Role of the kidneys in acid-base regulation and ammonia excretion in freshwater and seawater fish: implications for nephrocalcinosis

Maintaining normal pH levels in the body fluids is essential for homeostasis and represents one of the most tightly regulated physiological processes among vertebrates. Fish are generally ammoniotelic and inhabit diverse aquatic environments that present many respiratory, acidifying, alkalinizing, ionic and osmotic stressors to which they are able to adapt. They have evolved flexible strategies for the regulation of acid-base equivalents (H+, NH4+, OH− and HCO3−), ammonia and phosphate to cope with these stressors. The gills are the main regulatory organ, while the kidneys play an important, often overlooked accessory role in acid-base regulation. Here we outline the kidneys role in regulation of acid-base equivalents and two of the key ‘urinary buffers’, ammonia and phosphate, by integrating known aspects of renal physiology with recent advances in the molecular and cellular physiology of membrane transport systems in the teleost kidneys. The renal transporters (NHE3, NBC1, AE1, SLC26A6) and enzymes (V-type H+ATPase, CAc, CA IV, ammoniagenic enzymes) involved in H+ secretion, bicarbonate reabsorption, and the net excretion of acidic and basic equivalents, ammonia, and inorganic phosphate are addressed. The role of sodium-phosphate cotransporter (Slc34a2b) and rhesus (Rh) glycoproteins (ammonia channels) in conjunction with apical V-type H+ ATPase and NHE3 exchangers in these processes are also explored. Nephrocalcinosis is an inflammation-like disorder due to the precipitation of calcareous material in the kidneys, and is listed as one of the most prevalent pathologies in land-based production of salmonids in recirculating aquaculture systems. The causative links underlying the pathogenesis and etiology of nephrocalcinosis in teleosts is speculative at best, but acid-base perturbation is probably a central pathophysiological cause. Relevant risk factors associated with nephrocalcinosis are hypercapnia and hyperoxia in the culture water. These raise internal CO2 levels in the fish, triggering complex branchial and renal acid-base compensations which may promote formation of kidney stones. However, increased salt loads through the rearing water and the feed may increase the prevalence of nephrocalcinosis. An increased understanding of the kidneys role in acid-base and ion regulation and how this relates to renal diseases such as nephrocalcinosis will have applied relevance for the biologist and aquaculturist alike.publishedVersio

Heads and tails: The notochord develops differently in the cranium and caudal fin of Atlantic Salmon (Salmo salar, L.)

While it is well known that the notochord of bony fishes changes over developmental time, less is known about how it varies across different body regions. In the development of the Atlantic salmon, Salmo salar L., cranial and caudal ends of the notochord are overlaid by the formation of the bony elements of the neurocranium and caudal fin, respectively. To investigate, we describe how the notochord of the cranium and caudal fin changes from embryo to spawning adult, using light microscopy, SEM, TEM, dissection, and CT scanning. The differences are dramatic. In contrast to the abdominal and caudal regions, at the ends of the notochord vertebrae never develop. While the cranial notochord builds a tapering, unsegmented cone of chordal bone, the urostylic notochordal sheath never ossifies: adjacent, irregular bony elements form from the endoskeleton of the caudal fin. As development progresses, two previously undescribed processes occur. First, the bony cone of the cranial notochord, and its internal chordocytes, are degraded by chordoclasts, an undescribed function of the clastic cell type. Second, the sheath of the urostylic notochord creates transverse septae that partly traverse the lumen in an irregular pattern. By the adult stage, the cranial notochord is gone. In contrast, the urostylic notochord in adults is robust, reinforced with septae, covered by irregularly shaped pieces of cellular bone, and capped with an opistural cartilage that develops from the sheath of the urostylic notochord. A previously undescribed muscle, with its origin on the opistural cartilage, inserts on the lepidotrich ventral to it.publishedVersio

Detection of a nodavirus-like agent in heart tissue from reared Atlantic salmon Salmo salar suffering from cardiac myopathy syndrome (CMS)

The present study shows that a nodavirus-like agent is associated with the lesions of cardiac myopathy syndrome (CMS), a disease of unknown etiology which affects reared adult Atlantic salmon Salmo salar. In archive paraffin-embedded heart tissue from Atlantic salmon diagnosed as suffering from CMS, a distinct immunohistochemical reaction was observed when using a primary antibody against striped jack nervous necrosis virus (Nodaviridae). Immunolabeling was detected in the mesothelium and hypercellular lesions of the epicardium and in endothelial cells and myocytes within mild multifocal lesions of the atrial and ventricular trabeculae. Transmission electron microscopy, per formed on deparaffinized samples of the same tissue blocks, revealed substantial amounts of virus-like particles (VLP) in the cytoplasm of endocardial endothelium, in myocytes and in mesothelial cells of the epicardium. The VLP were isometric, spherical and unenveloped, with mean capsid diameters of approximately 25 nm, resembling a virus belonging to the Nodaviridae

Transcriptome sequencing of Atlantic salmon (Salmo salar L.) notochord prior to development of the vertebrae provides clues to regulation of positional fate, chordoblast lineage and mineralisation

Background: In teleosts such as Atlantic salmon (Salmo salar L.), segmentation and subsequent mineralisation of the notochord during embryonic stages are essential for normal vertebrae formation. However, the molecular mechanisms leading to segmentation and mineralisation of the notochord are poorly understood. The aim of this study was to identify genes/pathways acting in gradients over time and along the anterior-posterior axis during notochord segmentation and immediately prior to mineralisation of the vertebral bodies in Atlantic salmon. Results: Notochord samples were collected from unsegmented, pre-segmented and segmented developmental stages. In each stage, the cellular core of the notochord was cut into three pieces along the longitudinal axis (anterior, mid, posterior). RNA was sequenced (22 million pair-end 100 bp/library) and mapped to the salmon genome. 66569 transcripts were predicted and 55775 were annotated. In order to identify possible gradients leading to segmentation of the notochord, all 71 notochord-expressed hox genes were investigated, most of them displaying a typical anterior-posterior expression pattern along the notochord axis. The clustering of hox genes revealed a pattern that could be related to notochord segmentation. We further investigated how mineralisation is initiated in the notochord, and several factors related to chondrogenic lineage were identified (sox9, sox5, sox6, tgfb3, ihhb and col2a1), suggesting a cartilage-like character of the notochord. KEGG analysis of differentially expressed genes between stages revealed down-regulation of pathways associated with ECM, cell division, metabolism and development at onset of notochord segmentation. This implies that inhibitory signals produce segmentation of the notochord. One such potential inhibitory signal was identified, col11a2, which was detected in segments of non-mineralising notochord. Conclusions: An incomplete salmon genome was successfully used to analyse RNA-seq data from the cellular core of the Atlantic salmon notochord. In transcriptome we found; hox gene patterns possibly linked to segmentation; down-regulation of pathways in the notochord at onset of segmentation; segmented expression of col11a2 in non-mineralised segments of the notochord; and a chondroblast-like footprint in the notochord

Ion transporters and osmoregulation in the kidney of teleost fishes as a function of salinity

Euryhaline teleosts exhibit major changes in renal function as they move between freshwater (FW) and seawater (SW) environments, thus tolerating large fluctuations in salinity. In FW, the kidney excretes large volumes of water through high glomerular filtration rates (GFR) and low tubular reabsorption rates, while actively reabsorbing most ions at high rates. The excreted product has a high urine flow rate (UFR) with a dilute composition. In SW, GFR is greatly reduced, and the tubules reabsorb as much water as possible, while actively secreting divalent ions. The excreted product has a low UFR, and is almost isosmotic to the blood plasma, with Mg2+, SO42–, and Cl– as the major ionic components. Early studies at the organismal level have described these basic patterns, while in the last two decades, studies of regulation at the cell and molecular level have been implemented, though only in a few euryhaline groups (salmonids, eels, tilapias, and fugus). There have been few studies combining the two approaches. The aim of the review is to integrate known aspects of renal physiology (reabsorption and secretion) with more recent advances in molecular water and solute physiology (gene and protein function of transporters). The renal transporters addressed include the subunits of the Na+, K+- ATPase (NKA) enzyme, monovalent ion transporters for Na+, Cl–, and K+ (NKCC1, NKCC2, CLC-K, NCC, ROMK2), water transport pathways [aquaporins (AQP), claudins (CLDN)], and divalent ion transporters for SO42–, Mg2+, and Ca2+ (SLC26A6, SLC26A1, SLC13A1, SLC41A1, CNNM2, CNNM3, NCX1, NCX2, PMCA). For each transport category, we address the current understanding at the molecular level, try to synthesize it with classical knowledge of overall renal function, and highlight knowledge gaps. Future research on the kidney of euryhaline fishes should focus on integrating changes in kidney reabsorption and secretion of ions with changes in transporter function at the cellular and molecular level (gene and protein verification) in different regions of the nephrons. An increased focus on the kidney individually and its functional integration with the other osmoregulatory organs (gills, skin and intestine) in maintaining overall homeostasis will have applied relevance for aquaculture

Sulfate homeostasis in Atlantic salmon is associated with differential regulation of salmonid-specific paralogs in gill and kidney

Sulfate (SO2−4) regulation is challenging for euryhaline species as they deal with large fluctuations of SO2−4 during migratory transitions between freshwater (FW) and seawater (SW), while maintaining a stable plasma SO2−4 concentration. Here, we investigated the regulation and potential role of sulfate transporters in Atlantic salmon during the preparative switch from SO2−4 uptake to secretion. A prepara-tory increase in kidney and gill sodium/potassium ATPase (Nka) enzyme activity during smolt development indicate preparative osmoregulatory changes. In con-trast to gill Nka activity a transient decrease in kidney Nka after direct SW expo-sure was observed and may be a result of reduced glomerular filtration rates and tubular flow through the kidney. In silico analyses revealed that Atlantic salmon genome comprises a single slc13a1gene and additional salmonid- specific duplica-tions of slc26a1 and slc26a6a, leading to new paralogs, namely the slc26a1a and - b, and slc26a6a1 and - a2. A kidney- specific increase in slc26a6a1 and slc26a1a during smoltification and SW transfer, suggests an important role of these sul-fate transporters in the regulatory shift from absorption to secretion in the kid-ney. Plasma SO2−4 in FW smolts was 0.70mM, followed by a transient increase to 1.14±0.33mM 2days post- SW transfer, further decreasing to 0.69±0.041mM after 1month in SW. Our findings support the vital role of the kidney in SO2−4 ex-cretion through the upregulated slc26a6a1, the most likely secretory transport can-didate in fish, which together with the slc26a1a transporter likely removes excess SO2−4, and ultimately enable the regulation of normal plasma SO2−4 levels in SW

Sulfate homeostasis in Atlantic salmon is associated with differential regulation of salmonid-specific paralogs in gill and kidney

Sulfate (SO2−4) regulation is challenging for euryhaline species as they deal with large fluctuations of SO2−4 during migratory transitions between freshwater (FW) and seawater (SW), while maintaining a stable plasma SO2−4 concentration. Here, we investigated the regulation and potential role of sulfate transporters in Atlantic salmon during the preparative switch from SO2−4 uptake to secretion. A preparatory increase in kidney and gill sodium/potassium ATPase (Nka) enzyme activity during smolt development indicate preparative osmoregulatory changes. In contrast to gill Nka activity a transient decrease in kidney Nka after direct SW exposure was observed and may be a result of reduced glomerular filtration rates and tubular flow through the kidney. In silico analyses revealed that Atlantic salmon genome comprises a single slc13a1 gene and additional salmonid-specific duplications of slc26a1 and slc26a6a, leading to new paralogs, namely the slc26a1a and -b, and slc26a6a1 and -a2. A kidney-specific increase in slc26a6a1 and slc26a1a during smoltification and SW transfer, suggests an important role of these sulfate transporters in the regulatory shift from absorption to secretion in the kidney. Plasma SO2−4 in FW smolts was 0.70 mM, followed by a transient increase to 1.14 ± 0.33 mM 2 days post-SW transfer, further decreasing to 0.69 ± 0.041 mM after 1 month in SW. Our findings support the vital role of the kidney in SO2−4 excretion through the upregulated slc26a6a1, the most likely secretory transport candidate in fish, which together with the slc26a1a transporter likely removes excess SO2−4 , and ultimately enable the regulation of normal plasma SO2−4 levels in SW