Comparison of five assays for DNA extraction from bacterial cells in human faecal samples.

AIM: To determine the most effective DNA extraction method for bacteria in faecal samples. MATERIALS AND RESULTS: This study assessed five commercial methods, that is, NucliSens easyMag, QIAamp DNA Stool Mini kit, PureLink Microbiome DNA purification kit, QIAamp PowerFecal DNA kit and RNeasy PowerMicrobiome kit, of which the latter has been optimized for DNA extraction. The DNA quantity and quality were determined using Nanodrop, Qubit and qPCR. The PowerMicrobiome kit recovered the highest DNA concentration, whereby this kit also recovered the highest gene copy number of Gram positives, Gram negatives and total bacteria. Furthermore, the PowerMicrobiome kit in combination with mechanical pre-treatment (bead beating) and with combined enzymatic and mechanical pre-treatment (proteinase K+mutanolysin+bead beating) was more effective than without pre-treatment. CONCLUSION: From the five DNA extraction methods that were compared, the PowerMicrobiome kit, preceded by bead beating, which is standard included, was found to be the most effective DNA extraction method for bacteria in faecal samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The quantity and quality of DNA extracted from human faecal samples is a first important step to optimize molecular methods. Here we have shown that the PowerMicrobiome kit is an effective DNA extraction method for bacterial cells in faecal samples for downstream qPCR purpose.

Genetic evaluation for birth and conformation traits in dual-purpose Belgian Blue cattle using a mixed inheritance model.

The segregation of the causal mutation () in the muscular hypertrophy gene in dual-purpose Belgian Blue (dpBB) cattle is considered to result in greater calving difficulty (dystocia). Establishing adapted genetic evaluations might overcome this situation through efficient selection. However, the heterogeneity of dpBB populations at the locus implies separating the major gene and other polygenic effects in complex modeling. The use of mixed inheritance models may be an interesting option because they simultaneously assume both influences. A genetic evaluation in dpBB based on a mixed inheritance model was developed for birth and conformation traits: gestation length (GL), calving difficulty (CD), birth weight (BiW), and body conformation score (BC). A total of 27,362 animals having records were used for analyses. The total number of animals in the pedigree used to build the numerator relationship matrix was 62,617. Genotypes at the locus were available for 2,671 animals. Missing records at this locus were replaced with genotype probabilities. A total of 13,221 (48.3%) were registered as dpBB, 1,287 (4.7%) as beef Belgian Blue, and 12,854 (47.0%) were unknown. From those 13,221 dpBB animals, 650, 849, and 534 had double or single copies or no copy, respectively, of the causal mutation () in the muscular hypertrophy gene, whereas 11,188 had missing genotypes. This heterogeneity at the locus may be the reason for high variability in the studied traits, that is, high heritability estimates of 0.33, 0.30, 0.38, and 0.43 for GL, CD, BiW, and BC, respectively. In general, additive ( < 0.05) and dominance ( < 0.001) allele substitution for calves and dams had significant impact for all traits. The moderate coefficient of genetic variation (27.80%) and high direct heritability (0.28) for CD suggested genetic variability in dpBB and possible genetic improvement through selection. This variability has allowed dpBB breeders to successfully apply mass selection in the past. Genetic trend means from 1988 to 2016 showed that sire selection for CD within genotype was progressively applied by breeders. The selection intensity was more important for CD in double-muscled lines than in segregated lines. Our study illustrated the possible confusion caused by the use of major genes in selection and the importance of fitting appropriate models such as mixed inheritance models that combine polygenic and gene content information.

Integration of external estimated breeding values and associated reliabilities using correlations among traits and effects.

Based on a Bayesian view of linear mixed models, several studies showed the possibilities to integrate estimated breeding values (EBV) and associated reliabilities (REL) provided by genetic evaluations performed outside a given evaluation system into this genetic evaluation. Hereafter, the term "internal" refers to this given genetic evaluation system, and the term "external" refers to all other genetic evaluations performed outside the internal evaluation system. Bayesian approaches integrate external information (i.e., external EBV and associated REL) by altering both the mean and (co)variance of the prior distributions of the additive genetic effects based on the knowledge of this external information. Extensions of the Bayesian approaches to multivariate settings are interesting because external information expressed on other scales, measurement units, or trait definitions, or associated with different heritabilities and genetic parameters than the internal traits, could be integrated into a multivariate genetic evaluation without the need to convert external information to the internal traits. Therefore, the aim of this study was to test the integration of external EBV and associated REL, expressed on a 305-d basis and genetically correlated with a trait of interest, into a multivariate genetic evaluation using a random regression test-day model for the trait of interest. The approach we used was a multivariate Bayesian approach. Results showed that the integration of external information led to a genetic evaluation for the trait of interest for, at least, animals associated with external information, as accurate as a bivariate evaluation including all available phenotypic information. In conclusion, the multivariate Bayesian approaches have the potential to integrate external information correlated with the internal phenotypic traits, and potentially to the different random regressions, into a multivariate genetic evaluation. This allows the use of different scales, heritabilities, variance components, measurement units, or trait definitions for external and internal traits. However, one possible issue for implementing multivariate Bayesian approaches could be the availability or estimation of genetic correlations between external and internal traits.

Development and validation of an ultra-high performance liquid chromatography-tandem mass spectrometry method to measure creatinine in human urine.

Despite decades of creatinine measurement in biological fluids using a large variety of analytical methods, an accurate determination of this compound remains challenging. Especially with the novel trend to assess biomarkers on large sample sets preserved in biobanks, a simple and fast method that could cope with both a high sample throughput and a low volume of sample is still of interest. In answer to these challenges, a fast and accurate ultra-high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) method was developed to measure creatinine in small volumes of human urine. In this method, urine samples are simply diluted with a basic mobile phase and injected directly under positive electrospray ionization (ESI) conditions, without further purification steps. The combination of an important diluting factor (10(4) times) due to the use of a very sensitive triple quadrupole mass spectrometer (XEVO TQ) and the addition of creatinine-d3 as internal standard completely eliminates matrix effects coming from the urine. The method was validated in-house in 2012 according to the EMA guideline on bioanalytical method validation using Certified Reference samples from the German External Quality Assessment Scheme (G-Equas) proficiency test. All obtained results for accuracy and recovery are within the authorized tolerance ranges defined by G-Equas. The method is linear between 0 and 5 g/L, with LOD and LOQ of 5 × 10(-3) g/L and 10(-2) g/L, respectively. The repeatability (CV(r) = 1.03-2.07%) and intra-laboratory reproducibility (CV(RW) = 1.97-2.40%) satisfy the EMA 2012 guideline. The validated method was firstly applied to perform the German G-Equas proficiency test rounds 51 and 53, in 2013 and 2014, respectively. The obtained results were again all within the accepted tolerance ranges and very close to the reference values defined by the organizers of the proficiency test scheme, demonstrating an excellent accuracy of the developed method. The method was finally applied to measure the creatinine concentration in 210 urine samples, coming from 190 patients with a chronic kidney disease (CKD) and 20 healthy subjects. The obtained creatinine concentrations (ranging from 0.12 g/L up to 3.84 g/L) were compared, by means of a Passing Bablok regression, with the creatinine contents obtained for the same samples measured using a traditional compensated Jaffé method. The UHPLC-MS/MS method described in this paper can be used to normalize the concentration of biomarkers in urine for the extent of dilution.

AKI in early sepsis is a continuum from transient AKI without tubular damage over transient AKI with minor tubular damage to intrinsic AKI with severe tubular damage.

PURPOSE: The pathophysiology of septic acute kidney injury (AKI) is incompletely understood, and there is controversy on the role of renal hypoperfusion in early sepsis. We hypothesized that renal hypoperfusion plays a role in early sepsis and that there is a continuum between transient AKI without tubular damage, transient AKI with minor tubular damage, and intrinsic AKI. METHODS: A total of 107 consecutive patients with sepsis were included. Fractional excretion of sodium (FENa), urinary, and serum neutrophil gelatinase-associated lipocalin were measured at admission (T0) and 4 h (T4) and 24 h later (T24). Patients were classified according to FENa quartiles (FENaQ). Transient and intrinsic AKI were respectively defined as AKI that did or did not recover to no AKI in the following 5 days. RESULTS: A total of 57 developed transient AKI, 22 developed intrinsic AKI, and 28 did not have AKI. Of the ten patients with transient AKI classified in the two lowest FENa quartiles (FENa < 0.36 %) and without signs of local tubular damage, seven still did not show signs of tubular damage 24 h later. Also, 50 % of patients with intrinsic AKI classified in the same FENaQ did not show signs of local tubular damage at admission but did so 24 h later. CONCLUSIONS: There is a continuum between transient AKI without tubular damage, transient AKI with minor tubular damage, and intrinsic AKI in sepsis. Renal hypoperfusion seems to be the instigator for the development of AKI in the majority of patients with early sepsis. Other mechanisms in some patients cannot be excluded.

Genetic parameters for direct and maternal calving ease in Walloon dairy cattle based on linear and threshold models.

Calving ease scores from Holstein dairy cattle in the Walloon Region of Belgium were analysed using univariate linear and threshold animal models. Variance components and derived genetic parameters were estimated from a data set including 33,155 calving records. Included in the models were season, herd and sex of calf × age of dam classes × group of calvings interaction as fixed effects, herd × year of calving, maternal permanent environment and animal direct and maternal additive genetic as random effects. Models were fitted with the genetic correlation between direct and maternal additive genetic effects either estimated or constrained to zero. Direct heritability for calving ease was approximately 8% with linear models and approximately 12% with threshold models. Maternal heritabilities were approximately 2 and 4%, respectively. Genetic correlation between direct and maternal additive effects was found to be not significantly different from zero. Models were compared in terms of goodness of fit and predictive ability. Criteria of comparison such as mean squared error, correlation between observed and predicted calving ease scores as well as between estimated breeding values were estimated from 85,118 calving records. The results provided few differences between linear and threshold models even though correlations between estimated breeding values from subsets of data for sires with progeny from linear model were 17 and 23% greater for direct and maternal genetic effects, respectively, than from threshold model. For the purpose of genetic evaluation for calving ease in Walloon Holstein dairy cattle, the linear animal model without covariance between direct and maternal additive effects was found to be the best choice.

Review of protein-bound toxins, possibility for blood purification therapy.

Protein-bound uremic retention solutes, i.e. phenolic compounds, such as p-cresylsulfate, and indolic compounds, such as indoxyl sulfate, have been intensively studied in recent years and have been shown to be associated especially with cardiovascular toxicity and adverse outcomes in chronic kidney disease. In this review, we will focus on their toxicity and their removal by dialysis strategies, which is hampered due to their protein binding. Hemodiafiltration slightly improves the removal of protein-bound solutes as compared to hemodialysis, although the clinical benefit on outcomes still needs to be demonstrated. Removal by means of absorption and interference with intestinal generation or renal tubular excretion are interesting alternative strategies under investigation.

Uremic toxins inhibit renal metabolic capacity through interference with glucuronidation and mitochondrial respiration.

During chronic kidney disease (CKD), drug metabolism is affected leading to changes in drug disposition. Furthermore, there is a progressive accumulation of uremic retention solutes due to impaired renal clearance. Here, we investigated whether uremic toxins can influence the metabolic functionality of human conditionally immortalized renal proximal tubule epithelial cells (ciPTEC) with the focus on UDP-glucuronosyltransferases (UGTs) and mitochondrial activity. Our results showed that ciPTEC express a wide variety of metabolic enzymes, including UGTs. These enzymes were functionally active as demonstrated by the glucuronidation of 7-hydroxycoumarin (7-OHC; K(m) of 12±2µM and a V(max) of 76±3pmol/min/mg) and p-cresol (K(m) of 33±13µM and a V(max) of 266±25pmol/min/mg). Furthermore, a wide variety of uremic toxins, including indole-3-acetic acid, indoxyl sulfate, phenylacetic acid and kynurenic acid, reduced 7-OHC glucuronidation with more than 30% as compared with controls (p<0.05), whereas UGT1A and UGT2B protein expressions remained unaltered. In addition, our results showed that several uremic toxins inhibited mitochondrial succinate dehydrogenase (i.e. complex II) activity with more than 20% as compared with controls (p<0.05). Moreover, indole-3-acetic acid decreased the reserve capacity of the electron transport system with 18% (p<0.03). In conclusion, this study shows that multiple uremic toxins inhibit UGT activity and mitochondrial activity in ciPTEC, thereby affecting the metabolic capacity of the kidney during CKD. This may have a significant impact on drug and uremic retention solute disposition in CKD patients.

An update on uremic toxins.

In the last decade, uremic toxicity as a potential cause for the excess of cardiovascular disease and mortality observed in chronic kidney disease gained more and more interest. This review focuses on uremic toxins with known cardiovascular effects and their removal. For protein-bound solutes, for example, indoxylsulfate and the conjugates of p-cresol, and for small water-soluble solutes, for example, guanidines, such as ADMA and SDMA, there is a growing evidence for a role in cardiovascular toxicity in vitro (e.g., affecting leukocyte, endothelial, vascular smooth muscle cell function) and/or in vivo. Several middle molecules (e.g., beta-2-microglobulin, interleukin-6, TNF-alpha and FGF-23) were shown to be predictors for cardiovascular disease and/or mortality. Most of these solutes, however, are difficult to remove during dialysis, which is traditionally assessed by studying the removal of urea, which can be considered as a relatively inert uremic retention solute. However, even the effective removal of other small water-soluble toxins than urea can be hampered by their larger distribution volumes. Middle molecules (beta-2-microglobulin as prototype, but not necessarily representative for others) are cleared more efficiently when the pore size of the dialyzer membrane increases, convection is applied and dialysis time is prolonged. Only adding convection to diffusion improves the removal of protein-bound toxins. Therefore, alternative removal strategies, such as intestinal adsorption, drugs interfering with toxic biochemical pathways or decreasing toxin concentration, and extracorporeal plasma adsorption, as well as kinetic behavior during dialysis need further investigation. Even more importantly, randomized clinical studies are required to demonstrate a survival advantage through these strategies.

Advantages of new hemodialysis membranes and equipment.

Chronic kidney disease is characterized by the progressive retention of a number of compounds, several of which have the potential to cause cardiovascular damage. Many of these are difficult to remove by standard dialysis strategies. Removal of the larger middle molecules (mostly larger peptidic compounds) can be obtained by increasing dialyzer pore size and/or by applying convective strategies. For protein-bound solutes, convection (essentially hemodiafiltration) positively affects removal. The HEMO study demonstrated outcome superiority for the large-pore high-flux hemodialysis membranes in a number of subgroup analyses. Likewise, the Membrane Permeability Outcome study showed outcome superiority for high flux in patients with serum albumin <4 g/dl, the group for which the study had originally been designed. Apart from a small controlled trial, data suggesting superiority for convective strategies are all observational.

From uremic toxin retention to removal by convection: do we know enough?

The uremic syndrome is defined by a complex clinical picture, characterized by the dysfunction of most organs which are affected by the retention of multiple solutes. Recent research has helped to unravel the pathophysiology and to identify several as yet unknown responsible compounds. In this publication, we summarize which compounds play the most important pathophysiologic role, and which dialysis strategies can be considered to decrease their concentration and improve outcomes. The main pathophysiologic role is played by molecules which are so-called 'difficult to remove by dialysis'. Essentially observational studies have suggested that enhancement of removal of these molecules, by improving convection (hemodiafiltration), creates an improvement of survival. The knowledge of uremic toxicity is still far from complete however, and we need extra information about responsible compounds and mechanisms, eventually leading to a classification of the most important culprits, to allow the development of even more efficient or specific removal strategies.

Uremic toxins: do we know enough to explain uremia?

BACKGROUND: The uremic syndrome is characterized by a complex clinical picture, whereby the function of multiple organ systems is affected by the retention of a host of solutes. Recent research of the last decade has helped to unravel multiple pathophysiologic mechanisms and to identify as yet unknown responsible compounds. METHODS: The literature was screened to appreciate which compounds play the most important pathophysiologic role. RESULTS: The picture that ensues is that the main role is played by molecules which are so-called 'difficult to remove by dialysis'. The knowledge of uremic toxicity is still far from complete and we need extra information about responsible compounds and mechanisms, eventually leading to a classification of the most important culprits, to allow the development of efficient removal strategies and of pharmacologic methods to counteract pathophysiologic mechanisms. CONCLUSIONS: Uremic retention is a complex phenomenon and the most toxic compounds are difficult to remove by dialysis. Furthermore, our knowledge of the responsible pathways is still incomplete, and needs to be extended to develop new and more efficient treatment strategies.

Impact of hemodialysis duration on the removal of uremic retention solutes.

Several studies have stressed the importance of dialysis time in the removal of uremic retention solutes. To further investigate this, nine stable chronic hemodialysis patients were dialyzed for 4, 6, or 8 h processing the same total blood and dialysate volume by the Genius system and high-flux FX80 dialyzers. Inlet blood and outlet dialysate were analyzed for urea, creatinine, phosphorus, and beta2-microglobulin at various times. Total solute removal, dialyzer extraction ratios, and total cleared volumes were significantly larger during prolonged dialysis for urea, creatinine, phosphorus, and beta2-microglobulin. Reduction ratios increased progressively, except for phosphate and beta2-microglobulin, where the ratios remained constant after 2 h. In contrast, no significant difference was found for the reduction ratios of all solutes and Kt/V(urea) between the three different sessions. With longer dialyses, solutes are efficiently removed from the deeper compartments of the patient's body. Our study shows that care must be taken when using Kt/Vurea or reduction ratios as the only parameters to quantify dialysis adequacy.

Uremic toxins in chronic renal failure.

The uremic syndrome is a complex mixture of organ dysfunctions, which is attributed to the retention of a myriad of compounds that under normal conditions are excreted by healthy kidneys. During recent years major steps have been taken in the area of identification and characterization of uremic retention solutes and in the knowledge of their pathophysiological importance; however, our knowledge remains far from complete. In the present paper the general classification based on their molecular weight and on their protein-binding characteristics, with reflections on their removal, will be discussed. In addition, current knowledge about the main uremic retention products and their clinical and biological effects will be reviewed in detail.

New insights in uremic toxicity.

The uremic syndrome is characterised by the retention of a host of compounds that in healthy subjects are secreted by the kidneys into normal urine. These compounds disturb many physiologic functions, resulting in toxicity. Many of the responsible compounds remain unknown, however, as well as many patho-physiologic actions of the known retention solutes. In this publication, we review recent new information regarding uremic toxicity. Especially difficult to remove compounds, such as protein bound and larger molecules, seem to play a role. New strategies enhancing their removal might be highly useful.

Survival of hemodialysis patients and uremic toxin removal.

Uremic toxin removal based on diffusion and/or convection allows eliminating solutes with negative metabolic impact. Uremic solutes can be classified as small and water-soluble compounds, larger "middle" molecules, or protein bound solutes. The question arises whether more removal of each of these solute classes affects patient survival. Kt/V of urea is currently used as a surrogate for small water-soluble solute removal. There is ample evidence that Kt/V and survival are correlated, but the threshold Kt/V remains a matter of debate. Probably, the actually proposed threshold of 1.2 is too low. This impact of Kt/V is in contradiction with the low toxicity of urea and points to a role for other water-soluble solutes, e.g., potassium. More removal of middle molecules results in a lower morbidity and also in a lower mortality. In addition, a relationship has been demonstrated between the use of membranes with large pore size and a decrease of inflammatory status, by itself an important factor related to mortality. One of the problems is that large pore membranes are at the same time more biocompatible and reflect more dialysate impurities, compared to many small pore membranes, whereas they also reflect more dialysate impurities. It remains uncertain which one of these factors, if any, has a predominant effect. Recent studies point to a separate effect of pore size but await confirmation. Protein bound toxins inhibit several biochemical functions. Their removal pattern is totally different from that of classical markers such as urea. In analogy with drugs, it is essentially the free unbound fraction that exerts biological action; this free fraction is inversely related to serum albumin, another inflammatory marker related to survival. In a final section of this presentation, attention will be drawn to the relationship in uremic patients between inflammation, malnutrition, cardiovascular disease, and mortality, and some of the potential culprits are discussed. Virtually all of these molecules have a high molecular weight or are protein bound. It is concluded that both small and middle molecule removal have an impact on survival, so that more than urea removal alone should be pursued.

Blunted response to vitamin D in uremia.

It has been demonstrated that many chains in the vitamin D pathway are affected by uremia. Uremic solute retention is responsible for changes in calcitriol production, resulting in a net decrease of blood calcitriol levels. This effect contributes to the calcitriol deficiency currently observed in renal failure. Next to the altered production and metabolization of calcitriol, altered expression of the vitamin D receptor (VDR) and altered binding properties of the hormone receptor complex to the DNA could also contribute to the relative status of calcitriol resistance in renal failure. These alterations could finally result in an impairment of the end biological action of calcitriol.

The anti-proliferative effect of calcitriol on HL-60 cells is neutralized by uraemic biological fluids.

BACKGROUND: It has been demonstrated that uraemic serum/ultrafiltrate inhibits cell-mediated immune response in vitro, and that it suppresses calcitriol synthesis and its biological actions. METHODS: In the present in vitro study, the effect of calcitriol, uraemic ultrafiltrate (UUF) and a combination of both on the human promyelocytic leukaemia cell line, HL-60, was studied by evaluating bromodeoxyuridine (BrdU) incorporation into the DNA, luminol-amplified chemiluminescence (CL) production, expression of CD14, and levels of vitamin D receptor mRNA (VDR mRNA) and CD14 mRNA. RESULTS: The ability of calcitriol to block cell proliferation (37.4+/-5.4 to 30.5+/-5.6% cells incorporating BrdU, P:<0.01) was neutralized when UUF was applied together with calcitriol (53.4+/-21.3% cells incorporating BrdU, P:<0.01 vs calcitriol alone). Similarly to what was observed for BrdU incorporation, the CL production of HL-60 cells was enhanced in the presence of calcitriol (20126+/-10154 to 61528+/-24021 cpm, P:<0.01), and was suppressed again in the presence of calcitriol and UUF (20916+/-12075 cpm, P:<0.01 vs calcitriol alone); finally UUF also inhibited the calcitriol-induced CD14 expression (71.1+/-11.2 to 54.9+/-17.7% CD14 positive cells, P:<0.05). On the other hand, the calcitriol-induced CD14 mRNA levels were not significantly different in the presence of calcitriol and UUF compared to calcitriol alone. This points to an inhibition by UUF at a post-transcriptional level. Similar data were found for VDR mRNA levels. UUF was fractionated by HPLC in four fractions, hydrophilic uraemic solutes being eluted first (F1) and hydrophobic solutes being eluted last (F4); fractions 1, 2 and 3 simultaneously affected both BrdU incorporation and CL production in a significant way. CONCLUSIONS: It is concluded that UUF contains factors that impair calcitriol-activated function of HL-60 cells. Hence, the differentiation and immune response of these promyelocytic leukaemia cells, as induced by the supplementation of calcitriol, is neutralized in the presence of uraemic biological fluids. This may be of relevance for the propensity to infection and malignancy of the uraemic patient.