Dopamine Type 2 Receptor (D2r) Expression Modulates The Salt (Nacl) Induced Plasma Membrane Receptor Recruitment Of The Natriuretic Angiotensin Type 2 Receptor (At2r) In Renal Proximal Tubule Cells Obtained From The Urine Of Salt Study Participants.

The effect of D2R expression on the natriuretic AT2R plasma membrane recruitment in high (190 mM NaCl) and normal (140 mM NaCl) salt conditions was measured in human renal proximal tubule cells (RPTC) obtained from urine provided by salt study diet volunteers that were either salt resistant (SR) or inverse salt sensitive (ISS) (increased blood pressure on a low salt diet). Basal D2R expression was 36.9% ± 2.6% lower in ISS vs SR control n=5, p<0.01). D2R expression in the SR was reduced by siRNA (37.1% ± 2.0% reduction in ISS vs SR control, n=5, p<0.01, t-test). The ISS D2R expression was returned to normal levels by D2R overexpression using BacMam technology (22.5% ± 0.9% increase for ISS vs SR control siRNA, n=5, p<0.01, t-test). Culturing SR and ISS cell lines in normal salt (NS, 140 mM NaCl) and high salt (HS, 190 mM NaCl) leads to the recruitment of AT2R to the cell surface in the ISS cell line (ISS Control: NS 47978 ± 2728 RFU vs HS 74056 ± 3002 RFU, n=5, p<0.001, t-test), but not in the SR cell line. Knockdown of D2R using siRNA in the SR cell line altered the HS response in the SR cell line to that measured in the ISS cell line (SR D2R siRNA: NS 48514 ± 2560 RFU vs HS 82599 ± 1492 RFU, n=5, p<0.001, t-test), and the difference between SR HS control siRNA and SR HS D2R siRNA is also highly significant (SR HS: control siRNA 60154 ± 3347 RFU vs SR HS D2R siRNA 82599 ± 1492 RFU, n=5, p<0.001, t-test). Further lowering of D2R in ISS with D2R siRNA still showed a significant translocation of AT2R to the cell surface under HS (ISS D2R siRNA: NS 47953 ± 3058 RFU vs HS 80284 ± 2173 RFU, n-5, p<0.001, t-test) but not a further enhancement over SR siRNA. Overexpression of D2R in the ISS cell line completely blocked the HS AT2R cell surface recruitment, thus converting the ISS HS phenotype of ISS to a cell line that resembled a SR cell line. In conclusion, we have shown that a clear ISS phenotype of HS AT2R plasma membrane recruitment can be replicated in an SR cell line by reducing D2R expression using siRNA to levels seen in an ISS cell line. Similarly, overexpressing D2R in an ISS cell line to a level seen in SR cells reverts the ISS cells back to a SR associated sodium induced AT2R cell surface recruitment.