Periostin deficiency attenuates kidney fibrosis in diabetic nephropathy by improving pancreatic β-cell dysfunction and reducing kidney EMT

Figures

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  Figure 1. Periostin and tenascin C levels measured in urine in patients with diabetic nephropathy. (A) The concentration of POSTN in urine based on the UPCR value (N = 207). (B) POSTN urine concentration by CKD stage in DN patients (N = 199). Data are the mean ± SD. *p < 0.05, ***p < 0.001 (one-way ANOVA). (C) TNC urine concentration by CKD stage in DN patients (N = 207). Data are the mean ± SD. *p < 0.05, ***p < 0.001 (one-way ANOVA). (D) Correlation of POSTN/Cr and TNC/Cr. (E) Kaplan‒Meier patient survival curves for ESRD. (F) Kaplan‒Meier patient survival curves for death. (G) Kaplan‒Meier patient survival curves for composite.

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  Figure 2. Functional data of the mouse groups. (A) A graph of the weight changes by group for 24 weeks (WT Sham, WT STZ: N = 7/group; KO Sham, KO STZ: N = 4/group). (B) A comparison of creatinine measurements among groups of mice. (C) Comparative graph of the albumin/creatinine ratios among groups (WT Sham, WT STZ: N = 7/group; KO Sham, KO STZ: N = 4/group). Data are the mean ± SD. *p < 0.05 (Mann‒Whitney test). (D) Measurement of the shear-wave elastography (SWE) of WT Sham kidneys. (E) Shear-wave elastography (SWE) measurements of KO STZ kidneys. (F) A graph of elasticity (kPa) by group based on elastography (N = 3/group). Data are the mean ± SD. **p < 0.01 (Mann‒Whitney test).

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  Figure 3. Analysis of data related to fibrosis among the groups of mice. (A) The degree of fibrosis progression was compared between groups based on Sirius red staining (WT Sham, KO Sham: N = 3/group; WT STZ, KO STZ: N = 4/group). Data are the mean ± SD. *p < 0.05 (Mann‒Whitney test). (B) Results of POSTN IHC staining by group (WT Sham, KO Sham: N = 3/group; WT STZ, KO STZ: N = 4/group). Data are the mean ± SD. *p < 0.05 (Mann‒Whitney test). Bar = 500 μm. Bar = 100 μm. (C) Results of TNC IHC staining by group (WT Sham, KO Sham: N = 3/group; WT STZ, KO STZ: N = 4/group). Data are the mean ± SD. *p < 0.05 (Mann‒Whitney test). Bar = 500 μm. Bar = 100 μm. (D) The result of the concurrent confirmation of POSTN and TNC in the tubule of the kidney tissue is presented by immunofluorescence staining. (E) The degree of intraglomerular fibrosis progression between groups was investigated using Sirius red staining and IHC staining. The glomerular circumference of each group was measured and analyzed (WT Sham, KO Sham: N = 3/group; WT STZ, KO STZ: N = 4/group). Data are the mean ± SD. *p < 0.05 (Mann‒Whitney test). Bar = 100 μm. (F) A comparison of fibrosis marker expression levels by group as assessed…

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  Figure 4. The effect of periostin deficiency on the TGF-β pathway in a diabetic nephropathy model. The expression levels of TGF-β signaling-related markers differed among the study groups, as shown by the western blot results (WT Sham, KO Sham: N = 4/group; WT STZ, KO STZ: N = 8/group). Data are the mean ± SD. *p < 0.05, **p < 0.01 (Mann‒Whitney test). Original blots/gels are presented in Supplementary Fig. 2b, 3b.

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  Figure 5. TGF-β and periostin-induced fibrosis in HK-2 cells. (A) Morphological changes in HK-2 cells after treatment with TGF-β and rPOSTN. Bar = 200 μm. (B,C) Western blot results presenting a change in the expression of TNC and COL1A1 when HK-2 cells were treated with TGF-β and POSTN simultaneously or separately (N = 5/group). Data are the mean ± SD. *p < 0.05, **p < 0.01 (one-way ANOVA). Original blots/gels are presented in Supplementary Fig. 4b.

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  Figure 6. The role of POSTN in pancreatic function. (A) Changes in blood glucose levels by group observed during the 24-week study (WT Sham, WT STZ: N = 7/group; KO Sham, KO STZ: N = 4/group). Data are the mean ± SD. *p < 0.05, **p < 0.01 (Mann‒Whitney test). (B) A comparison of the blood insulin levels between groups (WT Sham, WT STZ: N = 6/group; KO Sham, KO STZ: N = 4/group). Data are the mean ± SD. *p < 0.05 (Mann‒Whitney test). (C) Morphological changes between groups of pancreatic islets as evidenced by PAS staining (WT Sham, KO Sham: N = 3/group; WT STZ, KO STZ: N = 4/group). The IHC staining results indicate differences between groups in insulin and glucagon expression. Data are the mean ± SD. *p < 0.05 (Mann‒Whitney test). Bar = 50 μm. (D) A comparison of POSTN, TNC, insulin, and glucagon levels in pancreatic tissue by group (WT Sham, KO Sham: N = 2/group; WT STZ, KO STZ: N = 4/group). Data are the mean ± SD. *p < 0.05 (Mann‒Whitney test). Original blots/gels are presented in Supplementary Fig. 5b. (E) An analysis of INS-1 cell viability when treated with STZ and neutralizing POSTN antibodies (N = 12/group). Data are the mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001 (one-way…

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  Figure 7. A schematic overview of the animal model. The diabetic nephropathy in vivo model was induced by streptozotocin (STZ) administration after unilateral nephrectomy (UNX). One week after UNX, the mice were injected with 50 mg/kg STZ intraperitoneally for 5 consecutive days. Tail vein blood glucose levels were measured to confirm diabetes (fasting blood glucose > 300 mg/dL). The mice were sacrificed at 24 weeks after induction of diabetes.