P-selectin blockade ameliorates lupus nephritis in MRL/lpr mice through improving renal hypoxia and evaluation using BOLD-MRI

Abstract

Background: Lupus nephritis is one of the most common and severe complications of systemic lupus erythematosus, of which poor prognosis is indicated by aggravated renal hypoxia and tubulointerstitial fibrosis. Cell adhesion molecules play a key role in the progression of lupus nephritis tubulointerstitial lesion, including P-selectin, which mediates the rolling of leukocytes and subsequent adhesion and infiltration and then initiates the inflammatory immune response and ischemia and hypoxia injury. However, the effects and mechanisms of P-selectin in lupus nephritis remain to be investigated, and a noninvasive measurement of lupus nephritis tubulointerstitial hypoxia and fibrosis remains to be explored.

Methods: Thirty-four MRL/lpr mice were randomly divided into the following three groups: MRL/lpr, saline, and anti-P-selectin, which consisted of no treatment, treatment with normal saline, and treatment with anti-P-selectin monoclonal antibody (mAb) from 12 to 16 weeks of age, respectively. Ten male C57BL/6 mice of the same age served as normal controls. 24-h urinary protein, urinary albumin-creatinine ratio, and periodic acid-Schiff were used to assess kidney damage; Western blot or immunohistochemical staining of the hypoxia probe Hypoxyprobe™-1, hypoxia-inducible factor 1α (HIF-1α), and CD31 were used to evaluate hypoxia in renal tissue; and NADPH oxidase subunit gp91phox and p22phox were used to examine renal oxidative stress. The correlation between kidney injury and blood oxygen level-dependent magnetic resonance imaging (BOLD-MRI) was calculated to assess the clinical value of BOLD-MRI.

Results: P-selectin is upregulated in lupus nephritis. Blocking P-selectin with mAb alleviated renal tubulointerstitial fibrosis, renal hypoxia, and peritubular capillary loss, without alteration of the levels of lupus activity indicators, anti-dsDNA antibody, or complement C3. BOLD-MRI showed that the reduced R2* values in the renal cortex and medulla of lupus mice were increased when treated with anti-P-selectin mAb as compared with those treated with normal saline, which were negatively correlated with Hypoxyprobe™-1 hypoxia probe and the expression of HIF-1α.

Conclusions: Early intervention of lupus nephritis with anti-P-selectin mAb can significantly improve the hypoxic state of the kidney and reduce the severity of tubulointerstitial lesions. BOLD-MRI techniques are noninvasive and can dynamically evaluate the changes in renal lesions and intrarenal oxygenation levels before and after treatment in lupus nephritis.

Keywords: BOLD-MRI; Hypoxic; Lupus nephritis; Tubulointerstitial fibrosis.

Fig. 1

Anti-P-selectin mAb treatment neutralized the increased P-selectin expression in lupus nephritis. a Representative immunostaining micrographs of P-selectin show increased P-selectin expression in the glomeruli and tubulointerstitium of MRL/lpr mice and the neutralization of P-selectin with anti-P-selectin mAb (magnification of ×400). b Western blot analysis was used to assess altered P-selectin expression in renal tissues from MRL/lpr mice. β-actin was used as a loading control. c The quantification of the average band intensity for P-selectin in b. The values of expression in the control group were set as 1. d The expression of P-selectin in the renal tissues of saline or anti-P-selectin mAb–treated MRL/lpr mice (the saline and anti-P-selectin groups) was determined by western blot analysis. e Densitometric analyses of the western blots in (D). The relative intensities of the bands were normalized to the intensities of the respective β-actin signal, and the value of the saline group was set as 1. The results are presented as the mean ± SD. *P < 0.05 between the two indicated groups, as analyzed by Student’s t test (n = 6–9 for each group)

Fig. 2

Anti-P-selectin mAb treatment ameliorated kidney injury in MRL/lpr mice. a Representative PAS staining micrographs of the glomerulus and tubulointerstitium in the C57BL/6, MRL/lpr, saline, and anti-P-selectin groups (magnification of ×400). b Semi-quantitative score of lesions in terms of kidney and tubulointerstitium. **P < 0.01 compared with C57BL/6 group; ^##P < 0.01 compared with saline group. c Urinary albumin-creatinine ratio of the MRL/lpr group compared with the C57BL/6 group at the age of 12 weeks. The results are presented as the medium (first quartile, third quartile). **P < 0.01 between the indicated two groups (n = 10–13 for each group). d Serum anti-dsDNA levels of each group. *P < 0.05 compared with the MRL/lpr, saline, and anti-P-selection groups as analyzed by ANOVA followed by Tukey’s multiple comparisons test. e Complement C3 levels of each group. No statistical significance among groups

Fig. 3

Anti-P-selectin mAb treatment ameliorated renal hypoxia in MRL/lpr mice. a Representative hypoxia probe in situ staining micrographs of by hypoxyprobe™-1 (magnification of ×400). b Immunohistochemistry staining of HIF-1α in the glomerulus and tubular interstitium (magnification of ×400). c Western blot analysis was used to assess altered HIF-1α expression in renal tissues from MRL/lpr mice. β-actin was used as a loading control. d The quantification of the average band intensity for HIF-1α in c. The values of expression in the control group were set as 1. e The expression of HIF-1α in the renal tissues of saline or anti-P-selectin mAb–treated MRL/lpr mice (the saline and anti-P-selectin groups) was determined by western blot analysis. f Densitometric analyses of the western blots in e. The relative intensities of the bands were normalized to the intensities of the respective β-actin signal, and the value of the saline group was set as 1. The results are presented as the mean ± SD. *P < 0.05 between the two indicated groups, as analyzed by Student’s t test (n = 6–9 for each group). g Peritubular capillary count (PTC) in the kidney of each group. ***P < 0.001 as analyzed by ANOVA followed by Tukey’s multiple comparisons test. h Immunohistochemistry staining of CD31 for PTC counting in the glomerulus and tubular interstitium (magnification of ×400)

Fig. 4

Anti-P-selectin mAb treatment ameliorated renal oxidative stress in MRL/lpr mice. a Representative immunohistochemistry staining micrographs of gp91phox in the C57BL/6, MRL/lpr, saline, and anti-P-selectin groups (magnification of ×400). b Western blot analysis was used to assess altered gp91phox and p22phox expression in renal tissues from MRL/lpr mice. β-actin was used as a loading control. c The quantification of the average band intensity for gp91phox and p22phox in b. The values of expression in the control group were set as 1. d The expression of gp91phox and p22phox in the renal tissues of saline or anti-P-selectin mAb–treated MRL/lpr mice (the saline and anti-P-selectin groups) was determined by western blot analysis. e Densitometric analyses of the western blots in d. The relative intensities of the bands were normalized to the intensities of the respective β-actin signal, and the value of the saline group was set as 1. The results are presented as the mean ± SD. *P < 0.05 between the two indicated groups, as analyzed by Student’s t test (n = 6–9 for each group)

Fig. 5

Evaluation of renal hypoxia in MRL/lpr mice through BOLD-MRI. a Representative T2* images of C57BL/6 and MRL/lpr mice. b Representative R2* images in the C57BL/6, MRL/lpr, saline, and anti-P-selectin groups. c Correlation between medullary ADC and urinary ACR in the C56BL/6 and MRL/lpr groups when b = 500 s/mm². d Correlation between medullary ADC and urinary ACR in the C56BL/6 and MRL/lpr groups when b = 800 s/mm². e Correlation between medullary R2* and tubulointerstitial scores in the C56BL/6 and MRL/lpr groups. f Correlation between medullary R2* and hypoxic probe score of glomeruli, tubules, interstitium, and arterioles, respectively, in the C56BL/6 and MRL/lpr groups. g Correlation between medullary R2* and HIF-1α score of glomeruli, tubules, interstitium, and arterioles, respectively, in the C56BL/6 and MRL/lpr groups. h Correlation between medullary R2* and hypoxic probe score of glomeruli, tubules, interstitium, and arterioles, respectively, in the saline and anti-P-selectin groups. g Correlation between the medullary R2* and HIF-1α score of glomeruli, tubules, interstitium, and arterioles, respectively, in the saline and anti-P-selectin groups