Sirt3 modulates fatty acid oxidation and attenuates cisplatin-induced AKI in mice

Abstract

Fatty acid oxidation (FAO) dysfunction is one of the important mechanisms of renal fibrosis. Sirtuin 3 (Sirt3) has been confirmed to alleviate acute kidney injury (AKI) by improving mitochondrial function and participate in the regulation of FAO in other disease models. However, it is not clear whether Sirt3 is involved in regulating FAO to improve the prognosis of AKI induced by cisplatin. Here, using a murine model of cisplatin-induced AKI, we revealed that there were significantly FAO dysfunction and extensive lipid deposition in the mice with AKI. Metabolomics analysis suggested reprogrammed energy metabolism and decreased ATP production. In addition, fatty acid deposition can increase reactive oxygen species (ROS) production and induce apoptosis. Our data suggested that Sirt3 deletion aggravated FAO dysfunction, resulting in increased apoptosis of kidney tissues and aggravated renal injury. The activation of Sirt3 by honokiol could improve FAO and renal function and reduced fatty acid deposition in wide-type mice, but not Sirt3-defective mice. We concluded that Sirt3 may regulate FAO by deacetylating liver kinase B1 and activating AMP-activated protein kinase. Also, the activation of Sirt3 by honokiol increased ATP production as well as reduced ROS and lipid peroxidation through improving mitochondrial function. Collectively, these results provide new evidence that Sirt3 is protective against AKI. Enhancing Sirt3 to improve FAO may be a potential strategy to prevent kidney injury in the future.

Keywords: Sirtuin3; acute kidney injury; cisplatin; fatty acid oxidation; mitochondrion.

Figure 1

Mice with cisplatin‐induced AKI exhibit fatty acid oxidation dysfunction and lipid deposition. A, PAS staining (scale bar, 100 µm) and oil red O staining (scale bar, 200 µm) of kidney tissue from wild‐type (WT) mice in the control group and the cisplatin group. B, Comparison of serum creatinine (Scr) levels between the control and cisplatin‐treated WT mice, n = 6, ***P < .001. C, Comparison of blood urea nitrogen (BUN) levels between the control and cisplatin‐treated WT mice, n = 6, ***P < .001. D, Quantitative analysis of necrotic tubules. Data are the mean ± SD of 20 random fields from each kidney, ***P < .001, n = 3. E, Comparison of the FFA concentrations in the control and cisplatin‐treated WT mice, n = 6, ***P < .001. F, Western blot and densitometric analysis of key fatty acid oxidation proteins in the kidney tissue of mice in the control group and cisplatin group, n = 4, **P < .01

Figure 2

Metabolic disorders and FFA‐related lipotoxicity increase the apoptosis of mRTEcs. A, Metabolomics analysis of the kidney cortex in the control group and the cisplatin group. Heat map showing the relative levels of metabolites in kidneys from mice in each group (n = 6). B‐D, LC‐MS detection of ATP, NAD+, NADP+ in the kidney cortex of mice in the control group and the cisplatin group, n = 6, *P < .05. E, LC‐MS detection of palmitic acid level in the kidney of mice in the control group and the cisplatin group, n = 6, ***P < .001. F, Comparison of ROS between the control and cisplatin‐treated WT mice, n = 3, *P < .05. G, Representative 4HNE immunohistochemical staining of mouse kidney sections from the control group and the cisplatin group and the semiquantitative positive 4HNE staining scores, scale bar, 100 µm. H, The apoptotic rates of mRTEcs in the control group and the palmitic acid group were detected by V‐FITC/PI staining, **P < .01, n = 3. I, Western blot and densitometric analysis of cleaved caspase‐3 in mRTEcs in the control group and the palmitic acid group, **P < .01, n = 4

Figure 3

Sirt3 knockout aggravates FAO dysfunction and kidney damage in mice with AKI. A, Pathological changes in kidney tissue sections from mice in each group, as shown by PAS staining, scale bar, 100 µm. B, Comparison of oil red O staining in kidney tissue sections from mice in different groups, scale bar, 200 µm. C, Comparison of serum creatinine levels in mice in each group, *P < .05, ***P < .001, n = 6. D, Comparison of blood urea nitrogen (BUN) levels in mice in each group, *P < .05, ***P < .001, n = 6. E, Quantitative comparison of the renal tubular necrosis score for each group with 20 HP field of vision, *P < .05, n = 3. F, Comparison of the FFA concentrations in kidney tissues from mice in each group, *P < .05, ***P < .001, n = 6. G, Western blot and densitometric analysis of CPT1A, ACADL in mice in different groups, *P < .05, **P < .01, n = 4. H, Western blot and densitometric analysis of PPARα in mice in different groups, *P < .05, **P < .01, n = 4. I, Comparison of ACADL activity in mice in each group, *P < .05, **P < .01, n = 4; J, TUNEL staining of kidney tissues from mice of each group, scale bar, 100 µm. Compared with TUNEL‐positive cells in each group, n = 10 HP visual field, **P < .01, ***P < .001

Figure 4

The up‐regulation of Sirt3 expression by HKL improves FAO dysfunction and renal function. A, Western blot (n = 4) and real‐time PCR analysis (n = 3) of Sirt3 in the renal tissue of control and cisplatin‐treated mice after saline or HKL administration, **P < .01. B, Comparison of renal function evaluated as serum creatinine and blood urea nitrogen in mice in each group, ***P < .001, **P < .01, n = 6. C, Comparison of renal histological changes evaluated as number of cast and necrotic tubules per HPF. ***P < .001, **P < .01. D, Oil red O staining showed lipid deposition in the kidneys of mice in each group, scale bar, 200 µm. E, Comparison of the FFA concentrations in kidneys from mice in each group, ***P < .001, n = 6. F, Western blot and densitometric analysis of key proteins related to FAO in the kidneys of mice in each group, **P < .01, ***P < .001, n = 4

Figure 5

Sirt3 may regulate FAO through the LKB1‐AMPK pathway. A, Western blot and densitometric analysis of P‐AMPKα and total AMPKα in the control and cisplatin groups of mRTECs, **P < .01, n = 3. B, Representative phospho‐AMPKα immunohistochemical staining of mouse kidney sections and semiquantitative positive scoring of phospho‐AMPKα staining among different groups, scale bar, 100 µm. C, Western blot and densitometric analysis of P‐AMPKα/total AMPKα and P‐ACC/total ACC in the kidneys of each group, *P < .05, **P < .01, n = 3. D, Immunoprecipitation and densitometric analysis of acetylated LKB1 in each group of mice, *P < .05, n = 3. E, Immunoprecipitation and densitometric analysis of acetylated LKB1 in vitro, **P < .01, n = 3

Figure 6

Sirt3 activation attenuates cisplatin‐induced mitochondrial damage and lipid peroxidation. A, Transmission electron microscopy showed the morphological mitochondrial structure in renal tubular epithelial cells from each group. Scale bar, 0.5 µm. B, Quantitative comparison of the transverse width of each group of mice. **P < .01, ***P < .001, n = 20. C, Comparison in the ATP levels between the groups of mice, *P < .05, **P < .01, ***P < .001, n = 5; D, Western blot and densitometric analysis of the mitochondrial complex I in each group, *P < .05, **P < .01, ***P < .001, n = 3. E, Representative confocal images and quantitative analysis of mitochondrial ROS detected by MitoSOX, scale bar, 10 µm. F, Representative 4HNE immunohistochemical staining of mouse kidney sections and semiquantitative positive 4HNE staining scores among the different groups, scale bar, 100 µm