Mesangioproliferative Kidney Diseases and Platelet-Derived Growth Factor-Mediated AXL Phosphorylation

Abstract

Rationale & objective: Immunoglobulin A nephropathy (IgAN) is a common glomerular disease, with mesangial cell proliferation as a major feature. There is no disease-specific treatment. Platelet-derived growth factor (PDGF) contributes to the pathogenesis of IgAN. To better understand its pathogenic mechanisms, we assessed PDGF-mediated AXL phosphorylation in human mesangial cells and kidney tissue biopsy specimens.

Study design: Immunostaining using human kidney biopsy specimens and in vitro studies using primary human mesangial cells.

Setting & participants: Phosphorylation of AXL was assessed in cultured mesangial cells and 10 kidney-biopsy specimens from 5 patients with IgAN, 3 with minimal change disease, 1 with membranous nephropathy, and 1 with mesangioproliferative glomerulonephritis (GN).

Predictor: Glomerular staining for phospho-AXL in kidney biopsy specimens of patients with mesangioproliferative diseases.

Outcomes: Phosphorylated AXL detected in biopsy tissues of patients with IgAN and mesangioproliferative GN and in cultured mesangial cells stimulated with PDGF.

Analytic approach: t test, Mann-Whitney test, and analysis of variance were used to assess the significance of mesangial cell proliferative changes.

Results: Immunohistochemical staining revealed enhanced phosphorylation of glomerular AXL in IgAN and mesangioproliferative GN, but not in minimal change disease and membranous nephropathy. Confocal-microscopy immunofluorescence analysis indicated that mesangial cells rather than endothelial cells or podocytes expressed phospho-AXL. Kinomic profiling of primary mesangial cells treated with PDGF revealed activation of several protein-tyrosine kinases, including AXL. Immunoprecipitation experiments indicated association of AXL and PDGF receptor proteins. An AXL-specific inhibitor (bemcentinib) partially blocked PDGF-induced cellular proliferation and reduced phosphorylation of AXL and PDGF receptor and the downstream signals (AKT1 and ERK1/2).

Limitations: Small number of kidney biopsy specimens to correlate the activation of AXL with disease severity.

Conclusions: PDGF-mediated signaling in mesangial cells involves transactivation of AXL. Finding appropriate inhibitors to block PDGF-mediated transactivation of AXL may provide new therapeutic options for mesangioproliferative kidney diseases such as IgAN.

Keywords: AXL; IgA nephropathy; Platelet-derived growth factor (PDGF); human mesangial cells; transactivation.

Graphical abstract

Figure 1

Phospho-AXL expression in glomeruli of kidney-biopsy specimens from patients with immunoglobulin A nephropathy (IgAN) and mesangioproliferative glomerulonephritis (MsPGN). Immunohistochemical staining of kidney biopsy specimens from patients with different glomerular diseases revealed that phospho-AXL is overexpressed in glomeruli of patients with IgAN (examples of glomeruli from 3 patients shown) and MsPGN but not in biopsy specimens from patients with minimal change disease (MCD) and membranous nephropathy (MN). Size bars (black lines) mark 50 μm.

Figure 2

Overexpressed phospho-AXL in human glomeruli in immunoglobulin A nephropathy (IgAN) biopsy samples in relation to markers for podocytes and endothelial cells. Immunofluorescence staining of sections of remnant frozen kidney biopsy specimens from patients with IgAN revealed a mesangial pattern of phospho-AXL staining. Phospho-AXL (green) did not colocalize with markers for podocytes (A) synaptopodin (red) and (B) endothelial cells, Ulex europeus lectin (red), indicating that phospho-AXL is not expressed to any great extent in those cells. Phospho-AXL had stronger expression in kidney biopsy specimens from patients with IgAN than in biopsy specimens from patients with minimal change disease (MCD). Images were taken with a ×40 objective.

Figure 3

AXL activation by PDGF. (A) Kinomic studies revealed that multiple protein-tyrosine kinases were activated in primary normal human mesangial cells (NHMC) during a 15-minute stimulation with platelet-derived growth factor (PDGF)-AB (10 ng/mL). Protein-tyrosine kinases identified by kinomic profiling and analyzed with protein-tyrosine kinase UpKin, version 8.0 (BioNavigator), software are listed on the y-axis with the normalized kinase statistic scores shown on the x-axis. The bar color of protein-tyrosine kinases indicates activities induced by PDGF-AB, showing highest activities in red. This kinomic profiling showed that the kinase activity of AXL, a member of TAM family, was top ranked. (B) Western blot analysis of cell lysates using antibodies against the 3 members of the TAM family, TYRO3, AXL, MERTK, revealed that AXL was the major protein of TAM family expressed in NHMC. Molecular weights of the standard proteins in kDa are shown on the side. (C) Immunofluorescence staining for AXL in primary human mesangial cells. AXL was expressed in NHMC as well as in mesangial cells isolated from kidney-biopsy specimens from patients with immunoglobulin A nephropathy (IgAN). Three samples were used in each group; representative images are shown. Negative control is without primary antibody; only nuclei are stained (blue).

Figure 4

Platelet-derived growth factor (PDGF) activation of AXL in primary human mesangial cells. Cell lysates prepared from normal human mesangial cells (NHMC) after a 15-minute stimulation with 10 ng/mL of PDGF-AB or mock-stimulation (Control) were analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) under reducing conditions followed by Western blotting with AXL- or phospho-AXL (P-AXL; Y779)-specific antibodies. (A) Typical example of a Western blot. Molecular weight of the standard protein (130 kDa) is shown on the side. These data were then evaluated by densitometry. (B) Individual data points and mean and standard deviation values calculated from 3 independent experiments (P = 0.01). Black dots are the results of groups treated with PDGF-AB. Blue dots are the results of groups without treatment. AXL was phosphorylated after a 15-minute stimulation of NHMC with PDGF-AB.

Figure 5

Platelet-derived growth factor (PDGF) activation of PDGF receptor (PDGFR)-β and cross-activation of AXL. Immunoprecipitation (IP) of cell lysates from normal human mesangial cells stimulated with PDGF-AB for 15 minutes. Antibodies specific for AXL or PDGFR-β were used for IP and the resultant material was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. The blots were probed with antibodies specific for PDGFR-β, P-PDGFR-β, AXL, and P-AXL. PDGFR-β and AXL were co-precipitated by antibodies specific for AXL or PDGFR-β, suggesting that a fraction of each protein was associated with the other protein. Furthermore, PDGF-AB activated PDGFR-β and cross-activated AXL, as evidenced by the reactivity with antibodies specific for phosphorylated forms of AXL (Y779) and PDGFR-β (Y751). Abbreviations: AXL, immunoprecipitations with AXL-specific antibody; IB, immunoblotting; IP: PDGFR-β, immunoprecipitations with PDGFR-β–specific antibody. Example of 1 of 2 independent experiments with similar results is shown. Molecular weights of the standard proteins (130 and 250 kDa) are shown on the side.

Figure 6

AXL inhibitor R428 partial inhibition of cellular proliferation of mesangial cells induced by platelet-derived growth factor (PDGF)-AB. AXL inhibitor R428 reduced in a dose-dependent manner the cellular proliferation of normal human mesangial cells (NHMC) induced by PDGF-AB (10 ng/mL; black bars). R428 did not alter the cellular proliferation of control NHMC (open bars). Results are shown as individual data points and mean and standard deviation values calculated from 3 independent experiments with duplicates. Blue dots are results from groups treated with R428. Black dots are results from groups treated with R428 and PDGF-AB. Statistical differences were determined by 1-way analysis of variance test (P = 0.002 for 0 vs 0.3 μmol/L of R428).

Figure 7

AXL inhibitor R428 inhibition of platelet-derived growth factor (PDGF)-induced phosphorylation of AXL and PDGF receptor (PDGFR)-β and the downstream signaling to AKT1 and ERK1/2. Our previous experiment showed that R428, an AXL inhibitor, partially inhibited PDGF-induced cellular proliferation. Here, we assessed the capacity of R428 to block cellular signaling induced by PDGF. Normal human mesangial cells were preincubated with AXL inhibitor R428 (0.3 μmol/L) for 1.5 hour and then stimulated with PDGF-AB (10 ng/mL) for 15 minutes. The solvent for R428, dimethyl sulfoxide (DMSO), was used as an additional control. Cell lysates were analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. R428 inhibited PDGF-induced phosphorylation of AXL as well as PDGFR-β. Downstream signaling to AKT1 and ERK1/2 was also inhibited by R428. White lines separate the noncontiguous parts of blots. Example of 1 of 2 independent experiments with similar results is shown. Molecular weights of the proteins are shown on the side (130, 180, 70, and 44/42kDa).