*2.3. Animals and Experimental Protocols*

Male Wistar rats weighing 150–200 g (National Laboratory Animal Center, Mahidol University, Salaya, Thailand) were housed in a controlled environment (24 ± 1 ◦C, 55 ± 5% humidity, 12 h light/dark cycle) with free access to food and water. All procedures complied with the guidelines established by the National Research Council of Thailand and were approved by the Animal Care and Use Committee of the Faculty of Medicine, Chiang Mai University (Project Number: 41/2559).

After acclimatization, rats were assigned to receive a normal diet (ND, *n* = 6) or a high-fat diet (HFD, *n* = 24). The diet composition is shown in the Appendix A (Table A1). Diabetes was induced in the HFD-fed rats two weeks later by a single intraperitoneal injection of streptozotocin (35 mg/kg in 0.1 M sodium citrate buffer, pH 4.5) [20], while the ND-fed rats received an equal amount of sodium citrate buffer. Two weeks after injection, rats with fasting blood glucose ≥250 mg/dL without hypoinsulinemia were considered diabetes and were included in the study.

Diabetic rats were randomly divided into 4 groups (*n* = 6 each): diabetic control group received vehicle (DMV), diabetic positive control group treated orally with metformin 50 mg/kg/day (DMM), diabetic group treated orally with PRHE 300 mg/kg/day (DME), and diabetic group treated with a combination of metformin and PRHE (DMME). The dose of PRHE was based on our preliminary results showing its efficacy in improving renal function together with lowering blood glucose and insulin resistance appropriately (Appendix A, Table A2). All diabetic rats were maintained on HFD for a further 12 weeks, whereas those in the nondiabetic group received a normal diet and vehicle (NDV). The 24 h urine samples were collected at the end of the study using metabolic cages, and blood and kidney tissues were collected thereafter under thiopental anesthesia (60 mg/kg, i.p.). Parts of the kidneys were rapidly taken for mitochondrial and histopathological studies. The remainders were snap-frozen in liquid nitrogen and stored at −80 ◦C for further analysis.

#### *2.4. Biochemical Assays*

#### 2.4.1. Determinations of Metabolic Indexes

Fasting blood glucose, cholesterol, and triglycerides were measured by enzymatic assay kits (ERBA Diagnostics Inc., Miami, FL, USA). Plasma insulin was quantified using ELISA kit (Millipore, Burlington, MA, USA). The Homeostasis Model Assessment for Insulin Resistance was calculated (HOMA-IR = fasting insulin level (ng/mL) × fasting glucose (mg/dL)/405.1) as described previously [21].
