Functional role of sodium glucose transporter in high glucose-mediated angiotensin type 1 receptor downregulation in human proximal tubule cells

Abstract

Previously, we have demonstrated human angiotensin type 1 receptor (hAT(1)R) promoter architecture with regard to the effect of high glucose (25 mM)-mediated transcriptional repression in human proximal tubule epithelial cells (hPTEC; Thomas BE, Thekkumkara TJ. Mol Biol Cell 15: 4347-4355, 2004). In the present study, we investigated the role of glucose transporters in high glucose-mediated hAT(1)R repression in primary hPTEC. Cells were exposed to normal glucose (5.5 mM) and high glucose (25 mM), followed by determination of hyperglycemia-mediated changes in receptor expression and glucose transporter activity. Exposure of cells to high glucose resulted in downregulation of ANG II binding (4,034 ± 163.3 to 1,360 ± 154.3 dpm/mg protein) and hAT(1)R mRNA expression (reduced 60.6 ± 4.643%) at 48 h. Under similar conditions, we observed a significant increase in glucose uptake (influx) in cells exposed to hyperglycemia. Our data indicated that the magnitude of glucose influx is concentration and time dependent. In euglycemic cells, inhibiting sodium-glucose cotransporters (SGLTs) with phlorizin and facilitative glucose transporters (GLUTs) with phloretin decreased glucose influx by 28.57 ± 0.9123 and 54.33 ± 1.202%, respectively. However, inhibiting SGLTs in cells under hyperglycemic conditions decreased glucose influx by 53.67 ± 2.906%, while GLUT-mediated glucose uptake remained unaltered (57.67 ± 3.180%). Furthermore, pretreating cells with an SGLT inhibitor reversed high glucose-mediated downregulation of the hAT(1)R, suggesting an involvement of SGLT in high glucose-mediated hAT(1)R repression. Our results suggest that in hPTEC, hyperglycemia-induced hAT(1)R downregulation is largely mediated through SGLT-dependent glucose influx. As ANG II is an important modulator of hPTEC transcellular sodium reabsorption and function, glucose-mediated changes in hAT(1)R gene expression may participate in the pathogenesis of diabetic renal disease.

Fig. 1.

A: hyperglycemic conditions inhibit ANG II type 1 receptor (AT₁R)-specific binding in human proximal tubule epithelial cells (hPTECs). A radioligand binding assay was done after 48-h 25 mM glucose (HG) treatment and losartan (Los) blockade. Cells were exposed to HG (25 mM) for 48 h, and [³H]ANG II binding was measured in the presence or absence of the AT₁R blocker losartan. NG, normal glucose. B: hyperglycemic conditions do not significantly affect cell viability for duration of binding studies. Trypan blue viability assay after exposure to 25 mM glucose. C: radioligand binding assay after 25 mM d-glucose (NG), l-glucose, or mannitol for 48 h. Values are means ± SE; n = 3 performed in triplicate. ***P < 0.001 vs. untreated control.

Fig. 2.

A: immunofluorescent study shows hAT₁R is downregulated in cells exposed to 25 mM glucose. Immunofluorescent staining used primary rabbit anti-hAT₁R IgG followed by secondary anti-rabbit IgG conjugated with Alexa Fluor 488. Nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI). Left: native hAT₁R in hPETCs in euglycemic condition (5.5 mM). Right: hAT₁R expression after treatment with 25 mM glucose for 48 h. B: Western blot analysis using total cell lysates indicates hAT₁R protein is downregulated during hyperglycemic conditions (HG). Total cell lysates were prepared from control (5.5 mM) and hyperglycemic (25 mM)-treated cells and immunoblotted with hAT₁R antibody (top). Blots were stripped and reprobed with anti-β-actin antibody to demonstrate equal loading (bottom). A representative blot is shown (n = 3). C: densitometric analysis of hAT₁R Western blots normalized to β-actin control (n = 3). Values are means ± SE. ***P < 0.001 vs. untreated control.

Fig. 3.

A: hyperglycemic conditions downregulate mRNA expression in hPETCs. Shown in a representative dual PCR-ethidium bromide gel of exposed cells that were euglycemic (NG; left) or hyperglycemic (HG; right) for 48 h. Bands were captured and quantified using Bio-Rad Quantity One software. hAT₁R bands are normalized to β-actin control. Bands detected are at 296 and 250 bp for β-actin and hAT₁R mRNAs, respectively. B: quantitation of hAT₁R mRNA expression determined by densitometric analysis after normalization to β-actin and data are expressed as means ± SE; n = 3. *P < 0.001 compared with untreated control.

Fig. 4.

Time- and dose-dependent effect of glucose uptake. A: time course of d-[³H]glucose uptake for 30 min in the presence of NG (◆) and HG (●). Data are expressed as nmol/mg protein; n = 3. B: d-[³H]glucose uptake studies with increasing concentration of glucose (from 0 to 25 mM) for 20 min. Data are expressed nmol/mg protein/20 min; n = 3.

Fig. 5.

Effects of sodium-glucose cotransporter (SGLT)- and glucose transporter (GLUT)-specific inhibitors in the presence of NG and HG. A: 20-min (time to reach maximum glucose uptake) d-[³H]glucose uptake study demonstrating SGLT and GLUT inhibition with phlorizin (Pzin) and phloretin (Ptin). B: [¹⁴C]methyl α-glucopyranoside (αMG) uptake studies with euglycemic and hyperglycemic conditions at 20 min. Values are means ± SE expressed as nmol·mg protein⁻¹·20 min⁻¹; n = 3. ***P < 0.001 compared with untreated control.

Fig. 6.

SGLT inhibition restores [³H]ANG II binding in cells exposed to hyperglycemic conditions. Radioligand binding assay was performed after 48-h 25 mM glucose (HG) treatment, HG with SGLT inhibition (0.5 mM Pzin), and HG with GLUT inhibition (0.15 mM Ptin). Values are means ± SE; n = 8. ***P < 0.001 compared with NG control.

Fig. 7.

hAT₁R-specific immunofluorescent studies in cells exposed to NG (A), HG (B), HG with 0.5 mM Pzin (C), and HG with 0.15 mM Ptin (D). Immunofluorescent staining was done using primary polyclonal anti-hAT₁R IgG followed by secondary IgG conjugated with Alexa Fluor 488. Nuclei were stained with DAPI. Images are representative of experiments performed; n = 3.

Fig. 8.

SGLT inhibition (Pzin) restores hAT₁R mRNA in hyperglycemic conditions (HG), while Ptin remains ineffective. hAT₁R expression was quantified by real-time PCR analysis after normalization to β-actin. Values are means ± SE relative to NG; n = 3. ***P < 0.001 compared with NG control.