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. Previously, we reported that renal proteasome function decreased after rat kidneys underwent CS followed by transplantation (CS + Tx). Here, we investigated the mechanism underlying proteasome dysfunction and the role of the proteasome in kidney graft outcome using a rat model of CS + Tx. We found that the key proteasome subunits ß5, α3, and Rpt6 are modified, and proteasome assembly is impaired. Specifically, we detected the modification and aggregation of Rpt6 after CS + Tx, and Rpt6 modification was reversed when renal extracts were treated with protein phosphatases. CS + Tx kidneys also displayed increased levels of nitrotyrosine, an indicator of peroxynitrite (a reactive oxygen species, ROS), compared to sham. Because the Rpt6 subunit appeared to aggregate, we investigated the effect of CS + Tx-mediated ROS (peroxynitrite) generation on renal proteasome assembly and function. We treated NRK cells with exogenous peroxynitrite and evaluated PAC1 (proteasome assembly chaperone), Rpt6, and ß5. Peroxynitrite induced a dose-dependent decrease in PAC1 and ß5, but Rpt6 was not affected (protein level or modification). Finally, serum creatinine increased when we inhibited the proteasome in transplanted donor rat kidneys (without CS), recapitulating the effects of CS + Tx. These findings underscore the effects of CS + Tx on renal proteasome subunit dysregulation and also highlight the significance of proteasome activity in maintaining graft function following CS + Tx.

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 [3H]WIN-55,212-2, and not [3H]CP-55,940, indicating that the expressed CBRs exhibit atypical binding properties. Furthermore, [3H]WIN-55,212-2 bound to a single site in all cancer cells with high affinity and varying densities. CBR characteristics were next compared between human prostate cancer cell lines expressing low (PC-3) and high (DU-145) CBR density. Although mRNA for canonical CBRs was detected in both cell lines, only 5 out of 15 compounds with known high affinity for canonical CBRs displaced [3H]WIN-55,212-2 binding. Functional assays further established that CBRs in prostate cancer cells exhibit distinct signaling properties relative to canonical Gi/Go-coupled CBRs. Prostate cancer cells chronically exposed to both CBR agonists and antagonists/inverse agonists produced receptor downregulation, inconsistent with actions at canonical CBRs. Treatment of DU-145 cells with CBR ligands increased LDH-release, decreased ATP-dependent cell viability, and produced mitochondrial membrane potential depolarization. In summary, several cancer cell lines express CBRs with binding and signaling profiles dissimilar to canonical CBRs. Drugs selectively targeting these atypical CBRs might exhibit improved anti-cancer properties.

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. Here, we elucidated the effects of cardiac IR injury on L-OPA1 cleavage and OMA1 activity. METHODS: Langendorff-mode perfused isolated rat hearts were subjected to 25-min of global ischemia followed by 90-min reperfusion in the presence or absence of XJB-5-131 (XJB, a mitochondria-targeting ROS scavenger) and sanglifehrin A (SfA, a permeability transition pore inhibitor). RESULTS: XJB in combination with SfA increased post-ischemic recovery of cardiac function and reduced mitochondrial ROS production at 30- and 60-min reperfusion and affected mitochondrial swelling. L-OPA1 levels were reduced in IR hearts; however, neither XJB, SfA, and their combination prevented IR-induced reduction of L-OPA1 cleavage. Likewise, IR increased the OMA1 enzymatic activity, which remained unchanged in the presence of XJB and/or SfA. CONCLUSION: IR-induced cardiac and mitochondrial dysfunctions are associated with OMA1 activation and L-OPA1 cleavage. However, XJB, SfA, and their combination do not prevent these changes despite improved heart and mitochondria function, thus, suggesting that different mechanisms can be implicated in L-OPA1 processing in response to cardiac IR injury.

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. Here, we used an in vivo syngeneic rat model of renal CS (18 h) followed by transplantation (24 h reperfusion) (CS + Tx) to similarly evaluate whether addition of a mitoBK activator to the CS solution can alleviate CS + Tx-induced renal injury. Western blots detected the pore-forming α subunit of the BK channel in mitochondrial fractions from rat kidneys, and mitoBK protein level was reduced after CS + Tx compared to sham surgery. The addition of the BK activator NS11021 (3 µM) to the CS solution partially protected against CS + Tx-induced mitochondrial respiratory dysfunction, oxidative protein nitration, and cell death, but not acute renal dysfunction (SCr and BUN). In summary, the current preclinical study shows that pharmacologically targeting mitoBK channels during CS may be a promising therapeutic intervention to prevent CS + Tx-induced mitochondrial and renal injury.

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. We exposed the kidneys of male Lewis rats (in vivo) and rat renal proximal tubular (NRK) cells (in vitro) to CS/Tx or rewarming (CS/RW), respectively. To compare CS-induced effects, in vivo kidney Tx without CS exposure (autotransplantation; ATx) was also used. Our study provides the first evidence that the chymotrypsin-like (ChT-L) peptidase activity of the proteasome declined only after CS/Tx or CS/RW, but not after CS or ATx. Interestingly, key mitochondrial proteins involved with respiration [succinate dehydrogenase complex, subunit A (SDHA), a complex II subunit, and ATP5B, an ATP synthase/complex V subunit] were detected in the detergent-insoluble fraction after CS/Tx or CS/RW, with compromised complex V activity. Pharmacological inhibition of ChT-L activity in NRK cells decreased the activity of mitochondrial complexes I, II, and V and also increased the levels of SDHA and ATP5B in the insoluble fraction. On the other hand, inhibiting mitochondrial respiration in NRK cells with antimycin A compromised ChT-L function and increased the amounts of SDHA and ATP5B in the insoluble fraction. Our results suggest that mitochondrial respiratory dysfunction during CS precedes compromised ChT-L function after CS/Tx and proteasome dysfunction further alters mitochondrial protein homeostasis and decreases respiration in the kidneys after CS/Tx. Therefore, therapeutics that preserve mitochondrial and proteasome function during CS may provide beneficial outcomes following transplantation.

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. CLP caused a time-dependent decrease in mitochondrial complex I and II/III respiration and reduced ATP. By 4 h after CLP, activity of manganese superoxide dismutase (MnSOD) was decreased by 50% and inhibition was sustained through 36 h. These events were associated with increased mitochondrial superoxide generation. We then evaluated whether the mitochondria-targeted antioxidant Mito-TEMPO could reverse renal mitochondrial dysfunction and attenuate sepsis-induced AKI. Mito-TEMPO (10 mg/kg) given at 6 h post-CLP decreased mitochondrial superoxide levels, protected complex I and II/III respiration, and restored MnSOD activity by 18 h. Mito-TEMPO also improved renal microcirculation and glomerular filtration rate. Importantly, even delayed therapy with a single dose of Mito-TEMPO significantly increased 96-h survival rate from 40% in untreated septic mice to 80%. Thus, sepsis causes sustained inactivation of three mitochondrial targets that can lead to increased mitochondrial superoxide. Importantly, even delayed therapy with Mito-TEMPO alleviated kidney injury, suggesting that it may be a promising approach to treat septic AKI.

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. Using newly characterized kidney-specific MnSOD knockout (KO) mice the extent of renal damage and oxidant production after I/R was evaluated. These KO mice (without I/R) exhibited low expression and activity of MnSOD in the distal nephrons, had altered renal morphology, increased oxidant production, but surprisingly showed no alteration in renal function. After I/R the MnSOD KO mice showed similar levels of injury to the distal nephrons when compared with wild-type mice. Moreover, renal function, MnSOD activity, and tubular cell death were not significantly altered between the two genotypes after I/R. Interestingly, MnSOD KO alone increased autophagosome formation, mitochondrial biogenesis, and DNA replication/repair within the distal nephrons. These findings suggest that the chronic oxidative stress as a result of MnSOD knockdown induced multiple coordinated cell survival signals including autophagy and mitochondrial biogenesis, which protected the kidney against the acute oxidative stress following I/R.

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). CLP serum produced a concentration-dependent increase in nitric oxide (NO) (nitrate + nitrite) release at 6 h and cytotoxicity (lactate dehydrogenase release) at 18 h compared with Sham serum treatment. Before cytotoxicity there was a decrease in mitochondrial membrane potential, which was followed by increased superoxide and peroxynitrite levels compared with Sham serum. The role of oxidants was evaluated by using the superoxide dismutase mimetic and peroxynitrite scavenger manganese(III)tetrakis(1-methyl-4-pyridyl)porphyrin tetratosylate hydroxide (MnTmPyP). MnTmPyP (10-100 µM) produced a concentration-dependent preservation of ATP and protection against cytotoxicity. MnTmPyP blocked mitochondrial superoxide and peroxynitrite generation produced by CLP serum but had no effect on NO levels. Although MnTmPyP did not block the initial CLP serum-induced fall in mitochondrial membrane potential, it allowed mitochondrial membrane potential to recover. Data from this in vitro model suggest a time-dependent generation of mitochondrial oxidants, mitochondrial dysfunction, and renal tubular epithelial cell injury and support the therapeutic potential of manganese porphyrin compounds in preventing sepsis-induced AKI.

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). In hepatocyte suspensions we reported that an inhibitor of neuronal nitric-oxide synthase (nNOS; NOS1), which has been reported to be in mitochondria, inhibited toxicity and protein nitration. We recently showed that manganese superoxide dismutase (MnSOD; SOD2) was nitrated and inactivated in APAP-treated mice. To understand the role of nNOS in APAP toxicity and MnSOD nitration, nNOS knockout (KO) and wild-type (WT) mice were administered APAP (300 mg/kg). In WT mice serum alanine aminotransferase (ALT) significantly increased at 6 and 8 h, and serum aspartate aminotransferase (AST) significantly increased at 4, 6 and 8 h; however, in KO mice neither ALT nor AST significantly increased until 8 h. There were no significant differences in hepatic GSH depletion, APAP protein binding, hydroxynonenal covalent binding, or histopathological assessment of toxicity. The activity of hepatic MnSOD was significantly lower at 1 to 2 h in WT mice and subsequently increased at 8 h. MnSOD activity was not altered at 0 to 6 h in KO mice but was significantly decreased at 8 h. There were significant increases in MnSOD nitration at 1 to 8 h in WT mice and 6 to 8 h in KO mice. Significantly more nitration occurred at 1 to 6 h in WT than in KO mice. MnSOD was the only observed nitrated protein after APAP treatment. These data indicate a role for nNOS with inactivation of MnSOD and ALT release during APAP toxicity.

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. We report that HL-1 cells subjected to 2.5 h of simulated ischemia displayed increased activity of OMA1 (the metalloprotease responsible for proteolytic processing of OPA1) during the initial 45 min following reoxygenation. This was accompanied by processing of mitochondrial OPA1 (i.e., cleavage to yield short-OPA1 peptides) and release of short-OPA1 into the cytosol. However, siRNA-mediated knockdown of OPA1 content did not exacerbate lethal IR injury, and did not attenuate the cardioprotection seen with IPC and a remote preconditioning stimulus, achieved by transfer of 'reperfusate' medium (TRM-IPC) in this cell culture model. Taken together, our results do not support the concept that maintenance of OPA1 integrity plays a mechanistic role in determining cell fate in the HL-1 cardiomyocyte model of lethal IR injury, or that preservation of OPA1 underlies the cardioprotection seen with ischemic conditioning.

The mitochondria-targeted antioxidant mitoquinone protects against cold storage injury of renal tubular cells and rat kidneys.

The majority of kidneys used for transplantation are obtained from deceased donors. These kidneys must undergo cold preservation/storage before transplantation to preserve tissue quality and allow time for recipient selection and transport. However, cold storage (CS) can result in tissue injury, kidney discardment, or long-term renal dysfunction after transplantation. We have previously determined mitochondrial superoxide and other downstream oxidants to be important signaling molecules that contribute to CS plus rewarming (RW) injury of rat renal proximal tubular cells. Thus, this study's purpose was to determine whether adding mitoquinone (MitoQ), a mitochondria-targeted antioxidant, to University of Wisconsin (UW) preservation solution could offer protection against CS injury. CS was initiated by placing renal cells or isolated rat kidneys in UW solution alone (4 h at 4°C) or UW solution containing MitoQ or its control compound, decyltriphenylphosphonium bromide (DecylTPP) (1 µM in vitro; 100 µM ex vivo). Oxidant production, mitochondrial function, cell viability, and alterations in renal morphology were assessed after CS exposure. CS induced a 2- to 3-fold increase in mitochondrial superoxide generation and tyrosine nitration, partial inactivation of mitochondrial complexes, and a significant increase in cell death and/or renal damage. MitoQ treatment decreased oxidant production ~2-fold, completely prevented mitochondrial dysfunction, and significantly improved cell viability and/or renal morphology, whereas DecylTPP treatment did not offer any protection. These findings implicate that MitoQ could potentially be of therapeutic use for reducing organ preservation damage and kidney discardment and/or possibly improving renal function after transplantation.

Acetaminophen-induced hepatotoxicity in mice occurs with inhibition of activity and nitration of mitochondrial manganese superoxide dismutase.

In overdose the analgesic/antipyretic acetaminophen (APAP) is hepatotoxic. Toxicity is mediated by initial hepatic metabolism to N-acetyl-p-benzoquinone imine (NAPQI). After low doses NAPQI is efficiently detoxified by GSH. However, in overdose GSH is depleted, NAPQI covalently binds to proteins as APAP adducts, and oxygen/nitrogen stress occurs. Toxicity is believed to occur by mitochondrial dysfunction. Manganese superoxide dismutase (MnSOD) inactivation by protein nitration has been reported to occur during other oxidant stress-mediated diseases. MnSOD is a critical mitochondrial antioxidant enzyme that prevents peroxynitrite formation within the mitochondria. To examine the role of MnSOD in APAP toxicity, mice were treated with 300 mg/kg APAP. GSH was significantly reduced by 65% at 0.5 h and remained reduced from 1 to 4 h. Serum alanine aminotransferase did not significantly increase until 4 h and was 2290 IU/liter at 6 h. MnSOD activity was significantly reduced by 50% at 1 and 2 h. At 1 h, GSH was significantly depleted by 62 and 80% at nontoxic doses of 50 and 100 mg/kg, respectively. No further GSH depletion occurred with hepatotoxic doses of 200 and 300 mg/kg APAP. A dose response decrease in MnSOD activity was observed for APAP at 100, 200, and 300 mg/kg. Immunoprecipitation of MnSOD from livers of APAP-treated mice followed by Western blot analysis revealed nitrated MnSOD. APAP-MnSOD adducts were not detected. Treatment of recombinant MnSOD with NAPQI did not produce APAP protein adducts. The data indicate that MnSOD inactivation by nitration is an early event in APAP-induced hepatic toxicity.

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). Here, we explored the role of OMA1-depedent OPA1 proteolytic processing in rat kidney proximal tubular epithelial (NRK) cells in an in vitro model of renal CS (18 h), followed by rewarming (6 h) (CS + RW). The involvement of mROS was evaluated by stably overexpressing manganese superoxide dismutase (MnSOD), an essential mitochondrial antioxidant enzyme, in NRK cells. Western blots detected rapid OPA1 proteolytic processing and a decrease in ATP-dependent cell viability in NRK cells subjected to CS + RW compared to control cells. Small interfering RNA (siRNA) knockdown of OMA1 reduced proteolytic processing of OPA1, suggesting that OMA1 is responsible for OPA1 proteolytic processing during CS + RW-induced renal injury. Overexpression of MnSOD during CS + RW reduced cell death, mitochondrial respiratory dysfunction, and ATP-dependent cell viability, but it did not prevent OMA1-dependent OPA1 processing. These data show for the first time that OMA1 is responsible for proteolytically cleaving OPA1 in a redox-independent manner during renal cell CS.

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. In this chapter, we provide additional insight into the OMA1 activity assay.

Reactive nitrogen species in acetaminophen-induced mitochondrial damage and toxicity in mouse hepatocytes.

Acetaminophen (APAP) toxicity in primary mouse hepatocytes occurs in two phases. The initial phase (0-2 h) occurs with metabolism to N-acetyl-p-benzoquinoneimine which depletes glutathione, and covalently binds to proteins, but little toxicity is observed. Subsequent washing of hepatocytes to remove APAP and reincubating in media alone (2-5 h) results in toxicity. We previously reported that the reincubation phase occurs with mitochondrial permeability transition (MPT) and increased oxidative stress (dichlorodihydrofluorescein fluorescence) (DCFH(2)). Since DCFH(2) may be oxidized by multiple oxidative mechanisms, we investigated the role of reactive nitrogen species (RNS) leading to 3-nitrotyrosine in proteins by ELISA and by immunoblots. Incubation of APAP with hepatocytes for 2 h did not result in toxicity or protein nitration; however, washing hepatocytes and reincubating in media alone (2-5 h) resulted in protein nitration which correlated with toxicity. Inclusion of the MPT inhibitor, cyclosporine A, in the reincubation media eliminated toxicity and protein nitration. The general nitric oxide synthase (NOS) inhibitor L-NMMA and the neuronal NOS (NOS1) inhibitor, 7-nitroindazole, added in the reincubation media decreased toxicity and protein nitration; however, neither the inducible NOS (NOS2) inhibitors L-NIL (N6-(1-iminoethyl)-L-lysine) nor SAIT (S-(2-aminoethyl)isothiourea) decreased protein nitration or toxicity. The RNS scavengers, N-acetylcysteine, and high concentrations of APAP, added in the reincubation phase decreased toxicity and protein nitration. 7-Nitroindazole and cyclosporine A inhibited the APAP-induced loss of mitochondrial membrane potential when added in the reincubation phase. The data indicate a role for RNS in APAP induced toxicity.

Delineating liver events in trichloroethylene-induced autoimmune hepatitis.

Exposure to the environmental pollutant trichloroethylene (TCE) has been linked to autoimmune disease development in humans. Chronic (32-week) low-level exposure to TCE has been shown to promote autoimmune hepatitis in association with CD4(+) T cell activation in autoimmune-prone MRL+/+ mice. MRL+/+ mice are usually thought of as a model of systemic lupus rather than an organ-specific disease such as autoimmune hepatitis. Consequently, the present study examined gene expression and metabolites to delineate the liver events that skewed the autoimmune response toward that organ in TCE-treated mice. Female MRL+/+ mice were treated with 0.5 mg/mL TCE in their drinking water. The results showed that TCE-induced autoimmune hepatitis could be detected in as little as 26 weeks. TCE exposure also generated a time-dependent increase in the number of antibodies specific for liver proteins. The gene expression correlated with the metabolite analysis to show that TCE upregulated the methionine/homocysteine pathway in the liver after 26 weeks of exposure. The results also showed that TCE exposure altered the expression of selective hepatic genes associated with immunity and inflammation. On the basis of these results, future mechanistic studies will focus on how alterations in genes associated with immunity and inflammation, in conjunction with protein alterations in the liver, promote liver immunogenicity in TCE-treated MRL+/+ mice.

Alteration of renal respiratory Complex-III during experimental type-1 diabetes.

BACKGROUND: Diabetes has become the single most common cause for end-stage renal disease in the United States. It has been established that mitochondrial damage occurs during diabetes; however, little is known about what initiates mitochondrial injury and oxidant production during the early stages of diabetes. Inactivation of mitochondrial respiratory complexes or alteration of their critical subunits can lead to generation of mitochondrial oxidants, mitochondrial damage, and organ injury. Thus, one goal of this study was to determine the status of mitochondrial respiratory complexes in the rat kidney during the early stages of diabetes (5-weeks post streptozotocin injection). METHODS: Mitochondrial complex activity assays, blue native gel electrophoresis (BN-PAGE), Complex III immunoprecipitation, and an ATP assay were performed to examine the effects of diabetes on the status of respiratory complexes and energy levels in renal mitochondria. Creatinine clearance and urine albumin excretion were measured to assess the status of renal function in our model. RESULTS: Interestingly, of all four respiratory complexes only cytochrome c reductase (Complex-III) activity was significantly decreased, whereas two Complex III subunits, Core 2 protein and Rieske protein, were up regulated in the diabetic renal mitochondria. The BN-PAGE data suggested that Complex III failed to assemble correctly, which could also explain the compensatory upregulation of specific Complex III subunits. In addition, the renal F0F1-ATPase activity and ATP levels were increased during diabetes. CONCLUSION: In summary, these findings show for the first time that early (and selective) inactivation of Complex-III may contribute to the mitochondrial oxidant production which occurs in the early stages of diabetes.

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. We used normal rat kidney proximal tubular epithelial (NRK) cells as an in vitro model of renal cold storage (18 h) and rewarming (2 h) (CS + RW). Western blots detected the pore-forming α subunit of the BK channel in mitochondrial fractions from NRK cells. The fluorescent K+-binding probe, PBFI-AM, revealed that isolated mitochondria from NRK cells exhibited mitoBK-mediated K+ uptake, which was impaired ~70% in NRK cells subjected to CS + RW compared to control NRK cells maintained at 37 °C. Importantly, the addition of 1 M NS11021 to CS solution prevented CS + RW-induced impairment of mitoBK-mediated K+ uptake. The NS11021-treated NRK cells also exhibited less cell death and mitochondrial injury after CS + RW, including mitigated mitochondrial respiratory dysfunction, depolarization, and superoxide production. In summary, these new data show for the first time that mitoBK channels may represent a therapeutic target to prevent renal CS-induced 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. Using a fluorescence-based reporter assay, we developed a sensitive method to measure OMA1 enzymatic activity in whole cell lysates.

Cold preservation mediated renal injury: involvement of mitochondrial oxidative stress.

Cold preservation has greatly facilitated the use of cadaveric kidneys for renal transplantation, but, clearly, damage occurs during both the preservation episode and the reperfusion phase (following transplantation). The aims of this study were twofold: to develop an in vivo model that was capable of evaluating renal function at early time points following cold preservation, and to evaluate the extent of renal mitochondrial damage that occurs following short periods of cold preservation in vivo. To accomplish these goals, we developed a novel rat model of in vivo renal cold ischemia followed by warm reperfusion (cold I/R) which avoided the complexity involved with transplantation. Briefly, after a right nephrectomy, cold I/R was initiated via pulsatile perfusion (40 minutes) of the left kidney with a cold University of Wisconsin solution followed by 18 hours of warm reperfusion. Cold I/R resulted in significant renal injury, nitrotyrosine production, and inactivation of the key mitochondrial antioxidant enzyme, manganese superoxide dismutase. Furthermore, the activities of the mitochondrial respiratory complexes were significantly reduced following cold I/R. In conclusion, short-term cold I/R results in inactivation of MnSOD, which may lead to the inhibition of mitochondrial complexes and subsequent renal injury. These data suggest that compounds designed to prevent early mitochondrial injury in kidneys that undergo cold preservation would significantly improve renal function and graft survival following transplantation.