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Contains fulltext : 244896.pdf (Publisher’s version ) (Closed access) BACKGROUND: Over the last decade, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, approximately 20% of all patients with tubulopathy lack a genetic diagnosis. METHODS: We performed whole-exome and -genome sequencing of a patient cohort with a novel, inherited, salt-losing tubulopathy; hypomagnesemia; and dilated cardiomyopathy. We also conducted subsequent in vitro functional analyses of identified variants of RRAGD, a gene that encodes a small Rag guanosine triphosphatase (GTPase). RESULTS: In eight children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients also had dilated cardiomyopathy and three underwent heart transplantation. We identified a heterozygous variant in RRAGD that segregated with the phenotype in eight members of a large family with similar kidney manifestations. The GTPase RagD, encoded by RRAGD, plays a role in mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron included the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro. CONCLUSIONS: Our findings establish a novel disease, which we call autosomal dominant kidney hypomagnesemia (ADKH-RRAGD), that combines an electrolyte-losing tubulopathy and dilated cardiomyopathy. The condition is caused by variants in the RRAGD gene, which encodes Rag GTPase D; these variants lead to an activation of mTOR signaling, suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function. 01 november 2021

Tubular ATP release is regulated by mechanosensation of fluid shear stress (FSS). Polycystin-1/polycystin-2 (PC1/PC2) functions as a mechanosensory complex in the kidney. Extracellular ATP is implicated in polycystic kidney disease (PKD), where PC1/PC2 is dysfunctional. This study aims to provide new insights into the ATP signaling under physiological conditions and PKD. Microfluidics, pharmacologic inhibition, and loss-of-function approaches were combined to assess the ATP release in mouse distal convoluted tubule 15 (mDCT15) cells. Kidney-specific Pkd1 knockout mice (iKsp-Pkd1(-/-)) and zebrafish pkd2 morphants (pkd2-MO) were as models for PKD. FSS-exposed mDCT15 cells displayed increased ATP release. Pannexin-1 inhibition and knockout decreased FSS-modulated ATP release. In iKsp-Pkd1(-/-) mice, elevated renal pannexin-1 mRNA expression and urinary ATP were observed. In Pkd1(-/-) mDCT15 cells, elevated ATP release was observed upon the FSS mechanosensation. In these cells, increased pannexin-1 mRNA expression was observed. Importantly, pannexin-1 inhibition in pkd2-MO decreased the renal cyst growth. Our results demonstrate that pannexin-1 channels mediate ATP release into the tubular lumen due to pro-urinary flow. We present pannexin-1 as novel therapeutic target to prevent the renal cyst growth in PKD.

Item does not contain fulltext Background: Mutations in the thyroid hormone (TH) transporter monocarboxylate transporter 8 (MCT8) cause MCT8 deficiency, characterized by severe intellectual and motor disability and abnormal serum thyroid function tests. Various Mct8 knock-out mouse models as well as mct8 knock-out and knockdown zebrafish models are used as a disease model for MCT8 deficiency. Although important for model eligibility, little is known about the functional characteristics of the MCT8 orthologues in these species. Therefore, we here compared the functional characteristics of mouse (mm) MCT8 and zebrafish (dr) Mct8 to human (hs) MCT8. Methods: We performed extensive transport studies in COS-1 and JEG-3 cells transiently transfected with hsMCT8, drMct8, and mmMCT8. Protein expression levels and subcellular localization were assessed by immunoblotting, surface biotinylation, and immunocytochemistry. Sequence alignment and structural modeling were used to interpret functional differences between the orthologues. Results: hsMCT8, drMct8, and mmMCT8 all facilitated the uptake and efflux of 3,3'-diiodothyronine (3,3'-T2), rT3, triiodothyronine (T3), and thyroxine (T4), although the initial uptake rates of drMct8 were 1.5-4.0-fold higher than for hsMCT8 and mmMCT8. drMct8 exhibited 3-50-fold lower apparent IC50 values than hsMCT8 and mmMCT8 for all tested substrates, and substrate preference of drMct8 (3,3'-T2, T3 > T4 > rT3) differed from hsMCT8 and mmMCT8 (T3 > T4 > rT3, 3,3'-T2). Compared with hsMCT8 and mmMCT8, cis-inhibition studies showed that T3 uptake by drMct8 was inhibited at a lower concentration and by a broader spectrum of TH metabolites. Total and cell surface expression levels of drMct8 and hsMCT8 were equal and both significantly exceeded those of mmMCT8. Structural modeling located most non-conserved residues outside the substrate pore, except for H192 in hsMCT8, which is replaced by a glutamine in drMct8. However, a H192Q substituent of hsMCT8 did not alter its transporter characteristics. Conclusion: Our studies substantiate the eligibility of mice and zebrafish models for human MCT8 deficiency. However, differences in the intrinsic transporter properties of MCT8 orthologues may exist, which should be realized when comparing MCT8 deficiency in different in vivo models. Moreover, our findings may indicate that the protein domains outside the substrate channel may play a role in substrate selection and protein stability.

BackgroundOver the last decaces, advances in genetic techniques have resulted in the identification of rare hereditary disorders of renal magnesium and salt handling. Nevertheless, ±20% of all tubulopathy patients remain without genetic diagnosis. Here, we explore a large multicentric patient cohort with a novel inherited salt-losing tubulopathy, hypomagnesemia and dilated cardiomyopathy (DCM).MethodsWhole exome and genome sequencings were performed with various subsequent functional analyses of identified RRAGD variants in vitro.ResultsIn 8 children from unrelated families with a tubulopathy characterized by hypomagnesemia, hypokalemia, salt-wasting, and nephrocalcinosis, we identified heterozygous missense variants in RRAGD that mostly occurred de novo. Six of these patients additionally suffered from DCM requiring heart transplantation in 3 of them. An additional dominant variant in RRAGD was simultaneously identified in eight members of a large family with a similar renal phenotype. RRAGD encodes GTPase RagD mediating amino acid signaling to the mechanistic target of rapamycin complex 1 (mTORC1). RagD expression along the mammalian nephron include the thick ascending limb and the distal convoluted tubule. The identified RRAGD variants were shown to induce a constitutive activation of mTOR signaling in vitro,ConclusionsOur findings establish a novel disease phenotype combining kidney tubulopathy and cardiomyopathy (KICA) caused by an activation of mTOR signaling suggesting a critical role of Rag GTPase D for renal electrolyte handling and cardiac function.SIGNIFICANCE STATEMENTHere, we report on heterozygous variants in RRAGD in patients with profound hypomagnesemia, renal salt wasting, nephrocalcinosis, and dilated cardiomyopathy. The identified RagD variants induce a constitutive activation of mTOR signaling in vitro. These findings not only establish a novel monogenic disorder of the kidney tubule, but demonstrate the essential role of mTOR signaling for distal tubular electrolyte handling and cardiac function.

The kidneys are essential for water and electrolyte homeostasis. This Review outlines the effect of tubular flow on renal electrolyte transport along the nephron and the current challenges in the emerging field of tubular flow dynamics in the kidney.The kidney is a remarkable organ that accomplishes the challenge of removing waste from the body and simultaneously regulating electrolyte and water balance. Pro-urine flows through the nephron in a highly dynamic manner and adjustment of the reabsorption rates of water and ions to the variable tubular flow is required for electrolyte homeostasis. Renal epithelial cells sense the tubular flow by mechanosensation. Interest in this phenomenon has increased in the past decade since the acknowledgement of primary cilia as antennae that sense renal tubular flow. However, the significance of tubular flow sensing for electrolyte handling is largely unknown. Signal transduction pathways regulating flow-sensitive physiological responses involve calcium, purinergic and nitric oxide signalling, and are considered to have an important role in renal electrolyte handling. Given that mechanosensation of tubular flow is an integral role of the nephron, defective tubular flow sensing is probably involved in renal disease. Studies investigating tubular flow and electrolyte transport differ in their methodology, subsequently hampering translational validity. This Review provides the basis for understanding electrolyte disorders originating from altered tubular flow sensing as a result of pathological conditions.

The kidney is a remarkable organ that accomplishes the challenge of removing waste from the body and simultaneously regulating electrolyte and water balance. Pro-urine flows through the nephron in a highly dynamic manner and adjustment of the reabsorption rates of water and ions to the variable tubular flow is required for electrolyte homeostasis. Renal epithelial cells sense the tubular flow by mechanosensation. Interest in this phenomenon has increased in the past decade since the acknowledgement of primary cilia as antennae that sense renal tubular flow. However, the significance of tubular flow sensing for electrolyte handling is largely unknown. Signal transduction pathways regulating flow-sensitive physiological responses involve calcium, purinergic and nitric oxide signalling, and are considered to have an important role in renal electrolyte handling. Given that mechanosensation of tubular flow is an integral role of the nephron, defective tubular flow sensing is probably involved in renal disease. Studies investigating tubular flow and electrolyte transport differ in their methodology, subsequently hampering translational validity. This Review provides the basis for understanding electrolyte disorders originating from altered tubular flow sensing as a result of pathological conditions.

Sea bream (Sparus aurata Linneaus) was acclimated to three salinity concentrations, viz. 5 (LSW), 38 (SW) and 55psμ (HSW) and three water temperatures regimes (12, 19 and 26 °C) for five weeks. Osmoregulatory capacity parameters (plasma osmolality, sodium, chloride, cortisol, and branchial and renal Na+,K+-ATPase activities) were also assessed. Salinity and temperature affected all of the parameters tested. Our results indicate that environmental temperature modulates capacity in sea bream, independent of environmental salinity, and set points of plasma osmolality and ion concentrations depend on both ambient salinity and temperature. Acclimation to extreme salinity resulted in stress, indicated by elevated basal plasma cortisol levels. Response to salinity was affected by ambient temperature. A comparison between branchial and renal Na+,K+-ATPase activities appears instrumental in explaining salinity and temperature responses. Sea bream regulate branchial enzyme copy numbers (Vmax) in hyperosmotic media (SW and HSW) to deal with ambient temperature effects on activity; combinations of high temperatures and salinity may exceed the adaptive capacity of sea bream. Salinity compromises the branchial enzyme capacity (compared to basal activity at a set salinity) when temperature is elevated and the scope for temperature adaptation becomes smaller at increasing salinity. Renal Na+,K+-ATPase capacity appears fixed and activity appears to be determined by temperature., This study was partly supported by grants AGL2007-61211/ACU (Ministerio de Innovación y Ciencia and FEDER, Spain) and Proyecto de Excelencia PO7-RNM-02843 (Junta de Andalucía) to JMM. L.V.C. is funded FONDAP-IDEAL 15150003.

Solute carrier family 41 member A1 (SLC41A1) has been suggested to mediate magnesium (Mg2+) transport by several in vitro studies. However, the physiological function of SLC41A1 remains to be elucidated. In this study, cellular Mg2+ transport assays combined with zebrafish slc41a1 knockdown experiments were performed to disclose SLC41A1 function and its physiological relevance. The gene slc41a1 is ubiquitously expressed in zebrafish tissues and is regulated by water and dietary Mg2+ availability. Knockdown of slc41a1 in zebrafish larvae grown in a Mg2+-free medium resulted in a unique phenotype characterized by a decrease in zebrafish Mg content. This decrease shows that SLC41A1 is required to maintain Mg2+ balance and its dysfunction results in renal Mg2+ wasting in zebrafish larvae. Importantly, the Mg content of the larvae is rescued when mouse SLC41A1 is expressed in slc41a1-knockdown zebrafish. Conversely, expression of mammalian SLC41A1-p.Asp262Ala, harboring a mutation in the ion-conducting SLC41A1 pore, did not reverse the renal Mg2+ wasting. 25Mg2+ transport assays in human embryonic kidney 293 (HEK293) cells overexpressing SLC41A1 demonstrated that SLC41A1 mediates cellular Mg2+ extrusion independently of sodium (Na+). In contrast, SLC41A1-p.Asp262Ala expressing HEK293 cells displayed similar Mg2+ extrusion activities than control (mock) cells. In polarized Madin-Darby canine kidney cells, SLC41A1 localized to the basolateral cell membrane. Our results demonstrate that SLC41A1 facilitates renal Mg2+ reabsorption in the zebrafish model. Furthermore, our data suggest that SLC41A1 mediates both Mg2+ uptake and extrusion. Electronic supplementary material The online version of this article (10.1007/s00424-018-2234-9) contains supplementary material, which is available to authorized users.

Magnesium (Mg2+) is an important cofactor of many enzymes crucial for life; therefore, maintaining a Mg2+ balance in the body is essential. In the kidney, the distal convoluted tubule (DCT) determines the final urinary Mg2+ excretion. The nephron is subjected to variable urinary flow, but little is known about the influence of flow on Mg2+ transport. Primary cilia, which are mechanosensory organelles that sense changes in flow, are expressed on tubular epithelial cells. This study aimed to elucidate whether urinary flow facilitates DCT Mg2+ transport. To this end, mouse DCT15 cells, with and without primary cilia, were exposed to physiologic fluid flow generating 0.3, 0.6, and 1.2 dyn/cm2 fluid shear stress (FSS). FSS stimulated Mg2+ uptake significantly. Net Mg2+ uptake ( i.e., the difference between static and FSS) followed a single component saturable first-order transport function and was independent of FSS magnitude and primary cilia. FSS did not affect the expression of magnesiotropic genes, including Cnnm2, Kcna1, Proegf, Trpm6, and Trpm7. Transient receptor potential cation channel subfamily melastatin (TRPM) member 7 (Trmp7) inhibition by 2-aminoethyl diphenyl borinate or knockout of TRPM6 did not alter net Mg2+ uptake, suggesting that TRPM6/TRPM7 homo/heterodimeric channels are not involved in FSS-activated Mg2+ transport. In summary, FSS generated by physiologic fluid flow is a new factor activating Mg2+ transport in DCT independent of primary cilia.-Verschuren, E. H. J., Hoenderop, J. G. J., Peters, D. J. M., Arjona, F. J., Bindels, R. J. M. Tubular flow activates magnesium transport in the distal convoluted tubule.

The PKD1 gene encodes polycystin-1 (PC1), a mechanosensor triggering intracellular responses upon urinary flow sensing in kidney tubular cells. Mutations in PKD1 lead to autosomal dominant polycystic kidney disease (ADPKD). The involvement of PC1 in renal electrolyte handling remains unknown since renal electrolyte physiology in ADPKD patients has only been characterized in cystic ADPKD. We thus studied the renal electrolyte handling in inducible kidney-specific Pkd1 knockout (iKsp- Pkd1−/−) mice manifesting a precystic phenotype. Serum and urinary electrolyte determinations indicated that iKsp- Pkd1−/−mice display reduced serum levels of magnesium (Mg2+), calcium (Ca2+), sodium (Na+), and phosphate (Pi) compared with control ( Pkd1+/+) mice and renal Mg2+, Ca2+, and Piwasting. In agreement with these electrolyte disturbances, downregulation of key genes for electrolyte reabsorption in the thick ascending limb of Henle's loop (TA;, Cldn16, Kcnj1, and Slc12a1), distal convoluted tubule (DCT; Trpm6 and Slc12a3) and connecting tubule (CNT; Calb1, Slc8a1, and Atp2b4) was observed in kidneys of iKsp- Pkd1−/−mice compared with controls. Similarly, decreased renal gene expression of markers for TAL ( Umod) and DCT ( Pvalb) was observed in iKsp- Pkd1−/−mice. Conversely, mRNA expression levels in kidney of genes encoding solute and water transporters in the proximal tubule ( Abcg2 and Slc34a1) and collecting duct ( Aqp2, Scnn1a, and Scnn1b) remained comparable between control and iKsp- Pkd1−/−mice, although a water reabsorption defect was observed in iKsp- Pkd1−/−mice. In conclusion, our data indicate that PC1 is involved in renal Mg2+, Ca2+, and water handling and its dysfunction, resulting in a systemic electrolyte imbalance characterized by low serum electrolyte concentrations.

Contains fulltext : 184156.pdf (Publisher’s version ) (Closed access) Magnesium (Mg(2+)) homeostasis is critical for metabolism. However, the genetic determinants of the renal handling of Mg(2+), which is crucial for Mg(2+) homeostasis, and the potential influence on metabolic traits in the general population are unknown. We obtained plasma and urine parameters from 9099 individuals from seven cohorts, and conducted a genome-wide meta-analysis of Mg(2+) homeostasis. We identified two loci associated with urinary magnesium (uMg), rs3824347 (P=4.4x10(-13)) near TRPM6, which encodes an epithelial Mg(2+) channel, and rs35929 (P=2.1x10(-11)), a variant of ARL15, which encodes a GTP-binding protein. Together, these loci account for 2.3% of the variation in 24-hour uMg excretion. In human kidney cells, ARL15 regulated TRPM6-mediated currents. In zebrafish, dietary Mg(2+) regulated the expression of the highly conserved ARL15 ortholog arl15b, and arl15b knockdown resulted in renal Mg(2+) wasting and metabolic disturbances. Finally, ARL15 rs35929 modified the association of uMg with fasting insulin and fat mass in a general population. In conclusion, this combined observational and experimental approach uncovered a gene-environment interaction linking Mg(2+) deficiency to insulin resistance and obesity. 01 januari 2018

Renal tubular cells respond to mechanical stimuli generated by urinary flow to regulate the activity and transcript abundance of important genes for ion handling, cellular homeostasis, and proper renal development. The primary cilium, a mechanosensory organelle, is postulated to regulate this mRNA response. The aim of this study is to reveal the transcriptome changes of tubular epithelia in response to fluid flow and determine the role of primary cilia in this process. Inner-medullary collecting duct (CD) cells were subjected to either static or physiologically relevant fluid flow (∼0.6 dyn/cm2). RNA-sequencing analysis of ciliated cells subjected to fluid flow showed up-regulation of 1379 genes and down-regulation of 1294 genes compared with static control cells. Strikingly, only 54 of these genes were identified as gene candidates sensitive to primary cilia sensing of fluid flow, of which 16 were linked to ion or water transport pathways in the CD. Validation by quantitative real-time PCR revealed that only the expression of transferrin receptor, which is involved in iron transport; and tribbles pseudokinase 3, which is involved in insulin signaling, were unequivocally regulated by primary cilia sensing of fluid flow. This study shows that the involvement of primary cilia in ion transport in the collecting duct is exceptionally specific.-Mohammed, S. G., Arjona, F. J., Verschuren, E. H. J., Bakey, Z., Alkema, W., van Hijum, S., Schmidts, M., Bindels, R. J. M., Hoenderop, J. G. J. Primary cilia-regulated transcriptome in the renal collecting duct.

Contains fulltext : 187812.pdf (Publisher’s version ) (Open Access)

In euryhaline teleosts, the hypothalamus-pituitary-thyroid and hypothalamus-pituitary-interrenal axes (HPT and HPI, respectively) are regulated in response to environmental stimuli such as salinity changes. However, the molecular players participating in this physiological process in the gilthead seabream (Sparus aurata), a species of high value for aquaculture, are still not identified and/or fully characterized in terms of gene expression regulation. In this sense, this study identifies and isolates the thyrotropin-releasing hormone (trh) mRNA sequence from S. aurata, encoding prepro-Trh, the putative factor initiating the HPT cascade. In addition, the regulation of trh expression and of key brain genes in the HPI axis, i.e., corticotrophin-releasing hormone (crh) and corticotrophin-releasing hormone-binding protein (crhbp), was studied when the osmoregulatory status of S. aurata was challenged by exposure to different salinities. The deduced amino acid structure of trh showed 65–81% identity with its teleostean orthologs. Analysis of the tissue distribution of gene expression showed that trh mRNA is, though ubiquitously expressed, mainly found in brain. Subsequently, regulation of gene expression of trh, crh, and crhbp was characterized in fish acclimated to 5-, 15-, 40-, and 55-ppt salinities. In this regard, the brain gene expression pattern of trh mRNA was similar to that found for the crh gene, showing an upregulation of gene expression in seabream acclimated to the highest salinity tested. Conversely, crhbp did not change in any of the groups tested. Our results suggest that Trh and Crh play an important role in the acclimation of S. aurata to hypersaline environments., This study was funded by project AGL2010-14876 from Ministerio de Ciencia e Innovación awarded to J. M. M. (Spain). This work was partially supported by Ph.D. scholarship from the University of Cadiz (UCA 2009-074-FPI) awarded to I. R-J.

Contains fulltext : 173023.pdf (Publisher’s version ) (Closed access) In kidney, transcellular transport of Ca2+ is mediated by transient receptor potential vanilloid 5 and Na+-Ca2+ exchanger 1 proteins in distal convoluted and connecting tubules (DCT and CNT, respectively). It is not yet understood how DCT/CNT cells can adapt to differences in tubular flow rate and, consequently, Ca2+ load. This study aims to elucidate the molecular mechanisms by which DCT/CNT cells sense fluid dynamics to control transepithelial Ca2+ reabsorption and whether their primary cilia play an active role in this process. Mouse primary DCT/CNT cultures were subjected to a physiologic fluid shear stress (FSS) of 0.12 dyn/cm2 Transient receptor potential vanilloid 5 and Na+-Ca2+ exchanger 1 mRNA levels were significantly increased upon FSS exposure compared with static controls. Functional studies with 45Ca2+ demonstrated a significant stimulation of transepithelial Ca2+ transport under FSS compared with static conditions. Primary cilia removal decreased Ca2+ transport in both static and FSS conditions, a finding that correlated with decreased expression of genes involved in transepithelial Ca2+ transport; however, FSS-induced stimulation of Ca2+ transport was still observed. These results indicate that nephron DCT and CNT segments translate FSS into a physiologic response that implicates an increased Ca2+ reabsorption. Moreover, primary cilia influence transepithelial Ca2+ transport in DCT/CNT, yet this process is not distinctly coupled to FSS sensing by these organelles.-Mohammed, S. G., Arjona, F. J., Latta, F., Bindels, R. J. M., Roepman, R., Hoenderop, J. G. J. Fluid shear stress increases transepithelial transport of Ca2+ in ciliated distal convoluted and connecting tubule cells.

Item does not contain fulltext Unique experimental advantages, such as its embryonic/larval transparency, high-throughput nature, and ease of genetic modification, underpin the rapid emergence of the zebrafish (Danio rerio) as a preeminent model in biomedical research. Particularly in the field of nephrology, the zebrafish provides a promising model for studying the physiological implications of human solute transport processes along consecutive nephron segments. However, although the zebrafish might be considered a valuable model for numerous renal ion transport diseases and functional studies of many channels and transporters, not all human renal electrolyte transport mechanisms and human diseases can be modeled in the zebrafish. With this review, we explore the ontogeny of zebrafish renal ion transport, its nephron structure and function, and thereby demonstrate the clinical translational value of this model. By critical assessment of genomic and amino acid conservation of human proteins involved in renal ion handling (channels, transporters, and claudins), kidney and nephron segment conservation, and renal electrolyte transport physiology in the zebrafish, we provide researchers and nephrologists with an indication of the possibilities and considerations of the zebrafish as a model for human renal ion transport. Combined with advanced techniques envisioned for the future, implementation of the zebrafish might expand beyond unraveling pathophysiological mechanisms that underlie distinct genetic or environmentally, i.e., pharmacological and lifestyle, induced renal transport deficits. Specifically, the ease of drug administration and the exploitation of improved genetic approaches might argue for the adoption of the zebrafish as a model for preclinical personalized medicine for distinct renal diseases and renal electrolyte transport proteins.

The aim of this study was to carry out a comparative analysis of the osmoregulatory properties and associated energy metabolism of euryhaline flatfish species that are cultured in the world. Culture of flatfish (pleuronectiformes) requires stage- and species-dependent osmotic conditions for rearing. Additionally, geographic origin of broodstock animals is another factor to be taken into account for the culture of pleuronectiformes. Larval and juvenile stages of many flatfish species are cultured in large nurseries situated in estuaries and shallow marine habitats, where the environmental salinity is close to the iso-osmotic point of their internal milieu. This fact implicates an advantage in terms of energy savings for osmoregulatory purposes. Thus, this ‘saved’ energy can be derived to other physiological processes, such as somatic growth. However, this scientific presumption does not always results in an optimal growth for many flatfish species. Indeed, iso-osmotic culture conditions can evoke a higher allostatic load than that in the usual hyper-osmotic environment where flatfish species live wildly. Optimization of flatfish culture thus requires adjustments of the osmotic culture conditions to the specific osmoregulatory and metabolic demands that eventually determine the allostatic load and consequently condition growth rates. In this sense, the geographical location of aquaculture farms and the osmoregulatory-based selection of the species to be cultivated in a particular area become critical factors to be considered for optimal flatfish culture.

Contains fulltext : 128576.pdf (Publisher’s version ) (Closed access) Introduction of zebrafish as a model for human diseases with symptomatic hypomagnesemia urges to identify the regulatory transport genes involved in zebrafish Mg(2+) physiology. In humans, mutations related to hypomagnesemia are located in the genes TRPM6 and CNNM2, encoding for a Mg(2+) channel and transporter, respectively; EGF (epidermal growth factor); SLC12A3, which encodes for the Na(+)-Cl(-) co-transporter NCC; KCNA1 and KCNJ10, encoding for the K(+) channels Kv1.1 and Kir4.1, respectively; and FXYD2, which encodes for the gamma-subunit of the Na(+),K(+)-ATPase. Orthologues of these genes were found in the zebrafish genome. For cnnm2, kcna1 and kcnj10, two conserved paralogues were retrieved. Except for fxyd2, kcna1b and kcnj10 duplicates, transcripts of orthologues were detected in ionoregulatory organs such as the gills, kidney and gut. Gene expression analyses in zebrafish acclimated to a Mg(2+)-deficient (0 mM Mg(2+)) or a Mg(2+)-enriched (2 mM Mg(2+)) water showed that branchial trpm6, gut cnnm2b and renal slc12a3 responded to ambient Mg(2+). When changing the Mg(2+) composition of the diet (the main source for Mg(2+) in fish) to a Mg(2+)-deficient (0.01 % (w/w) Mg) or a Mg(2+)-enriched diet (0.7 % (w/w) Mg), mRNA expression of branchial trpm6, gut trpm6 and cnnm2 duplicates, and renal trpm6, egf, cnnm2a and slc12a3 was the highest in fish fed the Mg(2+)-deficient diet. The gene regulation patterns were in line with compensatory mechanisms to cope with Mg(2+)-deficiency or surplus. Our findings suggest that trpm6, egf, cnnm2 paralogues and slc12a3 are involved in the in vivo regulation of Mg(2+) transport in ionoregulatory organs of the zebrafish model. 01 oktober 2013

Regulation of the body Mg2+ balance takes place in the distal convoluted tubule (DCT), where transcellular reabsorption determines the final urinary Mg2+ excretion. The basolateral Mg2+ extrusion mechanism in the DCT is still unknown, but recent findings suggest that SLC41 proteins contribute to Mg2+ extrusion. The aim of this study was, therefore, to characterize the functional role of SLC41A3 in Mg2+ homeostasis using the Slc41a3 knockout (Slc41a3−/−) mouse. By quantitative PCR analysis it was shown that Slc41a3 is the only SLC41 isoform with enriched expression in the DCT. Interestingly, serum and urine electrolyte determinations demonstrated that Slc41a3−/− mice suffer from hypomagnesemia. The intestinal Mg2+ absorption capacity was measured using the stable 25Mg2+ isotope in mice fed a low Mg2+ diet. 25Mg2+ uptake was similar in wildtype (Slc41a3+/+) and Slc41a3−/− mice, although Slc41a3−/− animals exhibited increased intestinal mRNA expression of Mg2+ transporters Trpm6 and Slc41a1. Remarkably, some of the Slc41a3−/− mice developed severe unilateral hydronephrosis. In conclusion, SLC41A3 was established as a new factor for Mg2+ handling.

In kidney, transcellular transport of Ca

Thyroid hormones are involved in many developmental and physiological processes, including osmoregulation. The regulation of the thyroid system by environmental salinity in the euryhaline gilthead seabream (Sparus aurata) is still poorly characterized. To this end seabreams were exposed to four different environmental salinities (5, 15, 40 and 55 ppt) for 14 days, and plasma free thyroid hormones (fT3, fT4), outer ring deiodination and Na+/K+-ATPase activities in gills and kidney, as well as other osmoregulatory and metabolic parameters were measured. Low salinity conditions (5 ppt) elicited a significant increase in fT3 (29%) and fT4 (184%) plasma concentrations compared to control animals (acclimated to 40 ppt, natural salinity conditions in the Bay of Cádiz, Spain), while the amount of pituitary thyroid stimulating hormone subunit β (tshb) transcript abundance remained unchanged. In addition, plasma fT4 levels were positively correlated to renal and branchial deiodinase type 2 (dio2) mRNA expression. Gill and kidney T4-outer ring deiodination activities correlated positively with dio2 mRNA expression and the highest values were observed in fish acclimated to low salinities (5 and 15 ppt). The high salinity (55 ppt) exposure caused a significant increase in tshb expression (65%), but deiodinase gene expression (dio1 and dio2) and activity did not change and were similar to controls (40 ppt). In conclusion, acclimation to different salinities led to changes in the peripheral regulation of thyroid hormone metabolism in seabream. Therefore, thyroid hormones are involved in the regulation of ion transport and osmoregulatory physiology in this species. The conclusions derived from this study may also allow aquaculturists to modulate thyroid metabolism in seabream by adjusting culture salinity., This work was partially supported by a Socrates/Erasmus Grant from the European Union and a Ph.D. scholarship from the University of Cadiz (UCA 2009-074-FPI) to I. R-J. It has been also supported by grants AGL2007-61211/ACU (Ministerio de Educación y Ciencia and FEDER, Spain) and Proyecto de Excelencia PO7-RNM-02843 (Junta de Andalucía) to J.M.M. BL (SFRH/BPD/89889/2012) and PISP (SFRH/BPD/84033/2012) were supported by the Science Foundation (FCT) of Portugal.

Item does not contain fulltext Senegalese sole (Solea senegalensis) is a eurytherm teleost that under natural conditions can be exposed to annual water temperature fluctuations between 12 and 26 degrees C. This study assessed the effects of temperature on sole metabolic status, in particular in what concerns plasma free amino acid changes during thermal acclimation. Senegalese sole maintained at 18 degrees C were acclimated to either cold (12 degrees C) or warm (26 degrees C) environmental temperatures for 21 days. Fish maintained at 18 degrees C served as control. Plasma concentrations of cortisol, glucose, lactate, triglycerides, proteins, and free amino acids were assessed. Cold acclimation influenced interrenal responses of sole by increasing cortisol release. Moreover, plasma glucose and lactate concentrations increased linearly with temperature, presumably reflecting a higher metabolic activity of sole acclimated to 26 degrees C. Acclimation temperature affected more drastically plasma concentrations of dispensable than that of indispensable amino acids, and different acclimation temperatures induced different responses. Asparagine, glutamine and ornithine seem to be of particular importance for ammonia detoxification mechanisms, synthesis of triglycerides that may be used during homeoviscous adaptation and, to a lesser extent, as energetic substrates in specimens acclimated to 12 degrees C. When sole is acclimated to 26 degrees C taurine, glutamate, GABA and glycine increased, which may suggest important roles as antioxidant defences, in osmoregulatory processes and/or for energetic purposes at this thermal regimen. In conclusion, acclimation to different environmental temperatures induces several metabolic changes in Senegalese sole, suggesting that amino acids may be important for thermal acclimation. 01 juli 2012

The thyroid gland in fish mainly secretes the thyroid prohormone 3,5,3 0 ,5 0 -tetraiodothyronine (T4), and extrathyroidal outer ring deiodination (ORD) of the prohormone to 3,5,3 0 -triiodothyronine (T3) is pivotal in thyroid hormone economy. Despite its importance in thyroid hormone metabolism, factors that regulate ORD are still largely unresolved in fish. In addition, the osmoregulatory role of T3 is still a controversial issue in teleosts. In this study, we investigated the regulation of the ORD pathway by cortisol and T3 in different organs (liver, kidney, and gills) of Solea senegalensis and the involvement of T3 in the control of branchial and renal Na C ,K C -ATPase activity, a prime determinant of the hydromineral balance in teleosts. Animals were treated with i.p. slow-release coconut oil implants containing cortisol or T3. Hepatic and renal ORD activities were up-regulated in cortisol-injected animals. T3-treated fish showed a prominent decrease in plasma-free T4 levels, whereas ORD activities did not change significantly. Branchial and renal Na C ,K C -ATPase activitieswerevirtually unaffected by T3, but were transiently up-regulated by cortisol. We conclude that cortisol regulates local T3 bioavailability in S. senegalensis via ORD in an organ-specific manner. Unlike T3, cortisol appears to be directly implicated in the up-regulation of branchial and renal Na C ,K C -ATPase activities.

The effects of prolonged feed deprivation were assessed on blood plasma metabolites and free amino acid levels in Solea senegalensis. Juvenile specimens were maintained at two experimental conditions (24 h fasted and 21 days feed-deprived). In feed-deprived fish, relative growth rate and hepatosomatic index as well as plasma triglycerides and proteins levels were significantly lower. However, plasma cortisol levels were higher in feed-deprived fish, while plasma glucose and lactate values were not significantly different between treatments. Furthermore, feed-deprived fish showed higher levels of total plasma free amino acids than 24 h fasted fish. In 21 days feed-deprived sole, an increase in plasma cortisol levels may suggest a functional role in mobilizing energy due to the catabolic action of this hormone in teleosts. Higher levels of glutamine, arginine and ornithine in 21 days feed-deprived fish may be indicative of a dual role for these amino acids: ammonia detoxification and carbon source for gluconeogenesis. The increased plasma glucogenic and branched-chain amino acid levels, together with the maintenance of plasma glucose in 21 days feed-deprived sole, suggest active liver gluconeogenic processes supported by tissue proteolysis.

We have investigated the regulation of thyroidal status and osmoregulatory capacities in juveniles from the teleost Solea senegalensis acclimated to different ambient temperatures. Juveniles, raised in seawater at 19°C, were acclimated for 3 weeks to temperatures of 12, 19 and 26°C. Since our preliminary observations showed that at 12°C feed intake was suppressed, our experimental design controlled for this factor. The concentration of branchial Na+,K+-ATPase, estimated by measurements of enzyme activity at the optimum temperature of this enzyme (37°C), did not change. In contrast, an increase in Na+,K+-ATPase activity (measured at 37°C), was observed in the kidney of 12°C-acclimated fish. In fish acclimated to 12°C, the hepatosomatic index had increased, which correlated with increased plasma levels of triglycerides and non-esterified fatty acids. Plasma cortisol levels did not differ significantly between the experimental groups. In liver and gills, the amount of iodothyronine deiodinases that exhibit thyroid hormone outer ring deiodination was up-regulated only when fish did not feed. When assayed at the acclimation temperature, kidney deiodinase activities were similar, indicating a temperature-compensation strategy. 3,5,3′-triiodothyronine (T3) tissue concentrations in gills and kidney did not differ significantly between experimental groups. However, at 12°C, lower T3 tissue levels were measured in plasma and liver. We conclude that S. senegalensis adjusts its osmoregulatory system to compensate for the effects of temperature on electrolyte transport capacity. The organ-specific changes in thyroid hormone metabolism at different temperatures indicate the involvement of thyroid hormones in temperature acclimation.

The gilthead sea bream Sparus aurata is a euryhaline and euritherm species with the capacity of living under different environmental conditions of salinity and temperature. The influence of acclimation to different environmental salinities (5, 38 and 55 per thousand) and temperatures (12 degrees , 19 degrees and 26 degrees C) for seven weeks was analyzed in plasma and tissues (liver, gills and kidney) of gilthead sea bream assessing levels of metabolites and enzyme activities related to energy metabolism. Changes observed in specimens acclimated to different environmental salinities agree with previous results reported for this species. The temperature alone did also affect metabolic parameters in a way similar to that previously described. A significant interaction of salinity with temperature was found in most parameters assessed in tissues suggesting that the metabolic effects of salinity are different depending on the temperature of acclimation. The interactions were different among tissues and parameters displaying different patterns of changes. In general, the acclimation to extreme temperatures (especially low) alters the metabolic responses to different salinities thus suggesting that the energy demand of increased osmoregulatory work is not so important under temperature conditions different from those commonly found in nature and in those used in culture.

Seasonal variations in osmoregulatory and metabolic parameters were assessed in juvenile gilthead sea bream (Sparus auratus) cultured in earthen ponds under a natural photoperiod and temperature. Specimens were sampled, and the plasma, gill, kidney and liver were collected during winter 2005 and 2006 (January), spring 2005 (April), summer 2005 (July) and autumn 2005 (October). Plasma osmoregulatory parameters showed higher values in summer, while metabolic parameters presented different patterns of variations. Gill Na+,K+-ATPase activity decreased significantly in winter, while gill metabolite levels showed different patterns of variations among seasons. The enzymatic activities tested did not present a clear pattern of variation [(glutamate dehydrogenase (EC 1.4.1.2) (GDH) and hexokinase (EC 2.7.1.11) (HK)] or significant differences along seasons [glucose 6-phosphate dehydrogenase (EC 1.1.1.49)]. Kidney Na+,K+-ATPase activity decreased during summer and autumn. Different patterns of variation were observed in kidney metabolite levels while all the enzymatic activities assessed [lactate dehydrogenase-oxidase (EC 1.1.1.27) (LDH-O), HK and GDH] presented the highest values during summer. In the liver, metabolite levels and enzymatic activities did not show significant variations or present clear patterns of variation along different seasons. These results indicated seasonal variations in the osmoregulatory and metabolic parameters of different organs (blood, gill, kidney and liver) in earthen pond-cultured gilthead sea bream (S. auratus), which could be mainly attributed to seasonal changes in temperature.

6 páginas, 4 figuras, 2 tablas., The seasonal variation of PRL, GH and SL gene and protein expression has been analyzed in gilthead sea bream (Sparus aurata) pituitaries using Real-Time Q-PCR and Western Blots, respectively. Animals were cultured in earthen ponds under natural photoperiod, temperature and salinity conditions. Samples were taken during winter 2005 (January), spring 2005 (April), summer 2005 (July) and autumn 2005 (October). β-actin, used as the housekeeping gene both for Q-RT-PCR and Western analysis, did not present significant differences among seasons. Higher expression was observed during spring and autumn for PRL, summer and winter for GH, and spring for SL. Expression of PRI, GH and SL, presented seasonal variation, suggesting that these hormones could play a role in the molecular signal transduction of environmental factors (especially of photoperiod and temperature) in eurythermal fish., This study was partly supported by grants AGL2007-61211/ACU (Ministerio de Educación y Ciencia and FEDER, Spain) and Proyecto de Excelencia PO7-RNM-02843 (Junta de Andalucía) to J.M.M. The authors wish to thank Cupimar S.L. (El Puerto de Santa María, Cádiz) for providing the experimental fish. L.V.C. is funded by the program Beca Presidente de la República de Chile and Comisión de Ciencia y Tecnología de Chile (CONICYT).

The effects of three different environmental salinities (15, 25 and 39‰) on growth, osmoregulation and energy metabolism of juvenile Senegalese sole ( Solea senegalensis Kaup, 1858) after a period of 10 weeks were investigated. Immature sole ( n = 150, 39 ± 1 g mean initial body weight) were randomly divided in 6 groups of 25 fish and reared under three different environmental salinities in an open system. Growth, weight gain, specific growth rate and estimated feed intake decreased in salinities lower than 39‰, with the most profound effects observed at 15‰. Branchial Na + ,K + -ATPase activity correlated positively with environmental salinity, while renal Na + ,K + -ATPase activity was not altered. Plasma electrolyte concentrations did not change in the salinity range tested, indicating that osmoregulatory capacities were unaffected. Plasma cortisol levels were higher in salinities different than 39‰ (15 > 25 > 39‰). Plasma glucose, non-esterified fatty acid, triglyceride, and protein levels were decreased in the lower salinities (25 and 15‰), whereas that of lactate was increased two-fold in the 15‰-exposed group only. Measured in tissues (liver, gill, kidney, white muscle), the most profound changes in metabolite levels were generally found in the group exposed to 15‰, compared to the 25‰- and 39‰-groups. A similar pattern was found for the activities of enzymes involved in energy metabolism. Taken together, our data suggest that the poor growth rate of 15‰-exposed fish is caused by decreased feed intake and the subsequent reallocation of energy sources that was non-sufficient to keep similar growth in fish exposed to 15‰ salinity compared with fish exposed to 25 and 39‰ salinity.

The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the two first reproductive cycles (RCs), in which the specimens develops as males. During the first RC (RC1), 11-ketotestosterone (11-KT) and testosterone (T), the main androgens in fish, play different and specific roles in the testicular physiology as they peak at different stages of RC1. Moreover, the profiles of T serum levels during the second RC (RC2) demonstrated that this androgen is not essential in the testicular regression process that occurs during this cycle. However, changes in serum levels suggest that 17beta-estradiol (E2) orchestrates this process during both RCs. Moreover, the E2 serum levels recorded during RC2 indicate that there is a threshold level that determines the initiation of ovarian development during this cycle without promoting complete feminization. We analysed triglyceride, protein, glucose and lactate serum levels in order to establish a relationship between the mobilization and transfer of nutrients, the hormonal changes that take place during the RC and body composition, finding that in vivo serum levels of metabolites change significantly throughout the first two RCs, although the physiological relevance of such changes is still unknown. Triglyceride levels seem to be affected by the beginning of ovary development during RC2 but not by testicular recrudescence during RC1. Moreover, glucose and lactate might be important sources of energy during the resting and testicular involution stages.

To assess the interaction between testosterone (T) treatment and acclimation to different salinities, seawater-acclimated gilthead sea bream (Sparus auratus) were implanted with slow-release coconut oil implants alone (control) or containing T (5 μg/g body mass). After 5 days, eight fish of control and T-treated groups were sampled. The same day, eight fish of each group were transferred to low salinity water (LSW, 6 ppt, hypoosmotic test), seawater (SW, 38 ppt, control test) and high salinity water (HSW, 55 ppt, hyperosmotic test) and sampled 9 days later. Gill Na+, K+-ATPase activity increased in HSW-acclimated fish with respect to SW- and LSW-acclimated fish in both control and T-treated groups. Kidney Na+, K+-ATPase activity was also enhanced in HSW-acclimated fish, but only in T-treated group. From a metabolic point of view, most of the changes observed can be attributed to the action of salinity and T treatment alone, since few interactions between T treatment and osmotic acclimation to different salinities were observed. Those interactions included in treated fish: in the liver, decreased capacity in using glucose in fish acclimated to extreme salinities; in the gills, decreased capacity in using amino acids in HSW; in the kidneys increased capacity in using amino acids in extreme salinities; and in the brain, decreased glycogen and acetoacetate levels of fish in LSW.

Contains fulltext : 36596.pdf ( ) (Open Access) The peripheral conversion of the prohormone 3,5,3',5'-tetraiodothyronine (T4) to the biologically active 3,5,3'-triiodothyronine (T3), via enzymatic deiodination by deiodinases, is an important pathway in thyroid hormone metabolism. The aim of this study was to test if thyroid hormones and cortisol, as well as the outer ring deiodination (ORD) metabolic pathway, are involved in the osmoregulatory response of Senegalese sole (Solea senegalensis, Kaup 1858). We measured osmoregulatory and endocrine parameters in immature juveniles S. senegalensis acclimated to seawater (SW, 38 per thousand) and that were transferred and allowed to acclimate to different salinities (5 per thousand, 15 per thousand, 38 per thousand and 55 per thousand) for 17 days. An adjustment and a chronic regulatory period were identified following acclimation. The adjustment period immediately follows the transfer, and is characterized by altered plasma osmolalities. During this period, plasma cortisol levels increased while plasma free T4 (fT4) levels decreased. Both hormones levels returned to normal values on day 3 post-transfer. In the adjustment period, renal and hepatic ORD activities had increased concomitantly with the decrease in plasma fT4 levels in fishes transferred to extreme salinities (5 per thousand and 55 per thousand). In the chronic regulatory period, where plasma osmolality returned to normal values, plasma cortisol had increased, whereas plasma fT4 levels decreased in animals that were transferred to salinities other than SW. No major changes were observed in branchial ORD activity throughout the experiment. The inverse relationship between plasma cortisol and fT4 suggests an interaction between these hormones during both osmoregulatory periods while ORD pathway can be important in the short-term adjustment period. 8 p.

Item does not contain fulltext Thyroid hormones, in particular 3,5,3'-triiodothyronine or T3, are involved in multiple physiological processes in mammals such as protein, fat and carbohydrate metabolism. However, the metabolic actions of T3 in fish are still not fully elucidated. We therefore tested the effects of T3 on Sparus aurata energy metabolism and osmoregulatory system, a hyperthyroid-induced model that was chosen. Fish were implanted with coconut oil depots (containing 0, 2.5, 5.0 and 10.0mug T3/g body weight) and sampled at day 3 and 6 post-implantation. Plasma levels of free T3 as well as glucose, lactate and triglyceride values increased with increasing doses of T3 at days 3 and 6 post-implantation. Changes in plasma and organ metabolite levels (glucose, glycogen, triglycerides, lactate and total alpha amino acid) and enzyme activities related to carbohydrate, lactate, amino acid and lipid pathways were detected in organs involved in metabolism (liver) and osmoregulation (gills and kidney). Our data implicate that the liver uses amino acids as an energy source in response to the T3 treatment, increasing protein catabolism and gluconeogenic pathways. The gills, the most important extruder of ammonia, are fuelled not only by amino acids, but also by lactate. The kidney differs significantly in its substrate preference from the gills, as it obtained metabolic energy from lactate but also from lipid oxidation processes. We conclude that in S. aurata lipid catabolism and protein turnover are increased as a consequence of experimentally induced hyperthyroidism, with secondary osmoregulatory effects. 01 januari 2016

The osmoregulatory and metabolic role of testosterone (T) in the euryhaline teleost Sparus auratus was examined. Fish were implanted with a slow-release coconut oil implant alone (control) or containing T (2 or 5microgg(-1) body weight) and sampled 1, 3, and 7 days after implantation. Gill Na(+),K(+)-ATPase activity increased in fish treated with the lower dose of T after 7 days of treatment. Kidney Na(+),K(+)-ATPase activity enhanced at first day post-implantation in the group treated with the higher dose of T but the values diminished by day 3. Plasma levels of metabolites (glucose, lactate, triglyceride, and protein) increased after T treatment. This higher availability of plasma metabolites was reflected in several metabolic changes within different tissues of T-treated fish such as (i) increased glycogen levels and capacity for gluconeogenesis, ketogenesis, glucose exporting, and amino acid catabolism in the liver, (ii) enhanced lipogenic capacity in the gills, (iii) increased glycogen levels and capacity for oxidizing amino acids in the kidney, and (iv) enhanced levels of glycogen, aceotacetate, glucose and triglycerides, and higher capacity of phosphorylating glucose in the brain. These results provide evidence regarding an osmoregulatory and metabolic role for T in S. auratus that could be related to changes in both processes during sexual maturation.

The gilthead sea bream ( Sparus auratus ) is an euryhaline fish where prolactin (PRL) and growth hormone (GH) play a role in the adaptation to different environmental salinities. To find out the role of these pituitary hormones in osmoregulation and energy metabolism, fish were implanted with slow release implants of ovine GH (oGH, 5 μg g − 1 body mass) or ovine prolactin (oPRL, 5 μg g − 1 body mass), and sampled 7 days after the start of the treatment. GH increased branchial Na + ,K + -ATPase activity and decreased sodium levels in line with its predicted hypoosmoregulatory action. GH had metabolic effects as indicated by lowered plasma protein and lactate levels, while glucose, triglycerides and plasma cortisol levels were not affected. Also, GH changed liver glucose and lipid metabolism, stimulated branchial and renal glucose metabolism and glycolytic activity, and enhanced glycogenolysis in brain. PRL induced hypernatremia. Furthermore, this hormone decreased liver lipid oxidation potential, and increased glucose availability in kidney and brain. Both hormones have opposite osmoregulatory effects and different metabolic effects. These metabolic changes may support a role for both hormones in the control of energy metabolism in fish that could be related to the metabolic changes occurring during osmotic acclimation.

The influence of acclimation to different environmental salinities (low salinity water, LSW; seawater, SW; and hyper saline water, HSW) and feeding conditions (fed and food deprived) for 14 days was assessed on osmoregulation and energy metabolism of several tissues of gilthead sea bream Sparus auratus. Fish were randomly assigned to one of six treatments: fed fish in LSW, SW, and HSW, and food-deprived fish in LSW, SW, and HSW. After 14 days, plasma, liver, gills, kidney and brain were taken for the assessment of plasma osmolality, plasma cortisol, metabolites and the activity of several enzymes involved in energy metabolism. Food deprivation abolished or attenuated the increase in gill Na+,K+-ATPase activity observed in LSW- and HSW-acclimated fish, respectively. In addition, a linear relationship between renal Na+,K+-ATPase activity and environmental salinity was observed after food deprivation, but values decreased with respect to fed fish. Food-deprived fish acclimated to extreme salinities increased production of glucose through hepatic gluconeogenesis, and the glucose produced was apparently exported to other tissues and served to sustain plasma glucose levels. Salinity acclimation to extreme salinities enhanced activity of osmoregulatory organs, which is probably sustained by higher glucose use in fed fish but by increased use of other fuels, such as lactate and amino acids in food-deprived fish.

SUMMARY Changes in different osmoregulatory and metabolic parameters over time were assessed in gills, kidney, liver and brain of gilthead sea bream Sparus auratus transferred either from seawater (SW, 38 p.p.t.) to hypersaline water (HSW, 55 p.p.t.) or from SW to low salinity water (LSW, 6 p.p.t.) for 14 days. Changes displayed by osmoregulatory parameters revealed two stages during hyperosmotic and hypo-osmotic acclimation: (i) an adaptive period during the first days of acclimation (1–3 days), with important changes in these parameters, and (ii) a chronic regulatory period (after 3 days of transfer) where osmotic parameters reached homeostasis. From a metabolic point of view, two clear phases can also be distinguished during acclimation to hyperosmotic or hypo-osmotic conditions. The first one coincides with the adaptive period and is characterized by enhanced levels of plasma metabolites(glucose, lactate, triglycerides and protein), and use of these metabolites by different tissues in processes directly or indirectly involved in osmoregulatory work. The second stage coincides with the chronic regulatory period observed for the osmoregulatory parameters and is metabolically characterized in HSW-transferred fish by lower energy expenditure and a readjustment of metabolic parameters to levels returning to normality,indicative of reduced osmoregulatory work in this stage. In LSW-transferred fish, major changes in the second stage include: (i) decreased glycolytic potential, capacity for exporting glucose and potential for amino acid catabolism in liver; (ii) enhanced use of exogenous glucose through glycolysis, pentose phosphate and glycogenesis in gills; (iii) increased glycolytic potential in kidney; and (iv) increased glycogenolytic potential and capacity for use of exogenous glucose in brain.

Intellectual disability and seizures are frequently associated with hypomagnesemia and have an important genetic component. However, to find the genetic origin of intellectual disability and seizures often remains challenging because of considerable genetic heterogeneity and clinical variability. In this study, we have identified new mutations in CNNM2 in five families suffering from mental retardation, seizures, and hypomagnesemia. For the first time, a recessive mode of inheritance of CNNM2 mutations was observed. Importantly, patients with recessive CNNM2 mutations suffer from brain malformations and severe intellectual disability. Additionally, three patients with moderate mental disability were shown to carry de novo heterozygous missense mutations in the CNNM2 gene. To elucidate the physiological role of CNNM2 and explain the pathomechanisms of disease, we studied CNNM2 function combining in vitro activity assays and the zebrafish knockdown model system. Using stable Mg2+ isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg2+-permeable cation channel TRPM7. In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg2+ uptake. Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg2+ absorption. These phenotypes were rescued by injection of mammalian wild-type Cnnm2 cRNA, whereas mammalian mutant Cnnm2 cRNA did not improve the zebrafish knockdown phenotypes. We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg2+ homeostasis. By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for monitoring their effects on the brain and kidney phenotype., Author Summary Mental retardation affects 1–3% of the population and has a strong genetic etiology. Consequently, early identification of the genetic causes of mental retardation is of significant importance in the diagnosis of the disease, as predictor of the progress of the disease and for the determination of treatment. In this study, we identify mutations in the gene encoding for cyclin M2 (CNNM2) to be causative for mental retardation and seizures in patients with hypomagnesemia. Particularly, in patients with a recessive mode of inheritance, the intellectual disability caused by dysfunctional CNNM2 is dramatically severe and is accompanied by severely limited motor skills and brain malformations suggestive of impaired early brain development. Although hypomagnesemia has been associated to several neurological diseases, Mg2+ status is not regularly assessed in patients with seizures and mental disability. Our findings establish CNNM2 as an important protein for renal magnesium handling, brain development and neurological functioning, thus explaining the physiology of human disease caused by (dysfunctional) mutations in CNNM2. CNNM2 mutations should be taken into account in patients with seizures and mental disability, specifically in combination with hypomagnesemia.

Contains fulltext : 133126.pdf (Publisher’s version ) (Closed access) Allan-Herndon-Dudley syndrome (AHDS) is an inherited disorder of brain development characterized by severe psychomotor retardation. This X-linked disease is caused by mutations in the monocarboxylate transporter 8 (MCT8), an important thyroid hormone transporter in brain neurons. MCT8-knockout mice lack the 2 major neurological symptoms of AHDS, namely locomotor problems and cognitive impairment. The pathological mechanism explaining the symptoms is still obscure, and no cure for this condition is known. The development of an animal model that carries MCT8-related neurological symptoms is warranted. We have employed morpholino-based gene knockdown to create zebrafish deficient for mct8. Knockdown of mct8 results in specific symptoms in the thyroid axis and brain. The mct8-morphants showed impaired locomotor behavior and brain development. More specifically, we observed maldevelopment of the cerebellum and mid-hindbrain boundary and apoptotic clusters in the zebrafish brain. The mRNA expression of zebrafish orthologs of mammalian TSH, thyroid hormone transporters, and deiodinases was altered in mct8 morphants. In particular, deiodinase type 3 gene expression was consistently up-regulated in zebrafish mct8 morphants. The thyroid hormone metabolite tetrac, but not T3, partly ameliorated the affected phenotype and locomotion disability of morphant larvae. Our results show that mct8 knockdown in zebrafish larvae results in disturbances in the thyroid axis, brain, and locomotion behavior, which is congruent with the clinical aspect of impaired locomotion and cognition in patients with AHDS. Taken together, the zebrafish is a suitable animal model for the study of the pathophysiology of AHDS. 01 juni 2014

We have investigated the regulation of thyroidal status and osmoregulatory capacities in juveniles from the teleost Solea senegalensis acclimated to different ambient temperatures. Juveniles, raised in seawater at 19°C, were acclimated for 3 weeks to temperatures of 12, 19 and 26°C. Since our preliminary observations showed that at 12°C feed intake was suppressed, our experimental design controlled for this factor. The concentration of branchial Na+,K+-ATPase, estimated by measurements of enzyme activity at the optimum temperature of this enzyme (37°C), did not change. In contrast, an increase in Na+,K+-ATPase activity (measured at 37°C), was observed in the kidney of 12°C-acclimated fish. In fish acclimated to 12°C, the hepatosomatic index had increased, which correlated with increased plasma levels of triglycerides and non-esterified fatty acids. Plasma cortisol levels did not differ significantly between the experimental groups. In liver and gills, the amount of iodothyronine deiodinases that exhibit thyroid hormone outer ring deiodination was up-regulated only when fish did not feed. When assayed at the acclimation temperature, kidney deiodinase activities were similar, indicating a temperature-compensation strategy. 3,5,3′-triiodothyronine (T3) tissue concentrations in gills and kidney did not differ significantly between experimental groups. However, at 12°C, lower T3 tissue levels were measured in plasma and liver. We conclude that S. senegalensis adjusts its osmoregulatory system to compensate for the effects of temperature on electrolyte transport capacity. The organ-specific changes in thyroid hormone metabolism at different temperatures indicate the involvement of thyroid hormones in temperature acclimation.

6 páginas, 6 figuras, 1 tabla., Temperature and salinity are important factors that affect several physiological processes in aquatic organisms, which could be produced by variation of certain hormones. In this study, the expression of pituitary hormones involved in the acclimation to different temperatures and salinities was examined in Sparus aurata, a euryhaline and eurytherm species, by Q-Real Time RT-PCR and Western blot analyses for mRNA and protein expression, respectively. Three different experimental conditions were designed with specimens (10 per treatment) acclimated to: a) low salinity water; b) sea water; and c) high salinity water. Additionally, fish under different salinities were acclimated to three different temperatures: 12, 19 and 26 °C. Animals were maintained seven weeks before sampling pituitary glands. Our results provided enough evidence for a differential expression of PRL, GH and SL in the pituitary of gilthead sea bream, under different temperature and salinity regimes., This study was partly supported by grants AGL2007-61211/ACU (Ministerio de Educación y Ciencia and FEDER, Spain) and Proyecto de Excelencia PO7-RNM-02843 (Junta de Andalucía) to JMM. Authors would like to thank Planta de Cultivos Marinos (CASEM, Universidad de Cádiz, Puerto Real, Cádiz, Spain) for providing experimental fish. LVC was founded by the program Beca Presidente de la República de Chile and Comisión de Ciencia y Tecnología de Chile (CONICYT).

The osmoregulatory response of Senegalese sole ( Solea senegalensis , Kaup 1858) to 14-day exposure and throughout 17-day exposure to different environmental salinities was investigated. A linear relationship was observed between environmental salinity and gill Na + ,K + -ATPase activity whereas kidney Na + ,K + -ATPase activity was unaffected. Two osmoregulatory periods could be distinguished according to variations in plasma osmolality: an adjustment period and a chronic regulatory period. No major changes in plasma osmolality and ions levels were registered at the end of the 14- to 17-day exposure period, indicating an efficient adaptation of the osmoregulatory system. Plasma levels of glucose and lactate were elevated in hypersaline water, indicating the importance of these energy substrates in these environments. Glucose was increased during hyper-osmoregulation but only in the adjustment period. Cortisol proved to be a good indicator of chronic stress and stress induced by transfer to the different osmotic conditions. This work shows that S. senegalensis is able to acclimate to different osmotic conditions during short-term exposure.

The role of sex-steroid hormones, testosterone (T) and 17beta-estradiol (E2), on the humoral immune parameters of the teleost gilthead seabream Sparus aurata was studied attempting to deepen on the knowledge of the immune-reproductive system interactions. Fish were injected intraperitoneally with coconut oil containing different dosages of T (0, 2, or 5 microg g(-1) body weight [bw]) or E2 (0, 1, or 2 microg g(-1) bw) and sampled 1, 3, and 7 days later. Hormonal levels and immune parameters (complement, peroxidase and antiprotease activities and IgM levels) were determined in plasma. Plasma hormone levels peaked at 1 day post-injection decreasing thereafter. Treatment with T significantly increased both complement and peroxidase activities after 3 days of injection but antiprotease activity and IgM levels remained unchanged. Treatment with E2 enhanced complement activity 1 day post-injection while decreased it after 3 and 7 days. However, peroxidase activity increased at 3 and 7 days post-injection while total IgM levels decreased. Implications of T and E2 in the immune-reproductive system interactions were discussed.

The influence of arginine vasotocin (AVT) on osmoregulation and metabolism in gilthead sea bream Sparus auratus was evaluated by two experimental approaches. In the first, seawater (SW, 36 ppt)-acclimatized fish were injected intraperitoneally with vehicle (vegetable oil) or two doses of AVT (0.5 and 1 microg/g body weight). Twenty-four hours later, eight fish from each group were sampled; the remaining fish were transferred to low saline water (LSW, 6 ppt, hypoosmotic test), SW (transfer control), and hypersaline water (HSW, 55 ppt, hyperosmotic test). After another 24h (48-h post-injection), fish were sampled. The only significant effect observed was the increase of sodium levels in AVT-treated fish transferred to HSW. In the second experiment, fish were injected intraperitoneally with slow-release vegetable oil implants (mixture 1:1 of coconut oil and seeds oil) alone or containing AVT (1 microg/g body weight). After 3 days, eight fish from each group were sampled; the remaining fish were transferred to LSW, SW, and HSW as above, and sampled 3 days later (i.e. 6 days post-injection). In the AVT-treated group transferred from SW to SW, a significant increase vs. control was observed in gill Na(+),K(+)-ATPase activity. Kidney Na(+),K(+)-ATPase activity decreased in the AVT-treated group transferred to LSW and no changes were observed in the other groups. These osmoregulatory changes suggest a role for AVT during hyperosmotic acclimation based on changes displayed by gill Na(+),K(+)-ATPase activity. AVT treatment increased plasma cortisol levels in fish transferred to LSW and HSW. In addition, AVT treatment affected parameters of carbohydrate, lipid, amino acid, and lactate metabolism in plasma and tissues (gills, kidney, liver, and brain). The most relevant effects were the increased potential of liver for glycogen mobilization and glucose release resulting in increased plasma levels of glucose in AVT-treated fish transferred to LSW and HSW. These changes may be related to the energy repartitioning process occurring during osmotic adaptation of S. auratus to extreme environmental salinities and could be mediated by increased levels of cortisol in plasma.