Fig 2: CLU expression is associated with decreased ratio of NOS 2+ (M1 marker)/ARG 1+ (M2 marker) cells at day 7 following renal IRI. ARG 1+ and NOS 2+ cells were identified by immunohistochemistry as described in the “Methods” section. (a) The numbers of ARG 1+ cells. *P = 0.0010 (WT versus KO, two-tailed t-test). (b) The numbers of NOS 2+ cells. **P = 0.0021 (WT versus KO, two-tailed t-test). (c) The difference in the M1/M2 ratio between the WT and CLU-KO groups was confirmed by the NOS 2+/ARG 1+ ratio in each kidney. ***P = 0.0179 (WT versus KO, two-tailed t-test). Data in a, b and c are representative of three independent experiments and presented as the mean ± s.d. (n = 3). (d) Representative images of three independent experiments. ARG 1+ (top 2) and NOS 2+ (bottom 2) cells (dark brown) in the tubulointerstitial area, the perivascular space and injured tubular epithelium of the renal cortical and medullary junction. Red arrowheads represent positively stained cells; Black arrowheads correspond to false-positive staining. Scale bar: 100 µm. ARG 1; arginase 1; CLU, clusterin; DT, distal convoluted tubule; G, glomerulus; IA, interlobular artery; KO, clusterin knockout; NOS 2, nitric oxide synthase 2; PT, proximal convoluted tubule; WT, wild type.

Fig 3: KORs regulate MI polarization state in CPB rats. (a) For detection of MI polarization state transitions, immunofluorescent counterstaining of iNOS/IBA-1 and arg-1/IBA-1 was carried out (scale bar = 50 µm). Accumulated IBA-1 expressions were detected when CPB occurred, and the MI polarization transition from M1 toward M2 phenotype was also observed when U50488H was applied to CPB rats. (b) Contents of inflammatory factors, M-CSF (pg/ml), IL-6 (pg/ml), and TNF-? (pg/ml), in rats' brain tissue samples were determined using ELISA. All data in charts were represented as mean ± standard deviation; “*” was labeled when the comparative significance between groups (p value) was less than 0.05.

Fig 4: CircZBTB44 promoted M2 polarization of macrophages by up-regulating HK3. (A) The co-culture system of the M0 macrophages with RCC cells. (B) Flow cytometry was applied to examine the ratio of CD86+CD206- macrophages and CD206+CD86- macrophages. (C) qRT-PCR and (D) Western blot were performed to evaluate the mRNA and protein expression of M1-assocaited genes (CD86, TNF-a) and M2-associated genes (CD206, ARG-1) in macrophages co-cultured with transfected RCC cells. *P < 0.05, **P < 0.01, ***P < 0.001