Fig 1: NEU1 is significantly upregulated in kidneys from patients with CKD.a Relative NEU1, NEU2, NEU3, and NEU4 mRNA levels. Data analysis from Nephroseq database (‘Ju CKD Tublnt’ dataset, median-centered log2). n = 31 samples in control group, n = 123 samples in CKD group. Unpaired t-test. ns, no significant difference. nd, not detected. b The expression of NEU1 (median-centered log2) in kidney specimens from control (n = 21 samples) and patients with chronic kidney disease (CKD, n = 149 samples). Unpaired t-test. Data analysis from Nephroseq database (‘Ju CKD Glom’ dataset). c The mRNA levels of NEU1 in kidney specimens of CKD (n = 53 samples) and control (n = 8 samples) in GSE66494 dataset (Probe ID: A_24_P394533). Unpaired t-test. d Tissue adjacent sections of kidney from patients with non-renal fibrosis or renal fibrosis by immunohistochemistry, Masson staining, and HE staining. Scale bar = 20 μm. NRF non-renal fibrosis, RF Renal fibrosis. n = 8 samples per group. e–g Quantification of NEU1 expression (e), fibrotic area (f), and score of kidney damage (g) based on immunohistochemistry, Masson, or HE staining in (d). Data were presented as mean ± SD. n = 8 samples per group. Unpaired two-tailed t-test. IOD: integrated optical density. h The correlation of NEU1 expression and degree of tubular degeneration (n = 16, Pearson χ2 test). i–k Pearson’s correlation of NEU1 with serum creatinine level (i), blood urea nitrogen (BUN) (j), and glomerular filtration rate (GFR) (k) (n = 16, Pearson χ2 test). l–n Representative images of co-immunofluorescence staining of NEU1 and KIM1 in kidney tissues of non-renal fibrosis and renal fibrotic patients (l). Fluorescence intensity of NEU1 and KIM1 in diagram k-up (m) and k-down (n), Image J software was used for statistics. Scale bars = 20 μm. n = 3 samples per group. a–c Data are presented as box-and-whisker plots, solid line inside box indicates the median, the bottom and top of box represent first and third quartiles, and the bottom and top whisker show the minimum and maximum, respectively. All tests were two-tailed.
Fig 2: TEC-specific deletion of Neu1 alleviates UUO-induced mouse renal fibrosis.a Scheme of the experimental approach. UUO, unilateral ureteral obstruction. b The gross appearance of kidneys (Scale bar, 1 mm), Hematoxylin and eosin (HE, Scale bar, 20 μm), Masson’s trichrome staining (Scale bar, 20 μm), immunohistochemistry staining of CD68, and p-NFκB (Scale bar, 50 μm) from control (Ctrl) and Neu1 CKO mice 10 days after UUO. The red arrow indicates positive cells. n = 3 mice per group. c The ratio of left renal weight to tibia length (TL). n = 12 mice per group. (d) Statistical results for interstitial collagen analyzed by Image Pro-Plus software. n = 3 mice per group. e, f Morphometric analysis, assessing percentage of tubular necrosis index (e) and tubulointerstitial inflammation index (f). n = 3 mice per group. g, h Quantitative results of CD68 (g) and p-NFκB (h). n = 3 mice per group. i Kim1 mRNA levels. n = 4 mice per group. j Images of immunofluorescence staining. Scale bars, 20 μm. k Statistical analysis of staining double-positive cells of E-cadherin and Vimentin. HPF, high power field. n = 3 mice per group. l, m EMT and extracellular matrix-associated gene mRNA levels. n = 3 mice per group. Gene expression levels were normalized to Gapdh. All statistic data were presented as mean ± SD, two-way ANOVA followed by Tukey’s multiple comparisons test.
Fig 3: NEU1 interacts with GS domain of ALK5.a Schematic representation of the full-length forms of transforming growth factor β family proteins (ALK1, ALK2, ALK3, ALK4, ALK5, ALK6, ALK7, AMHR2, ACVR2B, and TGRBR2 receptors). The red “+” indicates the combination with NEU1. ECD, extracellular domain; TM, transmembrane domain; GS, glycine-serine repeats; STK, serine/threonine kinase domain. b Co-immunoprecipitation of NEU1 with transforming growth factor β family proteins in HEK293T. Two independent experiments were performed. c Confocal images of NEU1 (red) and ALK5 (green) localization in the fibrotic kidney of patients. Scale bar, 20 μm. d, e Co-immunoprecipitation of NEU1 and ALK5 in HK-2 cells stimulated with TGFβ (10 ng/ml) for 24 h. Two independent experiments were performed. f Scheme of NEU1 and ALK5 fusion proteins used for bimolecular fluorescence complementation (BiFC) analysis. g, h BiFC signals were detected in HK-2 cells. Representative fluorescence images of HK-2 cells co-expression of NEU1-VC155 and ALK5-VN173 plasmid without (g) or with (h) TGFβ stimulation. Scale bar, 10 μm. The magnified image scale was 1 μm. i Schematic diagram of in situ proximity ligation assay (NEU1-ALK5 PLA). j Interaction between NEU1 and ALK5 (NEU1-ALK5, red arrow) was analyzed by PLA in the fibrotic kidney of patients. Scale bars, 10 μm. k The interaction between NEU1 and ALK5162-403aa tested by SPR. The frequency response and fitting curves were displayed, two-tailed Pearson’s test. l Co-immunoprecipitation of NEU1 and ALK5160-200aa in HEK293T cells. m Interaction between NEU1 and ALK5 (NEU1-ALK5, red) was analyzed by PLA in HK-2 cells transfected with ALK5160-200aa plasmid. Scale bars, 10 μm. n Co-immunoprecipitation of NEU1 and ALK5 in HEK293T cells transfected with ALK5160-200aa plasmid. c, g, h, j and l–n were repeated three times independently with similar results.
Fig 4: NEU1 mediates the renal protective effects of salvianolic acid B.a Scheme of the experimental approach. The Neu1 CKO mice were treated with salvianolic acid B (SaB) at the indicated doses for 10 continuous days after being subjected to UUO surgery. b Representative gross appearance of kidneys (Scale bar, 2 mm), kidney cross-sections stained with HE (Scale bar, 50 μm), Masson’s trichrome (Scale bar, 50 μm), immunohistochemical staining with E-Cadherin and Snail (Scale bar, 50 μm). n = 3 mice per group. The red arrow indicates positive area. c Statistical results for interstitial collagen in b analyzed by Image Pro-Plus software. n = 3 mice per group. d Kim1 mRNA level. n = 3 mice per group. e, f Quantification of staining of E-Cadherin (e) and Snail1 (f). n = 3 mice per group. g, h mRNA levels of the indicated genes in kidneys determined by qRT-PCR. n = 3 mice per group. Dotted line represents the expression in sham control tissue. Gene expression levels were normalized to Gapdh. i Representative image of immunohistochemical staining with p-ALK5 (ser165) (Scale bar, 50 μm). n = 3 mice per group. j Quantification of staining of p-ALK5 (ser165). n = 3 mice per group. All data were presented as mean ± SD, one-way ANOVA followed by Tukey’s multiple comparisons test. All tests were two-tailed.
Fig 5: Targeting NEU1 by salvianolic acid B alleviates UUO-induced renal fibrosis.a The interactions between 74 compounds with recombinant human NEU1 determined by surface plasmon resonance (SPR). KD, dissociation constant. The compound 3, 9, 11, 12, 18, 19, 20, 21, 41, 53, 58, 60, 61, 62, 67, 72, 73 have no KD value because they do not bind to NEU1. b, c The interaction between NEU1 and salvianolic acid B (SaB) (b) and salvianolic acid A (SaA) (c) was tested by SPR. The frequency response and fitting curves were displayed. Pearson’s test. d Scheme of the experimental approach. UUO, unilateral ureteral obstruction. e Hematoxylin and eosin (HE) and Masson’s trichrome staining from control (Ctrl) and SaA or SaB-treated mice 10 days after UUO. Scale bar, 50 mm. n = 3 mice per group. f Statistical results for interstitial collagen analyzed by Image Pro-Plus software. n = 3 mice per group. g–o the mRNA levels of kidney injury molecule 1 (Kim1, g), EMT associate genes (Snai1 and Snai2, h, i), inflammatory cytokines associate genes (Tnfα, Il1, and Il6, j–l) and extracellular matrix associate genes (Vim, Col1a1, and Col3a1, m–o) in kidney samples. All normalized to Gapdh. n = 6 samples per group. p Western blots (p, left panel) and quantitative results (p, right panel) of p-ALK5 (ser165), p-SMAD2/3, and SMAD2/3 in kidney from control (Ctrl) and SaA or SaB-treated mice 10 days after UUO. n = 3 mice per group. All statistic data were presented as mean ± SD, one-way ANOVA followed by Dunnett’s multiple comparisons test. All tests were two-sided.
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