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Published in final edited form as: Hypertension. 2010 Jul 26;56(3):505–511. doi: 10.1161/HYPERTENSIONAHA.110.152256

The HK-2 human renal proximal tubule cell as a model for GRK4-mediated dopamine-1 receptor uncoupling

John J Gildea 1, Ishan Shah 1, Ryan Weiss 1, Nicholas D Casscells 1, Helen E McGrath 1, Jin Zhang 2, Robin A Felder 1
PMCID: PMC3537327  NIHMSID: NIHMS229328  PMID: 20660820

Abstract

HK-2 human renal proximal tubule cells (RPTC) are commonly used in the in vitro study of “normal” RPTCs. We recently discovered that HK-2 cells are uncoupled from dopamine-1 receptor (D1R) adenylyl cyclase (AC) stimulation. We hypothesized that G protein coupled receptor kinase type 4 (GRK4) single nucleotide polymorphisms (SNPs) may be responsible for the D1R/AC uncoupling in HK-2. This hypothesis was tested by genotyping GRK4 SNPs, measuring D1-like receptor agonist (fenoldopam)stimulated cAMP accumulation, quantifying D1R inhibition of sodium transport, and testing the ability of GRK4 siRNA to reverse the D1R/AC uncoupling. We compared HK-2 to 2 normally coupled human RPTC cell lines (nRPTC) and 2 uncoupled RPTC cell lines (uRPTC). The HK-2 cell line was found to have 4 out of 6 potential GRK4 SNPs known to uncouple the D1R from AC (namely R65L, A142V, and A486V). AC response to fenoldopam stimulation was increased in the two nRPTC 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 two uncoupled or HK-2 cell lines. GRK4 siRNA 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 uRPTCs (N=6), but was observed in the two nRPTCs (−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.

Keywords: HK-2, renal proximal tubule, dopamine receptors, GRK4, NHE3, FRET, NaKATPase

Introduction

The HK-2 human renal proximal tubule cell (RPTC) has been the subject of research interest since it was first isolated and cultured in 1984,1 with over 170 publications using this cell line listed in PubMed. The relevance of using RPTCs of human origin to study human physiology and pathophysiology over using those derived from non-human sources, such as the MDCK (dog), LLC-PK (pig), LLC-RK1 (rabbit) and OK (opossum) can not be overemphasized. Initially, primary RPTC lines were used to study renal cellular physiology since they retained many of their in vivo characteristics in vitro.2 However, primary cell lines cannot be sustained in long-term culture, which makes inter-assay comparisons difficult.1, 3 Therefore, transformed cell lines were generated using immortalizing virus (e.g. SV40, human papilloma virus (HPV)) which extended their growth potential beyond the 8–15 passage limit of primary cells.4–5 Transformed RPTCs contain many of the functional and morphological characteristics of RPTCs in primary culture.5–6 There are instances where the HK-2 do not always accurately represent normal human RPTC physiology. For example, when cultured in a 3-dimensional matrix, HK-2 form aggregates or cysts similar to primary cultures of RPTCs from autosomal dominant polycystic kidney disease, as compared to primary cultures of RPTCs from normal kidney which form tubular structures.7

While using HK-2 cells as normal controls for human primary renal proximal tubule cells, we recently discovered that HK-2 cells are uncoupled from dopaminergic stimulation of adenylyl cyclase (AC). Because we have previously reported that the uncoupling of the dopamine-1 receptor (D1R) from AC is due to variants of single nucleotide polymorphisms (SNPs) of G protein-coupled receptor kinase type 4 (GRK4), we hypothesized that the uncoupling of D1R to AC in HK-2 cells may also be caused by GRK4 SNPs.8 This hypothesis was tested in HK-2 cells by genotyping GRK4 SNPs, measuring D1-like receptor agonist (fenoldopam) stimulated AC, quantifying D1R inhibition of sodium transport, and testing the ability of GRK4 siRNA to rescue the D1R/AC coupling. We compared the HK-2 RPTCs with human RPTCs that are normally coupled (nRPTC) or uncoupled (uRPTC) from AC in order to determine if HK-2 is suitable for the study of normal dopaminergic activity and cellular function.

Materials and Methods

(Please see http://hyper.ahajournals.org for Expanded Methods in Online Supplement)

Cell lines

Using previously published methods, we cultured HK-2 cells (ATCC®CRL-2190™), and compared them to 4 immortalized human RPTC lines routinely used in our laboratory (2 normally D1R/AC coupled nRPTC lines i14 and i16, and 2 uncoupled uRPTC lines i2 and i25).9–10 Our cell lines were obtained from IRB-approved normal tissue from nephrectomies in human subjects. Cell culture, characterization and immortalization procedures have been described.4, 8–9, 11–13 Each cell line has been genotyped for 3 single nucleotide polymorphisms (SNPs) to GRK4 (R65L (exon 3), A142V (exon 5), A486V (exon 14)) (Table 1) using established methods.14 In order to obviate the possibility that cell culture conditions were responsible for the aberrant behavior of the HK-2, we cultured HK-2 in keratinocyte-free media as suggested by the supplier, as well as in media used routinely in our laboratory for growing immortalized human renal proximal tubular cells.9, 12 Furthermore, we obtained fresh HK-2 cells from the ATCC (Manassas, VA) and compared them to our stock cells originally obtained from the ATCC several years ago.

Table 1.

Sequencing of Cell Lines for GRK4 SNPs

Cell Line R65L (exon 3) A142 (exon 5) A486 (exon 14)
HK-2 HETERO* HOMO HETERO
i2 (uRPTC) HOMO† HOMO WILD
i25 (uRPTC) HETERO HETERO HOMO
i14 (nRPTC) WILD‡ WILD WILD
i16 (NRPTC) WILD WILD WILD
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†

HOMO = homozygous variant,

*

HETERO = heterozygous variant,

‡

WILD = wild-type pmoles cAMP/mg Protein

Immunofluorescent Staining of HK-2 and Human RPTC for Proximal Tubule Characteristics

Markers of proximal tubules used were: Lotus tetragonolobus agglutinin (LTA), γglutamyl transpeptidase (GGT), megalin (MEG), aminopeptidase A (APA), aminopeptidase N (APN / CD13) and villin. Levels of genes expressed in proximal tubule as well as other segments include NHE3 (proximal tubule and thick ascending limb15 and caveolin-1 (proximal tubule, distal tubule, glomerulus and cortical collecting duct).16–17 Markers of cells from other nephron segments which served as negative controls were Tamm-Horsfall protein (THP) 18 and sodium chloride co-transporter (NCC).19 Controls for non-specific secondary antibody binding were performed for each cell type. See the Online Supplement and Table S1 for details about the antibodies and staining.

Determination of cAMP Accumulation

cAMP was measured both by a commercial ELISA (Cayman Chemical) and an intracellular real-time kinetic fluorescence resonance energy transfer (FRET) cAMP sensor (ICUE3) as previously described.10, 20 Agonists used were fenoldopam (FEN, 1 µmol/L, D1-like receptor agonist), and isoproterenol (ISO, 1 µmol/L, beta-adrenergic receptor agonist) in order to compare alternative adenylyl cyclase G protein coupled receptor pathways.

Rescue of cAMP Coupling following Transfection with GRK4 siRNA

100 nmol/L GRK4 siRNA (target sequence 5’ AATACAAAGAGAAAGTCAA 3’) or scrambled control (5’AGAAGATAAGAACAATAAC 3’) was transfected for 24 hours into cells by electroporation along with the ICUE3 cAMP biosensor plasmid. Details of transfection have been previously published. 10

Immunofluorescent Staining for GRK4 following Transfection with GRK4 siRNA

The cell lines were stained for GRK4 after being transfected with GRK4 siRNA or SCR control as stated above (without the ICUE biosensor). Immunofluorescent staining was performed as described above, using the Santa Cruz GRK4 antibody (SC-13079) at a 1:100 dilution. Identical exposures were taken at 100x magnification and quantitated using Slidebook v.4.2 software.

NHE3-mediated Sodium Accumulation Assay

NHE3 activity as a function of sodium accumulation was measured with modifications of our previous method.21 Details of our current method are in the Online Supplement. Briefly, cells were loaded with a sodium ion indicator, sodium benzofuran isophthalate (SBFI), and treated for 30 minutes with combinations of the following drugs: ouabain (OUB, 100 µmol/L, NaKATPase inhibitor), fenoldopam (FEN, 1 µmol/L, D1-like receptor agonist), LE300 (10 µmol/L, D1-like receptor antagonist), EIPA (10 µmol/L, NHE inhibitor), S3226 (10 µmol/L, NHE3 inhibitor) 22–23 and cariporide (HOE-642, 10 µmol/L, NHE1 inhibitor).24 The specificity of the assay was verified by the positive inhibition seen with S3226 (NHE3 selective) and negative inhibition seen with cariporide (NHE1 selective). Time-lapse ratiometric images were acquired every three minutes. Each intracellular sodium measurement (mmol/L) is derived from the emission at 510 nm when excited at 340 or 380 nm. Internal calibration of sodium concentration was performed according to the manufacturer’s instructions in Slidebook™ version 4.2, using calibration buffers described previously.21

NaKATPase-mediated Sodium Efflux Assay

The detailed method used to measure the rate of sodium efflux was perfomed simultaneously on the five cell lines as previously published.10 In brief, cells were cultured in glass bottom collagen-coated Matrical plates (Spokane, WA) and were serum-starved overnight prior to loading with a sodium ion indicator, sodium benzofuran isophthalate (SBFI, 5µM, Molecular Probes, OR). After a 30 minute recovery, cells were washed and incubated in potassium-free HEPES media (20 mM HEPES pH 7.4, 130 mM NaCl, 1 mM CaCl, 1 mM MgCl) to raise the internal sodium concentration. VEH, FEN, or OUB were added, then directly before imaging, EIPA (10 uM final concentration, a selective inhibitor of the Na+/H+ exchanger 3 (NHE3),) and KCl (2.7 mM final concentration) were added to all wells as 10X stock. Changes in intracellular sodium concentration were measured by live multiwell ratiometric fluorescence imaging of SBFI and internally calibrated using the ratio imaging module of Slidebook™ version 4.2.

Statistical Analysis

The data are expressed as mean ± SE. Comparisons within and among groups were made by repeated measures or factorial ANOVA, respectively, followed by Holm-Sidak or Duncan’s test. T-test was used for two-group comparisons. P values of <0.05 were considered significant.

Results

Our RPTC lines, as well as the HK-2 line, were characterized for their renal proximal tubule origin using a series of selective stains for proximal tubular proteins and carbohydrates (online supplement Figure S1). All of the primary and immortalized cell lines showed positive staining for the proximal tubule markers and were definitively negative for the markers for other nephron sites.

The HK-2 cells had virtually non-measurable expression of a proximal tubule specific marker, APN25, a markedly reduced expression of another proximal tubule specific marker, Lotus tetragonolobus agglutinin (LTA)26 and a slightly reduced expression of another proximal tubule specific marker, APA 27, when compared to our cell lines i2, i25, i14, and i16 (Figure S1, top panel).

In order to determine if the D1R were normally coupled to AC in HK-2 cells, we employed two methods for measuring intracellular cAMP.10 An established ELISA method to measure cAMP extracted from the cell cytoplasm (Figure 1) was compared to the intracytoplasmic fluorescent resonant energy transfer (FRET)-based sensor (Figure 2). We compared HK-2 with two normally coupled (nRPTC) and two uncoupled (uRPTC) lines, in their response to the dopamine D1-like receptor agonist fenoldopam (FEN, 1 µmol/L, 30 min), or DMSO vehicle (VEH) control. Figure 1 shows that there was no response to FEN stimulation in HK-2, which is similar to that seen in the uncoupled uRPTC lines (i2 and i25). However, there was a greater than 2-fold increase in cAMP accumulation (ELISA) following fenoldopam stimulation in nRPTCs (i14 and i16) (2.02 +/− 0.05 and 2.33 +/− 0.19-fold, respectively; P<0.001, N=4 vs VEH). This effect was blocked by the D1-like receptor antagonist, LE300 (10 µmol/L), indicating that the stimulatory effect was via D1-like receptors. LE-300 had no effect when added alone. The HK-2 cells showed a similar lack of AC stimulation by FEN when measured using a cAMP FRET Biosensor, ICUE3. D1-like stimulation with fenoldopam (FEN, 1 µmol/L, 30 min) caused a significant rise in intracellular cAMP levels in nRPTCs (i14, i16), but not in HK-2 cells or uRPTCs (i2, i25) (Figure 2).

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).

Figure 1

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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).

Figure 2

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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).

This lack of response to fenoldopam by the uncoupled cells was not due to an intrinsic defect in their Gs coupling, as is shown in the online supplement Figure S2. The beta-adrenergic receptor agonist isoproterenol (ISO, 1 µmol/L, 30 min) significantly increased cAMP accumulation in each of the cell lines (*P<0.001 vs VEH, N=10). In addition, the response of the uRPTCs i2 and i25 was significantly higher than that seen in their normally coupled counterparts, i14 and i16, or the HK-2 (**P<0.01, N=10 vs the other 3 cell lines, P< 0.001 from VEH).

We then investigated the role of GRK4 SNPs in the uncoupling of AC in the HK2, i2 and i25 cell lines by treating the cells with either scrambled (SCR) or siRNA to GRK4 (Figure 2) in addition to fenoldopam. siRNA to GRK4 rescued the uncoupling of AC in the HK-2, i2, and i25 cell lines (P<0.05, N=4 vs FEN + SCR), suggesting that GRK4 SNPs mediated the uncoupling of the D1-like receptor to AC. siRNA slightly decreased the FEN stimulation of AC in nRPTCs (i14 and i16), which is in agreement with our reports that wild-type GRK4 is needed for normal D1R function. 28–29

Figure 3 demonstrates that basal levels of GRK4 were similar among the 5 cell lines. GRK4 expression was quantified and equivalent immunoreactive staining was observed in all cell lines. This figure also demonstrates the efficacy of the GRK4 siRNA used in Figure 2. The cell lines showed a 63–83% reduction in GRK4 expression level after they were transfected with 100 nmol/L GRK4 siRNA for 24 hours, compared to the SCR transfected cells. Equivalent expression levels of GRK4 were seen between the SCR control cells and mock transfected cells without siRNA (data not shown).

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).

Figure 3

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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.

Since a normal action of dopamine is to inhibit sodium transport via NHE3,30 we investigated whether D1R/AC uncoupling decreased intracellular sodium accumulation in these cells. Figure 4 shows the effect of ouabain (OUB, 100 µmol/L) in increasing intracellular sodium concentration by preventing sodium efflux via inhibition of NaKATPase activity. This effect was reduced by FEN (10 µmol/L) in nRPTC lines i14 and i16 (P<.05 vs OUB + VEH, N=6 at the 21-, 24-, 27- and 30-minute time points). However, in the HK-2 cells or uRPTCs i2 and i25, FEN had no effect on the ouabainmediated increase in intracellular sodium. These results are summarized in the online supplement Figure S3, with the OUB + FEN data expressed as a percentage of the OUB + VEH control for each cell line at the 30-minute time point. At this time point, the inhibition in coupled RPTC lines was a 25.41±4.67% decrease for i14 and a 27.36±2.70% decrease for i16 (P<0.001 vs OUB + VEH, N=6).

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

Figure 4

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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.

Additional controls for the sodium influx assay are provided in the online supplement Figure S4. This figure depicts the change in intracellular sodium over the 30-minute time interval tested, in response to various pharmacological agents by themselves or in combination with OUB. FEN (1 µmol/L), cariporide (HOE, 10 µmol/L) and LE300 (10 µmol/L) by themselves had no effect on sodium influx over VEH alone. OUB (100 µmol/L) increased intracellular sodium while the NHE3 inhibitors EIPA (10 µmol/L) and S2336 (10 µmol/L) by themselves decreased intracellular sodium concentrations, relative to VEH. The combination of OUB + FEN inhibited sodium influx by 32+/−0.48% (P<0.05, N=6) compared to OUB alone. This effect of fenoldopam was reversed by LE300 (OUB + FEN + LE300), confirming the specificity of the FEN effect via D1-like receptors. The addition of either NHE3 inhibitor EIPA or S3226 almost completely blocked the OUB-mediated increase in intracellular sodium concentration. The addition of the NHE1 inhibitor, cariporide (HOE) did not reduce the OUB response, indicating that the increase in intracellular sodium concentration with OUB is not through NHE1 but rather through NHE3.

Since dopaminergic stimulation of RPTCs also causes an inhibition of NaKATPase activity,31 we compared these 5 cell lines in our NaKATPase-dependent sodium efflux assay. Fenoldopam inhibited sodium efflux to a greater extent in normally coupled renal proximal tubule cells compared to the uncoupled cell lines (Figure 5). The two adenylyl cyclase coupled cell lines i14 and i16 had a large decrease in sodium efflux upon agonist stimulation (**P<0.01 vs VEH, ANOVA, Holm-Sidak test, n=6/group) but this was not seen in the two uncoupled renal proximal tubule cells (i2 and i25). HK-2 cells displayed a fenoldopam-mediated reduction in sodium efflux, although it was significantly less than that seen in i14 and i16 (* P<0.05 vs VEH, i14 and i16, ANOVA, Holm-Sidak test, n=6/group).

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

Figure 5

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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).

The three GRK4 SNPs R65L, A142V, and A486V are associated with human essential hypertension or salt-sensitive hypertension 8, 14, 32–35, and cause the uncoupling of the D1R to AC. Table 1 shows that the HK-2 cell line is homozygous variant at exon 5, and heterozygous variant at exons 3 and 14, as compared to our control cell lines i14 and i16, which are wild-type at all 3 GRK4 exons and have normal adenylyl cyclase coupling.

Discussion

The HK-2 cell line has been relied on as a surrogate for “normal” renal proximal tubular physiology for over two decades. HK-2 cells have been reported to express parathyroid hormone receptor which participates in the proliferative response after energy depletion36, but other cellular parameters do not always show concordance with primary cultures of RPTCs (e.g., low basal angiotensinogen, basal NF-kB and STAT3 activities, and total protein expression).37 In the current study, we found that HK-2 cells had virtually non-measurable expression of APN25, and a reduced expression of the proximal tubule specific markers LTA27 and APA 25, when compared to our human RPTC lines i2, i25, i14, and i16. The differential expression of cell surface markers led us to examine dopamine-stimulated AC activity and sodium transport in HK-2 cells.

In the current studies, we demonstrate the novel finding that the D1-like stimulation in the HK-2 is uncoupled from D1-like stimulation of AC as well as D1-like stimulation of sodium influx (NHE3 activity), and displayed a blunted D1-like stimulated reduction in sodium efflux (NaKATPase activity). The fact that beta-adrenergic receptor stimulation of AC remains intact suggests that the uncoupling phenomenon is unique to the D1-like receptors. Similar to our previous findings, the siRNA silencing of GRK4 (which normally prevents the phosphorylation and inhibition of the D1-like receptors) restores D1-like coupling to AC in the uncoupled cells.

Zhang et al demonstrated normal coupling between D1R-like stimulation with fenoldopam and inhibition of NaKATPase activity in HK-2 cells .38 Their results suggest the presence of functional D1-like receptors in HK-2 cells, yet in our study HK-2 cells show a reduced inhibition of NaKATPase activity compared to normally coupled RPTC cells. Because GRK4 may not regulate D5R, the other D1-like receptor, 39 it is possible that the cell culture conditions of Zhang et al increased the expression of the D5R, overcoming the impaired D1R function, thus allowing the fenoldopam to work through the D5R. PMA, a known stimulator of the protein kinase C (PKC) pathway, has been shown to inhibit NaKATPase independently of cAMP.40 PTH has also been shown to inhibit NaKATPase independently of cAMP.41 However, the role of the D5R in the fenoldopam-mediated inhibition of NHE3 or NaKATPase in HK-2 cells remains to be further evaluated. One other possibility for our results differing from Dr. Zhang’s is that they may be using an entirely different subclone of HK-2 cells. 42, 43 The present findings demonstrate that GRK4 may be a potential therapeutic target, and that uncoupled cells may serve as a tool for screening for compounds with therapeutic potential. Moreover, there are significant cost and efficiency benefits to using cells expressing the intracellular cAMP sensor ICUE3 for high throughput screens. The D1R expressed in the HK-2 cell was uncoupled from AC similar to our two D1R uncoupled cell models.9, 11, 44 The presence of gene variants at three exons in GRK4 in the HK-2 cell line is in keeping with the known effects of GRK4 variants on uncoupling of the D1R to AC. We have previously demonstrated that variants R65L and A142V independently reduce the coupling of the D1R to AC by approximately 50%, while A486V is more potent at reducing D1R/AC coupling.8 When the expression of GRK4 was decreased with antisense oligonucleotides, normal coupling was restored between the D1R and AC.11 The fact that the uncoupling could also be reversed by the addition of siRNA to GRK4 in HK-2 cells suggests that both the HK-2 and our uncoupled cell lines possess similar mechanisms responsible for the uncoupling phenomenon.

We have reported that GRK4 gene variants are associated with an increased incidence of hypertension in several populations.14, 34, 45–46 These reports have been corroborated by others, in hypertensive Han Chinese32 and a white Australian cohort35 but not in an European cohort.47 However, in the same European population, polymorphisms in the GRK4 promoter were found to affect transcriptional activity.31, 48 The possibility that increased GRK4 expression may also be a cause of hypertension is supported by in vivo studies in rats: chronic renal silencing of GRK4 in spontaneously hypertensive rats (that over-express GRK4 but do not have an altered GRK4 genotype) resulted in about a 30% reduction in blood pressure.49 Thus, there is a need for human cell lines with various GRK4 polymorphisms to test the impact of these gene variants on RPTC function. Future studies will focus on the association between GRK4 genotype and the hypertension/salt sensitivity phenotype.

Supplementary Material

01

Perspectives.

Renal cell culture models must be used with caution since they may contain genes that are not associated with normal cell function. Moreover, the disease phenotypes that are encoded in the genome and possibly expressed, are usually not available for the various renal cell lines that are available to study. In these studies, we demonstrated that the widely used HK-2 cell line carries homozygous SNPs at one GRK4 allele, and heterozygous SNPs at 2 other alleles that are associated with either hypertension and/or salt sensitivity. The presence of these variants was associated with uncoupling of the D1R from AC, similar to that found in two human RPTC lines carrying more than three GRK4 variants. Two control cell lines that do not carry the GRK4 variants had normal D1R/AC coupling. These results suggest that the HK-2 cell line may be a good model for cellular physiology associated with GRK4 variants only when used in conjunction with additional cell models.

Acknowledgments

Sources of Funding: HL074940, HL23081, DK39308, HL68686, and HL092196.

Footnotes

Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Conflict of Interest/Disclosure: R.A.F. and Pedro A. Jose were awarded a US Patent (No. 6,660,474) on "GRK variants in essential hypertension" which has been assigned to Hypogen, Inc.

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