Third Party Funds Group - Sub project
Acronym: SFB TRR 374 A9-N
Start date : 01.01.2023
End date : 31.12.2026
The nephron consists of two structurally distinct compartments, the renal tubule and the corpuscle, both of which can develop different pathologies with different mechanisms. However, although glomerular disease originates in the glomerulus, concurrent damage to the tubule can contribute greatly to the overall deterioration of renal function. Conversely, severe glomerular damage can occur without simultaneously affecting the tubular systems. The tissue-level factors and molecular mechanisms responsible for cross-compartmental involvement are not yet known. Micro-RNAs (miRs) are short non-coding RNAs that regulate gene expression post transcriptionally by inhibiting mRNA translation or leading to mRNA degradation, with substantial impact on different physiological and pathological cell functions. Interestingly, miRs are involved in regulating extracellular matrix turnover, and their aberrant expression has been implicated in various kidney diseases. In particular, miRs have been shown to have functional effects on important intracellular signaling pathways such as autophagy. Critically, miRs can not only act within the same cell, but can be transported via exosomes, where they are protected from degradation, and can thus communicate with other cells or even different cell types. However, possible effects on glomerular-tubular crosstalk are not known. Exosomes may contain proteins and/or active enzymes derived from the cell they originate from, as well as adaptor proteins for specific delivery to recipient cells. This makes exosomes and exosome-mediated miR shuttles interesting targets for understanding and therapeutically modulating cell-cell communication and transcellular control of cell function.
Here we hypothesize that a previously unrecognized intercompartmental exchange between glomerular and tubule cells occurs via miR-loaded exosomes. In this way, the glomerulus could modulate tubular function, potentially by regulating autophagy pathways in tubule cells, with far-ranging implications for various renal diseases. In the proposed project, we aim to resolve tubular targets and uptake mechanisms of glomerular cell derived miRs using dynamic tracking of exosomes in vivo and determine the implications on cellular autophagy, inflammatory cascades as well as tissue recovery after tubular injury. As translational approach, urinary miR-containing exosomes from patients with membranous glomerulonephritis (MGN) will be evaluated as potential non-invasive biomarkers for tubular involvement.
Specifically, we will use human glomerular-tubular cell co-culture models, different transgenic zebrafish lines, as well as patient-derived material. We will perform dynamic live imaging and kidney compartment specific quantification of cell type-specific fluorescent exosomes containing miRs in co-culture and in our zebrafish model using 2-photon microscopy, light sheet and multiplex confocal microscopy as well as electron microscopy. Furthermore, we will perform functional analysis with the help of our established proteinuria- and tubular uptake assays and assess autophagy by specific flux assay and zebrafish reporter lines. We will further investigate signaling pathways of glomerular cell derived miRs in tubular cells with a special focus on autophagy and apoptosis. To detect miRs important for tubular injury, which might serve as novel non-invasive biomarkers for tubular injury in glomerular diseases, we will screen urinary samples from MGN patients, as well as healthy controls and correlate the results to clinical parameters and histological assessment of the tubular system. Furthermore, we will investigate the origin of urinary exosomes by flow cytometric analysis and proteomic analysis. The secretion of exosomes was originally proposed as a mechanism by which cells eliminate excess proteins. Recent studies have since shown that there is much more to it than just waste disposal and that exosomes may also play a key role in cellular communication. With the proposed work, we aim to gain insights into yet unknown signaling pathways and inter-compartment communication between the glomerulus and the tubular system, which are mediated by miR-containing exosomes and might play a role in modulating tubular injury in glomerular diseases.