Mice were placed in individual mouse metabolic cages (Tecniplast, Gazzada, Italy), with access to water and food for 24-h, to collect urine samples for subsequent analyses of albumin and creatinine concentrations. The presence of albuminuria (Albuwell M, Exocell, Philadelphia, PA, USA), urine creatinine levels (Abcam, Cambridge, UK), and serum creatinine concentrations (Abcam, Cambridge, UK) were determined using ELISA kits. Creatinine clearance was calculated using the following standard formula:
Mouse metabolic cages
Manufactured by Tecniplast
Sourced in Italy
Mouse metabolic cages are designed to monitor and collect data on various metabolic parameters of laboratory mice, such as food and water intake, urine, and fecal production. The cages allow for the controlled and isolated measurement of these parameters to support research in areas like energy metabolism, nutrition, and pharmacology.
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5 protocols using mouse metabolic cages
1
Measurement of Albuminuria and Creatinine Clearance in Mice
2
Assessing Renal Function in Mice
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Mice were placed in individual mouse metabolic cages (Tecniplast, Gazzada, Italy) with access to water and food for 24 h. Urine collection was done every four weeks and data from month 18 and 24 was used in this experiment. Albuminuria (Albuwell M, Exocell, Philadelphia, PA, USA) and urine creatinine concentration (The Creatinine Companion, Exocell, Philadelphia, PA, USA) were measured using ELISA kits. Serum creatinine concentrations and Blood urea nitrogen were measured using i-STAT system Cartridges (CHEM8+, Abbott Point of Care, Abbott Park, IL, USA). Creatinine clearance was calculated using a standard formula (urine creatinine (mg/dL) × urine volume (mL/24 h)/serum creatinine (mg/dL) × 1440 (min/24 h)).
3
Tracking Copper Biodistribution in Tumor-Bearing Mice
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BALB/c mice bearing EMT6 tumors in the right flank, with tumor sizes of 4–6 mm at day 0, were divided into three groups (n = 5). Mice in groups 1 and 2 were intratumorally injected with HCuSNPs-CpG (1 mg of Cu per mouse). Mice in group 1 were treated with laser (900 nm, 2.0 W/cm2, 40 s) at 30 min after injection and at day 6. Mice in group 3 without injection were used as control for endogenous Cu. All mice were transferred to mouse metabolic cages (Tecniplast) for urine and feces collection up to 14 days. At day 14, all mice were euthanized. The heart, liver, spleen, lung, kidney, tumor, and tumor-draining lymph nodes were collected. Tissue, urine, and feces samples were digested for the inductively coupled plasma mass spectrometer (ICP-MS) (model: X7, Thermo Electron Corporation) quantitative analysis of Cu according to our previously reported procedure.36 (link) Data of both HCuSNPs-CpG treatment groups were calculated by subtracting endogenous Cu content from the measured Cu values.
4
Metabolic Cage Studies of Mice
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Control and knockout mice from same litter were individually placed into mouse metabolic cages (Tecniplast, Buguggiate, Italy) during 6 days and fed with different salt diets (normal sodium 0.17%, sodium-deficient 0.02% and high sodium 3.5% diet, ssniff Spezialdiäten GmbH, Soest, Germany). During the experimentation, body weight, urine volume, water and food consumption were measured every 12 h at the end of the active night phase (6 a.m. local time) and at the end of the resting day phase (6 p.m. local time), for normal and high sodium diets. For the low sodium diet, metabolic parameters were measured each 6 h (time 0 and time 12 corresponding to 6 p.m. and 6 a.m. local time, respectively). The animals had free access to food and water. At the end of the experiment, mice were sacrificed and organs and blood were collected for further analyses. Supplementary Figure S2 details the used protocols. Each experiment was validated for GR expression by Western blot: only experiments showing at least a 50% GR reduction on total kidney lysates were considered for renal GR deletion. Unless indicated, samples collection was performed at the beginning of the resting light phase (8–10 a.m. local time).
5
Renal Homeostasis Under Dietary Salt
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Metabolic balance studies were performed to analyze the renal sodium, potassium and water homeostasis under normal sodium, sodium-deficient and high sodium and low potassium rescue diet. For time-course analyses, four-to six-week-old control and knockout mice from same litter were individually placed into mouse metabolic cages (Tecniplast, Buguggiate, Italy) and fed with different salt diets for 6 days. During the experimentation, body weight, urine volume, water and food consumption were determined and urine was collected every day at the same time. Experimental animals had free access to food and water during the experimentations in metabolic cages.
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