Dephosphorylation of AMP-activated protein kinase exacerbates ischemia/reperfusion-induced acute kidney injury via mitochondrial dysfunction

Kidney tubular epithelial cells are high energy-consuming epithelial cells that depend mainly on fatty acid oxidation for an energy supply. AMP-activated protein kinase (AMPK) is a key regulator of energy production in most cells, but the function of AMPK in tubular epithelial cells in acute kidney disease is unclear. Here, we found a rapid decrease in Thr172-AMPKα phosphorylation after ischemia/reperfusion in both in vivo and in vitro models. Mice with kidney tubular epithelial cell–specific AMPKα deletion exhibited exacerbated kidney impairment and apoptosis of tubular epithelial cells after ischemia/reperfusion. AMPKα deficiency was accompanied by the accumulation of lipid droplets in the kidney tubules and the elevation of ceramides and free fatty acid levels following ischemia/reperfusion injury. Mechanistically, ischemia/reperfusion triggered ceramide production and activated protein phosphatase PP2A, which dephosphorylated Thr172-AMPKα. Decreased AMPK activity repressed serine/threonine kinase ULK1-mediated autophagy and impeded clearance of the dysfunctional mitochondria. Targeting the PP2A-AMPK axis by the allosteric AMPK activator C24 restored fatty acid oxidation and reduced tubular cell apoptosis during ischemia/reperfusion-induced injury, by antagonizing PP2A dephosphorylation and promoting the mitophagy process. Thus, our study reveals that AMPKα plays an important role in protecting against tubular epithelial cell injury in ischemia/reperfusion-induced acute kidney injury. Hence, activation of AMPK could be a potential therapeutic strategy for acute kidney injury treatment. Kidney tubular epithelial cells are high energy-consuming epithelial cells that depend mainly on fatty acid oxidation for an energy supply. AMP-activated protein kinase (AMPK) is a key regulator of energy production in most cells, but the function of AMPK in tubular epithelial cells in acute kidney disease is unclear. Here, we found a rapid decrease in Thr172-AMPKα phosphorylation after ischemia/reperfusion in both in vivo and in vitro models. Mice with kidney tubular epithelial cell–specific AMPKα deletion exhibited exacerbated kidney impairment and apoptosis of tubular epithelial cells after ischemia/reperfusion. AMPKα deficiency was accompanied by the accumulation of lipid droplets in the kidney tubules and the elevation of ceramides and free fatty acid levels following ischemia/reperfusion injury. Mechanistically, ischemia/reperfusion triggered ceramide production and activated protein phosphatase PP2A, which dephosphorylated Thr172-AMPKα. Decreased AMPK activity repressed serine/threonine kinase ULK1-mediated autophagy and impeded clearance of the dysfunctional mitochondria. Targeting the PP2A-AMPK axis by the allosteric AMPK activator C24 restored fatty acid oxidation and reduced tubular cell apoptosis during ischemia/reperfusion-induced injury, by antagonizing PP2A dephosphorylation and promoting the mitophagy process. Thus, our study reveals that AMPKα plays an important role in protecting against tubular epithelial cell injury in ischemia/reperfusion-induced acute kidney injury. Hence, activation of AMPK could be a potential therapeutic strategy for acute kidney injury treatment. Translational StatementAcute kidney injury (AKI) is a significant health issue all over the world. We found renal ischemia/reperfusion (I/R) decreased fatty acid oxidation (FAO) by untargeted lipidomics and RNA sequencing. We developed tubular AMP-activated protein kinase (AMPK) α conditional knockout mice and confirmed AMPK plays a critical role in mitochondrial homeostasis in renal I/R injury. Furthermore, we proved that the AMPK activators C24 as well as metformin improve renal injury through the activation of AMPK and enhancing mitochondrial homeostasis and FAO. Our studies suggest that the AMPK allosteric activator might represent a novel strategy for the treatment of AKI and subsequent chronic kidney disease by targeting mitochondrial homeostasis. Acute kidney injury (AKI) is a significant health issue all over the world. We found renal ischemia/reperfusion (I/R) decreased fatty acid oxidation (FAO) by untargeted lipidomics and RNA sequencing. We developed tubular AMP-activated protein kinase (AMPK) α conditional knockout mice and confirmed AMPK plays a critical role in mitochondrial homeostasis in renal I/R injury. Furthermore, we proved that the AMPK activators C24 as well as metformin improve renal injury through the activation of AMPK and enhancing mitochondrial homeostasis and FAO. Our studies suggest that the AMPK allosteric activator might represent a novel strategy for the treatment of AKI and subsequent chronic kidney disease by targeting mitochondrial homeostasis. Acute kidney injury (AKI) is a common clinical syndrome characterized by rapid loss of kidney function. And, AKI affects ~15% of adults and 25% of children of all hospitalized patients.1Hoste E.A.J. Bagshaw S.M. Bellomo R. et al.Epidemiology of acute kidney injury in critically ill patients: the multinational AKI-EPI study.Intensive Care Med. 2015; 41: 1411-1423Crossref PubMed Scopus (1138) Google Scholar,2Ronco C. Bellomo R. Kellum J.A. Acute kidney injury.Lancet. 2019; 394: 1949-1964Abstract Full Text Full Text PDF PubMed Scopus (380) Google Scholar Unfortunately, no definite therapies are currently available to prevent or treat established AKI. The kidney is one of the most energy-demanding organs in the human body, especially in renal tubular cells.3Bhargava P. Schnellmann R.G. Mitochondrial energetics in the kidney.Nat Rev Nephrol. 2017; 13: 629-646Crossref PubMed Scopus (405) Google Scholar,4Wang Z.M. Ying Z. Bosy-Westphal A. et al.Specific metabolic rates of major organs and tissues across adulthood: evaluation by mechanistic model of resting energy expenditure.Am J Clin Nutr. 2010; 92: 1369-1377Crossref PubMed Scopus (233) Google Scholar Fatty acids have been identified as the major fuel in tubular cells, and fatty acid oxidation (FAO) is the most common adenosine triphosphate (ATP) production method.5Forbes J.M. Thorburn D.R. Mitochondrial dysfunction in diabetic kidney disease.Nat Rev Nephrol. 2018; 14: 291-312Crossref PubMed Scopus (169) Google Scholar FAO mainly occurs in mitochondria and involves a repeated sequence of reactions that result in the conversion of fatty acids to acetyl–coenzyme A (CoA). However, mitochondrial damage in tubular cells has been proven in AKI, including decreased mitochondrial abundance, swelling, and disruption of cristae.6Zsengellér Z.K. Ellezian L. Brown D. et al.Cisplatin nephrotoxicity involves mitochondrial injury with impaired tubular mitochondrial enzyme activity.J Histochem Cytochem. 2012; 60: 521-529Crossref PubMed Scopus (82) Google Scholar, 7Funk J.A. Schnellmann R.G. Persistent disruption of mitochondrial homeostasis after acute kidney injury.Am J Physiol Renal Physiol. 2012; 302: 853-864Crossref PubMed Scopus (148) Google Scholar, 8Zhang Q. Raoof M. Chen Y. et al.Circulating mitochondrial DAMPs cause inflammatory responses to injury.Nature. 2010; 464: 104-107Crossref PubMed Scopus (2297) Google Scholar The impairment of FAO is linked to ATP depletion-induced tubular apoptosis (AKI), lipotoxicity, and chronic kidney disease. Injured mitochondria not only reduce the ATP level in the cell but also are an important source of molecules that amplify injury, precipitate cell death, and induce inflammation.9Wan J. Kalpage H.A. Vaishnav A. et al.Regulation of respiration and apoptosis by cytochrome c threonine 58 phosphorylation.Sci Rep. 2019; 9: 1-16Crossref PubMed Scopus (8) Google Scholar,10Birk A.V. Chao W.M. Bracken C. et al.Targeting mitochondrial cardiolipin and the cytochrome c/cardiolipin complex to promote electron transport and optimize mitochondrial ATP synthesis.Br J Pharmacol. 2014; 171: 2017-2028Crossref PubMed Scopus (169) Google Scholar Structural disruption of mitochondria releases reactive oxygen species (ROS) and cytochrome c, the trigger of cell apoptosis, and mitochondrial DNA (mtDNA), which can serve as a proinflammatory danger signal.8Zhang Q. Raoof M. Chen Y. et al.Circulating mitochondrial DAMPs cause inflammatory responses to injury.Nature. 2010; 464: 104-107Crossref PubMed Scopus (2297) Google Scholar Targeting mitochondria to improve mitochondrial function is a potential strategy to prevent AKI. AMP-activated protein kinase (AMPK), a critical sensor of cellular energy status, plays an important role in energy production and the mitochondrial network.11Herzig S. Shaw R.J. AMPK: guardian of metabolism and mitochondrial homeostasis.Nat Rev Mol Cell Biol. 2018; 19: 121-135Crossref PubMed Scopus (1134) Google Scholar AMPK is activated by cellular stresses, particularly those involving ATP depletion and AMP elevation.12Lin S.C. Hardie D.G. AMPK: sensing glucose as well as cellular energy status.Cell Metab. 2018; 27: 299-313Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar Once activated, AMPK phosphorylates a myriad of downstream targets to promote ATP-generating catabolic processes and inhibit ATP-consuming anabolic processes.13Hardie D.G. Schaffer B.E. Brunet A. AMPK: an energy-sensing pathway with multiple inputs and outputs.Trends Cell Biol. 2016; 26: 190-201Abstract Full Text Full Text PDF PubMed Scopus (487) Google Scholar In renal ischemia-reperfusion (I/R), the energy stress state changes rapidly in both the ischemia period and the reperfusion period, which may cause a change in AMPK activity. During the ischemic phase, ATP production decreases because of oxygen insufficiency, whereas the AMP-to-ATP ratio increases, resulting in AMPK activation.14Mount P.F. Hill R.E. Fraser S.A. et al.Acute renal ischemia rapidly activates the energy sensor AMPK but does not increase phosphorylation of eNOS-Ser1177.Am J Physiol Renal Physiol. 2005; 289: F1103-F1115Crossref PubMed Scopus (61) Google Scholar However, the activity of AMPK after reperfusion has rarely been reported. In the present study, we found a significant accumulation of renal lipid droplets and free fatty acids (FFAs) after I/R, accompanied by a remarkable decrease in AMPK activity. Then, we confirmed that AMPK contributed to mitochondrial homeostasis via maintaining FAO in renal I/R by using tubular-specific AMPKα knockout mice. The AMPK allosteric activator C24 played a protective role during renal I/R by improving mitochondrial quality. Additional details for all methods are in the Supplementary Methods. The C57BL/6J mice were purchased from Vital River Laboratory Animal Corporation. Tubular-specific AMPKα1/α2 double-knockout mice (TAKO mice) were generated by crossing AMPKα1/α2-floxed mice with Cdh16-cre mice. Renal ischemia-reperfusion was induced in mice, as described in a previous article.15Wei Q. Dong Z. Mouse model of ischemic acute kidney injury: technical notes and tricks.Am J Physiol Renal Physiol. 2012; 303: F1487-F1494Crossref PubMed Scopus (186) Google Scholar The details of animal studies are provided in the Supplementary Methods. Primary renal tubular epithelial cells (RPTCs) were isolated according to the previous report.16Ding W. Yousefi K. Shehadeh L.A. Isolation, characterization, and high throughput extracellular flux analysis of mouse primary renal tubular epithelial cells.J Vis Exp. 2018; 2018: 1-10Google Scholar Detailed protocols are provided in the Supplementary Methods. Lipidomics and acyl-CoA analysis were performed by using an I-class ultra-high-performance liquid chromatograph coupled to a quadruple time-of-flight mass spectrometer. Detailed information is provided in the Supplementary Methods. Quantitative data were expressed as the mean ± SEM. Analytical details are provided in the Supplementary Methods. P < 0.05 was considered significantly different. Consistent with previous studies,17Tran M.T. Zsengeller Z.K. Berg A.H. et al.PGC1α drives NAD biosynthesis linking oxidative metabolism to renal protection.Nature. 2016; 531: 528-532Crossref PubMed Scopus (257) Google Scholar,18Zager R.A. Johnson A.C.M. Hanson S.Y. Renal tubular triglyercide accumulation following endotoxic, toxic, and ischemic injury.Kidney Int. 2005; 67: 111-121Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar Oil Red O staining revealed a significant lipid droplet accumulation in the renal tubules after I/R-induced AKI, indicative of lipid metabolic dysregulation (Figure 1a). Furthermore, the serum level of triglyceride and nonestesterified fatty acid decreased markedly after renal I/R (Supplementary Figure S1A). To better understand the mechanism of I/R-induced AKI, untargeted lipidomics was employed to elaborate the change pattern of the renal lipidome. Compared with the sham group, a total of 225 lipids were significantly increased, and 97 lipids were decreased in the I/R kidneys (Figure 1b). Among these lipids, the I/R group featured markedly elevated triglycerides, consistent with the observed lipid droplet accumulation following I/R-induced AKI. Moreover, fatty acyls (mainly FFAs) and sphingolipids (mainly ceramides) were remarkably increased after I/R (Figure 1c), demonstrating perturbations in fatty acid and sphingolipid metabolism. Notably, the variable importance in the projection score plot of the differentiated lipids indicated that ceramides were among the top 20 increased lipids (Figure 1d), which attracted our interest because ceramides have been identified as lipotoxins in the pathogenesis of many types of AKI.19Hao C.M. Breyer M.D. Physiologic and pathophysiologic roles of lipid mediators in the kidney.Kidney Int. 2007; 71: 1105-1115Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar Along with increased ceramides, the levels of most FFA species were higher in the I/R group, whereas long-chain acylcarnitines and acyl-CoAs were significantly reduced after I/R (Figure 1e), suggesting defective FA import into mitochondria and impaired FAO. And the level of the key enzymes in FAO (carnitine palmitoyltransferase 1a [CPT-1a], long-chain acyl-CoA dehydrogenase [LCAD], and medium-chain acyl-CoA dehydrogenase [MCAD]; Figure 1f) significantly decreased after I/R, indicating the insufficient FAO.Figure 1Renal ischemia-reperfusion (I/R) results in lipid accumulation and impairs fatty acid metabolism. (a) Oil Red O staining for lipids in sham and 24-hour post-ischemia 30-minute kidneys. Bar = 50 μm. (b) Heat map of differential lipids (variable importance in the projection [VIP] score > 1.2, fold change [FC] > 1.3, and P < 0.05). The VIP score was obtained through orthogonal partial least squares discrimination analysis (OPLS-DA) of untargeted lipidomics data between sham and I/R mice with total ion current (TIC) normalization. (c) The number of differential lipids in each lipid class. (d) VIP score plot of the top 20 significantly increased lipids induced by I/R. (e) Heat map of renal ceramides (Cer), free fatty acids (FFAs), acyl-carnitines, and acyl-CoAs. (b–e) n = 8. (f) Representative immunoblotting for the indicated proteins. n = 3. (g) Volcano plot of differentially expressed genes from renal RNA-sequencing data between sham and I/R mice (FC > 2, P-adjusted < 0.05). n = 5. (h) Top 20 upregulated and downregulated Kyoto Encyclopedia of Genes and Genomes pathway enrichments (false discovery rate < 0.05). (i) Schematic summary of the integrative lipidomics and transcriptomics analysis. The results of detected lipids and differentially expressed genes are shown, and red and blue colors indicate upregulation and downregulation, respectively. Akt, protein kinase B; ECM, extracellular matrix; MAPK, mitogen-activated protein kinase; PI3K, phosphatidylinositol-3′-kinase; PPAR, peroxisome proliferator–activated receptor; TG, triglyceride; TNF, tumor necrosis factor. 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) Furthermore, we examined the mechanisms underlying the association between I/R and dysregulated lipid metabolism by kidney RNA-sequencing analysis. Compared with the sham group, 1060 genes were significantly increased, and 595 genes were significantly decreased (Figure 1g). Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis revealed that the top upregulated genes were highly enriched in injury-related pathways. Moreover, lipid metabolism-related pathways were mainly downregulated, including peroxisome, peroxisome proliferator–activated receptor signaling, and fatty acid metabolism pathways (Figure 1h). Given the perturbed lipid metabolism observed through lipidomics and RNA-sequencing analysis, we mapped the related key gene change pattern after I/R (Figure 1i). Most of the genes in the lipid synthesis pathway were not significantly altered. However, the key genes involved in FAO, Cpt1b, Mcad, and acyl-CoA oxidase (Acox) were downregulated, which accounted for the decreased levels of acyl-carnitines and acyl-CoAs in the I/R group. Reverse transcription–polymerase chain reaction and immunohistochemistry results showed the same conclusion (Supplementary Figure S1B–E). Collectively, these findings suggest that impaired FAO within mitochondria might be the causative factor for lipid droplet accumulation in the I/R-induced AKI. A growing body of evidence indicates that AMPK specifically regulates lipid metabolism, mitochondrial biology, and homeostasis, including mitochondrial biogenesis and quality control.20Fullerton M.D. Galic S. Marcinko K. et al.Single phosphorylation sites in Acc1 and Acc2 regulate lipid homeostasis and the insulin-sensitizing effects of metformin.Nat Med. 2013; 19: 1649-1654Crossref PubMed Scopus (499) Google Scholar, 21Quirós P.M. Mottis A. Auwerx J. Mitonuclear communication in homeostasis and stress.Nat Rev Mol Cell Biol. 2016; 17: 213-226Crossref PubMed Scopus (364) Google Scholar, 22O’Neill H.M. Maarbjerg S.J. Crane J.D. et al.AMP-activated protein kinase (AMPK) β1β2 muscle null mice reveal an essential role for AMPK in maintaining mitochondrial content and glucose uptake during exercise.Proc Natl Acad Sci U S A. 2011; 108: 16092-16097Crossref PubMed Scopus (304) Google Scholar, 23Garcia-Roves P.M. Osler M.E. Holmström M.H. Zierath J.R. Gain-of-function R225Q mutation in AMP-activated protein kinase γ3 subunit increases mitochondrial biogenesis in glycolytic skeletal muscle.J Biol Chem. 2008; 283: 35724-35734Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar Therefore, we next examined the dynamic changes of AMPK activity after I/R. Plasma creatinine and urea increased dramatically from 6 to 24 hours after renal I/R (Supplementary Figure S2A and B). Western blot analysis showed that AMPKα activity significantly increased at the ischemia stage, whereas a dramatic decrease of AMPKα activity occurred as early as 1 hour after reperfusion, and pSer79–Acetyl-CoA carboxylase (ACC) showed a similar trend (Figure 2a and b). To further investigate the role of AMPKα in I/R, ATP D-R cell model was utilized for simulating renal I/R.24Tang C. Han H. Yan M. et al.PINK1-PRKN/PARK2 pathway of mitophagy is activated to protect against renal ischemia-reperfusion injury.Autophagy. 2018; 14: 880-897Crossref PubMed Scopus (131) Google Scholar,25Livingston M.J. Wang J. Zhou J. et al.Clearance of damaged mitochondria via mitophagy is important to the protective effect of ischemic preconditioning in kidneys.Autophagy. 2019; 15: 2142-2162Crossref PubMed Scopus (72) Google Scholar In immunoblot analysis, AMPKα activity showed extremely high after ATP depletion and decreased rapidly at 15 minutes after ATP repletion, whereas AMPK activity sustained at a low level within 2 hours after ATP repletion (Figure 2c). To determine the function of AMPKα in vitro, ATP D-R induced apoptosis model in RPTCs was performed. We knocked down AMPKα1 and AMPKα2 expression with specific small, interfering RNAs (Figure 2d). The results showed that AMPKα knockdown significantly increased the level of cleaved caspase-3 after ATP D-R (Figure 2e), suggesting that silencing of AMPK sensitized RPTC cells to ATP D-R induced apoptosis. Furthermore, we measured the FAO level by 14C–palmitic acid (PA) in ATP D-R. AMPKα silence cells showed a significant decrease in FAO in normal condition (Figure 2f). And there was a further decrease in AMPKα silence cells after ATP D-R. Then, we used AMPK allosteric activators to confirm the protective role of AMPK in vivo. Our results showed that AMPK activators A-769662, PF-06409577, C24,26Li Y.Y. Yu L.F. Zhang L.N. et al.Novel small-molecule AMPK activator orally exerts beneficial effects on diabetic db/db mice.Toxicol Appl Pharmacol. 2013; 273: 325-334Crossref PubMed Scopus (45) Google Scholar and metformin lowered apoptosis level significantly than control following ATP D-R (Supplementary Figure S2C), whereas C24 dose-dependently inhibited ATP D-R induced apoptosis (Supplementary Figure S2D). However, knockdown of AMPKα blocked the cytoprotective role of C24 (Figure 2g). These findings suggest that activation of AMPK plays a protective role in ATP D-R induced apoptosis. Mice with TAKO were generated to investigate the role of AMPK in renal injury. Littermate homozygous animals were considered controls. The transcript levels of AMPKα in whole kidney tissue of TAKO mice were reduced by 55% compared with control animals (Supplementary Figure S3A). Western blot results confirmed that the AMPKα levels were significantly reduced in the kidneys of TAKO mice compared with controls (Figure 3a). Under sham conditions, TAKO mice showed similar plasma creatinine and urea levels with control littermates (Figure 3b and c). After renal I/R, TAKO mice showed much higher levels of plasma creatinine and urea, indicating more severe renal impairment (Figure 3b and c). Furthermore, the mRNA levels of tubular injury marker (kidney injury molecule-1 [Kim-1] and Ngal) were significantly increased in TAKO mice after renal I/R (Figure 3d and e). Hematoxylin and eosin staining demonstrated that the kidneys of TAKO mice displayed extensive tubule injury in the renal cortex and outer medulla, including loss of the brush border, formation of tubular casts, and sloughing of cells into the tubular lumen after I/R (Figure 3f and g). Immunohistochemistry analysis also showed that renal I/R induced a significantly higher level of neutrophil gelatinase-associated lipocalin (NGAL) in TAKO mice than in controls (Figure 3f and h). We further evaluated tubular cell apoptosis by terminal deoxynucleotidyl transferase–mediated dUTP nick end-labeling (TUNEL) assay and cleaved caspase-3 immunohistochemistry. Quantitative analysis showed more TUNEL-positive tubular cells and cleaved caspase-3–positive tubular cells in TAKO mice after renal I/R (Figure 3f, i, and j). Inflammation plays an important role in the initiation and extension phases of AKI. Macrophages and neutrophils were analyzed by immunohistochemistry. Quantitative analysis revealed that renal I/R increased macrophages and neutrophils in the kidney. Notably, the TAKO mice showed more macrophage and neutrophil infiltration into kidney after renal I/R (Supplementary Figure S3B–D). And the transcript levels of proinflammatory cytokines Il1b and Il6 were significantly higher in TAKO mice than control mice after I/R (Supplementary Figure S3E). Taken together, these results suggest that tubular epithelial-specific deletion of AMPKα aggravated tubular injury during renal I/R. We examined lipid accumulation by Oil Red O staining in kidney slices, and the results showed that more positive staining occurred in TAKO mice after renal I/R (Figure 4a and b). Furthermore, most FFAs were elevated in the TAKO group (Figure 4c), indicative of more severe impairment of fatty acid metabolism. Meanwhile, TAKO mice showed a significant decrease in the levels of Cpt1a, Mcad, and Lcad compared with the control mice (Supplementary Figure S4A–C). LCAD immunohistochemistry staining showed a significant decrease in TAKO mice after renal I/R, suggesting a lower FAO level (Figure 4a and d). These data implied AMPKα deficiency induced mitochondria dysfunction during I/R. Next, mitochondrial morphology in tubular cells was evaluated by transmission electron microscopy. As shown in Figure 4a, many small mitochondria were observed in tubular cells of control mice after I/R, indicating mitochondria fragmentation. Notably, mitochondria in TAKO mice were more severely damaged, showing fragmentation, swelling, vacuoles in the mitochondrial matrix, and loss of cristae. Mitochondrial length data showed shorter mitochondria in TAKO mice after I/R (Figure 4e). Furthermore, mtDNA copy number was analyzed to examine mitochondrial content. The results showed that the TAKO mice had a greater reduction in mtDNA copy number compared with the control group after I/R (Figure 4f). Consistently, ATP content analysis also suggested that TAKO caused a more dramatic decrease in the levels of ATP production (Figure 4g). Autophagy/mitophagy has been proven to play a vital role in I/R-induced AKI model.24Tang C. Han H. Yan M. et al.PINK1-PRKN/PARK2 pathway of mitophagy is activated to protect against renal ischemia-reperfusion injury.Autophagy. 2018; 14: 880-897Crossref PubMed Scopus (131) Google Scholar,25Livingston M.J. Wang J. Zhou J. et al.Clearance of damaged mitochondria via mitophagy is important to the protective effect of ischemic preconditioning in kidneys.Autophagy. 2019; 15: 2142-2162Crossref PubMed Scopus (72) Google Scholar,27Kaushal G.P. Shah S.V. Autophagy in acute kidney injury.Kidney Int. 2016; 89: 779-791Abstract Full Text Full Text PDF PubMed Scopus (206) Google Scholar Immunohistochemistry staining of microtubule-associated protein 1A/1B-light chain 3 (LC3) revealed a granular LC3 staining of TECs in I/R kidneys, suggesting the formation of autophagosomes (Figure 4a). The quantified number of autolysosomes (colocation of LC3 and lysosomal-associated membrane protein 1 [LAMP1]) significantly decreased in TAKO mice compared with controls, indicating that AMPKα knockout in TECs inhibits autophagic flux (Figure 4h). Quantification analysis of mitolysosomes also revealed that there were significantly fewer mitolysosomes in renal tubules in TAKO mice after renal I/R (Figure 4a and i). Consistent with previous results,24Tang C. Han H. Yan M. et al.PINK1-PRKN/PARK2 pathway of mitophagy is activated to protect against renal ischemia-reperfusion injury.Autophagy. 2018; 14: 880-897Crossref PubMed Scopus (131) Google Scholar a significant increase in the fluorescence intensity of dihydroethidium (DHE) was detected in tubular cells of control mice after renal I/R, and the signal appeared stronger in renal tubule cells of TAKO mice (Figure 4a and j). Peroxisome proliferator-activated receptor-γ coactivator 1-α (PGC-1α), a master regulator of mitochondrial biogenesis, is necessary for renal recovery.28Tran M. Arany Z. Parikh S.M. et al.PGC-1α promotes recovery after acute kidney injury during systemic inflammation in mice.. 2011; 121: 4003-4014Google Scholar Mitochondrial transcription factor A (TFAM) is a key activator of mtDNA transcription and can be regulated by the PGC-1α signaling pathway. As shown in Supplementary Figure S4D through F, under sham condition, TAKO mice showed lower expression of PGC-1α and TFAM levels than control mice. After I/R, the PGC-1α and TFAM levels in TAKO mice were further decreased compared with controls, confirming the decreased mitochondrial biogenesis by AMPKα disruption in renal tubule cells. All these data demonstrate that AMPKα deficiency in tubular cells impaired β-oxidation in mitochondria, leading to disrupted mitochondrial homeostasis and increased lipid accumulation after renal I/R. The AMPK agonist C24 has been shown to reduce lipogenesis in the liver.29Sun S.-M. Xie Z.-F. Zhang Y.-M. et al.AMPK activator C24 inhibits hepatic lipogenesis and ameliorates dyslipidemia in HFHC diet-induced animal models.Acta Pharmacol Sin. 2021; 42: 585-592Google Scholar The effect of C24 on I/R-induced AKI was assessed, using AMPK indirect activator metformin as positive control. Both C24 and metformin significantly increased the pThr172-AMPKα level at 1 hour after reperfusion (Figure 5a and b). After 24 hours of reperfusion, C24 and metformin significantly decreased serum creatinine and urea levels (Figure 5c and d) and ameliorated tubular injury (Figure 5e and f). Both C24 and metformin significantly decreased NGAL levels in tubules after I/R (Figure 5e and g), and the mRNA levels of Ngal and Kim-1 showed the same results (Supplementary Figure S5A and B). Furthermore, C24 and metformin treatments resulted in fewer cleaved caspase-3– and TUNEL-positive cells in the kidney (Figure 5e, h, and i). The macrophage and neutrophil infiltration into the kidney after I/R was also reduced (Supplementary Figure S5C–E). Collectively, these results demonstrate that AMPK activation could rescue I/R-induced AKI. We further examined lipid accumulation in the kidney after C24 treatment. The Oil Red O data showed that C24 significantly decreased positive staining compared with the control group (Figure 5e and j). Meanwhile, the mRNA levels of Cpt-1α, Lcad, and Mcad were upregulated by C24 (Supplementary Figure S5G–I). However, metformin did not show the same phenotype, which may suggest the different mechanism of C24 and metformin. The effect of C24 on mitochondrial homeostasis was further examined. The results showed that C24 promoted the mtDNA-to–nuclear DNA (nDNA) and ATP levels in the kidney after I/R, suggesting that C24 treatment sustained mitochondrial function after I/R (Figure 6a and b). Furthermore, we found that C24 significantly increased autolysosomes and mito