Apocynin-treatment reverses hyperoxaluria induced changes in NADPH oxidase system expression in rat kidneys: a transcriptional study.

PURPOSE: We have previously shown that production of reactive oxygen species (ROS) is an important contributor to renal injury and inflammation following exposure to oxalate (Ox) or calcium-oxalate (CaOx) crystals. The present study was conducted, utilizing global transcriptome analyses, to determine the effect of Apocynin on changes in the NADPH oxidase system activated in kidneys of rats fed a diet leading to hyperoxaluria and CaOx crystal deposition. APPROACH: Age-, sex- and weight-matched rats were either fed regular rat chow or regular rat chow supplemented with 5% w/w hydroxy-L-proline (HLP). Half of the rats on the HLP diet were also placed on Apocynin-supplemented H(2)O. After 28 days, each rat was euthanized, their kidneys freshly explanted and dissected to obtain both cortex and medulla tissues. Total RNA was extracted from each tissue and subjected to genomic microarrays to obtain global transcriptome data. KEGG was used to identify gene clusters with differentially expressed genes. Immunohistochemistry was used to confirm protein expressions of selected genes. RESULTS: Genes encoding both membrane- and cytosolic-NADPH oxidase complex-associated proteins, together with p21rac and Rap1a, were coordinately up-regulated significantly in both renal medulla and cortex tissues in the HLP-fed rats compared to normal healthy untreated controls. Activation of NADPH oxidase appears to occur via the angiotensin-II/angiotensin-II receptor-2 pathway, although the DAG-PKC pathway of neutrophils may also contribute. Immuno histochemical staining confirmed up-regulated gene expressions. Simultaneously, genes encoding ROS scavenger proteins were down-regulated. HLP-fed rats receiving Apocynin had a complete reversal in the differential-expression of the NADPH oxidase system genes, despite showing similar levels of hyperoxaluria. CONCLUSIONS: A strong up-regulation of an oxidative/respiratory burst involving the NADPH oxidase system, activated via the angiotensin-II and most likely the DAG-PKC pathways, occurs in kidneys of hyperoxaluric rats. Apocynin treatment reversed this activation without affecting the levels of hyperoxaluria.

A structural element that facilitates proton-coupled electron transfer in oxalate decarboxylase.

The conformational properties of an active-site loop segment, defined by residues Ser(161)-Glu(162)-Asn(163)-Ser(164), have been shown to be important for modulating the intrinsic reactivity of Mn(II) in the active site of Bacillus subtilis oxalate decarboxylase. We now detail the functional and structural consequences of removing a conserved Arg/Thr hydrogen-bonding interaction by site-specific mutagenesis. Hence, substitution of Thr-165 by a valine residue gives an OxDC variant (T165V) that exhibits impaired catalytic activity. Heavy-atom isotope effect measurements, in combination with the X-ray crystal structure of the T165V OxDC variant, demonstrate that the conserved Arg/Thr hydrogen bond is important for correctly locating the side chain of Glu-162, which mediates a proton-coupled electron transfer (PCET) step prior to decarboxylation in the catalytically competent form of OxDC. In addition, we show that the T165V OxDC variant exhibits a lower level of oxalate consumption per dioxygen molecule, consistent with the predictions of recent spin-trapping experiments [Imaram et al. (2011) Free Radicals Biol. Med. 50, 1009-1015]. This finding implies that dioxygen might participate as a reversible electron sink in two putative PCET steps and is not merely used to generate a protein-based radical or oxidized metal center.

Gene expression profiling of early-phase Sjögren's syndrome in C57BL/6.NOD-Aec1Aec2 mice identifies focal adhesion maturation associated with infiltrating leukocytes.

PURPOSE: Despite considerable efforts, the molecular and cellular events in lacrimal gland tissues initiating inflammatory responses leading to keratoconjunctivitis sicca (KCS), autoimmunity, and Sjögren's syndrome (SjS) have yet to be defined. To determine whether altered glandular homeostasis occurs before the onset of autoimmunity, a temporal transcriptome study was carried out in an animal model of primary SjS. METHODS: Using oligonucleotide microarrays, gene expression profiles were generated for lacrimal glands of C57BL/6.NOD-Aec1Aec2 mice 4 to 20 weeks of age. Pairwise analyses identified genes differentially expressed, relative to their 4-week expression, during the development of SjS-like disease. Statistical analyses defined differentially and coordinately expressed gene sets. The PANTHER (Protein ANalysis THrough Evolutionary Relationships) classification system was used to define annotated biological processes or functions. RESULTS: Temporal transcript expression profiles of C57BL/6.NOD-Aec1Aec2 lacrimal glands before, or concomitant with, the first appearance of inflammatory cells revealed a highly restricted subset of differentially expressed genes encoding interactive extracellular matrix proteins, fibronectin, integrins, and syndecans. In contrast, genes encoding interepithelial junctional complex proteins defined alterations in tight junctions (TJ), adherens, desmosomes, and gap junctions, suggesting perturbations in the permeability of the paracellular spaces between epithelial barriers. Correlating with this were gene sets defining focal adhesion (FA) maturation and Ras/Raf-Mek/Erk signal transduction. Immunohistochemically, FAs were associated with infiltrating leukocytes and not with lacrimal epithelial cells. CONCLUSIONS: For the first time, FA maturations are implicated as initial biomarkers of impending autoimmunity in lacrimal glands of SjS-prone mice. Changes in TJ complex genes support an increased movement of cells through paracellular spaces.

EPR spin trapping of an oxalate-derived free radical in the oxalate decarboxylase reaction.

EPR spin trapping experiments on bacterial oxalate decarboxylase from Bacillus subtilis under turn-over conditions are described. The use of doubly (13)C-labeled oxalate leads to a characteristic splitting of the observed radical adducts using the spin trap N-tert-butyl-α-phenylnitrone linking them directly to the substrate. The radical was identified as the carbon dioxide radical anion which is a key intermediate in the hypothetical reaction mechanism of both decarboxylase and oxidase activities. X-ray crystallography had identified a flexible loop, SENS161-4, which acts as a lid to the putative active site. Site directed mutagenesis of the hinge amino acids, S161 and T165 was explored and showed increased radical trapping yields compared to the wild type. In particular, T165V shows approximately ten times higher radical yields while at the same time its decarboxylase activity was reduced by about a factor of ten. This mutant lacks a critical H-bond between T165 and R92 resulting in compromised control over its radical chemistry allowing the radical intermediate to leak into the surrounding solution.