Towards a Large-Scale Assessment of the Relationship between Biological and Chronological Aging: The INSPIRE Mouse Cohort.

Aging is the major risk factor for the development of chronic diseases. After decades of research focused on extending lifespan, current efforts seek primarily to promote healthy aging. Recent advances suggest that biological processes linked to aging are more reliable than chronological age to account for an individual's functional status, i.e. frail or robust. It is becoming increasingly apparent that biological aging may be detectable as a progressive loss of resilience much earlier than the appearance of clinical signs of frailty. In this context, the INSPIRE program was built to identify the mechanisms of accelerated aging and the early biological signs predicting frailty and pathological aging. To address this issue, we designed a cohort of outbred Swiss mice (1576 male and female mice) in which we will continuously monitor spontaneous and voluntary physical activity from 6 to 24 months of age under either normal or high fat/high sucrose diet. At different age points (6, 12, 18, 24 months), multiorgan functional phenotyping will be carried out to identify early signs of organ dysfunction and generate a large biological fluids/feces/organs biobank (100,000 samples). A comprehensive correlation between functional and biological phenotypes will be assessed to determine: 1) the early signs of biological aging and their relationship with chronological age; 2) the role of dietary and exercise interventions on accelerating or decelerating the rate of biological aging; and 3) novel targets for the promotion of healthy aging. All the functional and omics data, as well as the biobank generated in the framework of the INSPIRE cohort will be available to the aging scientific community. The present article describes the scientific background and the strategies employed for the design of the INSPIRE Mouse cohort.

Urinary peptidome analyses for the diagnosis of chronic kidney disease in dogs.

Chronic kidney disease (CKD) is clinically important in canine medicine. Current diagnostic tools lack sensitivity for detection of subclinical CKD. The aim of the present study was to evaluate urinary peptidome analysis for diagnosis of CKD in dogs. Capillary electrophoresis coupled to mass spectrometry analysis demonstrated presence of approximately 5400 peptides in dog urine. Comparison of urinary peptide abundance of dogs with and without CKD led to the identification of 133 differentially excreted peptides (adjusted P for each peptide <0.05). Sequence information was obtained for 35 of these peptides. This 35 peptide subset and the total group of 133 peptides were used to construct two predictive models of CKD which were subsequently validated by researchers masked to results in an independent cohort of 20 dogs. Both models diagnosed CKD with an area under the receiver operating characteristic (ROC) curve of 0.88 (95% confidence intervals [CI], 0.72-1.0). Most differentially excreted peptides represented fragments of collagen I, indicating possible association with fibrotic processes in CKD (similar to the equivalent human urinary peptide CKD model, CKD273). This first study of the urinary peptidome in dogs identified peptides that were associated with presence of CKD. Future studies are needed to validate the utility of this model for diagnosis and prediction of progression of canine CKD in a clinical setting.

A comparison between MALDI-MS and CE-MS data for biomarker assessment in chronic kidney diseases.

Non-invasive detection of diseases, based on urinary proteomics, is becoming an increasingly important area of research, especially in the area of chronic kidney disease (CKD). Different platforms have been used in independent studies, mostly capillary-electrophoresis coupled ESI-MS (CE-MS), liquid chromatography coupled mass spectrometry, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). We have compared the performance of CE-MS with MALDI-MS in detecting CKD, based on a cohort of 137 urine samples (62 cases and 75 controls). Data cross-talk between the two platforms was established for the comparison of detected biomarkers. The results demonstrate superior performance of the CE-MS approach in terms of peptide resolution and obtained disease prediction accuracy rates. However, the data also demonstrate the ability of the MALDI-MS approach to separate CKD patients from controls, at slightly reduced accuracy, but expected reduced cost and time. As a consequence, a practical approach can be foreseen where MALDI-MS is employed as an inexpensive, fast, and robust screening tool to detect probable CKD. In a second step, high resolution CE-MS could be used in those patients only that scored negative for CKD in the MALDI-MS analysis, reducing costs and time of such a program.

Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy.

Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.

A nest in renal fibrosis?

Sakairi and collaborators show that some tubular cells as well as some interstitial myofibroblasts express the intermediate filament protein nestin. These findings evoke questions about the origin and role of these nestin-positive cells in the development of tubulointerstitial fibrosis.

Gene expression profiling in the remnant kidney model of wild type and kinin B1 and B2 receptor knockout mice.

Angiotensin-converting enzyme inhibitors are the most efficient pharmacologic agents to delay the development of end-stage renal disease (ESRD). This is a multipharmacologic approach that inhibits angiotensin II formation while increasing kinin concentrations. Considerable attention has been focused on the role of decreased angiotensin II levels; however, the role of increased kinin levels is gaining in interest. Kinins affect cellular physiology by interacting with one of two receptors being the more inducible B1 and the more constitutive B2 receptors. This study utilizes the mouse remnant kidney of 20 weeks duration as a model of ESRD. Whole mouse genome microarrays were used to evaluate gene expression in the remnant kidneys of wild type, B1 and B2 receptor knockout animals. The microarray data indicate that gene families involved in vascular damage, inflammation, fibrosis, and proteinuria were upregulated, whereas gene families involved in cell growth, metabolism, lipid, and protein biosynthesis were downregulated in the remnant kidneys. Interestingly, the microarray analyses coupled to histological evaluations are suggestive of a possible protective role of kinins operating through the B2 receptor subtype in this model of renal disease. The results highlight the potential of microarray technology for unraveling complex mechanisms contributing to chronic renal failure.

Bradykinin-induced inhibition of cell proliferation and tyrosine kinase activity in rat mesangial cells.

The relationship between cell proliferation, protein tyrosine phosphorylation, phosphotyrosine kinase activity and bradykinin receptor activation in rat mesangial cells was investigated. We demonstrated that bradykinin (BK), through the B2 receptor, induced a dose-dependent inhibition of mesangial cell proliferation stimulated by fetal calf serum. We next found that BK induced a dose-dependent inhibition of phospho-tyrosine kinase activity. Treatments with pertussis-toxin, inhibition of phospholipase C and protein kinase C inhibitors and chelation of free cytosolic calcium did not change the bradykinin-induced inhibition of phosphotyrosine kinase. Western blot analysis of phosphotyrosinated proteins demonstrated that BK reduced tyrosine phosphorylation of several proteins among which we identified the 125-focal adhesion kinase. Taken together, these data suggest for the first time that, in proliferating rat mesangial cells, B2 receptor stimulation is able to induce, via a pertussis insensitive pathway, the inhibition of tyrosine kinase activity and mesangial cell proliferation.

Homologous and heterologous induction of the human bradykinin B1-receptor and B1-receptor localisation along the rat nephron.

The kinin B1-receptor which is absent or expressed at very low levels under physiological conditions is strongly induced under inflammatory conditions. It has been shown that B1-receptor induction during inflammation involves interleukin-1beta (IL-1beta) production and activation of nuclear factor-kappaB (NF-kappaB). Since bradykinin (BK), the B2-receptor agonist induces IL-1beta expression and activates NF-kappaB, we have analysed the effect of B2-receptor activation in cultured human lung fibroblasts cells on B1-receptor expression by a semiquantitative RT-PCR analysis. Treatment with BK resulted in a significant increase in the expression of B1-receptor mRNA which was abolished by a specific B2-receptor antagonist. This result suggests that B2-receptor activation can prime the expression of B1-receptors. Although the renal localisation of the B2-receptor has been thoroughly studied, nothing is known about the distribution of the B1-receptor in the kidney. Using a combination of microdissection and a semiquantitative RT-PCR/Southern blot analysis we showed the absence of B1-receptors under physiological conditions in 10 microdissected rat nephron segments. However, 18 h LPS-treatment induced significant expression of the B1-receptor in all, but one segment. These studies provide the first molecular basis for the observed changes in renal haemodynamics after B1-agonist infusion in animal kidney models.

Inhibition of cGMP accumulation in mesangial cells by bradykinin and tyrosine kinase inhibitors.

We examined the effect of bradykinin (BK) on the accumulation of cGMP of the mesangial cell (MC), a smooth muscle-like cell of the renal glomerulus. BK caused a time- and concentration dependent reduction of the cGMP concentration. In addition, BK inhibited total protein tyrosine kinase (PTK) activity. Two tyrosine kinase inhibitors (TKI) genistein and tyrphostin also reduced the cGMP concentration. The inhibition of BK and TKI were not additive. The inhibition of PTK by BK, mediated through activation of the B2-receptor, was unaffected by inhibitors of Gi/o proteins, phospholipase C, protein kinase C, cyclooxygenase and Ca2+ release from intracellular stores. Only IBMX a broad spectrum inhibitor of phosphodiesterases (PDE) and 8-methoxymethyl IBMX a specific type-1 PDE inhibitor prevented the inhibitory effects of BK and TKI indicating the involvement of type-1 PDE. In addition, BK had no effect on soluble guanylate cyclase (sGC) and nitric oxide synthase activity. In freshly isolated glomeruli, which represent the physiological environment of MC, BK also reduced the cGMP concentration. Like in MC, the inhibitory effect was suppressed by IBMX. These data demonstrate that BK suppresses a PTK-dependent pathway of cGMP production in rat MC at a level downstream of NO synthase and sGC. It is suggested that BK and TKI inhibitors decrease cGMP levels by preventing tyrosine phosphorylation of type-1 PDE activity, thereby leading to enzyme activation.

Renal bradykinin receptors: localisation, transduction pathways and molecular basis for a possible pathological role (review).

Kinins are biologically active peptides that exert their effects by activating two seven transmembrane G-protein coupled receptors termed B1 and B2 which have only about 36% of homology. The major kinin peptide under physiological conditions, bradykinin (BK), modulates renal haemodynamics and function. Under physiological conditions most BK effects involve bradykinin B2-receptors. Studies on the intra-cellular transduction pathways, the regulation of the expression and the localisation of these receptors along the nephron, as well as the first studies on transgenic mice models, have allowed to better define the role of these receptors under physiological and pathological conditions. The role of the renal B1-receptor, induced in a variety of pathologies related to inflammation, is poorly understood. Recent investigations on the molecular mechanism of B1-receptor induction and its detailed renal localisation have shown that under inflammatory conditions this kinin receptor might be of importance. B2-receptors are suggested to be involved in part of the renoprotective effects of angiotensin converting enzyme (ACE)-inhibitors in insulin-dependent diabetes. However, ACE-inhibitor treatment, resulting also in an increased B1-agonist concentration might result in homologous induction and activation of the B1-receptor.

Differential induction of functional B1-bradykinin receptors along the rat nephron in endotoxin induced inflammation.

BACKGROUND: Under physiological conditions, the effects of kinins in the kidney are mainly mediated by the bradykinin B2-receptor, whereas the kinin B1-receptor is strongly induced under inflammatory conditions in a variety of tissues. Knowledge of the distribution of the B1-receptor along the nephron is of importance since the B1-receptor might replace B2-receptors under these conditions. METHODS: Using a RT-PCR/Southern blot approach allowing relative quantification of mRNA levels, ten different microdissected rat nephron segments were analyzed for the presence of the B1- and B2-receptor before and after endotoxin treatment to induce experimental inflammation. The functionality of the expressed receptors was assessed by kinin-induced intracellular calcium ([Ca2+]i) mobilization in microdissected nephron segments. RESULTS: While under physiological conditions no B1-receptor mRNA could be detected, after 18 hours of treatment with bacterial lipopolysaccharide (LPS) the expression of B1-receptor mRNA was strongly induced in the efferent arteriole, the medullary and inner medullary thin limb, and in the distal tubule. Moderate expression was found in the glomerulus, proximal convoluted and straight tubules, and in the medullary thick ascending limb. Small but detectable expression was observed in the cortical collecting duct. The induction of B1-receptor mRNA expression resulted in functional receptor expression, since increases in [Ca2+]i were observed upon B1-agonist stimulation. LPS treatment also increased the expression of B2-receptor mRNA in all nephron segments except in the glomerulus, the inner medullary thin limb and the outer medullary collecting duct. However, no related changes in B2-agonist induced rises in [Ca2+]i were found. CONCLUSIONS: These studies show a functional induction of the B1-kinin receptor along the rat nephron, which should be taken in account to address the effects of kinins under inflammatory conditions in the kidney.

The B1-agonist [des-Arg10]-kallidin activates transcription factor NF-kappaB and induces homologous upregulation of the bradykinin B1-receptor in cultured human lung fibroblasts.

The bradykinin B1-receptor is strongly upregulated under chronic inflammatory conditions. However, the mechanism and reason are not known. Because a better understanding of the mechanism of the upregulation will help in understanding its potential importance in inflammation, we have studied the molecular mechanism of B1-receptor upregulation in cultured human lung fibroblasts (IMR 90) in response to IL-1beta and the B1-agonist [des-Arg10]-kallidin. We show that treatment of human IMR 90 cells by IL-1beta stimulates the expression of both B1-receptor mRNA and protein. The latter was studied by Western blot analysis using antipeptide antibodies directed against the COOH-terminal part of the human B1-receptor. We furthermore report the novel observation that the B1-receptor is upregulated by its own agonist which was completely blocked by the specific B1-antagonist [des-Arg10-Leu9]-kallidin, indicating an upregulation entirely mediated through cell surface B1-receptors. The increased population of B1-receptors was functionally coupled as exemplified by an enhancement of the B1-agonist induced increase in free cytosolic calcium. Upregulation by the B1-agonist was blocked by a specific protein kinase C inhibitor. B1-agonist-induced upregulation was correlated to the induction of transcription factor nuclear factor kappaB (NF-kappaB) which efficiently bound to the NF-kappaB-like sequence located in the promoter region of the human B1-receptor gene. This correlation was further confirmed by reporter gene assays which showed that this NF-kappaB-like sequence, in the B1-receptor promoter context, could contribute to IL-1beta and DLBK-induced B1-receptor transcription activation, and by the effect of NF-kappaB inhibitor pyrrolidinedithiocarbamate which diminished both B1-receptor upregulation and NF-kappaB activation. NF-kappaB is now recognized as a key inflammatory mediator which is activated by the B1-agonist but which is also involved in B1-receptor upregulation.

B2 kinin receptor upregulation by cAMP is associated with BK-induced PGE2 production in rat mesangial cells.

In the rat mesangial cell (MC), activation of the bradykinin B2 receptor (B2R) by bradykinin (BK) is associated with both phospholipase C (PLC) and A2 (PLA2) activities and with inhibition of adenosine 3',5'-cyclic monophosphate (cAMP) formation leading to cell contraction. Because cAMP plays an important role in the regulation of gene expression in general, we investigated the effect of increasing the intracellular cAMP concentration ([cAMP]i) in mesangial cells on the B2 mRNA expression, on the density of B2 receptor binding sites, on the BK-induced increase in both the free cytosolic Ca2+ concentration ([Ca2+]i), and in the prostaglandin E2 (PGE2) production. Forskolin, PGE2, and cAMP analog, 8-bromoadenosine 3',5'-cyclic monophosphate (8-BrcAMP), were used to increase [cAMP]i. Twenty-four-hour treatment with forskolin, PGE2, and 8-BrcAMP resulted in significant increases in B2 receptor binding sites, which were inhibited by cycloheximide. The maximum B2 receptor mRNA expression (160% above control) was observed in cells treated during 24 h with forskolin and was prevented by actinomycin D. In contrast, the D-myo-inositol 1,4,5-trisphosphate (IP3) formation and the BK-induced increase in [Ca2+]i, reflecting activation of PLC, were not affected by increased levels of [cAMP]i. However the BK-induced PGE2 release, reflecting PLA2 activity, was significantly enhanced. These data bring new information regarding the dual signaling pathways of B2 receptors that can be differentially regulated by cAMP.

Influence of mutations of Val226 on the catalytic rate of haloalkane dehalogenase.

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols. The 3D structure, reaction mechanism and kinetic mechanism have been studied. The steady state k(cat) with 1,2-dichloroethane and 1,2-dibromoethane is limited mainly by the rate of release of the halide ion from the buried active-site cavity. During catalysis, the halogen that is cleaved off (Cl alpha) from 1,2-dichloroethane interacts with Trp125 and the Cl beta interacts with Phe172. Both these residues have van der Waals contacts with Val226. To establish the effect of these interactions on catalysis, and in an attempt to change enzyme activity without directly mutating residues involved in catalysis, we mutated Val226 to Gly, Ala and Leu. The Val226Ala and Val226Leu mutants had a 2.5-fold higher catalytic rate for 1,2-dibromoethane than the wild-type enzyme. A pre-steady state kinetic analysis of the Val226Ala mutant enzyme showed that the increase in k(cat) could be attributed to an increase in the rate of a conformational change that precedes halide release, causing a faster overall rate of halide dissociation. The k(cat) for 1,2-dichloroethane conversion was not elevated, although the rate of chloride release was also faster than in the wild-type enzyme. This was caused by a 3-fold decrease in the rate of formation of the alkyl-enzyme intermediate for 1,2-dichloroethane. Val226 seems to contribute to leaving group (Cl alpha or Br alpha) stabilization via Trp125, and can influence halide release and substrate binding via an interaction with Phe172. These studies indicate that wild-type haloalkane dehalogenase is optimized for 1,2-dichloroethane, although 1,2-dibromoethane is a better substrate.

Kinetic characterization and X-ray structure of a mutant of haloalkane dehalogenase with higher catalytic activity and modified substrate range.

Conversion of halogenated aliphatics by haloalkane dehalogenase proceeds via the formation of a covalent alkyl-enzyme intermediate which is subsequently hydrolyzed by water. In the wild type enzyme, the slowest step for both 1,2-dichloroethane and 1,2-dibromoethane conversion is a unimolecular enzyme isomerization preceding rapid halide dissociation. Phenylalanine 172 is located in a helix-loop-helix structure that covers the active site cavity of the enzyme, interacts with the C1 beta of 1,2-dichloroethane during catalysis, and could be involved in stabilization of this helix-loop-helix region of the cap domain of the enzyme. To obtain more information about the role of this residue in dehalogenase function, we performed a mutational analysis of position 172 and studied the kinetics and X-ray structure of the Phe172Trp enzyme. The Phe172Trp mutant had a 10-fold higher Kcat/Km for 1-chlorohexane and a 2-fold higher Kcat for 1,2-dibromoethane than the wild-type enzyme. The X-ray structure of the Phe172Trp enzyme showed a local conformational change in the helix-loop-helix region that covers the active site. This could explain the elevated activity for 1-chlorohexane of the Phe172Trp enzyme, since it allows this large substrate to bind more easily in the active site cavity. Pre-steady-state kinetic analysis showed that the increase in Kcat found for 1,2-dibromoethane conversion could be attributed to an increase in the rate of an enzyme isomerization step that preceeds halide release. The observed conformational difference between the helix-loop-helix structures of the wild-type enzyme and the faster mutant suggests that the isomerization required for halide release could be a conformational change that takes place in this region of the cap domain of the dehalogenase. It is proposed that Phe172 is involved in stabilization of the helix-loop-helix structure that covers the active site of the enzyme and creates a rigid hydrophobic cavity for small apolar halogenated alkanes.

Specificity and kinetics of haloalkane dehalogenase.

Haloalkane dehalogenase converts halogenated alkanes to their corresponding alcohols. The active site is buried inside the protein and lined with hydrophobic residues. The reaction proceeds via a covalent substrate-enzyme complex. This paper describes a steady-state and pre-steady-state kinetic analysis of the conversion of a number of substrates of the dehalogenase. The kinetic mechanism for the "natural" substrate 1,2-dichloroethane and for the brominated analog and nematocide 1,2-dibromoethane are given. In general, brominated substrates had a lower Km, but a similar kcat than the chlorinated analogs. The rate of C-Br bond cleavage was higher than the rate of C-Cl bond cleavage, which is in agreement with the leaving group abilities of these halogens. The lower Km for brominated compounds therefore originates both from the higher rate of C-Br bond cleavage and from a lower Ks for bromo-compounds. However, the rate-determining step in the conversion (kcat) of 1, 2-dibromoethane and 1,2-dichloroethane was found to be release of the charged halide ion out of the active site cavity, explaining the different Km but similar kcat values for these compounds. The study provides a basis for the analysis of rate-determining steps in the hydrolysis of various environmentally important substrates.

Kinetics of halide release of haloalkane dehalogenase: evidence for a slow conformational change.

Haloalkane dehalogenase converts haloalkanes to their corresponding alcohols and halides. The reaction mechanism involves the formation of a covalent alkyl-enzyme complex which is hydrolyzed by water. The active site is a hydrophobic cavity buried between the main domain and the cap domain of the enzyme. The enzyme has a broad substrate specificity, but the kcat values of the enzyme for the best substrates 1,2-dichloroethane and 1,2-dibromoethane are rather low (3 and 3.5 s-1, respectively). Stopped-flow fluorescence experiments with substrate under single-turnover conditions indicated that halide release could limit the overall kcat. Furthermore, at 5mM 1,2-dibromoethane the observed rate of substrate binding to free enzyme was faster than 700 s-1 (within the dead time of the stopped-flow instrument) whereas displacement of halide by 5mM 1,2-dibromoethane occurred at a rate of only 8 s-1. The binding of bromide and chloride to free enzyme was also studied using stopped-flow fluorescence, and the dependence of kobs on the halide concentration suggested that there were two parallel routes for halide binding. One route, in which a slow enzyme isomerization is followed by rapid halide binding, was predominant at low halide concentrations. The other route involves rapid binding into an initial collision complex followed by a slow enzyme isomerization step and prevailed at higher halide concentrations. The overall rate of halide release was low and limited by a slow enzyme isomerization preceding actual release (9 and 14.5 s-1 for bromide and chloride, respectively). We propose that this slow isomerization is a conformational change in the cap domain that is necessary to allow water to enter and solvate the halide ion. A solvent kinetic isotope effect of 2H2O was found both on kcat and on the rate of halide release. 2H2O mainly affected the rate of the conformational change, which is in agreement with this step being rate-limiting and the overall stabilizing effect of 2H2O on the conformation of proteins.

Engineering proteins for environmental applications.

Recently, significant new insight has been obtained into the structure and catalytic mechanism of enzymes that convert environmental pollutants. Recent advances in protein engineering make it possible to use this information for improving the catalytic performance of such enzymes to achieve increased stability and expanded substrate range.

Construction of an expression and site-directed mutagenesis system of haloalkane dehalogenase in Escherichia coli.

Haloalkane dehalogenase from Xanthobacter autotrophicus was efficiently expressed in Escherichia coli BL21 (DE3) and E. coli JM101. After introduction of restriction sites by PCR the haloalkane dehalogenase gene (dhlA) was translationally fused behind the T7 (phi 10), trc, and tac promoters. This resulted in expression at 30 degrees C up to 38 and 18% of the total soluble cellular protein with the T7 and trc promoters, respectively. Dehalogenase expression under control of the tac promoter was below 1% of the soluble cell protein, however. Aggregation of haloalkane dehalogenase into inclusion bodies was found during growth at 37 degrees C but not at 30 degrees C. Aggregates were also formed from intact enzyme upon incubation at 37 degrees C of cells or crude extracts containing active mature dehalogenase. The high level of expression resulted in a short purification procedure in which 30-35 mg highly enriched haloalkane dehalogenase was obtained from an 0.51 culture. For the production of single-stranded DNA an f1(+) origin was introduced in the T7 expression system.