Phosphorylation of slit diaphragm proteins NEPHRIN and NEPH1 upon binding of HGF promotes podocyte repair

Abstract

Phosphorylation (activation) and dephosphorylation (deactivation) of the slit diaphragm proteins NEPHRIN and NEPH1 are critical for maintaining the kidney epithelial podocyte actin cytoskeleton and, therefore, proper glomerular filtration. However, the mechanisms underlying these events remain largely unknown. Here we show that NEPHRIN and NEPH1 are novel receptor proteins for hepatocyte growth factor (HGF) and can be phosphorylated independently of the mesenchymal epithelial transition receptor in a ligand-dependent fashion through engagement of their extracellular domains by HGF. Furthermore, we demonstrate SH2 domain–containing protein tyrosine phosphatase-2–dependent dephosphorylation of these proteins. To establish HGF as a ligand, purified baculovirus-expressed NEPHRIN and NEPH1 recombinant proteins were used in surface plasma resonance binding experiments. We report high-affinity interactions of NEPHRIN and NEPH1 with HGF, although NEPHRIN binding was 20-fold higher than that of NEPH1. In addition, using molecular modeling we constructed peptides that were used to map specific HGF-binding regions in the extracellular domains of NEPHRIN and NEPH1. Finally, using an in vitro model of cultured podocytes and an ex vivo model of Drosophila nephrocytes, as well as chemically induced injury models, we demonstrated that HGF-induced phosphorylation of NEPHRIN and NEPH1 is centrally involved in podocyte repair. Taken together, this is the first study demonstrating a receptor-based function for NEPHRIN and NEPH1. This has important biological and clinical implications for the repair of injured podocytes and the maintenance of podocyte integrity.

GDF-15 Suppresses Puromycin Aminonucleoside-Induced Podocyte Injury by Reducing Endoplasmic Reticulum Stress and Glomerular Inflammation

“…To look at the expression pattern of Gdf15 in podocytes during kidney injury, we made use of online available RNA sequencing data (GSE124622) and found that Gdf15 was induced by using PAN (100 µg/mL) treatment for 48 h (Figure 2A) [27,28]. Our in vitro experiments confirmed that the expression of Gdf15 in podocytes was low under physiological conditions.…”

A Review of Medicinal Plants with Renoprotective Activity in Diabetic Nephropathy Animal Models

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Using Drosophila Nephrocytes to Understand the Formation and Maintenance of the Podocyte Slit Diaphragm

“…Drosophila Enables Rapid, Scale-Able, in vivo Drug Screens Several studies have used Drosophila nephrocytes to complement a mammalian model system. One such study used a combination of in vitro podocyte and ex vivo nephrocyte models to investigate the phosphorylation of SD proteins Nephrin and NEPH1 in maintenance of the Actin cytoskeleton (Solanki et al, 2021). Fly nephrocytes were treated with hepatocyte growth factor (HGF) following chemically induced injury with protamine sulfate, which resulted in severe Actin cytoskeletal disorganization.…”