Chronic kidney disease may evoke anxiety by altering CRH expression in the amygdala and tryptophan metabolism in rats.

Chronic kidney disease (CKD) is associated with anxiety; however, its exact mechanism is not well understood. Therefore, the aim of the present study was to assess the effect of moderate CKD on anxiety in rats. 5/6 nephrectomy was performed in male Wistar rats. 7 weeks after, anxiety-like behavior was assessed by elevated plus maze (EPM), open field (OF), and marble burying (MB) tests. At weeks 8 and 9, urinalysis was performed, and blood and amygdala samples were collected, respectively. In the amygdala, the gene expression of Avp and the gene and protein expression of Crh, Crhr1, and Crhr2 were analyzed. Furthermore, the plasma concentration of corticosterone, uremic toxins, and tryptophan metabolites was measured by UHPLC-MS/MS. Laboratory tests confirmed the development of CKD. In the CKD group, the closed arm time increased; the central time and the total number of entries decreased in the EPM. There was a reduction in rearing, central distance and time in the OF, and fewer interactions with marbles were detected during MB. CKD evoked an upregulation of gene expression of Crh, Crhr1, and Crhr2, but not Avp, in the amygdala. However, there was no alteration in protein expression. In the CKD group, plasma concentrations of p-cresyl-sulfate, indoxyl-sulfate, kynurenine, kynurenic acid, 3-hydroxykynurenine, anthranilic acid, xanthurenic acid, 5-hydroxyindoleacetic acid, picolinic acid, and quinolinic acid increased. However, the levels of tryptophan, tryptamine, 5-hydroxytryptophan, serotonin, and tyrosine decreased. In conclusion, moderate CKD evoked anxiety-like behavior that might be mediated by the accumulation of uremic toxins and metabolites of the kynurenine pathway, but the contribution of the amygdalar CRH system to the development of anxiety seems to be negligible at this stage.

Newborn Screening for Vitamin B12 Deficiency in Germany-Strategies, Results, and Public Health Implications.

OBJECTIVE: To evaluate a systematic newborn screening (NBS) strategy for vitamin B12 deficiency. STUDY DESIGN: In a prospective single-center NBS study, a systematic screening strategy for vitamin B12 deficiency was developed and evaluated. Tandem-mass spectrometry screening was complemented by 2 second-tier strategies, measuring methylmalonic/3-OH-propionic/methylcitric acid, and homocysteine from dried blood spots. RESULTS: In a cohort of 176 702 children screened over 27 months, 33 children were detected by NBS in whom (maternal) vitamin B12 deficiency was confirmed. Homocysteine was the most sensitive marker for vitamin B12 deficiency, but only combination with a second-tier strategy evaluating methylmalonic acid allowed for detection of all 33 children. Mothers were of various ethnic origins, and 89% adhered to a balanced diet. Treatment in children was performed predominantly by oral vitamin B12 supplementation (84%), and all children remained without clinical symptoms at short-term follow-up. CONCLUSIONS: Vitamin B12 deficiency is a treatable condition but can cause severe neurologic sequelae in infants if untreated. The proposed screening strategy is feasible and effective to identify moderate and severe cases of vitamin B12 deficiency. With an incidence of 1:5355 newborns, vitamin B12 deficiency is more frequent than inborn errors of metabolism included in NBS panels. Treatment of vitamin B12 deficiency is easy, and additional benefits can be achieved for previously undiagnosed affected mothers. This supports inclusion of vitamin B12 deficiency into NBS but also stresses the need for increased awareness of vitamin B12 deficiency in caregivers of pregnant women.

Targeted cerebrospinal fluid analysis for inborn errors of metabolism on an LC-MS/MS analysis platform.

BACKGROUND: Laboratory investigations of cerebrospinal fluid (CSF) are essential when suspecting an inborn error of metabolism (IEM) involving neurological features. Available tests are currently performed on different analytical platforms, requiring a large sample volume and long turnaround time, which often delays timely diagnosis. Therefore, it would be preferable to have an "one-instrument" targeted multi-metabolite approach. METHOD: A liquid chromatography-tandem mass spectrometry (LC-MS/MS) platform, based on two different methods for analysing 38 metabolites using positive and negative electrospray ionisation modes, was established. To allow for platform extension, both methods were designed to use the same CSF sample preparation procedure and to be run on the same separation column (ACE C18-PFP). RESULTS: Assessment of the LC-MS/MS platform methods was first made by analytical validation, followed by the establishment of literature-based CSF cut-off values and reference ranges, and by the measurement of available samples obtained from patients with confirmed diagnoses of aromatic l-amino acid decarboxylase deficiency, guanidinoacetate methyltransferase deficiency, ornithine aminotransferase deficiency, cerebral folate deficiency and methylenetetrahydrofolate reductase deficiency. CONCLUSION: An extendable targeted LC-MS/MS platform was developed for the analysis of multiple metabolites in CSF, thereby distinguishing samples from patients with IEM from non-IEM samples. Reference concentrations for several biomarkers in CSF are provided for the first time. By measurement on a single analytical platform, less sample volume is required (200 µL), diagnostic results are obtained faster, and preanalytical issues are reduced. SYNOPSIS: LC-MS/MS platform for CSF analysis consisting of two differentially designed methods.

Role of the kisspeptin-KISS1R axis in the pathogenesis of chronic kidney disease and uremic cardiomyopathy.

The prevalence of chronic kidney disease (CKD) is increasing globally, especially in elderly patients. Uremic cardiomyopathy is a common cardiovascular complication of CKD, characterized by left ventricular hypertrophy (LVH), diastolic dysfunction, and fibrosis. Kisspeptins and their receptor, KISS1R, exert a pivotal influence on kidney pathophysiology and modulate age-related pathologies across various organ systems. KISS1R agonists, including kisspeptin-13 (KP-13), hold promise as novel therapeutic agents within age-related biological processes and kidney-related disorders. Our investigation aimed to elucidate the impact of KP-13 on the trajectory of CKD and uremic cardiomyopathy. Male Wistar rats (300-350 g) were randomized into four groups: (I) sham-operated, (II) 5/6 nephrectomy-induced CKD, (III) CKD subjected to a low dose of KP-13 (intraperitoneal 13 µg/day), and (IV) CKD treated with a higher KP-13 dose (intraperitoneal 26 µg/day). Treatments were administered daily from week 3 for 10 days. After 13 weeks, KP-13 increased systemic blood pressure, accentuating diastolic dysfunction's echocardiographic indicators and intensifying CKD-associated markers such as serum urea levels, glomerular hypertrophy, and tubular dilation. Notably, KP-13 did not exacerbate circulatory uremic toxin levels, renal inflammation, or fibrosis markers. In contrast, the higher KP-13 dose correlated with reduced posterior and anterior wall thickness, coupled with diminished cardiomyocyte cross-sectional areas and concurrent elevation of inflammatory (Il6, Tnf), fibrosis (Col1), and apoptosis markers (Bax/Bcl2) relative to the CKD group. In summary, KP-13's influence on CKD and uremic cardiomyopathy encompassed heightened blood pressure and potentially activated inflammatory and apoptotic pathways in the left ventricle.

Different administration schedules of darbepoetin alfa affect oxidized and reduced glutathione levels to a similar extent in 5/6 nephrectomized rats.

BACKGROUND: The development of erythropoiesis-stimulating agents (ESAs) with extended serum half-lives has allowed marked prolongation of the administration intervals. The level of oxidative stress is increased in chronic kidney disease, and is reportedly decreased after long-term ESA treatment. However, the effect of different dosing regimens of ESAs on oxidative stress has not been elucidated. METHODS: Five-sixths nephrectomized (NX) rats received either 0.4 µg/kg darbepoetin alfa (DA) weekly or 0.8 µg/kg DA fortnightly between weeks 4 and 10. NX animals receiving saline and a sham-operated (SHAM) group served as controls. The levels of oxidized and reduced glutathione (GSSG, GSH) were followed from blood samples drawn fortnightly. RESULTS: During the follow-up, the ratios GSSG/GSH showed similar trends in both DA groups, levels being significantly lower than those in the SHAM group at weeks 8 and 10. GSSG levels were lower than the baseline throughout the study in all groups except for NX controls. The GSH levels were increased in all three NX groups (weeks 6-10) compared with both the baseline and the SHAM group CONCLUSION: Our results suggest that the extent of oxidative stress is similar in response to different dosing regimens of DA in 5/6 NX rats when comparable hemoglobin levels are maintained. These findings remain to be confirmed in chronic kidney disease patients.

Neonatal Screening for Cystic Fibrosis in Hungary-First-Year Experiences.

The aim of this study is to evaluate the strategy of the cystic fibrosis newborn screening (CFNBS) programme in Hungary based on the results of the first year of screening. A combined immunoreactive trypsinogen (IRT) and pancreatitis-associated protein (PAP) CFNBS protocol (IRT/IRT×PAP/IRT) was applied with an IRT-dependent safety net (SN). Out of 88,400 newborns, 256 were tested screen-positive. Fourteen cystic fibrosis (CF) and two cystic fibrosis-positive inconclusive diagnosis (CFSPID) cases were confirmed from the screen-positive cases, and two false-negative cases were diagnosed later. Based on the obtained results, a sensitivity of 88% and a positive predictive value (PPV) of 5.9% were calculated. Following the recognition of false-negative cases, the calculation method of the age-dependent cut-off was changed. In purely biochemical CFNBS protocols, a small protocol change, even after a short period, can have a significant positive impact on the performance. CFNBS should be monitored continuously in order to fine-tune the screening strategy and define the best local practices.

Identification of potential interferents of methylmalonic acid: A previously unrecognized pitfall in clinical diagnostics and newborn screening.

OBJECTIVES: Determination of methylmalonic acid (MMA) from dried blood spots (DBS) is commonly performed in clinical diagnostics and newborn screening for propionic acidemia (PA) and methylmalonic acidemia. Isobaric compounds of MMA having the same mass can affect diagnostic reliability and quantitative results, which represents a previously unrecognized pitfall in clinical assays for MMA. We set out to identify interfering substances of MMA in DBS, serum and urine samples from confirmed patients with PA and methylmalonic acidemia. METHODS: Techniques included quadrupole time-of-flight high-resolution mass spectrometry (QTOF HR-MS), nuclear magnetic resonance (NMR) spectroscopy, liquid chromatography (LC) and tandem mass spectrometry (MS/MS). RESULTS: The five isobaric metabolites detected in DBS, serum and urine from PA and methylmalonic acidemia patients were confirmed as 2-methyl-3-hydroxybutyrate, 3-hydroxyisovalerate, 2-hydroxyisovalerate, 3-hydroxyvalerate and succinate using a series of experiments. An additional unknown substance with low abundance remained unidentified. CONCLUSIONS: The presented results facilitate the diagnostic and quantitative reliability of the MMA determination in clinical assays. Isobaric species should be investigated in assays for MMA to eliminate possible interference in a wide range of conditions including PA, methylmalonic acidemia, a vitamin B12 deficiency, ketosis and lactic acidosis.

Neuregulin-1ß Improves Uremic Cardiomyopathy and Renal Dysfunction in Rats.

Chronic kidney disease is a global health problem affecting 10% to 12% of the population. Uremic cardiomyopathy is often characterized by left ventricular hypertrophy, fibrosis, and diastolic dysfunction. Dysregulation of neuregulin-1ß signaling in the heart is a known contributor to heart failure. The systemically administered recombinant human neuregulin-1ß for 10 days in our 5/6 nephrectomy-induced model of chronic kidney disease alleviated the progression of uremic cardiomyopathy and kidney dysfunction in type 4 cardiorenal syndrome. The currently presented positive preclinical data warrant clinical studies to confirm the beneficial effects of recombinant human neuregulin-1ß in patients with chronic kidney disease.

Vitamin B12 (Cobalamin): Its Fate from Ingestion to Metabolism with Particular Emphasis on Diagnostic Approaches of Acquired Neonatal/Infantile Deficiency Detected by Newborn Screening.

Acquired vitamin B12 (vB12) deficiency (vB12D) of newborns is relatively frequent as compared with the incidence of inherited diseases included in newborn screening (NBS) of different countries across the globe. Infants may present signs of vB12D before 6 months of age with anemia and/or neurologic symptoms when not diagnosed in asymptomatic state. The possibility of identifying vitamin deficient mothers after their pregnancy during the breastfeeding period could be an additional benefit of the newborn screening. Vitamin supplementation is widely available and easy to administer. However, in many laboratories, vB12D is not included in the national screening program. Optimized screening requires either second-tier testing or analysis of new urine and blood samples combined with multiple clinical and laboratory follow ups. Our scope was to review the physiologic fate of vB12 and the pathobiochemical consequences of vB12D in the human body. Particular emphasis was put on the latest approaches for diagnosis and treatment of vB12D in NBS.

Comparison of different preparation techniques of dried blood spot quality controls in newborn screening for congenital adrenal hyperplasia.

In newborn screening, samples suspected for congenital adrenal hyperplasia (CAH), a potentially lethal inborn error of steroid biosynthesis, need to be confirmed using liquid chromatography-tandem mass spectrometry. Daily quality controls (QCs) for the 2nd-tier CAH assay are not commercially available and are therefore generally prepared within the laboratory. For the first time, we aimed to compare five different QC preparation approaches used in routine diagnostics for CAH on the concentrations of cortisol, 21-deoxycortisol, 11-deoxycortisol, 4-androstenedione and 17-hydroxyprogesterone in dried blood spots. The techniques from Prep1 to Prep5 were tested at two analyte concentrations by spiking aliquots of a steroid-depleted blood, derived from washed erythrocyte suspension and steroid-depleted serum. The preparation processes differed in the sequence of the preparation steps and whether freeze-thaw cycles were used to facilitate blood homogeneity. The five types of dried blood spot QCs were assayed and quantitated in duplicate on five different days using a single calibration row per day. Inter-assay variations less than 15% and concentrations within ±15% of the nominal values were considered acceptable. Results obtained by means of the four dried blood spot QC preparation techniques (Prep1, Prep2, Prep4 and Prep5) were statistically similar and remained within the ±15% ranges in terms of both reproducibility and nominal values. However, concentration results for Prep3 (spiking prior to three freeze-thaw cycles) were significantly lower than the nominal values in this setting, with differences exceeding the ±15% range in many cases despite acceptable inter-assay variations. These findings have implications for the in-house preparation of QC samples in laboratory developed tests for CAH, including 2nd-tier assays in newborn screening.