Fig 1: HPSE expression was upregulated in GEnCs of STZ-induced diabetic mice.A Quantitation of 24 h urine protein showed significant proteinuria at 4w and 8w respectively after the onset of diabetes in STZ-induced diabetic mice. B PAS staining exhibited dramatic mesangial matrix expansion in STZ-induced diabetic mice. Scale bar = 10 μm. C Immunohistochemical staining revealed the overexpressed HPSE in the glomeruli of diabetic mice. Scale bar = 50 μm. D The count of double positive immunofluorescence staining for CD31 and HPSE was markedly high in diabetic mice, while it was negative in sham animals. Scale bar = 10 μm. E HPSE expression was positively correlated with 24 h proteinuria (R = 0.8874, P = 0.0183) and glomerular mesangial matrix expansion (R = 0.8735, P = 0.023) respectively by Pearson correlation analysis. Each vertical bar represents the mean ± SD (n = 4~7) analyzed by one-way ANOVA or two-way ANOVA followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 2: HPSE had a positive correlation with EndMT of GEnCs in STZ-induced diabetic mice.The counts of double positive IF staining for CD31-α-SMA (A) and CD31-Snail1/2 (B) were markedly high in STZ-induced diabetic mice. Pearson correlation analysis exhibited that HPSE was positively correlated with the numbers of double positive staining for CD31-α-SMA (R = 0.8625, P = 0.0271) (C) or CD31-Snail1/2 (R = 0.8504, P = 0.0319) (D) respectively. Scale bar = 10 μm. Each vertical bar represents the mean ± SD (n = 4~6) analyzed by two-tailed t-tests test. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 3: High glucose promoted EndMT in cultured GEnCs through upregulating HPSE and p-ERK1/2.A High glucose significantly decreased the protein level of CD31 while increased the expressions of mesenchymal markers including α-SMA and Snail1/2. B The protein levels of HPSE and p-ERK1/2 were significantly upregulated by high glucose treatment. C The count of double positive IF staining for p-ERK1/2 and CD31 was dramatically increased in STZ-induced diabetic mice, compared with the negative double staining in sham animals. Scale bar = 10 μm. Each vertical bar represents the mean ± SD (n = 3) analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig 4: Bioinformatics analysis revealed a positive correlation between HPSE and ERK.The Pearson correlation analysis of DN patients’ data (n = 19) from GEO database (GSE14202) showed that nine related genes (A) had a significant positive correlation with HPSE, including MAPK1 (ERK2) (B) (R = 0.83, P = 0.041).
Fig 5: p-ERK1/2 was a vital signaling molecule in HPSE-mediated diabetic glomerular EndMT.A Under normal glucose or high glucose conditions, U0126, a specific MEK1/2 inhibitor, dramatically inhibited the expressions of p-ERK1/2 and the mesenchymal markers including α-SMA and Snail1/2, without affecting HPSE protein levels under the same treatments. Mannitol as osmotic control did not show different effects. B U0126 markedly blocked rmHPSE-induced expressions of p-ERK1/2, α-SMA and Snail1/2 at 12 h and 24 h, respectively. Each vertical bar represents the mean ± SD (n = 3) analyzed by one-way analysis of variance (ANOVA) followed by Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, and ***P < 0.001.
Supplier Page from R&D Systems, a Bio-Techne Brand for Recombinant Mouse Active Heparanase/HPSE Protein, CF