Emerging key roles for P2X receptors in the kidney

P2X ionotropic non-selective cation channels are expressed throughout the kidney and are activated in a paracrine or autocrine manner following the binding of extracellular ATP and related extracellular nucleotides. Whilst there is a wealth of literature describing a regulatory role of P2 receptors (P2R) in the kidney, there are significantly less data on the regulatory role of P2X receptors (P2XR) compared with that described for metabotropic P2Y. Much of the historical literature describing a role for P2XR in the kidney has focused heavily on the role of P2X1R in the autoregulation of renal blood flow. More recently, however, there has been a plethora of manuscripts providing compelling evidence for additional roles for P2XR in both kidney health and disease. This review summarizes the current evidence for the involvement of P2XR in the regulation of renal tubular and vascular function, and highlights the novel data describing their putative roles in regulating physiological and pathophysiological processes in the kidney

Current Perspective on the Location and Function of Gamma- Aminobutyric Acid (GABA) and its Metabolic Partners in the Kidney.

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter located in the mammalian central nervous system, which binds to GABAA and GABAB receptors to mediate its neurological effects. In addition to its role in the CNS, an increasing number of publications have suggested that GABA might also play a role in the regulation of renal function. All three enzymes associated with GABA metabolism; glutamic acid decarboxylase, GABA ?-oxoglutarate transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH) have been localised to the kidney providing the necessary machinery for localised GABA synthesis and metabolism. Moreover GABA receptors have been localised to both tubular and vascular structures in the kidney, and GABA is excreted in urine (~3 ?M) in humans. Despite the collective evidence describing the presence of a GABA system in the kidney, the precise function of such a system requires further clarification. Here we provide an overview of the current renal GABA literature and provide novel data that indicates GABA can act at contractile pericyte cells located along vasa recta capillaries in the renal medulla to potentially regulate medullary blood flow

Sympathetic nerve-derived ATP regulates renal medullary vasa recta diameter via pericyte cells: a role for regulating medullary blood flow?

Pericyte cells are now known to be a novel locus of blood flow control, being able to regulate capillary diameter via their unique morphology and expression of contractile proteins. We have previously shown that exogenous ATP causes constriction of vasa recta via renal pericytes, acting at a variety of membrane bound P2 receptors on descending vasa recta (DVR), and therefore may be able to regulate medullary blood flow (MBF). Regulation of MBF is essential for appropriate urine concentration and providing essential oxygen and nutrients to this region of high, and variable, metabolic demand. Various sources of endogenous ATP have been proposed, including from epithelial, endothelial, and red blood cells in response to stimuli such as mechanical stimulation, local acidosis, hypoxia, and exposure to various hormones. Extensive sympathetic innervation of the nephron has previously been shown, however the innervation reported has focused around the proximal and distal tubules, and ascending loop of Henle. We hypothesize that sympathetic nerves are an additional source of ATP acting at renal pericytes and therefore regulate MBF. Using a rat live kidney slice model in combination with video imaging and confocal microscopy techniques we firstly show sympathetic nerves in close proximity to vasa recta pericytes in both the outer and inner medulla. Secondly, we demonstrate pharmacological stimulation of sympathetic nerves in situ (by tyramine) evokes pericyte-mediated vasoconstriction of vasa recta capillaries; inhibited by the application of the P2 receptor antagonist suramin. Lastly, tyramine-evoked vasoconstriction of vasa recta by pericytes is significantly less than ATP-evoked vasoconstriction. Sympathetic innervation may provide an additional level of functional regulation in the renal medulla that is highly localized. It now needs to be determined under which physiological/pathophysiological circumstances that sympathetic innervation of renal pericytes is important

Inflammatory mediators act at renal pericytes to elicit contraction of vasa recta and reduce pericyte density along the kidney medullary vascular network

Introduction: Regardless of initiating cause, renal injury promotes a potent pro-inflammatory environment in the outer medulla and a concomitant sustained decrease in medullary blood flow (MBF). This decline in MBF is believed to be one of the critical events in the pathogenesis of acute kidney injury (AKI), yet the precise cellular mechanism underlying this are still to be fully elucidated. MBF is regulated by contractile pericyte cells that reside on the descending vasa recta (DVR) capillaries, which are the primary source of blood flow to the medulla. Methods: Using the rat and murine live kidney slice models, we investigated the acute effects of key medullary inflammatory mediators TNF-α, IL-1β, IL-33, IL-18, C3a and C5a on vasa recta pericytes, the effect of AT1-R blocker Losartan on pro-inflammatory mediator activity at vasa recta pericytes, and the effect of 4-hour sustained exposure on immunolabelled NG2+ pericytes. Results and discussion: Exposure of rat and mouse kidney slices to TNF-α, IL-18, IL-33, and C5a demonstrated a real-time pericyte-mediated constriction of DVR. When pro-inflammatory mediators were applied in the presence of Losartan the inflammatory mediator-mediated constriction that had previously been observed was significantly attenuated. When live kidney slices were exposed to inflammatory mediators for 4-h, we noted a significant reduction in the number of NG2+ positive pericytes along vasa recta capillaries in both rat and murine kidney slices. Data collected in this study demonstrate that inflammatory mediators can dysregulate pericytes to constrict DVR diameter and reduce the density of pericytes along vasa recta vessels, further diminishing the regulatory capacity of the capillary network. We postulate that preliminary findings here suggest pericytes play a role in AKI

A case controlled study examining the bladder microbiome in women with Overactive Bladder (OAB) and healthy controls

Objective: To characterise the microbiome in healthy women with no bladder symptoms and to compare this to the bladder microbiome in patients with overactive bladder syndrome (OAB).Study design: MSU specimens from 63 women with OAB were compared to urine from 35 controls. Urine was centrifuged and the resulting sediment pellet was re-suspended in supernatant and plated under aerobic conditions for 48 h and anaerobic conditions for 7 days. Each morphologically distinct colony was purity plated. Bacterial colonies were lysed and polymerase chain reaction undertaken to amplify the 16 s ribosomal RNA gene. This DNA was purified and sequenced allowing identification of bacterial genera.Results: The mean number of different bacterial genera was 5.0 in both controls and OAB patients (p = 0.99). The uropathogenic bacteria Proteus (P = 0.01) was more commonly isolated from women with OAB. The genus lactobacillus was present less commonly in urine from OAB patients when compared to urine taken from controls (p = 0.02). Overall the most commonly grown bacteria were staphylococcus (grown in 59% of samples), streptococccus (51%), corynebacterium (37%) and lactobacillus (28%). A total of 95 different genera were identified from the urine samples.Conclusion: The female human bladder has a diverse microbiome with stastistically significant differences between bacterial species present in OAB patients and controls

Age, Menopausal Status and the Bladder Microbiome

Objectives: The bladder is not sterile but contains a healthy community of microbes termed the microbiome. Alterations in the bladder microbiome have been demonstrated in disease states such as the overactive bladder. The microbiome in other anatomical niches is known to alter with age eg the vagina. The objective of this study was to identify if the bladder microbiome in healthy women varies with age and menopausal status. Study design: Urine from 79 healthy women attending secondary care gynaecological clinics with no urinary symptoms provided clean catch mid-stream urine specimens. Urine was centrifuged and the resultant pellet was re-suspended and inoculated onto chocolate agar plates and cultured under either aerobic or anaerobic conditions. Morphologically different colonies were purity plated and 16s rRNA gene sequencing was performed. A microbe genomic basic local alignment search tool (BLAST) was used to identify the genus of the bacteria. Results: There was no significant correlation between the age of a woman and the number of different genera identified (r=-0.034, p=0.79). There were few significant differences in the frequency with which the majority of organisms were found in pre and post-menopausal women. The exceptions however were lactobacillus, which was more common in pre-menopausal women (31 vs 3 p=0.002) and Mobiluncus, which was more common in post-menopausal women (0 vs 3 p=0.02). Conclusions: There was no significant correlation between patient age and diversity of the bladder microbiome but large numbers of different organisms were identified. Significant differences were however observed for Lactobacillus which is more common in pre-menopausal women and Mobiluncus which is more common in in post-menopausal women

Urinary ATP and visualization of intracellular bacteria: a superior diagnostic marker for recurrent UTI in renal transplant recipients?

Renal transplant recipients (RTR) are highly susceptible to urinary tract infections (UTIs) with over 50% of patients having at least one UTI within the first year. Yet it is generally acknowledged that there is considerable insensitivity and inaccuracy in routine urinalysis when screening for UTIs. Thus a large number of transplant patients with genuine urine infections may go undiagnosed and develop chronic recalcitrant infections, which can be associated with graft loss and morbidity. Given a recent study demonstrating ATP is released by urothelial cells in response to bacteria exposure, possibly acting at metabotropic P2Y receptors mediating a proinflammatory response, we have investigated alternative, and possibly more appropriate, urinalysis techniques in a cohort of RTRs.Mid-stream urine (MSU) samples were collected from 53 outpatient RTRs. Conventional leukocyte esterase and nitrite dipstick tests, and microscopic pyuria counts (in 1 ?l), ATP concentration measurements, and identification of intracellular bacteria in shed urothelial cells, were performed on fresh unspun samples and compared to ‘gold-standard’ bacterial culture results.Of the 53 RTRs, 22% were deemed to have a UTI by ‘gold-standard’ conventional bacteria culture, whereas 87%, 8% and 4% showed evidence of UTIs according to leukocyte esterase dipstick, nitrite dipstick, and a combination of both dipsticks, respectively. Intracellular bacteria were visualized in shed urothelial cells of 44% of RTRs, however only 1 of the 23 RTRs (44%) was deemed to have a UTI by conventional bacteria culture. A significant association of the ‘gold-standard’ test with urinary ATP concentration combined with visualization of intracellular bacteria in shed urothelial cells was determined using the Fisher’s exact test.It is apparent that standard bedside tests for UTIs give variable results and that seemingly quiescent bacteria in urothelial cells are very common in RTRs and may represent a focus of subclinical infection. Furthermore, our results suggest urinary ATP concentration combined with detection of intracellular bacteria in shed urinary epithelial cells may be a sensitive means by which to detect ‘occult’ infection in RTRs

Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC

Nucleotides and nucleosides are not only involved in cellular metabolism but also act extracellularly via P1 and P2 receptors, to elicit a wide variety of physiological and pathophysiological responses through paracrine and autocrine signalling pathways. For the first time, we have used an ion-pair reversed-phase high-performance liquid chromatography ultraviolet (UV)-coupled method to rapidly and simultaneously quantify 12 different nucleotides and nucleosides (adenosine triphosphate, adenosine diphosphate, adenosine monophosphate, adenosine, uridine triphosphate, uridine diphosphate, uridine monophosphate, uridine, guanosine triphosphate, guanosine diphosphate, guanosine monophosphate, guanosine): (1) released from a mouse renal cell line (M1 cortical collecting duct) and (2) in human biological samples (i.e., urine). To facilitate analysis of urine samples, a solid-phase extraction step was incorporated (overall recovery rate ? 98 %). All samples were analyzed following injection (100 ?l) into a Synergi Polar-RP 80 Å (250 × 4.6 mm) reversed-phase column with a particle size of 10 ?m, protected with a guard column. A gradient elution profile was run with a mobile phase (phosphate buffer plus ion-pairing agent tetrabutylammonium hydrogen sulfate; pH 6) in 2-30 % acetonitrile (v/v) for 35 min (including equilibration time) at 1 ml min(-1) flow rate. Eluted compounds were detected by UV absorbance at 254 nm and quantified using standard curves for nucleotide and nucleoside mixtures of known concentration. Following validation (specificity, linearity, limits of detection and quantitation, system precision, accuracy, and intermediate precision parameters), this protocol was successfully and reproducibly used to quantify picomolar to nanomolar concentrations of nucleosides and nucleotides in isotonic and hypotonic cell buffers that transiently bathed M1 cells, and urine samples from normal subjects and overactive bladder patients

Altered urothelial ATP signaling in a major subset of human overactive bladder patients with pyuria

Overactive Bladder (OAB) is an idiopathic condition, characterized by urgency, urinary frequency, and urgency incontinence, in the absence of routinely traceable urinary infection. We have described microscopic pyuria (?10 wbc/?l) in patients suffering from the worst symptoms. It is established that inflammation is associated with increased ATP release from epithelial cells, and extracellular ATP originating from the urothelium following increased hydrostatic pressure is a mediator of bladder sensation. Here, using bladder biopsy samples, we have investigated urothelial ATP signaling in OAB patients with microscopic pyuria. Basal, but not stretch-evoked, release of ATP was significantly greater from the urothelium of OAB patients with pyuria than from non-OAB patients or OAB patients without pyuria (<10 wbc/?l). Basal ATP release from the urothelium of OAB patients with pyuria was inhibited by the P2 receptor antagonist suramin and abolished by the hemichannel blocker carbenoxolone, which differed from stretch-activated ATP release. Altered P2 receptor expression was evident in the urothelium from pyuric OAB patients. Furthermore, intracellular bacteria were visualized in shed urothelial cells from ?80% of OAB patients with pyuria. These data suggest that increased ATP release from the urothelium, involving bacterial colonization, may play a role in the heightened symptoms associated with pyuric OAB patients

Murine Live Bladder Tissue Model to Study the Microvasculature Properties and Function In Situ

Regulation of microvascular blood flow in the bladder has historically been associated with regulation by pre-capillary arterioles and more recently to post-capillary venules. Although pericytes are known to regulate capillary vessel diameter in various capillary beds (e.g. brain, retina, heart and kidney), their role in regulating bladder capillary diameter remains controversial. We have developed a mouse full-thickness live bladder tissue model in which the microvasculature of the mucosa can be visualised in situ, enabling the regulation of suburothelial capillary diameter to be investigated. Bladder tissue viability was first investigated by fluorescence imaging of tissue labelled with Hoechst and propidium iodide to determine the live to dead cell ratio. Data indicated that tissue was viable in situ for up to 5 hours (>60%; P>0.05; n = 3 per time point). Real time DIC images of live tissue sections were collected for all experiments in which live tissue was exposed to vasoactive agents to investigate the pericyte-mediated regulation of capillary diameter. Images were analysed off line and vessel diameter was measured at both pericyte and non-pericyte sites along a capillary for each experiment. Angiotensin II (100 nM) and endothelin I (1 nM) evoked a pericyte-mediated vasoconstriction of suburothelial capillaries at pericyte sites (13.3 ± 5.8%, 22.0 ± 9.7%) that was significantly greater (p<0.05, n = 6 per drug) than that measured at non-pericyte sites (3.2 ± 2.7%, 6.0 ± 3.4%). Prostaglandin E2 (30 μM) evoked a pericyte-mediated dilation of capillaries (12.1 ± 2.6%) that was significantly greater than that measured at non-pericyte sites (2.3 ± 2.3%, p = 0.03, n = 3). In this study we therefore demonstrate that suburothelial capillary pericytes act to regulate capillary diameter in response to agents previously reported to regulate capillary beds of other organs. This indicates that in addition to smooth muscle-mediated regulation of upstream vessels in the bladder, spatial regulation of capillary diameter downstream of these vessels also occurs in the bladder