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1.  Role of mitochondrial-derived oxidants in renal tubular cell cold storage injury 
Free radical biology & medicine  2010;49(8):1273-1282.
Cold storage (CS) is regarded as a necessary procedure during donation of a deceased donor kidney that helps to optimize organ viability. Increased oxidant generation during both CS as well as during the reperfusion (or rewarming/CS.RW) phase have been suggested to be a major contributor to renal injury; although the source and/or biochemical pathways involved with oxidant production remain unclear. The purpose of this study was to determine if renal tubular mitochondrial superoxide is capable of inducing oxidant production and mitochondrial damage in response to a CS.RW insult. To test the role of mitochondrial superoxide in CS.RW injury, we used rat renal proximal tubular (NRK) cells overexpressing manganese superoxide dismutase (MnSOD), the major mitochondrial antioxidant. Oxidant production, mitochondrial membrane potential, respiratory complex function, and cell death were all altered following exposure of NRK cells to CS.RW. MnSOD overexpression or inhibition of nitric oxide synthase (NOS) provided significant protection against oxidant generation, respiratory complex inactivation, and cell death. These findings implicate mitochondrial superoxide, nitric oxide, and their reaction product, peroxynitrite, as key signaling molecules involved in CS.RW injury of renal tubular cells, and suggest that therapeutic inhibition of these pathways may protect the donor kidney.
PMCID: PMC3688469  PMID: 20659553
Cold preservation; cold storage; superoxide; nitric oxide; peroxynitrite; mitochondria; respiratory complexes
2.  Generation and characterization of a novel kidney-specific manganese superoxide dismutase knockout mouse 
Free radical biology & medicine  2011;51(2):406-416.
Inactivation of manganese superoxide dismutase (MnSOD), a mitochondrial antioxidant, has been associated with renal disorders and often results in detrimental downstream events that are mechanistically not clear. Development of an animal model that exhibits kidney-specific deficiency of MnSOD would be extremely beneficial in exploring the downstream events that occur following MnSOD inactivation. Using Cre-Lox recombination technology, kidney-specific MnSOD deficient mice (both 100% and 50%) were generated that exhibited low expression of MnSOD in discrete renal cell types and reduced enzymatic activity within the kidney. These kidney-specific 100% KO mice possessed a normal life-span, although it was interesting that the mice were smaller. Consistent with the important role in scavenging superoxide radicals, the kidney-specific KO mice showed a significant increase in oxidative stress (tyrosine nitration) in a gene-dose dependent manner. In addition, loss of MnSOD resulted in mild renal damage (tubular dilation and cell swelling). Hence, this novel mouse model will aid in determining the specific role (local and/or systemic) governed by MnSOD within certain kidney cells. Moreover, these mice will serve as a powerful tool to explore molecular mechanisms that occur downstream of MnSOD inactivation in renal disorders or possibly in other pathologies that rely on normal renal function.
PMCID: PMC3118857  PMID: 21571061
Cre-Lox technology; Kidney; MnSOD; Cre recombinase; Superoxide; Nitrotyrosine
3.  Peroxynitrite induced mitochondrial biogenesis following MnSOD knockdown in normal rat kidney (NRK) cells☆ 
Redox Biology  2014;2:348-357.
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. MnSOD is critical in maintaining the normal function of mitochondria, thus its inactivation is thought to lead to compromised mitochondria. Previously, our laboratory observed increased mitochondrial biogenesis in a novel kidney-specific MnSOD knockout mouse. The current study used transient siRNA mediated MnSOD knockdown of normal rat kidney (NRK) cells as the in vitro model, and confirmed functional mitochondrial biogenesis evidenced by increased PGC1α expression, mitochondrial DNA copy numbers and integrity, electron transport chain protein CORE II, mitochondrial mass, oxygen consumption rate, and overall ATP production. Further mechanistic studies using mitoquinone (MitoQ), a mitochondria-targeted antioxidant and L-NAME, a nitric oxide synthase (NOS) inhibitor demonstrated that peroxynitrite (at low micromolar levels) induced mitochondrial biogenesis. These findings provide the first evidence that low levels of peroxynitrite can initiate a protective signaling cascade involving mitochondrial biogenesis which may help to restore mitochondrial function following transient MnSOD inactivation.
Graphical abstract
•MnSOD knockdown in NRK cells results in a transient loss of MnSOD activity, increased nitrotyrosine and mitochondrial superoxide.•MnSOD knockdown in NRK cells results in a transient induction of mitochondrial biogenesis.•Nitric oxide synthase inhibition and Mitoquinone blocks mitochondrial biogenesis after MnSOD knockdown.•Low doses of peroxynitrite induce biogenesis in NRK cells.
PMCID: PMC3926114  PMID: 24563852
MnSOD; Peroxynitrite; siRNA; mtDNA; Respiration; Mitochondrial biogenesis
4.  Pharmacological targets in the renal peritubular microenvironment: implications for therapy for sepsis-induced acute kidney injury 
Pharmacology & therapeutics  2012;134(2):139-155.
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. This is due, at least in part, to the delay in establishing clinical evidence of an infection and the associated presence of the systemic inflammatory response syndrome and thus, a delay in initiating therapy. A second reason is a lack of understanding regarding the mechanisms leading to renal injury, which has hindered the development of more targeted therapies. In this review, we summarize recent studies, which have examined the development of renal injury during sepsis and propose how changes in the peritubular capillary microenvironment lead to and then perpetuate microcirculatory failure and tubular epithelial cell injury. We also discuss a number of potential therapeutic targets in the renal peritubular microenvironment, which may prevent or lessen injury and/or promote recovery.
PMCID: PMC3319265  PMID: 22274552
sepsis; acute kidney injury; microcirculation; oxidative stress; peritubular capillary; tubular epithelium
5.  MitoQ Blunts Mitochondrial and Renal Damage during Cold Preservation of Porcine Kidneys 
PLoS ONE  2012;7(11):e48590.
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. In order to better translate animal studies, the inclusion of large animal models is necessary to develop safe preclinical protocols. Therefore, we tested the hypothesis that addition of MitoQ to cold storage solution preserves mitochondrial function by decreasing oxidative stress, leading to less renal tubular damage during cold preservation of porcine kidneys employing a standard criteria donor model. Results showed that cold storage significantly induced oxidative stress (nitrotyrosine), renal tubular damage, and cell death. Using High Resolution Respirometry and fresh porcine kidney biopsies to assess mitochondrial function we showed that MitoQ significantly improved complex II/III respiration of the electron transport chain following 24 hours of cold storage. In addition, MitoQ blunted oxidative stress, renal tubular damage, and cell death after 48 hours. These results suggested that MitoQ decreased oxidative stress, tubular damage and cell death by improving mitochondrial function during cold storage. Therefore this compound should be considered as an integral part of organ preservation solution prior to transplantation.
PMCID: PMC3490900  PMID: 23139796
6.  Reactive Nitrogen Species in Acetaminophen-Induced Mitochondrial Damage and Toxicity in Mouse Hepatocytes 
Chemical Research in Toxicology  2010;23(7):1286-1292.
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) (DCFH2). Since DCFH2 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 (2–5h) 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.
PMCID: PMC3269780  PMID: 20578685
7.  Alteration of renal respiratory Complex-III during experimental type-1 diabetes 
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).
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.
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.
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.
PMCID: PMC2636815  PMID: 19166612
8.  Manganese Porphyrin Reduces Renal Injury and Mitochondrial Damage during Ischemia/Reperfusion ± 
Free radical biology & medicine  2007;42(10):1571-1578.
Renal ischemia/reperfusion (I/R) injury often occurs as a result of vascular surgery, organ procurement, or transplantation. We previously showed that renal I/R results in ATP depletion, oxidant production, and manganese superoxide dismutase (MnSOD) inactivation. There have been several reports that overexpression of MnSOD protects tissues/organs from I/R related damage, thus a loss of MnSOD activity during I/R likely contributes to tissue injury. The present study examined the therapeutic benefit of a catalytic antioxidant Mn(III) meso-tetrakis(N-hexylpyridinium-2-yl)porphyrin, (MnTnHex-2-PyP5+) using the rat renal I/R model. This was the first study to examine the effects of MnTnHex-2-PyP5+ in an animal model of oxidative stress injury. Our results showed that porphyrin pretreatment of rats for 24 hr protected against ATP depletion, MnSOD inactivation, nitrotyrosine formation, and renal dysfunction. The dose (50 μg/kg) used in this study is lower than doses of various types of antioxidants commonly used in animal models of oxidative stress injuries. In addition, using novel proteomic techniques, we identified ATP synthase- beta subunit as a key protein induced by MnTnHex-2-PyP5+ treatment alone, and complex V (ATP synthase) as a target of injury during renal I/R. These results showed that MnTnHex-2-PyP5+ protected against renal I/R injury via induction of key mitochondrial proteins that may be capable of blunting oxidative injury.
PMCID: PMC1924492  PMID: 17448904
kidney; ischemia/reperfusion; metalloporphyrin; proteomics; MnSOD; mitochondria; oxidants; nitrotyrosine; blue native polyacrylamide gel electrophoresis BN-PAGE; two dimensional fluorescence differential in gel electrophoresis (2D-DIGE)

Results 1-8 (8)