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Elimination of the fibrinogen integrin αMβ2-binding motif improves renal pathology in mice with sickle cell anemia

Md Nasimuzzaman, Paritha I. Arumugam, Eric S. Mullins, Jeanne M. James, Katherine VandenHeuvel, Marilou G. Narciso, Maureen A. Shaw, Sarah McGraw, Bruce J. Aronow and Punam Malik

Data supplements

Article Figures & Data

Figures

  • Figure 1.

    Relative ROS production of WBCs is reduced in Fibγ390-396ASS mice compared with the FibWTSS mice. Blood samples from the experimental mice were collected 9 months after BMT and stained with RBCs, WBCs, and platelet-specific antibodies and ROS detection reagent (CM-H2-DCFDA). (A) Relative RBC ROS. (B) Relative WBC ROS. (C) Relative platelets ROS. FibWT B6 (n = 12-14), Fibγ390-396A B6 (n = 11-14), FibWT SS (n = 12-14), and Fibγ390-396A SS (n = 12-14). Similar number of fully chimeric male and female mice were used in the experimental groups. Each symbol in the graph represents an individual mouse. The bars in the graph indicate the mean ± SEM. Statistical significance was determined using 1-way ANOVA followed by the Tukey's multiple comparison test; statistical significance is indicated as **P ≤ .01, ****P ≤ .0001. (A) For RBC ROS, the Dunn's test was used; ***P ≤ .001. ns, not significant.

  • Figure 2.

    Proinflammatory cytokines/chemokines are reduced in Fibγ390-396ASS mice compared with the FibWTSS mice. One year post-BMT, blood samples were collected from the experimental mice. Platelet-poor plasma samples were used for cytokine profiling by Luminex assay. (A) IL-6. (B) TNF-α. (C) KC. Similar number of fully chimeric male and female mice were used in the experimental groups. Each symbol in the graph represents an individual mouse. FibWT AA/B6 (n = 11-15), Fibγ390-396A AA/B6 (n = 8-12), FibWT SS (n = 17-22), and Fibγ390-396A SS (n = 17-22). The bars in the graph indicate the mean ± SEM. Statistical significance was determined using 1-way ANOVA followed by the Tukey's multiple comparison test; statistical significance is indicated as **P ≤ .01, *P ≤ .05.

  • Figure 3.

    Comparison of LV dimensions, systolic function, and mass. (A-B) LVID;d and LVID;s, respectively. (C) LV FS. (D) Echocardiographically derived LV mass is reduced in Fibγ390-396A SS compared with the FibWT SS mice. Echocardiograms were performed on mixed-sex chimeric mice 12 months after BMT. Data are presented as mean ± SEM. Statistical significance was determined using 1-way ANOVA followed by the Tukey's multiple comparison test; statistical significance is indicated as *P ≤ .05, **P ≤ .01, ***P ≤ .001, ****P ≤ .0001.

  • Figure 4.

    Fibγ390-396Aprotects SCA-associated glomerular pathology. Fully chimeric sickle mice were followed for 1 year posttransplant; kidney samples were fixed in formalin after the mice were euthanized, and stained with H&E and PAS reagent. Representative kidney sections of FibWT SS mice (left panel), Fibγ390-396A SS mice (middle panel) and semiquantitative scoring of histologic features (right panel) showing glomerulosclerosis (A), inflammatory cell infiltration (B), ischemic lesions (C), glomerular mesangial thickening (D), glomerular mesangial hypercellularity (E), and glomerular enlargement (F) are reduced in Fibγ390-396A SS mice compared with the FibWT SS mice. Histopathology scores ranged from 0 to 5, where 0 represents normal kidney morphology; 1, changes in <20% of glomerular area; 2, 21% to 40% of the glomerular area; 3, 41% to 60% of the glomerular area; 4, 61% to 80% of glomerular area; and 5, severe changes in almost all glomeruli. Similar number of fully chimeric male and female mice were used in the experimental groups. The bars in the graph indicate the mean ± SEM. Statistical analyses of the histologic scores were done by Mann-Whitney U test; statistical significance between FibWT SS (n = 7) and Fibγ390-396A SS (n = 8) mice is indicated as **P ≤ .01, *P ≤ .05.

  • Figure 5.

    Urine albumin, urine volume, and blood creatinine are reduced in Fibγ390-396ASS mice compared with the FibWTSS mice. One year after BMT, urine samples were collected for 24 hours from the experimental mice and albumin concentration was measured by the mouse albumin ELISA kit. (A) Urine albumin (normalized by urine creatinine) is reduced in Fibγ390-396A SS mice compared with the FibWT SS mice. (B) Urine volume is decreased in Fibγ390-396A SS mice compared with the FibWT SS mice. One year after BMT, blood samples were collected from the experimental mice. Platelet-poor plasma samples were used for urea nitrogen and creatinine measurement. (C) Blood creatinine is decreased in Fibγ390-396A SS mice compared with the FibWT SS mice. (D) Blood urea nitrogen shows a trend for reduction in Fibγ390-396A SS mice compared with the FibWT SS mice. (E) Urine osmolality is not improved in Fibγ390-396A SS mice compared with the FibWT SS mice. Similar number of fully chimeric male and female mice were used in the experimental groups. Each symbol represents an individual mouse. The bars in the graph indicate the mean ± SEM. Statistical significance was determined using 1-way ANOVA followed by the Tukey's multiple comparison test; statistical significance is indicated as ***P ≤ .001, **P ≤ .01, *P ≤ .05. (D) For blood urea nitrogen, the Dunn test were used.

  • Figure 6.

    Glomerular injury responses to SCA are strongly modified by the Fib γ390-396domain. RNA-sequencing analyses of glomerular fractions of kidney from the experimental mice 1 year posttransplant reveal chronic injury and loss of normal cellular programming in FibWT SS mice compared with the Fibγ390-396A SS mice. Heatmap data and gene-ontology pathways showing differentially expressed transcripts in FibWT SS (n = 3) vs Fibγ390-396A SS (n = 4) mice glomeruli. FibWT B6 (n = 2) and Fibγ390-396A B6 (n = 3) mice glomeruli were used as control. The most significant enrichments among each of the clusters are indicated by color codes (ToppGene functional enrichments were determined using the complete clusters as shown in supplemental Table 3) and indicate perturbation of the biological processes in the FibWT SS and/or the Fibγ390-396A SS group. Also see supplemental Figure 14 as well as supplemental Table 3 for expanded gene lists associated with each gene cluster. Differentially expressed genes were determined based on Welch ANOVA using a false discovery rate of 0.1 and fold-change >2 for any group comparison.

  • Figure 7.

    Immunofluorescence staining reveals reduced expression of podocyte marker, WT1 in FibWTSS compared with the Fibγ390-396ASS mice. Fully chimeric similar number of male and female mice were followed for 1 year posttransplant and kidney samples were fixed in formalin after the mice were euthanized, and stained with anti-WT1 antibody. The number of WT1+ podocytes were counted from 20 glomeruli of each kidney section and average number of WT1+ podocytes per glomerulus were calculated. (A) Kidney sections showing similar level of WT1 expression (inside the white dotted circle) in podocytes of glomeruli from FibWT B6 (left panel, n = 6) and Fibγ390-396A B6 (right panel, n = 6) mice. (B) WT1 expression (inside the white dotted circle) is depleted in podocytes of glomeruli of FibWT SS (n = 6) mice compared with the Fibγ390-396A SS (n = 6) mice. The bars in the graph indicate the mean ± SEM. Statistical analyses were done by Mann-Whitney U test and statistical significance are indicated as **P ≤ .01.

Tables

  • Table 1.

    Echocardiography analyses of the experimental mice

    ParameterFibWT AA/B6Fibγ390-396A AA/B6FibWT SSFibγ390-396A SS
    IVS;d, mm0.80 ± 0.050.84 ± 0.050.91 ± 0.050.83 ± 0.08
    LVPW;d, mm0.80 ± 0.040.81 ± 0.040.84 ± 0.040.77 ± 0.05
    LA, mm2.2 ± 0.62.5 ± 0.13.2 ± 0.2*2.8 ± 0.2
    MV E, cm/s645 ± 33645 ± 62730 ± 74689 ± 56
    MV LW E′, cm/s22 ± 321 ± 231 ± 620 ± 3
    MV E/LW E′32 ± 332 ± 431 ± 439 ± 8
    Heart rate, bpm344 ± 31330 ± 27372 ± 31383 ± 26
    • Echocardiograms were performed 12 months post-BMT. Similar numbers of fully chimeric male and female mice were used in the experimental groups. Data are presented as mean plus or minus SEM. Statistical significance was determined using 1-way ANOVA followed by the Tukey's multiple comparison test.

    • bpm, beats per minute; LW E′, lateral wall mitral valve tissue Doppler E wave; MV E, mitral valve early filling.

    • * P ≤ .01 vs FibWT AA/B6.

    • P ≤ .05 vs Fibγ390-396A AA/B6.