Gordon Ritchie

Mg2+/Ca2+sensing inhibits hormone-stimulated Mg2+ uptake in mouse distal convoluted tubule cells

The distal convoluted tubule plays a significant role in renal magnesium conservation. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. MDCT cells possess an extracellular polyvalent cation-sensing mechanism responsive to Mg2+, Ca2+, and neomycin. The present studies determined the effect of Mg2+/Ca2+ sensing on hormone-mediated cAMP formation and Mg2+ uptake in MDCT cells. MDCT cells were Mg2+ depleted by culturing in Mg2+-free media for 16 h, and Mg2+ uptake was measured by microfluorescence after placing the depleted cells in 1.5 mM MgCl2. The mean rate of Mg2+ uptake was 164 +/- 5 nM/s in control MDCT cells. Activation of Mg2+/Ca2+ sensing with neomycin did not affect basal Mg2+ uptake (155 +/- 5 nM/s). We have previously reported that treatment of MDCT cells with either glucagon or arginine vasopressin (AVP) stimulated Mg2+ entry. In the present studies, the addition of extracellular Mg2+ or Ca2+ inhibited glucagon- and AVP-stimulated cAMP formation and Mg2+ uptake in concentration-dependent manner with half-maximal concentrations of approximately 1.5 and 3.0 mM, respectively. Exogenous cAMP or forskolin stimulated Mg2+ uptake in the presence of Mg2+/Ca2+ sensing activation. We infer from these studies that Mg2+/Ca2+-sensing mechanisms located in the distal convoluted tubule may play a role in control of distal magnesium absorption.The distal convoluted tubule plays a significant role in renal magnesium conservation. An immortalized mouse distal convoluted tubule (MDCT) cell line has been extensively used to study the cellular mechanisms of magnesium transport in this nephron segment. MDCT cells possess an extracellular polyvalent cation-sensing mechanism responsive to Mg2+, Ca2+, and neomycin. The present studies determined the effect of Mg2+/Ca2+sensing on hormone-mediated cAMP formation and Mg2+ uptake in MDCT cells. MDCT cells were Mg2+ depleted by culturing in Mg2+-free media for 16 h, and Mg2+ uptake was measured by microfluorescence after placing the depleted cells in 1.5 mM MgCl2. The mean rate of Mg2+ uptake was 164 ± 5 nM/s in control MDCT cells. Activation of Mg2+/Ca2+sensing with neomycin did not affect basal Mg2+ uptake (155 ± 5 nM/s). We have previously reported that treatment of MDCT cells with either glucagon or arginine vasopressin (AVP) stimulated Mg2+ entry. In the present studies, the addition of extracellular Mg2+ or Ca2+ inhibited glucagon- and AVP-stimulated cAMP formation and Mg2+ uptake in concentration-dependent manner with half-maximal concentrations of ∼1.5 and 3.0 mM, respectively. Exogenous cAMP or forskolin stimulated Mg2+ uptake in the presence of Mg2+/Ca2+sensing activation. We infer from these studies that Mg2+/Ca2+-sensing mechanisms located in the distal convoluted tubule may play a role in control of distal magnesium absorption.

Glucagon and arginine vasopressin stimulate Mg2+ uptake in mouse distal convoluted tubule cells.

Glucagon and arginine vasopressin (AVP) enhance renal magnesium conservation through actions within the loop of Henle and the distal tubule. Studies were performed on an immortalized mouse distal convoluted tubule (MDCT) cell line to characterize the cellular actions of these hormones on Mg2+ transport in this segment of the distal tubule. Glucagon and AVP increased cellular cAMP concentrations by about fivefold above basal levels in normal and Mg(2+)-depleted cells. Intracellular free Mg2+ concentration ([Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg2+ uptake, MDCT cells were first Mg2+ depleted (0.22 +/- 0.01 mM) by culturing in Mg(2+)-free media for 16 h and then placed in 1.5 mM MgCl2, and the [Mg2+]i was determined. [Mg2+]i returned to basal levels, 0.53 +/- 0.02 mM, with a mean refill rate, d([Mg2+]i/dt, of 164 +/- 5 nM/s. Both glucagon and AVP stimulated Mg2+ uptake into MDCT cells, 196 +/- 11 and 189 +/- 6 nM/s, respectively, at concentrations of 3 x 10(-7) M and 10(-7) M, respectively. Enhanced Mg2+ uptake for each of the hormones was concentration dependent and inhibited by the channel blocker, nifedipine. Hormone stimulation of Mg2+ entry was not dependent on protein synthesis. 8-Bromo-cAMP, 10(-4) M, enhanced Mg2+ uptake (225 +/- 13 nM/s), whereas phorbol esters were without effect. Finally, protein kinase A inhibition prevented glucagon and AVP stimulation of Mg2+ uptake, supporting the notion that the cAMP pathway is important as expected in the hormone action. These studies demonstrate that glucagon and AVP stimulate Mg2+ uptake in MDCT cells and suggest that these hormones act to control magnesium conservation in the convoluted segment of the distal tubule.Glucagon and arginine vasopressin (AVP) enhance renal magnesium conservation through actions within the loop of Henle and the distal tubule. Studies were performed on an immortalized mouse distal convoluted tubule (MDCT) cell line to characterize the cellular actions of these hormones on Mg2+transport in this segment of the distal tubule. Glucagon and AVP increased cellular cAMP concentrations by about fivefold above basal levels in normal and Mg2+-depleted cells. Intracellular free Mg2+concentration ([Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To assess Mg2+ uptake, MDCT cells were first Mg2+depleted (0.22 ± 0.01 mM) by culturing in Mg2+-free media for 16 h and then placed in 1.5 mM MgCl2, and the [Mg2+]iwas determined. [Mg2+]ireturned to basal levels, 0.53 ± 0.02 mM, with a mean refill rate, d([Mg2+]i)/d t, of 164 ± 5 nM/s. Both glucagon and AVP stimulated Mg2+ uptake into MDCT cells, 196 ± 11 and 189 ± 6 nM/s, respectively, at concentrations of 3 × 10-7 M and 10-7 M, respectively. Enhanced Mg2+ uptake for each of the hormones was concentration dependent and inhibited by the channel blocker, nifedipine. Hormone stimulation of Mg2+ entry was not dependent on protein synthesis. 8-Bromo-cAMP, 10-4 M, enhanced Mg2+ uptake (225 ± 13 nM/s), whereas phorbol esters were without effect. Finally, protein kinase A inhibition prevented glucagon and AVP stimulation of Mg2+ uptake, supporting the notion that the cAMP pathway is important as expected in the hormone action. These studies demonstrate that glucagon and AVP stimulate Mg2+ uptake in MDCT cells and suggest that these hormones act to control magnesium conservation in the convoluted segment of the distal tubule.

Aldosterone potentiates hormone-stimulated Mg2+ uptake in distal convoluted tubule cells

The distal convoluted tubule reabsorbs significant amounts of filtered magnesium that is under hormonal control. In this study, we describe the effects of aldosterone on Mg2+ uptake in an immortalized mouse distal convoluted tubule (MDCT) cell line. Intracellular free Mg2+ concentration ([Mg2+]i) was determined on single MDCT cells using microfluorescence with mag-fura 2. To determine Mg2+ entry rate into MDCT cells, they were first Mg2+ depleted ([Mg2+]i, 0.22 +/- 0.01 mM) by culturing in Mg(2+)-free media for 16 h and then placed in 1.5 mM MgCl2. The rate of change in [Mg2+]i as measured as a function of time, d([Mg2+]i)/dt, was 164 +/- 5 nM/s in control cells. We have shown that glucagon or arginine vasopressin (AVP) stimulates Mg2+ entry by 63% and 15%, respectively. Incubation of MDCT cells with aldosterone for 16 h did not change the rate of Mg2+ uptake (172 +/- 8 nM/s). However, aldosterone potentiated glucagon- and AVP-stimulated Mg2+ uptake rate up to 330 +/- 39 and 224 +/- 6 nM/s, respectively. Aldosterone also potentiated glucagon- and AVP-induced intracellular cAMP accumulation in a concentration-independent manner. As cAMP stimulates Mg2+ entry in MDCT cells, it is inferred that aldosterone may stimulate Mg2+ uptake through intracellular signaling pathways involving cAMP. The actions of aldosterone were dependent on de novo protein synthesis, as pretreatment of the cells with cycloheximide inhibited aldosterone potentiation of hormone stimulation of Mg2+ uptake and cAMP accumulation. These studies with MDCT cells suggest that aldosterone may modulate the effects of hormones acting within the distal convoluted tubule to control magnesium absorption.

Some characteristics of sodium-independent phosphate transport across renal basolateral membranes

Sodium-independent phosphate transport was evaluated in porcine renal basolateral membrane vesicles. Phosphate uptake was saturable with an apparent Km 10.1 +/- 1.2 mM and Vmax 13.6 +/- 2.0 nmol (mg protein)-1 min-1, n = 5. Phosphate uptake was trans-stimulated with intravesicle phosphate and was enhanced with a positive transmembrane electrical potential. Arsenate and bicarbonate inhibited phosphate transport but other anions including sulfate and phosphonoformate were without effect. These studies indicate that phosphate uptake across basolateral membranes is present in the absence of sodium, is facilitated, and is specific for phosphate. The apparent affinity and rate of phosphate transport across the basolateral membrane is significantly higher than the respective parameters observed for the brush-border membrane.

Implementation of Next-Generation Sequencing for Hepatitis B Virus Resistance Testing and Genotyping in a Clinical Microbiology Laboratory

ABSTRACT Sanger sequencing or DNA hybridization have been the primary modalities for hepatitis B (HBV) resistance testing and genotyping; however, there are limitations, such as low sensitivity and the inability to detect novel mutations. Next-generation sequencing (NGS) for HBV can overcome these limitations, but there is limited guidance for clinical microbiology laboratories to validate this novel technology. In this study, we describe an approach to implementing deep pyrosequencing for HBV resistance testing and genotyping in a clinical virology laboratory. A nested PCR targeting the pol region of HBV (codons 143 to 281) was developed, and the PCR product was sequenced by the 454 Junior (Roche). Interpretation was performed by ABL TherapyEdge based on European Association for the Study of the Liver (EASL) guidelines. Previously characterized HBV samples by INNO-LiPA (LiPA) were compared to NGS with discordant results arbitrated by Sanger sequencing. Genotyping of 105 distinct samples revealed a concordance of 95.2% (100/105), with Sanger sequencing confirming the NGS result. Resistance testing by NGS was concordant with LiPA in 85% (68/80) of previously characterized samples. Additional mutations were found in 8 samples, which related to the identification of low-level mutant subpopulations present at <10% (6/8). To balance the costs of testing for the validation study, reproducibility of the NGS was investigated through an analysis of sequence variants at loci not associated with resistance in a single patient sample. Our validation approach attempts to balance costs with efficient data acquisition.

Development and Validation of a Pneumocystis jirovecii Real-time Polymerase Chain Reaction Assay for Diagnosis of Pneumocystis Pneumonia

Pneumocystis pneumonia is caused by Pneumocystis jirovecii, an opportunistic fungal pathogen. Presently, many clinical microbiology laboratories rely on direct microscopic detection of P jirovecii. The validation, and clinical and laboratory development of a qualitative P jirovecii real-time polymerase chain reaction assay for the rapid detection of Pneumocystis pneumonia is discussed by the authors. In addition, this new technique is compared with the existing gold-standard immunofluorescence assay.

Inhibition of sodium-phosphate cotransport in renal brush-border membranes with the stilbenedisulfonate, H2-DIDS

Membrane proteins involved with sodium/phosphate cotransport across the renal brush border provide the sensitive control for phosphate homeostasis. The present study describes the inhibition of sodium/phosphate cotransport with the stilbenedisulfonate derivatives, DIDS and H2-DIDS. Preincubation of the rat brush-border membrane vesicles with H2-DIDS led to the inhibition of sodium-dependent phosphate uptake with a half maximal concentration, IC50, of about 10 microM. The inhibition was irreversible supporting the notion that H2-DIDS forms covalent bonds with the cotransporter. The cotransporter could be protected by excess sodium phosphate but not sodium chloride, sodium sulfate, sodium succinate, sodium bicarbonate, nor sodium phosphonoformate. These observations suggest that the stilbenedisulfonates may be useful in labeling the sodium/phosphate cotransporter within renal brush-border membranes.

Reduction in hospital-associated methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus with daily chlorhexidine gluconate bathing for medical inpatients

Background: Daily bathing with chlorhexidine gluconate (CHG) is increasingly used in intensive care units to prevent hospital‐associated infections, but limited evidence exists for noncritical care settings. Methods: A prospective crossover study was conducted on 4 medical inpatient units in an urban, academic Canadian hospital from May 1, 2014‐August 10, 2015. Intervention units used CHG over a 7‐month period, including a 1‐month wash‐in phase, while control units used nonmedicated soap and water bathing. Rates of hospital‐associated methicillin‐resistant Staphylococcus aureus (MRSA) and vancomycin‐resistant Enterococcus (VRE) colonization or infection were the primary end point. Hospital‐associated S. aureus were investigated for CHG resistance with a qacA/B and smr polymerase chain reaction (PCR) and agar dilution. Results: Compliance with daily CHG bathing was 58%. Hospital‐associated MRSA and VRE was decreased by 55% (5.1 vs 11.4 cases per 10,000 inpatient days, P = .04) and 36% (23.2 vs 36.0 cases per 10,000 inpatient days, P = .03), respectively, compared with control cohorts. There was no significant difference in rates of hospital‐associated Clostridium difficile. Chlorhexidine resistance testing identified 1 isolate with an elevated minimum inhibitory concentration (8 &mgr;g/mL), but it was PCR negative. Conclusions: This prospective pragmatic study to assess daily bathing for CHG on inpatient medical units was effective in reducing hospital‐associated MRSA and VRE. A critical component of CHG bathing on medical units is sustained and appropriate application, which can be a challenge to accurately assess and needs to be considered before systematic implementation.