Proton/solute cotransport in rat kidney brush-border membrane vesicles: relative importance to both d-glucose and peptide transport

AbstractWe have determined the relative importance of the transmembrane proton electrochemical gradient to the transport of d-[ 14C]glucose and [ 14C]glycylsarcosine (gly-sar) in rat kidney brush-border membrane vesicles (BBMV) from superficial renal cortex. Electrogenic [14C]gly-sar transport was first optimised by imposing a pH gradient (pHo = 5.7, pHi = 8.4) and an interior negative p.d. (using outwardly directed K+ gradient plus valinomycin). Under identical conditions (pHo = 5.7, pHi = 8.4), an acceleration f of initial d-[14C]glucose (at 100 μM) transport by 2.0 ± 0.7-fold was observed compared to no proton gradient (pHo = 8.4, pHi = 8.4). This increase was due primarily to an effect of external protons, since acidic conditions (pHo = pHi = 5.7) also resulted in ann acceleration of d-glucose influx (2-fold). The increase ind-glucose transport in the presence of external acidity was reduced by the uncoupler FCCP, even in the absence of a proton gradient. Furthermore, the increased d-glucose transport with external acidity in the presence of a proton gradient was insensitive to a K+ gradient-driven diffusion potential in the presence of valinomycin. In no instance was an overshoot accumulation ofd-[14C]glucose observed in H+ gradient conditions. H+-stimulatedd-[14C]glucose transport showed a linear dependence on d-glucose concentration up to 20 mMd-glucose, unlike electrogenic Na+-dependentd-glucose transport, whose Km was 1.77 ± 0.35 mM. In contrast, the initial rate of [14C]gly-sar (100 μM) transport by the renal H+/di-tripeptide transporter was accelerated 15.7 ± 3.3-fold and stimulated a marked overshoot of 5.1 ± 0.4-fold over equilibrium values. Conversely, the electrogenic, Na+/glucose transporter could be readily demonstrated, whilst [14C]gly-sar transport could not be energised by an inward Na+ gradient. The absence of electrogenicd-glucose transport in H+ gradient conditions is clear evidence against H+/glucose cotransport in Na+-free conditions mediated by SGLT2 (sodium-glucose transporter, renal cortex). Furthermore, since a proton gradient does not increase brush-border membraned-glucose uptake in Na+-rich media, it is unlikely that in vivo renald-glucose transport mediated via SGLT2 may be energised by the transmembrane proton gradient

Farmers are not seeking help: what does service provision have to do with it?

Aim The rate of suicide in farmers is twice that of the general population. Help-seeking from health professionals, if this occurs in a timely manner, can significantly minimise the negative consequences of mental ill-health. However, it has now been demonstrated that farmers as a group are reluctant to seek help for mental ill-health, which likely contributes to this problem. Previous research has demonstrated that General Practitioners are the most commonly visited health professional in rural farming areas, however, they are under-utilised as a means of seeking help. This research aimed to examine the potential barriers and facilitators of mental health help-seeking in farmers, that relate to the provision of service from the perspective of farmers. Methods The present research draws on findings from semi‐structured interviews with 10 farmers residing in Queensland. The techniques of Braun and Clarke (2006) were used to guide the thematic analysis. Results Several key factors relating to services were identified as having the potential to directly or indirectly influence mental health help-seeking. These include: how services are marketed/packaged and delivered, availability and accessibility, continuity of care, having ‘knowledgeable bush practitioners’ as well as perception of good outcomes. Conclusion It is expected that this research will create a better understanding of the farmers’ perspective relating to service provision for the purpose of seeking help for mental health. The outcomes have implications for developing and providing interventions for farmers to promote services for the purpose of mental health help-seeking as well as create awareness in service provider and other stakeholders of issues that prevent timely help-seeking

Farmers tell us how to help improve their mental health help-seeking

Farmers’ mental health help-seeking is reported to be poor. Mental health help-seeking is seeking help from professionals such as GPs and psychologists for issues relating to distress or mental health (Rickwood & Thomas, 2012). Timely mental health help-seeking behaviour is important because it may minimise any negative outcomes. At present, there is no research that has identified how to target farmers to improve their mental health help-seeking. It is known that tailored approaches to intervention are superior and this is likely to be so for intervening with farmers whom have a strong culture and a vastly different situation than metropolitan populations. This study is part of a body of work exploring the factors that influence mental health help-seeking in farmers. Semi-structured interviews were conducted with 10 farmers (farming as their occupation) as well as 10 farmers’ partners (for their additional insight), all of whom resided/worked in Queensland. Braun and Clarke’s (2006) technique of thematic analysis was used to analyse the data. Based on the data three themes were developed regarding intervention: education (what needs to be taught and how to teach it), the medium (which mediums preferred and engagement with them) and multi-faceted programs (Many aspects important such as community input and role of GPs, family, and friends). This research improves the knowledge on how to target interventions, including the medium, specifically to farmers for mental health help-seeking. The findings could be utilised in the design process to create interventions that are more likely to have an impact, specific for farmers to improve their mental health help-seeking behaviour

Kinetic Measurements of Di- and Tripeptide and Peptidomimetic Drug Transport in Different Kidney Regions Using the Fluorescent Membrane Potential-Sensitive Dye, DiS-C3-(3).

Tri- and dipeptides are transported in the kidney by PEPT1 and PEPT2 isoforms. The aim of this study was to investigate differences in transport kinetics between renal brush border (BBMV) and outer medulla (OMMV) membrane vesicles (where PEPT1 and PEPT2 are sequentially available) for a range of di- and tripeptides and peptidomimetic drugs. This was accomplished through the use of the potential-sensitive fluorescent dye 3,3'-dipropylthiacarbocyanine iodide [DiS-C3-(3)]. BBMV and OMMV were prepared from the rat kidney using standard techniques. The presence of PEPT1 in BBMV and PEPT2 in OMMV was confirmed using Western blotting. Fluorescence changes were measured when extravesicular medium at pH 6.6 containing 0-1 mM substrates was added to a cuvette containing vesicles pre-equilibrated at pH 7.4 and 2.71 μM DiS-C3-(3). An increase in fluorescence intensity occurred upon substrate addition reflecting the expected positive change in membrane potential difference. Of the range of substrates studied, OMMV manifested the highest affinity to cefadroxil and valacyclovir (K m 4.3 ± 1.2 and 11.7 ± 3.2 µM, respectively) compared to other substrates, whilst the BBMV showed a higher affinity to Gly-His (K m 15.4 ± 3.1 µM) compared to other substrates. In addition, OMMV showed higher affinity and capacity to Gly-Gln (K m 47.1 ± 9.8 µM, 55.5 ± 2.8 ΔF/s/mg protein) than BBMV (K m 78.1 ± 13.3 µM and 35.5 ± 1.7 ΔF/s/mg protein, respectively). In conclusion, this study successfully separated the expression of PEPT1 and PEPT2 into different vesicle preparations inferring their activity in different regions of the renal proximal tubule