Curtiss, N., Balachandran, A., Krska, L., Peppiatt-Wildman, C., Wildman, S. and Duckett, J. (2018). Age, Menopausal Status and the Bladder Microbiome. European Journal of Obstetrics & Gynecology and Reproductive Biology [Online] 228:126-129. Available at: https://doi.org/10.1016/j.ejogrb.2018.06.011.
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.
Curtiss, N., Balachandran, A., Krska, L., Peppiatt-Wildman, C., Wildman, S. and Duckett, J. (2017). A case controlled study examining the bladder microbiome in women with Overactive Bladder (OAB) and healthy controls. European Journal of Obstetrics & Gynecology and Reproductive Biology [Online] 214:31-35. Available at: http://dx.doi.org/10.1016/j.ejogrb.2017.04.040.
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.
Gomez, I., Roach, A., Nakagawa, N., Amatucci, A., Johnson, B., Dunn, K., Kelly, M., Karaca, G., Zheng, T., Szak, S., Peppiatt-Wildman, C., Burkly, L. and Duffield, J. (2016). TWEAK-Fn14 Signaling Activates Myofibroblasts to Drive Progression of Fibrotic Kidney Disease. Journal of the American Society of Nephrology [Online] 27:3639-3652. Available at: http://doi.org/10.1681/ASN.2015111227.
The identification of the cellular origins of myofibroblasts has led to the discovery of novel pathways that potentially drive myofibroblast perpetuation in disease. Here, we further investigated the role of innate immune signaling pathways in this process. In mice, renal injury-induced activation of pericytes, which are myofibroblast precursors attached to endothelial cells, led to upregulated expression of TNF receptor superfamily member 12a, also known as fibroblast growth factor-inducible 14 (Fn14), by these cells. In live rat kidney slices, administration of the Fn14 ligand, TNF-related weak inducer of apoptosis (TWEAK), promoted pericyte-dependent vasoconstriction followed by pericyte detachment from capillaries. In vitro, administration of TWEAK activated and differentiated pericytes into cytokine-producing myofibroblasts, and further activated established myofibroblasts in a manner requiring canonical and noncanonical NF-?B signaling pathways. Deficiency of Fn14 protected mouse kidneys from fibrogenesis, inflammation, and associated vascular instability after in vivo injury, and was associated with loss of NF-?B signaling. In a genetic model of spontaneous CKD, therapeutic delivery of anti-TWEAK blocking antibodies attenuated disease progression, preserved organ function, and increased survival. These results identify the TWEAK-Fn14 signaling pathway as an important factor in myofibroblast perpetuation, fibrogenesis, and chronic disease progression.
Contreras-Sanz, A., Krska, L., Balachandran, A., Curtiss, N., Khasriya, R., Kelley, S., Strutt, M., Gill, H., Taylor, K., Mansfield, K., Wu, C., Peppiatt-Wildman, C., Malone-Lee, J., Duckett, J. and Wildman, S. (2016). Altered urothelial ATP signaling in a major subset of human overactive bladder patients with pyuria. American Journal of Physiology. Renal physiology [Online] 311:F805-F816. Available at: http://dx.doi.org/10.1152/ajprenal.00339.2015.
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.
Kennedy-Lydon, T., Crawford, C., Wildman, S. and Peppiatt-Wildman, C. (2015). Nonsteroidal anti-inflammatory drugs alter vasa recta diameter via pericytes. American Journal of Physiology - Renal Physiology [Online] 309:F648-F657. Available at: http://doi.org/10.1152/ajprenal.00199.2015.
We have previously shown that vasa recta pericytes are known to dilate vasa recta capillaries in the presence of PGE2 and contract vasa recta capillaries when endogenous production of PGE2 is inhibited by the nonselective nonsteroidal anti-inflammatory drug (NSAID) indomethacin. In the present study, we used a live rat kidney slice model to build on these initial observations and provide novel data that demonstrate that nonselective, cyclooxygenase-1-selective, and cyclooxygenase -2-selective NSAIDs act via medullary pericytes to elicit a reduction of vasa recta diameter. Real-time images of in situ vasa recta were recorded, and vasa recta diameters at pericyte and nonpericyte sites were measured offline. PGE2 and epoprostenol (a prostacyclin analog) evoked dilation of vasa recta specifically at pericyte sites, and PGE2 significantly attenuated pericyte-mediated constriction of vasa recta evoked by both endothelin-1 and ANG II. NSAIDs (indomethacin > SC-560 > celecoxib > meloxicam) evoked significantly greater constriction of vasa recta capillaries at pericyte sites than at nonpericyte sites, and indomethacin significantly attenuated the pericyte-mediated vasodilation of vasa recta evoked by PGE2, epoprostenol, bradykinin, and S-nitroso-N-acetyl-l-penicillamine. Moreover, a reduction in PGE2 was measured using an enzyme immune assay after superfusion of kidney slices with indomethacin. In addition, immunohistochemical techiques were used to demonstrate the population of EP receptors in the medulla. Collectively, these data demonstrate that pericytes are sensitive to changes in PGE2 concentration and may serve as the primary mechanism underlying NSAID-associated renal injury and/or further compound-associated tubular damage.
Kelley, S., Courtniege, H., Birch, R., Contreras-Sanz, A., Kelly, M., Durodie, J., Peppiatt-Wildman, C., Farmer, C., Delaney, M., Malone-Lee, J., Harber, M. and Wildman, S. (2014). Urinary ATP and visualization of intracellular bacteria: a superior diagnostic marker for recurrent UTI in renal transplant recipients?. Springerplus [Online] 3:1-1. Available at: http://dx.doi.org/10.1186/2193-1801-3-200.
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.
Wildman, S., Dunn, K., Peppiatt-Wildman, C. and Kelley, S. (2014). Current Perspective on the Location and Function of Gamma- Aminobutyric Acid (GABA) and its Metabolic Partners in the Kidney. Journal of Nephrology and Urology Research [Online] 2:47-57. Available at: http://dx.doi.org/10.12970/2310-984X.2014.02.02.5.
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.
Kelly, M., Crawford, C., Loo, R., Delaney, M., Farmer, C., Wildman, S. and Peppiatt-Wildman, C. (2013). Live kidney slices present a novel method for delineating the mechanisms of calcineurin inhibitor-mediated nephrotoxicity. The FASEB journal [Online] 27. Available at: http://dx.doi.org/10.1096/fasebj.27.1_supplement.646.8.
Cyclosporin A (CsA) and Tacrolimus (FK-506) are potent immunosuppressant drugs widely used to prevent allograft rejection in organ transplantation. They are calcineurin inhibitors (CNIs) and have limiting side effects including nephrotoxicity that impact upon long-term renal allograft and patient survival in other organ transplantation. The precise mechanisms of CNI-induced toxicity are unknown, although ischemic injury resulting from impairment of endothelial cell function, reduced production of endogenous renal vasodilators and concomitant release of vasoconstrictors resulting in net vasoconstriction have been described.
We have utilized the live kidney slice model, developed by our group , to investigate the mechanisms of CNI-induced nephrotoxicity in the renal medulla, a region of the kidney that is particularly sensitive to ischemia
Data presented here demonstrates both CsA (600 ng/ml) and FK-506 (800 ng/ml) cause vasoconstriction (9.9% ± 0.88% and 9.2% ± 0.63%, respectively) of vasa recta via their specific action at contractile pericytes. Tubule diameter was unaffected by bath application of both CsA and FK-506. Data suggest that CNI-induced constriction of vasa recta by pericytes may exacerbate renal medullary ischemia contributing to observed nephrotoxicity in solid organ transplantation.
Zerpa, H., Crawford, C., Knight, G., Fordham, A., Janska, S., Peppiatt-Wildman, C., Elliott, J., Burnstock, G. and Wildman, S. (2013). Extracellular ATP signaling in equine digital blood vessels. European Journal of Pharmacology [Online] 702:242-249. Available at: http://doi.org/10.1016/j.ejphar.2013.01.018.
The functional distribution of ATP-activated P2 receptors is well characterized for many blood vessels, but not in the equine digital vasculature, which is a superficial vascular bed that displays thermoregulatory functions and has been implicated in ischemia-reperfusion injuries of the hoof. Isolated equine digital arteries (EDA) and veins (EDV) were submitted to isometric tension studies, whereby electric field stimulation (EFS) and concentration-response curves to exogenously applied agonists were constructed under low tone conditions. Additionally, immunofluorescent localization of P2X and P2Y receptor subtypes was performed. EFS-induced constriction was abolished by tetrodotoxin (1 ?M, n=4). Endothelium denudation did not modify the EFS-induced constriction (n=3). The EFS-induced constriction in EDA was inhibited by phentolamine (67.7±1.8%, n=6; 10 ?M), and by the non-selective P2 receptor antagonist suramin (46.2±1.3%, n=6; 10 ?M). EFS-induced constriction in EDV was reduced by suramin (48.2±2.4%, n=6; 10 ?M), the P2 receptor antagonist pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (58.3±4.5%, n=6; 10 ?M), and phentolamine (23.2±2.5%, n=6; 10 ?M). Exogenous methoxamine and ATP mimicked EFS-induced constriction in EDA and EDV. Immunostaining for P2X1, P2X2 and P2X3, and, for P2X1 and P2X7 receptor subunits were observed in EDA and EDV smooth muscle and adventitia, respectively. ATP and noradrenaline are co-transmitters in sympathetic nerves supplying the equine digital vasculature, noradrenaline being the dominant agonist in EDA, and ATP in EDV. In conclusion, P2X receptors mediate vasoconstriction in EDA and EDV, although different P2X subunits are involved in these vessels. The physiological significance of this finding in relation to thermoregulatory functions and equine laminitis is discussed.
Birch, R., Schwiebert, E., Peppiatt-Wildman, C. and Wildman, S. (2013). Emerging key roles for P2X receptors in the kidney. Frontiers in Physiology [Online] 4:1-8. Available at: http://dx.doi.org/10.3389/fphys.2013.00262.
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.
Crawford, C., Wildman, S., Kelly, M., Kennedy-Lydon, T. and Peppiatt-Wildman, C. (2013). Sympathetic nerve-derived ATP regulates renal medullary vasa recta diameter via pericyte cells: a role for regulating medullary blood flow?. Frontiers in Physiology [Online] 4:1-8. Available at: http://dx.doi.org/10.3389/fphys.2013.00307.
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.
Crawford, C., Kennedy-Lydon, T., Sprott, C., Desai, T., Sawbridge, L., Munday, J., Unwin, R., Wildman, S. and Peppiatt-Wildman, C. (2012). An Intact Kidney Slice Model to Investigate Vasa Recta Properties and Function in situ. Nephron Physiology [Online] 120:17-31. Available at: http://doi.org/10.1159/000339110.
Background: Medullary blood flow is via vasa recta capillaries, which possess contractile pericytes. In vitro studies using isolated descending vasa recta show that pericytes can constrict/dilate descending vasa recta when vasoactive substances are present. We describe a live kidney slice model in which pericyte-mediated vasa recta constriction/dilation can be visualized in situ. Methods: Confocal microscopy was used to image calcein, propidium iodide and Hoechst labelling in ‘live’ kidney slices, to determine tubular and vascular cell viability and morphology. DIC video-imaging of live kidney slices was employed to investigate pericyte-mediated real-time changes in vasa recta diameter. Results: Pericytes were identified on vasa recta and their morphology and density were characterized in the medulla. Pericyte-mediated changes in vasa recta diameter (10–30%) were evoked in response to bath application of vasoactive agents (norepinephrine, endothelin-1, angiotensin-II and prostaglandin E2) or by manipulating endogenous vasoactive signalling pathways (using tyramine, L-NAME, a cyclo-oxygenase (COX-1) inhibitor indomethacin, and ATP release). Conclusions: The live kidney slice model is a valid complementary technique for investigating vasa recta function in situ and the role of pericytes as regulators of vasa recta diameter. This technique may also be useful in exploring the role of tubulovascular crosstalk in regulation of medullary blood flow.
Contreras-Sanz, A., Scott-Ward, T., Gill, H., Jacoby, J., Birch, R., Malone-Lee, J., Taylor, K., Peppiatt-Wildman, C. and Wildman, S. (2012). Simultaneous quantification of 12 different nucleotides and nucleosides released from renal epithelium and in human urine samples using ion-pair reversed-phase HPLC. Purinergic Signalling [Online] 8:741-751. Available at: http://doi.org/10.1007/s11302-012-9321-8.
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.
Kennedy-Lydon, T., Crawford, C., Wildman, S. and Peppiatt-Wildman, C. (2012). Renal pericytes: regulators of medullary blood flow. Acta Physiologica [Online] 207:212-225. Available at: http://dx.doi.org/10.1111/apha.12026.
Regulation of medullary blood flow (MBF) is essential in maintaining normal kidney function. Blood flow to the medulla is supplied by the descending vasa recta (DVR), which arise from the efferent arterioles of juxtamedullary glomeruli. DVR are composed of a continuous endothelium, intercalated with smooth muscle-like cells called pericytes. Pericytes have been shown to alter the diameter of isolated and in situ DVR in response to vasoactive stimuli that are transmitted via a network of autocrine and paracrine signalling pathways. Vasoactive stimuli can be released by neighbouring tubular epithelial, endothelial, red blood cells and neuronal cells in response to changes in NaCl transport and oxygen tension. The experimentally described sensitivity of pericytes to these stimuli strongly suggests their leading role in the phenomenon of MBF autoregulation. Because the debate on autoregulation of MBF fervently continues, we discuss the evidence favouring a physiological role for pericytes in the regulation of MBF and describe their potential role in tubulo-vascular cross-talk in this region of the kidney. Our review also considers current methods used to explore pericyte activity and function in the renal medulla.
Hall, A., Crawford, C., Unwin, R., Duchen, M. and Peppiatt-Wildman, C. (2011). Multiphoton Imaging of the Functioning Kidney. Journal of the American Society of Nephrology [Online] 22:1297-1304. Available at: http://dx.doi.org/10.1681/ASN.2010101054.
Translating discoveries made in isolated renal cells and tubules to the in vivo situation requires the assessment of cellular function in intact live organs. Multiphoton imaging is a form of fluorescence microscopy that is ideally suited to working with whole tissues and organs, but adequately loading cells with fluorescence dyes in vivo remains a challenge. We found that recirculation of fluorescence dyes in the rat isolated perfused kidney (IPK) resulted in levels of intracellular loading that would be difficult to achieve in vivo. This technique allowed the imaging of tubular cell structure and function with multiphoton microscopy in an intact, functioning organ. We used this approach to follow processes in real time, including (1) relative rates of reactive oxygen species (ROS) production in different tubule types, (2) filtration and tubular uptake of low-molecular-weight dextrans and proteins, and (3) the effects of ischemia-reperfusion injury on mitochondrial function and cell structure. This study demonstrates that multiphoton microscopy of the isolated perfused kidney is a powerful technique for detailed imaging of cell structure and function in an intact organ.
Crawford, C., Kennedy-Lydon, T., Callaghan, H., Sprott, C., Simmons, R., Sawbridge, L., Syme, H., Unwin, R., Wildman, S. and Peppiatt-Wildman, C. (2011). Extracellular nucleotides affect pericyte-mediated regulation of rat in situ vasa recta diameter. Acta physiologica (Oxford, England) [Online] 202:241-251. Available at: https://doi.org/10.1111/j.1748-1716.2011.02310.x.
We hypothesized that extracellular nucleotides, established as being released from renal tubular epithelial cells, act at pericytes to regulate vasa recta capillary diameter.
A rat live kidney slice model and video imaging techniques were used to investigate the effects of extracellular nucleotides on in situ (subsurface) vasa recta diameter at pericyte and non-pericyte sites. In addition, RT-qPCR was used to quantify P2 receptor mRNA expression in isolated vasa recta.
Extracellular ATP, UTP, benzylbenzyl ATP (BzATP) or 2-methylthioATP (2meSATP) evoked a significantly greater vasoconstriction of subsurface vasa recta at pericytes than at non-pericyte sites. The rank order of agonist potency was BzATP = 2meSATP > ATP = UTP. The vasoconstriction evoked at pericyte sites by ATP was significantly attenuated by the P2 receptor antagonists suramin, pyridoxal phosphate-6-azo(benzene-2,4-disulfonic acid) (PPADS) or Reactive Blue-2 (RB-2). UTP-evoked vasoconstriction at pericytes was attenuated by suramin or RB-2 but not PPADS. Interestingly, suramin or PPADS, when applied in the absence of a P2 receptor agonist, evoked a weak but significant vasoconstriction of vasa recta at pericyte sites, suggesting tonic vasodilation by nucleotides. Significant levels of P2X(1, 3 and 7) and P2Y(4 and 6) receptor mRNA were detected in vasa recta.
Extracellular nucleotides act at pericytes to cause vasoconstriction of in situ vasa recta. Pharmacological characterization, supported by RT-qPCR data, suggests that P2X(1 and 7) and P2Y(4) receptors mediate nucleotide-evoked vasoconstriction of vasa recta by pericytes. We propose that nucleotides released from renal tubular epithelial cells, in close proximity to vasa recta capillaries, are key in regulating renal medullary blood flow.
Howarth, C., Peppiatt-Wildman, C. and Attwell, D. (2009). The energy use associated with neural computation in the cerebellum. Journal of Cerebral Blood Flow & Metabolism [Online] 30:403-414. Available at: http://dx.doi.org/10.1038/jcbfm.2009.231.
The brain's energy supply determines its information processing power, and generates functional imaging signals, which are often assumed to reflect principal neuron spiking. Using measured cellular properties, we analysed how energy expenditure relates to neural computation in the cerebellar cortex. Most energy is used on information processing by non-principal neurons: Purkinje cells use only 18% of the signalling energy. Excitatory neurons use 73% and inhibitory neurons 27% of the energy. Despite markedly different computational architectures, the granular and molecular layers consume approximately the same energy. The blood vessel area supplying glucose and O2 is spatially matched to energy consumption. The energy cost of storing motor information in the cerebellum was also estimated.
Wildman, S., Boone, M., Peppiatt-Wildman, C., Contreras-Sanz, A., King, B., Shirley, D., Deen, P. and Unwin, R. (2009). Nucleotides downregulate aquaporin 2 via activation of apical P2 receptors. Journal of the American Society of Nephrology [Online] 20:1480-1490. Available at: http://dx.doi.org/10.1681/ASN.2008070686.
Vasopressin regulates water reabsorption in the collecting duct, but extracellular nucleotides modulate this regulation through incompletely understood mechanisms. We investigated these mechanisms using immortalized mouse collecting duct (mpkCCD) cells. Basolateral exposure to dDAVP induced AQP2 localization to the apical membrane, but co-treatment with ATP internalized AQP2. Because plasma membrane-bound P2 receptors (P2R) mediate the effects of extracellular nucleotides, we examined the abundance and localization of P2R in mpkCCD cells. In the absence of dDAVP, P2Y(1) and P2Y(4) receptors localized to the apical membrane, whereas P2X(2), P2X(4), P2X(5), P2X(7), P2Y(2), P2Y(11), and P2Y(12) receptors localized to the cytoplasm. dDAVP induced gene expression of P2X(1), which localized to the apical domain, and led to translocation of P2X(2) and P2Y(2) to the apical and basolateral membranes, respectively. In co-expression experiments, P2R activation decreased membrane AQP2 and AQP2-mediated water permeability in Xenopus oocytes expressing P2X(2), P2Y(2,) or P2Y(4) receptors, but not in oocytes expressing other P2R subtypes. In summary, these data suggest that AQP2-mediated water transport is downregulated not only by basolateral nucleotides, mediated by P2Y(2) receptors, but also by luminal nucleotides, mediated by P2X(2) and/or P2Y(4) receptors.
Wildman, S., Marks, J., Turner, C., Yew-Booth, L., Peppiatt-Wildman, C., King, B., Shirley, D., Wang, W. and Unwin, R. (2008). Sodium-dependent regulation of renal amiloride-sensitive currents by apical P2 receptors. Journal of the American Society of Nephrology [Online] 19:731-742. Available at: http://dx.doi.org/10.1681/ASN.2007040443.
The epithelial sodium channel (ENaC) plays a major role in the regulation of sodium balance and BP by controlling Na(+) reabsorption along the renal distal tubule and collecting duct (CD). ENaC activity is affected by extracellular nucleotides acting on P2 receptors (P2R); however, there remain uncertainties over the P2R subtype(s) involved, the molecular mechanism(s) responsible, and their physiologic role. This study investigated the relationship between apical P2R and ENaC activity by assessing the effects of P2R agonists on amiloride-sensitive current in the rat CD. Using whole-cell patch clamp of principal cells of split-open CD from Na(+)-restricted rats, in combination with immunohistochemistry and real-time PCR, we found that activation of metabotropic P2R (most likely the P2Y(2) and/or (4) subtype), via phospholipase C, inhibited ENaC activity. In addition, activation of ionotropic P2R (most likely the P2X(4) and/or (4/6) subtype), via phosphatidylinositol-3 kinase, either inhibited or potentiated ENaC activity, depending on the extracellular Na(+) concentration; therefore, it is proposed that P2X(4) and/or (4/6) receptors might function as apical Na(+) sensors responsible for local regulation of ENaC activity in the CD and could thereby help to regulate Na(+) balance and systemic BP.
Wildman, S., Marks, J., Churchill, L., Peppiatt-Wildman, C., Chraibi, A., Shirley, D., Horisberger, J., King, B. and Unwin, R. (2005). Regulatory interdependence of cloned epithelial Na+ channels and P2X receptors. Journal of the American Society of Nephrology : JASN [Online] 16:2586-97. Available at: http://dx.doi.org/10.1681/ASN.2005020130.
Epithelial Na+ channels (ENaC) coexist with a family of ATP-gated ion channels known as P2X receptors in the renal collecting duct. Although ENaC is itself insensitive to extracellular ATP, tubular perfusion of ATP can modify the activity of ENaC. To investigate a possible regulatory relationship between P2X receptors and ENaC, coexpression studies were performed in Xenopus oocytes. ENaC generated a persistent inward Na+ current that was sensitive to the channel blocker amiloride (I(am-s)). Exogenous ATP transiently activated all cloned isoforms of P2X receptors, which in some cases irreversibly inhibited I(am-s). The degree of inhibition depended on the P2X receptor subtype present. Activation of P2X2, P2X(2/6), P2X4, and P2X(4/6) receptor subtypes inhibited I(am-s), whereas activation of P2X1, P2X3, and P2X5 receptors had no significant effect. The degree of inhibition of I(am-s) correlated positively with the amount of ionic charge conducted by P2X receptor subtypes. ENaC inhibition required Na+ influx through I(am-s)-inhibiting P2X ion channels but also Ca2+ influx through P2X4 and P2X(4/6) ion channels. P2X-mediated inhibition of I(am-s) was found to be due to retrieval of ENaC from the plasma membrane. Maximum amplitudes of ATP-evoked P2X-mediated currents (I(ATP)) were significantly increased for P2X2, P2X(2/6), and P2X5 receptor subtypes after coexpression of ENaC. The increase in I(ATP) was due to increased levels of plasma membrane-bound P2X receptor protein, suggesting that ENaC modulates protein trafficking. In summary, ENaC was downregulated by the activation of P2X2, P2X(2/6), P2X4, and P2X(4/6) receptors. Conversely, ENaC increased the plasma membrane expression of P2X2, P2X(2/6), and P2X5 receptors.