Page 62 - Haematologica-5

A. Khaibullina et al.
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B
P=1x10-5
Figure 6. MSP1 increases glomerular perme-
ability in whole glomeruli assay. (A) P=0.008 Representative pictures of glomeruli isolated from control mice placed in isooncotic and hypooncotic solutions. Glomeruli were pre- treated with vehicle (PBS), or with MSP1 (1 μM) with or without RON inhibitor (RONi 200 nM). Bar size 40 μm. (B) Quantification of glomerular volume change in hypooncotic solution. CellSens Standard software was used for measurement of the glomeruli area and glomeruli volume was calculated. Percent of volume changes in isooncotic solution is shown. For quantification, graphs means are shown. Each dot represents a value obtained
from one glomerulus.
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were significantly increased after MSP1 treatment (Figure 5E-G). Phosphorylation of ERK and AKT was significantly reduced after treatment with RONi (Figure 5H-J). Taken together, MSP1 activated RON receptor signaling in cul- tured HGEC leading to the re-organization of F-actin and increasing cell motility and vWF expression.
MSP1 increases permeability in SCD mouse glomeruli
Glomerular hyperfiltration is an early stage manifesta- tion in the development of SCD glomerulopathy.34,35 To test whether MSP1 increases glomeruli permeability, we utilized a mouse whole glomeruli permeability assay.24 Renal glomerular permeability measurement was based on the determination of albumin permeability. In the absence of capillary filtration, the diffusional loss of albu- min from glomeruli capillary is negligible.24 Placement of intact glomeruli into the hypooncotic solution increases glomeruli volume due to water accumulation inside glomeruli according to oncotic gradient.24 Treatment of glomeruli with permeating agents that increase glomerular filtration significantly increases albumin loss and reduces oncotic gradient, leading to a reduced glomerular volume in the hypooncotic solution.24 Renal glomeruli were isolat- ed from control mice and treated with recombinant MSP1 (1 μM) in the presence or absence of RONi (200 nM) for 30 min (see Methods for details). PBS was used as a vehicle control. Placement of vehicle-treated glomeruli in hypoon- cotic solution significantly increased glomerular volume by more than 3.5-fold (Figure 6A and B). MSP1 treatment reduces glomerular volume, whereas RONi treatment restored the ability of glomeruli to enlarge in the hypoon- cotic solution (Figure 6A and B). Taken together, pre-treat- ment of mouse glomeruli with MSP1 significantly
increased glomerular permeability for albumin, and RONi reduced MSP1-associated glomerular permeability.
Treatment of SCD mice with RONi significantly ameliorates glomerular endothelial injury
To test whether RONi prevents development of glomerular endothelial injury in young SCD mice, 2- month old SCD mice were injected with either RONi (10 mg/kg of body weight in 2% DMSO) or vehicle (2% DMSO, n=6 per group) subcutaneously for 14 days. Control non-SCD mice were also injected with either RONi or vehicle (n=4 per group). We used younger (2- month old) mice to test whether RONi prevents develop- ment of glomerular disease, because older (4-month old) mice had already developed profound glomerular disease (Figures 1 and 2). Only 20% of 2-month old mice devel- oped microalbuminuria (1 of 5 mice), and 40% of 12-week old non-treated SCD mice developed microalbuminuria (2 of 5 mice) (data not shown). In contrast, we found that all non-treated mice had glomerular endothelia cell injury at 12 weeks of age. Because endothelial injury was detected before the onset of albuminuria, we focused on the role of RON signaling in the development of endothelial injury. Administration of RONi in SCD mice did not affect body weight (Figure 7A), but significantly reduced kidney size (Figure 7B and C). Moreover, administration of RONi in SCD mice significantly reduced glomerular hypertrophy and capillary congestion (Figure 8A and E and Online Supplementary Figure S5, H&E staining). Capillary dilation and thickening of capillary loops and glomerular basement membrane were significantly reduced in SCD mice treat- ed with RONi compared to mice with vehicle injection (Figure 8B and F and Online Supplementary Figure S5, PAS
haematologica | 2018; 103(5)