Background Mycobacterium tuberculosis remains one of the most significant causes of death from an infectious agent. Rapid and accurate diagnosis of pulmonary and extra-pulmonary tuberculosis (TB) is still a great challenge. The GeneXpert MTB/RIF assay is a novel integrated diagnostic system for the diagnosis of tuberculosis and rapid detection of Rifampin (RIF) resistance in clinical specimens. In 2012, the Arkansas Tuberculosis Control Program introduced GeneXpert MTB/RIF assay to replace the labour-intensive Mycobacterium Tuberculosis Direct (MTD) assay.Aims To rapidly diagnose TB within two hours and to simultaneously detect RIF resistance.Objectives 1. Describe the procedure used to introduce GeneXpert MTB/RIF assay in the Arkansas Tuberculosis Control Program. 2. Characterise the current gap in rapid M. tuberculosis diagnosis in Arkansas. 3. Assess factors that predict acid fast bacilli (AFB) smearnegative but culture-positive cases in Arkansas. 4. Illustrate, with two case reports, the role of GeneXpert MTB/RIF assay in reduction of time to confirmation of M. tuberculosis diagnosis in the first year of implementation.MethodBetween June 2012 and June 2013, all AFB sputum smearpositive cases and any others, on request by the physician, had GeneXpert MTB/RIF assay performed as well as traditional M. tuberculosis culture and susceptibilities using Mycobacteria Growth Indicator Tube (MGIT) 960 and Lwenstein-Jensen (LJ) slants. Surveillance data for January 2009-June 2013 was analysed to characterise sputum smear-negative but culture-positive cases.Results Seventy-one TB cases were reported from June 2012June 2013. GeneXpert MTB/RIF assay identified all culture-positive cases as well as three cases that were negative on culture. Also, this rapid assay identified all six smear-negative but M. tuberculosis culture-positive cases; two of these cases are described as case reports.Conclusion GeneXpert MTB/RIF assay has made rapid TB diagnosis possible, with tremendous potential in determining isolation of TB suspects on one hand, and quickly ruling out TB whenever suspected.

Tuberculosis (TB) is one of the leading causes of morbidity and mortality in the world. Its classic forms include cavitary and miliary TB. The non-specific clinical presentation of TB and similarity of its signs and symptoms with other pulmonary diseases makes its diagnosis difficult, especially in low burden settings like Arkansas. We emphasize the importance of early diagnosis and describe two cases where a delay resulted in complications and a prolonged treatment plan for the patients.

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). Nitric oxide can react with glutathione to generate a physiological molecule, S-nitrosoglutathione (GSNO). The impact GSNO has on mitochondrial function has been intensively studied in recent years, and several mitochondrial electron transport chain complex proteins have been shown to be targeted by GSNO. In this study we investigated the effect of GSNO on mitochondrial function using normal rat proximal tubular kidney cells (NRK cells). GSNO treatment of NRK cells led to mitochondrial membrane depolarization and significant reduction in activities of mitochondrial complex IV and manganese superoxide dismutase enzyme (MnSOD). MnSOD is a critical endogenous antioxidant enzyme that scavenges excess superoxide radicals in the mitochondria. The decrease in MnSOD activity was not associated with a reduction in its protein levels and treatment of NRK cell lysate with dithiothreitol (a strong sulfhydryl-group-reducing agent) restored MnSOD activity to control values. GSNO is known to cause both S-nitrosylation and S-glutathionylation, which involve the addition of NO and GS groups, respectively, to protein sulfhydryl (SH) groups of cysteine residues. Endogenous GSH is an essential mediator in S-glutathionylation of cellular proteins, and the current studies revealed that GSH is required for MnSOD inactivation after GSNO or diamide treatment in rat kidney cells as well as in isolated kidneys. Further studies showed that GSNO led to glutathionylation of MnSOD; however, glutathionylated recombinant MnSOD was not inactivated. This suggests that a more complex pathway, possibly involving the participation of multiple proteins, leads to MnSOD inactivation after GSNO treatment. The major highlight of these studies is the fact that dithiothreitol can restore MnSOD activity after GSNO treatment. To our knowledge, this is the first study showing that MnSOD activity can be reversibly regulated in vivo, through a mechanism involving thiol residues.

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.

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.

In overdose the analgesic/antipyretic acetaminophen (APAP) is hepatotoxic. Toxicity is mediated by hepatic metabolism to a reactive metabolite (NAPQI). Following low doses NAPQI is detoxified by glutathione (GSH); however, in overdose GSH is depleted, NAPQI covalently binds to proteins, and oxygen/nitrogen stress occurs. Toxicity is believed to occur by mitochondrial dysfunction. MnSOD inactivation occurs during many oxidant stress mediated diseases; thus, we examined MnSOD in APAP toxicity. MnSOD is a critical mitochondrial antioxidant enzyme in preventing oxygen/nitrogen stress within the mitochondria. Mice were treated with 300 mg/kg APAP. GSH was significantly reduced by 65% at 0.5 h and remained reduced from 1–4 h returning to control levels by 6 h. Serum ALT (hepatotoxicity) did not significantly increase until 4 h and was 2290 IU/L at 6 h. MnSOD activity was significantly reduced by 25% at 0.5 h and was 50% at 1 h and 2 h. In a dose‐response study performed at 1 h, GSH was significantly depleted by 62% and 80% with nonhepatotoxic doses of 50 and 100 mg/kg APAP, respectively. No further GSH depletion occurred with 200 and 300 mg/kg APAP. MnSOD activity was reduced by 28, 32 and 57% with 50, 100 and 300 mg/kg APAP respectively. Thus, MnSOD activity is dramatically decreased following toxic and nontoxic doses of APAP, and MnSOD inactivation precedes induction of hepatic toxicity. Supported by NIH grant 1 R01 DK079008.

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.

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 CS as well as during the reperfusion (or rewarming/CS.RW) phase has been suggested to be a major contributor to renal injury, although the source of and/or biochemical pathways involved in 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 after exposure of NRK cells to CS.RW. MnSOD overexpression or inhibition of nitric oxide synthase 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.

Inactivation of manganese superoxide dismutase (MnSOD), the major mitochondrial antioxidant, is a key issue in renal failure following transplantation or ischemia/reperfusion (IR) injury. Homozygous MnSOD knock out (KO) mice die shortly after birth, and heterozygous MnSOD KO mice have decreased MnSOD activity in all tissues/organs. To address the role MnSOD inactivation has on renal injury, we generated bi‐transgenic kidney specific MnSOD KO (50% and 100%) mice by breeding KidneyCre mice1 and floxed MnSOD mice2. Characterization of these KOs revealed that phenotypically the mice are smaller in size compared to littermate controls, but show no survival differences. Immunohistochemistry data showed that the Cre‐recombinase mediated MnSOD deletion occurred specifically in the kidney and was located predominantly in the medullary region. The kidney specific KOs exhibited histologic evidence of mild renal damage, increased tyrosine nitration, and renal cell death. KO mice renal function was not altered; however, more renal damage was observed following IR injury compared to littermate controls. In summary, we have developed a novel kidney specific MnSOD KO mouse model that will help define the molecular pathways that occur downstream of MnSOD inactivation during renal injury.Supported by NIH grant 1RO1DK0789361.

Cold preservation is a beneficial and necessary procedure used to preserve deceased donor kidneys prior to transplantation. However, extended preservation periods result in injury that can lead to poor graft function following transplantation. Thus, this study's purpose was to evaluate the early effects of cold storage (CS) alone and to determine if MitoQ, a mitochondrial targeted antioxidant, could offer protection during this stage. We hypothesize that CS increases oxidant production and mitochondrial dysfunction, and that adding MitoQ to cold preservation solutions will attenuate injury. Renal cells and rat kidneys were exposed to CS (UW‐Viaspan solution; 4°C) with or without MitoQ (1μM; 100μM respectively) and monitored for oxidants (O2·−, NO, ONOO−), mitochondrial function (respiratory complexes, mitochondrial membrane potential), and cell viability. Our cell model results suggest that CS first causes mitochondrial membrane depolarization, followed by oxidant production, then mitochondrial dysfunction, and modest cell death. MitoQ reduced oxidant production and protected against mitochondrial complex inactivation using both cellular and isolated rat kidney models. Excitingly, we have identified a potential therapeutic reagent that could be added to existing preservation solutions to reduce CS damage and possibly improve renal function following transplantation.

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.

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.