Rachel V. Floyd

Distribution of the AQP4 Water Channel in Normal Human Tissues: Protein and Tissue Microarrays Reveal Expression in Several New Anatomical Locations, including the Prostate Gland and Seminal Vesicles

Aquaporins facilitate osmotically driven water movement across cell membranes. Aquaporin 4 (AQP4) is a major water channel in the central nervous system where it participates in cerebral water balance. AQP4 is also present in basolateral membranes of lower respiratory tract airway and renal collecting duct epithelial cells, gastric parietal cells and skeletal muscle cells. However, the distribution of AQP4 in many other tissues is still unknown. The aim of this study was to determine the expression and relative abundance of AQP4 in human Tissue MicroArrays (TMAs) and human protein microarrays by immunohistochemistry and chemiluminescence. In the central nervous system AQP4 was abundantly expressed in the cerebral cortex, cerebellar cortex (purkinje/granular layer), ependymal cell layer, hippocampus and spinal cord. Lower levels were detected in choroid plexus, white matter and meninges. In the musculoskeletal system AQP4 was highly expressed in the sarcolemma of skeletal muscle from the chest and neck. In the male genital system AQP4 was moderately expressed in seminiferous tubules, seminal vesicles, prostate and epidiymis. In the respiratory system AQP4 was moderately expressed in lung and bronchus. AQP expression was abundant in the kidney. In the gastrointestinal system AQP4 was moderately present in basolateral membranes of parietal cells at the base of gastric glands. AQP4 was also detected in salivary glands, adrenals, anterior pituitary, prostate and seminal vesicles. Human protein microarrays verified the TMA data. Our findings suggest that AQP4 is expressed more widely than previously thought in human organs and may be involved in prostatic and seminal fluid formation.

Distribution, expression and functional effects of small conductance Ca-activated potassium (SK) channels in rat myometrium

Calcium-activated potassium channels are important in a variety of smooth muscles, contributing to excitability and contractility. In the myometrium previous work has focussed on the large conductance channels (BK), and the role of small conductance channels (SK) has received scant attention, despite the finding that over-expression of an SK channel isoform (SK3) results in uterine dysfunction and delayed parturition. This study therefore characterises the expression of the three SK channel isoforms (SK1-3) in rat myometrium throughout pregnancy and investigates their effect on cytosolic [Ca] and force and compares this with that of BK channels. Consistent expression of all SK isoform transcripts and clear immunostaining of SK1-3 was found. Inhibition of SK1-3 channels (apamin, scyllatoxin) significantly inhibited outward current, caused membrane depolarisation and elicited action potentials in previously quiescent cells. Apamin or scyllatoxin increased the amplitude of [Ca] and force in spontaneously contracting myometrial strips throughout gestation. The functional effect of SK inhibition was larger than that of BK channel inhibition. Thus we show for the first time that SK1-3 channels are expressed and translated throughout pregnancy and contribute to outward current, regulate membrane potential and hence Ca signals in pregnant rat myometrium. They contribute more to quiescence that BK channels.

Differential cellular expression of FXYD1 (phospholemman) and FXYD2 (gamma subunit of Na, K-ATPase) in normal human tissues: A study using high density human tissue microarrays

FXYD proteins have been proposed to function as regulators of Na, K-ATPase function by lowering affinities of the system for potassium and sodium. However, their distribution in normal human tissues has not been studied. We have therefore used immunohistochemistry and semi-quantitative histomorphometric analysis to determine the relative expression at the protein level and distribution of FXYD1 (phospholemman) and FXYD2 (gamma subunit of Na, K-ATPase) in human Tissue MicroArrays (TMAs). Expression of FXYD1 was abundant in heart, kidney, placenta, skeletal muscle, gastric and anal mucosa, small intestine and colon. Lower FXYD1 expression was detected in uterine, intestinal and bladder smooth muscle, choroid plexus, liver, gallbladder, spleen, breast, prostate and epididymis. The tissue distribution of FXYD2 was less extensive compared to that of FXYD1. There was an abundant expression in kidney and choroid plexus and moderate expression in placenta, amniotic membranes, breast epithelium, salivary glands, pancreas and uterine endometrium. Weaker FXYD2 expression was detected in the adrenal medulla, liver, gallbladder, bladder and pancreas. The common denominator in the distribution of FXYD1 and FXYD2 was expression in highly active transport epithelia of the kidney, choroid plexus, placenta and salivary glands. This study reveals, in human tissues, the specific expression of FXYD proteins, which may associate with Na, K-ATPase in selected cell types and modulate its catalytic properties.

Diabetes is associated with impairment of uterine contractility and high Caesarean section rate

Aims/hypothesisThe prevalence of births worldwide complicated by diabetes mellitus is increasing. In the UK, for example, <25% of diabetic women have a non-instrumental vaginal delivery. Strikingly, more than half the Caesarean sections (CS) in these patients are non-elective, but the reasons for this are not understood. We have tested the hypothesis that poor myometrial contractility as a consequence of the disease contributes to this high CS rate.MethodsWe compared spontaneous, high K depolarisation and oxytocin-induced contractions from diabetic and matched control patients having an elective CS. To investigate the mechanism of any differences we measured intracellular Ca, and performed western blotting and compared the tissues histologically.ResultsThere was significantly decreased contraction amplitude and duration in uteri from diabetic compared with control patients, even when possible confounders such as BMI were analysed. Reduced intracellular calcium signals and expression of calcium entry channels were found in uteruses from diabetic patients, which, along with a reduction in muscle content found on histological examination, could explain the reduced force. Myometrium from diabetic patients was responsive to oxytocin, but still did not reach the levels found in non-diabetic patients.Conclusions/interpretationsThese are the first data investigating myometrium in diabetic patients and they support the hypothesis that there is poorer contractility even in the presence of oxytocin. The underlying mechanism is related to reduced Ca channel expression and intracellular calcium signals and a decrease in muscle mass. We conclude that these factors significantly contribute to the increased emergency CS rate in diabetic patients.

Escherichia coli–Mediated Impairment of Ureteric Contractility Is Uropathogenic E. coli Specific

Background. Ureters are fundamental for keeping kidneys free from uropathogenic Escherichia coli (UPEC), but we have shown that 2 strains (J96 and 536) can subvert this role and reduce ureteric contractility. To determine whether this is (1) a widespread feature of UPEC, (2) exhibited only by UPEC, and (3) dependent upon type 1 fimbriae, we analyzed strains representing epidemiologically important multilocus sequence types ST131, ST73, and ST95 and non-UPEC E. coli. Methods. Contractility and calcium transients in intact rat ureters were compared between strains. Mannose and fim mutants were used to investigate the role of type 1 fimbriae. Results. Non-UPEC had no significant effect on contractility, with a mean decrease after 8 hours of 8.8%, compared with 8.8% in controls. UPEC effects on contractility were strain specific, with decreases from 9.47% to 96.7%. Mannose inhibited the effects of the most potent strains (CFT073 and UTI89) but had variable effects among other UPEC strains. Mutation and complementation studies showed that the effects of the UTI89 cystitis isolate were fimH dependent. Conclusions. We find that (1) non-UPEC do not affect ureteric contractility, (2) impairment of contractility is a common feature of UPEC, and (3) the mechanism varies between strains, but for the most potent UPEC type 1 fimbriae are involved.

Hypoxia and a hypoxia mimetic up-regulate matrix metalloproteinase 2 and 9 in equine laminar keratinocytes

The aim of this study was to determine if hypoxia and the hypoxia mimetic cobalt chloride regulate the activity of matrix metalloproteinase (MMP)-2 and -9 in cultures of equine hoof keratinocytes. These effects were assessed in primary cultures of laminar keratinocytes using gelatin zymography. Incubation of keratinocytes with cobalt chloride significantly increased the levels of active MMP-2 compared to untreated controls. Hypoxia significantly increased the expression of active MMP-2 and -9 in keratinocyte cultures. This up-regulation was observed after 6h and peaked at 24h. The study findings provide novel evidence of a potential link between hypoxia within the hoof and up-regulation of MMPs which may in turn result in damage to the lamellar basement membrane.

Morphology, calcium signaling and mechanical activity in human ureter

PURPOSE We determined the mechanisms of calcium signaling in the human ureter, and the relationship to peristaltic contractions and bundular structure in living tissue, thereby advancing the understanding of ureteral function in health and obstruction and reflux. MATERIALS AND METHODS Confocal imaging of 31 ureters was performed and simultaneous force and calcium measurements were made. Immunohistochemistry and Western blotting were also performed. RESULTS Confocal imaging showed a 3-dimensional network of smooth muscle bundles with no defined longitudinal or circular layers. Fast propagating Ca waves spread throughout the bundles, were closely associated with contraction and depended on L-type Ca channel entry. Immunohistochemistry and Western blotting demonstrated L-type Ca channels, Ca dependent K channels, sarcoplasmic reticulum Ca-adenosine triphosphatase isoforms 2 and 3, inositol triphosphate, and ryanodine receptors. Modulation of Ca and K channel activity was a potent mechanism for affecting Ca and force, whereas manipulation of the sarcoplasmic reticulum had little effect. CONCLUSIONS To our knowledge this study represents the first measurements of Ca signals in the human ureter obtained during phasic contractions and in response to agonists. Results show that it is controlled by fast propagating Ca waves, which spread rapidly between the muscle bundles, producing regular contractions, and drugs that interfere with excitability or Ca entry through L-type Ca channels have profound effects on Ca signaling and contractility. These data are discussed in relation to the treatment of patients with suspected ureteral dysfunction using Ca entry blockers.

Expression and Distribution of Na, K-ATPase Isoforms in the Human Uterus

Na, K-ATPase activity relies on the composition of its catalytic α, β, and FXYD constituents, all of which are expressed as multiple isoforms (4α, 4β, and 7 FXYD). We used reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry to study Na, K-ATPase expression in uterine samples from nonlaboring elective and laboring emergency caesarean sections (CSs). Transcripts of α1 to 3, β1 to 3, and FXYD1 isoforms were detected in all samples, but FXYD2 was only present in hysterectomy samples. Abundant immunoreactivity of α1 and moderate α2 was localized in myometrial smooth muscle and secretory glands of all groups. Smooth muscle and gland epithelia showed diffuse cytoplasmic α3 immunoreactivity. β isoforms were detected in all groups but β3 showed much denser immunoreactivity in myometrial samples taken from women in labor. In pregnancy, there was a switch in isoform expression, resulting in increased β3 and decreased FXYD2 at the protein and messenger RNA (mRNA) levels. Na, K-ATPase isoform alterations may modulate uterine contractility during labor.

Modulation of ureteric Ca signaling and contractility in humans and rats by uropathogenic E. coli

Ascending urinary tract infections, a significant cause of kidney damage, are predominantly caused by uropathogenic Escherichia coli (UPEC). However, the role and mechanism of changes in ureteric function during infection are poorly understood. We therefore investigated the effects of UPEC on Ca signaling and contractions in rat (n = 17) and human (n = 6) ureters. Ca transients and force were measured and effects of UPEC on the urothelium were monitored in live tissues. In both species, luminal exposure of ureters to UPEC strains J96 and 536 caused significant time-dependent decreases in phasic and high K depolarization-induced contractility, associated with decreases in the amplitude and duration of the Ca transients. These changes were significant after 3-5 h and irreversible over the next 5 h. The infection causes increased activity of K channels, causing inhibition of voltage-gated Ca entry, and K channel blockers could reverse the effects of UPEC on ureteric function. A smaller direct effect on Ca entry also occurs. Nonpathogenic E. coli (TG2) or abluminal application of UPEC did not produce changes in Ca signaling or contractility. UPEC exposure also caused significant impairment of urothelial barrier function; luminal application of the Ca channel blocker nifedipine caused a reduction in contractions as it entered the tissue, an effect not observed in untreated ureters. Thus, UPEC impairs ureteric contractility in a Ca-dependent manner, largely caused by stimulation of potassium channels and this mechanism is dependent on host-urothelium interaction.

Optimising the use of medicines to reduce acute kidney injury in children and babies

&NA; The majority of medications in children are administered in an unlicensed or off‐label manner. Paediatricians are obliged to prescribe using the limited evidence available. The 2007 EU regulation on the use of paediatric drugs means pharmaceutical companies are now obliged to (and receive incentives for) contributing to paediatric drug data and carrying out paediatric clinical trials. This is important, as the efficacy and adverse effect profiles of medicines vary across childhood. Additionally, there are significant age‐related changes in the pharmacodynamic and pharmacokinetic activity of many drugs. This may be related to physiological (differential expressions of cytochrome P450 enzymes or variable glomerular filtration rates at different ages for example) and psychological (increasing autonomy and risk perception in teenage years) changes. Increasing numbers of children are surviving life‐threatening childhood conditions due to medical advances. This means there is an increasing population who are at risk of the consequences of the long‐term, early exposure to nephrotoxic agents. The kidney is an organ that is particularly vulnerable to damage as a consequence of drugs. Drug‐induced acute kidney injury (AKI) episodes in children and babies are principally due to non‐steroidal anti‐inflammatory drugs, antibiotics or chemotherapeutic agents. The renal tubules are vulnerable to injury because of their concentrating ability and high‐energy hypoxic environment. This review focuses on drug‐induced AKI and the methods to minimise its effect, including general management plus the role of child‐specific pharmacokinetic data, the use of pharmacogenomics and early detection of AKI using urinary biomarkers and electronic triggers.