Qwin 3

Kidney tissues were cut into 4-μm paraffin sections and stained with Sirius red (Abcam, Cambridge, UK) to observe their histology. Deparaffinization and hydration were performed using xylene and ethanol, respectively. To block endogenous streptavidin activity, 3% hydrogen peroxide (H₂O₂, Sigma-Aldrich, St. Louis, MO, USA) was used. The deparaffinized sections were stained with anti-mouse TG2 antibody (Novus Biologicals, Centennial, CO, USA). These sections were then incubated with secondary antibodies, goat anti-rabbit (Vector Laboratories, Burlingame, CA, USA) and rabbit anti-rat IgG (Vector Laboratories, Burlingame, CA, USA). The sections were then counterstained with Mayer’s hematoxylin (Sigma-Aldrich) and visualized under a light microscope (DFC-295; Leica, Mannheim, Germany). For rodent and human kidney samples, at least five fields (magnification: ×100) were randomly selected, and Sirius red and brown-stained areas were quantified using computer-based morphometric analysis (Qwin 3; Leica).

The steps before blocking were the same as those described in the immunofluorescence section. Blocking reagent was added for 1 hour at room temperature. Images were obtained using a Leica TCS SP8 STED CW (Leica Microsystems). All morphometric parameters were determined using a microscope coupled to a computerized morphometry system (Qwin3, Leica). The antibodies used for immunofluorescence are listed in Supplemental Table 1.

Following cell culture on silicon‐based plates in serum‐free DMEM for 48 hrs, cardiomyocytes were incubated with anti‐α‐MHC (catalogue 05‐716, Upstate, Millipore Corporation, Billerica, Massachusetts, USA) and LC3b‐II (catalogue 2775; Cell Signaling Technology Inc.). Samples were then incubated with secondary antibodies conjugated with FITC or Alex (catalogue A21206; Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The surface areas of cardiomyocytes and LC3b‐II were determined using an image analysis software (Leica Qwin 3) and were calculated using the mean of 100 to 120 cells from randomly selected fields.

Eighty-three kidney tissue samples were processed for immunohistochemistry. Unstained tissue samples obtained from the study population were used. Paraffin-embedded kidney tissue samples were cut into 4-μm-thick sections, deparaffinized, and rehydrated using xylene and ethanol. After blocking the endogenous streptavidin activity using 3% hydrogen peroxide, the sections were stained with anti-CCL8 antibody (Ab) (Novus Biologics, Littleton, CO, USA) anti-CCR8 Ab (Invitrogen, Carlsbad, CA, USA) and anti-CCR2 Ab (Abcam, Cambridge, UK) and incubated at 4 °C overnight. Next, the samples were incubated with dextran polymer conjugated with horseradish peroxidase (GBI Labs, Bothel, WA, USA) for 5 min at room temperature. Finally, all sections were counterstained with Mayer’s hematoxylin (Sigma–Aldrich, St Louis, MO, USA) and examined by light microscopy (DFC-295; Leica, Mannheim, Germany). Quantification of CCL8 and CCR2-positive cells was performed using a computer-based morphometric analysis (Qwin 3 and LAS-4000, Leica, Mannheim, Germany). The intensity scoring was performed in a blinded manner by calculating the mean values of the positive areas (%). All analyses were reviewed and confirmed by the renal pathologist who had no knowledge of each experimental group.

After incubation for 48 hrs on silicon-based plates in serum-free DMEM medium, cardiomyocytes were incubated with the anti-α-MHC (catalogue no. 05-716; Upstate, Millipore Corporation, Billerica, MA, USA) and anti-LC3b-II (catalogue no. 2775; Cell Signaling Technology) antibodies. The samples were then incubated with secondary antibodies conjugated to FITC or Alexa (catalogue no. A21206; Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The surface area (SA) of cardiomyocytes and LC3b-II staining were determined using image analysis software (Leica Qwin3) and were calculated using the mean of 100 to 120 cells from randomly selected fields.

Masson’s trichrome staining (Sigma-Aldrich), and Sirius red staining (Abcam, Cambridge, UK) were performed to evaluate glomerular sclerosis. For the immunohistochemical assays, paraffin-embedded kidneys were cut into 4-μm-thick slices, deparaffinized, and hydrated using xylene and ethanol. Endogenous streptavidin activity was blocked using 3% hydrogen peroxide. The deparaffinized sections were stained with an anti-KLF2 antibody for kidneys and then incubated with horseradish-peroxidase-conjugated anti-mouse IgG (DAKO, Carpinteria, CA, USA, K3954). Next, 3,3′-diaminobenzidine tetrahydrochloride (Sigma-Aldrich) was used for immunohistochemical detection. Finally, all samples were counterstained with Mayer’s hematoxylin (Sigma-Aldrich) and evaluated under a light microscope (DFC-295; Leica, Mannheim, Germany). For each sample, five fields (X400) were randomly selected, and blue- (Masson’s trichrome staining) as well as brown-stained areas (immunohistochemistry) that reflected kidney fibrosis were quantified using computer-based morphometric analysis (Qwin 3; Leica). Scoring was performed in a blinded manner using the mean values of the positive areas (%).

Excised hearts were weighed, perfused with PBS and fixed with 4% polyformaldehyde for global morphometry and then with 10% formalin for further histological analysis. Paraffin-embedded hearts were sectioned at 4-μm thickness and stained with hematoxylin and eosin (H-E). Cardiomyocyte morphology and histology was visualized under a high magnification to assess cross-sectional area (CSA) using a video camera (Leica Qwin 3) attached to a micrometer. Twenty randomly chosen fields were evaluated from each cross section of the left ventricle (LV) free wall.