CypD Inhibitor Research Articles

Neuroprotective Effect of 2-(Benzyloxy)arylureas Is Not Related to CypD Inhibition nor Suppression of mPTP Opening.

Cyclophilin D (CypD) is a mitochondrial enzyme widely accepted as a regulator of the mitochondrial permeability transition pore (mPTP). Excessive opening of mPTP is associated with mitochondrial dysfunction and the development of various diseases; thus, suppression of mPTP opening through CypD inhibition presents a promising therapeutic approach. However, only a limited number of selective CypD inhibitors are currently available. In this study, 10 derivatives of 2-(benzyloxy)arylurea similar or identical to previously published CypD/mPTP inhibitors were synthesized. Unlike the original reports that assessed the opening of mPTP at the cellular level, the compounds were tested directly on the purified CypD enzyme to validate their putative mechanism of action. Additionally, the effect of the selected compounds was tested on isolated mitochondria. The obtained results show that the compounds are only weak inhibitors of CypD and mPTP opening, which is in contrast to previous conclusions drawn from the unspecific cellular JC-1 assay.

#855 Cyclophilin D in cholesterol crystal-induced acute kidney injury

Abstract Background and Aims Cholesterol crystal embolism (CCE) occurs when cholesterol crystal (CC) clots move from an atherosclerotic plaque in a large artery to a smaller artery, leading to obstruction, ischemia, and organ failure. CCE directly impairs blood flow by provoking a local inflammatory response and triggering arterial thrombus formation, consisting of collagen and fibrin mesh, platelet and neutrophil activation, and extracellular DNA trap formation. Activated platelets form a procoagulant surface that supports adhesion to collagen and fibrin mesh, promoting thrombus growth and the recruitment of immune cells. CCE often targets kidney tissue, leading to thrombotic angiopathy, acute loss of glomerular filtration rate (GFR), and necrosis in tubular cells. Cyclophilin D (CypD) plays a central role in regulating platelet procoagulant activity by triggering calcium bursts and mitochondrial depolarization. While the increased number of procoagulant platelets contributes to thrombosis, CypD-mediated procoagulant pathomechanisms in the kidney during CCE remain unknown. In our study presented here, we hypothesized that CypD deficiency might attenuate procoagulant platelet activity, thereby inhibiting CCE-induced kidney infarction and organ injury. Method 6-week-old wild-type (WT) and CypD-/- mice were administered 10 mg/kg CC through injections into the left kidney arteries in vivo. Kidneys were collected 24 hours later, and kidney function, infarct size, necroinflammation and kidney injury were evaluated using GFR measurements, TTC colorimetric assay, immunohistochemistry, and immunofluorescence microscopy. For in vitro studies, whole blood samples were isolated from WT and CypD-/- mice and perfused on a collagen-coated surface in the presence or absence of CC under arterial shear conditions of 1000s-1. Results After CC injection in mice, CypD deficiency ameliorated immunothrombosis, acute kidney injury, and organ failure. CypD-/- mice were protected from the sudden drop in GFR, i.e., acute kidney injury, which resulted from the reduced number of arteries obstructed by crystal clots. Kidney infarct size consistently decreased in CypD-/- mice. CypD-/- mice displayed reduced neutrophil recruitment to thrombotic microangiopathic lesions, indicating diminished immunothrombosis. Preemptive injection of the CypD inhibitor cyclosporin A could prevent CC-mediated vessel obstruction and infarct size, thereby improving kidney function. Interestingly, WT platelets showed increased platelet adhesion and phosphatidylserine (PS) exposure on a collagen-coated surface in the presence of CC under shear conditions in whole blood, suggesting increased procoagulant activity, which was strongly reduced in CypD-/- platelets. Conclusion During CCE-mediated kidney injury, CypD-mediated platelet activation and consequent procoagulant activity accelerate ischemic tissue necrosis and the recruitment of neutrophils. Inhibition of CypD-mediated signaling could be a potential therapeutic approach to inhibit acute kidney injury in patients with CCE.

Inhibition of mitochondrial cyclophilin D, a downstream target of glycogen synthase kinase 3α, improves sperm motility.

Cyclophilin D (CypD) negatively regulates ATP production by opening of the mitochondrial permeability transition pore. This study aimed to understand the role of CypD in sperm motility regulation. Changes in CypD during sperm capacitation and its interaction with glycogen synthase kinase 3α (GSK3α), a key kinase regulating sperm motility, were examined in mouse spermatozoa. The effects of CypD inhibitor cyclosporin A (CsA) and GSK3 inhibitor 6-bromo-indirubin-3'-oxime (BIO) on sperm motility, p-GSK3α(Ser21), mitochondrial permeability transition pore (mPTP), mitochondrial membrane potential (MMP), and ATP production were examined. The effect of proteasome inhibitor MG115 on the cellular levels of CypD was examined. In cauda epididymal spermatozoa, GSK3α was found in both cytosolic and mitochondrial fractions whereas CypD was primarily found in the mitochondrial fraction together with ATP synthase F1 subunit alpha (ATP5A), a mitochondrial marker. GSK3α and CypD were co-localized in the sperm midpiece. Interaction between GSK3α and CypD was identified in co-immunoprecipitation. CsA, a CypD inhibitor, significantly increased sperm motility, tyrosine phosphorylation, mPTP closing, MMP, and ATP levels in spermatozoa, suggesting that CypD acts as a negative regulator of sperm function. Under capacitation condition, both GSK3α and CypD were decreased in spermatozoa but ATP5A was not. The GSK3 inhibitor BIO markedly increased p-GSK3α(Ser21) and decreased CypD but significantly increased mPTP closing, MMP, ATP production, and motility of spermatozoa. This suggests that inhibitory phosphorylation of GSK3α is coupled with degradation of CypD, potentiating the mitochondrial function. Degradation of CypD was attenuated by MG115, indicative of involvement of the ubiquitin proteasome system. During sperm capacitation, CypD act as a downstream target of GSK3α can be degraded via the ubiquitin proteasome system, stimulating mitochondrial function and sperm motility.

New cyclophilin D inhibitor rescues mitochondrial and cognitive function in Alzheimer's disease.

Mitochondrial dysfunction is an early pathological feature of Alzheimer disease and plays a crucial role in the development and progression of Alzheimer's disease. Strategies to rescue mitochondrial function and cognition remain to be explored. Cyclophilin D (CypD), the peptidylprolyl isomerase F (PPIase), is a key component in opening the mitochondrial membrane permeability transition pore, leading to mitochondrial dysfunction and cell death. Blocking membrane permeability transition pore opening by inhibiting CypD activity is a promising therapeutic approach for Alzheimer's disease. However, there is currently no effective CypD inhibitor for Alzheimer's disease, with previous candidates demonstrating high toxicity, poor ability to cross the blood-brain barrier, compromised biocompatibility and low selectivity. Here, we report a new class of non-toxic and biocompatible CypD inhibitor, ebselen, using a conventional PPIase assay to screen a library of ∼2000 FDA-approved drugs with crystallographic analysis of the CypD-ebselen crystal structure (PDB code: 8EJX). More importantly, we assessed the effects of genetic and pharmacological blockade of CypD on Alzheimer's disease mitochondrial and glycolytic bioenergetics in Alzheimer's disease-derived mitochondrial cybrid cells, an ex vivo human sporadic Alzheimer's disease mitochondrial model, and on synaptic function, inflammatory response and learning and memory in Alzheimer's disease mouse models. Inhibition of CypD by ebselen protects against sporadic Alzheimer's disease- and amyloid-β-induced mitochondrial and glycolytic perturbation, synaptic and cognitive dysfunction, together with suppressing neuroinflammation in the brain of Alzheimer's disease mouse models, which is linked to CypD-related membrane permeability transition pore formation. Thus, CypD inhibitors have the potential to slow the progression of neurodegenerative diseases, including Alzheimer's disease, by boosting mitochondrial bioenergetics and improving synaptic and cognitive function.

In silico repurposing of CNS drugs for multiple sclerosis.

Multiple sclerosis (MS) is an autoimmune neurodegenerative disease affecting numerous people worldwide. While the relapsing subtypes of MS are to some extent treatable, the disease remains incurable leading to progressive disability. Limited efficacy of current small molecule drugs necessitates development of efficient and safe MS medications. Accordingly, drug repurposing is an invaluable strategy that recognizes new targets for known drugs especially in the field of poorly addressed therapeutic areas. Drug discovery largely depends on the identification of potential binding molecules to the intended biomolecular target(s). In this regard, current study was devoted to in silico repurposing of 263 small molecule CNS drugs to achieve superior binders to some MS-related targets. On the basis of molecular docking scores, thioxanthene and benzisothiazole-based antipsychotics could be identified as potential binders to sphingosine-1-phosphate lyase (S1PL) and cyclophilin D (CypD). Tightest interaction modes were observed for zuclopenthixol-S1PL (ΔGb -7.96kcal/mol) and lurasidone-CypD (ΔGb -8.84kcal/mol) complexes. Molecular dynamics (MD) simulations proved the appropriate and stable accommodation of top-ranked drugs inside enzyme binding sites during 100ns. Hydroxyethyl piperazine of zuclopenthixol and benzisothiazole of lurasidone flipped inside the binding pocket to interact with adjacent polar and apolar residues. Solvent accessible surface area (SASA) fluctuations confirmed the results of binding trajectory analysis and showed that non-polar hydrophobic interactions played significant roles in acquired stabilities. Our results on lurasidone binding pattern were interestingly in accordance with previous reports on X-ray structures of other norbornane maleimide derivatives as CypD inhibitors. According to this, Asn144, Phe102 and Phe155 served as important residues in providing stable binding pose of lurasidone through both exo and endo conformations. Although experimental results are necessary to be achieved, the outcomes of this study proposed the potentiality of some thioxanthene and benzisothiazole-based antipsychotics for binding to S1PL and CypD, respectively, as MS-related targets.

CypD-mediated mitochondrial dysfunction contributes to titanium ion-induced MC3T3-E1 cell injury

Titanium (Ti) ion can stimulate osteoblast apoptosis and therefore have a high potential to play a negative role in the aseptic loosening of implants. Mitochondrial abnormalities are closely related to osteoblast dysfunction. However, the mitochondrial molecular mechanism of Ti ion induced osteoblastic cell apoptosis is still unclear. This study investigated in vitro mitochondrial oxidative stress (mtROS) mediated mitochondrial dysfunction involved in Ti ion-induced apoptosis of murine MC3T3-E1 osteoblastic cells. In addition to reducing mitochondrial membrane potential (MMP) and decreasing adenosine triglyceride production, exposure to Ti ions increased mitochondrial oxidative stress. Moreover, mitochondrial abnormalities significantly contributed to Ti ion induction of osteoblastic cellular apoptosis. A mitochondria-specific antioxidant, mitoquinone (MitoQ), alleviated Ti ion-induced mitochondrial dysfunction and apoptosis in osteoblastic cells, indicating that Ti ion mainly induces mitochondrial oxidative stress to produce a cytotoxic effect on osteoblasts. Here we show that the primary regulator of mitochondrial permeability transition pore (mPTP), cyclophilin D (CypD), is involved in mitochondrial dysfunction and osteoblast cell apoptosis induced by Ti ion. Overexpression of CypD exacerbates osteoblast apoptosis and impairs osteogenic function. Moreover, detrimental effects of CypD were rescued by cyclosporin A (CsA), an inhibitor of CypD, which shows its protective effect on mitochondrial and osteogenic osteoblast functions. Based on new insights into the mitochondrial mechanisms underlying Ti ion-induced apoptosis of osteoblastic cells, the findings of this study lay the foundation for the clinical use of CypD inhibitors to prevent or treat implant failure.

Regulation of NETosis and Inflammation by Cyclophilin D in Myeloperoxidase-Positive Antineutrophil Cytoplasmic Antibody-Associated Vasculitis.

Antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is pathologically characterized by focal fibrinoid necrosis, in which ANCA-mediated neutrophil extracellular trap (NET) formation and subsequent endothelial cell necrosis occur. Cyclophilin D (CypD) plays an important role in mediation of cell necrosis and inflammation via the opening of mitochondrial permeability transition pores. This study was undertaken to examine the role of CypD in AAV pathogenesis. We assessed the role and mechanism of CypD in ANCA-stimulated neutrophils in vitro by immunostaining and electron microscopy observation. We performed a comprehensive RNA-sequencing analysis on ANCA-treated murine neutrophils. To investigate the role of CypD in vivo, we assessed disease features in CypD-knockout mice and wild-type mice using 2 different murine AAV models: anti-myeloperoxidase IgG transfer-induced AAV and spontaneous AAV. In vitro experiments showed that pharmacologic and genetic inhibition of CypD suppressed ANCA-induced NET formation via the suppression of reactive oxygen species and cytochrome c release from the mitochondria. RNA-sequencing analyses in ANCA-treated murine neutrophils revealed the involvement of inflammatory responses, with CypD deficiency reducing ANCA-induced alterations in gene expression. Furthermore, analyses of upstream regulators revealed the relevance of intracellular calcium (CypD activator) and cyclosporin (CypD inhibitor) in ANCA stimulation, indicating that the CypD-dependent opening of mitochondrial permeability transition pores is associated with ANCA-induced neutrophil activation and NETosis. In both AAV mouse models, the genetic deletion of CypD ameliorated crescentic glomerulonephritis via the inhibition of CypD-dependent neutrophil and endothelial necrosis. CypD targeting is a novel and specific therapeutic strategy for AAV via the resolution of necrotizing vasculitis.

Small-molecule inhibitors of cyclophilin D as potential therapeutics in mitochondria-related diseases.

Cyclophilin D (CypD) is a key regulator of mitochondrial permeability transition pore (mPTP) opening. This pathophysiological phenomenon is associated with the development of several human diseases, including ischemia-reperfusion injury and neurodegeneration. Blocking mPTP opening through CypD inhibition could be a novel and promising therapeutic approach for these conditions. While numerous CypD inhibitors have been discovered to date, none have been introduced into clinical practice, mostly owing to their high toxicity, unfavorable pharmacokinetics, and low selectivity for CypD over other cyclophilins. This review summarizes current knowledge of CypD inhibitors, with a particular focus on small-molecule compounds with regard to their in vitro activity, their selectivity for CypD, and their binding mode within the enzyme's active site. Finally, approaches for improving the molecular design of CypD inhibitors are discussed.

DEVELOPMENT OF NEW CYCLOPHILIN D RECEPTOR INHIBITORS FOR THE TREATMENT OF MULTIPLE SCLEROSIS

Objective: In this study, it was aimed to carry out computational studies for the development of new molecules for the inhibition of the cyclophilin D (CypD) receptor, which causes the disability of mitochondrial function in multiple sclerosis (MS) disease. Material and Method: Pharmacophore modeling study was applied via the PharmaGist Web server to the CypD inhibitors detected by the literature search. According to the best pharmacophore models from PharmaGist, 80 molecules were obtained from the ZINCPharmer database, and in silico ADME/Toxicology analysis was applied to these molecules. Then, molecular docking was performed with the ligands that gave the best results as a result of ADME/Tox analyses with the Autodock Vina program. Result and Discussion: ZINC00390492 molecule shows the best binding affinity and binding profile with both CypD, Sphingosine-1-phosphate receptor 1. It has been demonstrated that this molecule may be a lead molecule for the treatment of MS.

Abstract P455: Acetylated Cyclophilin D Regulates Mitochondrial Electron Transport Chain Activity In The Developing Heart

Background: Mitochondria generate energy in form of ATP in mature eukaryotic cells. The components of the electron transport chain (ETC) are expressed at embryonic (E) day 9.5 in mouse hearts, but ETC activity and oxidative phosphorylation (OXPHOS) are uncoupled, because the permeability transition pore (PTP) is open. In mouse hearts the mPTP closes around E11.5. Opening and closing of the PTP is regulated by cyclophilin D (CypD), but the regulatory mechanism are not understood. Hypothesis: Maturation of ETC activity is regulated by acetylated CypD. Methods: Cardiac tissue homogenates or isolated heart mitochondria from mice, ranging in age from E 9.5 to adult, were used to measure oxygen consumption and the calcium retention capacity. The expression of proteins of the ETC and their assembly into supercomplexes was followed by denaturing and native electrophoresis. The expression of CypD and its acetylation status were assessed by western blotting and densitometry. The enzymatic activity of the ETC complexes and CypD was measured using spectrophotometry. Results: In the heart of mouse embryos at E 9.5 mitochondrial ETC activity and OXPHOS are not coupled (respiratory control ratio (RCR) 1.48 ± 0.17, n=9). Addition of 1 μM cyclosporin A, an inhibitor of CypD and the PTP acutely increases the RCR to 3.69 ± 0.59 (n=5). At E13.5, the end of the embryonic period in the mouse, OXPHOS is coupled and not significantly different from the adult heart. The enzymatic activity of the ETC complexes I, II, III and V increases significantly from E9.5 to the adult heart and the assembly of mitochondrial supercomplexes begins at about E13.5. The total expression of CypD increases as the mouse heart matures, but the enzymatic activity of CypD decreases. In addition, the ratio of acetylated CypD to the total expressed CypD decreased from 1.1 ± 0.16 (n=3) at E9.5 to 0.54 ± 0.06 (n=3) in the adult heart. Conclusion: The activity of acetylated CypD regulates maturation of mitochondrial ETC activity in the developing mouse heart.

Identification of potent CypD inhibitors via pharmacophore based virtual screening, docking and molecular dynamics simulation

Cyclophilin D (CypD) is a mitochondrial matrix peptidyl prolidase that has been identified as a target for the treatment of acute pancreatitis. However, only a few compounds have been reported as CypD inhibitors. To discover novel CypD inhibitors, a ZINC database including 206,724 compounds was screened through 3D-QSAR pharmacophore model and molecular docking in sequence. After an in-depth analysis of the docking results obtained, ZINC161011368, ZINC664707610 and ZINC665010466 were selected for the molecular dynamics simulations and the binding free energy calculations. On this basis, ZINC161011368 was identified as having the potential of application as a novel CypD inhibitor. This study provides a basis for screening highly active CypD inhibitors for the treatment of acute pancreatitis.

Platelet Cyclophilin D-Dependent Events Limit Venous Thrombotic Occlusion and Platelet Accretion

The role of platelets in arterial thrombosis is well established. Recent findings have reinforced their importance in venous thrombosis as well. Which of the platelet's many functions mediate its effects in the venous setting remains unclear, and how procoagulant platelet formation regulates venous thrombosis has not been investigated. Cyclophilin D (CypD) is a mitochondrial peptidylprolylisomerase that has the key function of regulation of mitochondrial permeability transition pore (mPTP) formation. In the absence of CypD, mPTP and procoagulant platelet formation are markedly impaired in response to physiologic agonists. The functional consequence of CypD's absence in vivo has been examined in photochemical injury models in photochemical injury models of both the carotid and mesenteric arteries. In mice with platelet CypD-deficiency, in both of these high shear settings, thrombotic vasoocclusion was accelerated implicating procoagulant platelet fragility and loss of adhesiveness as a critical factor limiting thrombus growth. Whether CypD deficiency would similarly impact thrombus growth in a venous setting has not been previously examined.To investigate the role of CypD and procoagulant platelets in the low shear setting of venous thrombosis, we utilized the inferior vena cava (IVC) stenosis ligature murine model of deep vein thrombosis (DVT). This model was chosen as it utilizes mechanical narrowing to induce thrombosis in the setting of venous flow. 24 hours following IVC ligation, thrombi were significantly larger (1.83 fold) in CypD-deficient mice than wild type. (p<0.05). Similarly, in mice with platelet-specific CypD-deficiency, thrombi were 1.54 fold larger than those found in WT mice (p<0.05). To begin to investigate the mechanism by which CypD-deficiency resulted in increased thrombus size, the composition of the venous thrombi was analyzed. Western blots for markers of platelets (CD41), neutrophils (Ly6G), and fibrin (59D8) revealed that their relative levels in thrombi were unchanged, but indicated that red blood cell (spectrin α1) accumulation increased. Histologic appearance in both CypD-deficient and wild-type thrombi was similar with a consistent pattern observed. A white cap area dominated the thrombus closest to the site of ligation, while red thrombus was more common in the remaining thrombus area. To investigate whether the observed pattern is due to more rapid occlusion, ultrasound was utilized to investigate the time course of venous vasoocclusion following ligature placement. Preliminary ultrasound studies suggest that CypD-deficient mice develop vasoocclusion more rapidly, perhaps thus accounting for their larger size.Previous studies have delineated the role of CypD-dependent events in limiting platelet accretion in vitro in arterial, but not venous, flow conditions. In vitro studies utilizing whole blood from CypD-deficient mice showed increased accretion of platelets on collagen under venous flow conditions. Similarly, platelet accretion increased when human whole blood was treated with cyclosporin A, an inhibitor of CypD, and flowed over collagen at venous rates. Interestingly, washed platelets from CypD-deficient mice also showed increased accumulation on a von Willebrand factor (VWF) - coated surface under venous flow conditions, a condition in which neither collagen nor coagulation-mediated procoagulant platelet formation would be expected to occur. Thus, these studies have identified an important role for CypD-dependent platelet events in the regulation of platelet accumulation in vitro and thus slowing the early stages of thrombus growth in vivo . The results here, in venous settings, and previously in arterial settings are consistent with a limiting role for procoagulant platelet formation in thrombus growth. This regulatory feedback role should be taken into consideration prior to modulation of procoagulant platelet formation in the setting of thrombosis or inflammation. DisclosuresNo relevant conflicts of interest to declare.

Aceylated Cyclophilin D Regulates Mitochondrial Function in the Developing Mouse Heart

Mitochondria provide energy in form of ATP in mature eukaryotic cells. However, it is not clear, when and how during the embryonic cardiac development, mitochondria become able to regulate oxidative phosphorylation (OXPHOS) and prevent opening of the permeability transition pore (mPTP). Maturation of heart mitochondria is regulated by the activity of acetylated cyclophilin D (CypD). Cardiac tissue homogenates or isolated mitochondria ranging in age from embryonic (E) day 9.5 to adult wild-type mice were used to measure oxygen consumption, the expression of proteins of the electron transport chain and their assembly into supercomplexes was followed by denaturing and native electrophoresis. The expression of CypD and its acetylation status was assessed by western blotting and densitometry. n the heart of mouse embryos at E 9.5 mitochondrial electron transport chain (ETC) activity and OXPHOS are not coupled (respiratory control ratio (RCR) 1.48 ± 0.17, n=9). Addition of 1 µM cyclosporin A, an inhibitor of CypD D and the mPTP acutely increases the RCR to 3.69 ± 0.59 n=5). At E13.5, the end of the embryonic period in the mouse, OXPHOS is coupled and not significantly different from the adult heart. The enzymatic activity of the ETC complexes I, II, III and V increases significantly from E9.5 to the adult heart and the assembly of mitochondrial supercomplexes begins at about E13.5. Similarly, the expression of CypD increases as the heart matures. However, the detection of acetylated CypD decreases and the ratio of acetylated CypD to the total expressed CypD decreases from 1.1 ± 0.16 (n=3) at E9.5 to 0.54 ± 0.06 (n=3) in the adult heart. The activity of acetylated CypD regulates maturation of mitochondrial function in the developing heart.

Genetic Approach to Elucidate the Role of Cyclophilin D in Traumatic Brain Injury Pathology.

Cyclophilin D (CypD) has been shown to play a critical role in mitochondrial permeability transition pore (mPTP) opening and the subsequent cell death cascade. Studies consistently demonstrate that mitochondrial dysfunction, including mitochondrial calcium overload and mPTP opening, is essential to the pathobiology of cell death after a traumatic brain injury (TBI). CypD inhibitors, such as cyclosporin A (CsA) or NIM811, administered following TBI, are neuroprotective and quell neurological deficits. However, some pharmacological inhibitors of CypD have multiple biological targets and, as such, do not directly implicate a role for CypD in arbitrating cell death after TBI. Here, we reviewed the current understanding of the role CypD plays in TBI pathobiology. Further, we directly assessed the role of CypD in mediating cell death following TBI by utilizing mice lacking the CypD encoding gene Ppif. Following controlled cortical impact (CCI), the genetic knockout of CypD protected acute mitochondrial bioenergetics at 6 h post-injury and reduced subacute cortical tissue and hippocampal cell loss at 18 d post-injury. The administration of CsA following experimental TBI in Ppif-/- mice improved cortical tissue sparing, highlighting the multiple cellular targets of CsA in the mitigation of TBI pathology. The loss of CypD appeared to desensitize the mitochondrial response to calcium burden induced by TBI; this maintenance of mitochondrial function underlies the observed neuroprotective effect of the CypD knockout. These studies highlight the importance of maintaining mitochondrial homeostasis after injury and validate CypD as a therapeutic target for TBI. Further, these results solidify the beneficial effects of CsA treatment following TBI.

CaMKII as a key regulator of contrast-induced nephropathy through mPTP opening in HK-2 cells

Contrast-induced nephropathy (CIN), refers to acute kidney injury observed after administration of contrast media during angiographic or other medical procedures such as urography, and accounting for 12% of all causes of acute renal failure, but no specific prevention or treatment strategy exists for its obscure pathophysiology. The aim of our study was to explore the influence of calcium/calmodulin-dependent protein kinase II (CaMKII) in CIN by using HK-2 cells. Knockdown of CypD was achieved by lentivirus, and CaMKII overexpression by transfection with the plasmid. In this study, we have demonstrated that CypD-mediated mPTP opening triggered mitochondrial dysfunction and tubule cells apoptosis in CIN. We also found that iohexol treatment was associated with mitochondrial ROS overloading, ATP depletion and LDH release. Inhibition of CypD with the pharmacologic inhibitor or knockdown of CypD abrogated mPTP opening, oxidative stress, mitochondria damage, and cell apoptosis induced by iohexol. In addition, we found that inhibition of the CaMKII activity alleviated iohexol-induced CypD expression, whereas also decreased mPTP opening, oxidative stress, mitochondria damage, and cell apoptosis, similarly to the inhibition of CypD did. Moreover, CaMKII overexpression enhanced iohexol-induced mPTP opening, mitochondrial damage and renal tubular epithelial cells apoptosis. These findings first identified the novel role of CaMKII in iohexol-induced tubular cells apoptosis and delineated the CaMKII-CypD/mPTP pathway during contrast-induced tubular cell damage. Hence, these results could provide a new strategy for CIN protection.

Abstract 331: Impact of Acetylated Cyclophilin D on the Mitochondrial Permeability Transition Pore

Background: The mitochondrial matrix protein cyclophilin D (CypD) is a key regulator of mitochondrial function. CypD controls electron transport chain activity and ATP synthesis by regulating the permeability transition pore (PTP). The activity of CypD is regulated by several post-translational modifications including acetylation of lysine 166 in the mouse. Objective: To investigate how acetylation at lysine 166 of CypD specifically in the heart modifies its ability to regulate the PTP and the ATP synthase. Results: We generated a conditional cardiac knock-in mouse model where lysine 166 has been mutated into glutamine (CypD K166Q ) to mimic permanent acetylation of CypD. The mice were either +/+, +/- or -/- for the expression of native CypD. Results show that mitochondrial oxygen consumption was not affected by the expression of CypD K166Q . The calcium retention capacity (CRC) was measured with Arsenazo III and decreased significantly when CypD K166Q was expressed. The CypD inhibitor cyclosporine A significantly increased the CRC in WT mice. However, cyclosporine A was did not inhibit CypD in the hearts of mice expressing only CypD K166Q or in addition to wild-type CypD. The ability of the ATP synthase to create dimers or oligomers was assessed by western blotting and the hydrolysis of ATP in in-gel assays and shows that expression of CypD K166Q decreased the assembly of the ATP synthase into dimers or oligomers. Conclusions: Our data show that the expression of CypD K166Q increases the sensitivity of PTP opening to calcium and limits the assembly of ATP synthase into oligomers.

P0340CYPD DEFICIENCY AMELIORATES AUTOIMMUNE VASCULITIS IN MOUSE MODEL VIA THE INHIBITION OF PROGRAMMED NECROSIS

Abstract Background and Aims Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is a necrotizing vasculitis that affects systemic organs. Although the standard immunosuppressive therapy in AAV could control disease activity, these existing treatments have no effect to regulate the necrosis, which could cause the persistent organ damage. ANCA binds to neutrophils, leading to neutrophil extracellular (NETs) formation. The formed NETs induce vascular injury and become auto-antigens against ANCA, resulting in an auto-amplification loop of necrosis and inflammation. NETs work as innate immunity and simultaneously undergo programmed cell necrosis. However, the signaling pathway of ANCA-mediated NETs remains elusive. Cyclophilin D (CypD) regulates program necrosis via the opening of mitochondrial permeability transition pores (mPTP) with the consequence of mitochondrial ROS release. The Inhibition or genetic deletion of CypD has been reportedly to protect organ damage in brain and kidney ischemia-reperfusion injury mice. Herein, we hypothesized that CypD would be involved in ANCA-mediated NETs pathway and the pathogenesis of AAV. Method Animal experiment; Autoimmune vasculitis was induced in 10 week-old CypD(+/+) or CypD deficient (-/-) mice by the intravenous injection with anti-mouse MPO serum. At 24 hours after injection, we evaluated the renal and lung involvements. The severity of glomerulonephritis was assessed by the levels of BUN, haematuria, histological findings and TUNEL positivity. The lung injury was evaluated by histopathological alveolar haemorrhage scores and TUNEL positivity. The mPTP related necrosis in tissues was identified by immunostaining against cytchrome C. The NETs-forming neutrophils in peripheral bloods were detected as Sytox Green (SG)-positive neutrophils by flow cytometry (FCM). In vitro experiment; We investigated whether CypD inhibitor and genetic ablation suppresses ANCA-mediated NETs. Healthy human neutrophils were incubated with ANCA-IgG derived from AAV patients with pre-treatment of CypD inhibitor (cyclosporine A) or PBS for 4 hours. Next, WT or CypD (-/-) mice neutrophils were cultured with anti mouse MPO antibody for 4 hours. The NETs induction was assessed as Sytox green positive neutrophil using FCM and live imaging. Results The amount of haematuria, histological severity including glomerulonephritis and pulmonary haemorrhage in CypD(-/-) mice significantly decreased compared to those in CypD(+/+) mice. Furthermore, TUNEL and cytochrome C positivity of tissues in CypD(-/-) mice was lower than that of CypD(+/+) mice, respectively. The level of BUN in AAV-induced CypD (-/-) mice was significantly lower than that of AAV-induced CypD(+/+) mice (92.4 ± 41.8 vs 36.8 ± 21.5 mg/ dL, p &amp;lt;0.05). FCM analysis showed that circulating NETs and mitochondrial ROS in CypD(-/-) mice were lower than those in CypD(+/+) mice (NETs; 4.7 ± 2.4 vs 7.5 ± 2.6 %, p &amp;lt;0.05, ROS; 1.9 ± 0.38 vs 4.7 ± 0.78%, p &amp;lt;0.05). In vitro experiments, CypD inhibitor suppressed ANCA-induced NETs formation (Control; 0.45 ± 0.14 vs CypD inhibitor; 0.16 ± 0.04%, p &amp;lt;0.05). In addition, CypD(-/-) mice neutrophils-treated with ANCA showed lower NETs induction rate compared to CypD(+/+) neutrophils (CypD+/+: 4.27 ± 2.09 vs CypD-/-: 0.63 ± 0.35%, p &amp;lt;0.05). Conclusion Our data indicate that the inhibition of CypD might ameliorate autoimmune vasculitis via the suppression of programmed NETs formation.

Inhibition of the mitochondrial permeability transition improves bone fracture repair.

Bone fracture is accompanied by trauma, mechanical stresses, and inflammation - conditions known to induce the mitochondrial permeability transition. This phenomenon occurs due to opening of the mitochondrial permeability transition pore (MPTP) promoted by cyclophilin D (CypD). MPTP opening leads to more inflammation, cell death and potentially to disruption of fracture repair. Here we performed a proof-of-concept study and tested a hypothesis that protecting mitochondria from MPTP opening via inhibition of CypD improves fracture repair. First, our in vitro experiments indicated pro-osteogenic and anti-inflammatory effects in osteoprogenitors upon CypD knock-out or pharmacological inhibition. Using a bone fracture model in mice, we observed that bone formation and biomechanical properties of repaired bones were significantly increased in CypD knock-out mice or wild type mice treated with a CypD inhibitor, NIM811, when compared to controls. These effects were evident in young male but not female mice, however in older (13month-old) female mice bone formation was also increased during fracture repair. In contrast to global CypD knock-out, mesenchymal lineage-specific (Prx1-Cre driven) CypD deletion did not result in improved fracture repair. Our findings implicate MPTP in bone fracture and suggest systemic CypD inhibition as a modality to promote fracture repair.

T-2 toxin cytotoxicity mediated by directly perturbing mitochondria in human gastric epithelium GES-1 cells.

T-2 toxin is one of the most toxic trichothecenes and harmful to human health and animal husbandry. The mechanism underlying its growth suppression remains unclear, especially for mitochondrial damage in human gastric epithelial cells. In the present study, we investigated cell death caused by T-2 toxin in a human gastric epithelial cell line (GES-1) and the possible mechanism of T-2-induced cytotoxicity. T-2 strongly reduced the viability of GES-1 cells in a time- and dose-dependent manner within a small range of concentrations. However, when the concentrations of T-2 were >40 nM, there was no concentration dependence, only time dependence. Moreover, T-2 induced apoptosis, with the activation of caspase-3 in GES-1 and mitochondrial membrane potential (MMP) decrease and cytochrome c release. T-2 also resulted in the accumulation of reactive oxygen species (ROS) and DNA damage with a positive signal of p-H2A.X in GES-1 cells. While T-2 caused a MMP decrease, DNA damage and cell death were not blocked by pretreatment with 3 mM glutathione (GSH), a typical scavenger of ROS. The induction of mitochondrial permeability transition pore (mPTP) regulators voltage-dependent anion channel (VDAC1) and cyclophilin D (CypD) were also observed in T-2-treated cells. Interestingly, cyclosporine A (CsA), a CypD inhibitor, significantly reversed the drop in MMP and the DNA damage, as well as ROS accumulation caused by T-2. Additionally, GES-1 cell death could also be protected to some extent by 4, 4'-diisothiocyanatostilbene-2, 2'-disulfonic acid (DIDS), an inhibitor of VDAC1, especially the combination of CsA and DIDS, and 3 mM GSH could further enhance the effect of CsA + DIDS on cell viability. In conclusion, our present findings indicate that the T-2 induced MMP decrease, DNA damage and cell death, as well as ROS accumulation in GES-1 cells, starts with T-2 directly perturbing the mitochondria triggering ROS generation by acting on CypD and VDAC1. This study presents a new viewpoint for evaluating the toxicity of T-2 toxin.

Delineating a role for the mitochondrial permeability transition pore in diabetic kidney disease by targeting cyclophilin D.

Mitochondrial stress has been widely observed in diabetic kidney disease (DKD). Cyclophilin D (CypD) is a functional component of the mitochondrial permeability transition pore (mPTP) which allows the exchange of ions and solutes between the mitochondrial matrix to induce mitochondrial swelling and activation of cell death pathways. CypD has been successfully targeted in other disease contexts to improve mitochondrial function and reduced pathology. Two approaches were used to elucidate the role of CypD and the mPTP in DKD. Firstly, mice with a deletion of the gene encoding CypD (Ppif-/-) were rendered diabetic with streptozotocin (STZ) and followed for 24 weeks. Secondly, Alisporivir, a CypD inhibitor was administered to the db/db mouse model (5 mg/kg/day oral gavage for 16 weeks). Ppif-/- mice were not protected against diabetes-induced albuminuria and had greater glomerulosclerosis than their WT diabetic littermates. Renal hyperfiltration was lower in diabetic Ppif-/- as compared with WT mice. Similarly, Alisporivir did not improve renal function nor pathology in db/db mice as assessed by no change in albuminuria, KIM-1 excretion and glomerulosclerosis. Db/db mice exhibited changes in mitochondrial function, including elevated respiratory control ratio (RCR), reduced mitochondrial H2O2 generation and increased proximal tubular mitochondrial volume, but these were unaffected by Alisporivir treatment. Taken together, these studies indicate that CypD has a complex role in DKD and direct targeting of this component of the mPTP will likely not improve renal outcomes.