Cold Storage Disrupts the Proteome and Phosphoproteome Landscape in Rat Kidney Transplants.

Background: Prolonged cold storage (CS) of kidneys results in poor long-term outcomes after transplantation (Tx). We reported previously that CS of rat kidneys for 18 h before transplant impaired proteasome function, disrupted protein homeostasis, and reduced graft function. The goal of the present study was to identify the renal proteins, including phosphoproteins, that are dysregulated by this CS injury.

Differentiation activates mitochondrial OPA1 processing in myoblast cell lines.

Mitochondrial optic atrophy-1 (OPA1) plays key roles in adapting mitochondrial structure to bioenergetic function. When transmembrane potential across the inner membrane (Δψ) is intact, long (L-OPA1) isoforms shape the inner membrane through membrane fusion and the formation of cristal junctions. When Δψ is lost, however, OPA1 is cleaved to short, inactive S-OPA1 isoforms by the OMA1 metalloprotease, disrupting mitochondrial structure and priming cellular stress responses such as apoptosis.

Normal Proteasome Function Is Needed to Prevent Kidney Graft Injury during Cold Storage Followed by Transplantation.

Kidney transplantation is the preferred treatment for end-stage kidney disease (ESKD). However, there is a shortage of transplantable kidneys, and donor organs can be damaged by necessary cold storage (CS). Although CS improves the viability of kidneys from deceased donors, prolonged CS negatively affects transplantation outcomes.

Does Disruption of Optic Atrophy-1 (OPA1) Contribute to Cell Death in HL-1 Cardiomyocytes Subjected to Lethal Ischemia-Reperfusion Injury?

Disruption of mitochondrial structure/function is well-recognized to be a determinant of cell death in cardiomyocytes subjected to lethal episodes of ischemia-reperfusion (IR). However, the precise mitochondrial event(s) that precipitate lethal IR injury remain incompletely resolved. Using the in vitro HL-1 cardiomyocyte model, our aims were to establish whether: (1) proteolytic processing of optic atrophy protein-1 (OPA1), the inner mitochondrial membrane protein responsible for maintaining cristae junction integrity, plays a causal, mechanistic role in determining cardiomyocyte fate in cells subjected to lethal IR injury; and (2) preservation of OPA1 may contribute to the well-documented cardioprotection achieved with ischemic preconditioning (IPC) and remote ischemic conditioning.

Non-Canonical Cannabinoid Receptors with Distinct Binding and Signaling Properties in Prostate and Other Cancer Cell Types Mediate Cell Death.

Cannabinoids exert anti-cancer actions; however, the underlying cytotoxic mechanisms and the cannabinoid receptors (CBRs) involved remain unclear. In this study, CBRs were characterized in several cancer cell lines. Radioligand binding screens surprisingly revealed specific binding only for the non-selective cannabinoid [H]WIN-55,212-2, and not [H]CP-55,940, indicating that the expressed CBRs exhibit atypical binding properties.

Overexpression of MnSOD Protects against Cold Storage-Induced Mitochondrial Injury but Not against OMA1-Dependent OPA1 Proteolytic Processing in Rat Renal Proximal Tubular Cells.

Kidneys from deceased donors undergo cold storage (CS) preservation before transplantation. Although CS is a clinical necessity for extending organ quality preservation, CS causes mitochondrial and renal injury. Specifically, many studies, including our own, have shown that the triggering event of CS-induced renal injury is mitochondrial reactive oxygen species (mROS).

Fluorescence-Based Assay For Measuring OMA1 Activity.

Mitochondrial fusion depends on proteolytic processing of the dynamin-related GTPase protein, OPA1, which is regulated by the mitochondrial zinc metalloproteinase, OMA1. Last year we published a report describing a novel approach to directly measure the enzymatic activity of OMA1 in whole cell lysates. This fluorescence-based reporter assay utilizes an eight amino acid peptide sequence referred to as the S1 cleavage site where OMA1 cleaves within OPA1 and is flanked by a fluorophore and quencher.

Association Between L-OPA1 Cleavage and Cardiac Dysfunction During Ischemia-Reperfusion Injury in Rats.

Background/aims: Structural and functional alterations in mitochondria, particularly, the inner mitochondrial membrane (IMM) plays a critical role in mitochondria-mediated cell death in response to cardiac ischemia-reperfusion (IR) injury. The integrity of IMM can be affected by two potential intra-mitochondrial factors: i) mitochondrial matrix swelling, and ii) proteolytic cleavage of the long optic atrophy type 1 (L-OPA1), an IMM-localized dynamin-like GTPase engaged in the regulation of structural organization and integrity of the mitochondrial cristae. However, the relationship between these two factors in response to oxidative stress remains unclear.

The BK activator NS11021 partially protects rat kidneys from cold storage and transplantation-induced mitochondrial and renal injury.

Kidneys from deceased donors used for transplantation are placed in cold storage (CS) solution during the search for a matched recipient. However, CS induces mitochondrial and cellular injury, which exacerbates renal graft dysfunction, highlighting the need for therapeutic interventions. Using an in vitro model of renal CS, we recently reported that pharmacological activation of the mitochondrial BK channel (mitoBK) during CS protected against CS-induced mitochondrial injury and cell death.

Specific BK Channel Activator NS11021 Protects Rat Renal Proximal Tubular Cells from Cold Storage-Induced Mitochondrial Injury In Vitro.

Kidneys from deceased donors used for transplantation are placed in cold storage (CS) solution during the search for a matched recipient. However, CS causes mitochondrial injury, which may exacerbate renal graft dysfunction. Here, we explored whether adding NS11021, an activator of the mitochondrial big-conductance calcium-activated K (mitoBK) channel, to CS solution can mitigate CS-induced mitochondrial injury.

The first direct activity assay for the mitochondrial protease OMA1.

Mitochondria continually undergo fission and fusion which allow mitochondria to rapidly change their shape, size, and function throughout the cell life cycle. OMA1, a zinc metalloproteinase enzyme, is a key regulator of the mitochondrial fusion machinery. The paucity of information regarding OMA1 regulation and function largely stems from the fact that there is no direct method to quantitatively measure its activity.

Female mice exhibit less renal mitochondrial injury but greater mortality using a comorbid model of experimental sepsis.

Given the inherent heterogeneity of the septic patient population and possible comorbid conditions, it is not surprising that the influence of gender on incidence and outcomes are still unclear. The goal of this study was to use a clinically relevant murine model of sepsis, cecal ligation and puncture (CLP) in CD1 mice, with and without uniphrectomy as a comorbid condition to investigate possible gender differences in renal mitochondrial function and dynamics. High resolution respirometry on fresh kidney biopsies was used to measure renal respiratory complex activities.

Renal cold storage followed by transplantation impairs proteasome function and mitochondrial protein homeostasis.

Identifying pathways related to renal cold storage (CS) that lead to renal damage after transplantation (Tx) will help us design novel pathway-specific therapies to improve graft outcome. Our recent report showed that mitochondrial function was compromised after CS alone, and this was exacerbated when CS was combined with Tx (CS/Tx). The goal of this study was to determine whether the proteasome exacerbates mitochondrial dysfunction after CS/Tx.

Renal cold storage followed by transplantation impairs expression of key mitochondrial fission and fusion proteins.

Background: The majority of transplanted kidneys are procured from deceased donors which all require exposure to cold storage (CS) for successful transplantation. Unfortunately, this CS leads to renal and mitochondrial damage but, specific mitochondrial targets affected by CS remain largely unknown. The goal of this study is to determine whether pathways involved with mitochondrial fusion or fission, are disrupted during renal CS.

1,3-Butadiene-induced mitochondrial dysfunction is correlated with mitochondrial CYP2E1 activity in Collaborative Cross mice.

Cytochrome P450 2E1 (CYP2E1) metabolizes low molecular weight hydrophobic compounds, including 1,3-butadiene, which is converted by CYP2E1 to electrophilic epoxide metabolites that covalently modify cellular proteins and DNA. Previous CYP2E1 studies have mainly focused on the enzyme localized in the endoplasmic reticulum (erCYP2E1); however, active CYP2E1 has also been found in mitochondria (mtCYP2E1) and the distribution of CYP2E1 between organelles can influence an individual's response to exposure. Relatively few studies have focused on the contribution of mtCYP2E1 to activation of chemical toxicants.

Peroxynitrite induced mitochondrial biogenesis following MnSOD knockdown in normal rat kidney (NRK) cells.

Superoxide is widely regarded as the primary reactive oxygen species (ROS) which initiates downstream oxidative stress. Increased oxidative stress contributes, in part, to many disease conditions such as cancer, atherosclerosis, ischemia/reperfusion, diabetes, aging, and neurodegeneration. Manganese superoxide dismutase (MnSOD) catalyzes the dismutation of superoxide into hydrogen peroxide which can then be further detoxified by other antioxidant enzymes.

Inactivation of renal mitochondrial respiratory complexes and manganese superoxide dismutase during sepsis: mitochondria-targeted antioxidant mitigates injury.

Acute kidney injury (AKI) is a complication of sepsis and leads to a high mortality rate. Human and animal studies suggest that mitochondrial dysfunction plays an important role in sepsis-induced multi-organ failure; however, the specific mitochondrial targets damaged during sepsis remain elusive. We used a clinically relevant cecal ligation and puncture (CLP) murine model of sepsis and assessed renal mitochondrial function using high-resolution respirometry, renal microcirculation using intravital microscopy, and renal function.

The use of the Cre/loxP system to study oxidative stress in tissue-specific manganese superoxide dismutase knockout models.

Significance: Respiring mitochondria are a significant site for reactions involving reactive oxygen and nitrogen species that contribute to irreversible cellular, structural, and functional damage leading to multiple pathological conditions. Manganese superoxide dismutase (MnSOD) is a critical component of the antioxidant system tasked with protecting the oxidant-sensitive mitochondrial compartment from oxidative stress. Since global knockout of MnSOD results in significant cardiac and neuronal damage leading to early postnatal lethality, this approach has limited use for studying the mechanisms of oxidant stress and the development of disease in specific tissues lacking MnSOD.

Effect of S-nitrosoglutathione on renal mitochondrial function: a new mechanism for reversible regulation of manganese superoxide dismutase activity?

Mitochondria are at the heart of all cellular processes as they provide the majority of the energy needed for various metabolic processes. Nitric oxide has been shown to have numerous roles in the regulation of mitochondrial function. Mitochondria have enormous pools of glutathione (GSH≈5-10 mM).

Role of reduced manganese superoxide dismutase in ischemia-reperfusion injury: a possible trigger for autophagy and mitochondrial biogenesis?

Excessive generation of superoxide and mitochondrial dysfunction has been described as being important events during ischemia-reperfusion (I/R) injury. Our laboratory has demonstrated that manganese superoxide dismutase (MnSOD), a major mitochondrial antioxidant that eliminates superoxide, is inactivated during renal transplantation and renal I/R and precedes development of renal failure. We hypothesized that MnSOD knockdown in the kidney augments renal damage during renal I/R.

MitoQ blunts mitochondrial and renal damage during cold preservation of porcine kidneys.

Cold preservation has greatly facilitated the use of cadaveric kidneys for transplantation but damage occurs during the preservation episode. It is well established that oxidant production increases during cold renal preservation and mitochondria are a key target for injury. Our laboratory has demonstrated that cold storage of renal cells and rat kidneys leads to increased mitochondrial superoxide levels and mitochondrial electron transport chain damage, and that addition of Mitoquinone (MitoQ) to the preservation solutions blunted this injury.

Pharmacological targets in the renal peritubular microenvironment: implications for therapy for sepsis-induced acute kidney injury.

One of the most frequent and serious complications to develop in septic patients is acute kidney injury (AKI), a disorder characterized by a rapid failure of the kidneys to adequately filter the blood, regulate ion and water balance, and generate urine. AKI greatly worsens the already poor prognosis of sepsis and increases cost of care. To date, therapies have been mostly supportive; consequently there has been little change in the mortality rates over the last decade.

Role of mitochondrial oxidants in an in vitro model of sepsis-induced renal injury.

Oxidative stress has been implicated to play a major role in multiorgan dysfunction during sepsis. To study the mechanism of oxidant generation in acute kidney injury (AKI) during sepsis, we developed an in vitro model of sepsis using primary cultures of mouse cortical tubular epithelial cells exposed to serum (2.5-10%) collected from mice at 4 h after induction of sepsis by cecal ligation and puncture (CLP) or Sham (no sepsis).

Acetaminophen-induced hepatotoxicity and protein nitration in neuronal nitric-oxide synthase knockout mice.

In overdose acetaminophen (APAP) is hepatotoxic. Toxicity occurs by metabolism to N-acetyl-p-benzoquinone imine, which depletes GSH and covalently binds to proteins followed by protein nitration. Nitration can occur via the strong oxidant and nitrating agent peroxynitrite, formed from superoxide and nitric oxide (NO).