of clinically-relevant T cell populations against EBV.

immunology↗

bioRxiv2021

Super-resolved local recruitment of CLDN5 to filtration slits implicates a direct relationship with podocyte foot process effacement

Under healthy conditions, foot processes of neighboring podocytes are interdigitating and connected by an electron-dense slit diaphragm. Beside slit diaphragm proteins, typical adherens junction proteins are also found to be expressed at this cell-cell junction. It is therefore considered as a highly specialized type of adherens junction. During podocyte injury, podocyte foot processes lose their characteristic 3D structure and the filtration slits typical meandering structure gets linearized. It is still under debate how this change of structure leads to the phenomenon of proteinuria. Using super-resolution 3D structured illumination microscopy, we observed a spatially restricted up-regulation of the tight junction protein claudin 5 (CLDN5) in areas where podocyte processes of patients suffering from minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS) as well as in murine nephrotoxic serum (NTS) nephritis and uninephrectomy DOCA-salt hypertension models, were locally injured. CLDN5/nephrin ratios in human glomerulopathies and NTS-treated mice were significantly higher compared to controls. In patients, the CLDN5/nephrin ratio is significantly correlated with the filtration slit density as a foot process effacement marker, confirming a direct association of local CLDN5 up-regulation in injured foot processes. Moreover, CLDN5 up-regulation was observed in some areas of high filtration slit density, suggesting that CLND5 up-regulation preceded the changes of foot processes. Therefore, CLDN5 could serve as a biomarker predicting early foot process effacement. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=117 SRC="FIGDIR/small/429338v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@1d05e3aorg.highwire.dtl.DTLVardef@da61f4org.highwire.dtl.DTLVardef@154e7f1org.highwire.dtl.DTLVardef@147bc6b_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗