Calcium signalling and transport in the kidney.

The kidney plays a pivotal role in regulating calcium levels within the body. Approximately 98% of the filtered calcium is reabsorbed in the nephron, and this process is tightly controlled to maintain calcium homeostasis, which is required to facilitate optimal bone mineralization, preserve serum calcium levels within a narrow range, and support intracellular signalling mechanisms. The maintenance of these functions is attributed to a delicate balance achieved by various calcium channels, transporters, and calcium-binding proteins in renal cells. Perturbation of this balance due to deficiency or dysfunction of calcium channels and calcium-binding proteins can lead to severe complications. For example, polycystic kidney disease is linked to aberrant calcium transport and signalling. Furthermore, dysregulation of calcium levels can promote the formation of kidney stones. This Review provides an updated description of the key aspects of calcium handling in the kidney, focusing on the function of various calcium channels and the physiological stimuli that control these channels or are communicated through them. A discussion of the role of calcium as an intracellular second messenger and the pathophysiology of renal calcium dysregulation, as well as a summary of gaps in knowledge and future prospects, are also included.

Expression of the kidney anion exchanger 1 affects WNK4 and SPAK phosphorylation and results in claudin-4 phosphorylation.

In the renal collecting ducts, chloride reabsorption occurs through both transcellular and paracellular pathways. Recent literature highlights a functional interplay between both pathways. We recently showed that in polarized inner medullary collecting duct cells, expression of the basolateral kidney anion exchanger 1 (kAE1) results in a decreased transepithelial electrical resistance (TEER), in a claudin-4 dependent pathway. Claudin-4 is a paracellular sodium blocker and chloride pore. Here, we show that kAE1 expression in mouse inner medullary collecting duct cells triggers WNK4, SPAK and claudin-4 phosphorylation. Expression of a functionally dead kAE1 E681Q mutant has no effect on phosphorylation of these proteins. Expression of a catalytically inactive WNK4 D321A or chloride-insensitive WNK4 L319F mutant abolishes kAE1 effect on TEER, supporting a contribution of WNK4 to the process. We propose that variations of the cytosolic pH and chloride concentration upon kAE1 expression alter WNK4 kinase activity and tight junction properties.

Increased Slc34a2 expression and paracellular phosphate permeability contribute to high intestinal phosphate absorption in young mice.

AIM: Phosphorus is a critical constituent of bone as a component of hydroxyapatite. Bone mineral content accrues rapidly early in life necessitating a positive phosphorus balance, which could be established by a combination of increased renal reabsorption and intestinal absorption. Intestinal absorption can occur via a transcellular pathway mediated by the apical sodium-phosphate cotransporter, Slc34a2/NaPiIIb or via the paracellular pathway. We sought to determine how young mammals increase dietary phosphorus absorption from the small intestine to establish a positive phosphorus balance, a prerequisite for rapid bone growth. METHODS: The developmental expression profile of genes mediating phosphate absorption from the small intestine was determined in mice by qPCR and immunohistochemistry. Additionally, Ussing chamber studies were performed on small bowel of young (p7-p14) and older (8- to 17-week-old) mice to examine developmental changes in paracellular Pi permeability and transcellular Pi transport. RESULTS: Blood and urinary Pi levels were higher in young mice. Intestinal paracellular phosphate permeability of young mice was significantly increased relative to older mice across all intestinal segments. NaPiIIb expression was markedly increased in juvenile mice, in comparison to adult animals. Consistent with this, young mice had increased transcellular phosphate flux across the jejunum and ileum relative to older animals. Moreover, transcellular phosphate transport was attenuated by the NaPiIIb inhibitor NTX1942 in the jejunum and ileum of young mice. CONCLUSION: Our results are consistent with young mice increasing phosphate absorption via increasing paracellular permeability and the NaPiIIb-mediated transcellular pathway.

Maternal Epidermal Growth Factor Promotes Neonatal Claudin-2 Dependent Increases in Small Intestinal Calcium Permeability.

A higher concentration of calcium in breast milk than blood favors paracellular calcium absorption enabling growth during postnatal development. We aimed to determine whether suckling animals have greater intestinal calcium permeability to maximize absorption and to identify the underlying molecular mechanism. We examined intestinal claudin expression at different ages in mice and in human intestinal epithelial (Caco-2) cells in response to hormones or human milk. We also measured intestinal calcium permeability in wildtype, Cldn2 and Cldn12 KO mice and Caco-2 cells in response to hormones or human milk. Bone mineralization in mice was assessed by µCT. Calcium permeability across the jejunum and ileum of mice were 2-fold greater at 2 wk than 2 mo postnatal age. At 2 wk, Cldn2 and Cldn12 expression were greater, but only Cldn2 KO mice had decreased calcium permeability compared to wildtype. This translated to decreased bone volume, cross-sectional thickness, and tissue mineral density of femurs. Weaning from breast milk led to a 50% decrease in Cldn2 expression in the jejunum and ileum. Epidermal growth factor (EGF) in breast milk specifically increased only CLDN2 expression and calcium permeability in Caco-2 cells. These data support intestinal permeability to calcium, conferred by claudin-2, being greater in suckling mice and being driven by EGF in breast milk. Loss of the CLDN2 pathway leads to suboptimal bone mineralization at 2 wk of life. Overall, EGF-mediated control of intestinal claudin-2 expression contributes to maximal intestinal calcium absorption in suckling animals.

X-linked hypophosphatemia caused by a deep intronic variant in PHEX identified by PCR-based RNA analysis of urine-derived cells.

X-linked hypophosphatemia (XLH) is caused by dominant inactivating mutations in the phosphate regulating endopeptidase homology, X-linked (PHEX), resulting in elevated fibroblast growth factor 23 (FGF23), hypophosphatemia, rickets and osteomalacia. PHEX variants are identified in approximately 85 % of individuals with XLH, which leaves a substantial proportion of patients with negative DNA-based genetic testing. Here we describe a 16-year-old male who had typical features of XLH on clinical and radiological examination. Genomic DNA sequencing of a hypophosphatemia gene panel did not reveal a pathogenic variant. We therefore obtained a urine sample, established cell cultures and obtained PHEX cDNA from urine-derived cells. Sequencing of exon-spanning PCR products demonstrated the presence of an 84 bp pseudoexon in PHEX intron 21 due to a deep intronic variant (c.2147+1197A>G), which created a new splice donor site in intron 21. The corresponding PHEX protein would lack 33 amino acids on the C-terminus and instead include an unrelated sequence of 17 amino acids. The patient and his affected mother both had this variant. This report highlights that individuals with the typical clinical characteristics of XLH and negative genomic DNA sequence analysis can have deep intronic PHEX variants that are detectable by PCR-based RNA diagnostics.

Kidney stones, hypercalciuria, and recent insights into proximal tubule calcium reabsorption.

PURPOSE OF REVIEW: Most kidney stones are composed of calcium, and the greatest risk factor for kidney stone formation is hypercalciuria. Patients who form kidney stones often have reduced calcium reabsorption from the proximal tubule, and increasing this reabsorption is a goal of some dietary and pharmacological treatment strategies to prevent kidney stone recurrence. However, until recently, little was known about the molecular mechanism that mediates calcium reabsorption from the proximal tubule. This review summarizes newly uncovered key insights and discusses how they may inform the treatment of kidney stone formers. RECENT FINDINGS: Studies examining claudin-2 and claudin-12 single and double knockout mice, combined with cell culture models, support complementary independent roles for these tight junction proteins in contributing paracellular calcium permeability to the proximal tubule. Moreover, a family with a coding variation in claudin-2 causing hypercalciuria and kidney stones have been reported, and reanalysis of Genome Wide Association Study (GWAS) data demonstrates an association between noncoding variations in CLDN2 and kidney stone formation. SUMMARY: The current work begins to delineate the molecular mechanisms whereby calcium is reabsorbed from the proximal tubule and suggests a role for altered claudin-2 mediated calcium reabsorption in the pathogenesis of hypercalciuria and kidney stone formation.

Effects of parathyroid hormone on renal tubular calcium and phosphate handling.

Central to the maintenance of calcium homeostasis is the regulated reabsorption of calcium along the nephron. To this end, parathyroid hormone (PTH) is released from the parathyroid gland in response to lowered plasma calcium levels. This hormone acts through the PTH 1 receptor along the nephron to increase urinary phosphate excretion and decrease urinary calcium excretion. In the proximal tubule, PTH inhibits phosphate reabsorption by reducing the abundance of sodium phosphate cotransporters in the apical membrane. PTH likely decreases calcium reabsorption from the proximal tubule, by reducing the reabsorption of sodium, an event necessary for the paracellular movement of calcium across this segment. In the thick ascending limb (TAL), PTH increases calcium permeability and may increase the electrical driving force thereby increasing calcium reabsorption in the TAL. Finally, in the distal convolution, PTH acts to increase transcellular calcium reabsorption by increasing the activity and abundance of the apically expressed calcium channel TRPV5.

X-linked hypophosphatemia, fibroblast growth factor 23 signaling, and craniosynostosis.

This review summarizes the current knowledge of fibroblast growth factor 23 signaling in bone and its role in the disease pathology of X-linked hypophosphatemia. Craniosynostosis is an under-recognized complication of X-linked hypophosphatemia. The clinical implications and potential cellular mechanisms invoked by increased fibroblast growth factor 23 signaling causing craniosynostosis are reviewed. Knowledge gaps are identified and provide direction for future clinical and basic science studies.

Advancing Discovery Research in Nephrology in Canada: A Conference Report From the 2021 Molecules and Mechanisms Mediating Kidney Health and Disease (M3K) Scientific Meeting and Investigator Summit.

Purpose of conference: New discoveries arising from investigations into fundamental aspects of kidney development and function in health and disease are critical to advancing kidney care. Scientific meetings focused specifically on fundamental biology of the kidney can facilitate interactions, support the development of collaborative groups, and accelerate translation of key findings. The Canadian fundamental kidney researcher community has lacked such a forum. On December 3 to 4, 2021, the first Molecules and Mechanisms Mediating Kidney Health and Disease (M3K) Scientific Meeting and Investigator Summit was held to address this gap with the goal of advancing fundamental kidney research nationally. The meeting was held virtually and was supported by a planning and dissemination grant from the Canadian Institutes of Health Research. Attendees included PhD scientists, nephrology clinician scientists, engineers, industry representatives, graduate students, medical residents, and fellows. Sources of information: This report was prepared from the scientific program, registration numbers, and details obtained from the online platform WHOVA, and summaries written by organizers and participants of the 2021 meeting. Methods: A 21-person team, consisting of the organizing committee members and participants from the meeting, was assembled. Key highlights of the meeting and future directions were identified and the team jointly assembled this report. Key findings: Participation in the meeting was strong, with more than 140 attendees across a range of disciplines. The program featured state-of-the-art presentations on diabetic nephropathy, the immune system, kidney development, and fibrosis, and was heavily focused on trainee presentations. The moderated "Investigator Summit" identified key barriers to research advancement and discussed strategies for overcoming them. These included establishment of a pan-Canadian fundamental kidney research network, development of key resources, cross-pollination with clinical nephrology, better reintegration into the Canadian Society of Nephrology, and further establishment of identity and knowledge translation. Limitations and implications: The 2021 M3K meeting represented a key first step in uniting fundamental kidney researchers in Canada. However, it was universally agreed that regular meetings were necessary to sustain this momentum. The proceedings of this meeting and future actions to sustain the M3K Scientific Meeting and Investigator Summit are presented in this article.

Objectif de la conférence: De nouvelles découvertes découlant des enquêtes sur les aspects fondamentaux du développement et de la fonction des reins en santé ou malades sont essentielles pour faire progresser les soins rénaux. Les réunions scientifiques axées spécifiquement sur la biologie fondamentale du rein peuvent faciliter les interactions, appuyer le développement de groupes de collaboration et accélérer l'application des principaux résultats. La communauté canadienne des chercheurs fondamentaux en néphrologie a manqué d'un tel forum. Les 3 et 4 décembre 2021, le premier Sommet des chercheurs et la réunion scientifique M3K (Molecules and Mechanisms Mediating Kidney Health and Disease) sur les molécules et les médiateurs de la santé et des maladies rénales ont eu lieu pour combler cette lacune; l'objectif était de faire progresser la recherche fondamentale en néphrologie à l'échelle nationale. La réunion s'est tenue virtuellement et était financée par une subvention de planification et de diffusion des Instituts de recherche en santé du Canada. Des doctorants, cliniciens-chercheurs en néphrologie, ingénieurs, représentants de l'industrie, étudiants diplômés, résidents en médecine et en surspécialisation figuraient parmi les participants. Sources: Ce rapport a été préparé à partir du program scientifique, des informations et des numéros d'inscription tirés de la plateforme en ligne WHOVA, et des résumés rédigés par les organisateurs et les participants à la réunion de 2021. Méthodologie: Une équipe de 20 personnes composée de membres du comité organisateur et de participants à la réunion a été formée. Les principaux points saillants de la réunion et les orientations futures ont été déterminés, puis l'équipe a rédigé conjointement le présent rapport. Principaux résultats: La réunion s'est avérée un succès; plus de 140 personnes provenant d'un large éventail de disciplines y ont participé. Le program comprenait des présentations de pointe sur la néphropathie diabétique, le système immunitaire, le développement des reins et la fibrose, et était fortement axé sur des présentations par des stagiaires. Le « Sommet des chercheurs ¼, animé par un modérateur, a permis de déterminer les principaux obstacles à l'avancement de la recherche et de discuter des stratégies pour les surmonter. Ces dernières incluent notamment la création d'un réseau pancanadien de recherche fondamentale en néphrologie, le développement de ressources clés, la pollinisation croisée avec la néphrologie clinique, une « meilleure réintégration dans la Société canadienne de néphrologie ¼ et la poursuite de l'établissement de l'identité et de l'application des connaissances. Limites et implications: La réunion M3K de 2021 a constitué une première étape clé dans l'unification des chercheurs fondamentaux en néphrologie au Canada. On a cependant universellement convenu que des réunions régulières étaient nécessaires pour maintenir cet élan. Le compte rendu de cette réunion ainsi que les actions futures pour soutenir la réunion scientifique M3K et le Sommet des chercheurs sont présentés dans le présent article.

Involving Patient Partners in the KRESCENT Peer Review: Intent, Process, Challenges, and Opportunities.

Purpose of review: The Kidney Research Scientist Core Education and National Training (KRESCENT) is a national Canadian training program for kidney scientists, funded by the Kidney Foundation of Canada (KFOC), the Canadian Institutes of Health Research (CIHR), and the Canadian Society of Nephrology (CSN). We describe our first year of incorporating patient partners into a scientific peer-review committee, the 2017 committee to select senior research trainees and early-career kidney researchers for funding and training, in the hope that it will be helpful to others who wish to integrate the perspective of people with lived experience into the peer-review process. Sources of information: Other peer-review committees, websites, journal articles, patient partners, Kidney Foundation of Canada Research Council, Canadians Seeking Solutions and Innovations to Overcome Chronic Kidney Disease (Can-SOLVE CKD) Patient Council, participants in the 2017 Kidney Foundation of Canada KRESCENT peer-review panel. Methods: We describe our motivation, rationale, guiding principles, plans, feedback, implementation, and response. Key findings: We disseminated a "call for patient partners" 8 weeks before the meeting, seeking patients or their care givers to partner with the KRESCENT peer-review panel; we defined these people with lived experience of kidney disease as patient partners. Eight patient partners came forward and all participated as reviewers. Patient partners first participated in a webinar to learn about the function, structure, and processes of a peer-review committee. They practiced reviewing plain language summaries and giving feedback. In a subsequent teleconference, they shared and discussed their reviews. Plain language summaries were scored, overall, on the same 0-5 quality scale used by scientific reviewers. Three patient reviewers participated in some or all of the 6-hour meeting, which was conducted as usual, for this panel, by teleconference (initially audio only; from 2020 onwards by videoconference). In the meeting, the 2 assigned scientific reviewers first gave their scores, followed by the patient reviewers giving their scores, and discussion (mostly scientific, and conducted in usual scientific language). Scientific reviewers then negotiated a consensus score based on their initial scores, the discussion, patient reviewers' scores and statements, and the scientific officer's notes. Patient reviewers, scientific reviewers, and the Kidney Foundation of Canada (KFOC) were generally positive about the process. The increased length of the meeting (estimated at 1 hour) was generally thought to be acceptable. Patient reviewers also provided feedback on the methods used to incorporate patients into the research under review. These comments were concrete, insightful, and helpful. The patients did not uniformly recommend that basic scientists involve patients in their work. We did not detect bias against preclinical science, work that did not involve patients, or rarer diseases. Some patients found participation inspiring and enlightening. All participants appreciated the idea of patient partners as community witnesses to a group process committed to fairness and supportiveness. We discussed assigning formal meaningful weight to patient reviewers' assessments. Most, but not all, patients thought that the scientific reviewers were ultimately the best judges of the allocation of scarce research resources. Limitations: Patient participants tended to be Caucasian, middle class, and well educated. Because of the difficulties of travel for some people living with or supporting those living with kidney disease, our findings may not generalize fully to peer-review meetings that are conducted face to face. This is explicitly a supportive panel, committed to reviewing junior scientists with kindness as well as rigor; our findings may not generalize to panels conducted differently. We did not use formal qualitative methodology. Implications: Inclusion of patient partners as patient reviewers for the KRESCENT program peer-review panel was feasible, added value for scientific and patient reviewers, and for the funding stakeholders (CIHR, KFOC, and CSN). We were glad that we had taken this step and continue to refine the process with each successive competition.

Motif de la revue: Le KRESCENT (Kidney Research Scientist Core Education and National Training) est un programme national de formation pour les chercheurs en santé rénale financé par la Fondation canadienne du rein (FCR), les Instituts de recherche en santé du Canada (IRSC) et la Société canadienne de néphrologie (SCN). Nous décrivons notre première année d'intégration de partenaires patients dans un comité d'examen scientifique par les pairs, le comité de 2017, visant la sélection de stagiaires de recherche et de chercheurs en santé rénale en début de carrière pour le financement et la formation, dans l'espoir que cela sera utile à ceux qui souhaitent intégrer la perspective des personnes ayant une expérience vécue au processus d'examen par les pairs. Sources: Autres comités d'examen par les pairs, sites Web, articles de revues, partenaires patients, Conseil de recherche de la Fondation canadienne du rein, conseil des patients de Canadians Seeking Solutions and Innovations to Overcome Chronic Kidney Disease (CAN-SOLVE CKD), participants au comité d'examen par les pairs de la Fondation canadienne du rein de 2017. Méthodologie: Nous décrivons ce qui a motivé cette étude, notre raisonnement, nos principes directeurs, nos plans, la rétroaction, la mise en œuvre et les réponses. Principaux résultats: Nous avons diffusé un « appel à des partenaires patients ¼ huit semaines avant la réunion pour trouver des patients ou des soignants prêts à collaborer avec le comité d'examen par les pairs de KRESCENT; nous avons défini comme partenaires patients les personnes ayant une expérience vécue de maladie rénale. Huit partenaires patients ont répondu à l'appel et tous ont participé en tant qu'examinateurs. Les partenaires patients ont d'abord participé à un webinaire pour en apprendre davantage sur la fonction, la structure et les processus d'un comité d'examen par les pairs. Ils se sont ensuite entraînés à examiner des résumés en langage simple et à donner des commentaires. Lors d'une téléconférence ultérieure, ils ont partagé et discuté de leurs examens respectifs. Les résumés en langage clair ont été notés, dans l'ensemble, sur la même échelle de qualité de 0 à 5 utilisée par les examinateurs scientifiques. Trois patients examinateurs ont participé à une partie ou à la totalité de la réunion de 6 heures, qui s'est tenue comme d'habitude, pour ce panel, par téléconférence (initialement en audio seulement; par vidéoconférence à partir de 2020). Au cours de la réunion, les deux examinateurs scientifiques désignés ont d'abord donné leurs notes, puis les patients examinateurs ont donné leurs notes, et une discussion a suivi (principalement scientifique, et menée dans le langage scientifique habituel). Les examinateurs scientifiques ont ensuite négocié pour établir une note consensuelle en fonction de leurs notes initiales, de la discussion, des notes et des commentaires des patients examinateurs et des notes de l'agent scientifique.Les patients examinateurs, les examinateurs scientifiques et la Fondation canadienne du rein étaient généralement positifs à l'égard du processus. La durée accrue de la réunion (estimée à 1 heure) a généralement été jugée acceptable. Les patients examinateurs ont également fourni des commentaires sur les méthodes utilisées pour intégrer les patients à la recherche à l'étude. Ces commentaires étaient concrets, pertinents et utiles. Les patients ne recommandent pas uniformément que les scientifiques en recherche fondamentale impliquent les patients dans leur travail. Nous n'avons pas détecté de biais contre la science préclinique, les études qui n'impliquent pas de patients ou les maladies plus rares. Certains patients ont trouvé la participation inspirante et instructive. Tous les participants ont aimé l'idée des partenaires patients comme témoins communautaires d'un processus de groupe engagé dans l'équité et le soutien.Nous avons discuté de l'attribution d'un poids formel significatif aux évaluations des patients examinateurs. La plupart des patients, mais pas tous, étaient d'avis que les examinateurs scientifiques étaient en fin de compte les meilleurs juges de l'allocation des ressources limitées de la recherche. Limites: Les patients participants étaient pour la plupart de race blanche, de classe moyenne et bien éduqués. En raison des difficultés de déplacement pour certaines personnes vivant avec ou soutenant les personnes vivant avec une maladie rénale, nos résultats peuvent ne pas se généraliser entièrement aux réunions d'examen par les pairs menées en personne. Il s'agit essentiellement d'un groupe de soutien, qui s'est engagé à examiner les jeunes chercheurs avec bienveillance et rigueur; nos conclusions peuvent ne pas se généraliser à des groupes de travail menés différemment. Nous n'avons pas utilisé de méthodologie qualitative officielle. Résultats: L'inclusion de partenaires patients comme examinateurs dans un comité d'examen par les pairs du programme KRESCENT s'est avérée réalisable, et une valeur ajoutée pour les examinateurs scientifiques, les patients examinateurs et les parties responsables du financement (IRSC, FCR et SCN). Nous sommes heureux d'avoir franchi cette étape, nous continuons de raffiner le processus à chaque concours successif.

Molecular mechanisms underlying paracellular calcium and magnesium reabsorption in the proximal tubule and thick ascending limb.

Calcium and magnesium are the most abundant divalent cations in the body. The plasma level is controlled by coordinated interaction between intestinal absorption, reabsorption in the kidney, and, for calcium at least, bone storage and exchange. The kidney adjusts urinary excretion of these ions in response to alterations in their systemic concentration. Free ionized and anion-complexed calcium and magnesium are filtered at the glomerulus. The majority (i.e., >85%) of filtered divalent cations are reabsorbed via paracellular pathways from the proximal tubule and thick ascending limb (TAL) of the loop of Henle. Interestingly, the largest fraction of filtered calcium is reabsorbed from the proximal tubule (65%), while the largest fraction of filtered magnesium is reclaimed from the TAL (60%). The paracellular pathways mediating these fluxes are composed of tight junctional pores formed by claudins. In the proximal tubule, claudin-2 and claudin-12 confer calcium permeability, while the exact identity of the magnesium pore remains to be determined. Claudin-16 and claudin-19 contribute to the calcium and magnesium permeable pathway in the TAL. In this review, we discuss the data supporting these conclusions and speculate as to why there is greater fractional calcium reabsorption from the proximal tubule and greater fractional magnesium reabsorption from the TAL.

Mutations in CLDN2 Are Not a Common Cause of Pediatric Idiopathic Hypercalciuria in Canada.

Background: Hypercalciuria is the most common risk factor for kidney stone formation, including in pediatric patients. However, the etiology is often unknown and children are frequently diagnosed with idiopathic hypercalciuria. Nearly 50% of children with hypercalciuria have a first-degree relative with kidney stones, suggesting a strong genetic basis for this disease. A failure of calcium reabsorption from the proximal nephron is implicated in the pathogenesis of hypercalciuria. Claudin-2 is a tight junction protein abundantly expressed in the proximal tubule. It confers paracellular permeability to calcium that is essential for transport across the proximal tubule where the majority of filtered calcium is reabsorbed. Objective: Our objective was to examine the frequency of coding variations in CLDN2 in a cohort of children with idiopathic hypercalciuria. Design: Mixed method including retrospective chart review and patient interview, followed by genetic sequencing. Setting: Three tertiary care centers in Canada. Patients: Children (age 1-18 years) with idiopathic hypercalciuria. Patients with other causes of hypercalciuria were excluded. Methods: Data were collected from 40 patients with idiopathic hypercalciuria. Informed consent to collect DNA was obtained from 13 patients, and the final and only coding exon of CLDN2 was sequenced. Results: The majority of patients were male, white, and had a positive family history of kidney stones. Parathyroid hormone levels were significantly lower than the reference range (P < .001). The levels of 1,25-dihydroxyvitamin D were also significantly higher in our patient cohort, relative to the reference range (P < .001). Sequence analysis of CLDN2 did not identify any coding variations. Limitations: Sequencing analysis was limited to the final coding exon and small sample size. Conclusions: CLDN2 coding variations are not a common cause of idiopathic hypercalciuria in Canadian children. Further study is needed to determine the causes of hypercalciuria in pediatric patients and develop targeted therapies.

Contexte: L'hypercalciurie est le facteur de risque le plus courant pour la formation de calculs rénaux, y compris chez les patients pédiatriques. Son étiologie est cependant souvent inconnue et les enfants sont fréquemment diagnostiqués avec une hypercalciurie idiopathique. Près de 50 % des enfants atteints d'hypercalciurie ont un parent de premier degré souffrant de calculs rénaux, ce qui suggère une importante contribution génétique à cette maladie. Une atteinte de la réabsorption du calcium au niveau du néphron proximal est impliquée dans la pathogenèse de l'hypercalciurie. La claudine-2, une protéine de jonction abondamment exprimée dans le tubule proximal, confère une perméabilité paracellulaire au calcium, laquelle est essentielle pour le transport à travers le tubule proximal, où la majorité du calcium filtré est réabsorbée. Objectif: Étudier la fréquence des variations dans le codage de CLDN2 dans une cohorte d'enfants atteints d'hypercalciurie idiopathique. Conception de l'étude: Une méthode mixte, comprenant un examen rétrospectif des dossiers médicaux et un entretien avec les patients, suivie d'un séquençage génétique. Cadre: Trois centres de soins tertiaires au Canada. Sujets: Des enfants (1 à 18 ans) atteints d'hypercalciurie idiopathique. Les patients dont l'hypercalciurie avait une autre cause ont été exclus. Méthodologie: Les données proviennent de 40 patients atteints d'hypercalciurie idiopathique. Le consentement éclairé à la collecte d'ADN a été obtenu pour treize patients. L'exon final et le seul exon codant pour CLDN2, a été séquencé. Résultats: La majorité des sujets étaient des garçons d'origine caucasienne et avaient des antécédents familiaux de calculs rénaux. Les taux d'hormone parathyroïdienne étaient significativement plus faibles que les valeurs de référence (p < 0,001). Les taux de 1,25 dihydroxyvitamine D étaient significativement plus élevés dans notre cohorte de patients, par rapport à l'intervalle de référence (p < 0,001). Le séquençage de CLDN2 n'a pas révélé de variations dans le codage. Limites: L'étude porte sur un faible échantillon de patients et le séquençage s'est limité à l'exon final du gène. Conclusion: Les mutations du gène CLDN2 ne sont pas une cause fréquente d'hypercalciurie idiopathique chez les enfants canadiens. D'autres études sont nécessaires pour préciser la ou les causes de l'hypercalciurie chez les patients pédiatriques et développer des traitements ciblés.

Regulation of 1 and 24 hydroxylation of vitamin D metabolites in the proximal tubule.

Calcium and phosphate are critical for numerous physiological processes. Consequently, the plasma concentration of these ions are tightly regulated. Calcitriol, the active form of vitamin D, is a positive modulator of mineralization as well as calcium and phosphate metabolism. The molecular and physiological effects of calcitriol are well documented. Calcitriol increases blood calcium and phosphate levels by increasing absorption from the intestine, and resorption of bone. Calcitriol synthesis is a multistep process. A precursor is first made via skin exposure to UV, it is then 25-hydroxylated in the liver to form 25-hydroxyitamin D. The next hydroxylation step occurs in the renal proximal tubule via the 1-αhydroxylase enzyme (encoded by CYP27B1) thereby generating 1,25-dihydroxyvitamin D, that is, calcitriol. At the same site, the 25-hydroxyvitamin D 24-hydroxlase enzyme encoded by CYP24A1 can hydroxylate 25-hydroxyvitamin D or calcitriol to deactivate the hormone. Plasma calcitriol levels are primarily determined by the regulated expression of CYP27B1 and CYP24A1. This occurs in response to parathyroid hormone (increases CYP27B1), calcitriol itself (decreases CYP27B1 and increases CYP24A1), calcitonin (increases or decreases CYP24A1 and increases CYP27B1), FGF23 (decreases CYP27B1 and increases CYP24A1) and potentially plasma calcium and phosphate levels themselves (mixed effects). Herein, we review the regulation of CYP27B1 and CYP24A1 transcription in response to the action of classic phophocalciotropic hormones and explore the possibility of direct regulation by plasma calcium.

The contribution of regulated colonic calcium absorption to the maintenance of calcium homeostasis.

Calcium absorption and secretion can occur along the length of the small and large intestine. To date, the focus of research into intestinal calcium absorption has been the small intestine, the site contributing the majority of intestinal calcium absorption. However, evidence that the colon contributes as much as 10% of enteral calcium transport has been available for decades. Transcellular calcium absorption and bidirectional paracellular calcium flux contributing to either net absorption or secretion have been observed in the colon, depending on the physiological state. Moreover, the calcium transport pathways contributing to colonic absorption or secretion are regulated by a variety of hormones, including calcitriol, plasma calcium and dietary factors, including prebiotics. Herein we review historical and recent research highlighting the role of colonic calcium transport in overall maintenance of calcium balance, and suggest these data are consistent with the colon being a site of significant regulated transepithelial calcium transport.

Rituximab Use for the Treatment of Childhood Nephrotic Syndrome by Canadian Pediatric Nephrologists: A National Survey.

Background: There is known practice variation in the treatment of frequently relapsing, steroid-dependent, and steroid-resistant nephrotic syndrome in children. Rituximab is an emerging therapy for difficult-to-treat nephrotic syndrome; however, there are no clear treatment guidelines. We therefore hypothesized that a wide variety of approaches to this therapy exist. Objective: To evaluate when and how rituximab is used for the treatment of childhood nephrotic syndrome in Canada. Design and setting: An online survey was used. Participants: Canadian pediatric nephrologists. Methods: A cross-sectional survey was distributed across Canada through the Canadian Association of Pediatric Nephrologists (CAPN) to evaluate rituximab treatment practices. Results: Of a total of 20 responses, 19 (95%) use rituximab in the treatment of nephrotic syndrome, usually as a third or fourth agent. For the number of rituximab doses, the majority (68%) uses 2 doses each time they use it. Eighteen respondents (90%) measure B cells when using this medication, mostly monthly (50%) or every 3 months (39%). Respondents were administered additional doses of rituximab prophylactically (74%) or at first relapse (47%). Long-term drug safety and drug funding were identified as the main barriers to rituximab use. Limitations: This survey represents the practice styles of physicians in a single country, and there is a nonresponse bias of 63%. Also, associations were not calculated. Conclusions: Among Canadian pediatric nephrologists, rituximab use for nephrotic syndrome appears to be increasing, but significant practice variations remain, including approaches to B-cell monitoring. It is reserved mostly for second-line and third-line use due to cost, funding issues, and residual uncertainty regarding long-term safety. Understanding these critical areas of practice uncertainty is a first step to optimize treatment of nephrotic syndrome in children.

Contexte: Des variations sont connues dans les pratiques liées au traitement du syndrome néphrotique infantile, dépendant ou résistant aux stéroïdes, à récidives fréquentes. Le rituximab constitue une nouvelle approche thérapeutique pour soigner le syndrome néphrotique difficile à traiter. Il n'existe cependant aucune directive de traitement claire dans ce contexte. Nous avons donc émis l'hypothèse qu'il existait une grande variété d'approches dans l'utilisation de ce médicament. Objectifs: Examiner les pratiques d'utilisation (quand et comment) du rituximab dans le traitement du syndrome néphrotique infantile au Canada. Cadre et type d'étude: Étude menée par sondage en ligne. Participants: Des néphrologues pédiatriques canadiens. Méthodologie: Un sondage transversal a été distribué partout au Canada par l'entremise de l'Association canadienne des néphrologues pédiatriques (ACPN) afin d'examiner les pratiques de traitement au rituximab. Résultats: Parmi les 20 néphrologues ayant répondu au sondage, 19 (95 %) utilisent le rituximab dans le traitement du syndrome néphrotique, généralement comme 3e ou 4e agent, et la majorité d'entre eux (68 %) administre deux doses à chaque utilisation. Dix-huit répondants (90 %) mesurent les lymphocytes B lorsqu'ils emploient ce médicament, principalement tous les mois (50 %) ou tous les trois mois (39 %). Certains répondants avaient administré des doses additionnelles de rituximab à des fins prophylactiques (74 %) ou lors de la première rechute (47 %). Le recours au rituximab serait principalement freiné par des enjeux liés à son innocuité à long terme et au financement des médicaments. Limitations: Ce sondage représente les pratiques des médecins d'un seul pays et comporte un biais de non-réponse de 63 %. De plus, les associations n'ont pas été calculées. Conclusion: Chez les néphrologues pédiatriques canadiens, l'utilisation du rituximab dans le traitement du syndrome néphrotique semble augmenter; bien que des variations significatives demeurent dans la pratique, notamment en ce qui concerne les approches de surveillance des lymphocytes B. Actuellement, le rituximab est surtout utilisé comme thérapie de deuxième et de troisième ligne en raison de son coût, d'enjeux liés au financement et d'une incertitude résiduelle en ce qui concerne son innocuité à long terme. La compréhension de ces zones critiques d'incertitude dans les pratiques est une première étape pour optimiser le traitement du syndrome néphrotique chez les enfants.

Differential parathyroid and kidney Ca2+-sensing receptor activation in autosomal dominant hypocalcemia 1.

BACKGROUND: Parathyroid Ca2+-sensing receptor (CaSR) activation inhibits parathyroid hormone (PTH) release, while activation of renal CaSRs attenuates Ca2+ transport and increases expression of the pore-blocking claudin-14. Patients with autosomal dominant hypocalcemia 1 (ADH1), due to activating CASR mutations, exhibit hypocalcemia but not always hypercalciuria (elevated Ca2+ in urine). The latter promotes nephrocalcinosis and renal insufficiency. Although CaSRs throughout the body including the kidney harbor activating CASR mutations, it is not understood why only some ADH1 patients display hypercalciuria. METHODS: Activation of the CaSR was studied in mouse models and a ADH1 patient. In vitro CaSR activation was studied in HEK293 cells. FINDINGS: Cldn14 showed blood Ca2+ concentration-dependent regulation, which was absent in mice with kidney-specific Casr deletion, indicating Cldn14 is a suitable marker for chronic CaSR activation in the kidney. Mice with a gain-of-function mutation in the Casr (Nuf) were hypocalcemic with low plasma PTH levels. However, renal CaSRs were not activated at baseline but only after normalizing blood Ca2+ levels. Similarly, significant hypercalciuria was not observed in a ADH1 patient until blood Ca2+ was normalized. In vitro experiments indicate that increased CaSR expression in the parathyroid relative to the kidney could contribute to tissue-specific CaSR activation thresholds. INTERPRETATION: Our findings suggest that parathyroid CaSR overactivity can reduce plasma Ca2+ to levels insufficient to activate renal CaSRs, even when an activating mutation is present. These findings identify a conceptually new mechanism of CaSR-dependent Ca2+ balance regulation that aid in explaining the spectrum of hypercalciuria in ADH1 patients. FUNDING: Erasmus+ 2018/E+/4458087, the Canadian Institutes for Health research, the Novo Nordisk Foundation, the Beckett Foundation, the Carlsberg Foundation and Independent Research Fund Denmark.

Gentamicin Inhibits Ca2+ Channel TRPV5 and Induces Calciuresis Independent of the Calcium-Sensing Receptor-Claudin-14 Pathway.

BACKGROUND: Treatment with the aminoglycoside antibiotic gentamicin can be associated with severe adverse effects, including renal Ca2+ wasting. The underlying mechanism is unknown but it has been proposed to involve activation of the Ca2+-sensing receptor (CaSR) in the thick ascending limb, which would increase expression of claudin-14 (CLDN14) and limit Ca2+ reabsorption. However, no direct evidence for this hypothesis has been presented. METHODS: We studied the effect of gentamicin in vivo using mouse models with impaired Ca2+ reabsorption in the proximal tubule and the thick ascending limb. We used a Cldn14 promoter luciferase reporter assay to study CaSR activation and investigated the effect of gentamicin on activity of the distal nephron Ca2+ channel transient receptor potential vanilloid 5 (TRPV5), as determined by patch clamp in HEK293 cells. RESULTS: Gentamicin increased urinary Ca2+ excretion in wild-type mice after acute and chronic administration. This calciuretic effect was unaltered in mice with genetic CaSR overactivation and was present in furosemide-treated animals, whereas the calciuretic effect in Cldn14-/- mice and mice with impaired proximal tubular Ca2+ reabsorption (claudin-2 [CLDN2]-deficient Cldn2-/- mice) was equivalent to that of wild-type mice. In vitro, gentamicin failed to activate the CaSR. In contrast, patch clamp analysis revealed that gentamicin strongly inhibited rabbit and human TRPV5 activity and chronic gentamicin administration downregulated distal nephron Ca2+ transporters. CONCLUSIONS: Gentamicin does not cause hypercalciuria via activation of the CaSR-CLDN14 pathway or by interfering with proximal tubular CLDN2-dependent Ca2+ reabsorption. Instead, gentamicin blocks distal Ca2+ reabsorption by direct inhibition of the Ca2+ channel TRPV5. These findings offer new insights into Ca2+ wasting in patients treated with gentamicin.

Molecular mechanisms altering tubular calcium reabsorption.

The majority of calcium filtered by the glomerulus is reabsorbed along the nephron. Most is reabsorbed from the proximal tubule (> 60%) via a paracellular pathway composed of the tight junction proteins claudins-2 and -12, a process driven by sodium and consequently water reabsorption. The thick ascending limb reabsorbs the next greatest amount of calcium (20-25%), also by a paracellular pathway composed of claudins-16 and -19. This pathway is regulated by the CaSR, whose activity increases the expression of claudin-14, a protein that blocks paracellular calcium reabsorption. The fine tuning of urinary calcium excretion occurs in the distal convoluted and connecting tubule by a transcellular pathway composed of the apical calcium channel TRPV5, the calcium shuttling protein calbindin-D28K and the basolateral proteins PMCA1b and the sodium calcium exchanger, NCX. Not surprisingly, mutations in a subset of these genes cause monogenic disorders with hypercalciuria as a part of the phenotype. More commonly, "idiopathic" hypercalciuria is encountered clinically with genetic variations in CLDN14, the CASR and TRPV5 associating with kidney stones and increased urinary calcium excretion. An understanding of the molecular pathways conferring kidney tubular calcium reabsorption is employed in this review to help explain how dietary and medical interventions for this disorder lower urinary calcium excretion.