GLUT1 and prorenin receptor mediate differential regulation of TGF-β and CTGF in renal inner medullary collecting duct cells during high glucose conditions

Figures

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  Fig. 1. Characterization of mouse inner medullary collecting ducts (IMCD). IMCD cells grown in NG conditions and then treated with HG. A Light field microscopy image shows the characteristic shape and phenotype of the primary cultured IMCD cells (Scale bar: 100 μm). The zoom in image shows the cuboidal characteristic shape of primary cultures of IMCD cells after 6 d. B IMCD were characterized by the expression of collecting duct marker AQP-2 (green) in the plasma membrane (arrows) and cytoplasm (asterisk) of the principal cells. C IMCD cells were stained with PRR (green) showing some PRR-positive cells as well as negative cells (wide arrows) which are mainly principal cells. Scale bar for B and C: 20 μm. Co-localization experiments further demonstrate no colocalization of AQP-2 with GLUT1 (D) or PRR (E). However, GLUT1 colocalized with PRR (F). Staining of nucleus with DAPI. Scale bar for D, E and F: 20 μm. Immunofluorescence and phase contrast images were obtained using a Nikon Eclipse-50i immunofluorescence microscope (Nikon Eclipse-50i, Japan) and were digitalized using the NIS-Elements version 4.0 from Nikon

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  Fig. 2. GLUT1 expression following 48 h conditioning with NG, HG, or HG + GLUT1 inhibitor BAY 876 pretreatment. A mRNA levels of GLUT1 under treatment with HG alone were augmented almost twice that of controls. GLUT1 inhibition by BAY 876 (2 nM) for 48 h prevented the upregulation of GLUT1 (*p < 0.05, n = 5). B Similar results were observed when analyzing protein levels (*p < 0.05, n = 3). C Four representative immunofluorescence fields showing that GLUT1 abundance (red) was augmented after 48 h HG treatment, while HG + BAY 876 prevented this effect (nuclei in blue). Scale bar represents 20 µm

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  Fig. 3. HG conditions stimulate prorenin and renin release to the cell culture media, PRR upregulation, and activation of MAPK pathway; GLUT1 inhibition prevented these increases. A Prorenin and renin band detected by immunoblot in concentrated cell culture media. Intensity of prorenin-renin bands (together) was augmented in cell culture media in HG conditions but was prevented in cells pretreated with BAY 876. 40 μg total protein were loaded in each lane. B PRR was augmented in cell lysates of IMCD cells under both HG and HG + BAY 876 conditions, however, incubation with BAY 876 mitigated the extent of the response. C Phospho-ERK1/2 versus total ERK1/2 ratio was augmented after 48 h of HG; BAY 876 pretreatment impaired this effect. *p < 0.05 vs NG; **p < 0.01 vs NG

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  Fig. 4. IMCD cell protein and mRNA levels of TGF-β and CTGF following high glucose treatment and GLUT1 blockade. Immunoblot of IMCD cells measuring TGF-β and CTGF protein expression under the conditions of NG, HG, and HG plus the GLUT1 inhibitor BAY 876 for 48 h (HG + BAY) is shown along with fold change of treatment compared to NG expressed as the ratio of protein to housekeeping gene β-actin. The bottom-most plot shows the mRNA abundance as fold change of treatment compared to NG. mRNA levels were normalized using housekeeping gene β -actin. No effect was observed in NG + BAY 876 (not shown). *p < 0.05 versus NG, n = 3–4

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  Fig. 5. PRO20 prevented the induction of TGF-β but not CTGF after 48 h of HG treatment in IMCD cells. Protein and abundance levels of GLUT1, phosphorylated ERK, total ERK, TGF-β and CTGF during NG, HG, and HG + PRO20 conditions. Representative immunoblot (left) and protein abundances (right) quantified as the ratio protein versus β-actin. GLUT1, phosho ERK, CTGF and TGF-β abundances were augmented in HG conditions. Preconditioning with PRO20 prevented ERK phosphorylation and upregulation of TGF-β but not CTGF. No effect was observed in NG + PRO20 (not shown). *p < 0.05 versus NG, n = 3–4

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  Fig. 6. Intracellular ROS during HG conditions with or without GLUT1 blocker BAY 876, PRO20 and the effect of p-coumaric acid (PCA) on CTGF protein levels. A Significant increases in ROS (***p < 0.0001 vs NG, n = 8) was less evident in cells incubated with the GLUT1 specific inhibitor BAY 876 (*p < 0.05 vs NG, n = 8). PRR pharmacological blockade using PRO20 was not able to prevent the augmentation of ROS during HG (***p < 0.0001, n = 8). Co-treatment with ROS scavenger p-coumaric acid (PCA) at 10–7 M, was able to prevent ROS formation after 24 h HG treatment. ROS were measured by using L2',7'-dichlorodihydrofluorescein diacetate (H2DCFDA) which is intracellularly trapped by esterase activity that removes lipophilic blocking groups. Fluorescencence exhibited by dichlorofluorescien (DCF) resulting from the oxidation of the probe by ROS was normalized by micrograms of protein. B Representative immunoblotting showing that increased CTGF protein levels caused by HG were not observed with PCA treatment. *p < 0.05 vs NG, n = 3. C Intracellular TBARS was assessed in cell lysates after 1, 3 and 6 d of exposure to HG compared to controls. HG increased TBARS at day 3 and 6 while BAY876 slightly…

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  Fig. 7. IMCD cells incubated with HG upregulate GLUT1 enabling glucose uptake. HG increases PRR synthesis along with prorenin and renin secretion in cell culture mediate. The specific GLUT1 inhibitor, BAY 876, impairs the induction of PRR, prorenin, and renin. Increases in intracellular ROS during HG conditions are attenuated by GLUT1 blockade, while the induction of TGF-β and CTGF in HG is effectively blunted by BAY876. PRO20, a PRR blocker, impairs TGF-β induction but did not prevent increases in intracellular ROS and CTGF protein expression. Treatment with ROS scavenger p-coumaric acid impairs intracellular ROS concomitant with impairment of CTGF upregulation. Intracellular ROS formation from glucose exposure or uptake may likely mediate CTGF induction which also may be sustained by TGF-β receptor in the long term. Long term effects of HG may be also related to modification of the activity of NOX4 as suggested by our previous reports. Oxidative effects of HG-dependent uptake may be also related with other suggested transporters expressed in collecting duct cells (not studied here) such as GLUT12