Model 2002

Rats were placed in a stereotaxic frame (David Kopf Instruments; Tujunga, CA, USA) and the LHA was targeted bilaterally using a 25-gauge needle (Hamilton; Reno, NV, USA) using the following stereotaxic coordinates: 2.85 mm posterior to the bregma, ±2 mm lateral to the midline and 8.1 mm ventral [6 (link)]. One µl at each injection site of AAV vectors (1 × 10⁹ genomic copies µL⁻¹) encoding null or Opkr1 short-hairpin RNAs [6 (link),18 (link)] were delivered at a rate of 200 nL min⁻¹ for 5 min. Fourteen days after AAV injection, an osmotic minipump flow moderator (Model 2002, Alzet, DURECT Corporation; Cupertino, CA, USA) containing a daily dose of 4 mg/ kg of nicotine (nicotine–hydrogen–tartrate salt, Sigma-Aldrich; St Louis, MO, USA) or saline was implanted subcutaneously. Food and body weight were measured for 14 days.

All animal protocols conform to the National Institute of Health's Guide for the Care and Use of Laboratory Animals and were approved by the University of Pittsburgh IACUC. Male C57Bl/6 mice (Jackson Laboratories) were housed in a temperature‐controlled facility on a 12 h light/dark cycle. They were kept on standard laboratory chow, a 10% KCl diet (Envigo TD.09075) for 4 days, an 8% NaCl diet (Envigo TD.92012) for 4 weeks, or given aldosterone at a rate of 240 μg/kg/day via a subcutaneous minipump (DURECT Corporation, model 2002) for 2 weeks before sacrifice. Kidneys were collected and flash frozen or fixed in 4% paraformaldehyde for 24 h before being embedded in OCT.