Elevated Excretion Research Articles

OA13 Alkaptonuria: a big mimic

Abstract Background/Aims Alkaptonuria is a rare metabolic disorder, often misdiagnosed as spondyloarthritis or degenerative musculoskeletal disease. The population most affected are young males in their 30s with characteristic arthritis involving the spine and large joints. The patient may notice their urine turning dark on standing and pigmentation over the skin and connective tissues (ochronosis); however, these subtle features may sometimes go unnoticed. Methods A 38-year-old-male with long standing back pain presented with right knee swelling. With a diagnosis of spondyloarthritis, he was previously treated with DMARDS but did not have any relief. There was a history of urine turning dark on standing. On examination, there was subtle hyperpigmentation over left ear concha and a spot on the sclera. An x-ray of the thoracic spine showed flattening and calcification of intervertebral disc spaces at multiple levels. Aspiration of synovial fluid showed suspended fragments of articular cartilage. His inflammatory markers were negative. We performed gas chromatography mass spectrometry which showed elevated excretion of homogentisic acid (HGA). A homozygous pathogenic variant in the homogentisate 1,2-dioxygenase gene associated with alkaptonuria was reported on whole genome sequencing. Results Ochronotic arthropathy is defined as progressive degenerative joint disease mainly affecting the spine and large joints. Most patients require at least one joint replacement by age 55. Small joints of hands and feet and sacroiliac joints are usually spared. Arthroscopy of the joint may reveal dark brown to black pigmented cartilage defects. The synovium may show fragments of pigmented cartilage suspended in the fluid (ground pepper sign). Plain spine radiographs may show flattening and calcifications of the intervertebral discs with a characteristic vacuum phenomenon (radiolucent gas collection). Periarticular structures like tendons and ligaments are also affected, leading to rupture in around 20-30% of patients. Alkaptonuria is also associated with decreased bone mineral density and subsequent risk for fragility fracture. Conclusion Alkaptonuria often mimics ankylosing spondylitis. A triad of ochronosis, dark urine and ochronotic arthropathy is the key to diagnosis. Definitive diagnosis is through biochemical or genetic testing. Nitisinone has proved to be effective but shows significant side effects. Management mainly involves physiotherapy and palliative care. Disclosure M. Shah: None. S. Upadhyaya: None.

The arsenic-lowering effect of inulin-type prebiotics in end-stage renal disease: a randomized crossover trial.

Background: Circulatory imbalance of trace elements is frequent in end-stage renal disease (ESRD), leading to a deficiency of essential elements and excess of toxic elements. The present study aimed to investigate whether inulin-type fructans (ITFs) could ameliorate the circulatory imbalance by modulating gut microbiota and regulating the absorption and elimination of trace elements. Methods: Peritoneal dialysis patients were enrolled in a randomized crossover trial, undergoing interventions with ITFs (10 g d-1) and maltodextrin (placebo) over a 9-month period (with a 3-month washout). The primary outcomes included essential elements Mn, Fe, Co, Cu, Zn, Se, Sr, and Mo and potential toxic elements V, Cr, Ni, As, Cd, Ba, Tl, Pb, Th, and U in plasma. Secondary outcomes included the gut microbiome, short chain fatty acids (SCFAs), bile acids (BAs), and daily removal of trace elements through urine, dialysate and feces. Results: Among the 44 participants initially randomized, 29 completed the prebiotic, placebo or both interventions. The daily dietary intake of macronutrients and trace elements remained consistent throughout the study. The administration of 10 g d-1 ITFs significantly reduced plasma arsenic (As) by 1.03 μg L-1 (95%CI: -1.74, -0.33) (FDR-adjusted P = 0.045) down from the baseline of 3.54 μg L-1 (IQRs: 2.61-4.40) and increased the As clearance rate by urine and dialysis (P = 0.033). Positive changes in gut microbiota were also observed, including an increase in the Firmicutes/Bacteroidetes ratio (P = 0.050), a trend towards higher fecal SCFAs (P = 0.082), and elevated excretion of primary BAs (P = 0.035). However, there were no significant changes in plasma concentrations of other trace elements or their daily removal by urine, dialysis and feces. Conclusions: The daily administration of 10 g d-1 ITFs proved to be effective in reducing the circulating retention of As but demonstrated to be ineffective for other trace elements in ESRD. These sentences are ok to include but as "The clinical trial registry number is ChiCTR-INR-17013739 (https://www.chictr.org.cn/showproj.aspx?proj=21228)".

#4941 A METABOLOMIC FINGERPRINT PERSPECTIVE OF GUT-DERIVED METABOLITES ON EARLY DIABETIC KIDNEY DISEASE IN TYPE 2 DIABETES MELLITUS PATIENTS

Abstract Background and Aims Type 2 diabetes mellitus (T2DM) is the first cause of end stage renal disease worldwide. Metabolite profiling is an emerging field of great significance, as T2DM prevalence is continuously rising. The subtle dynamics of gut microbiota derived metabolites may have important implications in diabetic kidney disease (DKD) development. Nitrogen metabolic pathway and retinoic acid signaling pathway seem to be involved in early pathogenic mechanisms of DKD. The aim of study was to discover new putative gut-derived biomarkers that interfere with early physio-pathological mechanisms in DKD, regarding the podocyte, tubule, and the renal endothelium. Method The serum and urine of 90 T2DM patients (P group) and 20 healthy subjects (C group), were assessed in a cross-sectional study, by ultra-high-performance liquid chromatography coupled with electrospray ionization-quadrupole-time of flight-mass spectrometry (UHPLC-QTOF-ESI+-MS) techniques. P group was divided in 3 subgroups (normoalbuminuria-P1, microalbuminuria-P2, macroalbuminuria-P3). Statistical analysis was performed by untargeted multivariate (PLSDA, VIP scores, Random Forest, Biomarker analysis) and univariate (One Way ANOVA, Biomarker Analysis) methods. Results Multivariate analysis, by which the P group vs. C group were compared, displayed: (1) in serum samples: increased levels (P>C) of all-trans retinoic acid, cysteine-S sulfate, oleoylglicine, threonylglycine and decreased levels (P<C) of phenylalanine, tyrosine, kynurenic acid; (2) in urine samples, there were increased levels of: all-trans retinoic acid, indoxyl suflate, serotonin sulfate and glycylprolylarginine. Subsequently, by applying univariate analysis, the biomarkers derived from gut microbiota were selected: (1) in serum: all-trans retinoic acid (C<P1<P2<3), phenylalanine, tyrosine, and kynurenic acid with levels decreasing progressively from C to P1-P2-P3 (2) in urine: all trans retinoic acid, indoxyl sulfate, and serotonin sulfate, showing good differentiation between C and P1, and slight differences between P1∼P2∼P3 subgroups. The study suggests that ATRAs dynamic is disturbed in the normoalbuminuric stage of DKD. While its urinary levels correlated significantly with albuminuria, the changes observed in P1 subgroup, in serum and urine, may be correlated to the loss of renal tubular tight junction as a result of claudin downregulation. Phenylalanine and tyrosine, a part of nitrogen metabolic pathway, are indicators of the new-onset DKD. Their levels were upregulated in serum vs. urine, indicating their possible involvement in early DKD when comparing C vs. P1-P2-P3. Kynurenic acid and indoxyl-sulfate result from tryptophan metabolism. Low levels in serum of kynurenic acid may suggest its possible implication in incipient endothelial dysfunction in DKD. Indoxyl-sulfate (IS) urinary excretion is dependent on albuminuria. The study shows an elevated excretion of IS, in the normoalbuminuric group compared to controls and may suggest a tubular damage that precedes the glomerular modifications in DKD. Conclusion UHPLC-QTOF-ESI+-MS untargeted analysis reveals a particular metabolite fingerprint in early DKD. The study describes all-trans retinoic acid, phenylalanine, tyrosine, kynurenic acid, and indoxyl sulfate, as potential key biomarkers involved in early DKD pathogenesis, their levels being expressed in P1 subgroup compared with C group, and P2, P3 subgroups.

Bioavailability of orange juice (poly)phenols: β-glucan-rich oat bran decreases urinary excretion of flavanone phase II metabolites and enhances excretion of microbiota-derived phenolic catabolites.

The impact of β-glucan-rich oat bran on the bioavailability of orange juice (OJ) flavanones was investigated. Volunteers consumed 500mL of OJ with and without 22g of oat bran containing 6g of β-glucan (OB-6). Urine collected 12h prior to and over a 0-24h period post-supplementation was analysed by UHPLC-HRMS. Sixteen flavanone metabolites and thirty-nine colon-derived phenolic catabolites were identified and quantified. The major compounds were hesperetin-3'-glucuronide, along with hippuric acids and the C6-C3 phenolic acids 3-(3'-hydroxy-4'-methoxyphenyl)hydracrylic acid and 3-(4'-hydroxy-3'-methoxyphenyl)propanoic acid. A marked reduction in the 0-24h excretion of flavanone metabolites from 29.7μmol (9.3% recovery) to 9.3μmol (2.9% recovery), occurred following consumption of OB-6 compared to OJ. This appeared not to be an effect of fiber on the rate of transport in the upper gut. After consumption of OJ there was a 163±15μmol excretion of colon-derived phenolic catabolites, equivalent to 43% of (poly)phenol intake and following OB-6 intake there was a further significant 30% increase. The β-oat bran in OB-6 contained 5.8μmol of free and 52μmol of bound phenolic derivatives compared to 371μmol of OJ (poly)phenols. The elevated excretion of phenolics after OB-6 consumption appears not to be due to bound phenolics in the bran, rather it is consequence, principally, of a bran-mediated increase in the quantities of flavanones passing from the upper to the lower bowel where they were subjected to microbiota-mediated catabolism. CLINICAL TRIAL REGISTRATION NUMBER: This trial was registered at clinicaltrials.gov as NCT04867655.

Novel homozygous pathogenic mitochondrial DNAJC19 variant in a patient with dilated cardiomyopathy and global developmental delay.

BackgroundDilated cardiomyopathy with ataxia syndrome (DCMA) or 3‐methylglutaconic aciduria type V is a rare global autosomal recessive mitochondrial syndrome that is clinically and genetically heterogeneous. It is characterized by early‐onset dilated cardiomyopathy and increased urinary excretion of 3‐methylglutaconic acid. As a result, some patients die due to cardiac failure, while others manifest with growth retardation, microcytic anemia, mild ataxia, and mild muscle weakness. DCMA is caused by variants in the DnaJ heat shock protein family (Hsp40) member C19 gene (DNAJC19), which plays an important role in mitochondrial protein import machinery in the inner mitochondrial membrane.MethodsWe describe a single affected family member who presented with cardiomyopathy, global developmental delay, chest infection, seizures, elevated excretion of 3‐methylglutaconic acid, and 3‐methylglutaric acid in the urine.ResultsWhole‐exome sequencing followed by Sanger sequencing revealed a homozygous frameshift variant in the reading frame starting at codon 54 in exon 4 in the DNAJC19 gene (c.159del [Phe54Leufs*5]), which results in a stop codon four positions downstream. Quantitative gene expression analysis revealed that DNAJC19 mRNA expression in this patient was substantially reduced compared to the control.ConclusionsWe present a novel variant in the DNAJC19 gene that causes rare autosomal recessive mitochondrial 3‐methylglutaconic aciduria type V. By comparing the current case with previously reported ones, we conclude that the disease is extremely heterogeneous for reasons that are still unknown.

Primary Trophoblast Cultures: Characterization of HLA Profiles and Immune Cell Interactions

IntroductionTrophoblasts are essential in fetal-maternal interaction during pregnancy. The goal was to study HLA profiles of primary trophoblasts derived from placentas, and to investigate their usefulness in studying interaction with immune cells.MethodsAfter enzymatic digestion of first-trimester placental tissue from seven donors (6-9 weeks gestation) and trophoblast enrichment we cultured cytotrophoblasts (CTB) in stem cell medium. CTB were differentiated into EVT in a Matrigel-containing medium. A subset of CTB/EVT was profiled for microRNA levels. Expression of classical HLA molecules and of HLA-G was studied by flow cytometry, qPCR, and ELISA. Secondary trophoblast cell lines JAR and JEG-3 were studied as controls. Lymphocytes were investigated during co-culturing with EVT.ResultsThe trophoblasts could be easily maintained for several passages, upregulated classical trophoblast markers (GATA3, TFAP2C, chromosome-19 microRNAs), and upon differentiation to EVT they were selective in expressing HLA-C. EVT showed increasing expression of total HLA-G, an increasing proportion of HLA-G1 over G2- and G3 isoforms, and elevated excretion of soluble HLA-G. These features were distinct from those of the secondary trophoblast cell lines. TNF-α and IL-8 represented the most abundantly secreted cytokines by CTB, but their levels were minimal in EVT cultures. As proof of principle, we showed that EVT affect lymphocytes in three-day co-cultures (n=4) by decreasing activation marker HLA-DR.ConclusionWe verified the possibility culturing trophoblasts from first-term placentas, and their capability of differentiating to HLA-G expressing EVT. This culture model better represents the in-vivo situation than previously studied secondary trophoblast cell lines and enables mechanistic studies of fetal-maternal interactions.

EP018: Late-onset congenital erythropoietic porphyria associated with myeloid malignancy

Congenital erythropoietic porphyria (CEP, OMIM #263700) is a rare disorder of heme biosynthesis characterized biochemically by elevated excretion of type I porphyrin isomers in urine and feces. The disorder is caused by decreased uroporphyrinogen III synthase (UROS) activity, leading to an overproduction of the dead-end metabolites uro- and coproporphyrinogen I, primarily in the bone marrow, and the subsequent deposition of type I porphyrin isomers in tissues.

ANTIUROLITHIATIC ACTIVITY OF ETHANOLIC EXTRACT OF EICHHORNIA CRASSIPES (MART.) SOLMS WHOLE PLANT ON ETHYLENE GLYCOL-INDUCED UROLITHIASIS IN WISTAR RATS

Objective: In India Indigenous system of medicine, the plant Eichhornia crassipes (Mart.) Solms is claimed to be useful for different ailments. The purpose of this study is to investigate the significant activity of the ethanolic extract of E. crassipes (Mart.) Solms whole plant in the treatment of renal stone by ethylene glycol (EG)-induced urolithiasis in Wistar rats. Methods: Wistar rat will be divided into four groups. All the animals received EG 0.75%v/v by orally for 28 days. Group I was received 0.75%v/v EG for 28 days. Groups II, III, and IV were received cystone, ethanolic extract of E. crassipes (Mart.) Solms (200 mg/kg, and 400 mg/kg), respectively, administered orally from 15th to 28th day for curative regimen. Animals were housed in metabolic cages and collect the 24 h urine samples and serum samples on the 7th, 14th, 21th, and 28th day of the experiments. Under the microscopical study to observe the crystals in urine sample. The biochemical parameters monitored in the present study are calcium, oxalate, magnesium, phosphorus, urea, creatinine, uric acid, blood urea nitrogen (BUN), and lactate dehydrogenase (LDH) in urine and serum samples. Results: The EG feeding resulted in elevated renal excretion of calcium, phosphate, oxalate, and decreased renal excretion of magnesium as well-elevated serum BUN, creatinine, uric acid, urea, LDH levels, and decreased the urine pH and urine volume. Treatment of the ethanolic extract of E. crassipes (Mart.) Solms significantly reduced the elevated calcium, phosphate, oxalate in urine as well as BUN, creatinine, uric acid, urea, and LDH level in serum. It also restores the normal urine pH and promotes the urine output. Conclusion: Ethanolic extract of E. crassipes (Mart.) Solms exhibited significant anti-urolithiatic activity evident from urine calcium, phosphate, oxalate levels and serum BUN, creatinine, uric acid, urea and LDH levels.

Математический анализ индивидуальных профилей биомаркеров повреждения почек у детей с пузырно-мочеточниковым рефлюксом

Objective. To assess the severity of lesions to renal parenchyma in children with grade II–IV vesicoureteral reflux (VUR) using hierarchical clustering based on the analysis of individual profiles of urinary biomarkers. Patients and methods. In this study, we evaluated the excretion of urinary biomarkers of inflammation (IL-8, IL-18, MCP-1), angiogenesis (VEGF), and fibrogenesis (TGF-β1) and compared them with clinical and demographic parameters of 65 patients with grade II–IV VUR aged 1 to 17 years (mean age 4.9 ± 3.1 years) with a normal glomerular filtration rate (GFR). The control group comprised healthy children without kidney diseases (n = 25). We used hierarchical clustering (construction of dendrogram using the Ward's method) for all biomarkers. Results. All patients, regardless of their age, gender, and VUR severity demonstrated a pathological profile of urinary biomarkers excretion, including IL-8, IL-18, MCP-1, VEGF, TGF-β1 (p > 0.05). Using cluster analysis, we divided all patients into three subgroups (clusters) depending on the similarity of their individual biomarker profiles. We identified three main patterns of biomarkers that were associated with different severity of tubulointerstitial nephritis (TIN). The first pattern (I) with low levels of excretion of all biomarkers (p < 0.001) correlated with mild TIN and was observed in 61.5% of children. The second pattern (II) with average excretion of IL-8, IL-18, MCP-1, TGF-β1 (p < 0.01) and high VEGF excretion (p < 0.001) was associated with moderate TIN and was observed in 32.2% of patients. The third pattern (III) with elevated excretion of IL-8, IL-18, MCP-1 and VEGF (by 2.5–17 times compared to patterns I and II) (p < 0.01) correlated with severe TIN and was found in 6.1% of patients. Patients with the same VUR grades demonstrated different biomarker patterns. Conclusion. Hierarchical clustering enables comparison of individual profiles of urinary biomarkers with identified patterns, as well as their stratification into groups according to tubulointerstitial nephritis severity, which often remains undiagnosed with visualization techniques alone. Key words: children, cluster analysis, urinary biomarkers, vesicoureteral reflux, reflux nephropathy, tubulointerstitial nephritis

Differential Effect of Non-Purified and Semi-Purified Standard Diets on Kynurenine and Peripheral Metabolites in Male C57BL/6J Mice.

The kynurenine (Kyn) pathway plays crucial roles in several inflammation-induced disorders such as depression. In this study, we measured Kyn and other related molecules in the blood plasma, brain, and urine of male C57BL/6J mice (B6) fed non-purified (MF) and semi-purified (AIN-93G and AIN-93M) standard rodent diets. Mice fed MF had increased plasma Kyn levels compared with those on AIN93-based diets, as well as decreased hippocampal Kyn levels compared with those fed AIN-93G. Previous studies showed that branched chain amino acids (BCAAs) suppress peripheral blood Kyn transportation to the brain, but plasma BCAA levels were not significantly different between the diet groups in our study. Urine metabolome analysis revealed that feed ingredients affected the excretion of many metabolites, and MF-fed mice had elevated excretion of kynurenic and quinolinic acids, pivotal metabolites in the Kyn pathway. Collectively, the level of critical metabolites in the Kyn pathway in the central and peripheral tissues was strongly affected by feed ingredients. Therefore, feed selection is a critical factor to ensure the reproducibility of experimental data in studies involving rodent models.

Oral Treatment with Angiotensin-(1-7) Attenuates the Kidney Injury Induced by Gentamicin in Wistar Rats.

Acute Kidney Injury (AKI), a common disease of the urinary system, can be induced by high doses of gentamicin (GM). The renin-angiotensin system exerts a key role in the progression of the AKI since elevated intrarenal levels of Ang II, and ACE activity is found in this condition. However, it is unknown whether oral administration of angiotensin (Ang)-(1-7), a heptapeptide that evokes opposite effects of Ang II, may attenuate the renal injuries induced by gentamicin. To evaluate the effects of Ang-(1-7) on GM-induced renal dysfunction in rats. AKI was induced by subcutaneous administration of GM (80 mg/Kg) for 5 days. Simultaneously, Ang-(1-7) included in hydroxypropyl β-cyclodextrin (HPβCD) was administered by gavage [46 μg/kg HPβCD + 30 μg/kg Ang-(1-7)]. At the end of the treatment period (sixth day), the rats were housed in metabolic cages for renal function evaluation. Thereafter, blood and kidney samples were collected. Ang-(1-7) attenuated the increase of the plasmatic creatinine and proteinuria caused by GM but did not change the glomerular filtration rate nor tubular necrosis. Ang-(1-7) attenuated the increased urinary flow and the fractional excretion of H2O and potassium observed in GM rats but intensified the elevated excretion of sodium in these animals. Morphological analysis showed that Ang-(1-7) also reduced the tubular vacuolization in kidneys from GM rats. Ang-(1-7) promotes selective beneficial effects in renal injuries induced by GM.

Adrenal Steroid Metabolism and Blood Pressure in 5- to 7-Year-Old Children Born Preterm as Compared to Peers Born at Term.

Background: Previous studies indicated preterm birth to be a risk factor for hypertension in adolescence and adulthood. However, studies in children investigating the underlying mechanisms are scarce.Objective: We hypothesized children born preterm to have higher excretion of cortisol and/or androgen metabolites per day concomitantly with higher blood pressure as compared to peers born at term. We thus aimed to compare urinary steroid profiles and blood pressure between 5- to 7-year-old children born preterm and peers born at term. Furthermore, aldosterone precursor excretion per day was compared between both groups.Methods: Blood pressure was measured in 236 children (preterms n = 116; gestational age 29.8 ± 2.6 (30; 24–33) weeks [mean ± standard deviation (median; range)]) using an automatic oscillometric device. Urinary steroid profiles were determined in 24-h urine samples (preterms n = 109; terms n = 113) using gas chromatographic-mass spectrometric analysis. To assess excretion of cortisol and androgen metabolites per day, major cortisol and androgen metabolites were summed, respectively. To assess aldosterone excretion per day tetrahydrocorticosterone, 5α-tetrahydrocorticosterone, and tetrahydro-11-deydrocorticosterone were summed.Results: Multiple regression analyses showed prematurity to be associated with systolic but not with diastolic blood pressure. When adjusted for potential confounders (prematurity, gender, age at day of examination, being born small for gestational age, breastfeeding, accelerated weight gain during infancy, family history of cardiovascular disease, parental hypertension, and body mass index) prematurity was shown to be associated with an increase in systolic blood pressure by 2.87 mmHg (95% confidence interval 0.48–5.27; p = 0.02). Cortisol, androgen metabolite, and aldosterone precursor excretion per day were not higher in individuals born preterm. In contrast to our hypothesis, multiple regression analysis showed prematurity to independently decrease cortisol and aldosterone precursor excretion per day (p < 0.001 and 0.04, respectively).Conclusion: This study provides further evidence for systolic blood pressure to be higher after preterm birth as early as at the age of 5 to 7 years. However, this seems not to be explained by elevated excretion of cortisol and/or androgen metabolites.

GC-MS Analysis of Biological Nitrate and Nitrite Using Pentafluorobenzyl Bromide in Aqueous Acetone: A Dual Role of Carbonate/Bicarbonate as an Enhancer and Inhibitor of Derivatization.

Carbon dioxide (CO2) and carbonates, which are widely distributed in nature, are constituents of inorganic and organic matter and are essential in vegetable and animal organisms. CO2 is the principal greenhouse gas in the atmosphere. In human blood, CO2/HCO3− is an important buffering system. Inorganic nitrate (ONO2−) and nitrite (ONO−) are major metabolites and abundant reservoirs of nitric oxide (NO), an endogenous multifunctional signaling molecule. Carbonic anhydrase (CA) is involved in the reabsorption of nitrite and nitrate from the primary urine. The measurement of nitrate and nitrite in biological samples is of particular importance. The derivatization of nitrate and nitrite in biological samples alongside their 15N-labeled analogs, which serve as internal standards, is a prerequisite for their analysis by gas chromatography–mass spectrometry (GC-MS). A suitable derivatization reagent is pentafluorobenzyl bromide (PFB-Br). Nitrate and nitrite are converted in aqueous acetone to PFB-ONO2 and PFB-NO2, respectively. PFB-Br is also useful for the GC-MS analysis of carbonate/bicarbonate. This is of particular importance in conditions of pharmacological CA inhibition, for instance by acetazolamide, which is accompanied by elevated concomitant excretion of nitrate, nitrite and bicarbonate, as well as by urine alkalization. We performed a series of experiments with exogenous bicarbonate (NaHCO3) added to human urine samples (range, 0 to 100 mM), as well as with endogenous bicarbonate resulting from the inhibition of CA activity in healthy subjects before and after ingestion of pharmacological acetazolamide. Our results indicate that bicarbonate enhances the derivatization of nitrate with PFB-Br. In contrast, bicarbonate decreases the derivatization of nitrite with PFB-Br. Bicarbonate is not a catalyst, but it enhances PFB-ONO2 formation and inhibits PFB-NO2 formation in a concentration-dependent manner. The effects of bicarbonate are likely to result from its reaction with PFB-Br to generate PFB-OCOOH. Nitrate reacts with concomitantly produced PFB-OCOOH to form PFB-ONO2 in addition to the direct reaction of nitrate with PFB-Br. By contrast, nitrite does not react with PFB-OCOOH to form PFB-NO2. Sample acidification by small volumes of 20 wt.% aqueous acetic acid abolishes the effects of exogenous and endogenous bicarbonate on nitrite measurement.

P084 Recurrent fever in children: why not a mevalonate kinase deficiency?

Abstract Background Mevalonate kinase deficiency (MKD) is a rare autosomal recessive auto inflammatory disease. The clinical spectrum of this disease is a continuum ranging from the moderate form of Hyper-IgD syndrome (HIDS) to lethal forms of mevalonic aciduria (MA). An autoinflammatory disease should be considered in children with recurrent fever of unexplained origin. Case report M, 28 months old boy from a non-consanguineous marriage of Algerian parents, presented since the age of two months’ episodes of unexplained fever resistant to antibiotics and requiring several hospitalizations. Family history found that the mother and a maternal uncle experienced an unexplained recurrent fever. Personal history found episodes of fever lasting in average 6 days, with no obvious cause and recurring every 15–20 days; associated with bilateral cervical adenopathy, mouth ulcers, arthralgia, abdominal pain with vomiting and diarrhea. Clinical examination confirmed the fever with temperature of 38.7°, Chills and irritability, Multiple cervical adenopathy oral aphthae, tongue of geographical aspect, cervical pain and large joint arthralgia, tender abdomen with no organomegaly. Biologic workups found major inflammatory syndrome with hyper leucocytosis at 14X103/mm3 predominantly polynuclear, high CRP level, high ESR at 110 mm. Blood cultures and viral serologies were negative. Immunoelectrophoresis found elevated IgA, IgG and IgM with a normal IgD level. Chest X-ray and abdominal ultrasound were normal. The diagnosis of MKD in its moderate form was supported by elevated urinary mevalonic acid excretion to 5.1 mmol/mol during the febrile episode and subsequently confirmed by a 2nd contributory urine assay during another febrile episode. Conclusion MKD is a rare disease. The clinical spectrum of this condition is variable and could benefit of an effective treatment. Our patient developed a moderate form with a better long-term prognosis.

1046-P: Automated Early Flagging of Undiagnosed CKD Using Real-World Data

Aims: Early diagnosis and treatment of chronic kidney disease (CKD) may delay disease progression since 45% of patients are not diagnosed until stage 3. We examine the Kidney Disease Improving Global Outcomes (KDIGO) clinical guidelines that define rules based on the longitudinal progression of urine albumin creatinine ratio (UACR) and estimated glomerular filtration rate (eGFR) for flagging CKD in people with type 2 diabetes (T2-PwD). Methods: EHR data from over 400,000 T2-PwDs in the Indiana Network for Patient Care (INPC) database were used for this analysis. The KDIGO clinical practice guidelines defines CKD as persistently elevated urine albumin excretion (&amp;gt;=30 mg/g), or persistently reduced eGFR (&amp;lt; 60 ml/min per 1.73m2), or both, for more than 3 months. Our automated system scanned each patient’s longitudinal record and identified those patients with abnormal UACR and eGFR values in the cases where sufficient values were available. A flag associated with either UACR or eGFR or both and the date of flagging is indicated by the system if abnormal marker values persist for more than 3 months. CKD is indicated if one or both flags are triggered. Results: Out of 422,089 patients 35,573 (8%) were identified as diagnosed with CKD. Only 3% of these patients had at least 2 UACR readings and, therefore, the automated system considered primarily eGFR as the marker for flagging CKD. Out of 35,573 patients with CKD diagnosis, 16,342 (46%) had an eGFR warning flag that predated the CKD diagnosis date. The system flagged a potential identification as much as 599 days (median value) in advance of the diagnosis of CKD in the dataset. Conclusion: Our results show that earlier identification of CKD onset can be automated to flag patients wherein a positive diagnosis of CKD is likely. This flag can then prompt a confirmatory diagnosis of CKD, allowing better treatment interventions and management of CKD, reducing the burden of disease, and potentially decreasing costs associated with treatments in later CKD stages. Disclosure D. Malagarriga: None. S. Chittajallu: Employee; Self; Roche Diabetes Care. P. J. Galley: Employee; Self; Roche Diabetes Care. I. Singh: Employee; Self; Roche Diabetes Care. J. T. Odegard: Employee; Self; Roche Diabetes Care.

304-OR: Association of Eating Patterns and Diabetic Kidney Disease in Type 2 Diabetes

Evaluation of association of eating patterns with diabetes kidney disease (DKD) in patients with type 2 diabetes (T2DM) was performed. Cross-sectionally, T2DM patients underwent clinical, laboratory, and nutritional assessments. Dietary information was evaluated by a quantitative food frequency questionnaire validated for diabetes and converted into daily intakes. The eating patterns were identified by cluster analysis. DKD was defined as estimated glomerular filtration rate &amp;lt;60 mL/min/1.73m2 and persistently elevated urinary albumin excretion (≥14 mg/dL). A chi-square test, one-way analysis of variance (ANOVA/Bonferroni’s post hoc test) and Kruskal-Wallis test (Bonferroni post hoc correction) were used to test differences across the eating patterns. Poisson regression analysis was used to investigate the associations between eating patterns and DKD. IBM SPSS Statistics 25.0 (p&amp;lt;0.05). A total of 329 T2DM patients were evaluated: 10 years (5-19) diabetes duration; HbA1c=8.7±2.0%; 62±10 years old; BMI=30.9±4.2kg/m². Four eating patterns were identified: (1) high intake of dairy products, fruits, and vegetables; (2) ultra-processed foods; (3) high intake of refined carbohydrates, processed meats, and solids fats; and (4) high intake of red meat and sugary beverages. Age, sex, sedentary lifestyle, maximum doses of anti-glycemic agent in addition to fasting plasma glucose levels were different among the patterns. Poisson regression models confirmed the association of Pattern 2 (PR=1.48; 95% CI=1.10-1.99; P=0.010) and Pattern 4 (PR=1.93; 95% CI=1.29-2.89; P=0.001) with presence of DKD after adjusting for confounders. In conclusion, eating patterns with higher consumption of ultra-processed foods or red meat and sugary beverages were associated with the presence of DKD when compared to patterns rich in dairy products, fruits, and vegetables in this sample of T2DM outpatients. Disclosure C. C. R. Rodrigues: None. B. P. Riboldi: None. T. C. Rodrigues: None. R. A. Sarmento: None. J. P. Antonio: None. R. A. Borba: None. A. P. Vieira: None. I. S. Pasini: None. J. C. De almeida: None.

MO113CALCULATED URINARY CALCIUM-OXALATE SATURATION (ßCAOX) IS NOT SPECIFICALLY ELEVATED IN PATIENTS WITH PRIMARY HYPEROXALURIA

Abstract Background and Aims In the primary hyperoxalurias (PH; types 1-3) recurrent urolithiasis (UL) and/or progressive nephrocalcinosis (NC) are the clinical hallmarks. Three different enzyme defects lead to endogenous oxalate overproduction and to extremely elevated urinary oxalate excretion (UOx). Thus, it seems logical that urine is supersaturated for calcium-oxalate (CaOx). It was, hence, speculated that urinary CaOx saturation (ßCaOx), calculated by computed programs, is significantly higher as compared to that of patients with idiopathic CaOx stones. We now aimed to evaluate and calculate urinary ßCaOx in PH patients according to type, as well as in non-PH patients with UL or NC. Method The computed equilibrium program EQUIL2 was used for the calculation of ßCaOx. For this, 24 h urine specimen of 70 patients with non-PH NC (46 male, 24 female, median age 6.06 (range 0.3-31.4 years)), of 149 idiopathic CaOx UL (90/59 m/f, age 8.5 (0.1-68.6)), of 51 PH 1 patients (31/21, age 12.33 (0.8-63.8)), of 5 PH 2 patients (3/2, age 5.41 (4.3-12.9)) and of 14 PH 3 patients (8/6, age 8.5 (2.9-29.3)) were analyzed for all necessary components. All patients were in stable kidney function (eGFR &amp;gt; 45 ml/min). Results Uox was higher in the PH patients as compared to the non-PH UL or NC patients (p &amp;lt; 0.05). However, there was no statistical difference between the Uox in PH 1 vs PH 2 or PH 3 patients, although, a clear effect of B6 medication was visible in PH1 patients. Urinary calcium excretion was lower (not significant) in PH patients as compared to NC/UL. There was no difference in ßCaOx when PH were compared to non-PH patients and it mostly remained in the normal range. Conclusion Urine ßCaOx is similar in PH and non-PH stone formers. Therefore, calculation of ßCaOx using computed programs is not a reliable parameter to define the definitively extreme CaOx supersaturation of urine from PH patients. This miscalculation is related to a rather lowish urinary calcium excretion in PH as compared to other UL/NC patients. Therefore, we recommend not to use such programs to express the risk of recurrent stone disease or nephrocalcinosis in PH.

FC 111THE SOCIETAL IMPACT OF DELAYED DIALYSIS INITIATION ASSOCIATED WITH DAPAGLIFLOZIN BASED ON THE RESULTS OF DAPA-CKD

Abstract Background and Aims Chronic kidney disease (CKD) continues to be a growing health concern worldwide, with an estimated global prevalence of 9.1% in 2017. End-stage kidney disease (ESKD) is commonly treated with dialysis and is associated with a significant societal and economic impact. Approximately 34 – 45% of employed CKD patients will leave employment by the time of dialysis initiation. As such, there could be significant benefits associated with delaying onset of dialysis. Current treatment for CKD consists of ACE-inhibitors and Angiotensin Receptor Blockers, which delay kidney replacement therapy by approximately 6 months. DAPA-CKD is a randomised, double-blind, placebo-controlled trial in which patients with estimated glomerular filtration rate (eGFR) between 25-75mL/min/1.73m2 and elevated urinary albumin excretion were assigned to dapagliflozin or placebo in addition to standard of care. The trial ended prematurely due to overwhelming efficacy and showed a 36% reduction in the incidence of ESKD for patients treated with dapagliflozin. This analysis of the DAPA-CKD study data aimed to look at the societal benefit of delaying initiation of dialysis based on outcomes observed in DAPA-CKD. Method The incidence of ESKD was predicted for patients treated with dapagliflozin compared to placebo over a 10-year time horizon using data from DAPA-CKD. Parametric survival models were derived from trial results and extrapolated, with Weibull distributions fitted for the incidence of ESKD and mortality. The mean time to ESKD was estimated for patients treated with dapagliflozin in addition to standard of care or standard of care alone, and 62% of patients reaching ESKD were assumed to be treated with dialysis, based on results of the DAPA-CKD clinical trial. The benefit of delaying dialysis initiation from a societal perspective was calculated using the average annual salary in the US and the proportion of working time lost due to dialysis. Inputs were derived based on published US data, and costs were discounted at 3% per annum. Results Treatment with dapagliflozin was estimated to delay the mean time to ESKD by approximately 2 years when compared to placebo. Patients in the placebo arm progressed to ESKD at a mean time of 7.8 years, compared to a mean time of 9.9 years in the dapagliflozin arm, without adjustment for patient survival. Over a 10-year time horizon, a cohort of 5,000 patients treated with dapagliflozin in addition to standard of care were estimated to experience 2,508 ESKD events, a reduction of 519 events in comparison with standard of care alone. Additionally, treatment with dapagliflozin in addition to standard of care was estimated to prevent 198 renal deaths compared to treatment with standard of care alone. Delays in dialysis initiation associated with dapagliflozin treatment was estimated to translate to 10-year reduction in societal costs of $29.2 million per 5,000 treated patients in a cohort of CKD patients eligible for treatment with dapagliflozin. Conclusion Dapagliflozin is associated with a significant reduction in the mean time to ESKD, therefore delaying the onset of dialysis initiation. In addition to improving patient outcomes, this delay in progression to ESKD could have notable economic benefits from a societal perspective.

Urinary Excretion of Nadolol as a Possible In Vivo Probe for Drug Interactions Involving P-Glycoprotein.

Nadolol is a hydrophilic and nonselective β-adrenoceptor blocker with a bioavailability of 30%, relatively longer half-life, negligible metabolism, and predominant renal excretion. Previous studies have reported that nadolol is a substrate of P-glycoprotein, and the coadministration with itraconazole, a typical P-glycoprotein inhibitor, results in elevated plasma concentrations and cumulative urinary excretion of nadolol. In this study, we assessed whether measurements of urinary-excreted nadolol can be an alternative method of plasma pharmacokinetics for P-glycoprotein-mediated drug interactions in humans. We reanalyzed the pooled data set of plasma concentration and urinary excretion of nadolol from our previous clinical studies in a total of 32 healthy Japanese adults. The area under the plasma concentration-time curve from 0 to infinity (AUC0-∞ ) of nadolol in individual subjects was significantly correlated with the maximum plasma concentration (r = 0.80, P < .01) and the cumulative amount excreted into urine (Ae ) at 4 (r = 0.51, P = .01), 8 (r = 0.63, P < .01), 24 (r = 0.75, P < .01), and 48 (r = 0.77, P < .01) hours. Significant correlations were also observed between the AUC and Ae during the same respective periods. In the drug interactions of nadolol with itraconazole, rifampicin, a well-known P-glycoprotein inducer, or grapefruit juice, there were significant correlations between the differences in AUC0-48 and those in Ae, 0-48 from the controls in individual subjects. These results suggest that the measurements of urinary excretion of nadolol can be employed as a sensitive and reliable alternative to plasma pharmacokinetics for the evaluation of P-glycoprotein-mediated drug interactions.

Lysinuric protein intolerance: an overlooked diagnosis

BackgroundLysinuric protein intolerance (LPI) is an autosomal recessively inherited inborn error of metabolism (IEM) caused by the defect in the dibasic cationic amino acid transporter found on the basolateral membrane of the lung, small intestine, and kidney due to mutations in the SLC7A7 gene, which encodes the y+LAT1 protein. LPI may present as an acute hyperammonemic episode or as chronic symptoms. Major clinical symptoms are feeding problems, vomiting and diarrhea, failure to thrive, hepatosplenomegaly, and cytopenia. We present a delayed diagnosis of symptomatic LPI with a homozygous mutation in the SLC7A7 gene.Case presentationA 15-year-old girl was referred to our clinic due to growth retardation and diarrhea. Physical examination showed short stature, retarded puberty, and hepatosplenomegaly. Laboratory tests showed normal complete blood count and biochemical analyses except elevated aspartate aminotransferase, triglyceride, total cholesterol, and ferritin. Peripheral blood smear and hemoglobin electrophoresis were within normal limits. Bone marrow analysis showed hemophagocytic cells. Postprandial ammonium level was found elevated. Low lysine, arginine, and ornithine and elevated glycine and alanine in plasma amino acid analysis and high amount of lysine and slightly elevated arginine and ornithine excretion in urine were detected. Molecular genetic analysis of the SLC7A7 gene showed a previously reported homozygous mutation. Low protein diet, sodium benzoate, l-carnitine, low-dose l-citrulline, and calcium replacement were initiated. The patient is now in good condition still being followed up in our department.ConclusionsLPI is a metabolic disorder with multi-systemic involvement that may have severe consequences if left untreated. Initiation of early treatment is essential for the prevention of severe chronic complications. Also, confirmation of the genetic defect may provide the parents to have healthy offsprings in the future with the help of genetic counselling and preimplantation genetics.