Elisa kits specific for il 6

Supernatant, serum, or urine protein was analyzed by ELISA kits specific for IL-6 (BD Biosciences), ICAM-1 (R&D Systems), NGAL, and TREM-1 (Invitrogen, Thermo Fischer Scientific) according to manufacturers’ instructions. HRGEC were dissolved in 100 μL of in-house prepared RIPA lysis buffer. The kidney tissue was crushed in liquid nitrogen and ∼100 mg of powdered tissue was dissolved in 500 μl of in-house prepared RIPA lysis buffer. All samples were sonicated on ice and protein concentration was determined by Bradford protein assay reagent (Bio-Rad). 40 μg of protein from HRGEC was used for assessment of IL-6 and 50 μg of kidney protein was used for assessment of ICAM-1 using respective ELISA kits. Supernatant from HRGEC was analyzed for 105 cytokines using a proteome profiler cytokine array (R&D Systems) per manufacturer’s instructions and resultant pixel density was calculated using ImageJ software.