Acadian variant of Fanconi syndrome is caused by mitochondrial respiratory chain complex I deficiency due to a non-coding mutation in complex I assembly factor NDUFAF6.

The Acadian variant of Fanconi Syndrome refers to a specific condition characterized by generalized proximal tubular dysfunction from birth, slowly progressive chronic kidney disease and pulmonary interstitial fibrosis. This condition occurs only in Acadians, a founder population in Nova Scotia, Canada. The genetic and molecular basis of this disease is unknown. We carried out whole exome and genome sequencing and found that nine affected individuals were homozygous for the ultra-rare non-coding variant chr8:96046914 T > C; rs575462405, whereas 13 healthy siblings were either heterozygotes or lacked the mutant allele. This variant is located in intron 2 of NDUFAF6 (NM_152416.3; c.298-768 T > C), 37 base pairs upstream from an alternative splicing variant in NDUFAF6 chr8:96046951 A > G; rs74395342 (c.298-731 A > G). NDUFAF6 encodes NADH:ubiquinone oxidoreductase complex assembly factor 6, also known as C8ORF38. We found that rs575462405-either alone or in combination with rs74395342-affects splicing and synthesis of NDUFAF6 isoforms. Affected kidney and lung showed specific loss of the mitochondria-located NDUFAF6 isoform and ultrastructural characteristics of mitochondrial dysfunction. Accordingly, affected tissues had defects in mitochondrial respiration and complex I biogenesis that were corrected with NDUFAF6 cDNA transfection. Our results demonstrate that the Acadian variant of Fanconi Syndrome results from mitochondrial respiratory chain complex I deficiency. This information may be used in the diagnosis and prevention of this disease in individuals and families of Acadian descent and broadens the spectrum of the clinical presentation of mitochondrial diseases, respiratory chain defects and defects of complex I specifically.

The pesticide adjuvant, Toximul, alters hepatic metabolism through effects on downstream targets of PPARalpha.

Previous studies demonstrated that chronic dermal exposure to the pesticide adjuvant (surfactant), Toximul (Tox), has significant detrimental effects on hepatic lipid metabolism. This study demonstrated that young mice dermally exposed to Tox for 12 days have significant increases in expression of peroxisomal acyl-CoA oxidase (mRNA and protein), bifunctional enzyme (mRNA) and thiolase (mRNA), as well as the P450 oxidizing enzymes Cyp4A10 and Cyp4A14 (mRNA and protein). Tox produced a similar pattern of increases in wild type adult female mice but did not induce these responses in PPARalpha-null mice. These data support the hypothesis that Tox, a heterogeneous blend of nonionic and anionic surfactants, modulates hepatic metabolism at least in part through activation of PPARalpha. Notably, all three groups of Tox-treated mice had increased relative liver weights due to significant accumulation of lipid. This could be endogenous in nature and/or a component(s) of Tox or a metabolite thereof. The ability of Tox and other hydrocarbon pollutants to induce fatty liver despite being PPARalpha agonists indicates a novel consequence of exposure to this class of chemicals, and may provide a new understanding of fatty liver in populations with industrial exposure.

Two formulations of the agricultural pesticide adjuvant, Toximul, reduce the glycogen content of HepG2 cells.

Young mice exposed dermally to the Toximul (Tox) class of agricultural pesticide adjuvants have reduced levels of hepatic glycogen, a marker of subclinical toxicity. The present study determined whether these effects on glycogen also occurred in cultured HepG2 cells. Exposure (3 hr) to Tox resulted in significant, concentration-dependent glycogen reductions (up to 70%) relative to control values (76 +/- 3 microg glycogen/mg protein). These reductions did not appear to be due to loss of cell viability, and were reversible with Tox removal. Two different formulations of Tox (3409F and MP-A) differed significantly in the magnitudes of glycogen reduction in the HepG2 cells.

Paradoxical cyclosporine A requirements in pediatric renal transplants receiving high-dose steroids.

The potent immunosuppressant cyclosporine A (CyA) is a mainstay of treatment in the renal transplant population. During episodes of acute allograft rejection, therapy also includes the pulse administration of high-dose steroids such as prednisone or methylprednisolone. Both steroids and CyA are metabolized by the CYP3A4 isoenzyme of the cytochrome P450 catalytic system. On a theoretical basis, high steroid concentrations during a rejection episode could competitively inhibit CyA metabolism, increasing its systemic concentration and decreasing its dose requirements. A database was compiled consisting of pediatric patients who had undergone an acute renal rejection event during the years 1993 to 2003. The severity of rejection events, as well as the CyA and prednisone dosing regimens used during rejection, were assessed using a comprehensive chart analysis. The presence or absence of additional medications that could potentially interact with CyA was also examined. Although some patients responded in the predicted manner, the authors also found that a subgroup of pediatric patients placed on highdose pulse steroid therapy for acute graft rejection required increased amounts of CyA to maintain therapeutic concentrations. The authors recommend monitoring of patients on high-dose steroids for paradoxical CyA requirements intermittently during high-dose steroid treatment to individualize CyA therapy appropriately during renal allograft rejection and thereby maximize efficacy while minimizing potential toxic side effects of CyA such as under-immunosuppression and organ rejection.

A multicenter, open-label, pharmacokinetic/pharmacodynamic safety, and tolerability study of basiliximab (Simulect) in pediatric de novo renal transplant recipients.

BACKGROUND: Basiliximab (Simulect) has been shown to be safe and effective in adult renal transplant recipients, when used in combination with cyclosporine (Neoral) and corticosteroids. We report on the safety and preliminary efficacy of basiliximab in pediatric de novo renal transplant recipients. METHODS: This was an open-label, 12-month study of basiliximab in 41 patients (2 cohorts: <9 and 9 to <16 years). In phase 1, two intravenous (IV) bolus injections of basiliximab (12 mg/m ) were administered (before and 4 days postsurgery). In phase 2, two injections (<40 kg, 10 mg and > or =40 kg, 20 mg) were administered at the same time points. Most patients (26/41 [63%]) received cadaveric kidneys. Almost half of the patients had three human leukocyte antigen mismatches with the organ donors. Concurrent immunosuppression included Neoral and corticosteroids. Azathioprine was allowed after 28 days. RESULTS: All patients completed the 1-year study. The acute tolerability of basiliximab via IV bolus injection was good, without evidence of cytokine-release syndrome or acute local reactions. All patients experienced adverse events, but most (71%) were mild or asymptomatic. No deaths or malignancies occurred. The incidence and types of serious adverse events (59%) and serious infections (44%) were as expected in this patient population, and few were drug-related (7% and 5%, respectively). Thirty-eight patients (93%) had infections, mostly urinary tract infections, as expected for renal transplant patients. Six patients (15%) had drug-related adverse events. Biopsy-confirmed acute rejection episodes occurred in 6/41 (15%) of patients during the first 6 months posttransplantation and in 9/41 (22%) patients during the first 12 months. Five patients (12%) experienced graft loss, none of which were preceded by acute rejection episodes. CONCLUSIONS: Basiliximab is safe and well tolerated when administered by IV bolus injection in de novo pediatric renal transplant recipients. These preliminary data suggest that basiliximab, given in combination with cyclosporine and corticosteroids, is an effective immunosuppressive regimen for the prevention of acute rejection in pediatric renal transplantation.

Phenotypic heterogeneity in pediatric autosomal dominant polycystic kidney disease at first presentation: a single-center, 20-year review.

BACKGROUND: The presentation of autosomal dominant polycystic kidney disease (ADPKD) in childhood provides an insight into comorbidities and potential areas for interventions and investigation. METHODS: Phenotypic heterogeneity at the time of first presentation was studied with respect to age of diagnosis, mode of presentation, parental inheritance pattern, renal function, associated hypertension, and hyperlipidemia. Fifty-five children (median age of presentation, 8.7 years; 27% < 1 year) with ADPKD from 44 families followed up between March 1983 and March 2003 were reviewed. The diagnosis was based on family history and ultrasound confirmation of cysts. Progression of renal disease was followed over the study period (mean duration of follow-up, 4.9 years). RESULTS: A family history of ADPKD was known at presentation in 89%, which precipitated the screening diagnostic imaging in 59% of these children. Maternal inheritance was displayed in 51%, whereas 5% had no known family history of ADPKD. Bilateral renal findings were present in 78%. Hypertension (>95(th) percentile for age) was present in 22%, and hyperlipidemia was present in 54%. Renal function was not significantly diminished in 98% of patients with creatinine clearance > or =3rd percentile for age, and 7% had persistent proteinuria (>150 mg/d). No subjects had hepatic, splenic, or pancreatic cysts on ultrasound scan. A subpopulation of 10 patients had features of ADPKD dating back to prenatal ultrasound scans. All prenatal cases were characterized by bilateral renal findings, 90% had a known family history of ADPKD at the time of presentation, and 89% of these patients displayed maternal inheritance. Follow-up studies showed a persistence of hyperlipidemia despite pharmacotherapeutic treatment of hypertension, infrequent proteinuria, and sustained renal function in most patients. CONCLUSION: The results of this study show that many children at the time of first presentation have a significant prevalence of modifiable risk factors: hypertension, proteinuria, and hyperlipidemia, in the face of normal renal function. The results also show a unique presentation existing in prenatal subjects.

Effects of insulin-like growth factor-type 1 on weight gain and hepatic glycogen during early development in a surfactant/virus mouse model of acute liver failure: correlation with mortality.

Acute liver failure (ALF) was reproduced in young mice exposed daily for 12 days to the industrial surfactant, Toximul 3409F (Tox), and infected on postnatal day (P) 14 with sublethal doses of mouse-adapted human influenza B (Lee) virus (FluB). Combined Tox + FluB treatment potentiated mortality due to non-necrotic ALF. This study tested the hypothesis that mortality would decline if the known losses in energy production due to compromised fatty-acid beta-oxidation were compensated by pharmacological manipulation of hepatic glycogen stores. Glycogen levels, body weights, and mortality were determined without and with injections of insulin-like growth factor-1 (IGF-1). On P25, 13 days after Tox exposure ceased, glycogen levels (mg/100mg) were: 4.0 (control), 1.7 (Tox), 4.3 (FluB), and 2.9 (Tox + FluB). Corresponding cumulative mortalities were 0, 14, 2, and 38%. Following daily IGF-1 injections from P12 to P17, liver glycogen levels on P25 were: 3.5 (IGF-1), 3.9 (IGF-1 + Tox), 12.3 (IGF-1 + FluB), and 5.6 (IGF-1 + Tox + FluB). Unexpectedly, IGF-1 treatment increased mortality to 67% (IGF-1), 89% (IGF-1 + Tox), 63% (IGF-1 + FluB), and 81% (IGF-1 + Tox + FluB). For all groups there was a significant correlation between mortality and poor weight gain. This is the first report of persistent glycogen reductions after surfactant exposure and withdrawal. Their role in potentiating FluB-induced mortality remains to be established.

Suppression of early and chronic BK polyoma virus replication by mycophenolic acid in Vero cells.

Consensus is lacking about which immunosuppressant agents potentiate BK virus infection. The effects of mycophenolic acid (MPA) were investigated in BK virus (BKV)-infected Vero E6 cells. MPA (1-16 mg/l) exhibited a dose-dependent anti-viral effect (10(1)-10(4) fold reduction in BKV DNA copies/ml) in supernatant, similar to cidofovir (2.5-25 mg/l). This effect was observed for early and persistent infection, and infection with noncoding control region (NCCR) rearranged BKV. MPA reduced BKV DNA copies/ml by >1 log after day 14 in three patient isolates before and after NCCR rearrangement, and in cells. MPA reduced total cellular protein levels, consistent with an anti-metabolite effect without increased cytopathic activity. BKV infection was associated with a transient, significant reduction of collagen 1A1 on day 7 but not on days 14, 21, and 28 or in the presence of MPA. Reduction of alpha smooth muscle actin mRNA was observed only in the BKV + MPA group, and only on day 7. There was no significant alteration of heat shock protein 47 or transforming growth factor-beta mRNA expression. These in vitro data suggest that MPA may have a protective, anti-viral effect in BKV-infected renal tubular cells with an anti-viral response. Maintaining, or even increasing, the MPA dose should be evaluated for reduction of BKV viremia levels.

Pamidronate distribution in pediatric renal and rheumatologic patients.

OBJECTIVE: To evaluate the distribution and elimination of pamidronate in a population of pediatric patients with renal and rheumatologic disease. METHODS: Pamidronate whole blood levels were collected for the first 4 h after first exposure in 7 patients. The relationship between the rate of urinary excretion of pamidronate and bone formation or resorption was examined in 18 patients while receiving pamidronate at a total dose of 1 mg/kg/dose infused intravenously over a 4-h period. The urinary pamidronate clearances were correlated with renal function, calcium levels and measures of bone formation and resorption. RESULTS: Pamidronate levels reached steady state concentrations of 0.9-1.5 microg/ml within 30 min and the clearance of the drug (mean+/-SE) from blood was 180.0+/-64.2 ml/kg/h with an elimination half-life of less than 1 h. The mean urinary excretion of 31.5+/-2.2% of the administered dose indicated that about 68% of the drug was incorporated into bone, confirming the uptake of pamidronate into bone was similar in pediatric patients compared to that previously reported for adults. Bone specific alkaline phosphatase, which is a marker for bone growth and formation, had significant correlation with the uptake of pamidronate into bone (p=0.002). No correlation was demonstrated with a marker for bone resorption (urinary N-telopeptide/creatinine ratio), or with creatinine clearance or calciuria when assessed 2 months after treatment. CONCLUSION: Pamidronate at a dose of 1 mg/kg/dose every 2 months appears safe in the short term for pediatric patients, achieves relatively low whole blood pamidronate levels, and has similar skeletal uptake of pamidronate compared to adults.

Pamidronate treatment of pediatric fracture patients on chronic steroid therapy.

Pediatric nephrology and rheumatology patients with steroid-induced osteopenia are at risk of skeletal fracture. Bisphosphonate therapy has not been routinely advocated as a primary or secondary intervention for steroid-associated fractures in this population. This case control study evaluates the role of pamidronate therapy as a secondary fracture intervention. Children with symptomatic pathological fractures of the axial spine or ribs were treated with pamidronate 1 mg/kg/dose (n=17) IV at 60-day intervals for 1 yr (n=15) or 2 yr (n=2). Bone mineral density of L1-L4 (BMD) was assessed prior to treatment and at six-month intervals, and compared to 17 disease-age-gender-steroid dose-matched control patients. Alkaline phosphatase, calcium, phosphate, PTH, renal biochemistry, and 24-hr urine collections for CrCl, N-telopeptide/creatinine ratio, phosphate excretion, and calcium excretion were obtained every two months in the pamidronate population. Pamidronate caused a first exposure transient flu-like illness lasting <24 h in three patients and one patient had a new pathological fracture. No adverse events of hypocalcemia, allergic reaction or thrombophlebitis were noted. All patients reported improvement of skeletal pain. Despite ongoing steroid treatment, pamidronate significantly increased L1-L4 BMD Z-scores (mean+/-SE) relative to baseline (pamidronate vs control: 0-6 months: 0.27+/-0.14 vs -0.82+/-0.31; 0-12 months: 0.63+/-0.17 vs -0.46+/-0.27; 0-18 months: 0.55+/-0.32 vs 0.17+/-0.27; 0-24 months: 0.15+/-0.21 vs -0.23+/-0.22; 0-30 or 36 months: 0.77+/-0.71 vs -0.68+/-0.25) with repeated measures ANOVA assessment (F=11.27, p=0.0057). This study supports the safety and efficacy of pamidronate in steroid-induced fractures in pediatric nephrology and rheumatology patients.

A comparison of the pharmacokinetics of tacrolimus and microemulsified cyclosporin in paediatric renal transplant recipients.

OBJECTIVE: Our objective was to identify common factors that determine the dose of tacrolimus and microemulsified cyclosporin in paediatric renal transplant recipients. METHODS: The concentration profiles of tacrolimus and cyclosporin in blood were determined in 68 children who had received a renal transplant. To avoid disruption of therapy, measurements were made at 2-h intervals over an 8-h period during normal dosing regimens. Direct comparisons of the two drugs were made in 14 of the subjects who were switched from cyclosporin to tacrolimus. RESULTS: The ratio of peak to trough levels for tacrolimus was approximately twofold compared with over threefold for cyclosporin. Area under the curve (AUC) for tacrolimus remained relatively constant in each 2-h period of the dosage interval compared with the AUC for cyclosporin, which varied by over twofold in the same time period. In the 14 subjects who received both drugs, there was a poor correlation between C2/C0, C2, t(1/2) and AUC for tacrolimus and cyclosporin in the same individual. In a multivariate analysis, there were no significant associations for tacrolimus concentrations, AUC or C2/C0 with age, gender, calcium-channel blocker, quinolone or statin. For cyclosporin, there was some association for AUC with gender and quinolone use and a weak association with calcium-channel blocker or statin use. CONCLUSIONS: Tacrolimus and microemulsified cyclosporin display a wide intra- and inter-individual variation in pharmacokinetic properties in young subjects. In the case of absorption represented by the peak-trough ratios, the values for tacrolimus are significantly less than those obtained with cyclosporin. The pharmacokinetic parameters obtained for one of these agents is not predictive for the behaviour of the other in young renal transplant recipients.

Determination of pamidronate in human whole blood and urine by reversed-phase HPLC with fluorescence detection.

Pamidronate is a bisphosphonate that is effective in treating bone disease including osteopenia and osteoporosis in adults. A sensitive and reliable method for the analysis of pamidronate in whole blood and urine is key to the development of this drug for use in children. A previously described method for pamidronate analysis serum and urine did not consistently detect the drug at satisfactory levels in whole blood. The procedure involves co-precipitation of the bisphosphonates with calcium phosphate, pre-column derivitization with fluorescamine, HPLC utilizing a Nucleosil C(18) column, and fluorescence detection with excitation at 395 nm and emission at 480 nm. Changes to the original protocol included the use of a new internal standard (alendronate), the optimization of the concentration of ethylenediaminetetraacetic acid (EDTA) for dissolving the precipitate, and the elimination of the acidification step prior to deproteinization. The optimum EDTA concentration, which had a significant effect on the labeling capability of fluorescamine, was determined to be 20 mm.A good separation between pamidronate and alendronate was achieved using a heated (40 degrees C ) Nucleosil C(18), 10 micro m particle size column. The mobile phase was an aqueous solution of 1 mm Na(2)EDTA-methanol (97:3, v/v) adjusted to pH 6.5 using a fl ow-rate of 1 mL/min. Fluorescence detection was set at 395 nm for excitation and at 480 nm for emission. The limit of quantitation for pamidronate was 0.5 micro g/mL in whole blood and 0.1 micro g/mL in urine. The method was applied to both whole blood and urine samples from pediatric patients.

T-cell/periodic acid-Schiff stain: a useful tool in the evaluation of tubulointerstitial infiltrates as a component of renal allograft rejection.

Distinguishing between tubulitis and tubulointerstitial mononuclear cell infiltrates and determining the severity of tubulitis are critical components of diagnosing and grading renal allograft rejection using the 1993 Banff schema, the revised 1997 Banff schema, or the Cooperative Clinical Trials in Transplantation grading system. We describe a novel staining method, the T-PAS stain (CD3 and periodic acid-Schiff), which removes some of the subjectivity in the evaluation of tubulointerstitial infiltrates in renal allograft biopsies. The method simply combines two routine stains, immunoperoxidase staining for T cells (CD3) and periodic acid-Schiff (PAS) staining for tubular basement membrane, on the same section.

Neuropsychological outcome in children with acquired or congenital renal disease.

The neuropsychological abilities of children with congenital ( n=13) or acquired ( n=11) end-stage renal disease (ESRD) were compared. Patients were being treated with or being prepared for dialysis and were awaiting transplantation. None of the children had an identifiable syndrome with associated central nervous system (CNS) dysfunction or had exposure to drugs with known CNS toxicity. There were no group differences in intelligence, academic achievement, behavior, or immediate memory. Children with congenital ESRD had poorer fine motor coordination and more difficulty on tests of verbal and nonverbal long-term memory than children with acquired ESRD. However, the neuropsychological outcome for congenital ESRD is more favorable than previously described. Psychological and education treatment recommendations should be considered.