mTOR-Activating Mutations in RRAGD Are Causative for Kidney Tubulopathy and Cardiomyopathy.

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.

Pannexin-1 mediates fluid shear stress-sensitive purinergic signaling and cyst growth in polycystic kidney disease.

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.

Tubular flow activates magnesium transport in the distal convoluted tubule.

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.

Water temperature affects osmoregulatory responses in gilthead sea bream (Sparus aurata L.).

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.

SLC41A1 is essential for magnesium homeostasis in vivo.

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.

Primary cilia-regulated transcriptome in the renal collecting duct.

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.

Genome-Wide Meta-Analysis Unravels Interactions between Magnesium Homeostasis and Metabolic Phenotypes.

Magnesium (Mg2+) homeostasis is critical for metabolism. However, the genetic determinants of the renal handling of Mg2+, which is crucial for Mg2+ 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 Mg2+ homeostasis. We identified two loci associated with urinary magnesium (uMg), rs3824347 (P=4.4×10-13) near TRPM6, which encodes an epithelial Mg2+ channel, and rs35929 (P=2.1×10-11), 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 Mg2+ regulated the expression of the highly conserved ARL15 ortholog arl15b, and arl15b knockdown resulted in renal Mg2+ 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 Mg2+ deficiency to insulin resistance and obesity.

Polycystin-1 dysfunction impairs electrolyte and water handling in a renal precystic mouse model for ADPKD.

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 Pi wasting. 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.

Fluid shear stress increases transepithelial transport of Ca2+ in ciliated distal convoluted and connecting tubule cells.

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 (DCTs and CNTs, 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 DCTs/CNTs, 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.

Ion transport in the zebrafish kidney from a human disease angle: possibilities, considerations, and future perspectives.

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.

CNNM2 mutations cause impaired brain development and seizures in patients with hypomagnesemia.

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 Mg(2+) isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg(2+)-permeable cation channel TRPM7. In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg(2+) 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 Mg(2+) 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 Mg(2+) 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.

Energy metabolism of hyperthyroid gilthead sea bream Sparus aurata L.

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 10.0µg 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 α 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.

Tissue-specific expression and in vivo regulation of zebrafish orthologues of mammalian genes related to symptomatic hypomagnesemia.

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 γ-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.

Different environmental temperatures affect amino acid metabolism in the eurytherm teleost Senegalese sole (Solea senegalensis Kaup, 1858) as indicated by changes in plasma metabolites.

Senegalese sole (Solea senegalensis) is a eurytherm teleost that under natural conditions can be exposed to annual water temperature fluctuations between 12 and 26°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°C were acclimated to either cold (12°C) or warm (26°C) environmental temperatures for 21 days. Fish maintained at 18°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°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°C. When sole is acclimated to 26°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.

Sensing of tubular flow and renal electrolyte transport.

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.

Interactive effects of environmental salinity and temperature on metabolic responses of gilthead sea bream Sparus aurata.

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.

In Vitro Characterization of Human, Mouse, and Zebrafish MCT8 Orthologues.

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.

Identification of SLC41A3 as a novel player in magnesium homeostasis.

Regulation of the body Mg(2+) balance takes place in the distal convoluted tubule (DCT), where transcellular reabsorption determines the final urinary Mg(2+) excretion. The basolateral Mg(2+) extrusion mechanism in the DCT is still unknown, but recent findings suggest that SLC41 proteins contribute to Mg(2+) extrusion. The aim of this study was, therefore, to characterize the functional role of SLC41A3 in Mg(2+) 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 Mg(2+) absorption capacity was measured using the stable (25)Mg(2+) isotope in mice fed a low Mg(2+) diet. (25)Mg(2+) uptake was similar in wildtype (Slc41a3(+/+)) and Slc41a3(-/-) mice, although Slc41a3(-/-) animals exhibited increased intestinal mRNA expression of Mg(2+) transporters Trpm6 and Slc41a1. Remarkably, some of the Slc41a3(-/-) mice developed severe unilateral hydronephrosis. In conclusion, SLC41A3 was established as a new factor for Mg(2+) handling.