Rick G. Schnellmann

Objective: The mitogen-activated protein kinase/extracellular signal–regulated kinase signaling pathway is an essential component of innate immunity necessary for mediating proinflammatory responses in the setting of sepsis. We previously demonstrated that the mitogen-activated protein kinase 1/2 inhibitor trametinib prevents endotoxin-induced renal injury in mice. We therefore assessed efficacy of trametinib in a more clinically relevant experimental model of sepsis. Design: Controlled in vivo laboratory study. Setting: University animal research laboratory. Subjects: Male C57BL/6 mice. Interventions: Mice were subjected to cecal ligation and puncture to induce sepsis or underwent sham operation as controls. Six hours after cecal ligation and puncture, mice were randomized to four experimental groups as follows: 1) sham control; 2) sham control + trametinib (1 mg/kg, IP); 3) cecal ligation and puncture; and 4) cecal ligation and puncture + trametinib. All animals received buprenorphine (0.05 mg/kg, SC) and imipenem/cilastatin (14 mg/kg, SC) in 1.5 mL of warm saline (40 mL/kg) at the 6-hour time point. Mice were euthanized at 18 hours after induction of cecal ligation and puncture. Measurements and Main Results: Trametinib inhibition of mitogen-activated protein kinase/extracellular signal–regulated kinase signaling 6 hours after cecal ligation and puncture attenuated increases in circulating proinflammatory cytokines (tumor necrosis factor-α, interleukin-1β, interleukin-6, and granulocyte macrophage colony-stimulating factor) and hypothermia at 18 hours. Trametinib also attenuated multiple organ injury as determined by serum creatinine, alanine aminotransferase, lactate dehydrogenase, and creatine kinase. At the organ level, trametinib completely restored peritubular capillary perfusion in the kidney. Restoration of microvascular perfusion was associated with reduced messenger RNA expression of well-characterized markers of proximal tubule injury. mitogen-activated protein kinase/extracellular signal–regulated kinase blockade attenuated cecal ligation and puncture-mediated up-regulation of cytokines (tumor necrosis factor-α, interleukin-1β) and restored interleukin-6 to control levels in the renal cortex, indicating the protective effects on the proximal tubule occur primarily through modulation of the proinflammatory response in sepsis. Conclusions: These data reveal that the mitogen-activated protein kinase/extracellular signal–regulated kinase inhibitor trametinib attenuates systemic inflammation and multiple organ damage in a clinically relevant model of sepsis. Because trametinib has been safely used in humans, we propose that this drug might represent a translatable approach to limit organ injury in septic patients.

In acute organ injuries, mitochondria are often dysfunctional, and recent research has revealed that recovery of mitochondrial and renal functions is accelerated by induction of mitochondrial biogenesis (MB). We previously reported that the nonselective 5-HT₂ receptor agonist DOI [1-(4-iodo-2,5-dimethoxyphenyl)propan-2-amine] induced MB in renal proximal tubular cells (RPTCs). The goal of this study was to determine the role of 5-HT₂ receptors in the regulation of mitochondrial genes and oxidative metabolism in the kidney. The 5-HT_2C receptor agonist CP-809,101 [2-[(3-chlorophenyl)methoxy]-6-(1-piperazinyl)pyrazine] and antagonist SB-242,084 [6-chloro-2,3-dihydro-5-methyl-N-[6-[(2-methyl-3-pyridinyl)oxy]-3-pyridinyl]-1H-indole-1-carboxyamide dihydrochloride] were used to examine the induction of renal mitochondrial genes and oxidative metabolism in RPTCs and in mouse kidneys in the presence and absence of the 5-HT_2C receptor. Unexpectedly, both CP-809,101 and SB-242,084 increased RPTC respiration and peroxisome proliferator–activated receptor γ coactivator-1α (PGC-1α) mRNA expression in RPTCs at 1–10 nM. In addition, CP-809,101 and SB-242,084 increased mRNA expression of PGC-1α and the mitochondrial proteins NADH dehydrogenase subunit 1 and NADH dehydrogenase (ubiquinone) β subcomplex 8 in mice. These compounds increased mitochondrial genes in RPTCs in which the 5-HT_2C receptor was downregulated with small interfering RNA and in the renal cortex of mice lacking the 5-HT_2C receptor. By contrast, the ability of these compounds to increase PGC-1α mRNA and respiration was blocked in RPTCs treated with 5-HT_2A receptor small interfering RNA or the 5-HT_2A receptor antagonist eplivanserin. In addition, the 5-HT_2A receptor agonist NBOH-2C-CN [4-[2-[[(2-hydroxyphenyl)methyl]amino]ethyl]-2,5-dimethoxybenzonitrile] increased RPTC respiration at 1–100 nM. These results suggest that agonism of the 5-HT_2A receptor induces MB and that the classic 5-HT_2C receptor agonist CP-809,101 and antagonist SB-242,084 increase mitochondrial genes and oxidative metabolism through the 5-HT_2A receptor. To our knowledge, this is the first report that links 5-HT_2A receptor agonism to mitochondrial function.

Mitochondrial dysfunction is a key pathophysiological compofanent of many acute and chronic diseases. Maintenance of mitochondrial homeostasis through the balance of mitochondrial turnover, fission and fusion, and generation of new mitochondria via mitochondrial biogenesis is critical for tissue health. Pharmacological activation of mitochondrial biogenesis can enhance oxidative metabolism and tissue bioenergetics, and improve organ function in conditions characterized by mitochondrial dysfunction. However, owing to the complexity of mitochondrial assembly and maintenance, identification of specific activators of mitochondrial biogenesis has been difficult. This review provides an overview of the role of mitochondrial dysfunction in acute and chronic diseases, details the current state of therapeutics for the stimulation of mitochondrial biogenesis and their effects on disease outcomes, describes new screening methodologies to identify novel stimulators and noncanonical pathways of mitochondrial biogenesi...

While the importance of mitochondrial dysfunction in acute kidney injury (AKI) has been documented, non-invasive early biomarkers of mitochondrial damage are needed. We examined urinary ATP synthase subunit β (ATPSβ) as a biomarker of renal mitochondrial dysfunction during AKI. Mice underwent sham surgery or varying degrees (5, 10 or 15 min ischemia) of ischemia/reperfusion (I/R)-induced AKI. Serum creatinine, BUN and NGAL were elevated only in the 15 min I/R group at 24 h. Immunoblot analysis of urinary ATPSβ revealed two bands (full- length ~52 kDa and cleaved ~25 kDa), both confirmed as ATPSβ by LC-MS/MS, that increased at 24 h in 10- and 15-min I/R groups. These changes were associated with mitochondrial dysfunction evidenced by reduced renal cortical expression of mitochondrial proteins, ATPSβ and COX1, proximal tubular oxygen consumption, and ATP. Furthermore, in the 15-min I/R group urinary ATPSβ was elevated until 72 h before returning to baseline 144 h after reperfusion with recovery of renal function. Evaluation of urinary ATPSβ in a non-alcoholic steatohepatitis (NASH) model of liver injury only revealed cleaved ATPSβ, suggesting specificity of full-length ATPSβ for renal injury. Immunoblot analyses of patient urine samples collected 36h after cardiac surgery revealed increased urinary ATPSβ levels in patients with post-cardiac surgery-induced AKI. LC-MS/MS urinalysis in human subjects with AKI confirmed increased ATPSβ. These translational studies provide evidence that ATPSβ may be a novel and sensitive urinary biomarker of renal mitochondrial dysfunction and could serve as valuable tool for the testing of potential therapies for AKI and chemical-induced nephrotoxicity.

Although disruption of mitochondrial homeostasis and biogenesis (MB) is a widely accepted pathophysiologic feature of sepsis-induced acute kidney injury (AKI), the molecular mechanisms responsible for this phenomenon are unknown. In this study, we examined the signaling pathways responsible for the suppression of MB in a mouse model of lipopolysaccharide (LPS)-induced AKI. Downregulation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a master regulator of MB, was noted at the mRNA level at 3 hours and protein level at 18 hours in the renal cortex, and was associated with loss of renal function after LPS treatment. LPS-mediated suppression of PGC-1α led to reduced expression of downstream regulators of MB and electron transport chain proteins along with a reduction in renal cortical mitochondrial DNA content. Mechanistically, Toll-like receptor 4 (TLR4) knockout mice were protected from renal injury and disruption of MB after LPS exposure. Immunoblot analysis revealed activation of tumor progression locus 2/mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (TPL-2/MEK/ERK) signaling in the renal cortex by LPS. Pharmacologic inhibition of MEK/ERK signaling attenuated renal dysfunction and loss of PGC-1α, and was associated with a reduction in proinflammatory cytokine (e.g., tumor necrosis factor-α [TNF-α], interleukin-1β) expression at 3 hours after LPS exposure. Neutralization of TNF-α also blocked PGC-1α suppression, but not renal dysfunction, after LPS-induced AKI. Finally, systemic administration of recombinant tumor necrosis factor-α alone was sufficient to produce AKI and disrupt mitochondrial homeostasis. These findings indicate an important role for the TLR4/MEK/ERK pathway in both LPS-induced renal dysfunction and suppression of MB. TLR4/MEK/ERK/TNF-α signaling may represent a novel therapeutic target to prevent mitochondrial dysfunction and AKI produced by sepsis.

Skeletal muscle atrophy remains a clinical problem in numerous pathologic conditions. β₂-Adrenergic receptor agonists, such as formoterol, can induce mitochondrial biogenesis (MB) to prevent such atrophy. Additionally, atomoxetine, an FDA-approved norepinephrine reuptake inhibitor, was positive in a cellular assay for MB. We used a mouse model of dexamethasone-induced skeletal muscle atrophy to investigate the potential role of atomoxetine and formoterol to prevent muscle mass loss. Mice were administered dexamethasone once daily in the presence or absence of formoterol (0.3 mg/kg), atomoxetine (0.1 mg/kg), or sterile saline. Animals were euthanized at 8, 16, and 24 hours or 8 days later. Gastrocnemius muscle weights, changes in mRNA and protein expression of peroxisome proliferator–activated receptor-γ coactivator-1 α (PGC-1α) isoforms, ATP synthase β, cytochrome c oxidase subunit I, NADH dehydrogenase (ubiquinone) 1 β subcomplex, 8, ND1, insulin-like growth factor 1 (IGF-1), myostatin, muscle Ring-finger protein-1 (muscle atrophy), phosphorylated forkhead box protein O 3a (p-FoxO3a), Akt, mammalian target of rapamycin (mTOR), and ribosomal protein S6 (rp-S6; muscle hypertrophy) in naive and muscle-atrophied mice were measured. Atomoxetine increased p-mTOR 24 hours after treatment in naïve mice, but did not change any other biomarkers. Formoterol robustly activated the PGC-1α-4-IGF1–Akt-mTOR-rp-S6 pathway and increased p-FoxO3a as early as 8 hours and repressed myostatin at 16 hours. In contrast to what was observed with acute treatment, chronic treatment (7 days) with atomoxetine increased p-Akt and p-FoxO3a, and sustained PGC-1α expression and skeletal muscle mass in dexamethasone-treated mice, in a manner comparable to formoterol. In conclusion, chronic treatment with a low dose of atomoxetine prevented dexamethasone-induced skeletal muscle wasting and supports a potential role in preventing muscle atrophy.

While disruption of energy production is an important contributor to renal injury, metabolic alterations in sepsis-induced AKI remain understudied. We assessed changes in renal cortical glycolytic metabolism in a mouse model of sepsis-induced AKI. A specific and rapid increase in hexokinase (HK) activity (∼2-fold) was observed 3 h after LPS exposure and maintained up to 18 h, in association with a decline in renal function as measured by blood urea nitrogen (BUN). LPS-induced HK activation occurred independently of HK isoform expression or mitochondrial localization. No other changes in glycolytic enzymes were observed. LPS-mediated HK activation was not sufficient to increase glycolytic flux as indicated by reduced or unchanged pyruvate and lactate levels in the renal cortex. LPS-induced HK activation was associated with increased glucose-6-phosphate dehydrogenase activity but not glycogen production. Mechanistically, LPS-induced HK activation was attenuated by pharmacological inhibitors of the EGF receptor (EGFR) and Akt, indicating that EGFR/phosphatidylinositol 3-kinase/Akt signaling is responsible. Our findings reveal LPS rapidly increases renal cortical HK activity in an EGFR- and Akt-dependent manner and that HK activation is linked to increased pentose phosphate pathway activity.

Many acute and chronic conditions, such as acute kidney injury, chronic kidney disease, heart failure, and liver disease, involve mitochondrial dysfunction. Although we have provided evidence that drug-induced stimulation of mitochondrial biogenesis (MB) accelerates mitochondrial and cellular repair, leading to recovery of organ function, only a limited number of chemicals have been identified that induce MB. The goal of this study was to assess the role of the 5-hydroxytryptamine 1F (5-HT_1F) receptor in MB. Immunoblot and quantitative polymerase chain reaction analyses revealed 5-HT_1F receptor expression in renal proximal tubule cells (RPTC). A MB screening assay demonstrated that two selective 5-HT_1F receptor agonists, LY334370 (4-fluoro-N-[3-(1-methyl-4-piperidinyl)-1H-indol-5-yl]benzamide) and LY344864 (N-[(3R)-3-(dimethylamino)-2,3,4,9-tetrahydro-1H-carbazol-6-yl]-4-fluorobenzamide; 1–100 nM) increased carbonylcyanide-p-trifluoromethoxyphenylhydrazone–uncoupled oxygen consumption in RPTC, and validation studies confirmed both agonists increased mitochondrial proteins [e.g., ATP synthase β, cytochrome c oxidase 1 (Cox1), and NADH dehydrogenase (ubiquinone) 1β subcomplex subunit 8 (NDUFB8)] in vitro. Small interfering RNA knockdown of the 5-HT_1F receptor blocked agonist-induced MB. Furthermore, LY344864 increased peroxisome proliferator–activated receptor coactivator 1-α, Cox1, and NDUFB8 transcript levels and mitochondrial DNA (mtDNA) copy number in murine renal cortex, heart, and liver. Finally, LY344864 accelerated recovery of renal function, as indicated by decreased blood urea nitrogen and kidney injury molecule 1 and increased mtDNA copy number following ischemia/reperfusion-induced acute kidney injury (AKI). In summary, these studies reveal that the 5-HT_1F receptor is linked to MB, 5-HT_1F receptor agonism promotes MB in vitro and in vivo, and 5-HT_1F receptor agonism promotes recovery from AKI injury. Induction of MB through 5-HT_1F receptor agonism represents a new target and approach to treat mitochondrial organ dysfunction.

Mitochondrial biogenesis may be an adaptive response necessary for meeting the increased metabolic and energy demands during organ recovery after acute injury, and renal mitochondrial dysfunction has been implicated in the pathogenesis of AKI. We proposed that stimulation of mitochondrial biogenesis 24 hours after ischemia/reperfusion (I/R)-induced AKI, when renal dysfunction is maximal, would accelerate recovery of mitochondrial and renal function in mice. We recently showed that formoterol, a potent, highly specific, and long-acting β2-adrenergic agonist, induces renal mitochondrial biogenesis in naive mice. Animals were subjected to sham or I/R-induced AKI, followed by once-daily intraperitoneal injection with vehicle or formoterol beginning 24 hours after surgery and continuing through 144 hours after surgery. Treatment with formoterol restored renal function, rescued renal tubules from injury, and diminished necrosis after I/R-induced AKI. Concomitantly, formoterol stimulated mitochondrial biogenesis and restored the expression and function of mitochondrial proteins. Taken together, these results provide proof of principle that a novel drug therapy to treat AKI, and potentially other acute organ failures, works by restoring mitochondrial function and accelerating the recovery of renal function after injury has occurred.