MELATONIN ATTENUATES RENAL ISCHEMIA-REPERFUSION INJURY BY REGULATING MITOCHONDRIAL DYNAMICS AND AUTOPHAGY THROUGH AMPK/DRP1.

ABSTRACT: Ischemia-reperfusion injury (IRI) often stems from an imbalance between mitochondrial dynamics and autophagy. Melatonin mitigates IRI by regulating mitochondrial dynamics. However, the precise molecular mechanism underlying the role of melatonin in reducing IRI through modulating mitochondrial dynamics remains elusive. The objective of this study was to investigate whether pretreatment with melatonin before IRI confers protective effects by modulating mitochondrial dynamics and mitophagy. Melatonin pretreatment was administered to HK-2 cells and live rats before subjecting them to hypoxia-reoxygenation or IRI, respectively. Cells and rat kidney models were evaluated for markers of oxidative stress, autophagy, mitochondrial dynamics, and the expression of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and phospho-AMPKα (P-AMPK). After renal IRI, increased mitochondrial fission and autophagy were observed, accompanied by exacerbated cellular oxidative stress injury and aggravated mitochondrial dysfunction. Nevertheless, melatonin pretreatment inhibited mitochondrial fission, promoted mitochondrial fusion, and attenuated autophagy levels. This intervention was correlated with a notable reduction in oxidative stress injury and remarkable restoration of mitochondrial functionality. Ischemia-reperfusion injury led to a decline in P-AMPK levels, whereas melatonin pretreatment increased the level of P-AMPK levels. Silencing AMPK with small interfering RNA exacerbated mitochondrial damage, and in this context, melatonin pretreatment did not alleviate mitochondrial fission or autophagy levels but resulted in sustained oxidative stress damage. Collectively, these findings indicate that melatonin pretreatment shields the kidneys from IRI by mitigating excessive mitochondrial fission, moderating autophagy levels, and preserving appropriate mitochondrial fission, all in an AMPK-dependent manner.

Nuclear matrix protein 22 in bladder cancer.

Bladder cancer (BC) is ranked as the ninth most common malignancy worldwide, with approximately 570,000 new cases reported annually and over 200,000 deaths. Cystoscopy remains the gold standard for the diagnosis of BC, however, its invasiveness, cost, and discomfort have driven the demand for the development of non-invasive, cost-effective alternatives. Nuclear matrix protein 22 (NMP22) is a promising non-invasive diagnostic tool, having received FDA approval. Traditional methods for detecting NMP22 require a laboratory environment equipped with specialized equipment and trained personnel, thus, the development of NMP22 detection devices holds substantial potential for application. In this review, we evaluate the NMP22 sensors developed over the past decade, including electrochemical, colorimetric, and fluorescence biosensors. These sensors have enhanced detection sensitivity and overcome the limitations of existing diagnostic methods. However, many emerging devices exhibit deficiencies that limit their potential clinical use, therefore, we propose how sensor design can be optimized to enhance the likelihood of clinical translation and discuss the future applications of NMP22 as a legacy biomarker, providing insights for the design of new sensors.

Proteomic analysis of mitochondria associated membranes in renal ischemic reperfusion injury.

BACKGROUND: The mitochondria and endoplasmic reticulum (ER) communicate via contact sites known as mitochondria associated membranes (MAMs). Many important cellular functions such as bioenergetics, mitophagy, apoptosis, and calcium signaling are regulated by MAMs, which are thought to be closely related to ischemic reperfusion injury (IRI). However, there exists a gap in systematic proteomic research addressing the relationship between these cellular processes. METHODS: A 4D label free mass spectrometry-based proteomic analysis of mitochondria associated membranes (MAMs) from the human renal proximal tubular epithelial cell line (HK-2 cells) was conducted under both normal (N) and hypoxia/reperfusion (HR) conditions. Subsequent differential proteins analysis aimed to characterize disease-relevant signaling molecules. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was applied to total proteins and differentially expressed proteins, encompassing Biological Process (BP), Cell Component (CC), Molecular Function (MF), and KEGG pathways. Further, Protein-Protein Interaction Network (PPI) exploration was carried out, leading to the identification of hub genes from differentially expressed proteins. Notably, Mitofusion 2 (MFN2) and BCL2/Adenovirus E1B 19-kDa interacting protein 3(BNIP3) were identified and subsequently validated both in vitro and in vivo. Finally, the impact of MFN2 on MAMs during hypoxia/reoxygenation was explored through regulation of gene expression. Subsequently, a comparative proteomics analysis was conducted between OE-MFN2 and normal HK-2 cells, providing further insights into the underlying mechanisms. RESULTS: A total of 4489 proteins were identified, with 3531 successfully quantified. GO/KEGG analysis revealed that MAM proteins were primarily associated with mitochondrial function and energy metabolism. Differential analysis between the two groups showed that 688 proteins in HR HK-2 cells exhibited significant changes in expression level with P-value < 0.05 and HR/N > 1.5 or HR/N < 0.66 set as the threshold criteria. Enrichment analysis of differentially expressed proteins unveiled biological processes such as mRNA splicing, apoptosis regulation, and cell division, while molecular functions were predominantly associated with energy metabolic activity. These proteins play key roles in the cellular responses during HR, offering insights into the IRI mechanisms and potential therapeutic targets. The validation of hub genes MFN2 and BNIP3 both in vitro and vivo was consistent with the proteomic findings. MFN2 demonstrated a protective role in maintaining the integrity of mitochondria associated membranes (MAMs) and mitigating mitochondrial damage following hypoxia/reoxygenation injury, this protective effect may be associated with the activation of the PI3K/AKT pathway. CONCLUSIONS: The proteins located in mitochondria associated membranes (MAMs) are implicated in crucial roles during renal ischemic reperfusion injury (IRI), with MFN2 playing a pivotal regulatory role in this context.

Dual oxidase 2 bidirectional promoter polymorphisms confer differential immune responses in airway epithelia.

The dual oxidase enzymes, DUOX, localized to the respiratory tract epithelium, are important components of innate host defense against bacteria and virus. However, little is known regarding the regulation of DUOX transcription. To better understand DUOX2-mediated mechanisms of antiviral host defense in the airway epithelium, we designed a bidirectional promoter luciferase reporter system to identify important cis-regulatory regions in the human DUOX2/DUOXA2 promoter. In this report, we demonstrate that the genomic region between the translation start sites of DUOX2 and DUOXA2 functions as a bidirectional promoter in human airway tissue. We also identified key regulatory regions on the DUOX2/DUOXA2 promoter that were necessary for both bidirectional and unidirectional transcriptional activity. Importantly, we discovered two functionally important single-nucleotide polymorphisms (SNPs) within the promoter that differentially regulated DUOX2/DUOXA2 transcription in response to exogenous double-stranded DNA. One of these SNPs, rs269855 (enriched in people of African descent), conferred the highest level of DUOX2 promoter activity. The clinical sequelae for individuals who carry this polymorphism remain to be determined.

All-trans retinoic acid mediates DUOX2 expression and function in respiratory tract epithelium.

DUOX1 and DUOX2 are members of the NADPH oxidase family that are specifically regulated to produce hydrogen peroxide in epithelia of the thyroid, gastrointestinal tract, and respiratory tract. The determinants of DUOX1 or DUOX2 expression in various tissues have not been established. Using respiratory tract epithelial cells as a model, we investigated changes in DUOX mRNA and protein expression during the first 10 days of differentiation. By comparing a respiratory tract cell line, HBE1, with primary tracheobronchial epithelial (TBE) cells, we determined that DUOX2 was significantly expressed only in cell conditions that included all-trans retinoic acid (ATRA). In HBE1 cells, DUOX2 mRNA increased 6-fold after ATRA treatment. Similarly, ATRA induced a 19-fold increase in DUOX2 mRNA expression in primary TBE cells with parallel increases in DUOX protein and DUOX-mediated H(2)O(2) production as well. In addition, DUOX2 induction by rhinovirus required the presence of ATRA. ATRA had no effect on DUOX1 expression for all the conditions studied. Our data indicate that for respiratory epithelial cells, ATRA is important in the regulation of DUOX2 expression, function, and rhinovirus-mediated DUOX2 inducibility.

IFNgamma mediates DUOX2 expression via a STAT-independent signaling pathway.

The biological roles of the dual oxidases, DUOX1 and DUOX2, are dependent upon the tissue in which they are expressed. However, the mechanisms that control DUOX expression in these tissues are largely unexplored. Given the known role of DUOX for host defense in the gut and respiratory tract, we characterized potential mechanisms that control DUOX2 expression in response to interferon gamma (IFNgamma) in respiratory tract epithelium. We discovered that IFNgamma-mediated DUOX2 expression was regulated by a STAT-independent, JAK-independent pathway. These data provide insights into a novel IFNgamma signaling pathway with potential importance for regulation of host defense responses.

Duox2 exhibits potent heme peroxidase activity in human respiratory tract epithelium.

The dual oxidase isozymes Duox1 and Duox2 exhibit functional NADPH:O(2) oxidoreductase activity in thyroid and respiratory tract cells and are thought to be essential for H(2)O(2) generation in these tissues. However, it is not universally accepted that the heme peroxidase domains of the Duox isozymes are functional. To address this question, we modulated Duox2 expression in human tracheobronchial epithelial (TBE) cell culture systems and quantified peroxidase activity. We discovered that interferon-gamma (IFN-gamma) induced robust peroxidase activity in TBE cells that paralleled Duox2 expression. IFN-gamma-induced peroxidase activity was abolished in the presence of sodium azide, which implicated the activation of a heme peroxidase. IFN-gamma-induced peroxidase activity was abolished in TBE cell lines expressing anti-Duox2 short hairpin RNA transcripts. Together, these data unequivocally demonstrated that Duox2 contains a functional heme peroxidase in intact respiratory tract epithelium.

Differential regulation of dual NADPH oxidases/peroxidases, Duox1 and Duox2, by Th1 and Th2 cytokines in respiratory tract epithelium.

Partially reduced metabolites of molecular oxygen, superoxide (O2-) and hydrogen peroxide (H2O2), are detected in respiratory tract lining fluid, and it is assumed that these are key components of innate immunity. Whether these reactive oxygen species (ROS) are produced specifically by the respiratory epithelium in response to infection, or are a non-specific by-product of oxidant-producing inflammatory cells is not well characterized. Increasing evidence supports the hypothesis that the dual function NAD(P)H oxidases/peroxidases, Duox1 and Duox2, are important sources of regulated H2O2 production in respiratory tract epithelium. However, no studies to date have characterized the regulation of Duox gene expression. Accordingly, we examined Duox1 and Duox2 mRNA expression by real-time PCR in primary respiratory tract epithelial cultures after treatment with multiple cytokines. Herein, we determined that Duox1 expression was increased several-fold by treatment with the Th2 cytokines IL-4 and IL-13, whereas Duox2 expression was highly induced following treatment with the Th1 cytokine IFN-gamma. Duox2 expression was also elevated by polyinosine-polycytidylic acid (poly(I:C)) and rhinovirus infection. Diphenyleneiodonium (DPI)-inhibitable apical H2O2 production was similarly increased by the addition of Th1 or Th2 cytokines. These results demonstrate for the first time the regulation of Duox expression by immunomodulatory Th1 and Th2 cytokines, and suggest a mechanism by which ROS production can be regulated in the respiratory tract as part of the host defense response.

A reappraisal of the genomic organization of human Nox1 and its splice variants.

The recent discovery of non-phagocytic NAD(P)H oxidases belonging to the Nox family of enzymes sharing extensive homology to the leukocyte NAD(P)H oxidase has revolutionized our understanding of oxidative signaling related to fundamental biological processes and disease states. One form of this enzyme, Nox1, is a growth factor-responsive enzyme that catalyzes formation of the reactive oxygen species superoxide (O(2)(-)) and hydrogen peroxide (H(2)O(2)). Its expression is linked to a number of biological responses including cellular proliferation, angiogenesis, and activation of cellular signaling pathways. Whereas early published studies have described three distinct isoforms of Nox1, the current body of literature fails to adequately recognize this notion. Also, functional differences between isoforms remain relatively unexplored. Herein, we report that expression of human Nox1 is restricted to two distinct isoforms derived from a single gene; that is, the full-length gene product and a shorter spliced variant which lacks one of the NAD(P)H binding domains. We have developed PCR primer sets that distinguish between the two forms of Nox1 in several human cell lines. We could not find evidence for expression of the shortest reported form of Nox1 (NOH-1S), previously identified as a proton channel, and the absence of paired splice sites in the gene suggests that it represents a reverse transcriptase artifact. A survey of the scientific literature reveals that the majority of studies related to Nox1 do not utilize molecular strategies that would adequately discern between the two Nox1 variants. The current literature suggest the two identified isoforms of human Nox1 (which we have named Nox1-L and Nox1-S) may be functionally distinct. Future studies related to Nox1 will benefit from establishing the identity of the Nox1 isoform expressed and the functions attributed to each variant.

Identification of a novel MAGE D2 antisense RNA transcript in human tissues.

Using cDNA microarray analysis, we identified a cDNA clone, DD74, from primary human bronchial epithelial cells, which exhibits increased expression in vitro after treatment with all-trans retinoic acid. This clone corresponded to MAGE D2 mRNA, a gene previously identified to be upregulated in several cancer tissues. Surprisingly, in situ hybridization of lung tissue demonstrated positive hybridization signals with sense, but not antisense, MAGE D2-specific cRNA probes. Examination of several cell lines by Northern blot hybridization confirmed significant expression of two RNA bands. With strand-specific riboprobes, we identified a 2.0kb RNA transcript with the antisense probe as expected and identified a 4.1kb transcript by the sense probe. Further sequence analysis of the 4.1kb transcript revealed at least a 509 nucleotide sequence exactly complementary to the 2.0kb MAGE D2 mRNA sequence. This MAGE D2i sequence contains unique structural features not shared with those of previously described antisense transcripts. Identification of this transcript potentially has important implications for future studies examining MAGE D2 expression patterns in cancer and normal tissues.