Exercise-induced renal protection may be mediated by the irisin/AMPK axis in diabetic nephropathy
Animals and study design
All experiments were conducted in accordance with National Institutes of Health (NIH) guidelines for the care and use of laboratory animals, followed ARRIVE guidelines, and were approved by the NIH Research Ethics Board. State University of Campinas (UNICAMP) (protocol numbers: 3856-1 and 5569-1/2020). One hundred male Wistar Hannover (HanUnib) rats were obtained from the Multidisciplinary Center for Biological Research (CEMIB) and were used in this study. The rats were housed in groups of three in plastic cages at 21°C ± 2 with a light/dark cycle of 12 h.
In one experiment, diabetes was induced in eight-week-old male Wistar Hannover rats via a single intravenous injection of streptozotocin (STZ, Sigma-Aldrich®, St. Louis, MO; 60 mg/kg in citrate buffer of sodium, pH 4.5) through the tail vein. Control (non-diabetic) rats received only vehicle (citrate buffer). After 48 h, rats with fasting blood glucose ≥ 270 mg/dL were considered diabetic and eligible to participate in these experiments. Rats were divided into three groups: control (CT, non-diabetic), sedentary diabetic (DM), and diabetic animals subjected to a physical training protocol on a treadmill (DM + Exe) for eight weeks (Supplementary Fig. 1A) .
In another experiment, diabetes was induced in eight-week-old male Wistar Hannover rats as described above. The diabetic rats were divided into four groups: sedentary diabetic (DM), sedentary diabetic treated intraperitoneally with 1 mg/kg of αV integrin receptor inhibitorseven (CycloRGDyK – S7844, Selleck Chemicals®, Houston, TX, USA) five days a week (DM + CycloRGDyK), exercised diabetic animals (DM + Exe) and exercised diabetic rats treated intraperitoneally with 1 mg/kg CycloRGDyK five days per week (DM + Exe + CycloRGDyK) for eight weeks (Supplementary Fig. 1B). CycloRGDyK is an integrin class specific inhibitor23.24which is widely used in in vivo studies25.26.
After eight weeks of diabetes, the rats were euthanized by carbon dioxide (CO2) asphyxiation. Anesthesia procedures were used in full and every precaution was taken to ensure that animals did not suffer unduly during and after the experimental procedure. Renal cortex and skeletal muscle (gastrocnemius) were harvested and frozen at -80°C for future testing.
Incremental load test and aerobic physical training protocol
Rats were adapted to exercise on a motorized treadmill (AVS Projetos®, São Carlos, São Paulo, Brazil) for five days at 10 min/day, and the exercise intensity was 3 m/min . After two days, the animals were subjected to an incremental load test. The initial intensity of the test was 6 m/min at 0% incline, with increments of 3 m/min, every 3 min, until exhaustion. The exhaustion occurred after the animals reached the end of the treadmill five times. The rate of exhaustion (EV) was determined at the exhaustion point of the test, and 60% of the EV was then used as the physical training intensity27.28. Aerobic exercise training began after induction of diabetes and consisted of four weeks of running on a motorized treadmill for five days a week at 60% VE. Exercise volume was gradually increased (at 15, 30, 45 and 60 min) until the start of the fifth week. After that, the fourth-week exercise volume (60 min) was used over the next four weeks. To compensate for the performance gain during aerobic physical training, an incremental load test was performed before starting the exercise protocol (initial), after four weeks of exercise (4 weeks) and at the end of the aerobic training (8- week), adjusting the EV percentage for the eight weeks (Supplementary Figures 1A and B).
Cell culture and treatments
The human renal proximal tubular cell line (HK-2 cells) was obtained from ATCC (American Type Culture Collection®, Manassas, VA, USA) and maintained in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 1 g/L glucose supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 mg/mL streptomycin at 37°C in 5% CO2 atmosphere. Recombinant irisin was purchased from Adipogen Life Sciences® (San Diego, CA, USA – AG-40B-0136-C010). HK-2 cells from passages 8-12 were left on 1% FBS for 24 h and exposed to normal glucose (NG) 5.6 mmol/L, high glucose (HG) 30 mmol/L and HG plus 5, 15, and 30 ng/mL of recombinant irisin for 48 h. This duration (48 h) was chosen after preliminary testing of cell response to HG under various parameters (Supplementary Fig. 2).
We also investigated the effects of treating HK-2 cells cultured in HG with 4% human serum from non-diabetic, sedentary diabetic, and diabetic control patients undergoing a physical training program. For this purpose, we used five pools of the non-diabetic control group, five pools of sedentary diabetic patients and five pools of diabetic patients subjected to physical training. Each of these pools was prepared with serum from three different patients in each group. Sera from these subjects were provided by one of the authors of this manuscript (CRC). These samples were obtained from individuals enrolled in another study approved by the local institutional review board (Faculty of Medical Sciences (FCM) Research Ethics Board, UNICAMP, 1.597.626, 2.030.070 and 4,505,445). The last amendment to the protocol (4,505,445) allowed the use of the biological material collected for studies such as the current one. All patients signed informed consent prior to study enrollment. All procedures were performed in accordance with the guidelines set forth by the Declaration of Helsinki. The clinical characteristics of these subjects are shown in Supplementary Table 1.
systolic and diastolic blood pressure
After eight weeks of aerobic exercise, systolic and diastolic blood pressures were obtained with an automated optoelectronic device (BP-2000 Blood Pressure Analysis System, Visitech Systems®, Apex, NC). Each systolic and diastolic blood pressure value was taken as the average of three consecutive measurements obtained after stabilization of the readings.
Determination of albuminuria
Albumin concentration was determined using a sample from a 24 h urine collection by ELISA (Nephrat II®; Exocell, Philadelphia, PA). Urinary and serum creatinine was assessed by a commercially available enzymatic test (Labtest Diagnostica SA®, Minas Gerais, Brazil – MS 10009010237). Albuminuria was expressed as urinary albumin to creatine ratio (UACR).
Serum irisin concentration level was determined using a commercial ELISA kit (Phoenix Pharmaceuticals®, Burlingame, CA, USA – EK-067-29).
The kidneys were fixed with 10% buffered formalin and embedded in paraffin for sectioning. Five-micrometer kidney sections were obtained with a microtome (Leica® Microsystems RM2155) and stained with eosin and hematoxylin (HE stain). For the quantification of the glomerular surface, 35 glomeruli from each rat were obtained using a magnification of X630 on an optical microscope (Leica® – DMLB 100S). Quantification of glomerular area was performed using ImageJ Software® (National Institutes of Health, Bethesda, MD).
Immunohistochemistry was performed on 5 mm thick sections mounted on glass slides pre-coated with 2% silane, as previously described.29. The main antibodies used were: (type IV rabbit polyclonal anti-collagen 1:50 – abcam 6586 [Cambridge, UK); anti-rabbit polyclonal fibronectin 1:50 – abcam 2413; anti-mouse monoclonal TNF-α 1:25 – Santa Cruz Biotechnology sc-52746 [Santa Cruz, CA]; and monoclonal anti-mouse F4-80 1:50 Bio-Rad MCA497RT [Richmond, CA]) diluted in 1% milk. After that, 35 glomeruli from each rat were obtained using X630 magnification on an optical microscope (Leica® – DMLB 100S). Quantification of renal densities for type IV collagen, fibronectin, TNF-α, and F4/80 positivity in the zona glomerulus was performed using ImageJ Software® (National Institutes of Health, Bethesda, MD ).
Western blot analysis
Samples (renal cortex and gastrocnemius muscle) and Western blots were prepared and performed as previously described29.30. Protein quantification was determined using Bradford’s method31. Immunoreactive bands were visualized using the enhanced chemiluminescence method (Super Signal CL-HRP Substrate System, Pierce®, Rockford, IL). Protein load uniformity and transfer efficiency were assessed by rescanning the membranes for vinculin (rabbit anti-vinculin monoclonal antibody, 1:1000, Cell Signaling Technology®, Boston, USA) or GAPDH (#5174 ; 1:1000). Images were captured using a digital photodocumentator (ImageQuant LAS 500, GE Healthcare Life Sciences®, Boston, USA) and analyzed quantitatively using ImageJ® software (National Institutes of Health, USA). United). Three independent experiments were performed.
The antibodies used in our experiments are listed in Supplementary Table 2.
Measurement of muscle AMP and ATP levels
Adenosine triphosphate (ATP) and adenosine monophosphate (AMP) were determined in muscle tissue (gastrocnemius) homogenate using the HPLC method, as previously described.32.
Differences between groups were assessed using one-way ANOVA with post-test pairwise comparisons using Fisher’s method. Results are presented as means ± SE. A Pearson correlation coefficient (Pearson r) was used to estimate the correlation between muscle irisin expression and albuminuria, as well as between muscle irisin expression and glomerular expression of fibronectin and renal acetylation of NF-κB (p65). Calculations were performed using GraphPad Prism® 6 software. P values