Essential role of Gas6 for glomerular injury in nephrotoxic nephritis

Abstract

Growth-arrest specific gene 6 (Gas6) is a vitamin K-dependent growth factor for mesangial and epithelial cells. To investigate whether Gas6 is essential for progressive glomerular injury, we constructed Gas6 -/-mice and examined the role of Gas6 in accelerated nephrotoxic nephritis (NTN), a model of progressive glomerulonephritis. We found less mortality and proteinuria in Gas6 -/-mice than in wild-type mice following injection of nephrotoxic serum. Glomerular cell proliferation, glomerular sclerosis, crescent formation, and deposition of fibrin/fibrinogen in glomeruli were also reduced in Gas6 -/-mice. Furthermore, administering Gas6 -/-mice recombinant wild-type Gas6, but not Gas6 lacking a previously characterized N-terminal γ-carboxyl group, induced massive proteinuria, glomerular cell proliferation, and glomerulosclerosis, comparable to responses seen in wildtype mice. These data indicate that Gas6 induces glomerular cell proliferation in NTN and suggest that this factor contributes to glomerular injury and the progression of chronic nephritis. mice with heterologous nephrotoxic serum (NTS), which has reactivity to several glomerular cell and basement membrane antigens (10).In the studies reported here, we created Gas6 -/-mice and used them to examine the role of Gas6 in murine NTN. Our findings demonstrate that Gas6 is essential for the full expression of progressive glomerular injury in this model. MethodsConstruction of targeting vector. The mouse Gas6 gene was cloned from a 129/SvJ genomic library (Stratagene, La Jolla, California, USA) using a 0.2-kb cDNA fragment encompassing the initial ATG codon of mouse Gas6 as a probe (6). The 3.0-kb EcoRI-EcoRI and 3.5-kb BamHI-BamHI genomic fragments derived from the isolated clone were used for the construction of the targeting vector, along with a neomycin-resistance gene driven by the phosphoglycerate kinase-1 (Pgk-1) promoter (Pgk-neo r ) and a diphtheria toxin A-fragment gene driven by the MC1 promoter as positive and negative selection markers, respectively ( Figure 1) (11). Using this construct, homologous recombination results in the replacement of the EcoRI-BamHI genomic fragment that includes the translation starting codon in the Pgk-neo r cassette, resulting in abolition of Gas6 expression.Generation of Gas6 knockout mice. The embryonic stem cell line used in this study was E14 derived from 129/Ola mice. The targeting experiment and generation of mutant mice were performed as described previously (12). The germline chimera was backcrossed for 17 generations with C57BL/6 mice to obtain Gas6 +/-mice with a C57BL/6 background. The resulting Gas6 +/-F 17 mice were then intercrossed to generate the homozygous Gas6 -/-mice. Additional control inbred C57BL/6 mice were obtained from Shimizu Laboratory Animal Center (Hamamatsu, Japan). All mice were housed under specific pathogen-free conditions. All animal experiments were performed in accordance with institutional guidelines, and the Review Board of Kyoto University granted ethical permission to perform this...

Gas6 and protein S

“…Significantly, warfarin administration at subclinical doses inhibits these increases, further supporting the involvement of vitamin K (and Gas6) in the disease aetiology. In addition, Gas6 knockout mice were less susceptible to developing accelerated nephrotoxic nephritis than wild-type animals [32]. Gas6 up-regulation was also reported in conjunction with allograft rejection in a rat kidney transplant rejection model [33] as well as in dysfunctional human renal allografts [34].…”

Plasma sMer, sAxl and GAS6 levels correlate with disease activity and severity in lupus nephritis

“…13 In mouse model of glomerular nephritis, Yanagita and coworks, showed that, opposite to the function of Mer, ligand binding to Axl may contribute to glomerular injury and the progression of chronic nephritis. 20,26 Ekman et al found the plasma sAxl concentrations were significantly higher in patients with LN and correlated with SLEDAI, but this was not confirmed by others. 14,15,27 Given these findings, it seems likely that in humans, the Mer and Axl play an important role in the pathogenesis of LN.…”

Uterine sensitization-associated gene-1 (USAG-1), a novel BMP antagonist expressed in the kidney, accelerates tubular injury

“…Whole kidney protein was homogenized in RIPA buffer (50 mM Tris at pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.25% SDS, 1 mM Na3VO4, 2 mM EDTA, 1 mM PMSF, and 10 µg/ml aprotinin) and subjected to immunoblotting as described previously (49). Anti-E-cadherin antibody and anti-GAPDH antibody were from BD Biosciences -Transduction Laboratories and Research Diagnostic Inc., respectively.…”