Chemicals and antibodies
Reduced glutathione, ethanol, dimethyl sulfoxide (DMSO), xylene, thiobarbituric acid, 10% neutral buffered formalin (NBF) solution, protease and phosphatase inhibitor cocktails were purchased from Sigma Aldrich (St. Louis, MO, USA). Canagliflozin and isoprenaline were from Square Pharmaceutical Ltd. (Dhaka, Bangladesh) and Samarth Life Sciences Pvt. Ltd. (Mumbai, India), respectively. All standards and assay components for MDA, NO, APOP assays, H&E (Hematoxylin and Eosin) and picrosirius red staining were from Merck (Darmstadt, Germany). Standard and SOD assay components were bought from SR Group (Delhi, India). CK-MB assay kit was purchased from DCI Diagnostics (Budapest, Hungary). Antibodies raised to AMPK, p-AMPK (Thr172), Akt, p-Akt (Ser473), p-eNOS (Ser1177) and cleaved caspase-3 were purchased from Cell Signaling Technology (Danvers, MA, USA), and those for eNOS were from Abcam (Cambridge, UK) and BD Biosciences (San Jose, CA, USA). Antibodies for α-actin, iNOS, Bax and Bcl-2, HRP-conjugated anti-mouse and anti-rabbit secondary antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Phosphate-buffered saline (PBS, pH 7.4), radioimmunoprecipitation (RIPA) buffer, blot stripping buffer (Restore) were bought from Thermo Fisher Scientific (Waltham, MA, USA). DC Protein Assay Kit, tween 20, SDS-PAGE gel components, protein molecular weight marker, 10 × tris-buffered saline (TBS) and PVDF (polyvinylidene fluoride) membrane were bought from Bio-Rad (Hercules, CA, USA).
Animals and experimental design
The Ethics Committee of North South University approved all experimental protocols for animal care, handling and experimentation (AEC 005-2018). All experiments were performed in accordance with relevant guidelines and regulations. Twenty male Long Evans rats between 10 and 12 weeks of age were obtained from the Reproduction unit of the Animal House at North South University, Dhaka. All animals housed in individual cages in a temperature-controlled room (temperature 22 ± 2 °C; 55% humidity; 12-h light/dark cycles) had free access to standard chow diet and drinking water. The length of the experimental protocol was 2 weeks, where animals were randomly divided into four groups of five rats in each and treated as follows:
Group I: Control—only received standard chow diet for the entire period of 2 weeks.
Group II: Canagliflozin—given standard chow diet for the first week and then treated with canagliflozin at 5 mg/kg daily with chow diet for the second week30.
Group III: ISO—given subcutaneous injections of isoprenaline at 50 mg/kg twice a week for the first week to induce oxidative stress31. Animals in this group received standard chow diet throughout the duration of the experiment.
Group IV: ISO + canagliflozin—oxidative stress was induced first by subcutaneous injections of isoprenaline at 50 mg/kg twice a week for the first week and treated with canagliflozin at 5 mg/kg daily for the second week to study recovery.
Euthanasia and tissue harvesting
Animals were euthanized by intraperitoneal injection of ketamine/xylazine (500/50 mg/kg) followed by decapitation. For plasma biochemistry, blood was drawn from the hepatic portal vein and plasma separated by spinning samples at 8,000 rpm for 15 min at 4 °C. Biochemical analyses were conducted on fresh plasma or stored at − 80 °C for future experiments. Kidneys were collected, weighed and processed for biochemical, Western blotting and histological examination and the remaining samples stored at − 80 °C.
Uric acid and creatinine concentrations were determined using assay kits according to the manufacturer’s instructions (DCI Diagnostics, Budapest, Hungary).
Quantification of oxidative and nitrative stress markers: assays for malondialdehyde (MDA), nitric oxide (NO) and advanced protein oxidation products (APOP)
Roughly, 0.1 g of kidney tissue from each group was placed in 1 ml of phosphate buffer (pH 7.4) in an Eppendorf tube, homogenized and centrifuged at 10,000 rpm for 15 min at 4 °C. Supernatants were transferred into fresh tubes and used for the determination of MDA, NO and APOP. Lipid peroxidation was expressed in terms of MDA levels in kidney tissue homogenates using a colorimetric assay32. NO concentration was measured following the method of Tracey et al.33, and was calculated by using a standard curve and expressed as nmol/g of tissue. APOP concentration was measured according to a modified protocol by Witko-Sarsat et al.34 and Tiwari et al.35 and concentration expressed as nmol·ml−1 chloramine-T equivalents.
Determination of myeloperoxidase (MPO) activity
MPO activity was determined by an o-dianisidine-H2O2 method following protocols described previously by Rahman et al.21.
Estimation of endogenous antioxidant levels: measurement of catalase (CAT) and super oxide dismutase (SOD) activity and glutathione (GSH) concentration
CAT activities in plasma and kidney tissue homogenate were determined as described elsewhere36,37. Absorbance was read at 240 nm, and an absorbance change of 0.01 units/min change was counted as one unit of CAT activity. SOD activity was measured according to protocols described previously36,37. Absorbance of the reaction mixtures containing enzymes was read at 480 nm for one minute at 15-s intervals. A blank without samples was run in parallel. Epinephrine auto-oxidation present in the assay system was calculated and 50% inhibition of epinephrine auto-oxidation is taken as one unit of SOD activity. Reduced glutathione level was determined following protocols described previously38. Absorbance of the reaction mixture was promptly read at 405 nm with the development of the yellow chromophore and glutathione level expressed as ng/mg protein.
Histopathological examination of rat kidney sections
Kidney sections were fixed in 10% Neutral Buffered Formalin (NBF) followed by their treatment with graded ethanol and xylene. Sections were subsequently embedded into paraffin blocks and cut with a rotary microtome into 5 µm thin slices that were collected on fresh slides and stained with hematoxylin/eosin (H&E) to visualize immune cell infiltration in the tissue sections. Picrosirius red staining was also performed on the kidney sections in parallel to analyze the presence and extent of fibrosis. At the end of the staining procedure, slides containing stained sections were photographed and analyzed under a light microscope at 40 × magnification (Zeiss Axioscope)21. To quantify % of area fibrosis, ImageJ software (National Institutes of Health, Bethesda, MD) was used.
Cell culture and treatment
Human primary renal proximal tubule epithelial cells were purchased from ATCC (PCS-400-010) and cultured according to the manufacturer’s instructions. Briefly, cells were maintained in epithelial cell culture medium (PCS-400-030, ATCC) supplemented with epithelial cell growth kit (PCS-400-040, ATCC) containing fetal bovine serum (0.5%), triiodothyronine (10 nM), rhEGF (10 ng/ml), hydrocortisone hemisuccinate (100 ng/ml), rh insulin (5 µg/ml), epinephrine (1 µM), transferrin (5 µg/ml) and l-Alanyl-l-Glutamine (2.4 mM). Cells were grown to approximately 80% confluency in the culture medium at 37 °C in a 95% air:5% CO2 humidified atmosphere. For our experiments, renal proximal tubule epithelial cells were treated as follows:
Control: treated with vehicle (DMSO).
Canaglif: treated with 10 µM canagliflozin for 12 h25.
ISO: treated with 1 µM isoprenaline for 12 h39.
ISO + Canaglif: co-treated with 1 µM isoprenaline and 10 µM canagliflozin for 12 h.
ISO + Canaglif + DM: co-treated with 1 µM isoprenaline and 10 µM canagliflozin for 11 h, and then with 10 µM dorsomorphin40 for the last 1 h.
ISO + Canaglif + A-443654: co-treated with 1 µM isoprenaline and 10 µM canagliflozin for 11.5 h, and then with 5 µM A-44365441 for the last 30 min.
At the end of the treatment protocol, adherent cells were briefly washed with ice-cold PBS. Cells were then solubilized in modified radioimmunoprecipitation (RIPA) buffer (50 mM Tris–HCl, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 10 mM NaF, 10 mM Na2HPO4, pH 7.4) containing protease and phosphatase inhibitor cocktails. Cell lysates were processed for Western blotting as described below.
To extract proteins from kidney tissues, approximately 0.1 g of kidney tissue was minced in an Eppendorf tube and homogenized in modified RIPA buffer. Tissue homogenates were then centrifuged at 12,000 rpm for 10 min at 4 °C and supernatants transferred into fresh tubes. Protein concentration was standardized using DC Protein Assay Kit, and about 50 µg protein for each sample was boiled with 2 × SDS-sample buffer (Bio-Rad), resolved by SDS-PAGE and blotted onto PVDF membrane. Membranes were blocked with 5% milk solution in TBST (tris-buffered saline with 0.1% Tween 20) for 1 h at room temperature and the incubated with the following primary antibodies overnight: total eNOS (1:500 dilution), p-eNOS (1:200 dilution), iNOS (1:500 dilution), α-actin (1:5,000 dilution), p-Akt (1:400 dilution), total Akt (1:500 dilution), p-AMPK-α (1:400 dilution), total AMPK-α (1:500 dilution), Bax (1:200), Bcl-2 (1:200) and cleaved caspase-3 (1:200 dilution). Membranes were washed and incubated with HRP–conjugated secondary antibodies (1:5,000 dilution) for 1 h at room temperature. Following secondary antibody incubation, blots were washed three times and developed, and protein bands imaged using Gel Doc XR + System (Bio-Rad). Densitometric analysis of the protein bands were performed using ImageJ software (64-bit Java 1.8.0_112, National Institutes of Health, Bethesda, MD; URL: https://imagej.nih.gov/ij/download.html). Where needed, blots were stripped of IgG using Restore stripping buffer and reprobed.
Statistical analyses were carried out using OriginLab software v9.55 (2018b) (URL: https://www.originlab.com/index.aspx?go=SUPPORT&pid=3325). Values were expressed as mean ± standard error of mean (SEM). One-way analysis of variance (ANOVA) along with Newman–Keuls post-hoc test was used for multiple group comparisons. p < 0.05 (a priori) was considered statistically significant.