Fig 2: LTBP4 preserves mitochondrial functions in response to hypoxia and Ltbp4 deficiency contributes to fragmented mitochondria and aggravates oxidative stress in mice with unilateral ureteral obstruction (UUO).Seahorse XF Cell Mito Stress Test performed on LTBP4-overexpression human proximal tubule cells cultured under hypoxic conditions (H) for 48 h (H/48 h). A OCR profile plot, maximal respiration, ATP production and based glycolysis are higher in hypoxic LTBP4-overexpression cells (LTBP4) compared to hypoxic cells with normal LTBP4 expression (Mock). In vivo, tubulointerstitial fibrosis was induced by UUO for 5 days in wild-type (WT) and Ltbp4S knockout (Ltbp4S-/-) mice. These data were calculated from three independent experiments. Where indicated, O: oligomycin (1 µM), F: FCCP (1 µM) and R/A: rotenone/antimycin A (0.5 µM) were added. Each data point represents the mean ± SD. B Representative electron microscopic images of proximal tubular cells of the kidneys of WT and Ltbp4S-/- mice with UUO. White arrows indicate mitochondria. N indicates nuclei. Scale bars = 1 µm. Computer-assisted quantitative analyses of electron microscopic images for mitochondrial length (C) and number (D) are shown. E, F Assessment of reactive oxygen species by immunohistochemistry staining for 4-HNE in kidney sections. The kidney sections of Ltbp4S-/- mice with UUO showed strong positive staining for 4-HNE. Scale Bar = 100 µm. Data are presented as the mean ± SEM for each group of mice (n = 4–6 per group). *p < 0.05. 4-HNE: 4-hydroxynonenal. Results are from three independent experiments.

Fig 3: LTBP4 induces angiogenesis for kidney repair in mice subjected to unilateral ureteral obstruction (UUO).Tubulointerstitial fibrosis was induced by UUO for 5 days in wild-type (WT) and Ltbp4 knockout (Ltbp4S-/-) mice. A Representative histological images with Masson’s trichrome and immunofluorescence staining of kidneys for CD31 after UUO. Scale bars, 100 µm. B Quantitative results of vessels per HPF in the mouse kidneys. C Protein expression of VEGF and CD31 in the kidneys of mice with UUO, as detected by using immunoblotting. GAPDH served as an internal control. D, E Densitometric analyses of immunoblot images for VEGF (D) and CD31 (E) are shown. Data are presented as the mean ± SEM for each group (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. Results representative of three independent experiments performed in triplicate.

Fig 4: LTBP4 facilitates angiogenesis in HUVECS and HK-2 cells.Conditioned media (CM) collected from Mock (CM-Mock) or LTBP4-overexpressing HEK293T cells (CM-LTBP4) were used to treat human umbilical vein endothelial cells (HUVECs) and human proximal tubule HK-2 cells for 48 h, and tube areas and angiogenic factors were studied. A Representative images of tube formation in HUVECs. Control: HUVECs were treated with a regular growth medium. Scale bars, 400 µm. The results are from three independent experiments. B Densitometric analyses of tube areas in three groups are shown. C, D In proteomic profiling analysis, dot blot profiling for 55 angiogenesis-related proteins showed relatively increasing expression of proangiogenic factors, including VEGF, EGF, angiopoietin-2 and endothelin-1. E Relative expression of VEGFRs and VEGF in HUVECs treated with CM-Mock and CM-LTBP4. CM-LTBP4 stimulated VEGFRs and VEGFB expression. F, G Effect of overexpression of LTBP4 in HK-2 cells. In normoxia, LTBP4 promoted VEGFB expression while, in hypoxia, LTBP4 enhanced VEGFA expression. *p < 0.05, **p < 0.01, ***p < 0.001; VEGF vascular endothelial growth factor, EGF epidermal growth factor, VEGFA vascular endothelial growth factor A, VEGFB vascular endothelial growth factor B, VEGFR1 vascular endothelial growth factor receptor 1, VEGFR2 vascular endothelial growth factor 2.

Fig 5: Proposed model of LTBP4-mediated mechanisms in tubular interstitial fibrosis.In unilateral ureteral obstruction (UUO)-induced tubular damage, A LTPB4 provokes angiogenesis for tissue repair via VEGF and CD31 elevation and in addition to limiting mitochondrial shortness. B LTBP4 deficiency in proximal tubules causes a reduction in VEGF and CD31 expression, along with subsequent aberrant angiogenesis and severe mitochondrial shortness, which eventually accelerates tubular interstitial fibrosis.