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. 2025 Sep 25;15(1):32933.
doi: 10.1038/s41598-025-20600-x.

Effects of anserine on oxidative stress and on cell barrier integrity in methylmalonic aciduria

Felix Köpfer  1 , Maria Bartosova Medvid  1 , Thomas Fleming  2 , Tilman Pfeffer  1 , Jürgen Günther Okun  1 , Stefan Kölker  1 , Georg Friedrich Hoffmann  1 , Claus Peter Schmitt  1 , Marina Morath  3 , Verena Peters  1
Affiliations

Effects of anserine on oxidative stress and on cell barrier integrity in methylmalonic aciduria

Felix Köpfer et al. Sci Rep. .

Abstract (VSports手机版)

Kidney damage in individuals with methylmalonyl-CoA mutase deficiency (mut0) results from metabolic and oxidative stress, and disrupted mitochondrial homeostasis VSports手机版. Anserine, known for its antioxidant properties and protective effect on cell barrier integrity of endothelial and epithelial kidney and vascular cells, may offer therapeutic potential for chronic disorders. This study explored anserine's effects on immortalized kidney tubular epithelial cells (iKTEC) from mut0 patients. Compared to healthy controls, iKTEC from mut0 patients showed reduced cell viability, antioxidant capacity, oxygen consumption, and ATP production. Expression of the tight junction scaffolding protein zonula occludens-1 (ZO-1) was increased in mut0 cells compared to control while transepithelial resistance (TER), and dextran transport (10 kDa) remained unchanged. Anserine treatment restored antioxidant capacity and normalized ZO-1 expression but had no effect on TER or dextran transport. Additionally, cell viability, mitochondrial respiration, and ATP production were unaffected by anserine. Metabolic stress induced by high protein load or disease-associated branched-chain amino acids did not worsen mitochondrial dysfunction or epithelial integrity, and anserine exposure showed no further effects. In conclusion, anserine showed promise in restoring antioxidant capacity in iKTEC from mut0 patients, highlighting its potential as a therapeutic agent to mitigate ROS, although its effects on mitochondrial function and epithelial integrity warrant further investigation. .

Keywords: Anserine; Human immortalized kidney tubule cell; Metabolic cell stress; Methylmalonic aciduria; Oxidative stress. V体育安卓版.

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Conflict of interest statement

Declarations V体育ios版. Competing interests: All authors declare that they have no conflict of interest. Ethical approval: The clinical study was approved by the Institutional Ethics Committee of the University of Heidelberg (S-436/2016). Informed consent: All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2013. Informed consent was obtained from all patients for being included in the study.

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Fig. 1
Fig. 1
Effect of anserine on antioxidative capacity and cell viability in mut0 and control cells. (A) The presence of the genetic defect in immortalized kidney tubule epithelial cells (iKTEC) from three patients with clinically and molecular biologically confirmed loss of activity of methylmalonyl-CoA mutase (MMUT) was confirmed at protein level (by Western blot) before performing the experiments. C1-3 = Control patients; M1-3 = patients with mut0 defect. The figure is displayed as cropped version of the performed western blot, full-length gel is included in the supplementary information (Suppl. Fig. 1). (B) Antioxidative capacity, measured by ORAC, was reduced in iKTEC from 3 patients with methylmalonyl-CoA mutase deficiency (mut0) compared to immortalized cells from healthy individuals (Ctrl). Incubation with anserine (A, 2 mM) for 7 days increased the antioxidative capacity in cells from mut0 patients. (C) Cell viability (MTT assay) of iKTEC from patients was reduced in iKTEC from 3 mut0 patients. Incubation with anserine (2 mM) for 96 h had no influence on cell viability in cells from patients and controls. Statistical analysis was performed using a simple t-test for A and ordinary two-way ANOVA with Šídák’s multiple comparisons test for B and C.* = p < 0.05; ** = p < 0.01; **** = p < 0.0001. There is no significance between the groups, except for the ones indicated in the graphs. Antioxidant capacity and viability are shown as percentages relative to control cells in normal medium (dashed line = 100%). Symbols/lines in the violin plots represent the mean value for one patient analyzed in three independent experiments, each performed with 5–9 technical replicates.
Fig. 2
Fig. 2
Effect of anserine on mitochondrial energy production measured by real-time respirometry in mut0 and control cells. (A) Mitochondrial oxygen consumption rate (OCR) was lower in immortalized proximal tubule epithelial cells (iKTEC) from mut0 patients compared to control cells. Basal respiration, ATP production (area under the curve after oligomycin injection) and maximal respiratory rate (area under the curve after FCCP injection) was lower in mut0 cells compared to control cells. Incubation with anserine (A, 2 mM for 24 h) had no effect on ATP production in mut0 cells compared to control cells (p = 0.143 and p = 0.140). (B) Metabolic stress by High Protein load (HP) or branched-chain amino acids (IV, 1 mM isoleucine, 3 mM valine) did not further reduce mitochondrial oxygen consumption rate (OCR) compared to non-stressed iKTEC cells from mut0 patients. Incubation with anserine (A, 2 mM for 24 h) had no effect on the OCR in stressed cells. Statistical analysis was performed by using two-way ANOVA with Šídák’s multiple comparison test. * = p < 0.05; ** = p < 0.01 compared to control cells. Symbols/lines in the violin plots represent the mean value for one patient analyzed in three independent experiments, each performed with 5–9 technical replicates. There is no significance between the groups, except for the ones indicated in the graphs.
Fig. 3
Fig. 3
Effect of anserine on transepithelial resistance and ZO-1 localization and abundance. (A) Transepithelial resistance (TER), a measure of barrier integrity, was increased in mut0 cells compared to controls (Ctrl). Co-incubation with anserine (A, 2 mM) for 7 days had no effect on TER in cells from mut0 patients (upper panel). Metabolic stress by High Protein load (HP) or branched-chain amino acids (IV, 1 mM isoleucine, 3 mM valine) did further increase TER (lower panel). (B) Expression of the tight junction scaffolding protein zonula occludens-1 (ZO-1) was increased in mut0 cells compared to controls (Ctrl), expression could be normalized by addition of anserine for (A, 2 mM) in mut0 cells after an incubation period of 7 days. Metabolic stress by High Protein load (HP) or branched-chain amino acids (IV, 1 mM isoleucine, 3 mM valine) did not further increase ZO-1 expression. Analysis was performed after 5 days of growth. Grid line shows TER and ZO-1 levels of Ctrl in every graph. If not further explained, cells were treated with normal medium. Statistical analysis was performed by two-way ANOVA with Šídák’s multiple comparisons test. Symbols/lines in the violin plots represent the mean value for one patient analyzed in three independent experiments, each performed with 5–9 technical replicates. * = p < 0.05; ** = p < 0.01. There is no significance between the groups, except for the ones indicated in the graphs.
Fig. 4
Fig. 4
Effect of anserine on 10 kDa transport. Transport capacity for 10 kDa dextran was within the same range for mut0 and control cells (upper panel). Metabolic stress by High Protein load (HP) or branched-chain amino acids (IV, 1 mM isoleucine, 3 mM valine) had no additional effect on transport. Treatment with anserine for 7 days did not alter transport capacity. Dots in the violin plots represent the mean value for one patient analyzed in three independent experiments, each performed with 5–9 technical replicates. Statistical analysis was performed by two-way ANOVA with Šídák’s multiple comparisons test. There is no significance between the groups.
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References (VSports最新版本)

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