Gestation changes sodium pump isoform expression, leading to changes in ouabain sensitivity, contractility, and intracellular calcium in rat uterus.

Developmental and tissue-specific differences in isoforms allow Na+, K+-ATPase function to be tightly regulated, as they control sensitivity to ions and inhibitors. Uterine contraction relies on the activity of the Na+, K+ATPase, which creates ionic gradients that drive excitation-contraction coupling. It is unknown whether Na+, K+ATPase isoforms are regulated throughout pregnancy or whether they have a direct role in modulating uterine contractility. We hypothesized that gestation-dependent differential expression of isoforms would affect contractile responses to Na+, K+ATPase α subunit inhibition with ouabain. Our aims were therefore: (1) to determine the gestation-dependent expression of mRNA transcripts, protein abundance and tissue distribution of Na+, K+ATPase isoforms in myometrium; (2) to investigate the functional effects of differential isoform expression via ouabain sensitivity; and (3) if changes in contractile responses can be explained by changes in intracellular [Ca2+]. Changes in abundance and distribution of the Na+, K+ATPase α, ß and FXYD1 and 2 isoforms, were studied in rat uterus from nonpregnant, and early, mid-, and term gestation. All α, ß subunit isoforms (1,2,3) and FXYD1 were detected but FXYD2 was absent. The α1 and ß1 isoforms were unchanged throughout pregnancy, whereas α2 and α3 significant decreased at term while ß2 and FXYD1 significantly increased from mid-term onwards. These changes in expression correlated with increased functional sensitivity to ouabain, and parallel changes in intracellular Ca2+, measured with Indo-1. In conclusion, gestation induces specific regulatory changes in expression of Na+, K+ATPase isoforms in the uterus which influence contractility and may be related to the physiological requirements for successful pregnancy and delivery.

The contribution of Pseudomonas aeruginosa virulence factors and host factors in the establishment of urinary tract infections.

Pseudomonas aeruginosa can cause complicated urinary tract infections, particularly in people with catheters, which can lead to pyelonephritis. Whilst some subgroups appear more susceptible to infection, such as the elderly and women, the contribution of other host factors and bacterial virulence factors to successful infection remains relatively understudied. In this review, we explore the potential role of P. aeruginosa virulence factors including phenazines, quorum sensing, biofilm formation and siderophores along with host factors such as Tamm-Horsfall protein, osmotic stress and iron specifically on establishment of successful infection in the urinary niche. P. aeruginosa urinary tract infections are highly antibiotic resistant and require costly and intensive treatment. By understanding the infection dynamics of this organism within this specific niche, we may be able to identify novel therapeutic strategies to enhance the use of existing antibiotics.

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.

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.

Expression and distribution of Na, K-ATPase isoforms in the human uterus.

Na, K-ATPase activity relies on the composition of its catalytic alpha, beta, and FXYD constituents, all of which are expressed as multiple isoforms (4alpha, 4beta, 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 alpha1 to 3, beta1 to 3, and FXYD1 isoforms were detected in all samples, but FXYD2 was only present in hysterectomy samples. Abundant immunoreactivity of alpha1 and moderate alpha2 was localized in myometrial smooth muscle and secretory glands of all groups. Smooth muscle and gland epithelia showed diffuse cytoplasmic alpha3 immunoreactivity. beta isoforms were detected in all groups but beta3 showed much denser immunoreactivity in myometrial samples taken from women in labor. In pregnancy, there was a switch in isoform expression, resulting in increased beta3 and decreased FXYD2 at the protein and messenger RNA (mRNA) levels. Na, K-ATPase isoform alterations may modulate uterine contractility during labor.

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.

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.

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.