Fig 2: Immunohistochemical reactivities of integrin ß3, integrin avß3, HOXA10, LIF, and mucin 1 (MUC1) antibodies for all groups. (Streptavidin-peroxidase staining)

Fig 3: Neutralizing LIF antibody attenuated renal fibrosis induced by UUO. (a) Schematic diagram of the experimental design. (b and c) Representative images of H&E, Masson trichrome and Sirius red staining of renal cortex at day 14 after UUO in three groups (b) and quantitative analyses of fibrotic area (c). Scale bar, 50 µm. (d and e) Representative Western blot (d) and quantitative data (e) showed the protein levels of TNC, Fibronectin, COL1A1, a-SMA, EGR1and SHH at day 14 after UUO. (f and g) Representative Western blot (f) and quantitative data (g) showed the protein levels of p-ERK, ERK, p-STAT3 and STAT3 at day 14 after UUO. (h) Real-time PCR showed the mRNA levels of Il1ß, Il11, Ccl2 and Tnf at day 14 after UUO. Data were expressed as means ± SD. Student's t-test in (c) or one-way ANOVAs (Least-Significant Difference test or Dunnett's T3 test) in (e), (g) and (h) was used for determining the P values while comparing between each pair of groups, respectively, #P < 0.05, *P < 0.01 versus UUO14day-IgG (n = 6).

Fig 4: LIF expression in renal biopsies and urine from CKD patients. (a) The expression of LIF along with the decline of eGFR (n = 39). (b) The association between the log2 of LIF expression and eGFR (n = 39). (c) Differential expression of IL6 family cytokines between subjects with (n = 33) and without (n = 6) TIF. (d) The expression of LIF in different stages of Oxford-T grades of IgAN (n = 39). (e) Differential expression of IL6 family cytokines between CKD (n = 28) and healthy subjects (n = 9) from the ERCB Nephrotic Syndrome Data set. (f) The association between the log2 of LIF expression and eGFR in subjects (n = 33) from the ERCB Nephrotic Syndrome Data set. (g) Western blot analysis of LIF protein in 24 h-urine. (h) The amount of LIF protein in 24 h-urine was increased along with the stage of Oxford-T grades of IgAN (n = 50) and healthy controls (n = 8). The amount of LIF protein in 24 h-urine was measure by ELISA. (i) The amount of LIF protein in 24 h-urine negatively correlated with eGFR of IgAN patients (n = 50). (j–l) The level of uLIF at baseline predicts the risk of CKD progression to ESRD. Tertiles of uLIF-7 levels had a graded relationship with the risk of CKD progression to ESRD in total cohort (j), in subgroups of patients with eGFR <60 ml/min/1.73 m2 (k) and patients with proteinuria >1.0 g/d (l). Data in (a), (d) and (h) were represented as median ± interquartile range (IQR) and P values were determined by one-way ANOVA (Least-Significant Difference test). The Spearman correlation analysis was used in (b), (f) and (i). The R package limma (3.46.0) was used in (c) and (e) for gene differential expression analysis between two groups. Data in (j), (k) and (l) were generated using the Kaplan–Meier method and compared using the log-rank test.

Fig 5: LIF was upregulated in fibroblasts in mouse models of TIF induced by UUO and IRI. (a) The mRNA level of IL6 cytokines family at days 3, 7 and 14 after IRI (n = 6), normalized with gapdh. (b) The mRNA level of IL6 cytokines family at days 3, 7 and 14 after UUO (n = 6), normalized with gapdh. (c and d) Western blot analysis (c) and densitometric quantification (d) of LIF protein at days 3, 7 and 14 after UUO (n = 6). (e and f) Western blot analysis (e) and densitometric quantification (f) of LIF protein at day 14 after IRI (n = 6). (g) Dotplot illustrating expression of Lif and Il11 in single nucleus RNA sequencing (snRNA-seq) dataset of fibrotic kidney from mice 14 days after UUO. (h) Dotplot illustrating expression of Lifr, Il6st and Tgfb1 in single nucleus RNA sequencing (snRNA-seq) dataset of fibrotic kidney from mice 14 days after UUO. (i) Dotplot illustrating expression of Lif in a published Smart-Seq dataset of PDGFRß+ cells from mice 10 days after UUO. (j) Dotplot illustrating expression of Lifr, Il6st, pdgfrb and pdgfra in a published Smart-Seq dataset of PDGFRß+ cells from mice 10 days after UUO. (k) Representative LIF immunostaining in renal sections from sham mice (left panel) and UUO mice (middle and right panel). Magnification, ×400 (left and middle panel), ×1000 (right panel). Arrows indicate positive LIF expression. The boxed region was shown at higher magnification (right panels). Scale bar, 50 µm. (l) Representative LIFR immunostaining in renal sections from UUO 7 day mice. LIFR was detected in mesenchymal cells (Use triangle to indicate) and some tubular epithelial cells (Use arrows to indicate). Magnification, ×400. (m) Representative fluorescent images of coimmunostaining LIF (red) with FSP-1 (green) in renal sections from UUO mice. Arrows in magnified image indicate colocalization of LIF and FSP-1. The boxed region was showed at higher magnification. Scale bar, 50 µm. Data were expressed as means ± SD. #P < 0.05, *P < 0.01 versus sham mice. P values were determined by Student's t-test in (f) and one-way ANOVA (Least-Significant Difference test or Dunnett's T3 test) in (a), (b) and (d). Data in (g) and (h) referenced frome Wu, H., et al., 2019. Data in (i) and (j) referenced frome Kuppe, C., et al., 2021.