Renal function and cardiovascular risk markers in a remote Australian Aboriginal community

Background. Australian Aborigines living in remote areas have exceedingly high rates of renal failure together with increased cardiovascular morbidity and mortality. To examine the basis of this association, we studied markers of renal function and cardiovascular (CV) risk in a coastal Aboriginal community in a remote area of the Northern Territory of Australia. End-stage renal disease (ESRD) incidence rates in that community are 15 times the national non-Aboriginal rate and CV mortality rates in the region are increased 5-fold. Methods. A cross-sectional community survey was conducted. Markers of early renal disease examined included urine albumin/creatinine ratio (ACR), serum creatinine concentration and calculated glomerular filtration rate (GFR). CV risk markers included blood pressure as well as measures of glycaemia, diabetes and serum lipids. Results. The study group included 237 people, 58% of the adult population of the community. The crude prevalence of microalbuminuria (urine ACR: 3.4-33.9 g/mol, 30-299 mg/g) was 31% and of overt albuminuria (urine ACR: greater than or equal to34 g/mol, greater than or equal to300 mg/g), 13%. The prevalence of overt albuminuria increased with age, but the prevalence of microalbuminuria was greatest in the 45-54 year age group. Microalbuminuria was associated with increasing body mass index, whereas overt albuminuria was associated with increasing glycated haemoglobin (HbA1c) and systolic blood pressure and a history of diabetes. The prevalence of elevated serum creatinine concentration (greater than or equal to120 mumol/l) was 10%. GFR (calculated using the MDRD equation) was <60 ml/min/1.73m(2) in 12% and 60-79 ml/min/1.73 m(2) in a further 36% of the study population. Although many people with albuminuria had well preserved GFRs, mean GFR was lower in people with higher levels of albuminuria. Conclusions. The high prevalence of markers of renal disease in this community was consistent with their high rates of ESRD. The distribution of microalbuminuria suggested a 'cohort effect', representing a group who will progress to overt albuminuria. The powerful association of renal disease markers with CV risk factors confirms a strong link between renal and CV disease in the early, asymptomatic stages of each. Thus, pathologic albuminuria, in part, might be a manifestation of the metabolic/haemodynamic syndrome and both conditions might arise out of a common menu of risk factors. Hence, a single agenda of primary and secondary intervention may benefit both.

Angiotensin II facilitates autoregulation in the perfused mouse kidney: An optimized in vitro model for assessment of renal vascular and tubular function

Background and Aims: We have optimized the isolated perfused mouse kidney (IPMK) model for studying renal vascular and tubular function in vitro using 24-28 g C57BL6J mice; the wild type controls for many transgenic mice. Methods and Results: Buffer composition was optimized for bovine serum albumin concentration (BSA). The effect of adding erythrocytes on renal function and morphology was assessed. Autoregulation was investigated during stepped increases in perfusion pressure. Perfusion for 60 min at 90-110 mmHg with Krebs bicarbonate buffer containing 5.5% BSA, and amino acids produced functional parameters within the in vivo range. Erythrocytes increased renal vascular resistance (3.8 +/- 0.2 vs 2.4 +/- 0.1 mL/min.mmHg, P < 0.05), enhanced sodium reabsorption (FENa = 0.3 +/- 0.08 vs 1.5 +/- 0.7%, P < 0.05), produced equivalent glomerular filtration rates (GFR; 364 +/- 38 vs 400 +/- 9 muL/min per gkw) and reduced distal tubular cell injury in the inner stripe (5.8 +/- 1.7 vs 23.7 +/- 3.1%, P < 0.001) compared to cell free perfusion. The IPMK was responsive to vasoconstrictor (angiotensin II, EC50 100 pM) and vasodilator (methacholine, EC50 75 nM) mediators and showed partial autoregulation of perfusate flow under control conditions over 65-85 mmHg; autoregulatory index (ARI) of 0.66 +/- 0.11. Angiotensin II (100 pM) extended this range (to 65-120 mmHg) and enhanced efficiency (ARI 0.21 +/- 0.02, P < 0.05). Angiotensin II facilitation was antagonized by methacholine (ARI 0.76 +/- 0.08) and papaverine (ARI 0.91 +/- 0.13). Conclusion: The IPMK model is useful for studying renal physiology and pathophysiology without systemic neurohormonal influences.

Expression and function of neuronal nicotinic ACh receptors in rat microvascular endothelial cells

The expression and function of nicotinic ACh receptors (nAChRs) in rat coronary microvascular endothelial cells (CMECs) were examined using RT-PCR and whole cell patch-clamp recording methods. RT-PCR revealed expression of mRNA encoding for the subunits alpha(2), alpha(3), alpha(4), alpha(5), alpha(7), beta(2), and beta(4) but not beta(3). Focal application of ACh evoked an inward current in isolated CMECs voltage clamped at negative membrane potentials. The current-voltage relationship of the ACh-induced current exhibited marked inward rectification and a reversal potential (E-rev) close to 0 mV. The cholinergic agonists nicotine, epibatidine, and cytisine activated membrane currents similar to those evoked by ACh. The nicotine-induced current was abolished by the neuronal nAChR antagonist mecamylamine. The direction and magnitude of the shift in E-rev of nicotine-induced current as a function of extracellular Na+ concentration indicate that the nAChR channel is cation selective and follows that predicted by the Goldman-Hodgkin-Katz equation assuming K+/Na+ permeability ratio of 1.11. In fura-2-loaded CMECs, application of ACh, but not of nicotine, elicited a transient increase in intracellular free Ca2+ concentration. Taken together, these results demonstrate that neuronal nAChR activation by cholinergic agonists evokes an inward current in CMECs carried primarily by Na+, which may contribute to the plasma nicotine-induced changes in microvascular permeability and reactivity induced by elevations in plasma nicotine.

Disturbed microtubule function and induction of micronuclei by chelate complexes of mercury(II)

Interactions of mercury(II) with the microtubule network of cells may lead to genotoxicity. Complexation of mercury(II) with EDTA is currently being discussed for its employment in detoxification processes of polluted sites. This prompted us to re-evaluate the effects of such complexing agents on certain aspects of mercury toxicity, by examining the influences of mercury(H) complexes on tubulin assembly and kinesin-driven motility of microtubules. The genotoxic effects were studied using the micronucleus assay in V79 Chinese hamster fibroblasts. Mercury(II) complexes with EDTA and related chelators interfered dose-dependently with tubulin assembly and microtubule motility in vitro. The no-effect-concentration for assembly inhibition was 1muM of complexed Hg(II), and for inhibition of motility it was 0.05 muM, respectively. These findings are supported on the genotoxicity level by the results of the micronucleus assay, with micronuclei being induced dose-dependently starting at concentrations of about 0.05 muM of complexed Hg(II). Generally, the no-effect-concentrations for complexed mercury(II) found in the cell-free systems and in cellular assays (including the micronucleus test) were identical with or similar to results for mercury tested in the absence of chelators. This indicates that mercury(II) has a much higher affinity to sulfhydryls of cytoskeletal proteins than to this type of complexing agents. Therefore, the suitability of EDTA and related compounds for remediation of environmental mercury contamination or for other detoxification purposes involving mercury has to be questioned. (C) 2004 Elsevier B.V. All rights reserved.

Genotoxicity of inorganic mercury salts based on disturbed microtubule function

This study investigated the hypothesis that the chromosomal genotoxicity of inorganic mercury results from interaction(s) with cytoskeletal proteins. Effects of Hg2+ salts on functional activities of tubulin and kinesin were investigated by determining tubulin assembly and kinesin-driven motility in cell-free systems. Hg2+ inhibits microtubule assembly at concentrations above 1 muM, and inhibition is complete at about 10 muM. In this range, the tubulin assembly is fully ( up to 6 muM) or partially (similar to 6 - 10 muM) reversible. The inhibition of tubulin assembly by mercury is independent of the anion, chloride or nitrate. The no-observed-effect-concentration for inhibition of microtubule assembly in vitro was 1 muM Hg2+, the IC50 5.8 muM. Mercury(II) salts at the IC50 concentrations partly inhibiting tubulin assembly did not cause the formation of aberrant microtubule structures. Effects of mercury salts on the functionality of the microtubule motility apparatus were studied with the motor protein kinesin. By using a gliding assay'' mimicking intracellular movement and transport processes in vitro, HgCl2 affected the gliding velocity of paclitaxel-stabilised microtubules in a clear dose-dependent manner. An apparent effect is detected at a concentration of 0.1 muM and a complete inhibition is reached at 1 muM. Cytotoxicity of mercury chloride was studied in V79 cells using neutral red uptake, showing an influence above 17 muM HgCl2. Between 15 and 20 muM HgCl2 there was a steep increase in cell toxicity. Both mercury chloride and mercury nitrate induced micronuclei concentration-dependently, starting at concentrations above 0.01 muM. CREST analyses on micronuclei formation in V79 cells demonstrated both clastogenic (CREST-negative) and aneugenic effects of Hg2+, with some preponderance of aneugenicity. A morphological effect of high Hg2+ concentrations ( 100 muM HgCl2) on the microtubule cytoskeleton was verified in V79 cells by immuno-fluorescence staining. The overall data are consistent with the concept that the chromosomal genotoxicity could be due to interaction of Hg2+ with the motor protein kinesin mediating cellular transport processes. Interactions of Hg2+ with the tubulin shown by in vitro investigations could also partly influence intracellular microtubule functions leading, together with the effects on the kinesin, to an impaired chromosome distribution as shown by the micronucleus test.

Genotoxicity of inorganic lead salts and disturbance of microtubule function

Lead compounds are known genotoxicants, principally affecting the integrity of chromosomes. Lead chloride and lead acetate induced concentration-dependent increases in micronucleus frequency in V79 cells, starting at 1.1 μ M lead chloride and 0.05 μ M lead acetate. The difference between the lead salts, which was expected based on their relative abilities to form complex acetato-cations, was confirmed in an independent experiment. CREST analyses of the micronuclei verified that lead chloride and acetate were predominantly aneugenic (CREST-positive response), which was consistent with the morphology of the micronuclei (larger micronuclei, compared with micronuclei induced by a clastogenic mechanism). The effects of high concentrations of lead salts on the microtubule network of V79 cells were also examined using immunofluorescence staining. The dose effects of these responses were consistent with the cytotoxicity of lead(II), as visualized in the neutral-red uptake assay. In a cell-free system, 20-60 μ M lead salts inhibited tubulin assembly dose-dependently. The no-observed-effect concentration of lead(II) in this assay was 10 μ M. This inhibitory effect was interpreted as a shift of the assembly/disassembly steady-state toward disassembly, e.g., by reducing the concentration of assembly-competent tubulin dimers. The effects of lead salts on microtubule-associated motor-protein functions were studied using a kinesin-gliding assay that mimics intracellular transport processes in vitro by quantifying the movement of paclitaxel-stabilized microtubules across a kinesin-coated glass surface. There was a dose-dependent effect of lead nitrate on microtubule motility. Lead nitrate affected the gliding velocities of microtubules starting at concentrations above 10 μ M and reached half-maximal inhibition of motility at about 50 μ M. The processes reported here point to relevant interactions of lead with tubulin and kinesin at low dose levels. Environ. Mal. Mutagen. 45:346-353, 2005. © 2005 Wiley-Liss, Inc.

Age determination in individual wild-caught Drosophila serrata using pteridine concentration

Fluorescence spectrophotometry can reliably detect levels of the pteridine 6-biopterin in the heads of individual Drosophila serrata Malloch 1927. Pteridine content in both laboratory and field captured flies is typically a level of magnitude higher than the minimally detectable level (mean(lab)=0.54 units, mean(field)=0.44 units, minimum detectable level=0.01 units) and can be used to predict individual age in laboratory populations with high certainty (r(2)=57%). Laboratory studies of individuals of known age ( from 1 to 48 days old) indicate that while pteridine level increases linearly with age, they also increase in a linear manner with rearing temperature and ambient light levels, but are independent of sex. As expected, the longevity of laboratory-reared males ( at least 48 days) is higher than the range of predicted ages of wild-caught males based on individual pteridine levels (40 days). However, the predictive equation based on pteridine level alone suggested that a number of wild-caught males were less than 0 days old, and the 95% confidence for these predictions based on the inverse regression broad. The age of the oldest wild-caught male is to fall within the range of 2 to 50 days. The effects of temperature and light intensity determined in laboratory study (effect sizes omega(2)=14.3 and respectively) suggests that the calibration of the prediction equation for field populations would significantly improved when combined with fine scaled studies of habitat temperature and light conditions. ability to determine relative age in individual wild-caught D. serrata presents great opportunities for a variety evolutionary studies on the dynamics of populations.

Estimation of concentration-dependent diffusion coefficient in drying process from the space-averaged concentration versus time with experimental data

The estimation of a concentration-dependent diffusion coefficient in a drying process is known as an inverse coefficient problem. The solution is sought wherein the space-average concentration is known as function of time (mass loss monitoring). The problem is stated as the minimization of a functional and gradient-based algorithms are used to solve it. Many numerical and experimental examples that demonstrate the effectiveness of the proposed approach are presented. Thin slab drying was carried out in an isothermal drying chamber built in our laboratory. The diffusion coefficients of fructose obtained with the present method are compared with existing literature results.