Figure 1. Protein expressions of EMT biomarkers in NRK-52E cells treated by TGF-β and oxidized LDL. (A) Protein expression of phosphorylated (p)-Smad2, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (B) Protein expression of Snail, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (C) Protein expression of alpha smooth actin (α-SMA), * vs. other groups with different symbols (†, ‡, §), p < 0.001. (D) Protein expression of fibroblast-specific protein 1 (Fsp1), * vs. other groups with different symbols (†, ‡, §), p < 0.001. (E) Protein expression of E-cadherin (E-cad), * vs. other groups with different symbols (†, ‡, §), p < 0.001. (F) Protein expression of collagen type I (Coll-I), * vs. other groups with different symbols (†, ‡, §), p < 0.001. (G) Protein expression of laminin, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (H) Protein expression of elastin, * vs. other groups with different symbols (†, ‡, §), p < 0.001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 3 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level). TGF-β = transforming growth factor-beta; EMT = epithelial mesenchymal transition; LDL = low-density lipoprotein.

Figure 1. Protein expressions of EMT biomarkers in NRK-52E cells treated by TGF-β and oxidized LDL. (A) Protein expression of phosphorylated (p)-Smad2, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (B) Protein expression of Snail, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (C) Protein expression of alpha smooth actin (α-SMA), * vs. other groups with different symbols (†, ‡, §), p < 0.001. (D) Protein expression of fibroblast-specific protein 1 (Fsp1), * vs. other groups with different symbols (†, ‡, §), p < 0.001. (E) Protein expression of E-cadherin (E-cad), * vs. other groups with different symbols (†, ‡, §), p < 0.001. (F) Protein expression of collagen type I (Coll-I), * vs. other groups with different symbols (†, ‡, §), p < 0.001. (G) Protein expression of laminin, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (H) Protein expression of elastin, * vs. other groups with different symbols (†, ‡, §), p < 0.001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 3 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level). TGF-β = transforming growth factor-beta; EMT = epithelial mesenchymal transition; LDL = low-density lipoprotein.

Figure 2. Cellular levels of fibrosis/ECM and kidney injury biomarkers in NRK-52E cells treated by TGF-β and oxidized LDL. (A–D) Illustrating the immunofluorescent (IF) stain of microscopic finding (400×) for identification of cellular expression of laminin (green color). (E) Analytical result of number (%) of laminin+ cells, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (F–I) Illustrating the IF stain of microscopic finding (400×) for identification of cellular expression of fibronectin (green color). (J) Analytical result of number (%) of fibronectin+ cells, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (K–N) Illustrating the IF stain of microscopic finding (400×) for identification of the expression of collagen I (green color). (O) Analytical result of number (%) of collagen I+ cells, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (P–S) Illustrating the IF microscopic finding (400×) for identification of cellular expression of kidney injury molecule-1 (KIM-1) (green color). (T) Analytical result of number (%) of KIM-1+ cells, * vs. other groups with different symbols (†, ‡, §), p < 0.001. Scale bars in right lower corner represent 20 µm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 5 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level).

Figure 2. Cellular levels of fibrosis/ECM and kidney injury biomarkers in NRK-52E cells treated by TGF-β and oxidized LDL. (A–D) Illustrating the immunofluorescent (IF) stain of microscopic finding (400×) for identification of cellular expression of laminin (green color). (E) Analytical result of number (%) of laminin+ cells, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (F–I) Illustrating the IF stain of microscopic finding (400×) for identification of cellular expression of fibronectin (green color). (J) Analytical result of number (%) of fibronectin+ cells, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (K–N) Illustrating the IF stain of microscopic finding (400×) for identification of the expression of collagen I (green color). (O) Analytical result of number (%) of collagen I+ cells, * vs. other groups with different symbols (†, ‡, §), p < 0.001. (P–S) Illustrating the IF microscopic finding (400×) for identification of cellular expression of kidney injury molecule-1 (KIM-1) (green color). (T) Analytical result of number (%) of KIM-1+ cells, * vs. other groups with different symbols (†, ‡, §), p < 0.001. Scale bars in right lower corner represent 20 µm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 5 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level).

Figure 3. Impact of synergic effect of TGF-β and oxidized LDL on wound healing process and migratory assay of NRK-52E. (A–D) Illustrating the baseline (i.e., at 0 h) wound healing process. No difference in term of speed of the wound healing process. (E–H) Illustrating the 24 h morphological feature of wound healing process. (I) Analytical result of wound healing speed, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (J–M) Illustrating the microscopic finding (100×) for identification of cell migratory ability (pink-gray color) after 24 h incubation. (N) Analytical result of number of migratory cells, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. Scale bars in right lower corner represent 100 µm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 5 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level).

Figure 3. Impact of synergic effect of TGF-β and oxidized LDL on wound healing process and migratory assay of NRK-52E. (A–D) Illustrating the baseline (i.e., at 0 h) wound healing process. No difference in term of speed of the wound healing process. (E–H) Illustrating the 24 h morphological feature of wound healing process. (I) Analytical result of wound healing speed, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (J–M) Illustrating the microscopic finding (100×) for identification of cell migratory ability (pink-gray color) after 24 h incubation. (N) Analytical result of number of migratory cells, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. Scale bars in right lower corner represent 100 µm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 5 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level).

Figure 4. The mechanism of oxidized LDL boosting TGF-β on the EMT process in renal tubular cells. Based on the results of the in vitro studies, we schematically illustrate the underling mechanism of oxidized LDL boosting TGF-β on the EMT process in renal tubular cells, i.e., NRK-52E cells. Note that the upper panel (A) used the textual description to fully explain the pathological process of the renal tubular epithelial cells in the middle panel (B) after the oxidized LDL or TGF-β treatment. On the other hand, the lower panel (C) fundamentally concluded the final pathological outcomes of renal tubular epithelial cells after the oxidized LDL or TGF-β treatment. To illustrate this underlying mechanism of oxidized LDL or TGF-β induced renal tubular epithelial cells into EMT would lead to the reader easily understanding the impact of oxidized LD/TGF-β on the pathogenesis of EMT in the setting of CKD. A = upper panel; B = middle panel; C = lower panel.

Figure 4. The mechanism of oxidized LDL boosting TGF-β on the EMT process in renal tubular cells. Based on the results of the in vitro studies, we schematically illustrate the underling mechanism of oxidized LDL boosting TGF-β on the EMT process in renal tubular cells, i.e., NRK-52E cells. Note that the upper panel (A) used the textual description to fully explain the pathological process of the renal tubular epithelial cells in the middle panel (B) after the oxidized LDL or TGF-β treatment. On the other hand, the lower panel (C) fundamentally concluded the final pathological outcomes of renal tubular epithelial cells after the oxidized LDL or TGF-β treatment. To illustrate this underlying mechanism of oxidized LDL or TGF-β induced renal tubular epithelial cells into EMT would lead to the reader easily understanding the impact of oxidized LD/TGF-β on the pathogenesis of EMT in the setting of CKD. A = upper panel; B = middle panel; C = lower panel.

Figure 5. The time courses of circulating levels of BUN and creatinine and the ratio of urine protein to urine creatinine. (A) Baseline circulatory level of blood urine nitrogen (BUN), p > 0.5. (B) Baseline circulatory level of creatinine, p > 0.5. (C) Baseline ratio of urine protein to urine creatinine (RuPr/uCr), p > 0.5. (D) By day 14 after CKD induction, the circulatory BUN level, * vs. †, p < 0.0001. (E) By day 14 after CKD induction, the circulatory creatinine level, * vs. †, p < 0.0001. (F) By day 14 after CKD induction, the RuPr/uCr, * vs. †, p < 0.0001. (G) By day 28 after CKD induction, the circulatory BUN level, * vs. other groups with different symbols (†, ‡), p < 0.0001. (H) By day 28 after CKD induction, the circulatory creatinine level, * vs. other groups with different symbols (†, ‡), p < 0.0001. (I) By day 28 after CKD induction, the RuPr/uCr, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (J) By day 42 after CKD induction, the circulatory BUN level, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (K) By day 42 after CKD induction, the circulatory creatinine level, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (L) By day 42 after CKD induction, the RuPr/uCr, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level). SC = sham-operated control; CKD = chronic kidney disease; R = rosuvastatin.

Figure 5. The time courses of circulating levels of BUN and creatinine and the ratio of urine protein to urine creatinine. (A) Baseline circulatory level of blood urine nitrogen (BUN), p > 0.5. (B) Baseline circulatory level of creatinine, p > 0.5. (C) Baseline ratio of urine protein to urine creatinine (RuPr/uCr), p > 0.5. (D) By day 14 after CKD induction, the circulatory BUN level, * vs. †, p < 0.0001. (E) By day 14 after CKD induction, the circulatory creatinine level, * vs. †, p < 0.0001. (F) By day 14 after CKD induction, the RuPr/uCr, * vs. †, p < 0.0001. (G) By day 28 after CKD induction, the circulatory BUN level, * vs. other groups with different symbols (†, ‡), p < 0.0001. (H) By day 28 after CKD induction, the circulatory creatinine level, * vs. other groups with different symbols (†, ‡), p < 0.0001. (I) By day 28 after CKD induction, the RuPr/uCr, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (J) By day 42 after CKD induction, the circulatory BUN level, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (K) By day 42 after CKD induction, the circulatory creatinine level, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (L) By day 42 after CKD induction, the RuPr/uCr, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 8 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level). SC = sham-operated control; CKD = chronic kidney disease; R = rosuvastatin.

Figure 6. Protein expressions of EMT biomarkers in kidney parenchyma by day 42 after CKD induction. (A) Protein expression of phosphorylated (p)-Smad2, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (B) Protein expression of Snail, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (C) Protein expression of α-SMA, * vs. other groups with different symbols (†, ‡), p < 0.0001. (D) Protein expression of fibroblast-specific protein 1 (Fsp1), * vs. other groups with different symbols (†, ‡), p < 0.0001. (E) Protein expression of transforming growth factor (TGF)-1β, * vs. other groups with different symbols (†, ‡), p < 0.0001. (F) Protein expression of vimentin, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (G) Protein expression of E-cadherin, * vs. other groups with different symbols (†, ‡), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level).

Figure 6. Protein expressions of EMT biomarkers in kidney parenchyma by day 42 after CKD induction. (A) Protein expression of phosphorylated (p)-Smad2, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (B) Protein expression of Snail, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (C) Protein expression of α-SMA, * vs. other groups with different symbols (†, ‡), p < 0.0001. (D) Protein expression of fibroblast-specific protein 1 (Fsp1), * vs. other groups with different symbols (†, ‡), p < 0.0001. (E) Protein expression of transforming growth factor (TGF)-1β, * vs. other groups with different symbols (†, ‡), p < 0.0001. (F) Protein expression of vimentin, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (G) Protein expression of E-cadherin, * vs. other groups with different symbols (†, ‡), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level).

Figure 7. Protein expressions of apoptotic and oxidative stress biomarkers in kidney parenchyma by day 42 after CKD induction. (A) Protein expression of mitochondrial Bax (mit-Bax), * vs. other groups with different symbols (†, ‡), p < 0.0001. (B) Protein expression of cleaved caspase 3 (c-Casp3), * vs. other groups with different symbols (†, ‡), p < 0.0001. (C) Protein expression of cleaved poly (ADP-ribose) polymerase (c-PARP), * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (D) Protein expression of NOX-1, * vs. other groups with different symbols (†, ‡), p < 0.0001. (E) Protein expression of NOX-2, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (F) The oxidized protein expression, * vs. other groups with different symbols (†, ‡, §), p < 0.0001 (Note: the left and right lanes shown on the upper panel represent protein molecular weight marker and control oxidized molecular protein standard, respectively). M.W. = molecular weight; DNP = 1–3 dinitrophenylhydrazone. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level).

Figure 7. Protein expressions of apoptotic and oxidative stress biomarkers in kidney parenchyma by day 42 after CKD induction. (A) Protein expression of mitochondrial Bax (mit-Bax), * vs. other groups with different symbols (†, ‡), p < 0.0001. (B) Protein expression of cleaved caspase 3 (c-Casp3), * vs. other groups with different symbols (†, ‡), p < 0.0001. (C) Protein expression of cleaved poly (ADP-ribose) polymerase (c-PARP), * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (D) Protein expression of NOX-1, * vs. other groups with different symbols (†, ‡), p < 0.0001. (E) Protein expression of NOX-2, * vs. other groups with different symbols (†, ‡, §), p < 0.0001. (F) The oxidized protein expression, * vs. other groups with different symbols (†, ‡, §), p < 0.0001 (Note: the left and right lanes shown on the upper panel represent protein molecular weight marker and control oxidized molecular protein standard, respectively). M.W. = molecular weight; DNP = 1–3 dinitrophenylhydrazone. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level).

Figure 8. Protein expression of ECM in kidney parenchyma by day 42 after CKD induction. (A) Protein expression of matrix metalloproteinase (MMP)-2, * vs. other groups with different symbols (†, ‡), p < 0.0001. (B) Protein expression of MMP-9, * vs. other groups with different symbols (†, ‡), p < 0.0001. (C) Protein expression of laminin, * vs. other groups with different symbols (†, ‡), p < 0.0001. (D) Protein expression of fibronectin, * vs. other groups with different symbols (†, ‡), p < 0.0001. (E) Protein expression of collagen I, * vs. other groups with different symbols (†, ‡), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance for each other (at 0.05 level).

Figure 8. Protein expression of ECM in kidney parenchyma by day 42 after CKD induction. (A) Protein expression of matrix metalloproteinase (MMP)-2, * vs. other groups with different symbols (†, ‡), p < 0.0001. (B) Protein expression of MMP-9, * vs. other groups with different symbols (†, ‡), p < 0.0001. (C) Protein expression of laminin, * vs. other groups with different symbols (†, ‡), p < 0.0001. (D) Protein expression of fibronectin, * vs. other groups with different symbols (†, ‡), p < 0.0001. (E) Protein expression of collagen I, * vs. other groups with different symbols (†, ‡), p < 0.0001. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance for each other (at 0.05 level).

Figure 9. The histopathological analyses of kidney injury score and fibrosis in kidney parenchyma by day 42 after CKD induction. (A–D) Light microscopic findings (200×; H&E stain) showing significantly increased in loss of brush border in renal tubules (yellow arrows), tubular necrosis (green arrows), tubular dilatation (red asterisk), protein cast formation (black asterisk), and dilatation of Bowman’s capsule (blue arrows) in CKD + oxidized LDL group than in other groups. (E) Analytical result of kidney injury score, * vs. other group with different symbols (†, ‡), p < 0.0001. (F–I) Illustrating the microscopic finding (200×) of Masson’s stain for identification of fibrosis (blue color). (J) Analytical result of fibrotic area, * vs. other group with different symbols (†, ‡), p < 0.0001. (K–N) Illustrating the histological finding (200×) of Sirius red stain for identification of condensed collagen-deposition area in renal parenchyma (pink color). (O) Analytical result of condensed collagen-deposition area, * vs. other group with different symbols (†, ‡), p < 0.0001. Scale bars in right lower corner represent 50 µm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance for each other (at 0.05 level).

Figure 9. The histopathological analyses of kidney injury score and fibrosis in kidney parenchyma by day 42 after CKD induction. (A–D) Light microscopic findings (200×; H&E stain) showing significantly increased in loss of brush border in renal tubules (yellow arrows), tubular necrosis (green arrows), tubular dilatation (red asterisk), protein cast formation (black asterisk), and dilatation of Bowman’s capsule (blue arrows) in CKD + oxidized LDL group than in other groups. (E) Analytical result of kidney injury score, * vs. other group with different symbols (†, ‡), p < 0.0001. (F–I) Illustrating the microscopic finding (200×) of Masson’s stain for identification of fibrosis (blue color). (J) Analytical result of fibrotic area, * vs. other group with different symbols (†, ‡), p < 0.0001. (K–N) Illustrating the histological finding (200×) of Sirius red stain for identification of condensed collagen-deposition area in renal parenchyma (pink color). (O) Analytical result of condensed collagen-deposition area, * vs. other group with different symbols (†, ‡), p < 0.0001. Scale bars in right lower corner represent 50 µm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance for each other (at 0.05 level).

Figure 10. The histopathological analyses of kidney injury molecule and podocyte components in kidney parenchyma by day 42 after CKD induction. (A–D) Illustrating the immunofluorescent (IF) microscopic finding (200×) for identification of cellular expressions of kidney injury molecule (KIM)-1 (green color). (E) Analytical result of the expression of KIM-1, * vs. other group with different symbols (†, ‡), p < 0.0001. (F–I) Illustrating the IF microscopic finding (200×) for identification of cellular expressions of ZO-1 (green color). (J) Analytical result of expression of ZO-1, * vs. other group with different symbols (†, ‡), p < 0.0001. (K–N) Illustrating the IF microscopic finding (200×) for identification of cellular expression of synaptopodin (green color). (O) Analytical result of expression of synaptopodin, * vs. other group with different symbols (†, ‡), p < 0.0001. Scale bars in right lower corner represent 50 µm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance for each other (at 0.05 level).

Figure 10. The histopathological analyses of kidney injury molecule and podocyte components in kidney parenchyma by day 42 after CKD induction. (A–D) Illustrating the immunofluorescent (IF) microscopic finding (200×) for identification of cellular expressions of kidney injury molecule (KIM)-1 (green color). (E) Analytical result of the expression of KIM-1, * vs. other group with different symbols (†, ‡), p < 0.0001. (F–I) Illustrating the IF microscopic finding (200×) for identification of cellular expressions of ZO-1 (green color). (J) Analytical result of expression of ZO-1, * vs. other group with different symbols (†, ‡), p < 0.0001. (K–N) Illustrating the IF microscopic finding (200×) for identification of cellular expression of synaptopodin (green color). (O) Analytical result of expression of synaptopodin, * vs. other group with different symbols (†, ‡), p < 0.0001. Scale bars in right lower corner represent 50 µm. All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test (n = 6 for each group). Symbols (*, †, ‡) indicate significance for each other (at 0.05 level).