Neuraminidase 1 promotes renal fibrosis development in male mice

Abstract

The functions of the influenza virus neuraminidase has been well documented but those of the mammalian neuraminidases remain less explored. Here, we characterize the role of neuraminidase 1 (NEU1) in unilateral ureteral obstruction (UUO) and folic acid (FA)-induced renal fibrosis mouse models. We find that NEU1 is significantly upregulated in the fibrotic kidneys of patients and mice. Functionally, tubular epithelial cell-specific NEU1 knockout inhibits epithelial-to-mesenchymal transition, inflammatory cytokines production, and collagen deposition in mice. Conversely, NEU1 overexpression exacerbates progressive renal fibrosis. Mechanistically, NEU1 interacts with TGFβ type I receptor ALK5 at the 160-200aa region and stabilizes ALK5 leading to SMAD2/3 activation. Salvianolic acid B, a component of Salvia miltiorrhiza, is found to strongly bind to NEU1 and effectively protect mice from renal fibrosis in a NEU1-dependent manner. Collectively, this study characterizes a promotor role for NEU1 in renal fibrosis and suggests a potential avenue of targeting NEU1 to treat kidney diseases.

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. NEU1 is elevated in mouse fibrotic kidneys.

a NEU1 mRNA levels in kidneys of mice subjected to unilateral ureteral ligation (UUO) for 0, 3, 7, 10, and 12 days. n = 4 mice per group. Unpaired two-tailed t-test. Data were presented as mean ± SD. b Western blots of NEU1 levels in kidney from mice subjected to UUO for 0, 3, 7, 10, and 12 days or administered folic acid (FA, 250 mg/kg, i.p.) injection for 4 weeks. n = 3 mice per group. c, d Immunohistochemistry of kidney sections (c) and quantitative results (d) from mice after UUO surgery for 10 days or folic acid injection for 4 weeks. The NEU1 was determined with IOD by Image-Pro Plus 6.0. n = 3 mice per group. Scale bar, 20 μm. Data were presented as mean ± SD. Unpaired two-tailed t-test. IOD, integrated optical density. e–g Immunofluorescence images of NEU1 in kidney from mice subjected to UUO (e, f) or folic acid (g). Na⁺/K⁺-ATPase was used as tubular epithelial cell marker, CD68 was used as macrophage marker. n = 3 mice per group. Scale bar, 50 μm. h, i Neu1, Acta2, Vim, Col1a1, Col3a1, and Tgfβ mRNA level in kidney from mice after UUO surgery for 10 days (h) or folic acid injection for 4 weeks (i). n = 6 mice per group. Data were presented as mean ± SD. Unpaired two-tailed t-test. j–s Pearson’s correlation of NEU1 with Acta2, Vim, Col1a1, Col3a1, and Tgfβ mRNA. n = 12, two-tailed Pearson χ² test.

Fig. 3. 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. 4. TEC-specific deletion of Neu1 alleviates folic acid-induced renal fibrosis in mice.

a Scheme of the experimental approach. The mice were intraperitoneally injected with folic acid (250 mg/kg). b The gross appearance of whole kidneys from control (Ctrl) and Neu1 CKO mice 4 weeks after folic acid injection. Scale bar, 1 mm. c The ratio of left renal weight to body weight (BW). n = 6 mice per group. d, e Creatinine (d) and blood urea nitrogen (e) in serum measured by ELISA. n = 6 mice per group. f Kim1 mRNA levels. n = 4 mice per group. g Hematoxylin and eosin (HE), Masson’s trichrome staining, and immunohistochemistry staining of CD68 and p-NFκB in kidney sections from Ctrl and Neu1 CKO mice 28 days after folic acid administration. n = 3 mice per group. Scale bar, 50 μm. The red arrow indicates positive cells. h–k Quantification of tubular injury score (h), fibrosis area (i), staining of CD68 (j), and p-NFκB (k). n = 3 mice per group. l, m EMT and extracellular matrix associate gene mRNA level. n = 4 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. All tests were two-tailed.

Fig. 5. NEU1 overexpression aggravates UUO-induced renal fibrosis.

a Schematic diagram of NEU1 overexpression in mice. Mice were in situ injected with AAV9 encoding NEU1 or scramble. After the injection for 5 weeks, the mice were subjected to UUO surgery. b mRNA levels of Neu1 in the cortices of kidneys. n = 3 mice per group. c NEU1 protein levels in the kidneys of AAV9-Ctrl and AAV9-NEU1 mice. n = 2 mice per group. d The gross appearance of kidneys (Scale bar, 1 mm), kidney cross-sections stained with HE (Scale bar, 50 μm), and Masson’s trichrome (Scale bar, 100 μm). n = 6 mice per group. e The ratio of left renal weight to body weight (BW, mg/g). AAV9-Ctrl, n = 11; AAV9-NEU1, n = 10. f Statistical analysis of tubular injury score. n = 6 mice per group. g Statistical results for interstitial collagen in d analyzed by Image Pro-Plus software. n = 6 mice per group. h Kim1 mRNA level. n = 3 mice per group. i mRNA levels of the indicated genes in kidneys. n = 3 mice per group. Gene expression levels were normalized to Gapdh. j–n Images of immunohistochemical staining using indicated antibodies. Scale bars, 20 μm. n = 6 mice per group. o mRNA levels of the indicated genes in kidneys. n = 3 mice per group. All statistic data were presented as mean ± SD. Unpaired two-tailed t-test.

Fig. 6. 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 ALK5_162-403aa tested by SPR. The frequency response and fitting curves were displayed, two-tailed Pearson’s test. l Co-immunoprecipitation of NEU1 and ALK5_160-200aa in HEK293T cells. m Interaction between NEU1 and ALK5 (NEU1-ALK5, red) was analyzed by PLA in HK-2 cells transfected with ALK5_160-200aa plasmid. Scale bars, 10 μm. n Co-immunoprecipitation of NEU1 and ALK5 in HEK293T cells transfected with ALK5_160-200aa plasmid. c, g, h, j and l–n were repeated three times independently with similar results.

Fig. 7. NEU1 stabilizes ALK5 and enhances ALK5-SMAD2/3 signaling pathway.

a, b HK-2 cells transfected with siNEU1 (a) or NEU1 full-length plasmid (b) for 24 h and treated with TGFβ (10 ng/ml) for 24 h. Then the cells were incubated with cycloheximide (CHX, 20 μg/ml) for the indicated periods of time (0, 2, 4, 8, 12, 24 h) (left). Lysates were harvested from the cells and analyzed by western blots. Quantitation of ALK5 protein levels were shown in the right pane. n = 3 samples per group. Unpaired t-test. c–f Western blots (c, e) and quantitative results (d, f) of p-ALK5, ALK5, p-SMAD2/3, and SMAD2/3 in HK-2 cells transfected with siNEU1 or NEU1 plasmid for 24 h and treated with TGFβ (10 ng/ml) for 24 h. n = 3 samples per group. Relative protein levels were shown after normalization to GAPDH. One-way ANOVA followed by Tukey’s multiple comparisons test. g, h Western blots (g) and quantitative results (h) of p-ALK5, p-SMAD2/3, and SMAD2/3 in HK-2 cells transfected with NEU1 and ALK5_160-200 plasmid for 24 h and treated with TGFβ (10 ng/ml) for 24 h. n = 3 samples per group. Relative protein levels were shown after normalization to GAPDH. One-way ANOVA followed by Tukey’s multiple comparisons test. i Co-immunoprecipitation of NEU1 and ALK5 in HK-2 cells stimulated with TGFβ (10 ng/ml) for 24 h. λpp, Lambda Protein Phosphatase. Two independent experiments were performed. j Western blots of ALK5 in HK-2 cells. siALK5-1, siALK5-2, and siALK5-3 were 3 different siRNA sequences. HK-2 cells were transduced with siALK5 for 48 h. Two independent experiments were performed. k–m mRNA levels of KIM1 (k) and protein level of p-ALK5, p-SMAD2/3, and SMAD2/3 (l, m) in HK-2 cells transduced with NEU1 full-length plasmid and ALK5 siRNA for 48 h. KIM1 mRNA normalized to GAPDH. Relative protein levels were shown after normalization to GAPDH. k n = 3 samples per group. l, m n = 3 samples per group. Two-way ANOVA followed by Tukey’s multiple comparisons test. n The proposed mechanisms of NEU1-mediated renal fibrosis. All statistic data were presented as mean ± SD. All tests were two-tailed.

Fig. 8. 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). K_D, dissociation constant. The compound 3, 9, 11, 12, 18, 19, 20, 21, 41, 53, 58, 60, 61, 62, 67, 72, 73 have no K_D 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.

Fig. 9. 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.