HK-2 human renal proximal tubule cells as a model for G protein-coupled receptor kinase type 4-mediated dopamine 1 receptor uncoupling

Abstract

HK-2 human renal proximal tubule cells (RPTC) are commonly used in the in vitro study of "normal" RPTCs. We discovered recently that HK-2 cells are uncoupled from dopamine 1 receptor (D(1)R) adenylyl cyclase (AC) stimulation. We hypothesized that G protein-coupled receptor kinase type 4 (GRK4) single nucleotide polymorphisms may be responsible for the D(1)R/AC uncoupling in HK-2. This hypothesis was tested by genotyping GRK4 single nucleotide polymorphisms, measuring D(1)-like receptor agonist (fenoldopam)-stimulated cAMP accumulation, quantifying D(1)R inhibition of sodium transport, and testing the ability of GRK4 small interfering RNA to reverse the D(1)R/AC uncoupling. We compared HK-2 with 2 normally coupled human RPTC cell lines and 2 uncoupled RPTC cell lines. The HK-2 cell line was found to have 4 of 6 potential GRK4 single nucleotide polymorphisms known to uncouple the D(1)R from AC (namely, R65L, A142V, and A486V). AC response to fenoldopam stimulation was increased in the 2 normally coupled human RPTC cell lines (FEN: 2.02+/-0.05-fold and 2.33+/-0.19-fold over control; P<0.001; n=4) but not in the 2 uncoupled or HK-2 cell lines. GRK4 small interfering RNA rescued the fenoldopam-mediated AC stimulation in the uncoupled cells, including HK-2. The expected fenoldopam-mediated inhibition of sodium hydrogen exchanger type 3 was absent in HK-2 (n=6) and uncoupled RPTC cell lines (n=6) but was observed in the 2 normally coupled human RPTC cell lines (-25.41+/-4.7% and -27.36+/-2.70%; P<0.001; n=6), which express wild-type GRK4. Despite the fact that HK-2 cells retain many functional characteristics of RPTCs, they are not normal from the perspective of dopaminergic function.

Figure 1. Comparison of fenoldopam-stimulated coupling efficiency to adenylyl cyclase in HK-2 and immortalized human cell lines, uRPTC (i2, i25) and nRPTC (i14, i16)

cAMP accumulation was compared in HK-2, i2, i25, i14, and i16. Innate cAMP accumulation was similar in all cell lines (Vehicle (VEH)-treated cells). The D₁-like receptor agonist fenoldopam (FEN, 1 µmol/L, 30 min) stimulated cAMP accumulation in the coupled cell lines i14 and i16 (*P<0.001 vs others, N=6/group) but not in the uncoupled HK-2, i2, or i25 cells. The D₁-like receptor antagonist LE300 (10 µmol/L) had no effect alone, but reversed the FEN stimulation observed in i14 and i16.

Figure 2. Failure of FEN-stimulated cAMP accumulation in HK-2 cells can be reversed by the silencing of G protein-coupled receptor kinase type 4 (GRK4)

Intracellular cAMP accumulation was measured in real-time using a cAMP FRET Biosensor, ICUE3. Similar to cAMP accumulation demonstrated in Figure 1, FEN (1 µmol/L, 30 min) increased cAMP in i14 and i16 (*P<0.001 vs VEH, N=6/group), but not in HK-2, i2 or i25. Scrambled RNA control (SCR) had no effect on the FEN-stimulated cAMP accumulation in the coupled cell lines, i14 and i16. GRK4 siRNA rescued FEN-stimulated cAMP accumulation in HK-2, i2, and i25 cell lines, although not to control levels (#P<0.05 vs FEN + SCR, N=4/group).

Figure 3. GRK4 immunofluorescence in HK-2, uRPTC i2 and i25, and nRPTC i14 and i16, that were transfected with GRK4 siRNA or the scrambled control (SCR)

Cells were transfected with 100 nmol/L GRK4 siRNA or SCR and 24 hr later were stained for GRK4. GRK4 siRNA significantly knocked down GRK4 expression in each of the cell lines (P<0.001, N=6). Representative images of GRK4 expression with SCR or GRK4 siRNA are shown above each bar in the graph.

Figure 4. FEN reduces intracellular sodium accumulation in i14 and i16 but not in HK-2, i2, or i25 cell lines

Intracellular sodium concentration was determined using the sodium sensitive dye SBFI. The ouabain (OUB, 100 µmol/L plus vehicle, VEH)increased intracellular accumulation, by inhibition of sodium efflux via NaKATPase, was linear over a 30 minute time interval. OUB + FEN (1 µmol/L) reduced Na+ accumulation (*P<0.05, N=6) in only the i14 (□) and i16 (x) coupled cell lines.

Figure 5. Fenoldopam-mediated reduction in NaKATPase-dependent sodium efflux is greater in nRPTCs than uRPTCs

Cells labeled with the sodium sensitive dye SBFI were sodium loaded by incubating the cells in potassium-free media, and ratiometric fluorescence images were collected simultaneously on an automated confocal fluorescence microscope. The two adenylyl cyclase normally coupled cell lines i14 and i16 had a large decrease in sodium influx upon agonist stimulation (**P<0.01 vs VEH, ANOVA, Holm-Sidak test, n=6/group) but the two uncoupled renal proximal tubule cells (i2 and i25) had no decrease. HK2 cells displayed a small but significant fenoldopam-mediated reduction in sodium efflux although it was less than i14 and i16 (* P<0.05 vs VEH, i14 and i16, ANOVA, Holm-Sidak test, n=6/group).