Epigenetic transcriptional reprogramming by WT1 mediates a repair response during podocyte injury

Abstract

In the context of human disease, the mechanisms whereby transcription factors reprogram gene expression in response to injury are not well understood. This is particularly true in kidney podocytes, injury to which is the common and initial event in many processes that lead End Stage Kidney Disease. WT1 is a master regulator of gene expression in podocytes, binding nearly all genes known to be crucial for maintenance of the glomerular filtration barrier. Here, using purified populations of podocytes and glomeruli, we investigated WT1-mediated transcriptional reprogramming during the course of podocyte injury. Using the Adriamycin murine model of Focal Segmental Glomerulosclerosis, we discovered that podocyte injury led to increased intensity of WT1 binding and to the acquisition of new WT1 binding sites, both at previously identified target genes and at newly bound target genes, providing mechanistic insight on the transcriptional response to injury. We also observed a previously unrecognized transient increase in expression of WT1 target genes in both mice and human kidney organoids.Together, these features appear to constitute an attempt to repair the glomerular filtration barrier after podocyte injury. At later stages of injury, when proteinuria became severe, there was greatly decreased WT1 binding to most target genes.Furthermore, WT1 appeared to be required to maintain active chromatin marks at its target genes. These active marks were converted to repressive marks after loss of WT1 or Adriamycin-induced injury. This response to injury suggests that there may be a potential window of opportunity for repairing podocyte injury as a treatment for glomerular disease in humans. RESULTS Epigenetic regulation of podocyte gene expression by WT1WT1 has been identified as a key regulator of podocyte gene expression 3,4 and WT1 target genes are crucial for maintaining glomerular filtration barrier 4,11 . Two WT1 target genes were studied to elucidate the transcriptional response to injury.Nphs2 encodes Podocin, an essential component of the slit diaphragm and Synpo encodes Synaptopodin, an actin-associated protein important for maintaining the cytoskeleton integrity. To directly demonstrate WT1-dependent gene expression, Wt1 was conditionally inactivated in podocytes 12 of adult Nphs2-CreERT2, WT1fl/fl mice, leading to massive proteinuria (Fig. 1a). Kidneys appeared pale (Fig. 1b) with H&E and PAS staining showing protein casts, mesangial expansion and dilated tubules (Fig. 1c). WT1, Podocin and Synaptopodin transcript and protein levels were greatly reduced ( Fig. 1d, e). Tissue specific TFs activate gene expression in part by promoting histonemodifications that maintain open chromatin, such as H3K4me3 and H4K8ac. We used FACS-isolated podocytes to analyze the effect of WT1 inactivation on histone modifications during the course of injury at previously defined WT1 binding sites at the Nphs2 and Synpo genes 4 , here identified as Nphs2-1, Nphs2-2, Nphs2-3, Synpo-1, Synpo-2 and Synpo-3. Nphs2-1 and Nphs2-2 are located...

Ang II (Angiotensin II)–Induced FGFR1 (Fibroblast Growth Factor Receptor 1) Activation in Tubular Epithelial Cells Promotes Hypertensive Kidney Fibrosis and Injury

“…To determine which cell types are responding to infused Ang II by inducing FGFR1 phosphorylation, we colocalized FGFR1 with different cellular markers in mouse kidneys. Specifically, we marked renal epithelial cells with AQP1 (aquaporin 1), 17 podocytes with WT1 (Wilms tumor 1), 18 endothelial cells by VE-cadherin, and mesangial cells by SMA (smooth muscle actin). Our results show that FGFR1 immunoreactivity mainly colocalizes to AQP1-expressing cells (Figure 3A; Figure S2A).…”

Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process

“…Ildr2/ILDR2 is found to be specifically expressed in mouse and human podocytes as observed from publicly available databases including KIT (Wu et al, 2018;Wu et al, 2019), and MS identified it in proteomic analyses of whole podocytes (Rinschen et al, 2018). Ildr2 was also identified as a Wt1 transcriptional target in podocytes by ChIP-seq (Ettou et al, 2020). However, none of these studies noted the expression of Ildr2 or interrogated its localization within the kidney or podocytes.…”

Mapping of the podocin proximity-dependent proteome reveals novel components of the kidney podocyte foot process

“…44, 58 Ildr2 was also identified as a Wt1 transcriptional target in podocytes by ChIP-seq. 59 In early embryonic and postnatal mouse kidneys, Ildr2/ Ildr2 is expressed and localized to tubule cells and podocytes. However, in the adult kidney Ildr2 localization becomes restricted to podocytes.…”