Search

Abstract FAM111A gene mutations cause Kenney–Caffey syndrome (KCS) and Osteocraniostenosis (OCS), conditions characterized by short stature, low serum ionized calcium (Ca2+), low parathyroid hormone (PTH), and bony abnormalities. The molecular mechanism mediating this phenotype is unknown. The c‐terminal domain of FAM111A harbors all the known disease‐causing variations and encodes a domain with high homology to serine proteases. However, whether this serine protease domain contributes to the maintenance of Ca2+ homeostasis is not known. We hypothesized the disruption of the serine protease domain of FAM111A would disrupt Ca2+ homeostasis. To test this hypothesis, we generated with CRISPR/Cas9, mice with a frameshift insertion (c.1450insA) or large deletion (c.1253‐1464del) mutation in the Fam111a serine protease domain. Serum‐ionized Ca2+ and PTH levels were not significantly different between wild type, heterozygous, or homozygous Fam111a mutant mice. Additionally, there were no significant differences in fecal or urine Ca2+ excretion, intestinal Ca2+ absorption or overall Ca2+ balance. Only female homozygous (c.1450insA), but not heterozygous mice displayed differences in bone microarchitecture and mineral density compared to wild‐type animals. We conclude that frameshift mutations that disrupt the c‐terminal serine protease domain do not induce a KCS or OCS phenotype in mice nor alter Ca2+ homeostasis.

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.

Background: Acute kidney injury (AKI) in critically ill children is associated with increased risk for short- and long-term adverse outcomes. Currently, there is no systematic follow-up for children who develop AKI in intensive care unit (ICU). Objective: This study aimed to assess variation regarding management, perceived importance, and follow-up of AKI in the ICU setting within and between healthcare professional (HCP) groups. Design: Anonymous, cross-sectional, web-based surveys were administered nationally to Canadian pediatric nephrologists, pediatric intensive care unit (PICU) physicians, and PICU nurses, via professional listservs. Setting: All Canadian pediatric nephrologists, PICU physicians, and nurses treating children in the ICU were eligible for the survey. Patients: N/A. Measurements: Surveys included multiple choice and Likert scale questions on current practice related to AKI management and long-term follow-up, including institutional and personal practice approaches, and perceived importance of AKI severity with different outcomes. Methods: Descriptive statistics were performed. Categorical responses were compared using Chi-square or Fisher’s exact tests; Likert scale results were compared using Mann-Whitney and Kruskal-Wallis tests. Results: Surveys were completed by 34/64 (53%) pediatric nephrologists, 46/113 (41%) PICU physicians, and 82 PICU nurses (response rate unknown). Over 65% of providers reported hemodialysis to be prescribed by nephrology; a mix of nephrology, ICU, or a shared nephrology-ICU model was reported responsible for peritoneal dialysis and continuous renal replacement therapy (CRRT). Severe hyperkalemia was the most important renal replacement therapy (RRT) indication for both nephrologists and PICU physicians (Likert scale from 0 [not important] to 10 [most important]; median = 10, 10, respectively). Nephrologists reported a lower threshold of AKI for increased mortality risk; 38% believed stage 2 AKI was the minimum compared to 17% of PICU physicians and 14% of nurses. Nephrologists were more likely than PICU physicians and nurses to recommend long-term follow-up for patients who develop any AKI during ICU stay (Likert scale from 0 [none] to 10 [all patients]; mean=6.0, 3.8, 3.7, respectively) ( P < .05). Limitations: Responses from all eligible HCPs in the country could not obtained. There may be differences in opinions between HCPs that completed the survey compared to those that did not. Additionally, the cross-sectional design of our study may not adequately reflect changes in guidelines and knowledge since survey completion, although no specific guidelines have been released in Canada since survey dissemination. Conclusions: Canadian HCP groups have variable perspectives on pediatric AKI management and follow-up. Understanding practice patterns and perspectives will help optimize pediatric AKI follow-up guideline implementation.

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.

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.

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.

Summary: 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.

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 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.

Background: Women scientists are less likely to obtain Assistant Professorship and achieve promotion, and obtain less grant funding than men. Scientist/clinician-scientist training programs which provide salary awards as well as training and mentorship are a potential intervention to improve outcomes among women scientists. We hypothesized whether a programmatic approach to scientist/clinician-scientist training is associated with improved outcomes for women scientists in Canada when compared with salary awards alone. Trainees within the Kidney Research Scientist Core Education and National Training Program (KRESCENT), Canadian Child Health Clinician Scientist Program (CCHCSP), and the Canadian Institutes of Health Research (CIHR) salary award programs were evaluated. Objective: To examine whether the structured KRESCENT training program with salary support improves academic success for women scientists relative to salary awards alone. Design: Retrospective cohort study. Setting: Canadian national research scientist and clinician-scientist training programs and salary awards. Participants: KRESCENT cohort (n = 59, 2005-2017), CCHCSP cohort (n = 58, 2002-2015), and CIHR (n = 571, 2005-2015) Salary Awardees for postdoctoral fellows (PDF) and new investigators (NI). Measurements: National operating grant funding success, achieving an academic position as an Assistant Professor for PDF, or achieving promotion to Associate Professor for NI. Methods: The gender distribution of each cohort was determined using first name and NamepediA and was examined for PDF and NI, followed by a description of trainee outcomes by gender and training level. Results: KRESCENT and CIHR PDF were balanced (12/27, 44% men and 55/116, 47% women) while CCHCSP had a higher proportion of women (13/20, 65%). KRESCENT and CCHCSP NI retained women scientists (19/32, 59% and 22/38, 58% women), whereas CIHR NI had fewer women (165/455, 36% women vs 290/455, 64% men, P = 0.01). There was a high rate of NI operating grant success (91%-95%) with no gender differences in each cohort. There was a high proportion of CCHCSP PDF who achieved an Assistant Professorship (18/20, 90%) that may be due in part to a longer follow-up period (9.3 ± 3 years) compared with KRESCENT PDF (7/27, 26%, 0.88 ± 4.5 years), and these data were not available for CIHR PDF. Women KRESCENT NI showed increased promotion to Associate Professor ( P = 0.02, 0.25 ± 3.2 years follow-up) and CCHCSP NI had high promotion rates (37/38, 97%, 6.9 ± 3.6 years follow-up) irrespective of gender. There was an overall trend toward more men pursuing biomedical research. Limitations: KRESCENT and CCHCSP training program cohort size and heterogeneity; assigning gender by first name may result in misclassification; lack of data on the respective applicant pools; and inability to examine intersectionality with gender, ethnicity, and sexual orientation. Conclusion: Overall trainee performance across programs is remarkable by community standards regardless of gender. KRESCENT and CCHCSP training programs demonstrated balanced success in their PDF and NI, whereas the CIHR awardees had reduced representation of women scientists from PDF to NI. This exploratory study highlights the utility of programmatic training approaches like the KRESCENT program as potential tools to support and retain women scientists in the academic pipeline during the challenging PDF to NI transition period.

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease. Triacylglycerol accumulation in the liver is a hallmark of NAFLD. Metabolic studies have confirmed that increased hepatic de novo lipogenesis (DNL) in humans contributes to fat accumulation in the liver and to NAFLD progression. Mice deficient in carboxylesterase (Ces)1d expression are protected from high-fat diet-induced hepatic steatosis. To investigate whether loss of Ces1d can also mitigate steatosis induced by over-activated DNL, WT and Ces1d-deficient mice were fed a lipogenic high-sucrose diet (HSD). We found that Ces1d-deficient mice were protected from HSD-induced hepatic lipid accumulation. Mechanistically, Ces1d deficiency leads to activation of AMP-activated protein kinase and inhibitory phosphorylation of acetyl-CoA carboxylase. Together with our previous demonstration that Ces1d deficiency attenuated high-fat diet-induced steatosis, this study suggests that inhibition of CES1 (the human ortholog of Ces1d) might represent a novel pharmacological target for prevention and treatment of NAFLD.

Background & Aims: Intestinal Ca2+ absorption early in life is vital to achieving optimal bone mineralization. The molecular details of intestinal Ca2+ absorption have been defined in adults after peak bone mass is obtained, but they are largely unexplored during development. We sought to delineate the molecular details of transcellular Ca2+ absorption during this critical period. Methods: Expression of small intestinal and renal calcium transport genes was assessed by using quantitative polymerase chain reaction. Net calcium flux across small intestinal segments was measured in Ussing chambers, including after pharmacologic inhibition or genetic manipulation of TRPV6 or Cav1.3 calcium channels. Femurs were analyzed by using micro–computed tomography and histology. Results: Net TRPV6-mediated Ca2+ flux across the duodenum was absent in pre-weaned (P14) mice but present after weaning. In contrast, we found significant transcellular Ca2+ absorption in the jejunum at 2 weeks but not 2 months of age. Net jejunal Ca2+ absorption observed at P14 was not present in either Trpv6 mutant (D541A) mice or Cav1.3 knockout mice. We observed significant nifedipine-sensitive transcellular absorption across the ileum at P14 but not 2 months. Cav1.3 knockout pups exhibited delayed bone mineral accrual, compensatory nifedipine-insensitive Ca2+ absorption in the ileum, and increased expression of renal Ca2+ reabsorption mediators at P14. Moreover, weaning pups at 2 weeks reduced jejunal and ileal Cav1.3 expression. Conclusions: We have detailed novel pathways contributing to transcellular Ca2+ transport across the distal small intestine of mice during development, highlighting the complexity of the multiple mechanisms involved in achieving a positive Ca2+ balance early in life. Keywords: Calcium Channel, Pediatric, Bone, Development

Purpose of review: (1) To provide commentary on the 2017 update to the Kidney Disease Improving Global Outcomes (KDIGO) 2017 Clinical Practice Guideline Update for the Diagnosis, Evaluation, Prevention, and Treatment of Chronic Kidney Disease-Mineral and Bone Disorder (CKD-MBD); (2) to apply the evidence-based guideline update for implementation within the Canadian health care system; (3) to provide comment on the care of children with chronic kidney disease (CKD); and (4) to identify research priorities for Canadian patients. Sources of information: The KDIGO 2017 Clinical Practice Guideline Update for the Diagnosis, Evaluation, Prevention, and Treatment of CKD-MBD. Methods: The commentary committee co-chairs selected potential members based on their knowledge of the Canadian kidney community, aiming for wide representation from relevant disciplines, academic and community centers, and different geographical regions. Key findings: We agreed with many of the recommendations in the clinical practice guideline on the diagnosis, evaluation, prevention, and treatment of CKD-MBD. However, based on the uncommon occurrence of abnormalities in calcium and phosphate and the low likelihood of severe abnormalities in parathyroid hormone (PTH), we recommend against screening and monitoring levels of calcium, phosphate, PTH, and alkaline phosphatase in adults with CKD G3. We suggest and recommend monitoring these parameters in adults with CKD G4 and G5, respectively. In children, we agree that monitoring for CKD-MBD should begin in CKD G2, but we suggest measuring ionized calcium, rather than total calcium or calcium adjusted for albumin. With regard to vitamin D, we suggest against routine screening for vitamin D deficiency in adults with CKD G3-G5 and G1T-G5T and suggest following population health recommendations for adequate vitamin D intake. We recommend that the measurement and management of bone mineral density (BMD) be according to general population guidelines in CKD G3 and G3T, but we suggest against routine BMD testing in CKD G4-G5, CKD G4T-5T, and in children with CKD. Based on insufficient data, we also recommend against routine bone biopsy in clinical practice for adults with CKD or CKD-T, or in children with CKD, although we consider it an important research tool. Limitations: The committee relied on the evidence summaries produced by KDIGO. The CSN committee did not replicate or update the systematic reviews.

Abstract Tandem fluorescent protein timers are elegant tools to determine proteolytic stabilities of cytosolic proteins with high spatial and temporal resolution. In a new study published in BMC Biology, Kowalski et al. fused timers to precursors of proteins of the mitochondrial intermembrane space and found that they are under surveillance of the ubiquitin-proteasome system. Ubiquitination at lysine residues of these precursors directly inhibits their translocation into the intermembrane space and targets them for proteasomal degradation.

Forkhead Box O transcription factors play important roles in bone metabolism by defending against oxidative stress and apoptosis. FoxO3a is of special interest as it is the predominant isoform expressed in bone. In osteoblasts, the administration of 1,25 dihydroxyvitamin D3 (1,25D3) increases FoxO3a expression, and alters calcium handling. We therefore queried whether FoxO3a participates in vitamin D-mediated regulation of calcium transport pathways or matrix calcification, independent of reactive oxygen species (ROS) formation. To examine this possibility, we differentiated MC3T3-E1 cells into mature osteoblast-like cells over 7 days. This coincided with an increased ability to mineralize extracellular matrix. FoxO3a expression increased throughout differentiation. 1,25D3 enhanced both FoxO3a mRNA and protein expression. Immunofluorescence microscopy found increased FoxO3a nuclear localization with differentiation and after treatment with 1,25D3. Live cell ratiometric imaging with Fura-2AM identified significant L-type calcium channel mediated calcium uptake that was enhanced by 1,25D3. We observed expression of both Cav1.2 and Cav1.3, although expression decreased throughout differentiation and was not altered by 1,25D3 treatment. FoxO3a overexpression reduced calcium uptake and calcium deposition. FoxO3a overexpression also prevented alterations in calcium channel expression and the cell differentiation associated decrease in expression of Runx2 and increased expression of osteocalcin, findings consistent with a failure for the cells to differentiate. Based on both our expression and functional data, we suggest that high levels of FoxO3a prevent osteoblast differentiation and matrix calcification. Keywords: Forkhead Box O3, Osteoblast differentiation, Osteoblast mineralization, Matrix calcification, Calcium deposition, MC3T3-E1 cells

In-utero nutrition is an under-studied aspect of cognitive development. Fruit has been an important dietary constituent for early hominins and humans. Among 808 eligible CHILD-Edmonton sub-cohort subjects, 688 (85%) had 1-year cognitive outcome data. We found that each maternal daily serving of fruit (sum of fruit plus 100% fruit juice) consumed during pregnancy was associated with a 2.38 point increase in 1-year cognitive development (95% CI 0.39, 4.37; p

The renal proximal tubule (PT) is responsible for the reabsorption of approximately 65% of filtered calcium, primarily via a paracellular pathway. However, which protein(s) contribute this paracellular calcium pore is not known. The claudin family of tight junction proteins confers permeability properties to an epithelium. Claudin-12 is expressed in the kidney and when overexpressed in cell culture contributes paracellular calcium permeability (PCa). We therefore examined claudin-12 renal localization and its contribution to tubular paracellular calcium permeability. Claudin-12 null mice (KO) were generated by replacing the single coding exon with β-galactosidase from Escherichia coli. X-gal staining revealed that claudin-12 promoter activity colocalized with aquaporin-1, consistent with the expression in the PT. PTs were microperfused ex vivo and PCa was measured. PCa in PTs from KO mice was significantly reduced compared with WT mice. However, urinary calcium excretion was not different between genotypes, including those on different calcium containing diets. To assess downstream compensation, we examined renal mRNA expression. Claudin-14 expression, a blocker of PCa in the thick ascending limb (TAL), was reduced in the kidney of KO animals. Thus, claudin-12 is expressed in the PT, where it confers paracellular calcium permeability. In the absence of claudin-12, reduced claudin-14 expression in the TAL may compensate for reduced PT calcium reabsorption.

Background: Thiazide diuretics are commonly prescribed to prevent kidney stones. However, it is unclear whether higher doses confer greater benefit. Objective: To determine whether lower doses of thiazide diuretics confer a similar protective effect against kidney stone events as higher doses. Design: Population-based cohort study. Setting: Linked health administrative databases in Ontario, Canada. Patients: Older adults newly prescribed a thiazide diuretic between 2003 and 2014 were separated into 2 groups based on daily dose: low dose (⩽12.5 mg hydrochlorothiazide/chlorthalidone, or ⩽1.25 mg indapamide) or high dose. Measurements: The primary outcome was time to a kidney stone event, using diagnosis and procedure codes. A secondary outcome was kidney stone surgery. Methods: An association between thiazide diuretic dose and a kidney stone event was estimated using Cox proportional hazards regression. Results: A total of 536 of 105 239 patients (0.51%) experienced a kidney stone event. We did not detect a difference in kidney stone risk in the high-dose relative to the low-dose group (adjusted hazard ratio, 1.10; 95% confidence interval, 0.93-1.31). Results were similar when analysis was restricted to the more specific outcome of kidney stone surgery. Neither a history of prior kidney stones nor the type of thiazide diuretic modified the effect of diuretic dose on outcome. Limitations: Patients were >65 years old and we were unable to adjust for some potential confounders such as dietary factors. Conclusions: Lower dose thiazide diuretics appear to confer a similar protective effect as higher dose thiazides against the development of kidney stones.

Carbonic anhydrase II (CAII) is expressed along the nephron where it interacts with a number of transport proteins augmenting their activity. Aquaporin-1 (AQP1) interacts with CAII to increase water flux through the water channel. Both CAII and aquaporin-1 are expressed in the thin descending limb (TDL); however, the physiological role of a CAII-AQP1 interaction in this nephron segment is not known. To determine if CAII was required for urinary concentration, we studied water handling in CAII-deficient mice. CAII-deficient mice demonstrate polyuria and polydipsia as well as an alkaline urine and bicarbonaturia, consistent with a type III renal tubular acidosis. Natriuresis and hypercalciuria cause polyuria, however, CAII-deficient mice did not have increased urinary sodium nor calcium excretion. Further examination revealed dilute urine in the CAII-deficient mice. Urinary concentration remained reduced in CAII-deficient mice relative to wild-type animals even after water deprivation. The renal expression and localization by light microscopy of NKCC2 and aquaporin-2 was not altered. However, CAII-deficient mice had increased renal AQP1 expression. CAII associates with and increases water flux through aquaporin-1. Water flux through aquaporin-1 in the TDL of the loop of Henle is essential to the concentration of urine, as this is required to generate a concentrated medullary interstitium. We therefore measured cortical and medullary interstitial concentration in wild-type and CAII-deficient mice. Mice lacking CAII had equivalent cortical interstitial osmolarity to wild-type mice: however, they had reduced medullary interstitial osmolarity. We propose therefore that reduced water flux through aquaporin-1 in the TDL in the absence of CAII prevents the generation of a maximally concentrated medullary interstitium. This, in turn, limits urinary concentration in CAII deficient mice.

Calcium and phosphate are critical for a myriad of physiological and cellular processes within the organism. Consequently, plasma levels of calcium and phosphate are tightly regulated. This occurs through the combined effects of the phospho- and calciotropic hormones, parathyroid hormone (PTH), active vitamin D3, and fibroblast growth factor 23 (FGF23). The organs central to this are the kidneys, intestine, and bone. In the kidney, the proximal tubule reabsorbs the majority of filtered calcium and phosphate, which amounts to more than 60% and 90%, respectively. The basic molecular mechanisms responsible for phosphate reclamation are well described, and emerging work is delineating the molecular identity of the paracellular shunt wherein calcium permeates the proximal tubular epithelium. Significant experimental work has delineated the molecular effects of PTH and FGF23 on these processes as well as their regulation of active vitamin D3 synthesis in this nephron segment. The integrative effects of both phospho- and calciotropic hormones on proximal tubular solute transport and subsequently whole body calcium-phosphate balance thus have been further complicated. Here, we first review the molecular mechanisms of calcium and phosphate reabsorption from the proximal tubule and how they are influenced by the phospho- and calciotropic hormones acting on this segment and then consider the implications on both renal calcium and phosphate handling as well as whole body mineral balance.

The renal collecting duct is the nephron segment where the final urine content of acid equivalents and inorganic ions are determined. The role of two different cell types present in this nephron segment has been determined many years ago: principal cells that express the epithelial sodium channel ENaC and aquaporin 2, regulate electrolyte reabsorption, while intercalated cells, which express acid-base transporters and vacuolar H+-ATPase, maintain an apropriate acid-base balance. Recent evidence challenges this historical view. Rather than having independent and non-overlapping functions, the two cell types in the collecting duct appear to functionally cooperate to regulate acid-base and volume homeostasis via complex paracrine and endocrine interplay. This review summarizes these recent findings.

Background: Significant practice variation exists in Canada with respect to timing of dialysis initiation in children. In the absence of evidence to guide practice, physicians' perceptions may significantly influence decision-making. Objective: The objectives of this study are to (1) evaluate Canadian pediatric nephrologists' perceptions regarding dialysis initiation in children with chronic kidney disease (CKD) and (2) determine the factors guiding practice that may contribute to practice variation across Canada. Design: This study was a cross-sectional online survey. Setting: This study was done in academic pediatric nephrology centers in Canada. Participants: The participants of this study are pediatric nephrologists. Measurements and methods: An anonymous web-based survey was administered to pediatric nephrologists in Canada to evaluate perspectives and practice patterns regarding timing of dialysis initiation. We also explored the importance of estimated glomerular filtration rate (eGFR) vs. symptoms and the role of patient and provider factors influencing decisions. Results: Thirty-five nephrologists (59 %) completed the survey. Most respondents care for advanced CKD patients in a multidisciplinary clinic (86 %) and no centers have a formal policy on timing of dialysis initiation. Seventy-five percent of centers follow 30 patients. Discussions about dialysis initiation are generally informal (75 %) and the decision to start is made by the nephrologist (37 %) or a team (57 %). Fifty percent agreed GFR was important when deciding when to initiate dialysis, 41 % were neutral, and 9 % disagreed. Variability exists in the threshold that nephrologists considered early (vs. late) dialysis initiation: >20 (21 %), >15 (38 %), >12 (26 %), and >10 ml/min/1.73 m 2 (12 %). Practitioners however typically start dialysis in asymptomatic patients at eGFRs of 7–9 (9 %), 10–11 (41 %), 12–14 (38 %), and 15–19 (6 %) ml/min/1.73 m 2 . Patient factors important in the decision to start dialysis for >90 % of nephrologists were fatigue, >10 % weight loss, nausea, increasing missed school, and awaiting a pre-emptive transplant. Age was only a factor for 56 %. Limitations: This study has a 59 % response rate. Conclusions: Variability exists in Canada regarding the importance and threshold of eGFR guiding the decision as to when to start dialysis in children, whereas patient symptoms are almost universally important to pediatric nephrologists' decision-making. Additional studies evaluating outcomes of children starting dialysis earlier vs. later are needed to standardize decision-making and care for children with kidney failure.

Background: The incidence of kidney stones in children is increasing. While guidelines exist for acute surgical intervention, there is limited data to inform the decision as to when to intervene non-urgently. Objectives: To identify patient and stone characteristics predicting stone surgery in children. Design: Retrospective chart review. Setting: Stollery Children's Hospital, Edmonton, Alberta, Canada from 1990 to 2013. Patients: Sixty-three children aged 0–18 years old who presented with a total of 142 stones. Measurements: Patient's surgical history, demographics, metabolic measures, and stone number, type, and location. Methods: Univariate and multivariate analysis, controlling for presentation number and individual-level variation by repeated measures analysis were conducted to assess for patient and stone characteristics associated with surgical intervention. Results: Sixty-five percent (41/63) required surgery during a mean follow-up of 19 months. Stone characteristics associated with surgical intervention by multivariate analysis included larger stone size (>6 mm), and stone composition of calcium oxalate. Limitations: Single center study with a limited sample size and duration of follow up, thereby limiting predictive power. There were some missing data ( i,e . stone type was not always available). Despite this, stone type remained significant in multivariate modeling. Conclusion: Stone size > 6mm and composition with calcium oxalate but not patient age or symptoms associated with presentation predicted surgical intervention. These observations can be used to inform decisions as to whether urolithiasis should be surgically managed electively or observed.

Ras association domain family protein 1A (RASSF1A) is a tumor suppressor gene silenced in cancer. Here we report that RASSF1A is a novel regulator of intestinal inflammation as Rassf1a(+/-) , Rassf1a(-/-) and an intestinal epithelial cell specific knockout mouse (Rassf1a (IEC-KO) ) rapidly became sick following dextran sulphate sodium (DSS) administration, a chemical inducer of colitis. Rassf1a knockout mice displayed clinical symptoms of inflammatory bowel disease including: increased intestinal permeability, enhanced cytokine/chemokine production, elevated nuclear factor of kappa light polypeptide gene enhancer in B-cells (NFκB) activity, elevated colonic cell death and epithelial cell injury. Furthermore, epithelial restitution/repair was inhibited in DSS-treated Rassf1a(-/-) mice with reduction of several makers of proliferation including Yes associated protein (YAP)-driven proliferation. Surprisingly, tyrosine phosphorylation of YAP was detected which coincided with increased nuclear p73 association, Bax-driven epithelial cell death and p53 accumulation resulting in enhanced apoptosis and poor survival of DSS-treated Rassf1a knockout mice. We can inhibit these events and promote the survival of DSS-treated Rassf1a knockout mice with intraperitoneal injection of the c-Abl and c-Abl related protein tyrosine kinase inhibitor, imatinib/gleevec. However, p53 accumulation was not inhibited by imatinib/gleevec in the Rassf1a(-/-) background which revealed the importance of p53-dependent cell death during intestinal inflammation. These observations suggest that tyrosine phosphorylation of YAP (to drive p73 association and up-regulation of pro-apoptotic genes such as Bax) and accumulation of p53 are consequences of inflammation-induced injury in DSS-treated Rassf1a(-/-) mice. Mechanistically, we can detect robust associations of RASSF1A with membrane proximal Toll-like receptor (TLR) components to suggest that RASSF1A may function to interfere and restrict TLR-driven activation of NFκB. Failure to restrict NFκB resulted in the inflammation-induced DNA damage driven tyrosine phosphorylation of YAP, subsequent p53 accumulation and loss of intestinal epithelial homeostasis.

The sodium hydrogen exchanger isoform 3 (NHE3) mediates absorption of sodium, bicarbonate and water from renal and intestinal lumina. This activity is fundamental to the maintenance of a physiological plasma pH and blood pressure. To perform this function NHE3 must be present in the apical membrane of renal tubular and intestinal epithelia. The molecular determinants of this localization have not been conclusively determined, although linkage to the apical actin cytoskeleton through ezrin has been proposed. We set out to evaluate this hypothesis. Functional studies of NHE3 activity were performed on ezrin knockdown mice (Vil2(kd/kd)) and NHE3 activity similar to wild-type animals detected. Interpretation of this finding was difficult as other ERM (ezrin/radixin/moesin) proteins were present. We therefore generated an epithelial cell culture model where ezrin was the only detectable ERM. After knockdown of ezrin expression with siRNA, radixin and moesin expression remained undetectable. Consistent with the animal ultrastructural data, cells lacking ezrin retained an epithelial phenotype but had shortened and thicker microvilli. NHE3 localization was identical to cells transfected with non-targeting siRNA. The attachment of NHE3 to the apical cytoskeleton was unaltered as assessed by fluorescent recovery after photobleaching (FRAP) and the solubility of NHE3 in Triton X-100. Baseline NHE3 activity was unaltered, however, cAMP-dependent inhibition of NHE3 was largely lost even though NHE3 was phosphorylated at serines 552 and 605. Thus, ezrin is not necessary for the apical localization, attachment to the cytoskeleton, baseline activity or cAMP induced phosphrylation of NHE3, but instead is required for cAMP mediated inhibition.

Mutations in the SLC4A1 gene encoding the anion exchanger 1 (AE1) can cause distal renal tubular acidosis (dRTA), a disease often due to mis-trafficking of the mutant protein. In this study, we investigated whether trafficking of a Golgi-retained dRTA mutant, G701D kAE1, or two dRTA mutants retained in the endoplasmic reticulum, C479W and R589H kAE1, could be functionally rescued to the plasma membrane of Madin-Darby Canine Kidney (MDCK) cells. Treatments with DMSO, glycerol, the corrector VX-809, or low temperature incubations restored the basolateral trafficking of G701D kAE1 mutant. These treatments had no significant rescuing effect on trafficking of the mis-folded C479W or R589H kAE1 mutants. DMSO was the only treatment that partially restored G701D kAE1 function in the plasma membrane of MDCK cells. Our experiments show that trafficking of intracellularly retained dRTA kAE1 mutants can be partially restored, and that one chemical treatment rescued both trafficking and function of a dRTA mutant. These studies provide an opportunity to develop alternative therapeutic solutions for dRTA patients.