Bone marrow stromal cell antigen-1 deficiency protects from acute kidney injury

Abstract

This study aimed to investigate the role of bone marrow stromal cell antigen-1 (Bst1; also known as CD157) in acute kidney injury (AKI). Bst1 is a cell surface molecule with various enzymatic activities and downstream intracellular signaling pathways that modulate the immune response. Previous research has linked Bst1 to diseases such as ovarian cancer, Parkinson's disease, and rheumatoid arthritis. We used bilateral ischemia-reperfusion injury (IRI) as an AKI model and created bone marrow chimeric mice to evaluate the role of Bst1 in bone marrow-derived cells. We also used flow cytometry to identify Bst1/CD157 expression in hematopoietic cells and evaluate immune cell dynamics in the kidney. The findings showed that Bst1-deficient (Bst1^-/-) mice were protected against renal bilateral IRI. Bone marrow chimera experiments revealed that Bst1 expression on hematopoietic cells, but not parenchymal cells, induced renal IRI. Bst1 was mainly found in B cells and neutrophils by flow cytometry of the spleen and bone marrow. In vitro, migration of neutrophils from Bst1^-/- mice was suppressed, and adoptive transfer of neutrophils from wild-type Bst1^+/+ mice abolished the renal protective effect in Bst1 knockout mice. In conclusion, the study demonstrated that Bst1^-/- mice are protected against renal IRI and that Bst1 expression in neutrophils plays a crucial role in inducing renal IRI. These findings suggest that targeting Bst1 in neutrophils could be a potential therapeutic strategy for AKI.NEW & NOTEWORTHY Acute kidney injury (AKI), a serious disease for which there is no effective Federal Drug Administration-approved treatment, is associated with high mortality rates. Bone marrow stromal cell antigen-1 (Bst1) is a cell surface molecule that can cause kidney fibrosis, but its role in AKI is largely unknown. Our study showed that Bst1^-/- mice revealed a protective effect against renal bilateral ischemia-reperfusion injury (IRI). Adoptive transfer studies confirmed that Bst1 expression in hematopoietic cells, especially neutrophils, contributed to renal bilateral IRI.

Keywords: CD157; acute kidney injury; bone marrow stromal cell antigen-1; neutrophils.

Graphical abstract

Figure 1.

Bone marrow stromal cell antigen-1 (Bst1)-deficient mice are protected against renal ischemia-reperfusion injury (IRI). Bilateral IRI (26 min of ischemia and 24 h of reperfusion) was performed. The renal protective effect was observed in Bst1^−/− mice, as shown by plasma creatinine (A), tissue morphology [B; representative hematoxylin and eosin (H&E) staining of kidney sections], acute tubular necrosis (ATN; C; scored from H&E samples as the percentage of total surface area of the outer medulla occupied by injured tubules), and kidney injury markers (D; real-time PCR from the whole kidney). Injured tubules were identified based on the presence of cast formation, tubule dilation, and/or tubular epithelial denucleation. n = 6–8 mice each. Data in A, C, and D were analyzed with two-way ANOVA. Means were compared by a post hoc multiple-comparison test (Tukey’s). *P < 0.05, **P < 0.01, and ***P < 0.001. Scale bar = 100 μm in the main image; scale bar = 50 μm in the inset. Sham-operated mice underwent the same procedure as IRI except that the renal vessels were not clamped. Gapdh, glyceraldehyde 3-phosphate dehydrogenase; Havcr1 (Kim1), hepatitis A virus cellular receptor 1; Lcn2 (Ngal), lipocalin 2.

Figure 2.

Bone marrow stromal cell antigen-1 (Bst1) expression in hematopoietic cells is important for inducing renal ischemia-reperfusion injury (IRI). A: experimental design for the results shown in C. B: reconstitution rate of bone marrow chimeric mice. Seven to eight weeks after the generation of bone marrow chimeric mice, flow cytometry was performed to evaluate the reconstitution rate. The rate was calculated by dividing the number of CD45.1 cells or CD45.2 cells by the number of both CD45.1 and CD45.2 cells. C: bone marrow chimeric mice underwent bilateral IRI (biIRI; 26 min of ischemia and 24 h of reperfusion). Bst^+/+ mice with Bst1^−/−-derived bone marrow were protected from biIRI as shown by plasma creatinine. n = 3 mice in B; n = 4 or 5 mice in C. Data in C were analyzed with a Student’s t test (two tailed). *** P < 0.001.

Figure 3.

Bone marrow stromal cell antigen-1 (Bst1) is mainly expressed in B cells and neutrophils. A and B: gating strategy for bone marrow-derived cells and splenocytes. Single cell suspensions from the bone marrow or spleen were obtained from C57BL/6 wild-type (WT) mice and stained for flow cytometry as described in materials and methods. Debris was eliminated based on forward scatter (FSC) and side scatter (SSC). After gating on singlet and live CD45-positive cells, B cells were defined as CD19 positive, T cells as CD3-positive, and neutrophils as Ly6G and CD11b double positives. CD11b-positive Ly6G-negative cells were used for further monocyte gating, and F4/80 and CD11b double-positive cells were defined as monocytes. Bst1-positive cells were defined based on the fluorescence minus one (FMO) control. Numbers indicate the percentage of the gated population in each panel. FSCW, forward scatter width. C: ∼2% of bone marrow-derived cells and splenocytes were Bst1 positive as determined by flow cytometry. D: distribution of immune cells among Bst1-positive cells. E: normal distribution of immune cells in WT mice. After single cell suspensions from the bone marrow and spleen of C57BL/6 WT mice were obtained, flow cytometry was performed. This allowed us to compare the distribution of Bst1-positive cells with the normal distribution of total immune cells. Population distributions were expressed as a percentage of total live singlet cells in bone marrow and total CD45-positive cells in the spleen.

Figure 4.

Bone marrow stromal cell antigen-1 (Bst1) in the kidney is expressed in the descending thin limb and neutrophils. A: Bst1 was expressed in descending thin limb (DTL) segments 1–3. C57BL/6 mouse kidneys were stained with Alexa Fluor (A)488-Bst1 (a; green), A555-aquaporin 1 (Aqp1; b, red), and brilliant Pacific blue-CD13 (c; blue). The merged image is shown in d. Bst1 colocalized with either Aqp1 (arrows, yellow), indicating its expression in DTL2 and DTL3, or CD13 (arrowheads, cyan), which stains DTL1 in the medulla. Bst1 did not localize with CD13-stained proximal tubules in the cortex. Scale bar in d = 100 µm. B: Bst1 was expressed in the DTL but not in proximal tubules and distal tubules or collecting ducts. a–d: individual staining of Bst1 (a; green), Aqp1 (b; red), collectrin (c; blue), and epcam (d; blue). The merged image of sections labeled with Bst1, Aqp1, and collectrin is shown in e and with Bst1, Aqp1, and epcam in f. Bst1 colocalized partially only with Aqp1 in the medulla (e; yellow), indicating that it is expressed in DTL2 and DTL3. Bst1 labeling was not found in the cortex and was not localized with collectrin (f), which stains proximal tubule segments S1–S3. No Bst1 colocalization was found with epcam, which stains the thin ascending limb through the collecting ducts. Scale bar in d = 100 µm in a–d and scale bar in f = 100 µm in e and f. C: neutrophils expressed Bst1. Kidneys from C57BL/6 mice undergoing bilateral ischemia (26 min) were harvested 24 h after ischemia-reperfusion injury and fixed in p-formaldehyde-lysine-periodate as described in materials and methods. Sections were stained with A488-Bst1, A647-Ly6G (neutrophils), and A555-F4/80 (macrophages), and the single stains (a–c) and merged images (d) are shown. Neutrophils expressed Bst1 (arrows, yellow), but macrophages did not (arrowheads). Scale bar = 10 µm.

Figure 5.

Bone marrow stromal cell antigen-1 (Bst1) expression in neutrophils is important for inducing renal ischemia-reperfusion injury (IRI). A: migration of neutrophils from Bst1^−/− mice was suppressed compared with Bst1^+/+-derived neutrophils (photomicrograph of migrated cells, left; total number migrated from Transwell insert into tissue culture well, right). B: adoptive transfer of Bst1^+/+-derived neutrophils (intravenously) into Bst1^−/− mice abolished the renal protective effect in Bst1^−/− mice after bilaterial IRI (biIRI) in the acute injury model shown by plasma creatinine. n = 4 mice in A and B. C: the renal protective effect was observed in Bst1^−/−→ Bst1^−/− mice, as shown by tissue morphology [representative hematoxylin and eosin (H&E) staining of kidney sections] and acute tubular necrosis (ATN; scored from H&E samples as the percentage of the total surface area of the outer medulla occupied by injured tubules). D and E: composition of kidney cells from neutrophil transfer experiments after biIRI by flow cytometry analysis. D: no significant differences were found between Bst1^+/+ → Bst1^−/− and Bst1^−/− → Bst1^−/− mice in their respective immune cells including neutrophils. E: Bst1-positive cells were significantly increased in kidneys that received Bst1^+/+ neutrophils compared with kidneys that received Bst1^−/− neutrophils. All data were analyzed with a Student’s t test (two tailed). *P < 0.05; ***P < 0.001. Scale bar = 100 μm in the main image; scale bar = 50 μm in the inset. Euth, euthanize; ns, not significant.