RLDC triggered NETs formation.

The mice were injected weekly intraperitoneally with 7 mg/kg cisplatin for four weeks and analyzed by execution after 1 month (Cis group) or the untreated control (Veh group). Blood and kidney tissues were collected one week after the last cisplatin injection. A. Representative image of kidney size. B, C. Quantitative analysis of kidney weight and body weight (n = 6, **** p < 0.0001). D. Representative HE-stained images (bar = 100 μm). E. Pathological tubular atrophy score (n = 6, **** p < 0.0001). F, G. The concentrations of serum creatinine and blood urea nitrogen (n = 8, **p = 0.0083, and***p = 0.0008). H. Detection of CitH3 protein levels in the kidney via western blotting (n = 8, **p = 0.0026). I-K. The contents of H3Cit, NE, and cfDNA in plasma after intraperitoneal injection of cisplatin were evaluated via the H3Cit ELISA kit, NETosis assay, and dsDNA ELISA kit (n = 8, **p < 0.01). L, M. Blood flow in the lower limbs of the mice was measured at the start of cisplatin treatment and at the end of each week via a MoorFLPI2 blood flow scatter hemodynamometer. (n = 6, ****p < 0.0001, D28). N-O. NETs (confocal immunofluorescence microscopy images; stained for MPO, Cit H3, LTL, and DNA) visible in kidney samples from chemotherapeutic nephritic mice (n = 4, **p < 0.01, ***p < 0.001). Scale bar, 50 μm. Significant differences were revealed via one-way ANOVA vs. vehicle (A, B, E, F-K, M, and O-Q).

NETs mediate kidney injury.

A. Gross observation of kidneys from RLDC-induced WT or PAD4-/- mice. A representative image of kidney size is shown (n = 5). B. Trends in the body weights of the mice during the four weeks of modeling (n = 10). C-E. HE, PAS, and Masson’s trichrome staining of kidney slices (scale bars 50 μm, n = 6). F. Representative confocal images of KIM-1+ and LTL+ tubules (scale bars 50 μm, n = 6). G-I. Quantification of renal tubular damage and renal fibrosis in the mice in each group (n = 6, ****p < 0.0001). J, K. Quantification of KIM-1-positive and LTL-positive areas of the kidney (n = 6, ****p < 0.0001). L‒N. WB analysis and quantification of KIM-1 and BAX expression in the kidney in each group as indicated (n = 6, ***p < 0.0001, and ****p < 0.0001). Significant differences were revealed via one-way ANOVA vs. Vehicle/PAD4-/- (B, J-K, M, and N).

NETs activate the NLRP3 inflammasome and subsequent renal fibrosis.

A, B. Induction of CKD in WT and Pad4-/- mice treated with 7 mg/kg cisplatin for four weeks. Serum creatinine and blood urea nitrogen (n = 6, ***p < 0.0001, and **p = 0.0049). C-E. The contents of Cit H3, NE, and cfDNA in plasma after intraperitoneal injection of cisplatin were evaluated via the Cit H3 ELISA kit, NETosis assay, and dsDNA ELISA kit (n = 6, ****p < 0.0001). F‒L. Western blot analysis of NLRP3, Casp-1, IL18, IL-1β, IFNγ and Cit H3 in kidney tissues. β-actin was used as a loading control (n = 6, **p = 0.0029, ***p = 0.0004, ****p < 0.0001). M. Representative images of costaining for IFNγ (green), LTL (red), and DAPI (blue). Scale bar, 100 μm. N. Representative images of costaining for α-SMA (green), LTL (red), and DAPI (blue). Scale bar, 50 μm. O, P. Quantification of IFNγ-positive and α-SMA-positive areas in the kidney (n = 6, ****p < 0.0001). Significant differences were revealed via one-way ANOVA vs. vehicle/PAD4-/-(A-E, J-L, O and P).

NETs trigger thrombus formation, leading to local ischemia and hypoxia.

A-E. Western blot for HIF-1α, TF, MMP9 and TFPI in kidney tissues. β-actin was used as a loading control (n = 6, **p=0.0012, and ***p=0.0004, ****p < 0.0001). F. Confocal immunofluorescence microscopy images of kidney samples from chemotherapeutic nephritic mice (n = 4) stained for TF, Cit H3, LTL and DNA. Scale bar, 50 μm. G, H. Quantification of Cit H3-positive and TF-positive areas in the kidney (n = 4, ****p < 0.0001). I. Representative images of Ki67 immunofluorescence staining and costaining with LTL. Scale bar, 100 μm. K. Quantification of the Ki67 area in the kidney (n = 6, ****p < 0·0001). J, L. Measurement of lower limb blood flow in WT and PAD4-/- mice via a MoorFLPI2 blood flow scattering hemodynamometer (n = 6, ***p = 0.0003). Significant differences were revealed via one-way ANOVA vs. vehicle/PAD4-/-(B-E, G, H, K and L).

OPCs retain the integrity of the cisplatin-treated intestinal barrier.

A, B. Macroscopic images and the length of the colon from each group were measured (n = 6, ***p = 0·0005). C. HE staining of colon sections (scale bars 50 μm, n = 6). d. Colonic villus length (40 villi per group, ****p < 0.0001). E, F. Intestinal blood flow and perfusion indices were measured via a laser speckle blood flow analysis system (n = 6). G-I. Immunoblot analysis of tight junctions in the colons of cisplatin-treated mice (n = 6, ****p < 0.0001). J-M. The expression levels of the tight junction proteins ZO-1 and Occludin were observed via immunofluorescence (scale bars 100 μm, n = 6, ****p < 0·0001). N. LPS levels in the serum of the mice (n = 6, p < 0.0001). O. FITC-dextran distribution in the mice with colitis was observed via small animal imaging. P. Content of FITC-dextran in serum (n = 5, ***p = 0.0003). Significant differences were revealed via one-way ANOVA (B, D, F, H, I, K, M, N, and P).

Analysis of the mouse gut microbiota via 16S rDNA gene sequencing.

A-D. α-Diversity indicated by the Chao index, Shannon index, PD_whole_tree index and observed features (interquartile range, IQR, n = 8, *p < 0.05). E. Principal component analysis (PCA) revealed significant differences at the phylum level. F. Effect of OPCs on the β diversity of the gut microbiota assessed via principal coordinate analysis (PCoA). G, H. Relative abundance of Bacteroidetes and Firmicutes at the phylum level (n = 6, ***p = 0.0002). I. Abundances of the top 5 species at different taxonomic levels and absolute species abundance information on the basis of ASVs within samples. J. LDA analysis. K. LEfSe analysis. L, M. Classification of functions based on Tax4Fun and FAPROTAX analysis. N. Analysis of differences in KEGG metabolic pathways. (n = 8, *p < 0.05).

OPCs reduces kidney damage by inhibiting NETs.

The administration of OPCs (100 mg/kg, i.g./3 d) was conducted in conjunction with the initial intraperitoneal cisplatin injection. RLDC treatment was administered with or without OPCs treatment and was administered one week after the last cisplatin treatment. A‒M. Immunoblot analysis of HIF-1α, TF, MMP9, TFPI, BAX, NLRP3, Casp-1, IL18, IL1β, IFNγ, Cit H3 and KIM-1 in kidney tissues. For quantification, the protein was analyzed through densitometry and then normalized to β-actin (n = 6, **p < 0.01, ***p < 0.001, ****p < 0.0001). N-P. The content of Cit H3, NE, and cfDNA in plasma was evaluated via the Cit H3 ELISA kit, NETosis Assay, and dsDNA ELISA kit (n = 6, ***p < 0.001, ****p < 0.0001). Q, R. Serum creatinine (SCr) and blood urea nitrogen (BUN) levels (n = 6, ***p = 0.0002, ****p < 0.0001). S. Representative HE-stained kidney slices (scale bars = 50 μm). T. Quantification of renal tubular damage in the mice in each group (n = 6, ****p < 0.0001). U. Representative images of PAS-stained kidney slices (scale bars = 50 μm). V. Quantification of renal tubular damage in the mice in each group (n = 6, ****p < 0.0001). w. Masson’s trichrome staining of kidney cortex sections (scale bars = 50 μm). X. Quantification of the collagen-positive area according to Masson staining (n = 6, ****p < 0.0001). Significant differences were revealed via one-way ANOVA vs. Vehicle/Cis+OPCs (B-R, T, V, and X).

OPCs inhibit NETs production, with subsequent thromboembolism and inflammatory fibrosis.

A. NETs (confocal immunofluorescence microscopy images; stained for MPO, Cit H3, LTL and DNA) visible in kidney samples from chemotherapeutic nephritic mice (scale bar = 50 μm). B. Thrombus (confocal immunofluorescence microscopy images; stained for TF, Cit H3, LTL and DNA) visible in kidney samples from chemotherapeutic nephritic mice (scale bar = 50 μm). C-E. Quantification of renal Cit H3 positivity, MPO positivity and the colabeled area of the kidney (n = 4, ****p < 0.0001). F-H. Quantification of renal Cit H3 positivity, TF positivity and colabeled area of the kidney (n = 4, ****p < 0.0001). I. ROS analysis by DCFH-DA staining in neutrophils pretreated with OPCs for 1 h followed by cisplatin for 12 h. J. Quantification of DCFH-DA staining of neutrophils by luminescence zymography (n = 4, ****p < 0.0001). K. Serum SOD levels (n = 6, **p = 0.0015). L. Serum GSH levels (n = 6, *p = 0.0142). m. Representative images of KIM-1- and LTL–stained sections of mouse kidneys (scale bar = 50 μm). n, o. KIM-1-positive and LTL-positive areas of the kidney (n = 6, ****p < 0.0001, **p = 0.0077). P. Representative images of costaining for α-SMA (green), LTL (red), and DAPI (blue). Scale bar = 50 μm. Q. Quantification of the α-SMA-positive area of the kidney (n = 6, ****p < 0.0001). Significant differences were revealed via one-way ANOVA vs. Vehicle/Cis+OPCs (C-H, J-L, N, O, and Q).

OPCs inhibits the cisplatin- and LPS-induced release of NETs from neutrophils.

A, B. Immunostaining of mouse neutrophils cultured as indicated. Anti-MPO (purple), anti-Cit H3 (green), and DAPI (blue) staining was used to assess NETs formation. Scale bar = 100 μm (n = 3).

Schematic illustration showing that RLDC-induced NETs promote the development of CKD by disrupting the gut barrier and the therapeutic role of OPCs.

A. The combination of cisplatin and intestine-derived LPS induces NETs formation, leading to CKD via the gut‒kidney axis. B. Cisplatin-induced gut barrier dysfunction facilitates NETosis caused by both LPS and cisplatin, which disturb microcirculation. C. The inhibition of NETosis by OPCs is attributed to its anti-inflammatory and antioxidant activities and ability to maintain a balanced intestinal flora. D. NETs induce local ischemia and fibrosis, which are involved in the pathogenesis of kidney damage.