CRISPR Comparison Toolkit: Rapid Identification, Visualization, and Analysis of CRISPR Array Diversity.

CRISPR-Cas systems provide immunity against mobile genetic elements (MGEs) through sequence-specific targeting by spacer sequences encoded in CRISPR arrays. Spacers are highly variable between microbial strains and can be acquired rapidly, making them well suited for use in strain typing of closely related organisms. However, no tools are currently available to automate the process of reconstructing strain histories using CRISPR spacers. We therefore developed the CRISPR Comparison Toolkit (CCTK) to enable analyses of array relationships. The CCTK includes tools to identify arrays, analyze relationships between arrays using CRISPRdiff and CRISPRtree, and predict targets of spacers. CRISPRdiff visualizes arrays and highlights the similarities between them. CRISPRtree infers a phylogenetic tree from array relationships and presents a hypothesis of the evolutionary history of the arrays. The CCTK unifies several CRISPR analysis tools into a single command line application, including the first tool to infer phylogenies from array relationships.

Evaluation of bacterial attachment on mineralized collagen scaffolds and addition of manuka honey to increase mesenchymal stem cell osteogenesis.

The design of biomaterials to regenerate bone is likely to increasingly require modifications that reduce bacterial attachment and biofilm formation as infection during wound regeneration can significantly impede tissue repair and typically requires surgical intervention to restart the healing process. Further, much research on infection prevention in bone biomaterials has focused on modeling of non-resorbable metal alloy materials, whereas an expanding direction of bone regeneration has focused on development of bioresorbable materials. This represents a need for the prevention and understanding of infection in resorbable biomaterials. Here, we investigate the ability of a mineralized collagen biomaterial to natively resist infection and examine how the addition of manuka honey, previously identified as an antimicrobial agent, affects gram positive and negative bacterial colonization and mesenchymal stem cell osteogenesis and vasculature formation. We incorporate manuka honey into these scaffolds via either direct fabrication into the scaffold microarchitecture or via soaking the scaffold in a solution of manuka honey after fabrication. Direct incorporation results in a change in the surface characteristics and porosity of mineralized collagen scaffolds. Soaking scaffolds in honey concentrations higher than 10% had significant negative effects on mesenchymal stem cell metabolic activity. Soaking or incorporating 5% honey had no impact on endothelial cell tube formation. Although solutions of 5% honey reduced metabolic activity of mesenchymal stem cells, MSC-seeded scaffolds displayed increased calcium and phosphorous mineral formation, osteoprotegerin release, and alkaline phosphatase activity. Bacteria cultured on mineralized collagen scaffolds demonstrated surfaces covered in bacteria and no method of preventing infection, and using 10 times the minimal inhibitory concentration of antibiotics did not completely kill bacteria within the mineralized collagen scaffolds, indicating bioresorbable scaffold materials may act to shield bacteria from antibiotics. The addition of 5% manuka honey to scaffolds was not sufficient to prevent P. aeruginosa attachment or consistently reduce the activity of methicillin resistant staphylococcus aureus, and concentrations above 7% manuka honey are likely necessary to impact MRSA. Together, our results suggest bioresorbable scaffolds may create an environment conducive to bacterial growth, and potential trade-offs exist for the incorporation of low levels of honey in scaffolds to increase osteogenic potential of osteoprogenitors while high-levels of honey may be sufficient to reduce gram positive or negative bacteria activity but at the cost of reduced osteogenesis.

Prediction of Prophages and Their Host Ranges in Pathogenic and Commensal Neisseria Species.

The genus Neisseria includes two pathogenic species, N. gonorrhoeae and N. meningitidis, and numerous commensal species. Neisseria species frequently exchange DNA with one another, primarily via transformation and homologous recombination and via multiple types of mobile genetic elements (MGEs). Few Neisseria bacteriophages (phages) have been identified, and their impact on bacterial physiology is poorly understood. Furthermore, little is known about the range of species that Neisseria phages can infect. In this study, we used three virus prediction tools to scan 248 genomes of 21 different Neisseria species and identified 1,302 unique predicted prophages. Using comparative genomics, we found that many predictions are dissimilar from prophages and other MGEs previously described to infect Neisseria species. We also identified similar predicted prophages in genomes of different Neisseria species. Additionally, we examined CRISPR-Cas targeting of each Neisseria genome and predicted prophage. While CRISPR targeting of chromosomal DNA appears to be common among several Neisseria species, we found that 20% of the prophages we predicted are targeted significantly more than the rest of the bacterial genome in which they were identified (i.e., backbone). Furthermore, many predicted prophages are targeted by CRISPR spacers encoded by other species. We then used these results to infer additional host species of known Neisseria prophages and predictions that are highly targeted relative to the backbone. Together, our results suggest that we have identified novel Neisseria prophages, several of which may infect multiple Neisseria species. These findings have important implications for understanding horizontal gene transfer between members of this genus. IMPORTANCE Drug-resistant Neisseria gonorrhoeae is a major threat to human health. Commensal Neisseria species are thought to serve as reservoirs of antibiotic resistance and virulence genes for the pathogenic species N. gonorrhoeae and N. meningitidis. Therefore, it is important to understand both the diversity of mobile genetic elements (MGEs) that can mediate horizontal gene transfer within this genus and the breadth of species these MGEs can infect. In particular, few bacteriophages (phages) are known to infect Neisseria species. In this study, we identified a large number of candidate phages integrated in the genomes of commensal and pathogenic Neisseria species, many of which appear to be novel phages. Importantly, we discovered extensive interspecies targeting of predicted phages by Neisseria CRISPR-Cas systems, which may reflect their movement between different species. Uncovering the diversity and host range of phages is essential for understanding how they influence the evolution of their microbial hosts.

From Input to Output: The Lap/c-di-GMP Biofilm Regulatory Circuit.

Biofilms are the dominant bacterial lifestyle. The regulation of the formation and dispersal of bacterial biofilms has been the subject of study in many organisms. Over the last two decades, the mechanisms of Pseudomonas fluorescens biofilm formation and regulation have emerged as among the best understood of any bacterial biofilm system. Biofilm formation by P. fluorescens occurs through the localization of an adhesin, LapA, to the outer membrane via a variant of the classical type I secretion system. The decision between biofilm formation and dispersal is mediated by LapD, a c-di-GMP receptor, and LapG, a periplasmic protease, which together control whether LapA is retained or released from the cell surface. LapA localization is also controlled by a complex network of c-di-GMP-metabolizing enzymes. This review describes the current understanding of LapA-mediated biofilm formation by P. fluorescens and discusses several emerging models for the regulation and function of this adhesin.

MapA, a Second Large RTX Adhesin Conserved across the Pseudomonads, Contributes to Biofilm Formation by Pseudomonas fluorescens.

Mechanisms by which cells attach to a surface and form a biofilm are diverse and differ greatly among organisms. The Gram-negative gammaproteobacterium Pseudomonas fluorescens attaches to a surface through the localization of the large type 1-secreted RTX adhesin LapA to the outer surface of the cell. LapA localization to the cell surface is controlled by the activities of a periplasmic protease, LapG, and an inner membrane-spanning cyclic di-GMP-responsive effector protein, LapD. A previous study identified a second, LapA-like protein encoded in the P. fluorescens Pf0-1 genome: Pfl01_1463. Here, we identified specific growth conditions under which Pfl01_1463, here called MapA (medium adhesion protein A) is a functional adhesin contributing to biofilm formation. This adhesin, like LapA, appears to be secreted through a Lap-related type 1 secretion machinery, and its localization is controlled by LapD and LapG. However, differing roles of LapA and MapA in biofilm formation are achieved, at least in part, through the differences in the sequences of the two adhesins and different distributions of the expression of the lapA and mapA genes within a biofilm. LapA-like proteins are broadly distributed throughout the Proteobacteria, and furthermore, LapA and MapA are well conserved among other Pseudomonas species. Together, our data indicate that the mechanisms by which a cell forms a biofilm and the components of a biofilm matrix can differ depending on growth conditions and the matrix protein(s) expressed.IMPORTANCE Adhesins are critical for the formation and maturation of bacterial biofilms. We identify a second adhesin in P. fluorescens, called MapA, which appears to play a role in biofilm maturation and whose regulation is distinct from the previously reported LapA adhesin, which is critical for biofilm initiation. Analysis of bacterial adhesins shows that LapA-like and MapA-like adhesins are found broadly in pseudomonads and related organisms, indicating that the utilization of different suites of adhesins may be broadly important in the Gammaproteobacteria.

Ligand-Mediated Biofilm Formation via Enhanced Physical Interaction between a Diguanylate Cyclase and Its Receptor.

The bacterial intracellular second messenger, cyclic dimeric GMP (c-di-GMP), regulates biofilm formation for many bacteria. The binding of c-di-GMP by the inner membrane protein LapD controls biofilm formation, and the LapD receptor is central to a complex network of c-di-GMP-mediated biofilm formation. In this study, we examine how c-di-GMP signaling specificity by a diguanylate cyclase (DGC), GcbC, is achieved via interactions with the LapD receptor and by small ligand sensing via GcbC's calcium channel chemotaxis (CACHE) domain. We provide evidence that biofilm formation is stimulated by the environmentally relevant organic acid citrate (and a related compound, isocitrate) in a GcbC-dependent manner through enhanced GcbC-LapD interaction, which results in increased LapA localization to the cell surface. Furthermore, GcbC shows little ability to synthesize c-di-GMP in isolation. However, when LapD is present, GcbC activity is significantly enhanced (~8-fold), indicating that engaging the LapD receptor stimulates the activity of this DGC; citrate-enhanced GcbC-LapD interaction further stimulates c-di-GMP synthesis. We propose that the I-site of GcbC serves two roles beyond allosteric control of this enzyme: promoting GcbC-LapD interaction and stabilizing the active conformation of GcbC in the GcbC-LapD complex. Finally, given that LapD can interact with a dozen different DGCs of Pseudomonas fluorescens, many of which have ligand-binding domains, the ligand-mediated enhanced signaling via LapD-GcbC interaction described here is likely a conserved mechanism of signaling in this network. Consistent with this idea, we identify a second example of ligand-mediated enhancement of DGC-LapD interaction that promotes biofilm formation.IMPORTANCE In many bacteria, dozens of enzymes produce the dinucleotide signal c-di-GMP; however, it is unclear how undesired cross talk is mitigated in the context of this soluble signal and how c-di-GMP signaling is regulated by environmental inputs. We demonstrate that GcbC, a DGC, shows little ability to synthesize c-di-GMP in the absence of its cognate receptor LapD; GcbC-LapD interaction enhances c-di-GMP synthesis by GcbC, likely mediated by the I-site of GcbC. We further show evidence for a ligand-mediated mechanism of signaling specificity via increased physical interaction of a DGC with its cognate receptor. We envision a scenario wherein a "cloud" of weakly active DGCs can increase their activity by specific interaction with their receptor in response to appropriate environmental signals, concomitantly boosting c-di-GMP production, ligand-specific signaling, and biofilm formation.

A Multimodal Strategy Used by a Large c-di-GMP Network.

The Pseudomonas fluorescens genome encodes more than 50 proteins predicted to be involved in c-di-GMP signaling. Here, we demonstrated that, tested across 188 nutrients, these enzymes and effectors appeared capable of impacting biofilm formation. Transcriptional analysis of network members across ∼50 nutrient conditions indicates that altered gene expression can explain a subset of but not all biofilm formation responses to the nutrients. Additional organization of the network is likely achieved through physical interaction, as determined via probing ∼2,000 interactions by bacterial two-hybrid assays. Our analysis revealed a multimodal regulatory strategy using combinations of ligand-mediated signals, protein-protein interaction, and/or transcriptional regulation to fine-tune c-di-GMP-mediated responses. These results create a profile of a large c-di-GMP network that is used to make important cellular decisions, opening the door to future model building and the ability to engineer this complex circuitry in other bacteria.IMPORTANCE Cyclic diguanylate (c-di-GMP) is a key signaling molecule regulating bacterial biofilm formation, and many microbes have up to dozens of proteins that make, break, or bind this dinucleotide. A major open issue in the field is how signaling specificity is conferred in the unpartitioned space of a bacterial cell. Here, we took a systems approach, using mutational analysis, transcriptional studies, and bacterial two-hybrid analysis to interrogate this network. We found that a majority of enzymes are capable of impacting biofilm formation in a context-dependent manner, and we revealed examples of two or more modes of regulation (i.e., transcriptional control with protein-protein interaction) being utilized to generate an observable impact on biofilm formation.

Requirements for Pseudomonas aeruginosa Type I-F CRISPR-Cas Adaptation Determined Using a Biofilm Enrichment Assay.

CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR-associated protein) systems are diverse and found in many archaea and bacteria. These systems have mainly been characterized as adaptive immune systems able to protect against invading mobile genetic elements, including viruses. The first step in this protection is acquisition of spacer sequences from the invader DNA and incorporation of those sequences into the CRISPR array, termed CRISPR adaptation. Progress in understanding the mechanisms and requirements of CRISPR adaptation has largely been accomplished using overexpression of cas genes or plasmid loss assays; little work has focused on endogenous CRISPR-acquired immunity from viral predation. Here, we developed a new biofilm-based assay system to enrich for Pseudomonas aeruginosa strains with new spacer acquisition. We used this assay to demonstrate that P. aeruginosa rapidly acquires spacers protective against DMS3vir, an engineered lytic variant of the Mu-like bacteriophage DMS3, through primed CRISPR adaptation from spacers present in the native CRISPR2 array. We found that for the P. aeruginosa type I-F system, the cas1 gene is required for CRISPR adaptation, recG contributes to (but is not required for) primed CRISPR adaptation, recD is dispensable for primed CRISPR adaptation, and finally, the ability of a putative priming spacer to prime can vary considerably depending on the specific sequences of the spacer. IMPORTANCE: Our understanding of CRISPR adaptation has expanded largely through experiments in type I CRISPR systems using plasmid loss assays, mutants of Escherichia coli, or cas1-cas2 overexpression systems, but there has been little focus on studying the adaptation of endogenous systems protecting against a lytic bacteriophage. Here we describe a biofilm system that allows P. aeruginosa to rapidly gain spacers protective against a lytic bacteriophage. This approach has allowed us to probe the requirements for CRISPR adaptation in the endogenous type I-F system of P. aeruginosa Our data suggest that CRISPR-acquired immunity in a biofilm may be one reason that many P. aeruginosa strains maintain a CRISPR-Cas system.

Inhibition of the aryl hydrocarbon receptor prevents Western diet-induced obesity. Model for AHR activation by kynurenine via oxidized-LDL, TLR2/4, TGFß, and IDO1.

Obesity is an increasingly urgent global problem, yet, little is known about its causes and less is known how obesity can be effectively treated. We showed previously that the aryl hydrocarbon receptor (AHR) plays a role in the regulation of body mass in mice fed Western diet. The AHR is a ligand-activated nuclear receptor that regulates genes involved in a number of biological pathways, including xenobiotic metabolism and T cell polarization. This study was an investigation into whether inhibition of the AHR prevents Western diet-based obesity. Male C57Bl/6J mice were fed control and Western diets with and without the AHR antagonist α-naphthoflavone or CH-223191, and a mouse hepatocyte cell line was used to delineate relevant cellular pathways. Studies are presented showing that the AHR antagonists α-naphthoflavone and CH-223191 significantly reduce obesity and adiposity and ameliorates liver steatosis in male C57Bl/6J mice fed a Western diet. Mice deficient in the tryptophan metabolizing enzyme indoleamine 2,3-dioxygenase 1 (IDO1) were also resistant to obesity. Using an AHR-directed, luciferase-expressing mouse hepatocyte cell line, we show that the transforming growth factor ß1 (TGFß1) signaling pathway via PI3K and NF-κB and the toll-like receptor 2/4 (TLR2/4) signaling pathway stimulated by oxidized low-density lipoproteins via NF-κB, each induce luciferase expression; however, TLR2/4 signaling was significantly reduced by inhibition of IDO1. At physiological levels, kynurenine but not kynurenic acid (both tryptophan metabolites and known AHR agonists) activated AHR-directed luciferase expression. We propose a hepatocyte-based model, in which kynurenine production is increased by enhanced IDO1 activity stimulated by TGFß1 and TLR2/4 signaling, via PI3K and NF-κB, to perpetuate a cycle of AHR activation to cause obesity; and inhibition of the AHR, in turn, blocks the cycle's output to prevent obesity. The AHR with its broad ligand binding specificity is a promising candidate for a potentially simple therapeutic approach for the prevention and treatment of obesity and associated complications.

Contribution of Physical Interactions to Signaling Specificity between a Diguanylate Cyclase and Its Effector.

UNLABELLED: Cyclic diguanylate (c-di-GMP) is a bacterial second messenger that controls multiple cellular processes. c-di-GMP networks have up to dozens of diguanylate cyclases (DGCs) that synthesize c-di-GMP along with many c-di-GMP-responsive target proteins that can bind and respond to this signal. For such networks to have order, a mechanism(s) likely exists that allow DGCs to specifically signal their targets, and it has been suggested that physical interactions might provide such specificity. Our results show a DGC from Pseudomonas fluorescens physically interacting with its target protein at a conserved interface, and this interface can be predictive of DGC-target protein interactions. Furthermore, we demonstrate that physical interaction is necessary for the DGC to maximally signal its target. If such "local signaling" is a theme for even a fraction of the DGCs used by bacteria, it becomes possible to posit a model whereby physical interaction allows a DGC to directly signal its target protein, which in turn may help curtail undesired cross talk with other members of the network. IMPORTANCE: An important question in microbiology is how bacteria make decisions using a signaling network made up of proteins that make, break, and bind the second messenger c-di-GMP, which is responsible for controlling many cellular behaviors. Previous work has shown that a given DGC enzyme will signal for specific cellular outputs, despite making the same diffusible molecule as its sibling DGCs in the unpartitioned space of the bacterial cell. Understanding how one DGC differentiates its output from the dozens of other such enzymes in the cell is synonymous with understanding a large component of the bacterial decision-making machinery. We present evidence for a helix on a DGC used to physically associate with its target protein, which is necessary to achieve maximal signaling.

Chronic kidney disease, prevalence of premature cardiovascular disease, and relationship to short-term mortality.

BACKGROUND: Chronic kidney disease (CKD) is recognized as an independent cardiovascular disease (CVD) risk state, particularly in the elderly, and has been defined by levels of estimated glomerular filtration rate (eGFR) and markers of kidney damage. The relationship between CKD and CVD in younger and middle-aged adults has not been fully explored. METHODS: Community volunteers completed surveys regarding past medical events and underwent blood pressure and laboratory testing. Chronic kidney disease was defined as an eGFR <60 mL x min(-1) x 1.73 m(-2) or urine albumin-creatinine ratio (ACR) > or =30 mg/g. Premature CVD was defined as self-reported myocardial infarction or stroke at <55 years of age in men and <65 years of age in women. Mortality was ascertained by linkage to national data systems. RESULTS: Of 31 417 participants, the mean age was 45.1 +/- 11.2 years, 75.5% were female, 36.8% African American, and 21.6% had diabetes. A total of 20.6% were found to have CKD, with the ACR and eGFR being the dominant positive screening tests in the younger and older age deciles, respectively. The prevalences of premature myocardial infarction (MI), stroke, or death, and the composite were 5.3%, 4.7%, 0.8%, 9.2%, and 2.5%, 2.2%, 0.2%, 4.2% for those with and without CKD, respectively (P < .0001 for composite). Multivariable analysis found CKD (OR 1.44, 95% CI 1.27-1.63), age (OR 1.05 [per year], 95% CI 1.04-1.06), hypertension (OR 1.61, 95% CI 1.40-1.84), diabetes (OR 2.03, 95% CI 1.79-2.29), smoking (OR 1.91, 95% CI 1.66-2.21), and less than high school education (OR 1.59, 95% CI 1.37-1.85) as the most significantly associated factors for premature CVD or death (all P < .0001). Survival analysis found those with premature MI or stroke and CKD had the poorest short-term survival over the next 3 years after screening. CONCLUSIONS: Chronic kidney disease is an independent predictor of MI, stroke, and death among men and women younger than age 55 and 65 years, respectively. These data suggest the biologic changes that occur with kidney failure promote CVD at an accelerated rate that cannot be fully explained by conventional risk factors or older age. Screening for CKD by using both the ACR and eGFR can identify younger and middle-aged individuals at high risk for premature CVD and near-term death.

Independent components of chronic kidney disease as a cardiovascular risk state: results from the Kidney Early Evaluation Program (KEEP).

BACKGROUND: The relationships of anemia, microalbuminuria, and estimated glomerular filtration rate (eGFR) with cardiovascular disease (CVD) and subsequent death are not fully understood. We hypothesized that each of these chronic kidney disease-related measures would have an independent relationship with CVD. METHODS: A cohort of 37 153 persons screened in the National Kidney Foundation's Kidney Early Evaluation Program were followed up for a median of 16.0 months (range, 0.2-47.5 months). Participants were volunteers who completed surveys regarding past medical events and who underwent blood pressure and laboratory testing. Estimated glomerular filtration rate was computed using a 4-variable equation. Mortality was ascertained by linkage to national data systems. RESULTS: Of 37 153 persons, the mean +/- SD age was 52.9 +/- 15.9 years, and 68.7% were female. A total of 1835 (4.9%) had a self-reported history of myocardial infarction, 1336 (3.6%) had a history of stroke, and 2897 (7.8%) had a history of myocardial infarction or stroke. Multivariate analysis controlling for age demonstrated that the following were independently associated with CVD: male sex (odds ratio [OR], 1.64; P<.001), smoking (OR, 1.73; P<.001), body mass index (OR, 1.01; P = .03), diabetes mellitus (OR, 1.66; P<.001), hypertension (OR, 1.77; P<.001), eGFR of 30 to 59 mL/min per 173 m(3) (OR, 1.37; P = .001), hemoglobin level of 12.8 g/dL or less (OR, 1.45; P<.001), and microalbuminuria of greater than 30 mg/L (OR, 1.28; P = .01). Survival analysis found CVD (OR, 3.02; P = .003), chronic kidney disease (OR, 1.98; P = .05), and the combination (OR, 3.80; P<.001) to be independent predictors of mortality. Persons with a combination of all 3 chronic kidney disease measures (anemia, microalbuminuria, and eGFR of <60 mL/min per 1.73 m(2)) had the lowest survival of about 93% by the end of 30 months. CONCLUSION: Anemia, eGFR, and microalbuminuria were independently associated with CVD, and when all 3 were present, CVD was common and survival was reduced.

Octogenarians and nonagenarians starting dialysis in the United States.

BACKGROUND: The elderly constitute the fastest-growing segment of the end-stage renal disease (ESRD) population, but the epidemiology and outcomes of dialysis among the very elderly, that is, those 80 years of age and older, have not been previously examined at a national level. OBJECTIVE: To describe recent trends in the incidence and outcomes of octogenarians and nonagenarians starting dialysis. DESIGN: Observational study. SETTING: U.S. Renal Data System, a comprehensive, national registry of patients with ESRD. PARTICIPANTS: Octogenarians and nonagenarians initiating dialysis between 1996 and 2003. MEASUREMENTS: Rates of dialysis initiation and survival. RESULTS: The number of octogenarians and nonagenarians starting dialysis increased from 7054 persons in 1996 to 13,577 persons in 2003, corresponding to an average annual increase in dialysis initiation of 9.8%. After we accounted for population growth, the rate of dialysis initiation increased by 57% (rate ratio, 1.57 [95% CI, 1.53 to 1.62]) between 1996 and 2003. One-year mortality for octogenarians and nonagenarians after dialysis initiation was 46%. Compared with octogenarians and nonagenarians initiating dialysis in 1996, those starting dialysis in 2003 had a higher glomerular filtration rate and less morbidity related to chronic kidney disease but no difference in 1-year survival. Clinical characteristics strongly associated with death were older age, nonambulatory status, and more comorbid conditions. LIMITATIONS: Survival of patients with incident ESRD who did not begin dialysis could not be assessed. CONCLUSIONS: The number of octogenarians and nonagenarians initiating dialysis has increased considerably over the past decade, while overall survival for patients on dialysis remains modest. Estimates of prognosis based on patient characteristics, when considered in conjunction with individual values and preferences, may aid in dialysis decision making for the very elderly.

Relationship of phosphorus and calcium-phosphorus product with mortality in CKD.

BACKGROUND: Abnormalities of mineral metabolism are prevalent in patients with kidney failure and are associated with increased risk for cardiovascular events. There are limited data investigating relationships of phosphorus and calcium-phosphorus product with outcomes in patients with chronic kidney disease (CKD) stages 3 to 4. METHODS: Serum phosphorus and calcium were measured at baseline in 840 participants from the randomized cohort of the Modification of Diet in Renal Disease Study. Survival status until December 31, 2000, was obtained from the National Death Index. Cox models were performed to assess the relationship of serum phosphorus level and calcium-phosphorus product with all-cause and cardiovascular disease (CVD) mortality. RESULTS: Mean serum phosphorus level was 3.8 +/- 0.7 mg/dL (1.23 +/- 0.23 mmol/L), calcium-phosphorus product was 34.7 +/- 6.3 mg2/dL2, and glomerular filtration rate (GFR) was 33 +/- 12 mL/min/1.73 m2 (0.55 +/- 0.20 mL/s/1.73 m2). All-cause and CVD mortality rates were 25% and 15%. Serum phosphorus level was not related to all-cause mortality in multivariable models (P = 0.46). In unadjusted analysis, serum phosphorus level was associated with (hazard ratio [HR] per 1 mg/dL increase, 1.34; 95% confidence interval [CI], 1.05 to 1.71; P = 0.02) increased risk for CVD mortality, but this association was partly attenuated and not statistically significant after adjustment for GFR and other confounders (HR, 1.27; 95% CI, 0.94 to 1.73; P = 0.12). Calcium-phosphorus product was not associated with all-cause mortality in unadjusted (P = 0.23) or multivariate analysis (P = 0.35). Calcium-phosphorus product was related to CVD mortality in unadjusted (HR per 10 mg2/dL2 increase, 1.30; 95% CI, 1.01 to 1.69; P = 0.04) analysis, but this association was not statistically significant after adjustment for GFR and other confounders (HR, 1.22; 95% CI, 0.89 to 1.66; P = 0.23). CONCLUSION: In the Modification of Diet in Renal Disease Study cohort, serum phosphorus level and calcium-phosphorus product were not statistically associated with all-cause or CVD mortality after adjustment for GFR; however larger studies with additional statistical power are needed to evaluate these relationships, especially in the context of current practice patterns in patients with CKD.

C-reactive protein and albumin as predictors of all-cause and cardiovascular mortality in chronic kidney disease.

BACKGROUND: High C-reactive protein (CRP) and hypoalbuminemia are associated with increased risk of mortality in patients with kidney failure. There are limited data evaluating the relationships between CRP, albumin, and outcomes in chronic kidney disease (CKD) stages 3 and 4. METHODS: The Modification of Diet in Renal Disease (MDRD) Study was a randomized controlled trial conducted between 1989 and 1993. CRP was measured in frozen samples taken at baseline. Survival status and cause of death, up to December 31, 2000, were obtained from the National Death Index. Multivariable Cox models were used to examine the relationship of CRP [stratified into high CRP > or =3.0 mg/L (N= 414) versus low CRP<3.0 mg/L (N= 283)], and serum albumin, with all-cause and cardiovascular mortality. RESULTS: Median follow-up time was 125 months, all-cause mortality was 20% (N= 138) and cardiovascular mortality was 10% (N= 71). In multivariable analyses adjusting for demographic, cardiovascular and kidney disease factors, both high CRP (HR, 95% CI = 1.56, 1.07-2.29) and serum albumin (HR = 0.94 per 0.1 g/dL increase, 95% CI = 0.89-0.99) were independent predictors of all-cause mortality. High CRP (HR 1.94, 95% CI 1.13-3.31), but not serum albumin (HR 0.94, 95% CI 0.87-1.02), was an independent predictor of cardiovascular mortality. CONCLUSION: Both high CRP and low albumin, measured in CKD stages 3 and 4, are independent risk factors for all-cause mortality. High CRP, but not serum albumin, is a risk factor for cardiovascular mortality. These results suggest that high CRP and hypoalbuminemia provide prognostic information independent of each other in CKD.