Calprotectin

Fecal pH and organic acids were assessed at V1, V2, and V3 using pH-indicator paper (Merck, Darmstadt, Germany) and validated LCMS, respectively (23 (link)). ELISA kits were used to analyze fecal biomarkers at V1, V2, and V3 including secretory immunoglobulin A (sIgA), calprotectin (Immundiagnostik AG, Bensheim, Germany) and alpha-1-antitrypsin (AAT) (BioVendor – Laboratorni medicina a.s., Brno, Czech Republic). Fecal cytokines were quantified as previously published (24 (link)), diluted 1:2 in Meso Scale Discovery diluent (MSD; Rockville, MD, United States), using V-Plex Plus kits and U-plex according to manufacturer’s instructions, and a QuickPlex SQ 120 Imager (MSD; Rockville, MD, United States). Total protein content in fecal extracts was quantified using Pierce BCA protein assay kit (Thermo Scientific) and used for cytokine level normalization.

Commercially available ELISA kits were used to analyze fecal biomarkers at baseline, age 3, and 6 months, including secretory immunoglobulin A (sIgA), calprotectin (both Immundiagnostik AG, Bensheim, Germany), and alpha-1-antitrypsin (AAT; BioVendor – Laboratorni medicina a.s., Brno, Czech Republic).
Fecal pH and organic acids (including lactate, acetate, butyrate, isobutyrate, propionate, valerate, and isovalerate) were assessed at baseline, 3, and 6 months of age using pH indicator paper (pH range 1–10; Merck, Darmstadt, Germany) and validated liquid chromatography-tandem mass spectrometry according to a modified previously published method (34 (link)), respectively.

Fecal pH was assessed using an electrode-fitted pH meter after suspending 0.5 g (fresh weight) of fecal sample in 2 mL milliQ water. Organic acids (lactate, acetate, propionate, butyrate, isobutyrate, valerate, isovalerate) were determined by high performance anion‐exchange chromatography with UV and refractive index detection according to a modified and previously described method [40 (link)].
Commercially available ELISA kits were used to analyze fecal markers of intestinal immunity and health including secretory immunoglobulin A (sIgA), myeloperoxidase, calprotectin, human beta defensin (all Immundiagnostik AG, Bensheim, Germany) and neopterin (IBL, Hamburg, Germany).

Each participant (both patients and healthy controls) provided 3 samples of stool, which underwent spectrophotometric assessment of levels of calprotectin (Immundiagnostik AG, Bensheim, Germany) [7 (link)] and pyruvate kinase isoenzyme M2-PK (Schebo-Biotech, Giessen, Germany), after double reaction with monoclonal antibodies binding with specific epitopes of the enzymes (enzyme-linked immunosorbent assay, ELISA) [8 (link)]. Mean values from 3 measurements were calculated and used in further analyses.

Biochemical parameters (serum creatinine and C-reactive protein (CRP) levels, alanine aminotransferase (ALT), and gammaglutamyltranspeptidase (GGT) activity) were measured using standard laboratory methods. Kidney graft function was assessed using an estimated glomerular filtration rate (eGFR) calculated according to the Modification of Diet in Renal Disease formula.
Plasma concentrations of zonulin and calprotectin (Immundiagnostik AG, Bensheim, Germany) were assessed using commercially available ELISA kits, with the intra-assay and inter-assay coefficients of variability being <5 and <8.5% and <3.3 and <9.0%, respectively. Plasma levels of IL-6, CD-14 (R&D Systems, Minnesota, MN, USA), and bacterial lipopolysaccharides (Uscn Life Sciences Inc., Wuhan, China) were assessed by ELISA, with the intra-assay and inter-assay coefficients of variability being <7.2 and <7.8%, <6.4 and <7.4%, and <10 and <12%, respectively. Plasma FABP-2 (R&D systems, Inc., Minneapolis, MN, USA) and LBP levels (CloudClone Corp, Katy, TX, USA) were measured by ELISA kit with the intra-assay and inter-assay coefficients of variability being <4.1 and 11.1% and <10 and <12%, respectively.