2.1. Animals

In this study, wild-type littermates (WT) and global AT_1a receptor-deficient (Agtr1a^-/-) mice were used to test our hypothesis. Global Agtr1a^-/- mice were initially purchased from Jackson Labs and are currently maintained in this laboratory [22,31]. The animal studies using [¹²⁵I]-Ang II were previously approved by the Institutional Animal Care and Use Committees of Henry Ford Health System and the University of Mississippi Medical Center, whereas animal studies with the uptake of Alexa 488^®-labeled Ang II or with overexpression of intracellular cyan fluorescent Ang II fusion protein, ECFP/Ang II, in the proximal tubules of the kidney were approved by the Institutional Animal Care and Use Committees of the University of Mississippi Medical Center and Tulane University School of Medicine, respectively.

2.2. Mouse Proximal Tubule Cells with or without the Expression of AT₁ (AT_1a) Receptors

Wild-type (WT) mouse proximal tubule cell (mPCT) and Agtr1a^-/- mouse proximal tubule cell lines, as generated from adult male C57BL/6J and Agtr1a^-/- mice, were kindly provided by Dr. Ulrich Hopfer of Case Western Reserve University for the in vitro studies [37,38].

2.3. AT₁ (AT_1a) Receptor-Mediated Uptake of Systemically Infused [¹²⁵I]-Ang II by the Kidney Proximal Tubules in Mice

As a proof-of-concept experiment that circulating, systemic, or paracrine Ang II is taken up by mPCT cells to act as an intracellular peptide in the proximal tubules of the kidney, a radioligand [¹²⁵I]-Ang II was intravenously infused for two hours as we and others described previously [31,39]. Briefly, 2 groups (n = 8 per group) of adult male Sprague-Dawley rats (WT) and global AT_1a receptor-deficient mice (Agtr1a^-/-) were anesthetized with pentobarbital sodium (50 mg/kg, i.p.), and the left jugular vein was catheterized with a PE-10 catheter for intravenous infusion of [¹²⁵I]-Ang II (specific activity 2176 Ci/mmol, ~10 μCi/25 g body wt) for two hours. Generally, steady-state plasma levels of [¹²⁵I]-Ang II infusion were reached by 30 min, whereas steady-state tissue levels of [¹²⁵I]-Ang II in the kidney proximal tubules were reached within 60 min, as described previously [31,39]. At the end of the experiment, all animals were perfused with acidic buffered saline for 5 min to dissociate cell surface receptor-bound [¹²⁵I]-Ang II from cell membranes and wash out all extracellular [¹²⁵I]-Ang II. The kidneys were removed, [¹²⁵I]-Ang II activity was measured using a gamma counter, and kidneys were sectioned and exposed to X-ray films for 3 days for localization of the intracellular uptake of [¹²⁵I]-Ang II in the kidney, as we described previously [31,39].

2.4. AT₁ (AT_1a) Receptor-Mediated Uptake of Systemically Infused Alexa 488^®-Ang II by the Kidney Proximal Tubules in Mice

To further confirm whether circulating, systemic, or paracrine Ang II is taken up by mPCT cells in vivo, 2 groups (n = 8 per group) of WT and global AT_1a receptor-deficient mice (Agtr1a^-/-) were anesthetized with pentobarbital sodium (50 mg/kg, i.p.), and the left jugular vein was catheterized with a PE-10 catheter for intravenous infusion of fluorescent Alexa 488^®-Ang II (Invitrogen, Waltham, MA, USA, 10 ng/min, Cat: A13439; Lot: 828266) for two hours. As described for [¹²⁵I]-Ang II, steady-state plasma and proximal tubule uptake levels of Alexa 488^®-Ang II were reached by 30 and 60 min, respectively. At the end of the experiment, mice were perfused with acidic buffered saline for 5 min to dissociate cell surface receptor-bound Alexa 488^®-Ang II from cell membranes and wash out all extracellular Alexa 488^®-Ang II. The kidneys were removed, sectioned, and visualized for localization of the intracellular uptake of Alexa 488^®-Ang II in the kidney using an A1-HD25 Nikon Confocal Laser Scanning Microscopy System with a 488 filter [37].

2.5. Construction of Proximal Tubule Cell-Specific Intracellular Ang II Fusion Protein, Ad-Sglt2-ECFP/Ang II

It is difficult to completely separate the effects of intracellular Ang II mediated by cytoplasmic receptors from those of extracellular Ang II mediated by cell surface receptors in a practical manner. Therefore, we constructed a proximal tubule cell-specific, enhanced cyan fluorescent protein (EFCP)-tagged intracellular Ang II fusion protein for in vitro expression in mPCT cells or for in vivo expression in the proximal tubules of the mouse kidneys, as described by us and others previously [36,37,40]. The construct for this ECFP-tagged intracellular Ang II fusion protein, ECFP/Ang II, was kindly provided by Dr. Julia Cook of the Ochsner Clinic Foundation [36,40]. A proximal tubule-specific SGLT2 promoter, pGEM-sglt2-5pr-mut, was kindly provided by Drs. Isabelle Rubera and Michel Tauc of the Université Côte d’Azur, France [41]. An adenoviral construct encoding ECFP/Ang II and the SGLT2 promoter, Ad-Sglt2-ECFP/Ang II, was custom-designed, amplified, and purified by Vector BioLab for in vitro expression in mPCT cells or for in vivo expression in the proximal tubules of the mouse kidneys, as we described previously [22,37].

2.6. Adenovirus-Mediated Overexpression of Ad-Sglt2-ECFP/Ang II Selectively in the Proximal Tubules of WT Mouse Kidneys

Two groups (n = 8 each) of adult male WT littermates or global Agtr1a^-/- mice were infected without (time controls) or with adenovirus-mediated, proximal tubule-specific expression of Ad-Sglt2-ECFP/Ang II in the kidney for 2 weeks [22,37]. Briefly, animals were anesthetized with sodium pentobarbital (50 mg/kg, i.p.), and their renal arteries were exposed by a left and right flank incision, followed by temporarily clamping with a fine vessel clip, briefly interrupting blood flow to the kidney for 5 min. Ad-Sglt2-ECFP/Ang II was diluted 1:5 in sterilized phosphate-buffered saline and directly injected into the superficial cortex evenly in six different locations (20 μL each) [22,37]. The control groups of animals received only sham injections of saline instead. Renal blood flow was reestablished 5 min after injection of Ad-Sglt2-ECFP/Ang II or saline. Animals were allowed to recover from the surgery and express Ad-Sglt2-ECFP/Ang II for two weeks. At the end of the experiment, the in vivo expression of Ad-Sglt2-ECFP/Ang II in the proximal tubules of the kidney was visualized by an A1-HD25 Nikon Confocal Laser Scanning Microscopy System using a CFP filter [22,37]. These animal studies using an adenoviral construct were approved by the Institutional Recombinant DNA and Biosafety Committees of the University of Mississippi Medical Center and Tulane University School of Medicine, respectively.

2.7. Adenovirus-Mediated Expression of Ad-Sglt2-ECFP/Ang II in mPCT Cells with or without AT_1a Receptors

To determine whether intracellular Ang II induces important biological responses, Ad-Sglt2-ECFP/Ang II was expressed in mPCT cells (abbreviation: ECFP/Ang II). Briefly, WT and Agtr1a^-/- mPCT cells were split into 6-well plates and cultured to 80% confluency, and then transfected with Ad-Sglt2-ECFP/Ang II (~4 µg/well) for 48 h using a standard transfection protocol as previously described [22,37]. In another series of experiments, Agtr1a^-/- mPCT cells were transfected concurrently with Ad-Sglt2-ECFP/Ang II and a full-length mouse Agtr1a cDNA ORF clone (NM_177322, OriGene, Rockville, MD, USA) to rescue the AT_1a receptor expression in Agtr1a^-/- mPCT cells before they were treated with the inhibitors.

2.8. Pharmacological Inhibitors of AT₁ (AT_1a) or AT₂ Receptor-Mediated, MAP Kinase ERK1/2- and NF-κB-Dependent Signal Pathways

To determine the potential signaling mechanisms involved in Ad-Sglt2-ECFP/Ang II-induced biological responses, WT and Agtr1a^-/- mPCT cells expressing Ad-Sglt2-ECFP/Ang II were concurrently treated with the AT₁ receptor antagonist losartan (10 µM; Tocris, Minneapolis, MN, USA), the AT₂ receptor antagonist PD 123319 (10 µM; Tocris, Minneapolis, MN, USA), the MEK1/MEK2 kinase inhibitor U0126 (1 µM; Tocris, Minneapolis, MN, USA), the MEK inhibitor PD 980659 (1 µM; Tocris, Minneapolis, MN, USA), the NF-κB activation inhibitor RO 106–9920 (10 µM; Tocris, Minneapolis, MN, USA), and the p38 MAP kinase inhibitor SB202196 (10 µM; MCE, Belleville, NJ, USA).

2.9. Antibodies Targeting Signaling and Sodium Transporter or Cotransporter Proteins

The following antibodies were used in the present study to determine the expression of key Na⁺ transporter or cotransporter proteins or signaling pathway proteins. A mouse monoclonal antibody targeting a short amino acid sequence containing dually phosphorylated Thr 202 and Tyr 204 (pT202/pY204) of MAP kinases ERK1/2 of rat origin (sc-136521) and a rabbit polyclonal antibody targeting a short amino acid sequence containing phosphorylated Ser 276 of the NF-κB, p65 subunit of human origin (sc-101749) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). A rabbit polyclonal antibody targeting a synthetic peptide (KLH-coupled) in the C terminus of the rat MAP kinase ERK 1/2 (no. 9102) was purchased from Cell Signaling. A rabbit anti-Na⁺/HCO₃ cotransporter polyclonal antibody (AB3212), a monoclonal antibody targeting a fusion protein containing the C-terminal 131 amino acids of rabbit NHE3 (no. MAB3136), and a mouse monoclonal antibody targeting a synthetic peptide corresponding to human NF-κB, p65 subunit, anti-NF-κB, p65 subunit clone 12H11 (no. MAB3026) were purchased from Millipore, respectively.

2.10. Western Blot Analysis

At the end of the treatments, protein samples were extracted from all mPCT cells for Western blot analysis of the expressions. Protein concentrations were measured using a bicinchoninic acid protein assay kit (Pierce, Appleton, WI, USA) and GraphPad Prism 9.0 [15,37]. Briefly, 10 to 20 μg each of protein samples were electrophoretically separated on 8–16% Tris-glycine gels at 120 volts for 1.5–2.0 h and transferred to Millipore Immobilon-P membranes using a Bio-Rad Trans-Blot Semi-Dry system (25 V, 0.12 A, 1.5 h). All membranes were blotted overnight at 4 °C with 5% nonfat dry milk and incubated with a specific antibody as described above for 3 h at room temperature. The same membranes were then treated with a stripping buffer (Pierce) for 20 min, blotted with 5% nonfat dry milk, and re-probed with a mouse anti-β-actin monoclonal antibody at 1:2000 (Sigma-Aldrich, St. Louis, MO, USA) [15,37]. At least 6 wells/samples from untreated (controls) and inhibitor-treated wild-type or Agtr1a^-/- mPCT cells were included in all in vitro cell culture studies. Western blot signals for each signaling or transporter protein were detected using enhanced chemiluminescence (Amersham, Amersham, UK) and analyzed using a BIO-RAD ChemiDoc ^TM MP Imaging System and GraphPad Prism 9.5.1.

3.2. AT_1a Receptor-Mediated Uptake of Alexa 488-Labeled Ang II in Wild-Type and Agtr1a^-/- Mouse Kidneys

To further confirm and visualize whether the circulating Alexa 488^®-labeled Ang II is taken up by the proximal tubules of mouse kidneys, the intracellular uptake of Alexa 488^®-labeled Ang II in the kidney was directly compared between wild-type and Agtr1a^-/- mice (Figure 2). In Agtr1a^-/- mice, there was a minimal level of Alexa 488^®-labeled Ang II uptake by the glomeruli and the proximal tubules, suggesting that deletion of AT_1a receptors in mice blocked Alexa 488^®-labeled Ang II uptake by the kidney (Figure 2A). In wild-type mice, the uptake of Alexa 488^®-labeled Ang II was very intensive in the proximal tubules, as strong green fluorescence was observed over the proximal tubules in the superficial cortex (p < 0.01 vs. Agtr1a^-/- mice) (Figure 2B). However, the glomeruli and the cortical collecting tubules did not significantly take up systemically infused Alexa 488^®-labeled Ang II, suggesting that this response is a proximal tubule-specific phenomenon (Figure 2B).

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Figure 2

AT_1a receptor-mediated intracellular uptake of Alexa fluor 488^®-labeled Ang II in the proximal tubules of wild-type (WT) and global AT_1a receptor-deficient mice (Agtr1a^-/-). (A) Intracellular uptake of Alexa fluor 488^®-labeled Ang II in the proximal tubules of a representative adult male wild-type mouse (WT). (B) Intracellular uptake of Alexa fluor 488^®-labeled Ang II in the proximal tubules was blocked in a representative Agtr1a^-/- mouse. (C) Semiquantitative data on the intracellular uptake of Alexa fluor 488^®-labeled Ang II in the proximal tubules of WT and Agtr1a^-/- mice, expressed as arbitrary fluorescence intensity with WT mice at 100. G, glomerulus. PT, proximal tubule. Red represents DAPI-stained nuclei in the cortex, which were converted into RED for a better contrast with Alexa fluor 488^®-labeled Ang II. ** p < 0.01 vs. WT mice. Magnification: ×40.

3.3. Adenovirus-Mediated, Proximal Tubule-Specific Overexpression of Intracellular Ang II Fusion Proteins in Wild-Type and Agtr1a^-/- Mouse Kidneys

We then designed and constructed an adenovirus-mediated, proximal tubule-specific, intracellular Ang II fusion protein, Ad-Sglt2-ECFP/Ang II, for in vivo expression in wild-type mice, as described in [36,37,41]. Figure 3 shows that control mice showed minimal autofluorescence in the proximal tubules of the kidney (Figure 3A, C). By contrast, intensive cyan fluorescence (blue green) representing the expressed Ad-Sglt2-ECFP/Ang II was observed in the proximal tubules of the mouse kidney (p < 0.01 vs. control) (Figure 3B). No significant expression of Ad-Sglt2-ECFP/Ang II was visualized in the glomerulus (Figure 3B). These results demonstrated a proof-of-concept approach to expressing an intracellular Ang II fusion protein in the proximal tubules of the kidney using the sodium and glucose cotransporter 2 promoter (Sglt2) to study the effects and mechanisms of intracellular Ang II-induced expression of sodium transporters or cotransporters in mouse proximal tubule cells (see below).

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Figure 3

Adenovirus-mediated, proximal tubule-targeting expression of an intracellular cyan fluorescent Ang II fusion protein, Ad-Sglt2-ECFP/Ang II, in the proximal tubules of the mouse kidney. (A) A representative control mouse kidney without expressing Ad-Sglt2-ECFP/Ang II and only showing autofluorescence in the proximal tubules. (B) A representative mouse kidney expressing robust Ad-Sglt2-ECFP/Ang II in the proximal tubules. (C) Semiquantitative data on proximal tubule-specific expression of Ad-Sglt2-ECFP/Ang II in the proximal tubules, expressed as arbitrary fluorescence intensity. G, glomerulus. PT, proximal tubule. Cyan, Ad-Sglt2-ECFP/Ang II. Blue, nuclei. Red, apical. The basolateral membranes of the tubules as well as the glomeruli were stained by the high-affinity F-actin probe Alexa Fluor 568 phalloidin. ** p < 0.01 vs. control mice. Magnification: ×40.

3.4. Role of AT₁ (AT_1a) and AT₂ Receptors in Mediating Intracellular Ang II Fusion Protein-Induced NHE3 Expression in mPCT Cells

Figure 4 shows that the expression of an intracellular Ang II fusion protein (ECFP/Ang II) is biologically active in significantly inducing the expression of NHE3 by nearly 3-fold, one of the most important sodium transporters responsible for reabsorbing over 50% of the filtered sodium load by the glomeruli in WT mPCT cells (p < 0.01 vs. control) (Figure 4A). The effect of ECFP/Ang II on NHE3 expression was markedly attenuated by losartan, an AT₁ receptor blocker (p < 0.01 vs. ECFP/Ang II). In Agtr1a^-/- mPCT cells, however, the expression of ECFP/Ang II had a markedly smaller effect on NHE3 expression (n.s. vs. control) (Figure 4B). Both the AT₁ receptor blocker losartan and the AT₂ receptor blocker PD123319 had no significant effects on NHE3 expression in Agtr1a^-/- mPCT cells. These data confirm that proximal tubule-specific expression of intracellular Ang II fusion proteins induces NHE3 expression in wild-type but not Agtr1a^-/- mPCT cells.

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Figure 4

Effects of ECFP/Ang II on Na⁺/H⁺ exchanger 3 (NHE3) expression in wild-type and Agtr1a^-/- mouse proximal tubule cells (mPCT). Panel (A) shows that the expression of ECFP/Ang II as a surrogate of intracellular Ang II stimulated NHE3 protein expression in wild-type mPCT cells, and this response was significantly attenuated by losartan, an AT₁ receptor blocker, and PD123319, an AT₂ receptor antagonist. Panel (B) shows that the expression of ECFP/Ang II had insignificant effects in Agtr1a^-/- mPCT cells without or with losartan or PD123319 treatment. ** p < 0.01 vs. control; ⁺⁺ p < 0.01 vs. ECFP/ANG II.

3.5. Roles of the MAP Kinase ERK1/2, p38 MAPK, or NF-кB Signaling Pathways in Mediating Intracellular Ang II Fusion Protein-Induced NHE3 Expression in mPCT Cells

Figure 5 shows several potential downstream signaling mechanisms underlying AT₁ (AT_1a) receptor-mediated ECFP/Ang II-induced NHE3 expression in mPCT cells. In WT mPCT cells, the stimulated effect of ECFP/Ang II on NHE3 expression was markedly attenuated by U0126, a highly selective inhibitor of the MAP kinases MEK1 and MEK2 (p < 0.01 vs. control) (Figure 5A). Concurrent treatment of WT mPCT cells expressing ECFP/Ang II with Ro 106-9920, a cell-permeable inhibitor of NF-κB activation, also blocked the effect of ECFP/Ang II on NHE3 expression (p < 0.01 vs. control) (Figure 5A). However, concurrent treatment of WT mPCT cells expressing ECFP/Ang II with either PD980659 or SB202196, a selective p38 MAP kinase inhibitor, had no significant effect (n.s.) (Figure 5A). By comparison, U0126, Ro 106-9920, PD980659, and SB202196 did not have significant effects on ECFP/Ang II-induced NHE3 expression in Agtr1a^-/- mPCT cells (Figure 5B).

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Figure 5

The roles of the MAP kinase and NF-κB signaling pathways in mediating ECFP/Ang II-induced NHE3 expression in wild-type and Agtr1a^-/- mPCT cells. Panel (A) shows that in wild-type mPCT cells, ECFP/Ang II stimulated NHE3 expression significantly, and the response was attenuated by the MEK1/MEK2 kinase inhibitor U0126 and the NF-κB activation inhibitor Ro 106–9920, respectively. However, the MEK inhibitor PD 980659 and the p38 MAP kinase inhibitor SB202196 failed to attenuate the effect of ECFP/Ang II on NHE3 expression. Panel (B) shows that in Agtr1a^-/- mPCT cells, ECFP/Ang II failed to stimulate NHE3 expression, and the inhibitors of the MAP kinases and NF-κB signaling pathways had no significant effects on NHE3 expression. ** p < 0.01 vs. control WT mPCT cells. ⁺⁺ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

3.6. Role of AT₁ (AT_1a) and AT₂ Receptors in Mediating Intracellular Ang II Fusion Protein-Induced Na⁺/HCO₃^- Expression in mPCT Cells

The Na⁺/HCO₃^- cotransporter plays a key role in proximal tubular reabsorption of HCO₃^- and in maintaining intracellular pH. Figure 6 shows that the expression of intracellular ECFP/Ang II also significantly increased the expression of Na⁺/HCO₃^- cotransporter in WT mPCT cells (Figure 6A; p < 0.01 vs. control). This stimulatory response was markedly attenuated to a level below control by losartan, but PD123319 also attenuated the effect of ECFP/Ang II on Na⁺/HCO₃^- expression (p < 0.01 vs. ECFP/Ang II alone). The potential signaling mechanisms involved in ECFP/Ang II-induced Na⁺/HCO₃^- expression are shown in Figure 6B. The MAP kinase MEK1/MEK2 inhibitor U0126, the ERK1/2 inhibitor PD980659, as well as the inhibitor of NF-κB activation Ro-106 9920, all significantly attenuated ECFP/Ang II-induced Na⁺/HCO₃^- expression (Figure 6B; p < 0.01 vs. ECFP/Ang II). However, the p38 MAP kinase inhibitor SB202196 had no effect on ECFP/Ang II-induced Na⁺/HCO₃^- expression, suggesting that p38 MAP kinase may not be involved in this response.

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Figure 6

The roles of AT₁ and AT₂ receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced Na⁺/HCO₃^- cotransporter expression in wild-type mPCT cells. Panel (A) shows that ECFP/Ang II significantly increased Na⁺/HCO₃^- expression, and the response was attenuated by losartan but not by PD123319, suggesting a dominant role of AT₁ receptors in mPCT cells. Panel (B) shows that the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659 attenuated the effects of ECFP/Ang II on expression, but the p38 MAP kinase inhibitor SB202196 had no effect on Na⁺/HCO₃^- expression. ** p < 0.01 vs. control WT mPCT cells. ⁺⁺ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II.

3.7. Roles of AT₁ (AT_1a) and AT₂ Receptors in Mediating Intracellular Ang II Fusion Protein-Induced Sglt2 Expression in mPCT Cells

The sodium and glucose cotransporter 2 (Sglt2) is responsible for reabsorbing nearly all filtered glucose from the glomeruli and a small fraction of sodium in the proximal tubules of the kidney. Figure 7 shows that the expression of an intracellular Ang II fusion protein (ECFP/Ang II) significantly induced the expression of the sodium and glucose cotransporter 2 (Sglt2) by > 2-fold in WT mPCT cells (p < 0.01 vs. control) (Figure 7A). The effect of ECFP/Ang II on Sglt2 expression was markedly attenuated by losartan or PD123319 (p < 0.01 vs. ECFP/Ang II), suggesting that both AT₁ and AT₂ receptors are involved. In Agtr1a^-/- mPCT cells, ECFP/Ang II alone did not induce Sglt2 expression, but Sglt2 expression was increased significantly in the presence of losartan (p < 0.01 vs. ECFP/Ang II) and decreased significantly in the presence of PD123319 (p < 0.01 vs. ECFP/Ang II + Los). These data suggest that both AT₁ and AT₂ receptors are involved in ECFP/Ang II-induced Sglt2 expression in mPCT cells (Figure 7A, B).

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Figure 7

The roles of AT₁ and AT₂ receptors in mediating ECFP/Ang II-induced sodium and glucose cotransporter 2 (SGLT2) expression in wild-type and Agtr1a^-/- mPCT cells. Panel (A) shows that ECFP/Ang II significantly increased SGLT2 expression, and the response was attenuated by both losartan and PD123319, supporting an important role of AT₁ and AT₂ receptors in wild-type mPCT cells. Panel (B) shows that in the absence of AT_1a receptors, ECFP/Ang II had no effect on SGLT2 expression in Agtr1a^-/- mPCT cells. However, losartan increased SGLT2 expression, whereas PD123319 decreased SGLT2 expression in Agtr1a^-/- mPCT cells, suggesting an important role for AT₂ receptors in SGLT2 expression in Agtr1a^-/- mPCT cells. ** p < 0.01 vs. control WT or Agtr1a^-/- mPCT cells. ⁺⁺ p < 0.01 vs. WT or Agtr1a^-/- mPCT cells transfected with ECFP/Ang II and treated with losartan.

3.8. Roles of AT₁ (AT_1a) and AT₂ Receptors in Mediating Intracellular Ang II Fusion Protein-Induced NF-кB, p65 Expression in mPCT Cells

Nuclear factor NF-kappaB p65 subunit (NF-кB, p65) is an important transcription factor encoded by the RELA gene and plays a key role in NF-κB heterodimer formation, nuclear translocation, and activation [42]. Figure 8 shows that the expression of ECFP/Ang II in WT mPCT cells markedly increased NF-кB, p65 protein expression, and interestingly, this stimulatory response was blocked by concurrent treatment with losartan and PD123319 (Figure 8A), again suggesting that both AT₁ and AT₂ receptors are involved. This was supported by the lack of the NF-кB, p65 response to ECFP/Ang II expression in Agtr1a^-/- mPCT cells (Figure 8B), while concurrent treatment with losartan or PD123319 increased NF-кB, p65 expression in Agtr1a^-/- mPCT cells (Figure 8B). Further experiments showed that the MAP kinase MEK1/MEK2 inhibitor U0126, the MEK inhibitor PD980659, as well as the inhibitor of NF-κB activation Ro 106-9920, all markedly attenuated ECFP/Ang II-induced NF-кB, p65 expression (Figure 8C). However, the p38 MAP kinase inhibitor SB202196 again had no effect on ECFP/Ang II-induced NF-кB, p65 expression, suggesting that p38 MAP kinase may not be involved in this response (Figure 8C).

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Figure 8

The roles of AT₁ and AT₂ receptors, the MAP kinases, and NF-κB signaling pathways in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type and Agtr1a^-/- mPCT cells. Panel (A) shows that ECFP/Ang II increased NF-κB, p65 expression in wild-type mPCT cells, and the response was attenuated by both losartan and PD123319, supporting an important role of AT₁ and AT₂ receptors in mediating ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. Panel (B) shows that ECFP/Ang II alone had no significant effect on NF-κB, p65 expression in Agtr1a^-/- mPCT cells, but both losartan and PD123319 potentiated this response. Panel (C) shows that in wild-type mPCT cells, the effect of ECFP/Ang II on NF-κB, p65 expression was attenuated by the MEK1/MEK2 kinase inhibitor U0126, the NF-κB activation inhibitor Ro 106–9920, and the MEK inhibitor PD 980659, respectively. However, the p38 MAP kinase inhibitor SB202196 had no effect on ECFP/Ang II-induced NF-κB, p65 expression in wild-type mPCT cells. ** p < 0.01 vs. control WT or Agtr1a^-/- mPCT cells. ⁺⁺ p < 0.01 vs. WT mPCT cells transfected with ECFP/Ang II, or Agtr1a^-/- mPCT cells transfected with ECFP/ANG II.

3.9. Comparisons of Extracellular Ang II and Intracellular ECFP/Ang II-Induced MAP Kinases ERK1/2 Activation in WT and Agtr1a^-/- mPCT Cells

It is well known that extracellular Ang II can induce the activation of MAP kinases ERK1/2 through cell surface Ang II receptors in different cell types and tissues [20,24,25], but whether intracellular Ang II can also activate MAP kinases ERK1/2 through intracellular Ang II receptors remains poorly understood. Figure 9 shows that, as expected, extracellular Ang II induced MAP kinases ERK1/2 activation in WT mPCT cells in time- and concentration-dependent manners, with a maximal response at 5 min of stimulation by 10 nM Ang II (Figure 9A). Additionally, it is expected that extracellular Ang II-induced activation of the MAP kinases ERK1/2 in WT mPCT cells was markedly blocked by concurrent treatment with losartan or PD123319 (Figure 9B). Interestingly, the expression of intracellular ECFP/Ang II also markedly increased MAP kinases ERK1/2 expression in WT mPCT cells, and this response was blocked by losartan but not by PD123319 (Figure 9C), which was different from extracellular Ang II (Figure 9B). In Agtr1a^-/- mPCT cells, the expression of intracellular ECFP/Ang II did not increase MAP kinases ERK1/2 expression as in WT mPCT cells (Figure 10A), whereas the knock-in of AT_1a receptors in Agtr1a^-/- mPCT cells successfully rescued the stimulatory effect of ECFP/Ang II expression on MAP kinases ERK1/2 activation (Figure 10B). Taken together, these data provide proof-of-concept evidence that, similar to extracellular Ang II, intracellular Ang II may also activate the MAP kinases ERK1/2 to induce important biological and physiological effects and important signaling responses in mPCT cells.

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Figure 9

Comparisons of AT₁ and AT₂ receptor-mediated, extracellular Ang II- versus intracellular ECFP/Ang II-induced MAP kinases ERK1/2 activation in wild-type mPCT cells. (A) Time- and dose-dependent responses to extracellular Ang II stimulation with a peak response for 10 nM at 5 min. (B) The effect of losartan (an AT₁ blocker) and PD123319 (an AT₂ blocker). (C) The effect of losartan (an AT₁ blocker) and PD123319 (an AT₂ blocker) on intracellular ECFP/Ang II-induced MAP kinases ERK1/2 activation. These data suggest that both extracellular and intracellular Ang II activate MAP kinases ERK1/2 signaling in wild-type mPCT cells. ** p < 0.01 vs. control. ⁺⁺ p < 0.01 vs. ECFP/Ang II.

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Figure 10

AT₁ (AT_1a) receptor-mediated, intracellular ECFP/Ang II-induced activation of the MAP kinases ERK1/2 signaling pathway in Agtr1a^-/- mPCT cells without or with the knock-in of a full-length wild-type mouse AT_1a receptor. ** p < 0.01 vs. control; ⁺⁺ p < 0.01 vs. ECFP/Ang II + Los (A) or ECFP/Ang II (B), respectively.

We sincerely thank Julia L. Cook of Ochsner Clinic for providing a plasmid encoding an enhanced cyan fluorescent protein (ECFP)-tagged intracellular angiotensin II (Ang II) fusion protein, ECFP/Ang II; Isabelle Rubera and Michel Tauc of Université Côte d’Azur, France, for providing the Sglt2 promoter, and Ulrich Hopfer of Case Western Reserve University for providing wild-type and Agtr1a^-/- mouse proximal tubule cells for the present study. The portions of the present study were presented as conference abstracts in 2009 and 2010 American Society of Nephrology Annual Kidney Week [J. Am. Soc. Nephrol. 20(10): 2009. SA-FC413 & J. Am. Soc. Nephrol. 2010_TH-PO216), and American Heart Association Council for Hypertension Research 2014 Annual Research Conference (Hypertension, 2014; 64:A405), respectively.