All animal experiments were approved by the Regierung von Oberbayern (ROB-55.2–2532.Vet_02-19–163) at the Institute for Surgical Research at the Walter-Brendel-Centre of Experimental Medicine, Munich, Germany and were performed in accordance with the guidelines from Directive 2010/63/EU of the European Parliament on the protection of animals used for scientific purposes.

Induction of CKD

Sixteen German Landrace pigs of either sex were used in the experiment out of which 9 served as control (WT) and 7 of them underwent CKD induction.

At 12 weeks of age, swine underwent micro-embolization of afferent glomerular arterioles in both kidneys. The animals were sedated (ketamine 10% (15 mg/kg) (WDT, Garbsen, Germany), azaperone (2 mg/kg) (Stresnil, Elanco, Bad Homburg, Germany) and atropine sulfate (0.02 mg/kg) (Eifelfango, Neuenahr, Germany) against salivation i.m.), followed by Midazolam 15 mg/kg i.v.) (Ratiopharm-Teva, Ulm, Germany), intubated and artificially ventilated (Primus, Dräger, Lübeck, Germany) with a mixture of O2 and N2 (1:2). Anesthesia and analgesia were maintained by 1–2% (vol/vol) sevoflurane (Sevorane, AbbVie GmbH, Ludwigshafen, Germany) to ventilation and fentanyl (0.005 mg/kg/h i.v.) (Fentadon, Dechra, Aulendorf, Germany) respectively. Arterial access was obtained via a 9F sheath (Cordis, 504-609X) in the right carotid artery, allowing measurement of blood pressure and heart rate. A Swan-Ganz catheter (131F7, Edwards Lifesciences, Irvine, USA) was advanced sequentially in both renal arteries, and the balloon was inflated and 75 mg of polyethylene microspheres (38–42 μm, Cospheric, Santa Barbara, CA, USA) was infused separately into each kidney. Thereafter, the carotid was ligated and the wound was closed. Carprofen (4 mg/kg) was given against post-operative pain (Rimadyl, Zoetis, Berlin, Germany). Amoxicillin antibiosis was administered (Duphamox LA, Zoetis, Berlin, Germany) perioperatively and animals were subsequently allowed to recover.

Hemodynamic assessment

At 8 months of age, a terminal experiment was performed. Animals were sedated and pre-anesthetized as previously described. Anesthesia was maintained using propofol (7 mg/kg/h) and fentanyl (0.005 mg/kg/h). An echocardiography was performed (Esaote, MyLabX8vet, Neufahrn, Germany) to assess left ventricular (LV), right ventricular (RV) and left atrial (LA) dimensions, at systole and diastole. For right and left sided heart catherization, an 11F venous sheath (Terumo, RS*C11N10NR, Eschborn, Germany) and a 9F arterial sheath (Cordis, 504-609X, Miami Lakes, USA) were placed in the right external jugular vein and left internal carotid artery respectively. The latter was connected to a pressure transducer to continuously monitor arterial blood pressure and heart rate.

Under fluoroscopic guidance (Ziehm Vision, Nuremberg, Germany), a Swan-Ganz catheter (131F7, Edwards Lifesciences, Irvine, USA) was introduced and progressed into the pulmonary artery via the venous sheath to measure the pressure in pulmonary artery (PA), right ventricle (RV), and right atrium (RA) and to measure the pulmonary capillary wedge pressure (PCWP). The cardiac output was assessed by thermodilution.

Using the arterial access sheath, a pressure–volume loop catheter (FDH-7018B-E245A, Transonic, Ithaca, USA) was placed in the LV and PV loops were recorded using LabChart Pro (ADInstruments, Oxford, United Kingdom). Ventilation was briefly halted to obtain baseline PV loops as well as PV loops during preload reduction with a 14F Fogarty balloon (62080814F, Edwards Lifesciences, Irvine, USA) positioned in the inferior vena cava just below the diaphragm. Approximately 10 cardiac cycles recorded from the start of the occlusion were analyzed to obtain end-systolic and end-diastolic LV volumes (ESV, EDV) and pressures (ESP and EDP), and to calculate the end-diastolic pressure–volume relationship, end-systolic pressure–volume relationship, preload recruitable stroke work (slope of the relation between EDV and stroke work), arterial elastance (Ea, ratio of ESP and stroke volume (SV)), as well as cardiac efficiency (ratio of stroke work and pressure–volume area).

Subsequently, the thorax was opened, and a flow probe (3PSB, Transonic, Ithaca, USA) was placed around the proximal left anterior descending (LAD) coronary artery and connected to a computer using a perivascular flow module (TS420, Transonic, Ithaca, USA) and amplifier (16/35, ADInstruments, Oxford, United Kingdom). Baseline coronary blood flow was measured using LabChart Pro. An 6F angiocatheter (670–082-0E, Cordis, Miami Lakes, USA) was introduced into the LAD to infuse the vasodilator adenosine (50 μg/kg/min i.c., 01890, Merck, Taufkirchen, Germany) until maximum coronary blood flow was achieved. Upon completion of the experiments, ventricular fibrillation was induced using a 9 V battery on the surface of the heart, and the heart was excised. Samples were obtained from RA, RV, LA and LV, snap-frozen in liquid nitrogen and stored at -80 °C until further processing. In addition, a tissue block of the LV was processed to culture myocardial tissue slices.

Myocardial tissue slice cultivation

To assess the ex vivo contractile function, 300 μm thick living myocardial slices (LMS) were made and cultivated in biomimetic cultivation chambers (BMCCs) using the MyoDish cultivation set-up (InVitroSys, Gräfelfing, Germany). A posterior transmural LV sample (4 cm × 4 cm) was obtained and directly placed into cold (4 °C) cardioplegic buffer according to the LMS protocol developed by Fisher et. al. (2019) [14] and described in detail by Hamers et al. (2022) [16]. According to this protocol, myocardial LV slices were cut, prepared and hung into BMCCs in M199 medium (31,150–022, Thermo Fisher, Waltham, USA) (supplemented with 10% Penicillin–Streptomycin (100x) (P0781, Merck, Taufkirchen, Germany), 10% insulin-transferrin-selenium-X (100x) (51,500,056, Thermo Fisher, Waltham, USA), cort20 and β-mercaptoethanol (A1108.0100, AppliChem GmbH, Darmstadt, Germany). The LMS containing BMCCs were placed onto the specific cultivation rocker (60 rpm) in an incubator (21% O2, 5% CO2, 80% humidity) and electrically stimulated (30 bpm, 50 mA, 3 ms pulse duration) to contract. The twitch force of the LMS was continuously recorded. The cardiac slices equilibrated for 3 days in cultivation before experiments were started. Fresh cultivation medium was exchanged every other day, by removing the BMCCs from the rocker into a laminar flow hood, where medium was aspirated until approximately 0.8 mL remained. 1.6 mL of fresh medium was added to each dish.

After three days of cultivation, LMS (Con or CKD) were treated with 25 µM hydrogen peroxide, 25 µM Tempol or vehicle in order to study the relative contribution of oxidative stress to cardiac function. The treatment doses were chosen based on previous in vitro studies [8, 26]. Hydrogen peroxide/TEMPOL treatment was administered three times at 24 h intervals. The twitch force of all LMS was assessed at 60 min after each treatment, and normalized to the twitch force 24 h prior to the first treatment. After the third treatment, normalized twitch force over the course of the cultivation was compared between groups.

Real-time quantitative PCR

Subendocardial left ventricular tissue samples and LMS cultured for 7 days were snap-frozen in liquid N2. 30 mg of LV tissue or a single slice of 7 × 7 × 0.3 mm was homogenized and mRNA was extracted using the RNeasy Fibrous Tissue Mini kit, Qiagen, Hilden, Germany). cDNA was synthesized using 1000 ng of mRNA and a cDNA kit (M1661, Thermo Fisher, Waltham, USA). Target genes were normalized against HPRT and GAPDH using the StepOne software (Applied Biosystem CA, USA). Relative gene expression was calculated using the delta–delta Ct method. The primers are listed in Supplementary Table 6.

Western blots

Subendocardial left ventricular tissue samples were homogenized in RIPA buffer supplemented with protease and phosphatase inhibitor cocktail. 30 µg of protein lysates was loaded in precast protein gels (4–20% gradient gel, Bio-Rad) and transferred to a PVDF membrane (Trans-Blot Turbo Mini 0.2 µm PVDF Transfer Pack, Bio Rad). Membrane was blocked in 5% milk in PBST and then incubated with primary antibody overnight (eNOS and VEGF 1:1000; GAPDH – 1:10,000) and secondary antibody (1:5000) in 5% milk in PBST. The images were captured in iBright CL750 (Thermo Fisher Scientific, Waltham, USA) and quantification of bands by densitometry analysis was conducted in Image J Studio software. The antibodies are listed in Supplementary Table 6.

Enzyme-linked immunosorbent assays

To study the degree of kidney damage caused by renal embolization, neutrophil gelatinase-associated lipocalin (NGAL) levels were determined in urine samples obtained after sacrifice using ELISA (ab207924, Abcam, Berlin, Germany) per the manufacturer’s instructions. Oxidative stress was determined by measuring the 8-Hydroxy-2′-Deoxyguanosine (8-HDG) secreted in the urine samples using ELISA (ab201734 Abcam, Berlin, Germany) per the manufacturer’s instructions.

Colorimetric urine analysis

To determine the protein level in urine, urine samples obtained after sacrifice were tested using a colorimetric Coomassie protein assay kit according to the manufacturer’s instructions (23,200, Thermo Fisher, Waltham, USA). To assess total antioxidant capacity, 50 mg of LV sample (endocardium-anterior) was suspended in a lysing tube (845-CS-1140250, Innuscreen GmbH, Berlin, Germany) with 750 µL RIPA buffer (89,901, Thermo Fisher, Waltham, USA) and homogenized using a homogenizer (Speedmill Plus, Analytik Jena GmbH, Jena, Germany). Subsequently, a Trolox total antioxidant capacity (TAC) colorimetric assay (ab65329, Abcam, Berlin, Germany) was performed according to the manufacturer’s instructions.

Proteomics

Sample Preparation for Proteome Analysis: Frozen left subendocardial ventricular heart tissue samples were placed into precooled tubes and cryopulverized in a CP02 Automated Dry Pulverizer (Covaris, Woburn, MA, USA) with an impact level of 5 according to the manufacturer’s instructions. Tissue lysis was performed in 8 M urea/0.5 M NH4HCO3 with ultrasonication (18 cycles of 10 s) using a Sonopuls HD3200 (Bandelin, Berlin, Germany). Total protein concentration was quantified using a Pierce 660 nm Protein Assay (Thermo Fisher Scientific, Rockford, IL, USA). Fifty micrograms of protein was digested sequentially, first with Lys-C (FUJIFILM Wako Chemicals Europe GmbH, Neuss, Germany) for 4 h and, subsequently, with modified porcine trypsin (Promega, Madison, WI, USA) for 16 h at 37 °C.

Nano-Liquid Chromatography (LC)–Tandem Mass Spectrometry (MS) Analysis and Bioinformatics:

For LC–MS–MS analysis, an UltiMate 3000 nano-LC system connected online to a Q Exactive HF-X instrument (Thermo Fisher Scientific, Waltham, USA) was used. 1 μg of the digest was injected, transferred to a PepMap 100 C18 trap column (100 µm × 2 cm, 5 µM particles, Thermo Fisher Scientific) and then separated on an analytical column (PepMap RSLC C18, 75 µm × 50 cm, 2 µm particles, Thermo Fisher Scientific) at a flow rate of 250 nL/min with a gradient of 5–20% of solvent B for 80 min, followed by a ramp of 9 min to 40%. 0.1% formic acid in water made up solvent A, and 0.1% formic acid in acetonitrile made up solvent B. MS spectra were acquired with data independent acquisition using 50 12 m/z-wide isolation windows in the range of 400–1000 m/z. Protein identification and peptide quantification were carried out using DIAN-NN (1.8.1) [10] and the NCBI RefSeq Sus scrofa database (v.7–5-2020). All statistical analyses and data visualization were performed using R. Prior to statistical analysis, potential contaminants, only identified by site and reverse hits, were excluded. Proteins with at least two peptides detected in at least three samples of each condition were quantified using the MS-EmpiRe algorithm as previously described [2, 15]. The R script used for quantitative analysis is at https://github.com/bshashikadze/pepquantify. Proteins with a Benjamini–Hochberg-corrected p value ≤ 0.05 and fold change ≥ 1.3 were regarded as significantly altered for volcano plot and we used corrected p value ≤ 0.05 proteins for protein–protein interaction network. Preranked gene set enrichment analysis using STRING was employed to reveal biological processes related to differentially abundant proteins [48]. Signed (based on fold change) and log-transformed p values were used as ranking metrics and the false discovery rate was set to 1%. Raw mass spectrometry data and DIA-NN output data have been deposited to the ProteomeXchange Consortium via the PRIDE [38] partner repository with the dataset identifier PXD050994.

Histology and immunohistochemistry

LV anterior myocardial tissue and kidney samples were cut and fixated in Rotihistofix (Roth, P087.1) and transferred into 70% alcohol after 48 h. After that, the tissue was embedded in paraffin. 3 µm deparaffinized sections were stained with picrosirius red staining solution (Polysciences, Picrosirius Red Stain Kit#24,901) or Gomori silver stain (Abcam, #ab236473). The picrosirius red (PR) staining was analyzed under polarized light, and the amount of birefringence was quantified in ten randomly chosen nonoverlapping fields (× 200 magnification) using QuPath software. Cross-sectional areas of cardiomyocytes with visible nuclei were measured in Gomori staining. For immunostaining, 3 µm deparaffinized LV sections were boiled in Citrate Buffer, pH 6.0, for antigen retrieval. The sections were incubated with primary antibodies for 8-hydroxy-de-guanosine (1:1000 dilution: Abcam, #ab48508) CD31 (1:100 dilution: Thermo-scientific MA5-32,321) and WGA (1:1000 dilution: Thermo -scientific, W21404) overnight. On the following day, the sections were incubated in species specific secondary antibodies (1:500 dilution; Abcam—anti mouse or ab150113- anti rabbit-ab150080) for two hours and sections were mounted with DAPI for nuclear staining. The stained sections were quantified in ten randomly chosen nonoverlapping fields (× 400 magnification) using ImageJ software.

Statistical analysis

Statistical analysis was performed using a Student’s t-test or Analysis of Variance for Repeated Measurements as appropriate in Graphpad Prism (v10). All data passed the normality test using the Shapiro–Wilk test and data are shown as mean ± SEM.