Children with failed kidney transplant often become highly sensitized to human leukocyte antigens (HLA), limiting access to future transplantation. We report a successful accelerated peri-transplant desensitization protocol enabling HLA-incompatible deceased donor kidney transplantation in a highly sensitized pediatric recipient. A 14-year-old boy remained on hemodialysis for 11 years after failure of his first kidney transplant and had a calculated panel reactive antibody of 99.70%. Following an offer from a haplotype-matched deceased donor, he was found to have class I and class II donor-specific antibodies and a positive flow cytometry crossmatch. Accelerated desensitization over 6 days consisted of three plasmapheresis sessions followed by intravenous immunoglobulin and rituximab. Alemtuzumab was used as induction agent for transplant. Crossmatch reactivity decreased to an acceptable threshold, permitting transplantation. At 12 months, mixed T-cell- and antibody-mediated rejection occurred despite stable donor-specific antibodies. Graft function remained stable at 43 mL/min/1.73 m2 at 15 months post-transplant.

Abstract Spinal cord injury (SCI) disrupts innervation to the lower urinary tract, resulting in bladder dysfunction that predisposes to urinary infections and renal impairment. While inflammation is central to bladder pathology after SCI, the molecular events linking acute to chronic remodeling are poorly defined. We hypothesized that early treatment with pirfenidone, an anti-inflammatory and anti-fibrotic drug, would attenuate bladder pathology after SCI. Adult female C57BL/6J mice underwent contusive SCI or sham laminectomy, and bladders were collected at 2, 7, 16, and 45 days later. SCI induced bladder hypertrophy, edema, hemorrhage, neutrophil infiltration, cell proliferation and loss of voiding function in the first 48 hours. Transcriptomic profiling at this timepoint was characterized by activation of inflammatory and cytokine pathways including TNF-α, IL-6, the complement cascade, and TGFβ. Although bladder function partially recovered by day 7, inflammatory pathways persisted and extracellular matrix (ECM) remodeling programs emerged. By day 16, robust activation of ECM-remodeling pathways was evident in all bladders. Treatment with pirfenidone during the acute inflammatory phase (day 2–7) reduced bladder hypertrophy and suppressed expression of pro-fibrotic, inflammatory, and neuroplasticity-associated genes including Bdnf and Chrm2 that encodes muscarinic receptor 2 (M2). Mechanistically, pirfenidone attenuated TGFβ signaling as shown by downregulation of phosphoSmad2 protein in whole bladders and decreased M2 receptor expression in the urothelium. These molecular changes correlated with improved function in pirfenidone-treated mice as shown by fewer voiding events with larger urine volumes up until 45 days after SCI. Early treatment with pirfenidone limits inflammation and fibrosis, normalizes neural signaling, and improves bladder function after SCI. New & Noteworthy Using a mouse model of traumatic spinal cord injury, we showed that bladder inflammation and dysfunction precede fibrosis, with TGFβ serving as a key regulatory hub. Transient pirfenidone treatment during the acute inflammatory phase (days 2-7) attenuated bladder pathology, limited Tgfb2/3 , Bdnf , and Chrm2 expression, and reduced M2 receptor and phospho-Smad2 protein levels. These molecular events correlated with improved bladder storage function, highlighting an early therapeutic window and TGFβ signaling as a target for neurogenic bladder remodeling.

Background: Chronic kidney disease (CKD) affects over 10% of the global population. A genetic diagnosis can be identified in about 30% of pediatric and 10-30% of adults, informing treatment, prognosis, and family-based risk assessment. However, access to renal genetics services remains limited across many healthcare systems. Objectives: To characterize the clinical and genetic landscape of CKD in patients referred for genetic evaluation within a Canadian single-centre nephrology-genetics program, and to evaluate the diagnostic yield and clinical utility of an integrated renal genetics clinic. Methods: We conducted a retrospective study of 206 probands referred for suspected hereditary kidney disease to the McGill University Health Centre Renal Genetics Clinic between 2019 and 2024. Genetic testing was performed in accredited laboratories, predominantly through comprehensive multi-gene panels or phenotype-directed exome sequencing. All reported variants were classified according to the ACMG/AMP criteria, and variants of uncertain significance were reevaluated post hoc using standardized quantitative and evidence-tier frameworks to determine whether they trended toward "likely pathogenic" ("hot") or "likely benign" ("cold"), without implying formal reclassification. Results: A molecular diagnosis was established in 34.5% of probands (71/206), implicating pathogenic or likely pathogenic variants across 35 genes representing diverse monogenic kidney disease etiologies. The highest diagnostic yields were observed in cystic nephropathy (51.9%), tubulopathy (38.5%), and glomerulopathy (35.6%). Genetic results affected clinical management in 23.9% of diagnosed cases, leading to changes in treatment for 16.9%, modification of transplant management in 5.6%, informed living donor risk assessment in 14.1%, and facilitated cascade testing in 66.2% of families. CKD of unknown etiology comprised 28% of the cohort, with a genetic diagnosis reached in 25.9% of these cases. Variants of uncertain significance (VUS) were reported in 39.3% of probands, with higher overall variant burden and lower diagnostic yields among individuals of non-European ancestry. Post hoc internal reassessment stratified 67.4% of VUS as mid or lower confidence ("cold") and 31.4% as higher confidence ("hot") or likely pathogenic. Conclusions: In a diverse urban population, integration of a dedicated renal genetics service within nephrology care achieved high diagnostic yield, substantially influenced management, and facilitated family risk assessment. Structured referral pathways and multidisciplinary variant interpretation optimize the clinical utility of genetic testing in CKD. ### Competing Interest Statement All authors have completed the ICMJE uniform disclosure form and declare: support for the submitted work as follows: T. M. Kitzler reports institutional funding from the SickKids New Investigator Research Grant (NI21-1159); the following authors report financial relationships with organizations that might have an interest in the submitted work in the previous three years: A. Alam has served as site principal investigator on studies sponsored by Otsuka Canada and Vertex and received consulting fees and honoraria from Otsuka Canada; D. Blum has served on advisory boards for Otsuka and Bayer; M. Cantarovich has received honoraria from Sanofi for a speaker tour; T. Takano has served on an Otsuka Canada advisory board and received honoraria from Chugai Pharma; T. M. Kitzler received a one-time honorarium from Otsuka Canada; R. Sapir-Pichhadze reports institutional support to the MUHC Foundation from Paladin for a fellowship and honoraria from The Transplantation Society and the Banff Foundation; no other authors have financial relationships with organizations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work. ### Funding Statement This work was supported by a Fonds de recherche du Québec - Santé Junior 1 Clinician-Scientist Award to T.K. and the SickKids New Investigator Research Grant (NI21-1159). Z.S. received a Canadian Institutes of Health Research Doctoral Research Award. Dr. Sandal is supported by the Fonds de recherche du Québec - Santé Junior 1. The funders had no role in study design, data collection, analysis, interpretation, or manuscript preparation ### Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: McGill University Health Centre Research Ethics Board gave ethical approval for this work (Protocol 2025-10930). The same board gave ethical approval for participant reanalysis and expanded testing, and for the presentation of case-specific clinical information (Protocol 2023-9387). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes All non-identifiable participant data underlying the results are available from the corresponding author upon reasonable request

Millions of people are exposed to concentrations of arsenic that exceed the World Health Organization's limit of 10 μg/L. Inorganic arsenic (iAsIII) is metabolized by arsenic 3 methyltransferase (As3mt) that converts it to methylated arsenicals, mono and dimethyl arsonous acid (MMA and DMA). Chronic arsenic exposure is linked to an increased risk of chronic kidney disease (CKD), however, the effects of arsenic exposure on kidney development remain unclear. We hypothesized that exposure to arsenicals impairs nephron formation during mouse kidney development. Mouse embryonic kidney explants were treated with iAsIII and MMAIII (1.5 μM or 200 μg/L). iAsIII inhibited growth of kidney explants and ureteric bud branching morphogenesis at embryonic day 11.5 (E11.5) and E12.5, but not at E13.5. Similar effects were observed when kidney explants were treated with MMAIII. Additionally, iAsIII exposure increased apoptosis in the metanephric mesenchyme of E11.5 explants and decreased Gdnf transcription. To assess the impact of iAS exposure in utero and early postnatal life, female mice harboring a humanized version of AS3MT and wild-type mice with murine As3mt were exposed to iAsIII throughout gestation and weaning and their offspring were analyzed for kidney defects. Pups with human AS3MT exposed to 1.5 μM iAsIII in utero, showed a 20 % reduction in kidney weight normalized to body weight and a 28 % reduction in nephron number, compared to kidneys of wild-type mice. In conclusion, exposure to arsenicals during embryonic development impairs ureteric bud branching morphogenesis and decreases nephron endowment, which may predispose to CKD in adulthood.

The odd-skipped related family of proteins are evolutionarily conserved zinc finger transcription factors in bilaterian animals with essential roles in body segmentation, as well as gut, excretory system and appendage development. Although they are prognostic biomarkers in several cancers, their molecular function is poorly understood. To gain a deeper understanding of how this family of transcription factors is implicated in human disorders and cancer, we explore the functions of Odd-skipped related transcription factors as well as their invertebrate homologs during development. We found that vertebrate Osr1 binds to DNA targets within the WNT, BMP, HH, TGFβ, Notch and retinoic acid signaling pathways, suggesting that Osr genes coordinate and integrate multiple pathways during development, potentially by binding to heterochromatin as pioneer transcription factors. From a protein tree analysis of odd-skipped family orthologs and paralogs, it appears that vertebrate, nematode, and insect paralogues have arisen via independent gene duplication events, and that their common ancestor likely had a single odd-family gene. We hypothesize that the ancestral odd-family protein was required for gut and gut-derived structures and was subsequently co-opted to perform additional functions in other tissues as part of the evolution of organisms. These observations posit new uncharacterized functions for Osr genes in the development of bilaterian animals and in cancer models.

Bryophyllum pinnatum (BP) is a medicinal plant used to treat many conditions when taken as a leaf juice, leaves in capsules, as an ethanolic extract, and as herbal tea. These preparations have been chemically analyzed except for decoctions derived from boiled green leaves. In preparation for a clinical trial to validate BP tea as a treatment for kidney stones, we used NMR and MS analyses to characterize the saturation kinetics of the release of metabolites. During boiling of the leaves, (a) the pH decreased to 4.8 within 14 min and then stabilized; (b) regarding organic acids, citric and malic acid were released with maximum release time (tmax) = 35 min; (c) for glycoflavonoids, quercetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (Q-3O-ArRh), myricetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (M-3O-ArRh), kappinatoside, myricitrin, and quercitrin were released with tmax = 5–10 min; and (d) the total phenolic content (TPC) and the total antioxidant capacity (TAC) reached a tmax at 55 min and 61 min, respectively. In summary, 24 g of leaves boiled in 250 mL of water for 61 min ensures a maximal release of key water-soluble metabolites, including organic acids and flavonoids. These metabolites are beneficial for treating kidney stones because they target oxidative stress and inflammation and inhibit stone formation.

Claudins are a family of tight junction proteins expressed in epithelial tissues during development and in postnatal life. We hypothesized that claudins are required for branching morphogenesis in the developing chick lung. To test this hypothesis, we exposed cultured chick lung explants at embryonic day 5 to a truncated non-toxic form of the Clostridium perfringens enterotoxin known as C-CPE that removes C-CPE-sensitive claudins from tight junctions. Using in situ hybridization and immunofluorescence studies, we established that only one C-CPE-sensitive claudin, Claudin-3, was expressed in the chick lung at this stage. C-CPE treated lung explants did not exhibit any defect in lung branching compared to controls. However, they did exhibit a significantly smaller lumen area, suggesting that paracellular permeability was perturbed. The decrease in lumen area was associated with a loss of Claudin-3 expression within tight junctions of the respiratory epithelium and an increase in permeability of the respiratory epithelium. When C-CPE-treated lung explants were treated with forskolin, lumen area was restored. In summary, removal of a sealing claudin, Claudin-3, from tight junctions in embryonic lung epithelium results in a decrease in lumen area and in hydrostatic pressure needed for lung development.

The extracellular matrix of the bladder consists mostly of type I and III collagen, which are required during loading. During bladder injury, there is an accumulation of collagen that impairs bladder function. Little is known about the genes that regulate production of collagens in the bladder. We demonstrate that the transcription factor Odd-skipped related 1 (Osr1) is expressed in the bladder mesenchyme and epithelium at the onset of development. As development proceeds, Osr1 is mainly expressed in mesenchymal progenitors and their derivatives. We hypothesized that Osr1 regulates mesenchymal cell differentiation and production of collagens in the bladder. To test this hypothesis, we examined newborn and adult mice heterozygous for Osr1, Osr1+/−. The bladders of newborn Osr1+/− mice had a decrease in collagen I by western blot analysis and a global decrease in collagens using Sirius red staining. There was also a decrease in the cellularity of the lamina propria, where most collagen is synthesized. This was not due to decreased proliferation or increased apoptosis in this cell population. Surprisingly, the bladders of adult Osr1+/− mice had an increase in collagen that was associated with abnormal bladder function; they also had a decrease in bladder capacity and voided more frequently. The results suggest that Osr1 is important for the differentiation of mesenchymal cells that give rise to collagen-producing cells.

Objectives: Biallelic pathogenic variants in CYPA24A1 and SLC34A1 are causes of idiopathic infantile hypercalcemia. Pathogenic variants in both may also give rise to hypercalciuria with nephrocalcinosis or nephrolithiasis without previous hypercalcemia (renal group). Our objective was to examine the frequency of CYP24A1 or SLC34A1 variants in children with early hypercalcemia or late-onset hypercalciuria. Method: Forty-one children from 7 centers across Canada were recruited. Local investigations were undertaken. The serum was evaluated by liquid chromatography tandem-mass spectrometry for the ratio of 25-hydroxyvitamin D3 to 24,25-dihydroxyvitamin D3, (25-OH-D3:24,25-(OH)2D3), an elevation pathognomonic for the loss of function of the CYP24A1 enzyme. Mutational analyses were undertaken. Family cascade screening was performed if pathogenic variants were detected in probands. Results: Twenty-nine children had early-onset hypercalcemia; none had elevated 25-OH-D3:24,25-(OH)2D3 or variants. Interestingly, 2 of 12 in the renal group had elevated 25-OH-D3:24,25-(OH)2D3 and presented as preadolescents. In case 1, cascade testing revealed a sibling and parent with asymptomatic pathogenic variants in CYP24A1. Four CYP24A1 pathogenic variants were identified in these 2 probands: 3 have been described in European populations, and 1 is a rare variant in exon 7 (c931delC) that is likely pathogenic. No SLC34A1 pathogenic variants were detected. Conclusion: In Canada, pathogenic variants in CYP24A1 appear to manifest with late-onset hypercalciuria and its sequelae. The 25-OH-D3:24,25-(OH)2D3 ratio is an excellent tool for screening for biallelic pathogenic variants in CYP24A1. We confirm that cascade testing is important for these variants.

Congenital anomalies of the kidneys and urinary tracts (CAKUT) are disorders caused by defects in the development of the kidneys and their outflow tracts. The formation of the kidneys begins at week 3 and nephrogenesis continues until week 36, therefore, the kidneys and outflow tracts are susceptible to environmental risk factors that perturb development throughout gestation. Many genes have been implicated in kidney and outflow tract development, and mutations have been identified in patients with CAKUT. In severe cases of CAKUT, when the kidneys do not form, the fetus will not survive. However, in less severe cases, the baby can survive with combined kidney and outflow tract defects or they may only be identified in adulthood. In this review, we will cover the clinical presentation of CAKUT, its epidemiology, and its long-term outcomes. We will then discuss risk factors for CAKUT, including genetic and environmental contributions. Although severe CAKUT is rare, low nephron number is a much more common disorder with its effect on kidney function increasingly apparent as a person ages. Low nephron number appears to arise by the same mechanisms as CAKUT, but it differs in terms of the magnitude of the insult and the timing of when it occurs during gestation. By understanding the causes of CAKUT and low nephron number, we can begin to identify preventive treatments and establish clinical guidelines for how these patients should be followed.

Claudins are a family of tight junction proteins that are expressed during mouse kidney development. They regulate paracellular transport of solutes along the nephron and contribute to the final composition of the urinary filtrate. To understand their roles during development, we used a protein reagent, a truncated version of theClostridium perfringensenterotoxin (C-CPE), to specifically remove a subset of claudin family members from mouse embryonic kidney explants at embryonic day 12. We observed that treatment with C-CPE decreased the number and the complexity of ureteric bud tips that formed: there were more single and less bifid ureteric bud tips when compared to control-treated explants. In addition, C-CPE-treated explants exhibited ureteric bud tips with larger lumens when compared to control explants (p < .05). Immunofluorescent analysis revealed decreased expression and localization of Claudin-3, -4, -6, and -8 to tight junctions of ureteric bud tips following treatment with C-CPE. Interestingly, Claudin-7 showed higher expression in the basolateral membrane of the ureteric bud lineage and poor localization to the tight junctions of the ureteric bud lineage both in controls and in C-CPE-treated explants. Taken together, it appears that claudin proteins may play a role in ureteric bud branching morphogenesis through changes in lumen formation that may affect the efficiency by which ureteric buds emerge and branch.

Genome-wide association study (GWAS) loci for several immunity-mediated diseases (early onset asthma, inflammatory bowel disease (IBD), primary biliary cholangitis, and rheumatoid arthritis) map to chromosomal region 17q12-q21. The predominant view is that association between 17q12-q21 alleles and increased risk of developing asthma or IBD is due to regulatory variants. ORM sphingolipid biosynthesis regulator (ORMDL3) residing in this region is the most promising gene candidate for explaining association with disease. However, the relationship between 17q12-q21 alleles and disease is complex suggesting contributions from other factors, such as trans-acting genetic and environmental modifiers or circadian rhythms. Circadian rhythms regulate expression levels of thousands of genes and their dysregulation is implicated in the etiology of several common chronic inflammatory diseases. However, their role in the regulation of the 17q12-q21 genes has not been investigated. Moreover, the core clock gene nuclear receptor subfamily 1, group D, member 1 (NR1D1) resides about 200 kb distal to the GWAS region. We hypothesized that circadian rhythms influenced gene expression levels in 17q12-q21 region and conversely, regulatory elements in this region influenced transcription of the core clock gene NR1D1 in cis. To test these hypotheses, we examined the diurnal expression profiles of zona pellucida binding protein 2 (ZPBP2/Zpbp2), gasdermin B (GSDMB), and ORMDL3/Ormdl3 in human and mouse tissues and analyzed the impact of genetic variation in the ZPBP2/Zpbp2 region on NR1D1/Nr1d1 expression. We found that Ormdl3 and Zpbp2 were controlled by the circadian clock in a tissue-specific fashion. We also report that deletion of the Zpbp2 region altered the expression profile of Nr1d1 in lungs and ileum in a time-dependent manner. In liver, the deletion was associated with enhanced expression of Ormdl3. We provide the first evidence that disease-associated genes Zpbp2 and Ormdl3 are regulated by circadian rhythms and the Zpbp2 region influences expression of the core clock gene Nr1d1.

Bacterial urinary tract infections (UTIs) are one of the most common reasons for children to be admitted to hospital. Bacteria infect and invade the bladder (the lower urinary tract) and if the infection disseminates to the upper urinary tract, significant inflammation in the kidneys may arise. Inflammation is a double-edged sword: it is needed to clear bacteria, but if excessive, kidney tissue is injured. During injury, nephrons are destroyed and replaced with deposition of extracellular matrix and a renal scar. In this review, we explore the pathogenesis of UTIs and discuss the risk factors that result in dissemination of bladder infection to the kidneys. Three major risk factors predispose to kidney infections: the presence of vesicoureteric reflux, the presence of bladder and bowel dysfunction, and defects in the ability of the host immune response to clear bacteria. In this review, we will discuss these factors, their relationship to renal scarring, and potential treatments that might be beneficial to prevent renal scar formation in children.

Kidney stones affect 10% of the population. Yet, there is relatively little known about how they form or how to prevent and treat them. The claudin family of tight junction proteins has been linked to the formation of kidney stones. The flavonoid quercetin has been shown to prevent kidney stone formation and to modify claudin expression in different models. Here we investigate the effect of quercetin on claudin expression and localization in MDCK II cells, a cation-selective cell line, derived from the proximal tubule. For this study, we focused our analyses on claudin family members that confer different tight junction properties: barrier-sealing (Cldn1, -3, and -7), cation-selective (Cldn2) or anion-selective (Cldn4). Our data revealed that quercetin’s effects on the expression and localization of different claudins over time corresponded with changes in transepithelial resistance, which was measured continuously throughout the treatment. In addition, these effects appear to be independent of PI3K/AKT signaling, one of the pathways that is known to act downstream of quercetin. In conclusion, our data suggest that quercetin’s effects on claudins result in a tighter epithelial barrier, which may reduce the reabsorption of sodium, calcium and water, thereby preventing the formation of a kidney stone.

INTRODUCTIONTo consider alternative mechanisms that give rise to a refluxing uretero-vesical junction (UVJ), we hypothesized that children with a common heritable urinary tract defect, vesico-ureteric reflux (VUR), may have a defect in the extracellular matrix composition of the UVJ and other tissues that would be revealed by assessment of the peripheral joints. Hypermobile joints can arise from defects in the extracellular matrix within the joint capsule that affect proteins, including Tenascin XB.METHODSWe performed an observational study of children with familial and non-familial VUR to determine the prevalence of joint hypermobility, renal scarring, and DNA sequence variants in Tenascin XB.RESULTSThe majority of children, 27/44, exhibited joint hypermobility using the Beighton scoring system. This included 15/26 girls (57.6%) and 12/18 boys (66.6%), which is a significantly higher prevalence for both sexes when compared to population controls (p<0.005). We found no association between joint hypermobility and renal scarring. Seven of 49 children harbored rare pathogenic sequence variants in TNXB, and two also exhibited joint hypermobility. No sequence variants in TNXB were identified in 25/27 children with VUR and joint hypermobility. Due to the observational design of the study, there was missing data for joint hypermobility scores in six children and for DMSA scans in 17 children.CONCLUSIONSWe observed a high prevalence of VUR and joint hypermobility in children followed within a tertiary care pediatric urology clinic. While mutations in TNXB have been reported in families with VUR and joint hypermobility, we identified only two children with these phenotypes and pathogenic variants in TNXB. We, therefore, speculate that VUR and joint hypermobility may be due to mutations in other extracellular matrix genes.

Although sequence variants in CD2-associated protein (CD2AP) have been identified in patients with focal segmental glomerulosclerosis (FSGS), definitive proof of causality in human disease is meager. By whole-exome sequencing, we identified a homozygous frame-shift mutation in CD2AP (p.S198fs) in three siblings born of consanguineous parents who developed childhood-onset FSGS and end stage renal disease. When the same frameshift mutation was introduced in mice by gene editing, the mice developed FSGS and kidney failure. These results provide conclusive evidence that homozygous mutation of CD2AP causes FSGS in humans.