Immortalized Mouse Renal Proximal Tubule Cells

D₁R Mutants Mistarget to the Non-Raft Domains

Dopamine receptors, notably the D₁ receptor (D₁R), are pivotal in promoting sodium excretion and blood pressure regulation by inhibiting sodium transporters in renal cells. These receptors localize in lipid rafts/caveolae, specialized microdomains within cell membranes enriched with cholesterol and sphingolipids. Understanding the localization and structural requirements of D₁R in these microdomains is essential for elucidating their function in physiological processes such as sodium balance and blood pressure regulation.

Site-directed mutagenesis at ΔD₁R-347S did not affect the receptor's targeting to the plasma membrane, as previously reported. Tiu et al. confirmed this by expressing D₁R-wild-type (WT) and mutants (ΔD₁R-347A, ΔD₁R-351A, ΔD₁R-218S) in human renal proximal tubule cells (hRPTCs), where they localized in the plasma membrane fraction marked by gamma-glutamyl transferase (GGT) (Fig. 1A). Confocal microscopy also verified their colocalization with the plasma membrane (Fig. 2B). In contrast, the ΔD₁R-LL mutant was mainly cytoplasmic. They further assessed the segregation of D₁R-WT and mutants into lipid rafts. Expressed in hRPTCs and separated by sucrose gradient ultracentrifugation, D₁R-WT was present in both lipid raft and non-raft domains, more prominently in the latter (Fig. 2). Conversely, the D₁R mutants were confined to non-raft domains.

Fig. 1. D₁R mutants are targeted to the plasma membrane but not the lipid raft (Tiu AC, Yang J, et al., 2020).

Fig. 2. Lipid raft distribution of D₁R-WT and mutants (Tiu AC, Yang J, et al., 2020).

Gastrin, GLP-1, or Gastrin plus GLP-1 Negatively Regulates the Sodium Transporter/Exchanger/Pump in Human Renal Proximal Tubule Cells (hRPTCs)

Excessive sodium intake can lead to hypertension due to ineffective renal sodium excretion, while efficient sodium excretion maintains normal blood pressure. The gastrointestinal tract regulates sodium balance through hormone secretion, including gastrin and GLP-1. Fan's team investigated their effect on SGLT2, Na-K/ATPase, and NHE3 expressions, and NHE3 phosphorylation in immortalized human renal proximal tubule cells (hRPTCs). After 3 hours of incubation, GLP-1 inhibited SGLT2 expression by 45%, while gastrin had no effect but did not alter GLP-1's inhibition (Fig. 3A). Gastrin decreased Na-K/ATPase expression by 34%, with GLP-1 enhancing this effect (Fig. 3B). Both hormones inhibit NHE3 to induce natriuresis. Gastrin and GLP-1 increased the phosphorylation of NHE3 at serine 552 individually and synergistically (Fig. 3D and E), but only their combination increased phosphorylation at serine 605 (Fig. 3E). These results suggest GLP-1 and gastrin have complementary effects on sodium transport.

Fig. 3. Effect of gastrin and GLP-1 on sodium transporter/pump/exchanger in human renal proximal tubule cells (hRPTCs). The hRPTCs were treated with gastrin (100 nM), GLP-1 (10 nM), or gastrin plus GLP-1 for 3 h (Fan C, Asico LD, et al., 2021).