Nuclear farnesoid X receptor attenuates acute kidney injury through fatty acid oxidation

Acute kidney injury (AKI) is a life-threatening condition that is one of most common side effects of cisplatin therapy. Fatty acid oxidation (FAO) is the main source of energy production in kidney proximal tubular epithelial cells (PTECs) but it is inhibited in AKI. Recent work demonstrated that activation of the farnesoid X receptor (FXR) protects against AKI, but the underlying mechanism remains elusive. Using a model of cisplatin-induced AKI, we found that FXR and FAO-related genes were remarkably downregulated while kidney lipid accumulated. Proximal tubule-specific or whole body FXR knockout worsened, while pharmacological activation attenuated these effects. Conversely, FXR knockout in non-proximal tubules did not. RNA-sequencing of PTECs demonstrated increased transcripts involved in metabolic pathways in cells overexpressing FXR versus control after cisplatin treatment, specifically transcripts associated with FAO and peroxisome proliferator-activated receptor-γ (PPARγ) signaling. Furthermore, FXR overexpression or activation improved FAO and inhibited intracellular lipid accumulation in cisplatin-treated cells. In vivo studies have shown that pharmacological activation of PPARγ can prevent cisplatin-induced lipid accumulation, kidney tubule injury and kidney function decline. However, inhibition of PPARγ eliminated the protective effects of FXR compared to control mice during the cisplatin treatment phase and after ischemia-reperfusion injury. Consistent with findings in vivo, FXR/PPARγ reduced lipid accumulation by improving FAO in cisplatin-treated cells. Furthermore, the inhibition of carnitine palmitoyltransferase 1α abolished the protective effect of FXR in cisplatin-treated mice. Thus, FXR improves FAO and reduced lipid accumulation via PPARγ in PTECs of the kidney. Hence, reconstruction of the FXR/PPARγ/FAO axis may be a novel therapeutic strategy for preventing or treating AKI. Acute kidney injury (AKI) is a life-threatening condition that is one of most common side effects of cisplatin therapy. Fatty acid oxidation (FAO) is the main source of energy production in kidney proximal tubular epithelial cells (PTECs) but it is inhibited in AKI. Recent work demonstrated that activation of the farnesoid X receptor (FXR) protects against AKI, but the underlying mechanism remains elusive. Using a model of cisplatin-induced AKI, we found that FXR and FAO-related genes were remarkably downregulated while kidney lipid accumulated. Proximal tubule-specific or whole body FXR knockout worsened, while pharmacological activation attenuated these effects. Conversely, FXR knockout in non-proximal tubules did not. RNA-sequencing of PTECs demonstrated increased transcripts involved in metabolic pathways in cells overexpressing FXR versus control after cisplatin treatment, specifically transcripts associated with FAO and peroxisome proliferator-activated receptor-γ (PPARγ) signaling. Furthermore, FXR overexpression or activation improved FAO and inhibited intracellular lipid accumulation in cisplatin-treated cells. In vivo studies have shown that pharmacological activation of PPARγ can prevent cisplatin-induced lipid accumulation, kidney tubule injury and kidney function decline. However, inhibition of PPARγ eliminated the protective effects of FXR compared to control mice during the cisplatin treatment phase and after ischemia-reperfusion injury. Consistent with findings in vivo, FXR/PPARγ reduced lipid accumulation by improving FAO in cisplatin-treated cells. Furthermore, the inhibition of carnitine palmitoyltransferase 1α abolished the protective effect of FXR in cisplatin-treated mice. Thus, FXR improves FAO and reduced lipid accumulation via PPARγ in PTECs of the kidney. Hence, reconstruction of the FXR/PPARγ/FAO axis may be a novel therapeutic strategy for preventing or treating AKI. Translational StatementFatty acid oxidation (FAO) is the main energy source for proximal tubular epithelial cells. Acute kidney injury (AKI) causes FAO disorders. Here, we demonstrate that the nuclear receptor farnesoid X receptor (FXR) specifically in the proximal tubule protects against AKI and attenuates lipid accumulation. We further show that these correlate with FAO; FXR and peroxisome proliferator–activated receptor-γ may regulate key enzymes required for FAO. Our results provide new insights into the role of FXR in the prevention or treatment of AKI. Fatty acid oxidation (FAO) is the main energy source for proximal tubular epithelial cells. Acute kidney injury (AKI) causes FAO disorders. Here, we demonstrate that the nuclear receptor farnesoid X receptor (FXR) specifically in the proximal tubule protects against AKI and attenuates lipid accumulation. We further show that these correlate with FAO; FXR and peroxisome proliferator–activated receptor-γ may regulate key enzymes required for FAO. Our results provide new insights into the role of FXR in the prevention or treatment of AKI. Acute kidney injury (AKI) is a life-threatening condition with high morbidity and mortality, occurring in approximately 10%–15% of hospitalized patients, while its incidence has been reported in more than 50% of patients in intensive care admitted to hospital.1Ronco C. Bellomo R. Kellum J.A. Acute kidney injury.Lancet. 2019; 394: 1949-1964Google Scholar, 2Bellomo R. Kellum J.A. Ronco C. Acute kidney injury.Lancet. 2012; 380: 756-766Google Scholar, 3Rewa O. Bagshaw S.M. Acute kidney injury-epidemiology, outcomes and economics.Nat Rev Nephrol. 2014; 10: 193-207Google Scholar The major causes of AKI include nephrotoxicity, ischemia/reperfusion (I/R), sepsis, and contrast media.3Rewa O. Bagshaw S.M. Acute kidney injury-epidemiology, outcomes and economics.Nat Rev Nephrol. 2014; 10: 193-207Google Scholar, 4Jang H.R. Rabb H. Immune cells in experimental acute kidney injury.Nat Rev Nephrol. 2015; 11: 88-101Google Scholar, 5Basile D.P. Anderson M.D. Sutton T.A. Pathophysiology of acute kidney injury.Compr Physiol. 2012; 2: 1303-1353Google Scholar Of note, nephrotoxicity is a dose-limiting factor of the chemotherapeutic agent cisplatin. The proximal tubular epithelial cells (PTECs) are the major target for the toxic effects of cisplatin that lead to AKI.6Ozkok A. Edelstein C.L. Pathophysiology of cisplatin-induced acute kidney injury.Biomed Res Int. 2014; 2014: 967826Google Scholar Injured proximal tubular cells suffer from significant changes in metabolic pathways, cellular signaling, and cell cycle.6Ozkok A. Edelstein C.L. Pathophysiology of cisplatin-induced acute kidney injury.Biomed Res Int. 2014; 2014: 967826Google Scholar, 7Liu B.C. Tang T.T. Lv L.L. Lan H.Y. Renal tubule injury: a driving force toward chronic kidney disease.Kidney Int. 2018; 93: 568-579Google Scholar, 8Perazella M.A. Drug-induced acute kidney injury: diverse mechanisms of tubular injury.Curr Opin Crit Care. 2019; 25: 550-557Google Scholar, 9McSweeney K.R. Gadanec L.K. Qaradakhi T. et al.Mechanisms of cisplatin-induced acute kidney injury: pathological mechanisms, pharmacological interventions, and genetic mitigations.Cancers (Basel). 2021; 13: 1572Google Scholar However, there is currently no effective therapeutic option to treat AKI, whose development is therefore crucial.9McSweeney K.R. Gadanec L.K. Qaradakhi T. et al.Mechanisms of cisplatin-induced acute kidney injury: pathological mechanisms, pharmacological interventions, and genetic mitigations.Cancers (Basel). 2021; 13: 1572Google Scholar Although kidneys are not classified as metabolic organs, metabolism plays a key role in the kidneys.10Li X. Zheng S. Wu G. Amino acid metabolism in the kidneys: nutritional and physiological significance.Adv Exp Med Biol. 2020; 1265: 71-95Google Scholar,11Piret S.E. Attallah A.A. Gu X. et al.Loss of proximal tubular transcription factor Kruppel-like factor 15 exacerbates kidney injury through loss of fatty acid oxidation.Kidney Int. 2021; 100: 1250-1267Google Scholar Renal PT cells have a high energy demand, mainly provided by fatty acid oxidation (FAO) and glycolysis.12Kang H.M. Ahn S.H. Choi P. et al.Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development.Nat Med. 2015; 21: 37-46Google Scholar Notably, FAO in the mitochondria and the peroxisomes are major energy sources, essential for supporting cells with high energy demands.13Nieth H. Schollmeyer P. Substrate-utilization of the human kidney.Nature. 1966; 209: 1244-1245Google Scholar, 14Console L. Scalise M. Giangregorio N. et al.The link between the mitochondrial fatty acid oxidation derangement and kidney injury.Front Physiol. 2020; 11: 794Google Scholar, 15Portilla D. Energy metabolism and cytotoxicity.Semin Nephrol. 2003; 23: 432-438Google Scholar During AKI, FAO is inhibited, leading to lipotoxicity and energy deprivation, which is characterized by intracellular lipid accumulation and a decrease of renal adenosine triphosphate (ATP) level, renal tubular cell injury, and death.16Wei Q. Xiao X. Fogle P. Dong Z. Changes in metabolic profiles during acute kidney injury and recovery following ischemia/reperfusion.PLoS One. 2014; 9e106647Google Scholar, 17Yu X. Xu M. Meng X. et al.Nuclear receptor PXR targets AKR1B7 to protect mitochondrial metabolism and renal function in AKI.Sci Transl Med. 2020; 12eaay7591Google Scholar, 18Jang H.S. Noh M.R. Jung E.M. et al.Proximal tubule cyclophilin D regulates fatty acid oxidation in cisplatin-induced acute kidney injury.Kidney Int. 2020; 97: 327-339Google Scholar The carnitine palmitoyltransferase (CPT1) inhibitor etomoxir can exacerbate this process in cisplatin-induced kidney injury.12Kang H.M. Ahn S.H. Choi P. et al.Defective fatty acid oxidation in renal tubular epithelial cells has a key role in kidney fibrosis development.Nat Med. 2015; 21: 37-46Google Scholar,18Jang H.S. Noh M.R. Jung E.M. et al.Proximal tubule cyclophilin D regulates fatty acid oxidation in cisplatin-induced acute kidney injury.Kidney Int. 2020; 97: 327-339Google Scholar Conversely, the upregulation of FAO attenuated kidney injury. Recent studies have shown that the activation of peroxisome proliferator–activated receptor-γ (PPARγ) and PPAR coactivator-1α increases FAO to reduce the accumulation of free fatty acids (FAs).19Soliman E. Elhassanny A.E.M. Malur A. et al.Impaired mitochondrial function of alveolar macrophages in carbon nanotube-induced chronic pulmonary granulomatous disease.Toxicology. 2020; 445: 152598Google Scholar, 20Legchenko E. Chouvarine P. Borchert P. et al.PPARgamma agonist pioglitazone reverses pulmonary hypertension and prevents right heart failure via fatty acid oxidation.Sci Transl Med. 2018; 10eaao0303Google Scholar, 21Zhao F. Xiao C. Evans K.S. et al.Paracrine Wnt5a-beta-catenin signaling triggers a metabolic program that drives dendritic cell tolerization.Immunity. 2018; 48: 147-160.e7Google Scholar, 22Du Q. Tan Z. Shi F. et al.PGC1alpha/CEBPB/CPT1A axis promotes radiation resistance of nasopharyngeal carcinoma through activating fatty acid oxidation.Cancer Sci. 2019; 110: 2050-2062Google Scholar However, there are no reports showing the activation of PPARγ increases FAO in AKI. Nuclear receptors are transcription factors activated by ligands and play vital aspects in the context of renal physiology and pathophysiology.23Ruan X.Z. Varghese Z. Powis S.H. Moorhead J.F. Nuclear receptors and their coregulators in kidney.Kidney Int. 2005; 68: 2444-2461Google Scholar Many nuclear receptors are involved in a large number of metabolic processes, such as lipids, bile acids, glucose, cholesterol metabolism, and drug clearance.23Ruan X.Z. Varghese Z. Powis S.H. Moorhead J.F. Nuclear receptors and their coregulators in kidney.Kidney Int. 2005; 68: 2444-2461Google Scholar Farnesoid X receptor (FXR) is a member of the nuclear receptor family activated by endogenous bile acids and functions as an intracellular sensor.24Shapiro H. Kolodziejczyk A.A. Halstuch D. Elinav E. Bile acids in glucose metabolism in health and disease.J Exp Med. 2018; 215: 383-396Google Scholar Except for the liver and small intestine, FXR is highly expressed in the kidneys, especially in the proximal tubules (PTs).25Zhang X. Huang S. Gao M. et al.Farnesoid X receptor (FXR) gene deficiency impairs urine concentration in mice.Proc Natl Acad Sci U S A. 2014; 111: 2277-2282Google Scholar FXR is associated with the process of energy metabolism via the transcriptional regulation of downstream target genes.26Appelman M.D. van der Veen S.W. van Mil S.W.C. Post-translational modifications of FXR; implications for cholestasis and obesity-related disorders.Front Endocrinol (Lausanne). 2021; 12: 729828Google Scholar In the liver, which uses FAO as their cellular ATP production, acetylation of FXR attenuates dyslipidemia and hepatic steatosis by promoting FAO.27Schug T.T. Li X. Sirtuin 1 in lipid metabolism and obesity.Ann Med. 2011; 43: 198-211Google Scholar FXR activation through its ligands was proposed as a potential strategy to treat insulin resistance and type 2 diabetes.28Ma K. Saha P.K. Chan L. Moore D.D. Farnesoid X receptor is essential for normal glucose homeostasis.J Clin Invest. 2006; 116: 1102-1109Google Scholar Earlier studies have illustrated the protection of FXR in kidney disease, particularly in acute injury.29Bae E.H. Choi H.S. Joo S.Y. et al.Farnesoid X receptor ligand prevents cisplatin-induced kidney injury by enhancing small heterodimer partner.PLoS One. 2014; 9e86553Google Scholar, 30Gai Z. Chu L. Xu Z. et al.Farnesoid X receptor activation protects the kidney from ischemia-reperfusion damage.Sci Rep. 2017; 7: 9815Google Scholar, 31Zhu J.B. Xu S. Li J. et al.Farnesoid X receptor agonist obeticholic acid inhibits renal inflammation and oxidative stress during lipopolysaccharide-induced acute kidney injury.Eur J Pharmacol. 2018; 838: 60-68Google Scholar However, the potential role of FXR in regulating FAO in AKI is yet unknown. In this study, we hypothesized that FXR improves PPARγ expression and promotes renal FAO in the context of cisplatin AKI. Overall, establishing a mechanism by which FXR regulates FAO is important. Pharmacologic activation of FXR is a potential strategy for the treatment of AKI. Additional details for methods are provided in the Supplementary Methods. All experiments were approved by the Animal Care and Use Committee of Fudan University and were performed in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory. FXR knockout (KO), Kap-Cre, Cdh16-Cre, and FXRflox/flox mice were purchased from the Jackson Laboratory. The details of animal studies are provided in the Supplementary Methods. Oxygen consumption rate (OCR) was measured using a Seahorse Bioscience XFe96 extracellular flux analyzer by cells cultured in an XF96 cell culture microplate (102601, Agilent). Detailed protocols are provided in the Supplementary Methods. All data were expressed as mean ± SEM and were analyzed using the Prism software package (GraphPad Software). Analytical details were provided in the Supplementary Methods. A value of P < 0.05 was considered statistically significant. To investigate the dynamic changes in the context of cisplatin AKI, cisplatin-treated mice were examined at different times (0, 24, 48, and 72 hours). As previously reported,6Ozkok A. Edelstein C.L. Pathophysiology of cisplatin-induced acute kidney injury.Biomed Res Int. 2014; 2014: 967826Google Scholar,32Kim J. Long K.E. Tang K. Padanilam B.J. Poly(ADP-ribose) polymerase 1 activation is required for cisplatin nephrotoxicity.Kidney Int. 2012; 82: 193-203Google Scholar cisplatin resulted in significant renal functional and histologic damage (Supplementary Figure S1A–D); the expression of proinflammatory cytokine interleukin-6 (IL-6) and cell apoptosis increased significantly at 72 hours (Supplementary Figure S1E and F). Because AKI is associated with FAO deficiency,6Ozkok A. Edelstein C.L. Pathophysiology of cisplatin-induced acute kidney injury.Biomed Res Int. 2014; 2014: 967826Google Scholar our study showed that kidney ATP level was significantly declined at 72 hours after cisplatin administration (Supplementary Figure S2A). We further investigated the relationship between AKI and lipid accumulation. The kidney and serum triglyceride levels were significantly increased at 72 hours after cisplatin administration (Supplementary Figure S2B–E). In addition, electron microscopy demonstrated that the number of lipid droplets increased drastically at 72 hours (Supplementary Figure S2C). Lipid droplet generation is a multifactorial and complex process not yet completely understood. Here, we selected perilipin 2 (Plin2), a lipid droplet surface protein, to investigate the effects of lipid droplets. Co-staining of Plin2 and proximal renal tubular marker Lotus lectin indicated that Plin2 was mainly expressed in the proximal tubules (Supplementary Figure S3A); furthermore, co-staining for Plin2 and bodipy demonstrated that the expression of Plin2 was increased after I/R and cisplatin AKI (Supplementary Figure S3B–F). Previous studies have shown that FAO is inhibited during AKI, leading to intracellular lipid accumulation and renal tubular cell death.33Desvergne B. Michalik L. Wahli W. Transcriptional regulation of metabolism.Physiol Rev. 2006; 86: 465-514Google Scholar It is worth noting that the expressions of FAO-related genes were significantly decreased at cisplatin AKI (Supplementary Figure S4A–E).34Houten S.M. Violante S. Ventura F.V. Wanders R.J.A. The biochemistry and physiology of mitochondrial fatty acid beta-oxidation and its genetic disorders.Annu Rev Physiol. 2016; 78: 23-44Google Scholar Similarly, lipid droplets increased in a dose- and time-dependent manner (Supplementary Figure S5A–F), whereas FAO-related genes decreased after cisplatin treatment (Supplementary Figure S5E and F). Collectively, these data indicate that lipid accumulation and FAO disruption are associated with the development of cisplatin-induced renal injury. Various transcriptional factors play a pivotal role in the regulation of lipid metabolism.33Desvergne B. Michalik L. Wahli W. Transcriptional regulation of metabolism.Physiol Rev. 2006; 86: 465-514Google Scholar As previous studies reported, the FXR is a transcriptional factor that regulates renal lipid metabolism.35Jiang T. Wang X.X. Scherzer P. et al.Farnesoid X receptor modulates renal lipid metabolism, fibrosis, and diabetic nephropathy.Diabetes. 2007; 56: 2485-2493Google Scholar,36Wang X.X. Jiang T. Shen Y. et al.The farnesoid X receptor modulates renal lipid metabolism and diet-induced renal inflammation, fibrosis, and proteinuria.Am J Physiol Renal Physiol. 2009; 297: F1587-F1596Google Scholar It is accepted that the localization of transcriptional factor is important. Therefore, we explored the distribution of FXR in the kidneys. As shown in Supplementary Figure S6A and B, FXR was highly expressed in the epithelium of PTs; the expression of FXR was significantly reduced in a dose- or time-dependent manner (Supplementary Figure S7A–E). Furthermore, the reduction of FXR is mainly located in the nucleus (Supplementary Figure S7F and G). These data indicate that cisplatin reduced the nuclear expression of FXR. To further clarify the role of FXR in cisplatin AKI, we generated FXR KO mice (Supplementary Figure S8A and B). The ablation of FXR was confirmed by immunofluorescence and immunohistochemistry (Supplementary Figures S6A, B and S8C). Although FXR deficiency exacerbated serum creatinine (Figure 1a), it did not show significantly exacerbated tubular injury after cisplatin AKI (Figure 1b). In addition, FXR deficiency upregulated neutrophil gelatinase–associated lipocalin, Kim-1, IL-6 expression, and the number of apoptotic cells in the kidneys of FXR-deficient mice treated with cisplatin (Supplementary Figure S9A–E). As shown in Figure 1c, the ATP level was decreased in FXR-deficient mice compared with wild-type (WT) mice after cisplatin treatment. The lipid levels in circulation and the kidney were increased in FXR-deficient mice compared with WT mice (Figure 1d and e). In addition, the expression of lipid droplet surface protein Plin2 in FXR-deficient cisplatin-treated mice was increased compared with that in WT mice (Figure 1f, Supplementary Figure S10A and C). FAO is the main energy source of renal tubular epithelial cells, mainly occurring in the peroxisomes and mitochondria. Our study found that the expressions of renal FAO-related genes, such as CPT1α, PGC1α, Pecr, Crot, Ehhad, and SLC27a2, were significantly decreased in FXR-deficient mice compared with those in WT mice after cisplatin treatment (Figure 1g, Supplementary Figures S10B–E and S11). In addition, transporters Ctr1 and OCT2, the nuclear receptor RXR, and IR-1 were decreased in FXR-deficient mice (Supplementary Figure S12). Taken together, these results indicate that FXR deficiency may exacerbate cisplatin-induced lipid accumulation via FAO inhibition. We further determined the potential impact of FXR pharmacologic activation on cisplatin AKI and administered GW4064 to mice before cisplatin treatment. Contrary to the results in FXR-deficient mice, FXR activation significantly ameliorated renal functional and histologic impairment, and reduced the expression of proinflammatory mediator IL-6 (Supplementary Figure S13A–E). As shown in Supplementary Figure S13F, GW4064 restored the renal ATP level after cisplatin exposure. In addition, GW4064 treatment markedly attenuated lipid accumulation in the circulation and kidneys of cisplatin-treated mice, with more preserved FAO genes (Supplementary Figures S14A–G and S15A–E). This upregulation in response to GW4064 was confirmed for FXR, CPT1a from microdissected S2/S3 PT segments (Supplementary Figure S16). Collectively, these data demonstrate that FXR activation attenuates cisplatin-induced lipid accumulation and FAO dysregulation in PTs of the kidneys. We have demonstrated that the kidney exhibits abundant FXR expression in the epithelium of PTs. To investigate the specific role of FXR in the PTs in the context of cisplatin-induced AKI, we crossed FXRflox/flox mice (FXRf/f) with Kap-Cre expressing mice to generate PT-FXR-KO mice (Supplementary Figure S17A and B), which allows the deletion of FXR in PT segments, as confirmed by immunofluorescence (Supplementary Figure S18A and B). Consistent with the results from cisplatin-treated FXR-deficient mice, PT-FXR-KO exacerbated the cisplatin-induced AKI, as evidenced by renal histologic damage and dysfunction (Figure 2a and b, Supplementary Figure S19A). In addition, the intrarenal and plasma lipid content of cisplatin-treated PT-FXR-KO mice was higher than that of cisplatin-treated FXRf/f mice (Figure 2c–e); moreover, the expression of FAO-related genes in cisplatin-treated PT-FXR-KO mice was markedly downregulated compared with that in FXRf/f mice (Figure 2f–h , Supplementary Figure S19B).Figure 3Farnesoid X receptor (FXR) overexpression attenuates cisplatin (CP)-induced lipid accumulation and improved fatty acid oxidation in proximal tubular epithelial cells (PTECs). PTECs were infected with adenovirus (Ad)-FXRα2 (25 multiplicity of infection [MOI]) or Ad-VP16 (25 MOI). After 36 hours, the cells were challenged with CP (50 μM) for 24 hours. (a) Kyoto encyclopedia of genes and genomes (KEGG) pathway enrichment analysis of differentially genes upregulated in Ad-VP16 versus Ad-FXRα2 PTECs treated with CP. (b) Representative immunofluorescent staining of FXR (red) in PTECs. The nuclei were visualized via 4′,6-diamidino-2-phenylindole (DAPI; blue). Bar = 10 μm. (c) Western blot analyses of FXR in Ad-VP16 or Ad-FXRα2 PTECs treated with CP. β-Actin was used as the loading control. (d) Cell viability was measured using the CCK8 assay (n = 10–12). ∗P < 0.05, ∗∗∗P < 0.001. (e) Cellular adenosine triphosphate (ATP) content was evaluated using an ATP colorimetric assay kit (n = 6–11). ∗P < 0.05, ∗∗∗P < 0.001. (f,g) Seahorse 96xf were used to detect the basal oxygen consumption rate (OCR) and spare respiratory (n = 10). ∗∗∗P < 0.001. (h) Representative boron-dipyrromethene (bodipy) staining to detect lipid accumulation (green). The nuclei were visualized via DAPI (blue) staining. Bar = 10 μm. (i) Quantitative reverse transcription–polymerase chain reaction analysis of FXR, perilipin 2 (Plin2), peroxisome proliferator–activated receptor-γ (PPARγ), PPAR coactivator-1α (PGC1α), and carnitine palmitoyltransferase-1α (CPT1α) mRNA expression (n = 3–8). ∗P < 0.05, #P < 0.05, ∗∗∗P < 0.001. ∗P < 0.05 versus the Ad-VP16-NS group, #P < 0.05 versus Ad-VP16 or Ad-VP16+CP. FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Figure 4Farnesoid X receptor (FXR) attenuates cisplatin (CP)-induced lipid accumulation and improved fatty acid oxidation (FAO) in proximal tubular epithelial cells (PTECs). FXR was activated in PTECs using GW4064 (5 μM) for 1 hour and then incubated with CP (50 μM) for 24 hours. (a) Cell viability was measured using the cell counting kit 8 (CCK8) assay (n = 10–12). (b) Cellular adenosine triphosphate (ATP) content was evaluated using an ATP colorimetric assay kit (n = 6). (c,d) Seahorse 96xf were used to detect basal oxygen consumption rate (OCR) and spare respiratory (n = 10). (e) Representative boron-dipyrromethene (bodipy) staining to detect lipid accumulation (green). The nuclei were visualized via 4′,6-diamidino-2-phenylindole (DAPI; blue) staining. Bar = 10 μm. (f–h) Western blot analysis of perilipin 2 (Plin2), peroxisome proliferator–activated receptor coactivator-1α (PGC1α), and carnitine palmitoyltransferase-1α (CPT1α) expressions in CP-treated PTECs with or without GW4064 administration (n = 4–5). β-Actin was used as the loading control. (i) OCR in PTECs treated with Z-guggulsterone (20 μM) for 1 hour, then incubated with CP (50 μM) for 24 hours, and added with FAO substrate BSA or palmitate (Palm). OCR was measured initially and after the addition of etomoxir (Eto) and oligomycin (oligo). Quantification of OCR due to FAO. (j) Representative bodipy staining to detect lipid accumulation (green) in CP- and/or oleic acid (OA)–treated PTECs with or without GW4064 administration. The nuclei were visualized via DAPI (blue) staining. Bar = 15 μm. (k) Western blot analysis of Plin2 in CP- and/or OA-treated PTECs with or without GW4064 administration (n = 4). β-Actin was used as the loading control. Data are presented as mean ± SEM. ∗P < 0.05, ∗∗∗P < 0.001. BSA, bovine serum albumin; DMSO, dimethylsulfoxide; FCCP, carbonyl cyanide 4-trifluoromethoxyphenylhydrazone; NC, negative control. To optimize viewing of this image, please see the online version of this article at www.kidney-international.org.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To further examine the effect of FXR on cisplatin-induced AKI, we crossed FXR-floxed mice with kidney-specific cadherin (Cdh16-cre) mice to investigate whether FXR in other tubular segments has a significant effect on AKI (Supplementary Figure S17A and B). Our results showed that other tubules-FXR (OT-FXR) deficiency did not significantly aggravate cisplatin-induced renal histologic damage and dysfunction (Supplementary Figure S20A and B); moreover, the renal and plasma lipid levels in OT-FXR-KO mice were not higher than those in cisplatin-treated FXRf/f mice (Supplementary Figure S20C–E). Interestingly, the expression of PPARγ in cisplatin-treated OT-FXR-KO mice was downregulated compared with that in cisplatin-treated FXRf/f mice (Supplementary Figure S20F). However, the cisplatin-induced inhibition of FAO-related genes was not significantly exacerbated in the kidneys of OT-FXR-KO mice (Supplementary Figure S20F–H). Collectively, these results further prove that FXR plays a critical role in cisplatin-induced AKI by improving FAO in PTs (not in other tubules). We have confirmed that FAO is involved in the protective effect of FXR on mice. Consistent with the in vivo results, mRNA sequencing analysis showed that FXR overexpression regulates FAO-related pathways in the context of cisplatin-treated PTECs (Figure 3a). The expression of FXR, Plin2, PGC1α, and CPT1α was further confirmed (Figure 3b and c, Supplementary Figure S21A–E). In addition, the overexpression of FXR prevented the reduction of cell viability induced by cisplatin; of note, under normal conditions, there was almost no effect on cell viability (Figure 3d), and a similar trend was observed in ATP synthesis (Figure 3e). Then we examined the OCR using seahorse; FXRα2 adenoviruses (Ad-FXR) reversed the decreased OCR and maximal respiratory capacity of cisplatin-treated PTECs (Figure 3f and g). Moreover, FXR overexpression reversed the dysregulation of lipid accumulation and FAO-related genes (Figure 3h and i, Supplementary Figure S21F and G). It has been suggested that FXR activation plays a vital role in renal epithelial cells.30Gai Z. Chu L. Xu Z. et al.Farnesoid X receptor activation protects the kidney from ischemia-reperfusion damage.Sci Rep. 2017; 7: 9815Google Scholar Based on the findings observed in FXR overexpression, FXR activation prevented the reduction of cisplatin-treated PTECs (Supplementary Figure S22A). In addition, FXR activation ameliorated the reduction in cell viability induced by cisplatin, reversing the alterations of ATP production and reduction of OCR and maximal respiratory capacity (Figure 4a–d). Furthermo