Abstract
Podocytes are a key component of the glomerular filtration barrier, and its dysfunction and eventual loss drive glomerular disease progression. Recent research has demonstrated the importance of podocyte cross-talk with other glomerular cells, such as glomerular endothelial cells (GECs), in both glomerular homeostasis and in disease settings. However, how GECs are affected globally by podocyte injury and loss in disease settings remains unclear. Therefore, to characterize the molecular changes occurring in GECs in response to the podocyte loss, we performed the transcriptomic profiling of isolated GECs after diphtheria toxin (DT)-mediated podocyte depletion in transgenic mice with podocyte-specific human DT receptor and endothelial-specific enhanced yellow fluorescent protein (EYFP) expression. DT administration led to nearly 40% of podocyte loss with the development of glomerulosclerosis. Differential gene expression analysis of isolated GECs in the diseased mice showed significant changes in pathways related to cell adhesion and actin cytoskeleton, proliferation, and angiogenesis, as well as apoptosis and cell death. However, quantification of EYFP + GECs indicated that there was a reduction in GECs in the diseased mice, suggesting that despite the ongoing proliferation, the concomitant injury and the activation of cell death program results in their overall net loss. The upstream regulator analysis strongly indicated the involvement of p53, TGF-β1, and TNF-α as key mediators of the molecular changes occurring in GECs in the diseased mice. Our findings demonstrate significant molecular changes in GECs as a secondary consequence of podocyte loss and provide a valuable resource for further in-depth analysis of potential glomerular cross-talk mediators.
Highlights
Podocyte dysfunction and loss are major causes of proteinuria and are associated with the the progression of many glomerular diseases [1, 2]
In this study, we undertook an unbiased approach of transcriptomic analysis of isolated glomerular endothelial cells (GECs) in mice expressing nuclear enhanced yellow fluorescent protein (EYFP) transgene expression under the control of Flk-1 element for effective isolation of GECs [13,14,15] and podocyte-specific human diphtheria toxin (DT) receptor transgenes for effective dosedependent podocyte depletion upon DT administration as a wellestablished model of focal segmental glomerulosclerosis (FSGS) [4, 16]
DT-mediated podocyte depletion and glomerulosclerosis in Flk1-EYFP transgenic mice To facilitate the isolation of GECs, we utilized the Flk1-EYFP transgenic mice that express the nuclear-localized EYFP under the regulatory elements of Flk1 gene [13,14,15] that was crossed with transgenic mice with podocyte-specific human DT receptor expression (Pod-DTR) to generate the performed using the following antigenerate iDTR;Nphs2-Cre;Flk1-EYFP (Pod-DTR);Flk1-EYFP mice
Summary
Podocyte dysfunction and loss are major causes of proteinuria and are associated with the the progression of many glomerular diseases [1, 2]. In this study, we undertook an unbiased approach of transcriptomic analysis of isolated GECs in mice expressing nuclear enhanced yellow fluorescent protein (EYFP) transgene expression under the control of Flk-1 element for effective isolation of GECs [13,14,15] and podocyte-specific human diphtheria toxin (DT) receptor transgenes for effective dosedependent podocyte depletion upon DT administration as a wellestablished model of focal segmental glomerulosclerosis (FSGS) [4, 16] Using this mouse model, we provide the transcriptomic analysis of molecular changes occurring in GECs in response to podocyte dysfunction and loss in experimental FSGS
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