BASIC RESEARCH www.jasn.org Sirtuin 5 Regulates Proximal Tubule Fatty Acid Oxidation to Protect against AKI Takuto Chiba,1 Kevin D. Peasley ,1 Kasey R. Cargill,1 Katherine V. Maringer,1 Sivakama S. Bharathi,1 Elina Mukherjee,1 Yuxun Zhang ,1 Anja Holtz ,2 Nathan Basisty ,2 Shiva D. Yagobian,1 Birgit Schilling,2 Eric S. Goetzman,1 and Sunder Sims-Lucas1 1Department of Pediatrics, University of Pittsburgh School of Medicine, University of Pittsburgh Medical Center Children’s Hospital of Pittsburgh, University of Pittsburgh, Pittsburgh, Pennsylvania; and 2Buck Institute for Research on Aging, Novato, California ABSTRACT Background The primary site of damage during AKI, proximal tubular epithelial cells, are highly metabol- ically active, relying on fatty acids to meet their energy demands. These cells are rich in mitochondria and peroxisomes, the two organelles that mediate fatty acid oxidation. Emerging evidence shows that both fatty acid pathways are regulated by reversible posttranslational modifications, particularly by lysine acylation. Sirtuin 5 (Sirt5), which localizes to both mitochondria and peroxisomes, reverses post- translational lysine acylation on several enzymes involved in fatty acid oxidation. However, the role of the Sirt5 in regulating kidney energy metabolism has yet to be determined. Methods We subjected male Sirt5-deficient mice (either +/2 or 2/2) and wild-type controls, as well as isolated proximal tubule cells, to two different AKI models (ischemia-induced or cisplatin-induced AKI). We assessed kidney function and injury with standard techniques and measured fatty acid oxidation 14 14 by the catabolism of C-labeled palmitate to CO2. 2/2 Results Sirt5 was highly expressed in proximal tubular epithelial cells. At baseline, Sirt5 knockout (Sirt5 ) mice had modestly decreased mitochondrial function but significantly increased fatty acid oxidation, 2/2 which was localized to the peroxisome. Although no overt kidney phenotype was observed in Sirt5 2/2 mice, Sirt5 mice had significantly improved kidney function and less tissue damage compared with controls after either ischemia-induced or cisplatin-induced AKI. This coincided with higher peroxisomal 2/2 fatty acid oxidation compared with mitochondria fatty acid oxidation in the Sirt5 proximal tubular epithelial cells. Conclusions Our findings indicate that Sirt5 regulates the balance of mitochondrial versus peroxisomal fatty acid oxidation in proximal tubular epithelial cells to protect against injury in AKI. This novel mecha- nism might be leveraged for developing AKI therapies. JASN 30: 2384–2398, 2019. doi: https://doi.org/10.1681/ASN.2019020163 Received February 18, 2019. Accepted August 29, 2019. Proximal tubular epithelial cells (PTECs) are highly T.C., K.D.P., and K.R.C. contributed equally to this work. sensitive to damage after AKI largely because of its 1,2,3 Published online ahead of print. Publication date available at high metabolic rate. To meet energy demands, www.jasn.org. PTECs rely on fatty acid b-oxidation (FAO). Paral- lel FAO pathways exist in mitochondria and in per- Correspondence: Dr. Sunder Sims-Lucas or Dr. Eric S. Goetzman, Department of Pediatrics, University of Pittsburgh School of Medicine, oxisomes, both of which are notably abundant in UPMC Children’s Hospital of Pittsburgh, University of Pittsburgh, 4401 PTECs. Mitochondrial FAO directly links to the Penn Avenue, Pittsburgh, PA 15224. E-mail: [email protected] or mitochondrial electron transport chain and is an [email protected] oxygen-intensive source of energy. Concomitant Copyright © 2019 by the American Society of Nephrology 2384 ISSN : 1046-6673/3012-2384 JASN 30: 2384–2398, 2019 www.jasn.org BASIC RESEARCH with oxygen utilization is the formation of reactive oxygen Significance Statement species (ROS), which are thought to be particularly damaging during ischemic- and cisplatin-induced AKI.4,5 Limiting mito- Proximal tubular epithelial cells, a primary site of injury in AKI, are chondrial FAO limits ischemic injury in the heart6,7 and kidney.8 rich in mitochondria and peroxisomes, the two organelles that Sirtuin 5 Sirt5 In contrast to mitochondria, little is known about the role of mediate fatty acid oxidation. Deletion of ( )reverses posttranslational lysine acylation of several enzymes involved in peroxisomes in AKI. Although peroxisomes do not produce fatty acid oxidation. The authors demonstrate that mice lacking energy, they metabolize long-chain fatty acids to acetate and Sirt5 had significantly improved kidney function and less tissue other short-chain products. Because of their hydrophilicity, damage following either ischemia-induced or cisplatin-induced the metabolites can cross plasma membranes to either leave AKI compared with wild-type mice. These differences coincided the cell or enter mitochondria to be oxidized to completion. with higher peroxisomal fatty acid oxidation compared with mi- tochondria fatty acid oxidation in the Sirt5-deficient proximal tu- Peroxisomes have been observed to physically interact with bular epithelial cells. Their findings indicate that Sirt5 regulates the mitochondria and ablation of peroxisomal function has sec- balance of mitochondrial versus peroxisomal fatty acid oxidation in ondary effects on mitochondrial function.9 Peroxisomes may proximal tubular epithelial cells to protect against injury in AKI. serve as a sink for mitochondrially produced ROS because This novel mechanism has potential therapeutic implications for of the abundance of catalase and other ROS-neutralizing en- treating AKI. zymes in the peroxisome.10,11 Peroxisomes may also serve to 2 2 2 protect PTECs from the accumulation of toxic long-chain together to produce WT (Sirt5+/+), Sirt5+/ ,andSirt5 / mu- fatty acids.12 tants. Age-matched 10- to 14-week-old male mice were used Emerging evidence shows both FAO pathways are regulated throughout the study. The University of Pittsburgh Institu- by reversible post-translational modifications (PTMs), in par- tional Animal Care and Use Committee approved all experi- ticular by lysine acylation and the sirtuin deacylase enzymes ments (approval no. 16088935). that remove these PTMs.13,14 Mammalian sirtuins Sirt1–Sirt7 differ in their subcellular localization and substrate specificity. Ischemic and Cisplatin AKI Models in Mice The nuclear/cytosolic sirtuin Sirt1 has been shown to protect Ischemic AKI was induced by a renal ischemia-reperfusion against AKI by maintaining peroxisomes, upregulating cata- injury (IRI) model as previously described,23 with modifica- lase, and eliminating renal ROS.15 The mitochondrial sirtuin tions. Briefly, mice were anesthetized with inhalant 2% Sirt3 is also renoprotective through its role in improving mi- isoflurane. Core body temperature of the mice was moni- tochondrial dynamics.16 Although its role in regulating kidney tored with a rectal thermometer probe and was maintained FAO is not clear, Sirt3 promotes mitochondrial FAO in liver at 36.8–37.2°C throughout the procedures with a water-heating and heart.14,17 Sirt5, which is unique among the sirtuins in its circulation pump system (EZ-7150; BrainTree Scientific) and substrate preference for succinyllysine, malonyllysine, and an infrared heat lamp (Shat-R-Shield). Buprenorphine (Par glutaryllysine,18,19 also promotes mitochondrial FAO in liver Pharmaceutical) was administered for pain control (0.1 mg/kg and heart.13,20 Intriguingly, Sirt5 was recently shown to local- body wt, administered subcutaneously). With aseptic tech- ize to peroxisomes. In direct contrast to its effect on mito- niques, a dorsal incision was made to expose the kidney, and chondrial FAO, Sirt5 was observed to suppress peroxisomal renal ischemia for 22 minutes was induced by unilateral clamp- FAO in vitro and in rodent liver.21 However, the role of Sirt5 in ing of the left kidney pedicle with a nontraumatic microvascular 2/2 the kidney has not yet been studied. Here, we report that Sirt5 clamp (18055–04; Fine Science Tools). Renal reperfusion was mice are protected against ischemic and cisplatin AKI. visually verified. Delayed contralateral nephrectomy of the right Although mitochondrial function is moderately suppressed kidney was performed at day 6.23 Mice were euthanized at day 7 2 /2 in Sirt5 kidney, peroxisomal function is enhanced to harvest blood and the injured kidney. Serum was separated both before and after AKI. These results indicate that Sirt5 from the blood and analyzed by the Kansas State Veterinary Di- regulates the balance of mitochondrial versus peroxisomal agnostic Laboratory for levels of creatinine and BUN. FAO in PTECs to protect against PTEC injury in AKI. This To induce cisplatin AKI, mice were given a single dose of novel mechanism may be leveraged for developing future 20 mg/kg intraperitoneal cisplatin (Fresenius Kabi) or vehicle AKI therapies. control of normal saline as described previously.24 Mice were euthanized on day 3 to harvest blood and the kidneys. METHODS Cultured PTECs Primary mouse PTECs were isolated from single-cell kidney 2 2 2 Animals suspensionof10- to 14-week-old maleSirt5 / , Sirt5+/ ,B6/129 2 2 Homozygous global Sirtuin 5 knockout mice (Sirt5 / )22 were strain WT, or 129 strain WT mice by Dynabeads FlowComp obtained from the Jackson Laboratory (B6;129-Sirt5tm1Fwa/J, Flexi Kit (Thermo Fisher Scientific) conjugated to lotus tetra- stock no. 012757). B6/129S F2 strain wild-type (WT) mice gonolobus lectin (LTL) (L-1320; Vector Laboratories). Passage were used as controls. To produce appropriate littermate con- 3–6 PTECs were