Nirmala Parajuli

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.

Lapatinib and doxorubicin enhance the Stat1‐dependent antitumor immune response

The dual erbB1/2 tyrosine kinase inhibitor lapatinib as well as the anthracycline doxorubicin are both used in the therapy of HER2‐positive breast cancer. Using MMTV‐neu mice as an animal model for HER2‐positive breast cancer, we observed enhanced tumor infiltration by IFN‐γ‐secreting T cells after treatment with doxorubicin and/or lapatinib. Antibody depletion experiments revealed a contribution of CD8+ but not CD4+ T cells to the antitumor effect of these drugs. Doxorubicin treatment additionally decreased the content of immunosuppressive tumor‐associated macrophages (TAMs) in the tumor bed. In contrast, Stat1‐deficient mice were resistant to tumor growth inhibition by lapatinib and/or doxorubicin and exhibited impaired T‐cell activation and reduced T‐cell infiltration of the tumor in response to drug treatment. Furthermore, Stat1‐deficiency resulted in reduced expression of the T‐cell chemotactic factors CXCL9, CXCL10, and CXCL11 in the tumor epithelium. The inhibition of TAM infiltration of the tumor by doxorubicin and the immunosuppressive function of TAMs were found to be Stat1 independent. Taken together, the results point to an important contribution toward enhancing T‐cell and IFN‐γ‐based immunity by lapatinib as well as doxorubicin and emphasize the role of Stat1 in building an effective antitumor immune response.

Interaction and functional interference of glucocorticoid receptor and SOCS1.

Cytokine and glucocorticoid (GC) hormone signaling act in an integrated fashion to control inflammation and immune response. Here we establish a new mode of interaction of these two pathways and propose Suppressor of Cytokine Signaling (SOCS)-1 as an essential player in mediating cross-talk. We observed that glucocorticoid receptor (GR) and SOCS1 form an intracellular complex through an interaction, which required the SH2 domain of SOCS1 and the ligand binding domain of GR. Furthermore, GC stimulation was found to increase the nuclear level of SOCS1. SOCS1 binding to the GR did not require ligand binding of the receptor; however, it was abolished after long term GC stimulation, suggesting a functional role of the interaction for the early phase of GC action. The interaction between GR and SOCS1 appeared to negatively influence the transcription of the two GR-regulated genes, FKBP5 and MKP1, because the GC-dependent expression of these genes was inhibited by the SOCS1 inducer IFNγ and enhanced in SOCS1-deficient murine embryonic fibroblasts as compared with IFNγ treated wild-type cells. Our results suggest a prominent role of SOCS1 in the early phase of cross-talk between GR and cytokine signaling.

Generation and characterization of a novel kidney-specific manganese superoxide dismutase knockout mouse

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.

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-derived oxidants in renal tubular cell cold-storage injury

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.

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. 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.

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. To circumvent this problem, a number of investigators have employed the Cre/loxP system to precisely knockout MnSOD in individual tissues. RECENT ADVANCES Multiple tissue and organ-specific Cre-expressing mice have been generated, which greatly enhance the specificity of MnSOD knockout in tissues and organ systems that were once difficult, if not impossible to study. CRITICAL ISSUES Evaluating the contribution of MnSOD deficiency to oxidant-mediated mitochondrial damage requires careful consideration of the promoter system used for creating the tissue-specific knockout animal, in addition to the collection and interpretation of multiple indices of oxidative stress and damage. FUTURE DIRECTIONS Expanded use of well-characterized tissue-specific promoter elements and inducible systems to drive the Cre/loxP recombinational events will lead to a spectrum of MnSOD tissue knockout models, and a clearer understanding of the role of MnSOD in preventing mitochondrial dysfunction in human disease.

Replenishment of the B cell compartment after doxorubicin-induced hematopoietic toxicity is facilitated by STAT1

STAT1 serves as an important regulator in the response to pathogens, oncogenic transformation, and genotoxic insults. It exerts these effects by shaping the innate and adaptive immune response and by participating in genotoxic stress pathways, leading to apoptosis and inhibition of cell proliferation. We have investigated the role of STAT1 in hematopoietic toxicity induced by doxorubicin in STAT1‐proficient and ‐deficient mice. Whereas the early genotoxic effect of doxorubicin did not depend on STAT1, expression of STAT1 was required for efficient B lymphocyte repopulation in the recovery phase. We found a lower abundance of lymphocyte precursors in the BM of STAT1‐deficient animals, which was particularly evident after doxorubicin‐induced hematopoietic toxicity. In accordance, colony‐forming assays with STAT1‐deficient BM cells revealed a decreased number of pre‐B colonies. Differentiation from the pro‐B to the pre‐B stage was not affected, as demonstrated by unaltered differentiation of purified B cell precursors from BM in the presence of IL‐7. With the exception of Sca‐1, expression of genes implicated in early lymphocyte development in pro‐B cells did not depend on STAT1. Our findings indicate a specific requirement for STAT1 in lymphoid development before differentiation to pre‐B cells, which becomes particularly apparent in the recovery phase from doxorubicin‐induced hematopoietic toxicity.

Infiltrating CD11b+CD11c+ cells have the potential to mediate inducible nitric oxide synthase‐dependent cell death in mammary carcinomas of HER‐2/neu transgenic mice

The development of autochtonous mammary tumors in HER‐2/neu transgenic mice is facilitated by immune tolerance to the neu‐transgene. However, appropriate vaccination strategies can initiate immune system‐mediated antitumor response by a process that requires IFN‐γ. We investigated the role of inducible nitric oxide synthase (iNOS) induction by IFN‐γ to promote tumor cell apoptosis. Tumors from FVBN202 mice expressing the normal neu gene under the control of the MMTV‐LTR were treated in slice cultures with IFN‐γ for up to 24 hr. Apoptosis was induced, which depended on iNOS enzymatic activity. iNOS expression was predominantly found in infiltrating CD11b+CD11c+ myeloid cells and at much lower levels in the tumor epithelium. By contrast, IFN‐γ treatment of explant cultures of tumor epithelial cells was not sufficient to efficiently induce iNOS, emphasizing an important role of the integrity of tumor tissue architecture, which was preserved in the slice cultures. This notion was further supported by the upregulation of iNOS costimulatory cytokines TNF‐α and IL‐1β in slice cultures but not in explants and the capability of purified CD11b+CD11c+ cells to enhance iNOS expression of tumor cells in cocultures. The findings suggest that tumor‐infiltrating myeloid cells in immuno‐tolerant HER‐2/neu transgenic mice possess tumor killing ability via induction of iNOS and underline the capacity of antitumor strategies designed to stimulate infiltrating myeloid cells.