Ethics

All animal procedures were approved by the Institute of Animal Care and Use Committee at Kaohsiung Chang Gung Memorial Hospital (Affidavit of Approval of Animal Use Protocol No. 2021062210) and performed in accordance with the Guide for the Care and Use of Laboratory Animals.

Cell culture and in vitro studies

The rat renal proximal tubular cell line (NRK-52E) procured from Bioresource Collection and Research Center (BCRC) was grown at 37 °C under 5% CO2 in culture dishes containing Dulbecco’s modified Eagle’s medium 11965-084, GIBCO) supplemented with 10% fetal bovine serum, 1% Non-Essential Amino Acids (NEAA), 100 µg/ml streptomycin, and 100 units/ml penicillin. Additionally, H9C2 cells from BCRC cardiomyoblasts were maintained as NRK-52E condition. The p-Cresol was purchased from Sigma and utilized to treat the cells with different concentrations at 48 h.

In our in vitro studies, the rat renal tubular epithelial cells were categorized into four groups, i.e., A1 (NRK-52E only), A2 [NRK-52E + p-Cresol (50 µM, cultured for 48 h)], A3 [NRK-52E + p-Cresol (50 µM) + Empagliflozin (EMPA) (50 μm, cultured for 48 h)] and A4 [NRK-52E + p-Cresol (50 µM) + EMPA (100 μm, cultured for 48 h)].

Consistently, the cardiomyoblasts were categorized into four groups, i.e., B1 (H9C2 only), B2 [H9C2 + p-Cresol (50 μm, cultured for 48 h)], B3 [H9C2 + p-Cresol (50 μm + EMPA (50 μm, cultured for 48 h)], and B4 [H9C2 + p-Cresol (50 μm) + EMPA (100 μm, cultured for 48 h)]

Measuring the levels of reactive oxygen species (ROS) in culturing NRK-52E and H9C2 cells

Total intracellular ROS of were measured by the H2DCF-DA oxidation method. Cells were incubated in HEPES buffer supplemented with H2DCF-DA for 30 min. H2DCF-DA is a cell-permeable probe that is oxidized by intracellular ROS to generate fluorescent DCF. The fluorescent DCF could be detected by flow cytometry or immunofluorescent microscopy.

Flow cytometric analysis for apoptosis detection

The percentages of viable and apoptotic cells were determined by flow cytometry using double staining of annexin V and propidium iodide (PI). The early phase of apoptosis was defined as annexin V+/PI- whereas the late phase of apoptosis was defined as annexin V+/PI+.

CRS model by 5/6 subtotal nephrectomy for induction of CKD, and doxorubicin (DOX) induced dilated cardiomyopathy (DCM) with feeding high-protein diet (HPD)

The procedure and protocol of induction of CRS has been described in our report (Yeh et al. 2021). Briefly, pathogen free, adult male SD rats, weighing 285–325 g (Charles River Technology, BioLASCO Taiwan Co., Ltd., Taiwan), age of 9–12 weeks, after 7-day adaption period, were randomly allocated to different groups. The animal model of CRS was defined as: (1) DCM induced for the first 20 days, followed by (2) CKD model 2 weeks (total 35 days needed, equal to day 0 of CRS). DCM was first induced, followed by CKD induction in the same animal to finally become as the CRS model. To provide a more reliable experimental model mimicking the clinical setting of CRS patients, all CRS animals were fed with a high-protein diet (HPD) [5TJR-High-Protein Ketogenic Diet (Rodent) (TestDie Company)] since day 1 after CKD induction to increase the burden on the kidney and the accumulation of uremic toxins in the blood.

The animals were randomly and equally divided into four groups, i.e., group 1 [sham-operated control (SC)], group 2 (SC + HPD since day 1 after DCM induction), group 3 [CRS (i.e., DCM + CKD) + HPD], group 4 [CRS + HPD + EMPA (20 mg/kg/day since day 35 after DCM induction) by intraperitoneal administration since day 35 after CRS induction)]. Animals in each group were euthanized by day 63 after CRS induction (i.e., total four week’s EMPA treatment).

Animal model of dilated cardiomyopathy (DCM)

The procedure and protocol as well as the optimal dosage of doxorubicin to induce DCM have been reported in our previous study in detail (Yeh et al. 2021). Briefly, each animal in DCM group was treated by an accumulated dosage of 7.5 mg/kg doxorubicin (DOX) (i.e., intraperitoneal administration once at each 5-day’s interval within 20 days, i.e., at 4 separated time points). Transthoracic echocardiography specific for animal study was used for assessing the successful conduction of a rat DCM model in the present study.

Animal model of CKD

The protocol and procedure of creation of CKD were based on our previous report (Yeh et al. 2021). Briefly, by day 20 after DCM induction, all animals were anesthetized with 2.0% inhalational isoflurane for midline laparotomies. The sham-operated control (SC) group received laparotomy only, followed by closure of the muscle and skin layers, while CKD induction was performed in the remaining groups. CKD was conducted by right nephrectomy plus arterial ligation of upper and middle thirds of blood supplies to the left kidney, thereby creating a 5/6 nephrectomy model with limited renal function (refer to supplementary Fig. 1). In this way, combination of DCM and CKD was defined as the animal model of CRS in rat for the purpose of the current study.

To assess the serial changes of renal artery resistive index (RARI)

The RARI is an essential and reliable factor for assessing the renal arterial resistance in various kidney diseases, such as CKD. By using the ultrasound machine (Vevo 2100, Visualsonics), an animal ultrasound specialist measured two parameters of renal artery, including (1) peak systolic blood velocity (PSV) and (2) lowest diastolic blood velocity (LDV) by day 0 prior to and by days 35 and 60 after DCM induction. Thus, the calculation formula was defined as RI = (PSV-LDV)/PSV.

Serial collection of the blood samples for assessment of renal function

In the present study, serial blood samples for measuring the circulating levels of blood urine nitrogen (BUN) and creatinine were drawn at day 0 prior to and by day 35, 42 and 63 after CRS induction. Three mL of peripheral blood from tail vein was collected at each time interval, followed by adequate fluid supply, i.e., 6 mL normal saline, was intraperitoneally administered each time after blood sampling.

Serial collection of the 24h urine for assessment of the ratio of urine protein to urine creatinine (RUp/Uc)

The procedure and protocol have been reported in our previous studies (Yang et al. 2019; Yang et al. 2019b). Briefly, for the collection of 24 h urine in individual study, each animal was put into a metabolic cage [DXL-D, space: 190 × 290 × 550 mm3, Suzhou Fengshi Laboratory Animal Equipment Co. Ltd., China] for 24 h with free access to food and water. Urine in 24 h was collected in all animals prior to and by days 35, 42 and 63 after CRS induction for determining the (RUp/Uc).

Histopathology scoring of kidney injury in CKD rodent

The scoring system of kidney injury has been described in our previous studies (Yang et al. 2019; Yang et al. 2019b; Huang et al. 2015). Briefly, the kidney and heart specimens were fixed in 10% buffered formalin prior to being embedded in paraffin blocks from which 5 μm sections were obtained and stained with hematoxylin and eosin (H&E) for microscopic examination. The grading of tubular necrosis, loss of brush border, cast formation, and tubular dilatation was scored in 10 randomly chosen, non-overlapping microscopic fields (200x) as follows: 0 (none), 1 (≤ 10%), 2 (11–25%), 3 (26–45%), 4 (46–75%), and 5 (≥ 76%). Additionally, the grading of fibrotic area in LV myocardium under microscopic fields (200x) was expressed as follows: 0 (none), 1 (≤ 10%), 2 (11–25%), 3 (26–45%), 4 (46–75%), and 5 (≥ 76%).

Histological study of fibrosis areas

The procedure and protocol were based on our previous reports (Huang et al. 2015; Yang et al. 2019b; Yang et al. 2019). In detail, Masson’s trichrome was utilized for assessing fibrosis in kidney parenchyma and left ventricular (LV) myocardium. Three 4 μm thick serial sections of kidney and LV myocardium were prepared by Cryostat. The integrated area (µm2) of fibrosis in each section was calculated using Image Tool 3 (IT3) image analysis software. The mean integrated area (µm2) of fibrosis in kidney and in LV myocardium per HPF was obtained using a conversion factor of 19.24 (1 µm2 corresponded to 19.24 pixels).

Western blot assay

The procedure and protocol were based on our previous reports (Huang et al. 2015; Yang et al. 2019b; Yang et al. 2019). Briefly, primary antibodies against the specific proteins were used. Additionally, anti-rabbit IgG horseradish peroxidase–conjugated antibodies (1:2000, Cell Signaling Danvers, MA, USA) were utilized. Furthermore, the results were normalized to beta-actin (1:10000, Merck) expression.

Immunohistochemical (IHC) and immunofluorescent (IF) staining by day 42 after CKD induction

The procedure and protocol for IHC and IF staining have been described in our previous reports(Huang et al. 2015; Yang et al. 2019b; Yang et al. 2019). Sections were incubated with primary antibodies specifically against xanthine oxidase (ab109235, 1:500, Abcam), CD45 (ab10558, 1:400, Abcam), H2DCFDA (D6883, Sigma), CD68 (ab31630, 1:500, Abcam), CHAC1 (15207-1-AP, 1:100, Proteintech) and CHAC2 (16304-1-AP, 1:100, Proteintech), while sections incubated with the use of irrelevant antibodies served as controls. Three sections of kidney specimen and quadriceps muscle from each rat were analyzed.

Statistical analysis

Quantitative data were expressed as mean ± SD. Statistical analysis was adequately performed by ANOVA followed by Bonferroni multiple-comparison post hoc test. Statistical analysis was performed using SPSS statistical software for Windows version 22 (SPSS for Windows, version 22; SPSS, IL, USA). A value of p < 0.05 was considered as statistically significant.