Fig 1: Properdin expression and localization in kidneys post IR at 72 h, as well as its co localization with F4/80+ macrophages. (A) The expression of properdin mRNA in kidneys analyzed by qPCR was raised by IR in contrast to the sham control in WT mice. (B) The expression of properdin protein in kidney homogenates detected by Western blot was increased by IR in WT mice but was not seen in PKO mice (sham: n = 3–4; IR: n = 7). Data were shown as means ± SEMs. Significance was assessed by unpaired t-test for qPCR result and one-way ANOVA and LSD test for Western blots. *p < 0.05. (C) Representative images of properdin immunostaining in the cortex of WT sham and IR kidneys, with the boxed area enlarged and properdin staining on the apical surface of tubular cells was pointed by an arrow. All scale bar represent 50 µm. (D) Properdin and F4/80 double-stained cells were visualized in tubular areas, interstitial areas, and tubular lumina (pointed by arrows), with the boxed area enlarged. Most of double staining positive cells had typical apoptotic features including reduced cell volume, and the cellular membrane becoming ruffling and blebbing were also shown. Scale bar: 50 µm (upper row) and 20 µm (lower row).
Fig 2: Properdin contributes to the phagocytic efficacy of primary isolated TECs. (A) Flow cytometry analysis of phagocytosed FITC-labeled E. coli by primary isolated WT and PKO TECs with or without H2O2 treatment for 24 h (n = 3). The fluorescent intensity of FITC conjugated E. coli reflected the phagocytic function of TECs. (i) The fold change of the average intensity of FITC fluorescence among total cells, calculated against the control (-E. coli) group, was increased in WT TECs but decreased in PKO TECs by H2O2. (ii) The fold change of E. coli positive cell counts (P1 area) in each group against the control (-E. coli) group was increased in WT TECs by H2O2 but stayed low in PKO TECs of either the control or H2O2 groups compared to corresponding WT groups. (B) Representative images of apoptotic WT and PKO TECs detected by ISEL (ISEL+ cells indicated by arrows, n = 3). Scale bar: 50 µm. (C) Healthy WT TECs approaching, partially engulfing, and phagocytosing adjacent ISEL+ cells were demonstrated and indicated by arrows. Scale bar: 5 µm. (D) Semiquantitative analysis showed that the percentage of ISEL+ cells against the total number of cells was increased by PKO regardless of serum conditions, while the percentage of phagocytosed ISEL+ cells against the total number of ISEL+ cells was decreased by PKO after H2O2 treatment for 24 h (n = 3). Data were shown as means ± SEMs and analyzed by one-way ANOVA and LSD test. *p < 0.05; **p < 0.01.
Fig 3: Properdin tagged apoptotic TCMK-1 cells and inhibiting properdin increased apoptotic cells and HMGB1 expression in H2O2-treated TCMK-1 cells. (A) Properdin protein was visualized in TCMK-1 cells after H2O2 treatment for 24 h (indicated by a circle and two arrows nearby), in which the staining of properdin was negatively controlled by normal rabbit IgG. ISEL+ apoptotic TCMK-1 cells and its colocalization with properdin protein were also seen (pointed by another two arrows, n = 3). Scale bar: 5 µm. (B) The expression of properdin and HMGB1 detected by Western blot was increased by H2O2 treatment for 24 h, whereas siP reduced properdin expression but increased HMGB1 (n = 3). (C) The early and late apoptotic TCMK-1 cells detected by flow cytometry analysis were both increased by H2O2 treatment for 24 h and furthered by siP (n = 3). FITC fluorescein was tagged to Annexin V, thus can reveal the binding of Annexin V to the cellular surface of apoptotic cells that exposed phosphatidylserine (PS). Propidium iodide (PI) passed leaky necrotic cells to stain the DNA. Data were shown as means ± SEMs and analyzed by one-way ANOVA and LSD test. siP, siRNA targeting properdin; siNC, negative control siRNA. *p < 0.05; **p < 0.01.
Fig 4: Properdin mediated and enhanced phagocytosis of TECs. Schematic illustration of the role of properdin in the phagocytosis of living TECs from WT and PKO mice. Properdin produced by living TECs tags adjacent apoptotic TECs to facilitate their uptake by neighbor living TECs, promoting damage reduction and inflammatory clearance. Lack of properdin impaired TECs phagocytosing damaged cells and resulted in more severe inflammation and tissue damage and compromised repair activities. Thus, properdin associated phagocytic function of TECs facilitates the balance between injury and repair of the kidney.
Supplier Page from Abcam for Anti-Properdin/PFC antibody