Tissue analysis.

Renal inner medullary tissue was immediately harvested after the second 30-min urine collection period, and total RNA was isolated using Purelink Mini RNA extraction kit (Ambion) according to the manufacturer's instructions. Then, the isolated RNA was reverse transcribed using QuantiTect Reverse Transcription kit (Qiagen). Finally, the resulting cDNA was used to quantify ET-1 mRNA by Real Time-PCR (CFX96 Real-Time System, Biorad) using TaqMan primer gene expression assays with ET-1 (catalog no. Rn01775763_g1), P2Y₂ receptor (catalog no. Rn02070661_s1), P2Y₄ receptor (catalog no. Rn02133903_s1), P2Y₆ receptor (catalog no. Rn02134326_s1), and GAPDH (catalog no. Rn01775763_g1) primers. We assessed the expression of these three P2Y receptors because UTP can directly activate both P2Y₂ and P2Y₄ and may be converted to UDP that is an agonist for P2Y₆ receptors (11). ET-1 and P2Y receptor gene expression was quantified relative to GAPDH using 2^−ΔΔCt method.