Animals and housing
Male Sprague-Dawley rats (150-180g) bred and housed in the Biomedical Research Unit (BRU) of the University of KwaZulu-Natal were used in the study. The animals were maintained under standard laboratory conditions of temperature in the range of (22±2 °C), CO2 content (<5000 p.m.), relative humidity (55±5%), and illumination (12 h light/dark cycle, lights on at 07h00). The noise level was maintained at less than 65 decibels as described by Luvuno et al., 2020[50]. The animals were allowed access to food and fluids ad libitum. All animal experimentation was approved by the Animal Research Ethics Committee of the University of KwaZulu-Natal (ETHICS #: AREC/024/0180). The animals were allowed to acclimatize to their new environment for 1 week while consuming standard rat chow and tap water before exposure to the experimental diets [50]. Procedures involving animal care were conducted in conformity with the institutional guidelines for animal care of the University of KwaZulu-Natal in conjunction with the ARRIVE guidelines.
Induction of prediabetes
The animals were randomly assigned to the following diet groups: standard rat chow with normal drinking water (NPD, n=6) and high-fat high-carbohydrate (HFHC) diet with drinking water supplemented with 15% fructose (HFHC+Fructose, n=6) (AVI Products (Pty) Ltd, Waterfall, South Africa). Prediabetes was induced by allowing the animals to feed on the HFHC and fructose diet for 20 weeks, as previously described by Luvuno et al., 2018[8]. After 20 weeks, the American Diabetes Association criteria were used to diagnose prediabetes whereby the criteria to define prediabetes include impaired fasting glucose (IFG) with fasting plasma glucose levels of 5.6 to 6.9 mmol/L, impaired glucose tolerance (IGT) with plasma glucose levels of 7.8 to 11.0 mmol/L 2-hour postprandial, or glycated hemoglobin (HbA1c) of 5.7 to 6.4%. The animals that were fed the standard diet were also tested at week 20 and were found to be normoglycemic and without prediabetes.
OGTT response
The NPD and the PD groups were exposed to an 18-hour fasting period. At the end of the fasting period, their blood glucose was measured (time 0) immediately followed by loading with a monosaccharide syrup (glucose; 0.86 g/kg, p.o.) by oral gavage using an 18-gauge gavage needle that is 38 mm long curved, with a 21/4 mm ball end (Able Scientific, Canning Vale, Australia)[51]. Blood was collected via the tail pick method using a OneTouch select glucometer (Lifescan, Mosta, Malta, United Kingdom) to determine the blood glucose concentration at times 0, 15, 30, 60, 90, and 120 minutes.
Blood pressure measurements
The systolic, diastolic, and mean arterial blood pressure (MAP) was measured at week 20 using the non-invasive tail-cuff method with photoelectric sensors (IITC Model 31 Computerised Blood Pressure Monitor, Life Sciences, Woodland Hills, California, USA) as previously described by Gamede et al., 2019[52]. The equipment was calibrated each day prior to measurements. The animals were kept warm at ± 30°C in an enclosed chamber (IITC Model 303sc Animal Test Chamber, IITC Life Sciences, Woodland Hills, California, USA) for 30 minutes before blood pressure recording. All measurements were conducted at 09h00 as previously described[52].
Blood collection and tissue harvesting
At the end of the experimental period, all animals were anesthetized with isoflurane (100 mg/kg) (Isofur, Safeline PharmAceuticals (Pty) Ltd, Roodeport, South Africa) via a gas anesthetic chamber (Biomedical Resource Unit, UKZN, Durban, South Africa) for 3 minutes. While rats were unconscious, blood was collected by cardiac puncture and then injected into individual pre-cooled heparinized containers. The blood was then centrifuged (Eppendorf centrifuge 5403, Germany) at 4°C, 503 g for 15 minutes. Plasma was collected and stored at -70 °C in a Bio Ultra freezer (Snijers Scientific, Holland) until biochemical analysis as previously described by Luvuno et al., 2020[50].
Biochemical analysis
The kidney NADPH oxidase, SOD, GPX1, and Kim1 as well as plasma insulin, Ang II, and aldosterone concentration were measured using separate, specific ELISA kits according to the manufacturer's instructions (Elabscience and Biotechnology, Wuhan, China). MDA concentration was determined using a previously described protocol by Mkhwanazi et al.,
2014[53].
Quantitative real-time PCR
RNA extraction in the kidney was performed as per the ReliaPrep miRNA Cell and Tissue Miniprep System (Promega, USA). Synthesis of cDNAs was performed by reverse transcription reactions with 2 μg of total RNA using GoTaq® 2-Step RT-qPCR System as a cDNA synthesis kit (Promega, USA) as described by the manufacturer.
The ROCHE light cycler SYBR Green I master mix was used for amplification according to the manufacturer's instructions on the ROCHE light cycler system. The primer sequences (Metabion, Germany) used in this study can be found in Table 1 below. The cycling conditions were: Pre-incubation was carried out at 95℃ for 60s, followed by a 3-step amplification of 45 cycles at 95℃ for 15s, 60℃ for 30s, and 72℃ for 30s. Melting was effectuated at 95℃ for 10s, 65℃ for 60s and 97℃ for 1s. Furthermore, cooling was achieved at 37℃ for 30s. Glyceraldehyde-phosphate dehydrogenase (GAPDH) as an internal control was used as the housekeeping gene. Gene expression values are represented using the 2-ΔΔCt relative quantification method.
Table 1: List of primers used in this study
Table 1: List of primers used
Gene of interest
|
Sequence
|
renin
|
Forward: 5’-GAGG-CCTTCCTTGACCAATC- 3’
Reverse: 5′- TGT-GAATCCCACAAGCAAG-3′
|
angiotensinogen
|
Forward: 5′- GAGTGGGAGAGGTTCTC-AA-3′
Reverse: 5′- TCGTAGATGCGAACAG-GA-3′
|
ACE
|
Forward: 5′- CCATCTGCTAGGGAA-CATGT-3′
Reverse: 5′- GTGTCCATCCCTG-CTTTATCA-3′
|
AT1R
|
Forward: 5’- GCTCACGTG-TCTCAGCAT-3’
Reserve: 5’- TTGGCCAC-CAGCATCGT-3’
|
GAPDH
|
Forward: 5’-AGTGCCAGCCTCGTCTCATA-3’
Reserve: 5’-GATGGTGATGGGTTTCCCGT-3’
|
The urine creatinine ratio was determined using IDEXX vetlab station (USA IDEXX Laboratories, Inc). One IDEXX Drive Westbrook, ME 04092 USA 1-800-548-6733.
Analysis of data
All data were expressed as means ± S.E.M. Statistical comparisons were performed with Graph Pad instat Software (version 5.00, GraphPad Software, Inc., San Diego, California, USA) using Student's unpaired two-sided t-test. A value of p< 0.05 was considered statistically significant.