Montmorillonite-Illite clay minerals were refined and provided by FIM Biotech GmbH (Berlin, Germany). Technical processing steps were elutriation, fine grinding, and calcination which resulted a in processed clay mineral (pClM). Ground lanthanum carbonate (LaC) tablets (Fosrenol® 750 mg, Shire Pharmaceuticals, Hampshire, Great Britain) were used as reference and positive control.
Male Wistar rats, weighing 200 to 220 g, received a stepwise 5/6 nephrectomy by removing one kidney and one week later 2/3 of the second kidney to induce chronic renal failure (n = 26), or sham surgery (n = 8) (Charles River Laboratories, Germany). Rats were allowed free access to rat chow and tap water during routine husbandry in a 12-h dark/light cycle at 21–22 °C. Untreated chronic renal failure (CRF, n = 12) and sham-operated rats (sham) received a high phosphate (disodium phosphate 1.2%, d/w) and calcium (1.2%, d/w) diet. Both nephrectomized treatment groups also received a phosphate-rich (disodium phosphate 1.2%, d/w) and calcium-rich (1.2%, d/w) diet in combination with either 2% (d/w) of a processed clay mineral (pClM, n = 6) or lanthanum carbonate (LaC, n = 8). Samples of urine and feces were collected using metabolism cages for 24 h fortnightly. After 12 weeks, rats were weighed and euthanized by an overdose of i.p. injected ketamine-xylazine (100/25 mg/kg BW) followed by retrobulbar blood collection and subsequent transcardial perfusion with PBS. Tissue samples were either drop-fixed in 4% paraformaldehyde or snap-frozen in liquid nitrogen.
All experimental procedures were approved by and conducted in accordance with the guidelines of the State Department of Agriculture, Food Security and Fisheries Mecklenburg-Western Pomerania (Section 6/Department 600, Protocol Number: TV 7221.3–1.1-005/13). The study was carried out in compliance with the ARRIVE guidelines. Rats were weighed weekly and health conditions were checked daily with regard to their fur (smooth), eyes (clean and open) and posture (normal). Animals that lost more than 20% of their body weight or showed abnormal behavior and signs of severe pain were excluded from the experiment.
Phosphate measurement via ICP-OES
For the analysis of phosphate in feces of rats, phosphate was extracted according to the International Organization for Standardization DIN EN 16174 . Total phosphate of solid samples was measured by inductively coupled plasma optical emission spectrometry (ICP-OES).
Analytical chemistry and glomerular filtration rate (GFR)
Levels of uremic toxins in blood and urine were quantitatively determined using a wet-chemical colorimetric method (Cobas Mira Plus, Roche, Germany). Blood samples were centrifuged and plasma concentrations of urea, uric acid, and creatinine were determined. Phosphate concentration was determined in serum. The uremic retention solutes urea, uric acid, creatinine, and phosphate were measured in 24 h urine collection samples. The volume of excreted urine was documented for calculation of GFR and 24 h phosphate excretion and is shown as such. The GFR, creatinine clearance and blood urea nitrogen (BUN) clearance were calculated as values per animal according to S. Pestel et al. .
Histochemistry and immunofluorescence
Paraffin-embedded tissues were cut into 4 μm thick histological sections using a microtome and subsequently deparaffinized and rehydrated before histochemical staining with hematoxylin and eosin dye (Medite, Burgdorf, Germany), or Masson’s Goldner trichrome (Sigma-Aldrich, Germany) according to the manufacturers protocol. For immunofluorescence labelling, antigen retrieval was performed after deparaffinization and rehydration in heated trisodium citrate buffer (pH 6), followed by incubation in blocking solution (4% BSA, 1x PBS, 0.1% Tween) for 1 h. The primary antibody against α-smooth muscle actin (α-SMA, Sigma-Aldrich, Munich, Germany) was incubated overnight and detected with Alexa Fluor® 594 anti-mouse IgG (Life Technologies, Ober-Olm, Germany). Fluorescence intensity and collagen content was imaged using a Nikon Eclipse Ti-E microscope (Nikon GmbH, Düsseldorf, Germany).
Quantitative histological analyses
For quantification of α-SMA in aortic arch sections, five α-SMA positive areas were randomly selected and the average intensity was measured. Analyses of vascular fibrosis and ventricular hypertrophy from cross-sectional sections of paraffin-embedded hearts were performed according to the protocol described by Finch et al. with minor modifications . In brief, after Masson’s Goldner trichrome staining (i) medial area to luminal area ratio and (ii) perivascular collagen area to luminal area ratio were determined to quantify the severity of cardiac fibrosis. Histological sections were analyzed using NIS-Elements AR software (Nikon GmbH, Düsseldorf, Germany).
Total protein concentrations were determined using a BCA™ protein assay kit (Pierce, part of Thermo Fisher Scientific, Rockford, USA). Proteins of tissue homogenates were separated by SDS-PAGE using 20 μg total protein per lane. After blotting, membranes were blocked with 1% BSA and initially incubated overnight with primary antibody against α-SMA (1:1000). Blots were then stripped (24 mM glycine, 2% SDS, aqua dest, pH 2.0) at 65 °C, blocked with 1% BSA, and incubated overnight with primary antibody against GAPDH as a loading control (1:1500, Biomol, Hamburg, Germany). HRP-conjugated secondary antibodies (anti-mouse and anti-rabbit 1:10,000, GE Healthcare, Buckinghamshire, UK) were used as detection antibodies. Target proteins were visualized and quantified by Fusion Capt Advance FX7 detection software (Vilber Lourmat GmbH, Eberhardzell, Germany).
Data are expressed as box plots with median (min to max). All data were tested for normal distribution using Kolmogorov-Smirnov normality test and analyzed by one-way ANOVA. P values p < 0.05 were considered as statistically significant. Levels of significance were determined as follows: *p < 0.05, **p < 0.01 and ***p < 0.001. All statistical calculations were performed using GraphPad Prism 6 (GraphPad Software Inc., San Diego, CA, USA).