Cinacalcet for managing secondary hyperparathyroidism in dialysis patients in clinical practice in Belgium: a 16-month observational study (ECHO-B).

In Belgium, the calcimimetic cinacalcet is initially reimbursed for < or = 4 months in dialysis patients with secondary hyperparathyroidism (SHPT) and intact parathyroid hormone (iPTH) > or = 800 pg/mL, or iPTH 300-800 pg/ mL and Ca x P > 55 mg 2/dL2 despite > or = 6 months' optimal treatment with vitamin D sterols and/or phosphate binders. The Belgian, multicentre, observational study ECHO-B evaluated cinacalcet in such patients. Patients who began cinacalcet treatment after March 1, 2007 were eligible. Data were collected retro/prospectively from 6 months before until 16 months after starting cinacalcet (whether or not cinacalcet was continued). Median iPTH was markedly elevated (816 [IQR 551-991] pg/mL) at baseline (the time of starting cinacalcet), but decreased continuously over the course of the study, reaching a value of 414 pg/mL (IQR 240-641; median change -41%) at 4 months, 335 pg/mL (IQR 159-616; -60%) at 12 months and 250 pg/mL (IQR 172-436; -64%) at 16 months. Reductions in serum calcium (-7%) and phosphorus (-13%) were already (near) maximal at 4 months. The primary outcome (iPTH 150-300 pg/mL and/or a > or = 30% reduction within 4 months of starting cinacalcet; criterion for continued reimbursement in Belgium) was achieved in 65/81 patients (80%; 95% CI 72-89%). Results show that in dialysis patients with SHPT in real-life clinical practice, mineral metabolism improves after starting cinacalcet: our study findings suggest that PTH levels may continue decreasing after 12 months' treatment in this setting.

[Experimental nephrology unit: overview and research projects]. / Unité de néphrologie expérimentale facultaire: etat des lieux et projets d'avenir.

After a short historical background of the Laboratory, the main research topics--renal toxicology, physiopathology of renal interstitial fibrosis and hormonology--are described in the perspective of a partnership between research clinicians and full time scientists. National as well as international scientific collaborations underline the need of combining expertises, stimulating also the training of youngest colleagues to the experimental approach of their future discipline.

Aristolochic acid induces proximal tubule apoptosis and epithelial to mesenchymal transformation.

Aristolochic acid contamination in herbal remedies leads to interstitial fibrosis, tubular atrophy, and renal failure in humans. To study the cellular mechanisms contributing to the pathophysiology of this renal disease, we studied Wistar rats treated with aristolochic acid and measured tubular and interstitial cell proliferation, epithelial/mesenchymal cell marker expression, tubular membrane integrity, myofibroblast accumulation, oxidative stress, mitochondrial damage, tubular apoptosis, and fibrosis. Oxidative stress, a loss of cadherin concomitant with vimentin expression, basement membrane denudation with active caspase-3 expression, and mitochondrial injury within tubular cells were evident within 5 days of administration of the toxin. During the chronic phase, interstitial mesenchymal cells accumulated in areas of collagen deposits. Impaired regeneration and apoptosis of proximal tubular cells resulted in tubule atrophy with a near absence of dedifferentiated cell transmembrane migration. We suggest that resident fibroblast activation plays a critical role in the process of renal fibrosis during aristolochic acid toxicity.

[Nephropathy caused by Chinese plants and aristolochic acids: from clinical observation to experimental model]. / Néphropathie aux plantes chinoises et acides aristolochiques : de l'observation clinique au modèle expérimental.

In 1992, a new type of progressive renal fibrosis was reported in patients after the intake of weight-reducing pills containing a Chinese herb (Aristolochia fangchi) rich of nephrotoxic and carcinogenic aristolochic acids (AA). Up to now, Chinese herb nephropathy (CHN), also called aristolochic acid nephropathy (AAN), has been observed in more than 100 patients in Belgium, but also in numerous patients all around the world. The main histological characteristics of CHN are tubular atrophy and interstitial fibrosis leading to severe renal functional impairment and end-stage renal failure. Urothelial carcinomas were also found in about 50% CHN patients suffering from end-stage renal failure. Experimentally, the main features of CHN were successfully reproduced after 35 days of daily AA injections to rats. Starting from this model, we demonstrated that other potential nephrotoxic substances (dexfenfluramine, diuretics) also contained in the weight-reducing pills, did not enhance the renal toxicity of AA. Interestingly, the inhibition of renin angiotensin system did not prevent the development of renal lesions, suggesting that, in contrast with other animal models, physiopathological mechanisms leading to renal fibrosis might be largely independent of angiotensin II. From clinical observations to experimental studies, we currently increased our knowledge in the understanding of pathophysiological mechanisms of nephropathies of toxic origin. Botanicals which are known or suspected to contain AA are still sold on Web sites or over-the-counter markets. It is not surprising then to find new AAN cases reported in the medical literature. Therefore, further studies are needed to elucidate pathways of AA-related nephrotoxicity and carcinogenicity in order to develop therapeutic strategies.

Nod genes and Nod signals and the evolution of the Rhizobium legume symbiosis.

The establishment of the nitrogen-fixing symbiosis between rhizobia and legumes requires an exchange of signals between the two partners. In response to flavonoids excreted by the host plant, rhizobia synthesize Nod factors (NFs) which elicit, at very low concentrations and in a specific manner, various symbiotic responses on the roots of the legume hosts. NFs from several rhizobial species have been characterized. They all are lipo-chitooligosaccharides, consisting of a backbone of generally four or five glucosamine residues N-acylated at the non-reducing end, and carrying various O-substituents. The N-acyl chain and the other substituents are important determinants of the rhizobial host specificity. A number of nodulation genes which specify the synthesis of NFs have been identified. All rhizobia, in spite of their diversity, possess conserved nodABC genes responsible for the synthesis of the N-acylated oligosaccharide core of NFs, which suggests that these genes are of a monophyletic origin. Other genes, the host specific nod genes, specify the substitutions of NFs. The central role of NFs and nod genes in the Rhizobium-legume symbiosis suggests that these factors could be used as molecular markers to study the evolution of this symbiosis. We have studied a number of NFs which are N-acylated by alpha,beta-unsaturated fatty acids. We found that the ability to synthesize such NFs does not correlate with taxonomic position of the rhizobia. However, all rhizobia that produce NFs such nodulate plants belonging to related tribes of legumes, the Trifolieae, Vicieae, and Galegeae, all of them being members of the so-called galegoid group. This suggests that the ability to recognize the NFs with alpha-beta-unsaturated fatty acids is limited to this group of legumes, and thus might have appeared only once in the course of legume evolution, in the galegoid phylum.

Unusual methyl-branched alpha,beta-unsaturated acyl chain substitutions in the Nod Factors of an arctic rhizobium, Mesorhizobium sp. strain N33 (Oxytropis arctobia).

Mesorhizobium sp. strain N33 (Oxytropis arctobia), a rhizobial strain isolated in arctic Canada, is able to fix nitrogen at very low temperatures in association with a few arctic legume species belonging to the genera Astragalus, Onobrychis, and Oxytropis. Using mass spectrometry and nuclear magnetic resonance spectroscopy, we have determined the structure of N33 Nod factors, which are major determinants of nodulation. They are pentameric lipochito-oligosaccharides 6-O sulfated at the reducing end and exhibit other original substitutions: 6-O acetylation of the glucosamine residue next to the nonreducing terminal glucosamine and N acylation of the nonreducing terminal glucosamine by methyl-branched acyl chains of the iso series, some of which are alpha,beta unsaturated. These unusual substitutions may contribute to the peculiar host range of N33. Analysis of N33 whole-cell fatty acids indicated that synthesis of the methyl-branched fatty acids depended on the induction of bacteria by plant flavonoids, suggesting a specific role for these fatty acids in the signaling process between the plant and the bacteria. Synthesis of the methyl-branched alpha,beta-unsaturated fatty acids required a functional nodE gene.

The common nodulation genes of Astragalus sinicus rhizobia are conserved despite chromosomal diversity.

The nodulation genes of Mesorhizobium sp. (Astragalus sinicus) strain 7653R were cloned by functional complementation of Sinorhizobium meliloti nod mutants. The common nod genes, nodD, nodA, and nodBC, were identified by heterologous hybridization and sequence analysis. The nodA gene was found to be separated from nodBC by approximately 22 kb and was divergently transcribed. The 2. 0-kb nodDBC region was amplified by PCR from 24 rhizobial strains nodulating A. sinicus, which represented different chromosomal genotypes and geographic origins. No polymorphism was found in the size of PCR products, suggesting that the separation of nodA from nodBC is a common feature of A. sinicus rhizobia. Sequence analysis of the PCR-amplified nodA gene indicated that seven strains representing different 16S and 23S ribosomal DNA genotypes had identical nodA sequences. These data indicate that, whereas microsymbionts of A. sinicus exhibit chromosomal diversity, their nodulation genes are conserved, supporting the hypothesis of horizontal transfer of nod genes among diverse recipient bacteria.

Structure of the Mesorhizobium huakuii and Rhizobium galegae Nod factors: a cluster of phylogenetically related legumes are nodulated by rhizobia producing Nod factors with alpha,beta-unsaturated N-acyl substitutions.

Rhizobia are symbiotic bacteria that synthesize lipochitooligosaccharide Nod factors (NFs), which act as signal molecules in the nodulation of specific legume hosts. Based on the structure of their N-acyl chain, NFs can be classified into two categories: (i) those that are acylated with fatty acids from the general lipid metabolism; and (ii) those (= alphaU-NFs) that are acylated by specific alpha,beta-unsaturated fatty acids (containing carbonyl-conjugated unsaturation(s)). Previous work has described how rhizobia that nodulate legumes of the Trifolieae and Vicieae tribes produce alphaU-NFs. Here, we have studied the structure of NFs from two rhizobial species that nodulate important genera of the Galegeae tribe, related to Trifolieae and Vicieae. Three strains of Mesorhizobium huakuii, symbionts of Astragalus sinicus, produced as major NFs, pentameric lipochitooligosaccharides O-sulphated and partially N-glycolylated at the reducing end and N-acylated, at the non-reducing end, by a C18:4 fatty acid. Two strains of Rhizobium galegae, symbionts of Galega sp., produced as major NFs, tetrameric O-carbamoylated NFs that could be O-acetylated on the glucosamine residue next to the non-reducing terminal glucosamine and were N-acylated by C18 and C20 alpha,beta-unsaturated fatty acids. These results suggest that legumes nodulated by rhizobia synthesizing alphaU-NFs constitute a phylogenetic cluster in the Galegoid phylum.

The metabolic fate of long-term inhaled nitric oxide.

PURPOSE: The fate of inhaled nitric oxide (NO) has not been precisely defined in critically ill patients. This study aimed at defining the effects of long-term NO inhalation on circulating NO byproduct levels. MATERIAL AND METHODS: During NO therapy, plasma and urine from 13 critically ill patients were sampled daily for determination of the stable byproducts of NO (nitrite [NO2-] and nitrate [NO3-]. Routine monitoring data included inhaled NO concentration, hemodynamic parameters, arterial blood gases, creatinine clearance, and C-reactive protein. RESULTS: For the first 24 hours of NO inhalation (6.3+/-1.1 ppm), NO3- plasma concentration increased (from 13.3+/-5.4 to 52.3+/-17.6 micromol/L), but NO2- plasma concentration was not affected. The NO3- plasma concentration was correlated with the C-reactive protein level, the inhaled NO concentration. Renal excretion of NO metabolites was unaltered by NO inhalation. The NO3 concentrations returned to baseline when NO therapy was discontinued. CONCLUSION: Long-term NO inhalation was associated with a consistent increase in the NO3- plasma concentration. NO byproducts may be implicated in the systemic effects associated with this treatment.

The common nodABC genes of Rhizobium meliloti are host-range determinants.

Symbiotic bacteria of the genus Rhizobium synthesize lipo-chitooligosaccharides, called Nod factors (NFs), which act as morphogenic signal molecules on legume hosts. The common nodABC genes, present in all Rhizobium species, are required for the synthesis of the core structure of NFs. NodC is an N-acetylglucosaminyltransferase, and NodB is a chitooligosaccharide deacetylase; NodA is involved in N-acylation of the aminosugar backbone. Specific nod genes are involved in diverse NF substitutions that confer plant specificity. We transferred to R. tropici, a broad host-range tropical symbiont, the ability to nodulate alfalfa, by introducing nod genes of R. meliloti. In addition to the specific nodL and nodFE genes, the common nodABC genes of R. meliloti were required for infection and nodulation of alfalfa. Purified NFs of the R. tropici hybrid strain, which contained chitin tetramers and were partly N-acylated with unsaturated C16 fatty acids, were able to elicit nodule formation on alfalfa. Inactivation of the R. meliloti nodABC genes suppressed the ability of the NFs to nodulate alfalfa. Studies of NFs from nodA, nodB, nodC, and nodI mutants indicate that (i) NodA of R. meliloti, in contrast to NodA of R. tropici, is able to transfer unsaturated C16 fatty acids onto the chitin backbone and (ii) NodC of R. meliloti specifies the synthesis of chitin tetramers. These results show that allelic variation of the common nodABC genes is a genetic mechanism that plays an important role in signaling variation and in the control of host range.

The NodA proteins of Rhizobium meliloti and Rhizobium tropici specify the N-acylation of Nod factors by different fatty acids.

Rhizobia synthesize mono-N-acylated chitooligosaccharide signals, called Nod factors, that are required for the specific infection and nodulation of their legume hosts. The biosynthesis of Nod factors is under the control of nodulation (nod) genes, including the nodABC genes present in all rhizobial species. The N-acyl substitution can vary between species and can play a role in host specificity. In Rhizobium meliloti, an alfalfa symbiont, the acyl chain is a C16 unsaturated or a (omega-1) hydroxylated fatty acid, whereas in Rhizobium tropici, a bean symbiont, it is vaccenic acid (C18:1). We constructed R. meliloti derivatives having a non-polar deletion of nodA, and carrying a plasmid with either the R. meliloti or the R. tropici nodA gene. The strain with the R. tropici nodA gene produced Nod factors acylated by vaccenic acid, instead of the C16 unsaturated or hydroxylated fatty acids characteristic of R. meliloti Nod factors, and infected and nodulated alfalfa with a significant delay. These results show that NodA proteins of R. meliloti and R. tropici specify the N-acylation of Nod factors by different fatty acids, and that allelic variation of the common nodA gene can contribute to the determination of host range.

Rhizobium lipo-chitooligosaccharide nodulation factors: signaling molecules mediating recognition and morphogenesis.

Rhizobia elicit on their specific leguminous hosts the formation of new organs, called nodules, in which they fix nitrogen. The rhizobial nodulation genes specify the synthesis of lipo-chitooligosaccharide signals, the Nod factors (NFs). Each rhizobial species has a characteristic set of nodulation genes that specifies the length of the chitooligosaccharide backbone and the type of substitutions at both ends of the molecule, thus making the NFs specific for a given plant host. At extremely low concentrations, purified NFs are capable of eliciting on homologous legume hosts many of the plant developmental responses characteristic of the bacteria themselves, including cell divisions, and the triggering of a plant organogenic program. This review summarizes our current knowledge on the biosynthesis, structure, and function of this new class of signaling molecules. Finally we discuss the possibility that these signals could be part of a new family of plant lipo-chitooligosaccharide growth regulators.

Rhizobium meliloti lipooligosaccharide nodulation factors: different structural requirements for bacterial entry into target root hair cells and induction of plant symbiotic developmental responses.

Rhizobium meliloti produces lipochitooligosaccharide nodulation NodRm factors that are required for nodulation of legume hosts. NodRm factors are O-acetylated and N-acylated by specific C16-unsaturated fatty acids. nodL mutants produce non-O-acetylated factors, and nodFE mutants produce factors with modified acyl substituents. Both mutants exhibited a significantly reduced capacity to elicit infection thread (IT) formation in alfalfa. However, once initiated, ITs developed and allowed the formation of nitrogen-fixing nodules. In contrast, double nodF/nodL mutants were unable to penetrate into legume hosts and to form ITs. Nevertheless, these mutants induced widespread cell wall tip growth in trichoblasts and other epidermal cells and were also able to elicit cortical cell activation at a distance. NodRm factor structural requirements are thus clearly more stringent for bacterial entry than for the elicitation of developmental plant responses.

Role of the Rhizobium meliloti nodF and nodE genes in the biosynthesis of lipo-oligosaccharidic nodulation factors.

Rhizobia nodulation (nod) genes are involved in the synthesis of symbiotic signals, the Nod factors, which are mono-N-acylated chito-oligosaccharides. Nod factors elicit, in a specific manner, various plant responses on legume roots. In this report we address the question of the role of nodFEG genes in the synthesis of the acyl moiety of Rhizobium meliloti Nod factors. In a Nod factor-overproducing strain with the wild-type nod region, in addition to the delta 2,9-C16:2 and delta 2, 4,9-C16:3 acyl groups already described, delta 9-C16:1 was also found, together with a series of C18 to C26 (omega-1)-hydroxylated fatty acids. A deletion of nodE resulted in the absence of C16:2 and C16:3 fatty acids, which were replaced by vaccenic acid (delta 11-C18:1), but did not change the proportion of (omega-1)-hydroxylated fatty acids. A nodF deletion, non-polar with respect to nodE, resulted in the same alterations in the Nod factor N-acyl composition, showing that both nodF and nodE are required for the synthesis of the C16 polyunsaturated chains. In contrast, nodG mutations did not result in a detectable change in the Nod factor N-acyl moiety. When a plasmid carrying the nodFE genes of Rhizobium leguminosarum bv. viciae was introduced into R. meliloti nodFE- and nodFEG-deleted strains, Nod factors with polyunsaturated C18 fatty acids (C18:2, C18:3, and C18:4) could be detected. These results provide evidence that the molecular basis of allelic variation between the R. meliloti and R. leguminosarum bv. viciae host range nodFE genes lies in the fact that the two nodFE alleles specify the synthesis of unsaturated fatty acid substituents with a different carbon length.

The Rhizobium, Bradyrhizobium, and Azorhizobium NodC proteins are homologous to yeast chitin synthases.

The nodABC genes of rhizobia are essential for the synthesis of lipo-oligosaccharidic (N-acylated chitin oligomers) nodulation signals. nodC gene products from Rhizobium, Bradyrhizobium, and Azorhizobium exhibit extensive homology with chitin synthases, suggesting that the NodC proteins are involved in the synthesis of the chitin oligomer backbone by catalyzing the beta-1,4-linkage between N-acetyl-D-glucosamine residues.

Signaling and host range variation in nodulation.

Rhizobium, Bradyrhizobium, and Azorhizobium strains, collectively referred to as rhizobia, elicit on their leguminous hosts, in a specific manner, the formation of nodules in which they fix nitrogen. Rhizobial nod genes, which determine host specificity, infection, and nodulation, are involved in the exchange of low molecular weight signal molecules between the plant and the bacteria as follows. Transcription of the nod operons is under the control of NodD regulatory proteins, which are specifically activated by plant flavonoid signals. The common and species-specific structural nod genes are involved in turn in the synthesis of specific lipo-oligosaccharides that signal back to the plant to elicit root-hair deformations, cortical-cell divisions, and nodule-meristem formation.

Molecular basis of symbiotic host specificity in Rhizobium meliloti: nodH and nodPQ genes encode the sulfation of lipo-oligosaccharide signals.

The symbiosis between Rhizobium and legumes is highly specific. For example, R. meliloti elicits the formation of root nodules on alfalfa and not on vetch. We recently reported that R. meliloti nodulation (nod) genes determine the production of acylated and sulfated glucosamine oligosaccharide signals. We now show that the biochemical function of the major host-range genes, nodH and nodPQ, is to specify the 6-O-sulfation of the reducing terminal glucosamine. Purified Nod factors (sulfated or not) from nodH+ or nodH- strains exhibited the same plant specificity in a variety of bioassays (root hair deformations, nodulation, changes in root morphology) as the bacterial cells from which they were purified. These results provide strong evidence that the molecular mechanism by which the nodH and nodPQ genes mediate host specificity is by determining the sulfation of the extracellular Nod signals.

Role of the nodD and syrM genes in the activation of the regulatory gene nodD3, and of the common and host-specific nod genes of Rhizobium meliloti.

To analyse the regulation of the nodulation (nod) genes of Rhizobium meliloti RCR2011 we have isolated lacZ gene fusions to a number of common, host-range and regulatory nod genes, using the mini-Mu-lac bacteriophage transposon MudII1734. Common (nodA, nodC, nod region IIa) and host-range (nodE, nodG, nodH) genes were found to be regulated similarly. They were activated (i) by the regulatory nodD1 gene in the presence of flavones such as chrysoeriol, luteolin and 7,3',4'-trihydroxyflavone, (ii) by nodD2 in the presence of alfalfa root exudate but not with the NodD1-activating flavones, and (iii) by the regulatory genes syrM-nodD3 even in the absence of plant inducers. Thus common and host-range nod genes belong to the same regulon. In contrast to the nodD1 gene, the regulatory nodD3 gene was not expressed constitutively and exhibited a complex regulation. It required syrM for expression, was activated by nodD1 in the presence of luteolin and was positively autoregulated.

Interference between Rhizobium meliloti and Rhizobium trifolii nodulation genes: genetic basis of R. meliloti dominance.

Transfer of an IncP plasmid carrying the Rhizobium meliloti nodFE, nodG, and nodH genes to Rhizobium trifolii enabled R. trifolii to nodulate alfalfa (Medicago sativa), the normal host of R. meliloti. Using transposon Tn5-linked mutations and in vitro-constructed deletions of the R. meliloti nodFE, nodG, and nodH genes, we showed that R. meliloti nodH was required for R. trifolii to elicit both root hair curling and nodule initiation on alfalfa and that nodH, nodFE, and nodG were required for R. trifolii to elicit infection threads in alfalfa root hairs. Interestingly, the transfer of the R. meliloti nodFE, nodG, and nodH genes to R. trifolii prevented R. trifolii from infecting and nodulating its normal host, white clover (Trifolium repens). Experiments with the mutated R. meliloti nodH, nodF, nodE, and nodG genes demonstrated that nodH, nodF, nodE, and possibly nodG have an additive effect in blocking infection and nodulation of clover.