Oxidized low-density lipoprotein potentiates angiotensin II-induced Gq activation through the AT1-LOX1 receptor complex: Implications for renal dysfunction
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eLife assessment
This study provides useful in vitro evidence to support a mechanism whereby dyslipidemia could accelerate renal functional decline through the activation of the AT1R/LOX1 complex by oxLDL and AngII. As such, it improves the knowledge regarding the complex interplay between dyslipidemia and renal disease and provides a solid basis for the discovery of novel therapeutic strategies for patients with lipid disorders. The methods, data, and analyses support the presented findings, although the observed variability and need for further in vivo validation require additional research in this key area.
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Abstract
Chronic kidney disease (CKD) and atherosclerotic heart disease, frequently associated with dyslipidemia and hypertension, represent significant health concerns. We investigated the interplay among these conditions, focusing on the role of oxidized low-density lipoprotein (oxLDL) and angiotensin II (Ang II) in renal injury via G protein αq subunit (Gq) signaling. We hypothesized that oxLDL enhances Ang II-induced Gq signaling via the AT1 (Ang II type 1 receptor)-LOX1 (lectin-like oxLDL receptor) complex. Based on CHO and renal cell model experiments, oxLDL alone did not activate Gq signaling. However, when combined with Ang II, it significantly potentiated Gq-mediated inositol phosphate 1 production and calcium influx in cells expressing both LOX-1 and AT1 but not in AT1-expressing cells. This suggests a critical synergistic interaction between oxLDL and Ang II in the AT1-LOX1 complex. Conformational studies using AT1 biosensors have indicated a unique receptor conformational change due to the oxLDL-Ang II combination. In vivo, wild-type mice fed a high-fat diet with Ang II infusion presented exacerbated renal dysfunction, whereas LOX-1 knockout mice did not, underscoring the pathophysiological relevance of the AT1-LOX1 interaction in renal damage. These findings highlight a novel mechanism of renal dysfunction in CKD driven by dyslipidemia and hypertension and suggest the therapeutic potential of AT1-LOX1 receptor complex in patients with these comorbidities.
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eLife assessment
This study provides useful in vitro evidence to support a mechanism whereby dyslipidemia could accelerate renal functional decline through the activation of the AT1R/LOX1 complex by oxLDL and AngII. As such, it improves the knowledge regarding the complex interplay between dyslipidemia and renal disease and provides a solid basis for the discovery of novel therapeutic strategies for patients with lipid disorders. The methods, data, and analyses support the presented findings, although the observed variability and need for further in vivo validation require additional research in this key area.
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Reviewer #1 (Public Review):
Summary:
This study demonstrates a key role of oxLDL in enhancing Ang II-induced Gq signaling by promoting the AT1/LOX1 receptor complex formation. Importantly, Gq-mediated calcium influx was only observed in LOX1 and AT1 both expressing cells, and AT1-LOX1 interaction aggravated renal damage and dysfunction under the condition of a high-fat diet with Ang II infusion, so this study indicated a new therapeutic potential of AT1-LOX1 receptor complex in CKD patients with dyslipidemia and hypertension.
Strengths:
This study is very exciting and the work is also very detailed, especially regarding the mechanism of LOX1-AT1 receptor interaction and its impact on oxidative stress, fibrosis, and inflammation.
Weaknesses:
The direct evidence for the interaction between AT1 and LOX1 receptors in cell membrane …
Reviewer #1 (Public Review):
Summary:
This study demonstrates a key role of oxLDL in enhancing Ang II-induced Gq signaling by promoting the AT1/LOX1 receptor complex formation. Importantly, Gq-mediated calcium influx was only observed in LOX1 and AT1 both expressing cells, and AT1-LOX1 interaction aggravated renal damage and dysfunction under the condition of a high-fat diet with Ang II infusion, so this study indicated a new therapeutic potential of AT1-LOX1 receptor complex in CKD patients with dyslipidemia and hypertension.
Strengths:
This study is very exciting and the work is also very detailed, especially regarding the mechanism of LOX1-AT1 receptor interaction and its impact on oxidative stress, fibrosis, and inflammation.
Weaknesses:
The direct evidence for the interaction between AT1 and LOX1 receptors in cell membrane localization is relatively weak. Here I raise some questions that may further improve the study.
Major points:
(1) The authors hypothesized that in the interaction of AT1/LOX1 receptor complex in response to ox-LDL and AngII, there should be strong evidence of fluorescence detection of colocalization for these two membrane receptors, both in vivo and in vitro. Although the video evidence for AT1 internalization upon complex activation is shown in Figure S1, the more important evidence should be membrane interaction and enhanced signal of intracellular calcium influx.
(2) Co-IP experiment should be provided to prove the AT1/LOX1 receptor interaction in response to ox-LDL and AngII in AT1 and LOX1 both expressing cells but not in AT1 only expressing cells.
(3) The authors mentioned that the Gq signaling-mediated calcium influx may change gene expression and cellular characteristics, including EMT and cell proliferation. They also provided evidence that oxidative stress, fibrosis, and inflammation were all enhanced after activating both receptors and inhibiting Gq was effective in reversing these changes. However, single stimulation with ox-LDL or AngII also has strong effects on ROS production, inflammation, and cell EMT, which has been extensively proved by previous studies. So, how to distinguish the biased effect of LOX1 or AT1r alone or the enhanced effect of receptor conformational changes mediated by their receptor interaction? Is there any better evidence to elucidate this point?
(4) How does the interaction between AT1 and LOX1 affect the RAS system and blood pressure? What about the serum levels of rennin, angiotensin, and aldosterone in ND-fed or HFD-fed mice?
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Reviewer #2 (Public Review):
Individuals with chronic kidney disease often have dyslipidemia, with the latter both a risk factor for atherosclerotic heart disease and a contributor to progressive kidney disease. Prior studies suggest that oxidized LDL (oxLDL) may cause renal injury through the activation of the LOX1 receptor. The authors had previously reported that LOX1 and AT1 interact to form a complex at the cell surface. In this study, the authors hypothesize that oxLDL, in the setting of angiotensin II, is responsible for driving renal injury by inducing a more pronounced conformational change of the AT1 receptor which results in enhanced Gq signaling.
They go about testing the hypothesis in a set of three studies. In the first set, they engineered CHO cell lines to express AT1R alone, LOX1 in combination with AT1R, or LOX1 with …
Reviewer #2 (Public Review):
Individuals with chronic kidney disease often have dyslipidemia, with the latter both a risk factor for atherosclerotic heart disease and a contributor to progressive kidney disease. Prior studies suggest that oxidized LDL (oxLDL) may cause renal injury through the activation of the LOX1 receptor. The authors had previously reported that LOX1 and AT1 interact to form a complex at the cell surface. In this study, the authors hypothesize that oxLDL, in the setting of angiotensin II, is responsible for driving renal injury by inducing a more pronounced conformational change of the AT1 receptor which results in enhanced Gq signaling.
They go about testing the hypothesis in a set of three studies. In the first set, they engineered CHO cell lines to express AT1R alone, LOX1 in combination with AT1R, or LOX1 with an inactive form of AT1R and indirectly evaluated Gq activity using IP1 and calcium activity as read-outs. They assessed activity after treatment with AngII, oxLDL, or both in combination and found that treatment with both agents resulted in the greatest level of activity, which could be effectively blocked by a Gq inhibitor but not a Gi inhibitor nor a downstream Rho kinase inhibitor targeting G12/13 signaling. These results support their hypothesis, though variability in the level of activation was dramatically inconsistent from experiment to experiment, differing by as much as 20-fold. In contrast, within the experiment, differences between the AngII and AngII/oxLDL treatments, while nominally significant and consistent with their hypothesis, generally were only 10-20%. Another example of unexplained variability can be found in Figures 1g-1j. AngII, at a concentration of 10-12, has no effect on calcium flux in one set of studies (Figure 1g, h) yet has induced calcium activity to a level as great as AngII + oxLDL in another (Figure 1i). The inconsistency of results lessens confidence in the significance of these findings. In other studies with the LOX1-CHO line, they tested for conformational change by transducing AT1 biosensors previously shown to respond to AngII and found that one of them in fact showed enhanced BRET in the setting of oxLDL and AngII compared to AngII alone, which was blocked by an antibody to AT1R. The result is supportive of their conclusions. Limiting enthusiasm for these results is the fact that there isn't a good explanation as to why only 1 sensor showed a difference, and the study should have included a non-specific antibody to control for non-specific effects.
The authors then repeated similar studies using publicly available rat kidney epithelial and fibroblast cell lines that have an endogenous expression of AT1R and LOX1. In these studies, oxLDL in combination with AngiI also enhanced Gq signaling, while knocking down either AT1R or LOX1, and treatment with inhibitors of Gq and AT1R blocked the effects. Like the prior set of studies, however, the effects are very modest and there was significant inter-experimental variability, reducing confidence in the significance of the findings. The authors then tested for evidence that the enhanced Gq signaling could result in renal injury by comparing qPCR results for target genes. While the results show some changes, their significance is difficult to assess. A more global assessment of gene expression patterns would have been more appropriate. In parallel with the transcriptional studies, they tested for evidence of epithelial-mesenchymal transition (EMT) using a single protein marker (alpha-smooth muscle actin) and found that its expression increased significantly in cells treated with oxLDL and AngII, which was blocked by inhibition of Gq inhibition and AT1R. While the data are sound, their significance is also unclear since EMT is a highly controversial cell culture phenomenon. Compelling in vivo studies have shown that most if not all fibroblasts in the kidney are derived from interstitial cells and not a product of EMT. In the last set of studies using these cell lines, the authors examined the effects of AngII and oxLDL on cell proliferation as assayed using BrdU. These results are puzzling---while the two agents together enhanced proliferation which was effectively blocked by an inhibitor to either AT1R or Gq, silencing of LOX1 had no effect.
The final set of studies looked to test the hypothesis in mice by treating WT and Lox1-KO mice with different doses of AngII and either a normal or high-fat diet (to induce oxLDL formation). The authors found that the combination of high dose AngII and a high-fat diet (HFD) increased markers of renal injury (urinary 8-ohdg and urine albumin) in normal mice compared to mice treated with just AngII or HFD alone, which was blunted in Lox1-KO mice). These results are consistent with their hypothesis. However, there are other aspects of these studies that are either inconsistent or complicating factors that limit the strength of the conclusions. For example, Lox1- KO had no effect on renal injury marker expression in mice treated with low-dose AngII and HFD. It also should be noted that Lox1-KO mice had a lower BP response to AngII, which could have reduced renal injury independent of any effects mediated by the AT1R/LOX1 interaction. Another confounding factor was the significant effect the HFD diet had on body weight. While the groups did not differ based on AngII treatment status, the HFD consistently was associated with lower total body weight, which is unexplained. Next, the authors sought to find more direct evidence of renal injury using qPCR of candidate genes and renal histology. The transcriptional results are difficult to interpret; moreover, there were no significant histologic differences between groups. They conclude the study by showing the pattern of expression of LOX1 and AT1R in the kidney by immunofluorescence and conclude that the proteins overlap in renal tubules and are absent from the glomerulus. Unfortunately, they did not co-stain with any other markers to identify the specific cell types. However, these results are inconsistent with other studies that show AT1R is highly expressed in mesangial cells, renal interstitial cells, near the vascular pole, JG cells, and proximal tubules but generally absent from most other renal tubule segments.
In sum, this study tackles an important clinical issue and provides some in vitro evidence to support a mechanism whereby dyslipidemia could accelerate renal functional decline through activation of the AT1R/LOX1 complex by oxLDL and AngII.
However, a very high degree of variability in the results, modest within-experiment differences, some internal inconsistencies that aren't explained, and the lack of compelling and strongly supportive in vivo results suggest this is still more a hypothesis than an established likely mechanism.
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