Dialysis and transplantation among Aboriginal children with kidney failure

Background:

Relatively little is known about the management and outcomes of Aboriginal children with renal failure in Canada. We evaluated differences in dialysis modality, time spent on dialysis, rates of kidney transplantation, and patient and allograft survival between Aboriginal children and non-Aboriginal children.

Methods:

For this population-based cohort study, we used data from a national pediatric end-stage renal disease database. Patients less than 18 years old who started renal replacement treatment (dialysis or kidney transplantation) in nine Canadian provinces (Quebec data were not available) and all three territories between 1992 and 2007 were followed until death, loss to follow-up or end of the study period. We compared initial modality of dialysis and time to first kidney transplant between Aboriginal children, white children and children of other ethnicity. We examined the association between ethnicity and likelihood of kidney transplantation using adjusted Cox proportional hazard models for Aboriginal and white children (data for the children of other ethnicity did not meet the assumptions of proportional hazards).

Results:

Among 843 pediatric patients included in the study, 104 (12.3%) were Aboriginal, 521 (61.8%) were white, and 218 (25.9%) were from other ethnic minorities. Hemodialysis was the initial modality of dialysis for 48.0% of the Aboriginal patients, 42.7% of the white patients and 62.6% of those of other ethnicity (p < 0.001). The time from start of dialysis to first kidney transplant was longer among the Aboriginal children (median 1.75 years, interquartile range 0.69–2.81) than among the children in the other two groups (p < 0.001). After adjustment for confounders, Aboriginal children were less likely than white children to receive a transplant from a living donor (hazard ratio [HR] 0.36, 95% confidence interval [CI] 0.21–0.61) or a transplant from any donor (HR 0.54, 95% CI 0.40–0.74) during the study period.

Interpretation:

The time from start of dialysis to first kidney transplant was longer among Aboriginal children than among white children. Further evaluation is needed to determine barriers to transplantation among Aboriginal children.Compared with non-Aboriginal people, Aboriginal adults with end-stage renal disease in Canada have lower rates of kidney transplantation, the optimal treatment for renal failure.^1–4 Most studies to date that have examined health outcomes among Canadian Aboriginal people with kidney disease have focused on adults.^1–8 Relatively little is known about the outcomes among Aboriginal children with renal failure. A single-centre cohort study from the province of British Columbia reported that Aboriginal children who received a kidney transplant had similar short-term, but poorer long-term allograft survival than white children.⁹ No further studies have examined differences in modality of renal replacement treatment or the likelihood of kidney transplantation among Aboriginal children with renal failure.We performed an observational cohort study of children beginning renal replacement treatment in Canada. We compared differences in dialysis modality, time spent on dialysis, rates of kidney transplantation, and graft and patient survival between Aboriginal children, white children and children of other ethnicities.     

Topological effects on the electrophoretic mobility of rigid rodlike DNA in polyacrylamide gels

The electrophoretic migration of rigid rodlike DNA structures with well defined topologies has been investigated in polyacrylamide (PA) hydrogels prepared by copolymerization of acrylamide and N, N'-methylenebisacrylamide. Previous studies have reported structural and dynamic characteristics of linear and branched DNA during electrophoresis in PA gels using a variety of experimental parameters. However, a thorough investigation aimed at establishing specific relationships between topological features of rigid rodlike DNA structures and their electrophoretic behavior is still needed. In order to study these topological effects on mobility, an intensive examination of the electrophoretic mobility of small linear and starlike DNA was performed. A series of model DNA structures with well-defined branched topologies were synthesized with varying molecular parameters, such as number of arms surrounding the branch point and arm length. The electrophoretic mobility of these structures was then contrasted with a series of data obtained using linear DNA of comparable molecular size. When large DNA stars (M >/= 60 bp) were compared with linear DNA of identical molecular weight, the Ferguson plots were quite different. However, small DNA stars (24-32 bp) and linear analogues had identical Ferguson plots. This indicates that a different motional mode or greater interaction with the gel exists for the larger DNA stars. When the total molecular weight of the DNA stars was held constant and the number of arms varied, the Ferguson plots for all the stars were identical. Additionally, a critical pore size was reached when the ratio of linear DNA mobility to star DNA mobility increased dramatically. Thus, while the incorporation of a single branch point can produce a large reduction in mobility, above a critical molecular size, the incorporation of additional branch points does not appear to provide further reduction in mobility. This finding is consistent with the transport properties of large synthetic star polymers, where a large reduction in their diffusion coefficient is observed when a single branch is added. When additional arms are incorporated, large synthetic stars do not display an appreciable further reduction in diffusion coefficient. The effect of arm length on mobility for rigid rod DNA stars was also studied. For four-arm DNA stars, the mobility was found to scale as an exponential function of the arm length. Finally, a recently proposed phenomenological model was used to successfully fit the mobility data for linear rigid rod DNA at various concentrations of PA.     

Tyrosinase activity in the larva of the fleshfly, Sarcophaga barbarta

When plasma from third instar larvae of the fleshfly, Sarcophaga barbarta, was diluted tenfold with distilled water, lipoproteins precipitated out. After centrifuging, the water supernatant was rendered 30, 50, and 65% to ammonium sulphate, and it was found that the 50% fraction contained 95% of the tyrosinase activity in all the fractions, the enzyme being present in its inactive form or proenzyme. The proenzyme was activated by mixing it with activator isolated from the larval cuticle. After addition of activator there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme but was independent of the activator concentration.The level of proenzyme in the plasma rose steadily throughout the third larval instar reaching a maximum in 7 day larvae, formation of the puparium commencing about 24 hr later, the rounded-off white stage (r.o.). At the r.o. and golden-brown stage (1 hr later) the level was still maximal, but 12 hr later at the dark-brown puparial stage no proenzyme was isolatable from the plasma, all the enzyme at this stage behaving as active enzyme.The vast majority (95%) of the proenzyme isolated from plasma in the larval stages and at the r.o. white stage was present in the 50% ammonium sulphate fraction, whereas 1 hr later at the golden-brown stage only 33% of the proenzyme was found in the 50% fraction, 62% now being found in the 65% fraction. At the dark-brown puparial stage 12 hr later, not only was there a further redistribution, but all the enzyme behaved as active enzyme. It is suggested that these changes in the distribution and behaviour of the proenzyme indicate that, in vivo, activation of the enzyme in the blood has taken place over the period r.o. white to the golden-brown to dark-brown puparial stage.     

Cdc42p Is Activated during Vacuole Membrane Fusion in a Sterol-dependent Subreaction of Priming

Cdc42p is a Rho GTPase that initiates signaling cascades at spatially defined intracellular sites for many cellular functions. We have previously shown that Cdc42p is localized to the yeast vacuole where it initiates actin polymerization during membrane fusion. Here we examine the activation cycle of Cdc42p during vacuole membrane fusion. Expression of either GTP- or GDP-locked Cdc42p mutants caused several morphological defects including enlarged cells and fragmented vacuoles. Stimulation of multiple rounds of fusion enhanced vacuole fragmentation, suggesting that cycles of Cdc42p activation, involving rounds of GTP binding and hydrolysis, are required to propagate Cdc42p signaling. We developed an assay to directly examine Cdc42p activation based on affinity to a probe derived from the p21-activated kinase effector, Ste20p. Cdc42p was rapidly activated during vacuole membrane fusion, which kinetically coincided with priming subreaction. During priming, Sec18p ATPase activity dissociates SNARE complexes and releases Sec17p, however, priming inhibitors such as Sec17p and Sec18p ligands did not block Cdc42p activation. Therefore, Cdc42p activation seems to be a parallel subreaction of priming, distinct from Sec18p activity. Specific mutants in the ergosterol synthesis pathway block both Sec17p release and Cdc42p activation. Taken together, our results define a novel sterol-dependent subreaction of vacuole priming that activates cycles of Cdc42p activity to facilitate membrane fusion.     

Stimulation of actin polymerization by vacuoles via Cdc42p-dependent signaling

We have previously shown that actin ligands inhibit the fusion of yeast vacuoles in vitro, which suggests that actin remodeling is a subreaction of membrane fusion. Here, we demonstrate the presence of vacuole-associated actin polymerization activity, and its dependence on Cdc42p and Vrp1p. Using a sensitive in vitro pyrene-actin polymerization assay, we found that vacuole membranes stimulated polymerization, and this activity increased when vacuoles were preincubated under conditions that support membrane fusion. Vacuoles purified from a VRP1-gene deletion strain showed reduced polymerization activity, which could be recovered when reconstituted with excess Vrp1p. Cdc42p regulates this activity because overexpression of dominant-negative Cdc42p significantly reduced vacuole-associated polymerization activity, while dominant-active Cdc42p increased activity. We also used size-exclusion chromatography to directly examine changes in yeast actin induced by vacuole fusion. This assay confirmed that actin undergoes polymerization in a process requiring ATP. To further confirm the need for actin polymerization during vacuole fusion, an actin polymerization-deficient mutant strain was examined. This strain showed in vivo defects in vacuole fusion, and actin purified from this strain inhibited in vitro vacuole fusion. Affinity isolation of vacuole-associated actin and in vitro binding assays revealed a polymerization-dependent interaction between actin and the SNARE Ykt6p. Our results suggest that actin polymerization is a subreaction of vacuole membrane fusion governed by Cdc42p signal transduction.     

Haemolymphal activation of protyrosinase and the site of synthesis of haemolymph protyrosinase in larvae of the fleshfly, Sarcophaga barbata

When whole blood from 5 day third instar larvae of the fleshfly, Sarcophaga barbata was incubated under nitrogen at 25°C for 16 hr in the presence of salivary glands there was an increase in its protyrosinase content, which amounted to 53% of that which occurs in vivo over the same period. The protyrosinase in ammonium sulphate fractions of haemolymph that were allowed to stand at 4°C for 24 hr following the incubation at 25°C was found to have autoactivated. Analysis of all these fractions revealed the presence of a protyrosinase activator in the 30% saturated ammonium sulphate fraction. When proenzyme and haemolymph activator were mixed there followed a lag period before the rapid phase of activation, the duration of the lag being dependent upon the concentration of both proenzyme and activator. The final activity attained was dependent upon the concentration of proenzyme, but was independent of the activator concentration and was comparable to that obtained using the cuticle activator. The level of activator in the haemolymph increased as larvae aged from 4 to 7 days.The effect of several compounds on the catecholase activity of the activated haemolymph protyrosinase and on the cuticle enzyme is reported and the significance of haemolymphal activation of protyrosinase is discussed.     

Epidemiology of severe trauma among status Aboriginal Canadians: a population-based study

Background

Aboriginal Canadians are considered to be at increased risk of major trauma. However, population-based studies characterizing the distribution, determinants and outcomes of major trauma in this group are lacking. We sought to measure the impact of ethnicity, as reflected by Aboriginal status, on the incidence of severe trauma and to broadly define the epidemiologic characteristics of severe trauma among status Aboriginal Canadians in a large health region.

Methods

This population-based, observational study involves all adults (people ≥ 16 years) resident in the Calgary Health Region between Apr. 1, 1999, and Mar. 31, 2002. Stratification of the population into status Aboriginal Canadians and the reference population was performed by Alberta Health and Wellness using an alternate premium arrangement field within the personal health care number. Injury incidence was determined by identifying all injuries with severity scores of 12 or greater in the Alberta Trauma Registry, regional corporate data and the Office of the Medical Examiner.

Results

Aboriginal Canadians were at much higher risk than the reference population in the Calgary Health Region of sustaining severe trauma (257.2 v. 68.8 per 100 000; relative risk [RR] 3.7, 95% confidence interval [CI] 3.0–4.6). Aboriginal Canadians were found to be at significantly increased risk of injuries resulting from motor vehicle crashes (RR 4.8, 95% CI 3.5–6.5), assault (RR 11.1, 95% CI 6.2–18.6) and traumatic suicide (RR 3.1, 95% CI 1.4–6.1). A trend toward higher median injury severity scores was observed among Aboriginal Canadians (21 v. 18, p = 0.09). Although the case-fatality rate among Aboriginal Canadians was less than half that in the reference population (14/93 [15%] v. 531/1686 [31%], p < 0.0001), population mortality was almost 2 times greater (RR = 1.8, 95% CI 1.0–3.0, p = 0.046).

Interpretation

Severe trauma disproportionately affects Aboriginal Canadians.In Canada, injury is the leading cause of death among people under the age of 45 and the leading cause of potential years of life lost.¹ Although difficult to quantify, the cost of injury was estimated to be at least $12.7 billion in 1998.² Trauma has been known, even in industrialized countries, to disproportionately affect the most marginalized members of society.³ Aboriginal Canadians are considered to be particularly at risk, and data showing alarming patterns of trauma mortality in this group are beginning to emerge. Unfortunately, the number of studies looking at injury risk among Aboriginal Canadians is small,⁴ and little attention has been paid to quantifying the risk of nonfatal injury. Better understanding of the nature of trauma risk and outcome among Aboriginal Canadians could lead to more effective prevention and treatment strategies.In this study, we used a population-based design in an attempt to quantify the impact of injury, both fatal and nonfatal, on the Aboriginal community in a large, heterogeneous Canadian region with over 1 million urban and rural inhabitants. We sought to measure the impact of ethnicity (defined by registered status within the definition of the Indian Act⁵) on the incidence of severe trauma and to broadly define the epidemiologic characteristics of severe trauma among status Aboriginal Canadians.     

T4 DNA ligase is more than an effective trap of cyclized dsDNA

T4 DNA ligase is used in standard cyclization assays to trap double-stranded DNA (dsDNA) in low-probability, cyclic or highly bent conformations. The cyclization probability, deduced from the relative yield of cyclized product, can be used in conjunction with statistical mechanical models to extract the bending stiffness of dsDNA. By inserting the base analog 2-aminopurine (2-AP) at designated positions in 89bp and 94bp dsDNA fragments, we find that T4 DNA ligase can have a previously unknown effect. Specifically, we observe that addition of T4 ligase to dsDNA in proportions comparable to what is used in the cyclization assay leads to a significant increase in fluorescence from 2-AP. This effect is believed to originate from stabilization of local base-pair opening by formation of transient DNA-ligase complexes. Non-specific binding of T4 ligase to dsDNA is also confirmed using fluorescence correlation spectroscopy (FCS) experiments, which reveal a systematic reduction of dsDNA diffusivity in the presence of ligase. ATP competes with regular DNA for non-covalent binding to the T4 ligase and is found to significantly reduce DNA-ligase complexation. For short dsDNA fragments, however, the population of DNA-ligase complexes at typical ATP concentrations used in DNA cyclization studies is determined to be large enough to dominate the cyclization reaction.