Bone alkaline phosphatase in CKD-mineral bone disorder.

Overall and cardiovascular mortality in patients with chronic kidney disease (CKD) is greatly increased, without obvious current effective treatments. Mineral and bone disorder (MBD) is a common manifestation of CKD and contributes to the high risk of fracture and cardiovascular mortality in these patients. Traditionally, clinical management of CKD-MBD focused on attenuation of secondary hyperparathyroidism due to impaired renal activation of vitamin D and phosphate retention, although recently, adynamic forms of renal bone disease have become more prevalent. Definitive diagnosis was based on histologic (histomorphometric) analysis of bone biopsy material supported by radiologic changes and changes in levels of surrogate laboratory markers. Of these various markers, parathyroid hormone (PTH) has been considered to be the most sensitive and currently is the most frequently used; however, the many pitfalls of measuring PTH in patients with CKD increasingly are appreciated. We propose an alternative or complementary approach using bone alkaline phosphatase (ALP), which is directly related to bone turnover, reflects bone histomorphometry, and predicts outcomes in hemodialysis patients. Here, we consider the overall merits of bone ALP as a marker of bone turnover in adults with CKD-MBD, examine published bone histomorphometric data comparing bone ALP to PTH, and discuss possible pathogenic mechanisms by which bone ALP may be linked to outcomes in patients with CKD.

PTH--a particularly tricky hormone: why measure it at all in kidney patients?

Plasma parathyroid hormone (PTH) concentrations are commonly measured in the context of CKD, as PTH concentration elevation is typical in this clinical context. Much has been inferred from this raised PTH concentration tendency, both about the state of skeletal integrity and health and also about the potential clinical outcomes for patients. However, we feel that reliance on PTH concentrations alone is a dangerous substitute for the search for, and use of, more precise and reliable biomarkers. In this article, we rehearse these arguments, bringing together patient-level and analytical considerations for the first time.

Bone-specific alkaline phosphatase concentrations are less variable than those of parathyroid hormone in stable hemodialysis patients.

Abnormalities of bone mineral metabolism and vascular calcification are prevalent in patients with kidney failure. Clinical management is based on biochemical targets, in particular parathyroid hormone (PTH) concentrations, but this has many limitations including high biological variation. A possible alternative is bone-specific alkaline phosphatase (ALP); therefore, we evaluated the biological variation of this marker in patients undergoing hemodialysis. Bone ALP was measured in non-fasting serum samples taken twice a week over a 6-week period in 22 stable hemodialysis patients and 12 healthy volunteers. The within-individual coefficients of variance were calculated and used to derive the critical difference required to be certain that an observed change was significant. The coefficient of variance for bone ALP was significantly higher in hemodialysis patients compared to healthy individuals. Seven samples were required to estimate the homeostatic set point of bone ALP, within 10%, in a hemodialysis patient. The concentration of serial bone ALP measurements would need to change by 36% between any two measurements before it can be considered a significant change. Since the biological variation of bone ALP is less than half that reported for PTH, our study provides further support for the use of bone ALP as an alternative marker of bone mineral metabolism in the setting of chronic kidney disease-mineral and bone disorder.

Characterization of a two-component signal transduction system that controls arsenite oxidation in the chemolithoautotroph NT-26.

NT-26 is a chemolithoautotrophic arsenite oxidizer. Understanding the mechanisms of arsenite signalling, tolerance and oxidation by NT-26 will have significant implications for its use in bioremediation and arsenite sensing. We have identified the histidine kinase (AroS) and the cognate response regulator (AroR) involved in the arsenite-dependent transcriptional regulation of the arsenite oxidase aroBA operon. AroS contains a single periplasmic sensory domain that is linked through transmembrane helices to the HAMP domain that transmits the signal to the kinase core of the protein. AroR belongs to a family of AAA+ transcription regulators that interact with DNA through a helix-turn-helix domain. The presence of the AAA+ domain as well as the RNA polymerase σ(54) -interaction sequence motif suggests that this protein regulates transcription through interaction with RNA polymerase in a σ(54) -dependent fashion. The kinase core of AroS and the receiver domain of AroR were heterologously expressed and purified and their autophosphorylation and transphosphorylation activities were confirmed. Using site-directed mutagenesis, we have identified the phosphorylation sites on both proteins. Mutational analysis in NT-26 confirmed that both proteins are essential for arsenite oxidation and the AroS mutant affected growth with arsenite, also implicating it in the regulation of arsenite tolerance. Lastly, arsenite sensing does not appear to involve thiol chemistry.

Structural insights into the catalytic mechanism of Trypanosoma cruzi GPXI (glutathione peroxidase-like enzyme I).

Current drug therapies against Trypanosoma cruzi, the causative agent of Chagas disease, have limited effectiveness and are highly toxic. T. cruzi-specific metabolic pathways that utilize trypanothione for the reduction of peroxides are being explored as potential novel therapeutic targets. In the present study we solved the X-ray crystal structure of one of the T. cruzi enzymes involved in peroxide reduction, the glutathione peroxidase-like enzyme TcGPXI (T. cruzi glutathione peroxidase-like enzyme I). We also characterized the wild-type, C48G and C96G variants of TcGPXI by NMR spectroscopy and biochemical assays. Our results show that residues Cys48 and Cys96 are required for catalytic activity. In solution, the TcGPXI molecule readily forms a Cys48-Cys96 disulfide bridge and the polypeptide segment containing Cys96 lacks regular secondary structure. NMR spectra of the reduced TcGPXI are indicative of a protein that undergoes widespread conformational exchange on an intermediate time scale. Despite the absence of the disulfide bond, the active site mutant proteins acquired an oxidized-like conformation as judged from their NMR spectra. The protein that was used for crystallization was pre-oxidized by t-butyl hydroperoxide; however, the electron density maps clearly showed that the active site cysteine residues are in the reduced thiol form, indicative of X-ray-induced reduction. Our crystallographic and solution studies suggest a level of structural plasticity in TcGPXI consistent with the requirement of the atypical two-cysteine (2-Cys) peroxiredoxin-like mechanism implied by the behaviour of the Cys48 and Cys96 mutant proteins.

A GAF domain in the hypoxia/NO-inducible Mycobacterium tuberculosis DosS protein binds haem.

The majority of the Mycobacterium tuberculosis response to hypoxia and nitric oxide is through the DosRS (DevRS) two-component regulatory system. The N-terminal input domain of the DosS sensor contains two GAF domains. We demonstrate here that the proximal GAF domain binds haem, and identified histidine 149 of DosS as critical to haem-binding; the location of this histidine residue is similar to the cGMP-binding site in a crystal structure of cyclic nucleotide phosphodiesterase 2A. GAF domains are frequently involved in binding cyclic nucleotides, but this is the first GAF domain to be identified that binds haem. In contrast, PAS domains (similar to GAF domains in structure but not primary sequence) frequently use haem cofactors, and these findings further illustrate how the functions of these domains overlap. We propose that the activation of the DosS sensor is controlled through the haem binding of molecular oxygen or nitric oxide.