Patients

Serum albumin data from two cohorts of patients with advanced chronic liver disease (ACLD) were analyzed, one cohort with 663 ACLD patients (Hartl et al. 2021a) and a second cohort with 168 ACLD patients (Costa et al. 2021). Albumin concentrations of patients with ACLD were normalized to data of 1812 individuals (never smokers) randomly drawn from one general practice in each of 24 towns in England, Wales, and Scotland (Phillips et al. 1989).

Mice

Six-to-eight-week-old albumin knockout C57BL/6 J-Albem8Mvw/MvwJ (#025,200; The Jackson Laboratory) and corresponding C57BL/6 J wild-type (Janvier Labs, France) mice were used. Based on genotyping results, both homozygous (albumin−/−) and heterozygous (albumin±) albumin-deficient mice were included in the study. The mice were fed ad libitum on a regular chow diet (Ssniff) with free access to drinking water and were housed under standard light and temperature conditions. All experiments were approved by the local animal welfare committee (#81–02.04.2020.A304; LANUV, North Rhine-Westphalia, Germany).

Ochratoxin A toxicity experiment

To study the role of albumin in OTA toxicity, 8-week-old female homozygote and heterozygote albumin-deficient mice, and corresponding C57BL/6 J wild-type controls were challenged intravenously with 10 mg/kg OTA (#10470691, Fisher Scientific) dissolved in 0.1 M NaHCO3, pH 7.4, with an application volume of 2 ml/kg. On day 1 after OTA injection, heart blood samples were collected, and plasma was separated according to a standard protocol (Gianmoena et al. 2021). In addition, tissue specimens were collected from the left liver lobe and kidneys, fixed for 2 days in 4% paraformaldehyde (#P087-5, Roth), and finally embedded in paraffin for histopathology and immunohistochemistry analyses (Schneider et al. 2021b).

Biochemical analysis of mouse plasma

Plasma concentration of albumin, creatinine, and blood urea nitrogen (BUN), as well as activities of alanine transaminase (ALT), and aspartate transaminase (AST) were analyzed by the Piccolo Xpress Chemistry Analyzer (Hitado, Germany) using the Piccolo general chemistry 13 panel (#AB-114-400-0029, Hitado, Germany).

Histopathology

Hematoxylin and eosin (H&E) staining was performed in 4 µm-thick paraffin-embedded liver and kidney tissue sections as previously described (Campos et al. 2020).

Immunostaining

Visualization of albumin protein expression in the liver tissue, and immune cell infiltration in the liver and kidney tissues was done by immunohistochemistry using an autostainer (Discovery Ultra Automated Slide Preparation System, Roche, Germany) (Ghallab et al. 2021b). For this purpose, 4 µm-thick paraffin-embedded liver or kidney tissue sections were incubated with primary antibodies against albumin (# ab192603, abcam), CD45 (a pan leukocyte marker; #550,539, BD Bioscience), and FSP1 (a marker of inflammatory subpopulation of macrophages (Österreicher et al. 2011) (#ab197896, Abcam). Dilutions of the primary antibodies as well as the used secondary antibodies are described in Table 2. Following staining, entire slides were scanned (Axio Scan.Z1, Zeiss, Germany) and representative images are shown in the results section.

Table 2 Setup for immunohistochemistry and intravital imagingIntravital imaging of ochratoxin A transport in the liver and kidneys

Intravital imaging of OTA transport in the livers and the kidneys of anesthetized wild-type and albumin knockout mice was done using an inverted two-photon microscope (LSM MP7, Zeiss, Germany). Details of the surgical preparation of the mice and exposure of the liver and the kidneys for imaging were previously described (Ghallab et al. 2021a; Reif et al. 2017). Tissue morphology was visualized by intravenous administration of the mitochondrial membrane potential marker tetramethylrhodamine ethyl ester (TMRE) (#T669, ThermoFisher Scientific) approximately 10 min before recording (Table 2). To image its transport kinetics in the liver and kidneys, a bolus of OTA (5 mg/kg) was administered via a tail vein catheter (SAI-infusion, IL, USA) within approximately 20 s after the onset of recording; the first few frames recorded prior to the OTA administration served to detect the basal tissue auto-fluorescence (Table 2). The mice were kept under controlled ambient temperature conditions of 36 °C, with exposure to a maintenance isoflurane inhalation anesthesia during the entire recording session.

Image analysis

During image processing, rigid body registration was performed using StackReg (Thévenaz et al. 1998) to compensate for tissue movement in the recorded time series. From these stabilized videos, 2D projections were generated using average and standard deviation operators. The pixel classification and autocontext workflows of the interactive image segmentation software ilastik (version 1.3.3post1) (Berg et al. 2019) were used for compartment segmentation in the 2D projections of liver and kidney. The compartments considered are sinusoids, hepatocyte cytoplasm and bile canaliculi in the liver, as well as inter-tubular capillaries, renal tubular lumen, and the tubular epithelial cells in the kidney. Mean raw OTA intensities were measured per compartment and frame. In addition, for the kidney, mean OTA intensities per tubule were measured based on the signal in the TMRE positive cell compartment, and tubules were subsequently grouped into two sets using k-means clustering based on their maximum mean OTA intensity over time.

Calculation of elimination half-life

For the calculation of the elimination half-life, Microsoft excel was used. First, the decadic logarithm of the intensity was plotted against the time. Then, the linear declining portion of the curve was selected manually and used for construction of a trend line. Next, the slope of the trendline was multiplied by 2.303 to convert the decadic to the natural logarithm and enable usage of the following formulas:

(1)

kel = -slope (Certara knowledgebase)

(2)

T ½ = ln (2) /kel (Certara knowledgebase)

(https://www.certara.com/knowledge-base).

Statistical analysis

Data analysis was done using GraphPad Prism version 9.3.1 (GraphPad Software, Inc., La Jolla, CA). Significance level is indicated as *p value ≤ 0.05, **p value ≤ 0.01, ***p value ≤ 0.001 using Dunnett's or Tukey's multiple comparisons test.