A Nuclear Magnetic Resonance (NMR)- and Mass Spectrometry (MS)-Based Saturation Kinetics Model of a Bryophyllum pinnatum Decoction as a Treatment for Kidney Stones.

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.

Claudin-3 regulates luminal fluid accumulation in the developing chick lung.

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.

Osr1 Is Required for Mesenchymal Derivatives That Produce Collagen in the Bladder.

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.

The pathogenesis and management of renal scarring in children with vesicoureteric reflux and pyelonephritis.

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.

Recessive mutation in CD2AP causes focal segmental glomerulosclerosis in humans and mice.

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.

Heterozygous loss-of-function mutation in Odd-skipped related 1 (Osr1) is associated with vesicoureteric reflux, duplex systems, and hydronephrosis.

Odd-skipped related 1 (Osr1) is a transcriptional repressor that plays critical roles in maintaining the mesenchymal stem cell population within the developing kidney. Here, we report that newborn pups with a heterozygous null mutation in Osr1 exhibit a 21% incidence of vesicoureteric reflux and have hydronephrosis and urinary tract duplications. Newborn pups have a short intravesical ureter, resulting in a less competent ureterovesical junction which arises from a delay in urinary tract development. We describe a new domain of Osr1 expression in the ureteral mesenchyme and within the developing bladder in the mouse. OSR1 was sequenced in 186 children with primary vesicoureteric reflux, and 17 have single nucleotide polymorphisms. Fifteen children have a common synonymous variant, rs12329305, one child has a rare nonsynonymous variant, rs3440471, and one child has a rare 5'-UTR variant, rs45535040 The impact of these SNPs is not clear; therefore, the role of OSR1 in human disease remains to be elucidated. Osr1 is a candidate gene implicated in the pathogenesis of vesicoureteric reflux and congenital abnormalities of the kidney and urinary tract in mice.

Rac1 activation in podocytes induces the spectrum of nephrotic syndrome.

Hyper-activation of Rac1, a small GTPase, in glomerular podocytes has been implicated in the pathogenesis of familial proteinuric kidney diseases. However, the role of Rac1 in acquired nephrotic syndrome is unknown. To gain direct insights into this, we generated a transgenic mouse model expressing a doxycycline-inducible constitutively active form of Rac1 (CA-Rac1) in podocytes. Regardless of the copy number, proteinuria occurred rapidly within five days, and the histology resembled minimal change disease. The degree and severity of proteinuria were dependent on the transgene copy number. Upon doxycycline withdrawal, proteinuria resolved completely (one copy) or nearly completely (two copy). After one month of doxycycline treatment, two-copy mice developed glomerulosclerosis that resembled focal segmental glomerulosclerosis (FSGS) with urinary shedding of transgene-expressing podocytes. p38 MAPK was activated in podocytes upon CA-Rac1 induction while a p38 inhibitor attenuated proteinuria, podocyte loss, and glomerulosclerosis. Mechanistically, activation of Rac1 in cultured mouse podocytes reduced adhesiveness to laminin and induced redistribution of ß1 integrin, and both were partially reversed by the p38 inhibitor. Activation of Rac1 in podocytes was also seen in kidney biopsies from patients with minimal change disease and idiopathic FSGS by immunofluorescence while sera from the same patients activated Rac1 in cultured human podocytes. Thus, activation of Rac1 in podocytes causes a spectrum of disease ranging from minimal change disease to FSGS, due to podocyte detachment from the glomerular basement membrane that is partially dependent on p38 MAPK.

Vesicoureteral reflux and the extracellular matrix connection.

Primary vesicoureteral reflux (VUR) is a common pediatric condition due to a developmental defect in the ureterovesical junction. The prevalence of VUR among individuals with connective tissue disorders, as well as the importance of the ureter and bladder wall musculature for the anti-reflux mechanism, suggest that defects in the extracellular matrix (ECM) within the ureterovesical junction may result in VUR. This review will discuss the function of the smooth muscle and its supporting ECM microenvironment with respect to VUR, and explore the association of VUR with mutations in ECM-related genes.

Rare variants in tenascin genes in a cohort of children with primary vesicoureteric reflux.

BACKGROUND: Primary vesicoureteral reflux (PVUR) is the most common malformation of the kidney and urinary tract, and reflux nephropathy is a major cause of chronic kidney disease in children. Recently, we reported mutations in the tenascin XB gene (TNXB) as a cause of PVUR with joint hypermobility. METHODS: To define the role of rare variants in tenascin genes in the etiology of PVUR, we screened a cohort of patients with familial PVUR (FPVUR) and non-familial PVUR (NFPVUR) for rare missense variants inTNXB and the tenascin C gene (TNC) after excluding mutations in ROBO2 and SOX17. RESULTS: The screening procedure identified 134 individuals from 112 families with PVUR; two families with mutations in ROBO2 were excluded from further analysis. Rare missense variants in TNXB were found in the remaining 110 families, of which 5/55 (9%) families had FPVUR and 2/55 (4%) had NFPVUR. There were no differences in high-grade reflux or renal parenchymal scarring between patients with and without TNXB variants. All patients with TNXB rare variants who were tested exhibited joint hypermobility. Overall we were able to identify causes of FPVUR in 7/57 (12%) families (9% in TNXB and 3% in ROBO2). CONCLUSIONS: In conclusion, the identification of a rare missense variant in TNXB in combination with a positive family history of VUR and joint hypermobility may represent a non-invasive method to diagnose PVUR and warrants further evaluation in other cohorts.

Loss of Rho-GDIα sensitizes podocytes to lipopolysaccharide-mediated injury.

Nephrotic syndrome is a disease of glomerular permselectivity that can arise as a consequence of heritable or acquired changes to the integrity of the glomerular filtration barrier. We recently reported two siblings with heritable nephrotic syndrome caused by a loss of function mutation in the gene ARHGDIA, which encodes for Rho guanine nucleotide dissociation inhibitor-α (GDIα). GDIs are known to negatively regulate Rho-GTPase signaling. We hypothesized that loss of GDIα sensitizes podocytes to external injury via hyperactivation of Rho-GTPases and p38 MAPK. We examined the response of cultured podocytes with and without knockdown of GDIα to LPS injury by assessing the levels of phospho-p38 as well as the degree of synaptopodin loss. GDIα knockdown podocytes showed more pronounced and sustained p38 phosphorylation in response to LPS compared with control podocytes, and this was blunted significantly by the Rac1 inhibitor. In LPS-treated control podocytes, synaptopodin degradation occurred, and this was dependent on p38, the proteasome, and cathepsin L. In GDIα knockdown podocytes, the same events were triggered, but the levels of synaptopodin after LPS treatment were significantly lower than in control podocytes. These experiments reveal a common pathway by which heritable and environmental risk factors converge to injure podocytes, from Rac1 hyperactivation to p38 phosphorylation and synaptopodin degradation via the ubiquitin-proteasome pathway and cathepsin L.

Vesicoureteric reflux and reflux nephropathy: from mouse models to childhood disease.

Vesicoureteric reflux (VUR) is a common congenital urinary tract defect that predisposes children to recurrent kidney infections. Kidney infections can result in renal scarring or reflux nephropathy defined by the presence of chronic tubulo-interstitial inflammation and fibrosis that is a frequent cause of end-stage renal failure. The discovery of mouse models with VUR and with reflux nephropathy has provided new opportunities to understand the pathogenesis of these conditions and may provide insight on the genes and the associated phenotypes that need to be examined in human studies.

TNXB mutations can cause vesicoureteral reflux.

Primary vesicoureteral reflux (VUR) is the most common congenital anomaly of the kidney and the urinary tract, and it is a major risk factor for pyelonephritic scarring and CKD in children. Although twin studies support the heritability of VUR, specific genetic causes remain elusive. We performed a sequential genome-wide linkage study and whole-exome sequencing in a family with hereditary VUR. We obtained a significant multipoint parametric logarithm of odds score of 3.3 on chromosome 6p, and whole-exome sequencing identified a deleterious heterozygous mutation (T3257I) in the gene encoding tenascin XB (TNXB in 6p21.3). This mutation segregated with disease in the affected family as well as with a pathogenic G1331R change in another family. Fibroblast cell lines carrying the T3257I mutation exhibited a reduction in both cell motility and phosphorylated focal adhesion kinase expression, suggesting a defect in the focal adhesions that link the cell cytoplasm to the extracellular matrix. Immunohistochemical studies revealed that the human uroepithelial lining of the ureterovesical junction expresses TNXB, suggesting that TNXB may be important for generating tensile forces that close the ureterovesical junction during voiding. Taken together, these results suggest that mutations in TNXB can cause hereditary VUR.

A single-center cohort of Canadian children with VUR reveals renal phenotypes important for genetic studies.

BACKGROUND: Many genes and loci have been reported in genetic studies of primary vesicoureteral reflux (VUR), but few have been reproduced in independent cohorts, perhaps because of phenotype heterogeneity. We phenotyped children with VUR who attended urology clinics so we could establish criteria to stratify patients based on the presence or absence of a renal malformation. METHODS: History, chart review, and DNA were obtained for 200 children with VUR from 189 families to determine the grade of VUR, the mode of presentation, and the family history for each child. Kidney length measured on ultrasound (US) and technetium dimercaptosuccinic acid (DMSA) scans at the time of VUR diagnosis were used to establish the presence of a concurrent renal malformation and identify the presence of renal scarring. RESULTS: There was an even distribution of girls and boys, and most patients were diagnosed following a urinary tract infection (UTI). Thirty-four percent of the children had severe VUR, and 25 % had undergone surgical correction. VUR is highly heritable, with 15 % of the families reporting multiple affected members. Most patients had normally formed kidneys as determined by US and DMSA imaging. Of the 93 patients who underwent DMSA imaging, 17 (18 %) showed scarring, 2 (2 %) showed scarring and diffuse reduction in uptake, and 13 (14 %) showed an isolated diffuse reduction in uptake. CONCLUSION: Prospective long-term studies of patients with primary VUR combined with renal phenotyping using US and DMSA imaging are needed to establish the presence of a renal malformation. The majority of patients in our study had no renal malformation. This cohort is a new resource for genetic studies of children with primary VUR.

Submandibular gland epithelial development and the importance of junctions.

Salivary glands consist of highly specialized epithelial cells that secrete the fluid, saliva, and/or transport saliva into the oral cavity. Saliva is essential to lubricate the oral cavity for food consumption and to maintain the hygiene of the oral cavity. In this review, we will focus on the formation of the epithelial cell lineage and the cell junctions that are essential for formation of saliva and maintenance of the epithelial barrier between the ducts that transport saliva and the extracellular environment.

Case Report of Renal Calculi in a Child Receiving Imatinib for Acute Lymphoblastic Leukemia.

Rationale: Imatinib is used in the treatment of Philadelphia chromosome positive (Ph+) leukemias and has been reported to have a direct effect on bone physiology. Presentation: To report on a child with Ph+ acute lymphoblastic leukemia who presented with bilateral flank pain and gross hematuria. Diagnosis: She was diagnosed with obstructive kidney stones 101 days after commencing daily oral imatinib. Stone analysis revealed the presence of calcium phosphate. Interventions and outcome: The patient passed the stones spontaneously with medical therapy that included the use of thiazide, allopurinol, and potassium citrate, but she required temporary insertion of a double-J stent to relieve an obstruction. Novel findings: Imatinib inhibits receptor tyrosine kinases and stimulates the flux of calcium from the extracellular fluid into bone, resulting in hypocalcemia with a compensatory rise in parathyroid hormone that may result in phosphaturia and the formation of calcium phosphate stones. Given that kidney stones are rare events in children, we believe that monitoring for kidney stone formation needs to be performed in children receiving imatinib.

Justification: L'imatinib est utilisé dans le traitement des leucémies à chromosome Philadelphie (Ph+) et a été décrit comme ayant un effet direct sur la physiologie osseuse. Présentation du cas: Une enfant atteinte d'une leucémie lymphoblastique aiguë à Ph+ présentant des douleurs lombaires bilatérales et une hématurie macroscopique. Diagnostic: La patiente a reçu un diagnostic de calculs rénaux obstructifs 101 jours après avoir commencé la prise quotidienne d'imatinib par voie orale. L'analyse des calculs a révélé la présence de phosphate de calcium. Interventions et résultats: La patiente a éliminé spontanément ses calculs grâce à un traitement médical qui comprenait un diurétique thiazidique, de l'allopurinol et du citrate de potassium, mais on a dû lui insérer temporairement une endoprothèse double J pour traiter une obstruction. Nouveaux enseignements: L'imatinib inhibe les récepteurs de la tyrosine kinase et favorise le flux du calcium du liquide extracellulaire vers les os, ce qui entraîne une hypocalcémie avec élévation secondaire de l'hormone parathyroïdienne pouvant provoquer une phosphaturie et la formation de calculs de phosphate de calcium. Puisque la formation de calculs rénaux est rare chez les enfants, nous pensons qu'elle devrait faire l'objet d'une surveillance chez les enfants qui reçoivent de l'imatinib.

The tight junction protein claudin-3 shows conserved expression in the nephric duct and ureteric bud and promotes tubulogenesis in vitro.

The claudin family of proteins is required for the formation of tight junctions that are contact points between epithelial cells. Although little is known of the cellular events by which epithelial cells of the ureteric bud form tubules and branch, tubule formation is critical for kidney development. We hypothesize that if claudin-3 (Cldn3) is expressed within tight junctions of the ureteric bud, this will affect ureteric bud cell shape and tubule formation. Using transmission electron microscopy, we identified tight junctions within epithelial cells of the ureteric bud. Whole mount in situ hybridization and immunoassays were performed in the mouse and chick and demonstrated that Cldn3 transcript and protein were expressed in the nephric duct, the ureteric bud, and its derivatives at critical time points during tubule formation and branching. Mouse inner medullary collecting duct cells (mIMCD-3) form tubules when seeded in a type I collagen matrix and were found to coexpress CLDN3 and the tight junction marker zonula occludens-1 in the cell membrane. When these cells were stably transfected with Cldn3 fused to the enhanced green fluorescent protein reporter, multiple clones showed a significant increase in tubule formation compared with controls (P < 0.05) due in part to an increase in cell proliferation (P < 0.01). Cldn3 may therefore promote tubule formation and expansion of the ureteric bud epithelium.

Vesico-ureteric reflux: using mouse models to understand a common congenital urinary tract defect.

Vesico-ureteric reflux (VUR) is a common congenital urinary tract defect in which urine flows retrogradely from the bladder to the kidneys because of an abnormally formed uretero-vesical junction. It is associated with recurrent urinary tract infections, renal hypo/dysplasia, reflux nephropathy, hypertension, and end-stage renal disease. In humans, VUR is genetically and phenotypically heterogeneous, encompassing diverse renal and urinary tract phenotypes. To understand the significance of these phenotypes, we and others have used the mouse as a model organism and this has led to the identification of new candidate genes. Through careful phenotypic analysis of these models, a new understanding of the genetics and biology of VUR is now underway.

CYP24A1 and SLC34A1 Pathogenic Variants Are Uncommon in a Canadian Cohort of Children with Hypercalcemia or Hypercalciuria.

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.

The C3H/HeJ inbred mouse is a model of vesico-ureteric reflux with a susceptibility locus on chromosome 12.

Vesico-ureteric reflux is the most common congenital anomaly of the urinary tract, characterized by a defective uretero-vesical junction with retrograde urine flow from the bladder toward the kidneys. Because there is strong evidence for a genetic basis for some cases of vesico-ureteric reflux, we screened 11 inbred mouse strains for reflux and kidney size and identified one strain, C3H/HeJ, that has a 100 percent incidence of vesico-ureteric reflux with otherwise normal kidneys at birth. These mice are predisposed to reflux as a result of a defective uretero-vesical junction characterized by a short intravesical ureter. This defect results from a delay in urinary tract development initially manifested by a ureteric bud arising from a more caudal location along the mesonephric duct. In contrast, C57BL/6J mice (resistant to reflux at birth) have long intravesical ureters, normally positioned ureteric buds, and no delay in urinary tract development. Genome-wide and additional fine mapping of backcross mice, derived from C3H/HeJ and C57BL/6J crosses, identified a significant reflux susceptibility locus, Vurm1, on chromosome 12 (peak logarithm of the odds=7.39). The C3H/HeJ mouse is a model of vesico-ureteric reflux without renal malformation, and further characterization of this model will allow for the identification of a pathway important for urinary tract development, a finding that will serve as a model for the human disorder.