Creatinine assay

Urine albumin and creatinine concentrations were measured using a mouse albumin ELISA assay (Bethyl Laboratories) and a creatinine assay (R&D systems), respectively, according to the manufacturer’s directions. Urine albumin excretion was expressed as the ratio of urine albumin to creatinine (ACR).

MicroRNA was reverse transcribed into cDNA using the TaqMan MicroRNA Reverse Transcription kit (Applied Biosystems, Paisley, UK) and messenger RNA (mRNA) was reverse transcribed using the High Capacity Reverse Transcription kit (Applied Biosystems) according to manufacturer's instructions. Relative microRNAs and mRNA levels were determined by real time, quantitative PCR using TaqMan Fast Universal Master Mix (Applied Biosystems). Quantitative PCR was performed in triplicate on the Applied Biosystems 7,500 Fast System. All PCR and RT probes were obtained from Applied Biosystems. Urinary microRNA expression was first normalized to creatinine to correct for diuretic effect (Creatinine Assay, R & D Systems, Abingdon, UK) and then normalized to cel. miR-39. Cellular microRNA expression following HK-2 transfections was normalized to U6 small nuclear RNA and mRNA expression was normalized to GAPDH, a frequently used housekeeping gene. Relative expression was calculated using the 2^−ΔCt method (33 (link)).

Urine was collected and the albumin concentration was measured with a mouse albumin ELISA (Bethyl laboratories, Montgomery, TX). A creatinine assay (R&D systems, Minneapolis, MN) was used to determine urine creatinine concentrations. Results are expressed as ACR in μg of albumin per mg of creatinine.