Shp2 Associates with and Enhances Nephrin Tyrosine Phosphorylation and Is Necessary for Foot Process Spreading in Mouse Models of Podocyte Injury

Abstract

bIn most forms of glomerular diseases, loss of size selectivity by the kidney filtration barrier is associated with changes in the morphology of podocytes. The kidney filtration barrier is comprised of the endothelial lining, the glomerular basement membrane, and the podocyte intercellular junction, or slit diaphragm. The cell adhesion proteins nephrin and neph1 localize to the slit diaphragm and transduce signals in a Src family kinase Fyn-mediated tyrosine phosphorylation-dependent manner. Studies in cell culture suggest nephrin phosphorylation-dependent signaling events are primarily involved in regulation of actin dynamics and lamellipodium formation. Nephrin phosphorylation is a proximal event that occurs both during development and following podocyte injury. We hypothesized that abrogation of nephrin phosphorylation following injury would prevent nephrin-dependent actin remodeling and foot process morphological changes. Utilizing a biased screening approach, we found nonreceptor Src homology 2 (sh2) domain-containing phosphatase Shp2 to be associated with phosphorylated nephrin. We observed an increase in nephrin tyrosine phosphorylation in the presence of Shp2 in cell culture studies. In the human glomerulopathies minimal-change nephrosis and membranous nephropathy, there is an increase in Shp2 phosphorylation, a marker of increased Shp2 activity. Mouse podocytes lacking Shp2 do not develop foot process spreading when subjected to podocyte injury in vivo using protamine sulfate or nephrotoxic serum (NTS). In the NTS model, we observed a lack of foot process spreading in mouse podocytes with Shp2 deleted and smaller amounts of proteinuria. Taken together, these results suggest that Shp2-dependent signaling events are necessary for changes in foot process structure and function following injury. P odocytes are highly differentiated epithelial cells with membrane extensions that arborize over the basement membrane in a highly polarized manner. The terminal branches of these actin-rich membrane extensions, called foot processes, interdigitate with each other, forming specialized intercellular junctions called slit diaphragms. Podocytes undergo flattening of the foot processes, or effacement, in most forms of glomerular diseases that present with protein leaks in the urine. Foot process effacement correlates with failure of the filtration barrier and development of proteinuria in both human diseases and animal models of podocyte dysfunction. The strong correlation between foot process morphological changes and failure of the filtration barrier suggests that prevention or reversal of effacement would be beneficial.Nephrin is a transmembrane protein of the immunoglobulin superfamily that is located at the slit diaphragm (1). Nephrin's ability to regulate actin dynamics in a phosphorylation-dependent manner has been demonstrated by us and other investigators (2-6). A critical role for nephrin is suggested by the lack of normal foot process development in mice lacking nephrin or humans born with nephrin mutations ...

A Review of Podocyte Biology

“…Functional integrity of the glomerular filter cannot be assessed with the protamine sulfate model of podocyte injury, as the animals are sacrificed following the experiment. SHP-2-deleted mouse podocytes subjected to the nephrotoxic serum demonstrated that proteinuria is reduced in addition to the absence of foot process effacement [25]. Similar observations have been made when focal adhesion regulatory proteins, such as focal adhesion kinase (FAK), Crk, and uPAR, are deleted in the mouse [24,36,37].…”

“…Both are seen markedly less in FSGS compared with membranous nephropathy and minimal change disease, suggesting that there may be important differences in their signaling processes [24]. Lower levels of phospho-SHP-2 staining were observed in the glomerulus from biopsy samples from FSGS patients compared with those from patients with membranous and minimal change disease [25]. The discrepancy could be explained by a decrease in the number of podocytes in FSGS at the time of biopsy; alternatively, there could be distinct signaling pathways involved in the different proteinuric glomerular diseases.…”

“…Efforts to identify a process to downregulate nephrin signaling during podocyte injury have focused on SHP-2, a phosphatase that is found within the cytoplasm, and is known to be associated with phosphorylated nephrin during podocyte development and injury. However, when nephrin and SHP-2 were put together in vitro, they were found to upregulate nephrin phosphorylation rather than decrease it, suggesting a role in the activation of Src kinases [25]. Furthermore, mice models with SHP-2 deleted from the podocytes were protected from injury with protamine sulfate [25].…”

“…However, when nephrin and SHP-2 were put together in vitro, they were found to upregulate nephrin phosphorylation rather than decrease it, suggesting a role in the activation of Src kinases [25]. Furthermore, mice models with SHP-2 deleted from the podocytes were protected from injury with protamine sulfate [25]. Functional integrity of the glomerular filter cannot be assessed with the protamine sulfate model of podocyte injury, as the animals are sacrificed following the experiment.…”

“…Nephrin can also become tyrosine phosphorylated in cases of podocyte injury (e.g., protamine sulfate exposure), allowing the podocyte foot processes to remodify their actin structure to some extent [24,25]. While it is known that there is an upregulation of tyrosine phosphorylation in glomerular injury, the identity of the other tyrosine kinases in addition to Src kinases that might be involved remains unclear.…”