Oxidized LDL potentiates Ang II-induced Gq signaling and calcium influx in a LOX-1-dependent manner

a Dose-dependent response of IP1 concentration by the activation of Gq signaling in response to oxLDL and Ang II in CHO-LOX-1-AT1 cells. Cells were treated with oxLDL and Ang II at the concentrations described in the figure. (n=4 for each oxLDL concentration). *P=0.0004 for 0 μg/mL vs 20 μg/mL, †P=0.0003 for 5 g/mL vs 20 μg/mL, ‡P=0.0020 for 0 μg/mL vs 10 μg/mL, and§P=0.0015 for 5 μg/mL vs 10 μg/mL at 10−9 M Ang II; ||P=0.0051 for 0 μg/mL vs 10 μg/mL, P=0.0004 for 0 μg/mL vs 20 μg/mL at 10−8 M Ang II.

b IP1 concentration in response to vehicle, native LDL (nLDL 10 μg/mL), and oxLDL (10 μg/mL) in the combination of Ang II (10−8 M) in CHO-LOX-1-AT1 cells (n=5 for each group)

c IP1 concentration in response to vehicle, oxLDL (10 μg/mL) in the combination of Ang II (10−8M) in CHO-LOX-1-AT1 and CHO-AT1 cells (n=5 for each group).

d IP1 concentration in response to vehicle, oxLDL (10 μg/mL), BSA (10 or 100 μg/mL), BSA-conjugated AGE (10 or 100 μg/mL) in the combination of Ang ll (10−8 M) in CHO-LOX-1-AT1 cells.

e IP1 concentration in response to vehicle, oxLDL (10 μg/mL) in the combination of Ang II (10−8 M) in genetically engineered CHO cells with or without intact β-arrestin binding domain (n=5 for each group). AT1mg indicates AT1 a mutant AT1 lacking a functional β-arrestin binding domain but retaining G-protein-biased signaling capability.

f IP1 concentration in response to oxLDL (10 μg/mL) in the combination of Ang II (10−8 M) and additional effect of PTX, a Gi inhibitor, YM-254890, a Gq inhibitor, and RKI-1448, a downstream Rho kinase inhibitor targeting G12/13 signaling, in CHO-LOX-1-AT1 cells (n=5 for each group).

g Intracellular calcium dynamics measured using Fura 2-AM by the ratio of the mission signals at excitation wavelength 340 nm and 380 nm in response to oxLDL (5 μg/mL), Ang II (10−12 M), and their combination (for each agonist, 4-7 regions of interest were selected). Addition of these agonists is marked with arrows on the timeline of the assay.

h Percentage changes from baseline in the ratio of emission signals (F340/F380) measured by Fura 2-AM were quantified following treatment with oxLDL and Ang II at specified concentrations in CHO-LOX-1-AT1 cells as detailed in the figure (n=4-8).

i Percentage change from baseline in the ratio of emission signals (F340/F380) measured by Fura 2-AM after stimulation with oxLDL (5 μg/mL), Ang II (10−12 M), and YM-254890, a Gq inhibitor, in CHO-LOX-1-AT1 cells.

j Percentage changes from baseline in the ratio of emission signals (F340/F380) measured by Fura 2-AM were quantified following treatment with oxLDL (5 μg/mL) and Ang II at specified concentrations in CHO-AT1 cells, as detailed in the figure.

Data are represented as mean ± SEM. Differences were determined using one-way ANOVA, followed by Tukey’s multiple comparison test for (a-f) and (h-j).

Co-treatment of oxLDL with Ang II induces conformational change of AT1 different from each treatment alone

a Change in green puncta (AT1-eGFP) and red puncta (LOX-1-mScarlet) by the treatment with vehicle, oxLDL (10μg/mL), Ang II (10−5M), and the combination of oxLDL and Ang II in CHO cell overexpressing these fluorescent protein-conjugated receptors (n=10-11 for each group). The puncta were manually counted by a blinded observer and the number of puncta at 0 and 3 min was determined (N0 and N3, respectively). The change in puncta was calculated as (N0-N3)/N0.

b-d Changes in BRET signals were monitored in CHO-LOX-1 cells expressing the following conformational biosensors: AT1-C-tailP1 (b) and AT1-ICL3P3 (c, d) bearing FlAsH insertion at the cytoplasmic-terminal tail (C-tailP1) and the third intracellular loop (ICL3P3) of AT1, respectively, that interact with Renilla luciferase at the end of the cytoplasmic tail. Cells were subjected to treatments with vehicle, oxLDL (10 μg/mL), Ang II (10−5M), the combination of Ang II and oxLDL, and the combination of AngII, oxLDL and LOX-1 antibody. The BRET ratios were calculated every 16 seconds for a total of 320 seconds and the relative change in intramolecular BRET ratio (ΔBRET) was calculated by subtracting the average BRET ratio measured for cells stimulated with vehicle at each time point. Lower panels indicate average ΔBRET of all the time points during measurement.

Data are represented as mean ± SEM. Differences were determined by one-way ANOVA, followed by Tukey’s multiple comparison test (a-d).

Oxidized LDL potentiates Ang II-induced Gq-calcium signaling in renal cells

a, b IP1 concentration in response to Ang II (10−7 M), oxLDL (10 μg/mL), and the combination of both with or without YM-254890, Gq inhibitor, in NRK52E (a) and NRK49F cells (b) (n=5 for each group).

c, d IP1 concentration in response to Ang II (10−7 M) and oxLDL (10 μg/mL) in the combination of Ang II (10−7 M) and the additional effect of siRNA-mediated knockdown of AT1a or LOX-1 in NRK52E (c) and NRK49F cells (d) (n=5 for each group).

e Intracellular calcium concentration in NRK49F cells using Fura 2-AM and dual-excitation microfluorometry. Changes in the fluorescence intensity ratio (F340/F380) served as an index of the calcium dynamics. Cells were exposed to Ang II (10−7 M), oxLDL (2 μg/mL), and a combination of both agents. Addition of these agonists is marked with arrows on the timeline of the assay. Data acquisition and analysis were performed using a digital image analyzer to monitor real-time calcium flux (n=4-9).

f Percentage changes from baseline in the ratio of emission signals (F340/F380) measured by Fura 2-AM were quantified following treatment with Ang II (10−7 M) and oxLDL at the concentrations detailed in the figure in NRK49F cells (n=5 for each group).

g Impact of siRNA-mediated knockdown AT1 or LOX-1 on the percentage changes from baseline in the ratio of emission signals (F340/F380) measured by Fura 2-AM were quantified following treatment with Ang II (10−7 M) and oxLDL (2 μg/mL) in NRK49F cells (n=3-7).

g Impact of co-treatment of YM-254890, Gq inhibitor, or Irbesartan, an angiotensin receptor blocker (ARB), on the the percentage changes from baseline in the ratio of emission signals (F340/F380) measured by Fura 2-AM were quantified following treatment with Ang II (10−7 M) and oxLDL (2 μg/mL) in NRK49F cells (n=4 for each group).

Data are represented as mean ± SEM. Differences were determined using one-way ANOVA, followed by Tukey’s multiple comparison test for (a-d) and (f-h).

Co-treatment of oxLDL and AII enhanced cellular response upon Gq activation in renal cells

a,b Quantitative real-time PCR analysis was performed to measure the gene expression of NADPH oxidase subunits (p67phox and p91phox), fibrosis markers (fibronectin, collagen-1, collagen-4, and TGFβ), and inflammatory cytokines (TNFα, IL1β, IL-6, and MCP-1) in NRK49F cells (a) and NRK 52E cells (b). Gene expression levels were normalized to those of GAPDH. Cells were stimulated by oxLDL (5 μg/mL), Ang II (10−7 M) or their combination (n=4 for each group).

c, d Cells were pre-treated with vehicle or Gq inhibitor (YM-254890, Gqi), followed by treatment with vehicle or the combination of oxLDL (5 μg/mL) and Ang II (10−7 M) in NRK49F cells (c) and NRK 52E cells (d) (n=4 for each group).

e, f Cells were pre-treated with vehicle or ARB (Irbesartan, Arb), followed by treatment with vehicle or the combination of oxLDL (5 μg/mL) and Ang II (10−7 M) in NRK49F cells (e) and NRK 52E cells (f) (n=4 for each group).

Data are represented as mean ± SEM. Differences were determined by one-way ANOVA, followed by Tukey’s multiple comparison test (a-f).

Oxidized LDL enhanced Ang II-induced epithelial mesenchymal transition in NRK52E and NRK49F cells

a, b Left: Western blot analysis of α-smooth muscle actin (α-SMA), a marker of epithelial–mesenchymal transition (EMT), in NRK49F (a) and NRK52E (b) cells. Cells were stimulated with oxLDL (5 μg/mL), Ang II (10−7 M), and TGF-β (10 ng/mL), with TGF-β serving as a well-known EMT inducer. Right: Densitometric analysis of α-SMA protein expression normalized to α-Tubulin (n=3 for each group).

c, d Left: Western blot analysis of α-SMA in NRK49F (c) or NRK52E (d) after stimulation with oxLDL (5 μg/mL), Ang II (10−7 M), and their combination. Right: Densitometric analysis of α-SMA protein expression normalized to α-tubulin (n=3 for each group).

e, f Left: Western blot analysis of α-SMA in NRK49F(e) or NRK52E (f) after treatment with a combination of oxLDL (5 μg/mL) and Ang II (10−7 M). Prior to this treatment, the cells were pre-treated with either a vehicle or a Gq inhibitor (YM-254890, Gqi). Right: Densitometric analysis of α-SMA protein expression normalized to α-Tubulin (n=3 for each group).

g, h Left: Western blot analysis of α-SMA in NRK49F (e) or NRK52E (f) after treatment with a combination of oxLDL (5 μg/mL) and Ang II (10−7 M). Prior to treatment, cells were pre-treated with either vehicle or ARB (Irbesartan, Arb). Right: Densitometric analysis of α-SMA protein expression normalized to α-tubulin (n=3 for each group).

Data are represented as mean ± SEM. Differences were determined by one-way ANOVA, followed by Tukey’s multiple comparison test (a-f).

Oxidized LDL enhanced Ang II-induced renal fibroblast proliferation via AT1-Gq signaling and LOX-1-dependent manner

a Proliferative activity assessed by BrdU incorporation into NRK49F cells. Cells were pretreated with vehicle, YM-254890, or ARB, Irbesartan, followed by the treatment with oxLDL (5 μg/mL), Ang II (10−7 M), or their combination. (n=5 for each group).

b NRK49F cells were subjected to siRNA-mediated knockdown using specific siRNAs for AT1a (siAT1) or LOX-1 (siLOX-1). Following knockdown, cells were treated with either vehicle, oxLDL (5 μg/mL), Ang II (10−7 M), or their combination. Proliferative activity was assessed by measuring the BrdU levels (n=5 for each group).

Data are represented as mean ± SEM. Differences were determined using one-way ANOVA, followed by Tukey’s multiple comparison test for (a) and (b).

Oxidized LDL-inducible diet exacerbates Ang II-induced renal dysfuntion in wildtype mice, but not in LOX-1 knockout mice

a A Schematic protocol for the animal experiments. Eight-week-old male WT mice and male LOX-1 KO mice were fed either an ND or an HFD for 6 weeks. After 10 weeks of age, the mice were treated over a 4-week period with infusions of either saline or Ang II. Ang II was administered at two dosage levels: a subpressor dose of 0.1 γ and a pressor dose of 0.7 γ, delivered via subcutaneously implanted osmotic pumps. At the end of the infusion period, urine was collected, the animals were sacrificed, and comprehensive tissue analysis was conducted to evaluate the renal effects of the treatments.

b Average systolic blood pressure (SBP) measured at half-week intervals in WT and LOX-1 KO mice during the 4-week infusion period.

c, d Urine 8-OHDG concentrations (mg/g creatinine [Cr]) (c) and urine albumin concentrations (mg/g creatinine [Cr]) (d) in WT and LOX-1 KO mice at the conclusion of the 4-week infusion period.

Data are represented as mean ± SEM. Differences were determined by one-way ANOVA, followed by Tukey’s multiple comparison test (a-d).

A high-fat diet enhanced Ang II-induced renal injury-related gene expression in the kidney in a LOX-1-dependent manner.

Quantitative real-time PCR analysis for gene expression of NADPH components (p67phox and p91phox), inflammatory cytokines (IL-6, TNFα, IL1β, and MCP-1), and fibrosis markers (TGFβ, fibronectin, collagen-1a, and collagen-4a) in the kidney harvested from WT and LOX-1 KO mice.

The experimental procedures, including the dietary regimen and Ang II administration, are detailed in Fig. 7a.

Data are represented as mean ± SEM. Differences were determined using one-way ANOVA, followed by Tukey’s multiple comparison test for (a) and (b).

LOX-1 and AT1a were predominantly co-localized in renal tubules

a, b Representative images depicting staining for LOX-1 (a) and AT1 (b) in the renal cortex tissues from wildtype mice (WT) and LOX-1 knockout mice (LOX-1 KO). Nuclei are stained blue with DAPI. Green and red signals indicates AT1, while the red signal indicates LOX-1. Overlay images demonstrate the merged visualization of AT1 or LOX-1 with DAPI, highlighting the predominant colocalization of LOX-1 and AT1 in renal tubules as opposed to the glomerulus.

Schematic overview of the AT1 and LOX-1 Interaction dynamics in renal cells

This schematic summary illustrates the predicted structure-activation relationship of the AT1 receptor within the LOX-1-AT1 complex in renal component cells. This highlights how the simultaneous binding of Ang II to AT1 and oxLDL to LOX-1 induces conformational changes in AT1. These changes were more pronounced than those triggered by the individual ligands. Such structural alterations have been proposed to amplify Gq signaling pathway activation, subsequently leading to renal damage.

Live-imaging analysis of membrane LOX-1 and AT1 in response to the co-treatment of oxLDL with AngII.

Real-time membrane imaging of CHO cells co-transfected with LOX-1-mScarlet and AT1-eGFP in response to oxLDL (10μg/ml) in the combination of Ang II (10−7M) (Supplemental Video). A count of puncta was performed using separate images visualizing LOX-1-mScarlet (red puncta) and AT1-eGFP (green puncta) immediately before and 3 min after ligand application.

Oxidized LDL in combination with Ang II do not increase cellular IP1 content in human umbilical vein endothelial cells and bovine vascular endothelial cells, human aortic vascular smooth muscle cells, and rat macrophages

IP1 concentration in response to oxLDL (10μg/ml) in the combination of Ang II (10−7M) in HUVECs (human umbilical vein endothelial cell), BAECs (bovine aortic endothelial cell), HAVSMCs (human aortic vascular smooth muscle cell), and A10 cells (rat macrophages).

Data are represented as mean ± SEM. Differences were determined using one-way ANOVA, followed by Tukey’s multiple comparison test (n=5 for each group).

Efficiency of siRNA-mediated knockdown for AT1a and LOX-1 in NRK52E and NRK49F cells

NRK52E and NRK49F cells were transfected with siRNAs against scrambled siRNA, AT1a or LOX-1. The efficiency of siRNA-mediated gene silencing was quantified by assessing AT1a and LOX-1 expression levels using quantitative real-time PCR.

Data are represented as mean ± SEM. Differences were determined using one-way ANOVA, followed by Tukey’s multiple comparison test.

Calcium influx was not induced by either the combination treatment of Ang II or oxLDL or each treatment alone in NRK52E cells

Percentage changes from baseline in the ratio of emission signals (F340/F380) measured by Fura 2-AM were quantified following treatment with Ang II (10−7M) and oxLDL at the concentrations detailed in the figure for NRK49E cells (n=5-7 for each group).

Data are represented as mean ± SEM. Differences were determined using one-way ANOVA, followed by Tukey’s multiple comparison test.

High Fat Diet used in the study prominently increased plasma LOX-1 ligand concentration

Plasma LOX-1 ligand concentration of 14-week-old wildtype mice upon ND and HFD for 6 weeks. From 10 weeks of age, these mice also received concurrent 4-week infusions of Ang II at a pressor dose of 0.7 γ, administered through subcutaneously implanted osmotic pumps.

Data are represented as mean ± SEM. Differences were determined using Student’s t-test (n=7 for each group).

Impact of Diet and Ang II Infusion on Body Weight and Systolic Blood Pressure in Mice

a Final Body weight: This figure shows the body weights of WT and LOX-1 KO mice at the end of the 4-week infusion period, highlighting the effects of diet and pharmacological treatment.

b, c Serial Body Weight Changes: These graphs depict the progression of body weight over time in WT and LOX-1 KO mice, illustrating the impact of the dietary regimen and Ang II infusion on weight dynamics.

d, e Systolic Blood Pressure Trajectory: Serial measurements of systolic blood pressure (SBP) obtained using the tail-cuff method are presented for WT and LOX-1 KO mice.

The figures show the changes in SBP over the course of the study, corresponding to the administration of a pressor dose of 0.7 γ (d) and a subpressor dose of 0.1 γ (e) of Ang II, respectively.

f Final SBP: This figure shows SBP of WT and LOX-1 KO mice at the end of the 4-week infusion period, highlighting the effects of diet and pharmacological treatments.

Beginning at 8 weeks of age, mice were fed either an ND or an HFD for 6 weeks. From 10 weeks of age, coinciding with the 2-week time point in the figure, the rats underwent a 4-week period of infusion with either vehicle or Ang II. The infusion was delivered at specific dosage levels through subcutaneously implanted osmotic pumps.

Data are represented as mean ± SEM. Differences were determined using one-way ANOVA, followed by Tukey’s multiple comparison test for (a) and (b).

No significant difference was found in plasma aldosterone concentration between a normal diet and a high fat diet-fed wildtype mice with a pressor dose of Ang II

Plasma aldosterone concentration in 14-week-old wild-type mice fed an ND or an HFD for 6 weeks. From 10 weeks of age, these mice also received concurrent 4-week infusions of Ang II at a pressor dose of 0.7 γ, administered through subcutaneously implanted osmotic pumps.

Data are represented as mean ± SEM. Differences were determined using Student’s t-test (n=7 for each group).

A high-fat diet enhanced Ang II-induced renal injury-related gene expression in the kidney in a LOX-1-dependent manner.

Quantitative real-time PCR analysis for gene expression of NADPH components (p40phox and p47phox), inflammatory gene (COX-2), fibrosis markers (αSMA and vimentin), epithelial markers (E-cadherin and cadherin-16), tubular marker (NAGL), AT1a, AT1b, and LOX-1 in the kidney harvested from WT and LOX-1 KO mice.

The experimental procedures, including the dietary regimen and Ang II administration, are detailed in Fig. 7a.

Data are represented as mean ± SEM. Differences were determined using one-way ANOVA, followed by Tukey’s multiple comparison test for (a) and (b).

The treatment with Ang II, a High Fat Diet, or their combination for 4 weeks did not induce any histological changes indicative of renal injury

a Left: Representative histological images of Masson-Trichrome staining used to detect fibrosis in renal tissues harvested from WT and LOX-1 KO mice. Right: Quantitative analysis by Masson-Trichrome staining.

Data are represented as mean ± SEM. Differences were determined using one-way ANOVA, followed by Tukey’s multiple comparison test.

b Representative histological images of renal tissues harvested from WT and LOX-1 KO mice stained for PAS to assess mesangial expansion and glomerular area, providing insight into the structural integrity of the glomeruli.

Eight-week-old mice were fed an ND or an HFD for 6 weeks. From 10 weeks of age, these mice were concurrently treated for 4 weeks with infusions of vehicle or Ang II (a subpressor dose of 0.1 γ or a pressor dose of 0.7 γ) through subcutaneously implanted osmotic pomps.