A mutant fibrinogen that is unable to form fibrin can improve renal phenotype in mice with sickle cell anemia

Abstract Sickle cell anemia (SCA) causes nephropathy which may progress to kidney failure. To determine if soluble fibrinogen (FibAEK) can prevent kidney damage in mice with SCA, we performed bone marrow transplantation (BMT) of Berkeley sickle mice into wild‐type fibrinogen (FibWT), and FibAEK mice that bear a germ‐line mutation in fibrinogen Aα chain at thrombin cleavage site which prevents fibrin formation. We found improved albuminuria in SS FibAEK mice compared with SS FibWT mice at 12 months post‐BMT due to the reduced kidney fibrosis, ischemic lesions, and increased survival of podocytes in the glomeruli, but did not improve urine concentrating defect. Therefore, our study clarifies the distinct role of fibrinogen and fibrin in the renal pathology of SCA.


Measurement of urine albumin and osmolality
24-hours urine samples were collected from the experimental mice using metabolic cages. A mouse albumin ELISA kit (Bethyl Laboratories, Montgomery, TX) was used to determine the concentration of urine albumin which was normalized to 24 h urine volume. Urine osmolality of the same samples was measured using Vapro Pressure Osmometer 5600 (Wescor Biomedical Systems, Logan, UT). 3

Histopathology analyses of organ samples
All of the mice were weighed before being euthanized. Kidneys were fixed in 10% buffered formalin and embedded in paraffin. Sections (4 µm) were stained with hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and Masson's trichrome and evaluated by a pathologist blinded to the animal genotypes. 3

Immunofluorescent staining of kidney sections for podocyte marker, Wilms tumor (WT) 1
Formalin-fixed, paraffin-embedded kidney sections (4 µm) were used for immunofluorescent staining of Wilms tumor 1 (WT1). Antigen retrieval was done in 10 mM sodium citrate buffer (pH 6.0) for 20 min by heating in a microwave oven. The section was incubated with blocking buffer (Santa Cruz Biotechnology, Dallas, TX) for 1 to 2 h at ambient temperature. The sections were stained with a primary antibody, rabbit antimouse Wilms tumor 1 (WT1) (Santa Cruz Biotechnology) overnight at 4°C. The following day, the slides were washed with PBS-Tween 20 (0.1%) and incubated with a secondary antibody, goat anti-rabbit Alexa-Fluor-594 (Invitrogen, San Diego, CA). Diamino-2phenylindole (DAPI) (Southern Biotechnology, Birmingham, AL) was used for nuclear counterstaining. 20 glomeruli of each kidney section were counted and the average number of WT1 + podocytes are shown in the graph. 3

Measurement of Blood fibrinogen
Mouse blood samples were collected in the presence of sodium citrate from the inferior vena cava and platelet-poor plasma was used for measuring fibrinogen concentrations using the Mouse Fibrinogen ELISA Kit (Innovative Research, Novi, MI).

Statistical analyses
Data were analyzed using GraphPad Prism software version 8. For two groups, parametric Student T-test or non-parametric Mann-Whitney U test were used for analyses of statistical significance. One-way ANOVA followed by Tukey's or Dunn's multiple comparison test for four groups of parametric or non-parametric data. Values were expressed as the mean ± standard error of the mean (SEM).

Parameter BoyJ Fib WT BoyJ Fib AEK SS Fib WT SS Fib AEK
Hemoglobin (g/dL) 11.8 ± 0.4 11.1 ± 0.5 8.0 ± 0.2**** 7.8 ± 0.2**** RBC (10 6 /µL) 8.5 ± 0.1 7.8 ± 0.3 6.2 ± 0.1**** 5.6 ± 0.2**** Reticulocytes (%) 4.0 ± 0. Each kidney section was entirely examined and scored. Histopathology scores are ranged from 0 to 5, where 0 is the normal kidney morphology; 1 is the pathology in less than 20% the kidney sections; 2, is the pathology in 21% to 40% the kidney sections; 3, is the pathology in 41% to 60% the kidney sections; 4 is the pathology 61% to 80% the kidney sections. 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 SS Fib WT (n=10) and SS Fib AEK (n=9) mice is indicated as ns: not significant. ). Histopathology scores are ranged from 0 to 5, where 0 is the normal kidney morphology; 1 is the pathology in less than 20% the kidney sections; 2, is the pathology in 21% to 40% the kidney sections; 3, is the pathology in 41% to 60% the kidney sections; 4 is the pathology 61% to 80% the kidney sections. 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 SS Fib WT (n=10) and SS Fib AEK (n=9) mice is indicated as ns, not significant.