Investigation of juxtaglomerular structure and function

Renin is the initiator and rate-limiting factor of the renin-angiotensin system, a major mechanism of blood pressure regulation. The synthesis and secretion of this active circulatory enzyme is confined exclusively to the dense core granules of kidney juxtaglomerular (JG) cells where its precursor prorenin is packaged, cleaved to the active form and stored for release on a regulated pathway. Given its importance, surprisingly little is known about this process, in part due to the difficulty in culturing primary JG cells in vitro and the lack of reliable cell lines. The initial aim of the current work was to investigate renin-containing granule dynamics in live JG cells. To achieve this, I attempted to derive novel cell lines from triple transgenic mouse models comprising immortalised granulated or non-granulated JG cells. Due to the nature of JG cells in culture, the use of these cell lines to investigate granulation was not feasible; therefore the culture of primary JG cell culture was modified and enhanced to visualise granule dynamics in live, primary JG cells for the first time. By isolating cells using a Percoll gradient and plating them on fibronectin-coated dishes, rapid and full adhesion of JG cells was achieved, as well as prolonged expression of renin from 3 days to up to 8 days post-isolation. Using this protocol to isolate JG cells from RenGFP renin reporter mice and identifying granules using the acidotropic fluorophore Lysotracker, granule dynamics were investigated in primary JG cells. High resolution, rapid image acquisition was performed using widefield and total internal reflection microscopy, showing that dense core granules respond dynamically to the β-adrenergic agonist isoproterenol, a known renin secretory stimulus. Two different pools of granules of varying granule diameters and dynamic parameters were identified optically, suggesting that separate granule pools were being identified. Mice null for the Ren-1d gene lack renin storage granules in their JG cells, however granulation was restored in Ren1d-null mice carrying a transgene encompassing the human renin (hRen) locus. Therefore in order to investigate the relationship between renin expression and the amount of granulation in JG cells, mice expressing human renin were used. To dissect the granulation phenotype in detail, 2D electron micrographs were taken of JG cells, which were immunogold stained to confirm renin content, and reconstructed in 3D. Female hRen mice showed a significantly higher volume of granulation and an increased granule number compared to males, a finding consistent with the sexually dimorphic expression of the transgene, supporting the hypothesis that granulation in JG cells is dependent on the level of renin expression. The macula densa (MD) is a critical sensor of flow and salt content in the blood; through extensive tubulo-vascular crosstalk known as tubuloglomerular feedback (TGF), it releases key signalling factors stimulating and inhibiting renin synthesis and secretion from JG cells. Ren-1d-/- mice showed a hypercellular and columnar MD plaque, which was not restored by the introduction of the hRen transgene, indicating that TGF may be impaired in these mice. Using an isolated, perfused juxtaglomerular apparatus model it was shown that high salt- and increased flow-induced TGF functioned effectively in Ren1d-/- and huRen+/-Ren1d-/- mice, although animals on a Ren1d-/- background showed decreased sensitivity of glomerular tuft contraction and abnormal calcium signalling within macula densa cells. In conclusion, an appropriate in vitro model was developed for investigating granule dynamics in JG cells, using which granule motion was visualised and quantified for the first time in these cells. Although JG cell granulation is required for normal MD morphology, it was shown to not affect JGA function

FK506 regulates IP3 evoked Ca2+ release independently of FKBP in endothelial cells

Background and Purpose FK506 and rapamycin are modulators of FK-binding proteins (FKBP) that are used to suppress immune function after organ and hematopoietic stem cell transplantations. The drugs share the unwanted side-effect of evoking hypertension that is associated with reduced endothelial function and nitric oxide production. The underlying mechanisms are not understood. FKBP may regulate IP3 and ryanodine receptors to alter Ca2+ signalling in endothelial cells. Experimental Approach We investigated the effects of FK506 and rapamycin on Ca2+ release via IP3 and ryanodine receptors in large numbers of endothelial cells in intact arteries. Key Results While confirmed to be present, FKBP modulation with rapamycin did not alter IP3-evoked Ca2+ release. Conversely, FK506, which modulates FKBP and additionally blocks calcineurin, increased IP3-evoked Ca2+ release. Inhibition of calcineurin (using okadiac acid or cypermethrin) also increased IP3-evoked Ca2+ release and blocked FK506 effects. Indeed, when calcineurin was inhibited with okadiac acid, FK506 reduced IP3-evoked Ca2+ release. These findings suggest that FKBP does not modulate IP3-evoked Ca2+ release and FK506 increased IP3-evoked Ca2+ release by calcineurin inhibition. FK506 and rapamycin are also unlikely to mediate their effects via RyR. The RyR activator caffeine and ryanodine itself failed to evoke Ca2+ changes suggesting that RyR is not functional in native endothelium. Conclusion and Implications The hypertensive effects of the immunosuppressant drugs FK506 and rapamycin, while mediated by endothelial cells, do not appear to be exerted at documented cellular targets of the drugs on Ca2+ release and altered FKBP binding to IP3 and RyR

Investigating the RAS can be a fishy business: Interdisciplinary opportunities using Zebrafish

The renin-angiotensin system (RAS) is highly conserved, and components of the RAS are present in all vertebrates to some degree. Although the RAS has been studied since the discovery of renin, its biological role continues to broaden with the identification and characterization of new peptides. The evolutionarily distant zebrafish is a remarkable model for studying the kidney due to its genetic tractability and accessibility for in vivo imaging. The zebrafish pronephros is an especially useful kidney model due to its structural simplicity yet complex functionality, including capacity for glomerular and tubular filtration. Both the pronephros and mesonephros contain renin-expressing perivascular cells, which respond to RAS inhibition, making the zebrafish an excellent model for studying the RAS. This review summarizes the physiological and genetic tools currently available for studying the zebrafish kidney with regards to functionality of the RAS, using novel imaging techniques such as SPIM microscopy coupled with targeted single cell ablation and synthesis of vasoactive RAS peptides

Imaging the Developing Heart: Synchronized Timelapse Microscopy During Developmental Changes

How do you use imaging to analyse the development of the heart, which not only changes shape but also undergoes constant, high-speed, quasi-periodic changes? We have integrated ideas from prospective and retrospective optical gating to capture long-term, phase-locked developmental time-lapse videos. In this paper we demonstrate the success of this approach over a key developmental time period: heart looping, where large changes in heart shape prevent previous prospective gating approaches from capturing phase-locked videos. We use the comparison with other approaches to in vivo heart imaging to highlight the importance of collecting the most appropriate data for the biological question.Comment: Carl J. Nelson and Charlotte Buckley and John J. Mullins and Martin A. Denvir and Jonathan Taylor, "Imaging the Developing Heart: Synchronized Timelapse Microscopy During Developmental Changes", Proc. SPIE (10499), 10499-41 (2018). Copyright 2018 Society of Photo Optical Instrumentation Engineers (SPIE

MiTiSegmenter: Software for high throughput segmentation and meshing of microCT data in microtiter plate arrays

Lab-based microCT is a powerful means of visualising the internal structure of physical specimens deployed across the physical sciences, engineering and the arts. As its popularity has grown, demand for bulk digitisation of multiple samples within a single scan has increased. High throughput workflows can increase sample sizes and reduce scan time, yet downstream segmentation and meshing remain a bottleneck. We present MiTiSegmenter as a new tool for the bulk archiving of valuable zooarchaeological and palaeontological remains. We foresee MiTiSegmenter as particularly useful when incorporated into workflows that ultimately require the destructive testing of specimens, including sampling for ancient DNA and proteomics. The software may also play an important role in national museums' ongoing mass digitisation efforts, facilitating the high-speed archiving of specimen 3D morphology across extensive collections with very minimal user intervention or prior training. - We present MiTiSegmenter, a software package for semi-automated image processing and segmentation of array-based batch microCT data. - Implemented in Python, MiTiSegmenter expedites cropping, meshing and exporting samples within stacked microtiter plates, facilitating the rapid digitisation of hundreds-thousands of samples per scan. - We illustrate MiTiSegmenter's capabilities when applied to bulk archiving of valuable zooarchaeological and palaeontological remains

Visual Advantage in Deaf Adults Linked to Retinal Changes

The altered sensory experience of profound early onset deafness provokes sometimes large scale neural reorganisations. In particular, auditory-visual cross-modal plasticity occurs, wherein redundant auditory cortex becomes recruited to vision. However, the effect of human deafness on neural structures involved in visual processing prior to the visual cortex has never been investigated, either in humans or animals. We investigated neural changes at the retina and optic nerve head in profoundly deaf (N = 14) and hearing (N = 15) adults using Optical Coherence Tomography (OCT), an in-vivo light interference method of quantifying retinal micro-structure. We compared retinal changes with behavioural results from the same deaf and hearing adults, measuring sensitivity in the peripheral visual field using Goldmann perimetry. Deaf adults had significantly larger neural rim areas, within the optic nerve head in comparison to hearing controls suggesting greater retinal ganglion cell number. Deaf adults also demonstrated significantly larger visual field areas (indicating greater peripheral sensitivity) than controls. Furthermore, neural rim area was significantly correlated with visual field area in both deaf and hearing adults. Deaf adults also showed a significantly different pattern of retinal nerve fibre layer (RNFL) distribution compared to controls. Significant correlations between the depth of the RNFL at the inferior-nasal peripapillary retina and the corresponding far temporal and superior temporal visual field areas (sensitivity) were found. Our results show that cross-modal plasticity after early onset deafness may not be limited to the sensory cortices, noting specific retinal adaptations in early onset deaf adults which are significantly correlated with peripheral vision sensitivity

“Captain of All These Men of Death”: An Integrated Case Study of Tuberculosis in Nineteenth-Century Otago, New Zealand

The South Island of New Zealand saw several major waves of migration in the mid-nineteenth century, predominantly from Europe but also with an ethnically distinct Chinese presence. The rural community of Milton, Otago, was a settler community established primarily by immigrants from the United Kingdom in search of a better quality of life. However, these settlers faced unique challenges related to surviving in an isolated location with very little infrastructure compared to their origin populations. In 2016, excavation was undertaken at St. John’s burial ground, Milton, with the object of using bioarchaeological methods to elucidate the lived experience of the first organized European settlement of this region, particularly in terms of health and disease. Here we present a case study of Burial 21 (B21), a male individual of known identity and a documented cause of death. We use biochemical and paleopathological methods to ground-truth his written history, which includes a period of invalidism due to tuberculosis, and discuss the implications of our findings for the community, provision of care, and quality of life in rural colonial New Zealand. He maha tonu ngā hekenga tāngata ki Te Waka a Māui i ngā tau kei waenga pū o te rau tau 1800, ko te nuinga nō Ūropi, heoi he tokomaha tonu nō Haina. Nā ngā manene nō Peretānia te hapori o Milton i whakatū ki Tokomairaro, i Ōtākou, i tō rātou hiahia ki tētehi oranga kounga ake i tō rātou oranga i Peretānia. Heoi, ko ētehi o ngā wero nui i tau ki ngā manene nei i ahu mai i te noho pūreirei ki tētehi wāhi kāore rawa ngā ratonga i rite ki ngā wāhi i ahu mai ai rātou. I te tau 2016, i hahu kōiwi i te urupā o Hato Hone, i Milton, hei whakamātau i te kaha o te ora me ngā momo mate i pā atu ki ngā tāngata whai i noho i te rohe nei. Nei rā he ripoata mō tētehi kua hahua, kua tapaina ko B21, he tāne ia, ko tōna ingoa kua mōhiotia, ko tōna mate kua āta tuhia. Kua āta whakamātauria ōna kōiwi me ōna toenga kiko mō ngā tohu ora me ngā tohu mate, kia mārama ai mena rānei e hāngai ana ngā tuhinga rongoā mōna, ngā mea i tuhia nōna e takatū ana, tae atu ki te wā i tūroro ia i te mate kohi, ki ngā tohu e puta ana i te mātauranga Rongoā-Koiora ō nāianei. Ka matapakina ngā hīrautanga o ngā kitenga me te māramatanga kua puta i tēnei rangahau e pā ana ki te hapori, ki ngā ratonga hauora, me te kounga o te oranga mō te hunga noho tuawhenua i tērā wā i Aotearoa

Carbenoxolone and 18β-glycyrrhetinic acid inhibit inositol 1,4,5-trisphosphate-mediated endothelial cell calcium signalling and depolarise mitochondria

Background and Purpose: Coordinated endothelial control of cardiovascular function is proposed to occur by endothelial cell communication via gap junctions and connexins. To study intercellular communication, the pharmacological agents carbenoxolone (CBX) and 18β-glycyrrhetinic acid (18βGA) are used widely as connexin inhibitors and gap junction blockers. Experimental Approach: We investigated the effects of CBX and 18βGA on intercellular Ca 2+ waves, evoked by inositol 1,4,5-trisphosphate (IP 3) in the endothelium of intact mesenteric resistance arteries. Key Results: Acetycholine-evoked IP 3-mediated Ca 2+ release and propagated waves were inhibited by CBX (100 μM) and 18βGA (40 μM). Unexpectedly, the Ca 2+ signals were inhibited uniformly in all cells, suggesting that CBX and 18βGA reduced Ca 2+ release. Localised photolysis of caged IP 3 (cIP 3) was used to provide precise spatiotemporal control of site of cell activation. Local cIP 3 photolysis generated reproducible Ca 2+ increases and Ca 2+ waves that propagated across cells distant to the photolysis site. CBX and 18βGA each blocked Ca 2+ waves in a time-dependent manner by inhibiting the initiating IP 3-evoked Ca 2+ release event rather than block of gap junctions. This effect was reversed on drug washout and was unaffected by small or intermediate K +-channel blockers. Furthermore, CBX and 18βGA each rapidly and reversibly collapsed the mitochondrial membrane potential. Conclusion and Implications: CBX and 18βGA inhibit IP 3-mediated Ca 2+ release and depolarise the mitochondrial membrane potential. These results suggest that CBX and 18βGA may block cell–cell communication by acting at sites that are unrelated to gap junctions