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Renal accumulation of MSP1 in sickle cell disease
Six SCD and 4 control mice were injected with RONi. Similar groups were injected with 2% DMSO (vehicle).
Immunohistochemistry
Paraffin embedded tissue sections were used for immunostain- ing with rat anti-mouse F4/80 (AbD Serotec), rabbit anti-vWF (Dako), rat anti-mouse ICAM (BioLegend), rat anti-mouse CD34 (Cedarline), and mouse anti-MSP1 (R&D Systems). AEC and DAB kits were obtained from Vector Laboratory. Images were acquired with an Olympus 1x51 microscope with an Olympus DP 72 cam- era. Quantification of positive staining was performed using ImageJ Fiji version and CellSens Standard (Olympus) software.
Cell culture
Human glomerular endothelial cell line (HGEC) was generated as described22 and maintained in DMEM media supplemented with 10% fetal bovine serum (FBS) and antibiotics (all from Thermo-Fisher). THP1 human monocytic cell line was purchased from ATCC and maintained in RPMI-1640 media supplemented with 10% FBS, antibiotics and 2 μM β-mercaptoethanol (Sigma- Aldrich). THP-1 cells were differentiated into macrophages by treatment with 10 nM phorbol 12-myristate 13 acetate (PMA, Sigma-Aldrich) for 48 hours (h).
Treatment of THP-1 cells with hemin and RBC lysate
Hemin was obtained from Frontier Scientific. Blood samples were collected from healthy control subjects. RBCs were isolated by centrifugation, frozen at -80°C for 15 min, and then thawed for lysis. Cellular debris was pelleted by centrifugation, and the hemolysates were stored at -80°C. Differentiated THP-1 cells were treated either with different concentrations of hemin or hemolysates for 18 h.
Immunofluorescent staining and western blots
Rabbit MT-SP1 (Calbiochem) antibody was used for immunos- taining and western blot (WB) of THP1 cells. HGEC were treated with 1 μM of human recombinant MSP1 (R&D Systems) with or without RON inhibitor (200 nM) for varying times. WB analysis was performed with rabbit anti-p44/p42 MAPK (Erk1/2), rabbit anti-phospho-p44/p42 Erk1/2, rabbit anti-pan-Akt, and rabbit anti-phospho-Akt antibodies (all from Cell Signaling Technology). Mouse anti-β-actin antibodies and phalloidin-FITC conjugate were obtained from Sigma-Aldrich.
Wounding migration assay
The HGEC monolayers were wounded by strokes across the diameter of the well with 2.5-mm-wide pipet and medium with MSP1 (1 μM) was added. The width of the wound was visualized with pictures taken at pre-treatment and 5 h post treatment. A total of 20 random measurements were analyzed for each wound at each time point.
Isolation of mouse renal glomeruli and glomeruli permeability assay
Mouse renal glomeruli were isolated from control mice using a sieving technique.23 Glomerular permeability was measured by a determination of albumin permeability, as described previously24 with slight modification (Online Supplementary Methods).
Statistical analysis
Results were expressed as mean±Standard Deviation. Differences between two groups were compared by unpaired parametric t-test, between multiple groups by one-way ANOVA (GraphPad software). Differences between groups were consid- ered significant at P<0.05.
Results
SCD mouse kidneys have increased macrophage infiltration and MSP1 accumulation in the glomeruli
Kidneys were collected from 4-month old SCD and con- trol mice (n=5 per group) and renal injury was evaluated. SCD mice develop RBC sickling, anemia, leukocytosis, behavioral changes, and multi-organ pathology character- istic of SCD patients.21,25,26 Glomerular abnormalities in 4- month old SCD mice were characterized by: glomerular hypertrophy [Figure 1A, B, and E, hematoxylin and eosin (H&E) staining]; expansion of mesangium [Figure 1C and D, periodic acid Schiff (PAS) staining, and Online Supplementary Figure S1, PCNA staining]; capillary dilation, and thickening of capillary loops and glomerular basement membrane (Figure 1C, D and F, PAS staining). Glomerular capillaries, peritubular cortical capillaries, and capillaries in the medulla and papilla were markedly congested, and sickling of RBCs was observed especially in the medulla and papilla (Online Supplementary Figure S2, H&E staining). Capillary congestion and dilation together with hemolysis might induce endothelial cell injury and secretion of von Willebrand Factor (vWF) into the surrounding blood and sub-endothelium.27,28 Indeed, glomerular capillary expres- sion of vWF was significantly increased in SCD mice (Figure 2A, B and E, immunostaining, red). Murine glomerular capillary showed positive immunostaining for endothelial marker CD3429 that was significantly increased in SCD mice (Figure 2C, D and F, immunostaining, red). Because CD34 is also expressed on hematopoietic progen- itors cells which are increased in the circulation in SCD mice30 these cells may also be a source of increased CD34 staining in the congested capillary. We also observed a sig- nificant renal glomerular and interstitial infiltration of acti- vated macrophages in SCD mice (Figure 3A, B and E, F4/80 immunostaining, arrows, and Online Supplementary Figure S3). Glomerular infiltrated macrophages were nega- tive for the inducible nitric oxide synthase (iNOS), a mark- er of M1 pro-inflammatory macrophage (Online Supplementary Figure S4). MSP1 was accumulated in SCD renal glomerular capillary (Figure 3C, D and F, immunohis- tostaining, brown). High levels of MSP1 accumulation were found in 46±8% of glomeruli in SCD mice. In con- trast, less than 20% of glomeruli demonstrated low levels of MSP1 accumulation in control mice. Together, these findings show that renal disease in SCD mice is associated with significant endothelial injury, macrophage infiltra- tion, and accumulation of MSP1 in the glomerular capillar- ies.
Hypoxia, hemin, and red blood cell lysate stimulate expression of matriptase 1 in human macrophages
MSP1 is produced by the liver and secreted into circula- tion where it is activated by proteolytic cleavage.18 Macrophage membrane-bound matriptase 1 (MT-SP1) is one of the proteases which activate MSP1 protein.16,31 We tested whether renal hypoxia and RBC hemolysis increased expression of MT-SP1. Meta-analysis of the NCBI Geo database demonstrated a 2-fold increase in MT- SP1 expression in human monocyte-derived differentiated macrophages compared to non-differentiated monocytes (Figure 4A). Meta analysis also showed that MT-SP1 expression was further increased in macrophages that were cultured under hypoxia (Figure 4B). Non-differentiat- ed human pro-monocytic cell line (THP-1 cells) expressed
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