Spot urine was collected and 2 μL of each urine sample, and BSA standards at 0, 1, 3, and 10 μg, were separated by SDS‐PAGE on a NuPAGE Novex 4–12% Bis‐Tris gel (Thermo Fisher Scientific). After electrophoresis, the gel was immersed in fixing solution (50% (v/v) methanol (Fisher Scientific), 10% (v/v) acetic acid (Acros)) for 1 h, incubated for 20 min with agitation in staining solution (0.1% (w/v) Comassie brilliant blue R‐250 (Bio‐Rad), 50% (v/v) methanol, 10% (v/v) acetic acid), and finally incubated several times in destaining solution (40% (v/v) methanol, 10% (v/v) acetic acid) until destained.
Urinary albumin content in spot urine was assessed using Albuwell M indirect ELISA (Exocell) and urinary creatinine content was measured, using Creatinine Companion kit (Exocell). Both measurements were performed according to manufacturer′s instructions. Albumin:creatinine ratio was calculated by dividing μg albumin/mL urine with mg creatinine/mL urine, getting the ratio μg albumin/mg creatinine.
Urinary albumin content in spot urine was assessed using Albuwell M indirect ELISA (Exocell) and urinary creatinine content was measured, using Creatinine Companion kit (Exocell). Both measurements were performed according to manufacturer′s instructions. Albumin:creatinine ratio was calculated by dividing μg albumin/mL urine with mg creatinine/mL urine, getting the ratio μg albumin/mg creatinine.