High molecular weight kininogen contributes to early mortality and kidney dysfunction in a mouse model of sickle cell disease

Abstract

Background: Sickle cell disease (SCD) is characterized by chronic hemolytic anemia, vaso-occlusive crises, chronic inflammation, and activation of coagulation. The clinical complications such as painful crisis, stroke, pulmonary hypertension, nephropathy and venous thromboembolism lead to cumulative organ damage and premature death. High molecular weight kininogen (HK) is a central cofactor for the kallikrein-kinin and intrinsic coagulation pathways, which contributes to both coagulation and inflammation.

Objective: We hypothesize that HK contributes to the hypercoagulable and pro-inflammatory state that causes end-organ damage and early mortality in sickle mice.

Methods: We evaluated the role of HK in the Townes mouse model of SCD.

Results/conclusions: We found elevated plasma levels of cleaved HK in sickle patients compared to healthy controls, suggesting ongoing HK activation in SCD. We used bone marrow transplantation to generate wild type and sickle cell mice on a HK-deficient background. We found that short-term HK deficiency attenuated thrombin generation and inflammation in sickle mice at steady state, which was independent of bradykinin signaling. Moreover, long-term HK deficiency attenuates kidney injury, reduces chronic inflammation, and ultimately improves survival of sickle mice.

Keywords: anemia; blood coagulation; high molecular weight; inflammation; kidney disease; kininogen; sickle cell.

FIGURE 1

Levels of cleaved high molecular weight kininogen (HK) are higher in sickle patients than in healthy controls. Plasma from HbAA (n = 23) and HbSS (n = 54) patients was analyzed for (A) cleaved (cHK) and (B) intact HK (iHK). C, The ratio of cHK/iHK was also calculated. Data are presented as mean ± standard deviation. *P < .05 versus HbAA by Mann-Whitney test

FIGURE 2

High molecular weight kininogen (HK) contributes to thrombin generation and inflammation in sickle cell disease. Wild type (WT) and HK^−/− mice were lethally irradiated and transplanted with bone marrow from AA (n = 15–22) and SS mice (n = 25–28). Four months later plasma was collected for analysis of (A) thrombin-antithrombin (TAT), (B) interleukin 6 (IL-6), and (C) soluble vascular cell adhesion molecular (sVCAM). Data are presented as mean ± standard error of the mean and were analyzed by two-way analysis of variance and Tukey’s multiple comparison post-hoc analysis. *P < .05 and ***P < .001 versus AA/WT. Asterisks above lines indicate significance between SS/HK^−/− and SS/WT

FIGURE 3

Long-term high molecular weight kininogen (HK) deficiency prolongs survival and reduces inflammation in sickle mice. Two-month-old wild type (WT) and HK^−/− mice were lethally irradiated and transplanted with bone marrow from AA and SS mice. A, Kaplan-Meier survival curve of SS/WT (gray, n = 27 at day 0) and SS/HK^−/− (blue, n = 21 at day 0) mice after bone marrow transplantation, and analyzed by Mantel-Cox test, with a significant difference in survival of **P < .01. After 8 months, blood and was collected for analysis of (B) neutrophil-lymphocyte ratio. *P < .05 and **P < .01 versus AA/WT; asterisks above lines indicate difference from SS/WT

Figure 4:

Sickle mice develop mild cardiovascular dysfunction. Echocardiography of the LV was performed to evaluate (A) LV Mass, (B) ejection fraction, LV volume at (C) diastole and (D) systole, LV internal diameter at (E) diastole and (F) systole, and IVS distance at (G) diastole and (H) systole. Data are presented as mean ± SEM and analyzed by Two-way ANOVA with post-hoc analysis by Tukey’s multiple comparison test. *p<0.05, **p<0.01, and ***p<0.001 versus AA/WT or AA/HK^−/−.

FIGURE 5

Long-term high molecular weight kininogen (HK) deficiency protects sickle mice from glomerular injury. Kidneys were collected and weighed, and (A) the ratio of kidney to body weight was calculated. Urine was collected for 24 hours and analyzed for (B) albumin normalized to creatinine. Data are presented as mean ± standard error of the mean (SEM) and analyzed by two-way analysis of variance and Tukey’s multiple comparisons post hoc analysis. Formalin-fixed and paraffin-embedded kidneys were analyzed for (C) glomerular size, presented as mean area of glomeruli (µm²) ± SEM. D, Quantification of glomerulosclerosis represented as sclerosis index score and (E) representative Masson trichrome-stained sections of glomeruli from all four groups of mice. Original magnification ×120; scale bar = 50 µm. F, Quantification of mesangial expansion score and (G) representative periodic acid Schiff-hematoxylin-stained sections of kidneys from all four groups of mice. Original magnification ×120; scale bar = 50 µm. H, Quantification of WT-1⁺-stained glomerular sections, represented as number of WT-1⁺-stained cells per glomerular area and (I) representative WT-1 staining from all four groups of mice. Original magnification ×120; scale bars = 50 µm. All data are presented as mean ± SEM. Data were analyzed by two-way analysis of variance; *P < .05, **P < .01, ****P < .0001 versus AA/WT. Asterisks above lines indicate difference from SS/WT

FIGURE 6

Long-term high molecular weight kininogen (HK) deficiency attenuates tubular injury in sickle mice. Urine was collected for 24 hours from AA/WT, AA/KO, SS/WT, and SS/KO mice 240 days after bone marrow transplant and analyzed for (A) osmolality and (B) kidney injury marker-1 (KIM-1) levels normalized to creatinine. Data are presented as mean ± standard error of the mean (SEM). C, Quantification of interstitial fibrosis index score and (D) representative images of Picro Sirius-red stained sections. Original magnification ×20; scale bar = 100 µm. E, Quantification of F4/80⁺-stained area and (F) representative images of F4/80⁺-stained glomerular sections. Original magnification ×40; scale bar = 50 µm. G, Quantification of brush border loss index score and (H) representative images from hematoxylin and eosin-stained sections. Original magnification ×120; scale bar = 50 µm. (I) Quantification of Prussian blue staining representing iron deposition and (J) representative images of Prussian blue stained sections. Original magnification ×40; scale bar = 50 µm. Data are presented as mean ± SEM, and analyzed by two-way analysis of variance. *P < .05, **P < .01, ***P < .001 and ****P < .0001 versus AA/WT. Asterisks above lines indicate difference from SS/WT