Dapagliflozin-entresto protected kidney from renal hypertension via downregulating cell-stress signaling and upregulating SIRT1/PGC-1α/Mfn2-medicated mitochondrial homeostasis

Abstract

This study tested whether combined dapagliflozin and entresto would be superior to mere one therapy on protecting the residual renal function and integrity of kidney parenchyma in hypertensive kidney disease (HKD) rat. In vitro results showed that the protein expressions of oxidative-stress/mitochondrial-damaged (NOX-1/NOX-2/oxidized-protein/cytosolic-cytochrome-C)/apoptotic (mitochondrial-Bax/cleaved caspeases 3, 9)/cell-stress (p-ERK/p-JNK/p-p38) biomarkers were significantly increased in H₂O₂-treated NRK-52E cells than those of controls that were reversed by dapagliflozin or entresto treatment. Adult-male SD rats (n = 50) were equally categorized into group 1 (sham-operated-control), group 2 (HKD by 5/6 nephrectomy + DOCA-salt/25 mg/kg/subcutaneous injection/twice weekly), group 3 (HKD + dapagliflozin/orally, 20 mg/kg/day for 4 weeks since day 7 after HKD induction), group 4 (HKD + entresto/orally, 100 mg/kg/day for 4 weeks since day 7 after HKD induction), and group 5 (HKD + dapagliflozin + entresto/the procedure and treatment strategy were identical to groups 2/3/4). By day 35, circulatory levels of blood-urine-nitrogen (BUN)/creatinine and urine protein/creatinine ratio were lowest in group 1, highest in group 2, and significantly lower in group 5 than in groups 3/4, but no difference between groups 3/4. Histopathological findings showed the kidney injury score/fibrotic area/cellular expressions of oxidative-stress/kidney-injury-molecule (8-OHdG+/KIM-1+) exhibited an identical trend, whereas the cellular expressions of podocyte components (synaptopodin/ZO-1/E-cadherin) exhibited an opposite pattern of BUN level among the groups. The protein expressions of oxidative stress/mitochondrial-damaged (NOX-1/NOX-2/oxidized protein/cytosolic-cytochrome-C/cyclophilin-D)/apoptotic (mitochondrial-Bax/cleaved-caspase 3)/mitochondrial-fission (PINK1/Parkin/p-DRP1)/autophagic (LC3BII/LC3BI ratio, Atg5/beclin-1)/MAPK-family (p-ERK/p-JNK/p-p38) biomarkers displayed a similar pattern, whereas the protein expression of mitochondria-biogenesis signaling (SIRT1/PGC-1α-Mfn2/complex I-V) displayed an opposite pattern of BUN among the groups. In conclusion, combined dapagliflozin-entresto therapy offered additional benefits on protecting the residual kidney function and architectural integrity in HKD rat.

Keywords: Chronic kidney disease; dapagliflozin; entresto; hypertension; kidney ultrastructure; renal function.

Figure 1.

Cell viability and protein expressions of MAPK family signaling, apoptosis and cytochrome C in cytosol and mitochondria and flow cytometric analysis for identification of early and late apoptosis. (A) Cell viability by 24 h, * versus †, P < 0.001. (B) Cell viability by 48 h, * versus †, P < 0.001. (C) Cell viability by 72 h, * versus †, P < 0.001. n = 6 for each group (i.e. A to C). (D) Protein expression of phosphorylated (p)-ERK1/2, * versus other group with different symbols (†, ‡), P < 0.001. (E) Protein expression of p-JNK, * versus other group with different symbols (†, ‡), P < 0.001. (F) Protein expression of p-p38, * versus other group with different symbols (†, ‡), P < 0.001. (G) Protein expression of cleaved caspase 9 (c-Casp9), * versus other groups with different symbols (†, ‡), P < 0.001. (H) Protein expression of cytosolic cytochrome C (cyt-CytoC), * versus other groups with different symbols (†, ‡), P < 0.001. (I) Protein expression of mitochondrial cytochrome C (mit-CytoC), * versus other groups with different symbols (†, ‡), P < 0.001. n = 3 for each group (i.e. D to I). (J to M) Illustrating the flow cytometric analysis for determining the early and late apoptosis. (N) Flow cytometric result of early (AN-V+/PI-) apoptotic cells, * versus other group with different symbols (†, ‡, §), P < 0.0001. Flow cytometric result of late (AN-V+/PI+) apoptotic cells, * versus other group with different symbols (†, ‡, §), P < 0.0001. n = 6 for each group (i.e. J and K). All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡) indicate significance for each other (at 0.05 level). G1 = NRK-52E; G2 = NRK-52E cells + H₂O₂; G3 = NRK-52E cells + H₂O₂ + dapagliflozin; G4 = NRK-52E H₂O₂ + sacubitril/valsartan.

Figure 2.

Protein expressions of oxidative stress and apoptosis in NRK-52E cells undergoing the two components of oxidative-stress stimulation. (A) Protein expression of mitochondrial Bax (mit-Bax), * versus other group with different symbols (†, ‡, §), P < 0.0001. (B) Protein expression of cleaved caspase 3 (c-Casp3), * versus other group with different symbols (†, ‡, §), P < 0.0001. (C) Protein expression of Bcl-2, * versus other group with different symbols (†, ‡, §), P < 0.0001. (D) Protein expression of NOX-1, * versus other group with different symbols (†, ‡, §, ¶), P < 0.0001. (E) Protein expression of NOX-2, * versus other group with different symbols (†, ‡, §, ¶), P < 0.0001. (F) The oxidized protein expression, * versus other groups with different symbols (†, ‡, §), P < 0.001 (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. n = 3 for each group (i.e. A to F). (G) Flow cytometric analysis for identification of mitochondrial reactive oxygen species (ROS) (i.e. mean fluorescent intensity of mitoSOX [AU]), * versus other group with different symbols (†, ‡, §), P < 0.0001 (n = 6 for each group). All statistical analyses were performed by one-way ANOVA, followed by Bonferroni multiple comparison post hoc test. Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level). G5 (NRK-52E cells only); G6 (NRK-52E cells + H₂O₂ + p-Cresol); G7 (NRK-52E cells + H₂O₂ + p-Cresol + dapagliflozin); G8 (NRK-52E + H₂O₂ + p-Cresol + sacubitril/valsartan); G9 (NRK-52E + H₂O₂ + p-Cresol + combined dapagliflozin; and sacubitril/valsartan).

Figure 3.

Time courses of circulatory levels of BUN and creatinine and R^uPr/uCr and femoral arterial blood pressure and kidney injury score by day 35 after CKD induction. (A to C) Baseline circulating levels of BUN (A) and creatinine (B) and ratio of urine protein to urine creatinine (R^uPr/uCr) (C) did not differ among the groups, P > 0.5. (D to F) By day 35 after CKD induction: circulating levels of BUN (D) and creatinine (E) and R^uPr/uCr (F), * versus other groups with different symbols (†, ‡, §), all P values < 0.0001. (G) By day 35 after CKD induction, the right femoral arterial systolic blood pressure (RFASBP), * versus other groups with different symbols (†, ‡, §), P < 0.0001. (H to L) 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 group than in other groups. (M) Analytical result of kidney injury score, * versus other group 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 creatine and BUN level of each group and n = 6 for evaluation of kidney injury score each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level). SC: sham control; HKD: hypertensive kidney disease; DAPA: dapagliflozin; En: entresto.

Figure 4.

Impact of dapagliflozin and entresto on protein levels of oxidative stress, apoptosis and cell-stress/death signaling by day 35 after CKD induction. (A) Protein expressions of NOX-1, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (B) Protein expression of NOX-2, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (C) Protein expression of mitochondrial Bax (mit-Bax), * versus other groups with different symbols (†, ‡, §, ¶), P < 0.0001. (D) Protein expression of cleaved caspase 3 (c-Casp3), * versus other groups with different symbols (†, ‡, §), P < 0.0001. (E) Protein expression of Bcl-2, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (F) Protein expression of phosphorylated (p)-ERK1/2, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (G) Protein expression of p-JNK, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (H) Protein expression of p-p38, * versus 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). SC: sham control; HKD: hypertensive kidney disease; DAPA: dapagliflozin; En: entresto.

Figure 5.

Schematically illustrated the proposed underlying mechanism of DAPA-entresto therapy on protecting the renal tubular cells and integrity of kidney parenchyma against HKD through downregulating oxidative-stress and cell-stress signaling-medicated mitochondrial dynamic hemostasis.

Figure 6.

Impact of dapagliflozin and entresto on protein levels of electron transport chain, autophagy, and mitochondrial damage by day 35 after CKD induction. (A) Protein expression of complex I, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (B) Protein expression of complex II, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (C) Protein expression of complex III, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (D) Protein expression of complex V, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (E) Protein expressions of ratio of LC3B-II/LC3B-I, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (F) Protein expression of Atg5, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (G) Protein expression of beclin 1, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (H) Protein expression of cytosolic cytochrome C (cyt-CytoC), * versus other groups with different symbols (†, ‡, §), P < 0.0001. (I) Protein expression of cyclophilin D (cyc-D), * versus other groups with different symbols (†, ‡, §), P < 0.0001. (J) Protein expression of DRP1, * versus 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). SC: sham control; HCKD: hypertensive chronic kidney disease; DAPA: dapagliflozin; En: entresto.

Figure 7.

Impact of dapagliflozin and entresto on protein expressions of mitochondrial integrity, mitophagy regulators and mitochondrial dynamic biogenesis by day 35 after CKD induction. (A) Protein expression of mitochondrial cytochrome C (mit-CytoC), * versus other groups with different symbols (†, ‡, §), P < 0.0001. (B) Protein expression of protein expressions of PGC-1α, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (C) Protein expression of Mfn2, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (D) Protein expression of PINK1, * versus other groups with different symbols (†, ‡, §), P < 0.0001. (E) Protein expression of Parkin, * versus 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). SC: sham control; HKD: hypertensive kidney disease; DAPA: dapagliflozin; En: entresto.

Figure 8.

Impact of dapagliflozin and entresto on cellular levels of fibrosis and collagen deposition by day 35 after CKD induction. (A to E) Illustrating the microscopic finding (100×) of Masson’s trichrome stain for identification of fibrotic area (blue color). (F) Analytical result of fibrotic area, * versus other group with different symbols (†, ‡, §), P < 0.0001. (G to K) Illustrating the microscopic finding (100×) of Sirius red stain for identification of collagen-deposition area (pink). (L) Analytical result of collagen-deposition area, * versus other group with different symbols (†, ‡, §), P < 0.0001. Scale bar in right lower corner represents 100µm. 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). SC: sham control; HCKD: hypertensive chronic kidney disease; DAPA: dapagliflozin; En: entresto.

Figure 9.

Impact of dapagliflozin and entresto on cell-cell contact component in renal tubules and small vessel density in kidney parenchyma by day 35 after CKD induction. (A to E) Illustrating the microscopic finding (200×) of immunohistochemical (IHC) stain for identification of cellular expression of the E-cadherin (gray color). (F) Analytical result of score expression of E-cadherin, * versus other group with different symbols (†, ‡, §, ¶), P < 0.0001. (G to K) Illustrating the microscopic finding (200×) of alpha-smooth muscle actin stain for identification of expression of small vessels (i.e. ⩽ 25μM of diameter) (gray color). (L) Analytical result of small number of vessels, * versus other group with different symbols (†, ‡, §), P < 0.0001. Scale bar in right lower corner represents 50µm. 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). SC: sham control; HKD: hypertensive kidney disease; DAPA: dapagliflozin; En: entresto.

Figure 10.

Impact of dapagliflozin and entresto on cellular expressions of podocyte components in glomeruli of kidney parenchyma by day 35 after CKD induction. (A to E) Showing the microscopic finding (200×) of immunofluorescent (IF) stain for identification of cellular expression of ZO-1 (green color). (F) Analytical result of fluorescent intensity of ZO-1, * versus other group with different symbols (†, ‡, §), P < 0.0001. (G to K) Showing the IF microscopic finding (200×) for identification of cellular expression of synaptopodin (green color). (L) Analytical result of fluorescent intensity of synaptopodin, * versus other group with different symbols (†, ‡, §), P < 0.0001. Scale bar in right lower corner represents 50µm. 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). SC: sham control; HKD: hypertensive kidney disease; DAPA: dapagliflozin; En: entresto.

Figure 11.

Impact of dapagliflozin and entresto on cellular expressions of renal tubular and oxidative DNA damage in kidney parenchyma by day 35 after CKD induction. (A to E) Illustrating the microscopic finding (200×) for identification of cellular expression of 8-hydroxy-2′-deoxyguanosine (8-OHdG), i.e. an oxidative DNA damage marker (gray color). (F) Analytical result of score expression of 8-OHdG, * versus other group with different symbols (†, ‡, §), P < 0.0001. (G to K) Illustrating the immunofluorescent microscopic finding (200×) for identification of cellular expression of kidney injury molecule (KIM)-1. (L) Analytical result of score expression of KIM-1,* versus other group with different symbols (†, ‡, §), P < 0.0001. Scale bar in right lower corner represents 50µm. 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). SC: sham control; HKD: hypertensive kidney disease; DAPA: dapagliflozin; En: entresto.

Figure 12.

Impact of dapagliflozin and entresto on cellular level of mitochondrial cytochrome C expression in NRK-52E cells. (A to D) Illustrating the immunofluorescent (IF) microscopic finding (400×) for identification of mitochondrial cytochrome C + cells (green color) in NRK-52E cells. (E to H) Illustrating IF microscopic finding (400×) for identification of heat shock protein 60 (HSP60) + cells (red color) in NRK-52E cells. (I to L) Illustrating IF microscopic finding (400×) of merged cytochrome C and HSP60 positively stained cells (i.e. double stain) for identification of cytochrome C in mitochondria (i.e. green-red color). Blue color indicated nuclei stained by DAPI. (M) Analytical result of number of mitochondrial cytochrome C + cells in NRK-52E cells, * versus other group with different symbols (†, ‡), P < 0.001. Scale bars in right lower corner represent 20µm. 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). G1 = NRK-52E; G2 = NRK-52E cells + H₂O₂; G3 = NRK-52E cells + H₂O₂ + dapagliflozin; G4 = NRK-52E H₂O₂ + sacubitril/valsartan.

Figure 13.

Impact of dapagliflozin and entresto on cellular level of mitochondrial cytochrome C expression in renal tubular cells of rat kidney. (A to E) Illustrating the immunofluorescent (IF) microscopic finding (800×) for identification of mitochondrial cytochrome C + cells (green color) in renal tubular cells. (F to J) Illustrating IF microscopic finding (800×) for identification of heat shock protein 60 (HSP60) + cells (red color) in renal tubular cells. (K to O) Illustrating IF microscopic finding (800×) of merged cytochrome C and HSP60 positively stained cells (i.e. double stain) for identification of cytochrome C in mitochondria (i.e. green-red color). Blue color indicated nuclei stained by DAPI. (P) Analytical result of number of mitochondrial cytochrome C + cells in renal tubular cells, * versus other group 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 = 6 for each group). Symbols (*, †, ‡, §) indicate significance for each other (at 0.05 level). 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). SC: sham control; HKD: hypertensive kidney disease; DAPA: dapagliflozin; En: entresto.