*4.1. Study Design*

This study was conducted in compliance with established guidelines and the prevailing protocol (S01754) as approved by the Institutional Animal Care and Use Committee (IACUC) at the University of California, San Diego, in accordance with the National Institutes of Health. Recombinant human GH was kindly provided by Genetech (South San Francisco, CA, USA). Six-week-old male c57BL/6J mice were purchased from the Jackson Laboratory (strain: 000664) (Bar Harbor, ME, USA) and used for this study. CKD in mice were induced by 2-stage 5/6 nephrectomy while a sham operation was carried out in control mice [13]. Individual mice were housed in each cage in 12:12 hour light–dark

cycles with ad libitum access to mouse diet 5015 (LabDiet, St. Louis, MO, USA, catalog 0001328, with a metabolizable energy value of 3.59 kcal/g) and water prior to the initiation of the experiment. We performed the following two studies. Study 1: We evaluated the dietary effects of GH in CKD and sham mice. CKD and sham mice were administrated with GH (5 mg/kg/day or 10 mg/kg/day, intraperitoneal) or vehicle (normal saline), respectively. The study period was 42 days, and all mice were fed ad libitum. We measured caloric intake and accompanying weight change in CKD and sham mice. The caloric intake for each mouse was calculated by multiplying total mouse diet consumption during the 42 days (in grams) with the metabolizable energy value of the diet (3.59 kcal/g). Average daily energy intake in mice was expressed as kcal/mouse/day. Study 2: We evaluated the effects of GH in CKD mice beyond nutritional stimulation by employing a diet-restrictive strategy. CKD and sham mice were given GH (10 mg/kg/day, intraperitoneal) or vehicle for 42 days. Each mouse was individually housed during the study period. CKD mice treated with vehicle were fed ad libitum. We measured caloric intake in vehicle-treated CKD mice by multiplying total mouse 5015 diet consumption during the 42 days (in grams) with the metabolizable energy value of the diet (3.59 kcal/g). The average daily energy intake for vehicle-treated CKD mice was calculated and expressed as kcal/mouse/day. We then fed the same amount of mouse 5015 diet based on the recorded average daily energy intake for vehicle-treated CKD mice to other groups of mice, i.e., CKD mice treated with GH (10 mg/kg/day, intraperitoneal) as well as sham mice treated with GH (10 mg/kg/day, intraperitoneal) or vehicle. We fed the mice daily during the daytime (0900-1200). We measured weekly weight change for each mouse. The schematic study plan for the ad libitum and diet-restrictive study is illustrated in Figure 1A,D, respectively.
